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Full text of "Report of program activities : National Institute of Dental Research"

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us NATIONAL INSTITUTE OF DENTAL RESEARCH 
ANNUAL REPORT 
Jiily 1, 1967 - June 30, 1968 . . 

CONTENTS 

PAGE 
Office of the Director 

Report of the Director 1 

Dental Research Institutes 2 

Program Planning Office 3 

Information Office. h 

Collahorative Research Office 8 

Research Contracts 10 

Intramural Research 

Report of the Director of Intramural Research 21 

Physiology Section 

Simimary Statement ■• , 27 

Project Reports: ■' 

No. Investigator Title 

1. R. Dubner Functional Organization of the Trigeminal 

Brainstem Nuclei in the Cat 30 

2. R. Duhner Interaction of Sensory Stimuli in 

Association Areas of Cerebral Cortex in 

the Cat 33 

3. M. I. Krichevsky . The Biochemistry of the Differentiating 

Cellular Slime Mold, Dictyostelium 

discoideum 36 

k. J. M. Tanzer Energy Dependent Phosphate Accumulation 

by Streptococci Implicated in Smooth 
Surface Caries ■ 39 



ft 



CONTENTS 

PAGE 
Physiology Section (Continued) 

Project Reports: 

No. Investigator TITLE 

5. J. M. Tanzer The Metabolic Fate of Glucose Catabolized 

by Stationary Phase Streptococci Implicated 

in Smooth Surface Caries ^3 

6. B. M. Chassy Growth and Metabolism of Cariogenic 

Streptococci ^6 

7. J. M. Tanzer Selection of Plaque -Forming Mutants 

of Streptococci ^8 

8. J. J. Wilson Laboratory Information System 50 

Laboratory of Microbiology 

R iiTTim ary Statement 53 

Project Reports: 

No. Investigator TITLE 

9. R. J. Fitzgerald Gnotobiotic Studies of Problems Relating 

to Oral Disease 6I 

10. E. G. Hampp Studies of Culture Media for the Mass 

Cultivation of Representatives of the 
Genera Treponema and Borrelia . The Use of 
Bovine Serum Fraction for Growth Initiation 
of Spirochetes 65 

11. H. V. Jordan Relationship of Specific Oral Bacteria to 

Dental Caries and Periodontal Disease 69 

12. R. H. Larson Role of Genetic and Environmental Factors 

in Experimental Dental Caries T^ 

13. J. P. London Physiology and Regulation of Metabolic 

Processes in Lactic Acid Bacteria T8 

±h. S. E. Mergenhagen Immiinological Mechanisms in Oral and 

Systemic Disease 82 

15. A. L. Notkins Recurrent and Persistent Viral Infections.. 88 



II 



CONTENTS 

PAGE 
Laboratory of Microbiology (Continued) 

Project Reports: 

No. Investigator Title 

16. A. A. Rizzo The Mechanisms by Which Bacterial Products 

may Cause Destruction in Human Periodontal 
Disease 93 

17. M. Rogosa Systematic Microbiological Taxonomic 

Studies 97 

18. C.L. Wittenberger Studies on the Regulation of Lactic Acid 

Production by Microorganisms 101 

Laboratory of Biochemistry 

S\immary Statement IO5 

Project Reports: ■ . 

Ho. Investigator Title 

19. K. A. Piez Aiialytical and Structural Studies on 

Collagen 110 

20. K. A. Piez Factors Influencing Resorption and Cell 

Growth in Collagen Implants Formed by 
■ Thermal Gelatin in vivo 113 

21. E. J. Miller The Chemistry of Bone and Cartilage 

Collagens II6 

22. G. R. Martin The Chemistry and Biosynthesis of Elastin... 120 
S. R. Pinnell 

23. E. Schiffmann Calcification of Organic Matrices 123 

D. R. Lavender 

2i|. E. Schiffmann . Long-Term Effects of Water Fluoridation in 

Grand Rapids , Michigan 128 

25. J. E. Folk Chemistry and Mechanism of Action of 

(1) Chymotrypsin C and (2) Transglutaminase 130 

26. A. J. Steffek Prenatal Developmental Factors Influencing 

Oral Disease 13^ 

27. A. J. Steffek Study of Teratogenesis and Organogenesis in 

the Non-Human Primate (Contract J+3-66-U57, 
Hazelton Laboratories, Falls Church, Va.)... 1^5 



III 



CONTENTS 

PAGE 

Laboratory of Biochemistry (Continued) 

Project Reports: . 

No. Investigator Title 

28. H. L. Cooper Chromosome and Cell Growth Studies in 

Normal and Abnormal Subjects IU9 

Human Genetics Branch 

Summary Statement 1 57 

Project Reports: 

No. Investigator Title 

29. K. S. Brown Discrimination and Segregation Analysis of 

Hereditary Deafness in the Students of the 
Clarke School for the Deaf 16O 

30. K. S. Brown Environment and Genetic Factors in Taste 

and Smell Abilities l6k 

31. D. W. Runck Genetic Studies of Oral Diseases, Anomalies 

J . D . Niswander and Development • , 167 

32. P. L. Workman Theoretical and Applied Analyses in Human 

Populations with Particular Emphasis on the 
Study of Genetic Variation 1T2 

33. J. W. Graef Studies on Site of Action of Phytohemag- 

glutinin on Circulating Human Lymphocytes... 175 

3^. K. S. Brown Developmental Processes in Genetically 

Controlled Traits I77 

35- D. R. Bergsma A Study of the Relationships Between Genetic 

Factors , Exposure to Vitamin D iri_ utero , and 
Buphthalmos in the Rabbit I80 

36 . R . . Wolf Saliva Study ' ' I83 

37- J. D. Niswander Genetic Studies of Oral Clefts and Other 186 

Major Congenital Malformations 

Laboratory of Histology and Pathology 

Summary Statement ]_q3 



IV 



CONTENTS 



PAGE 



Laboratory of Histology and Pathology (Continued) 



Project Reports: 

No. Investigator 

38. M. U. Nylen. 

39. M. U. Nylen 

UO . J . Waerhaug 

kl. P. D. Frazier 

it2. S. Gobel" 

i+3. H. A. Bladen, Jr. 

kk. G. J. Hageage 

U5. R. C. Thompson 

k6. R. C. Thompson 

1+7. H. M. Fiollmer 

48. W. A. Gibson 



k9. J. F. Go 



ggms 



50. G. S. Lazarus 



51. E. D. Eanes 



Title 

Experimentally Induced Enamel Defects 199 

Collaborative Projects and Training 

Activities 202 

Studies on Bone Resorption and Collagen 
Degradation Due to Pressure 206 

X-Ray Diffraction Studies on the Effect of 

Fluoride on Bones, Teeth and Synthetic 

Compounds 209 

Fine Structural Studies of the Main Sensory 
Nucleus of the Trigeminal Nerve 212 

Bacterial , Viral and Macromolecular 
Structure-Function Relationships 215 

The Relationship Between Function and 

Structure in Microorganisms 219 

Electron Microscopic Radioautography : 

Response Characteristics of Nuclear Emulsion 

to High Energy Beta Particle Irradiation.... 223 

Uranium Tracer System Employing Nuclear 

Fission Reaction 226 

Studies of Hioman Collagenase 229 

Histochemical and Chemical Studies of 

Connective Tissues 233 

Histochemical and Chemical Studies of 

Connective Tissues and Teeth. . 237 

Collagenase Activity of Human Normal and 
Diseased Tissues 2^+0 

X-Ray Diffraction Studies on Fibrous 

Proteins ' 2^13 



V 



CONTENTS 
LaTjoratory of Histology and Pathology (Continued) 
Project Reports: 



PAGE 



No. 



52. 



Investigator 
B. 0. Fowler 



53. 



5^. 



P. H. Keyes 



J . Waerhaug 



Title 

Infrared Absorption Spectrophotometric and 
X-Ray Diffraction Studies of the Inorganic 
Portion of Teeth and Bones and Related 
Synthetic Compounds 2il6 

Odontopathic Bacterial Plaques: Etiologic 
Factors, Pathological Sequelae, Therapeutic 
Measures 251 

Fate of Microorganisms Inserted into 
Healthy Gingival Pockets 



Biometry and Field Investigations Branch 
Summary Statement 



255 
259 



Project Reports: 
No. Investigator 
J. P. Carlos 



55. 
56. 

57. 
58. 

59. 
60. 
61. 



H. R. Englander 



H . R . Englander 



H. R. Englander 



H . R . Englander 



N. W. Littleton 



L. F. Mills 



Title 

Studies on the Design and Analysis of Dental 
Clinical Trials 265 

Clinical Anti-Caries Effect of Repeated 
Topical Sodium Fluoride Applications by 
Mouthpieces 267 

Anti-Caries Effect of Repeated Topical 
Fluoride Applications in a Fluoridated 
Community 270 

Anti-Caries Effect of Repeated Topical 

Fluoride Treatments on the Deciduous 

Dentition 273 

Experimental Dental Caries in the Syrian 
Hamster 276 

Studies of Oral Health in Persons Nourished 

by Stomach Tube 279 

Production of "Elfin" Facies and Abnormal 
Dentition by Vitamin D2 during Pregnancy: 
Relationship to the Supravalvular Aortic 
Stenosis Syndrome 283 



VI 



CONTENTS 

PAGE 
Biometry and Field Investigations Branch (Continued) 

Project Reports: 

No. Investigator Title 

62. L. F. Mills Epidemiological Studies of Malocclusion 285 

63. L. F. Mills Serial Extraction Study on Preadolescent 

Children Having Crowded Class I Occlusion... 286 

6k. L. F. Mills Clinical Trial of a Dentifrice Containing 

Phosphate and Fluoride 290 

Report of the Clinical Director 292 

Oral Medicine and Surgery Branch 

Summary Statement 295 

Project Reports: 

No. Investigator Title 

65. H. 0. Archard Histopathology of Oral Mucous Memhrane 305 

66. H. 0. Archard Clinical and Morphologic Studies of the 

Human Dentition in Metabolic Diseases, 

Either Acquired or Inherited 308 

67. P. N. Baer Studies on the Etiology and Treatment of 

Periodontal Diseases in Children and 

Adolescents 311 

68. P. N. Baer Studies on Experimental Calculus Formation.. 313 

69. W. A. Bell The Effects of Blastomycosis on Oral 

Tissue 316 

TO. W. A. Bell The Staining Morphology of Various Dental 

Materials in Tissue 318' 

71. A. F. Binderman A Measure of the Effectiveness of Root 

Amputation as a Means of Slowing Pocket 
Progression . 320 

72. A. F. Binderman Studies in the Healing of Alveolar Bone 

in Dogs 322 

73- N. A. Cummings Study of Behcet's Syndrome 325 

VII 



Oral Medicine and Surgery 



Project Reports: 

No. Investigator 

Tk. N. A. Cummings 

75. E. J. Driscoll 

76. T. C. Francis 

77. G. E. Garrington 

78. G. E. Garrington 
79- G. E. Garrington 

80. S. A. Geis 

81. E. A. Graykowski 



CONTENTS 
Branch (Continued) 



PAGE 



82. 


J. 


E. Hamner, III 


83. 


J. 


E. Hamner, III 


81+. 


J. 


E. Hamner, III 


85. 


s. 


Kakehashi 


86. 


s. 


Kakehashi 




N. 


W. Littleton 


87. 


P. 


M. Lightbody 



89. 



P. M. Lightbody 



P. M. Lightbody 



Title 

Mechanisms of Cryoprecipitation in 

Cryoglobulins 328 

General Anesthesia on Ambulatory Dental 

Patients 33I 

Immunologic , Biochemical and Micro- 
biological Studies in Recurrent Aphthous 
Stomatitis 335 

Osteosarcoma and Chondrosarcoma of the 

Jaws 338 

Leprosy Involving the Dental Pulp 3I+1 

Dilantin Gingival Hyperplasia 3l;3 

Microbiological Phase of Rampant Caries 

Study 3I+6 

Immunological Studies in Recurrent Aphthous 
Stomatitis 350 

Betel Quid Carcinogenesis 353 

Benign Fibro-Osseous Lesions of the Maxilla 

and Mandible 3^5 

Submucous Fibrosis 350 

Dental Pulp and Periodontal Studies in 

Germfree and Conventional Laboratory Rats... 351 

A Long-Term Study of Periodontal Disease in 

a Stable , Adult , Male Population 363 

Post-Surgical Tissue Healing. 355 

Sectional Roentgenographic Study of the 
Temporomandibular Joint Following Bilateral 
Osteotomy of the Ramus of the Mandible...... 367 

Evaluation of Premedication in Conjunction 
with Local Anesthesia in Oral Surgical 
Procedures 3^0 

VIII 



CONTENTS 
Oral Medicine and Surgery Branch (Continued) 



PAGE 



r-roject Reports: 

No. Investigator 

90. T. Lundy 

91. T. Lundy 

92. H. R. Stanley 

93. H. R. Stanley 
9h. R. M. Stephan 



95- J. F. Bosma 



96. J. F. Bo 



sma 



97. J. F. Bosma 



98. R. D. Christensen 



99- R. D. Christensen 



100. R. L. Christiansen 



101. R. L. Christiansen 



102. P. J. Coccaro 



103. P. J. Coccaro 



Title 

Effects of Oral Fluids on the Dental Pulp.... 371 

Histopathology of the Periodontal Ligament 

and Alveolar Bone Following Endontic 

Treatment 37I; 

Histopathology of the Human Dental Pulp 377 

Autogenous Replantation of Hiiman Teeth 382 

Studies on the Etiology and Control of 
Rampant Dental Caries : Clinical and 
Experimental Animal Studies on the 
Differentiation of Cariogenic and Non- 
Cariogenic Foods 385 

Studies of Oral and Pharyngeal Form and 
Function in Infants 389 

Studies of Development of the Head Skeleton 

of the Rat .39I 

Studies of Sensory and Motor Functions in 
Subjects Impaired "by Malformations of 
Neurological Disease 393 

Serial Extraction Study on Preadolescent 
Children Having Crowded Class I Occlusion. ... 39Y 

Comparative Skeletal and Soft Tissue 
Cephalometric Analysis of Acromegalic and 
Normal Human Adults I+OO 

Study of Taste Thresholds, Tastebud 
Distribution, and Associated Dentofacial Form 1| 02 

Study of Oral Area Motor Mechanisms by Use of 
Pressure Transducers UoU 

Restitution of Mandibiilar Form After Condylar 
Injury U07 

Clinical and Roentgenographic Analysis of 
Orthodontics (on a Continuing Basis) in Cleft 
Palate Habilitation l^io 

IX 



DENTAL 

PAGE 
Oral Medicine and Surgery Branch (Continued) 

Project Reports: 

No. Investigator Title 

10^+. L. A. Krames Prenatal Development of the Larynx; Human 

and Comparative Investigation lH3 

Dental Services Branch 

Summary Statement Il25 

Project Reports: 

No. Investigator Title 

105. H. Swerdlow Reaction of the Human Dental Pulp to Cavity 

Preparations and Filling Materials 1i3_q 

106. B. Goldman Palatal Vault Measurements in Patients with 

Congenital Heart Disease }l2l+ 



X 



REPORT OF THE DIRECTOR 
THE NATIONAL INSTITUTE OF DENTAL RESEARCH 
July 1, 1967 - June 30, 1968 
by 
Seymour J. Kreshover 

The dental research institutes program, for which grant support was first 
provided in FY I96T, has maintained steady progress. It has stimulated 
planning and development of broad, interdisciplinary, university-based 
centers of excellence in the sciences related to oral health. In the process, 
these institutes have attracted to the dental environment and to the solution 
of oral health problems the knowledge and skills of investigators in disciplines 
not heretofore intimately involved in dental research. Dental research 
institutes are in varying stages of development at the following universities : 
Alabama, Michigan, North Carolina, Pennsylvania, and Washington. Strong 
interest in establishing such interdisciplinary centers has been evidenced by 
four other universities or university consortia. 

The report of the National Advisory Commission on Health Manpower included a 
number of observations and recommendations on dental education and research. 
In order to obtain a more adequate background on, and a better understanding 
of, the report, top Institute staff met with the two prominent dental educator- 
spokesmen who served as members of the Commission's Education and Supply Panel. 
The ensuing discussion focussed on the role of research and research training 
in dental education and developed possible approaches whereby NIDR might take 
a leadership role. It is anticipated that the reorganization of health 
activities within DHEW when fully implemented will permit NIDR to effectively 
provide leadership in this and other areas of dental education. 

While the National Institute of Dental Research has had a long history of 
collaboration with the National Bureau of Standards, it has become increasingly 
evident that refinements in this interrelationship were mutually desirable. 
Accordingly, several meetings were held by top NIDR and NBS staff to explore 
possible new collaborative efforts and to define the role of each of the two 
participating institutions. It is expected that these meetings will result 
in a new agreement giving special emphasis to the research efforts at NBS 
supported by NIDR. 

The organizational structure of the Institute continued to develop and evolve 
in a more meaningful fashion and to more adequately give visibility to its 
broad-ranging efforts and activities. Accordingly, the equivalent of branch 
status was authorized for the four extramural categorical program areas; i.e.. 
Dental Caries and Hard Tissues Program, Periodontal Diseases and Soft Tissues 
Program, Oral-facial Growth and Development Program, and Materials Science and 
Special Clinical Studies Program. In order to emphasize the significance 
of applied studies, a new Biometry and Field Investigations Branch, reporting 



directly to the Office of the Director and consisting of three sections 
(Biometry, Epidemiology, and Clinical Trials), was established. New sections 
in the intramural research area were set up as follows : a Virology Section in 
the Laboratory of Microbiology, and a Population Genetics Section and a 
Developmental Genetics Section in the Human Genetics Branch. The Institute 
became the first of the NIH complex to have its own personnel office, as a 
result of an experimental decentralization of the central NIH personnel 
management branch . 

In cooperation with the NIH Management Policy Branch, an intensive study was 
made of work-flow, records, and other administrative procedures in the 
extramural programs area. This has resulted in a number of changes leading 
to greater efficiency and effectiveness. 

The Institute and the dental scientific community were saddened by the death, 
in December, I967, of Dr. Francis A. Arnold, Jr., former NIDR Director and a 
pioneer in fluoridation research on the prevention of dental caries. Institute 
personnel changes included the retirement of Dr. F. Earle Lyman from the 
position of Associate Director for Special Programs, the designation of 
Dr. Clair L. Gardner as the new Associate Director for Special Programs, and 
the transfer of Dr. Aaron Ganz from the Office of the Director, NIH, to serve 
as the NIDR Chief of the Office of Program Planning and Evaluation. 

Dental Research Institutes 

Designed to accelerate the development of scientific knowledge as an essential 
base for the advancement of dental research, dental education, and dental 
care, the program to establish dental research institutes in university 
settings is unique in drawing on total institutional resources. Its immediate 
aims are to facilitate the cooperation of biological, physical, and social 
scientists in the study of oral health problems of common interest and to have 
them interact with the educational programs of the parent universities by also 
f-unctioning as members of academic departments. Additionally, the institutes 
will align themselves wherever practicable with other existing regional 
resources, including educational and research institutions, hospitals, computer 
facilities, Medlars regional centers, and primate centers. 

Implemented one year ago, institutional grants have now been awarded to five 
universities: (l) the University of Alabama, (2) the University of Michigan, 
(3) the University of North Carolina, {k) the University of Pennsylvania, and 
(5) the University of Washington. Several other individual universities and 
groups of universities are currently developing proposals for the establishment 
of dental research institutes. 

Since the Dental Research Institutes program represents, in the national 
interest , a unique concept of grant support involving rather large expenditures 
of public funds, specific guidelines for continuing evaluation by the granting 
agency and recipient institutions have been tailored to its specific require- 
ments. This assessment is directed at assuring the orderly development of 
broadly based complexes bringing together a new mix of scientific and 
professional personnel with firm adherence to high standards of scientific merit 
and fiscal accountability. 



Although sufficient time has not yet elapsed to expect substantive progress in 
meeting objectives, there have been indications that the participating dental 
schools are already benefiting from the rich cross-fertilization made possible 
by this new program. 

Program Planning Office 

This report encompasses the activities of the Program Planning Office, 
including those of the Reports and Analysis Section, during fiscal year I968. 

Analytical staff support was provided on a continuing basis to the Director, 
the Associate Director for Extramural Programs, the Associate Director for 
Special Programs, and the four extramural categorical program chiefs. In 
addition, a number of special projects were undertaken, as follows: 

(1) An estimate of dental research manpower requirements for 1975 was 
prepared, based on the anticipated manpower needs of the following six 
categories of institutions that conduct dental research: dental schools, 
dental research institutes, the Federal establishment, other non-profit dental 
institutions, other non-profit, non-dental institutions, and private industry. 

(2) A detailed analysis of the FY I96T grants and activities of the 
Materials Science and Special Clinical Studies Program was prepared for 
presentation by the Area Chief to the Program Planning Committee and to the 
National Advisory Dental Research Council. 

(3) A comprehensive analysis of how the ^9 operating U.S. dental schools 
have utilized their General Research Support funds during the period I962 
through 1966 was completed. In addition to nine tables of basic data 
prepared for each of these institutions, summary tables and a commentary were 
also provided. This analysis was utilized to determine the impact of proposed 
GRS modifications on dental schools. 

{k) Completed during the year was a major project analyzing the total 
NIDR extramural project awards made from FY I967 funds for research and training 
purposes. This report provides the most comprehensive listing and analysis of 
these programs to date and will serve as a prototype and basis for regular 
annual publications in future years. 

(5) Efforts continued to identify and obtain essential data on all 
current dental research activities being conducted in the United States and 
around the world. This kind of information is necessary to place NIDR's 
efforts in proper perspective by assessing the nature and magnitude of other 
related programs, thus providing a better basis for avoiding unnecessary 
duplication, improving coordination, and developing opportunities for various 
collaborative efforts. Broad coverage has already been obtained of such 
related activities of other NIH, PHS , and DHEW units. Cooperation has also 
been forthcoming from the Department of Defense, the Veterans Administration, 
and the Atomic Energy Commission. Major central resources such as the 
Scientific Information Exchange, the National Library of Medicine, and the 
Biomedical Research Information Service of the World Health Organization have 
been utilized in efforts to obtain a better picture of dental research 

o 



activities on a world-wide basis. In addition, special efforts have teen 
devoted to a critical evaluation of the progress of the ITIDR-supported Dental 
Research Information Center (DRIC) , operated hy the American Dental Association. 
This activity, established to provide a comprehensive source of information 
concerning the total U.S. involvement with dental research, is about to 
launch a major resurvey to obtain current data concerning the Ajnerican dental 
research investigator community and its ongoing research activities. As a 
consequence of the importance of making this resurvey effort more directly 
responsive to NIDR's needs for reliable, overall information of this type, 
the Chief of the Program Planning Office has participated in a major way in 
redefining the objectives of DRIC and restructuring the original investigator 
and project questionnaires so that these key instruments can be more 
effectively utilized in obtaining the necessary information. 

The Program Planning Office continued to serve in a liaison capacity between 
NIDR and related DRFR and DDH activities such as General Research Support, 
and Health Research and Health Educational Facilities construction programs. 
Contributions were made by this office to an intensive evaluation of the 
implications to NIDR of the April 1 reorganization of the health functions of 
DHEW. 

Finally, a detailed evaluation was undertaken of the role and functions of 
the Program Planning Committee in order to achieve a more effective degree of 
utilization of Council members and other consultants in identifying and dealing 
with major problem areas that transcend the activities of the specific task- 
oriented committees of NIDR. 

Information Office 

The public's view of the dentist as an artisan-technician not only does an 
injustice to dentistry but also affects dental research. A major 
responsibility of the Information Office of the Dental Institute is to change 
this image. A changed attitude would not only invite more attention to oral 
disorders but would also create a greater public sensitivity to their 
importance and the need for preventive measures. By substituting a hope born 
of knowledge for current attitudes of indifference, apathy, and outright 
fatalism about tooth loss , we can greatly raise the level of oral health of 
the Nation. 

Dental research is exceedingly diversified, current Institute-supported 
studies embracing a majority of the basic and clinical sciences. Reporting of 
research reflecting such a broad base of science helps to erode the common 
misconception of the narrowness of this field. Every opportimity is therefore 
vigorously sought to tell the story to the general public, the practicing 
dentist, dental educators, and the scientific community. 

In vying for media attention, dental diseases patently are at a competitive 
disadvantage against such dramatic problems as heart disease and cancer; 
moreover, their relation to general health is not clearly perceived. 
Nonetheless, efforts this year have been successful in achieving greater 
coverage in the radio and television media as well as mass periodicals. One 
step in this direction has been to provide film clips on newsworthy research. 
For example, TV film clips and movies were prepared, in cooperation with 



Merck Sharpe and Dohme, to publicize the encouraging experimental results 
with dextranase. This research report received wide publicity, including 
coverage on the Walter Cronkite Evening News Program. Also, news items 
appeared not only in the daily press, but in national publications such as 
T his Week , Business Week , and Current Science . 

For the first time this year, the Information Office took advantage of the 
opportunity offered by National Children's Dental Health Week, sponsored by 
the American Dental Association. This year's theme was "grow up smiling." 
NIDR provided the mass media with a feature story and photographs illustrating 
the role played by children in "research for the smile of health." 

In another new service introduced this year, a feature article was prepared 
and mailed to approximately 3,000 company house organs throughout the country. 
Clippings now being received indicate good usage of this material. 

National magazine queries, resulting in several articles about the Institute's 
research program, continue to be stimulated by the quarterly mailing of "NIDR 
Research Capsules," prepared for selected science writers. Time , Newsweek , 
Reader's Digest , Parade , Project Engineering , U. S. News and World Report . 
Consumer's Digest . U. S. Medicine , and Knight Newspaper Syndicate (which 
reaches 1.8 million readers) were among those reporting on dental research. 
Articles also appeared in the foreign press, including British, French, 
Italian, and Australian journals, and several Latin American publications. 
In the dental press, too, numerous articles appeared as a result of the monthly 
mailing of "research news from NIDR." 

New opportunities to reach various segments of the public with information on 
research progress against oral disorders are also sought continuously. This 
year, for example, copies of the Institute's pamphlets on tooth decay and oral 
ulcerations were sent to several thousand teacher members of the American 
Association for Health, Physical Education, and Recreation. The pamphlets 
were also published in their entirety in TIC , a periodical found in the 
reception room of dental offices. 

Related to the narrow image of dental research is the serious shortage of 
dental research personnel. A major effort has been undertaken to acquaint 
young science students, as well as science teachers and guidance counselors, 
with the variety of career opportunities in this field. The principal segment 
of this program is the production, under grant, of a motion picture and career 
brochure, illustrating the broad diversity of the dental sciences and focusing 
on the oral cavity as an integral system of the body. 

Particular emphasis was also placed this year on the showing of a specially 
designed exhibit on the spectrum of careers in dental research. Although plans 
for showing the exhibit were modified due to budget restrictions, it was shown 
at four national and regional meetings of science students and teachers. A new 
brochure published this year, illustrating the wide scope of Institute grant- 
supported research, was made available at the exhibit. In addition to the large 
volume of material distributed from the exhibit, several thousand copies of the 
brochure, "The Expanding World of Dental Research" were sent to members of high 
school science clubs throughout the United States. 



As part of the effort to present career opportunities, a member of the 
Information Office staff attended a Future Health Careers Day at St. Clair 
Shores, Michigan. Approximately 1,600 high school students attended to 
discuss health careers with representatives from many associations and 
agencies. In addition to the pertinent NIDR literature distributed at the 
meeting, arrangements were made to supply the Michigan State Health Council 
with literature and display material for a permanent exhibit panel for the 
Michigan State Health Careers Mobile Unit. This is a 50-foot trailer which 
travels throughout the state visiting senior high schools and junior colleges 
encouraging interest in the health careers. 

A related service was introduced following a survey to identify appropriate 
honorary scientific societies to elicit membership interest in Institute 
programs. As part of this new continuing service, 7,260 pieces of literature, 
including leaflets on training and fellowship opportunities , as well as the two 
Institute brochures, were sent to the executive officers of 25 societies for 
distribution to their membership. Also, 700 pieces of training and fellowship 
literature were sent to 350 selected dental school faculty members, and former 
NIDR trainees and fellows. 

The observance of the Institute's 20th Anniversary provided the Information 
Office an excellent opportunity to publicize progress of the past and plans 
for the future. In this connection, considerable encouragement and assistance 
was provided to outside authors for preparation of major articles for possible 
publication in early issues of the Journal of the American Dental Association , 
Science , Reader's Digest , and Today's Health . Other feature articles, with 
photos, were prepared by the Information Office for the NIH Feature News 
Service, NIH Photo Features, NIH Record, PHS World, Research for Health Column, 
News from NIDR and NIDR Capsules. A photo montage and selected individual 
photographs, together with a fact sheet and additional background material, 
were widely distributed to other audiences and science writers. This material 
was also sent to the deans of all dental schools and to the public relations 
officers of all NIDR grantee institutions with an accompanying letter requesting 
their cooperation in publicizing 20 years of progress against the oral disorders. 
This activity resulted in the mailing of 2,541 pieces of material to 1,596 
addressees. 

Responding to their expressed need for obtaining more information about NIH 
research programs, the NIDR Information Office mailed two of its principal 
brochures to each member of Congress. The publications, "The Expanding World 
of Dental Science" and "A Spectrum of Dental Research," excited wide interest 
and comment. Additional requests were received from members of Congress for 
an additional 2,420 copies of each publication for mailing to their 
constituents . 

Due to budget restrictions, only one new scientific exhibit was developed by 
the Information Office this year. Designed in cooperation with the staffs of 
the Oral and Pharyngeal Development Section and the NIH Medical Arts and 
Photography Branch, the exhibit has created considerable interest. In 
competition with 80 other scientific exhibits, it was awarded second prize 
at the annual meeting of the American Dental Association. Although numerous 
requests for showing the exhibit were received, it was shown only at the 

r 

6 



American Dental Association, American Association for the Advancement of 
Science, and the Federation of American Societies for Experimental Biology 
meetings. Altogether, the NIDR exhibited at nine meetings during FY 1968. 

Another voluminous mailing of Institute literature was made this year to 
officers and research coordinators in universities, institutions, hospitals, 
and other organizations who might not be aware of the increasingly extended 
range of dental research. The 1,522 persons selected from certain mailing 
keys of the Division of Research Grants were sent 16,975 pieces of literature. 

The Information Office recently revised four leaflets relating to the extra- 
mural programs. One was revised to inform dental students about the Institute- 
supported summer research opportunities in 1968. In this connection, an 
editorial was prepared for publication in the February issue of the Dental 
Student's Magazine , announcing these opportunities, and listing the schools 
providing the training. Sixty-thousand pieces of training and fellowship 
literature were mailed in February to the college addresses of over 10,000 
dental school students. 

In another effort to further acquaint the academic community with the scope 
of the extramural programs, the Information Office developed and made extensive 
distribution of the following informational items: Research and Research 
Training Supported by the NIDR; Directory of Resource Persons by Program 
Responsibility; Procedural Flow Charts for Research Grant Applications and 
Training Grant and Fellowship Applications; Statement of Function for the 
NIDR Extramural Programs, and Basic Science — The Core of Dental Research. 
Mailings of 2,055 of these items were made to 685 NIDR grantees, research 
coordinators and other selected university personnel. A total of approximately 
161,000 pieces of literature was distributed this fiscal year, a 50 percent 
increase over last year. 

The Institute's visitor program was especially active this year as a result of 
the "Open House" arranged in connection with the annual American Dental 
Association Meeting held in Washington, D.C. In addition, orientation programs 
and tours were arranged for approximately 357 visitors to the Institute. These 
programs are especially designed to meet the particular interest of each indivi- 
dual or group. 

The Information Office also provided staff assistance in the Press Room at the 
American Dental Association Meeting and made necessary arrangements for four 
NIDR investigators to appear on the ADA closed circuit TV programs. 

As the clearance center for manuscripts produced by Institute scientists, the 
Information Office processed 104 manuscripts and 53 abstracts during this year. 
The Office also prepared the NIDR component of the NIH annual bibliography, and 
the "Professional Staff of NIDR." 

Internal reports prepared in whole or in part by the Information Office during 
FY 1968 numbered 116. Such documents include weekly reports to the Director 
of NIH of selected dental research advances and program developments, various 
annual reports, and revision of Departmental documents. Special material for 
budgetary and appropriation hearings prepared by the Information Office included 

7 



the Director's Opening Statement, narratives for the annual National Science 
Foundation report for Federal Funds for Science Series and the annual NSF 
Survey of Scientific Information, highlights of research progress, and other 
Congressional reports. Also, six speeches were prepared for key personnel of 
the Government. 

In addition to the anniversary articles mentioned above, 39 articles were 
prepared for the NIH Record, PHS World, Research for Health, and Magazine 
Memo. Also, 22 press releases, summaries and announcements were produced. 

Reaction to the Institute's first press conference, held in June, was 
enthusiastic, further attesting to the newsworthiness of present-day dental 
research. The Institute's Director and two members of the staff discussed new 
findings and new approaches to the prevention of dental caries. The Information 
Office provided press kits and TV film clips for media representatives in 
attendance. 

Information plans for Fiscal Year 1969 will continue to emphasize recruitment. 
The motion picture now under production will be extensively promoted. Through 
widespread dissemination of research findings, using NIH information channels 
and direct media contacts, continued intensive efforts will be made to increase 
public understanding of the oral disorders and the hope held out through 
research. 

Collaborative Research Office 

The Collaborative Research Program continues its primary activity in support 
of research in the area of biomaterials . In addition, various intramural 
collaborative research activities are being supported by contract, and an 
initial step has been made toward extending the use of the contract, as a 
means of research support, to NIDR extramural programs. 

Approximate expenditures for FY 1968 were: biomaterials research - $346,000; 
intramural collaborative studies - $160,000; interagency research agreements - 
$221,000; and extramural study in the oral-facial growth and development 
program - $8, 777. 

Biomaterials Research Program 

This program is directed toward the development of new and more effective 
dental restoratives and other biomaterials for use in dentistry, including 
dental adhesive materials, characterization of the tooth and its function 
within the oral environment, and testing and evaluating new materials that 
may contribute to major advances in the practice of prosthetic and restorative 
dentistry. This effort is programmed by the multidisciplinary Biomaterials 
Research Advisory Committee; additional consultants are used as necessary. 

The Committee does much of its work in programming at biannual meetings but, 
in addition, frequently reviews program elements through the year. 

A systematic program to develop an adhesive dental filling material has been 
underway since 1964, and now shows increasing promise of success. Many 



scientific disciplines are involved with the nature of adhesion mechanisms, 
of tooth surface and filling materials, the ultrastructure of enamel and 
dentin, the physical character of the cavity surface after drilling, improve- 
ment of existing dental materials and the fabrication of synthetic materials 
which relate to this program as well as evidencing useful application to other 
dental problem areas. 

As a result of an Ad Hoc Workshop held in FY 1967, the investigation of 
barnacle cement was initiated to learn the composition and setting mechanism 
and to apply this knowledge to the development of an adhesive dental filling 
material. Experiments have shown that the barnacle cement adheres to all 
known materials except those containing copper. A substance which appears to 
be uncured cement has been extracted and chemical analysis of the cured cement 
has been initiated. To accelerate research interest in this material a 
pamphlet has been prepared describing the anatomy and physiology of barnacles, 
methods of growing barnacles in aquaria and means of harvesting the uncured 
cement. This pamphlet will be widely distributed to researchers and educators. 

An Ad Hoc Workshop on Tooth Implants 

An Ad Hoc Workshop was held at the NIDR to examine the method of replacing a 
tooth immediately after its extraction with a replica in plastic inserted 
immediately into the socket. The participants of the meeting represented the 
several disciplines which would be involved in a comprehensive targeted program 
to investigate this procedure which could be taken up by dentistry as part of 
its method of treatment. The group recommended that an aggressive program 
effort be initiated by the NIDR to develop that additional information which 
is needed to determine if this is a reliable clinical procedure. 

Some additional comments were made concerning a wider application — such as 
those needing alveolar ridge reconstruction and support of periodontally 
involved teeth. It was agreed to consider only the single tooth implant at 
this time. It was suggested that a special advisory committee be used to 
guide this targeted program as this method would be effective in starting 
the program, monitoring it, and evaluating results. 



Contract Narratives 

Collaborative Research Office 
Fiscal Year 1968 

ALPHA RESEARCH & DEVELOPMENT, INC. (PHU3-6U-^02 ) 

Title : "Investigations Leading to Improved Dental Restorative Materials" 

Contractor's Project Di rector: Dr. Robert L. Patrick 

Project Officer (NIDR) : Dr. Robert J. Nelsen 

Objectives : (l) To investigate the adhesion of rubbery polymers to 
hydroxyapatite , enamel, dentin and tough restorative materials, and to 
determine the degree of irreversible adsorption of a series of polymers 
to the various substrates; and (2) To investigate the permeability of 
restorative systems to water and diffusion of water through the rubbery 
interlayer. 

Methods Employed and Major Findings : Four general systems were considered. 
The unlined model restoration; an acrylic rubber liner; a silane liner; 
and a dual-film acrylic rubber silane liner. Synergism was clearly shown 
in the case of the dual-film liner. 

The stereoscanning electron microscope was utilized to evaluate film 
thicknesses in the model restorations. In addition, the surfaces of the 
model preparations were examined prior to application of liners and 
restorative resin. 

Significance to NIDR Prot;rain: This investigation indicates that the use 
of a rubbery interliner acts as a sealant to the diffusion of fluids at 
the interface of the tooth filling and that the use of silane increases 
the effectiveness of this seal. There are also definite indications that 
the bond between the tooth and bulk restorative material is improved. 

Proposed Course of Project: Scheduled for termination in FY 1969 . 

Current Annual Level: $28,131. 



10 



Contract Narratives 
Collaborative Research Office 
Fiscal Year I968 



AMERICM DENTAL ASSOCIATION (PHl| 3-66-1127) 

Title : "Preparation and Editing of a Dental Science Handbook" 

Contractor's Project Director : Dr. Lon W. Morrey 

Project Officer (NIDR ) : Dr. Robert J. Nelsen 

Objectives : This dental science handbook is "designed for use by dental 
researchers, students and educators and will contain, in condensed form, 
the total spectrum of dental science and art including, but not limited 
to, the categorical areas of morphology, function, growth and develop- 
ment, treatment, materials, environmental factors of the mouth, pathology, 
public health and socio-economic statistics." 

Each categorical area is being edited by a scientist of acknowledged 
leadership. He will select the text, illustrations, charts, etc., for 
his area from the literature. The project director is Editor Emeritus 
of the Journal of the American Dental Association . It is anticipated 
that this will be published in 1969- 

Proposed Course of Project : Scheduled for completion in calendar year 
1968. 

Date Contract Initiated : June 21, 1966. 

Current Annual Level: $l6,^02 (estimate). 



11 



Contract Narratives 
Collatorative Research Office 
Fiscal Year 1968 

THE EPOXYLITE CORPORATION (PHU3-6U-5i48) 

Title : "A Study of Epoxy Resin Dental Materials" 

Contractor's Project Director : Dr. Henry Lee 

Project Officer (NIDR ) : Dr. Herbert Swerdlow 

Objectives : This contract is directed toward the study of epoxy resins 
for possible use as dental restorative materials. 

Methods Employed : (l) Synthesis of specific resins customed for dental 
applications; (2) Measurements of physical properties of new resins, 
especially the reaction rate, water sorption, adhesive properties, color 
stability, hardness, and adhesion. 

Major Findings : A number of filling materials which are free flowing, 
harden at tolerable t emper attire s , and have low curing shrinkage, are 
tough, hard and moisture resistant, have been developed. With these 
epoxy resins, the best adhesion to dentin is obtained with a citric acid - 
organo-fimctional silane - butanol pre-treatment of the tooth substrate. 
These materials are filled with glass beads, or aluminum, to increase 
their strength to approximate thermal expansion of the tooth. It has 
been found in this program that the bovine tooth is suitable as a test 
surface . 

Significance to NIDR Program : The epoxy resin system appears to present 
one of the most lucrative means of developing a suitable adhesive dental 
restorative material. 

Proposed Course of Project : This project will be continued at least until 
late FY 1969 , and may be considered for an additional year. 

Date Contract Initiated : April 28, 196U. 

Current Annual Level: $53,500 



12 



Contract Narratives 
Collaborative Research Office 
Fiscal Year I968 

HA P.aiS RESEARCH LABORATORIES (PHU3-6U-$30) 

Title : "An Investigation of the Mechanism of Adhesion to Teeth" 

Contractor's Project Director : Dr. Anthony Schwartz 

Project Officer (NIDR ) : Dr. Robert J. Nelsen 

Objectives : To develop a dental restorative system that seals the tooth 
cavity completely and which provides a monolithic structure with respect 
to the strength of the restored teeth: Experiments directed toward the 
discovery of intermediately placed materials to bond acrylic to tooth 
substance have formed the central core of this project. 

Major Findings : Polyurethane type compositions have been developed as 
adhesive liners for amalgam and self-curing acrylic restorations. Several 
polyglycol-diisocyanate compositions exhibited adhesion to human dentin 
when applied to moist teeth in_ vitro . Conventional acrylic or amalgam 
restorations became durably bound when applied on these liners within 
thirty minutes after they had been applied to the teeth. The composite 
restoration was water resistant and performed well in tensile, compressive 
and thermal shock adhesive tests. 

The first experiments with the proposed standard test for adhesion were 
very promising. While some of the apparatus and a few manipulative pro- 
cedures could be improved, a period of test procedure development should 
precede the introduction of formal changes . 

Significance to NIDR Program : This contract is part of the total program 
directed toward an adhesive dental restoration. Specifically, it is 
mainly concerned with the use of conditioning agents and adhesive liners. 
Success in the development of an intermediate material which both the 
filling material and the tooth will bond is more likely to occur than 
will the development of a filling material that is itself directly 
adhesive to the tooth. 

Proposed Course of Project : To be continued to FY I969, and tentatively 
scheduled for expiration in April I969. 

Date Contract Initiated : April 29, 196^1. 

Current Annual Level : $32,Ul7. 



13 



Contract Narratives 

Collaborative Research Office 

Fiscal Year I968 

UNIVERSITY OF AKRON (PHU3-67-II72 ) 

Title : "A Study of Barnacle Cement" 

Contractor's Project Director : Dr. Roger F. Keller 

Project Officer (NIDR) : Dr. Robert J. Nelsen 

Objectives : (l) To obtain imcured barnacle cement for study aimed at 
assessing its value as a dental adhesive; (2) To analyze barnacle cement 
in terms of its chemical structure with a view to developing commercial 
m-ethods of synthesizing the cement in large quantities. The synthesis may 
be of the total cement or of its adhesively fianctional subgroups. 

Methods Employed and Major Findings : Work progressed in several distinct 
areas: (l) Anatomical, to further elucidate the cement flow path, isolate 
the responsible secreting gland, and prove that there are no glandular 
structures within the shell wall or base plate responsible for creation 
of cement; (2) Histological, aimed at the sectioning of the cement gland 
and identification of its fimctional parts; and also to examine the nature 
of the cement bond to human teeth; (3) Isolation and analysis of cured 
cement . 

Future Work : It is anticipated that the uncured cement will be extracted 
and purified and the chemical structure will be determined through instru- 
mental and other techniques. Studies will be made of the cured condition, 
the chemistry of curing and the rate of curing. In addition, the continu- 
ation of histological studies, both of the glandular secretions and of 
the barnacle tooth bonds is being made. This investigation will determine 
the feasibility of synthesizing barnacle cement which could prove to be 
a significant breakthrough in the field of adhesive dental materials, and, 
quite likely, may have application to the general area of tissue adhesives. 

Sip:nificance to the NIDR Program : This investigation will determine the 

feasibility of artificially producing barnacle cement which could prove 

to be a significant breakthrough in the field of adhesive dental materials. 

Proposed Course of Project : The project is scheduled to run through FY I969 
and will be considered for renewal on the basis of findings up until that 
time . 

Current Annual Level : $3h ,6kO . 



^ 



Contract Narratives 

Collaborative Research Office 

Fiscal Year I968 



IMIVERSITY OF WASHINGTON 

Title: "Histologic Comparison of Human and Swine Teeth for Operative 
Dentistry" 

Contractor's Project Director : Dr. Gerald D. Stibbs 

Project Officer (NIDR) : Dr. Herbert Swerdlow 

Objectives : The purpose of this project is to evaluate the feasibility 
of using Hanford Miniature Swine for future testing of dental materials 
and restorative procedures by operating on and maiing direct comparisons 
between human and swine teeth under identical or equivalent conditions. 

Methods Employed : Recognizing the need to test new materials and pro- 
cedures in other than human teeth, this program will compare the response 
of human teeth to swine teeth as they are exposed to conventional and 
experimental materials and procedures. Procedures for human subjects and 
the miniature swine will be the same. Hioman teeth to be removed for 
orthodontic reasons will be subject to the test procedure or material. 
The same operator will repeat the procedures on the miniature swine teeth. 
All subjects will be anesthetized during all operations. The response of 
the tissues of the human teeth will be compared to the response of the 
miniature swine and the observations will be correlated. After the inter- 
pretation and cross extrapolation of the results, it is expected that a 
model system will be developed for possible evaluation of new dental 
restorative materials utilizing miniature swine as a stand-in for man. 
Preliminary studies indicate the existence of a strong correlation between 
human and swine teeth in their response to dental treatment procedures . 

Significance to NIDR Program : It is necessary to establish baseline in- 
formation on the morphologic and histologic similarities and differences 
between human and swine teeth before swine can be used as experimental 
subjects for dental operations, although preliminary studies indicate the 
existence of strong correlation. 

Proposed Course of Project : Scheduled to run through FY 1969. At the end 
of one year the project may be considered for renewal depending on results. 

Date Contract Initiated : June 1968 (estimated). 

Current Annual Level: $i+5,llT (estimated). 



15 



Contract Narratives 
Collaborative Research Office 
Fiscal Year I968 



MOIiSMTO RESEARCH CORPORATION 

Title : "Surface Coatings to Produce Permanent Silicate Cement Restorations" 

Contractor's Project Director : E. A. McElhill 

Project Officer (NIDR) : Dr. Robert J. Nelsen 

Objectives : There is good evidence that exposure of the surface of dental 
restorative materials to the mouth environment contributes heavily to 
their disintegration. This project is designed to determine the feasibility 
of protecting these surfaces from exposure to saliva and other fluids with 
a coating so that the integrity of the restoration may be maintained for 
a longer period of time. 

Methods : Resins will be used as a coating material that have been developed 
as dental restorative materials. These resins have two of the required 
properties, namely, good mechanical toughness and good color stability. 
In addition, techniques are known for curing them in the environment of 
the mouth. 

To obtain a strong bond of these materials to the surface of the silicate 
restoration, coupling agents developed in the aerospace industry for 
coupling polymers to glass surfaces will be used. These coupling agents 
are dual -functional and couple the resin directly to silicate surfaces. 

The two major types of resinous dental restorative materials, namely, the 
methacrylate type and the epoxy type will be used as coating materials. 

The restorations with coatings will be tested for solubility, discoloration, 
dehydration, compression resistance and tensile strength. 

Significance to NIDR Program : If this project is successful it will allow 
an immediate and significant improvement of "on the shelf" materials. 

Proposed Course of Project : This is a feasibility study to be started in 
late FY 1968 and be continued through FY I969. It will not be continued 
longer than one year unless results warrant it. 

Date Contract Initiated : June I968 (estimate). 

Current Annual Level : $21,000 (estimate). 



16 



Contract Narratives 

Collaborative Research Office 

Fiscal Year I968 



UN T^yERSITY OF TENNESSEE 

Title : "Development of Standardized Toxicity Testing for Dental Materials 
and Products" 

Contractor's Project Director : Dr. John Autian 

Project Officer (NIDR) : Dr. Herbert Swerdlow 

Objectives : (l) To develop aji "initial" or "preliminary" testing protocol 
for dental materials and products; (2) To develop a scoring system which 
may be helpful in delineating the degree of toxicity; (3) To perform 
toxicity tests on selected candidate materials and products which are 
being developed through contracts and grants from NIDR. 

Methods Employed : Presently, the most perplexing problem in regard to the 
toxic potential of dental materials and products is the lack of a uniform 
manner in rating or scoring a toxic response. This program is designed to 
develop preliminary testing protocols for dental materials and products 
which will make use of a scoring system to delineate the degree of toxicity. 
When the scoring system has been defined, toxicity tests on selected candi- 
date materials which are being developed in other programs of the NIDR 
will be performed as a means of screening. 

Significance to NIDR Program : The experimental and testing procedures sug- 
gested in this proposal will, in effect, be the first systematic approach 
to the evaluation of acute toxicity of dental materials. 

In the last several years, NIDR has encouraged and supported research in 
the development of new materials which may become useful as adhesives in 
one or more dental applications. Up to this date a number of candidate 
materials have evolved which are now being evaluated by physical, chemical 
and mechanical means. Results of research on implantable plastic teeth 
have also opened up a new avenue of possible dental treatment promising 
benefits to a large group of ovir population. These very recent develop- 
ments add further support to the concept that the time has arrived for a 
systematic evaluation of dental materials and products. 

Proposed Course of Project : Project is scheduled for implementation in 
late FY 1968 and will be considered for renewal dependent upon results. 

Date Contract Initiated : June I968 (estimated). 

Current Annual Level: $2i+,8l0 (estimated). 



17 



Contract Narratives 
Collaborative Research Office 
Fiscal Year 1^ 



MONSMTO RESEARCH CORPORATION (PH43-66-889) 

Title : "Research on Composite Silicate Dental Cements" 

Contractor's Project Director : Dr. Thomas Rockett 

Project Officer (NIDR) : Dr. Robert J. Nelsen 

Objectives : Plastics and polymers offer promise of a new restorative 
material with improved properties, but there are still many unsolved 
problems associated with their use. This project is an attempt to im- 
prove existing dental materials by designing new compositions of silicate 
dental cements. To do this, cement- forming characteristics of a wide 
range of compositions are being studied. 

Methods Employed : Frit composition is being varied systematically within 
a mult i component oxide and fluoride phase equilibria system. 

Major Findings : Several very promising compositions were discovered 
during the past work, and recently several more very interesting composi- 
tions were found. In addition to compositional work, the setting of 
commercially available materials and experimental cements are being 
studied by X-ray diffraction and electron microscope techniques in order 
to understand the setting mechanism of these cements. This information 
is being used to guide systematic variation of composition. 

Last year several contributions were reported by this contractor. These 
included the identification of crystalline phases in the matrix of the 
silicate cements, development of a theory of setting mechanisms of cements 
and a ten-fold improvement of strength properties over some commercially 
available cements. 

Accomplishment of the following tasks is planned for the coming year: 

1. Complete screening of CaO-Al203-P205-Si02 frits. 

2. Complete the refined tests on most of the superior experiments. 

3. Supply to in vivo testing group at least two experimental cements. 
h. Begin work on fluorine concentration 

5. Begin work on additive materials. 

Significance to NIDR Programs : This project is designed to help assure 
maximum benefit from current restorative systems until such time as 
better ones are developed and become universally available. In addition, 

18 



improved existing cements may well become part of the total dental 
restorative system of the future. 

Proposed Course of Project : Scheduled for continuance through FY I969, 
at which time the continuance will be determined based on results. 

Date Contract Initiated : June 1, 1966. 

Current Annual Level : $6l,355 (estimate). 



19 



Contract Narratives 
Collaborative Research Office 
Fiscal Year I968 



TYCO LABORATORIES 



Title : "Non-Noble Metal Alloys for Dental Purposes" 

Contractor's Project Director : Mr. Fritz Wald 

Project Officer (NIDR) : Dr. Robert J. Nelsen 

Objectives : This project is directed toward determining the applicability 
of the copper-nickel-manganese system to dental purposes. The alloys to 
be investigated are used industrially as electrical resistance alloys; 
therefore, if they are suitable for dental use, they would be immediately 
available . 

Methods: This program has been initiated to investigate the resistant 
alloys of copper, nickel and manganese. The physical property of this 
alloy system indicates that it may be quite possible to develop casting 
alloys which have the physical properties of the presently used gold 
alloys but without a high intrinsic cost. 



Significance to NIDR Program : The successful development of an adhesive 
dental material will increase significantly the use of castable alloys in 
treatment procedures. Some of the newer techniques of prosthetic treat- 
ment now make use of cast posterior chewing surfaces. The presently avail- 
able - gold and cobalt - chrome alloys are costly, difficult to cast or 
too hard and brittle, and thus have limited usefulness. In addition, this 
metal may become too costly for dental use. 

Proposed Course of Project : This is a six-month feasibility study. Any 
consideration of renewal will be based on results. 

Date Contract Initiated : June 1968 (estimate). 

Current Annual Level : $2i+,i+60 (estimate). 



9'' 

20 



THE REPORT OF THE DIRECTOR OF INTRAMURAL RESEARCH 
THE NATIONAL INSTITUTE OF DENTAL RESEARCH 
July 1, 1967 - June 30, 1968 
by 
Dr. Richard C. Greulich 



Significant advances during the course of the past year have been achieved 
with respect to the two fundamental aspects of direct operating programs, 
namely program development and program execution. With respect to the latter, 
continuing and enthusiastic exploitation of individual and collaborative re- 
search efforts has provided additional evidence of the efficacy of the Insti- 
tute's broadly based research approach, which includes a balanced spectrum of 
basic, clinical and applied studies. Highlights of the year's research pro- 
gress are described below in terms of program execution, and are detailed, 
together with other items of progress, in the summary reports of NIDR's 
Laboratory and Branch Chiefs appended to this brief review. 

Program Development 

A variety of administrative changes have been implemented during the past year, 
calculated in the aggregate to provide a more conducive environment within 
NIDR for systematic pursuit and application of new knowledge relating to oral 
function and oral disease. Although several years' experience will be required 
to permit valid assessment of the efficiencies which these organizational 
changes are intended to foster, preliminary scrutiny of the research activities 
initiated during the past year inclines the writer to view them already as 
progressive steps. Hence, they will be described in this report. 

Prominent among reorganizational changes made were those relating to the Insti- 
tute's direct programs in the areas of epidemiology and biometry. In recog- 
nition of the essentiality of expanded capabilities for experimental design, 
automatic data processing and statistical analysis, and of the corollary 
essentiality of strengthening the Institute's expertise in the design and 
execution of field studies, a Biometry and Field Investigations Branch was 
established. This new research unit supplants the Institute's pre-existing 
Epidemiology and Biometry Branch, whose functions and personnel have largely 
been absorbed within it. Subsequently, three distinct sectional entities 
have been designated within the new Branch, namely the Biometry Section, the 
Clinical Trials Section and the Epidemiology Section. As its summary report 
will indicate, the activities of the Biometry and Field Investigations Branch 
have already been much expanded and refined as compared to immediately previous 



z: 



years. A soundly-based field studies approach, backed by a high degree of 
competence in biometry, now permits systematic exploration of dental and oral 
problems in terms both of new modes of prevention and therapy, as well as of 
geographic pathology. 

Other sections have been created within the framework of the Institute's 
laboratory research programs, notable amongst which are the Enzyme Chemistry 
Section, the Connective Tissue Section and the Cell Biology Section, all 
established during the year within the Laboratory of Biochemistry. Initiation 
of the Connective Tissue Section provides a functional visibility for the pre- 
existing excellence of individual and team efforts within the Laboratory in 
the area of mineralization, viewed as a normal - and all too frequently ab- 
normal - characteristic of mesenchymal ly-derived tissue systems. Similarly, 
identification of an enzyme-oriented group serves to acknowledge pre-existing 
expertise in the area, and to emphasize the Institute's commitment to explor- 
ation of the fundamental nature of enzyme action. Systematically derived 
knowledge of this process is now generally conceded to be essential to gaining 
a full understanding of normal and abnormal biological function. 

With respect to the Cell Biology Section, it may be noted that this group was 
previously attached to NIDR's Human Genetics Branch, as the Cellular Biology 
and Cytogenetics Section. Logical evolution of the research interests of this 
group, however, had been such as to turn its attention away from cellular 
inheritance, ultimately to focus on biochemical parameters of genetic expres- 
sion in isolated cell populations. Conceptually as well as practically, 
therefore, it was deemed appropriate to merge this group's future activities 
with those of the Laboratory of Biochemistry. 

Consequent to the loss of the Cytogenetics Section, careful scrutiny was also 
given to the research program of the Human Genetics Branch. Review of pro- 
grammatic needs and individual talents within the Branch revealed the feasi- 
bility of designating two new sectional enterprises, namely the Population 
Genetics Section and the Developmental Genetics Section. These organizational 
titles accurately portray the clear recognition by the Branch of its continuing 
responsibility to expand its already rewarding program relating to genetic 
influences on human oral disease, expressed at the population level. Equally, 
there is implicit recognition of the need to explore to the fullest those 
aspects of genetics which can best be approached by analysis at the laboratory 
level, using animal models. In this context, promising emphasis is now being 
directed toward elucidation of genetic mechanisms governing organ differentia- 
tion in several mammalian systems. 

The role of viral vectors in oral disease has long been a major area of re- 
search interest within the Laboratory of Microbiology. Demonstration by 
laboratory scientists during 1966 that a virus can combine with its antibody 
without losing infectivity provided even greater impetus to the study of 
virology, both with respect to the nature of the viral neutralization reaction, 
and to the role of viral infection in modulating immunological response. In 
concert with these research developments, and in recognition of the growing 
relevance of virology to our overall health mission, a Virology Section was 
established within the Laboratory of Microbiology during the past year. 






Another significant administrative development pertained not to definition 
of a new organizational unit, but rather to reimplementation of an already 
existing one. Thus, personnel attrition having over the previous few years 
greatly reduced our capacity for research programs in X-ray crystallography, 
the Institute was faced with the need to assess the current and projected 
relevance of this technology to its mission. Accordingly, an intramural 
workshop was organized on the subject of "Crystallographic Concepts in Dental 
Research: Retrospect and Prospect." This informal, two-day symposium, the 
membership of which included experts in crystal structure drawn from Federal 
and academic sectors, as well as from NIH, concluded that many problems of 
great relevance to dental research remain to be explored, and strongly recom- 
mended that NIDR's intramural resources be geared to accommodate them. Re- 
cruitment of a new chief for the Crystal Chemistry Section in the Laboratory 
of Histology and Pathology was therefore undertaken, and, as the appended 
project reports will reveal, new and exciting dimensions of creativity are 
already evident. 

In last year's summary report of intramural activities, the writer attempted 
to give visibility to a variety of individual research projects which, in the 
aggregate, focused on the oral environment. More specifically these dealt 
with selected aspects of endogenous and exogenous control of the oral apparatus. 
The writer employed this reportorial technique to emphasize his conviction 
that, to achieve better balance within the Institute's direct operating pro- 
grams, establishment of an administrative unit concerned with oral physiology 
was both timely and appropriate. Accordingly, initial steps in this direction 
were taken during the year just past, in that a Physiology Section was organ- 
ized, operating within the Office of the Director of Intramural Research. A 
significant increment in research creativity is well documented in the appended 
project reports from this Section, and augurs well for the future success of 
the unit. It is fully intended that, as budgetary circumstances will permit, 
this unit will ultimately evolve into a full-fledged Laboratory of Physiology. 

Program Execution 

Any effort to utilize this summary report as a vehicle for highlighting sig- 
nificant research advances is fraught with difficulty, if not danger. Thus, 
it seems prudent to preface any identification of specific research accomplish- 
ments with the caution that, whether the findings described below do indeed 
constitute significant steps forward in our quest for understanding of dental 
disease, will be determined only by the passage of time. 

Without question, a greater effort than ever before has been devoted this past 
year to a concerted attack on the problem of dental caries. For prior re- 
search successes at NIDR and elsewhere, now clearly permit the development 
of rationally-based approaches to caries control. Particular emphasis has 
been concentrated on the natural history of a distinctive group of anaerobic 
streptococci which in animal model systems have been irrevocably implicated 
in smooth-surface dental caries. Evolving from previously accumulated evidence, 
exciting recent experiments have focused on a singular metabolic characteristic 
of these organisms, namely their production of an extracellular coating of a 



2-^ 



polysaccharide derived from dietary sucrose. This coating, a long-chain dex- 
tran, appears to facilitate adherence of the organisms to the tooth surface 
as dental plaque. On the assumption that the adhesiveness of this gummy coat- 
ing uniquely facilitates the apposition of organisms to the tooth surface, 
thereby leading to carious destruction, laboratory experiments have tested 
the effects of a dextran-hydrolyzing enzyme (dextranase) on plaque formation 
and carles induction. Incorporation of dextranase in both in vivo and in vitro 
systems has now been shown to prevent plaque accumulation and to inhibit the 
development of caries. These most promising laboratory findings suggest a 
potentially significant approach to the control of dental caries in man. 
Consequently, clinical field trials are to be undertaken very shortly. 

Related studies of these plaque-forming streptococci have recently provided a 
deeper insight into their metabolic characteristics, particularly as they may 
specifically relate to the biochemistry of caries formation. Although re- 
quiring further experimental confirmation, results to date suggest that phos- 
phate in the immediate environment enters these organisms very rapidly but 
does not leak out. Thus the cariogenic plaque effectively constitutes a 
metabolic sink for phosphate ion, much of which may well be derived from the 
subjacent enamel surface. Further evidence has been adduced which suggests 
that the intracellular phosphates are sequestered as negatively charged poly- 
phosphates. Presence of the latter within the dental plaque may effectively 
convert it into a potent medium for cation exchange. It is tempting to specu- 
late that this unique environmental situation contributes significantly to 
decalcification of the tooth in the caries process, and/or to mineralization 
of the plaque as it becomes converted to calculus. 

Another type of microorganism, namely a filamentous diphtheroid ( Odontomyces 
v iscosus ) , has been implicated by past Institute studies in the etiology of 
periodontal disease. A potentially important advance in our capability to 
undertake systematic scrutiny of this disease entity has been achieved during 
the past year. Thus, after many unsuccessful attempts, cervico-radicular 
plaque formation and associated periodontal disturbances have been induced in 
hamsters by feeding them a diet containing starch, and by inoculating their 
mouths with diphtheroids of human origin. 

Research on the natural economy of connective tissue continues to comprise a 
major endeavor within the Institute, in keeping with the clear-cut involvement 
of connective tissues in so many aspects of oral disease. New information of 
a basic character, relating to the mechanisms involved in fibrillogenesis of 
collagen and elastin, has recently been revealed. Prior study at NIDR had 
established that frank fibrillogenesis occurs as a consequence of extensive 
cross-linking of protofibrils, a mechanism known to involve conversion of 
lysyl residues in the polypeptide. An enzyme has now been identified in ex- 
tracts of connective tissue that converts lysyl residues to allysine, presuma- 
bly a first step in the cross-linking process. The activity of this enzjrme is 
inhibited in vitro by treatment with a lathyrogen, at dose levels which pre- 
viously have been shown to block collagen and elastin cross-linking in vivo 
and in vitro . Paradoxically, penicillamine, which also inhibits cross-linking, 
does not inhibit the enzyme, and so apparently exerts its deleterious effect 
at some stage in the process after allysine has been formed. 



2k 



The effects of lathyrogens have also been explored with respect to their 
capacity for inducing abnormalities of growth and development in utero . These 
substances have now been shown to interfere with normal closure and fusion of 
the palatal shelves, so producing cleft palate. Thus, though presumably ex- 
erting their damaging effects through a mechanism related to their action on 
protein cross-linking, the lathyrogens join a long list of unrelated pharma- 
cological agents which can induce palatal malformation. 

Also within the area of connective tissue studies, research has been pursued 
and expanded with respect to identification and characterization of endogenous 
enzyme systems, which are assumed to play an important role in the formation, 
maintenance, and quite probably also, the destruction of various connective 
tissue components. In this context, emphasis has continued to be applied to 
characterization of collagenase which has been isolated from connective tissues 
under both normal and abnormal conditions. Of particular interest has been the 
recent demonstration that granulocytic leukocytes possess high levels of col- 
lagenase activity, probably associated with their complement of cytoplasmic 
granules. Evidence has now been developed which suggests that the collagenase 
is released to the external environment in response to a variety of stimuli, 
including tissue inflammation, and that only following release from the cell 
is the enzyme active in collagenolysis. In parallel studies, endogenous hya- 
luronidase has been identified in alveolar macrophages, and the environmental 
optima for its lytic action on hyaluronic acid have been defined. 

Although an integral facet of NIDR's research mission, disorders of oral-facial 
development, including malocclusion, have long constituted a difficult area 
for systematic study. The prime hindrance to an orderly exploration of facial 
growth stems from the lack of reliable animal models or systems. A promising 
development in this regard has been the recent discovery that high doses of 
Vitamin D administered to pregnant rabbits will regularly induce character- 
istic facial defects in the offspring. While this study is still in its 
initial phase, there is some reason to believe that the skeletal and oral 
defects will be sufficiently reproducible as to make this system highly useful 
for investigation of facial growth. Thus, detailed comparison of normal and 
abnormal rabbits may indeed provide leads to understanding of the growth 
mechanisms involved, and in turn these may ultimately be extrapolatable to 
the human situation. 

Turning from research highlights, it is to be emphasized that, as in past 
years, all intramural professional staff have continued to acquit themselves 
with distinction in the generation and execution of research. Over 100 papers 
documenting their activities have appeared in the scientific literature during 
the past year. Moreover, in concert with their growing prominence in the 
scientific community at large, most of the professional staff have availed 
themselves of other avenues for communication, including presentation of newer 
research findings at national and international scientific meetings. 

Reciprocally, as an ongoing aspect of NIDR's institutional responsibility for 
providing an environment conducive to creative scholarship, the weekly seminar 
program continued to provide a platform for the report of current research by 
scientists from NIDR, other NIH Institutes and Divisions, as well as from 59 



i_ .^ 



other speakers, including 18 from foreign countries. As in past years also, 
this formally-structured program of seminars was complemented by weekly 
clinical conferences, and by a multitude of more program-oriented meetings 
at the Laboratory, Branch, or Section levels. 

It is a matter of considerable pride to report that collaborative research 
activities have continued to play a major role in the execution of our re- 
search mission. Review of individual projects provides tacit evidence that 
the ramifications of dental research extend far beyond parochial and artificial 
boundaries. Thus, specific research projects were in progress or were com- 
pleted during the past year involving active interplay between all of the 
Laboratories and Branches of NIDR, all of the categorical Institutes and re- 
search-related Divisions of NIH, several other research arms of the Federal 
government, and nearly two dozen institutions of higher learning in this 
country and abroad. 

During the past year also the Institute has maintained its support of the 
activities of trainees and fellows including 7 Research Associates, 3 Clinical 
Associates, 3 Staff Fellows, 1 Postdoctoral Fellow and 1 graduate student in 
out-of-service training. On the international level, NIDR has also played 
host to 1 Visiting Scientist, 2 Visiting Associates and 1 Visiting Fellow 
whose individual research interests related to our activities in Microbiology, 
Histology and Pathology, Biochemistry, and Human Genetics. 

A particular debt of gratitude is owed by the Dental Institute, and especially 
by the writer, to its Board of Scientific Counsellors. As in previous years, 
the Board's dedicated interest, enthusiastic support and wise counsel have 
provided further incentive for achievement, and have encouraged a profitable 
interchange of ideas relating to long-range program planning. 



Report of the Physiology Section 
National Institute of Dental Research 
Summary Statement 

The Physiology Section was established to fill a need within the Institute 
for investigations into systems of controlled and uncontrolled interaction 
between the organism and its environment, as related to oral biology. In 
this context are studies of neurophysiological mechanisms and pathways orga- 
nized for processing of somatic sensation from the oro-facial region into 
the higher brain centers. This incoming sensory information is interacted 
with input from other modalities (e.g. visual, auditory, etc.) in associa- 
tion areas of the cerebral cortex and the response properties of nerve cells 
in such polysensory regions also is under investigation. 

Another level on which organisms interact with each other as well as with 
their environment is during growth and development. The environment of a 
cell, particularly its biochemical environment, is an overriding determinant 
in deciding the type of development that it will undergo. A model for the 
environmental biology of development is found in the studies of the external 
control of the rate of differentiation and/or growth of the amoeboid sline 
mold, Dictyostelium discoideum . 

The least organized bio-system under study in the Physiology Section is the 
response of caries-conducive streptococci to environmental parameters. A 
multiparametric approach is being adopted. Ongoing studies include: the 
dynamics of growth of these bacteria; the sequestration of phosphate (a main 
constituent of teeth) into the bacterial cell substance; the determinants of 
acquisition of capability to adhere to surfaces; and, related to this last, 
the dynamics of accumulation of cell-associated polysaccharides. 

An area of investigation and service which interacts with all the foregoing 
areas of research is the application of computer science to the flow and 
control of laboratory information and experiments. This encompasses such 
diverse areas as on-line data reduction, investigations into better mathe- 
matical models for laboratory computations, and information retrieval. 

Specific accomplishments in these several areas for the past year just passed 
are described in the following paragraphs. 

Neurophysiological studies have been concerned with the functional properties 
of single cells in two central areas of the brain — trigeminal brainstem 
nuclei and the cerebal cortex — and how these properties relate to the 
response or reaction to painful sensations or experience. The data indicate 
that considerable modification of somatic input occurs at the first central 
relay nucleus in the trigeminal system. The masking of one sensation by 
another is related to the interaction between sensory stimuli and appears to 
involve the selection and filtering of input at many levels of the neuraxis . 
Modification of oro-facial pain involves such sensory interactions in the 
trigeminal system and the present studies offer physiological data that may 
explain these phenomena. 



Other neurophysiological studies suggest that specific visual input is pro- 
cessed in middle suprasylvian gyrus, an association or polysensory area of 
cat cerebral cortex. The affective component of pain derived from the inter- 
action of past painful experience and the perception of new pain is probably 
mediated in part by an alerting system projecting through medial thalamic 
structures and association areas of the cerebral cortex. This interaction 
requires that specific information about a painful stimulus, such as its 
shape, location in space, etc., be retained in polysensory areas of the 
central nervous system. 

In investigating the biochemical factors initiating and controlling morpho- 
genetic development of Dlctyostelium discoideum , the main effort is concerned 
with those parameters affecting the rate of differentiation but not the 
quality. Based on the work to date it seems very likely that the ribose 
mononucleotides which accumulate in the cells only at the onset of differ- 
entiation provide a triggering message which starts the process. The mecha- 
nism by which this effect is mediated is currently under investigation. The 
demonstrated role of steroid metabolism in controlling the rate of morpho- 
genesis also requires further clarification. 

The multifaceted study of the environmental control of the cellular physiology 
of the caries-conducive streptococci has been quite fruitful. A coupling of 
phosphate accumulation to glucose fermentation and the control of this coup- 
ling by the environmental hydrogen ion concentration represents a new funda- 
mental physiological phenomenon never described previously. Another unstis- 
pected finding is the demonstration of massive inorganic polyphosphate 
accumulation by these bacteria. This last finding has important implications 
for any model to be constructed of the dynamics of caries production. The 
bacteria may represent an open-ended compartment for the sequestration of 
tooth phosphate. 

Research on parameters of carbohydrate utilization in these streptococci are 
relevant to understanding the propagation of the carious lesion in two ways. 
The finding that glucose is predominantly metabolized to lactic acid by 
stationary plaque-forming streptococci establishes one of the critical 
chemical conditions which are part of the micro-environment of the tooth 
surface which contributes to the local dissolution of the tooth. The tech- 
nological expertise gleaned in this study will be applied directly to the 
studies on growth and metabolism of these streptococci using the uniquely- 
labeled, high-specific activity sucrose we have synthesized. The sucrose 
will allow the study of the second parametric area of carbohydrate metabolism 
which is the polymerization of a portion of the sucrose into the adherent 
polysaccharide cell coating. It is apparently this dextran coating which 
allows the cells to adhere locally to the tooth surface, where the acid pro- 
duced from the carbohydrate promotes tooth dissolution. The ability to 
select a population of adherent cells from normally non-cariogenic strepto- 
cocci may indicate that the potential for adherence is possessed by a 
variety of oral microbiota. 

Many of the above studies have been substantially aided and some even made 
possible by the application of computer technology and systems analysis to 



O 



'8 



the tasks of acquiring and analyzing the data. New methods have had to be 
devised as required. The major effort has been in the area of calculations 
of liquid scintillation counting data for radioactive tracer studies. 



7'- 



Serial No. NIDR-1 (66) 



1. Office of the Director 
of Intramural Research 

2. Physiology Section 

3. Bethesda, Maryland 



PHS -NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Functional Organization of the Trigeminal Brainstem 
Nuclei in the Cat. 

Previous Serial Number: NIDR-82 

Principal Investigator: Dr. R. Dubner 

Other Investigators: None 

Cooperating Units: None 

Man Years : 

Total: 1 3/4 
Professional: 1 
Other: 3/4 

Project Description: 

Objectives ; 

Somatic sensation from the oro-facial region reaches higher brain 
centers in the thalamus and cerebral cortex via pathways distinct 
from somatosensory projections from all other parts of the body. 
These pathways comprise the central connections of the trigeminal 
system which are also unique in their close proximity to the 
reticular formation of the brainstem. The present studies were 
designed to investigate the functional properties of cell groups 
within trigeminal brainstem nuclei and to study the modifying in- 
fluences of central and peripheral inputs on these cells. 

Methods Employed ; 

Cellular activity in trigeminal brainstem nuclei was studied 
utilizing extra-cellular microelectrode recording techniques in 
cats anesthetized with chloralose or nembutal. Peripheral recep- 
tive fields were located with natural stimuli such as a fine camel 
hair brush, were then stimulated electrically, and the cellular 
responses interacted with peripheral input (flash or click) and 
central input (cerebral cortex) and thalamic stimulation. Precise 



3r: 



Part A (continued) Serial No. NIDR-1 (66) 

electrode localization was determined histologically with the aid 
of a dye-marking technique. The excitability of primary afferent 
trigeminal fibers was tested by placing a stainless steel micro- 
electrode at the junction of the trigeminal nerve and the main 
sensory nucleus in the brainstem. The antidromic compound action 
potential evoked by stimulation of this site was recorded from the 
cut ends of the infraorbital or mental nerve. Conditioning visual, 
auditory, somatic or central stimuli were employed to determine 
the presence of primary afferent depolarization evoked by such 
stimuli. 

Major Findings : 

Excitability changes in the terminals of trigeminal afferent fibers 
were produced by cortical stimulation and by electrical stimulation 
of the face or trigeminal peripheral nerve strands. Light flash 
stimuli produced small and variable excitability changes in these 
terminals. However, stimulation of the optic disc resulted in con- 
sistent excitability changes with a time course similar to the 
other conditioning stimuli. Stimulation of the dura mater or pia 
vessels on the cortical surface also produced primary afferent 
depolarization. Primary afferent depolarization still was evoked 
by stimulation of the optic disc, or the somatosensory areas of 
the cerebral cortex, after contralateral trigeminal nerve section 
proximal to the Gasserian ganglion. 

Significance to Dental Research : 

The data indicate that considerable modification of somatosensory 
input occurs at this first central relay nucleus in the trigeminal 
system. The masking of one sensation by another is related to the 
interaction between sensory stimuli and appears to involve the 
selection and filtering of input at many levels of the neuraxis, 
including the first synapse. Modification of oro-facial pain (e.g. , 
audio analgesia, the rubbing of a painful area, etc.) involves such 
sensory interactions in the trigeminal system; the present studies 
offer physiological data that may explain these phenomena. 

Proposed Course of Study : 

Further research will include studies on pathways of visual and 
auditory input to trigeminal brainstem nuclei. In addition, pat- 
terns of activity initiated by large and small fiber components 
will be studied utilizing computer methodology and the responses 
to these stimuli will be interacted with nonsomatosensory input. 



Part B . Publications Serial No. NIDR-1 (66) 

1. Dubner, R. : Interaction of Peripheral and Central Input in the 
Main Sensory Trigeminal Nucleus of the Cat. Exptl. Neurol . 17: 
186-202, 1967. 



32 



Serial No. NIDR-2 (64) 



1. Office of the Director 
of Intramural Research 

2. Physiology Section 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Interaction of Sensory Stimuli in Association Areas 
of Cerebral Cortex in the Cat 

Previous Serial Number: NIDR-83 

Principal Investigator: Dr. R. Dubner 

Other Investigators: Dr. B. Dow and Mr. F. J. Brown 

Cooperating Units: None 

Man Years : 

Total: 2 1/2 
Professional: 2 
Other: 1/2 

Project Description: 

Objectives ; 

The association areas or "non-primary" projection areas of the cat 
cerebral cortex are responsive to stimuli of polysensory origin 
(e.g. visual, auditory, somatosensory, etc.), and exhibit response 
characteristics different from those of primary sensory areas of 
the cerebral cortex. Our previous studies indicate that cells in 
these association areas, are unequally responsive to different 
stimuli, and that cellular mechanisms are available for the inter- 
action and processing of sensory-specific information. Other 
investigators, however, have suggested that these association areas 
are merely concerned with the "arousal" and "orientation" of the 
animal and do not receive modality-specific input. The present 
studies were designed to investigate further the response character- 
istics of neurons in one association area (middle suprasylvian 
gyrus) to discrete visual stimuli. 

Methods Employed : 

Cellular activity in the middle suprasylvian gyrus of the cerebral 
cortex was studied with microelectrodes in cats anesthetized with 



o -J 



Part A (continued) Serial No. NIDR-2 (64) 

chloralose. Receptive fields and response characteristics of these 
cells were ascertained using moving and stationary light sources 
projected on a tangent screen. Stimuli included circular spots 
(1-8° in diameter) and variously oriented rectangular bars (1/4-2° 
width, 2-6° length), as well as straight edges of variable length 
(up to 20 degrees). 

Major Findings : 

Nearly three fourths of the cells encountered in middle suprasylvian 
gyrus show specific responses to visual stimuli. Three distinct 
response patterns are apparent: 1) Edge detector cells give brisk 
responses to specifically oriented edges moved in preferred. direc- 
tions across sharply delimited positions in their receptive fields, 
and respond poorly to stationary stimuli. 2) Spot detection (? 
light detector) cells fail to respond to moving stimuli, but give 
strong off responses (and sometimes weak on responses as well) to 
properly positioned large spots. Field sizes for these cells 
range from as small as 3-5 degrees up to 20 degrees or more. Small- 
er spots produce weaker (but similar) responses over the same 
receptive field. Binocular facilitation is common, with off 
responses from one eye combining with on/off responses from the 
other eye to give a strongly off field with scattered weaker on 
responses overlapping in the center. 3) Complex center cells give 
nonspecific responses to moving edges, most typically to edges of 
any orientation moving towards the receptive field center (i.e. 
centripetally) . Stationary stimuli produce strong on/off responses 
in the receptive field center (5-20 degrees) and somewhat weaker 
off responses over a more extensive surrounding area (greater than 
40 degrees in some cases). Optimal stimuli have yet to be deter- 
mined for this latter group of cells, but variously oriented bars 
tend to be more effective than spots. 

Significance to Dental Research : 

These data suggest that sensory specific visual input reaches 
middle suprasylvian gyrus, a "non-primary" or association area of 
cat cerebral cortex. The results are at a variance with the 
hypothesis that all the association areas of cat cerebral cortex 
receive identical inputs and are devoid of sensory specificity. 
From our previous results, it is known that neurons in these areas 
respond differentially to multiple sources of input and exhibit 
cellular synaptic patterns which may be important in sensory 
integration. 

The response or reaction to pain appears to involve many levels and 
many parts of the central and peripheral nervous systems. It has 
been suggested that the affective component of pain derived from 
the interaction of past painful experience and the perception of 
new pain is mediated in part by an alerting system projecting 
through medial thalamic structures and association areas of the 



Part A (continued) Serial No. NIDR-2 (64) 

cerebral cortex. This interaction requires that specific 
information about a "painful stimulus" such as its shape, location 
in space, etc., be retained in the central nervous system. The 
present studies indicate that one association area of the cat 
cerebral cortex, the middle suprasylvian gyrus, receives specific 
visual input (and possibly specific auditory and somatosensory 
input) as well as non-specific input form medial thalamic 
structures. This polysensory convergence from multiple central 
and peripheral sources provides the association cortex with inte- 
grative capabilities necessary for participation in sensory 
responses such as pain, taste, and olfaction, where affective 
phenomena play a prominent role. 

Proposed Course of Study : 

Further clarification of the response properties of these cells is 
planned. Efforts will be made to establish the distribution and 
interaction of cells in this cortical region, as well as to deter- 
mine the nature of the input from thalamus, primary visual cortex, 
and corpus callosum. 

Computer methods will be employed to analyze time distributed 
cellular events occurring under conditions of unaltered and altered 
input to association cortex. Plans include "on-line" operation 
using a computer-controlled light source, with new stimulus 
parameters being determined on the basis of feed-back from previous 
responses. 



Part B . Publications 

1. Dubner, R. and Brown, F. J.: Response of cells to restricted 
visual stimuli in an association area of cat cerebral cortex. 
Exptl. Neurol . 20:70-86, 1968. 



Serial No. NIDR-3 (60) 

1. Office of the Director 
of Intramural Research 

2. Physiology Section 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: The Biochemistry of the Differentiating Cellular Slime 
Mold, Dictyostelium discoideum 

Previous Serial Number: NIDR-6 

Principal Investigator: Dr. M. I. Krichevsky 

Other Investigators: Dr. B. M. Chassy 

Cooperating Units: None 



ivfen Years : 




Total : 


2 1/2 


Professional: 


1 1/4 


Other: 


1 1/4 



Project Description: 

Objectives : 

To investigate the biochemical factors initiating and controlling 
morphogenesis of Dictyostelium discoideum . Presently the main 
effort is concerned with the factors affecting the rate of differ- 
entiation but not the quality. Special emphasis is being placed on 
the earliest biochemical changes observable with the onset of the 
morphogenetic process, since the nutritional factors increasing the 
rate of differentiation can be shown to do so during the initial 
period under study. 

Methods Employed : 

All are standard techniques routine to the types of studies herein 
described. 



3B 



Part A (continued) Serial No. NIDR-3 (60) 

Major Findings : 

Ribose mononucleotides accumulate in the cellular slime mold at the 
onset of differentiation (and only then). These materials are 
stimulatory to the rate of differentiation as are the 5' -esters 
when supplied exogenously. The specificity of the mononucleotide 
stimulation of the rate of differentiation in the cellular slime 
mold was found to be complete for the whole molecule. That is, the 
stimulant must consist of purine or pyrimidine base, pentose moiety, 
and a phosphate group. Changing the nature of the purine or pyrim- 
idine or substituting deoxyribose for ribose or moving the phosphate 
from the 5' -position to 3' affected the stimulation only quantita- 
tively. Leaving off one or more parts of the tripartite molecule 
resulted in either loss of stimulation or actual inhibition of the 
rate of differentiation. 

A compound which is known to inhibit steroid biosynthesis in 
mammals was found to inhibit the rate of differentiation in D. 
discoideum . The inhibition could slow the completion of differen- 
tiation from 1.5 days to as much as 7 days. Analysis of cells 
entering the differentiation stage by gas chromatography indicated 
that they already contained high levels of steroid. Therefore, the 
same assay was used to investigate the ability to arrest growth and 
steroid biosjmthesis. It was found that growth was inhibited less 
stringently than is steroid biosjmthesis. The steroid is formed 
after the majority of growth takes place. 

Significance to Dental Research ; 

The phenomena of nongenetic functional changes in cell types such 
as embryological differentiation, microbial spore formation, 
induced enzyme formation, cancerous de-differentiation, aging, etc. , 
are an integral and fundamental part of the life history of all 
organisms. It is desirable to have information regarding the 
biochemical factors influencing the initiation and course of such 
changes, since many aspects of medical phenomena are intimately 
affected by these functional differences among cells and tissues. 
. An example of anomalous differentiation of concern in dental 
research is the cleft palate syndrome. 

Proposed Course of Study : 

Experiments will be performed to determine whether the 2'-, 3'- 
mononucleotides are converted to the 5' -mononucleotides during 
differentiation. Adenosine-5' -monophosphate metabolism will be 
studied to gain insight as to the mechanism of action of the 5'- 
esters. 



-^ 7 



Part B. Publications Serial No. NIDR-3 (60) 

1. Krichevsky, M, I.: Some uses of computers in chromatographic 
technology, present and future. In Automation in Analytical 
Chemistry . New York, N. Y. , I^diad Inc., 1967, 453-455pp. 

2. Krichevsky, M. I. and Love, L. L.: Accumulation of mononucleotides 
in washed suspensions of myxamoebae of Dictyostel ium discoideum . 

J. Gen. Microbiol . 50:15-21 (1968). 

3. Krichevsky, M. I., Zaveler, S. A., and Bulkeley, J.: Computer-aided 
single or dual isotope channels ratio quench correction in liquid 
scintillation counting. Anal. Biochem. 22_:442-464, 1968. 

4. Krichevsky, M. I. and Keyes , P. H. : Considerations in designing a 
system for developing models. Currents in Modern Biology . In press. 



•to 



Serial No. NIDR - 4 (66) 

1. Office of the Director 
of Intramural Research 

2. Physiology Section 

3. Bethesda, Maryland 



PHS -NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Energy Dependent Phosphate Accumulation by 

Streptococci Implicated in Smooth Surface Caries 

Previous Serial No. : NIDR-29 

Principal Investigator: Dr. J. M. Tanzer 

Other Investigators: Dr. M. I. Krichevsky 

Cooperating Units: None 

Man Years : 

Total: 1/2 

Professional: 1/4 

Other: 1/k 

Project Description: 

Objectives : 

The overall goal of this project is an understanding of the 
regulation of the interplay of PO^ metabolism and the formation of 
acid and polysaccharide from sugar by plaque-forming streptococci 
implicated in smooth surface caries. 

The immediate goal of the present phase of study is to establish 
whether these plaque-forming streptococci are capable of forming 
polyphosphates. The conditions which regulate the formation of 
polyphosphates by these cells will be delineated in respect to the 
critical variables of in^ vivo plaque. 

Methods Employed : 

Streptococcal cultures were maintained, grown, harvested, and 
experimentally incubated as stationary phase cells. Cells were 
exposed to o--^2pQ challenged with glucose, and the accumulation 
of P-^^ monitored. These techniques were previously detailed 
(Tanzer, Annual Report 1967). 



Part A (continued) Serial No. NIDR-4 (66) 

Two basic experimental stratagems were employed: 

1. In order to determine whether phosphate accumulation was 
directly coupled to the accumulation of exogenous carbon- 
containing fragments, cells were challenged with glucose-U-^^C 
and the simultaneous uptake of -^^P and ■'■ C was monitored. 

2. In order to determine whether phosphate was accumulated into 
a pre-existing carbon-containing cellular pool, cellular 
carbon-containing pools were prelabeled by growing the cells in 
glucose-U-l^C. Then 32p uptake was monitored when cells were 
challenged with carrier glucose. 

After the period of phosphate uptake in response to challenge 
by exogenous glucose, cells were either sequentially extracted 
with cold and hot HClOi^^ or they were extracted with NaOH. 

Cell extracts were analyzed by column chromatography on 
Sephadex G-25 and on Dowex-1 -formate. Fractions eluted from 
the columns were counted for 32p and l^C radioactivity. '^P 
counts were resolved from l^C counts by a computerized quench 
correction technique. 

A thin layer chromatographic technique was developed which 
permitted separation of polyphosphates of different size. 
Techniques were developed which permitted elution of -^^p 
containing fragments independent of ^^C containing fragments. 

Maior Observations: 



Li 



1. About 50% of the phosphate accumulated by the organism under 
study is not extractable in cold HCIO/^. 

2. Most P incorporation into cells appears not to require 
simultaneous incorporation of ■'•^C into the same cellular pools. 

3. A large fraction of the P incorporated into cells passes into 
a pool which contains no pre-existing l^C. Observations 2. and 
3. are consistent with the hypothesis that this caries-active 
streptococcus is capable of synthesizing a large quantity of 
polyphosphate -- a polymer of phosphoryl residues, free of 
carbon. 

4. By thin layer chromatography approximately 20% of the total 
■^^P incorporated by the cells behaves as if it were poly- 
phosphate of 16 phosphoryl residues or greater in dimension. 
This permits the tentative conclusion that 20% of the 32p 
accumulated exists in phosphate poljmiers of at least 1500 
molecular weight units. 



/. n 

'7 U 



Part A (continued) Serial No. NIDR-4 (66) 

5. No polyphosphate appears to be exclijded from the void volume 
of Sephadex G-25. Hence, no polyphosphates are apparent with 
a greater molecular size than about 5000 molecular weight units. 

Conclusions 2. through 5. must be viewed as tentative and subject 
to confirmation by subsequent experimentation. 

Significance to Dental Research ; 

Streptococci of the variety under study have been shown to form 
plaque on the smooth surfaces of teeth. Smooth surface carious 
lesions occur only under such bacterial growths. What are the 
aspects of metabolic behavior of the plaque -forming microorganisms 
which allow their establishment, survival and growth on the tooth 
surface, and their invasion of the tooth substance? Does the tooth 
modify the metabolic activity of these etiologic agents? These 
issues have come into focus. If answers can be achieved in respect 
to the demands, capacities and regulation of metabolic activity of 
these cells, one might be able to more fully comprehend plaque- 
associated disease and design more meaningful interceptive measures. 
This work is addressed to these ultimate goals. 

Some specific questions and points of significance can now be 
crystallized. From previous data (Tanzer, Annual Report 1967) it 
was seen that these plaque-forming streptococci accumulate phosphate 
at very high rates. We now know that the flux of phosphate across 
the cell membranes of these cells is unidirectional. Hence, phos- 
phate in the environment of these cells not only moves into the 
cells at very high rates but it does not leak out. These cells, 
living on the surface of enamel, therefore, constitute a sink, 
continuously renewed, for the movement of phosphate. 

Thus, a critical question arises in view of the avidity of these 
cells for phosphate and the unidirectional ity of phosphate movement: 
Mist these cells draw upon the phosphate pool of the tooth in order 
to meet their demand for phosphate? 

The tentative establishment of the synthesis of polyphosphate by 
these cells directs attention to at least two points. 

1. Cellular synthesis of polyphosphates is classically an event 
which occurs only in non-growing (stationary) cell populations. 
The synthesis of polyphosphate allows cells to accumulate 
phosphate throughout their life cycle. 

2. The presence of the highly negatively charged polyphosphate 
within the plaque cell may effectively convert it into a potent 
cation exchange resin. This effect could be important in 
either of the processes of decalcification of the tooth in 
caries or in mineralization of the plaque in calculus formation. 



Part A (continued) Serial No. NIDR-4 (66) 

Proposed Course of Study : 

The project to date has employed a washed non-growing cell system. 
This work will be continued in this model and extended in a 
direction such that the behavior of the intact plaque resident on 
the tooth surface will be approximated. Preliminary studies have 
investigated the feasibility of proceeding in this direction. 

Preliminary experiments: 

1. Growth of intact plaque. Quantitative measurements of in vitro 
plaque growth on a smooth inert surface have been shown 
feasible. Under the conditions employed growth of plaque is 
linear rather than exponential and is at a rate of 5 mg wet 
weight/day/cm^ surface area. Since the dry weight of this 
plaque and its total DNA content increase in parallel with the 
wet weight, there appears to be no change in the plaque 
composition with time. The linear rather than exponential 
growth of this system attests to the diffusion limitation of 
behavior of intact plaque. 

2. Fowler has produced easily manipulated disks of hydroxyapatite 
which can serve as conveniently symmetrical "teeth" of known 
composition. Plaque growth on such disks is rapid and produces 
demineralization of the disk. We therefore seem to be in a 
position in which we can characterize the differences, if any, 
of plaque growth on an inert surface in contrast to plaque 
growth on a "tooth". We are also in a position in which we can 
define the dissolution rate of "tooth" under plaque and the 
associated rates of acid production and PO^ accumulation by the 
plaque. 



Part B not included 



kl 



Serial No. NIDR-5 (67) 

1. Office of the Director 
of Intramural Research 

2. Physiology Section 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: The Metabolic Fate of Glucose Catabolized by Stationary 
Phase Streptococci Implicated in Smooth Surface Caries 

Previous Serial No. : None 

Principal Investigator: Dr. J. M. Tanzer 

Other Investigators: Dr. M. I. Krichevsky and Dr. P. H. Keyes 

Cooperating Units: None 

Man Years : 

Total : 1/4 . 

Professional: 1/4 

Other: 

Project Description: 

Objectives : 

It seems unlikely that frequently ingested sugar solutions would 
support a significant rate of plaque growth. It is also unlikely 
that oral fluids would supply nutrients required for rapid plaque 
growth. Hence, it is probable that plaque in vivo is growing at 
very slow rates. Some indirect experimental data support this 
assumption. 

Strangely, almost no data is available concerning the metabolic 
fate of catabolized sugar by plaque-forming streptococci. The 
information which is available, however, describes rapidly growing 
cells. In view of the purported role of acid products of bacterial 
metabolism in the etiology of caries, the goal of this project was 
a definition of the acid products of sugar fermentation by station- 
ary phase plaque-forming streptococci. 



Li3 



Part A (continued) Serial No. NIDR-5 (67) 

Methods Employed : 

Streptococcal cultures were maintained, grown, harvested and 
experimentally incubated with glucose-l^C as stationary phase 
cells. These techniques were previously described (Tanzer, Annual 
Report, 1967). The products of fermentation were separated by 
Dowex-1 -formate anion exchange chromatography and by partition 
chromatography upon Celite-535 columns. Fractions eluted were 
assessed for ^ C radioactivity. 

Major Findings : 

1. Lactic acid comprises about 90% of the fermentation products 
of glucose. 

2. Acetic acid and CO2 are virtually absent as fermentation 
products. 

3. About 67o of the metabolic products of fermentation are 
represented by a carboxylic acid(s) of 3C or greater chain 
length. 

^■. No appreciable ^ C becomes incorporated into cell substance' 
other than as mono and diphosphate esters of the Embden- 
>fyerhof path or intracellular amylopectin-type polysaccharide. 
This datum supports the conclusion, reported independently 
(NIDR-4) in this document, that these cells form polyphosphates. 

Significance to Dental Research ; 

This work establishes that the acid produced by stationary plaque- 
forming streptococci is predominantly lactic acid. It thereby 
establishes one of the critical chemical conditions which exists on 
the surface of the tooth. It is a condition which is germane to 
the definition of the environment contributing to dissolution of 
the tooth. 

One bonus results from showing that 907o of the products of sugar 
fermentation by plaque-forming streptococci is lactic acid. Since 
it is well established that 2 net moles of ATP are produced in the 
conversion of 1 mole of glucose to 2 moles of lactic acid, at least 
1.8 moles of ATP are produced per mole of glucose catabolized by 
these cells. If one knows the rate of acid production by such 
cells, that rate is numerically equal to the number of net ATP 
moles produced in fermentation. Thus, by measuring the acid 
production rate, one can evaluate the energetic concomitant of any 
energy consuming process under investigation. 



't^ 



Part A (continued) Serial No. NIDR-5 (67) 

Proposed Course of Study : 

It is felt that the goal of this project has been achieved. Thus, 
there are no present plans for continuation of this study. 



Part B not included- 



45 



Serial No. NIDR-6 (67) 

1. Office of the Director 
of Intramural Research 

2. Physiology Section 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Growth and lyfetabolism of Cariogenic Streptococci 

Previous Serial No. : None 

Principal Investigator: Dr. Bruce M. Chassy 

Other Investigators: Dr. M. I. Krichevsky and Dr. J. M. Tanzer 

Cooperating Units : None 

Man Years : 

Total : 1 1/4 
Professional: 1 
Other: 1/4 

Project Description: 

Objectives : 

The overall goal of this project is to provide an understanding of 
the relationship of diet, growth, and culture conditions in the 
formation of plaque and caries by human oral cavity streptococci. 

Specific goals of this project are 1) to synthesize (glucose-l^C) 
sucrose and (f ructose--'-^C) sucrose 2) determine the fate of sucrose 
(using the isotopically labeled sucroses synthesized above) in 
cariogenic streptococci under a variety of growth conditions (i.e. 
under varying conditions of pH, concentration of substrate, temper- 
ature, time of incubation, etc.) and 3) to evaluate the effect of 
exogenous ly supplied dextranase on sucrose utilization. 

Methods Employed : 

Sucrose (isotopically labeled in either the glucose or fructose 
residue) is synthesized from uniformly labeled sucrose by a combina- 
tion of chemical and enzymatic methods. These specifically labeled 
sucroses have heretofore not been available at high purity and 
specific activity. 



^6 



Part A (continued) Serial No. NIDR-6 (67) 

Bacterial growth experiments, on a pure strain of cariogenic 
streptococci, have been performed in a temperature controlled pH- 
stat. Uptake of ^^po^^ production of DNA, acid, and polysaccharide 
has been followed by chemical and instrumental methods. 

Major Findings ; 

1. Synthesis of labeled sucrose has been improved over older 
methods . 

2. It is possible to use controlled growth instrumentation as a 
tool in investigating the growth of cariogenic streptococci. 

3. A dextran-producing cariogenic streptococcus has been found to 
grow linearly rather than exponentially on sucrose. This 
organism grows in the expected exponential manner on other 
carbohydrates including mixtures of glucose and fructose. 

Significance to Dental Research ; 

An approach to effective control of the causes of smooth surface 
caries necessitates a basic understanding of metabolism, growth and 
plaque formation by caries specific bacteria. An examination of 
the unique role of sucrose in its effect upon the growth character- 
istics, dextran (polysaccharide) formation, and plaque formation 
should suggest fundamental methodology for oral hygiene as well as 
contribute to our understanding of the complex interaction of 
various components present in the oral cavity. 

Proposed course of Study : 

1. To delineate the sucrose metabolism of a cariogenic 
streptococci by existing methodology. 

2. Study the effect of various factors on growth and development 
of cariogenic streptococci (i.e., dextranase, pH, inhibitors). 

3. Develop an automated, on line, computer controlled evaluation 
system that will monitor and control various experimental 
parameters. Such a system, applicable to bacterial growth and 
enzymology, will allow rapid and precise experimentation on 
some of the factors mentioned above. The system will be used 
to develop a mathematical model to help understand linear 
growth as well as growth and acid production under the conditions 
found in the mouth. The introduction of modern data gathering 
and reduction techniques will not only greatly facilitate this 
study but should be of general interest to the biomedical 
research community. 



Part B not included 



i*7 



Serial No. NIDR-7 (67) 

1. Office of the Director 
of Intramural Research 

2. Physiology Section 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Selection of Plaque-Forming Mutants of Streptococci. 

Previous Serial Number : None 

Principal Investigator: Dr. J. M. Tanzer 

Other Investigators: Mr. R. M. McCabe 

Cooperating Units : None 

Man Years: 

Total: 1/4 

Professional: 1/4 

Other: 

Project Description: 

Objectives : 

How do people become infected by caries-active plaque-forming 
streptococci? Infection could conceivably occur from the external 
environment of the host. It could also occur via mutation of 
caries-inactive streptococci to caries-active variants. This 
study's goal was to attempt to select plaque-forming mutants from 
a previously non-plaque-forming population of cells. 

Methods Employed : 

A number of strains of plaque-forming and non-plaque-forming strep- 
tococci were grown in broth containing sucrose. A steel wire was 
introduced to the broth and the wire was transferred daily to a 
tube of uninoculated culture broth. Plaque growth was assessed by 
a semi-quantitative visual method at the end of 3 series of 5-day 
wire transfers. After 3 series of transfers, the microorganisms 
were cultured in broth free of sucrose. 

On return to broth containing sucrose one could test whether experi- 
mentally observed changes were heritable or merely a result of 
enzymatic inductions in the presence of sucrose. 



if8 



Part A (continued_ Serial No. NIDR-7 (67) 

Major Observations : 

1. With non-plaque-formlng bacterial strains 2M2, 2M2R, and 903- 
1600, the passage of a bacteria-covered wire from tube to tube 
In sucrose-containing broth selects the most adhesive fraction 
of a population of cells. 

2. This results In a population with a greater ability to colonize 
on a smooth surface, i.e., to form plaque. 

3. Two conditions appear Important for the selection process: 
the presence of sucrose and a means of preferential transfer 
of more adhesive cells. 

Significance to Dental Research : 

These experiments suggest a possible means whereby plaque-forming 
strains evolve from non-plaque-formlng inhabitants of oral cavity, 
i.e., by natural selection for growth on the tooth's smooth sur- 
faces of more adhesive microorganisms under the influence of sucrose. 

Proposed Course of Study : 

If one were to implant a tagged non-plaque-formlng strain in an 
experimental animal and show that one could select mutants which 
were sufficiently adhesive to form plaque and cause carles — then 
the origin of caries-active microorganisms from previously caries- 
inactive ones would have been demonstrated. 



Part B . Publications 

Tanzer, J. M. and McCabe, R. M. Selection of Plaque-Forming Streptococci 
by the Serial Passage of Wires Through Sucrose Containing Broth. Arch. 
Oral Biol. 13:139-143, 1968. 



h3 



Serial No. NIDR-8 

1. Office of the Director 
of Intramural Research 

2. Physiology Section 

3. Bethesda, Maryland 



PHS-NIH 

Individual Project Report 

July 1, 1967 through June 30, 1968 



Part A 



Project Title: Laboratory Information System 

Previous Serial Number: None 

Principal Investigator: Mr. John J. Wilson 

Other Investigators: None 

Cooperating Units: Division of Computer Research and Technology 

Man Years : 

Total: 1 

Professional: 1 

Other: 

Project Description: 

Objectives ; 

1. To design and develop a computer system capable of on line 
instrument monitoring and data processing. 

2. To investigate the mathematical properties of laboratory 
experiments and develop computer solutions. 

3. To design an automated information retrieval system to allow 
for the formation and utilization of the data bank on microbial 
taxonomy now being developed. 

Methods Employed : 

The techniques of systems analysis and computer programming normal 
to these needs. 

Major Accomplishments : 

1. Computer Time Sharing. 

A study was conducted of the available computer time sharing 
services. The features and costs of each system were evaluated 



50 



Part A (continued) Serial No. NIDR-8 

with respect to the requirements of the Institute. Recommenda- 
tions were made to the Scientific Director for initiation of 
two of the services. These services should be in operation at 
NIDR in May, 1968. 

2. Scintillation Counting. 

A series of programs were designed and written to provide com- 
puter processing of the data from two Packard Tri-Carb scintil- 
lation counters which were acquired last year. 

The programs were written to determine the disintegration per 
minute of one or two isotopes counted in a scintillation cock- 
tail. The programs were written in Fortran IV and are being 
run on the IBM 360/50 computer system, Division of Computer 
Research and Technology, NIH. 

3. Phosphate Uptake. 

A program was written in Fortran IV to determine the phosphate 
uptake and acid production rate for hamster-type plaque forming 
caries-active streptococcus. 

A. Information Retrieval. 

A computerized system was designed to provide personnel budget 
allocation by project number. The program was written by DCRT 
personnel according to design specifications. 

The background obtained in this effort is directly applicable 
to the microbial taxonomy problem. 

5. On Line Computer. 

Process control computer characteristics were investigated. 
The laboratory experiments and instruments to be interfaced 
with an on-line computer were studied. Working with DCRT engi- 
neers and appropriate investigators, the specifications for an 
on-line system were established. A Request for Proposals was 
drawn up and sent to interested computer manufacturers. The 
responses from the manufacturers are now being evaluated. 

Proposed Course of Study ; 

1. On Line Applications. 

Systems are being designed for closed loop experimentation. 
Two areas are receiving immediate attention: 

a) Computer control of visual stimuli in neurophysiological 
experiments for determining optimum responses during 
micro electrode recordings. 

b) Computer control of multiparametric growth experiments 
with cariogenic streptococci. 

51 



Part A (continued) Serial No. NIDR-8 

2. Experimental Data Processing. 

Further applications and improvements of computerized mathe- 
matical techniques to processing experimental data will be 
investigated. More sophisticated mathematical models will be 
developed to improve liquid scintillation counting programs. 
The investigation of better methods of resolving overlapping 
peaks in chromatographic data will be continued in collabora- 
tion with mathematicians of the Division of Computer Research 
and Technology. 

3. Information Retrieval. 

A system of computer programs is being developed to handle 
taxanomic data or general administrative information reports. 



Part B not included 



52 



Report of the Laboratory of Microbiology 
National Institutes of Dental Research 
Summary Statement 

Contemplation of shifting fashions in dental research indicates that it is 
timely to recollect continuing problems of the first importance, which have 
justified the principle premises on which programs of the Laboratory of 
Microbiology have contributed consistently and meaningfully during the past 
decade. Dental caries and chronic periodontitis continue throughout the 
world to constitute by far the major problems of dentistry. Despite much 
progress toward understanding their mechanisms, and despite development of 
at lease partially effective control measures, their elimination does not 
seem close to hand. As infectious diseases, both of these conditions result 
from complex interactions between an indigenous oral microbiota, the reac- 
tivity of the host tissues as influenced by innate and acquired resistance, 
and the external environment as exemplified by macro- and micronutrients 
and socio-economic parameters. A major commitment to oral microbiology 
therefore continues to be an essential component of dental research. But 
to be meaningful, study of oral microorganisms per se must be integrated 
into its ecological context, that is, the complex of host-parasite inter- 
actions. Such a comprehensive approach prospers best when infectious 
disease processes are investigated in organizational and geographic propin- 
quity to such cognate areas as systematic microbiology, microbial physiology, 
immunology, and virology. Applied programs in collaboration with clinical, 
pathological, and epidemiological units are an essential component. But 
distinction between basic and applied studies eludes definition; they form 
a continuum, and dichotomous arrangements disadvantage both. 

Our programs therefore have comprised studies directly concerned with 
mechanisms of experimental and clinical dental caries and periodontal dis- 
ease; taxonomic studies aimed at better definition of members of the oral 
microbiota, to facilitate their identification and study of their ecologic 
habits; physiological studies focused on regulatory mechanisms controlling 
phenotypic expression of genotypic microbial potentialities, particularly 
intermediary carbohydrate metabolism ending in lactic and other acids, 
which are generally conceded to be the demineralizing agents in dental 
caries; immunological studies ultimately relevant primarily to periodontal 
disease, concerned on the one hand with local defenses peculiar to the 
oral cavity and on the other hand with immunopathological reactions 
affecting the oral tissues; and virological studies centering on factors 
influencing latent, persistent, and recurrent infections of the oral soft 
tissues. These investigations have entailed numerous collaborative projects 
with other laboratories and branches. 

Dental Caries . Evidence continues to accumulate that in normal germbearing 
hamsters and rats the predominant cariogenic bacteria comprise a distinc- 
tive group of anaerobic streptococci. This year attention has been con- 
centrated on the production by these bacteria of extracellular polysaccha- 
ride (dextran) specifically from sucrose. This polysaccharide seemed to 
contribute to development of caries because it was responsible for adherence 



53 



of the streptococci to teeth in the form of what is called dental plaque. 
Accordingly, it seemed logical to expect that application of a dextran- 
hydrolyzing enzyme (dextranase) should prevent plaque formation, remove 
preformed plaque, and reduce the initiation of caries by thus reducing 
the accumulation of cariogenic bacteria. Purified dextranase derived from 
a mold (Penicillium funiculosum ) was made available through the cooperation 
of scientists at Merck and Company. When added to an ±n vitro culture 
system, the dextranase preparations prevented plaque accumulation and 
disintegrated preformed plaque. When the dextranase was incorporated in 
the diet and drinking water of hamsters on a cariogenic regimen, plaque did 
not accumulate and caries did not develop. These observations are most 
encouraging for a new approach to control of dental caries in humans. Field 
trials are pending to measure prevention of both plaque and caries by use 
of dextranase preparations. 

Available evidence indicates that plaque accumulation requires dextran of 
high molecular weight. For example, incorporation of low molecular weight 
dextran in the diet and drinking water of hamsters prevents plaque and 
caries by what amounts to competitive or feed-back inhibition of the dextran- 
sjmthesizing enzyme, whereby production of high molecular weight dextran is 
suppressed. Likewise many dextran- forming strains of streptococci, and 
similar organisms of the genus Leuconostoc , are noncariogenic presumably 
because their dextran is not of the high molecular weight, insoluble, 
adherent type. These observations may point the way to an alternative 
method of caries control by preventing dextran synthesis. 

Investigators of experimental caries in rats have long been puzzled, and 
often hampered, by the necessity of using only weanling animals, for the 
adults become insufficiently susceptible. A possible explanation now 
comes from collaborative studies with the University of Zurich on the teeth 
of gnotobiotic rats. When newly erupted, these teeth have hypomineralized 
areas at the bottom of the sulci where caries develops best in rats. As 
germfree rats mature, these areas become normally mineralized, and suscep- 
tibility to caries decreases. In rats monoinfected with a cariogenic 
streptococcus, however, normal mineralization of these areas does not occur; 
instead, progressive demineralization develops. 

Use of a recently developed special transport medium, which keeps bacteria 
viable for many days but does not permit their multiplication, has greatly 
extended the range of our studies in epidemiological bacteriology. Samples 
of plaque were collected from groups of about 100 children each in (a) 
Cheektowaga, New York, a low-fluoride area with average caries incidence; 
(b) Charlotte, North Carolina, which has adequately fluoridated water and 
low caries incidence, and (c) two villages in Colombia, South America, with 
low fluoride water and a high caries rate. The samples were analyzed in 
Bethesda for cariogenic- type streptococcio The results confirmed previous 
preliminary findings indicating that the streptococci are more numerous and 
widespread where caries is more prevalent. However, repeated topical 
application of a fluoride gel by means of a mouthpiece during a two-year 
period (Cheektowaga study) did not reduce the streptococcal census. 



5^ 



Apparently these bacteria decline in a community only after a prolonged 
period of low-caries incidence. 

This year the interplay of genetic and dietary factors in caries was 
studied in the classic Hunt-Hoppert caries-resistant and caries-susceptible 
strains of rats. These strains have been genetically stabilized by 25 
years of selection and inbreeding. In these experiments, the microbial 
factor was equalized as well as possible by housing caries-active rats in 
the same cages with test animals. Differences in caries incidence could 
therefore be attributed to genetic and dietary factors respectively. 
Surprisingly, the Hunt-Hoppert strains have been classified as "resistant" 
or "susceptible" using only diets conducing to caries in the sulci of the 
teeth. When tested on a diet conducing to caries both in sulci and on 
smooth surfaces, the "resistant" strain did indeed develop few sulcal 
lesions, but it was highly susceptible to smooth surface caries. Paradox- 
ically, the "susceptible" strain on the same diet developed less smooth 
surface caries than did the "resistant" strain. The total scores (sulcal 
plus smooth surface lesions) were the same in the two strains, but the 
ratios of the two tj^es of lesion differed. At least part of these results 
can be explained by the fact that the diet conducing to smooth surface 
does so primarily by conducing to accumulation of plaque on the sm.ooth 
surfaces, thereby in effect enhancing the bacterial challenge and also 
facilitating transmission of cariogenic organisms from animal to animal. 
On diets where the caries is confined to the sulci, the bacteria tend to 
remain impacted, and transmissibility is greatly reduced. Clearly resist- 
ance and susceptibility to caries are not absolute genetically determined 
attributes of the host but must be defined relative to the bacterial 
challenge and the modifying effects of different diets. 

Periodontal Disease. Previous studies had shown that antigens, _i.e. , 
substances of high molecular weight, could be absorbed from the normal 
gingival pocket of rabbits, inducing antibody formation systemically and, 
locally, an allergic inflammatory reaction histologically very similar to 
that seen in human periodontal disease. It was suspected, however, that 
antigen absorption required a preliminary superficial break in the integ- 
rity of the crevicular epithelium. Using an improved irrigation procedure 
that does not per se irritate the pocket tissues, it has been shown that 
endotoxins and other antigens, which would be expected from bacteria in 
a periodontal pocket, do indeed not penetrate intact crevicular epithelium. 
When the pocket lining was ulcerated preliminarily, however, prolonged 
irrigation with concentrations of endotoxin as low as 0.01 |-ig per milliliter 
resulted in sufficient penetration to initiate inflammation in adjacent 
lymph nodes, with subsequent appearance of antibody- forming cells. Further 
study using direct injection of known quantities of endotoxin showed that 
with minute doses intramuco sally, antibody- forming cells appeared only in 
the regional lymph nodes. Following similar minute doses intravenously, 
antibody- forming cells were found only in the spleen. With sufficiently 
large doses of endotoxin by either route, immune response was general and 
antibody appeared in the serum. These results suggest that with the small 
doses of bacterial antigens that are estimated to be available from bacteria 
in the gingival sulcus, both antibody response and immunopathological reac- 
tions may remain quite local. 

3 55 



Search for bacterial products that might initiate superficial alteration 
of crevicular epithelium continued to focus on ammonia and hydrogen sulfide. 
Detailed histopathological and electron microscopic description has been 
made of the effects of these substances on corneal epithelium. Using the 
innocuous irrigation system mentioned previously, it was found that irri- 
gation of rabbit gingivae and lip mucosa with hydrogen sulfide in con- 
centrations as low as 0.01 molar produced edema, erythema, and increased 
vascular permeability. Histopathological changes resembled those observed 
previously in cornea. 

A filamentous bacterium designated as Odontomyces viscosus is the principal 
etiological agent of a periodontal pathosis in hamsters, which has been 
studied extensively in this laboratory and in the Laboratory of Histology 
and Pathology. A key feature of this pathosis is the accumulation subgingi- 
vally of large masses of gelatinous plaque. At least part of this plaque 
seems to be attributable to an extrabacterial polysaccharide (levan, 
comparable to the dextran produced by cariogenic streptococci) , but the 
evidence indicated that something else was involved. Since in certain 
culture media 0. viscosus produces a ropy, slimy growth not due to levan, 
such growth was analyzed for mucinoid factors, which have been identified 
as a complex of ribonucleic acid and protein. The possible role of such 
complexes in plaque prodromal to both periodontal disease and dental caries 
merits further study. 

A number of bacterial strains resembling 0. viscosus have been isolated from 
human gingival sulcal accumulations, but it has proved difficult to implant 
them in the mouths of hamsters, as a test of their ability to induce perio- 
dontal disease. Attention was turned to possible dietary parameters 
affecting implantation. Initially it was thought that a diet high in 
sucrose was essential for the hamster pathosis, but it has been found that 
other carbohydrates, such as glucose and starch, work as well. Preliminary 
data indicate that a starchy diet conduces to implantation of 0. viscosus 
of human origin in hamsters. On this regimen, some of these strains have 
produced the characteristic periodontal pathosis in hamsters. This system 
promises to facilitate greatly a long-term study in progress of oral 
filamentous organisms and periodontal disease in a stable institutional 
population, where parameters such as diet and oral hygiene can be controlled. 

Systematic Bacteriology . Last year we reported initiation of a long-term 
project in numerical taxonomy, primarily of oral bacteria, in collaboration 
with the Division of Computer Research and Technology, NIH, the trustees 
of Bergey's Manual of Determinative Bacteriology and microbiologists at 
Georgetown University, University of Maryland, and the American Type Culture 
Collection. Word of this project seems to have spread spontaneously through 
the bacteriological community and it has aroused what might well be called 
an exponentially rising curve of interest. With a view to making this 
program as comprehensive as possible, therefore, this year has been devoted 
to perfection of a data protocol defining some 250 parameters applicable 
to any of the known groups of bacteria. This phase of such a program is 
most critical, for if the most meaningful parameters are not selected, if 

' 56 



the questions are not phrased unambiguously, and if test conditions are not 
defined precisely, a consensus of respondents can not be obtained and 
mathematical analysis of the information is futile. From present indica- 
tions, it does not seem fanciful to envision NIDR and NIH as a world center 
for general microbial taxonomy. 

This laboratory has a continuing interest in species of Veillonella . a 
genus of gram negative anaerobic cocci, because they are the second most 
numerous bacteria in the human oral cavity. Study has now been essentially 
completed of organisms officially designated as Veillonella renif ormis , but 
anomolous in a number of respects. Briefly, these organisms stain variably 
by Gram's method and they can utilize carbohydrate, though to a limited 
extent. Primarily they are fermenters of amino acids, principally glutamic 
acid, which they convert mostly to butyrate, acetate, carbon dioxide, 
hydrogen, and ammonia. When these data were assembled, it became evident 
that they fit equally well a group of presumably gram positive bacteria 
officially recognized as Peptococcus (Micrococcus ) aerogenes. Tests of a 
number of strains of the latter, provided by other investigators, confirmed 
this similarity. The question of reaction to the Gram stain was resolved 
by electron microscopy in collaboration with the Laboratory of Histology 
and Pathology. These organisms have the multilayered outer cell wall 
characteristic of gram negative bacteria. Definitive taxonomic disposition 
of these bacteria is under negotiation by the several investigators concerned. 

Bacterial Physiology . It is now possible to present a quite complete 
formulation of how lactic acid production is regulated in one species that 
has been under study, namely, Butyribacterium rettgeri . Regulation in 
this bacterium is essentially repressive, that is, it depends on the fact 
that the chief energy mediator of the cell (adenosine triphosphate, ATP) 
inhibits the enzyme lactic dehydrogenase (LDH) , which irreversibly converts 
pyruvate to lactate. This inhibition provides the cell a mechanism to 
modulate the flow of carbon compounds according to the demand for biosyn- 
thesis and the available supply of ATP needed to energize biosynthesis. 
Briefly, when ATP is low, carbohydrate is glycolyzed to lactate with con- 
current increase in ATP but not of pyruvate for biosynthesis. With 
accumulation of ATP, LDH is inhibited, and continued glycolysis supplies 
pyruvate rather than lactate. Pyruvate and ATP are then consumed in the 
synthesis of amino acids and other structural components, glycolysis to 
lactate is resumed, and so on. Experiments with purified LDH from B. 
rettgeri , too detailed to recount here, have made possible formulation 
of a molecular model explaining the inhibition of LDH by ATP. Essentially, 
the catalytic function of this enzyme is regulated by means of alterations 
of its three-dimentional structure which are mediated by interactions with 
ATP or its coenzyme. In the normal enzymically active configuration, the 
enzyme is in reversible combination with the necessary coenzyme and can 
effect conversion of pyruvate to lactate. In allosteric configuration, 
the enzyme combines r ever sibly with ATP, and combination with coenzyme is 
impossible. 



57 



In streptococci, the regulation of lactate production is positive in 
character. That is, LDH in these organisms is normally inactive and requires 
specific activation by critical levels of fructose diphosphate (FDP) , an 
early intermediate in the glycolytic scheme. (coincidentally, FDP represses 
a lactate degrading system.) Present evidence indicates that FDP combines 
with LDH and induces a configurational change to create a site on the LDH 
molecule suitable for combination both with coenzyme and substrate (pyruvate). 

Rather different regulatory mechanisms seem to operate in streptococci 
grown on substrates other than glucose, notably malate. In this case, the 
malic enzjrme, which initiates utilization of malate by the so-called shunt 
pathway, is inhibited by ATP, FDP, and 6-phospho-gluconate. Though many 
details need to be determined, these facts indicate that in this case 
regulation is essentially repressive, as it is in B. rettgeri . 

Immunology. Research in the Immunology Section concentrated mainly on the 

role of serum complement (G ' ) as a mediator of biological effects of 

bacterial endotoxins and on factors controlling the phenomenon of immuno- 
logical tolerance. 

Collaborative studies with investigators at the Johns Hopkins Medical 
School defined the reactions of endotoxin with C in normal sera and 
showed that at least two of the biological actions of endotoxin are mediated 
by this reaction. Classically, C has been considered as an adjuvant to 
the combination of antigen with antibody, with "fixation" of C' and sub- 
sequent manifestations such as lysis of cells bearing the antigen, enhance- 
ment of phagocytosis, and production of anaphylactic reactions. Recent 
studies, however, have shown that complement mediates a variety of physio- 
logical phenomena not involving any antigen-antibody reaction. In the 
case of endotoxin, incubation with fresh normal sera results in the util- 
ization of components C '3, 5, 6, 7, 8 and 9 but not of C'1,4, and 2. In the 
clasical reaction with antigen-antibody complexes, fixation of C'1,4, and 
2 is a necessary forerunner of the reactions with the remaining 6 components, 
in the order indicated. The consumption of (C '3 ,5 ,6,7 ,8, and 9) by 
endotoxin is paralleled by a release of anaphylatoxin, a factor which 
in vivo brings about smooth muscle contraction and increased capillary 
permeability, presumably by releasing histamine. The reaction of endotoxin 
with normal serum also liberates a chemotactic factor which causes poly- 
morphonuclear leukocytes to migrate into its vicinity. These several 
reactions are directly applicable to explain the production of acute 
inflammation by endotoxin, which is the ultimate concern here. It remains 
to ascertain whether under these conditions anaphylatoxin and chemotactic 
factor result from breakdown of endotoxin by complement components, or 
vice versa. 

Increasing general interest in the feasibility of tooth transplantation 
prompted initial studies of some transplantation phenomena. As is well 
known, unless the transplanted tissue is autogenous or isogeneic, the 
recipient rejects it sooner or later by an immunological response of the 
delayed, cell-mediated hypersensitivity type. Accordingly, much effort 
has gone into the use of immunosuppressive agents, of which antilymphocyte 



58 



serum (ALS) currently seems promising. Unfortunately, ALS itself is foreign 
to the recipient and soon removed by immune elimination. The question 
was asked whether the immunosuppressive action of ALS could be prolonged 
if the recipient had been made immunologically tolerant. ALS was prepared 
by injecting mouse lymphocytes into rabbits. This ALS was administered 
(a) to untreated mice and (b) to mice rendered immunologically tolerant 
by prior injections of normal rabbit gamma globulin. Skin horaografts 
were then applied to the mice. Those on the immunologically tolerant 
animals persisted much longer, thus confirming the test hypothesis. 

Previous collaborative studies with the Virology Section had shown that 
chronic virus infection, using lactic dehydrogenase virus (LDV) , acted 
as an immunological adjuvant in mice, for it raised the normal gamma 
globulin level and enhanced the antibody response to a heterologous 
antigen. Extension of the experiments has shown that such virus infection 
has the opposite effect on a cell-mediated immunological reaction of the 
delayed hypersensitivity type, namely, the so-called graft vs. host reaction. 
In this experiment, cells extracted from mouse spleens are injected into 
other mice. Ordinarily, the recipients develop a marked enlargement of 
the spleen within about 48 hours. The reaction can be quantitated simply 
by removing and weighing the spleens. In mice infected with LDV, such 
splenomegaly did not develop, indicating that the virus acted as an 
immunosuppressive agent. 

Virology. As a corollary to the foregoing experiments, the effect of 
virus infection on immunological tolerance was studied. Mice were inoc- 
ulated with doses of human gamma globulin (HGG) such that (a) they did 
not develop antibody to HGG and (b) they did not develop antibody to 
subsequent doses of HGG which would induce antibody in untreated mice. 
That is, the mice were rendered immunologically tolerant to HGG. When 
the mice were subsequently infected with LDV, however, this tolerance 
was "broken" and antibody was produced forthwith. Other means, such as 
injection of endotoxin, have been shown to break tolerance but this is 
the first time a virus infection has been shown to do so. These obser- 
vations raise the question whether some virus infections might not convert 
the host's normal tolerance of his own tissues to auto-antibody formation, 
and thus play a trigger role in some auto- immune diseases. 

Study of the paradoxical coexistence of infective virus and antiviral 
antibody in the serum during LDV infection has established the principle 
that a virus can remain infective in a virus-antibody complex, that is, 
some antibody combines with virus without neutralizing it. Such virus 
is said to be sensitized, and it is demonstrated by the fact that it is 
neutralized by anti-gamma-globulin homologous to the antiviral antibody 
(by anti-antibody, as it were). On the other hand, sensitized virus is 
markedly resistant to neutralization by addition of more antiviral anti- 
body. This phenomenon affords an explanation for a number of viral 
infections that persist or recur in the face of antibody, such as human 
serum hepatitis, lymphocytic choriomeningitis, and herpes labialis. Viral 
sensitization seems to be a general phenomenon. We demonstrated it also 
with herpes simplex virus (HSV) and a number of laboratories subsequently 
have demonstrated in both _in vitro and _in vivo with a variety of viruses. 

5S 



We have directed attention also to refinement of the "anti-gamma" 
neutralization test vjith sensitized virus with a view of its use as a 
highly specific, highly sensitive serological tool. Details are given 
in the project report of the Virology Section. Briefly, conditions have 
been defined for optimal degree of sensitization, for appropriate concen- 
trations of anti-gamma, and for removal of excess gamma globulin in the 
virus-antivirus mixture. A modification of the standard serum-dilution 
neutralization test permits quantitation of the resistance of sensitized 
virus to further neutralization by antiviral antibody. With a virus that 
can be quantitated by a plaque technique, such as HSV, such resistance to 
neutralization can be measured precisely by the reduction in its rate of 
neutralization (neutralization inhibition kinetics). Using these methods, 
it was shown with both LDV and HSV that resistance to further neutralization 
by antiviral antibody correlates closely with degree of sensitization. 

With these methods it has become feasible to use sensitized virus as an 
indicator for detecting specifically and titrating different classes of 
immunoglobulin, such as gamma G, M, or A, allotypic immunoglobulin, and 
active fragments of immunoglobulin derived enzymically. For example, 
using HSV sensitized with human sera and applying the anti-gamma test 
with anti-human gamma G and anti-human gamma M, respectively, it has been 
found so far that the anti-HSV is always of the Gamma G type, though the 
antibody formed first after a primary infection was expected to be of the 
gamma M class. Conversely, virus sensitized with antiviral antibody of 
a given immunoglobulin class can be used to detect and titrate homologous 
normal anti-immunoglobulin. 

Aside from its potentiality for a renaissance in the theory of the viral 
neutralization reaction, the possible practical application of viral 
sensitization and of the anti-gamma neutralization test are too numerous 
to detail here. One of the most obvious is the diagnostic detection and 
titration of anti-viral antibody in weak sera, or in sera whose antibody 
sensitizes but does not neutralize. Traditionally, neutralization of 
virus by dilutions of serum less than about 1:4 has been regarded as non- 
specific and insignificant. The anti-gamma test in effect increases the 
titer of a serum by as much as 64-fold. Workers elsewhere are already 
utilizing this leverage to demonstrate antibodies to adenoviruses and 
arboviruses in sera previously diagnosed as negative. 



SO 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Serial No. NIDR-9 (59) 

1. Microbiology 

2. Gnotobiotic 

3. Bethesda, Md. 



Project Title: Gnotobiotic Studies of Problems Relating to 
Oral Disease 

Previous Serial Number: NIDR-2 



Principal Investigator: 
Other Investigators: 

Cooperating Units: 



Dr. R. J. Fitzgerald 

Dr. R. H. Larson 

Dr. P. H. Keyes 

Dr. R. J. Gibbons (v.s.) 

Dental Research Institute, University of 
Zurich; Microbiology Research Laboratory , 
Merck Institute 



Man Years: 

Total: 6-1/4 
Professional: 1 
Other: 5-1/4 

Project Description: 

Objectives ; 

This activity is a continuing project in which germfree and 
gnotobiotic animals are used in the study of diseases of the 
oral cavity. 

Methods Employed; 

Microbiological and biochemical laboratory technics are used to 
study microorganisms suspected of involvement in oral diseases 
Germfree, conventional and limited flora animals are used to 
investigate the role of these organisms in disease processes. 



61 



Part A (continued) Serial No. NIDR-9 (59) 

Major Findings; 

1. Maturation of hypomineralized areas in molars of gnotobiotic 
rats (with Dr. Konig, University of Zurich). Newly erupted 
rat raolars show hypomineralized areas of enamel, particularly 
at the base of the fissures, the site most susceptible to 
caries. In germfree animals it was found that the degree of 
hjrpomineralization detectable by stainability with silver 
nitrate begins to decrease about 20 days after eruption of the 
tooth and maturation is essentially complete after 60 days. 

In gnotobiotic rats monoinfected with a cariogenic strepto- 
coccus and maintained on a high sucrose diet, maturation of 
the hypomineralized areas was completely prevented due to 
the superimposed effects of carious demineralization. The 
results are consistent with the clinical impression that 
newly erupted teeth are especially susceptible to caries 
and suggest that one reason for this may be the incompletely 
mineralized state of the enamel in some areas of these teeth. 
The observation that mineralization increases with exposure 
to a non-caries conducive oral environment may explain why 
teeth become more resistant to caries the longer they exist 
in such an environment. 

2. Inhibitions of plaque formation and caries in animals treated 
with dextranase (with Dr. Keyes and Dr. Stoudt, Merck and Co.). 
The streptococci of human or animal origin which have been 
found to induce caries in rodents, characteristically produce 
large amounts of extracellular insoluble dextran when metab- 
olizing sucrose. This dextran has been associated with the 
ability of these organisms to form microbial plaque deposits 

on teeth of humans or animals. If the hypothesis is correct 
that dextran mediated plaque deposition is a key factor in 
caries induction by these organisms, then agents which can 
destroy or prevent the formation of dextran could be effective 
in caries and plaque control. One such agent, the enzyme 
dextranase was administered to albino hamsters which had been 
infected with a cariogenic dextran-producing streptococcus 
and maintained on a high sucrose diet. Depending on the time 
at which dextranase was administered either the complete 
dentition or those teeth which erupted after dextranase 
administration began could be protected from caries. Coronal 
dental plaque deposits were removed and the progress of caries 
which had begun in teeth prior to dextranase administration 
was retarded. 



G2 



Part A (continued) Serial No. NIDR-9 (59) 

3. Inhibition of plaque formation and caries in animals by 
low molecular weight dextrans (Dr. Gibbons and Dr. Keyes) . 
It has been found that low molecular weight dextrans 

(ca 20,000 M.W.) will inhibit the synthesis of insoluble high 
molecular weight dextrans by the enzyme dextran-sucrase of 
cariogenic streptococci. If high molecular v/eight dextrans 
are responsible for plaque formation by cariogenic strepto- 
cocci then conceivably the administration of low molecular 
weight dextran to animals infected with these microorganisms 
should inhibit both plaque formation and caries. It was 
found that both plaque formation and the development of caries 
could be prevented in hamsters infected with dextran-producing 
cariogenic streptococci when the high sucrose diet on which 
they were maintained was supplemented with 16% of dextran of 
molecular weight of 20,000, 

4. Dextran-producing streptococci and caries in gnotobiotic rats> 
The ability to produce extracellular dextrans from sucrose 

is a property which is common to several species of strepto- 
cocci including Leuconostoc mesenteroides , Streptococcus 
sanguis and Streptococcus bovis as well as the cariogenic 
streptococcal strains isolated in our laboratory. Several 
strains of the above mentioned species have now been implanted 
in gnotobiotic rats but have failed to induce progressive 
dental caries lesions. Since both S. bovis and ^. sanguis 
have approximately the same acid producing potential as the 
cariogenic strains, other factors in addition to acid production 
and dextran production appear to be essential for cariogenicity. 
One possibility which is now under study is that tjrpes of 
dextran produced by the organisms may vary in molecular size 
and adhesive properties from strain to strain with consequent 
effects on their ability to form plaque deposits on teeth. 

Significance to Dental Research: 

Caries susceptibility in the rodent model system which we 
employ may be influenced on the part of the host by the 
degree of maturation of the enamel. As far as the attacking 
organisms are concerned acidogenisis and dextran production 
from sucrose are common characteristics of all cariogenic 
streptococci, but some organisms which are not cariogenic 
also exhibit these properties. Since the enz5mie dextranase, 
which specifically attacks dextran, can inhibit plaque 
formation and smooth surface caries in hamsters, dextran must 
now be accorded a key role in plaque formation by these 
cariogenic streptococci. The possibility therefore arises 
that cariogenic streptococci may produce types of dextrans 
which differ in physical or chemical properties from those 
elaborated by non-cariogenic dextran formers. 



6 



Par t A (continued) Serial No. NTDR-9 ('^9^ 

Prop osed Course of Project: 

Future studies will be conducted in the three areas. 

1. Testing of dextranase for possible utility in controlling 
plaque and caries in humans. 

2. Basic studies to determine the metabolic basis for the 
cariogenic ability of the caries conducive streptococci. 

3. Epidemiological studies to determine the geographic 
distribution of potentially cariogenic streptococci in 
humans . 



Part B; 



Publications: 



1. Fitzgerald, R. J., Spinnell, D. M. , and Stoudt, T. H: 
Enzymatic removal of artificial plaques. Arch, oral 
Biol. JJ: 75, Jan. 1968. 

2. Fitzgerald, R. J., Keyes, P. H„ , Stoudt, T. H. , and 
Spinell, D. M; The effects of a dextranase preparation 
on plaque and caries in hamsters, a preliminary report. 
Jo Amer. Dent. Assn. 76: 301, Feb. 1968. 



S^ 



Serial No. NIDR-IQ (63) 
1. Microbiology 
3. Bethesda, Md. 



Part A 



Project Title: 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Studies of Culture Media for the Mass Cultivation of 
Representatives of the Genera Treponema and Borrelia . 
The Use of Bovine Serum Fraction for Growth Initiation 
of Spirochetes. 



Previous Serial Number : NIDR-4 
Principal Investigator: Dr. E. G. Hampp 
Other Investigators: None 



Cooperating Units ; 
Man Years : 



American Dental Association 



Total: 


1-1/4 


Professional: 


1 


Other: 


1/4 



Project Description: 
Objectives : 

1. To determine the factors present in ascitic fluid and serum 
products that are essential for growth of spirochetes in a 
medium that is otherwise nutritionally complete. 

2. To study the various commercially available components of 
bovine serum for growth enhancement of the spirochetes in broth 
media. 

3. Comparison of ascitic fluid, bovine serum albumin, Dubos oleic 
acid complex and Middlebrooks OADC enrichment for growth initia- 
tion of the spirochetes. 

Methods Employed : 

A medium prepared from dehydrated products and designated as BHI was 
used in previous studies as an agar medium both for isolation of oral 
spirochetes and also for the cultivation of new and old strains of a 
variety of these microorganisms. The BHI agar medium containing 
0.1% reduced glutathione was also employed previously to assay the 
growth potential of bovine serum fractions for certain strains of 
spirochetes ; it was ideal for this purpose since no growth occurred 



S5 



Part A (continued) Serial No. NIDR-10 (6 3) 

unless ascitic fluid or serum was incorporated in the finished medium. 
During this period, the BHI medium was prepared as a broth containing 
0.1% glutathione and was used for the evaluation of serum fractions 
as well as other substance for initiation of spirochetal growth. In 
these experiments two strains of oral treponemes (FM, MRB) , two strains 
of Borrelia vincentii (CON, N9) and the Reiter treponeme were used as 
representatives of the spirochetal collection. These organisms had 
been previously adapted to grow in Huntoon's hormone agar containing 
0.1% glutathione but no ascitic fluid or serum supplement. As a 
source of inoculum for broth culture experiments, the organisms were 
grown free of ascitic fluid in Huntoon's broth containing 0.1% gluta- 
thione. One ml quantities spirochetal containing broth culture was 
used for an inoculum in 18 ml experimental medium in screwtop test 
tubes. The bovine serum fractions (NBC) were used in 0.1% final 
concentration in the experimental medium and included beta lipopro- 
teins FR. Ill 0, FR IV 1 and FR IV 4, also, glycoprotein FR VI, beta 
globulin FR III, gamma globulin FR II, globulin FR IV 7, alpha •-•lobulin 
FR IV 1, and beta globulin FR IV 4. Other test substances included 
hog gastric mucin (Wilson, 701W) , sodium glucuronate , D(+) glucosa- 
mine-free base, acetyl glucosamine and D(+) glucosamine; these were 
employed in a final concentration of 0.4% in the finished BHI medium. 
Each of these substances was filter sterilized and autoclaved (10 ml 
amounts, 3 min. 121°C) . However, autoclaved samples of glycoprotein 
FR VI and gamma globulin FR II could not be used since they were coagu- 
lated by heat. In addition, bacteriologic grade bovine serum albumin 
FR V, crystalline bovine albumin FR V, Dubos oleic acid albumin complex 
and Middlebrook's OADC enrichment were used individually in 0.5% 
concentration in the BHI medium. These serum products were obtained 
commercially in a sterile condition with the exception of the crystal- 
line albumin, and it was filter sterilized in the laboratory. Control 
cultures consisted of BHI medium with and without an asitic fluid 
supplement. Growth estimation was determined by cell enumeration 
using a Petrof f-Hauser counting chamber. 

Major Findings : 

The spirochetes are fastidious microorganisms requiring a complex 
medium for growth, and, in addition, having an absolute requirement 
for ascitic fluid or serum products which further complicates their 
study. However, certain strains of spirochetes may be adapted to 
grow to a limited extent in Huntoon's agar and broth in the absence 
of a serum additive. This finding may in part be due to the presence 
of an alcohol precipitable substance that was previously demonstrated 
in a low heat veal heart infusion and was shown to substitute to a 
certain degree for ascitic fluid. Recent studies demonstrated that 
spirochetes could be grown in BHI agar only if it contained ascitic 
fluid, serum products or fractions of bovine serum. Therefore, it 
was advisable to pursue further these findings in BHI broth medium. 
When grown in the experimental medium with ascitic fluid, the average | 



G6 



Part A (continued Serial No. NIDR-10 (63) 

counts for the five strains of spirochetes were as follows: FM, 
1.35 X 10^; ^, 1.5 X 10 ; N9, 6 X 10 ; CON, 4. A x 10 , and 
ER 5.35 X 10 . The BHI broth control cultures without ascitic fluid 
did not show growth of any of the spirochetes. However, when the 
beta lipoproteins FR III 0, FR IV 1 and FR IV 4 , both filtered and 
autoclaved, were used in the basic medium in a final concentration 
of 0.1%, it was found that all strains of spirochetes grew at a level 
between 40-60% of the ascitic fluid controls. Glycoprotein FR VI 
initiated growth of all strains of spirochetes but it was 20-30% 
of that of the control cultures. In this experiment, only the filter 
sterilized material was employed since heat coagulated the solution 
to be tested, l^en the filter sterilized and autoclaved solutions 
of bovine globulins, onsisting of beta globulin FR III, gamma 
globulin FR II, globulin FR IV 7, alpha globulin FR IV 1, and beta 
globulin FR IV 4 were employed in the BHI broth medium at a final 
concentration of 0.1%, they elicited a growth response of all the 
strains of spirochetes that varied between 50-70% of that of the 
control cultures. The autoclaved sample of gamma globulin FR II 
was not used since it coagulated on autoclaving. When gastric mucin 
was employed at 0.4% concentration in the BHI broth it supported the 
growth of all 5 strains of spirochetes at about 20-30% of that of the 
control cultures. However, when sodium glucuronate, D(+) glucosamine 
free base, acetyl glucosamine and D(+) glucosamine were employed at 
the 0.4% level in the growth medium, both filter sterilized and auto- 
claved, they had no effect on growth initiation of any of the 5 strains 
of spirochetes. It was also found that the bacteriologic ^rade of 
bovine serum albumin FR V was superior to the highly purified crys- 
talline bovine serum albumin FR V as well as Dubbs oleic acid complex 
and Middlebrook's OADC enrichment. Also, compared with the ascitic 
fluid controls, the bacteriologic grade serum albumin was 70-80% 
as effective in growth initiation of the spirochetal strains. The 
mode of action of the bovine serum fractions for spirochetal growth 
is unknown and needs further study. As previously pointed out, the 
lipoproteins of servmi contain considerable amounts of cholesterol, 
phosphatids and fatty acids and it is possible that the protein 
moiety may detoxify certain of the lipids and release them as needed 
by the spirochetes. Gyma et al. (J. Bacteriol. 65_'. 1953) have shown 
that serum albumin serves the sole purpose of detoxification of oleic 
acid and releases the material as needed by the Reiter treponeme. The 
bovine globulin fractions also contain varying amounts of lipid, with 
the exception of beta globulin IV 4, which is virtually free of this 
material. Despite its freedom from lipid, however, the later fraction 
still elicited good growth of the various spirochetes. There is also 
a possibility that traces of serum albumin are carried over in the 
lipoproteins and the globulins, which may in part account for their 
mode of action. 



67 



Part A (continued) Serial No. NIDR-10 (6 3 ) 

Significar'^-. ■ ■ v sntal R e search ; 

The oral spirochetes constitute a complex group of microorganisms 
about which little is known concerning their metabolic requirements. 
They will only grow in complex media and require as well supplements 
of ascitic fluid or serum products which further complicates the 
exploration of their biochemical processes. The lack of knowledge 
concerning the mode of action of ascitic fluid has hampered progress 
on the development of a chemically defined growth medium and a means 
of identification and classification of these microorganisms on a 
sound basis. It is foreseeable when these obstacles are surmounted 
that such studies may contribute to the elucidation of their rela- 
tionship to acute necrotizing ulcerative gingivitis since they have 
long been implicated in this disease process. 

Proposed Course of Project : 

1. Sfzudies will be continued on the absolute requirement of the 
spirochetes for ascitic fluid or serum products for growth 
initiation in complex media. 

2. Since serum fractions have been shown to initiate growth of the 
spirochetes in place of ascitic fluid, a variety of similar 
materials such as myosinogen, oxoglobulin, edestin, araadin, legu- 
min, excelsin, protosoy, zein, and other purified proteins may 

be useful in gaining some insight into this problem. 

3. Tlie bovine serum fractions may possibly be further purified by 
charcoal treatment at low pH and with virtually complete removal 
of fatty acids (Chen, J. Biol. Chem. , 242 : 1967) for further 
study of their effect on growth initiation of the spirochetes. 

4. A partially defined medium (Nevin and Hampp , J. Bacteriol. 78 : 
1959) may be of use in these studies with serum fractions since 
it will permit addition and deletion of certain defined components 
of the medium. 

Part B Not included 






68 



Serial No. NIDR-11 (65) 

1. Microbiology 

2. Gnotobiotic 

3. Bethesda, Md. 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project' Title: Relationship of Specific Oral Bacteria to Dental 
Caries and Periodontal Disease 

Previous Serial Number: NIDR-5 

Principal Investigator: Dr. H. V. Jordan 

Other Investigators: Dr. P. H. Keys, Dr. H. R. Englander 

Cooperating Units: None 

Man Years: 

Total: 2-1/4 
Professional: 1 
Other: 1-1/4 

Project Description: 

Objectives : 

1. To study the relationship between certain oral streptococci 
and dental caries in experimental animals and to apply this 
knowledge to a study of the disease in humans. 

2. To investigate the mechanism by which the filamentous micro- 
organism Odontomyces viscosus induces periodontal disease in 
experimental animals and to extend the studies in organisms 
of this type to periodontal disease in humans. 

Methods Employed : 

Specific strains of bacteria are studied using standard bacterio- 
logical and biochemical techniques. Certain aspects of the 
diseases are simulated in a laboratory model under controlled 
conditions. Included are in vitro models, as well as gnotobiotic 
rats in the experimental hamster model. 

Extensive use is made of infecting bacteria "tagged" with 
antibiotic resistance in order to follow their implantation and 
colonization in animal studies. 



63 



Serial No. NIDR-II (65) 

Part A (continued) 

Major Findings : 

1. Dental caries and specific streptococci . Microbiological surveys 
were conducted on a number of population groups in order to 
establish the presence and distribution of caries inducing 
streptococci in humans. Relative numbers of streptococci similar 
to known caries inducing types were recorded in plaque samples 
from school children, aged 12-14 years using the method described 
in the 1967 project report. Four population groups were sampled: 
111 children in Cheektowaga, N. Y. (0.3 ppm NaF) with an average 
of 6.5 DMFT; 97 children in Charlotte, N. C. (1.2 ppm NaF) with 
an average of 2.3 DMFT and 104 children from 2 different villages 
in Colombia, South America with averages of 12.4 and 4.1 DMFT. 
Approximately 60 percent of the children in the Cheektowaga and 
Charlotte groups received daily applications of a NaF gel applied 
topically to the teeth in fitted mouthpieces. The Cheektowaga 
children had received over 200 fluoride treatments over a period 
of 19 months and plaque samples were taken 11 months after the 
treatments had stopped. The Charlotte children had received a 
total of 59 treatments over a period of 5 months and the experi- 
ment was still in progress when the plaque samples were taken. 

The Colombian populations were of interest because of a known 
difference in caries experience which could not be explained on 
the basis of dietary differences or fluoride intake. 

Specific caries-inducing streptococci were detected in 70 percent 
of the Cheektowaga subjects, in 30 percent of the Charlotte 
subjects, and in 69 and 59 percent of the subjects respectively, 
in the high and intermediate caries groups in Colombia. In the 
Cheektowaga and Charlotte groups the incidence and numbers of 
the specific streptococci were essentially the same for the 
fluoride-treated children and the untreated controls. 

Ninety-nine strains of the caries-inducing types of streptococci 
were isolated from the 4 population groups and characterized 
biochemically. All fermented mannitol and most strains fermented 
sorbitol. All strains formed a very adherent growth when cultured 
in sucrose broth. One hundred and two strains representing all 
other types of streptococci cultured from the plaque were univer- 
sally negative in these characteristics. Dental caries was 
induced in gnotobiotic rats and in the conventional hamster 
model using 8 representative strains of the caries-inducing types. 

It was concluded that these specific bacterial types are wide- 
spread in various human populations , but they may occur in high 
numbers sometimes constituting 50 percent or more of the plaque 
streptococci, and their presence correlates with caries experience 

2 y ?■> 



( 



Serial No. NIDR-11 (65 ) 

Part A (continued) 

on a group basis. Caries-free individuals were usually free 
of these types of bacteria. In a selected group of school 
children observed over a one year period, a strong correlation 
was noted between the presence and numbers of the caries- 
inducing streptococcal types and the appearance of a new smooth 
surface carious lesions . Repeated topical applications of a 
NaF gel had little immediate influence on the presence or 
relative numbers of caries-inducing streptococci in dental 
plaque. 

In cooperation with the Epidemiology Group at NIDR a project 
was initiated using personnel at the Coast Guard Station on 
Governors Island in New York. The bacteriological aspects of 
this study are designed to determine the incidence of the 
specific caries-inducing streptococci in nursery school 
children, 2-4 years of age. Since a large percentage of these 
children are caries-free and their oral flora may be less complex 
than that of adults, this population is considered an excellent 
model in which to study the bacteriology of dental caries. 
Preliminary studies have indicated that the specific organisms 
are present in only 30 percent of these children. Bacterio- 
logical examinations will be conducted periodically in order 
to correlate caries onset and progression with the appearance 
of caries-inducing streptococci. The population is composed 
of a large number of sibling groups which provides an opportunity 
to observe transmission patterns of the organisms within family 
groups . 

2. Relationship of Odontomyces viscosus and other filament -forming 
bacteria to periodontal disease. Original studies which estab- 
lished the etiologic relationship between 0. viscosus and 
periodontal disease in hamsters were carried out using a sucrose- 
containing diet. As described in the 1967 progress report 0. 
viscosus produces an extracellular levan from sucrose which may 
be of some significance when the organisms colonize the oral 
cavity of hamsters fed a sucrose diet. However, previous work 
has shown that the sucrose component of the diet is not vital 
to the initiation and progress of the disease. The infecting 
organism becomes implanted and induces the typical disease 
syndrome when the hamsters are fed a diet containing rice flour 
or corn starch in place of sucrose. Studies are now underway 
which will attempt to define the dietary conditions controlling 
the implantation and colonization of 0. viscosus and related 
bacteria. Groups of hamsters infected with a streptomycin- 
resistant strain of 0. viscosus are fed a diet in which the 
sucrose is replaced with different monosaccharides. Periodic 
cultures are taken to follow the course of the infection. 



Serial No. NIDR-11 (65) 
Part A (continued) 

0. vlscosus grows as a stringy viscous culture in the presence 
of glucose. A high molecular weight material can be collected 
by alcohol precipitation of viscous broth cultures. Chemical 
analysis indicates the presence of a nucleic acid-protein 
complex in this material. Continuing studies will investigate 
the relationship between this phenomenon and the plaque forming 
ability of the organism. 

Cooperative studies have recently been initiated with personnel 
at the Lincoln State School, Lincoln, Illinois, an institution 
for mental defectives. The high prevalence of periodontal 
problems in this population makes it an ideal source of material 
to study bacteriological factors in periodontal disease. Samples 
are collected at the Lincoln School and sent to Bethesda for 
examination, using the transport medium and method described in 
last year's report. A variety of filamentous bacteria have been 
isolated and are currently being tested for their ability to 
form gingival plaque and periodontal pathology in hamsters and 
gnotobiotic rats. 

Signific ance to Dental Research ; 

This work should be considered as an attempt to project the knowl- 
edge gained from animal experiments to a study of dental caries 
and periodontal disease at the human level. Population studies 
of the type described will yield necessary information about the 
relative proportions of the caries-inducing streptococci in 
different kinds of plaque, their correlation with different types 
of dental caries and their distribution in different population 
groups. 

Studies on periodontal disease are expected to describe factors 
of importance which influence the ecology of the oral flora as it 
relates to periodontal disease. Present studies are centered on 
the filaments because they are known to colonize in the gingival 
crevice. Current studies with filaments isolated from humans and 
tested under different dietary conditions may lead to a useful 
animal model system for selecting organisms involved in human 
periodontal disease. Studies of extracellular materials produced 
by these organisms will help to explain some of the mechanisms 
of plaque formation. 

Proposed Course of the Project : 

Distribution patterns of the caries-inducing streptococci in 
various human populations around the world hdve been established 
as a result of the epidemiological surveys described. Future 
studies of this type will be concerned with establishing the 



Serial No. NIDR-11 (65) 

Part A (continued) 

origin and transmission of these organisms within population 
groups. Studies of a more intensive nature on selected populations 
will be designed to examine the role of these specific bacteria 
in the initiation and development of particular types of carious 
lesions. The influence of certain dietary factors on the implanta- 
tion and persistence of these organisms will also be investigated. 
Certain aspects of these studies on human populations, such as 
dietary variables will be duplicated in animal models for study 
under controlled conditions. 

Continuing studies with the filament- forming bacteria will be 
oriented toward experiments on the mechanism by which these 
organisms become established in the gingival sulcus and induce 
periodontal disease. The parameters governing this pathogenicity 
are not well defined as are the disease parameters of dental caries. 
Future studies will attempt to describe dietary factors of this 
type. Isolation and testing of filamentous bacteria from the human 
oral cavity will continue in order to establish the pathogenic 
potential of organisms of this type in periodontal disease. One 
of the objectives will be to determine if many types or a single 
specific type of filament -forming organism is involved. Future 
studies will be broadened to include other organisms in addition 
to the filament. 



Part B Publications 

1. Howell, A., Jr. and Jordan, H. V.: Production of an extra- 
cellular levan by Odontomyces viscosus . Arch. Oral Biol . , 
12: 571-573, 1967. 

2. Jordan, H. V., and Krasse, B. : A method for sampling human 
dental plaque for caries-inducing streptococci. Arch. Oral 
Biol . , in press . 

3. Krasse, B. , Jordan, H. V., Edwardsson, S. , Svensson, I., and 
Trill, L: The occurence of caries -inducing streptococci in 
human dental plaque material. With special reference to the 
frequency of caries in selected groups of persons. Arch. Oral 
Biol . , in press. 



■ O 



Serial No. NIDR-12 (61) 

1. Microbiology 

2. Gnotoblotic 

3. Bethesda, Md. 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Role of Genetic and Environmental Factors in 
Experimental Dental Caries 

Previous Serial Number: NIDR-7 

Principal Investigator: Dr. R. H. Larson 

Other Investigators: Dr. R. J. Fitzgerald 

Cooperating Units: University of ZUrich, ZUrich, Switzerland 

University of Hawaii, Honolulu, Hawaii 

Man Years: 

Total: 2-1/4 
Professional: 1 
Other: 1-1/4 

Project Description: 

Objectives : 

This is a continuing project to evaluate the separate and inter- 
related roles of host animal, oral flora and dietary challenge 
on the development of caries in laboratory rodents. 

Methods Employed : 

Conventional methods for animal experimentation have been used. 
Both the levels and patterns of caries activity have been studied 
for several strains of rats and mystromys with variations in oral 
flora and dietary challenge. 

Major Findings : 

Studies of the interrelationship between host factors, diet and 
bacterial infection have been continued. Animals which had origi- 
nally been considered caries resistant were shown to be highly 
caries active when challenged by certain combinations of diet and 
bacterial infection. 



T>. 



Serial No. NIDR- 12 (61) 
Part A (continued) 



A. Hunt-Hoppert rats . For the past 25 years workers at Michigan 
State University have bred and studied two strains of rats which 
they designated as caries-susceptible (Ca-S) and caries-resistant 
(Ca-R) . Throughout these studies the diet used was of the type 
which is associated with caries in the sulci only. Under these 
conditions, there was no increase in caries as a result of trans- 
mission of flora, and throughout the years of study the two strains 
of rats continued to show the widely different levels of caries 
development. 

When different dietary-bacterial challenges were provided, a better 
understanding of the host differences became evident. (1) When 
Ca-S and Ca-R rats were challenged with a coarse particle corn diet, 
caries development was essentially the same as that in the Michigan 
studies. The total number of carious enamel areas (CEA) was twice 
as high for the Ca-S (33.0) as for the Ca-R (16.7), the lesions 
were almost exclusively in the sulci and the infected group showed 
no increase over the uninfected. (2) The animals fed a fine 
particle, high sucrose Diet 2000, developed lesions on all surfaces 
of the teeth. The uninfected Ca-S animals developed twice as many 
CEA (51.5) as the Ca-R (22.4), with 38.6 vs. 18. 6 in the sulci and 
12.5 vs. 3.8 on the smooth surfaces. Both infected groups showed 
increased activity over the controls and the Ca-S and Ca-R developed 
an equally high total number of CEA (71.2 vs. 71.4). However, the 
infected CA-S developed almost twice as many CEA in the sulci as 
the Ca-R (42.7 vs. 23.7) and only half as many on the smooth 
surfaces (28.5 vs. 47.7). 

The results of these studies show that the Ca-R animals are not 
actually caries resistant, but were caries inactive in early studies 
because the challenge was limited to the sulci only. When a suitable 
combination of diet and flora was provided, the Ca-S and the Ca-R 
animals appeared to be equally susceptible, but the pattern of 
lesion distribution differed for the two strains. 

B. The white-tailed rat (Mystromys albicaudatus) . It was suggested 
by Ockerse in 1953 that the white-tailed rat is a suitable animal 
for experimental caries studies. However, the only animals in which 
he reported the development of lesions were offspring of mothers 
which were maintained on a high sucrose diet during pregnancy and 
lactation. The role of an organism, not native to this species, 

was clearly demonstrated in an experiment in which they were fed 
Diet 2000. The uninfected control animals averaged less than 4 
carious lesions each, whereas those infected with a streptococci 
of human origin (Kl-R) averaged 27 lesions each. This organism 
was already known to be caries conducive in rats, hamsters and 
gerbils. 



■■' c: 



Serial No. NIDR-12 (61) 

Part A (cor-tinue'l) 

This study shows again that animals may be caries inactive only 
because the specific type of organisms necessary for the caries 
process is not present. Since this work was done in conventional 
animals it is not known what other organisms, native to the host, 
may have participated in the caries process. 

Significance to D ent al Research : 

Any advancement in the knowledge of factors associated with 
experimental caries should lead to a better understanding of the 
human disease process and to the development of methods for its 
control. 

Proposed Course of Project : 

With each advance in the understanding of the influence of various 
factors in the experimental caries process it becomes more evident 
that the effect is mediated by its influence on the bacterial 
challenge. Thus, it would appear that any real advance in the 
eradication of this disease will require a more complete knowledge 
of the microbiota involved in the process. The inability, so far, 
to produce the rampant disease in gnotobiotes which can be produced 
in conventional animals suggests that a combination of several 
organisms may be required for the process. However, the finding 
that streptococci Kl-R had an almost catalytic effect in the 
development of caries in several species of animals suggests the 
possibility that a limited number of organisms may play an essential 
role. If such is the case, the identification of these organisms, 
their nutritional requirements , and a knowledge of their metabolic 
processes and byproducts should lead to a means by which the disease 
could be radically reduced. 



Part B Publications: 

1. Larson, R. H. and Keyes, P. H. : The influence of reduced 
salivary flow on the intensity of the cariogenic challenge. 
Helv. odont. Acta , 11: 36-43, 1967. 

2. Fitzgerald, R. J. and Larson, R. H. : Age and caries suscepti- 
bility in gnotobiotic rats. Helv. odont. Acta , 11: 49-52, 1967. 

3. Larson, R. H. , Theilade, E. , and Fitzgerald, R. J.: The inter- 
action of diet and microflora in experimental caries in the 
rat. Arch, oral Biol . . 12: 663-668, 1967. 



i 



76 



Serial No. NIDR-12 (61) 



Part B (continued) 



4. Chung, C. S. and Larson, R. H. Factors and inheritance of 
dental caries in the rat. J. Dental Res. , 46: 559-564, 1967. 

5. Larson, R. H. and Goss, B. J.: Diet as a limiting factor in 
the transmissibility of caries activity between rats of 
different strains. Arch, oral Biol . ,12: 1085-1094, 1967. 

6. Frostell, G. , Keyes, P. H. and Larson, R. H. : Effect of 
various sugars and sugar substitutes on dental caries in 
hamsters and rats. J. Nutrition , 93: 65-76, 1967. 

7. Chung, C. S., Larson, R. H. , and Goss, B. J.: Perinatal and 
growth associated factors influencing dental caries in rats. 
J. Dental Res. , 47: 139-141, 1968. 

8. Larson, R. H. , Keyes, P. H. and Goss, B. J. The development 
of caries in the Hunt-Hoppert caries-susceptible and caries- 
resistant rats under different experimental conditions. 

J. Dental Res . , in press. 

9. Larson, R. H. and Fitzgerald, R. J.: Caries development in the 
African white-tailed rat (Mystromys albicaudatus ) infected 

a streptococcus of human origin. J. Dental Res . , in press. 

10. Larson, R. H.: Tooth age and caries susceptibility. In 
Harris, R. E. (Ed.): The Art and Science of Dental Caries 
Research . New York, N. Y. , Academic Press, in press. 



Serial No. NIDR-13 (66) 'jj 
1. Microbiology 
3. Bethesda, Md. 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Physiology and Regulation of Metabolic Processes in 
Lactic Acid Bacteria 

Previous Serial Number: NIDR-8 

Principal Investigator: Dr. J. P. London 

Other Investigators: None 

Cooperating Units: American Dental Association 

Man Years: 

Total: 2-1/4 
Professional: 1 
Other: 1-1/4 

Project Description: 

Objectives : 

The objective of the present research project is to provide 
fundamental information relating to the regulation of catabolic 
processes in homofermentative streptococci and other groups of 
microorganisms. The immediate problems involve (1) a biochemically- 
oriented characterization of inducible enzymes responsible for 
the dissimilation of lactic and malic acid and (2) an understanding 
of the mechanisms employed by the microorganisms to control the 
synthesis and function of these enzymes. 

Methods Employed: 

All microbiological and biochemical procedures used in this study 
were adopted directly or derived from conventional techniques. 

Major Findings: 

1. The physiological conditions controlling the synthesis of a 
flavin-linked lactate oxidase system was the subject of last 
year's report. Since then, a preliminary characterization of 
the enzjrme (s) responsible for the oxidation of lactate has 



Part A (continued) Serial No. NIDR-13 (66) 

been made. The system is particulate in nature and is probably 
an integral part of the cell envelope. Attempts to solubilize 
the particle with detergents or surface-active agents have 
resulted in complete inactivation of the enzyme complex. 
However, the enzyme can be solubilized through extensive ultra- 
sonic disruption. The flavin moiety was removed by chemical 
treatment and shown to be flavin mononucleotide; in the absence 
of the flavin cof actor the apoenzyme was rapidly inactivated. 

2, The ability of homofermentative streptococci to grow at the 
expense of a variety of substrates including carbohydrates, 
polyalcohols, mono-and dicarboxylic acids is a well documented 
but unappreciated trait of this group of organisms. Many of 
the enzymes which catalyze the dissimilation of these organic 
substrate are apparently inducible and at the present time 
virtually nothing is knovm about the regulation of synthesis 
or function of this class of enzjmies. One such enzyme has 
been studied this past year. 

The group D streptococci are capable of growing aerobically 
and anaerobically on L(+) malic acid. It was shown, that in 
the presence of malate, a "malic enzyme" is induced which 
converts the substrate to pyruvate and C0„. The fate of 
pyruvate produced from malate differs radically from that 
produced during glucose catabolism. The latter is converted 
entirely to lactic acid while the former appears as acetate, 
ethanol and C0„. This divergence in end products is a direct 
result of a requirement by lactate dehydrogenase for fructose-1, 
6-diphosphate (FDP) which serves as an activator for the enz5rme. 
Since FDP is not a direct intermediate product of malate catabo- 
lism, it does not accumulate in significant quantities and 
cannot activate the lactate dehydrogenase. Hence, pyruvate is 
diverted away from lactic acid towards acetate and ethanol. 

It has been demonstrated that the malic enzyme is not subject 
to catabolite repression by intermediate products of glycolysis 
and is synthesized during growth on glucose in a medium which 
also contains malate. However, despite the production and 
accumulation of malic enzyme in growing cultures containing 
glucose and malate, the latter was not utilized until the 
exogenous supply of glucose had disappeared. These observations 
prompted a thorough study of the factors influencing the regula- 
tion of enz3nne activity. 

A procedure was developed which resulted in a 40-fold purifi- 
cation of the malic enzyme. The enzyme was subsequently 
characterized biochemically and physically. A specificity for 
malate as substrate and nicotine adenine dinocleotide (NAD) 



Part A (continued) Serial No. NIDR-13 (66) 

as cof actor was demonstrated. Manganous and magnesium ion 
activate the enz3mie; however, the former is far more effective. 
The enzyme has a molecular weight of between 85-95,000 and an 
optimal pH of 8.6. The equilibrium of the reaction is strongly 
in the direction of pyruvate and it is doubtful that the enzyme 
takes part in the biosynthesis of 4-carbon dicarboxylic acids. 

A variety of compounds act as inhibitors of the purified enzyme. 
Oxaloacetate, a structural analogue of malate, is a competitive 
inhibitor. This was not unexpected and is a trait shared by 
all malic enzymes derived from a variety of sources. However, 
nucleotide triphosphates are also effective inhibitors as are 
6-phosphogluconate (6-PG) and FDP. Through thermal inactivation 
and p-chloromercuriphenol sulfonic acid inactivation studies 
in direct evidence was obtained for the presence of inhibitor 
sites on the enzyme. Depending on the treatment, the enzyme 
could be desensitized to the respective inhibitors. 

The roles played by the various inhibitors is not difficult to 
visualize. The inhibition by ATP and other nucleotide triphos- 
phates provides a means for regulating the rate of malate 
dissimilation in a dynamic system. Any accumulation of ATP 
would cause a decrease in the rate of reaction, conversely, a 
decrease in the ATP pool would increase the reaction rate of 
the enzjnne. The inhibition produced by FDP and 6PG would 
prevent malate dissimilation during aerobic or anaerobic 
glucose utilization. 

A dissimilation of glucose and malate occurring simultaneously 
could easily produce an imbalance in growth by virtue of 
(1) a rapid accumulation of pyruvate which could produce a 
substrate inhibition of pyruvate-utilizing enzyme, or (2) 
by shifting the equilibrium between NAD and NADH far in the 
direction of reduced pyridine nucleotide. Either situation 
could potentially produce unbalanced growth and result in 
a grossly inefficient utilization of carbon and energy, or 
in cellular lysis. 

Significance to Dental Research: 

Knowledge of the physiology and regulation of metabolic processes 
in lactic acid bacteria is needed for better appreciation of the 
biological potentialities of oral streptococci and for understanding 
their behavior in the various environments where they are found. 
The present phase of the projects is of immediate significance for 
the problem of dental caries, since it attempts to define biochemical 
processes responsible for the production, accumulation, and subse- 
quent utilization of lactic acid by homofermentative organisms. 



^n 



<( 



Part A (continued) Serial No. NIDR-13 (66) 

Proposed Course of Project: 

1. A further characterization of the lactate oxidase will be 
undertaken to elucidate the mode of catalysis. In addition, 
attempts will be made to demonstrate flavin- linked oxidative 
phosphorylation. 

2. The study of the malic enzyme is near completion. However, 
some ancillary problems have arisen which require attention. 
Most pressing at this time is a clarification of the pathway 
by which energy is obtained and ethanol produced during malate 
fermentation. At present there is no known biochemical system 
which can catalyze such a reaction. 

3. A variety of inducible enzymes will be studied on a comparative 
basis to learn whether regulation of this class of enz3mies is 
achieved by a limited, select group of glycolytic intermediates, 
namely, fructose-6-phosphate and fructose-1, 6-diphosphate. 



Part B: 



Publications : 



1. London, J. P., and Rittenberg, S. C: Thiobacillus perometabolis 
nov. sp. A non-autotrophic thiobacillus. Arch, fur Mikrobiol . 
59: 218-225, 1967. 

2. Smith, A., London, J. P., and Stanier, R. Y, : Biochemical 
basis of obligate autotrophy in blue-green algae and thiobacilli. 
J. Bacteriol . 94: 972-983, 1967. 

3. Cohen-Bazire, Go, and London, J. P.: Basal organelles of 
bacterial flagella. J. Bacteriol . 94: 458-465, 1967. 

4. London, J. : Regulation and function of lactate oxidation in 
Streptococcus faecium. J. Bacteriol . 95: 1380-1387, 1968. 



Serial No. NIDR-14 (65) 

1. Microbiology 

2. Immunology 

3. Bethesda, Md. 



PHS-NIH 

Individual Project Report 

July 1, 1967 through June 30, 1968 



Part A 



Project Title: Immunological Mechanisms in Oral and Systemic Disease 

1. Significance of the Complement System to the Mechanism 
of Action of Endotoxin. 

2. Studies on Biological Effectors of Immune Response and 
Immunological Tolerance. 

Previous Serial Number: NIDR-9 

Principal Investigator: Dr. S„ E. Mergenhagen 

Other Investigators: Dr. H. Gewurz , Dr. S. E. Berglund, 

Dr. R. Snyderman and Dr. R. J. Howard 



Cooperating Units; 



Department of Microbiology, Temple University 
School of Medicine; Department of Microbiology, 
Johns Hopkins University School of Medicine; 
Department of Pediatrics and Microbiology, 
University of Minnesota Medical School; Max- 
Planck Institute for Immunobiology, Freiburg, 
Germany. 



Man Years; 



Total: 8-1/2 
Professional: 5 
Other: 3-1/2 

Project Description: 

Objectives: 

1. To investigate immunological mechanisms by which oral and 
other microbial antigens incite tissue damage: mechanism 

of action of endotoxin through activation of the complement 
system. 

2. To investigate the influence of viral and other biological 
effector systems on the immune mechanism. 



8-2 



Part A (continued) 

Methods Employed; 



Serial No. NIDR-14 (65) 



Immunological analyses of bacterial and other antigens are 
carried out by immunoelectrophoresis, disc gel electrophoresis, 
Ouchterlony analysis, and passive hemagglutination techniques. 
Complement fixation and complement (C) component assays are 
performed with purified C components in collaboration with 
Dr. Manfred Mayer and his colleagues at Johns Hopkins University. 
A modification of localized hemolysis in gel (Jerne technique) 
is employed for detection of antibody by lymphoid cells. Iso- 
topically (I-'--^-'- and ll25) labelled proteins are used in immune 
elimination studies. Column chromatography and sucrose gradient 
ultracentrifugation have been used for separation of serum 
antibodies and biologically-active polypeptides generated in 
serum with endotoxin. The Boyden chamber technique and the 
Schultz-Dale apparatus were used to study chemotaxis-generation 
and anaphylatoxin-generation _in vitro. 

Major Findings; 

1. Interaction of the complement (C) system with endotoxic 
lipopolysaccharides (LPS) . As previously reported (Bladen 
et al., J. Exp. Medicine, 125 , 767, 1967), the C system, 
by analogy with hemolytic C, exerts its maximum or nine- 
component effect upon interaction with LPS from gram negative 
bacteria. Unlike antigen-antibody precipitates, LPS incubated 
in fresh mammalian serum interacts most efficiently with the 
terminal components of C (C'3, 5, 6, 7, 8, 9) with little 
fixation of C'l, 4, 2. This sequence of events is known to 
include as bj^roducts the promotion of phagocytosis and 
aggregation phenomenon, the generation of anaphylatoxin 
and production of factors chemotactic for neutrophils. It 
has long been known that endotoxin injection into the mammalian 
host induces aggregation of platelets and polymorphonuclear 
leukocytes, along with neutrophil chemotaxis and alterations 
of vascular permeability. During the past year we have per- 
formed experiments which support the hypothesis that endotoxin 
induced inflammation is mediated via the complement system. 

One such study sought to determine whether the generation of 
chemotactic factor by endotoxin in serum was dependent upon 
complement system activation. The Boyden chamber employing 
rabbit poljmiorphonuclear leukocytes jji vitro was used for 
this study. Pre-heating serum, incubating at 0°C , or in- 
cubating in the presence of EDTA all prevented chemotactic 
factor generation as well as C fixation by endotoxin. 
"Endotoxoids" deficient in C- fixing activity were also 
deficient in chemotactic factor generation. Chemotactic 



83 



Part A (continued) Serial No. NIDR-14 (65) 

factor could not be generated by endotoxin sera of mice 
congenitally deficient in the C'5 component of complement 
while chemotactic factor was generated by endotoxin in the 
sera of coisogenic mice with normal C' levels for that species. 
The chemotactic factor induced by endotoxin was heat stable 
and non-dialyzable. Molecular sieve chromatography and sucrose 
density gradient ultracentrifugation demonstrated that the 
chemotactic factor was a relatively low molecular weight 
product (20,000-30,000) and as such differed from previously 
described C system derived chemotactic factors. These 
experiments lend further support to the hypothesis that 
endotoxin- induced inflammation is dependai t upon C' system 
activation. Furthermore, the relatively low molecular weight 
of this factor suggests that it might be derived from 
activation of a single complement component (perhaps C'5) 
rather than from complexing of multiple complement components. 

In addition to our studies on chemotaxis, we have found that 
when endotoxin is added to normal undiluted guinea pig, rat 
or pig serum, a substance is generated with the character- 
istics of anaphylatoxin: i.e., it contracts guinea pig ileum 
and produces tachyphylaxis; its activity is blocked by anti- 
histamines; and is not produced in heated serum or in the 
presence of EDTA, or at 0°C. Production of anaphylatoxin 
by endotoxin in guinea pig serum occurs with little con- 
sumption of C'l, 4, or 2, but correlates with consumption 
of C'3 and C'5. The relationship of the anaphylatoxin 
generating factor to the chemotactic factor is under 
investigation. 

2. Immunoglobulin deficient sera and the role of antibody in 
endotoxin- complement interactions . Attempts to deplete 
antibodies to endotoxin by selective absorptions proved 
noncritical, in part because of solubilization of endotoxin. 
Therefore, endotoxin -C ' interactions were investigated in 
certain agammaglobulinemic porcine, bovine and human sera. 
Endotoxins were reacted with pre-colostral piglet serum 
containing < 2.5 x lO'^mg % gamma globulin and with sow 
serum (500 mg % gamma globulins) derived from pathogen free 
Minnesota miniature pigs. Comparable C fixation was 
observed in both groups of specimens. Over 80% of the 
piglet C was fixed, neutrophil chemotaxis and anaphylatoxin 
were produced and characteristic C- mediated lesions 
appeared on the endotoxin. Similar results were obtained 
with other immunoglobulin-def icient sera. The only human 
immune deficiency sera showing a lowered reactivity with 
endotoxin came from individuals with Swiss, type lymphopenic 
agammaglobulinemia. However, unlike other human specimens, 
these sera were markedly deficient in the C ' 1 component 



8k 



Part A (continued) Serial No. NIDR-14 (65) 

of C and in "properdin". Further studies on the pathway 
for C activation by endotoxin are under investigation. 

3. Studies on effectors of the immune response. 

(A) Ant i lymphocyte serum. Antilymphocyte serum (ALS) is a 
potent immunosuppressive agent. It has been found that mice 
readily form antibodies to the yG fraction of ALS. This 
prompted us to investigate whether a state of immunological 
tolerance to rabbit gamma globulin could alter the effec- 
tiveness of ALS. The prolongation of skin homografts by 

ALS is normal and tolerant mice was studied. Animals that 
were tolerant to rabbit gamma globulin, showed homograft 
survival times for longer than normal animals. These results 
have been attributed to a decreased rate of elimination of 
the active component of ALS in mice rendered tolerant to RGG. 

(B) Influence of virus infection on the immune response. 
Our prior work showed that infection of mice with the lactic 
dehydrogenase virus (LDV) enhances the antibody response in 
mice and converts toleragenic doses of human gamma globulin 
into immunizing stimuli. During the past year a study was. 
undertaken to determine the effect of LDV infection on 
cellular immune reactions by testing the ability of mice 
infected with LDV to reject skin homografts. In brief, median 
homograft survival times were significantly prolonged in 

mice infected with LDV. Thus, while LDV infection facilitates 
the production of humoral antibody and acts like an immuno- 
logic adjuvant, the same virus infection depresses cellular 
immunity. It is obvious that such studies have far reaching 
implications in tumor immunology and autoimmunity. 

(C) Cellular and humoral antibody response to submucosally 
administered antigen in rabbits. Indirect evidence suggests 
that bacteria and their products which reside in diseased 
periodontal tissues stimulate an immune response. Our 
results have shown that small doses of bacterial antigens 
injected into the oral mucosa stimulate a marked immune 
response as indicated by increasing numbers of antibody- forming 
cells in regional Ijrmph nodes and by increased serum antibody 
titers. Concomitantly, no detectable immune response occurs 

in the spleen or bone marrow. These studies suggest that 
low doses of bacterial antigens which gain access to perio- 
dontal tissues are adequate to stimulate an immune response 
which resides predominately in the regional lymph nodes and 
at local inflammatory sites. 



65 



Part A (continued) Serial No. NIDR-14 (65) 

Significance to Dental Research : 

The role of immunological reactions in oral and systemic 
health and disease is a question of major concern to our 
understanding of disease pathogenesis. Studies concerned 
with interaction of microbial antigens and the immune mechanism 
of the host contribute to a better understanding of the inflam- 
matory response and could clarify the role of bacterial products 
in oral and systemic disease. Studies of viral and other 
biological effectors of the immune response is important to 
an understanding of mechanisms of humoral and cellular immune 
reactions in the host. 

Proposed Course of Project; 

To continue our investigations as outlined above with particular 
emphasis on humoral and cellular mechanisms of host resistance. 



Part B 



Honors and Awards: 

Dr. S. E. Mergenhagen: Recipient of the lADR Award for basic 
research in Oral Science, 1966. Drs. H, Gewurz , R. Snyderman, 
H. So Shin, L. Lichstein, and S. E. Mergenhagen, paper entitled 
"Complement consumption by endotoxic lipopolysaccharide in 
immunoglobulin-def icient sera". Selected for presentation at 
the Plenary Session of the American Society for Clinical Investi- 
gations meeting held in Atlantic City, May 5, 6, 1968. Dr. 
H. Gewurz, invited participant at the Sannibel Research 
Conference on Developmental Immunology, Sannibel Island, Florida, 
February, 1968, paper entitled "Interactions of complement with 
endotoxic lipopolysaccharide". 

Publications ; 

1. Bladen, H. A., Gewurz, H. , and Mergenhagen, S. E. : Inter- 
actions of the complement system with the surface and 
endotoxic lipopolysaccharide of Veillonella alcalescens . 
J. E2C£. Med., 125: 767-786, 1967. 

2. Mergenhagen, S. E., Notkins, A. L. , and Dougherty, S. F.. : 
Adjuvanticity of lactic dehydrogenase virus: Influence of 
virus infection on the establishment of immunologic tolerance 
to a protein antigen in adult mice. J„ Immunol . 99: 576-581, 
1967. 

3. Berglund, S. E. , Markey, P„ A., and Mergenhagen, S. E.: 
Observations on the kinetics of the hemolytic antibody 
response by localized hemolysis in gel over frozen sections 

of mouse spleen. Proc . Soc. Exp . Biol . Med., 126: 84-88, 1967, 



Part B (continued) Serial No, NIDR-14 (65) 

4. Gewurz, H. : The immunologic role of complement. Hospital 
Practice , 2: 44-56, 1967. 

5. Mergenhagen, S. E. , Gewurz, H. , Bladen, H. A., Nowotny, A., 
Kasai, N. , and Luderitz, 0.: Interactions of the complement 
system with endotoxins from a Salmonella minnesota mutant 
deficient in 0-polysaccharide and heptose. J. Immunol . , 100 : 
227-229, 1968. 

6. Gewurz, H. , Mergenhagen, S. E. , Nowotny, A., and Phillips, 
J. K. : Interactions of the complement system with native 
and chemically modified endotoxins, J. Bacterid . 95: 
397-405, 1968. 

7. Howard, R. J„, Dougherty, S. F., and Mergenhagen, S. E.: 
Prolongation of skin homografts by rabbit anti-mouse 
lymphocyte serum in mice rendered tolerant to rabbit gamma 
globulin. J. Immunol , , in press, 1968. 

8. Gewurz, H. , Pickering, R. J., Christian, C« L. , Snyderman, R. , 
Mergenhagen, S. E. , and Good, R. A.: Decreased C'l protein 
concentration and agglutinating activity in agammaglobulinemia 
sjmdromes: An inborn error involving the complement system. 
Clinical and Experimental Immunology , in press, 1968. 



87 



^ 



Serial No. NIDR-15 (67) 

1. Microbiology 

2. Virology 

3. Bethesda, Md 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Recurrent and Persistent Viral Infections 

Previous Serial Number: NIDR-10 

Principal Investigator: Dr, A. L. Notkins 

Other Investigators: ^/pc . W, K. Ashe 

Dr. C. A. Daniels 
Dr. S. E. Mergenhagen 

Cooperating Units: National Cancer Institute 

Man Years: 

Total: 7-1/2 
Professional: 3 
Other: 4-1/2 

Project Description: 

Objectives: 

This project concerns recurrent and persistent viral infections, 
the mechanism of virus sensitization and neutralization, the 
effect of virus infections on the immune system of the host, 
and the mechanism of action of lactic dehydrogenase virus (LDV) . 

Methods Employed : 

The methodology was described in previous reports and new methods 
will be discussed under Major Findings. 



Major Findings ; 



Sensitized Virus as an Indicator System for Studying Antiviral 
Immunoglobulins. Previous experiments showed that antiviral 
antibody could attach to herpes simplex virus (HSV) in the form 
of an infectious-virus antibody complex (sensitized virus) and 
that sensitized virus, in contrast to unsensitized virus, could 
be readily neutralized by anti-7-globulin. Over the past year 



88 



Part A (continued) Serial No. NIDR-15 (67) 

we have studied a number of the factors and conditions involved 
in the neutralization of sensitized virus by anti-y-globulin. 
Maximum neutralization of sensitized virus was found to depend 
upon: 1) the degree of sensitization; 2) the amount of free 
or unattached 7-globulin in the reaction mixture; 3) the con- 
centration of anti-7-globulin; 4) the nature of the sensitizing 
immunoglobulin; and 5) the specificity of the anti-7-globulin. 
In addition, we found that the interaction between 7-globulin 
and anti-7-globulin made it possible to use sensitized virus 
as a highly sensitive indicator system for studying antiviral 
immunoglobulins. Whereas the detection of a particular immuno- 
globulin by the immuno-dif fusion method requires a sufficiently 
high concentration of antigen and antibody to produce a visible 
precipitate, a single sensitized virus particle can be detected 
as a result of replication and plaque formation. Neutralization 
of sensitized virus by specific an ti- immunoglobulins thus makes 
it possible to detect and characterize the minute amount of anti- 
viral immunoglobulin which is attached to the sensitized virion. 
In addition, we found the sensitized virus could be used to study 
and titer specific anti-immunoglobulins. Experiments in progress 
suggest that this technique might rival the hemagglutination 
system which is considered one of the most sensitive tools in 
immunology. 

In addition, the anti-7-globulin technique is proving to be an 
extremely useful serologic tool for demonstrating otherwise 
undetectable or low levels of antiviral antibody. For example, 
our experiments showed that the neutralization endpoint of certain 
antiviral sera was increased by as much as 64- fold when anti-7- 
globulin was added to the reaction mixture. We hope that this 
technique will prove useful in the early serologic detection of 
certain viral infections. 

Neutralization Inhibition Kinetics and Chronic Viral Infections . 
The anti-7-globulin technique also has proved valuable in studying 
the kinetics of virus sensitization. For example, incubation of 
HSV with anti-HSV for 2.5 minutes resulted in little if any 
neutralization, but rendered 75% of the surviving virus neutral- 
izable by anti-7-globulin. The degree of sensitization increased 
with time and at 20 minutes over 99.8% of the surviving virus had 
become sensitized. Further experiments showed that sensitized 
virus was neutralized at a slower rate by antiviral antibody than 
unsensitized virus. The relationship between sensitization and 
inhibition of neutralization was studied by a technique which 
we refer to as neutralization inhibition kinetics. Basically, 
the procedure involves sensitizing the virus with different 
concentrations of anti-HSV and then determining the neutralization 



89 



Part A (continued) Serial No. NIDR-15 (67) 

rate constant of the sensitized virus by a second incubation 
with anti-HSV. Our data showed that as the degree of sensitization 
increased from to 99.9%, the neutralization rate constant 
decreased from 14 for unsensitized virus to 1.8 for highly 
sensitized virus. This represents an 87% reduction in the rate 
of neutralization. The positive correlation between the degree 
of sensitization and the reduction in the neutralization rate 
constant supports the contention that the initial sensitization 
of the virus, sterically or otherwise, hinders the attachment 
of additional antiviral antibody and thereby retards or prevents 
further virus neutralization. Additional support for the argu- 
ment that steric hinderance is responsible for the reduction in 
the neutralization rate constant comes from experiments in which 
we used papain digested antibody fragments. These experiments 
showed that sensitization of the virus with undigested anti-HSV 
7-globulin reduced the neutralization rate constant by 53% when 
tested with undigested anti-HSV anti-globulin, but resulted in only 5% 
reduction in the neutralization rate constant when tested with 
the smaller univalent (Fab) antibody fragments. The demonstration 
by neutralization inhibition kinetics that sensitization with 
undigested anti-HSV inhibited the subsequent rate of neutralization 
by undigested anti-HSV but did not appreciably decrease the rate 
of neutralization by the smaller univalent Fab fragments points 
to the possibility that univalent antibody fragments might prove 
useful in neutralizing highly sensitized and otherwise resistant 
virus. The data from the HSV experiments and our previous 
findings with LDV also suggest that sensitization may play an 
important role in accounting for the chronic nature of certain 
virus infections. 

Recurrent and Persistent Viral Infections of the Oral Cavity . 
Studies performed by Ashe and Rizzo on inapparent HSV infections 
in rabbits were published this year. In brief, they found that 
the higher the level of serum antibody the less susceptible was 
the oral mucosa to experimental infection with HSV. The severity 
of the oral lesions and the ability to culture virus from these 
lesions was found to be inversely related to the level of serum 
antibody. However, despite the presence of neutralizing antibody 
in the blood, infectious virus could at times be recovered from 
the saliva, even many months after the animals had been inoculated 
with the virus. In light of our recent findings with sensitized 
virus, it would be of interest to see whether the virus in the 
saliva is sensitized and if so whether it is more resistant to 
neutralization by antiviral antibody than unsensitized virus. 

Over the past year, studies were continued on the biological 
and physical properties of the hemagglutinin associated with the 
rat submaxillary gland virus and the anti-hemagglutinin found in 



90 



Part A (continued) Serial No, HIDR-15 (67) 

the serum of rats. These experiments showed that the hemagglu- 
tinin was not found in the salivary glands of young rats but 
appeared at about 2 months and increased in titer with age. 
Further studies showed that an anti-hemagglutinin was present 
in the serum of young rats and that the titer of the anti- 
hemagglutinin increased with age and roughly paralleled the rise 
of the hemagglutinin. The data suggest that the early anti- 
hemagglutinin might be of maternal origin. Studies pertaining 
to the transmission of the rat submaxillary gland virus and the 
effect of surgical removal of the submaxillary gland on the 
titer of the hemagglutinin are in progress. 

Effect of Viral Infections on the Immune System of the Host. One 
aspect of our studies on the effect of virus infections on the 
immune system of the host was brought to completion. We found 
that an acute virus infection (lactic dehydrogenase virus) could 
stimulate antibody production against a foreign protein (unaggre- 
gated human 7-globulin) that otherwise induces immunologic 
tolerance. In addition, our data suggest that one of the 
mechanisms of action of an immunologic adjuvant may be its ability 
to convert the tolerance- inducing components of a particular 
antigenic preparation into an immunogenic stimulus. Experiments 
in progress indicate that a virus infection also can effect the 
ability of the host to reject skin grafts. These and previously 
reported findings show that a virus infection can greatly in- 
fluence the immune response of the host and points to the possi- 
bility that viruses also might play an important role in auto- 
immune diseases. 

Significance to Dental Research ; 

A number of laboratories are now using the anti-7-globulin 
technique for detecting sensitized virus, enhancing virus 
neutralization, and measuring low or otherwise undetectable 
levels of antiviral antibody. The use of sensitized virus as 
an indicator system for studying antiviral immunoglobulins 
should give new insight into the nature and properties of anti- 
viral immunoglobulins in the saliva. 

The development of the neutralization inhibition test has made 
it possible to study the effect of sensitization on virus 
neutralization. Our experiments showed that sensitized virus 
was neutralized at a slower rate than unsensitized virus. These 
findings suggest that sensitization might contribute to the 
chronic nature of certain virus infections and points to the 
possibility that herpes simplex virus might exist in saliva in 
the sensitized state. 



31 



Part A (continued) Serial No. NIDR-15 (67) 

Proposed Course of Project; 

Present experiments are concerned with (1) the development and 
application of the anti-7-globulin technique for the detection 
of sensitized virus; (2) the use of sensitized virus as an 
indicator system for detecting and characterizing antiviral 
immunoglobulins; (3) the extension of the neutralization inhibition 
test to other viruses and classes of immunoglobulins; (4) the 
effect of virus infections on the immune system of the host; and 
(5) the detection and characterization of antiviral antibody in 
human saliva and attempts to recover sensitized virus from the 
oral cavity. 

Part B Publications: 

1. Ashe, W. Ko , and Rizzo, A. A.: Inapparent herpes simplex 
virus infection in inoculated rabbits. Proc. Soc. Exptl . 
Biol. Med ., 124: 1150-1154, 1967. 

2. Rizzo, A. A., and Ashe, W. K. : The influence of different 
levels of serum antibody on the susceptibility of rabbit 
oral mucosa to experimental herpes simplex virus infection,. 
Arch. Oral Biol .. 12: 933-936, 1967. 

3. Mergenhagen, S. E., Uotkins, A. L. , and Dougherty, S. F.r 
Adjuvant icity of lactic dehydrogenase virus; Influence of 
virus infection on the establishment of immunologic tolerance 
to a protein antigen in adult mice, J. Immunol ., 99: 576-581, 
1967. 

4. Ashe, Wo K, and Notkins, A. L. : Kinetics of sensitization 

of herpes simplex virus and its relationship to the reduction 
in the neutralization rate constant. Virology . 33: 613-617, 
1967. 

5. Notkins, A. Lo , Mage, M. , Ashe, W. K. , and Mahar, S. : 
Neutralization of sensitized lactic dehydrogenase virus by 
anti-7-globulin. J. Immunol ., 100: 314-320, 1968. 

6. Hampar, B. , Notkins, A. L. , Mage, M. , and Keehn, M. A.: 
Heterogeneity in the properties of 7S and 19S rabbit 
neutralizing antibodies to herpes simplex virus. J. Immunol ., 
100: 586-593, 1968. 



92 



Serial No. NIDR-16 (64) 
1. Microbiology 
3. Bethesda, Md. 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: The Mechanisms by which Bacterial Products May Cause 
Destruction in Human Periodontal Disease 

Previous Serial Number: NIDR-11 

Principal Investigator: Dr. A. A. Rizzo 

Other Investigators: None 

Cooperating Units: None 

Man Years : 

Total: 2-1/4 
Professional: 1 
Other: 1-1/4 

Project Description: 

Objectives: 

1. To investigate the dif fusibility of endotoxins and antigens 
into rabbit gingival pocket tissues. 

2. To investigate the Jjl vivo toxicity of bacterial products to 
rabbit corneal and gingival tissues under conditions simu- 
lating those of the human mouth. 

3. To determine whether hydrogen sulfide production in human 
periodontal pockets is related to periodontal disease. 

Methods Employed: 

Much of the methodology used in these studies has been described 
in previous annual reports (NIDR-12, 1967 ; NIDR-12, 1966). One 
method used this year, but not previously employed, included the 
use of a vascular labelling technique to show that topically 
applied bacterial agents such as hydrogen sulfide can cause an 
increase in vascular permeability in oral tissue. Another mod- 
ification applied this year was to use as a test tissue for 



93 



Part A (continued) Serial No. NIDR-16 (64) ' ' 

topical toxicity experiments an area of lip mucosa which resembles 
gingival sulcus tissue, but which can be irrigated without any 
mechanical trauma. 

Major Findings; 

1. Gingival absorption of endotoxins and antigens . 

Using topical administration by means of locally placed cotton 
wicks containing endotoxins and antigens, rabbit ingival 
pocket tissues were tested for absorption of these agents. 
Such tests, reported in a previous annual report (NIDR-12, 
1967) , indicated that these high molecular weight substances 
would not penetrate an intact epithelial sulcular lining. 
This year these results have been confirmed and extended by 
using prolonged irrigation of the gingival tissues with 
high concentrations of endotoxins and antigens. When the 
pocket lining was ulcerated, prolonged irrigation with con- 
centrations of endotoxin as low as 0.01 [ig/ml resulted in 
sufficient penetration to evoke an antibody response in 
regional Ijmiph nodes. 

2,, In vivo toxicity of bacterial products. 

a. Electron microscopy of rabbit corneas irrigated with 
neutral solution of ammonia has elucidated the nature 
of the toxic effect of ammonia on the anterior epi- 
thelium of this organ. The principal morphologic 
alterations were the presence of intracytoplasmic 
vacuoles up to 10-15 microns in diameter and nuclear 
shrinkage. There was no apparent disruption of cyto- 
plasmic, nuclear, or basement membranes, nor of 
desmosomes, and no alteration in the underlying 
structures of the stroma, 

b. Microscopic studies of corneas irrigated with neutral 
solutions of hydrogen sulfide have revealed changes 
throughout the epithelium. Surface cells were 
observed to degenerate and become stripped off, and 
basal nuclei showed obvious swelling. Superficial 
cells had lost their cytoplasm, and the nuclei had 
undergone swelling before becoming exfoliated. The 
process of degeneration and detachment resulted in 
formation of a shallow crater in the surface outline 
and a thinner integument. 

c. Findings not mentioned previously in relation to 
sulfide irrigation of eyes included evidence that the 

gas might be diffusing through the cornea into the aqueous 



9^ 



Part A (continued) Serial No. NIDR-16 (64) 

humor and iridial tissues. Sulfide treated eyes showed 
slower and diminished pupillary constriction to light 
compared to controls, and the iridial vasculature exhibited 
an increase in permeability when tested by the vascular 
labelling method. Specific chemical tests indicated that 
hydrogen sulfide appeared in the aqueous humor very soon 
after irrigation with relatively low concentrations of 
sulfide had begun. 

d. Irrigation of rabbit gingiva and lip mucosa with hydrogen 
sulfide produced gross edema, and erythema which was quite 
dramatic at higher concentrations, but was still evident at 
concentrations as low as 0.01 molar. Increased vascular 
permeability was demonstrated in such specimens by means of 
the vascular labelling technique. Preliminary histologic 
examination of hydrogen sulf ide-treated gingival specimens 
showed epithelial changes similar to those observed in cornea. 
In addition, widened intercellular spaces were observed in 
both deep and superficial areas of the lining epithelium. 

Significance to Dental Research: 

A serious obstacle to disease-oriented research in the periodontal 
field has been the lack of suitable test systems to study directly 
the effects upon tissues of the many substances chronically in 
contact with the human gingival tissues. The methods which have 
been developed and applied in the present studies have provided 
meaningful information on _in vivo local toxicity of ammonia and 
hydrogen sulfide under conditions simulating those of the human 
moutho Since both of these substances are known to be produced 
by bacteria in the human mouth, the demonstration that low levels 
of these agents can produce epithelial damage in a neutral, 
isotonic milieu may be of considerable importance in the initi- 
ation of periodontal disease. It is of possible greater signif- 
icance that short-term exposure to neutral solutions of hydrogen 
sulfide not only causes epithelial alterations, but also induces 
gingival edema and erythema, two well known clinical signs of 
human periodontal disease. 

Tests on the absorption of an antigenic protein and of bacterial 
endotoxin indicate that these agents can diffuse into gingival 
tissues and cause alterations only after ulceration has taken 
place. Thus, the role of these non-enzymic, high molecular 
weight substances may be more important in the progression, 
rather than in the initiation of periodontal disease. 



So 



Part A (continued) Serial No. NIDR-16 (64) 

Proposed Course of Project: 

1, To define further the pathologic alterations induced by 
ammonia and hydrogen sulfide in experimental systems. These 
studies are to include light and electron microscopic studies 
of eye and oral specimens exposed to ammonia, hydrogen sulfide, 
and to mixtures of these agents. 

2. To compare the pathologic alterations in inflammed human gin- 
gival tissues to the alterations induced by ammonia and 
hydrogen sulfide in experimental tissues, using criteria 
developed in the proposed studies described above (Item 1). 

3o (a) To carry out additional studies on hydrogen sulfide pro- 
duction in human periodontal pockets to determine whether or 
not the production of this gas shows a clear-cut association 
with periodontal disease. 

(b) To estimate the concentrations of hydrogen sulfide which 
actually develop in healthy and diseased periodontal sites 
in humans. 

4. To test ammonia and hydrogen sulfide as vehicles for aiding 
in the transmission into gingival tissues of high molecular 
weight bacterial products such as antigens and endotoxins 
and of bacterial cells themselves. 



Part B: 



Publications ; 



1. Rizzo, A. A.: The possible role of hydrogen sulfide in 
human periodontal disease. I. Hydrogen sulfide production 
in periodontal pockets. Periodontics 5: 233-236, 1967. 

2. Rizzo, A. A.: Rabbit corneal irrigation as a model system 
for studies on the relative toxicity of bacterial products 
implicated in periodontal disease. The toxicity of 
neutralized ammonia solutions. J. Periodontol . , 38: 
491-499, 1967. 

3. Rizzo, A. A.: Absorption of bacterial endotoxin into rabbit 
gingival pocket tissue. Periodontics , 6: 65-70, 1968. 

4. Rizzo, A. A. : Summary of papers on biochemistry, physiology 
and microbiology of the periodontium, in Proceedings of the 
International Symposium on Oral Diseases at the University 
of Alabama, 1968. Ala. J. Med. Sci . In press. 



9S 



Serial No. NIDR-17 (67) 
1. Microbiology 
3. Bethesda, Md. 



PHS-NIH 

Individual Project Report 

July 1, 1967 through June 30, 1968 



Part A 



Project Title: Systematic Microbiological Taxonomic Studies 

Previous Serial Number: NIDR-13 

Principal Investigator: Mr. Morrison Rogosa 

Other Investigators: Dr. M. I. Krichevsky 

Dr. J. P. London 

Cooperating Units: Division of Computer Research and Technology, N.I.H. 

University of Maryland 
American Type Culture Collection 
Georgetown University 
Sergey's Manual Trust 
International Subcommittee on Lactobacilli 

and Related Organisms 
International Subcommittee on the Bifid Bacteria 

Man Years: 

Total: 2-1/4 
Professional: 1 
Other: 1-1/4 

Project Description: 

Objectives : 

To determine the systematic taxonomic relationships of microbial 
organisms, with particular reference to members of the oral 
microbiota. 

Methods Employed: 

Sophisticated modern techniques are applied in the study of 
morphological, culture, biochemical, genetic, immunological, 
ecological, and pathogenic characteristics of microorganisms. 
Computer technology is employed to store, retrieve, and assess 
the significance of data obtained in this laboratory, as well 
as information from other world laboratories, in the synthesis 
of a comprehensive systematic microbiological taxonomy of global 
usefulness. 



97 



Part A (continued) Serial No. NIDR-17 (67) 

Major Findings ; 

The anaerobic gram negative organisms, whose nutritional 
characteristics were described previously were found to be 
amino acid fermenters, particularly of glutamic acid, with 
the production of 2 moles acetic/1 mole butyric acid, plus 
C0„. A relatively simple gas chromatographic method, developed 
in this laboratory, was employed for the quantitiative detection 
of the lower fatty acid homologues. Organisms previously studied 
by others as Micrococcus aerogenes appear biochemically similar 
to our isolates. Electron micrographs indicate both groups of 
organisms have characteristic gram negative structure. Accordingly, 
they appear to be members of the genus Peptococcus. Thus through 
our work, the delineation of Peptococcus has been now achieved. 

The studies on the utilization of malate and citrate by Lacto - 
bacillus casei and Lactobacillus Plantarum are continuing. The 
studies of cell wall and somatic antigens in lactobacilli, in 
cooperation with C. A. Mills and P. A. Hansen of the American 
Type Culture Collection, have shown that agglutinating antigens 
are present in cell wall and somatic teichoic acids, and that 
group precipitating antigens may be associated with the cell 
wall teichoic acids only. A manuscript describing these findings 
is being prepared. 

As Chairman of the Bergey's Manual Committee on non-spore-forming 
gram-positive rods, the principal investigator conducted a meeting 
of the European members in London, England on March 1-3, 1968 
inclusive. Twenty bacterial genera, including a number of oral 
importance, were discussed and plans made for their improved 
definition in the forthcoming edition of Bergey's Manual. This 
meeting was mandatory. Further European meetings, one to be 
held this summer in England, are indispensable for the successful 
conduct of the work. 

A comprehensive questionnaire, to be answered by world specialists 
in various genera, was prepared in this laboratory with the 
cooperation of Dr. Krichevsky and Dr. Colwell of Georgetown 
University. Dr. Colwell is an acknowledged world expert on the 
use of computers in bacterial taxonomy. This questionnaire is 
being submitted to the world scientific community for consideration. 
The questionnaire is designed so that answers can be computer 
coded, making it possible for the data to be stored, retrieved 
and assessed with computer aid. Through this program the 
National Institutes of Health has a unique opportunity to render 
an invaluable service to world academic science and clinical 
diagnosis. 



93 



Part A (continued) Serial No, NT.DR-17 (67) 

Significance to Dental Research ; 

Many problems exist in the characterization of indigenous oral 
microorganisms and their natural relationships with other 
organisms. Systematic taxonomic studies are indispensable for 
the advancement of academic and clinical science. 

Proposed Course of Project ; 

1. Utilization of computer technology to store, retrieve and 
analyze data of characteristics of 265 genera of bacteria, 

in cooperation with Drs. Krichevsky and Colwell, the Bergey's 
Manual Trust, and the International Committees concerned 
with the definition of various genera. 

2. Studies of the genetic relatedness of lactobacilli, and their 
macromolecular biology; continued analysis of their antigenic 
relatedness; studies of their malate and citrate metabolism; 
investigations of their potential aerobic or nonglycolytic 
metabolism. 

3. Continued studies of utilization of lactate and other metabo- 
lizable substrates by Veillonella species and related organisms; 
studies of gluconeogenesis in Veillonella species. 



Part B 



Honors ; 



Member of the Subcommittees on lactobacilli and related organisms; 
Neisseriaceae ; and Bifidobacterium of the International Committee 
on Bacteriological Nomenclature. Author of Research Proposal 
accepted by the Subcommittee on Bifidobacterium . 

Member of the merican Society for Microbiology Committee on 
Lactobacilleae and Propionibacteriaceae . 

Appointed Chairman of the Bergey's Manual Committee on Gram- 
Positive Non-Sporulating Rods. 

Appointed by the Bergey's Manual Committee on Gram-Negative 
Anaerobic Bacteria to rewrite the description of the genus 
Veillonella. 

Appointed to the Board of Editors of Bacteriological Reviews. 



39 



^ / ^- A\ Serial No, NIDR-17 (67) 
part B (continued) seridj. inu, 1__^ 



Publications : 



Rogosa, M. and L. L. Love: Direct quantitative gas chromato- 
graphic separation of C -C fatty acids, methanol, and ethanol 
in aqueous microbial fermentation media. Applied Microbiology , 16; 
285-290, 1968. 



1 fif^ 



Serial No. NIDR-18 (61) 
1. Microbiology 
3. Bethesda, Md. 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Studies on the Regulation of Lactic Acid Production 
by Microorganisms. 

Previous Serial Number: NIDR-14 

Principal Investigator: Dr. C. L. Wittenberger 

Other Investigators: None 

Cooperating Units: American Dental Association 

Man Years: 

Total: 3-1/4 
Professional: 1 
Other: 2-1/4 

Project Description: 

Objectives : 

It is the general purpose of this program to examine mechanisms 
by which the biochemical activities of the microbial cell are 
regulated, and to delineate, where possible, the molecular basis 
for such regulation. The specific biochemical process currently 
under investigation is that of anaerobic lactic acid metabolism. 
One organism employed in these studies, Butyribacterium rettgeri , 
can ferment glucose with the formation of lactic acid or, under 
appropriate conditions, it can utilize lactate as a substrate 
for growth. This single organism, therefore, provides an excel- 
lent model system for studying various biochemical aspects of 
both lactate formation and lactate degradation. In addition, 
studies have been initiated to ascertain what factors may operate 
to regulate the formation of lactic acid in members of the genus 
Streptococcus . 

Methods Employed : 

All are standard techniques routine to the tj^je of studies herein 
described. 






Part A (continued) Serial No. NIDR-18 (61) 

Maior Findings; 

Prior studies relating to the regulation of lactic acid metabolism 
in Butyribacterium rettgeri dealt with a detailed analysis of 
certain factors which influence the catalytic activity of the 
enzyme (lactate dehydrogenase) involved in the conversion of 
pyruvic acid to lactic acid. It was established that this enzyme 
possessed two binding sites for its substrate, pyruvate, and a 
site for binding adenosine triphosphate (ATP) which was separate 
and distinct from any occupied by the other ligands involved in 
the reaction. Interaction of the enzyme with ATP resulted in an 
inhibition of its catalytic function. This inhibition of the 
conversion of pyruvate to lactate by ATP provides the cell with 
a means by which it may free pyruvate carbon for utilization in 
biosynthetic pathways when sufficient energy (ATP) is available 
for such endergonic processes. 

One phase of our present investigation has been oriented toward 
resolving the mechanism by which ATP inhibits the E. rettgeri 
lactate dehydrogenase (LDH) . This is part of a general effort 
to understand, at the molecular level, those processes which 
operate within the cell to regulate its biochemical activities. 
The results from these studies have led to the formulation of 
a model for the LDH which appears to explain satisfactorily its 
various physical and kinetic properties. 

The proposed model predicts that interactions of the enzyme with 
ATP or NADH result in a partial restriction at the site for the 
unbound ligand. That such ligand-protein interactions do indeed 
result in conformational alterations of the protein is indicated 
by the fact that ATP confers a marked thermal stability to the 
enzjmie while NADH reverses the protective effect of ATP. The 
conf igurational changes which accompany binding of the coenzyme 
or nucleotide inhibitor, appear to involve alterations in the 
tertiary structure of the protein rather than alterations of its 
quaternary structure. For example, no significant change in the 
molecular weight of the LDH can be detected by either gel filtra- 
tion or sucrose density gradient centrifugation as a result of its 
interaction with either of the ligands. 

Lactic acid formation has also been studied in members of the 
genus Streptococcus . In _S. faecium , the LDH requires fructose-1, 
6-diphosphate (FDP) for activation. The enzyme is virtually 
completely inactive in the absence of FDP and the requirement for 
FDP as the activating ligand is highly specific. A variety of 
metabolic intermediate compounds have been tested for their 
ability to activate the LDH without success. 



Ij2 



Part A (continued) Serial No. NIDR-18 (61 ) 

The LDH has been purified about 100-fold over the present in 
cell-free extracts of S_. faecium and this purified enzyme has 
been used to study the mechanism of FDP activation. Preliminary 
results suggest that FDP activates the ensyme by mediating a 
conformational alteration of the LDH which makes both the coenzyme 
(NADH)- and substrate (pyruvate) -binding sites more accessible 
to the respective reactants. It has been shown, for example, 
that FDP lowers the apparent K^ for both NADH and pyruvate. 

Finally, it has long been known that the streptococci produce 
more lactate from glucose at an acid pH than at an alkaline pH. 
We have found that the S. faecium LDH binds its activator (FDP) 
very well at an acid pH but very poorly at an alkaline pH. This 
probably provides a mechanistic explanation for the established 
physiological relationship between pH and lactic acid production. 

Significance to Dental Research: 

It is anticipated that information derived from these studies 
on biochemical control mechanisms will have general significance 
in advancing the knowledge of cell physiology. Such knowledge 
is of fundamental importance in formulating rigorous and system- 
atic approaches to such diverse problems as cell reproduction, 
chemotherapy, and viral alteration of host metabolism. Present 
work on the regulation of microbial lactic acid production should 
further contribute specifically to a more comprehensive under- 
standing of the factors involved in dental caries. 

Proposed Course of the Project; 

Present studies on the regulation of lactic acid production in 
streptococci will be continued. Special emphasis will be 
given to establishing the precise mechanism by which the lactate 
dehydrogenase from these organisms is activated by fructose-1, 
6-diphosphate. Other studies with the streptococci will also be 
initiated to determine whether intermediate compounds from one 
catabolic pathway can influence other pathways of catabolism. 

Part B; Publications 

1. Wittenberger , C. L. : Kinetic studies on the inhibition of a 

D(-) -specific lactate dehydrogenase by adenosine triphosphate. 
Jo Biol. Chem. , 1968, In press. 



10 3 



Report of the Laboratory of Biochemis try- 
National Institute of Dental Research 
S-unUnary Statement 

With the creation of the Connective Tissue Section, the appointment of a 
Chief for the Enzyme Chemistry Section, and the relocation (from Human 
Genetics Branch) and renaming of the Cell Biology Section, all laboratory 
personnel are now assigned to one of five sections which reflect the major 
programs of the laboratory. Since the sections are all located on one floor, 
there is considerable interchange and collaboration. 

These administrative and program changes mark the close of all studies on 
fluoride metabolism and experimental caries in this laboratory with the ex- 
ception of one study on the long-term effects on bone and aorta of water 
f loridation in Grand Rapids . This study is being continued under contract 
and should be completed by the end of I968. The present efforts of the 
laboratory represent broadly based and fundamental biochemical programs 
designed to have relevance to the modem concept of dental research. 

In, all of its programs, the laboratory continues to depend upon approximately 
equal proportions of senior research staff and postdoctoral personnel here 
under one of the several NIH programs or as guest workers. Postdoctoral 
training is, therefore, an important byproduct of the laboratory operation 
as well as an important source of scientific talent. 

For purposes of summarization, the program of the laboratory is divided 
according to the section designations. 

Protein Chemistry Section 

The major effort for sane years has been the study of the structure of col- 
lagen in an attempt to understand the factors involved in stabilization of 
molecular structure, fibril formation (aggregation) and maturation (covalent 
cross -linking). The large size of the molecule reqiiires a stepwise "taking 
apart" of the molecule to smaller pieces amenable to chemical and physical 
chemical studies. The three o; chains can be isolated chrcmatographically 
and the a chains can be cleaved chemically at specific positions (methionyl 
residues) with CNBr to yield a relatively simple mixture of unique peptides. 
In addition to the al chain of rat skin collagen which has been studied in 
this way, the 02 chain of rat skin collagen and the ai and 02 chains of 
chick bone collagen are now being investigated. The results of these studies 
plus results on other collagens being studied elsewhere peimit some important 
conclusions. First, collagens from different species differ but there is 
close homology. Comparisons show what portions of the amino acid sequence 
are critical and provide a basis for relating structure to function. Second, 
collagens from different tissues of one species may have identical sequences. 
For example, the nonhelical cross-link region of the al chain, which pre- 
sumably has a critical role in maturation and aggregation, is identical in 
chick bone and skin collagen and in rat skin and tail tendon collagen but 
the rat and chick collagens differ from each other in one residue of the 

105 



( 



nineteen in the region. Human skin collagen differs in several positions <, 
The apparent identity of the primary structixre of the two collagens from 
rat and of the two collagens from chick suggests that collagens in different 
tissues of one animal may arise from a single gene (or group of genes). 
Differences in fimction must then be ascribed to differences between the 
collagens introduced after polypeptide chain assembly (such as the degree 
of hydroxylation ajid of aldehyde formation) or to other tissue components o 

The chemistry of the cross-links in collagen is under f^orther investigation. 
Chick bone collagen has been shown by chemical and x-ray diffraction studies 
to have a high degree of intermolecular cross -linking. Preliminary results 
suggests it is a suitable tissue for study to deteimine the sites and mode 
of intermolecular cross-linking. 

The amino acid sequence of 5^ residues at the amino terminal end of the al 
chain of rat skin and rat tail tendon collagen are now known. Studies in 
progress here and in laboratories outside NIH are increasing this number 
with the hope that eventually the entire sequence of about 1000 amino acids 
can be determined. 

The availability of peptides from collagen of known sequence provides an 
usual opportunity to study collagen helix formation in a well-defined system. 
Studies in progress show that a random coil peptide can convert in a complete- 
ly reversible reaction to the collagen helix by aggregation to a trimer 
producing a triple-chain helix. The kinetic and thermodynamic parameters of 
the process can be calculated. 

The same process of reversible helix formation has been utilized -under other 
conditions in a mechanochemical study. Solutions of certain salts denature 
or shrink (loss of helix) collagen fibers. If the fibers are stretched under 
force, helix is regained. It was shown that interaction with the salt is 
greater in the shrunken than in the stretched state. A theoretical model 
was derived for the process and shown to be consistent with the experimental 
data. 

Connective Tissue Section 

Ctirrent studies are largely in two areas--cross-linking of collagen and elastin 
and mineralization of connective tissue. Previous studies established that 
the initial step in the cross-linking of collagen and elastin is identical. 
Lysine in peptide linkage is converted to a-amino adipic- - semialdehyde 
(allysine)o A major breakthrough was made with the finding of an enzyme in 
extracts of connective tissue that carries out this reaction. The enzyme is- 
inhibited in vitro by levels of a lathyrogen which previously have been shown 
to block collagen and elastin cross-linking in vivo and in ctilture. Penicil- 
lamine, another compound inhibiting the cross-linking of collagen and elastin, 
has been found to block the cross-linking of elastin after the formation of 
allysine. in the presence of penicillamine an altered elastin, rich in 
allysine but deficient in cross-links, accimulates. 

Elastin from aorta and nuchal ligament was used in an in vitro system to , 
study mineralization. The amino acids located at the nucleating site of t 

lOS 



mineralizing elastin were isolated by using elastase to remove nonmineralized 
matrix. The mineralized organic residue following digestion was found to be 
rich in cysteine and dicarboxylic amino acids. Fe was found to be coij,cen- 
trated at this site. Model studies showed that a ternary complex of Fe , 
phosphate and ccaaplexing agent is the nucleating species. Presumably, in 
elastin, Fe reacts with cysteine, and phosphate fills the unoccupied co- 
ordination positions of this complex. This local concentration of oriented 
phosphates acts as the seed upon which calcium and other phosphates ions 
are deposited o 

A second system to study mineralization is provided by embryonic chick bone. 
This tissue mineralizes when maintained under proper conditions in vitro. 
Bones obtained from animals just prior to mineralization require embryo 
extract for mineralization and to promote a high growth rate and differentia- 
tion. However, bones obtained after mineralization has started will con- 
tinue to mineralize in the absence of embryo extract in a manner that is 
largely independent of growth or further differentiation. In the yoimg, 
nonmineralized bones, only two cell types (cartilage and fibroblasts), can 
be obtained in cell culture after trypsinization. A third type of cell is 
obtained from the shaft of mineralizing bones. This cell, appearing at the 
site and time of mineral deposition, apparently initiates this process. 

Enzyme Chemistry Section 

Studies carried out in this section have been primarily focused on the basic 
mechanisms by which enzymes function as organic catalysts. Two enzymes that 
catalyze very different biological reactions, porcine pancreatic chymotryp- 
sin C and guinea pig liver transglutaminase, have been under intensive study. 
A sequence of twelve amino acids surrounding the essential histidine of the 
chymotrypsin C active site has been elucidated. Certain differences between 
this sequence and those in other chymotrypsins of other mammals may reflect 
species differences and determine specificity. A kinetic mechanism for 
transglutaminase action, consistent with all of the experimental observations, 
has been formulated. This mechanism, wherein a common acyl enzyme intermedi- 
ate is formed, takes into consideration the metal ion activation of the 
enzyme and explains the varied calcium ion concentration dependency for the 
several reactions catalyzed by transglutaminase. There appear to be two 
separate metalbinding sites and a conformational change accompanies the 
binding. 

Pharmacology Section 

The emphasis of recent studies has been on drug- induced fetal malformations ■ 
with a particular interest in cleft palate. Normal mammalian palatogenesis 
consists of horizontal rotation of the platal shelves and subsequent fusion. 
Prior to and during the process there is a gradual reduction in the flexure 
of the cranial base. It is proposed that this straightening results in an 
"internal shelf force" which raises and rotates the palatal shelves. 

Failure to effect closiure co-uld result if the shelves do not rotate or if 
fusion fails even though rotation is normal. The process of fusion was 
studied by histochemical techniques. Enzyme changes associated with epithelial 
breakdown at the point of ftision were observed. 



107 



In view of the complexity of the process, it is not surprising that pala- 
togenesis can be effected by many routes. A new example was provided by 
the observation that lathyrogens, which have as their prijmry effect the 
inhibition of cross-linking of collagen and elastin, can produce cleft palate » 

A new approacVi to the study of teratogens was developed „ Techniques were 
devised whereby a drug or control compound in a millipore filter can be 
applied directly to the embryo reducing the contribution of maternal and 
placental factors o 

The study of teratogenesis in the primate has been conducted under contract o 
Thalidcimide J a known teratogen, was used as a positive control and produced 
malformations in the Rhesus monkey. If given at the appropriate time, very 
low doses were active in producing a variety of malformations. 

Cell Biology Section 

Current investigations are aimed at an imderstanding of the mechanisms 
bywhich genetic information contained within the cell functions in the 
regulation of normal and abnormal cell growth and cellular differentiation. 
The major effort consists of a multidirectional approach to the question 
of lymphocyte growth. In this unique system, normal human cells can be 
studied in an essentially physiological resting state in vitro , and then 
are induced to enter a state of rapid growth and division by contact with a 
variety of stimulating agents, particularly phytohemagglutinin (EHA) frou 
kidney beans. 

As background to detailed studies, quantitative and kinetic data have been 
obtained, determining the optimal dose of growth- stimulating agents, the time 
course of the growth response, and the interactions between cell growth and 
cell death which occur at excessive doses of stimulating agent. Current 
evidence suggests that the same mechanism which stimulates the lymphocyte 
to grow may, when carried to extremes, kill the stimulated cell. 

Studies with polycations (DEAE-dextrans ) and polyanions (heparin, dextran 
sulfate) suggest that an important event in initiating and permitting con- 
tinuation of cell growth occurs at the cell surface, and have f\irther shown 
that the surfaces of malignant lymphoid cells differ from those of normal 
lymphocytes in their reactivity with polyions. Some growth stiiiiulating 
agents, particularly specific antigens, were shown to require an interaction 
between lymphocy-tes and macrophages in order to produce effective growth 
stimulation. This finding is consistent with the widely held notion that 
macrophages must process antigens in order to make them recognizable by the 
lymphocyte o 

Immediately after stimulation of lymphocytes by FHA, RKA synthesis increases. 
At least three steps which govern the production of various classes of RM 
have been identified. Controls at each step are altered after growth stimu- 
lation directing a specific pattern of changes which characterize the shift 
of resting cells to a growing state. A large increase in the rate of riboso- 
mal RM synthesis accounts in part for the progressive cytoplasmic accumula- 
tion of ribosomes, increased protein synthesis, and cellular enlargement 
found in growing cells. 

L loa 



Ihe evidence indicates that the non-growing lymphocyte contains a limiting 
amount of protein (or proteins) required for ribosomal RMA. synthesis. It 
fiirther suggests that this protein must be constantly produced to allow the 
increased ribosomal RM. synthesis that follows growth stimulation. The 
availability of this protein may therefore constitute a control mechanism 
in governing lymphocyte growth. 

The pre-existing ribosomes of the resting lymphocyte were found to be capable 
of increased protein synthetic activity, implying that the availability of 
ribosomes is not a limiting factor in determining cell protein synthesis in 
the resting state » The resting cell thus has the capacity to increase the 
production of 'proteins needed for ribosome and ribosomal RM synthesis with- 
out any need for new ribosomes. However, one new ribosomes are available, 
the synthesis ©f .the various products needed for multiplication can proceed. 

In separate experiments, a hitherto unreported double- stranded RNA. form has 
been found in animal cells by the use of new analytical techniques. This 
material was found in small amounts in lymphocytes, but in much larger 
quantity in malignant lymphoma cells. 



lOS 



Serial No„ KIDR-19 (^2) 

1. Biochemistry 

2. Protein Chemistry 
3„ Bethesda, Mi. 



PHS-NIH 

Individual Project Report 

July 1, 1967 through June 30, I968 



Part A 



Project Title: Analytical and Structural Studies on Collagen 

Previous Serial Wumber: WIDR-30 

Principal Investigator: Dr. K. A., Piez 

Other Investigators: Dr. P. Fietzek, Dr. Jo Daniels, Dr. £» Schiffraann 

and Dr. M« Rubin 

Cooperating Units: Dr. Po Bornstein (UniVo of Washington, Seattle), 

Dr. ¥„ Butler (Univ. of Alabama, Birmingham), 
Drc A„ Kang (Massachusetts General Hospital, Boston) 

Man Years: 

Total: 5 1/2 
Professional: k 
Other: 1 l/2 

Project Description: 

Objectives ; 

It is the long range purpose of this project to study the chemistry 
and structure of collagen as it relates to function. The immediate 
probleins are: (l) characterization of the a chains of collagen, 
including limited amino acid sequencing; (2) the chemistry and 
biosynthesis of covalent cross-links in collagen; and (3) the 
renaturation of collagen polypeptide chains into the collagen 
triple-helical structure. 

Methods Employed : 

Usual laboratory techniques. 

Major Findings ; 

The characterization of the a chains of collagen has been approach- 
ed by utilizing a specific reagent for cleavage (CWBr). Eight 
peptides accounting for the entire al chain have been isolated. 
The order of these peptides has now been determined. This informa- 
tion is necessary to assemble amino acid sequences of the individual 
peptides as they become available. 

ilO 

- 1 



Part A (continued) Serial Noc MIDR-I9 {'^2) 



Amino acid analyses and partial sequences are becoming available 
for several of the peptides from several different collagens. The 
sequence around the cross-link region of Qfl. is identical in chick 
skin and chick bone collagens and in rat skin and tail tendon 
collagens. The chick and rat collagens differ, but only in a 
single amino residue in a total of 10 residues. This pattern 
holds for other peptides suggesting that different connective 
tissue collagens in the same animal are derived from, the same 
gene. If this finding is confirmed as a more complete comparison 
is made, tissue differences must be ascribed to differences in the 
collagen that are introduced after polypeptide synthesis (such as 
in the degree of hydroxylation or of aldehyde formation prior to 
cross -linking) or to other tissue components. 

Specific cleavage of the 02 chain of rat skin collagen with CHBr 
produces five or six unique peptides. These have been isolated 
and partially characterized. 

The CKBr peptide (Qa.-CB6) from the carboxyl terminal end of al 
from rat skin collagen, which has a molecTzlar weight of about 
16,000, is being studied to see if it has any unusual features 
analagous, or complementary, to the amino terminal end where 
cross-links originate. Cleavage of al-CB6 with chymotrypsin 
jrields three peptides which have been isolated and will be 
characterized. 

The chemistry of the intramolecular cross-link in rat skin collagen 
is being pursued. Indirect evidence has suggested that two lysine- 
derived aldehydes (allysine) condense to form analdol product. 
Asstmiing this to be correct, a series of organic reactions has been 
devised to degrade the cross-link and yield specific products which 
will prove the structure. Preliminary results are consistent with 
the proposed structure. 

The natiire of the forces that hold the three chains of collagen 
together in a triple chain helix is not well understood. This 
problem has been approached by using a small peptide (36 amino 
acid residues) to study helix formation and denaturation. It has 
been found that completely reversible helix formation occurs and 
the kinetic and thermodynamic parameters of the process can be 
calculated. This simple system serves as a model for the more 
complex a chains which have about 1000 residues and form a perfect 
triple helix in vivo . 

The collagen fiber has been used as a model to study the effect of 
environment on conformation. In the presence of certain aqueous 
salt solutions the shrinkage temperature (a measure of loss of 
conformation) of a collagen fiber is markedly lowered. This 
phenomenon is an example of a mecha.nochemical process. Although 



111 



Fart A (continued) Serial Ro. KEDR-l^ (^2) 

this particular process is not utilized by living organisms to do 
work, it is analogous to living processes and has the advantage 
of being amenable to study. The parameters of work (measured as 
stretching force or tension on the fiber), conformation (measured 
as length of the fiber) and salt (LiBr) concentration were related 
in a theoretical model. Experimental data were obtained consistent 
with the model. It was found that the interaction of salt and 
collagen was force and conformation dependent. 



Part B Publications : 

Bernstein, P.: The incomplete hydroxylation of individual prolyl 
residues in collage. J. Biol. Ghem, , 2lf2:2572-257l|, May I967. 

Piez, K„ A.: Soluble collagen and the components resulting from 
it denaturation. In Raraachandran, G. N„ (Ed.): Treatise on 
Collagen, Vol. I, Chemistry of collagen. Academic Press, I967, 
pp 207-252. 

Piez, K. A., Bomstein, P., and Kang, A. H.: The chemistry and 
biosynthesis of interchain cross-links in elastin. In Fibrous 
Proteins. Butterworths , in press. 

Bomstein, Po: Comparative sequence studies of rat skin and tendon 
collagen. I. Evidence for incomplete hydroxylation of individual 
prolyl residues in the normal proteins. Biochemn stry 6, 3082-3093? 
Oct. 1967. 

Piez, K. A.: Molecular weight determination of random coil peptides 
from collagen by molecular sieve chromatography. Anal. Eiochem. , in 
press. 



112 



Serial No. NIDR-20 (66) 



PHS-WIH 
Individual Project Report 
July 1, 1967 through June 30, I968 



Part A 



Project Title: Factors Influencing Resorption and Cell Growth in 

Collagen Itaplants Fozmed by Thermal Gelation iji vivo 

Previous' Serial Nuniber: BIIDR-32 

Principal Investigator: Dr. K. A. Piez 

Cooperating Units: None 

Man Years: None 

Project Description: 

Objectives; 

This project was established under contract as an extension of 
intramural metabolic and chemical studies on the maturation and 
chemistry of collagen. The immediate obj'ective is to obtain 
information concerning the fate of collagen reconstituted in vivo 
with particular interest in its resorption and maturation through 
cross -linking. A longer term objective is to determine the poten- 
tial of reconstituted collagen as a filling material for excisional 
wounds in hard and soft tissue. 

Methods Employed: 

This project is conducted largely by Dr. S. Shoshan, Hadassah 
School of Dental Medicine, Jerusalem, Israel, under contract 
utilizing PL ij-80 funds. (Research Agreement No. 6^51^1 in the 
amo-unt of 2^9,7^1 Israeli Pounds for the period December 15, 19^5 
to December 15, I968.) The project employs half the time of Dr. 
Shoshan and 2-l/2 man years by supporting personnel. 

The project first requires an in vitro study of the conditions 
under which ptirified collagen can be reconstituted to native type 
fibrils. Using the established conditions, collagen is recon- 
stituted in vivo in experimental animals in such a way that the 
implant can be removed at a later time and examined by histological 
and chemical methods. Studies during the current year utilized 
diffussion chambers containing purified reconstituted collagen 
prepared in various ways and placed under the skin of gimea pigs. 
In this way a sample could be pretreated, placed in the animal, 
and then reisolated at a later time for analysis. 

113 



Part A (continued) Serial No. HIDR-20 (66) 



Major Findings : 

Chemical studies on collagen have previously shown that cross- 
linking is initiated in a specific region of the collagen molecule „ 
A lysyl residue in a non helical region is enzymatically converted 
to an aldehyde (allysine) which reacts with other functional groups 
on adjacent polypeptide chains to form covalent cross-links. Since 
the region is nonhelical and at the amino terminal ends of the a 
chains of collagen, it can be removed by enzymatic treatment. 
Chemical studies have also suggested that collagen from lathyritic 
animals (fed p-arainopropionitrile) differs from normal collagen 
only in that the conversion of lysine to allysine did not occur. 
To confirm and extend these results the following experiments 
were done, (l) Normal collagen was implanted in normal animals. 
(2) Collagen from lathyritic animals was implanted in normal 
animals. (3) Normal collagen was implanted in lathritic animals 
(k) Chymotrypsin- treated collagen was implanted in normal animals. 
After removal and examination for changes in cross- linking, it 
was found that normal as well as lathyritic collagen in normal 
animals continued to cross-link in an essentially normal fashion 
while normal collagen in lathyritic animals did not cross-link. 
These results show that cross-linking can proceed in a diffusion 
chamber where cells, but not enzymes, would be excluded. The 
defect in lathyrisin is clearly at the enzymatic step in allysine 
formation. JESnzymatic removal of a specific site, previously de- 
fined chemically, prevented cross -linking. It is concluded that 
this site is the major or sole site of cross-link formation during 
early maturation of collagen. 

Significance to Dental Research 

The maturation of collagen is fundamental property of collagen 
necessary for the proper function of connective tissues. These 
implant studies using carefully controlled conditions will lead 
to a better understanding of the process and its control. The 
basic findings will hopefully be useful in determining conditions 
under which collagen can be used as an implant to promote healing 
after operative procedures such as tooth extraction. 

Proposed CoxxTse of Project ; 

The current studies should be completed within the contract period 
(Ending December I968). It is hoped to extend the contract an 
additional year rbo study resorption of collagen using the same 
techniques. 



rih 



Part B Publications: Serial No. WIDR-20 (66) 

Shoshan, S., and Finkelstein, S.: Cell growth promoting effect 
of enriched collagen solutions thermally gelled in vivo . Israel 
J. Med. Sciences 3, 755-58, Sept-Oct. 1967. 

Shoshan, S., and Finkelstein, S.: In vivo studies on collagen 
cross -linking. Biochim. Biophys . Acta , i^k, 26I-63, Jan. I966, 



115 



Serial Noo NIDR-21 (62) 

1. Biochemistry 

2. Protein Chemistry 

3. Bethesda, Md. 



PHS-NIH 
Individual Project Report 
July 1, 1967 throTigh June 30, I968 



Part A 



Project Title: The Chemistry of Bone and Cartilage Collagens 

Previous Serial Number: WIDR-31 

Principal Investigator: Dr. E. J. Miller 

Other Investigators: Dr. J. M. Lane, Dr. G. R. Martin, and 

Dr. K„ A. Piez 

Cooperating Units: Laboratory of Experimental Pathology, NIAMD, 

Dr. L. Sokoloff 

Laboratory of Histology and Pathology, NIDR, 
Dr. E. D. Eanes 

Man Years : 

Total: h 
Professional: 2 
Other: 2 

Project Description: 

Objectives ; 

To study the biosynthesis of bone and cartilage collagens; specifi- 
cally, to determine their primary structure, mode of cross-linking, 
and relationships with other connective tissue components, to study 
alterations of these parameters in patliological states. 

Methods Employed ; 

Usual laboratory procedures of biochemistry. 

Major Findings: 

An investigation of the primary structure of collagen in a normally 
calcifying tissue (chick bone) has been initiated using al and 02 
chains isolated from acetic acid- soluble collagen of the femora 
and tibiae of lathyritic chicks. Acid-soluble collagen was used 
exclusively in these studies since previous results indicated that 

116 



Serial No. NIDR-21 (62) 

Part A (continued) 

extraction of bone collagen in denaturing solvents led to degrada- 
tion of extracted components. Cleavage of the al chain (containing 
nine methionyl residues) at methionyl residues with cyanogen bromide 
gives rise to ten peptides which have been separated by ion exchange 
and molecular sieve chromatography o The ten peptides obtained in 
this manner each constitute a -unique portion of the al chain as 
demonstrated by chromatographic properties, amino acid compositions 
and -molecular weight determinations. In addition, the isolated 
peptides account for all of the amino acids in the al chain and 
their molecular weights totaled 9^,000 (as deteimined by molecular 
sieve chromatography) in good agreement with 955OOO found by 
sedimentation studies on al. These peptides, therefore, represent 
convenient starting materials for further studies of the primary 
structure of the al chain. Although these results indicate that 
the al fraction of this collagen is a single molecular species, 
it was found that some heterogeneity is introduced as a result of 
variable hydroxy lation of lysyl residues. Variation in the degree 
of hydroxylation of lysyl residues was evident from amino acid 
analyses of most of the' peptides containing both lysine and 
hydroxy lysine. For instance, al-CB3 was comprised of 1U9 amino 
acids and contained k.3 lysyl residues and O.5 hydroxylysyl residues. 
This phenomenon was further investigated in one of the smaller 
peptides, al-CBl. The latter peptide contained 17 amino acids, 
was derived from a nonhelical portion of the al chain (since it 
contained only 3 glycyl residues), and appeared to be homologous 
to al-CBl previously isolated from rat skin collagen. Like al-CBl 
of rat skin collagen, al-CBl from chick bone collagen contained one 
lysyl residue, but in the latter case it was consistently observed 
that the lysyl residue was approximately 50^ hydroxy lated . Diges- 
tion of al-CBl with trypsin and isolation of the resulting peptides 
showed that lysine and hydroxylysine occupied identical positions in 
the peptide. Preliminary results from the studies on bone collagen 
suggest that intermolecular cross-linking predominates in bone col- 
lagen and that al-CBl is somehow involved in their formation, 
presumably through the lysyl residue. The significance of the 
partial hydroxylution of the lysyl residue in al-CBl of chick bone 
collagen is, at present, -unknown. 

In collaboration with Dr. Eanes, NIDR, a study of the x-ray dif- 
fraction properties of bone collagen has been completed. These 
studies were designed to shed light upon the forces responsible 
for the relative insolubility of bone collagens. The x-ray dif- 
fraction data indicates that bone collagen fibrils are much less 
highly oriented than the fibers of tendon, a result to be expected 
in view of the random orientation of osteons in secondary bone and 
the different orientations of fibers within a given osteon. Al- 
though helical structure is maintained when the bone collagen is 

117 



Serial No. WIDR-21 (6?.) 

Part A (continued) 

allowed to equilibrate with water or dilute acid solutions, a 
reversible lateral separation of adjacent molecules does occur as 
indicated by a shifty of the equatorial reflection from a spacing 
of llA. (dry) to I6.5A (wet). When the bone collagen is in contact 
with denaturing solvents (5 M guanidine hydrochloride or 5 M lithium 
chloride), a collagen x-ray diffraction pattern is no longer ob- 
tained indicating a loss of helical structure and disruption of 
lateral molecular aggregation. When bone collagen is washed free 
of denaturing solvents, the characteristic collagen diffraction 
pattern is restored with no net change in intensity of the lines or 
degree of fiber orientation. In contrast, rat tail tendon sub- 
jected to the same treatment exhibits an irreversible loss of 
helical structure and intermolecular spatial relationships. The 
tail tendon will, however, display a stability similar to bone 
collagen if it is pretreated with formalin solution. These results 
indicate that bone collagen molecules are stabilized by a high 
degree of intermolecular cross-linking which provides a nimber 
of fixed points regulating the movement of molecules dviring de- 
naturation and allows a rapid restoration of the spatial relation- 
ships between individual chains and molecules during renaturation 
after removal of the denaturing solvent. 

In collaboration with Dr. Sokoloff , WIAMD, studies on human costal 
and articular cartilage were continued. Specifically, the current 
studies were designed with a view to characterizing the pigment 
associated with connective tissues in aging. The yellowish pigment 
is prominent in cartilaginous tissues such as rib cartilage and has 
previously been thought to bind to collagen fibers making them more 
and more insoluble as a function of age. It was found that the 
pigment resisted extraction in a wide variety of aqueous and organic 
solvents, but could be brought into solution during proteolytic 
digestion of aged rib cartilage. Following short periods of pro- 
teolytic digestion the pigment remained nondialysable, but was 
partially dialysable after long term digestion with large quantities 
of enzyme. These results suggested that the pigment was indeed 
associated with protein. Moreover, isolation of the acidic poly- 
saccharides from the digestion mixture demonstrated that the pigment 
was not associated with that fraction of the tissue. Further 
studies involving trichloroacetic acid fractionation and amino acid 
analyses of the proteolytic digests demonstrated that the pigment 
was associated exclusively with non- collagenous protein. It is 
interesting to note that rib cartilage which displays an unusual 
amount of pigmentation with age, also accumulates a high proportion 
of noncollagenous protein as a function of age. On the other hand, 
articular cartilage which retains a high proportion (approximately 
90^) of its protein as collagen throughout life, exhibits very little 
pigmentation. 

118 



Serial Wo. inDR-21 (62) 

Fart A ( c ontinued ) 

Significance to Dental Research ; 

The collagens of bone and cartilage comprise highly significant 
proportions of the organic material in these tissues. Moreover, 
it can be assumed that the collagens in these tissues play a role 
in normal development and fimction of the tissues. The characteri- 
zation of bone collagen at the molecular level is expected to shed 
light upon the possible role of collagen in mineral deposition and 
similar studies on the collagen in various cartilages will lead 
to an understanding of the manner in which collagen and protein- 
polysaccharide interact to provide a highly elastic tissue yet 
one which is capable of withstanding considerable stress. 

Proposed Course of Pro.iect ; 

These studies will be continued with a view toward chemical and 
structural characterization of bone and cartilage collagen at the 
molecular level. 



Part B Publications: 

Miller, E. J., Martin, G. P., Piez, K. A., and Powers, M. J.: 
Characterization of chick bone collagen and compositional changes 
associated with maturation. J. Biol. Chem. 2^2: 5i|-8l-5U89, Dec. 
1967. 

van der Korst, J. K. , Sokoloff, L., and Miller, E. J.: Senescent 
pigmentation of cartilage and degenerative Joint disease. A.M.A. 
Arch. Pathol ., in press, I968. 

Miller, E. J., and Martin, G. R.: The collagen of bone. Clinical 
Orthopaedics and Related Research , in press, I968. 



119 



k 



Serial No. NIDR-22(62) 

1. Biochemistry 

2. Connective Tissue 

3. Bethesda, Md. 

PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 

Part A 

Project Title: The Chemistry and Biosynthesis of Elastin 

Previous Serial Number: NIDR-37 

Principal Investigators: Dr. G. R. Martin and Dr. S. R. Pinnell 

Other Investigators: Dr. E. J. Miller, Dr. E. Schiffmann and Dr. E. R. 

Goldstein 

Cooperating Units: None 

Man Years: 

Total : 4 and 1/4 

Professional: 3 

Other : 1 and 1/4 

Project Description: 

Objectives ; 

Collagen and elastin, the major connective tissue proteins, depend 
on cross-linking for their structural integrity. The purpose of 
this project is to (1) identify and study the enzjmie responsible 
for initiating the cross- link process and (2) study the biosynthesis 
of cross-linking by chemically interfering at various levels of the 
process. 

Methods Employed : 

Usual laboratory techniques. 

Major Findings ; 

Earlier work in this laboratory established that the initial step 
in the cross- linking of both collagen and elastin is identical. 
Lysine in peptide-linkage is converted to alpha-amino adipic-^- 
semialdehyde. For the sake of brevity, we have given this compound 
the trivial name allysine to indicate that it is an aldehyde derived 
from lysine. The enzyme responsible for this conversion has been 
detected with the aid of two different assays utilizing an elastin 
substrate. (1) Elastin labelled with lysine-6- H releases tritium 



120 



part A (Continued) Serial No. NIDR-22(62) 

when lysine is converted to allysine. The tritium released is 
measured as tritium water following distillation of the water in 
the reaction mixture. (2) l^C-allysine produced from elastin 
labelled with lysine-l-^C can be determined by amino acid analysis 
of its oxidized derivative l^C-a-aminoadipic acid. With the aid 
of these two assays, enzyme has been detected in homogenates of 
skin, aorta and bone. The enzyme is soluble in phosphate-buffered 
saline, requires no readily dialyzable cofactors, and is inhibited 
by physiologically active levels of such lathyrogens as p-amino- 
propionitrile and semicarbazide. The activity is reduced or absent 
in extracts prepared from the bones of lathyritic chick embryos. 
The enzyme is distinct from such previously described enzymes as 
mono or diamine oxidase. The enzyme is specific in that it converts 
lysine in peptide linkage to allysine but does not act on free 
lysine. 

Previous work has established that p-aminopropionitrile prevents 
the cross-linking of collagen and elastin at the step where certain 
lysines in peptide linkage are converted to allysine. Now we have 
found a compound that inhibits cross- linking after the lysine- 
allysine conversion. 

Penicillamine, a compound previously shown to inhibit desmosine 
biosynthesis in elastin, has recently been shown to interfere with 
collagen cross-linking. In an effort to determine the nature of 
penicillamine interference with cross-link biosynthesis, the effect 
of penicillamine was studied on the formation of allysine in 
elastin. Unlike BAPN which interferes with allysine formation, 
penicillamine interferes with desmosine formation after allysine 
is formed, causing the accumulation of an elastin with a high 
aldehyde content. 

Significance to Dental Research ; 

Collagen and elastin, the major structural proteins of the body, 
constitute an important part of the tissues of the oral cavity. 
Their structural integrity depends largely upon proper cross-link- 
ing. Further knowledge of connective tissue protein cross- link 
formation may contribute to our understanding of pathological 
conditions involving connective tissue. 

Proposed Course of Project ; 

Studies on the enzyme responsible for allysine formation from 
lysine are just beginning. Attempts will be made to purify the 
enzyme, identify precisely its substrate, determine any co- 
factor('s), and identify tissue levels in normal and pathological 
tissues. The effect of penicillamine on collagen cross-link 
biosynthesis will be studied to determine if the collagen effect 

121 



Part A (Continued) Serial No. NIDR-22(62) 



is similar to the effect in elastin- 



Part _B Publications: 

Pinnell, S. R. , Martin, G. R. , Miller, E. J.: The nature of the 
inhibition of desmosine biosynthesis by D-penicillamine. Science . 
In press. 

Pinnell, S. R. , Martin, G. R. : Cross-linking of collagen and 
elastin: the enzymatic conversion of lysine in peptide linkage to 
a-aminoadipic- ^-semialdehyde by an extract from bone. Proc . 
Natl. Acad. Sci. U. S . In press. 

Miller, E. J., Martin, G. R. , Piez, K. A., and Powers, M. J.: 
Characterization of chick bone collagen and compositional changes 
associated with maturation. J. Biol. Chem . 242:5481-5489, Dec. 
1967. 

Miller, E. J. and Martin, G. R. : The collagen of bone. Clinical 
Orthopaedics and Related Research , 1968. In press. 



Serial No. NIDR-23(62) 

1. Biochemistry 

2. Connective Tissue 

3. Bethesda, Md, 
PHS-NIH 

Individual Project Report 
July 1, 1967 through June 30, 1968 

Part A 

Project Title: Calcification of Organic Matrices 

Previous Serial Number: NIDR-34 

Principal Investigators: Dr. E. Schiffmann, Dr. D. R. Lavender 

Other Investigators: Dr. E. J. Miller and Dr. G. R. Martin 

Cooperating Units: None 

Man Years: 

Total: 4 
Professional: 2 
Other: 2 

Project Description: 

Objectives : 

To study basic mechanisms whereby organic matrices calcify. The 
systems under investigation are: 1. mineralizing elastin-contain- 
ing tissue in vitro . 2. calcification in embryonic chick bone 
rudiments. 3. cultures of cells from osteoid and related tissue. 

Methods Employed : 

1) Elastic Tissue 

Nuchal ligament preparations, a material rich in elastin, were 
obtained from exhaustive extraction of the bovine tissue with 
guanidine to remove much collagenous component. Mineralization of 
this matrix was accomplished in calcifying media in the presence of 
Ca^5 and/or P-^^ at 38°. Extent of calcification was determined by 
isotopic assay on aliquots of acidic extracts of the mineralized 
matrix. Measurement of mineral formation in the absence of matrix 
was by assaying the radioactivity of aliquots of an acidic solution 
of mineral that had been collected on a Millipore filter. Labelling 
matrix with Fe^^ was accomplished by incubating sulfhydryl-treated 
elastin in the presence of 0.01 mM Fe^Sci . Incubation of matrix 
with elastase was carried out in Tris at 38°, pH 7.5. Standard 
procedures were used to determine amino acid contents of samples. 



123 



Part A (Continued) Serial No. NIDR-23(62) 

2) Chic k Bone Rudiment Calcification . 

Chick tibias v^ere isolated from embryos of varying ages and placed 
in organ culture dishes together with tissue culture media under a 
957„ air-57o CO2 gas phase. Ca^5 and proline-H^ were added as needed 
to medium. Calcium uptake by rudiments was determined by both a 
fluorometric method for calcium and by assaying radioactivity of 
acid extracts of the rudiments. Proline incorporation was measured 
by isotopic assay. 

3) Bone Cell Culture 

Cells were obtained from embryonic chick bones, rat and human bones 
after trypsinization. These were grown in tissue culture medium 
under 957„ air -5% CO2. 

Ma i or Findings ; 

1) Blast in- containing tissue 

It has been shown that Fe^"*", complexed to sulfhydryl groups of the 
matrix, participates in the nucleation of an elastin matrix. Studies 
on the characterization of amino acids at or near the nucleation 
site have been carried out, and it has been determined to which 
residues Fe3+ is bound. In addition some information has been 
obtained on the nature of the nucleating complex in a calcifying 
medium in the absence of a matrix. 

a) Amino acids at the nucleating site. 

Proteolytic enzymes have been used as probes to determine those 
amino acids concentrated at the nucleation site on mineralizing 
elastin. We have assumed that the deposition of mineral at specific 
sites in elastin would prevent the enzymatic degradation of these 
regions by proteases. Elastase was found to remove up to 97% of 
the organic phase of mineralizing elastin without appreciable 
solubilization of calcium. The composition of the organic material 
associated with the mineral phase was quite distinct from the 
original elastin as well as residues remaining after the digestion 
of non-mineralized samples. It was found that calcified residues 
were greatly enriched in cysteine as well as dicarboxylic amino 
acids. These studies indicate that only a small portion of this 
matrix is associated with mineral. The portion of the matrix 
associated with the mineral phase is distinct from the rest of the 
matrix and is rich in cysteine which was shown previously to be 
involved with nucleation. 



12i^ 



Part A (Continued) Serial No. NIDR-23(62) 

3+ 

b) Fe binding specificity. 

The amount of Fe^"^ bound as measured by the use of Fe^^ was greater 
in calcified residues compared to that in uncalcified residues, and 
this increased binding could be accounted for by ascribing it to 
sulfhydryl groups. It is estimated that 1 mole of Fe is bound per 
mole of sulfhydryl, and less than 0.05 mole of Fe is bound per mole 
of other amino acids. 

Therefore, evidence is presented for the presence of both cysteine 
and Fe^''' at the nucleating site in mineralizing elastin. 

c) The nucleating complex in solution. 

It was conceivable that a complexed metallic cation nucleated by 
binding phosphate ions to its unfilled coordination positions. In 
a calcifying medium, calcium ions might then be attracted to the 
complex, initiating nucleation. The formation of a ternary complex 
between a cation (Fe2+), a complexing agent (norepinephrine), and 
phosphate was studied by two methods. Potentiometric titration of 
solutions of cation and complexing agent and solutions containing 
cation, ligand, and phosphate indicated that in both cases the, 
cation- ligand complex was present. Also a typical color formation 
indicated the formation of this complex. In the solution without 
phosphate, the amount of complex that precipitated was greater than 
in the solution that contained phosphate. This was demonstrated by 
absorption spectrophotometry of the supernatents of the two solutions. 
The results suggested that phosphate solubilized to some extent the 
cation-ligand complex perhaps by forming a ternary complex. It was 
also shown that accelerated formation of a mineral phase occurred at 
a much greater rate in calcifying solutions containing both cation 
and ligand than in media with either cation or ligand alone. The 
evidence suggests that a ternary complex is involved in nucleation 
of a mineral phase from solution. 

2) Mineralization of embryonic chick bone rudiments. 

The normal pattern of growth and mineralization of the long bones of 
the chick embryo has been determined as a basis of comparison for the 
bones in vitro . Little calcium is found in the chick bone prior to 
the eighth day, after which the total calcium increased markedly. 
Yet there is no abrupt change in concentration of calcium in the bone 
as rapid bone growth accompanies mineralization. However, some 
important morphological alterations occur in the chick bone as the 
bone differentiates from a cartilaginous to a true bone structure. 
This latter change seems to be correlated with cell type as well as 
mineralization. 



In vitro the 9 or 10 day old chick tibia undergoes rapid growth. 



12 



Part A (Continued) Serial No. NIDR-23(62) 



greater growth rate and a higher level of calcium are found in the 
9 day old bone grown in medium supplemented with chick embryo 
extract. In the 10 day old bone, growth is again stimulated by the 
medium containing embryo extract. However, an equivalent uptake of 
calcium occurs in bones at lower growth rates in media lacking 
embryo extract. The levels of calcium accumulating in the 9 day bone 
grown in the embryo extract-fortified medixjm or in the 10 day bones 
grown with or without embryo extract are similar to the levels found 
in developing bones _in vivo . 

3) Bone cell culture . 

Two distinct types of cells have been obtained in culture from 8 or 
9 day old bone, fibroblasts, and cartilage cells. An additional 
cell type occurs in cultures obtained from 10 day old bones <> These 
cells arise from the shaft rather than from the ends of the bones. 
This cell type also can be isolated and cultured from younger bones 
grown in culture for a few days. 

These findings indicate that mineralization occurs as a result of 
differentiation. A cell distinct in morphology from fibroblast or 
cartilage cell arises at the site and at the time of mineralization. 

Significance to Dental Research; 

The processes whereby osseous and non-osseous tissue mineralize are 
not yet understood. Studies on systems which calcify ±n vitro , such 
as elastin-containing tissue on one hand and chick bone rudiments 
as well as isolated cells from the latter source on the other hand 
may contribute to understanding pathological and normal calcification 
respectively. 

Proposed Course of Project ; 

1) Elastin . The nucleating properties of peptide material from the 
nucleating site will be studied. It is of interest to know the 
role, if any, of other functional groups besides sulfhydryl in 
nucleation and/or crystal growth. With reference to this it may be 
possible to relate composition of acids at the nucleating site with 
changes in the composition of the mineral phase. 

2. Chick bone rudiments . The ±n vitro mineralizing chick bone 
system will be used to define the factors involved in mineralization. 

3. Bone cell culture . The ability to culture cells from bone will 
permit the measurement of cellular activities such as collagen 
synthesis and cross-linking in cells from normal and diseased tissue. 



126 



Part B Serial No. NIDR-23(62) 

Honors and Awards: 

Dr. Dick R. Lavender. Edward H. Hatton Award, lADR, 1968, 1st 
Place, Graduate Division. 

Publications: 

Schif fmann, E. : Remarks in First Conference on Biology of Hard 
Tissues , June 1965. New York Academy Science 1967. 

Schif fmann, E. , Martin, G. R. , and Miller, E. J.: Matrices that 
calcify in Biological Calcification , Ed. R. Schraer, Appleton- 
Century-Crof ts. In press. 



IZl 



Serial No. NIDR-24(61) 

1. Biochemistry 

2. Connective Tissue 

3. Bethesda, Md. 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Long Term Effects of Water Fluoridation in Grand Rapids, 
Michigan 

Previous Serial Number: NIDR-40 

Principal Investigator: Dr. E. Schiffmann 

Other Investigators: Dr. F. J. McClure and Dr. G. R. Martin 

Cooperating Units: Albert Einstein College of Medicine, Yeshiva Univer- 
sity - Dr. E. F. Geever 

Grand Rapids -Kent County Health Dept, Grand Rapids, 
Michigan - Mr. Harold E. Samuelson 

Man Years : 

Total: 1 
Professional: 
Other: 1 

Project Description: 

Obiectives ; 

To determine long term effects of fluoridation by histological and 
chemical examination of rib, vertebra and aorta of individuals re- 
siding for at least 20 years in Grand Rapids, Michigan. 

Methods Employed ; 

Grand Rapids, Michigan has been fluoridated since January 1945. To 
evaluate the long-term effects of fluoride, rib, vertebra and aorta 
are being collected from the following age categories of postmortem: 
0-20, 21-40, 41-60, 61-80 and above 80 years of age. Specimens are 
sent in fixative to Dr. E. F. Geever of the Albert Einstein College 
of Medicine, New York, N. Y. , for histological examination (Contract 
PH43-66-941 with Albert Einstein College of Medicine of Yeshiva 
University, New York, N. Y. ; approximate cost $9,500). Portions 
of the same specimens will be analyzed for fluoride, calcium, 
phosphorus and ash content. Control specimens, are being obtained 
from New York City and Albany, New York. 



128 



Part A (Continued) Serial No. NIDR-24(61) 



Major Findings ; 

Sample collection and histological examination is well advanced and 
chemical analysis has begun. However, data are not yet sufficient 
for conclusions to be made. 

Proposed Course of Project ; 

The current contract runs to July, 1968, but will be extended for 
six months to one year to complete study. 

Significance to Dental Research ; 

Although the long-term effects of fluoride naturally present in 
water have been studied, the availability of a population receiving 
controlled amounts of fluoride for more than 20 years offers an 
unusual opportunity to demonstrate the safety of water containing 
fluoride at an optimal level for caries inhibition. 



Part B: Not included. 



12S 



Serial No„ NIDR-25 (52 ) 

1 o Biochemistry 

2o Enzyme Chemistry 



3. Bethesda, Md< 



PHS-NIH 

Individual Project Report 

July 1, 1967 through June 30, 1968 



Part A 



Project Title: Chemistry and Mechanism of Action of (1) Chymotrypsin C 
and (2) Transglutaminase 

Previous Serial Number: NIDR-36 

Principal Investigator: Dr. J. E. Folk 

Other Investigators: Dro H. Kato and Dr. Ro Lc Boothe 

Cooperating Units: Biometrics Research Branch, NHI, Dr. J, P. Mullooly 

Man Years: 

Total: 4 1/2 
Professional: 3 
Other: 1 1/2 

Project Description: 

Objectives : 

The purpose of these studies is to examine the basic mechanisms of 
enzyme action. These include the order of substrate addition and 
product release, the role of metals in activation, the specific 
functional groups involved in substrate binding and in catalytic 
modification, the nature of the amino acid sequence around 
functional groups, and the effect of protein modification on 
activity. 

Methods Employed : 

1. Chymotrypsin C has been labeled at the "active site" histidine. 
residue by means of the specific irreversible inhibitor, ■'■^C- 
tosyl-L-leucyl chloromethylketone, which was synthesized in 
this laboratory. The labeled enzyme has been subjected to 
digestion by pepsin. The resulting labeled peptide has been 
separated and the amino acid sequence analysis has been carried 
out by conventional methods. 

2. The enzymatic activity of purified transglutaminase has been 



130 



Part A (Continued) Serial No„ NIDR-25 (52 ) 

studied by kinetic procedures with particular emphasis on 
the role of metal ion in the hydrolysis reaction. The enzyme 
has been subjected to inactivation by trace quantities of 
copper ion. The mechanism of inactivation has been studied 
using sulfhydryl titration technics and ■'•'^C- labeling methods » 
A number of peptide derivatives containing methyl glutamine 
residues have been prepared in an effort to examine the steric 
arrangement of groups in the substrate binding site of trans- 
glutaminase. The enzjnne has been subjected to N- terminal and 
C-terminal analysis with the hope of determining the number of 
polypeptide chains in the molecule. 

Major Findings ; 

lo The complete sequence of 12 amino acids around the active 
histidine of chymotrypsin C has been elucidated. This 
sequence, Ala-Ala-His-Cys-Ile-Asp-Ser-Gly-Thr-Ser-Arg-Thr, 
shows certain differences from those around active histidines 
in chymotrypsins A and B. 

2. Kinetic and chemical studies strongly support a premise that 
the divalent cations, calcium and strontium, activate trans- 
glutaminase by binding at two separate sites in the enz5aiie 
molecule and that a conformational change in the enzjmie protein 
accompanies the metal ion activation. These studies also form 
the basis for a mechanism of metal ion and substrate additions, 
termed a "ping pong" mechanism, wherein a common acyl enzyme 
intermediate is formed during the hydrolysis or transfer 
reactions and where water or acceptor amine, respectively, add 
after the release of ammonia. 

The findings that copper ion-catalyzed inactivation of trans- 
glutaminase requires high levels of calcium ion, results in 
essentially no binding of copper, but causes the formation of 
two disulfide bonds suggests that inactivation occurs only in 
the conformationally changed enzyme molecule and is a result of 
limited oxidization of 4-sulfhydryl groups. Preliminary studies 
also suggest that the so-called "active site" sulfhydryl group 
is not involved in disulfide bond formation during this inacti- 
vation. The fact that this inactivation is readily reversed by 
reducing agents is consistent with the above findings. 

Preliminary studies show that the length of the methylene chain 
in transglutaminase substrates is not limited to that of 
glutamine - the a-amino-adipamyl derivative is a substrate for 
the enzjmie. First findings with N-terminal and C-terminal 
amino acid methods show that this enzyme molecule of 90,000 
molecular weight has available terminal groups. The type and 
number of residues have not been assigned as yet. ^ -^ i 



Part A (Continued) Serial No. NIDR-25 (52 ) 

Significance to Dental Research ; 

Understanding of the mechanisms of action of enzymes, the use of 
these enz3mies for protein modifications and structure studies, and 
a knowledge of the functions and interrelationships of these 
enzymes contribute to the resolution and understanding of basic 
biochemical processes. 

Proposed Course of Project ; 

lo Further studies of the primary structure of chymotrypsin C and 
comparisons of this structure with those in other proteolytic 
enzymes are underway. The proposed course of investigation is 
a study of zjmiogen activation with particular emphasis on 
structural changes. 

2. Future studies of transglutaminase will entail an examination 

of the location and spacial arrangement of the sulfhydryl groups 
that are involved in disulfide formation during copper ion- 
catalyzed inactivation. The object of this investigation is 
to obtain more information concerning the calcium ion-induced 
conformational rearrangements in the molecule. 

Stereochemical substrates and irreversible inhibitors are in 
preparation. A study of the action of transglutaminase on 
these substrates and inhibition by these inhibitors should 
provide information as to the type of amino acid side chains 
in or near the binding site of the enzyme and the orientation 
of the substrate on the enzyme surface. 

Studies are in progress on the number and type of terminal 
residues in transglutaminase. Various reagents are under test 
as possible chemical modifying agents. 



Part B Publications; 

Folk, J. E., Cole, P. W., and Millooly, J. P.: Mechanism of action 
of guinea pig liver transglutaminase IV . The trimethylacyl 
enzyme. J. Biol. Chem . 242; 4329-4333, October 1967. 

Folk, J. E., Cole, P. W., and Mullooly, J. P.; Mechanism of action 
of guinea pig liver transglutaminase V. The hydrolysis reaction. 
J. Biol. Chem . 243; 418-427, Jan. 1968. 

Tobita, T., and Folk, J. E,: Chymotrypsin C III. Sequence of 
amino acids around an essential histidine residue. Biochim. et 
Biophys. Acta 147: 15-25, July 1967. 



132 



Part B (Continued) Serial No. NIDR-25 (52 ) 

Folk, Jo Eo: Carboxypeptidase A (bovine pancreas),, In 
Specifications and Criteria for Biochemical Compounds , (Ed. 2), 
Natlo Acad, of Sciences, 1967, pp. 233-234. 

Folk, Jo E.: Carboxypeptidase B (porcine pancreas). In 
Specifications and Criteria for Biochemical Compounds , (Ed. 2), 
Natl. Acad, of Sciences, 1967, pp. 235-236. 

Folk, J, E.: Chjnnotrypsin A (bovine pancrease). In Specifications 
and Criteria for Biochemical Compounds , (Ed. 2), Natl. Acad, of 
Sciences, 1967, pp. 237-238, 

Folk, J. E.: Trypsin (bovine pancreas). In Specifications and 
Criteria for Biochemical Compounds , (Ed. 2), Natl. Acad, of 
Sciences, 1967, pp. 261-262. 

Folk, J. E„: Transglutaminase (guinea pig liver). In Tabor, H., 
and Tabor, C. W. (Eds.): Metabolism of Amino Acids and Amines . 
In press. 



133 



Serial No. NIDR-26(63) 
1. Biochemistry 
2- Pharmacology 
3. Bethesda, Md. 
PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Prenatal Developmental Factors Influencing Oral Disease 

Previous Serial Number: NIDR-37 

Principal Investigator: Dr. A. J. Steffek (ADA Research Associate) 

Other Investigators: Dr. M. V. Barrow and Dr. A. C. Verrusio 

Cooperating Units: Office of International Research, NIH, Rio de 

Janeiro, Brazil, Dr. C. T. G. King 

Man Years : 

Total: 7 and 1/4 

Professional: 3 

Other: 4 and 1/4 

Project Description: 

The Pharmacology Section, LB, NIDR, has been involved during recent 
years with investigations related to the mechanisms involved in 
experimentally-produced congenital malformations with particular 
emphasis directed toward the pathogenesis of cleft palate. Specific 
teratogens such as chlorcyclizine and related structural analogues. 
Vitamin A, 6-aminonicotinamide and more recently lathyrogenic agents 
have been implemented in various laboratory animals in an attempt 
to define the etiological factors related to the production of 
these malformations. The results of these investigations are 
presented as follows: 

Subproject A : 

Mechanisms involved in normal development of the secondary palate. . 

Objectives : 

Current theories of cleft palate pathogenesis hold that either the 
palatal shelves fail to assume the horizontal position, or, that 
having done so, they fail to fuse. Thus it may be of great import- 
ance to understand the biological processes underlying both the 
"rotation" of the shelves to the horizontal position and the 
subsequent fusion of the shelves. Since the two processes do not. 

'i2M 



Part A (continued) Serial No. NIDR-26(63) 



occur simultaneously, they can theoretically be considered separate 
phases of palate development --each subject to experimental study 
and manipulation. The tongue may also play a role in palate 
closure once it has been displaced from between the shelves, by 
aiding fusion (flattening the shelves and bringing their free 
edges together). 

Methods Employed ; 

Embryos undergoing palatogenesis were obtained by laparotomy from 
pregnant rodents (gestational days 14-17). The embryonic heads 
were then subsequently prepared for conventional histological and 
histochemical techniques. 

Ma jor Findings ; 

Palatal Closure; 

Prior to the initiation of palate closure in the rodent the cranial 
base is flexed in the area of the craniopharyngeal canal (day 13 
1/2 in the mouse and day 15 1/2 in the rat). As the process of 
palate closvire progresses, there is a gradual reduction in the 
flexure of the cranial base until the palatine processes are 
horizontal, at which time the cranial base is straight. Rapid 
growth and straightening of the cranial base might play a role in 
palate closure by providing the "internal shelf force." Several 
pieces of evidence support this hypothesis: rat embryos possess 
a considerable flexure, and their palatal development is not 
affected by cortisone; the angle of flexure is smaller in C57BL 
mice than in rats and 17- 18% of C57BL embryos have cleft palate 
after cortisone treatment; and finally, embryos of the A/j strain 
have almost no range of movement because the cranial base is almost 
fully extended before palatal closure commences and as a result 
they have a cleft palate frequency of 100% after cortisone treat- 
ment. 

Several C57BL/6 embryos have been observed with spontaneously 
occurring severe micrognathia and microglossia. In those animals 
with large remnants of the tongue, the palate is cleft and the 
small tongue extends upwards between the palatine shelves. However, 
in a few cases the mandible is reduced to the point of being non- 
existent and there is no tongue. In these animals the palate is 
fused--although it could hardly be called a "normal" palate. This 
indicates that the palate can form, even in the absence of the 
tongue. 

Palatal Fusion; 

The hydrolytic enzyme acid phosphatase was assayed histochemically 

J35 



Pare A (continued) Serial No. NIDR-26(63) 

in the midpalatal oral-facial region of normal A/j mice and 
Sprague-Dawley rats during fusion of the palatal processes. Enzyme 
activity was associated with the degenerative epithelium of the mid- 
palatal region in normal 15 and 16 day A/j mouse embryos. The 
palatal midline epithelium and underlying mesenchj^e in normal 16 
day rat embryos showed minimal amounts of acid phosphatase. Great- 
est enzyme activity occurred on the 17th gestational day, associated! 
with degenerative changes in the midline epithelium. By the 18th 
day, the midline seam was virtually absent and the enzyme activity 
was principally associated with palatal osteogenesis. These results 
show that acid phosphatase is present in the midpalatal epithelium 
during normal palatal fusion of rodent embryos and participates in 
the breakdown of these opposing epithelium. 

Signif icance to Dental Research ; 

These investigations have attempted to elaborate mechanisms involved i 
in normal palatal development and have yielded information relevant 
to other structures of the craniofacial complex, which might 
potentially participate in the production of experimental cleft 
palate. 

Proposed Course of Project ; 

Sagittal sections of rat embryos treated with 80 mg/kg chlorcycli- 
zine on days 10-15 of gestation will be examined and the angle 
of flexure of the cranial base will be compared with that of normal, 
untreated animals before, during, and after palatal closure to 
ascertain if the cranial base is affected by this teratogen. Mice 
of the A/j and C57BL strains will be treated with 19 mg/kg of 6-AN 
on day 13 1/2 of gestation and then compared with normal embryos 
to see if cranial base morphology is affected in both these strains — 
the resistant C57BL and the susceptible A/j. 

Subproject _B; 

Teratogenic agents and congenital oral-facial malformations in 
mammalian embryos. 

Objectives ; 

To investigate possible mechanisms involved in the production of 
oral-facial lesions by specific teratogenic agents in various 
mammalian species. 

Methods Employed ; 

Fetuses obtained from pregnant mice, rats and rabbits treated with 
lathyrogens or 6-aminonicotinamide during organogenesis were 

136 



Part A (continued) Serial No. NIDR-26(63) 

observed for gross congenital malformations. The heads were 
subsequently removed, fixed, embedded and sectioned by conventional 
techniques for the histological characterization of the oral-facial 
lesions. 

Major Findings: 

A) Lathyrogenic agents 

The maternal ingestion of a 50 percent ground Lathyrus odoratus 
pea seed diet by pregnant Sprague-Dawley rats' from the 10th through 
20th days of gestation resulted in resorptions and multiple fetal 
malformations of the limbs, vertebral column and spinal cord and 
maxillofacial regions including micrognathia and cleft palate. 
Various degrees of edema were also exhibited in these fetuses. 
Pregnant mice of the A strain maintained on the same diet from the 
9th to the 18th days of gestation produced young with cleft palate 
and malformations of the vertebral column and spinal cord. The 
lathyrus factor, p-aminopropionitrile (BAPN) was also checked for 
teratogenic activity in the rat. Oral administration of this 
agent (200 mg from the 12th to 19th gestational days) produced a 
high incidence of resorptions (95%). The viable young that were 
obtained by this procedure showed edema, cleft palate and vertebral 
and limb malformations. As the oral dosage of this compound was 
increased to 500 mg and restricted to the 15th day of gestation, 
the number of resorptions was markedly reduced to 9%, while cleft 
palate was observed in 95% of the fetuses accompanied by a variable 
incidence of edema and limb malformations. 

The susceptibility of the rabbit embryo to the teratogenic action 
of BAPN was also investigated and gross malformations of the limbs 
and oral-facial region including palatal clefts were again demon- 
strated. This latter finding indicates that the teratogenic 
susceptibility of the compound is not limited to the rodent species. 

The oral administration of other lathyrogenic agents (D-penicill- 
amine, aminoacetonitrile and semicarbazide) to pregnant rats during 
the period of organogenesis also resulted in the production of 
congenital malformations involving the oral-facial regions. Cleft 
palate was observed in a high incidence of the viable young obtained 
from pregnant rats treated with 400 mg of D-penicillamine from the 
10th through 15th days of gestation. The oral administration of 
aminoacetonitrile in half the amount (200 mg) of either BAPN or 
D-penicillamine at comparable times of gestation resulted in 
maternal deaths indicating a greater toxicity for this compound. 
As the dosage was reduced to 50 mg and administered only on the 
15th day of gestation (period of palatal closure) the viable young 
obtained from this procedure all demonstrated clefts of the secondary 
palate. Virtually no resorption sites were noted during this 

^ 137 



Part A (continued) Serial No. NIDR-26(63) 



regime. Semicarbazide administration at the same dose as amino- 
acetonitrile (50 mg) to pregnant rats from the 10th through 16th 
gestational days also resulted in the production of cleft palate 
in the offspring obtained from this treatment. 

The histopathological characterization of the oral-facial lesion 
of rat embryos observed after these treatments were as follows: 

1) The palatal processes retained their vertical orientation and 
exhibited a moderate degree of hypoplasia, although in some in- 
stances, horizontal transmigration of the palatal shelves had been 
effected. In the instance in which the vertical orientation of 
the palatal processes persisted, no histological evidence of glosso- 
palatal fusion was observed. Another histological feature of the 
oral-facial lesion obtained with these treatments was a mass of 
well defined heterotopic cartilage associated with Meckel's 
cartilage and directed medially near the base of the tongue. This 
finding shows that lathyrogenic agents have the capacity of produc- 
ing heterotopic cartilage in the developing oral-facial regions of 
rat embryos. 

B) 6-Aminonicotinamide 

A colony of mice was established to investigate a cytoplasmically 
transmitted difference in response to the teratogenic effects of 
6-aminonicotinamide (6-AN) in the C57BL and A/j strains. The 
frequency of cleft palate produced by maternal treatment on day 
13 1/2 of gestation with 19 mg/kg 6-AN is lower in C57BL (20%) 
than in A/ J (100%) mice. The frequency was found to be significant- 
ly lower in the offspring of C57BL females maintained on Purina 
Lab Chow (20%) than on Breeder Chow (70%). A/j females do not show 
the effect of diet. There is a matroclinous reciprocal cross 
difference in the frequency of induced cleft palate which persists 

in the back cross, but only when the F mothers are maintained on 

1 
Lab Chow. Since mitochondria are transmitted through the egg 

cytoplasm, and 6-AN forms an inactive NAD analogue that interferes 

with oxidative phosphorylation in mitochondria, it is reasonable 

to postulate that the cytoplasmic factor is associated with a 

difference in the mitochondria of the two strains. 

The important difference between the two diets used seems to be the 
higher niacin content in Lab Chow, since niacin counteracts the 
effect of 6-AN. Because there were other differences between the 
diets and commercial diets are highly variable in content, the 
experiments are currently being repeated using a defined diet 
supplemented with two different levels of niacin (45.4 mg/lb and 
20 mg/lb). Preliminary results show that the cytoplasmic effect is 
still present on these diets and can be directly related to the 



<l 



ue 



Part A (continued) Serial No. NIDR-26(63 ) 

level of niacin in the diet. 

Significance to Dental Research ; 

These results extend out studies in the pathogenesis of experi- 
mentally-induced cleft palate. The ability to produce predictively 
specific types of palatal defects in various manunalian species 
offers an excellent technique for investigating drug-induced oral- 
facial malformations in particular and gene-environment inter- 
actions in general. 

Proposed Course of Research: 

A) Lathyrogens 

Quantitative biochemical investigations will be undertaken in an 
attempt to relate the palatal and cartilaginous lesions with 
defects of connective tissue metabolism. 

B) 6-Aminonicotinamide 

The mitochondrial difference described above will be further in- 
vestigated by an electron microscopic study of the mitochondria of 
the two strains before and after treatment with 6-AN. It is hoped 
that the mitochondria of the two strains will present a different 
morphological reaction to the administered teratogen, and that this 
response will help elucidate the nature of the mitochondrial 
difference. 

Subproject C: 

Intrauterine application of teratogens. 

Objectives ; 

To develop a technique for intrauterine application of compounds 
for assessing the role of maternal and placental metabolism, the 
teratogenic compound, and the critical time in the production of 
fetal rat malformations by benzhydrylpiperazine compounds. 
Additionally, structural analogues of chlorcyclizine (relatively 
unaltered by maternal metabolism) were presented to the developing 
embryo to investigate the minimum structure eliciting cleft palate 
and syndactyly. 

Methods Emp 1 oyed ; 

Pregnant Sprague-Dawley rats (11th to 16th days of gestation) were 
anesthetized, a mid- line abdominal incision made and the uterus 
was exposed. A small cut was made in the uterine wall and a 



133 



_jPart_ A (continued) Serial No. NIDR-26(63) 

Millipore filter (0.2 cm x 0.2 cm) impregnated with 50 |xg chlor- 
cyclizine HCl, 50 |ig norchlorcyclizine HCl, or a HCl control was 
placed on either the intact amniotic sac (over the fetus) or the 
placenta. The uterus was returned to the abdominal cavity, the 
incision closed, and gestation continued to the 20th day. At this 
time the maternal animal was killed and the fetuses were examined. 
After insertion each compound was traced by radioactive (chlor- 
cyclizine) or spectrophotometric (norchlorcyclizine) assay of fetal, 
placental or maternal tissues. The other structural analogues 
chemically related to chlorcyclizine tested for their teratogenic 
potential by this technique were by generic name: homochlorcycli- 
zine, norhomochlorcyclizine, chlorcyclizine-N-oxide, cyclizine, 
norcyclizine, and others by formula name, piperazine, 4 chlorbenz- 
hydrol, (4 chlorphenyl-1-methyl) N-piperazine, (2 methy phenyl 
phenyl methyl) piperazine, (2 methyl phenyl phenyl methyl) N methyl 
N piperazine, and (4 chloro diphenyl methyl) tris methyl methyl 
piperazine. 

Ma ior Findings ; 

Filters containing the HCl control, those inserted on the placenta 
and those implanted other than day 13 or 14 produced no cleft, 
palate. A specific left fore limb syndactly was produced when 
filters impregnated with norchlorcyclizine, and to a lesser degree 
chlorcyclizine, were inserted centrally over the fetus on days 11 
through 16. Left hind limb syndactly was seen when norchlorcycli- 
zine filters were placed off center over the hind limb on the 14th 
day. 

When the filters were inserted on the amnion, levels of 20-40 iJ,g/g 
chlorcyclizine or norchlorcyclizine were found in the fetus, but 
when they were placed on the placenta only 5-10 ^ig/g were found in 
the fetus. These concentrations were maintained for two hours, but 
after eight hours neither compound could be detected in the uterine 
area. Inserted chlorcyclizine was not demethylated to norchlor- 
cyclizine by the fetus or by the placenta. These data suggest that 
norchlorcyclizine is the more potent teratogen, the critical time 
for cleft palate production is the 13th or 14th day (11th through 
16th days of gestation for syndactly production), and that these 
compounds act directly on the fetus in producing these malformations. 

The analogs that produced cleft palate were: norhomochlorcyclizine 
(at a rate of 33%), (2 methyl phenyl phenyl methyl) N piperazine 
(137„), homochlorcyclizine (10%), norcyclizine (7%) and (4 chloro 
diphenyl methyl) tris methyl methyl N piperazine (2%). These 
compounds also caused specific limb syndactly in parallel percentages, 
In addition, the compounds cyclizine and (2 methyl phenyl phenyl 
methyl) N methyl N piperazine produced 2% and 4% syndactly 
respectivel}'. 



140 



Part A (continued) Serial No. NIDR-26(63) 



Significance to Dental Research ; 

Direct access to the developing fetus with small quantities of 
teratogens and the elimination of the factors of maternal metabolism 
and excretion of these compounds can greatly aid the investigation 
of the pathogenesis of cleft palate. Minimal structural activity 
in eliciting the teratogenic response can also be assessed by this 
technique. 

Proposed Course of Research ; 

An attempt to alter the teratogenic response of norchlorcyclizine 
will be made by co-inserting possible competitively binding non- 
teratogenic compounds such as structural analogs or Ca salts. 

Previous in vitro studies have shown that norchlorcyclizine binds 
stoichiometrically to bovine nasal septvmi cartilage. When calcium 
concentration is high, norchlorcyclizine is displaced completely 
whereas if the calcium concentration is low norchlorcyclizine 
preferentially binds to the cartilage. These rn vitro cartilage 
binding studies will be expanded to include all the mentioned 
analogs and an analysis of their degree of binding with norchlor- 
cyclizine and calcium displacement will be undertaken. This data 
will indicate if a correlation exists between this binding affinity 
of cartilage and expression of teratogenicity. 

Subproject D; 

Different effects of chlorcyclizine in the Sprague-Dawley and Long- 
Evans strains of rats. 

Objectives ; 

To compare the teratogenic effect of chlorcyclizine in the Long- 
Evans and Sprague-Dawley strain of rat. 

Methods Emp 1 oyed ; 

Pregnant Long-Evans and Sprague-Dawley rats were treated with either 
40 mg/kg, 60 mg/kg or 80 mg/kg of chlorcyclizine on days 12 through 
15 of gestation. On day 20 mothers were killed and fetuses examined 
grossly for edema and cleft palate. Each litter was divided and 
half the fetuses placed in Bouin's fluid for later internal examina- 
tion by razor blade hand sections and half in alcohol for clearing 
and alizarin staining. 

Ma ior Findings i 

At a dose of 40 mg/kg the main differences in the response of the 



8 1^1 



Part A (continued) Serial No. NIDR-26(63) 



two strains was a 20% incidence of cleft palate in the Sprague- 
Dawley (SD) versus a 5% incidence in the Long-Evans (LE) with half 
of the SD clefts being fused to the tongue. Differences became 
more apparent at a dose of 60 mg/kg where hydrocephalus and 
hydronephrosis were more common in the SD strain. In addition 
decreased or absent sternal and central vertebral body calcification 
and mild edema occurred only in the Sprague-Dawley strain. At 80 
mg/kg severe edema, all fused cleft palates, absent sternal, central 
vertebral body and sacral calcification occurred only in the 
Sprague-Dawley strain whereas minimal edema, unfused cleft palate 
and mostly decreased rather than absent central vertebral body 
calcification was noted in the Long-Evans group. 

These results indicate a definite difference in response to 
chlorcyclizine with the Sprague-Dawley strain being more sensitive 
to the drug. 

Proposed Course of Project ; 

Comparative metabolism studies in both strains will be carried out 
to elucidate whether the difference in response is due to a more 
rapid turnover of chlorcyclizine, less production of norchlor- 
cyclizine, the active metabolite, or some other mechanism. 

Significance to Dental Research ; 

This study will further delineate the differences that are manifest 
in the oral regions of two strains of rats subjected to the same 
teratogen. 

Subproject E: 

Prolonged gestation in the rat as an aid in teratological research. 

Objectives ; 

To evaluate whether prolonging gestation in rats may be helpful in 
elucidating certain teratogenic mechanisms. 

Methods Employed; 

Groups of pregnant rats received orally one of the following 
regimens: chlorcyclizine, 80 mg/kg, gestational days 10-15; 
Vitamin A, 100,000 units 10-15; p-aminopropionitrile 200 mg days 
10-16 or Lathyrus odoratus ground seed in the diet days 10-20. 
Litters were killed on day 20 or on day 23 after prolonging gesta- 
tion with progesterone (10 mglM, days 20-22). Untreated animals 
served as controls. 



1^2 



Part A (continued) Serial No. NIDR-26(63) 

Major Findings ; 

Observations concerning teratogenic effects of several compounds 
have been made in rat litters after prolonged gestation. In the 
23 day chlorcyclizine group, limb abnormalities, edema and 
hydronephrosis were more marked than in the 20 day groups. 
Vertebral calcification usually not present on day 20 had occurred 
by day 23 whereas cryptorchidism persisted. After Vitamin A limb 
and ear abnormalities were more easily identified and prognathism 
was more severe on day 23. BAPN and Lathyrus induced limb mal- 
formations noted on day 20 tended to progress in severity by day 
23. Prolonging gestation avoids early death of deformed fetuses, 
permits fuller development of certain induced abnormalities and 
allows ambiguous findings to be further clarified. It also may 
help determine whether an unusual observation is the result of 
delayed development and therefore transient or is a more permanent 
defect. 

Significance to Dental Research ; 

This study can investigate "postnatal" changes in the oral-facial 
region due to specific teratogens by the prolongation of gestation. 
Specifically, the extensive chondrogenesis present throughout the 
oral-facial regions can be observed by prolongation of gestation 
for potential ossification by maintaining the fetus _in utero . 

Proposed Course of Research ; 

Detailed measurements of the mandibular growth after Vitamin A 
administration in excess and after prolonged gestation will be 
made; in addition further clarification of delay or absence of 
calcification of the central vertebral bodies after chlorcyclizine 
will be attempted by evaluating the degree of calcification on days 
20, 21, 22 and 23. 



Part B Publications: 

Steffek, A. J., King, C. T. G. and Wilk, A. L.: Abortive effects 
and comparative metabolism of chlorcyclizine in various mammalian 
species. J. of Teratology . In press. 

Koziol, C. A. and Steffek, A. A.; Acid phosphatase activity in 
palates of developing normal and chlorcyclizine treated rodents. 
Archives of Oral Biology . In press. 

Fraser, F. C. , Chew, D. and Verrusio, A. C: Oligohydramnios and 
cortisone- induced palate in the mouse. Nature 214:417-418, 1967. 



10 1^3 



part B (Continued) Serial No. NIDR-26(63) 

Verrusio, A. C, Pollard, D. R., and Fraser, F. C: A cyto- 
plasmically transmitted diet-dependent, difference in response 
to the teratogenic effects of 6-aminonicotinamide in the mouse. 
Science 160:206-207, 1968. 



1 ^ ^1 

11 



Serial No. NIDR-27(66) 

1. Biochemistry 

2. Pharmacology 

3. Bethesda, Md. 
PHS-NIH 

Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Study of Teratogenesis and Organogenesis in the Non- 
human Primate (Contract 43-66-457, Hazelton Laboratories, 
Falls Church, Virginia) 

Previous Serial Number: NIDR-39 

Principal Investigator: Dr. A. J. Steffek (ADA Research Associate) 

Other Investigators: Dr. K. A. Piez, Dr. M. V. Barrow 

Cooperating Units: Office of International Research, NIH, Rio de 

Janeiro, Brazil, Dr. C. T. G. King 

Man Years: 

None 
Project Description: 
Subproject A: 

Thalidomide syndrome in Rhesus monkeys (Macaca mulatta ). 
Objectives : 

To study the response of the Rhesus monkey to thalidomide and 
further delimit the teratogenic time period. 

Methods Employed ; 

This project is conducted by Contract PH43-66-457, 1967, to 
Hazelton Laboratories, Falls Church, Virginia. The principal in- 
vestigators at Hazelton Laboratories are Dr. William M. Reese, Jr., 
and Mr. Howard Feinman. The Project Officer is Dr. K. A. Piez and 
the cost of the contract is 37,000. The Rhesus monkeys were main- 
tained at the Hazelton Facility and the fetuses are obtained by 
Caesarean Section. , The experimental procedures are performed at 
Hazelton Laboratories and subsequent examination and analysis of 
the specimens is executed at NIDR. 



L^ 



Part A (Continued) Serial No. NIDR-27(66) 



Ma i or Findings ; 

Three pregnant Rhesus monkeys (Macaca mulatta) were treated with 
100 mg of thalidomide by intubation on the following schedules: 
mother 286L - days 24, 25, 26 of gestation; mother 197N - 
gestational days 26, 27, 28; and mother 140R - gestational day 
30 only. The fetus from mother 286L showed severe phocomelia of 
all extremities, dislocated shoulder and hip joints, rudimentary 
digits, micrognathia, a double gall bladder, malrotation of the 
gut and a double right coronary artery. Mother 197N's fetus showed 
only a missing first toe of the right foot and an extra digit of 
the left. The fetus from mother 140R was normal. 

Not only is administration during a critical and early time period 
necessary for the development of severe and complete phocomelia in 
the Rhesus monkey but an impressively low dosage of the compound 
is needed to achieve this end. During days 24 to 26 of gestation, 
no or very early limb buds are present and treatment during this 
stage of development results in the full phocomelia syndrome. 
Treatment later (days 26-28) resulted in only posterior limb 
anomalies indicating a cephalocaudal gradient of susceptibility. 
By day 30, still before the appearance of finger rays thalidomide 
was no longer effective under the conditions of our experiment. 

Thus the primary site of action on the extremities must lie in its || 
effect on early mesenchymal tissue destined to become or influence 
the proximal portions of the limbs. 

Soft tissue malformations both typical (gut malrotation) and 
atypical (a double gallbladder rather than an absent one) for 
thalidomide were also demonstrated. 

Significance to Dental Research ; 

The oral-facial embryogenesis of the non-human primate simulate 
more closely the human as compared to rodents. This fact could 
allow a more valid extrapolation of teratogenic effects based on 
human experience, including those malformations of the oral-facial 
regions. 

Proposed Course of Project ; 

Additional investigations evaluating the non-human primate as a 
possible model system in the assessment of other potential human 
teratogens will be initiated with particular direction towards the 
production of cleft palate in this species. 

Subproject B; 

Effects of pancreatectomy in the Rhesus monkey. 2^C 



Part A (Continued) Serial No. NIDR-27(66) 

Objectives ; 

This was a preliminary study to evaluate the effects of pancrea- 
tectomy in mature female Rhesus monkeys and to provide background 
information to enable evaluation of the effects on organogenesis 
at a later date. Two monkeys were initially used. 

Ma jor Findings ; 

A near total pancreatectomy (90%) was performed on the first monkey. 
This animal died within eight hours of surgery. The second monkey 
also received a 907o pancreatectomy. After an initial stormy post- 
operative course with wide variations in blood sugars from the low 
to high range requiring frequent monitoring and insulin. The 
animal gradually stabilized and over several weeks the insulin 
dosage was gradually decreased and later discontinued. After 
several weeks of no insulin therapy the animal was eating and 
reacting normally; blood studies and chemistries were generally 
normal except for a mild anemia and abnormal blood glucoses indicat- 
ing definite diabetes. The blood sugars ranged near 100 in the 
morning and between 250 to 300 in the mid-afternoon (normal 75-100 
during this time) . 

This preliminary study indicates not only the feasibility of using 
the subhuman primate as an experimental model for diabetes mellitus 
but also the potential use of these animals in sludying the effect 
of diabetes on growth and development. 

Proposed Course of Project ; 

More pancreatectomies are anticipated in non-pregnant and later in 
pregnant and non-pregnant, but actively breeding, animals to further 
study the production of experimental diabetes in the monkey and its 
effects on growth and development. 

Subpro ject C: 

Aminopterin toxicity in the Rhesus monkey. 

Objective ; 

To evaluate any adverse effect of aminopterin and to help determine 
a useful dosage for teratogenic studies in the non-human primate. 

Methods Employed : 

Two adult female Rhesus monkeys were used. One was given amino- 
pterin orally by intubation at a dose level of 1 mg/kg and one at 
10 mg/kg. Both were treated for five consecutive days. Observations 

3 IL^I 



Fart A (Continued) Serial No. NIDR-27(66) 

were recorded daily including body weights and pertinent clinical 
laboratory studies, including blood parameters, liver and renal 
functions, were carried out. Necropsy was conducted on deceased 
animals and on survivors at the termination of the experiment. 

Ma i or Findings ; 

The monkey treated with 10 mg/kg became ill on the final day of 
treatment, developed vomiting and weakness and died four days later. 
Four days prior to death the hematocrit white blood count was 
decreasing but still within normal limits. Blood chemistries were 
norma 1 . 

The second monkey was treated with 1 mg/kg for 5 days. This monkey 
did not become ill and all bleeding studies and chemistries remained 
within normal limits. On the basis of these studies it was elected 
to use doses of 0.25 to 1 mg/kg to begin teratogenic studies since 
this was approximately one tenth the lethal dose. 

Proposed Course of . Project : 

Teratogenic studies using aminopterin will be carried out in . 
pregnant monkeys and a critical dosage and time period established. 



Part B: Not included. 



m^ 



Serial No, NIDR-28 (62 ) 

1„ Biochemistry 

2, Cell Biology 

3. Bethesda, Md. 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Chromosome and Cell Growth Studies in Normal and 
Abnormal Subjects 

Previous Serial Number: NIDR-89 (c) 

Principal Investigator: Dr., Ho L. Cooper 

Other Investigators: Dr. J. Jo Oppenheim, Dr. Ro Stem, Dr» S, Handmaker, 

Dr. J. Eo Kay, and Dr. J. Graef (Human Genetics 
Branch, NIDR) o 

Cooperating Units: National Cancer Institute, Dr. Ro Friedman and Dr. 

B. Leventhal 

Man Years : 

Total: 10 1/2 
Professional: 5 1/4 
Other: 5 1/4 

Project Description: 

Objectives : 

lo An intensive study of biochemical processes involved in the 

transition of non-growing human Ijnnphocytes to a state of active 
growth leading to DNA replication and division. Special 
emphasis is placed on regulation of RNA and protein synthesis, 
and on ribosome function. 

2o A study of the relationship of lymphocyte growth response in 
vitro to aspects of immunological phenomena in vivo. 

3o A study of a hitherto undiscovered form of RNA found in animal 
cells, and its possible relevance to the question of the viral 
etiology of neoplasia or abnoirmal cell proliferation. 

4. A study of the molecular organization of certain intermediate 
forms of viral RNA found during replication of an RNA virus 
in animal cells. (In collaboration with Dr. R. Friedman, NCI). 



1^9 



Part A (Continued) Serial No, KIDR-28 (62 ) 

Methods Etaployed ; 

Lymphocytes for studies of ribonucleic acid metabolism were 
obtained from the peripheral blood of normal human volunteers. 
Ljnnphocytes were separated from the remainder of the blood cells 
by a differential adsorption technique. 

The cells were stimulated to grow by addition either of phyto- 
hemagglutinin (an extract of the kidney bean) or of specific 
antigens. Metabolic changes were studied during the course of the 
subsequent cell enlargement and division. Radioactive precursors ■ 
and metabolic inhibitors were used in various combinations to ^ 
explore different aspects of cell growth stimulation. Ribonucleic 
acid sjmthesis in stimulated cells was studied by a variety of 
biochemical and physical means: RNA was extracted and purified by 
the phenol-sodium dodecyl sulfate method, its components separated 
by ultracentrifugation, and the behavior of individual components 
during different experiments was followed by assay of incorporation 
of radioactive precursor using liquid scintillation spectrometry. 
Components of RNA were also analyzed by their behavior during gel 
filtration and during chromatography on benzoylated DEAE cellulose 
columns. 

Growth of lymphocytes in response to various agents was also 
measured by uptake of tritiated thjnnidine into DNA. A variety of 
agents known to stimulate l3miphocyte growth, including purified 
phytohemagglutinins, specific antigens, ant i- leukocyte antisera 
and antisera to specific cell products, are added to cultures under 
a variety of conditions, to investigate the dynamics of the lympho- 
cyte response. 

In some investigations, Ijmiphocytes obtained from the Ijmiph nodes 
of small mammals were employed. 

Patient Material : 

Lymphocytes for studies of normal cells were obtained from the blood 
of normal volunteers, collected in the Clinical Center Blood Bank 
by the usual procedures employed there. Nothing was done to any 
normal volunteer other than removal of blood. Blood specimens 
were also obtained from a variety of patients in the Clinical Center, 
admitted by other units and cared for by them. No drugs or other 
procedures beyond drawing of blood were used for our studies. No 
patient risk was incurred. 

Major Findings : 

1. a. A detailed study of the kinetics of the lymphocyte growth 
response to phytohemagglutinin (PHA) was completed. This 

150 



Part A (Continued) Serial No„ NIDR-28 (62 ) 

study used a combination of biochemical radioautographic and 
cytological techniques to determine the time-dose interrelationship 
between lymphocyte growth and optimal, sub-optimal and supra- 
optimal doses of PHA. It was shown that the time-course of stimula- 
tion of DNA S3mthesis was identical with different doses of PHA, 
Sub-optimal doses of PHA stimulated a smaller proportion of cells 
to respond, while supra-optimal doses stimulated a greater 
proportion of cells to respond, but also induced cell death » 
The overall effect of supra-optimal doses was to diminish DNA 
synthesis in the entire population because of reduced cell survival. 
Now under study is the question of whether the toxicity of supra- 
optimal doses of PHA is due to excessive activity of the same type 
as that involved in growth stimulation, or whether two simultaneous 
activities are present, affecting different aspects of cell 
metabolism. Dr. Handmaker was in charge of this investigation, 
in which Dr. B. Leventhal of NCI also collaborated. 

b. In a study carried out by Dr. Kay, with Dr. Leventhal 
collaborating, the effects of inhibition of ribosome synthesis 
on protein and DNA synthesis by PHA-stimulated Ijmphocytes was 
examined. It was found that, when ribosome s3iTithesis was abolished 
with low doses of Actinomycin-D, protein synthesis could still be 
enhanced by treatment with PHA. Protein synthesis increased until 
the point at which DNA S3nithesis usually begins in such cells, and 
these increased no further. Normal DNA synthesis did not begin. 

It was concluded that the ribosomes of resting lymphocytes are not 
functioning optimally in protein sjmthesis, and that their activity 
could be enhanced by PHA. It was suggested that the onset of DNA 
sjmthesis may be geared to the rate of ribosome sjmthesis or the 
number or density of cytoplasmic ribosomes. The onset of DNA 
synthesis may be further involved in the stimulation of synthesis 
of a variety of proteins. 

c. Preliminary stages have been completed in establishing the 
methodology for a study of the RNA synthesis of isolated Ijmiphocyte 
nuclei and their response to mitogenic agents either directly or as 
mediated through various subcellular fractions. This project is 
being carried out by Dr. Graef . 

d. A detailed study has been completed of the early changes in 
the production of certain classes of RNA by lymphocytes as they 
shift from the resting to the growing state. Resting Ijmphocytes 
synthesize predominantly heterogeneous -sized RNA molecules which 
are rapidly degraded within the nucleus. Only 3-47. of the RNA 
sjmthesized during any short interval is stable ribosomal RNA. 
Upon treatment with the growth stimulant, PHA, there is an 
acceleration of all RNA synthesis, but ribosomal RNA synthesis 
increases disproportionately. After 24 hours of growth, overall 
RNA sjmthesis has increased over 20-fold, while ribosomal RNA 



151 



Part A (Continued) Serial No, NlDR-28 (62 ) 

synthesis has increased 75 to 100-fold , This increase accounts 
for the marked accujnulation of cytoplasmic ribosomes which 
distinguishes growing lymphocytes from resting ones. Several 
steps have been identified in RNA synthesis at which control 
mechanisms may exist which can be altered by a growth stimulant. 
These are: 

i) Activity of pre-existing RNA polymerase . Pre-existing, 
apparently inactive, lUSIA polymerase can be activated to increase 
production of heterogeneous, but not of ribosomal RNA, without a 
requirement for new protein S5mthesis. 

ii) Synthesis of large molecular precursor of ribosomal RNA 
(45s molecule ). Continued synthesis of this molecule in resting 
lymphocytes requires continuous protein S3nithesis. Synthesis of 
ribosomal precursor RNA (unlike heterogeneous RNA) cannot be 
increased by PHA in the absence of protein sjmthesis. Thus, a 
critical requirement exists in the lymphocyte for a protein (or 
proteins) whose absence specifically prevents ribosomal RNA 
synthesis. This protein(s) may play a physiological role in cell 
growth regulation by fixing the rate at which ribosomal RNA may 
be produced, 

iii) Conversion of ribosomal precursor RNA (45s) to an inter- 
mediate precursor molecule (32s). After addition, of PHA, this 
conversion was shown to increase with different kinetics than the 
acceleration of 45s synthesis, and therefore may be subject to 
separate control. 

This study was carried out by Dr, Cooper. 

2. We are investigating the induction and immunological relevance of 
in vitro Ijmiphocyte proliferation with antigen, nonspecific 
stimuli, and antisera in normal humans and other mammalian species. 
Variations from the normal in vitro response are found with 
lymphocytes from patients with immunological abnormalities such as 
Wiskott-Aldrich syndrome, ataxia-telangiectasia, aphthous stomatitis 
and hypogammaglobulinemia. 

We are studying the intercellular relationship of macrophages and 
lymphocytes which we have found promote lymphocyte proliferation 
in vitro . We have also found that macromolecular polycations 
block lymphocytes in vitro. This block can be overcome by poly- 
anions, which at times can enhance the Ijnnphocyte growth. The 
effects of these synthetic polymers parallel those of soluble anti- 
immunoglobulin: immunoglobulin complexes which have a nontoxic 
inhibitory effect on the lymphocytes in vitro and may reflect a 
feedback control mechanism of in vivo Ijrmphocyte response to anti- 
genic stimuli. This work is being carried out by Dr. Oppenheim. 

152 



Part A (Continued) Serial No, NIDR-28 (62 ) 

3o In order to improve our ability to separate and study- 
various types of RNA produced by the cell, a chromatographic 
technique was developed by Dr, Stern, employing benzoylated 
DEAE cellulose and a variety of eluting gradients. RNA 
molecules are distinguished by their degrees of secondary 
structure. Using this technique, an unusual form of RNA was 
isolated from cultured Burkitt lymphoma cells which has 
characteristics resembling those of a viral intermediary form. 
This material is now under intensive study. 

4, In collaboration with Dr. R. Friedman, NCI, Dr. Stem has 
verified the multi-stranded nature of the viral replicative 
intermediate form of Semi iki-f ore st virus. Replication of the 
nucleic acid of this RNA virus appears to take place on a 
double stranded RNA template, analogous to the normal trans- 
cription of RNA on double stranded DNA in the cell nucleus. 

Significance to Dental Research ; 

The studies performed in this project are aimed at an understanding 
of the mechanisms by which genetic information contained within 
the cell functions in the regulation of normal and abnormal cell 
growth and cell differentiation. Such knowledge is of basic ' 
importance in the dental and medical fields, where disordered 
cellular differentiation may result in congenital malformations, 
and where control of cell growth may become disordered with 
resultant neoplasia. 

Major problems to be surmounted in this field include those 
preventing successful transplantation of teeth and progress in the 
field of histocompatibility t3rping would certainly be a major step 
to achieving this goal. 

There are some oral cavity disorders which are related to contact 
hypersensitivity, "autoimmunity" and allergic reactions. 
Elucidation of these cellular responses involved may lead to 
improved therapeutic approaches for these conditions. 

Proposed Course of Project ; 

Research will continue primarily in the area of cell growth 
regulation, as studied in peripheral blood lymphocytes which may 
be stimulated to shift from a resting state to active growth. It 
is hoped that the mechanisms concerned with maintaining the 
resting state, and the alterations which trigger the onset of cell 
growth will be further elucidated. 

An understanding of the cellular mechanisms which regulate the 
synthesis of messenger RNA is another area of study to be pursued. 



15^3 



Part A (Continued) Serial No. NIDR-28 (62 ) 

This information will be important for an understanding, not only 
of control of cell growth, but also of cellular differentiation. 
The interaction between lymphocytes and macrophages will be studied 
to learn how the cell types communicate, and how this may be 
achieved synthetically. Increasing emphasis will be placed on 
phenomena occurring at the cell surface during growth stimulation, 
as part of the continuing effort to elucidate the initiating 
events in the onset of cell growth. 

Unusual RNA forms found in normal and malignant cells will be 
studied in greater detail in an effort to understand their role 
in cell function. 



Part B Publications: 

Friedman, R., and Cooper, H. : Stimulation of interferon production 
in human lymphocytes by mitogens. Proc. Soc. Exp. Biol, and Med . 
125: 901-905, July 1967. 

Cooper, H. L.: Studies on early biochemical changes in phyto- 
hemagglutinin-stimulated lymphocytes. In Rieke, W. 0. (Ed.): 
Proc. 3rd Annual Conference on Leukocyte Culture . Apple ton- 
Century-Crofts. In press, 1968. 

Cooper, H. L.: Alterations in RNA metabolism in lymphocytes during 
the shift from, resting state to active growth. In Nat. Cancer 
Inst. Monograph . In press, 1968. 

Bradley, J., and Oppenheim, J. J.: The in vitro proliferation of 
lymphocytes from patients with hypogammaglobulinaemia. Clin, and 
Exper. Immunology 2: 549-557, 1967. 

Oppenheim, J. J.: The relationship of in vitro Ijrmphocyte trans- 
formation to delayed hypersensitivity in guinea pigs and man. 
Fed. Proc . 27: 21-28, Jan-Feb. 1968. 

Leventhal, B. G., and Oppenheim, J. J.: Effect of cell density on 
the rate and degree of response of leukocyte and purified lympho- 
cyte cultures. In Rieke, W. 0. (Ed,): Proc. 3rd Annual Conference 
on Leukocyte Culture . Appleton-Century-Crofts. In press, 1968. 

Francis, T. C, Oppenheim, J. J., and Barile, M. F.: Lymphocyte 
transformation by streptococcal antigens in guinea pigs and man. 
In Rieke, W. 0. (Ed.): Proc. 3rd Annual Conference on Leukocyte 
Culture. Appleton-Century-Crofts. In press, 1968. 



ISk 



^^^^ ^ (Continued) Serial No. NIDR-28 (62 ) 

Knight, So, Bradley, J., Oppenheim, Jo Jo, and Ling, N. Roi 
The in vitro proliferation of human thymoma, normal rabbit and 
guinea pig thymocytes, Clin, and Exper, Immunol . In press. 

Oppenheim, J. J., Leventhal, B., and Harsh, E. M. : The trans- 
formation of column purified Ijrmphocytes with nonspecific and 
specific antigenic stimuli. J. Immun . In press. 

Handmaker, S. D., Leventhal, B. G., and Cooper, H. L.: The 
kinetics of PHA-stimulation of human lymphocytes. In Rieke, W. 0< 
(Ed.): Proc. 3rd Annual Conference on Leukocyte Culture . 
Appleton-Century-Crofts. In press, 1968. 

Kanfer, J. N., Richards, R. , Kampine, J. P., Handmaker, S., and 
Yankee, R. A.: Alteration of the sphingolipid content in 
leukocytes from patients with Chediak-Higashi syndrome. Life Sci. 
6: 2661-2664, 1967. 



1j5 



Annual Report of the Human Genetics Branch 
National Institute of Dental Research 
Summary Statement 



The Human Genetics Branch has been divided into two sections which cover 
the two major areas of research being done in the Branch. The Population 
Genetics Section carries out field studies on the variations and defects 
of oral structures in human populations and the relationship to the genetic 
structure and mating systems of different population groups. The Developmental 
Genetics Section uses a variety of laboratory methods to study the genetic 
mechanisms controlling the development of the structure and function, and 
to study the effects of genes involved in developmental defects of laboratory 
animals. The functions of the two sections compliment each other in the 
combination of laboratory and population techniques used to study specific 
defects, such as cleft palate, and specific genes such as those causing 
Waardenburg Syndrome in both men and animals. 

MALOCCLUSION 

Two studies are being conducted in this area; the first is a study of 
racial variation with emphasis on the effects of racial crossing. This 
study is being conducted in collaboration with the School of Public Health 
at the University of Hawaii. The past year has been spent almost entirely 
in data collection, with examinations completed on approximately 19,000 
high school students. Other studies of malocclusion are being conducted in 
the Papago Indians. Although the results are quite preliminary, comparison 
of present Papagos with ancestral skeletal remains suggest a marked increase 
in the frequency of malocclusion in the period since Spanish contact. It 
is believed that these dental changes are the result of changing culture 
and dietary patterns rather than of genetic origin. 

CLEFT LIP AND CLEFT PALATE 

Continued utilization is being made of data collected in past studies. A 
significant finding has been the failure to confirm previous reports of 
minor anomalies in clinically normal relatives of oral cleft patients using 
laminographic x-ray procedures. Previous studies have been without benefit 
of controls; hence, the present results are considered more reliable. 
Another finding has been the demonstration of mid-facial changes (relative 
hypertelorism) in patients with cleft lip. Normal relatives of these 
patients do not show the same changes, suggesting that this is a phenomenon 
secondary to the cleft lesion itself, rather than a basic genetic morphological 
or growth difference. Collaborative arrangements have been made for the 
use of two large bodies of data on oral clefts. These are being utilized 
for studies of genetic segregation and for analysis of geographic and temporal 
clustering. 



157 



AMERICAN INDIAN CONGENITAL MALFORMATION 

To date approximately 28,000 consecutive newborn records have been obtained 
from the Division of Indian Health hospitals. Significant has been the 
finding that for certain malformations (cleft lip and spina bifida) the 
American Indian shows frequencies intermediate between Caucasian and other 
Mongoloid populations. For other defects (isolated cleft palate and 
anencephaly) Indians still retain frequencies quite similar to other Mongoloids. 
Indian newborn data are also being used for the studies of birth weight. The 
most significant result is the development of a mathematical model, which 
shows promise of considerable utility in epidemiological studies of prenatal 
factors affecting birth weight. 

POPULATION GENETICS 

These studies include theoretical approaches involving the development of 
mathematical models and computer simulation as well as collection and analysis 
of data from human populations. Methods are being developed for the simulta- 
neous analysis of two or more genetic loci. Human data is invariably collected 
in this form but is rarely analysed for more than one locus at a time. A 
multi-locus approach should provide a great deal more information on population 
variation than consideration of loci individually. 

STUDIES OF HUMAN CHILDHOOD DEAFNESS 

The study of the medical histories of the past and present pupils of the 
Clarke School for the Deaf has shown that the major exogenous causes of 
deafness in early childhood are infection of the central nervous system 
in infancy or in utero (rubella), and severe prematurity with birth weights 
under four pounds „ These conditions account for less than 157o of childhood 
deafness. About three-fourths of all childhood deaf do not have any history 
of disease in the newborn or prenatal periods. Statistical and genetic 
data suggests that about 40% of childhood deafness results from simple 
recessive genes and 15% from dominant genes. The remaining 20% are undiffer- 
entiated, but may be either of complex genetic origin or the result of 
undiagnosed diseases. 

SALIVA STUDY 

The etiology of polymorphism in the recently discovered isoamylases of 
human parotid is under continued study. Family populations including twins 
are being utilized to discover genetic control. Isolated disease states 
are being analysed for possible environmental effects. A method for the 
study of isoenzymes of lysozyme as well as amylase is available for serum, 
urine and saliva. 

A small molecular weight protein-like substance has also been identified. 
This was found to be more prevelant in parotid saliva of those persons 
having high rates of calculus formation. Further studies of this substance 
are in progress. 



158 



STUDIES OF TASTE AND SMELL 

Variations in the ability to taste specific chemical compounds, such as 
phenylthiourea and anetholetrithione, have been shown to be under genetic 
control. The classical view that the various taste receptors are restricted 
to the tongue has recently been abandoned, since the palate and pharynx are 
now known to possess taste receptors. Their stimulation appears to occur 
consequent to a loose coupling of the effector substance with proteins in 
the taste buds. 

Preliminary studies of twins have shown that identical twins are much more 
alike in taste responses than are like-sex fraternal twins, but that the 
relationship of a olfactory response is more complex. A specific chemical 
odor perception defect for isovaleric acid is being analyzed by family 
study for mode of inheritance, while other analogous odor blindnesses are 
being sought by screening populations. 

EXPERIMENTAL STUDIES OF GENETICALLY CONTROLLED 
DEVELOPMENTAL MALFORMATIONS 

The control of the defect of hair formation in the downless mouse is being 
studied in a tissue culture system, in which the effects of the mesoderm 
as an inducer of differentiation of ectoderm will be determined in normal 
and genetically defective mice. The downless state is of interest because 
it appears histologically to result from a failure in the initiation of 
hair follicle formation. Since the teeth develop as downgrowths into under- 
lying mesoderm as do hair follicles, follicular growth may provide a 
convenient analog of tooth development. A specific mutant in the mouse, 
whose genetic defects include amelogenesis imperfecta and abnormal hair 
development, has also been identified. This mutant is now also being studied 
both genetically and histologically. 

The hereditary defects of the domestic cat produced by a single dominant 
gene have been studied in detail. Defects include hearing loss, as well as 
ocular and pigmentary anomalies. Hearing loss varies in degree and may be 
unilateral or bilateral. This is apprently secondary to a failure in the 
last stages of maturation of the cochlea, particularly in the production of 
endolymph, and is manifested by varing degrees of collapse of the membranous 
labyrinth. Depending on the localization of the collapse the hearing deficit 
is variable. The defect in the formation of the eye of these cats is 
independent of the ear anomaly. Congenital absence of the tapetum, when 
it occurs, is complete within an affected eye. Even when the mutant gene 
is present, however, the tapetal anomaly may occur in both, one or neither 
eye . 



159 



Serial No. NIDR-29 (62) 

1. Human Genetics 

2. Developmental Genetics 

3. Bethesda, Maryland 



PHS -NIH 

Individual Project Report 

July 1, 1967 through June 30, 1968 



Part A 



Project Title: Discrimination and Segregation Analysis of Hereditary 
Deafness in the Students of the Clarke School for the 
Deaf. 

Previous Serial Number: NIDR-86 (c) 

Principal Investigator: Dr.. K. S. Brown 

Other Investigators: None 

Cooperating Units: The Clarke School for the Deaf, Northampton, 

Massachusetts; National Institute of Neurological 
Diseases and Blindness; School of Public Health, 
University of Hawaii. 

Man Years ; 

Total: 1 
Professional: 1/4 
Other: 3/4 

Project Description: 

Oblectives : 

1. To discriminate the various types of deaf people into groups 
based on the characteristics of their conditions, including 
patterns of audiogram, physical findings and laboratory values. 

2. To analyze the patterns of genetic segregation among the 
various types of hereditary deafness in order to estimate 
the number and kind of genetic factors causing deafness. 

Methods Employed : 

Personal examination of about 300 deaf people and 700 of their 
hearing relatives, including parents, grandparents, and siblings, 
has been completed. The examination has been limited to the head 
and neck, except for blood pressure and cardiac auscultation. 
It has included caloric and turning-chair vestibular examinations, 
oral examination with DMF, otoscopic and ophthalmoscopic 

160 



Serial No. NIDR-29 (62) 

examinations of 170 deaf children who are currently attending the 
Clarke School, and a similar examination lacking the caloric and 
dental examinations, but including a pure tone audiogram for the 
hearing relatives. All but the youngest children were given a 
PTC taste test, a routine microscopic urinalysis, and urine analysis 
for glucose, protein, and phenylketone-like substances. 

Blood samples were drawn for genotyping and laboratory studies of 
the serum. These include protein and lipoprotein electrophoresis, 
total-protein, total lipid, and cholesterol determinations. All 
individuals showing clinical goiter, together with their sibs and 
parents, have been examined for protein bound iodine. A contract 
has been completed by Bionetics Research Laboratories, Inc., under 
NINDB support to carry out the serum studies exclusive of the 
electrophoresis . 

The pedigree and history material collected by the Clarke School 
has been examined, and a questionnaire form to supplement and up-, 
date it has been prepared (Budget Bureau No. 68-6229), and over 
2,000 copies have been distributed. The resultant data are being 
coded by employees of the Clarke School under NIDR direction on 
two computer coding sheets prepared for this purpose (PHS-T-96, 
PHS-T-97)o This work is being done under a contract between the 
Clarke School and NINDB. The resulting coded data is being processed 
at the NIH computer center, utilizing the SEGRAN program, developed 
by Dr. Chung. 

Patient Material : 

Patient material consists of the students at the Clarke School for the 
Deaf and their siblings and parents or other relatives, if indicated. 
The alumni of the Clarke School and their families, where possible, 
will be examined, including as many of the graduates since 1930 as 
are still available. An estimated 1,500 people are available of 
whom about 700 are deaf. 

Major Findings ; 

Genetic information has been coded on 5,553 persons related to 
Clarke School pupils of whom, 1,471 are deaf. This included over 
99% of pupils attending Clarke School since 1930, 96% of those 
attending since 1920 and 47%, of all pupils who have ever attended 
Clarke School. The processing of the records of the genetic data 
has been completed and the analysis is in progress at the computer 
center, School of Public Health, University of Hawaii and in the 
Human Genetics Branch, NIDR. 

Evaluation of the medical records and physical findings on the 
present and former pupils of Clarke School shows that about 20% 
of the childhood deaf were the result of classifiable disease. 



161 



Serial No, NIDR-29 (62) 

accident or toxic condition. An additional 47o resulted from 
unclassif iable, but probably extrinsic causes, while 76?o were the 
result of hereditary factors or undetected disease. Genetic 
analysis further suggests that undetected disease accounts for 
187o of the total, so that 587o of the defects observed have a simple 
hereditary basis. 

Among the acquired cases, the major cause of deafness (327o) is 
acute CNS disease, such as meningitis or encephalitis of which 
measles is the most common cause. 

The second most common cause of extrinsic childhood deafness (26%) 
is undifferentiated severe prematurity with birth weights under 
5 1/2 pounds. Most of these are severe prematurity under 4 pounds 
at birth but there is a less severe group that is possibly confounded 
with prenatal rubella which was not diagnosed. 

Gregg's syndrome due to prenatal rubella is the third most common 
extrinsic cause of deafness and accounts for 147o of the classifiable 
cases. 

Other prominant extrinsic causes, each producing just over 57o of 
the total classifiable cases, are suppurative otitis, neonatal 
jaundice, and acute febrile illness without CNS history. 

Genetic analysis shows that simple recessive mendelian factors 
account for about 407. of childhood deafness. Several methods 
suggest that there may be between 50 and 100 individual genes 
involved, although there may be more very rare genes that also 
cause deafness. About 157. of deafness is due to dominant genes, 
also of many types. The selective disadvantage of deafness, suggests 
the probability that recurrent mutation is the source of genes 
causing deafness. 

Laboratory study of the genetics, physiology, and anatomy of the 
congenital hearing loss that may occur in the domestic cat is being 
carried out as an extension of our interest in the Waardenburg' s 
syndrome of man. Affected cats, like Waardenburg' s syndrome people, 
show variable degrees of hearing loss and lack of normal pigment 
in the hair and eye. A colony of these animals is being bred for 
study of the physiological and anatomical characteristics associated- 
with varying degrees of handicap. 

Significance to Dental Research ; 

Development of diagnostic criteria which allow the separation of 
various groups of congenital and non-congenital deafness is basic 
to the understanding of possible etiologic factors involved. 



1G2 



Serial No. NIDR-29 (62) 

Development of genetic information about a complex congenital 
malformation of known genetic origin may have a guiding role in 
the analysis of the complex genetic factors in such oral malforma- 
tions as cleft palate and malocclusion. 

Proposed Course of Project ; 

The data collection phase of the study of the Clarke School pupils 
and relatives has been completed. The statistical and genetical 
studies on the data are in progress and this work will continue 
for at least one additional year. The analysis of the segregation 
patterns will be the basis for genetic conclusions regarding the 
quality and quantity of simple genetic factors involved in the 
production of childhood and adult deafness. Further detailed 
examination of those family records in which there is interaction 
of environment and genetic factors will be undertaken. 

The study of anatomical and physiological characters of the white 
cat with hearing loss will be continued. The electrophysiology 
of the cochlea in these defective animals will be evaluated in 
relation to normal controls. The genetics of the defect will be 
studied by continued breeding experiments. 



Part B 



Publications: 



Brown, K. S., Hopkins, M. S . , and Hudgins, R„ B. : Causes of 
childhood deafness. In: Proceedings of International Conference 
on Oral Education of the Deaf -- (Ed. 1), by the Alexander Graham 
Bell Assoc, for the Deaf, Inc., 1967, Vol. 1, pp. 77-107. 



163 



Serial No. NIDR-30 (54) 

1. Human Genetics 

2. Developmental Genetics 

3. Bethesda, Maryland 



PHS -NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Environment and Genetic Factors in Taste and 
Smell Abilities 

Previous Serial Number: NIDR-88 (c) 

Principal Investigator: Dro K. S. Brown 

Other Investigators: Mr. C. J. MacLean, Mr. W. C. Leyshon 

Cooperating Units: None 

Man Years: 



Total: 


1 1/4 


Professional: 


3/4 


Other: 


1/2 



Project Description: 
Objectives : 

1. To describe variation between individuals in the ability to 
detect the odors and taste of specific chemical compounds. 

2. To evaluate the nature of the relationships between the 
thresholds for taste and smell of different compounds in the 
same individual. 

3. To evaluate the genetic and non-genetic contributions to the 
absolute threshold for specific compounds. 

4. To evaluate the genetic and non-genetic contributions to the 
relationships between the thresholds for different compounds 
in the same person. 

Methods Employed : 

Serial dilutions, in order of increasing concentration of the specific 
compounds, are presented to the subjects who state whether they can 
or cannot detect any odor or taste. The level of first detection 
is the threshold which is used as the data base for analysis. 



iGk 



Serial No. NIDR-30 (54) 
The subjects include: . 

1„ Sixty young adults without known illness for a study of the 
interactions of eight odors. 

2. Two thousand school children for a study of six odors and the 
ability to taste phenylthiocarbamide (PTC). 

3. Eighty pairs of parents and their children in a study of two 
odors and ability to taste PTC. 

4. One hundred and fifty students in a study of the correlation 
between ability to taste PTC and the ability to taste 
anetholetrithione. 

5. Ten pairs of same sex twins in a study of PTC, anetholetrithione 
(ATTH), and isobutyric acid. 

Major Findings ; 

The threshold for the ability to smell most chemical compounds is 
controlled by general sensitivity factor and by special factors 
related to the specific chemical properties of the particular 
odorants. Preliminary evidence has identified two of these factors 
as being related to the capacity of the odorant to undergo molecular 
ionization as an acid or as a base. A third factor is related to 
molecular size or weight. 

The threshold for the smell of cyanide ion appears to be controlled 
by different factors, and the smell may be related to metabolic 
properties of cyanide rather than to its molecular properties as an 
odorant in the usual sense. 

Family studies of the threshold for the odor of cyanide have not 
supported the published claims that the threshold varies as a 
simple genetic trait. 

The ability to taste PTC has been long recognized as a simple genetic 
trait. ATTH is a compound of very different molecular structure, 
which has been reported to have a taste threshold inherited as a 
genetic trait independent of PTC. Our study showed that there is 
a high correlation between the ability to taste these two compounds. 

Twin studies are not conclusive, because of small numbers, but they 
suggest that PTC and ATTH are genetically independent. 

Significance to Dental Research ; 

The senses of taste and smell are two of the least known senses. 

The mechanisms involved are unknown and only a few studies of genetic 



165 



Serial No. NlDR-30 (54) 

aspects of those abilities have been attempted. Application of 
genetic and statistical techniques to quantitative threshold data 
on these abilities may produce increased understanding of these two 
special senses involving the oral cavity. 

Proposed Course of Project ; 

Further study of data already collected may suggest direction for 
new experiments. The immediate goal is to further evaluate the 
nature of the factors controlling receptor thresholds. This will 
involve tests on a series of subjects with a wide variety of compounds 
showing relationship to those already tested. 

Search for odorants showing specific variations in different 
individuals will also be continued. These will be analyzed by 
both family and twin study techniques. 



Part B 



Publications: 



Brown, K. S. and Robinette, R. R. : No simple pattern of inheritance 
in ability to smell solutions of cyanide. Nature 215 (5099): 
406-408, July 1967. 



1G6 



Serial No. NIDR-31 (63) 

1. Human Genetics 

2. Population Genetics 

3. Bethesda, Maryland 



PHS -NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Genetic Studies of Oral Diseases, 
Anomalies and Development 

Previous Serial Number: NIDR-90 (c) 

Principal Investigator: Dr. D. W. Runck 

Dr. J. D. Niswander 

Other Investigators: Mr. C. J. MacLean 

Cooperating Units: School of Public Health, University of Hawaii 

Man Years: 

Total: 3 
Professional: 2 
Other: 1 

Project Description: 

Objectives : 

1. The purpose of the study is to clarify the relative role of 
genetic and environmental factors in certain oral conditions. 

a. To determine the effects of inbreeding and hybridization 
on malocclusion and dental caries. Specifically; do we 
obtain an Fj^ effect in the frequencies of these conditions, 
is there evidence of a recombination effect on dental 
characteristics. 

b. To assess the role of environment in malocclusion-- 
specifically has the frequency, type and severity of 
malocclusion changed over time, and if so, can the 
changes be related to socioeconomic status and changing 
cultural patterns. 

c. To assess the correlation between relatives in malocclusion. 

d. Elucidate genetic mechanisms involved in morphological 
variations of the teeth. 



167 



Serial No. NIDR-31 (63) , 

2. Utilize the teeth and oral cavity in the study of basic genetic 
phenomena particularly in the field of quantitative genetics. 
It is relatively easy to obtain large amounts of data, particu- 
larly in family groupings. Also, good methods exist for 
permanent capture of the raw data (casts and X-rays). Therefore, 
the teeth and oral cavity offer an excellent area for the study 
of biometrical genetics. 

Methods Employed : 

Effort during the last year has been focused in two areas: 

(1) survey of oral conditions of high school children in Hawaii, 

and (2) studies of malocclusion in the Papago Indians. 

The Hawaiian project is designed to study the causes of racial 
variation in dental characteristics, particularly malocclusion, 
with emphasis on the effects of racial crossing. The past year 
has been spent in data collection which is expected to be completed 
in the summer of 1968. At that time approximately 20,000 individuals 
will have been examined, the data processed and ready for analysis. 

This study concerns itself primarily with a population sample 
ranging in age from 12 to 17, i.e. individuals born between 1949 
and 1954. Reasons for selecting this group of children are as 
follows: Firstly, the individuals of this age group are in the 
intermediate schools (grades 7, 8, and 9) or the high schools 
(grades 10, 11, and 12) so that examination of subjects can be done 
readily through the public school systems. Secondly, this age 
group has well-developed permanent dentition, as well as readily 
discernible malocclusion, when this defect is present. 

The examination includes the following items: (1) evaluation of 
occlusions based on the Angle classification, and on other criteria 
by visual inspection; (2) simple functional evaluation of malocclusion; 
(3) intra-oral measurement of mesio-distal diameter of the upper 
central incisors; (4) the number of teeth, noting congenitally 
missing teeth; (5) scoring of periodontal disease based on Russell's 
method (1965); (6) status of oral hygiene; (7) scoring of dental 
caries (DMFS , DMF) ; (8) presence or absence of cleft palate and/or 
harelip; (9) presence or absence. of other oral anomalies, and (10) 
anthropometrics including height, weight, and cephalic measurements. 

Automated linkage of recorded data will include coupling of preexist- 
ing information on the birth certificates (already on tape) with the 
dental characteristics obtained by the present examinations. In 
progress is an extensive genetic study with record linkage by 
Drs . Mi and Morton of the Department of Genetics of the University 
of Hawaii. They are collecting biological, medical and sociological 
information on all individuals involved in marriage, birth, and 
death in Hawaii since 1942. This enormous file will be a great 

^ 168 



Serial No. NIDR-31 (63) 

potential source of genetic and epidemiological studies of medical 
and dental health. 

Additionally, since 1930, the Strong-Carter Dental Clinic of 
Honolulu has been offering annual free dental service to over 
4,000 children of "indigent" families. The eligibility requirement 
for enrollment with the Clinic is such that 177o of the students in 
the public schools are in this category at the present time. As 
each child registers with the Clinic, he is given a general dental 
examination, including posterior radiographs. Information on race, 
sex, birth date, general health condition, and sociological status 
is also collected. Thereafter, until age 12, regular annual 
examination and treatment continue. Thus, these data, though biased 
with respect to the socioeconomic status of the family, will 
supplement the first source of data in the studies of tooth abnor- 
malities, particularly as regards missing teeth and dental caries. 
These records will be linked by the computer with other sources of 
data to supplement epidemiological information and to eliminate 
duplications. The obstetric and pediatric information from the 
Kapiolani Maternity Hospital will also be used in the record linkage 
work. 

The Papago studies have involved the field examination of approxi- 
mately 300 school children and the collection of dental casts on 
about 140. Primary concern is the evaluation of tooth alignment 
and occlusion. An attempt is being made to collect similar data 
from skeletal remains of ancestors of the present Papagos. Suitable 
skeletal material is quite scarce, with only about a dozen specimens 
examined to date. Arrangements have been made to accompany 
archeologists from the Amerind Foundation on their next excavation. 
It is hoped that considerably more material will be obtained through 
this source. 

Serological genotypes are being determined for the purpose of 
defining biological subgroups of the Papago. Approximately 15 
red cell and serum markers are being evaluated. Certain oral 
conditions (primarily occlusion and dental morphology) are being 
evaluated simultaneously. If clear-cut genetic differences within 
the tribe can be established as a consequence of the blood studies, 
these genetic subgroups can then form the basis for study of subtle 
differences in dental and oral conditions. 

Major Findings ; 

The studies in Hawaii have to date involved only data collection. 
There are therefore no major findings to report. Frequencies of 
the four major classes of malocclusion, based on molar relationship, 
are shown below for the Papago and other groups examined by identical 
methods . 



163 



Serial No. NIDR-31 (63) 





Papago 


Bak 


arl 


Xavante 










Indians 


Ind 


ians 


Indians 


Japanese 


Caucasi; 


No mal- 
















occlusion 


.33 




.55 


.95 




.41 


.36 


Class I 


.48 




.31 


.05 




.44 


.30 


Class II 


.14 




.07 







.12 


.24 


Class III 


.05 




.07 


-- - 




.03 


.10 



The Xavante represent an essentially untouched tribe in central 
Brazil, whereas the Bakari, although presently residing in close 
proximity to the Xavante, have been in permanent contact with 
modern Brazilians for over 50 years, and have undergone considerable 
acculturation. Among the Indian groups there is a striking associa- 
tion between the degree of acculturation and the frequency as well 
as severity of malocclusion, the Papago data closely approximating 
those for modern Japanese. To date only 10-12 intact early Papago 
dentitions have been examined. These date roughly from a period 
between 1300 and 1700 AD. All have shown nearly perfect occlusioni 

These findings, together with previous results, suggest that while 
recessive genes play a role in the development of malocclusion, 
environmental factors are probably of most importance. Studies of 
siblings suggest that these environmental factors are correlated 
within families, and genetic factors are probably more of a 
predisposing than causative nature. 

Although these data do not allow very definitive conclusions by 
themselves, they are consistent with the other results and add 
considerable strength to the interpretation that environmental 
factors are of great importance in the etiology of malocclusion. 

Significance to Dental Research ; 

A great deal of speculation presently exists as to whether the high 
frequency of malocclusion in civilized man represents the untoward 
results of changing diet (either nutritional or functional), the 
results of evolution (relaxation of natural selection against 
occlusal disharmonies), or the result of admixture of diverse 
physical types. The majority of evidence seems to indicate the 
fact that primitive populations in general are characterized by 
lower frequency of malocclusion. There is also evidence to suggest 
the same trend applies to periodontal disease. 

The present studies are designed to yield information on these 
questions and are, therefore, of significance for understanding 
the etiology of oral developmental anomalies and malocclusion. 



no 



Serial No. NIDR-31 (63) 

Such knowledge should have eventual application in prevention and 
treatment. 

Proposed Course of Project ; 

To continue collecting data on "primitives" as opportunities are 
available and to further search out and examine groups for which 
skeletal material exists, and otherwise fulfill sufficiently 
the requirements outlined at the beginning, and to collect data 
on oral conditions in living North American Indian groups with 
particular reference to malocclusion. The Papago Indians of 
Arizona appear to fulfill many of these requirements. It is 
planned to continue collecting data on this group. 

To proceed with analysis of the data presently being collected in 
Hawaii. 



Part B 



Publications: 



Bailit, H. L. , Thompson, L„ A., and Niswander, J. D. : Dental 
eruption and hypodontia. J. Dento Res. , July 1968 (in press). 



rn 



^ 



Serial No. NIDR-32 

1. Human Genetics 

2. Population Genetics 

3. Bethesda, Maryland 



Part A 



Project Title: 



PHS -NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Theoretical and Applied Analyses in Human Populations 
with Particular Emphasis on the Study of Genetic 
Variation. 



Previous Serial Number: None 

Principal Investigator: Dr. P. L. Workman 

Other Investigators: Mr„ C. J. MacLean, Mr. W. C. Leyshon 

Cooperating Units: None 

Man Years: 

Total: 2 1/2 
Professional: 1 1/4 
Other: 1 1/4 

Project Description: 



Objectives 
1. 



3. 



To determine which particular factors are influencing the 
pattern of genetic variation in any population at a given 
time. Of especial interest are such factors as assortative 
mating, selection, population intermixture, and population 
size. 

To develop suitable methods for the analysis of intra vs 
inter population differences. Particularly important are 
those techniques which permit an assessment of the relative 
stability of genotypic distributions over space and time. 
This work will concentrate on the differences within and 
between North and South American Indian populations. 

To assess which indicators of developmental instability in 
humans are useful for comparing populations under genetic 
stress (severe inbreeding for example) or environmental stress 
(poor nutrition, high disease rates). Two particular measures 
of instability are being analyzed in different populations: 
(1) the asymmetry of fingerprint ridge count, and (2) the 



172 



Serial NOo NIDR-32 

asymmetry with respect to measurement characters of homologous 
teeth. 

4. A theoretical analysis of the manner in which dominance 

variation has evolved, with special reference to the evolu- 
tionary origin of stable buffered developmental pathways. 

Methods : 

Methodology in this area involves both data analysis and more 
theoretical mathematical procedures, ranging from model building 
to computer simulation. The major source of data presently being 
used Is the genetic information obtained from field studies of the 
Papago Indians in conjunction with the social demographic and 
genealogic information available in the Papago tribal register. 

Major Findings ; 

1. The determination of the appropriate methods for separating 
the effects of intermixture between populations from changes 
in the genetic structure due to selection has shown that for 
several human polymorphisms, in particular populations, 
selective forces can be demonstrated. Since the forces are 
generally not large, this method, aimed at assessing the 
cumulative effects of the separate forces over a period of 
several generations, may be extremely useful for detecting the 
existence of selective mechanisms. 

2. The analysis of human population data in terms of two or more 
loci, considered simultaneously, is shown to provide far more 
information, on the long term pattern of variation in a population 
than that normally obtained by a consideration of one locus at 

a time. Further evidence is provided to show that single-locus 
analyses only reveal the most extreme imbalances in population 
structure. 

Significance to Dental Research ; 

In addition to developing the theoretical framework for population 
analysis, the results should indicate which populations might be 
most profitably studied in great detail, particularly with respect 
to demonstration of selective forces, and determination of the 
biological forces underlying them. 

Proposed Course of Project ; 

Continuation of same program for another year. 



17: 



Serial No. NIDR-32 

Part B 

Publications: 

Workman, P. L.: Gene Flow and the Search for Natural Selection 
in Man. Human Biology . May, 1968 (in press). 



Ilk- 



Serial No. NIDR-33 
L. Human Genetics 
3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Studies on Site of Action of Phytohemagglutinin 
on Circulating Human Lymphocytes 

Previous Serial Number: None 

Principal Investigator: Dr. J. W. Graef 

Other Investigators: Dr. H. L. Cooper, Laboratory of Biochemisty 

Cooperating Units: None 

Man Years: 

Total: 3/4 
Professional: 1/2 
Other: 1/4 

Project Description: 

Objectives : 

1. To determine those portions of the cell required for in vitro 
activation of RNA polymerase in human lymphocytes. 

2. To elucidate the mechanism of stimulation of the human lymphocyte 
by phytohemagglutinin (PHA). 

Methods Employed : 

Blood is drawn from normal, healthy volunteer donors and the 
lymphocytes purified by standard techniques. These are placed in 
culture in vitro for varying lengths of time from 16-48 hours and 
are harvested. Cells are disrupted and preparations of nuclei are 
obtained with an average purity of 70-80 percent. These are incubated 
in the presence of labeled RNA precursors and necessary cofactors 
and uptake of label is considered a measure of activation of the 
RNA polymerase enzjrme reflecting the potential "transforming" and 
growth activity of the particular culture. The nuclei are incubated 
with and without phytohemagglutinin and various cell fractions are 
added to the system both from cells previously exposed to phytohemagg- 
lutinin and from resting cells to determine those fractions most 
greatly enhancing phytohemagglutinin activity. 

ITS 



Serial No. NIDR-33 

Patient Material ; 

Normal volunteers. 

Major Findings ; 

Nuclear preparations of 70-80 percent purity, in the absence of 
cytoplasmic additions are not stimulated by phytohemagglutinin. 
If anything, phytohemagglutinin appears to interfere with the uptake 
of labeled RNA precursors in the resting nuclei. 

Significance to Dental Research ; 

The phytohemagglutinin mitogen offers an experimental model for the 
study of cell division and the basic events involved in the transition 
of cells from resting to active. Because the human lymphocyte's 
specialized functions effect a wide range of human disease and, in 
general, are involved to some extent in virtually every inflammatory 
condition known, including periodontitis, aphthous stomatitis and 
resistance to herpetic infection, elucidation of mechanisms whereby 
this cell is stimulated is of fundamental importance. It also offers 
a model for the study of similar activity in other cell types not 
so easily obtained in vitro . 

Proposed Course ; 

The initial experiments involving the bio-assay for nuclear activity 
and base-line measurements of this activity, both with and without 
PHA, have been completed with what appears to be reliable, reproduc- 
ible data. It is proposed to move on to the major portion of the 
project involving measurement of the effect of different cell frag- 
ments on the nuclear system. Problems anticipated are technical and 
involve the difficulty of preparing biologically active fragments in 
amount and concentration necessary to effect the bio-assay system. 

Part B not included. 



ITS 



Serial No. NIDR-34 

L. Human Genetics 

2o Developmental Genetics 

3« Bethesda, Maryland 



PHS-NIH 

Individual Project Report 

July 1, 1967 through June 30, 1968 



'art A 



Project Title: Developmental Processes in Genetically Controlled 
Traits 

Previous Serial Number: None 

Principal Investigator: Dro K. S, Brown 

Other Investigators: Dr, D. Ro Bergsma, Dr. J. A. Sofaer 

Cooperating Units: Dr. E. L. Eagles, NINDB 

Man Years: 

Total: 2 
Professional: 1 1/2 
Other: 1/2 

Project Description: 

Objectives : 

1„ To describe the development of e:q)erimental animals with 

genetically produced congenital malformations, particularly 
those of dentition and the oral region. 

2. To examine the interaction between the tissues of these 

animals in the developmental processes in order to determine 
the nature of the mechanism producing the genetic defect. 

Methods Employed : 

1. A survey of mouse mutants is being undertaken to evaluate their 
oral structures in relation to those of the lines from which 
they arose. 

2. Mutants of specific interest are being collected, and colonies 
of these animals are to be developed as a basis for experimental 
study. 

3. Timed matings are being made to produce animals of known 
gestational age. Serial sacrifice and histological study will 



177 



Serial No. NIDR-34 

produce systematic documentation of the developmental processes. 

4. Organ culture is being undertaken of genetically defective skin 
and related tissues. Comparative studies of the development of 
mutant ectoderm with normal mesoderm and vise versa are being 
made to assess the nature of the developmental defect. 

5o In the cat, the dominant genes producing defects of pigmentation, 
hearing and eye color are being studied in a colony of 80 
defective animals being maintained by NINDB. Histological and 
physiological studies of these animals are being made. Studies 
of the function and survival of these animals in nature as 
compared with the colony are also being made in collaboration 
with the Animal Quarantine Station at Poolesville. 

Major Findings ; 

Two mutants showing dental defects have been identified and detailed 
studies are underway. One of these occurs in the mutant (dn) down- 
less, in which both hair and tooth development are defective in 
their Initiation. Another mutant exhibits recurrent alopecia, and 
appears to have an amelogenesis imperfecta -like dental condition. 
Several other mutants have been examined and rejected for further 
study. 

The mutant downless tissues have been chosen for the first organ 
culture studies, and cultures of separated ectoderm from early 
embryonic developmental stages have been made. These cultures have 
dononstrated the ability to separate the germ layers and maintain 
growth. Experimental cultures are just being started. They will 
attempt to localize the site of the specific defect and also to see 
if the site changes with the stage of development of the tissue. 

Studies of fetal histologic material from kittens with the dominant 
gene or genes producing the syndrome of deafness, white fur and 
heterochromia, or bilateral blue iris, have been carried out using 
serial section mapping of the membranous part of the inner ear. The 
anatomic defect does not appear in the stages before birth, but is 
only visible histologically in the early neonatal period, when the 
inner ear is undergoing its last differentiation and beginning to 
function. Correlated with these changes in the normal kitten are 
changes of the sulcus cells of the organ of Corti. In the abnormal 
animals, there appears to be a lack of production or regression in 
the production of endolymph. The Reisners membrane is partly or 
completely collapsed onto the organ of Corti. Hyalinized bodies 
occur in the stria vascularis in older animals. A comparable 
finding has been reported in viral disease, but its occurrence in 
genetic disease suggests that it is a common degenerative result 
rather than specific to any one etiology. 



179 



Serial No„ NIDR-34 

Studies of the histology of the iris and retina in genetically 
defective white cats suggest that there is an absence of certain 
differentiating stimuli. The anterior layer of the iris, which 
normally contains melanocytes, is not pigmented. The tapetum, a 
special structure occurring in the pigment cell layer of the retina 
is not developed although some pigment is formed. The rest of the 
retina is physiologically and histologically normal. Because the 
tapetal defect can occur unilaterally, the opportunity exists to 
test several theories of its function. Genetic studies have shown 
that the eye and ear defects are not localized to the same side on 
any one animal. They are probably examples of the complete penetrance 
of a pleotropic gene or gene complex. Breeding experiments are 
underway to determine whether one or many genes are involved in this 
trait. 

Significance to Dental Research : 

Early development of teeth and hair both result from the down- 
growth of localized ectoderm into underlying mesoderm.. The 
analysis of the interactions of ectoderm and mesoderm is fundamental 
to the understanding of the development of teeth. The use of animals 
in which specific genes produce defects in the normal processes will 
allow analysis of the mechanism of interaction and its genetic control. 

Neural crest cells are a part of the early ectoderm that gives rise 
to pigmentation and to the sensory neurones. The generalized but 
incomplete expression of the anatomic defect in the cochlea of the 
"deaf white cat" suggests that the syndrome is a result of a defect 
in the embryology of the neural crest cells. If the hypothesis 
of a neural crest defect can be tested, it may have implications 
for the development of the facial complex in Waardenburg' s syndrome 
of man, a human analog or homolog of the cat syndrome. The genetics 
of the cat syndrome may also suggest the nature of the genetics in 
man. 

Proposed Course of Project ; 

Further surveys of mutant mouse lines with respect to dental defects 
will be undertaken. Colonies of specific mutants will be established 
and culture studies of the development of the defect undertaken. 
Genetic, embryological and histological approaches to the mechanism • 
of genetic defects will be undertaken along the same lines already 
in progress. 



Part B 



Publications: 



Brown, K. S,, Wakeford, 0. S., and Binder, P. A.: Knife-wetting 
device; wet celloidin technique. Arch. Otolaryn , 87: 131, April 1968. 

3 173 



Serial No, NIDR-35 (66) 

L. Human Genetics 

3. Bethesda, Maryland 



PHS-NIH 

Individual Project Report 

July 1, 1967 through June 30, 1968 



Part A 



Project Title: A Study of the Relationships between Genetic 
Factors, Exposure to Vitamin D in utero , and 
Buphthalmos in the Rabbit, 

Previous Serial Number: Related to NIDR-52 

Principal Investigator: Dr, D. R. Bergsma 

Other Investigators: Dr. L„ F„ Mills 

Cooperating Units: This project is an outgrowth of another 

NIH project, namely Serial No. NIDR-52 (66); 
"Production of 'Elfin' Facies and Abnormal 
Dentition by Vitamin D2 during Pregnancy: 
Relationship to the Supravalvular Aortic 
Stenosis Syndrome." 

Principal Investigator: Dr. L. F. Mills, NIDR 
Cooperating Unit: Dr, W, F. Friedman, 

National Heart Institute, Cardiology Branch 

Man Years: 

Total: 1/2 
Professional: 1/2 
Other: 

Project Description: 

Objectives : 

1. To differentiate the rates of spontaneous (primarily genetic) 
buphthalmos in rabbits and that caused by exposure to vitamin D 
in utero (effects may be cumulative), 

2. To establish the dosage levels of vitamin D required to produce 
this defect. 

3. To correlate these findings with those of Drs. Mills and Friedman 
defining the relationship of vitamin D to elfin facies, abnormal 
dentition and the supravalvular aortic stenosis syndrome, see 
above. 



180 



Serial No. NIDR-35 (66) 

Methods Employed ; 

One normal male and one buphthalmlc male were obtained as stud sires. 
Four normal females and two buphthalmlc females were obtained as 
young breeders. Three major genetic types of animals are being 
produced: (1) those with both parents normal, (2) those with one 
parent affected, and (3) those with both parents affected. After 
a sufficient number of genetically defined animals have been produced 
in each group to serve as controls, selected females will be given 
progressively higher doses of vitamin D during pregnancy. Rates 
of buphthalmos and other abnormalities such as premature closure of 
fontanelles, malocclusion, and mortality rate will be compared for 
each group with appropriate controls. 

Major Findings ; 

1. In November, 1967, Dr. Mills observed that three New Zealand 
white male rabbits, who had been exposed to high doses of 
vitamin D^ in utero , had unusually large eyes. Further examina- 
tion revealed that each of these animals (who all had different 
mothers) had elevated intraocular pressure. Although enlarged 
eyes with elevated intraocular pressure ("juvenile glaucoma" or 
"buphthalmos") does occur as an irregular recessive trait ariiong 
NIH rabbits, the high incidence in those exposed to the vitamin D 
and its absence in a small control group was considered suggestive 
enough to warrant a controlled study. That study is this project. 

2. To date the project is still in the preliminary stage of produc- 
ing an adequate number of genetically defined animals. No 
exposure to vitamin D has been attempted. Therefore there are 
no new major findings. 

Significance to Dental Research ; 

The relationship between exposure to vitamin D and genetic background 
in producing the several abnormalities mentioned above is being 
studied in rabbits as an experimental model of these abnormalities 
in the human. For example, there is some evidence that increased 
vitamin D intake during pregnancy may be partially responsible for 
the increased incidence of the supravalvular aortic stenosis syndrome 
observed in humans in the past 25 years. Moreover, the causes of 
buphthalmos in humans are undefined, but there is evidence that both 
this and other types of glaucoma have a genetic component. Finally, 
vitamin D has been shown to produce dental malocclusion in rabbits. 
Therefore, this project combines study of the dental and genetic 
programs of NIDR. 



181 



Serial No. NIDR-35 (66) 

Proposed Course of Project ; 

Initially problems of offspring survival delayed the project. 
The major factors adversely influencing offspring survival have 
been identified and eliminated. It is anticipated that the project 
will reach the experimental level described under methods during 
the coming fiscal year. 

Part B not included. 



182 



Serial No, NIDR-36 (62) 
1„ Human Genetics 
2o Developmental Genetics 
3. Bethesda, Maryland 



PHS -NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Saliva Study 
Previous Serial Number: NIDR-93 (c) 
Principal Investigator: Dro Ro 0„ Wolf 
Other Investigators: Mr. Lo L. Taylor 
Cooperating Units: None 



Man Years: 




Total: 2 


1/4 


Professional: 1 




Other: 1 


1/4 


Project Description: 




Obiectives: 





lo To use various analytical techniques which have inherently 
varying resolving powers to determine and define salivary 
cOTtiponentSo 

2. To define normal variation of salivary components in normal 
individuals, as related to environmental conditions at the 
time of collection. 

3. To relate salivary components to genetic control. This includes 
genetic systems, such as secretor factor, as well as other, 
unknown but suspected, factors influencing salivary proteins. 

4. After methods are perfected and normal bases are established, 
the salivary components of selected disease entities will be 
studied. <i 

5. Determine if human salivary isoamylases are under genetic and/or 
physiological control. 

6. Study possible genetic /environmental control of muramidase in 
saliva and other body fluids. 



18 



'^ 



Serial No, NIDR-36 (62) 

7a Further investigation of factors in parotid saliva which 
correlate positively with the rate of calculus formation. 

8. Continue development of salivary isoenzyme detection techniques. 

Methods Employed ; 

1„ Amyloclastic method of saliva serum and urine isoamylase 

detection after polyacrylamide gel electrophoretic separation. 

2. Muramidase isoenzyme detection in saliva, serum and urine after 
disc electroporetic separation. 

3. Centrifugal ultrafiltration for separation of dental calculus- 
correlated parotid biuret positive material. 

4. Saliva fractionation by column chromatography and gel filtration. 

5. Schneyer segregators and Carlson-Crittenden cups are used for 
the collection of segregated saliva. 

Patient Material : 

1„ Normal control patients of the Clinical Center (NIH permission 
for the study of Normal Control Patients has been obtained), 

2= Selected Clinical Center patients. 

3„ Individuals from genetically defined populations. 

4. Outpatient volunteer families (obtained and administered 
through the Normal Volunteer Patient Section). 

Major Findings ; 

Human parotid and whole saliva have been shown to be polymorphic 
and attempts to discover the control of the variation are underway 
with the major emphasis on the genetic control. 

The isoenzyme technique utilized for the demonstration of amylase 
isozymes has been further developed for the demonstration of lysozyme 
isoenzymes. A study of the polymorphism of both amylase and lysozyme 
are in progress for the human body fluids: saliva, serum and urine. 

An ultrafilterable biuret positive material was reported found in 
human parotid saliva which was positively correlated with the rate 
of dental calculus formation. 



ISlf 



Serial No. NIDR-36 (62) 

Significance to Dental Research ; 

Two new areas for dental genetic research are being utilized in 
the study of the control of the polymorphic states of isoamylases 
and isolysozymes. A hint that a small protein-like molecule in 
human parotid saliva has something to do with dental calculus 
formation. Further elucidation of salivary components and their 
relationships to oral health. 

Proposed Course of Project ; 

1. To detect and define possible gene-controlled salivary components 
which, in turn, may lead to the definition of new metabolic 
pathways or corroborate known ones. 

2. To investigate batch separation of saliva proteins by continuous 
flow electrophoresis and gel filtration techniques. 

3. Conduct further genetic/environmental studies on human salivary 
isoamylases and isolysozymes. 

4. Corroborate and study the calculus correlated component of 
human parotid saliva. 



Part B 



Publications: 



Wolf, R. 0. and Taylor, L. L.; Isoenzyme demonstration technique. 
Am. J. Clino Path. , June 1968 (in press). 



185 



Serial No„ NIDR-37 (58) 

1. Human Genetics 

2. Population Genetics 

3. Bethesda, Maryland 



ras -NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Genetic Studies of Oral Clefts and Other 
Major Congenital Malformations 

Previous Serial Number: NIDR-91 (c) 

Principal Investigator: Dr. J. D. Niswander 

Other Investigators: Mr. C. J. MacLean 

Cooperating Units: PHS Division of Indian Health; Lancaster Cleft 

Palate Clinic, Lancaster, Pennsylvania; 
Epidemiology Branch, NCI; School of Public 
Health, University of Hawaii, 

Man Years: 

Total: 3 1/4 
Professional: 3/4 
Other: 2 1/2 

Project Description: 

Objectives : Subproject A 

lo To determine if there are physical stigmata associated with 
cleft palate, familial in nature, that may be useful in: 

a. defining etiologically different defects or syndromes now 
lumped together. 

b. clarifying the role of genetic (and/or environmental) 
factors in the genesis of certain clefts. Further, to 
determine the ccmplexity of the genetic systems involved, 
e.g., monomeric or polygenic, and to study inheritance of 
malformations by the use of segregation analysis. 

2. To study genetic "fitness" associated with oral clefts through: 

a. reproductive performance of couples who have produced one 
or more malformed children. 



186 



Serial No. NIDR-37 (58) 

bo study of the survival of children with clefts. An associated 
objective is to identify the causes of death among those 
affected individuals who have died and to compare these with 
the general population. (This aspect of the project has 
been inactive over the last year due to a one year leave of 
absence of Dr. Stark.) 

3o To determine the frequency of oral clefts in various population 
groups, as well as genetic and environmental factors important 
in their etiology. 

Methods Employed ; 

Detailed genetic, social, medical and reproductive history is being 
obtained on 100 families with oral clefts and 100 controls in 
Lancaster, Pennsylvania. All family members receive physical 
examination (primarily limited to head and neck region), tests of 
visual and auditory acuity and a mid-facial laminograph. Dermato- 
glyphic patterns and dental casts are also obtained. These data 
are being analyzed from the standpoint of answering a number of 
specific questions encompassed under objectives la, lb and 2a. 

All children born in Michigan between 1950 and 1960 are being 
followed to determine the fact of death, age and cause. This aspect 
involves mainly the use of birth and death certificates and will 
provide information pertinent to the mortality component of fitness 
of individuals with clefts. 

Data for all cleft births occurring in Montana over a ten year 
period have been obtained. Analysis will be directed toward the 
identification of temporal and spatial clustering of cleft births. 
Also available is data on over 400 families with HL + CP from Utah. 
These data are being used for genetic segregation analysis. 

Major Findings ; 

lo Significantly increased inner canthal distance among affected 
individuals from families with multiple cases of HL + CP. No 
significant changes in interocular dimensions were found, 
however, among individuals without clefts in these families. 

2. No significant increase in minor abnormalities of the palate 
and nasal cavity were found in relatives of cleft patients 
using frontal roentgenograph ic laminography = This finding is 
contradictory to other reports in the literature. 

3o Dermatoglyphic patterns show greater asymmetry among familial 
case of HL + CP than the control, isolated cleft palate, or 
sporadic harelip. 



181 



Serial No. NIDR-37 (58) 

4. There is a similar increase in dental asymmetry among the 
familial cases of harelip + cleft palate. 

5. Analysis of pedigrees shows a significant increase in the 
frequency of other major congenital anomalies among relatives 
of children with harelip and/or cleft palate in contrast to 
the control families. These findings are in contrast to the 
considerable data which shows a higher frequency of associated 
anomalies in individuals with isolated cleft palate compared 
to individuals with harelip. 

6. Segregation analysis of harelip + cleft palate pedigrees 
suggests that as many as 60% of the cases may be strongly 
genetic in origin. 

Significance to Dental Research ; 

Better definition of genetic factors in oral facial defects would 
be useful for counseling and predictive purposes. Also those 
entities which are non-genetic or have a large environmental 
component would define a group in which immediate measures aimed 
at prevention could be instituted. 

Better knowledge of associated disease offers hope for better 
understanding of etiology and should prove of value in medical 
management of affected children. 

Proposed Course of Project ; 

Complete analysis of data from Michigan regarding causes of 
mortality in children with oral clefts. Cluster analysis of the 
Montana data and additional segregation studies of the Utah data. 

Objectives ; Subproject B 

The purpose of this investigation is to ascertain the total (at 
birth) frequency of congenital malformation among American Indians 
as well as frequencies of specific major defects. We will then 
compare these frequencies to comparable data for other Mongoloid 
and Caucasian populations, as well as certain primitive Indian 
groups. A further objective is to determine what differences in 
malformation rates exist between the major linguistic and tribal 
groups of American Indians. Other factors being assessed include: 
degree of Indian blood, parental age, parity, season of birth, 
geography and certain socioeconomic and cultural variables. These 
variables are to be related not only to "total" malformation rate 
but also to certain specific anomalies. An additional objective of 
this project is to serve as a screening procedure to define unique 
and specific problems of genetic interest for which further detailed 
studies may be designed. Studies of factors influencing variation 



ise 



Serial No. NIDR-37 (58) 

in birth weight are also being conducted as part of this project. 

Methods Employed ; 

The major portion and nucleus of this study involves the use of 
birth and pediatric records of the various Indian hospitals. 
Through the cooperation of the Division of Indian Health, copies 
of these records are available for all Indian births. 

A large body of data exists for comparison purposes on congenital 
malformations in Japanese. These data have been collected by the 
Department of Human Genetics at the University of Michigan. This 
group is presently conducting further studies in Japan to supplement 
these data as well as to collect other material of genetic signifi- 
cance which can be utilized for comparison. More directly applicable 
are the studies on primitive Indians being undertaken by the 
Department of Human Genetics. 

Field studies among the Papago Indians have continued. One objective 
is to examine at one year of age all infants on whom we have birth 
records. This study will provide information on the adequacy of 
the newborn examination for detection of congenital malformation. 
In addition, genetic studies (pedigree analysis) will be undertaken 
for selected malformations encountered among the Papago. Micropth- 
almia is one such anomaly. 

Major Findings ; 

1. The frequency of cleft lip with or without cleft palate in 
American Indians is intermediate between Caucasian and other 
Mongoloid populations, suggesting a relatively simple effect 
of genetic admixture. Isolated cleft palate frequencies in 
the Indian closely approximate those of present Japanese, This 
finding is not easily explained by any simple biological hypoth- 
esis. 

2. Anencephaly and spina bifida are similar, in that American 
Indian frequencies for spina bifida are intermediate between 
Caucasian and Japanese, whereas anencephaly in the Indian remains 
close to Japanese in frequency. 

3. Variation in birth weight among various Indian tribes correlates 
well with adult weight and stature. This variation can be 
related to precolumbian food procurement patterns. 

4. Analysis of low birth weight in the Indian suggest that this 
group can be divided into two groups; (1) "Ordinary" low 
birth weight, i.e. the lower tail of the normal distribution 
of birth weight and (2) "Deviant" low birth weight, i.e. those 
babies whose low weight represents the expression of an abnormal 



183 



Serial No, NIDR-37 (58) 

developmental process. Demonstration of this phenomenon is 
significant to clinical medicine in refining criteria for 
prematurity and in establishing guidelines for the management 
of low weight infants. The method used also presents a model 
which should prove of value in population studies of factors 
affecting birth weight. 

Significance to Dental Research ; 

lo Comparison of the data with material being collected on Indians 
will provide measures of racial variation in human populations. 
Studies of this nature are of importance in defining the relative 
role of genetic and environmental factors in the etiology of 
malformation and, hence, ultimately contribute to the knowledge 
necessary for control. 

2. These data will have further bearing on certain theoretical 

genetic questions of relevance to the etiology of malfonnations. 

3o Specific oral-facial anomalies will be studied in light of 

the total picture. Their study, not only as isolated entities 
but in the framework of malformation in general, may lead to 
clearer understanding of the genetic factors involved. 

Proposed Course of Prolect ; 

To continue collection of Indian birth data on a semi-permanent 
basis. Starting from experience and knowledge gained in this 
project to extend to other more exhaustive studies of dental and 
physical conditions in American Indians. 

To initiate specific studies on diseases and traits of genetic 
interest as the specific problems become defined. 



Part B 



Publications: 



Adams, M. S. and Niswander, Jo D.: Birth weight of American Indian 
tribes. Human Biology (in press) 1968., 

Adams, M. S. and Niswander, J. D.: Developmental "Noise" and a 
Congenital Malformation. Genetical Research 10: 313-317, 1967. 

Adams, M. S., MacLean, C. J. and Niswander, J. D.: Discrimination 
between Deviant and Ordinary Low Birth Weight: American Indian 
Infants Growth. Pediatrics (in press) 1968. 

Adams, M. S. and Niswander, J. D. : Health of the American Indian - 
Congenital Defects. Eugenic Quar . (in press) 1968. 

190 



Serial Noo NIDR-37 (58) 

Niswander, J. D.: Laminographic X-ray Studies in Families with 
Cleft Lip and Cleft Palate„ Archives of Oral Biol , (in press) 1968, 

Niswander, J, D. and Adams, M. S,: Major Malformations in the 
Relatives of Oral Cleft Patients, Acta Genet . 18: 229-240, 1968. 

Niswander, J. D, and Adams, M„ S.: Oral Clefts in the American 
Indian.. Pub. Health Rep . 82: 807-812, 1967. 



191 



Report of the Laboratory of Histology and Pathology 
National Institute of Dental Research 
Summary Statement 

The past year has resulted in steady progress in most areas. The most 
noticeable advance has been the revitalization of the Section of Crystal 
Chemistry through appointment of a new chief. On the other hand, the program 
on the morphology of mineralized tissue has suffered through the permanent 
loss of one and the temporary absence of a second key investigator. An improve- 
ment is not expected in the immediate future, although every effort will be made 
to reinforce this vital area. 

For the purposes of the present report the activities of the Laboratory 
of Histology and Pathology are summarized according to several areas of general 
interest. The projects from which the report have been gathered together are 
carried out by staff members alone or jointly, and often in collaboration with 
workers from other laboratories and institutes. The specialized fields repre- 
sented include electron microscopy and electron diffraction, microradiography, 
autoradiography, histochemistry, x-ray diffraction, infrared spectrophotometry 
and experimental pathology. 

Cellular and Extracellular Morphology . 

This year all the efforts which have involved the use of the electron 
microscope and microradiographic methods are described under this heading. 
Most of the projects represent a continuation of previous studies although in 
several instances the investigations have progressed to a new level. This 
year's research into the ultras tructure of the central nervous system, for 
example, has been directed at the main sensory nucleus of the trigeminal 
nerve. This nucleus is an amportant link in the chain of events through which 
complex movements such as mastication, deglutition and those involved in speech 
are effected. Some of the morphological findings are unique relative to other 
regions of the central nervous system. It is speculated that they represent 
part of the morphological basis for the neurophysio logic findings of presynaptic 
inhibition which characterize this region. One important aspect of these studies 
has been the perfection of a perfusion technique which has made it possible to 
obtain satisfactory fixation of the trigeminal nuclear complex routinely. 

With minor variation the same perfusion technique has been employed in a 
study of bone resorption and collagen degradation in the periodontal membrane 
of various animals exposed to extreme stress. In this investigation, however, 
the resulting fixation has not been entirely satisfactory, although part of the 
difficulties may be associated with the handling of the specimens subsequent to 
perfusion. Several possible sources of error have been explored and it is ex- 
pected that the studies will result in the development of a reproducible tech- 
nique for preparing such tissues for electron microscopy. 



133 



Most previous work on the ultras true cure of periodontal membrane has 
been restricted, for reasons of poor fixation, to the examination of remnants 
on extracted teeth. While this type of specimen has limited value, it has 
been used successfully in this laboratory in previous ultrastructural studies 
of epithelial rests from normal periodontal membrane. The studies suggested 
that although the epithelial cells were relatively undifferentiated they pos- 
sessed the potential for assuming a more active role. Subsequent histochemical 
and autoradiographic studies- showed that this could be achieved by explanting 
pieces of human periodontal ligament in a suitable medium. Electron micro- 
scopic examination of the explant specimens revealed that the epithelial cells 
were much more highly differentiated judging from the appearance and organiza- 
tion of their cytoplasmic organelles and inclusions. The demonstration that 
these cells may respond to environmental stimuli throws new light on their 
possible role in the formation of odontogenic tumors and cysts. 

The ultrastructure of the accessory boring organ (ABO) from drilling 
muricid gastropod molluscs has been investigated in detail. Although of 
epithelial origin the secretory cells of the ABO, as are osteoclasts, appear 
to be involved in hard tissue destruction. The electron micrographs showed 
that the secretory epithelium was penetrated by a complex system of capillaries, 
muscles and nerves , which was much more extensive than indicated by optical 
microscopy. The presence of nerve endings on both muscles and epithelial cells 
suggested the existence of afferent as well as efferent pathways. Comparison 
of ABO's from actively drilling and inactive snails failed to demonstrate any 
major cytological differences. However, more detailed information may be 
forthcoming from cytochemical studies which have been initiated recently. 

The studies on microbial morphology which constitute an important program 
area within the overall objectives of the laboratory have shown steady progress. 
One project, the determination of the site of action of complement in the com- 
plement dependant serum bactericidal reaction has been concerned primarily with 
the chemical rather than the structural aspects during the past year. This 
development has followed logically on last year's demonstration that the endo- 
toxic lipopolysaccharide (LPS) was the site of action in at least 2 gram nega- 
tive microorganisms. Consequently LPS was isolated from one of the 2 and split 
into a lipid and a carbohydrate fraction, each of which was interacted with 
complement. While some technical difficulties have been encountered, the 
results so far suggest that the uptake of complement by LPS is mediated by 
the lipid fraction. This conclusion if substantiated will lead to a fuller 
comprehension of serimi-cell interactions. 

A second project concerned the formation, localization and nature of 
sulfur inclusions in certain gram negative bacteria. A combination of methods 
including autoradiography and freeze etching was utilized. The results indi- 
cated that the inclusions actually formed outside the cytoplasm while the 
enzyme(s) responsible for the oxidation of the sulfides resided in the cyto- 
plasmic membrane. This type of study in which mem.brane function is correlated 
with morphological entities is important in the overall understanding of cell- 
environment relationships under investigation in this laboratory. 



ISk 



other studies carried out by this group , yet not concerned with micro- 
bial morphology, have involved localization of at least 2 peptides in the al 
polypeptide of collagen. The al polypeptide chain concains S peptides which 
can be separated chem ically and purified. Solutions of al chains and of 3 
of the 8 peptides were prepared and ATP was added. The al chains readily 
converted into Segmented Long Spacings (SLS) under these conditions. Peptides 
CB 6 and CB 8 also formed SLS or rather fragments of SLS. Both exhibited 
characteristic cross banding patterns which were sufficiently precise to allow 
identification with similar patterns in the al SLS. The third peptide tested 
formed only poorly identifiable SLS fragments in the initial experiments. 
While a distribution of the 8 peptides has been proposed on the basis of bio- 
chemical considerations, the morphological findings will provide more accurate 
data. Knowing the exact location of the various peptides will contribute con- 
siderably to an understanding of various normal and pathologic processes. An 
example of the latter would be identification of the exact site of action of 
collagenase, another major concern of the laboratory. 

Today radioactive tracers are used extensively for identification of many 
substances at the ultrastructural level. The parameters for the use of low 
energy isotopes are well defined while certain discrepancies have been observed 
in studies involving high energy isotopes. Since bone seeking isotopes possess 
a high energy, knowledge of the practical resolution and sensitivity character- 
istics of such a system is necessary for valid data interpretation. In experi- 
mental studies with P , sensitivity values were found to be higher than those 
predicted theoretically and they deviated more the smaller the angle of inci- 
dence. It was concluded that scattering events were responsible for the re- 
sponsible for the results and that good correspondence could be obtained be- 
tween experimental and theoretical data by correcting the theoretical distri- 
bution curve for the influence of angle of incidence on emulsion sensitivity. 

Instead of isotopes, fissionable elements may be used as tracers. The 
resolution which can be obtained with such a tracer system is considerably 
better than that provided by radioautographic nethods . In addition it is 
possible to calculate accurately the amount of fissionable material present 
from the number of tracks . Certain advances were made toward making this 
approach a useful biological tool. Methods for registration of the tracks 
were refined and standardized. Numerous tracks were recorded after bombard- 
ment of a suspension of uranium containing hydroxy apatite crystals. On the 
other hand no tracks were observed over uranium containing bone sections , a 
failure which may be due to the density of the bone tissue. Work is presently 
under way aimed at circumventing this difficulty. 

Histochemistry . 

The major concern in this area continues to be the definition of meta- 
bolic parameters in oral and other connective tissues. Particular emphasis 
is placed on enzymes associated with the breakdown of these tissues; namely 
collagenase and hyaluronidase . The data have remained consistent with the 
assumption that the collagenase detected is the one concerned with the normal 
turnover of collagen, and that increased amounts are associated with patho- 
logic conditions. For example, greater than normal amounts of collagenase 

3 ^:^5 



tvere detected in culture fluids of (1) skins from individuals with amyotrophic 
lateral sclerosis and certain other neuromuscular diseases and (2) synovia 
from patients with rheumatoid activity. In the latter instance the amount of 
collagenase detected correlated with the clinical severity of the disease. 
Several additional sites of collagenase activity in normal tissue were demon- 
strated in the past year. Thus the enzyme was detected in culture fluids of 
normal human articular and growth plate cartilage and bone. Likewise a spe- 
cific collagenase was found in the granule fraction of human neutrophilic 
leukocytes. This discovery explains how these cells may contribute to the 
destruction of collagen in many pathologic states. Further characterization 
of the enzyme indicated that it is active at pH 7-9 and that it is completely 
inhibited by EDTA and partially by cystein. Electron microscopic studies 
coupled with data from acrylamide gels showed that the human collagenase 
cleaved the collagen molecule into a 3/4 and 1/4 piece, an action which is 
similar to that of tadpole derived collagenase, but distinct from that of the 
bacterial enzyme. 

The studies on hyaluronidase involved detection of the enzyme in alveolar 
macrophages in rabbits. Here as in human gingivae the enzyme manifested a pH 
optimum different from that of bacterial hyaluronidases . The detection of 
hyaluronidase in various tissues explains how hyaluronic acid, chondroitin 
sulfate A and chondroitin sulfate C may be degraded in normal and pathologic 
tissues . 

Histochemical studies of enzymes with activities less directly identi- 
fiable with specific cellular functions were also continued. These enzymes 
which are concerned with the production of energy, and fatty acid, carbohydrate 
and mucopolysaccharide metabolism include the phosphates, the est 
numerous dehydrogenases, and the glycosidases , Both esterase and selected 
glycosidase activity appeared to be correlated with cellular activity asso- 
ciated with active remodelling of bone and cementum. Most of the esterase 
activity in rat jaws was demonstrated to be non-specific in nature, the bulk 
being a B type esterase while only a small amount of C type esterase was 
identified. 

Crystal Chemistry 

The activities of the Crystal Chemistry Section can be conveniently 
categorized into three areas of interest. Two of these areas represent new 
directions of study for the section. 

New research has been initiated in the areas of diffraction of x-rays by 
biofibers. The broad objective in undertaking these studies is to collect 
diffraction data from biological fibers under a wide variety of experimental 
situations and attempt to interpret the x-ray findings in terms of those 
structural parameters which may be of particular relevance to understanding 
their biologic role. During the past year this objective has been pursued 
through two specific lines of investigation. First, a comparative study of 
the x-ray diffraction properties of hard and soft tissue collagens was under- 
taken. From data accumulated in this study to date, advances in understanding 
the role covaient crosslinks may play in defining collagen structure have 



been made. Of particular significance was the finding that the ability of 
chemically denatured collagen to renature was dependent upon the presence of 
intermolecular covalent cross-links. Bone collagen, which is crosslinked was 
found to completely renature under conditions where tendon collagen, devoid of 
cross-links , showed no signs of renaturing. 

A second specific line of investigation undertaken was a diffraction 
study aimed at delineating the structure of the protein components of amyloid 
tissue. Previous studies in this laboratory and elsewhere have demonstrated 
that amyloid deposits contain 2 morphologically different structures which do 
not seem to be interconvertible. One of these appears as a periodic rod, the 
other and ma jot component is a non-periodic fibril. The discovery that this 
latter amyloid protein exhibited an x-ray pattern of the cross-p type may have 
particular importance to dental research. The only other cross-p protein re- 
portedly found in human tissue comprises the major component of the matrix of 
enamel. The periodic rods proved to have an "amorphous " wide-angle x-ray dif- 
fraction pattern. Their structure as observed in the electron microscope 
suggests, however, that this material may have a small-angle diffraction pat- 
tern, a possibility which will be further investigated. 

The Crystal Chemistry Section has maintained a long standing interest in 
biological and synthetic calcium phosphates. This interest, however, has taken 
a new direction with attention shifting away from crystalline salts and, in- 
stead, focusing on amorphous phases. Previous reported studies have demonstrated 
that the mineral investing hard tissue contains, in addition to crystalline hy- 
droxyapatite , an amorphous calcium phosphate as a major phase. Much of what is 
known, however, about this amorphous salt has been deduced by inference from 
its synthetic analogue. Unfortunately, the diffuseness of the diffraction 
pattern has placed definite limitations on the ability of x-ray analysis to 
demonstrate whether this synthetic amorphous calcium phosphate is a unique 
phase or is a cryptocrystalline form of one of the known crystalline calcium 
phosphates , A new experimental program is being undertaken with the express 
purpose of more clearly defining the synthetic amorphous salt by employing 
thermochemical techniques . 

Another area of continuing interest to the section has been infrared 
absorption spectrophotometry of hydroxyapatite and related compounds. During 
the past year, studies on isotopically substituted apatites have led to some 
major reassignments of absorption bands in the IR spectra of hydroxyapatite. 
Another important advancement in the study of the IR spectra of apatite has 
been the development of a hydrothermal procedure for isotopically labeling 
PO4 groups in hydroxyapatite with 0^°. This procedure should not only prove 
invaluable in the future study of the IR spectra of apatites but also in ex- 
panding the capability of IR spectroscopy for the study of reaction mechanisms 
and thermal diffusion in apatites, A study was also initiated on characterizing 
the IR spectra of strontium and various apatites to aid in the assignment of 
some low frequency bands in the hydroxyapatite spectrum. The object of all 
these studies is not just to understand the IR spectrum of hydroxyapatite for 
its own intrinsic interest but to develop this understanding to the point 
where IR spectroscopy can be of invaluable assistance in the important problem 
of obtaining information on the effects impurities have on the chemical and 
physical properties of biological apatites. 



197 



Experimental Pathology 

This year's studies as were those of the preceding years have been aimed 
at gaining greater insights into the components of the dento-bacterial plaques, 
their pathogenic potential, and methods for controlling the deposits and their 
toxic by-products. Special emphasis has been placed on understanding the 
mechanism of adhesion between the microorganisms and the surfaces of the teeth. 
It appears that this, in large measure, is effected through the production of 
extracellular polysaccharides. Streptococci that produce abundant amounts of 
dextran and high concentrations of intraplaque acids are conducive to rampant 
dental decay. When dextranase was added to the diet and /or water of hamsters 
affected with dental plaque infections produced by dextranogenic streptococci, 
this type of deposit did not collect on smooth surfaces. As a result dental 
caries did not occur in these highly vulnerable areas. However, this enzjrme 
did not affect plaque deposits which resulted from the interaction of levano- 
genic diphteroids and various starches and sugars. Nevertheless preventive 
treatment aimed at the dispersion by chemical means of adherent plaques on the 
surfaces of teeth represents a new and possibly major advance in the fight to 
achieve plaque control. 

The importance of appropriate dieto-bacterial challenges in the induction 
of plaque infections was demonstrated again in studies of experimental caries 
and periodontal syndromes. After many unsuccessful attempts, cervico-radicular 
plaque formation and associated periodontal disturbances were induced in ham- 
sters by feeding them a diet containing starch and by inoculating their mouths 
with human diphteroids . An infectious component was also found to be involved 
in experimental periodontal pathosis in rats. However, attempts to colonize 
an established hamster strain of plaque forming microorganism in dogs failed 
even under favorable dietary conditions. In contrast a high degree of caries 
activity was induced in rats once considered to be resistant to cavitation by 
an appropriate dieto-bacterial challenge. 

While it may become possible to control smooth surface caries through 
the use of plaque dispersing agents , prevention of fissure caries may depend 
on other approaches. One that could be of great value in preventing this type 
of lesion is the use of sealing agents to seal vulnerable retention sites. 
Initial experiments with materials containing n-methyl cyanoacrylate were only 
partly successful because the adhesive did not possess adequate bio-stability. 
In vitro experiments indicated, however, the effectiveness of this approach. 
At present other sealing materials are being tested in vitro and in animals 
before undergoing clinical trials. 



198 



Serial No, NIDR-38 (63) 

1. Histology and Pathology 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Experimentally Induced Enamel Defects 

Previous Serial Number: NIDR-15 

Principal Investigator: Dr , M. U. Nylen 

Other Investigators: None 

Cooperating Units: Dr. K.-A, Omnell, Royal Dental School, 

Malmo, Sweden 



Man Years: 




Total: 


1 


Professional: 


1/4 


Other : 


3/4 



Project Description: 

Objectives : 

Previous experiments have shown that intraperitoneal injections of 
tetracycline hydrochloride (TC) in rats result in both hypoplastic 
and hypomineralized enamel defects. The purpose of the continued 
studies was: 

1. to compare the effect of oxytetracycline (OTC) with that 
of TC. 

In the course of these experiments the enamel was found to be labeled 
by the antibiotic in an unusual yet consistent manner. Because of 
these observations new experiments were designed, the purpose of which 
was: 

2. to investigate in more detail the relative staining behavior 
of normal and pathologic enamel. 

Methods Employed : 

1. Twenty Sprague-Dawley rats, approximately 75 days old, were used 
as experimental animals. Half of the rats were given 25 mg/kg body 



193 



Serial No. NIDR-38 (63) 

weight of TC on day 0, an equivalent dose mol per mol of OTC on day 5, 
followed by 150 mg/kg body weight of TC on day 10 and an equivalent 
dose of OTC on day 15, In the remaining animals the sequence of admin- 
istration was reversed with a low dose OTC starting the injection series. 
All the rats were sacrificed 10 days after the. last injection and 50 
micron midsagittal ground sections were prepared from the upper incisors 
and exam.ined under ultraviolet light and microradiographically. 

2. Sprague-Dawley rats, approximately 75 days old, were injected sub- 
cutaneous ly with NaF in doses ranging between 80 and 125 mg/kg body 
weight. Intraperitoneal injections of 125 mg/kg body weight TC were 
administered 5 and 10 days later. The animals were sacrificed 1 , 5 or 
10 days after the TC injection and upper incisors were prepared for 
examination under UV light and for microradiography as described under 1, 

Major Findings ; 

Neither TC nor OTC produced enamel defects consistently at the lower 
dose levels. On the other hand both were capable of producing enamel 
defects at the higher dose levels. On the basis of differences in the 
severity of the response the lesions were divided into 3 groups. Group 
1 comprised the least severe lesions, i.e., those in which the incre- 
mental band constituted the only "structural" disturbance. Group 3, on 
the other hand, exhibited lesions including gross hypoplastic defects, 
in addition to the incremental line. A comparison of the effect of the 
high doses of TC and OTC on the enamel showed that while 15 of the total 
20 animals developed group 3 malformations following the TC administra- 
tion, only 1 such lesion was associated with the OTC injections. In 
contrast only 1 TC , but 17 OTC lesions, were classed in group 1, 

Under U,V, light, 4 fluorescent lines, each representing one injection 
were present in the dentin. The color of the fluorescence varied; the 
OTC bands yielding a greenish yellow, and the TC bands a golden yellow 
fluorescence. In the enamel, the incremental bands were generally non- 
fluorescent. Of the gross hypoplastic lesions, only those associated 
with the first high dose injection were labeled, while those caused by 
the second administration were non-fluorescent. Although all of the 
labeled lesions were the result of a TC injection, the color of the 
fluorescence seemed greenish rather than golden yellow. 

These findings suggested that preexisting lesions were labeled perma- 
nently by subsequent administrations of tetracycline while the initial 
label was lost as it is in normal developing enamel. Since the hypo- 
plastic lesions are markedly hypomineralized and contain large inter- 
crystalline spaces, as shown by electron microscopy, they are readily 
accessible to the antibiotic at a level of development when the sur- 
rounding normal enamel is too highly mineralized. Why the label should 
be retained permanently at this stage of development and not initially 
remains to be explained, although it may be related to crystal growth 
phenomena . 

2 200 



Serial No. NIDR-38 (63) 

The ability of the fluorphor to locate permanently in areas of defective 
enamel was borne out by the 2nd series of experiments. In these studies 
it was found that gross hypoplastic lesions , created by subcutaneous 
injections with high concentrations of NaF, were the only portions of 
the enamel labeled permanently by a subsequent administration of tetra- 
cycline . 

Significance to Dental Research ; 

The tetracyclines are some of the most commonly used antibiotics. 
Previous investigators have shown that although all of the commercially 
available products may discolor the developing teeth permanently, the 
effect of oxytetracycline is somewhat less severe. The present findings 
that oxytetracycline also is less apt to produce hypoplastic enamel 
lesions give added significance to the previous observations. 

The findings that defective enamel may be labeled during later adminis- 
tration of tetracycline while tetracycline induced defects lose their 
initial label are of importance in determining the origin of enamel 
lesions. Only those associated "geographically" with fluorescent dentin 
lines may be attributed to the antibiotic. These findings further indi- 
cate that incorporation of tetracycline in the enamel is not limited to 
the formative stage, but may occur during the entire pre-eruptive phase 
provided defective zones are present. 

Proposed Course of Project ; 

The study has been completed and is in the process of being written up. 

Part B 

Publications ; 

o 

1. Lofgren, C.-J., Omnell, K.-A., and Nylen, M. U.; Effect of 
Intraperitoneal Injections of Tetracycline Hydrochloride and 
Oxytetracycline on Forming Enamel of Rat Incisors, Accepted for 
publication in Calcified Tissue Research. 

2. Nylen, M. U.: Recent electron microscopic and allied investiga- 
tions into the normal structure of human enamel. Int. Dent. J. 
17; 719-733, 1967. 



201 



Serial No. NIDR-39 (59) 

1. Histology and Pathology 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, -1967 through June 30, 1968 



Part A 



Project Title: Collaborative Projects and Training Activities 
Previous Serial Number: NIDR-16 
Principal Investigator: Dr. M. U, Nylen 
Other Investigators: see below 
Cooperating Units: see below 



Years: 




Total: 


2 1/4 


Professional: 


1/2 


Other : 


1 3/4 



Project Description: 

Objectives, Methods Employed, and Major Findings ; 

Over the years a number of collaborative projects involving the use 
of biophysical methods have been undertaken upon the request of investi- 
gators in other laboratories. Many of the problems have been directly 
related to the general program of this laboratory and have constituted 
a sizeable portion of our activity. The principal projects are listed 
below. 

Collaborative Studies ; 

1. Studies of calcareous corpuscles in tapeworms. X-ray diffraction 
of corpuscles from different tapeworm species after various degrees 
of heating. In addition the uptake of phosphate by isolated corpuscles 
is investigated using x-ray diffraction methods. Uninterrupted heating 
of calcareous corpuscles of Taenia tacniaformis for 180 days at 150 C 
resulted in a very faint pattern possibly indicating the formation of 
dolomite. A much more distinct pattern was visible when the temperature 
was increased to 180 C, the heating period lasting from 4 to 5 days. 
At 300 C dolomite was formed after 15 minutes and at 450 C after 5 



202 



Serial No, NIDR-39 (59) 



minutes although some decomposition took place at the latter temperature 
as indicated by the slow appearance of calcium carbonate and magnesium 
oxide patterns. Relatively large amounts of phosphate were incorporated 
in calcareous corpuscles incubated j^ vitro in a phosphate solution. 
The diffraction studies indicated that the phosphate gave rise to apa- 
tite formation. With Dr. T. von Brand, Laboratory of Tropical Disease, 
NIAID, To be continued. 

2. Electron microscopic studies of the accessory boring organ of 
molluscs. This past year the work has been concerned largely with 
studying the relationship of the epithelial cells to each other and to 
the muscles, blood vessels and nerves, which permeate the tissues. The 
epithelial cells are arranged in groups, each of which is surrounded by 
a basement membrane. The cells are very irregular especially in the 
basal half where they interdigitate profusely with each other. Numerous 
pericyte- lined vessels are located between the epithelial cell groups. 
They are most prominent basally but can be followed almost to the distal 
microvillar zone, where they appear to be open to the interstitial 
spaces. As a consequence hemocyanin is found not only inside the ves- 
sels but also in all the spaces between the vessels and the epithelial 
cells, A much greater number of hemocyanin molecules is seen in the 
active than in the inactive organs, probably reflecting the greater 
demand for oxygen in the former instance. Muscles too are found between 
the epithelial groups where they frequently assume an orientation per- 
pendicular to the long axis of the secretory cells. Groups of nerves 
seem to follow the muscles into the tissue while others appear to be 
located on the epithelial side of the basement membrane surrounding the 
epithelial cell groups. Axons of the latter can be traced distally to 
the immediate vicinity of the microvillar border. 

Apart from mapping this complex tissue, additional information has been 
obtained concerning the cytoplasmic organization of the epithelial cells. 
Of particular interest has been the finding of numerous large bodies 
which appear to be autophagic in nature , In order to identify these 
bodies better, cytochemical studies have been initiated, and the con- 
tinuation of the project will center around this aspect. With Dr , M. R, 
Carriker, Marine Biological Laboratories, Woods Hole, Massachusetts, 
and Dr. V. Provenza, Maryland University Dental School, Baltimore, 
Maryland . 



3. Electron microscopic studies of explanted epithelial rests from 
normal human periodontium. Small root fragments with adherent perio- 
dontal membrane were explanted in a medium described by McCoy et al and 
were harvested at various incubation intervals ranging from 5-12 days 
after explantation. Experimental and control specimens were fixed in 
glutaraldehyde followed by osmic acid and embedded in Maraglas . Exami- 
nation of lead stained thin sections revealed many distinct differences 
between the cultured and the control material. 



203 



serial No. NIDR-39 (59) 

The cytoplasm is more abundant in the in vitro cells and contains an 
increased quantity of better defined organelles. Thus the mitochondria 
are larger with more distinct outer membranes and internal cristae. 
The Golgi complex which is missing in the control is very prominent in 
the experimental material. The endoplasmic reticulum is also enhanced. 
The profiles, however, are only partially covered by ribosomes suggest- 
ing a transitional stage between smooth and rough surfaced endoplasmic 
reticulum. Free ribosomes as well as polyribosomal aggregates are also 
more numerous in the in-vitro cells. Of the inclusions lysosomes and 
lipid bodies are more frequent in the explants than in the resting cells 
while the opposite is true of glycogen and tonof ilaments . Additional 
evidence of an increased metabolic activity is presented by the change 
in cell contour. In the cultured cells the plasma membranes feature 
numerous microvillar projections which interdigitate with those of the 
adjacent cells while those in the control are much more regular. 

Since both histochemical and autoradiographic studies have indicated 
that the epithelial rests assume a more active state following ex- 
plantation, the morphological changes are not unexpected. The findings 
serve to emphasize, however, once more the relationship between cell 
activity and cytological differentiation. The fact that these cells 
have the capacity to become more active is of interest to considerations 
of their possible contribution to the formation of cyst linings and 
even to odontogenic tumors. The project which has been carried out. in 
collaboration with Dr. H. A. Zander and Dr. H. E. Grupe , Eastman Dental 
Center, Rochester, New York, is completed and the results are in the 
process of being written up. 



4, A microradiographic study of the zone of calcification which in man 
attaches the articular cartilage to bone. Variations in this interface 
with age, species and disease as well as in experimentally induced re- 
modeling are being investigated. Preliminary results have indicated 
that in man the calcified layer is more highly mineralized than the 
subchondral bone. With Dr. E. D. Eanes , LHP, NIDR and Drs . L. Sokoloff 
and W. Green, LEP, NIAMD . To be continued. 



Training Activities ; 

The following persons have received training in electron microscopy and 
associated techniques. 

Dr. Lenore Disher , School of Dentistry, University of California, San 
Francisco Medical Center and a U.S.P.H.S. post-doctoral fellow has been 
a guest worker in the laboratory since February 1, 1968. 

Dr. A. Carl Verrusio, Section of Pharmacology, Laboratory of Biochemistry, 
N.I.D.R,, who has been with the laboratory as a trainee since January 1968. 



20k 



Serial No. NIDR-39 (59) 



Significance to Dental Research ; 



The importance of the collaborative and training efforts is self-evident, 
Through the collaborative projects, the laboratory staff gains a broader 
experience in the general field of biophysical instrumentation, which 
together with the accumulated data, frequently serve as a basis for new 
experimental approaches to problems more directly related to the oral 
tissues , 

Proposed Course of Project ; 

As indicated above, collaborative work will continue on numerous prob- 
lems. Training activities will also be continued. 



Part B: 



Publications; 



von Brand, T., Nylen, M, U. , Martin, G. N,, and Churchwell, F. K.: 
Composition and crystallization patterns of calcareous corpuscles 
of cestodes grown in different classes of hosts. J. Parasitol. 
53:683-687, August 1967. 



205 



Serial No. NIDR-40 (67) 

1. Histology and Pathology 

3, Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Studies on Bone Resorption and Collagen Degradation 
due to Pressure 

Previous Serial Number: None 

Principal Investigator: Dr. Jens Waerhaug 

Other Investigators: Dr. M. U. Nylen 

Cooperating Units: Dr. W. Titus, Laboratory Aids Branch, Division 

of Research Services 

Man Years: 

Total: 1 
Professional: 3/4 
Other: 1/4 

Project Description: 

Objectives : 

To study the changes which occur in the periodontal membrane of teeth 
exposed to extreme stress. 

Destruction of the bone that supports the teeth is one of the most 
spectacular features of periodontal disease. However, until about a 
decade ago the mechanism by which bone was resorbed was highly specu- 
lative. The application of electron microscopy to the study of osteo- 
clastic resorption has contributed substantially to our general under- 
standing of this process , but it still remains to be shown to what 
extent the same series of reactions take place in the periodontal mem- 
brand under varying degrees of stress. The purpose of the present 
experiments is to create conditions in the periodontal membrane of 
experimental animals that correspond to the concept of extreme trauma 
from occlusion in man. 

Methods Employed : 

Dogs, cats, and rats were used as experimental animals. In the larger 
animals trauma to the periodontal membrane was created by cementing a 
high crown onto the lower first molar on one side, allowing the anta- 
gonists to occlude with a shelf on the crown. In the rats all the 



206 



Serial No. NIDR-40 (67) 

upper molars on one side and one or two molars on the other side were 
extracted on the assumption that the remaining molars which still had 
antagonists would be subjected to excessive stress. 

The experimental period varied from 2 weeks in the dogs and cats to 
5 days in the rats. On the day of sacrifice the animals were anaesthe- 
tized, their thoracic cavity opened, and a canula was inserted through 
the left ventricle into the ascending aorta. In the larger animals the 
subclavian arteries and the descending aorta were ligated immediately 
prior to insertion of the canula. In the rats the descending aorta was 
clamped with a hemostat. In all animals the right atrium was opened as 
soon as the canula was inserted to allow free drainage of the fluid. 

The perfusate consisted in most instances of phosphate buffered glutar- 
aldehyde in amounts which depended on the size of the animal. One 
animal, however, was perfused with phosphate buffered osmic acid only 
and another one was perfused first with glutaraldehyde and subsequently 
with osmic acid . 

Immediately after perfusion the stressed teeth were excised and cut into 
smaller pieces either using a rotating saw blade or a water cooled, ro- 
tating rubber banded carbide wheel. In all instances final fixation 
was effected by immersion of the small pieces in phosphate buffered 
osmic acid over night. 

Dehydration and embedding in epon followed standard procedures for hard 
tissues. Thick sections were cut with a diamond knife, stained with 
toluidine blue and examined under the optical microscope. Thin sections 
were likewise cut with a diamond knife from selected areas, picked up on 
carbon covered substrates, stained with lead citrate and examined in a 
Siemens Elmiskop lA 6B electron microscope. 

Major Findings : 

Thick sections revealed the presence of numerous osteoclasts and dis- 
appearance of collagen structure. Unfortunately the results at the 
ultras tructural level have been disappointing in that most of the 
samples failed to exhibit satisfactory fine structural details. It 
seems obvious that perfusion alone rarely is sufficient to achieve a 
satisfactory degree of fixation. To what extent the damage is a result 
of the treatment which followed perfusion and/or failure of the fixative 
to reach the pertinent areas during the subsequent immersion phase are 
being investigated. Since it has been shown that glutaraldehyde fixed 
tissues remain osmotically active, the cutting-up of the specimens may 
well be the critical phase. 



207 



Serial No. NIDR-40 (67) 

Significance to Dental Research ; 

A further clarification of the mode of action of bone resorbing cells 
has considerable didactic importance. Bone resorption has been and 
still is, a poorly understood biological process. In addition very 
little is known about the fine structural changes which occur in the 
periodontal membrane under pathologic conditions. The development of 
useful preparatory techniques which will allow such studies to be carried 
out in a reproducible and meaningful manner is a prerequisite for any 
advances in this area. 

Proposed Course of Project : 

The investigator's tenure of appointment ceases in August 1968. The 
experimental work will be continued to that date, while examination of 
the material and analysis of the results will go on past that date. 

Part B: not included 



206 



Serial No, NIDR-41 (64) 

1. Histology and Pathology 

2. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: X-ray Diffraction Studies on the Effect of Fluoride 
on Bones , Teeth and Synthetic Compounds 

Previous Serial Number: NIDR-19 

Principal Investigator: Dr. Paul D. Frazier 

Other Investigators: None 

Cooperating Units: Dr. J. E. Seegmiller, NIAMD , NIH; Dr. P. Carbone , 

NCI, NIH; Dr. H. Schraer , Penn State University, 
University Park, Penna. 

Man Years: 

Total: 1 
Professional: 1 
Other : 

Project Description: 

Objectives : 

These studies are being conducted to determine more completely the 
effect of fluoride on crystal chemistry of bones and teeth. The 
influence of fluoride on nucleation, size, shape, strain and lattice 
constants of apatite crystal systems are also being investigated. 

Methods Employed : 

Wide-angle X-ray diffraction techniques are employed using film and 
electronic detectors. Electron microscopy and contact microradiography 
were also used. 

Major Findings ; 

The wide variations in crystallite size of enamel reported in the 
literature suggested that the breadth of the diffraction profiles 
used to make the calculations was affected by preparation techniques. 
A study was therefore conducted to determine the influence of prepara- 
tion methods on profile breadth. Broadening was observed in enamel 

203 



serial No. NIDR-41 (64) 



ground with high and low speed dental instruments when compared to 
counter-part enamel ground under controlled conditions in a ball mill. 
Prolonged ball grinding also caused severe line broadening. A single 
crystal of mineral hydroxyapatite was not damaged when it was prepared 
by grinding with low speed dental instruments. The ease with which 
enamel is damaged may be related to its composite structure as opposed 
to the mineral apatite. Electron microscopy studies of the damaged 
enamel indicated that 1) there is a significant number of broken crys- 
tallites , 2) the number increases as does line broadening with increased 
grinding and 3) the size of unbroken clumps of crystals was far smaller 
than individual particles of mineral hydroxyapatite prepared under iden- 
tical conditions. 

X-ray diffraction studies of human enamel containing varying amounts of 
fluoride indicate that there is a measurable decrease in line breadth 
of the 002, 211, and 300 sets of planes as fluoride content increases. 
The correlation coefficients for 002, 211, and 300 were -0.9, -0.7 and 
-0.7 respectively. The highest degree of association between fluoride 
and change in line breadth was for the 002 peak; earlier investigations 
in this laborator^y have indicated no such relationship for bone. 

Electron probe analysis of incipient carious lesions has shown that the 
probe is a useful instrument for detecting Ca to P ratios on a micro- 
scopic level, Demineralized areas in the lesion have been observed 
with higher than normal Ca/P ratios; there are also hypermineralized 
areas with lower than normal Ca/P ratios. 

Three methods of measuring changes in bone crystallinity have been 
developed which will be more universally applicable than the template 
method originally developed in this laboratory. The primary advantages 
of these techniques are all based on direct measurements or data taken 
directly from the instrument. This eliminates assumptions needed for 
constructing the original template and the data can be fed directly 
into a computer if desired. 

Significance to Dental Research ; 

Enamel crystallite size values obtained from severely damaged enamel 
were 1/4 that of the a-axis dimension and up to 1/6 that obtained for 
the c-axis of non-damaged counter-part enamel. 

These findings support the argument that the wide variation in crystal- 
lite size values in the literature could be related to variations in 
sample preparation. Many investigations are using these same grinding 
and collecting techniques for other physical studies. However, investi- 
gations have not yet been made which might associate the "damaged" 
fraction of enamel observed by X-ray diffraction and electron microscopy 
with variation in 1) chemical composition, 2) density, 3) and/or other 
physical properties. 



210 



Serial No. NIDR-41 (64) 

The finding that increased fluoride incorporated into bone mineral 
improves its crystallinity has been extended to human enamel. The 
complete significance of these findings cannot be determined until 
experimental evidence is obtained separating the influence of strain 
and size on line broadening. 

The results of the electron probe study of carious lesions indicate 
that future studies in conjunction with electron microscopy will give 
information concerning the chemical as well as morphological changes 
within the lesion. 

The new methods developed for studying bone will facilitate rapid 
accurate studies of the age effect on crystallinity in high and low 
fluoride areas. In addition the technique can be applied to animal 
studies where age and fluoride feeding are controlled. 

Proposed Course of Project ; 

Principal investigator has been attending Graduate School at the 
University of Washington, Seattle, Washington, since September 1967. 
The project is discontinued for the duration of training period except 
for writing up some of the data. 



Part B: 



Publications: 



1. Frazier, P. D, Adult human enamel. Ill An electron microscopic 
study of crystallite size and morphology. Accepted for publication 
in Ultrastructure Research. 

2. Frazier, P. D. and Wong, V. G. Cystinosis: Histologic and crystal- 
lographic examination of crystals in eye tissues. Accepted for 
publication in Archives of Opthalmology . 



21 



Serial No. NIDR-42 (66) 

1, Histology and Pathology 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Fine Structural Studies of the Main Sensory Nucleus 
of the Trigeminal Nerve 

Previous Serial Number: NIDR-21 

Principal Investigator: Dr , Stephen Gobel 

Other Investigators: Dr , Ronald Dubner , Section of Physiology, NIDR 

Cooperating Units: None 

Man Years: 

Total: 2 
Professional: 1 
Other : 1 

Project Description: 

Objectives : 

1, To determine the normal morphological characteristics of the 
neurons and glial cells comprising the main sensory nucleus, 

2. To determine the normal synaptic relationships between the neurons 
of the main sensory nucleus and the axons entering this nucleus. 

Methods Employed : 

A procedure for fixation of the brain stem of adult cats- by vascular 
perfusion has been developed. Before opening the thoracic cavity 
ligatures are placed around the subclavian arteries distal to the 
emergence of the vertebral arteries and around both common carotid 
arteries. Just prior to perfusion the above arteries are tied off. 
A canula is then inserted through the left ventricle into the ascending 
aorta and tied in place. The right atrium is opened to allow for out- 
flow of the perfusate and the descending aorta distal to the emergence 
of the left subclavian artery is clamped off. The perfusion of the 
brain stem takes place through the vertebral arteries. By restricting 
the flow of perfusate to the vertebral arteries it has been possible to 

212 



serial No. NIDR-42 (66) 



obtain consistently preparations of the trigeminal nuclear complex 
suitable for fine structural studies. The perfusate consists of phos- 
phate buffered glutaraldehyde followed by dichromate buffered OSO4 or 
dichromate buffered OsO^ alone. After fixation the pons is divided 
into right and left halves. Each half is cut anteroposterior ly into 
1 mm blocks and flat embedded in an epoxy resin. One micron sections 
are cut from these blocks in order to locate the main sensory nucleus. 
The nucleus is then divided into 4 quadrants, reembedded and subsequently 
prepared for electron microscopical study. 

In an auxiliary technique formalin or glutaraldehyde fixed material is 
impregnated with AgNOo by the rapid Golgi method. Thick sections (lOOu) 
are cut on a freezing microtome. Material treated in this way is used 
to provide information on the extent of branching and dispersion of the 
dendrites in the main sensory nucleus. 

Major Findings ; 

The neurons of the main sensory nucleus are the second neuron in a 
chain of three leading from the orofacial area to the cerebral cortex. 
Their cell bodies range from 10-30 u id diameter and are apposed by 
dendrites, myelinated and unmyelinated axons, astrocytic processes and 
cell bodies of neurons, astrocytes and oligodendrocytes. The density 
of axonal boutons synapsing on the surface of these cell bodies is 
relatively low. A few spines protrude from the cell bodies and usually 
have synapses on them. 

The neurons of the main sensory nucleus contain 3 or more main dendrites. 
After a short distance they branch secondarily. Some of these secondary 
branches extend more than half the width of the nucleus. On some proxi- 
mal branches the density of axonal boutons can be quite dense; four or 
five adjacent boutons may synapse on them. Occasional short spines 
occur along the dendrites and protrude into axons with which they synapse , 

Several distinct morphological types of axons have been identified. 
One type, having large synaptic segments which contain large diameter 
synaptic vesicles, forms numerous axodendritic and axo-axonic synapses. 
The synaptic segments of this axon may have 2 or more axo-axonic synapses 
on them as well as sending out projections which form synapses by invagi- 
nating into other axonal types. Two types of axons which consist of 
smaller synaptic segments and contain smaller diameter synaptic vesicles 
enter into axo-axonic synapses with each other and with large diameter 
axons previously described. Axo-axonic synapsis is often made by inter - 
digitation of small spine-like projections. The involvement of three 
axons in axo-axonic synapses by means of projecting into one another 
has not been observed previously in the central nervous system. This 
series of axo-axonic synapses probably represents a part of the morpho- 
logic basis for the neurophysio logic findings of presynaptic inhibition 
which characterize this region of the central nervous system. 



213 



Serial No. NIDR-42 (66) 



Significance to Dental Research 

The trigeminal nerve conveys to the central nervous system information 
pertaining to tactile discrimination, pain and temperature sensation 
from the teeth, periodontal ligament, oral cavity and anterior two 
thirds of the face as veil as proprioceptive information from the perio- 
dontal ligament and muscles of mastication. In the main sensory nucleus 
as well as other parts of the trigeminal nuclear complex such information 
is passed on to a set of neurons which serve as relays to the cerebral 
cortex via the thalamus. However, before this information leaves the 
trigeminal nuclear complex it is modified and integrated with information 
coming from diverse parts of the central nervous system, i.e., cerebral 
cortex, spinal cord, reticular formation and different parts of the tri- 
geminal system. It is through such integration of information that 
complex movements such as mastication, deglutition, turning of the head 
and those involved in speech can be effected. An appreciation of the 
fine structural basis for this integration is essential for comprehend- 
ing orofacial function. 



Proposed Course of Project ; 

A. Separate lesions will be made in the trigeminal nerve, spinal 
nucleus of V and the cerebral cortex. The interrupted axons will 
be allowed to degenerate. By identifying electron microscopically 
the degenerated axonal boutons it will be possible to conclusively 
demonstrate the manner in which axons from the above sources synapse 
with cells of the main sensory nucleus and whether they form axo- 
axonic synapses with each other. It is planned to establish whether 
there is synaptic interaction between axons from the above-mentioned 
sources . 

B. Long range plans involve extending fine structural studies to the 
spinal nucleus of the V, motor nucleus of the V, and the mesen- 
cephalic nucleus as well as the mode of origin of the trigeminal 
nerve in relation to odontoblasts. 



Part B: 



Publications ; 



1. Gobel, S.: Electron microscopical studies of the cerebrellar 
molecular layer. J. Ultras true ture Research, 21:430, 1968. 



21if 



Serial No, NIDR-43 (62) 

1. Histology and Pathology' 

3, Bethesda, Maryland 



PHS-NIH 

Individual Project Report 

July 1, 1967 through June 30, 1968 



Part A 



Project Title: Bacterial, Viral and Macromolecular Structure-function 
Relationships 

Previous Serial Number: NIDR-22 

Principal Investigator: Dr. H. A. Bladen, Jr , 

Other Investigators: Dr. G. Hageage , Dr. K. A. Piez 

Cooperating Units: None 

Man Years: 

Total: 2 3/4 
Professional: 1 
Other: 1 3/4 

Project Description: 

Objectives : 

As in the past , studies have been primarily an attempt to determine 
possible relationships between form and function of biological materials. 
Recent interests in complex biological problems have dictated the neces- 
sity for utilizing techniques in disciplines other than electron micros- 
copy. These include biochemistry, immunology and microbiology. The 
problems presently under investigation in this laboratory are: 

1. To determine the chemical as well as morphological site of 
action of serum complement on the bacteria cell and its endotoxic LPS. 

2. To determine the morphological relationship of specific col- 
lagen peptides to the collagen molecule itself. 

Methods Employed ; 

1. Endotoxic lipopolysaccharide (LPS) of V. alcalescens was 
isolated by phenol-water extraction of whole cells. Final lyophilization 
of the water phase resulted in LPS which had a characteristic structure 
when negatively stained. 



9 



15 



serial No. NIDR-43 (62) 

The llpoidal moiety of the LPS, designated as Lipid A, was pre- 
pared by refluxing LPS with 0.1 N HCl for 1 hour. The milky reaction 
mixture was then extracted several times with chloroform, washed with 
water, and finally extracted with hot acetone. This was dried to give 
a brown, waxy material which was lipid A. 

Utilization of serum complement by V. alcalescens LPS was demon- 
strated by initially reacting known amounts of LPS with complement, 
then determining residual complement by the usual hemolytic assay 
procedure . 

Complement utilization by Lipid A was difficult to quantitate due 
to the insolubility of lipid in non-oirganic solvents. The best results 
were obtained by sonication of the lipid fraction followed by filtration 
through an 8 u milipore filter. The relatively uniform suspension of 
Lipid A which resulted was then reacted with a known amount of serum 
complement and, after an incubation period, residual complement was 
determined by hemolytic assay. 

2. Purified collagen peptides, the amino acid composition of which 
was known were supplied by the Laboratory of Biochemistry, NIDR. Seg- 
mented Long Spacings (SLS) were prepared from these specimens by dialy- 
zing against 0,05M acetic acid and then reacting with ATP, The reaction 
mixtures became turbid after intervals of time ranging from 3 to 30 
minutes. Occasionally SLS were formed by dialyzing the specimens in 
acetic acid against ATP, After formation of SLS, preparations were 
positively or negatively stained with phosphotungstic acid and examined 
in an AEI 6B electron microscope. 

Major Findings ; 

1, Previous studies in this laboratory have demonstrated that 
guina pig serum produces lesions approximately 90 A in diameter on the 
cell surface of E. coli and V. alcalescens , These lesions are identical 
in morphology to lesions present on erythrocyte membranes after action 
of complement. 

Similarly, the endotoxic LPS of these organisms reveal lesions 
after reaction with serum complement. Continued experimentation demon- 
strated these lesions to be dependent on the presence of serum complement, 

In the present phase of the study, LPS was fractionated into the 
lipoidal and carbohydrate fractions. Mild hydrolysis of LPS yielded a 
carbohydrate moiety as well as a lipid, termed lipid A. Initial studies 
which were concerned with the carbohydrate moiety demonstrated that 
30-407o of the available complement was taken up by the carbohydrate. 
However, chemical analysis showed that the carbohydrate obtained still 
had considerable lipid present. Strenuous hydrolysis of LPS eventually 
resulted in a carbohydrate fraction in which lipid could not be detected 

2 216 



serial No. NIDR-43 (62) 



by methods used. This carbohydrate showed negligible complement uptake. 
It should be noted, however, that such harsh treatment of the carbohy- 
drate most certainly hydrolyzed it, so that it bore little resemblance 
to the carbohydrate moiety of the parent LPS . 

Initial experiments with the Lipid A fraction were rather erratic 
since the Lipid was not soluble in the buffer system used. Sonication 
resulted in a particulate suspension which was difficult to test quanti- 
tatively for complement uptake Some lipid suspensions resulted in 
practically total uptake of complement while others gave only about 
20-307o uptake. For instance, when relative volumes of Lipid A were 
increased, no increase over 20% utilization was observed, suggesting 
that the amount of lipid present was very small, Centrifugation of 
sonicated lipid suspensions also yielded preparations which did not 
utilize complement quantitatively. This was probably due to the disper- 
sal of existing clumps of lipid. However, sonication followed by filtra- 
tion through an 8 u milipore filter resulted in rather reasonably straight 
line relationships. In one experiment, it was determined that 60 ug , 
Lipid A utilized approximately 507o of the available complement. This 
figure is quantitatively similar to the utilization by 100 ug LPS, 60% 
of which is lipid. 

In another experiment, 100 ug Lipid A was placed on a TLC plate, 
separated into several spots and eluted as a whole, before being tested 
for uptake of complement. Approximately 40-457o uptake was determined. 
These results suggest that all of the uptake of complement by LPS may 
be due to the lipid fraction. 

2. At the present phase of this study, three different peptides, 
isolated from the ccl polypeptide chain of collagen, have been converted 
into Segmented Long Spacings (SLS) by the addition of ATP. The structural 
integrity, i.e. the arrangement of bands at various intervals, along 
peptides CB6 and CBS is sufficiently precise as to allow localization 
of these peptides in SLS prepared from the intact cul polypeptide. 

Peptide CBS is composed of at least 9 resolvable bands which are 
identical in width, intensity and separation to 9 bands present in the 
parent al polypeptide SLS. The CBS peptide begins approximately 3S0A 
from the A end of the parent al SLS and, with a total length of 870A, 
comprises 27% of it. 

Peptide GB6 has at least six identifiable bands which correspond 
to six bands with a location near the B end. It is approximately 700 
A in length and occupies 227o of the total length of the molecule. 

The third peptide investigated is termed Peptide CB7, This 
peptide, at present, has been only poorly resolved. However, some 
information has been obtained indicating that it is approximately 
5S0 A long, has at least 5 separable less dense bands, and makes up 



in 



Serial No. NIDR-43 (62) 



nearly 18% of the total SLS molecule. Placement of this peptide along 
the parent SLS molecule is somewhat questionable. However, initial 
observations suggest that Peptide CB7 is probably adjacent to Peptide 
CB6. 

Since the three peptides, GB6 , CB7 and DBS comprise approximately 
677o of the parent SLS collagen molecule, the other 5 known peptides 
present in collagen should be located within the remaining 337o of the 
molecule . 



Significance to Dental Research 

The knowledge that complement utilization is mediated by a par- 
ticular substrate may eventually lead to a fuller comprehension of 
serum-cell interactions. This may include certain host reactions to 
endotoxic LPS which are initiated via the complement system. Continua- 
tion of these studies may lead to a further understanding of fundamental 
problems relating to oral microbiota and various pathological conditions. 

Collagen is an important component of all connective tissues. 
Knowledge concerning its structural morphology in relation to its 
chemical composition would contribute considerably to an understanding 
of many normal and pathological processes. 

Proposed Course of Projects : 

1. Future work in this area will be basically a continuation of 
the present study. This will include an attempt to separate the various 
lipids present in the LPS and to determine which, if any, is responsible 
for the reaction between serum complement and endotoxic LPS. Other 
endotoxins and membranes will be examined in a similar manner. 

2. Work is continuing in collaboration with the Laboratory of 
Biochemistry, NIDR, in an effort to morphologically map the collagen 
molecule with respect to known peptide sequences . Future studies may 
involve investigation of pathological collagen conditions with respect 
to possible variations in collagen structure at the peptide level. 



Part B 



Publications: 



Mergenhagen, S. E., Gewurz , H., Bladen, H. A., Nowotny, A., 
Kasai, N. and Luderitz, 0. Interactions of the complement system 
with endotoxins from A Salmonella Minnesota mutant deficient in 
0-polysaccharide and heptore. J. Immunol. 100:1, 1968. 



218 



1 



Serial No. NIDR-44 (66) 

1. Histology and Pathology 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: The Relationship Between Function and Structure in 
Microorganisms 

Previous Serial Number: NIDR-23 

Principal Investigator: Dr. G. J. Hageage 

Other Investigators: Dr. H. A. Bladen, Dr. E. D. Eanes 

Cooperating Units: Dr. R, Gherna (American Type Culture Collection); 

Dr. R, Steere (USDA) 

Man Years : 

Total: 2 
Professional: 1 
Other: 1 

Project Description: 

Objectives : 

The studies involving structure -function relationships under investi- 
gation in this laboratory have been concentrated in the -area of mem- 
brane systems. Generally, the problems involve the morphological and 
chemical structure, the nature of the enzymes, and the functions asso- 
ciated with the total membrane fraction of gram negative bacteria. 
Specific objectives of the present study are three fold: 

(1) To determine the site of sulfide oxidation in gram-negative 
bacteria 

(2) To determine what role, if any, the cytoplasmic membrane plays 
in the oxidation of sulfides, and 

(3) To determine the nature of the sulfur deposits derived from the 
oxidation of sulfides. 



219 



Serial No, NIDR-44 (66) 



Me thods Employed ; 

Chroma tium and Thiospirlllum species were grown in the laboratory of 
Dr. R.'cherna In screw top bottles containing a synthetic mineral 
medium, Vitamin B^-, and sodium sulfide. The bottles were illuminated 
with continuous light from a 25 Watt bulb. 

Cells to be examined by electron microscopy were either negatively 
stained with phosphotungstic acid or were fixed, dehydrated, and 
embedded in Vestopal W according to the procedure of Kellenberger . 
Thin sections were cut and stained with uranyl acetate and lead citrate. 

Autoradiography for electron microscopy were performed on cells grown 
in the presence of NaoS . Grids containing thin sections were attached 
to glass slides, looped after the procedure of Caro with Ilford L-4 
emulsion and stored in the dark for 2 weeks. For development the grids 
were immersed in Microdol-X for four minutes at 20°; washed in distilled 
water, fixed in an acid fixer for four minutes and finally washed in two 
changes of distilled water. Lead citrate was used to remove the gelatin 
as well as stain the sections. 

Cells to be freeze-etched were glycerinated in 107o glycerin for 8 hours 
prior to freezing. Samples were placed on copper rivets and frozen in 
liquid freon 22, Following this treatment the specimens were fractured ( 
under liquid nitrogen in a vacuum evaporator with a pre-cooled scalpel. 
After etching at -100°C for one minute the specimens were shadowed with j 
carbon-platinum and backed with carbon. The organic matter was digested 
with chromic acid and the fragments of replica rinsed in distilled water 
prior to being picked up on formvar- carbon backed grids. 

The sulfur globules were isolated from Chromatium cells by osmotic j 
lysis of spheroplasts prepared by lysozyme-EDTA treatment. The sulfur 
globules were collected by differential centrifugation and washed sev- 
eral times with distilled water. X-ray diffraction patterns of freshly 
isolated, wet sulfur packed in a 0,5 mm capillary tubes were taken with 
a 57.3 mm diameter powder camera at exposure times of one hour. Indi- 
vidual samples were then dried by vacuum for 30 minutes without removing 
the capillaries from their brass mounting rods. Consequently it was 
possible to examine approximately the same cross-sectional area of the 
same sample in both the wet and dry state. 

Major Findings : 

Where Chromatium or Thiospirlllum cells devoid of sulfur inclusions 
were exposed to hydrogen sulfide and incubated in the presence of light 
they immediately began to accumulate round, refractile inclusion which 
increased in size until they occupied most of the cell volume. This 
event took 10-20 minutes. Electron autoradiography of cells exposed to 



( 



220 



serial No. NIDR-44 (66) 

35 
Na2S revealed developed silver grains on or in close proximity to 

these inclusions. When viewed through crossed Nicol prisms, the sulfur 

inclusions, whether in the cell or isolated in a pure, wet state, had a 

characteristic maltese cross appearance. Rotation of the mount did not 

change the orientation of the arms possibly suggesting a symmetrical 

radial arrangement of the birefringent units. Drying of isolated sulfur 

inclusions resulted in the formation of birefringent crystals. 

X-ray patterns of isolated wet sulfur inclusions gave two^broad and 
diffuse diffraction rings with maxima at 3.75 A and 4.84 A. This 
pattern closely resembles the diffraction pattern of liquid sulfur. 
When dried, the same sample gave diffraction lines that agree with 
those of Orthorhombic sulfur as given in the ASTM powder diffraction 
file. In addition five diffraction lines (d= 5.98, 5.35, 4.55, 3.70, 
2.96) were observed which could not be indexed as orthorhombic, rhom- 
bohedral or monoclinic sulfur. 

Thin sections revealed that the cell envelope of Chromatium , like that 
of other gram-negative organisms, consisted of a highly convoluted outer 
membrane-like structure overlying a thin electron dense layer. The 
cytoplasm contained a membrane system which appeared as connected vesi- 
cles and bulged tubes. Work performed in other laboratories has shown 
that these structures (chromatophores) originate as tubular or vesicular 
invaginations of the cytoplasmic membrane. In freshly fed cells the 
greater part of the cytoplasm was occupied by sulfur inclusions. These 
inclusions, encompassed by an electron-dense border, had a density 
similar to that of the cytoplasm. In sections heavily stained with lead, 
cords of slightly denser material appeared to radiate from the center 
of the inclusions forming a stellate pattern. 

Freeze-etched preparations revealed that the sulfur inclusions were 
enclosed not only by the border seen in thin sections but also by the 
same membrane which invaginates to form the chromatophores . This indi- 
cated that (1) the inclusions are not intracellular per se but are 
actually outside the cytoplasm, and (2) the enzyme(s) for the oxidation 
of sulfides reside on the cytoplasmic membrane. The latter conclusion 
is supported biochemically by the formation and accumulation of sulfur 
globules around Chromatium spheroplast and isolated membrane fragments 
after exposure of these entities to hydrogen sulfide . 

Significance to Dental Research ; 

Studies concerning the relationship between structure and function, 
especially as concerned with the biologically universal "cell membrane" 
may lead to a further understanding of the fundamental problems relating 
to oral microbiota and various pathologic conditions. 



221 



Serial No, NIDR-44 (66) 
Proposed Course of Project ; 

Studies concerned with the role of the cytoplasmic membrane in the 
oxidation of hydrogen sulfide and the nature of the sulfur formed will 
be continued. 

Part B 

Publications ; 

1. Hageage, G. J. Observations on the fine structure and cell surface 
of flexibacter species. Bacteriological Proceedings 1967. P. 25, 

2. Doetsch, R, N. and Hageage, G, J. Motility in procaryotic organisms; 
problems, points of view, and perspectives. Biological Reviews, 

In press . 



9 



11 



Serial No. NIDR-45 (67) 

1. Histology and Pathology 

3. Bethesda, Maryland 



PHS-NIH 

Individual Project Report 

July 1, 1967 through June 30, 1968 



Part A 



Project Title: Electron Microscopic Radioautography: Response 

Characteristics of Nuclear Emulsion to High Energy 
Beta Particle Irradiation 

Previous Serial Number: None 

Principal Investigator: Dr. Robert C. Thompson 

Other Investigators: None 

Cooperating Units: IBM 360/50 Computing Facility, DCRT, NIH 

Man Years: 

Total: 3/4 
Professional: 1/2 
Other: 1/4 

Project Description: 

Objectives : 

Numerous investigators have stressed the importance of beta particle 
energy and range in relation to the resolution and sensitivity character- 
istics of electron microscopic radioautography. In particular it has 
been pointed out that the predicted density distribution of developed 
emulsion grains about a point source of radioactivity should follow a 
Cos-^ 9 curve for very low energy particles, and a Cos function for 
high energy isotopes. Extensive data exists for tritium confirming 
the predicted Cos 9 distribution, but limited data available from 
high energy studies using phosphorous -3 2 show a much broader distri- 
bution than that predicted. 

It is probable that characteristics of the emulsion itself are 
responsible for the observed discrepancies, and this project concerns 
the experimental measurement of the response of nuclear emulsion mono- 
layers to high energy beta particle irradiation. 



99 



Serial No. NIDR-45 (67) 

Methods Employed : 

Ilford L.4 nuclear emulsion was dissolved in water at 50 C and applied 
to carbonized Formvar coated grids using a standard looping technique 
described in the literature. Representative randomly chosen grids 
were observed with the electron microscope to confirm a monolayer grain 
distribution. 

A point deposit of phosphorus -32 on the surface of a thin polypropylene 
film was mounted at the center of a large metal bell jar and emulsion 
coated grids were positioned a short distance away oriented at various 
angles to the direct path line from the source. After evacuation of 
the chamber the source was uncovered for the desired irradiation time 
period using a remote control operated motor to appropriately position 
a lead shield. Control grids in the chamber protected from direct rays 
were used to assay scattered background radiation, and other grids com- 
pletely protected from beta radiation by a heavy clear glass container 
served to control emulsion background and stray light exposure. 

Following irradiation the grids were developed, fixed, and dried using 
a standardized procedure, and were then observed with the electron 
microscope. Grain counting was performed over randomly chosen fields 
of each grid and the results analyzed using standard statistical tech- 
niques , 

Certain mathematical computations of this study were of necessity 
executed on the digital computer. 

Ma j or F ind ings ; 

Very high energy beta particles passing through thin films are known 
to suffer negligible fractional energy loss and little or no scattering 
in direction; under such conditions the number of exposed grains pro- 
duced per unit of particle path length is a constant, and this is an 
underlying assumption of the Cos 9 distribution derivation. Such a 
particle striking a uniform emulsion layer at an angle of 9 from the 
normal is expected to yield secant 9 times the number of exposed grains 
produced by the particle incident normally. 

However, the results of our experimental studies using phosphorus -32 
clearly showed a stronger dependency on irradiation angle than secant 9 
for certain angles; sensitivity values at 35° incidence and 70° inci- 
dence were respectively 1.9 and 1.3 times higher than predicted based 
on the above theories (P=0.02). It was concluded that scattering 
events are probably responsible for the increased sensitivity and that 
these effects feegin to become important at emulsion path lengths as 
short as 2000 A for particles from phosphous -32. 



22^ 



Serial No. NIDR-45 (67) 

2 
A new predicted curve was constructed by plotting the product of Cos 9 

and the corresponding sensitivity ratio for that 9 value against tangent 
9. The resulting curve was found to match the broad shape of the pub- 
lished experimental data referred to earlier in this report. 

It was concluded that modification of the theoretical distribution 
curve to include the influence of angle of incidence on emulsion 
sensitivity as measured in these studies will largely eliminate the 
discrepancies between theory and certain experiments noted previously 
in the literature . 

A paper concerning these findings is in preparation. 

Significance to Dental Research ; 

Extension of the system of electron microscopic radioautography to 
include bone seeking isotopes of high energy is currently of great 
interest and knowledge of the practical resolution and sensitivity 
characteristics of the system under these conditions is required for 
valid data interpretation. 

Proposed Course of Project : 

This investigator's tenure of appointment ceases on June 30, 1968. 
This work will be continued at another institution. 

Part B: not included 



225 



Serial No. NIDR~46 (66) 
1, Histology & Pathology 
3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Uranium Tracer System Employing Nuclear Fission 
Reaction 

Previous Serial No: NIDR-24 

Principal Investigator: Dr. Robert C. Thompson 

Other Investigators: None 

Cooperating Units: Nuclear Reactor Facility of the Armed Forces 

Radiobiology Research Institute, Bethesda, Md . 

Man Years: 

Total: 1/2 
Professional: 1/2 
Other : 

Project Description 

Objectives : 

A previous report of this project described the basic characteristics 
of tracks in various thin films resulting from fission of uranium atoms 
deposited on the films. 

The purpose of current studies of this high resolution tracer system 
is threefold: a) to refine and standardize experimental methods of 
producing registration films; b) to investigate uranyl ion-calcium ion 
exchange sites on the surface of synthetic hydroxy apatite crystals, 
and c) to investigate uranium deposition in vivo in long bones of rats 
given injections of uranium. 

Methods Employed : 

Electron microscope grids covered with collodion or Formvar films were 
coated with thin evaporated films of chromium, silicon monoxide, or 
boron carbide in different experiments. Chromium films were produced 
by evaporation from a tungsten wire loop previously electroplated with 
a known weight of chromium. Careful evaporation of a known weight of 



22S 



Serial No. NIDR-46 (66) 



silicon monoxide from a tungsten wire basket was found to be suitable 
for producing films of this material. Boron carbide was evaporated 
from a cavity in a carbon rod heated by passing very high current 
through the rod, or from a special carbon arc constructed with a hollow 
center in one carbon rod packed with boron carbide. Structural analysis 
of the evaporated films was attempted using x-ray and electron diffrac- 
tion techniques . 

Synthetic hydroxy apatite was prepared by a precipitation reaction of 
calcium nitrate and ammonium phosphate carried out at pH 10 and in a 
CO2 free environment. The precipitate was allowed to mature 3 days at 
100°C and pH 10. After washing with distilled water the product was 
stored wet for several months prior to use. X-ray diffraction, electron 
microscopy, and infrared spectroscopy analyses were consistent with an 
hydroxy apatite of low carbon dioxide content and small crystal size. 

Exchange reactions were carried out for 15 minutes at room temperature 
in filtered aqueous solutions of uranyl acetate containing either natural 
uranium or enriched U-235, 

Using centrifugation, the exchanged hydroxy apatite was washed succes- 
sively in water, twice with acetone, and isoamyl acetate, and suspended 
in a final solution of 207o collodion in isoamyl acetate. Thin films of 
this material prepared by the casting on water technique were found to 
provide satisfactory dispersion and fixation of the crystals on the 
electron microscope grids. 

For the short term animal studies rats were given pharmacologic doses 
of uranyl or sodium acetate and were sacrificed 3 hours later. Both 
femurs and tibiae were cleaned and immediately freeze-dried. Small 
portions of the shaft of the bones were embedded directly and thin 
sections taken for electron microscopy. 

Grids containing the samples were irradiated in the AFFRI nuclear 
reactor facility and following a suitable cooling-off period were 
observed with the electron microscope. 

Major Findings ; 

Thin collodion membranes without additional evaporated supporting 
films are unstable and are destroyed in the nuclear reactor. Light 
carbon evaporated onto such films provides only minimal protection, 
but in areas of membrane that do happen to survive irradiation, tracks 
appear as extremely wide linear gaps in the membrane. Carbonised 
formvar membranes were found to be more durable. Silicon monoxide 
films are highly membrane protective, but also appear to obscure or 
reduce the damage to the membrane so that tracks are difficult to dis- 
tinguish from general background. Boron carbide or boron-carbon films 
if thick tend to "decorate" tracks with dark material located along 



4.^ 



:7 



serial No. NIDR-46 (66) 



the axis. Lighter films of the same material appear to produce a I 
stippling effect of separate black dots throughout the area of the 
track. Chromium films provide good membrane protection and also 
produce a narrower track of higher contrast. All types of films can 
completely obscure any tracks if too thick a layer is used. 

X-ray and electron diffraction studies of boron containing films reveal | 
a predominantly amorphous pattern; only when the material evaporated was 
highly crystaline could any consistent lines be obtained from the re- , 
suiting films. It is believed that films produced by evaporating pow- 
dered boron carbide or a boron- graphite mixture from a carbon rod are 
probably a mixture of amorphous boron carbide and carbon. 

Tracks associated with hydroxy apatite crystals from a uranium exchange 
reaction were seen only rarely, and none were seen in areas devoid of 
visible crystals. The limited number of tracks prohibited any conclu- 
sions about the distribution of the uranium. No change in crystal mor- 
phology was noted, and therefore one could elect to irradiate for a 
longer period to produce more tracks. Alternatively the use in a current 
experiment of enriched uranium-235 uranyl acetate is expected to yield 
130 times as many tracks for the same amount of material and duration 
of irradiation. 

Bone tissue density and background variation seemed to be responsible | 
for the failure to observe tracks in these sections; occasional holes 
in the membranes and section cannot clearly be called tracks. Distinct 
morphological differences were apparent between sections from experi- 
mental and control animals, but no visible areas of increased electron 
density suggestive of uranium deposition were noted. 

Significance to Dental Research 

The development of this tracer system using fissionable "bone seeking" 
elements may provide a useful new method of extremely high resolution 
for ultrastructural studies of the mineralization process and surface 
exchange reactions of mineral crystals. 

Proposed Course of Project ; 

The investigator's tenure of appointment ceases on June 30, 1968. 
This work will be continued at another institution. 

Part B: not included 



228 i 



Serial No. NIDR-47 (55) 

1. Histology and Pathology 

2. Histochemistry 

3. Bethesda, Maryland 



PHS -NTH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Studies of Human Collagenase 

Previous Serial Number: NIDR-25 

Principal Investigator: Dr. H. M. Fullmer 

Other Investigators: Dr. W. A. Gibson, Dr. J. F. Goggins , 

Dr . G . S . Lazarus 

Man Years: 

Total: 2 1/4 
Professional: 13/4 
Other: 1/2 

Project Description: 

Objectives : 

A. To produce collagenase by cultures of human gingivae in sufficient 
quantities to permit isolation and purification. The purified enzyme 
can be used for a number of studies including (a) if antigenic, for the 
production of antisera which can be conjugated with a fluorescent tag 
to be employed for the determination of the cell types producing the 
enzyme (b) for the characterization of the action of collagenase on the 
collagen molecule, (c) for characterization of optimal conditions of 
enzymatic activity as well as determination of its inhibitors and 
activators . 

B. To determine the amount of collagenase detectable from (a) certain 
body fluids, particularly spinal fluid, urine and blood, and (b) cultures 
of certain tissues, particularly skin, bone synovia, brain, spinal cord 
and muscle and the relationship of these amounts to certain connective 
tissue and neuromuscular diseases such as amyotrophic lateral sclerosis, 
scleroderma, rheumatoid arthritis , etc. 



229 



Serial No, NIDR-47 (55) 



Methods Employed ; 



1. Tissue culture: Gingival samples, excised in the course of treat- 
ment of various diseases, are provided by local dentists. After cleans- 
ing and mincing, the specimens are cultured in Tyrode's solution with 
antibiotics at 37°C in a CO2 incubator supplying 5°L CO2 in air. Culture 
fluid is collected and replenished every 2 days for three harvests. 
Cells and sediments are separated from culture fluids by centrifugation 
at 18,000 rpm, Collagenase is obtained by purification of the culture 
fluid. 

Various other tissues are obtained from surgery or at autopsy. They 
are likewise cleansed, minced into small pieces, and cultured. Culture 
fluids are assayed for collagenase. 

2. Separation methods: Culture fluids have been submitted to electro- 
phoresis on the Brinkman Model FF apparatus. Good separation has been 
achieved utilizing tris buffer pH 8 with 0.12 M on the electrode and 
0.04 M on the curtain. Column chromatography utilizing Sephadex G200 
has provided further separation. Use of acrylamide gels aids determi 
nation of the degree of purification achieved during separation pro- 
cedures . 

3. Collagenase assay methods: 

A. Gel inhibition: Collagen solutions brought to 37 C form a gel. 
Addition of collagenase to the collagen solution prevents gelation, and 
the degree of prevention of gelation is a function of the amount present 
per unit of time. The degree of prevention of gelation is a function 
of the amount present per unit of time , The degree of prevention of 
gelation is read in a Klett photometer as units of opacity. This method 
has frequently been found to be unreliable. 



B, Viscometry: Collagen solutions manifest a characteristic 
viscosity at 20 C . Addition of collagenase to collagen solutions 
results in a reduction of viscosity measured in time. 



14 
C. Release of radioactive degradation products: C-glycine has 

been administered to young growing rats that incorporate the isotope 
into collagen. The collagen is extracted, purified and used as a sub- 
strate for collagenase. The release of radioactive degradation products 
from purified collagen per unit of time is a measure of collagenase 
activity, A liquid scintillation counter is utilized. 

Major Findings ; 

In 1965 we demonstrated the existence of collagenase in man. Prior to 
this , collagenase had been detected only in cultures of certain micro- 
organisms , and of tadpole tails--particularly during the ppocess of 
metamorphosis. Our observations indicated that cultures of gingivae 
that had been excised for the treatment of periodontal diseases produce 
a collagenase. ^ O'kVi 



Serial No. NIDR-47 (55) 

Collagenase has now een detected in cultures of normal human skin, and 
increased amounts were detected in culture fluids of skins taken from 
individuals with certain neuromuscular diseases; namely, amyotrophic 
lateral sclerosis, progressive spinal muscular atrophy, myotonic dys- 
trophy, occulopharyngeal neuromuscular disease, Parkinsonism periodic 
paralysis, myasthenia gravis and polymyositis. These findings are in 
accord with our 1960 observations which reported the existence of a 
connective tissue disorder, demonstrable morphologically and histo- 
chemically, in the dermis of 60% of individuals with amyotrophic lateral 
sclerosis. Collagenase was also found in the culture fluids of skins 
taken from Individuals with scleroderma, dermatomyositis , and certain 
other connective tissue diseases provided the individual was not on 
prednisone or prednisilone therapy. Our data is consistent with the 
hypothesis that the collagenase detected is the one concerned with the 
normal metabolic turnover of collagen, and that something happens during 
certain diseases which permits detection of increased amounts of the 
enzyme in cultures of skin. 

Collagenase has been detected in culture fluids of articular and growth 
plate cartilates of man, goat and rabbit and in culture fluids of verte- 
brae, mandible, maxilla and pieces from long bones of man and goat. 

Data from acrylamide gels and electron microscopy indicates that the 
collagenase from gingivae, bone, and granules of leukocytes acts on the 
collagen molecule in a fashion identical to the collagenase derived from 
tadpoles; that is, the collagen molecule is cleaved only once resulting 
in a 3/4 piece and a 1/4 piece. Subsequently, other enzymes in culture 
fluids and in the granules of leukocytes act on the cleaved collagen 
molecules resulting in numerous digestion products. 

preliminary data indicates human collagenase has a broad range of 
activity from pH 7-9, and scant activity below pH 7. It is completely 
inhibited by EDTA, and partially by cysteine. It has an absolute re- 
quirement for calcium in low concentrations and is inhibited by high 
concentrations of calcium. It has the capacity to reduce the viscosity 
of 0.20% solutions of collagen as much as 35-557o in 20 hours at 20°C as 
compared to 2% for control solutions. 

Significance to Dental Research ; 

The consequence of periodontal diseases is the loss of periodontal 
fibers, principally collagen, and bone of which collagen is the major 
organic constituent. It is difficult to conceive of any oral disease 
in which collagen, the substrate of collagenase, is not involved. We 
have a system which is able to assay the catabolism of this very 
important protein. Utilization of this system will provide greater 
understanding of connective tissues in health and disease. 

231 



serial No, NIDR-47 (55) 



Proposed Course of Project : 

The major effort will be directed toward the production, purification 
and characterization of collagenase. Further attempts will also be 
made toward elucidation of the relationship of detectability of col- 
lagenase in cultures of skin, bones, synovial membranes, brains, spinal 
cords and muscles to neuromuscular and other diseases. 



Part B: 



Publications: 



Fullmer, H. M. The development of oxytalan fibers, "Mechanisms of 
tooth support" A Symposium Oxford 6-8 July, 1965, John Wright and 
Sons, 1967, pp 72-75. 

Fullmer, H, M, A decalcification technic for enzyme preservation, 
"Symposium on the dental pulp" J. Dent. Res. (In press). 

Fullmer, H. M. , Lazarus, G. S., Stam, A. C, Gibson, W. A. 

Collagenase in neuromuscular disease. "First National Symposium on | 

amyotrophic lateral sclerosis" 1967. (In press). ■ 

Fullmer, H. M. , Lazarus, G. S. Collagenase in human, goat and rat 
bone. Israel J. Med. Sci. 3:758-761, 1967. 



232 



serial No. NIDR-48 (64) 

1. Histology and Pathology 

2. Histochemistry 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Histochemical and Chemical Studies of Connective Tissues 

Previous Serial Number: NIDR-26 

Principal Investigator: Dr. W. A. Gibson 

Other Investigators: Dr. H. Fullmer, Dr. J. Goggins 

Cooperating Units: 

Man Years: 

Total: 2 3/4 
Professional: 2 1/4 
Other: 1/2 

Project Description: 

Objectives ; 

A. To assay various histochemically detectable substances in normal and 
diseased tissues through the use of existing techniques, by modification 
of existing techniques, or through the development of new techniques. 

B. To determine various metabolic parameters of normal and diseased 
tissues, and to use and develop techniques for the detection and assay 
of the factors involved. 

C. To develop defined in vitro systems of tissues and cells in continuous 
as well as primary cultures and to utilize such systems for histochemical 
and biochemical studies of normal and diseased tissues. 

Methods Employed : 

The cells and tissues utilized in these studies include those from oral 
and other regions obtained from human and animal sources. 



233 



Serial No, NIDR-48 (64) 



Histochemical : 



Since enzymes are the substances of interest in the current studies, 
tissue specimens are handled in a manner to minimize the loss of enzyme 
activity. This includes freezing and sectioning in a cryostat. Prior 
demineralization of mineralized tissues is accomplished by a process 
devised by Balogh, and Fullmer and Link. The resulting sections are 
subjected to various staining procedures to determine the site and 
quantity of particular enzymes . 

Modification of existing techniques and the development of new 
techniques involve qualitative and quantitative changes in substrates, 
cof actors , dyes, activators and inhibitors and conditions such as pH 
and temperature . 

B. Biochemical: 

Quantitative assays of several selected enzymes are being performed 
to evaluate the effects of tissue processing, especially the deminerali- 
zation procedure currently employed on the accuracy and reliability of 
the histochemical methods. 

Electrophoretic separations and identification of various enzymes 
and their molecular variants are being performed to supplement informa- 
tion from histochemical studies as well as to evaluate histochemical 
methods . 

C. Cell and Tissue Culture: 

Standard and original techniques are being utilized in current 
studies of various cells and tissues in primary culture. 

Quantitative studies of primary human and animal oral cells grown 
in vitro have been hindered by the lack of methods of growing large 
numbers of uniform cultures partially because of damaging disaggregation 
technics used. Classical technics such as trypsin disaggregation have 
proven useless because of cell injury. Technics utilizing other enzymes 
such as collagenase, elastase and hyaluronidase are being tested. 

Major Findings : 

A, The continued histochemical study of the periodontal ligament and 
alveolar bone has revealed widespread and impressive amounts of various 
enzymes. Non specific esterase activity was correlated with the cellular 
activity associated with active remodeling. Osteoclasts stained intensely 
in all sections while the staining of osteoblasts and cementoblasts ranged 
from little or none in quiescent areas to intense in areas of active bone 
and cementum formation respectively. Fibroblasts stained most intensely 
in areas of active remodeling. 



23h 



Serial No. NIDR-48 (64) 



In sections stained for cholinesterase activity, Schwann cells, 
red blood cells, megakaryocytes and muscle motor end plates stained 
intensely. True acetyl-cholinesterase activity was demonstrated in 
red blood cells, megakaryocytes and muscle motor end plates, but no 
nerve associated true acetylcholinesterase was detected in the rat 
periodontal ligament. 

In sections stained for a number of selected glycosidases , osteoclasts 
stained intensely for ^-glucuronidase , p-N-acetylglucosaminidase , p-gal- 
actosidase and p-blucosidase. Osteoblasts and cementob lasts in areas of 
bone and cementum formation as well as fibroblasts, superficial osteocytes 
and cementocytes in areas of active remodeling were similarly but less 
intensely stained for the same enzymes. Little or no staining occurred 
for any of the enzymes studied in cells located in more quiescent areas, 

B. Zymograms of the rat jaw esterases consistently demonstrated 6 major 
bands of activity. When 10-^ eserine sulfate was incorporated into the 
incubation media the total esterase activity was diminished only slightly. 
However, band number 4 was reduced to a trace. This confirms the histo- 
chemical finding that most of the esterase activity of the rat jaw is 
non-specific in nature. When 10"% E-600 was incorporated into the 
incubation media all but band number 2 and a portion of band number 3 
were eliminated. This confirms the histochemical finding that the bulk 
of the non-specific esterase activity is E-600 sensitive or B type 
esterase, while a small but definite amount of E-600 resistant or C type 
esterase is present in the rat jaw tissue. 

C. A technique devised by M. Neiders for growing human gingival epi- 
thelial cells in spinner culture is being adapted in this laboratory 
for routine plate cultures. The isolation of the epithelial cells is 
obtained by first separating the epithelium from the dermis by elastase 
treatment. The cells are then dispersed by treatment with solutions 
containing low concentrations of trypsin: the yields of uniform viable 
cells are greater than that obtained with any other method used to date. 

Significance to Dental Research ; 

The overall objectives of the project are designed to lead to a further 
understanding of the physiologic and pathologic processes occurring in 
oral tissues. 

Proposed Course of Project ; 

Histochemical, biochemical, and cell and tissue culture investigations 
of connective tissues will continue. The areas of specific interest 
will be the metabolic analysis of normal and diseased tissues as re- 
vealed by qualitative and quantitative enzyme histochemistry. The 
modification of techniques and the development of new techniques and 
tools of research will be no small part of the developing program of 
research. 



235 



serial No. NIDR-48 (64) 



Part B 



Publications; 



1, Gibson, W, A, and Fullmer, H. M.: Demonstration of 5 '-nucleotidase 
activity in decalcified bones and teeth. J. Histochem. Cytochem. 
14:934-935, 1967. 

2, Gibson, W. A. and Fullmer, H. M.: Histochemistry of the periodontal 
ligament: II. The phosphatases. Periodontics 5:226-232, 1967. 

3, Gibson, W. A. and Fullmer, H. M.: Histochemistry of the periodontal 
ligament: III. The esterases. Periodontics. 6:71-77, 1968. 



236 



Serial No. NIDR-49 (66) 

1. Histology and Pathology 

2. Histochemistry 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Histochemical and Chemical Studies of Connective Tissues 
and Teeth 

Previous Serial Number: NIDR-27 

Principal Investigator: Dr. J. F. Goggins 

Other Investigators: Dr. H. M. Fullmer, Dr. W. A. Gibson, Dr. G. S. Lazarus 

Cooperating Units: None 

Man Years: 

Total: 1 1/4 
Professional: 1 
Other: 1/4 

Project Description: 

Objectives : • 

A. To determine various metabolic parameters of normal and diseased 
tissues and to use and develop techniques for the detection and assay 
of the factors involved. 

B. To relate these factors to the tissue physiology of pathology by 
the development and application of qualitative and quantitative histo- 
chemical and biochemical methods , 

C. To determine the feasibility of preserving viability and enzyme 
activity in oral tissues after freezing and prolonged storage. 

Methods Employed : 

A. Histochemical: 

Since enzymes are the substances of current interest tissue 
specimens are handled in a manner to minimize the loss of enzyme 
activity. This includes freezing and sectioning in a cryostat. The 



237 



Serial No. NTDR-49 (66) 



resulting sections are subjected to various staining procedures to 
determine the site of particular enzymes. Enzyme activity is related 
to cell type, function, age, differentiation, etc. 

B. Biochemical: 

Hyaluronidase activity in human gingival tissues and rab it alveolar 
macrophages is being investigated. The ability of various tissue prepa- 
rations to degrade hyaluronic acid is assayed by determination of the 
release of N-acetylglucosamine reactive groups by the method of Reissig, 
Strominger and Leloir. The method has been adapted for micro determi- 
nations. The effects of pH, enzyme concentration and other factors on 
hyaluronidase activity are being studied. Products of hyaluronic acid 
released by hyaluronidase action are being isolated with the aid of ion 
exchange chromatography. 

C. Cryobiological: 

Existing methods of controlled freezing have been applied to oral 
tissues in conjunction with the use of protective agents. After storage 
for various periods of time the tissues are cultured in vitro to deter- 
mine viability. Some are analyzed histochemically . 

Major Findings : 

A. Hyaluronidase has been detected in isolated alveolar macrophages. 
The enzyme manifests a pH optimum between 3.9 and 4.2 as does gingival 
hyaluronidase. No activity was detected above pH 5. With increasing 
time, progressively smaller oligosaccharides were detected. Several 
bacterial hyaluronidases were inactive under conditions of the assay. 

B. Experiments indicate that human gingival tissue can be successfully 
frozen and stored with preservation of enzyme activity and viable cells 
for up to at least 14 months. Viability was demonstrated by cell out- 
growth. Histochemically, enzyme activity was demonstrated for 10 
oxidative and 3 hydrolytic enzymes after 14 months storage. The distri- 
bution was similar to that of fresh frozen tissue and enzyme activity 
was demonstrable within incubation periods usual for fresh-frozen tissue, 

Significance to Dental Research : 

The overall objectives of the histochemical and biochemical enzyme 
studies are to lead to a better understanding of the physiological 
processes occurring in oral tissues and to relate these to any changes 
found in pathological conditions . 



238 



Serxai iio . NIoR-49 (66) 



Proposed Course of Project : 



Histochemical and biochemical investigations of enzymes in connective 
tissues will continue. A particular field of study will be the corre- 
lation of data from utilization of the Lowry quantitative microchemical 
methods with that from histochemical methods to obtain reliable informa- 
tion about the metabolic status of small regions. Qualitative assays 
suggest wide variations of enzyme activity in various regions of perio- 
dontal membranes, for example, depending on the function of the cells 
at any particular time. 



Part B 



Publications; 



1. Goggins , J. F. and Fullmer, H. M. , Hydrolytic enzyme histochemistry 
of the rat molar pulp. Arch, oral Biol., 12:639-644, 1967. 

2. Goggins, J. F., Fullmer, H. M. and Steffek, A. J., Hyaluronidase 
activity of human gingiva. Arch. Path., 85:272-274, 1968. 

3. Goggins, J. F. and Gibson, W. A., Histochemistry of viable frozen- 
stored human gingiva. J. Invest. Derm., (In press). 



239 



Serial No, NIDR-50 (67) 

1. Histology and Pathology 

2. Histochemistry 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Collagenase Activity of Human Normal and Diseased 
Tissues 

Previous Serial Number: None 

Principal Investigator: Dr. Gerald S. Lazarus 

Other Investigators: Dr. H. M. Fullmer, Dr. John Daniels 

Dr. J. F. Goggins , Dr. H. A. Bladen 

Cooperating Units: Dr. John Decker, NIAMD; Dr. Robert Brown, NCI; 

Dr. Cap Oliver, GWU Hospital; Dr. Carter Multz, 
WRAMC; Dr. Werner Barth, NIAMD 



Man Years: 




Total: 

Professional: 
Other : 


2 1/4 

1 

1 1/4 



Project Description: 

A. To purify, characterize and elucidate the action of collagenase on 
the collagen molecule. 

B. To study the role of collagenase activity in synovia of patients 
with rheumatoid arthritis, and to attempt characterization of the patho- 
physiology of rheumatoid arthritis. 

C. To define the role of granulocyte collagenase in inflammation. 
Methods Employed : 

A. Biochemical 

Human granulocytes were extracted and the collagenase purified by 
ion exchange chromatography on DEAE , The enzyme was characterized by 
viscometry, acrylamide gel electrophoresis, polarimetry and radioactive 



2^0 



Serial No. NIDR-50 (67) 



reconstituted collagen fibril assays. In addition, inhibitors and 
activators of collagenase activity were studied using previously 
described technics. Similar technics were employed with rheumatoid 
collagenase. 

B. Morphological 

The morphology of segment long spacing collagen (SLS) which has been 
digested with collagenase has been studied by electron microscopy. 

Major Findings ; 

A specific collagenase was found in the granule fraction of human 
granulocytes. The enzyme cleaves the collagen molecule into two discrete 
products. These pieces, representing 3/4 and 1/4 of the molecule appear 
identical to those produced by tadpole and synovial collagenases . The 
enzyme is specifically inhibited by glutathione and cysteine. 

Increased collagenase activity was found in cultures of synovia of 
patients with rheumatoid activity. The amount of collagenase detected 
correlates with the clinical severity of the disease. This suggests that 
collagenase activity may play a significant role in the disease process. 

Significance to Dental Research : 

An understanding of collagenase metabolism gained from the granulocyte 
and synovial studies will increase our appreciation of patholophysiology 
of connective tissues everywhere. In addition, technics utilized are 
being applied to collagenase derived from other sources. 

Proposed Course of Project ; 

Collagenase studies will continue within this section and in Boston 
where I begin July 1, 1968. 



Part B 



Publications: 



1. Lazarus, G. S. and Mowry, F. M. Endocarditis localized to the 
tricuspid valve; report of a case. Michigan Medical Center 
Journal. 33; 219, 1967. 

2. McDonald, F, D,, Lazarus, G, S., and Campbell, W. L. Phenylbutazone 
anuria: Southern Medical Journal 60:1318, 1967. 

3. Lazarus, G. S,, Brown, R. B., Daniels, J. D., and Fullmer, H. M. 
Human granulocyte Collagenase, Science 159:1483, 1968. 



Serial No. NIDR-50 (67) 



4. Brown, R. B., Lazarus, G. S., and Fullmer, H, M, Collagenolotic 
activity of human granulocytes. Blood. 3^:856, 1967. 

5. Lazarus, G. S., Daniels, J. D,, Brown, R, B., Bladen, H. , and 
Fullmer, H. M. Mechanism of destruction of collagen by human 
white cells. Journal of Clinical Investigation. (In press). 



2^2 



Serial No, NIDR-51 (67) 

1. Histology and Pathology 

2. Crystal Chemistry 

3. Be the s da, Maryland 



PHS-NIH 

Individual Project Report 

July 1, 1967 through June 30, 1968 



Part A 



Project Title: X-ray Diffraction Studies on Fibrous Proteins 

Previous Serial Number: None 

Principal Investigator: Dr. E. D. Eanes 

Other Investigators: Dr. E. J. Miller 

Cooperating Units: Dr. G. G. Glenner, NIAMD-LEP 

Man Years: 

Total: 2 3/4 
Professional: 1 1/2 
Other 11/4 

Project Description: 

Objectives : 

The continuing objective of this project is to study the x-ray diffraction 
properties of fibrous proteins and to relate these properties to struc- 
tural parameters of interest to hard tissue biology. During the year 
covered by this report, two studies were undertaken with the following 
more specific objectives in mind: 

1. To compare the crystallographic properties of bone and soft tissue 
collagens under a variety of experimental situations with specific intent 
of establishing the relationship between structural stability in these 
collagens and the degree of covalent cross-linking, 

2, To determine the crystallographic features underlying the structure 
of the fibrous protein component of amyloid tissue. 

Methods Employed : 

The principal technique employed in both studies was wide-angle x-ray 
diffraction. Collagenous material suitable for diffraction analysis 
was obtained from demineralized tibia of 3-week-old chicks and from 

2^3 



Serial No. NIDR-51 (67) 



the tail tendon of rats . Suitably cut segments were mounted in glass 
capillaries and x-ray diffraction diagrams taken with a Ches ley-Philips 
microcamera. The collagen segments were examined either dried or in 
contact with one of the following test solutions: distilled H2O, 0.5M 
acetic acid, 5M guanidine hydrochloride. 

Amyloid tissue was obtained from the organs of human patients and experi- 
mental animals afflicted with amyloidosis. Both fresh tissue and purified 
fibrous extracts were studied. The material was examined by essentially 
the same diffraction procedures as employed in the collagen study as well 
as with standard powder dif fractometry techniques. 

Major Findings ; 

1. Collagen study : The diffraction patterns from dried tendon and dried 
bone collagen were alike in all fundamental respects except the arcing 
of the diffracting lines from the bone collagen was more extensive. This 
finding demonstrates that the monomeric units of these two collagens have 
the same basic crystallographic properties but that the alignment of the 
fibers at a tissue level of organization is less ordered in the bone 
collagen than in the tendon. 

The diffraction data further demonstrated that the two tissue col- 
lagens were also alike in their response to the test solutions. In H2O 
and 0,5M acetic acid, no breakdown in intramolecular triple helical 
structure took place for either collagen, even though extensive (>5A) 
lateral separation of adjacent molecular units did occur. When in con- 
tact with 5M guanidine, on the other hand, both collagens became com- 
pletely denatured. 

Differences in diffraction properties reflecting important distinc- 
tions in the two collagens at an intermolecular level of structural 
organization did not become evident until attempts were made to re-nature 
guanidine denatured material. When washed free of guanidine and dried, 
the diffraction pattern of bone collagen was identical to that of unde- 
natured material. In contrast, the tendon pattern following the same 
re-naturation procedure showed no signs of native collagen structure. 
These results indicate that bone collagen can elastically renature 
under conditions where the monomers of tendon collagen cannot reestablish 
their helical structure and lateral relationships. Native collagen 
structure was observed, however, in formalin-fixed rat tail tendon follow- 
ing subjection to the same denaturation-renaturation routine. 

The above experimental findings can be interpreted in terms of the 
degree to which covalent cross- linking between adjacent monomers is 
known to occur in these collagens. In bone collagen, the presence of 
intermolecular cross-links prevent random dissociation of adjacent 
monomers during denaturation even though the individual polypeptide 
chains become randomly coiled. This preservation of interchain association 

2 2kk 



I 



Serial No. NIDR-51 (67) 



apparently assures reassembly into native configurations upon renatura- 
tion. The absence of such cross-links in tendon collagen results in the 
loss of all spatial associations during denaturation with the consequence 
that upon attempted renaturation irregular non-helical associations form 
preferentially. Confirmation of this interpretation was found in the 
fact that covalently cross- linking tendon by formalin fixation enabled 
this collagen to become more like bone collagen in its ability to recover 
from guanidine denaturation. 

2. Amyloid study ; The position of the diffraction lines in unoriented 
x-ray patterns obtained from fibrous amyloid protein classified this 
material as of a p-type. The polypeptide chains giving rise to such 
patterns are in a pleated sheet configuration in which adjacent chains 
are in an antiparallel arrangement. Further, a cross-p pattern was 
obtained from samples in which fiber orientation had been induced by 
mechanical means . This latter finding suggests that the chain axes are 
directed perpendicular to the fiber direction. The p-type pattern was 
produced by all amyloid laden tissue studied which included material 
from mouse and duck as well as from human. In addition, the p-type 
pattern obtained from fresh wet tissue was unaltered by subsequent drying 
and purification. It appears, then, that the pleated sheet structure is 
a natural and stable configuration for amyloid protein fibers. 

Significance to Dental Research : 

1. Since collagen is an important component in all connective tissue 
including dentin, knowledge concerning the physical and chemical proper- 
ties cross-links impart to this fibrous protein would further contribute 
to an understanding of its role in tissue structure and function. 

2. The amyloid fiber and enamel matrix are the only naturally occurring 
cross-p proteins in human tissue reported to date. Studies on fibrous 
amyloid protein, therefore, may have particular relevance to dental 
research in that it may contribute to a more complete understanding of 
the nature and function of the protein matrix in enamel tissue. 

Proposed Course of Project ; 

The wide-angle x-ray diffraction phase of these projects is essentially 
completed. Collagen, however, has a well developed pattefn in the 
small-angle region of x-ray scatter. Studies will be conducted to 
explore the effect cross-linking may have on this small-angle diagram 
of collagen. The amyloid fiber also appears to scatter x-rays at small 
angles, and studies will be undertaken to accurately record these small- 
angle diffraction lines in the expectation of obtaining additional infor- 
mation on the structure of the amyloid fiber . 



Part B: Not included 



2^S 



Serial No, NIDR-52 (63) 

1. Histology & Pathology 

2. Crystal Chemistry 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Infrared Absorption Spectrophotometric and X-ray 
Diffraction Studies of the Inorganic Portion of 
Teeth and Bones and Related Synthetic Compounds 

Previous Serial Number: NIDR-20 

Principal Investigator: Mr, B. 0, Fowler 

Other Investigators: None 

Cooperating Units: None 

Man Years: 

Total: 1 
Professional: 1 
Other: 

Project Description: 

Objectives : 

The main objective is to define more clearly the composition of the 
inorganic phase of teeth and bones. The reactions of various synthetic 
calcium phosphates with different substances under varying experimental 
conditions are being determined and the results related to biological 
mineral systems. Additional objectives involve studies to ascertain the 
chemical bonding occurring between the organic and inorganic components 
in hard tissue. 

Methods Employed : 

The primary methods used are infrared spectroscopy and x-ray diffraction. 
Specialized infrared techniques involve utilizing reflectance, polari- 
zation, low and high temperature, and high pressure devices in obtaining 
spectra. In addition, physical-chemical techniques for synthesis and 
purification of hydroxyapatite , f luorapatite , carbonate apatite, and 
related calcium phosphates are employed. High temperature and pressure 
apparatus is designed and constructed in order to synthesize certain 



2^6 



Serial wo, NIDR-52 (63) 

substituted calcium phosphates in well-crystallized form, and study 
their reactions in vacuum or with carbon dioxide and /or steam under 
pressure at temperatures to 1100°C. The techniques are supplemented by 
chemical analyses to determine calcium, phosphorous, fluoride, carbonate, 
and trace elements. 

Major Findings ; 

The OH stretching and librational infrared absorption bands of hydroxy- 
fluorapatite (HFA) have previously been characterized and assigned. In 
order to obtain additional characterization, the HFA spectra have been 
examined down to 200 cm"-'-, the frequency limit of the available instru- 
mentation. One HFA band in this region was found to shift as a function 
of fluoride content. A study was made to establish the reason(s) for 
this band shift, in order to further characterize the structural features 
and infrared spectra of this biologically important compound. 

The fundamental infrared absorption bands in hydroxyapatite (HA) arising 
from internal vibrations (stretching and bending modes) of the phosphate 
groups and the OH stretching and the OH librational modes have been 
given assignments. The OH stretching and librational modes have been 
assigned with certainty as based on shifts upon deuteration. Thel^^) 
i\,2^i^, and 2^2 phosphate bands have been primarily assigned on the basis 
of (a) the theoretically predicted bands arising from the spatial geometry 
of the phosphate group and (b) previously recorded Raman spectra of the 
phosphate group. The Z^^ , 2^]^ , and 2-'^ vibrational assignments of the 
phosphate group are most probably correct. The bands which have been 
assigned to the 1^2 vibration by several investigators is now questionable 
as a result of information obtained in the present studies which suggests 
a new assignment for the Z^ band and other low-frequency bands. 

Three major HA bands occur in the low-frequency region at 342,280 and 230 
cm"-*-. The band at 342 has a shoulder at about 355 cm"-'- and the band at 
280 cm"l splits into a well-resolved doublet at liquid-nitrogen tempera- 
ture. The major bands at 342 and 280 cm"-'- have been assigned by several 
investigators to theJ^ components of the phosphate groups. The band at 
230 cm~l has apparently not been reported. 

Three approaches utilizing (a) band intensity, (b) isostructural analogues, 
and (c) isotopic substitution were employed to assign and /or reassign 
bands in the low-frequency region. 

Band Intensity: the "^i and the ^^2 modes of the undistorted tetrahedral 
phosphate group are infrared inactive; however, these modes may become 
active when the phosphate group is distorted to lower symmetry. To 
establish the i^i, 1^2 band intensity relationship compounds containing 
HPO4 groups, for which both the i^-j^ and 1^2 modes are active, were examined. 
A series of HPO4 salts containing cations with increasing mass, CaHP04, 



Ikl 



Serial No. NIDR-52 (63) 



SrHPO^ and BaHP04 was chosen in order that lattice vibrations would be 
shifted to lower frequencies and as a consequence not introduce inter- 
fering absorption in the V2 region. 

Two major bands at 400 and 265 cm"-'- were observed for CaHP04 in the 
450-200cm"l region; SrHP04 and BaHP04 each have only one band at about 
415 cm-1. The bands at 400 and 415 cm-1 are most probably the "2^2 
components and the 'K2 band intensity is about one-half the intensity 
of the 2^]^ band. By analogy the intensity of the 2^2 band in HA should 
be less than the intensity otT^\. However, the intensity of the bands 
in HA which have been assigned tolJ^2 previously are approximately 
twenty times the intensity of thej^i vibration in HA. 

Isostructural Analogues: Since there are three major bands in the 
400-200 cm"l region of HA with two bands showing additional structure, 
the weak 2^2 bands, which would comprise only about one-twentieth of the 
area of these bands, could be superimposed on the major bands and there- 
fore not easily detected. In order to shift interfering bands from this 
region, the isostructural analogues of HA, strontium apatite (SrA) and 
barium apatite (BaA) were prepared and examined in this region. Lattice 
vibrations arising from the isostructural analogues with heavier cations, 
i.e. Sr and Ba should show greater shifts to lower frequencies than the 
fundamental phosphate modes of these analogues. 

SrA has two bands in this region at 322 and 235 cm"-*-. The SrA band at 
235 cm"l is too low in frequency to be a component of 2^2- The intensity 
of the 322 cm"-*- band is about four times the intensity of the z/. band 
and it shifts by about 10 cm"-*- on deuteration. The intensity and shifts 
on deuteration indicate that this band is not 1^2 ^^^ rather arises from 
translational motion of the OH groups. 

Only preliminary results are available on BaA in this region; however, 
only one definite band is observed in the 400-210 cm"^ region at 280 
cm"^. Both the intensity of this band, which is several tiroes that of 
the 2^1 band and its low-frequency position indicate that this band is 
not 2^2 • 

The low-frequency bands in HA at 280 and 230 cm"-*- are apparently lattice 
vibrations judging from the shifts of the corresponding bands in the 
Sr and Ba apatite analogues. 

Isotopic Substitution: The bands in HA (342), HFA (355-345) and SrA 
(322 cm"l), suspected of arising from the OH groups, were found to shift 
by approximately the expected amount on deuteration for translational 
motion of the OH groups. Attempts were made to further characterize 
these bands by mass dependancy utilizing 0^° . 

Samples were prepared by treating dehydrated samples of HA or other 
Ca0-P205 mixtures of the proper proportions with H20IS contained in 
sealed vycor tubes at pressures from 5 to 20 atmospheres in the 

3 2k^ 



Serial No. NIDR-52 (63) 

temperature range 500 to 1100°C. At 500°C apatite samples were prepared 
which contained approximately 40% of the total OH groups replaced by 
olSg groups; however, the samples were too poorly crystallized to defi- 
nitely establish a shift in the 342 cm~l band. Samples heated with 
H20^^ (5 atm) at 500°C for five days not only showed 0^% substitution, 
but in addition, the O''-^ from the water had exchanged with the oxygens 
of the orthophosphate groups . That had exchanged with O^^ in the 
phosphate groups was established by both new and shifted bands that 
appeared in the spectra of these samples. 

This exchange was fortuitous , in that the vibrational modes of the 
phosphate groups could be established unequivocally due to the mass 
effect as a result of O-*-^. Attempts were made to exchange 90% of the 
apatite 0^° by treating the samples with excess H20-'-^ at higher tempera- 
tures and pressures. The choice of variables, temperature, pressure and 
time to effect this exchange was dictated and limited by the physical 
properties and chemical composition (Si02) of the vycor tubing used for 
the reaction chamber . The maximum O^^ exchange obtained thus far , about 
50%, was effected by treating a 3 to 7 molar mixture of Ca(P03)2 and " 
CaC03 at 20 atmospheres H2O pressure at 900° for two hours. The total 
qI^ to 0^8 proportion was 1 to 5. Treatment at higher temperature, 
pressure and longer time did not increase the qIo exchange; lack of 
increased exchange under these conditions may be due to 0^° exchange 
of the H2OI8 with Q-'-^ of the vycor (Si02) tubing. An inert tube liner 
will be used in effort to increase the 0^° exchange , 

The infrared spectra of the sample containing about one-half of the 
total oxygen as showed distinct band shifts which confirm that 
the previous assignments for the ^3, 2^ -j^, andii'4 phosphate modes are 
correct. However, the weak HA band at 472 cm-1, which has been assigned 
to ai^S -^^4 difference tone by several investigators, shifts on 0^° 
substitution by the calculated amount for the 2^2 phosphate vibration. 

The band intensity, deuteration and preliminary Q-'^" data indicate that 
the weak HA and HFA bands at 472 cm"l are the 2^2 phosphate mode, and 
that the bands at 342 cm"l in HA and at 355 to 345 cm"! in HFA arise 
from translation motion of the OH groups. 

The isostructural analogues of HA, SrA and BaA were prepared and examined 
by infrared spectroscopy to aid in the assignments of the. HA low-frequency 
bands. It was necessary, therefore, to assign the SrA and BaA absorption 
bands. The major phosphate bands were assigned for both compounds and 
the OH modes for SrA. The OH modes of the BaA have not been unequivocally 
assigned due to the low intensity of the bands. The OH motions are of 
primary importance, particularly the OH librational motion which has 
been shown to be very sensitive to its molecular environment. The 
phosphate bands in SrA shift about 12 cm" relative to the corresponding 
phosphate bands in HA whereas the OH stretching band shifts 20 cm'-'- and 

2^9 



serial No. NIDR-52 (63) 

the OH librational band shifts 93 ctn-l. The about eight-fold shift 
of the OH librational band shift relative to the phosphate band shift 
further illustrates the sensitivity of this mode to its molecular 
environment . 

Significance to Dental Research ; 

Characterization and assignment of the infrared absorption bands of 
pure synthetic apatites and band changes caused by the presence of, for 
example fluoride, chloride and carbonate, are essential in better 
defining the composition of the inorganic phase(s) of teeth and bones. 

The amount of carbonate present in enamel has long been thought to 
have some bearing on relative susceptibility of teeth to caries Carbo- 
nate has also been labelled by some investigators as essential for 
apatite formation. The exact location of carbonate within the crystal 
lattice and verification of the role of carbonate in apatite formation 
are essential to an understanding of its influence on caries suscepti- 
bility. 

Some of the postulated reactions of fluoride with enamel, such as 
replacement of hydroxyl and carbonate groups, have not been clearly 
established by chemical experiments. Knowledge of the exact reaction(s) 
of fluoride, which accompany reduction in caries, would enable better 
selection of chemical conditions to maximize the reaction(s) . 

Proposed Course of Project ; 

Completion of 0^^ isotopic exchange experiments in order to assign 
with certainty the low-frequency apatite infrared absorption bands . 
Characterization of bands in this region will give additional informa- 
tion to aid in interpreting the structural features of apatites con- 
taining biologically important anions, such as, fluoride and carbonate. 

Completion of the infrared studies of hydroxy-f luorapatite and carbonate 
containing apatite. 

Part B: not included 



250 



Serial No, NIDR-53 (59) 

1. Histology and Pathology 

3. Bethesda, Maryland 



PHS-NIH 

Individual Project Report 

July 1, 1967 through June 30, 1968 



Part A 



Project Title: Odontopathic Bacterial Plaques: Etiologic Factors, 
Pathological Sequelae, Therapeutic Measures 

Previous Serial Number: NIDR-28 

Principal Investigator: Dr , P. H. Keyes 

Other Investigators: Dr. R. J. Fitzgerald, Dr, H. V, Jordan, 

Dr. R. J. Gibbons, Dr. R, H, Larson, 
Dr, A, J, Gwinnett, Dr. P, N, Baer 



Cooperating Units: 


None 


Man Years: 




Total: 

Professional: 

Other: 


4 1/4 
2 1/4 
2 


Objectives: 





The objectives of the work described in this project still remains: 
(1) to attain a better understanding of dental plaque infections. 
Answers are needed to questions regarding the etiological factors invol- 
ved, pathological features, and therapeutic measures of prevention and 
cure , 

Methods Employed ; 

Animal model systems which use Syrian hamsters have been and continue 
to be of value. In vitro methods are also employed for assessing 
factors conducive to plaque formationand its control. 

Experiments and Major Findings : 

1, Studies in collaboration with Robert J. Fitzgerald demonstrated that 
dextranase products produced by colleagues at Merck and Company will 
prevent the accumulation of coronal plaque and smooth surface caries in 
animals subjected to appropriate dietobacterial challenges, i.e., sucrose 
and dextranogenic streptococci, «^ r < 



Serial No. NIDR-53 (59) 



2. Studies in collaboration with Harold V. Jordan demonstrated that 
important differences in the disease response of hamsters followed the 
interactions between diets containing various amounts of sucrose and 
starch and plaque -forming bacteria, namely detranogenic streptococci 
and levanogenic diphtheroids. As the sucrose content of the diet de- 
creased and the starch content increased, the population of streptococci 
fell and that of diphtheroids increased; caries activity and severe 
ulcerative gingival disease decreased; progressive periodontal pathosis 
became the only detectable disturbance. 

Until recently it has not been possible to induce periodontal syndromes 
by inoculating human diphtheroids into the mouths of hamsters, although 
the isolates used appeared to be very similar to the Odontomyces viscosus , 
previously described as having the potential to form plaque and perio- 
dontal pathosis in this species. Therefore it has been encouraging to 
find that hamsters fed a diet containing starch have developed typical 
cervicoradicular plaque, gingival distortion, and alveoloclasia follow- 
ing the inoculation of human diphtheroids into their mouths. 

3. Collaborative studies with Ronald J, Gibbons have demonstrated that 
in hamsters the addition of dextran to diets containing sucrose reduced 
plaque formation and caries activity. 

4. Studies in collaboration with Rachel H. Larson revealed that rats 
designated by researchers at Michigan State University as caries-resistant 
were not resistant to either crevice decay, plaque formation, or to smooth 
surface decay when subjected to appropriate dietobacterial challenges. 

5. Methyl cyanoacrylate was mixed with several inorganic salts, some 
of which contained fluoride, and applied to extracted human teeth and to 
the teeth of Syrian hamsters. A. John Gwinnett examined sections of 
these human teeth and found satisfactory penetration of the sealant into 
the enamel surface. No decalcification of surfaces treated in this manner 
occurred while such teeth were incubated in culture medium containing 
caries conducive streptococci. The same findings were noted in the teeth 
of hamsters . However the material tended to soften and not to retain its 
bonding with the surface . Work with this compound stopped , after it was 
learned that this isomer of cyanoacrylate hydrolyses and is not bio- 
stable . 

6. Studies in collaboration with Paul N. Baer showed that an infectious 
and transmissible flora was responsible for plaque and calculus formation 
in rats . 

Significance to Dental Research ; 

1. Any substances which will assist in the dispersion of adherent 
pathogenic plaques on the surfaces of teeth will be of value in the 
prevention and cure of dental plaque infections. Clinical trials 

2 252 



Serial No. NIDR-53 (59) 



with dextranase are now anticipated. It may be possible to supplement 
the antiplaque benefits of such enzymes with antibacterial agents , if 
the clinical trials prove beneficial. 

2. The importance of dietary residues in the etiology of periodontal 
syndromes has not been generally recognized. The finding that starch 
enhances the periodontal syndrome in hamsters appears to be in coincidence 
with the observations that human populations which consume large quanti- 
ties of cereal products may have serious root surface disease, i. e., 
radicular infections, and associated periodontal detachment. The suc- 
cessful implantation of human filament forming bacteria in mouths of 
hamsters and the subsequent development of periodontal pathosis may 

lead to animal model systems for assessing the pathogenic potential 
of human bacteria isolated from human patients with cervico-radicular 
infections . 

3 . The observation that additions of dextran to the diet can attenuate 
plaque forming reaction with sucrose suggests that food additives might 
be found which would be somewhat beneficial in man. 

4. The use of sealing agents to seal vulnerable occlusal crevices in 
teeth (pits and fissures) could be of great value in preventing carious 
lesions in retention sites on teeth. Limited observation in vitro 
suggests that these products are highly effective, and clinical observa- 
tions elsewhere are in agreement. However, real progress in this area 
will have to await the development of sealing agents which are bio-staple 
and thus adhere to enamel surfaces for long periods of time. 

Proposed Course of Project ; 

1. Dextranase will be assessed in clinical trials. Human volunteers 
who harbour dextran producing streptococci will dissolve cubes of 
sucrose periodically during the day for several days. Dextranase will 
be administered in various formulations during or after the experimental 
period to determine its potential either to prevent or to displace 
plaque accumulations . 

2. Tests in hamsters will be used to gather further insight into dieto- 
bacterial factors associated with periodontal pathosis. If bacterial 
isolates from humans can be shown to colonize on the cervico radicular 
surfaces of teeth and thereby induce gingivitis and periodontal syndromes , 
significant progress may be made in our understanding of "periodontal 
disease". Efforts are now in progress to let a contract with the Lincoln 
State School, Lincoln, Illinois, whose staff members are interested in 
defining the plaque infection status of mentally retarded persons under 
their care. Cultures from the mouths of these patients will be assessed 
in hamsters and in vitro. 



253 



Serial No, NIDR-53 (59) 



3, A new sealing material has been prepared by Ethicon Inc. (sole 
distributors of Eastman Kodak's cyanoacrylates) for experimental trials 
in animals and in vitro . Isobutyl 2-Qyanoacrylate and a special filler 
will be applied to extracted human teeth, which will then be sectioned 
and examined histologically (in collaboration with A. John Gwinnett, 
Dalhousie University, Halifax). These specimens will also be incubated 
with streptococcal cultures to determine whether the material will 
prevent decalcification of the coated surface. It will also be applied 
to the teeth of hamsters subjected to caries conducive conditions. 

4. In vitro methods are being developed which may permit meaningful 
assays of the ability of antibacterial formulations to retard plaque 
formation and the potential of fluoride preparations to attenuate demin- 
eralization of enamel surfaces. 



Part B 



Publications: 



1. Englander, H, R, , Keyes , P. H. , and Gestwicki , M. Clinical anti- 
caries effect of repeated topical sodium fluoride applications by 
mouthpieces. J. Am. Dent . Assoc . 75:638-644, 1967. 

2. Prevention and conservation in dentistry: a symposium. Bull. Acad. 
Med. N. J. 13:160-166, 1967. 

3. Keyes, P. H. Odontopathic infections in the golden hamster: its 
biology and use in medical research. Eds. R. A. Hoffman, P. F, 
Robinson, and H. Magalhaes . Iowa State Press. Ames. 1968. 

4. McCabe, R. M. , Keyes, P. H., and Howell, A., Jr. An in vitro 
method for assessing the plaque forming ability of oral bacteria. 
Arch. Oral Biol. 12:1653-1656, 1967. 

5. Fitzgerald, R. J., Keyes, P. H. , Stoudt , T. H. , Spinnell, D. M. 
The effects of a dextranase preparation on plaque and caries in 
hamsters. A preliminary report. J. Am. Dent. Assoc. 76:301-304, 
1968. 

6. Keyes, P. H. A review of dental caries. J. Am. Dent. Assoc. 76: 
June 1968. 

7. Gwinnett, A. J. and Matsui , A. A study of enamel adhesives. The 
physical relationship between enamel and adhesive. Archs . Oral 
Biol. 12:1615-1620, 1967. 

8. Gwinnett, A. J. Scandium as a target material for microradiography. 
J. Dent. Research. 46:1479, 1967. 

2Sk 



Serial No. NIDR-54 (67) 

1. Histology and Pathology 

3. Bethesda, Maryland 



PHS-NIH 

Individual Project Report 

July 1, 1967 through June 30, 1968 



Part A 



Project Title: Fate of Microorganisms Inserted into Healthy Gingival 
Pockets . 

Previous Serial Number: None 

Principal Investigator: Dr. Jens Waerhaug 

Other Investigators: Dr. Harold Jordan and Dr. Paul Keyes 

Cooperating Units: Dr. W. Titus, Laboratory Aids Branch, Division 

of Research Services 

Man Years: 

Total: 3/4 

Professional: 1/2 
Other: 1/4 

Project Description: 

Objectives : 

To study the fate of bacteria inserted into the potential space between 
the tooth and its normal epithelial cuff. 

It has become increasingly clear that periodontitis is a bacterial 
disease and that the point of attack of the bacteria is at the gingival 
margin or in the gingival pocket. According to criteria outlined by one 
of the investigators (Dr. Jens Waerhaug) a healthy gingival pocket is 
one in which the normal epithelial cuff (attachment) adheres to the tooth 
in its full width without interjacent plaque. There are extreme differ- 
ences of opinion, however, concerning the presence or absence of bacteria 
in this potential pocket. One group maintains that the healthy gingival 
pocket is essentially sterile and that bacteria which are introduced 
mechanically are eliminated. The other group holds that all such pockets 
always harbor bacteria. 



255 



Serial N , NIDR-54 (67) 



Methods Employed and Major Findings ; 

The dog was chosen as the experimental animal partly because the anatf-my 
of the gingival area is very similar to that of humans, and partly b ■ 
cause the dog is handled rather easily v/ithout anesthesia. 

In order to differentiate experimentally introduced microorganisms from 
those normally present on the teeth and their gingivae it is necessary 
to implant microorganisms which have been made resistant to a certain 
antibiotic. By culturing subsequent samples in media which contain 
sufficiently high concentrations of the antibiotic, growth of all but 
the resistant microorganisms is prevented. 

It was hoped originally that one of the established hamster or rat strains 
of plaque forming bacteria could be used for experimental implantation 
in the dogs. As a consequence, in the first experiments the capacity of 
one such strain (T6 1600) which had been made resistant to streptomycin, 
to colonize in the experimental animal was investigated. The tongue and 
gingival areas of one dog was swabbed with 48 hr . cultures of T6 1600 
for 10 min. and in addition 5 ml of the broth was added to the drinking 
water. Samples taken from the dog 1 and 4 days after inoculation showed 
no growth. 

Since sucrose favors growth of T6 1600 in rodents , a soft diet was pre- 
pared by mixing equal amounts of laboratory chow pellets and confection- 
ers sugar with a suitable amount of water. Two dogs were fed this diet 
for 10 days before inoculation with T6 1600 according to the technique 
previously described. The inoculation was repeated 2 days later. Again 
samples taken after 1 and 4 days showed no growth on the streptomycin 
containing medium. 

The inability of the T6 1600 to colonize in the oral cavity of the dog 
made it useless for the present purpose and it was necessary to find a 
microorganism which was indigenous to the dog. Samples were collected 
from a fairly old dog with ample amounts of supra-and small amounts of 
subgingival plaque i A number of filament -forming bacteria and diphtheroids 
were isolated from the dog samples . However , it has been very difficult 
to subculture these initial isolates, a necessary step in the development 
of streptomycin resistant strains. pH determinations suggest that the 
saliva of the dog has a much higher pH than that of the rodents , a factor 
which may explain some of the difficulties. 

Significance to Dental Research ; 

The establishment of whether or not known plaque-forming microorganisms 
are eliminated when introduced mechanically into the normal epithelial 
cuff is of the utmost importance for a correct appreciation of the patho- 
genesis of gingivitis and periodontal destruction. 



256 



Serial No, NIDR-54 (67) 



Proposed Course of Project ; 

The investigator's tenure of appointment ceases in August 1968. A 
continuation of the program past this date is not planned. 

Part B not included 



257 



Report of the Biometry and Field Investigations Branch 
National Institute of Dental Research 
S\immary Statement 



In the past year, the existing Epidemiology and Biometry Branch was 
reorganized into the Biometry and Field Investigations Branch. This 
reorganization included the establishment of three sections: Biometry, 
Clinical Trials, and Epidemiology. Drs. James P. Carlos, Harold R. Englander, 
and Norman W. Littleton, respectively, were appointed chiefs of these newly 
established sections. This reorganization provides the basis for imple- 
menting a broad, well-balanced program of field studies of oral diseases. 
In addition, biomctric capabilities have been strengthened. Emphasis is now 
being given to the development of a computer-oriented data processing system. 
This system also will be used to investigate the methodology of design, 
estimation and analytical techniques in clinical trials. 

Dxiring the year, research activities of the branch included field studies of 
dental caries, periodontal disease, and malocclusion. Some of these studies 
were initiated during the current year; others represent a continuation and 
expemsion of productive efforts which began the previous year. In addition 
to direct research activities , members of the branch participated in 
collateral projects which included consultations on the design aind conduct 
of field studies being undertaken by others and biometric services to the 
professional staff of the Institute. 

Dental Caries 



Dental caries activity in hamsters can be prevented completely by a fluoride 
containing gel topically applied to the teeth by use of a fitted vinyl mouth- 
piece. A field study was undertaken to test the effects of a similar regimen 
on dental caries activity in children living in an area with a fluoride 
deficient water supply. Water soluble gels containing either 1.1 percent 
sodium fluoride and 0.1 M phosphate (pH U.5), or 1.1 percent sodium fluoride 
(pH T.O) were used. These gels were topically applied to the teeth of 
children for six minutes per day by use of fitted vinyl mouth applicators. 

After 21 months, comparisons with an untreated control group indicated that 
dental caries increments were importantly lower in children receiving either 
type of gel. Differences in the increments of DMF teeth averaged about 
65 percent lower while differences in DMF tooth surfaces averaged 75 to 
80 percent lower in treated children. Eleven months after fluoride gel 
applications had been stopped, dental caries increments were still importantly 
lower for treated children than for those in the control group. But 
differences in dental caries increments at this examination were not as great 
as those observed during the 21-month period of fluoride application. This 
may indicate that there is a falling off of protection after repeated 
topical fluoride therapy is discontinued. Children in this study will be 
examined again to determine if this trend continues. 

259 



Analyses of exfoliated deciduous teeth showed that the oute layers of 
enamel of teeth from treated children acquired significantl more fluoric ■* 
than was found in the enamel of teeth from untreated chilu .n. Six to 9 
months after fluoride applications were stopped, the oute layers of enamel 
still contained high concentrations of fluoride (2800 ppt y . 

This project has demonstrated the value of a dental caries preventive 
technique that can "be used in conjunction with other e^ ropriate caries 
inhibitory measures. It has also raised the question x" the minimum concen- 
tration of fluoride in enamel necessary for an optimur anti-caries effect. 

Another study of the anti-caries effects of repeated opical fluoride gel 
applications (3 times per week) was initiated in a fj oridated community in 
November 1966. This project will demonstrate whether additional caries 
inhibition can be obtained from repeated topical fluoride therapy in children 
using fluoridated water continuously from birth. Initial dental examinations 
for this study were completed in November-December I966. Children in the 
experimental and control groups had 2.7 and 2.6 DMF teeth, respectively. 

Deciduous teeth treated with sodium fluoride gel prior to exfoliation 
progressively acquired more fluoride as the number of treatments increased. 
Most of the acquired fluoride was restricted to the outer 20 microns of 
enamel. For example, teeth receiving an average of 56 topical sodium 
fluoride treatments had 1,785 ppm in the outermost enamel layer as compared 
to 922 ppm fluoride for xantreated control teeth from children consuming only 
fluoridated water. Follow-up examinations of children in this study are 
scheduled in May I968. 

A third study of daily topical fluoride gel applications has been initiated 
in preschool children who are dependents of U.S. Coast Guard personnel. The 
primary purpose of this study is to determine if such treatment can maintain 
the deciduous dentition of these children free from the development of smooth 
surface lesions. Children participating in this study were first examined in 
November I967, and are to be reexamined in June I968. 

Oral microorganisms (dental plaque) and intra-oral, food residues have been 
implicated in the etiology of dental caries. Studies of the interaction of 
these factors in persons nourished by stomach tube were continued during the 
year. Plaque material from tube-fed persons was found to be much less acidic 
when suspended in 10 percent solutions of sucrose, glucose, fructose, invert 
sugar or starch than plaque material from persons fed by mouth. Bacterio- 
logical study showed that total streptococcus counts were lower in plaque 
from tube-fed persons. Filamentous bacteria, lactobacillus and streptococcus 
salivarius types were isolated less frequently from either plaque or from 
swabs of the oral mucosa obtained from tube- fed persons. Comparisons between 
groups clearly indicated that the acidogenic properties and certain bacterial 
components of plaque obtained from tube-fed persons differed from plaque 
obtained from persons who were fed by mouth. However, within group comparisons 
failed to establish any consistent relationship between these variables. 

260 



Another study was undertaiken in this population to determine the effects of 
sucrose, invert sugar and starch on the acidogenic properties and certain 
bacterial components of dental plaque when these carbohydrates were given 
orally to persons who had been nourished previously via stomach tube. 

A substantial increase in the acidic potential of plaque material from tube 
fed persons was observed 30 days following oral administration of these 
carbohydrates. Exposure to sucrose or invert sugar, however, seemed to 
enhance the acidic properties of plaque to a greater extent than starch. 
Thirty days after the cessation of oral carbohydrate supplementation, the 
acidogenic capacity of plaque taken from these persons reverted to pre-test 
levels. Oral administration of these csurbohydrates, however, had little 
effect on total counts of either streptococci or lactobacilli in plaque 
material obtained from these tube-fed persons. 

The availability for study of persons nourished solely by stomach tube 
provides a \inique opportunity to investigate the dynamic interactions 
between oral microorganisms and intra-oral food residues in the etiology of 
oral diseases. Plans have been made to expand these initial studies of the 
effects of selected carbohydrates on the oral microflora when these foods 
are introduced orally under well controlled, experimental conditions to 
persons nourished by stomach tube. 

Certain strains of streptococci are known to cause dental caries in 
experimental animals. Strains have been isolated from humans which are cario- 
genic when implanted binder proper conditions within the mouths of either rats 
or hamsters. Distinctive cultxiral characteristics of these types of 
streptococci have been identified. As a result, it seems expeditious to 
undertake epidemiological investigations of the occurrence of these micro- 
organisms in human populations, and to determine if the observed distributions 
relate to dental caries activity. 

A study of the distribution of streptococci which have cultural characteristics 
of microorganisms that are caries-conducive in animals is included in the 
fluoride-gel studies. Such streptococci were found to be widely distributed 
in these populations; they were recovered in high niombers from dental plaque; 
and their presence was correlated with the past dental caries experience. 

Caries-conducive streptococci were not recovered from 96 percent of the caries- 
free two-year old children included in one of these studies. 

Plans are being made to extend investigations of the occxirrence of these 
"caries-conducive" streptococci to other populations. 

Periodontal Disease 

Field studies conducted by this- branch show that a spectrum of clinical signs 
indicative of periodontal disease are intimately associated with intra-oral 
irritants such as plaque and calculus deposits. But the role of these and 
other local irritants in the etiology sind pathogenesis of periodontal disease, 
a chronic and progressive process, is not adequately understood. 

261 



A long-term investigation of periodontal disease has been undertaken in 
collaboration with the Oral Medicine and Surgery Branch to study the influence 
of various intra-oral irritants on the initiation and progression of 
periodontal disease in a stable, adult male population. Base line data for 
this study have been collected from about 600 adult males living in 
Metropolitan Washington, D.C. Computer programs for the analysis of these 
data are being developed. A second series of examinations in this popula- 
tion is planned for September I968. 

The brajich also is participating in a study of the effects of periodic dental 
prophylaxis and instruction in oral hygiene in the initiation and progression 
of periodontal disease. This program is being conducted in collaboration 
with the Dental College and Hospital, Lucknow, India, and is supported by 
PL UQO funds. Initial examinations have been obtained from about 2500 
persons, and appropriate treatment regimens are in progress. It is estimated 
that the first meaningful findings will be available in I969. 

Occlusion 

Recent investigations indicate that a derangement in vitamin Dg metabolism 
during pregnancy may be responsible for the cardiovascular anomalies of the 
supravalvular aortic stenosis syndrome (SASS), especially when associated 
with idiopathic infantile hypercalcemia. Peculiar "elfin" facies and dental 
anomalies, especially malocclusion, are reported to be common features of 
SASS. A study was undertaken to determine if SASS, including the 
characteristic facial and dental features, could be produced in offspring by 
exposing pregnant rabbits to large doses of vitamin Dp. 

Through a series of teat dosages it was determined that about 750,000 units 
of the vitamin is sufficient to produce most of the sinomalies characteristic 
of SASS. Various developmental defects of the cranio-facial complex were 
observed in the offspring of rabbits given large doses of vitamin Do. Under- 
development of the mandible resulted in dysgnathia and dental cross-bite. 
The abnormal osseous development responsible for the malocclusion occurred in 
65 percent of these offspring. Differences were noted in the timing of 
suture and fontanel closure. Many of the bones of the cranio-facial complex 
were smaller, malformed, and had altered contour. Enamel hypoplasia of the 
central incisors was noted in 95^ of the test offspring. None of these 
dental and facial abnormalities were noted in the offspring of rabbits in the 
control groups. 

Experimental findings in the vitamin D treated rabbits resemble anatomically 
the skeletal and orthodontic manifestations of SASS that have been reported 
in humans. Further use of this experimental technique may lead to a better 
understanding of the effects of hypervitaminosis D on growth and development. 

Orthodontic care claims a considerable share of funds spent to improve dental 
health, but the natviral history of occlusion has received little emphasis in 
epidemiological study. A serious deterrent to progress in the epidemiologic 
study of malocclusion has been the lack of a comprehensive, valid and reliable 
index of occlusal disorders for use in field investigations of large 
populations. 

262 



Recently a new index was proposed which is based upon sound statistical and 
orthodontic principles and may be useful for the study of the distribution 
of occlusal deviations from an ideal "standard." In collaboration with 
dental personnel from the Division of Health Examination statistics. 
National Center for Health Statistics, an investigation has been initiated 
to determine the reproducibility of the TPI (Treatment Priority Index) and 
the level of agreement as to the severity of malocclusion attained when 
index distributions are compared with the clinical judgment of orthodontists. 

A long-term study of occlusion also is in progress and it involves the 
reexamination of children at ages of critical tooth emergence and jaw 
development. To date, the results of this study indicate that a parabola 
computed from direct measurements of arch length and breadth adequately 
describes the size and shape of the dental arches. Malalignment of the 
dentition seems to be inversely related to arch breadth, but unrelated to 
either tooth size or arch length. Relationships established during the course 
of this study may prove valuable in understanding the sequence of events 
that lead to the various occlusal forms. 

Clefts of the lip and/or palate frequently show familial predisposition; 
the majority of cases, however, do not show clear-cut evidence of a simple 
genetic mode of transmission. It has been reported that certain oro-facial 
aberrations occur with greater frequency and consistency in the near kindred 
of cleft propositi. It has been suggested that such aberrations may be minor 
expressions of congenital oro-facial clefts. In cooperation with Human 
Genetics Branch, a study was done to determine if selected dental and oral 
morphological aberrations occurred with greater frequency in the near kindred 
of cl/cp individuals when compared with a control population. Examinations 
were conducted on a total of 93 families with one or more cl/cp propositi. 
Results were compared with those obtained from 82 control (non-cleft) 
families. With the exception of a greater niimber of minor palatal defects 
in the index families, no significant differences were observed between 
these populations. At the present time, the usefulness of including these 
defects in genetic and epidemiological studies of cl/cp seems quite limited. 

Biometric and Collateral Activities 

Considerable effort has been given by personnel of the Biometry Section to 
the design and development of a computer-oriented data system to produce 
faster and more detailed analyses of data gathered in the clinical testing 
of potential caries preventatives, and in epidemiologic surveys. This ^stem 
is based upon the field collection of data in a form suitable for automatic 
production of punched cards by an Optical Mark Page Reader. Data reduction 
and analysis is accomplished on the NIH IBM 360/50 computer using algorithmic 
language programs developed by Section personnel. The system is expected to 
be fully operative by July, 1968 and will be available for use by 
investigators conducting such studies. 

The Biometry Section has also begun a broad program of investigation into the 
methodology of optimum design, estimation and analytical techniques in dental 
trials. 

263 



In addition to direct research activiti-s and biometric ^rvice? to o^ir 
professional staff, a considerable amoi it of time was de ited to cons' Itation 
in the design, conduct, data processin , and analyses . the res '.ts ol* fie i 
studies undertaken by others. 

During the year, plans have been made t> offer residency programs in 
Biometry and Field Investigations to he! > meet the ne .-d for adc'itionaQ 
trained personnel in this area. Applici iion has bee made to t le American 
Dental Association for designation of t? is program iS an appro ed residen-.y. 
It is anticipated that the first reside; :s will be appointed d iring the 
coming year. 



26/f 



Serial No. NIDR-55 (68) 

1. Biometry and Field 

Investigations Br. 

2. Biometry Section 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A: 



Project Title: Studies on the Design and Analysis of Dental Clinical 

Trials 

Previous Serial Number: None 

Principal Investigator: Dr. James P. Carlos 

Other Investigator: Mr. Rickley S. Senning 

Cooperating Units: None 



Years 




Total: 


1/2 


Professional: 


1/4 


Other: 


1/4 



Project Description: 
Objectives : 

1. To develop a rationale for optimum allocation of subjects in 
dental caries clinical trials by studying the quantitative effects 
of various predictor variables on the incidence of caries. 

2. To investigate unbiased methods of estimating the true increment 
of new carious lesions, when the clinical observations are subject 
to error. 

3. To study the power and efficiency of various statistical tests of 
hypotheses in analyzing data from caries clinical trials. 

Methods Employed : 

Several mathematical models of dental caries clinical trials have 
been developed and are under study to determine their predictive 
validity in terms of data actually observed. With these models, 
the effect of misclassif ication error in the data can be simulated, 
and its consequences in tests of hypotheses observed, when various 
procedures are used to estimate the true incidence of caries during 
a study. 



2SS 



Serial No. NIDR-55 (68) 

Part A (continued) 

Standard multivariate statistics and simulation techniques are 
used to evaluate the relative efficiency of matched, balanced 
and random designs in dental clinical trials and the appropriate- 
ness of alternative significance tests of the data. 

Major Findings : 

Using one of the models referred to above, it has been demon- 
strated that estimation procedures conventionally used in dental 
trials are biased and usually result in overly conservative tests 
of significance with increased probability of failure to recognize 
an effective agent. 

Procedures for computing unbiased estimates of the parameters of 
this model have been developed. 

Preliminary data suggest that matched and balanced designs common- 
ly used in dental trials have little theoretical foundation and 
probably result in reduced experimental efficiency. 

Proposed Course of Project : 

The project is continuing. 



Part B: 



Publications: 



Carlos, J. P. and Senning, R. S. Error and bias in dental 
clinical trials. J. Dent . Res. 47:142-148; Jan-Feb, 1968. 



266 



Serial No. NIDR-56 (64) 

1. Biometry and Field 

Investigatioiis Br, 

2. Clinical Trials Sec. 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A: 



Project Title: Clinical Anti-Caries Effect of Repeated Topical 
Sodium Fluoride Applications by Mouthpieces 

Previous Serial Number: NlDR-45 

Principal Investigator: Dr. H. R. Englander 

Other Investigators: Drs. James P. Carlos and Harold V. Jordan, Jr, 

Cooperating Units: Cheektowaga Central School District No. 1; 

James R. Mellberg, Research Division of the 
Kendall Company, Barrington, Illinois 



Man Years : 




Total: 


1-1/4 


Professional: 


3/4 


Other: 


1/2 


Obiectives: 





The first two years of this project were supported by an NIDR 
Contract No. PH 43-64-944. The original purpose of this project 
was to test the anti-caries effect of water soluble gels con- 
taining 1.1 per cent sodium fluoride when the gels were applied 
daily for six minutes in mouth applicators to the teeth of child- 
ren living in fluoride-deficient Cheektowaga, New York. 

The acquisition of permanently bound fluoride by enamel of de- 
ciduous teeth from the topical applications, and the occurrence 
and distribution of caries-conducive streptococcal strains has 
also been studied, 

sodium fluoride 
The children in the study have not applied/in mouth applicators 
during the last two years. Therefore, additional data have been 
collected to determine the residual anti-caries effect of the 
fluoride applications after they had been discontinued. 



2&7 



Serial No. NIDR-56 (64) 

Part A (Continued) 

Furthermore, the data from the entire series of examinations are 
being analyzed to observe the effect on caries experience in 
clinical trials of the level of community dental care, the desig- 
nation of questionable carious lesions in the examination tech- 
nique, and the possible influence of positional errors on the 
interpretation of relative caries increments. In the future 
the data will be analyzed so that it will be possible to separate 
smooth surface from pit and fissure caries increments. 

Methods Employed : 

Clinical examination of approximately 500 children, initially 
aged 11-14 years, who were randomly assigned to groups receiv- 
ing the sodium fluoride gels and a control group not receiving 
the gels. Statistical evaluation of data collected during the 
21-month period in which the gels were applied, and of data 
collected 11 and 23 months after the gels had been discontinued. 
Chemical analysis of the fluoride concentration in the outer 
enamel layers of exfoliated deciduous and extracted permanent 
teeth, and bacterial culture of dental plaque. 

Major Findings : 

After 21 months, children applying an average of 245 repeated 
fluoride gel applications developed about four-fifths fewer DMFS 
than children in the control group not applying the fluoride gels. 

Eleven months after the repeated topical fluoride gel applications 
had been discontinued, dental caries increments were importantly 
lower in the children that had applied the fluoride gels than in 
the controls. However, the differences in increm.ents between the 
fluoride gel and control groups were not as great when compared 
with the differences obtained during the 21-month period of the 
repeated applications. 

During the 21-month period of fluoride gel applications, the 
amount of permanently bound fluoride acquired by deciduous enamel 
increased with the number of repeated fluoride applications re- 
ceived before exfoliation. 

The outermost layers of enamel of deciduous teeth exfoliated 
6-9 months after the fluoride applications had been stopped 
contained high concentrations of fluoride (about 2800 ppm) . 
This indicated that very little fluoride had been lost from 
enamel during the period that fluoride was not applied. 

No differences could be found in the ability to recover caries- 
conducive streptococci from the dental plaques of children 
applying and not applying the fluoride gels. 2S8 



Serial No. NIDR-56 (64) 

Part A (Continued) 

Significance to Dental Research : 

This project has demonstrated the striking anti-caries benefits 
that can be achieved with repeated topical fluoride treatments 
by mouth applica,tors. Furthermore, it has demonstrated the im- 
portance of considering the fluoride uptake of teeth in the 
evaluation of clinical trials with fluorides. 

The data on dental caries increments after the fluoride appli- 
cations had been discontinued do not indicate that there is 
a falling off of protection during this period, and may lead 
to the recommendation that repeated topical fluoride appli- 
cations can be discontinued in the treatment of rampant caries 
cases in order to obtain optimum anti-caries benefits when the 
enamel has acquired 2000-3000 ppm fluoride from topical appli- 
cations. 

Proposed Course of Project : 

The children will be examined in May 1968 to evaluate the 
residual anticaries effect 23 months after the discontinuance 
of the fluoride applications. 



Part B 



Publications; 



Englander, H. R. , P. H. Keyes and M. Gestwicki: Clinical 
anti-caries effect of repeated topical sodium fluoride 
applications by mouth pieces. JADA 75: 638, Sept. 1967. 

Mellberg, J. R. , H. R. Englander, and C. R. Nicholson: 
Acquisition of fluoride in vivo by deciduous enamel from 
daily topical fluoride applications. J. Oral Ther. & Pharm . 3: 
330, March 1967. 

Mellberg, J R. , H. R. Englander, and C. R. Nicholson: 
Acquisition of fluoride in vivo by deciduous enamel from 
topical fluoride applications over 21 months. Arch. Oral Biol . 12: 
1139, Oct. 1967 

Englander, H. R. : Views on the rationale of topical fluoride 

therapy. JACD 35: 15, Jan. 1968. 

Jordan, H. V., H. R. Englander and S. Lim: The presence of 
potentially cariogenic streptococci in various population groups. 
Preprinted Abstracts (370) lADR 46th Gen. Meeting, March, 1968. 



269 



Serial No. NIDR-57 (66) 

1. Biometry and Field 

Investigations Br. 

2. Clinical Trials Sec. 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: 



Anti-Caries Effect of Repeated Topical Fluoride 
Applications in a Fluoridated Community 



Previous Serial Number: NIDR-46 

Principal Investigator: Dr. Harold R. Englander 

Other Investigators: Dr. Harold V. Jordan and Dr. James P. Carlos 



Cooperating Units: 



Man Years 



Total: 

Professional: 

Other: 



Central Piedmont Community College, Charlotte, 
North Carolina, Drs. Barry G. Miller and 
Harry Snyder; Mecklenburg County Health Depart- 
ment, Charlotte, North Carolina, Dr. Luby Sherrill; 
The Research Division of the Kendall Company, 
Harrington, Illinois, Mr. James R„ Mellberg. 



■1/4 
3/4 
1/2 



Objectives : 



This study is being supported by contract number PH 43-67-60. 
The principal purpose of the project is to determine whether 
frequently repeated topical treatments with a concentrated 
sodium fluoride gel in mouth applicators can further reduce 
the low dental caries activity in children consuming fluori- 
dated water continuously from birth. Such applications will 
be made three times weekly for three minutes over a period of 
three years. 

Exfoliated deciduous teeth are also being collected from the 
children in order to determine how much additional fluoride can 
be acquired by enamel from the topical treatments over that 
acquired from consuming fluoridated water alone. 

. . 270 



Serial No. NIDR-57 (66) 

Part A (Continued) 

The occurrence and distribution of caries -conducive streptococcal 
strains in the dental plaque of children in Charlotte is being 
determined as part of an epidemiologic survey. The effect of 
the fluoride gel applications on the prevalence of this strain 
in the plaque will be studied. 

The usefulness of radiographs in addition to clinical examinations 
in the conduct of clinical trials will be evaluated. 

Methods Employed : 

Clinical and radiographic examinations of approximately 900 child" 
ren, aged 11-14 years, randomly assigned to one group applying a 
gel containing 1.1 per cent sodium fluoride or to another group 
(control) not applying the gels. Statistical analysis and inter- 
pretation of data collected initially and at periodic intervals. 
Fluoride analysis of the outer enamel layers of exfoliated de- 
ciduous teeth, storing them in Jordan's transport medium, and 
plating and identifying the streptococci on mitis-salivarius 
agar. 

Major Findings : 

Initial dental examinations were completed in November-December 
1966. Children in the experimental and control groups had 2.7 
and 2.6 DMF teeth, respectively, and the status of gingival health 
and oral hygiene was similar for both groups. 

The teeth treated with the sodium fluoride gel progressively 
acquired more fluoride as the number of treatments increased. 
Most of the acquired fluoride was restricted to the outer 
20 microns of enamel. For example, teeth receiving an average 
of 56 topical sodium fluoride treatments had 1,785 ppm fluoride 
in the outermost enamel layer as compared to 922 ppm fluoride 
for untreated control teeth from children consuming only 
fluoridated water. 

About 70 percent of the children in this fluoridated coromunity 
did not harbor caries -conducive streptococcal strains on their 
teeth. The repeated fluoride applications did not alter the 
occurrence and distribution of these microorganisms. 

Significance to Dental Research : 

The p^roject will demonstrate whether an additional anti-caries 
benefit can be obtained from repeated topical fluoride therapy 
in children consuming fluoridated water continuously .'Irom birth. 
If so, this technique could be recommended in fluoridated areas 
for the management of caseg of rampant dental caries., 

2 



Serial No. NIDR-57 (66) \ 

Part A (Continued) 

The fluoride analysis of teeth and the microbiological phases 
of this study will provide valuable information on the mechanism 
of fluoride action and the epidemiology of the caries -conducive 
streptococci. 

Proposed Course of Project : 

The thrice weekly gel applications will continue in school until 
June 1969. The follow-up clinical and radiographic examinations 
to determine the effect of the repeated fluoride applicators 
after 18 months will be conducted in May 1968. 

Deciduous teeth will continue to be collected and analyzed for 
fluoride. 

Urine specimens will be analyzed for fluoride to monitor possible 
fluoride ingestion. 

Further plaque samples will be collected for culturing in May 1968. 



Part B 



Publications 



1. Mellberg, J. R. , Nicholson, C. R. , Miller, B. G. , and 
Englander, H. R. : Acquisition of fluoride by enamel from 
repeated topical sodium fluoride applications in a fluori- 
dated area. J. Dent. Res . (In press). 

2. Jordan, H. W. , Englander, H. R. , and Lim, S.: The presence 
of potentially cariogenic streptococci in various population 
groups. lADR Preprinted Abstracts (370) 46th Gen. Meeting, 
March 1968. 



2T2 



Serial No. NIDR-58 (68) 

1. Biometry and Field 

Investigations Br. 

2. Clinical Trials Sec. 
3o Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Anti-Caries Effect of Repeated Topical Fluoride 
Treatments on the Deciduous Dentition 

Previous Serial Number: None 

Principal Investigator: Dr, Harold R. Englander 

Other Investigators: Dr. Harold V. Jordan, Jr. , Dr. James P. Carlos, 

and Dr. Peter J. Coccaro 

Cooperating Units: United States Coast Guard, Governors Island, 

New York, New York, Captain William 0. Engler, 
and Commander Edward D. Woolrldge 

Man Years 

Total: 1-1/4 
Professional: 3/4 
Other: 1/2 

Objectives : 

The principal purpose of this project is to determine whether 
frequently repeated daily topical applications of a concentrated 
sodium fluoride gel can maintain the initially caries-free de- 
ciduous dentition of pre-school children free from the initiation 
of smooth surface dental caries. The population selected for 
this study are children, aged 2-7 years, who are dependents of 
U.S. Coast Guard personnel. 

Exfoliated deciduous teeth are also being collected from the 
children applying the fluoride and placebo gels in mouthpieces 
in order to estimate .how much fluoride is acquired by enamel 
from the topical treatments, and to estimate the fluoride levels 
in the surface enamel of teeth of children who remain caries - 
free in the fluoride group and in the enamel of. those who 
develop caries in the placebo tieated group. 

273 



Serial No. NIDR-58 (68) 

Part A (Continued) 

The distribution of cariogenic streptococci in the dental plaque 
of caries free 2 and 3 year-olds is being follov^ed to study the 
relationship between the occurrence of these bacteria and the 
development of pit and fissure and smooth surface caries. 

Methods Employed : 

Clinical and radiographic examinations of approximately 400 
children, aged 2-7 years, randomly assigned to one group applying 
a gel containing 1.1 per cent sodium fluoride and 0.1 molar sodium 
phosphate (pH 4.5) and to another group applying a neutral placebo 
gel. Statistical evaluation of data collected initially and at 
six -month intervals. Chemical analysis of the fluoride concen- 
tration in enamel from exfoliated teeth. Collecting plaque 
samples from each child and culturing for streptococci, lacto- 
bacilli, and other microbiota. Measurement of dental casts of 
the children for growth and development studies. 

Major Findings : 

The children were examined for dental caries, gingival health 
(PI) and oral cleanliness (OHI) in November 1967. Plaque was 
also obtained from a sample of the children. 

Children in the gel and control group had 4.7 and 4,8 def teeth, 
respectively, and about 85 children in each group were caries- 
free. Group PI and OHI scores were similar. Ninety-six percent 
of the caries-free 2-year olds did not harbor cariogenic strepto- 
cocci. 

Significance to Dental Research : 

The use of the deciduous rather than the permanent teeth for 
evaluating the potential of an anti-caries technique has seldom 
been used in field testing, but it has certain advantages. Inas- 
much as the pits and fissures of deciduous molars are usually not 
as deep or as defective as those found in the permanent molars 
and proximal lesions are more readily detectable clinically, 
examiner error and bias is greatly reduced. Furthermore, since 
the carious lesions affecting deciduous molars initiate primarily 
on smooth surfaces, the value of the topical treatments in pre- 
venting smooth surface caries can be evaluated with greater 
facility. 

A comparison of the fluoride levels in the teeth of children 
remaining caries-free with those control children developing 
extensive caries will provide information concerning the optimum 
fluoride concentration necessary for a maximum anti-caries effect. 



271* 



Serial No. NIDR-58 (68) 
Part A (Continued) 

Studies on the plaque microflora will provide insight into the 
relations between the cariogenic streptococci and smooth surface, 
and pit and fissure decay. 

Proposed Course of Project : 

The repeated topical fluoride applications will continue for 
2.5 more years. The next clinical examination will be conducted 
in May 1968. Deciduous teeth from each child will be collected 
for fluoride analysis. Further plaque samples will be collected 
for culturing. 



Part B Not included. 






Serial No. NIDR-59 (63) 

1. Biometry and Field 

Investigations Br. 

2. Clinical Trials Sec. 

3. Bethesda, Maryland 



PHS -NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Experimental Dental Caries in the Syrian Hamster 

Previous Serial Number: NIDR-48 

Principal Investigator: Dr. Harold R. Englander 

Other Investigators: Dr. Paul H. Keyes 

Cooperating Units: None 

Man Years 

Total: 1 
Professional: 1/2 
Other: 1/2 

Project Description: 

Objectives : 

The anti-caries effect of dietary supplements with organic and 
inorganic phosphates has been evaluated. In other e^cperiments 
the anti-caries effect of prophylactic pastes containing various 
fluoride formulations have been assayed. 

Methods Employed : 

1. A caries-conducive ration has been supplemented with either 
monosodium phosphate, diammonium phosphate, sodium trimeta- 
phosphate, or sodium phytate. 

2. Infected animals fed a diet containing a high concentration 
of sucrose were given prophylaxes with silex or with abrasive 
pastes containing stannous fluoride or sodium-silico-f luoride. 

Major Findings : 

1. Significantly less cavitation developed in hamsters fed diets 

supplemented with the phosphates at a concentration of 2 percent 

276 



Part A (Continued) , Serial No. NIDR-59 (63) 

Dental caries scores were lowest for the animals given the 
trimetaphosphate. When the phosphate compound concentrations 
were reduced to 0.5 percent only the scores for the trimeta- 
phosphate group were significantly less than the controls. 
No anti-caries effect was observed when dietary supplementation 
with the phosphate compounds (0.5 percent) was made thrice 
weekly. 

2. Animals given one-minute prophylaxes with abrasive pastes 

containing stannous fluoride plus zircate or sodium fluoride at 
comparable fluoride concentrations developed less cavitation in 
the maxillary second molars than control animals receiving a 
prophylaxis with fluoride-free pumice. However the zircate 
paste did not have a greater anti-caries effect than the sodivim 
fluoride paste. 

Significance to Dental Research ; 

1. When phosphate compounds are administered intermittently in a 
caries-test ration, their anti-caries effect tends to disappear. 
It is therefore \inlikely they would have any importsmt potential 
as an anti-caries agent when consumed intermittently by hximan 
beings. 

2. Since the zircate paste containing stannous fluoride did not 
have a greater anti-caries effect than a paste containing 
sodium fluoride and there have been reports of its toxicity, it 
is doubtful that it should be recommended for clinical practice. 

Proposed Course of Project ; 

The anti-caries effect of polyphosphates will be studied. 

Evaluation of prophylactic pastes containing other fluoride 
formiilations will continue together with a fluoride analysis of 
hamster teeth receiving varying degrees of protection with fluoride. 



Part B 



Publications 



Englander, H. R. and Keyes, P. H. : Acid production in the dental 
plaque of hamsters protected from dental C8u:ies with sodium fluoride. 
J. Oral Thera. and Pharm. U: 382, March 1968. 



2T7 



Part B (Continued) Serial No. NIDR-59 (63) 

Keyes , P. H., Rowberiy, S. H. , Englander, H. R. , and Fitzgerald, 
R, J.: Bioassays of medicoments for the control of dentobacterial 
plaque, dental caries, and periodontal lesions in Syrian hamsters. 
J. Oral Thera. and Pharm. 3.: 157, Nov. I967. 



278 



Serial No. NIDR-60 (65) 

1. Biometry and Field 

Investigations Br. 

2. Epidemiology Section 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Studies of Oral Health in Persons Nourished by 

Stomach Tube 

Previous Serial Number: NIDR-44 

Principal Investigator: Dr. N. W. Littleton 

Other Investigator: Mr. R. M. McCabe 

Cooperating Units: Sunland Hospital at Orlando, Orlando, Florida 

Man Years 

Total: 1-3/4 
Professional: 1-1/2 
Other: 1/4 

Project Description: 

Objectives : 

Oral microorganisms (dental plaque) and intra-oral, food residues 
have been implicated in the etiology of oral diseases. The ob- 
jectives of this study are: 

1. To compare oral health status in persons nourished solely by 
stomach tube for long periods with that of persons who were fed 
orally. 

2. To compare plaque material from tube-fed persons with that 
obtained from persons fed orally in regard to: 

a. Acidogenic properties 

b. Selected bacterial components 

3. To compare the recovery of lactobacilli and streptococcus 
salivarius types from swabs of the oral mucosa obtained from 
tube-fed persons and persons fed orally. 

4. To investigate the effects of sucrose, invert sugar and starch 
on the acidogenic properties and selected bacterial components of 
dental plaque when these carbohydrates are given orally l:o persons 
nourished by stomach tube. 



Serial No. NIDR-60 (65) 

Part A (continued) 

Methods Employed : 

The oral tissues of about 400 mentally retarded, physically handi- 
capped persons were examined clinically. This population included 
about 75 persons who had been nourished by stomach tube for periods 
ranging from a few days to more than five years. Plaque material 
and swabs of oral mucosa were obtained from a subsample of these 
tube-fed persons. The pH of plaque material was determined in 
vitro with and without the addition of a 10 percent solution of 
sucrose, glucose or fructose. Plaque material, after appropriate 
serial dilutions, and swabs of the oral mucosa were plated onto 
selective culture media. Following incubation under 95 percent 
nitrogen and 5 percent carbon dioxide, growth of lactobacilli, 
filamentous bacteria and streptococci was estimated. Results on 
samples obtained from tube-fed persons were compared with those 
from similar samples obtained from a subgroup of persons fed orally. 

In addition, a single carbohydrate--sucrose, invert sugar, or 
starch--was given orally for 30 days to persons fed by stomach 
tube. Plaque samples were obtained from these persons before, 
during and after oral supplementation. The effects of the oral 
administration of these carbohydrates on the acidic potential 
and bacterial composition of plaque obtained from tube-fed persons 
was investigated. 

Major Findings : 

Evidence of dental caries activity in this population was limited 
almost entirely to missing teeth. Teeth with either open carious 
lesions or restorations were observed rarely. Gingival and perio- 
dontal disease was widespread and severe, but clinical evidence of 
disease was particularly overt in tube-fed persons. Plaque was 
present in the mouths of all persons in this study, but chess 
deposits were less extensive in persons fed by stomach tube than 
in persons who were fed by mouth. Abundant deposits of calculus 
were observed about the teeth of persons in both groups „ 

The pH remained above 6.0 over a one-hour experimental period 
following suspension of plaque material from tube-fed persons in 
10 percent solutions of sucrose, glucose, fructose, invert sugar, 
or starch. Under similar conditions the pH of plaque suspensions 
from persons fed by mouth decreased substantially and reached 
levels in the range of 4.3 to 4.8 within 5 to 10 minutes. Limited 
bacteriological study indicated that total streptococcus counts 
were lower in plaque from tube-fed persons. Filamentous bacteria, 
lactobacilli and streptococcus salivarius types. were isolated 
frequently from either plaque or from swabs of the oral mucosa 



280 



Serial No. NIDR-60 (65) 
P art A (continued) 

obtained from tube-fed persons. Between group comparisons clearly 
indicated that the acidogenic properties and certain bacterial 
components of plaque obtained from tube-fed persons differed from 
plaque obtained from persons who were fed by mouth. However, 
within group comparisons failed to establish any consistent re- 
lationships between these variables, 

A substantial increase in the acidogenic potential of plaque 
material was observed 30 days following oral administration of 
sucrose, invert sugar or starch to persons nourished by stomach 
tube. Exposure to sucrose and invert sugar, however, seemed to 
enhance the acidic properties of plaque to a greater extent than 
starch. Thirty days after the cessation of oral carbohydrate 
administration, the acidogenic capacity of plaque taken from 
these persons reverted to pre-test levels. Oral administration 
of these carbohydrates, however, had little effect on total 
counts of either streptococci or lactobacilli in plaque material 
from these tube-fed persons. 

In collaboration with Dr. Harold Jordan plaque material from a 
small sample of these children was examined for the presence of 
extracellular polysaccharide producing streptococci. These 
microorganisms were recovered from children eating by mouth, 
but they were not recovered from any of the 12 plaque samples 
obtained from tube-fed persons. 

Significance to Dental Research : 

The availability for study of persons nourished solely by stomach 
tube provides a unique opportunity to investigate the dynamic 
interactions between oral microorganisms and intraoral food 
residues in the etiology of oral diseases. 

Proposed Course of Project : 

Plans have been made to extend the study of oral diseases in this 
population. These plans include further study of the effects of 
selected carbohydrates on the oral microflora when these foods 
are introduced orally under well controlled, experimental con- 
ditions to persons nourished by stomach tube. 



281 



Serial No. NIDR-60 (65) 

Part B 

Publications: 

Littleton, N. W. , McCabe, R. M. and Carter, C. H. : Studies of 
oral health in persons nourished by stomach tube. II. Acidogenic 
properties and selected bacterial components of plaque material. 
Arch. Oral Biol , (in press). 



282 



Serial No. NIDR-61 (68) 

1. Biometry and Field 

Investigations Tr. 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through Jvine 30, I968 



Part A 



Project Title: Production of "Elfin" Facies and Abnormal Dentition by 
Vitamin D2 during Pregnancy: Relationship to the 
Supravalvular Aortic Stenosis Syndrome 

Previous Serial Niomber: None 

Principal Investigator: Dr. Loren F. Mills 

Other Investigator: None 

Cooperating Unit: National Heart Institute, Cardiology Branch, 

Dr. William F. Friedman 



Man Years 




Total: 


1-1/4 


Professional: 


1/2 


Other: 


3/I4 



Project Description: 

Objectives : 

Recent investigations have suggested that a derangement in vitamin 
D2 metabolism during pregnancy may be responsible for the cardio- 
VEiscular anomalies of the supravalvular aortic stenosis syndrome 
(SASS), especially when the latter are associated with idiopathic 
infantile hypercalcemia. Peculiar "elfin" facies and dental 
anomalies, especially malocclusion, are reported to be common 
features of SASS. 

The present study was undertaken to fiirther investigate the 
characteristic facial and dental features of SASS. 

Methods Employed : 

Through a series of test dosages of vitamin D it was determined 
that approximately 750,000 units of the vitamin is sufficient to 
produce most of the anomalies characteristic to SASS. Histologic 
and macro and microscopic techniques are being used for observation 
of the various teratologic al effects. Blood and bone samples have 
been submitted for calcium and phosphorus determinations . 

283 



Serial No. NIDR-61 (68) 

Part A (Continued) 

Major Findings : 

Various developmental defects of the cranio-f acial complex were 
observed in vitamin D offspring when compared to controls.. Under- 
development of the mandible resulted in dysgnathia and dental 
cross-bite. The abnormal asseous development responsible for 
the malocclusion occurred in 65 percent of the offspring. Differ- 
ences were noted in the timing of suture and f ortanel closure. 
Many of the bones of the cranio-facial complex were smaller, 
malformed, and had altered contour. Enamel hypoplasia of the 
central incisors was noted in 957o of the test offspring. None 
of these dental and facial abnormalities were noted in control 
offspring. 

Significance to Dental Research : 

The experimental findings in rabbits resemble anatomically the 
skeletal and orthodontic manifestations of SASS that have been 
reported in humans. Further use of this experimental technique 
may lead to a better understanding of the effects of hyper- 
vitaminosis D on growth and development. 

Proposed Course of Project : 

Further investigations are to be undertaken to (1) determine 
minimal dosage necessary to produce SASS (2) determine if defects 
of the craniofacial complex can be produced by hypervitaminosis D 
without SASS. 



Part B 



Publications 



Friedman, William and Mills, L. F. : The relationship between 
vitamin D and the cranio-facial and dental anomalies of the SASS, 
(Submitted to Pediatrics Feb., 1968). 



28«t 



Serial No. NIDR-62 (62) 
1. Biometry and Field 

Investigations Br. 
3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report ■ 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Epidemiological Studies of Malocclusion 

Previous Serial Number: NIDR-49 

Principal Investigator: Dr. Loren F. Mills 

Other Investigators: None 

Cooperating Units: Board of Education, Prince Georges County, Maryland; 

West Liberty State College, West Liberty, West 
Virginia; United States Naval Academy, Annapolis, 
Maryland; Cleft Palate Clinic, Lancaster, Pennsylvania 

Man Years 

Total: 1-1/4 
Professional: 1/2 
Other : 3/4 

Project Description: 

Objectives : 

To derive a direct and reproducible method for determining the 
size and shape of dental arches; 

To develop criteria for assessing the prevalence and severity of 
occlusal anomalies in populations; 

To apply these criteria in attempts to elucidate possible 
relationships between the factors under study. 

Methods Employed : 

By direct observation each individual in this study is examined 
for the following variables: 1) alignment of teeth; 2) maxillo- 
mandibular relationship; 3) length and breadth of the dental arches; 
4) size and morphology of teeth; and 5) height,' weight and other 
anthropometric characteristics. 

Z2S 



Serial No. NIDR-62 (62) 

Part A (Continued) 

These methods are being applied to populations to obtain both long- 
term and cross-sectional data. The initial step in this investi- 
gation of malocclusion was a pilot study conducted in about 500 
Midshipmen examined at the United States Naval Academy. The study 
was extended this year to include examinations of about 1000 college 
students, aged 18 to 24 years, at West Liberty State College. In 
addition, yearly examinations are obtained from about 300 school 
children, aged 6 to 18 years. 

Ma.ior Findings : 

Data obtained during the course of this study has indicated that a 
parabola computed from direct measurements of arch length and 
breadth adequately describes the size and shape of the dental 
arches. Tabulations indicate that malalignment of the dentition 
was inversely related to arch breadth. Size of teeth and arch 
length were apparently unrelated to the occurrence of malalign- 
ment. Results also indicate that in these samples the space 
occupied by the teeth in the dental arches decreases with age. 
This is consistent with the generally accepted concepts of 
approximal wear, mesial drift of teeth and difference in size 
between deciduous molars and bicuspids. 

Significance to Dental Research : 

Criteria developed during the course of this study could prove 
valuable in the investigation of orthodontic problems in popu- 
lations. These criteria could aid in detecting the need for 
early preventive orthodontic care. In population studies of 
malocclusion obtaining dental casts is time consuming and ex- 
pensive and therefore prohibitive in studies of large groups. 
This technique may obviate the need for dental casts in popu- 
lation studies concerning: (1) the effects of race, nutrition, 
climate, geographic location, or other environmental factors 
on the size and shape of the jaws, (2) malocclusion studies 
which estimate the space available for teeth and the effects 
of faulty mouth habits on jaw size, and (3) predetermining the 
appropriate size of the maxilla before cleft palate repairs 
are made by the plastic surgeon. 

Proposed Course of Project : 

Continue to analyze these data, and to test these findings in 
longitudinal study to evaluate their predictive potential. 
The future course of this study will be developed as the re- 
sults of the data are analyzed and interpreted. 



286 



Serial No. NIDR-62 (62) 



Part B 

Publications 



Mills, L F.Nis wander, J. D. , Mazaheri, M. , and Brunelle, J. 4 
Minor oral and facial defects in relative of oral cleft patients: 
(Accepted for publication). Angle Orthodontist, Dec. 2, 1967. 



287 



Serial No. NIDR-63 (66) 
1. Biometry and Field 

Investigations Br, 
3. Bethesda, Maryland 



PHS-NIH 

Individual Project Report 

July 1, 1967 through June 30, 1968 



Part A 



Project Title: Serial Extraction Study on Preadolescent Children Having 
Crowded Clsiss I Occlusion 

Previous Serial Number: NIDR-51 

Principal Investigator: Dr. Loren F. Mills 

Other Investigators: Dr. Richard Christiansen 

Cooperating Units: Board of Education, Prince Georges County, Maryland 



Man 


Years 
Total: 




1 






Profess 


iional: 




3/it 




Other: 






1/h 



Project Description: 
Objectives : 

To compare the changes which occur within the jaws of subjects who 
undergo serial extraction and subjects with a similar diagnosis who 
do not receive extractions. Also to compare the degree and direc- 
tion of the development of the facial bones. 

Methods Employed : 

Final screening of approximately 56 subjects will be conducted at 
the National Institutes of Health. The initial records secured on 
the 56 project subjects ultimately selected will include the 
following: (a) medical and dental histories; (b) detailed mouth 
examinations; (c) five cephalometric radiographs; (d) radiographs 
of individual teeth; (e) orthodontic models; (f) facial and intra- 
oral photographs; and (g) medical examination. The sample will be 
divided into two groups of 28 subjects, each comprised of Ik boys 
and lit girls. 



288 



Part A (Continued) 

Ma.1or Findings ; 

In May and October of 1967, approximately 1500 children, between 
the ages of 6-12 years, were examined in four schools in Prince 
Georges Covinty. Approximately k2 of these children fit the 
criteria for the study. 

Significance to Dental Research ; 

Serial extraction procedure is designed to anticipate and 
hopefully prevent the development of a fully matured deformity in 
the permanent dentition. The procedure of serial extraction is 
used by some to help guide the eruption of permanent teeth into 
more favorable positions in the arches. This study will try to 
dociiraent the concomitant changes that occur dentally and 
skeletally and their frequency of occurrence. 

Proposed Course of Project ; 

The data gathered to date will be utilized for a futvire publication. 
Plajis for the future coiirse of this study await necessary personnel 
and equipment. Reevaluation of diagnostic criteria is in progress. 



Psurt B not included. 



2SS 



Serial No. NIDR-64 (61) 
1. Biometry and Field 

Investigations Br. 
3. Sethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Clinical Trial of a Dentifrice Containing Phosphate 
and Fluoride 

Previous Serial Number: NIDR-53 

Principal Investigator: Dr. L. F. Mills 

Other Investigators: Dr. F. J. McClure and Mr. C. L. White 

Cooperating Units: Lancaster Cleft Palate Clinic and Milton J. Hershey 

School 

Man Years 

Total: 1 
Professional: 1/2 
Other: 1/2 

Project Description: 

Objectives : 

To determine if a dentifrice containing a soluble phosphate and 
sodium fluoride will reduce the incidence of dental caries. 

Methods Employed : 

A dentifrice containing 10 per cent soluble phosphate and 0.25 per 
cent sodium fluoride is being tested in a double blind study at an 
institution with 900 boys residing in approximately 50 cottages. 
The cottages were matched by caries prevalence and equal numbers 
were randomly assigned to study and control groups. The control 
dentifrice is similar but contains no soluble phosphate or fluoride. 
A base line of caries prevalence was established by a bite wing 
radiograph and visual dental examination. Caries incidence will 
be determined from periodic repetition of the bite wing and visual 
examination. Mean incidence of carious teeth and tooth surfaces 
are to be compared for study and control groups. 



290 



Serial No. NIDR-64 (61) 

Part A (Continued) 

Major Findings : 

Preliminary tabulations and analyses of the data failed to 
demonstrate any dental caries inhibiting effects of a tooth- 
paste containing sodium fluoride and 10 per cent soluble 
phosphate. Dental caries increments over a period of 36 months 
were essentially the same in both test and control groups. 

Significance to Dgntal Research : 

Dietary control of caries is impractical for large groups. 
Water fluoridation is not readily available to those who are 
not served by a communal water supply and has been denied to 
many others by the action of antif luoridationists or by the 
inaction of local authorities. It would be desirable to have 
available other effective methods for the mass control of 
dental caries to supplement fluoridation. Because tooth- 
brushing ia a widespread established habit available to all, 
therapeutic dentifrices offer promise as a means of inhibiting 
caries. There is abundant evidence from animal experimentation 
that a soluble phosphate in the diet is capable of inhibiting 
caries. Animal experiments further indicate that the cario- 
static actip^ of a soluble phosphate may be topical and hence 
its incorporation in a dentifrice to assure appreciable contact 
with tooth surface. Also, it has been reported that a topical 
solution copt^ining fluoride and phosphate, is more cariostatic 
than fluoride alone. 

Proposed Course Qf Project : 

This project was terminated in September 1967. Final analysis 
of the data is. in progress. Even though the results were nega- 
tive, many interesting aspects of the study, heretofore unpublished, 
have merit and Will be reported. 



Part B not included. 



/". 



91 



Report of the Clinical Director 
National lastitute of Dental Research 
Summary Statement 



In our struggle to preserve and maintain the maximum degree of health 
and function of the oral cavity, certain trends in our clinical dental 
research program are becoming apparent. 

The ideal of a dental researcher is to reach eventually a plateau of 
knowledge J understanding and control of dental problems that first the 
oral surgeon, then the endodontist, and then the periodontist will be 
eliminated as specialists. Certainly the day is on the horizon when the 
choice to extract a tooth will be as rare as the amputation of an infected 
limb and considered almost as primitive. 

Through our many human pulp studies standards have been established 
that define the tolerance level of survival for the dental pulp. With 
these standards, the clinicians now have an understanding as to which 
procedures are best for the dental pulp and can provide a better quality 
of clinical practice. Empiricism is losing ground in the field of restora- 
tive dentistry. As this area of research continues, more and more "wives' 
tales" will be gradually eliminated from dental curriculae and hopefully 
forgotten in the world's dental literature. 

In the same sense, similar standards of treatment need to be developed 
in the field of periodontology. Despite accepted periodontal treatment 
procedures, the frustrating problem of progrtission of periodontal disease 
is still too frequently with us. The knowledge to be gained from such 
extreme approaches as replantation and root resection will help us zoom 
in on less obvious, more subtle but critical insidious events. 

It is obvious in dealing with the areas of stomatitis and oral cancer 
that the discipline of immunology is looked upon as our greatest helping 
hand to solve some of our most difficult problems. At least if etiology 
is not soon revealed, a greater understanding of many of these odd 
oral diseases will be helpful in directing; future research. Repeatedly 
some aspect of immunology is reflected in many of the annual reports to 
follow. 

As replantation is a study in extreme to help us gain fundamental knowledge 
concerning periodontal disease, so in the study of Behcet's syndrome hope- 
fully knowledge will be gained to help us comprehend and treat realistically 
the less severe diseases such as aphthous stomatitis and periadenitis. 



292 



In all our efforts there is an undercurrent of the importance of the 
"normal.' Not only in many dental problems, pulp biology and periodonto- 
logy, but also in the oral mucosa, the pharynx, larynx, tongue, and 
temporomandibular joint, studies defining the range of normal are underway. 
Our profession so frequently has been misled by incorrect published 
information or the lack of adequate information concerned with the normal 
aspect of some portion of the head and neck area that such emphasis is duly 
warranted. It is a very healthy sign that among our staff so much 
attention is being given to the "normal," indicating a level of suspicion 
and uncomfortableness concerning our present level of knowledge and 
understanding. 

Certainly from the technical side of dentistry, our clinicians justifiably 
take their position alongside all the other specialties. In working with 
cancer, heart, and arthritic patients, the dental clinician has become 
a recognized member of a team of practitioners who is consulted not only 
after surgery but prior to and during the procedure in order to provide the 
best and most satisfying result for the suffering individual, not only from 
the functional but cosmetic point of view. 

In describing our program in a less specific manner, this stunmary has been 
held to a few pages. Although every effort has been made to recognize 
all projects equally, no doubt some bias could have arisen due to the 
author's emphasis in areas of greatest personal experience. 



293 



Report of the Oral Medicine and Surgery Branch 
National Institute of Dental Research 
Summary Statement 

With gradual expansion of the Oral Medicine and Surgery Branch we have 
attempted to acquire staff that would give emphasis to all the main 
problem areas of clinical dentistry--caries, periodontal disease, aphthous 
stomatitis, orthodontics, prosthodontics, endodontics, oral surgery and 
oral pathology. Although some areas are still under-staffed because of 
recruitment problems and space we feel that we are finally on the way, 

A. Biology of the Human Dental Pulp : In this project there continues 
an evaluation of the response of the human dental pulp to changes induced 
by dental drilling procedures and by various restorative and related 
materials, such as cavity liners. This study has furnished the dental 
profession with considerable practical information on operative proceduresj 
particularly in regard to optimal cutting speeds, the proper use of coolants, 
and modifications in technic such as employing liners and varnishes 
necessary for the safe placement of toxic restorative materials. 

Since pulp inflammation following high speed cutting technics is minimal 
as compared to low speed cutting technics, the incidence of reparative dentin 
formation is also reduced. Thus, cut dentinal tubules remain completely 
patent permitting the toxic or irritating products of cements, silicates, 
and epoxy resins to permeate to the pulp tissue and cause unnecessary and 
frequently irreversible damage. This lack of reparative dentin formation is 
creating a formidable problem in restorative dentistry, especially in the 
field of full mouth rehabilitation where often the entire coronal dentin 
becomes exposed through full-crown preparation. Experimental drugs designed 
to reduce sensitivity of teeth (i.e., corticosteroid compounds) and to more 
effectively seal the dentinal tubules are being sought, as well as drugs and 
technics to increase the incidence of reparative dentin formation. 

When prepared cavities are washed with a steroid formula, containing 
1% prednisolone in a vehicle of parachlorophenol, cresatin and gum camphor, 
before restoration with zinc oxide and eugenol, the pulp response to the 
cavity preparation is considerably minimized. When the prednisolone is used 
without the vehicle, the inflammatory response is minimized only 12 days. 
Also when the same formula is applied to the cavity preparation several days 
after the full potential of the response has occurred, the resolution period 
is still shortened. 

In an attempt to find more ideal restorative materials, collaborative research 
is being conducted with various dental manufacturers and the National Bureau 
of Standards who provide us with experimental restorative materials. Some 
of the experimental adhesive materials have the potential of supplanting the 



295 



conEionljf used anterior restorative materials such as silicate and acrylic i 
resin» A new alloy composed of gallium- tin-palladium is supposedly superior 
to dental amalgam. Also a new temporary protective packing (Pharmatec) , 
designed to function both as a temporary filling material and a provisional 
adhesive agent for crowns and bridges, is being evaluated. 

Because the Clinical Center can supply only about 300 human teeth per year 

for such studies it is necessary to supplement our needs with contracts I 

with several universities and other government facilities. 

(See Methods Employed and Major Findings) | 

In order to better correlate clinical symptoms of pulpitis with histo- 
pathologic pulp changes a means of standardizing clinical symptoms with 
intentional salivary contamination of a cavity preparation has been developed. 
This will greatly increase the diagnostic ability of the clinician. Another 
study to determine the rate of movement of bacteria in exposed dentinal tubules 
is in progress, an area of dental research that has been surprisingly 
neglected. 

Interestingly enough it has become apparent that the leprosy bacillus has 
a proclivity for the dental pulp of maxillary anterior teeth and can 
represent the first signs of re-activation of the disease. Such cases of 
leprosy are being pursued. 

Other clinical studies just underway involve the response of the pulp to the ( 
removal of cementum and the response of the periodontal ligament and alveolar 
bone to intentional perforation of previously intact root by endodontic 
procedures. 

In germfree rats several studies have been completed. The healing of 
experimentally exposed pulps in rodents is primarily dependent on the absence 
of a microbial flora. This healing process appears not to be altered 
substantially by the application of conventional or steroid drugs. 

B. Clinical and Morphologic Studies of Human Dentition : To characterize 
the clinical and microscopic manifestations of the deciduous and peruianent 
dentitions in inherited or acquired metabolic disorders has revealed several 
interesting findings. In hereditary hypophosphatemia, dentinal defects 
and tubular tracts in the pulp horn region by which microorganisms gain 
access to the pulp have been demonstrated. 

Premature exfoliation of deciduous or permanent teeth has taken on added 
significance as an ominous diagnostic sign for the physician, pediatrician, 
and dentist. 



29G ^ 



The metabolic defect of the odontoblast is assumed to be similar to the 
defect in metabolic transport proposed for the proximal renal tubular 
cell such that phosphorus cannot be incorporated into the apatite salts of 
the dentin. This clearly demonstrates the significance of incorporation 
of phosphorus into the calcifying dentin, an observation that may be most 
important from the standpoint of strengthening the tooth substrate and 
possibly increasing the resistance of the tooth to caries attack by the 
prophylactic addition of dietary phosphate supplements. 

C. Dental Caries ; From a research standpoint, rampant caries offers 
a most favorable opportunity to study the basic factors which activate or 
control the caries process because the usually prolonged time element in the 
development of carious lesions is reduced to a minimum. 

Clinical studies have indicated that the frequent eating of foods containing 
sucrose or other fermentable carbohydrates is a very important factor in the 
etiology of rampant caries. But during the past year an attempt has been 
made to find foods containing considerable amounts of fermentable carbohydrates 
but not cariogenic. Although fish protein concentrate contains from 100 to 
250 ppm Fluoride, this fluoride probably accounts for only part of the 
anticariogenic effect, mainly due to the relatively high level of calcium, 
phosphorus and basic amino acids. 

There is a need to develop more non- cariogenic snacks. The demonstration 
that fish protein concentrates exert a great anticariogenic effect on 
sucrose suggests that this material may be helpful in not only overcoming 
human malnutrition but also in the control of caries. 

The fluorescent antibody technique enables the microbiologist to examine 
the actual bacterial flora of dental plaque and tooth sections rather than 
having to rely on what organisms grow out (and out-grow others) in various 
artificial media. Since the plaque smears and paraffin tissue sections 
are stable for staining at a much later date, strict time elements can be 
eliminated from sample procurement. 

D. Studies of Soft Tissue Lesions ; 

1. Precancerous changes: Human buccal mucosa, although appearing 
clinically normal, may undergo various changes with the increasing age of the 
patient. Since the buccal site is frequently biopsied, standards need to be 
established on normal mucosa to eliminate errors in diagnosis due to the age 
factor. There is a need for correlation of clinical and histologic changes 
in the oral mucosa resulting from intrinsic or extrinsic factors. Particular 
interest is directed towards the keratinizing lesions of a premalignant 
or closely related nature. 



297 



Preliminary studies using tritiated thymidine on oral mucosa involved in 
verrucous carcinoma have revealed epithelial turnover rates remarkably 
similar to those believed to be found in normal oral mucosa. Similar 
studies have been performed on the oral mucosa of patients with Darier's 
disease. Preliminary results reveal a need for further utilization of this 
technique in the study of normal as well as disease states involving the 
oral mucosa. 

Detailed clinical and morphologic studies of leukoedema have been carried 
out with verification of the existence of this clinical entity originally 
described by Sanstead and Lowe in 1953. Comparison and analysis of histo- 
logic materials from their original study in 1953 with our cases revealed 
the histologic nature of the lesion to be chiefly one of parakeratosis and 
acanthosis; however, our studies gave no indication that the lesion was, as 
originally stated, of nutritional origin or that it was in any way premalignant. 

A previously undescribed change in the superficial epithelial layers of 
chronic hyperplastic oral mucosa has been studied. It occurs chiefly in 
specimens of epulis fissuratum though it may also be seen in irritation 
fibroma. On the basis of its morphology and histochemical finding of -SH 
groups, it has been referred to as "keratin pools." This change may 
be related to a particular environmental agent and may represent an allergic 
or toxic manifestation. 

2. Recurrent aphthous stomatitis: Investigations continue to I 
determine whether a relationship exists between the L-form of an alpha 
streptococcus in recurrent aphthous ulcers and the etiology of this disease 
or whether the disease is one of hypersensitivity. 

The immunology of these streptococci, streptococcal antigens, streptococcal 
immune complexes, and antistreptococcal sera are studied in both human 
and animal cell culture systems. Also, the histocompatibility of aphthous 
and nonaphthous cells in cell culture using radiated and nonradiated cells 
are investigated, as well as the role of lymphocytes in host-defense 
mechanisms. The successful treatment of a patient with immuno- suppressive 
drug indicates that the etiology of this disease is in part an immune 
mechanism. 

An attempt is being made to develop an accurate diagnostic test based on the 
presence of specific hypersenstivity in aphthous patients and determine if 
immunization would be an effective means of treatment. 

The laboratory experimental model for aphthous stomatitis is the hyper- 
sensitized albino female guinea pig. Hartley strain. Intradermal skin tests 
with aphthous streptococcus antigen indicate the degree of hypersensitivity 
and the effectiveness of desensitization procedures. 

The repeated intravenous injection of the Eli Lilly Streptococcal 
vaccine into hypersensitized guinea pigs reduces the skin reaction to the 
streptococcal cell wall antigen (CHO) indicating some degree of desensitiza- g 
tion. Similarly in certain patients the vaccine produced a gradual decrease " 
in frequency and intensity of the disease. 

298 

4 



Also an effort will be directed toward demonstrating the specific anti- 
bodies against the aphthous streptococcus 2A in the circulating lyiiiphocytef; 
of aphthous patients. 

3. The Study of Behcet's Syndrome ; In this newly initiated program 
it is very necessary to study the natural history of this syndrome, in an 
attempt to more closely define its nature, extent and course and at the 
same time derive information concerning possible etiology and a realistic 
therapeutic regime for these patients. 

In studying these patients emphasis will be placed on lymphocyte transformations 
antibodies to various oral and ocular antigens, flocculation and other sero- 
logic reactions, cryoglobulins, and immunoglobulins of various body fluids 
and cultures for microorganisms. 

Many serious diseases, often of the connective tissue type, have important 
oral manifestations. Behcet's syndrome is such an example, since 80 percent 
of these patients present with aphthous stomatitis. As an adjunct to the 
above program investigations will proceed on the mechanisms of cryoprecipita- 
tion in cryoglobulins, to determine the conformational and other physico- 
chemical and immunochemical properties that affect cryoprecipitation, to 
find specific causative determinants of cryoprecipitation factors affecting 
protein solubility and structural aspects of immunoglobulins, and to find 
a more rational therapeutic approach to cryoglobulinemia. 

The isolated cryoglobulin has the solubility characteristics of a euglobulin. 
Its intrinsic viscosity increases as temperatures increase. Reduction stops 
cryoprecipitability; reoxidation results in regaining this property but 
alkylation destroys cryoprecipitability irreversibly. 

The study of the immunologic behavior of protein in a wide variety of 
connective tissue diseases, such as S.L.E. and rheumatoid arthritis brings 
many important aspects to oral medicine as well. 

E. Clinical periodontal studies ; 

1, By studying the response of the periodontal tissues to tooth 
reimplantation it is hoped that some knowledge relative to the factors 
permitting the differentiation of fibroblasts into cementoblasts will be 
obtained. After reimplantation, the teeth become firmly attached with new 
alveolar bone formation. In the absence of continued cementum production, 
periodontal fibers lose their root attachment with eventual exfoliation. 
New functioning cementoblasts have not been found earlier than 29 days. 
Similar studies are being carried out on baboons with the intention 
eventually to substitute artificial teeth for natural teeth. 

2. In the treatment of the osseous defects in periodontosis, 
preliminary results indicate that the autogenous transplantation of developing 
third molars into the first molar sites can be an effective way of inducing 
healing of the alveolar bony lesions and in restoring periodontal health. 



23S 



4 



3. A long-term study of periodontal disease in a stable, adult 
male population has completed its first year. The occurrence of destructive 
periodontal disease is being assessed by direct observation on such a 
population. Examinations will be repeated biannually. A series of 
preliminary field examinations have been conducted to test the adequacy and 
reproducibility of the clinical criteria developed for use in this study. 
The initial series of examinations have been completed but the statistical 
analysis is incomplete. 

4. Children V7ith bizarre gingival lesions not corresponding to any 
human gingival disease or oral manifestations of any systemic disease are beini 
studied. Their lesions are characterized by severe gingival recession about 
the labial surfaces of one or more maxillary teeth. The gingival margins in 
the affected areas are usually ragged and bleeding. 

Prior to onset of oral problems, such children experience a sense of deprivation 
Much anxiety is expressed by the parents as such lesions become the center of 
family attraction. Gingival self-mutilation becomes a way of handling the 
anxiety generated by experiencing a loss of dependency needs. 

5. The staining morphology of various dental materials in tissue 

Problems of identification exist when dental materials are found in oral ^ 
and superficial gingival tissues microscopically. By intentionally I 

placing various materials into the rat dermis and running a series of 
staining reactions, identification of some of these extraneous materials 
when encountered will be more easily recognized. 

6. A study to determine the effectiveness of root amputation 
procedures to prevent progression of pocket formation is underway. The 
controversy of root amputation versus root planing and curettage has been 
debated for a number of years. Thus far, teeth treated by root amputation 
seem to be maintained easier. than those in which curettage is employed. 
This is a major problem facing the therapist. Many conflicting opinions have 
been voiced but no attempt at a controlled study has been carried out. 

7. The presence of dense cortical plate of bone lining most 
long-standing infra-bony defects has been implicated in the lack of success 
of new attachment procedures utilized in clinical periodontal therapy. 
Some therapists puncture or remove this bony plate but no evidence exists 
as to whether this exposure of marrow spaces would speed up bone formation 
with attachment in mind. To study the quantitative and qualitative aspects 
of healing of bony defects when the cortical plate covering such a defect 
has been removed is presently in progress. 



300 



E. Ardical periodontal studies : 

It appears that differences in the chemical constituents of saliva 
can influence calculus formation. The saliva of Holtzman rats fed either 
a high fat or a high protein diet for 30 days reveals more acid and alkaline 
phosphatase activity and an increased protein content of the whole mixed 
saliva. 

8. Epileptics since the late 1930 's have experienced the side 
effect of dilantin gingival hyperplasia. Years ago at NIDR considerable 
research was carried out on rats with no positive results. 

Since cats are now recognized as the most suitable animal for producing 
gingival dilantin hyperplasia, a renewed effort will be made to determine 
its mode of action. 

F. Oral surgery studies ; 

1. General anesthesia on ambulatory dental patients. 

Nitrous oxide was the first general anesthetic used by 
dentists. Because of its relative safety, no reliable records were kept 
on morbidity and mortality. 

With the advent of sodium pentothal and other intravenous drugs, which were 
more potent, it became important to study the changes in normal physiology 
associated with their use. We are constantly improving and refining present 
methods of anesthesia as well as laying a sound foundation for further 
research. Our studies have indicated the importance of nitrous oxide and 
oxygen supplementation and demerol premedication to assist in the control 
of adverse rises in blood pressure and tachycardia. 

2. Post-surgical tissue healing. 

To eliminate localized osteitis, the most encountered complica- 
tion of third molar surgery, neospirin powder is placed in the socket. At 
this point the efficacy of neospirin has not been exciting. 

3. Sectional roentgenographic study of the temporomandibular 
joint following bilateral osteotomy of the ramus of the 
mandib le . 

What happens in the TMJ? Does relapse of the newly 
established occlusion occur? This investigation will delineate any 
subjective or objective changes of the condylar head in the glenoid 
fossa. 



301 



4. Evaluation of premedication in conjunction with local 
anesthesia in oral surgical procedures 

Hospitals today are too overcrowded to admit patients 
just for the benefit of general anesthesia; therefore, different types 
of premedication drugs are evaluated. Each procedure is done \^ath a 
different intravenous premedication. It is hoped that the oral surgeon 
will be another tool for effective pain control. 

G. Oral pathology investigations ; 

1. Submucous fibrosis 

Submucous fibrosis is a disease very prevalent in India of 
unknown etiology but associated with a very high prevalence of oral cancer. 
It is the intent of this study to determine if chili powders can produce 
submucous fibrosis in the buccal pouch of hamsters. 

2. Betel quid carcinoma 

In a study of hamsters' pouches to determine the individual 
and/or combined roles that calcium hydroxide, tobacco and gambler might 
play in causing betel quid induced carcinomas, it was found that: 

a. Calcium hydroxide caused chemical burns with necrosis 
and ulceration followed gy regeneration. Three of these animals had 
atypical epithelial lesions, resembling focal leukoplakia in man. The 
affected pouches of hamsters treated with calcium hydroxide showed one or 
more of the following lesions: deposits of calcium, inflammation, fibro- 
blastic proliferation, ulceration, atrophy, hyperplasia, hyperkeratosis, 
parakeratosis, acanthosis, and cellular atypia. 

No squamous cell carcinomas were produced in any of the groups. But it has 
not yet been ascertained whether the lesions that developed were the final 
phase of the reaction of the treatment with calcium hydroxide or whether 
they had the potential of progression to neoplasia. No changes were noted 
in cheek pouches treated with snuff or starch powder alone. Several 
hamsters treated with gambier developed minute ulcers with inflammation. 

Distinguishing the causative carcinogenic agent in the betel quid 

chew is important to geographic pathology because of the high incidence 

of oral carcinoma in the area of the world where quid chewing is prevalent.- 

Until the causative factor is determined public health measures cannot be 

realistically investigated. 

In an effort to modify the epithelium of the hamsters' pouch, two experiments 
have been initiated to induce liver cirrhosis. Once sufficient liver damage 
is induced, the cheek pouches will be retreated to determine whether the 
prevalence for neoplastic change is increased. More and more evidence is 
accumulating to justify the belief that alcoholics are more prone to oral 
cancer. 

302 

8 



( 



Oral Pharyngeal Development Section : The activities of this Section contl-v.:c 
in diversification appropriate to the background and interests of investi- 
gators, but each project is oriented to an aspect of development of form 
and /or function of the mouth, pharynx and larynx. 

A new extension is that into embryology, as L. Krames is engaged in study 
of the neural determinants of the laryngeal skeleton, employing isotope 
labelling of elements of the neural plate which migrate to form, the larynx. 

Studies of postnatal development of the rat head skeleton are in stages of 
publication by M. Baer, J.F. Bosma, and J. Ackerman. 

The sequential development of the dental alignment and the facial skeleton 
in childhood is under epidemiological study by R.D, Christensen, L.F, Mills, 
and R.L. Christiansen, with observation of differential effects of serial 
dental extraction upon crowding of incisor teeth. R.D. Christensen, in 
collaboration with endocrinologists J. Roth and P. Gordon is specifying 
the acromegalic distortions of facial skeleton and associated soft tissue 
per cephalometric radiographs. The contribution of orthodontics to 
correction of skeletal deformities and dental alignment is under demonstra- 
tion by P. Coccaro in cleft palate children and in a child who had suffered 
fracture of a mandibular condyle in infancy. 

Correlated studies of form and of functions of sensation and of the 
separate motor actions of feeding and of speech have further evidenced 
aspects of the intimate association (or relation) betv/een somatic form, 
and neurally governed performance. Utilization of these composite criteria 
of form, sensation and movements, makes possible a more comprehensive 
definition of disorders or abnormalities. In some clinical circumstances 
such as that of "facial hypoplasia" the abnormalities of sensation afford 
clearest definition of the entity. In others, such as amyotrophic lateral 
sclerosis, the utilization of dual criteria of motion performance provide 
useful clues to available or potential motor resources. 

The correlations of form and function occasion new questions for investigation. 

Such, for instance, as the relation between abnormalities of facial form 

and disorders of smell and taste. Appropriate study is directed toward 

the neural determinants of somatic form in the facial area, but also to the 

possibility that both the facial skeleton and the chemosensory mechanisms 

of this area may reflect (evidence) a primary endocrine determinant. 

It is clear that the development of form is itself a function, but a function 

which reflects a composite or cynosure of multiple factors or influencing 

mechanisms, which contribute in a succession or temporally ordered array 

in embryonic, fetal, postnatal, and postmature development. 

This composite approach of clinical description gives us opportunity 
of definition of some of the facially malformed children and ad^ilts. But 
these criteria are not yet appropriate to the study of orally, facially, 
pharyngeally malformed infants, because of lack of anatomical cr-lteria of 
form, of criteria of sensory-guided motor function, and of criteria of 
subjective indications of, or response to sensory stimuli. 

301 



ThuSj at present, we are limited to the retrospections of the clinical 
histories of the disabilities in infancy of feeding and of respiratory 
functions in children, who may be identified syndromically. And, 
reciprocally, we must study the dysphagic and respiratory-impaired infants 
by standard routines of still and cine photography and radiography at 
appropriate sequences during their development until defined by criteria 
which are identified in older children as known or specific syndromes of 
disorders. 



20k 



{ 



10 



Serial No. NIDR-65 (c) (63) 
1. Oral Medicine and Surgery 
3. Bethesda, Maryland 



PHS -NIH 
Individual Project Report- 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Histopathology of Oral Mucous Membrane 

Previous Serial Number: NIDR-54 (c) 

Principal Investigator: Dr. H. 0. Archard 

Other Investigators: None 

Cooperating Units: National Cancer Institute, Dermatology Branch 

Man Years: 

Total: 3/4 
Professional: 3/4 
Other: 

Project Description: 

Objectives : 

1. Correlation of clinical and histologic changes in oral mucosa 
resulting from intrinsic or extrinsic factors. Particular interest 
is directed towards the keratinizing lesions of a premalignant 

or closely related nature. 

2. Identification of significant variables related to these clinical 
and morphologic changes. 

Methods Employed : 

1. Patients with oral keratinizing lesions are screened and a 
detailed environmental history obtained. This data is supplemented 
with clinical photographic records where possible. 

2. Representative bioffsy material is obtained and processed- by routine 
histologic techniques. Repeat biopsies are obtained where possible 

to delineate the pathogenesis of the disease process. 



305 



Serial No. NIDR-65 (c) (63) ( 
Major Findings : 

1. Leukoedema , as originally described by Sandstead and Lowe in 
1953, has been re-defined in the light of careful clinical and 
morphologic studies of some of Sandstead 's original material and 
several additional cases followed at the Clinical Center for a period 
of time. There was no indication that the lesion was, as originally 
proposed, of nutritional origin or that it was premalignant. 

2. A previously undescribed change in the superficial epithelial 
layers of chronic hyperplastic oral mucosa has been studied clinically, 
morphologically, and histochemically. It occurs chiefly in specimens 
of epulis fissuratum though it may also be seen in irritation fibromas 
and probably other pathologic mucosal tissue. On the basis of its 
morphology and the histochemical finding of -SH groups, it has been 
referred to as "kertin pools" and appears most commonly in chronically 
inflamed hyperplastic mucosa, particularly that associated with 
dentures (i.e., epulis fissuratum). 

Significance to Dental Research : 

1. It is now possible to separate histologically the condition 
known as leukoedema of the oral mucosa from otherclinical white 
lesions, some of which are of a premalignant or malignant nature. | 

2. The identification of the unusual "keratin pooling" in the 
superficial epithelium in chronic hyperplastic oral mucosa has 
considerable significance since this change may be related to 

a particular environmental agent (e.g., acrylic material of dentures) 
and may represent an allergic or toxic manifestation. 

3. Biodynamic studies of oral mucosa in various reactive and 
neoplastic (premalignant and malignant) states are potentially 
most significant. Such studies will provide information regarding 
the cellular activity of these various human oral lesions and 
indicate which morphologic changes have greatest prognostic value. 

4. The identification of specific morphologic alterations of the 
human oral mucosa in relation to particular environmental agents 
still occupies a pre-eminent position in applied clinical research. 
Prevention of most of these mucosal diseases is predicated on the 
identification and control of these environmental factors. 

Proposed Course of Project : 

1. The spectrum of keratinizing human oral lesions should be 
studied longitudinally for both the clinical and light microscopic 

306 ^ 



Serial No. NIDR-65 (c) (63) 

changes which occur. Each case should be approached from the 
standpoint of obtaining a detailed environmental analysis in 
conjunction with a properly documented record of the morphologic 
changes in order to demonstrate the clinicopathologic progression 
of the disease. Such studies, however, will require enhanced 
support in the form of improved technical services and facilities. 

2. Biodynamic studies of selected cases with keratinizing hyper- 
plastic or neoplastic oral lesions should be undertaken to assess 
the kinetics of these lesions. A sufficient number of cases will 
be needed to establish a significant norm for each of the various 
types of keratinizing oral lesions. 

3. Appropriate correlative studies of these lesions should be 
pursued employing such established techniques as electron micros- 
copy and/or histochemistry. 



Part B 



Publications : 



1. Archard, H. 0. , Carlson, K. P. , and Stanley, H.R. : "Leukoedema 
of the human oral mucosa." Oral Surg . 25: 717-728, May, 1968. 

2. Archard, H.O;: Chapters for "Dermatology in General Medicine" 
edited by Fitzpatrick, Van Scott, et al: Chap. 28) "Biology of 
the human oral integument" (accepted for publication). Chap. 29) 
"Common stomatologic disorders" (in preparation). Chap. 30) "Oral 
manifestations of systemic diseases" (in preparation) . 



307 



Serial No. NIDR-66 (c) (66) 
1. Oral Medicine and Surgery 
3. Bethesda, Maryland 



PHS -NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Clinical and morphologic studies of the human dentition 
in metabolic diseases, either acquired or inherited. 

Previous Serial Number: NIDR-55 (c) 

Principal Investigator: Dr. H. 0. Archard 

Other Investigators: None 

Cooperating Units: Section of Child Neurology, National Institute of 

Neurological Diseases and Blindness; Metabolic 
Diseases Branch, National Institute of Arthritis 
and Metabolic Diseases; Clinical Endocrinology 
Branch, National Heart Institute 



Man Years: 




Total: 


1/4 


Professional: 


1/4 


Other: 





Project Description 




Obiectives: 





1. To characterize the clinical manifestations of the deciduous 
and permanent dentitions in inherited or acquired metabolir dis- 
orders, particularly those currently under investigation by the 
various Institutes of the National Institutes of Health. 

2. To characterize the gross and light microscopic alterations in 
the deciduous and permanent teeth from patients with inherited or 
acquired metabolic disorders. 

Methods Employed : 

1. Patients under treatment for metabolic disorders are frequently 
seen in the Dental Services Branch of the Clinical Center. With the 
cooperation of the attending physician and dentist such patients are 
seen on a referral basis, and details of the clinical history, oral 
examination, and dental roentgenographic studies are incorporated 
into the patients' permanent records. 

308 



Serial No. NIDR-66 (c) (66) 

2. Arrangements are made for the proper histologic study of any 
teeth that are to be removed or are indicated for eventual removal. 
The teeth may be studied by routine decalcification, sectioning, 
and staining, or by preparation of ground' sections, depending on 
the nature of the metabolic disease process or the requirements of 
the tissue to be studied. 

Major Findings : 

1. Certain known inherited metabolic disorders have recently been 
recognized to present characteristic clinical and morphologic 
dental manifestations. We have characterized in detail both the 
clinical and morphologic features of the deciduous teeth in 
hereditary hypophosphatemia (vitamin D-resistant rickets) and clearly 
demonstrated previously undescribed dentinal defects and tubular 
tracts in the pulp horn region by which microorganisms gain access 

to the pulp of otherwise normal appearing teeth. 

2. Teeth from various metabolic diseases such as hypophosphatasia, 
pseudohypoparathyroidism, Hurler's syndrome, diabetes insipidus, 
etc., are in process of being examined, and clinical and morphologic 
changes will be reported when sufficient case material is available. 

Significance to Dental Research : 

1. The characterization of the morphologic changes in the dentin 
of the deciduous teeth of patients with hereditary hypophosphatemia 
has provided a basis for understanding the unusual clinical dental 
manifestations in this disease. 

2. The metabolic defect of the odontoblast in hypophosphatemia is 
assumed to be similar to the defect in metabolic transport proposed 
for the proximal renal tubular cell such thiat phosphorus cannot be 
incorporated into the apatite salts of the dentin. This clearly 
demonstrates the significance of incorporation of phosphorus into 
the calcifying dentin, an observation that may be most important 
from the standpoint of strengthening the tooth substrate. 

3. Premature exfoliation of deciduous or permanent teeth has taken 
on added significance as an ominous diagnostic sign for the 
physician, pediatrician, and dentist. 

Proposed Course of Project : 

1. Further collection of tooth specimens (both deciduous and per- 
manent) from patients with known metabolic diseases will be under- 
taken. 

309 



Serial No. NIDR-66 (c) (66) 
Part B 

Publications : 

1. Archard, H. 0. and Witkop, C.J.: "Hereditary hypophosphatemia 
(vitamin D-resistant rickets) presenting primary dental manifesta- 
tions. Oral Surg . 22: 184-193, August, 1966. 



310 < 



Serial No„ NIDR-67 (c) (59) 
1. Oral Meaicine and Surgery 
3. Bethegda, Maryland 



PH3 -NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Studies on the Etiology and Treatment of Periodontal 
Diseases in Children and Adolescents 

Previous Serial Number: NIDR-56 (c) 

Principal Investigator: Dr. P.N. Baer 

Other Investigators: None 

Cooperating Units: Dr. H. Hoffman, National Institute of Mental Health 

Man Years: 

Total: 1 
Professional: 3/4 
Other: 1/4 

Project Description: 

Objectives : 

1. To investigate the etiology of bizarre gingival lesions which 
occur in young children. The lesions are characterized by severe 
gingival recession about the labial surfaces of one or more maxillary 
teeth. The gingival margins in the affected areas are ragged and 
bleeding. 

Methods Employed : 

1. Children with bizarre gingival lesions which do not correspond 
to any known gingival disease or oral manifestation of any known 
systemic disease were studied. These patients were examined by a 
physician who, with the aid of proper laboratory tests ruled out the 
presence of a systemic disease. The patients and their parents 
then receive psychiatric interviews. Emphasis was placed on inter- 
viewing the parents as a couple, and, when indicated, individaally. 



311 



Serial No, NIDR-67 (c) (59) 
Major Findings ; 

1. Prior to the onset of the oral problems, each of the affected 
children was experiencing a sense of deprivation. 

2. In all cases a documented organic gingival lesion occurred 
during the time each child was experiencing the stress associated 
with the frustration of his dependency needs. 

3. A marked shift in the family dynamics occurred. Much anxiety 
was generated by the occurrence of oral pathology and each child 
became the center of his environment. 

4. It was hypothesized that at a time of extreme stress these 
children had organic oral pathology which significantly altered 
their environment. Their dependency needs were then met, albeit in 
a regressive way. They aggravated these lesions by excoriating 

and denuding their gingiva. This habit of gingival mutilation 
became a way of handling the anxiety generated by experiencing a 
loss of dependency needs. 

Significance to Dental Research ; 

Studies of this nature may provide new insights into the etiology 
of certain types of periodontal lesions. 

Proposed Course of Project ; 

Further investigations into other etiologic factors which may play 
a role in periodontal diseases in children is being pursued. 

Part B not included 



312 



Serial No. NIDR-68 (c) (66) 
1. Oral Medicine and Surgery 
3. Bethesda, Maryland 



PHS -NIH 
Individual Project Report • 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Studies on Experimental Calculus Formation 

Previous Serial Number: NIDR-58 (c) 

Principal Investigator: Dr. P.N. Baer 

Other Investigators: Dr. P.H. Keyes, Dr. I. Zipkin, Mr. C. White, Mr. N. 

Mantel, and Mr. G.R. Hawkins 

Cooperating Units: None 

Man Years: 

Total: 1-1/4 
Professional: 1/4 
Other: 1 

Project Description: ^ 

Subproject A : 

Objectives : 

To investigate whether the differences noted in calculus formation 
between Holtzman rats obtained from two different sources could be 
explained, in part, by the differences in the chemical constituents 
of the saliva. 



Methods Employed : 



Forty -four female, weanling Holtzman rats, 22 from the NIH breeding 
colony and 22 from the Holtzman Laboratories, Madison, Wisconsin, 
were used in this study. Each group was divided and fed either a 
high fat or a high protein diet, known to encourage calculus 
formation. At the end of 30 days, saliva was collected and chemi- 
cally analyzed for acid phosphatase, alkaline phosphatase, protein, 
total carbohydrate,' phosphorus, calcium, and rate of flow. The 
animals were then sacrificed, decapitated, the heads defleshed, 
and the molar teeth scored for calculus. 



313 



Serial No. NIDR-68 (c) (66) 
Major Findings : 

1. Calculus deposition was greater in the Holtzman rat obtained 
from the NIH colony than from the colony obtained from Madison, 
Wisconsin. 

2. The NIH Holtzman rats maintained on high fat and high protein 
diets showed more acid and alkaline phosphatase activity and an 
increased protein content of the whole mixed saliva, as compared 

to the Holtzman rat fed the same diet from the Holtzman Laboratories, 
Madison, Wisconsin. 

Subpro.iect B : 

Objectives : 

To investigate the effect on calculus formation of: (a) antibiotic 
administration, (b) housing a substrain that is relatively 
susceptible to calculus formation with one that is relatively 
resistant, and (c) diet. 

Methods Employed : 

To investigate the effects of an antibiotic and intermixing of the i 
two substrains on calculus formation, 15 NIH and 15 Wisconsin weanling 
female rats were caged separately, two animals per cage, while 
another 15 NIH and 15 Wisconsin rats were housed together, two of 
each strain to a cage. They were fed a high protein calculus- 
inducing diet for 30 days. In addition, 15 NIH and 15 Wisconsin 
rats, housed separately, were fed a high protein diet which con- 
tained 100 mg. of penicillin G per kilogram of diet. To investi- 
gate the effect of composition and consistency of diet on calculus 
formation, five groups, consisting of 15 animals per group of wean- 
ling female NIH Holtzman rats, were placed on the following dietary 
regimes: (1) a high protein diet for 60 days; (2) a high protein 
diet for 30 days and then Purina chow soaked in water, for 30 days; 
(3) a high protein diet for 30 days and Purina chow, pellet form, 
for 30 days; (4) Purina chow, pellet form, for 60 days; (5) Purina 
chow, mush consistency for 60 days. At the end of the experimental 
periods the animals were sacrificed, and the molar teeth scared for 
calculus. 

Major Findings : 

1, Factors involved in calculus formation were shown to be 
transmissible in rats fed a high protein, calculus -inducing 
diet. 



31ii 



i 



Serial No. NIDR-68 (c) (66) 

2. Penicillin incorporated in the calculus -inducing diet 
significantly reduced the amount of calculus formed. 

3. Housing a strain of rat which was relatively resistant to 
calculus formation with one that was relatively susceptible 
significantly increased the amount of calculus deposition in the 
more resistant strain. 

4. A nutritionally balanced, commercially available diet 
significantly reduced calculus formation to even a greater 
degree than penicillin. 

Significance to Dental Research : 

Calculus is believed to be an important etiologic factor in perio- 
dontal disease; all knowledge which can be obtained concerning its 
formation should be of major interest. 

Proposed Course of Project : 

Further studies on the effects of nutritional and transmissibility 
factors as they affect calculus formation will be pursued. 



Part B 



Publications: 



1. Baer, P.N. , Hawkins, G.R. , Wells, H. , Mantel, N. , and Zipkin, I.: 
Studies on Experimental Calculus Formation in the Rat. XI, Relation 
to Diet and Selected Salivary Constituents. J. Periodont. 38: 
323-329, July-August, 1967. 

2. Baer, P.N. , KeyeS, P. H. , and White, C.L. : Studies on Experimental 
Calculus Formation in the Rat. XII. On the Transmissibility of 
Factors Affecting Calculus. J. Periodont . (in press) 

3. Baer, P.N. : Use of Laboratory Animals for Calculus Studies. 
Proc. N. Y. Acad. Sciences (in press) 



315 



Serial No. NIDR-69 (c) (68) 
1. Oral Medicine and Surgery 
3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: The Effects of Blastomycosis on Oral Tissue 

Previous Serial Number: None 

Principal Investigator: Dr. W. A. Bell 

Other Investigators: Drs. G. E. Garrington and J. W. Gamble 

Cooperating Units: None 

Man Years : 

Total: 1/2 
Professional: 1/2 
Other: 

Project Description: 

Objectives : 

1. To study the disease pattern of North American blastomycosis in 
oral structures and differentiate it from those of other oral 
granuloma. 

2. To evaluate histologically the tissue penetration pattern of the 
blastomycosis organism. 

3. To evaluate treatment methods used on the patients reviewed. 
Methods Employed : 

Patient records from the National Institutes of Health of patients 
with oral blastomycosis are being reviewed along with submitted 
histologic material. 

Major Findings : 

The clinical and histologic material is still being gathered and 
evaluated at this time. 



316 



Serial No. Nn)R-69 (c) (68) 

Significance to Dental Research : 

1. Large numbers of case reports are needed to properly evaluate 
diagnosis, treatment, and probable disease pattern. 

2. Mycotic diseases of the mouth and para-oral structures are seen 
with increasing frequency by pathologists. 

3. The importance of patient histories will enable the practicing 
physician or dentist to possibly link environmental or occupational 
hazards . 

Proposed Course of the Project : 

It is intended to have at least eight or nine cases with documented 
follow-up. New cases will be added to the above group as they are 
submitted. 

Part B not included 



317 



Serial No. NIDR-70 (c) (68) 
1. Oral Medicine and Surgery 
3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: The Staining Morphology of Various Dental Materials in 
Tissue 

Previous Serial Number: None 

Principal Investigator: Dr. W. A. Bell 

Other Investigators: Dr, G. E. Garrington 

Cooperating Units: None 

Man Years : 



Total: 


1/2 


Professional: 


1/2 


Other: 






Project Description: 
Objectives : 

1. To provide a histologic demonstration of the various dental 
materials as seen when surgically introduced into the dermis. 

2. To evaluate tissue response for each given substance. 

3. To evaluate the staining characteristics for determining the 
identity of the embedded substance. 

Methods Employed : 

Various materials have been gathered which include silver amalgam 
particiles, gold dust, gutta percha, carborundum dust, zinc phosphate 
cement dust, Kerr's root canal sealer, silicate cement, enamel dust, 
steel filings, calculus particles, diamond dust particles, and pumice, 



318 



Serial No. NIDR-70 (c) (68) 

Major Findings ; 

The materials needed for this project are being gathered at this 
t ime . 

Significance to Dental Research ; 

1. When evaluating autoradiography studies with hemotoxylin and 
eosin-staining techniques, some foreign-body material can be 
accidentally introduced. This study would possibly help ideuiiify 
some of these extraneous materials. 

2. When evaluating dental or para-oral structures histologically, 
pigments are often encountered whose exact nature cannot be deter- 
mined morphologically. 

Proposed Course of Project : 

Various dental materials will be surgically placed in the extremi- 
ties of experimental animals and the staining characteristics of 
each substance will be noted. 

Part B not included 



319 



Serial No. NIDR-71 (c) (68) 
1. Oral Medicine and Surgery 
3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: A Measure of the Effectiveness of Root Amputation as a 
Means of Slowing Pocket Progression 

Previous Serial Number: None 

Principal Investigator: Dr. A.F. Binderman 

Other Investigators: Dr. T. Lundy 

Cooperating Units: None 

Man Years : 



Total: 


3/4 


Professional: 


1/2 


Other: 


1/4 


Project Description 


• 


Objectives : 





1. To determine the effectiveness of root amputation procedures in 
the long-term change in rate of progression of pocket formation in- 
to the furcation areas of teeth, 

2. To determine, from clinical staining procedures, differences in 
abilities to maintain root-amputated teeth hygienically, in compari- 
son to a group of root-planed, curetted teeth with similar involve- 
ment in the same patient. 

Methods Employed ; 

1. During a two-year period of time, patients exhibiting similar 
involvements of furcation areas on a molar on both the right and 
left sides of the arch have been utilized. 

2. On one side, dependent upon which furcations are involved, under 
xylocaine anesthesia, a root is removed. On the other side, also 
under xylocaine anesthesia, thorough root planing and curettage are 
accomplished. Both sides have a dressing placed for two weeks. At 
this time the dressings are removed and both sides are cavitroned on 
exposed tooth surface to remove all particles of pack, desquamated 
epithelial cells, etc. 

320 



Serial No. NIDR-71 (c) (68) 

3. Previous to the above, all teeth have been thoroughly scaled and 
the patients instructed on home care procedures (modified Stillman's 
technique utilizing a Broxodent electric toothbrush, Stim-U-Dents, 
and use of a Lactona interdental stimulator) , and have had a care- 
ful occlusal adjustment. Thus, each patient commences with only the 
variable of home maintenance to act on the rate of pocket formation. 

4. At two-month intervals, radiographs and clinical photos of the 
operated areas, after the patient has utilized a crest dye tablet, 
are taken. Plaque formation around the two teeth is noted in this 
manner, and pocket depth is recorded. Patients will remain under 
observation for a period of two years. 

Major Findings : 

1. Thus far, teeth treated by root amputation seem to be more easily 
maintainable than those in which curettage is employed. These are 
only, however, very short-term observations, and inasmuch as the 
study is a long-term result, are, as yet, of little value. 

Significance to Bental Research : 

1. No long-term studies of tooth survival after varying therapeutic 
regimes have been done in bi- and tri-furcation involvements. This 
is a major problem facing the therapist. Many conflicting opinions 
have been voiced regarding root amputations as a long-term solution 
to this problem, but no attempt at a controlled study has been yet 
made. This study will be important, therefore, in evaluating long- 
term prognosis, on the basis of a two-year maintenance trend, and 
thus help to determine if root amputation is actually an effective 
procedure. 

Proposed Course of Project : 

Dependent upon results of this study, if root amputation is deter- 
mined to be a valid therapeutic procedure, the study will branch into 
the restorative aspect of the question. Series of teeth with one or 
two roots removed will be restored with full crowns, giving them 
proper contour relationships with the hard and soft supporting tissues, 
These will be compared to unrestored, root -amputated teeth, in terms 
of plaque deposits, pocket formation, and mobility, over a course 
of two years . 

Part B not included 



321 



Serial No. NIDR-72 (c) (68) 
1, Oral Medicine and Surgery 
3. Bethesda, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1957 through June 30, 1968 

Part A 

Project Title: Studies in the Healing of Alveolar Bone in Dogs. 

Previous Serial Number: None 

Principal Investigator: Dr. A.F. Binderman 

Other Investigators: Drs. P.M. Lightbody and P.N. Baer 

Cooperating Units : None 

Man Years: 

Total: 3/4 
Professional: 1/2 
Other: 1/4 

Project Description: 

Objectives : 

1. To study the quantitative and qualitative aspects of the heal- 
ing of a bony defect when the cortical plate covering such a defect 
has been removed. 

2. To compare thea qualitative and quantitative aspects with those 
of defects in which the cortical covering has not been removed. 

3. To determine the causes of variations in the healing rates in the 
two aforementioned variable conditions. 

Methods Employed : 

1. Six dogs, each placed under IV nembutal anesthesia, were sub- 
jected to the extraction of maxillary and mandibular left and right 
second bicuspids. The right side remained as a control. 

On the left side, utilizing a high speed #2 round bur, numerous per- 
forations were made through the cortical plate of the socket, into 
the adjacent cancellous bone, in order to expose and give access to 



322 



Serial No. NIDR~72 (c) (68) 

the marrow cavities within this bone, effectively reaiovirig the co, 
tical plate. The sockets on both the left and right sides were 
closed by the drawing over and suturing of the overlying soft tis- 
sues. The animals were then placed on a liquid diat for one 'sjesk, 

2. At intervals of 3, 5, 7, 15, 30, and 90 days, the dogs were, 
and are, being sacrificed and four block sections are taken, .f>',e3e! 
will include the extraction site and one adjacent tooth on either 
side. 

3. Radiographs are then taken on occlusal films as one means of 
comparison of new bone formation. 

4. The block sections are then prepared for sectioning and stain- 
ing ( H&E, Mallory trichrome) . 

There is no patient material. 

Major Findings ; 

1. Examinations of radiographs show them to be not sensitive 
enough to pick up slight variations in degrees of bony fill and 
calcification, at least to the extent that variations exist within 
the framework of this study. 

2. Histologic sections are being prepared at the present time. 

Significance to Dental Research ; 

The presence of a dense, cortical plate of bone lining most long- 
standing, infra-bony defects has been implicated in the lack of 
success of new attachment procedures utilized in clinical perio- 
dontal therapy. In order to more predictable achieve their desired 
results, many therapists have been puncturing or removing this bony 
plate as a part of their procedure. No evidence exists, however, 
as to \^ether this exposure of marrow spaces would speed up bone 
formation rate, or would, in fact, retard it, because of the in- 
flammatory reaction that could be caused by the manipulation of the 
boiE. This study will measure the relative responses accompanying 
such a therapeutic variable, determining whether it is justified or 
not. 

Proposed Course of Project ; 

Dependent upon histologic results, clinical application of this de- 
corticalization will be evaluated in patients undergoing periodon- 
tal therapy in the Clinical Center. 

In a series of lesions specifically indicated for new attachment 

323 



Serial No. NIDR-72 (c) (68) 

procedures, decor ticalization will be applied. After following 
their course by monthly radiographs taken with utilisation of the 
x-ray grid over the film (to measure degree of new bony attachment) , 
at six months, the areas will be re-entered and re- photographed to 
determine what their course has been. (re-entry will be necessary 
anyway, inasmuch as recontouring of bone to its desired architec- 
tural form must be done in re-attachment procedures in the vast 
majority of cases). 

A trend in clinical predictability may then be offered by study of 
these cases; clinical comparisons are at best unlikely in the 
same arch, as two lesions of comparable architectural arrangement 
are rarely found in one mouth. 



Part B not included 



321* 



i 



Serial No. NIDR-73 (c) (68) 
1. Oral Medicine and Surgery 
3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Study of Behcet's Syndrome 

Previous Serial Number : None 

Principal Investigator: Dr. Norman A. Cummings 

Other Investigators: Dr. Thomas Francis, Dr. Donald Bergsma, Dr. John 

Graef, Dr. Thomas Mlnas, Dr. Vernon Wong, Dr. John 
Decker, Dr. Daryll DeVivo and Dr. Richard Epstein 

Cooperating Units: NIAMD, Arthritis and Rheumatism Branch; NINDB, 

Ophthamology Branch; NINDB, Medical Neurology Branch; 
NIMH, Laboratory of Clinical Science; NCI, Dermatology 
Branch 



Man 


Years: 








Total: 




1/2 




Profess 


ional: 


1/4 




Other : 




1/4 


Pro. 


ject Description 




Obi( 


actives : 







1. To recruit and select patients for clinical study v/ith Behcet's 
Syndrome and related systemic diseases with aphthous stomatitis or 
other serious oral manifestations. 

2. To study the natural history of these syndromes, in an attempt 
to more closely define their nature extent and course. 

3. To correlate histopathologic, cultural and chemical findings 
with clinical observations in order to derive information concerning 
possible etiology of these conditions. 

4. To derive a successful and clinically substantiated therapeutic 
regime for these patients. 



325 



Serial No. NIDR-73 (c) (68) f 
Methods Employed : 

1. Patients are screened for admission by outpatient examination 
or perusal of referral records. 

2. Admitted patients have detailed history and physical examinations 
performed; tests on serum and cerebrospinal fluid are carried out 
for a variety of studies. Included are: (1) lymphocyte transformation, 
(2) antibodies to various oral and ocular antigens, (3) flocculation 
and other serologic reactions, (4) cultures for bacteria, fungi, 

PPLO and viruses, (5) cryoglobulins, and (6) immunoglobulins of 
various body fluids. 

3. All patients have careful attention paid to nervous system, ocular, 
and psychiatric status. 



4. Tissue biopsy is performed, with cultures, of indicated oral, 
skin, eye and genital lesions. 

5. X-ray examination of sacro-iliac joints is carried out. 

6. Other clinical and laboratory parameters as indicated are obtained 
when necessary. 



Major Findings : 

This phase has just begun in late 1967. Careful correlation and 
evaluation of data is not yet ready, and would be premature. 

Significance to Dental Research : 

Many serious diseases, often connective tissue diseases, have impor- 
tant manifestations in the mouth. Behcet's Syndrome is a major 
example, since 807o of these patient's present with aphthous stomatitis 
This syndrome is common in Mediterranean countries and may be more 
prevalent in the U.S.A. than previously thought. The Syndrome itself 
can be malignant and may carry a mortality of over 107o. It can 
result in blindness, neurologic disability, and long years of pain 
and illness. 

Besides studying the course, etiology and potential therapy of 
Bulicet's Syndrome, this project attempts to consider those broader 
aspects of serious disease states which have important oral manifes- 
tations. This seems particularly true of connective tissue diseases 
(such as Behcet's), and thus their study may serve as a link between 



I 

[ 

I 



511,1 



hTsic research and clinical aspects of both internal medicine J 
dentistry. " 



32B 



Serial No. NIDR-73 (c) (68) 
Proposed Course of Project ; 

As above, under Objectives and Methods Employed. 
Part B not included 



327 



Serial No. NIDR-74 (c) (68) 
1. Oral Medicine and Surgery 
3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Mechanisms of Cryoprecipitation in Cryoglobulins 

Previous Serial Number: None 

Principal Investigator: Dr. Norman A. Cummings 

Other Investigators: Drs. Martin D. Lidsky and Henry Metzger 

Cooperating Units: Houston Veteran's Administration Hospital, Baylor 

University College of Medicine, Arthritis Research 
Section; NIAMD, Arthritis and Rheumatism Branch 

Man Years; 

Total: 1 
Professional: 3/4 
Other: 1/4 

Project Description: 

Objectives : 

1. To determine the conformational and other physiochemical and 
immunochemical properties that affect cryoprecipitation, and to 
study analytically what departure from normal amino acid content or 
arrangement leads to this change. 

2. To find specific causative determinants of cryoprecipitation, and 
compare these with information from parallel studies on cryoglobulins 

derived from other species. 

3. To interpret the evidence in terms of factors affecting protein 
solubility and structural aspects of immunoglobulins. 

4. To gain better insight into the possible relationship between 
protein solubility and certain connective tissue diseases. 

5. To lead to a more rational therapeutic approach to cryoglob- 
ulinemia. 



328 



Serial No. NIDR-74 (c) (68) 

Methods Employed ; 

1. Cryoglobulins are isolated from the sera of affected patients by 
repeated cold precipitation and washing. Purity is evaluated by 
analytical ultracentrifugal, electrophoretic, and solubility studies, 
as well as immunochemical techniques. 

2. The isolated cryoglobulins are examined in different media to 
study conformational changes at different temperatures. Experiments 
are performed using viscometry, titration, diffusion, optical 
rotatory dispersion, ultra-violet spectroscopy, and analytical 
ultracentrifugation. 

3. In parallel experiments, the globulin molecule is digested into 
various fragments and chains in order to more closely approximate 
that portion of the molecule wherein the properties responsible for 
cryoprecipitability reside. 

4. Peptide mapping of smaller fragments are compared to analogous 
molecular fragments from normal IgG. Amino acid composition by 
spetrophotometric and chemical techniques is determined. 

5. Relationships between structure and solubility is considered in 
light of the above data, with regards to (a) cryoprecipitability; 
(b) cryoglobulins from other species; (c) structure and function of 
immunoglobulins; (d) problems of protein solubility in terms of 
thermodynamic parameters, and (e) possible relationship to connective 
tissue diseases. 

Major Findings : 

1. The isolated cryoglobulin has the solubility characteristics of 

a euglobulin, and satisfies criteria of purity Immunoelectrophoresis, 
cellulose acetate electrophoresis, gel filtration, and analytical 
ultracentrifugation. It has and S25,w of 6.61; solubility is a 
linear function of both temperature and ionic strength. 

2. The characteristics of molecules precipitating at various 
temperatures are the same by all physicochemical criteria determined. 

3. Intrinsic viscosity of the cryoglobulin increases significantly 
as temperatures increase from 25° to 37° C. in 1.0/4 buffer; similar 
changes do not occur in normal IgG. No aggregation is detected under 
these conditions. 

4. Reduction stops cryoprecipitability; reoxidation results in 
regaining this property. Alkylation after reductions destroys 
cryoprecipitability irreversibly. 



329 



Serial No. NIDR-74 (c) (68) 

5. Peptic digestion results in a 5S fragment which retains over 30% 
of cryoprecipitability, implying that the C-terminal half of the 
heavy chains are not necessary. 

Further reduction and alkylation reveals that the light chains do 
not cryoprecipitate; the N-terminal half of the heavy chains are 
insoluble in aqueous media. 

6. Spectrophotometric titration data indicate a decrease in the 
tyrosine content of cryoglobulin compared to normal IgG, while 
tryptophan is unchanged. These data are confirmed by standard amino 
acid analyses. 

Significance to Dental Research : 

This work studies the relationshop between protein solubility and 
structure with regards to temperature. It offers an opportunity to 
explore the phenomenon of solubility as well as the primary to 
tertiary structure and immunochemical behavior of normal and patho- 
logic immunoglobulins. 

In addition to gaining further insight into immunologic behavior of 
proteins, a subject in itself important with respect to many aspects 
of oral medicine, the project deals with a protein found to be present 
in a wide variety of connective tissue diseases. These diseases, 
such as S.L.E. , often have important oral manifestations; the 
relationship of protein precipitates to pathological lesions, such as 
found in rheumatoid arthritis, etc., is yet to be fully explored from 
a physicochemical point of view, whether those lesions are generalized 
or located specifically in the mouth. 

Proposed Course of Project : 

As outlined above under Objectives and Methods. 

Part B 

Publications: 



1. Cummings, N.A. , Kuff, E.L. , and Sober, H.A. : Examination of 
Magnesium Binding to Serum Proteins by Ultracentrifugal Analysis. 
Analytical Biochemistry 22: 108, 1968. 



330 



I 



Serial No. NIDR-75 (c) (58) 
1. Oral Medicine and Surge^'y 
3. Bethesdaj J4aryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: General Anesthesia on Ambulatory Dental Patients 

Previous Serial Number: NIDR-56 (c) 

Principal Investigator: Dr. E.J. Driscoll 

Other Investigators: Dr. P. Lightbody 

Cooperating Units: Dr. C.L. Hebert, Anesthesiology Department, 

Clinical Center, Dr. C. Battig, Biomedical 
Engineering and Instrumentation Branch, DRS 

Man Years : 

Total: 2 
Professional: 1 
Other : 1 

Project Description : 

This is a report of a recently reactivated project. However, 
since almost all of the personnel, both professional and ancillary 
had to be trained or retrained and since the research endeavor 
has only recently assumed a normal operating facility, this report 
is necessarily concise and sketchy. 

Objectives : 

To study the changes in normal physiology which are caused by 
or associated with the general anesthetics which are used by 
oral surgeons on ambulatory dental patients. 

Our original study became operational in 1958 and was temporarily 
interrupted for 18 months in 1966. 



331 



Serial No. NIDR-75 (c) (58) 



Me thods Employed : 



1. Full mouth extractions are selected rather than random 
oral surgical procedures, so that the surgical trauma 
could be standardized. The teeth are extracted in 
quadrants, and the entire operation is systematized for 
over-all time of procedure and traumatic experience. 

2. A multi-channel polygraph is employed and the physiologic 
data being recorded are: pulse, blood pressure, arterial 
0„ satxiration, respiratory phenomena, cortical brain 
activity and the electrical activity of the heart (EKG) , 
Likewise recorded are such important technical data as 
amount of drug used in units of time, length of operation, 
duration of sleep, mental alertness on awakening, age, 
race, sex, emotional status in relation to the conduct of 
anesthesia and post-anesthetic sequelae (headache, nausea 
and vomiting, depression, and hiccoughs). 

Patient Material : 

So far we have reported on over 1200 operations in utmost detail. 
We have no significant numbers to add to this total in this past 
year's limited experience. 

Si gnificance to Dental Research : 

1. One of the first surgical procedures ever performed under 

general anesthesia was a dental extraction. The short dental 
procedure was found to be very suitable to the early attempt 
to accomplish painless surgery on the unconscious subject. 
Dentistry was, therefor^ closely identified historically with 
the early development of general surgical anesthesia. 

Nitrous Oxide was the first general anesthetic used by dentists 
and it actually survived the early "trial and error" experience. 
In the early 20th Century it was the only general anesthetic 
used by the majority of dental specialists for procedures on 
ambulatory patients. 

Very little was known about the physiological behavior 
associated with the use of this gas; but, because of its 
relative safety and the fact that practically no reliable 
records were kept on morbidity and mortality, the true story 
was never known, fully appreciated or for that matter even 
questioned. 



332 



Serial No. NIDR-75 (c) (58) 

2„ With the advent in the late 1930' s of Sodium Pentothal and 

other intravenous drugs, which were also found very applicable 
to dental anesthesiology, a change in philosophy was inevitable. 
The drugs were much more potent and since they could easily 
kill or cause serious morbidity, a much more cautious attitude 
was necessary. 

3. Our research team as well as others interested in this area 
of investigation are diligently searching the literature and 
other sources for information regarding newly discovered 
anesthetic agents which might be safer and more effective. 
These drugs are being and will continue to be produced; but, 
without these baseline data on physiologic response, which 
are the results of our previous studies, it would be difficult 
to impossible to evaluate them. 

4. We are constantly studying the possibility for improvement 
and refinement of present methods of anesthesia, as well as 
laying a sound foundation for future anesthesiology research. 
An example of refinement in methods is indicated in our studies 
of N2O and O2 supplementation and demerol premedication, both 
of which have been shown to assist in the control of adverse 
rises in blood pressure and to a lesser degree tachycardia. 

Proposed Course of Project : 

We have established the broad baselines of performance on all 
anesthetic agents and combinatiois thereof, which are presently 
utilized by oral surgeons and we are now ready to examine some 
of these physiological parameters in detail and to attempt 
practical clinical interpretations. 

Changes of significant proportions have been shown to occur in 
such vital physiological areas as blood pressure and pulse, and 
it is, therefore, extremely important that the causes of these 
peripheral cardiovascular responses be investigated in depth. 
For example, if an increase in cardiac output is found to attend 
these blood pressure and pulse elevations, this is a far more 
favorable finding in terms of patient safety, than if a decrease 
in cardiac output is demonstrated. If the latter is the case, 
then the pulse and pressure elevations are potentially a far 
more serious finding. 

Other very fertile areas for future investigation stem naturally 
from our past demonstration of deviations from normal physiology 
in the anesthetized state. Various abnormalities in the EKG 
have been consistently shown to occur in this type of anesthesia. 
A more detailed investigation is planned to study these changes 
in the electrical conduction system of the heart. 



Part B included: Yes 



333 



Serial No, NIDR-75 (c) (58) 

Part B 

Honors and Awards relating to this project : 

1. Horace Wells Award 196/ - For outstanding research in 
dental anesthesiology. 



33ii < 



Serial No. NIDR-7& (c) (66) 
1. Oral Medicine and Surger;;' 
3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Immunologic, Biochemical and Microbiological Studies in 
Recurrent Aphthous Stomatitis 

Previous Serial Number: NIDR-59 (c) 

Principal Investigator: Dr. T.C. Francis 

Other Investigators: Dr. H.R. Stanley, Dr. J.F. Bosma, Dr. E.A. Graykowski 

Cooperating Units: Division of Biologies Stand£.rds, Dr. M. F. Barile 

Man Years: 

Total: 2 
Professional: 1 
Other: 1 

Project Description: 

Objectives : 

1. To study the clinical aspects of aphthous stomatitis. 

2. To study the microbiologic and immunologic relationship of 
Streptococcus sanguis and other related streptococci in this disease. 

3. To determine if a relationship exists between the presence of the 
L-form of an alpha streptococcus in recurrent aphthous ulcers and the 
etiology of this disease. 

4. To study the immunology of these streptococci, streptococcal 
antigens, streptococcal immune complexes, and antistreptococcal 
sera in both human and animal cell sulture systems. 

5. To study the histocompatibility of aphthous and nonaphthous 
cells in cell sulture using radiated and nonradiated cells, 

6. To investigate the role of the lymphocyte in host-defense 
mechanisms . 



335 



Serial No. NIDR-76 (c) (66) 

7. To record daily long term records on these aphthous patients 
in order to evaluate the frequency, severity of lesions and, 
their possible relation to stress, trauma, drug therapy, or other 
factors. 

8. To collaborate with Dr. Cummings in the study of Behcet's 
Disease. 

Methods Employed : 

1. The following tests are performed: red blood count, white 
blood count, the differential, Wintrobe indices, sedimentation rate, 
complement fixation, urinalysis, serum iron, total serum proteins, 
C-reactive protein, anti-streptolysin titers. Strep. MG titers. 

2. The oral lesions of all patients are biopsied, part of this 
tissue is sent to the histopathology laboratory and part is prepared 
for bacterial and viral studies. 

3. Thoroughness of examination of the microbiologic and histo- 
pathologic aspects in a few selected patients are emphasized. 

4. Lymphocyte transformation studies are now in progress to 
determine the in vitro effect of strep antigens on human peripheral 
lymphocytes and animal lymphoid cells. This will include patient 
groups consisting of: (1) normals, (2) Aphthous Stomatitis, (3) 
Behcet's Disease, (4) post streptococcal illness, and (5) other. 

5. The evaluation of our long term patient records (kept daily by 
the patients) has been analyzed by computer analysis. The results 
indicate that the frequency of lesion occurrance, the number of 
lesions, and their severity is a completely random process and is 
not correlated to any other clinical variable recorded. 

Patient Material : 

Patients are selected from those on record from the earlier stomatitis 
studies of the Dental Institute and those being referred at the 
present time to the Dental Services Branch, NIDR, About 40 patients 
comprise the study group. 

Major Findings : 

1. Final findings indicate that peripheral leucocytes from aphthous 
and normal patients show no significant difference in their response 
to the streptococcus sanguis 2A isolated by Dr. Graykowski. 



336 



Serial No. NIDR-76 (c) (66) 

2. A more basic finding is that human pathogenic streptococci 
stimulate human cell cultures significantly more than non patho- 
genic strains and conversly, animal pathogenic streptococci stimulate 
animla lymphocytes significantly more than* either non pathogens or 
human pathogenic strains. 

3. The treatment of a single aphthous patient with an immuno- 
suppressive drug resulted in a period of remission from lesions. 

Significance to Dental Research: 

The study attempts to explain the etiology of Aphthous Stomatitis 
and possibly discover more suitable methods of controlling the 
disease. Other allied projects investigate the nature of strep- 
tococcal hypersensitivity in humans, and the nature of recurrent 
ulcerative stomatitis as seen in Behcet's Disease. 

Proposed Course of Project : 

The general course of the project will continue along the lines of 
investigation already outlined in this report. 

The treatment strongly indicated that (1) the etiology of this disease 
is in part an immune mechanism and (2) based on this and other data, 
a broader project study may be indicated to further evaluate the 
therapeutic efficacy of these drugs. Several other patients treated 
in this manner in other clinics have produced similar results. 

Beginning in September, the prinicpal investigator. Dr. Francis, 
will begin a two year period of training. Arrangements have been 
made to maintain routine folow-up care for the clinical patients 
on this study. It is hoped that many of the laboratory projects 
already in progress can also be continued. 



Part B 



Publications : 



1. Francis, T.C. , and Archard, H. 0. : Nasopalatine duct cyst with 
epidermoid features; report of case, J. Oral Surg . , 25: 1967. 

2. Barile, M. F. and Francis, T.C. : Streptococcus sanguis in the 
pathogenesis of recurrent aphthous stomatitis. In Protoplasts, 
Spheroplasts, and L Forms. Guze, L. (Ed.) Williams and Wilkins Co. ^ 
Baltimore. In press. 

3. Francis, T.C, Oppenheim, J.J. , Barile, M.F.: Lymphocyte trans- 
formation by streptococcal antigens in guinea pigs and man. In Pro- 
ceedings of the Third Annual Leukocyte Conference, 1967. Riecke, W. 
(Ed.) (Appleton-CenCury, Crofts, N„ x. ) In press. 



O 



37 



Serial No. NIDR-77 (c) (66) 
1. Oral Medicine and Surgery 
3. Bethesda, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 

Part A 

Project Title: Osteosarcoma and Chondrosarcoma of the Jaws 

Previous Serial Number: NIDR-60 (c) 

Principal Investigator: Dr. G.E. Garrington 

Other Investigators: None 

Cooperating Units: Armed Forces Institute of Pathology 

Man Years: 

Total: 3/4 
Professional: 1/2 
Other: 1/4 

Project Description: 

Objectives : 

1. To define the behavioral patterns of these tumors in the jaws 
and compare this to behavior in other bones. 

2. To better define the clinical and radiographic character of 
these tumors, thereby aiding in earlier diagnosis, 

3. To evaluate therapeutic measures. 

4. To determine prognosis in relation to therapy. 

Methods Employed : 

Fifty-six cases of osteosarcoma and 36 cases of chondrosarcoma 
primary in the maxilla or mandible were selected because they 
appeared to satisfy the established criteria of these tumors. 
Complete follow-up information including hospital and clinic records, 
dental and skeletal roentgenograms and clinical photographs, was 
obtained. Additional and more current information was obtained from 
the patient's physician and/or dentist, from patients still living, 



338 



Serial No. NIDR-77 (c) (66) 

and from death certificates and autopsy protocols of those patients 
who died. Histologic slides were carefully examined, and the 
collected materials were evaluated and subjected to statistical 
analysis leading to a correlation of clinical, histologic and radio- 
graphic findings with diagnosis as well as to a correlation of therapy 
to survival. 



Major Findings : 

1. Osteosarcoma and Chondrosarcoma do apparently behave somewhat 
differently in the jaws in relation to each other than they do 

in other bones. Specifically, Osteosarcoma appears to have a better 
prognosis and Chondrosarcoma a worse prognosis in the jaws than in 
other bones. The survival rate for Chondrosarcoma in other bones is 
much better than for Osteosarcoma; this appears to be reversed for 
the tumors in the jaws. 

2. Radical resection appears to offer the best hope for cure for 
either tumor. 

3. Tumors in the symphysis of the mandible have the best prognosis 
and those in the maxillary antrum have the worst prognosis. 

4. An important early finding is roentgenographic evidence of a 
symmetrically widened periodontal membrane space with maintenance 
of an intact lamina dura. 

5. The histologic degree of malignancy does not appear to be 
related to prognosis. 

6. Prognosis is apparently somewhat better than has been 
previously reported. 

Significance to Dental Research : 

1. This study should provide better definition of a rather poorly 
characterized group of tumors. 

2. The differences in behavior and prognosis that are pointed out 
should provide the clinician with a firmer basis for deciding what 
therapy to use. 

3. There are some subtle indications in this study that there may 

be a sex difference in the distribution at least of the osteosarcomas, 
If this is true, it should provide another bit of data for the 
building of a case for hormonal influences on tumor igenes is and 
control. 



339 



Serial No. NIDR-77 (c) (66) 
Proposed Co urse of Project : 

The collection and evaluation of materials on osteosarcoma and 
chondrosarcoma have been completed. It is now intended to begin 
study of another group of poorly understood primary bone tumors: 
the myxomas and myxocarcomas . The present intent is to follow 
basically the same pattern of study that was used for the osteosarcomas 
and chondrosarcomas; to try to determine whether the myxomatous group 
of tumors is in fact an entity or is perhaps a more primitive form 
of the osteogenic group; and to determine whether the myxomatous 
tumors primary in bone are similar to, or different from, the 
odontogenic myxomatous tumors. 



Part B 



Publications; 



1. Garrington, G.E. , Scofield, H.H. , Cornyu, J. C. and Hooker, S.P. : 
Ostoesarcoma of the Jaws. Cancer 20: 377-391, March, 1967. 



3kQ i 



Serial No. NIDR-78 (c) (67) 
1. Oral Medicine and Surgery 
3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Leprosy involving the Dental Pulp 

Previous Serial Number: None 

Principal Investigator: Dr. G. E, Garrington 

Other Investigators: Dr. M. C. Crump 

Cooperating Units: U.S.P.H.S. Hospital, Carville, La.; U.S.P.H.S. Hos- 
pital, San Francisco, Calif.; U.S.P.H.S. Outpatient 
Clinic, San Pedro, Calif.; Leonard Wood Memorial 
(Cebu City, Phillipines, Leprosy Laboratory) 

Man Years: 

Total: 1/2 
Professional: 1/4 
Other: 1/4 

Project Description: 

Objectives : 

1. To determine the morphologic manifestations of dental pulpal in- 
fection due to leprosy. 

2. To establish a basis for determination of the best form of ther- 
apy (endodontic versus exodontic) . 

Methods Employed : 

Extracted teeth from patients with leprosy have been collected from 
the National Institutes of Health, the Carville Hospital, and the 
San Pedro Outpatient Clinic. These have been evaluated for pulpal 
infection by leprosy bacilli. 

They are presently being evaluated for viability of organisms by 
personnel of the Leprosy Service at the San Francisco Hospital who 
have devised a method of determining viability. of leprosy bacilli 



3h 



Serial No. NIM-78 (c) (67) 

in paraffin sections. Contact has been made with the Leprosy Lab- 
oratory at Cebu City in the Phillippines , through Dr. Chapman 
Binford, Director of the Leonard Wood Memorial. The dentist at 
Cebu City has consented to submit extracted teeth from there. This 
should provide a source of teeth from persons with tuberculoid le- 
prosy, which is desirable, because most of the teeth obtained from 
this country are from patients with lepromatous leprosy. The teeth 
and any surrounding soft or bony tissues removed are to be studied 
histologically and histochemically for infection and alterations. 

Major Findings : 

1. In the teeth examined thus far, it is apparent that leprosy can 
and does involve the dental pulp to the point of producing pulp de- 
generation and dental symptoms. 

2. It appears that pulpal involvement may be a manifestation of 
active disease. 

3. Mycobacterium leprae can invade the dentinal tubules of unres- 
tored, caries-free teeth. 

Significance to Dental Research : 

The effect of pulpal infection by leprosy bacilli is not presently 
known. This study will define these effects and provide for better 
clinical evaluation of dental pain in leprosy patients. 

Proposed Course of Project ; 

Teeth will continue to be collected, principally from Cebu City, in 
order to determine the extent of pulpal involvement due to tubercu- 
loid leprosy as compared to lepromatous leprosy. An effort will be 
made to assess the relative merits of extraction versus endodontics 
in cases of pulpitis due to leprosy. 



Part B 



Publications ; 



1. G. E. Garrington and M. C. Crump: Pulp Death in a Patient with 
Lepromatous Leprosy, Oral Surg., Oral Med., Oral Path. ; 25:427-434, 
1968. 



3^2 



Serial No. NIDR-79 (c) (67) 
1. Oral Medicine and Sur^^^rv 
3. Bethesda, Maryland 



PHS -NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Dilantin Gingival Hyperplasia 

Previous Serial Number: None 

Principal Investigator: Dr, G.E. Garrington 

Other Investigators: Dr. Harold Fullmer 

Cooperating Units: V. A. Hospital, Washington, D.C. ; U.S. Army Institute 

of Dental Research; possibly, the Johns Hopkins Hos- 
pital; Epilepsy Clinic and Parke-Davis Company will 
be involved, but this is not yet definite. 

Man Years : 

Total: 1/4 
Professional: 1/4 
Other: 

Project Description: 

Objectives : 

1. To determine the mechanism by which dilantin causes gingival 
hyperplasia, 

2. To study the morphologic and developmental character of the 
process in humans and in experimental animals, 

3. To assay enzymatic activity in dilantin hyperplastic tissue 
and compare it with the activity in non-hyperplastic tissue from 
epileptics on dilantin, with tissue from epileptics not on dilantin, 
with tissues from normal patients and with animal tissue from cats ' 
medicated with dilantin. 

4. To determine whether there are changes in vascularity or gingi- 
val circulation in dilantin hyperplastic tissues as opposed to nor- 
mal tissues, 

5. To assay dilantin levels in gingival tissue. 

2k3 



Serial No. NIDR-79 (c) (67) 

Methods Employed : 

The project is in the period of planning and Initiation, now. The 
following methods are anticipated: 

1. Approximately 50 patients, principally from the V. A. Hospital, 
with dilantin gingival hyperplasia of varying degree will be biop- 
sied. A sm^ll group of patients on dilantin with no clinical hyper- 
plasia will be biopsied. A small group of epileptic patients not on 
dilantin will be biopsied. And a small group of non-epileptic pat- 
ients will be biopsied. Portions of the excised tissue will be fix- 
ed in formalin for routine histology and special histochemistry. 
Another portion of excised tissue will be immediately frozen and 
used for enzyme studies. A third portion is tentatively proposed 
for use in assaying dilantin levels in the tissue itself. This lat- 
ter is dependent upon the cooperation of Parke-Davis Company in per- 
forming the assays. 

2. Dilantin will be administered to cats to promote gingival hyper- 
plasia and basically the same studies will be done that are proposed 
in (1) above to determine the comparability of dilantin gingival hy- 
perplasia in cats to that in humans. 

3. A small number of cats will be injected with a silicone rubber 
preparation that is useful for tracing circulation. This method has 
been found useful at the U.S. A, I. D. R. for demonstrating microvascu- 
lature. The object is to compare minute circulation in the hyper- 
plastic tissues with that of non-hyperplastic basically normal tis- 
sues. 

Major Findings : 

The project is in the initial stages at present. 

Significance to Dental Research : 

1. As currently projected, the major significant finding is hoped 
to be a clue as to what initiates the hyperplasia. This is intend- 
ed as a step toward determining what leads to tissue proliferations 
including tumors. 

2. The study of enzymatic activity in the gingival specimens should 
add to the rather meager current knowledge on that subject. It may 
be that this will add to knowledge of periodontal disease as well. 
One specific enzyme to be assayed is collagenase, thereby perhaps 
shedding some light on the relationship it may have to gingival di- 
sease. 



3kk 



Serial No. NIDR-79 (c) (67) 

Proposed Course of Project : 

As stated previously, the study is now in the initial stages. The 
course proposed is to follow the methods outlined in the "Methods 
Employed" section above. Following completion of the study it is 
proposed to follow-up any leads that may be developed regarding tis- 
sue proliferation and tumorigenesis. 



Part B 



Not included. 



3^5 



Serial No. NIDR-80 (c) (62) 
1. Oral Medicine and Surgery 
3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July I, 1967 through June 30, 1968 



Part A 



Project Title: Microbiological Phase of Rampant Caries Study 

Previous Serial Number: NIDR-61 (c) 

Principal Investigator: Mrs. S. A. Geis 

Other Investigators: Drs. R. M. Stephan and R. J, Fitzgerald 

Cooperating Units: None 



Man Years: 




Total: 

Professional: 

Other: 


1-1/4 
1 

1/4 



Project Description: 

Objectives : 

The objective of this project is to identify and classify human 
oral streptococci and to determine whether or not specific strains 
of streptococci can be identified as etiologic agents in dental 
caries. The flourescent antibody technique is employed to identify 
specific streptococci strains in histologic sections of carious 
teeth and in the mixed culture of dental plaque smears. 

Methods Employed : 

Laboratory research into possible streptococcal causes of rampant 
caries may be classified into three phases: 

1. The production, testing and purification of diagnostic 
antisera to oral streptococci, 

2. Testing streptococcal antisera to cultures, plaque smears 
and histologic sections of teeth taken from patients. 

3. Animal studies. 

Rabbits already highly immunized to human oral streptococci were 
kept on antisera production with weekly booster shots of heat-killed 



SltG 



i 



Serial No. NIDR-80 (c) (62) 

vaccines and periodic bleedings to provide a constant source of 
fresh antisera. New antisera were developed to four organisms 
freshly isolated from new rampant caries patients. All of the 
organisms possessed metabolic indicators of possible cariogenic 
activity; they produced dextran from sucrose, produced iodophyllic 
intracellular polysaccharide and fermented mannitol and sorbitols 
One had colony morphology like the cariogenic rat streptococcus s train 
FAl. One was serologically related to Dr. Zinner's cariogenic str«.i.n 
BHT (which is supposed to be closely related to rat strain FAl) as 
well as our own strains PH4, 2KD, IDD, BY and a Lancefield Group A. 
This cross reaction necessitated careful investigation and 
absorption of several antisera. 

The crude and absorbed antisera were tested against samples of dental 
plaque smears in an effort to obtain a pattern of antigenic impor- 
tance of the streptococcal components of plaque and to ascertain 
the relative presence of these organisms in plaque from persons with 
and without severe dental caries problems. 

Streptococcus strain RC2 was introduced into a tank of germ-free 
rats to study its cariogenic ability. The organism was isolated 
from a child with rampant caries and is serologically reciprocally 
cross-reactive with the Zinner strain BHT. 

Two studies on the effects of feeding germ-free rats diets 585V and 
2000V were done in order to have control comparison animals for our 
gnotobiotic studies. 

Tissue studies were completed on the germ-free experiment involving 
mono-infection of rats with human Streptococcus mitis strain PH4. 
The results were analyzed and reported. 

Major Findings : 

Interesting serological cross reactions were found to exist between 
streptococci isolated from rampant caries patients and some of the 
streptococci isolated by Dr. Zinner in Miami. Since the Zinner BHT 
organism is cariogenic and related to the rat cariogenic streptococcus 
strain FAl it was necessary to determine the relationship, natural 
occurrence and cariogenicity of the serologically related strain 
RC2. With careful absorptions it was possible to remove cross 
reactions from the RC2 serum and from the BHT, PH4, 2KDs IDD and BY 
sera. The absorbed RC2 serum was reactive with 20 to 507„ of the 
cocci in the plaque smears tested, which was a lower reactivity than 
the crude serum. The absorbed BHT serum had a much lower reactivity 
with plaque cocci than the crude serum. Many plaque samples that 
were cultured on mitis-salivarius agar and smeared on slides for the 



3^7 



Serial No. NIDR-80 (c) (62) 

fluorescent antibody stain had many cocci reacting to BHT serum but 
failed to grow out either BHT or RC2 type colonies in culture. 

The Rampant caries streptococcus strain KBl isolate that has colony 
morphology similar to BHT and FAl colony morphology is not sero- 
logically related to either strain. Reactivity of cocci in plaque 
smears to KBl antiserum varies widely. 

Streptococcus strain RC2 was allowed to become established in the 
mouths of 7 germ-free rats for three months. Gross examination at 
sacrifice and examination of the 1/2 of each head defleshed and 
dried revealed minimal, caries formation. Present evidence indicates 
that streptococcus strain RC2 is not cariogenic. 

Tissue sections of gnotobiotic rats infected with streptococcus strain 
PH4 failed to explain the small white lesions present on the lung 
surfaces of the rats at autopsy. All the rats had emphysema and 
calcification in the kidneys but the PH4 streptococcus was not im- 
plicated in the pathology. The PH4 gnotobiotic rats had minimal 
caries formation. 

Two germ- free control studies were done but examination of only one 
has been completed. All six rats on diet 585V and and all six rats 
on diet 2000V had fractures of the molars but no caries. Although 
the large contaminating bacillus was present in the sulci among food 
particles there was no invasion of the fractures or the dentin 
tubules. The bacillus was present in the lumena of the stomach and 
intestines but was absent in all other tissues. All of the rats on 
both diet 585V and 2000V had emphysema and at autopsy one rat on diet 
585V and one rat on diet 2000V had tiny, white lesions on the surface 
of the lungs. The contaminating bacillus was not implicated in the 
lesions or the emphysema. The rats on diet 585V did not have any 
areas of calcification in the kidneys but two of the six rats on 
diet 2000V had small, scattered areas of calcification. Only gross 
autopsy data is available for the uncontaminated germ- free control 
study. No lung lesions were present in the four rats on diet 585V; 
no gross fractures or caries were seen in the teeth. All five rats 
on diet 2000V had small, white nodules on the surfaces of the lungs, 
none of the rats on diet 2000V had gross fractures or carious lesions 
in the teeth. 

Significance to Dental Research : 

The fluorescent antibody technique developed in this study enables the 
microbiologist to examine the actual bacterial flora of dental plaque 
and tooth sections rather than having to rely on what organisms grow 
out (and out-grow others) in various artificial media. This can be 

1^8 



Serial No. NIDR-80 (c) (62) 

of major significance in epidemiological studies of dental caries. 
Since the plaque smears and paraffin-fixed tissue sections are stable 
for staining at a much later date, strict time elements can be 
eliminated from sample procurement. 

Proposed Course of Project : 

It is planned to continue to develop purified antisera to additional 
strains of streptococci isolated from carious areas of human teeth. 
The fluorescent antibody technique, using new and presently developed 
antisera, will be applied to future clinical and animal studies and 
in epidemiological surveys of the caries problem. Attempts will be 
made to recognize cultural and metabolic characteristics of cariogenic 
streptococci in addition to those factors already theorized to be 
associated with cariogenic activity. Work will continue on the 
problem of establishing probable cariogenic human streptococci in 
test animals. 

Part B not included 



2k3 



Serial No. NIDR-81 (c) (65) 
1« Oral Medicine and Surgery 
3. San Francisco J California 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Immunological Studies in Recurrent Aphthous Stomatitis 

Previous Serial Number: NIDR-62 (c) 

Principal Investigator: Dr. E. A. Graykowski 

Other Investigators: None 

Cooperating Units: None 

Man Years : 

Total: 1 
Professional: 1 
Other: 

Project Description: 

Objectives : 

1. To develop further evidence that recurrent aphthous stomatitis 
is a hypersensitivity disease. 

2. To develop an immunologic test for diagnosing aphthous patients 
in an inactive period. 

3. To determine if immunization is an effective means of treatment 
in this disease. 

Methods Employed : 

1. The following tests are performed on aphthous patients admitted 
to the study: Red blood cell count, hemoglobin, hematocrit, sedi- 
mentation rate, VDRL complement fixation test, total serum proteins 
and albumin-globulin ratio, serum electrophoresis, and urinalysis. 

2. The oral lesions are biopsied; part of the tissue specimen is 
sent for histopathologic diagnosis, part is utilized in procedures 
to detect cellular antibodies. 



350 



se 



Serial No. NIDR-81 (c) 

3. Blood is drawn from patients at periodic intervals in the coir. 
of their disease to obtain circulating lymphocytes for use in the 
Jerne plaque test which was designed to detect lymphocyte antibodies 
against the aphthous streptococcus 2A. 

4. The laboratory experimental model for aphthous stomatitis is the 
hypersensitized albino female guinea pig. Hartley strain. Various 
desensitization procedures are performed utilizing these animals, 
such as the injection of intravenous streptococcal vaccines and 
immunosuppressant drugs. Intradermal skin tests with the aphthous 
streptococcus antigens are used to indicate the degree of hyper- 
sensitivity and the effectiveness of desensitization procedures. 

5. A detailed analysis of the aphthous streptococcus antigens is 
being performed to determine the specific antigenic component for 
use in diagnostic tests and possibly the treatment of patients. 

6. Patients are selected from those referred to the NIDR Unit at 
the PHS Hospital in San Francisco from military bases and the private 
practitioners of medicine and dentistry in the area. A study group 
of approximately 50 patients is planned. 

Major Findings : 

1. Animal studies have indicated that hypersensitivity to the anti- 
gens of the alpha streptococcus isolated from recurrent aphthae is 
an important factor in the development of these lesions. Positive 
skin tests (delayed type hypersensitivity) to these antigens are 
obtained in patients with aphthous stomatitis but not in control 
individuals. The degree of the skin test reaction is directly 
proportionate to the severity of the disease in the patient tested. 

2. The injection of the Eli Lilly intravenous streptococcal vaccine 
into hypersensitized guinea pigs over a period of several weeks 
reduces the skin reaction to the streptococcal cell wall antigen 
(CHO), indicating some degree of desensitization. 

3. The use of the Lilly intravenous streptococcal vaccine in two 
patients with the severe form of recurrent aphthous stomatitis, 
periodenitis, produced a gradual decrease in the frequency and in- 
tensity of the mucosal lesions with complete cessation of the 
lesions after a latent period of approximately 6 months after the 
last injection of the vaccine. One patient has remained free of 
S3miptoms for over 2 years and the other for 1 year. 



351 



Serial No. NIDR-81 (c) 

Significance i:o Dental Research : 

The presence of transitional L-forms of an alpha streptococcus in 
large numbers and in pure culture from numerous lesions, their 
persistence in lesions in one patient for at least five months, the 
associated bacteremia during exacerbation, and the recovery of a 
stable L-form from tissue during remission may be significant. 
These findings suggest that a relationship exists between the L-form 
of the streptococcus and the pathogenesis of recurrent aphthae. The 
finding of a skin hypersensitivity to the suspected causative agent 
of aphthous stomatitis (L-form of an alpha streptococcus) may prove 
to be a very important factor in establishing the etiology of this 
condition and in differentiating it from the other stomatitides. 

The apparent desensitization or immunization of two patients pre- 
venting the recurrence of mucosal lesions for a long period of time 
indicates that this may be an effective long- lasting treatment of 
this disease. 

Proposed Course of Project ; 

A major effort will be made to develop an accurate diagnostic test 
for aphthous patients. The investigation will be directed toward 
demonstrating the presence of specific antibodies against the I 
aphthous streptococcus 2A in the circulating Ijrmphocytes of aphthous 
patients. 

An attempt will be made to find a more acceptable means of immunizing 
patients other than the intravenous administration of a vaccine. The 
role of the immunosuppressant drugs in creating tolerance to bacterial 
antigens will be investigated. 



Part B 



Publications : 



1, Graykowski, E.A. : Treatment of Oral Ulcerations, M odern Treat - 
ment 4:555-571, May 1967. 

2. Barile, M.F., Francis, T.C., and Graykowski, E.A.: Streptococcus 
sanguis in the Pathogenesis of Recurrent Aphthous Stomatitis. In 
Guze, L., (Ed.): Protoplasts. Spheroplasts and L-forms , Baltimore, 
Md., Williams & Wilkins Co., 1968, pp. 444-456. 



352 



<l 



Serial No. NIDR-82 (c) (66) 
1. Oral Medicine and Surgery 
3. Be the s da, Maryland 



PHS-NIH 

Individual Project Report 

July 1, 1967 through June 30, 1968 



Part A 



Project Title: Betel Quid CarGinogenesis 

Previous Serial Number: NIDR-63 (c) 

Principal Investigator: Dr.- J. E. Hamner, III 

Other Investigators: None 

Cooperating Unit: Dr. L. J. Dunham, National Cancer Institute 

Man Years : 

Total: 1 
Professional: 1/2 
Other: 1/2 

Project Description: 

Objectives ; 

1. To determine the individual and/or combined roles that calcium 
hydroxide, tobacco, and gambler may play in causing betel quid 
induced carcinomas, 

2. To induce squamous cell carcinoma in the hamster cheek pouch, 
using various betel quids. 

3. To determine which of the betel quid ingredients are the 
causative agent. 

4. To relate these findings to the high incidence of oral squamous 
cell carcinoma in the betel quid chewing areas of the world. 

Methods Employed : 

Eight groups of male and female hamsters totaling 54 animals were 
given one of the following treatments: (a) gambler alone, (b) snuff 
alone, (c) starch powder alone, (d) calcium hydroxide alone, 
(e) equal parts of calcium hydroxide and gambler, (f) equal parts 



353 



Serial No. NIDR-82 (c) (66) 

of calcium hydroxide and snuff, (g) calcium hydroxide in the morning 
and snuff in the afternoon, and (h) calcium hydroxide in the morning 
and starch powder in the afternoon. Hamsters were 3-1/2 to A-1/2 
weeks old when treatment began. A Vienna nasal speculum was used 
for the applications which were placed in the check pouches five 
days each week. The hamsters were sacrificed when moribund, or 
were taken for examination after spontaneous death. Autopsies were 
performed on each animal and histologic sections prepared. 

Major Findings : 

1. Calcium hydroxide caused chemical burns with necrosis and 
ulceration followed by regeneration. Three of these animals had 
atypical epithelial lesions, resembling focal leukoplakia in man. 
The affected pouches of hamsters treated with calcium hydroxide 
showed one or more of the following findings: deposits of calcium, 
inflammation, giant cells, and fibroblastic proliferation in the 
lamina propria; and inflammation, ulceration, atrophy, hyperplasia, 
hyperkeratosis, parakeratosis, acanthosis, and cellular atypia in 
the epithelium. 

2. No squamous cell carcinomas were produced in any of the groups. 

3. No changes were noted in the cheek pouches treated with snuff 
or starch powder alone. 

4. Two of 14 hamsters treated with gambler developed minute ulcers 
with inflammation. 

Significance to Dental Research : 

1. Distinguishing the causative carcinogenic agent in the betel 
quid chew is important to geographical pathology because of the 
high incidence of oral carcinoma in the area of the world where 
betel quid chewing is so prevalent, namely southeast Asia and India. 

2. Until the causative factor or factors are determined. Public 
Health measures and population education cannot be instigated to 
reduce the high mortality from oral squamous cell carcinoma in 
southeast Asia. 

Proposed Course of Project : 

It cannot be ascertained from this experiment whether the lesions 
that developed were the final phase of the reaction to the treatment 
with calcium hydroxide, or whether they had the potential of pro- 
gression to neoplasia. Studies are planned to increase the contact 



35L» 



Serial No. NIDR-82 (c) (66) 

with the calcium hydroxide and to modify the environment afforded 
by the hamster. 

In an effort to modify the epithelium of the hamsters' buccal pouch 
mucosa, two experiments have been started to gradually induce 
cirrhosis. Twenty hamsters have been maintained on lb°L ethylaicohol 
in their drinking water for 4 months. Twenty other hamsters have 
had 0.5 ml. injections of 10% CCI4 in olive oil intradermally fc?: 
4 months (1 injection per week). These will continue for 6 monthSj 
then liver biopsies will be done to ascertain the presence of 
cirrhosis. 

Once sufficient liver damage is induced, the cheek pouches will be 
treated with calcium hydroxide, similar to the original experiment. 



Part B 



Publications : 



1. Dunham, L.J., Muir, C.S. and Hamner, J.E., III; Epithelial 
atypia in hamster cheek pouches treated repeatedly with calcium 
hydroxide. Brit. J. Cancer 20:588-593, Sept. 1966. 



355 



Serial No. NIDR-83 (c) (66) 
1. Oral Medicine and Surgery 
3. Bethesda, Maryland 



PHS -NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Benign Fibro-osseous Lesions of the Maxilla and Mandible 

Previous Serial Number: NIDR-64 (c) 

Principal Investigator: Dr. J.E. Hamner, III 

Other Investigators: None 

Cooperating Units: Captain H. H. Scofield (DC) USN, Armed Forces Insti- 
tute of Pathology 

Man Years : 

Total: 1/2 
Professional: 1/4 
Other: 1/4 

Project Description: 

Objectives : 

1. To devise a classification system for benign fibro-osseous lesions 
found in the jaws from the vast wealth of case material in the files 
of the A. F.I. P., using a thorough clinico-pathological correlation 
and accurate follow-up of patients. 

2. To correlate the pathogenesis, radiographic picture, clinrcai 
behavior, and microscopic features of these lesions (ossifying fi- 
broma, cementifying fibroma, cemento-ossifying fibroma, central 
fibroma, desmo-plastic fibroma, active ossifying fibroma, giant cell 
tumor, aneurysmal bone cyst, hyperparathyroidism lesion, monostotic 
fibrous dysplasia, and familial fibrous dysplasia) by a detailed 
study. 

3. To determine the tissue of origin of these lesions. 
Methods Employed : 

A complete listing of cases with the above diagnoses was compiled 



356 



Serial No. NIDR-83 (c) (66) 

by the A. F.I. P. Case histories, radiographs, and microscopic mater- 
ial on each case was reviewed. Special stains were used when indi- 
cated, and all slides were examined under polarized light. Follow- 
up forms were mailed to both the attending dentist or physician and 
the patient. This major project is still in progress and is quite 
lengthy to complete. 

One portion of the project was a histochemical study to determine 
the tissue of origin of these lesions. Forty-two cases diagnosed 
as either hyperparathyroidism, aneurysmal bone cyst, giant cell tu- 
mor, familial fibrous dysplasia, monostotic fibrous dysplasia, ce- 
mentifying fibroma, ossifying fibroma, cemento-ossifying fibroma, or ac- 
tive ossifying fibroma were chosen from the main A. F.I. P. listing 
of these cases. Sections were cut and stained by the following 
methods: (a) Hematoxylin and eosin, (b) monopersul fate -aldehyde 
fuchsin-Halmi for oxytalan fibers, (c) Rinehart for mucopolysaccha- 
ride and collagen fibers, and (d) Taenzer-Unna orcein for elastic 
fibers. The H and H sections were also studied under polarized 
light. 

Major Findings : 

1. Ossifying fibroma, cementifying fibroma, and cemento-ossifying 
fibroma arise from the periodontal membrane. 

2. Ossifying fibroma can also arise from medullary bone, as do the 
remainder of the fibro-osseous lesions of the jaws. 

3. Oxytalan fibers may occur in most benign fibro-osseous lesions 
of the jaws, regardless of their tissue of origin, provided that ma- 
ture collagen fibers are present in the lesion. 

4. Inasmuch as oxytalan fibers and pre-elastic fibers cannot be dis- 
tinguished with present histochemical methods, the demonstration of 
fibrous elements stained with the oxytalan fiber method does not con- 
stitute conclusive evidence of odontogenic origin of the tumor. 

5. The birefringence pattern under polarized light does serve as an 
excellent differential for diagnosis. Fibrous dysplasia gives a ran- 
dom irregular birefringence, indicative of woven bone, whereas; the 
other fibro-osseous lesions manifested birefringence as parallel 
light and dark bands, indicative of the varying degrees of lamellar 
bone formation. 

Significance to Dental Research : 

1. Fibro-osseous lesions may arise from the periodontal membrane 



357 



Serial No. NIDR-83 (c) (66) 
and the medullary bone when found in the jaws. 

2. Oxytalan fibers have again been found in a pathologic condition 
and have been shown to have a direct relationship to mature collagen 
fibers. 

3. Polarized light has been confirmed as an excellent tool for dif- 
ferential diagnosis for the pathologist. 

Proposed Course of Project : 

The major portion of this project involving review of the listed 
cases is almost complete. Approximately 625 cases are well docu- 
mented and acceptable to be included in the final analysis. The 
follow-up information requires time to complete. When this part 
of the project is completed, the final conclusions can be drawn. 



Part B 



Publications: 



1. Hamner, J.E. , III and Fullmer, H, M. : Oxytalan fibers in benign 
fibro-osseocs jaw lesions, Arch. Psth . 82: 35-39, July, 1966. 

2. Hamner, J.E. , III, Scofield, H.H. , and Conryn, J.: Benign 

f ibro-osseous jaw lesion of periodontal membrane origin: An analysis 
of 249 cases, Cancer (accepted for publication). 



358 



< 



Serial No. NIDR-34 (c) (68) 
1. Oral Medicine and Surgery 
3. Bethesda, Maryland 



PHS -NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Submucous Fibrosis 

Previous Serial Number: None 

Principal Investigator: Dr. James E. Hamner, III 

Other Investigator: None 

Cooperating Units: None 

Man Years : 

Total: 1/2 
Professional: 1/4 
Other: 1/4 

Project Description: 

Objectives : 

1. To determine if chili powders can produce submucous fibrosis 
in the buccal pouch of hamsters. 

2. To compare the effects of chili powders from two states in India 
(Kerala and Gujarat) . 

Methods Employed : 

Twenty hamsters were given Kerala chili powder via a Vienna nasal 
speculum in their right buccal cheek three times per week. Biopsies 
were taken after 1, 2, 4, 6, 8, and 12 months of the treatment, and 
the tissue specimens were stained with hematoxylin and eosin, 
Rinehart's stain and van Gieson's stain. 

Major Findings : 

The experiment is in progress. No findings yet. 



35S 



Serial No. NIDR-84 (c) (68) 



Significance to Dental Research : 

Submucous fibrosis is an oral disease peculiar to India. The fibrosis 
affects the buccal mucosa, tongue, palate, and pharynx, causing extreme 
difficulty in swallowing and eating. Severe cases can be fatal. There 
is a strong possibility that this is a premalignant condition. Capsai- 
cin (in chili powder) has been suggested as the possible etiologic 
factor. It is hoped to cause this disease in animals to prove its 
etiology and give a model for its continued study. 

Proposed Course of Project : 

The project is now at its halfway mark toward completion. Biopsies 
of the chili-treated cheek pouches will continue to be taken at the 
specified intervals. Beginning at the six month mark, tissue 
specimens will be studied using the electron microscope, as well as 
the light microscopy procedures. 

Of no changes are evident in the pouch mucosa after twelve months 
treatment, the same treatment will be continued for another year. 

Part B not included 



3G0 



< 



Serial No. NIDR-85 (c) (66) 
1. Oral Medicine and Surgery 
3. Be the s da, Maryland 



PHS-NIH 

Individual Project Report 

July 1, 1967 through June 30, 1968 



Part A 



Project Title: Dental Pulp and Periodontal Studies in Germfree and 
Conventional Laboratory Rats 

Previous Serial Number: NIDR-65 (c) 

Principal Investigator: Dr. S. Kakehashi 

Other Investigators: Drs. H. R. Stanley and R. J. Fitzgerald 

Cooperating Units: None 

Man Years : 

Total: 1/4 
Professional: 1/4 
Other: 

Project Description: 

Objectives : 

To study the comparative healing of pulp and surgical wounds of 
periodontal tissues in germfree and conventional rats and how the 
healing potential can be modified. 

Methods Employed : 

A total of 56 Fisher rats, consisting of germfree and conventional 
control animals were surgerized to expose the previously intact 
pulp of the maxillary first molars. The exposed pulps were treated 
with corticosteroid and/or a mixture of prednisolone, camphor, 
methyl cresyl acetate and parachlorophenol which was sealed in 
place with a temporary filling material. Appropriate control groups 
were established. Animals were killed at intervals from 1 to 72 
days postoperatively. Serial sections of the surgerized tooth 
were stained with hematolylin and eosin, Masson's trichrome, 
Giemsa and Brown and Brenn stains. 



3S1 



Serial No. NIDR-85 (c) (66) 
Major Findings : 

1. The conventional animals, regardless of treatment, showed an 
immediate, severe inflammatory response which quickly led to total 
pulpal necrosis. 

2. The germfree animals showed a minimal inflammatory response 
followed by a reparative process with complete dentinal bridging. 
The several modalities of treatment used appeared to be of no 
value. 

Significance to Dental Research : 

This study indicates that healing of experimentally exposed dental 
pulps in rodents is primarily dependent on the absence of a 
microbial flora. This healing process appears not to be altered 
by corticosteroid treatment. 

Proposed Course of Project : 

The effects of surgical wound healing are being studied in a germ- 
free system. 



Part B 



Publications : 



1. Kakehashi, S., Stanley, H.R., and Fitzgerald, R.J.: The 
Exposed Germfree Pulp - The Effects of Topical Corticosteriod 
Medication and Restoration. In press. 



362 



Serial No. NIDR-86 (c) (6G) 
1. Oral Medicine and Surgei 
3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: A Long Term Study of Periodontal Disease in a Stable ^ 
Adult, Male Population 

Previous Serial Number: NIDR-66 (c) 

Principal Investigators: Drs . S. Kakehashi and N. W. Littleton 

Other Investigators: None 

Cooperating Units: District of Columbia Fire Department 

Man Years : 

Total: 1-3/4 
Professional: 1 
Other: 3/4 

Project Description: 

Objectives : 

The purpose of this investigation is to study the initiation and 
progression of periodontal disease on a long term basis in a stable, 
adult, male population. The occurrence of periodontal disease is 
to be studied with regard to selected intraoral factors of suspected 
etiologic importance. 

Methods Employed : 

The occurence of destructive periodontal disease is being assessed 
by direct observation of volunteers of the D. C. Fire Department. 
Examinations are to be repeated biannually and changes in the 
initiation and progression of the disease are to be related to the 
following factors: (1) Gingivitis, (2) Debris, (3) Calculus, 
(4) Overhangs and Caries, (5) Interproximal Contacts, (6) Mobility, 
and (7) Occlusion, (a) Centric Permaturities, (b) Balancing Contacts, 



3&3 



Serial No. NIDR-86 (c) (66) 



Major Find?lngs : 



The initial series of examinations conducted on 581 volunteers 
from the District of Columbia Fire Department have been completed. 
To date, the statistical analysis of the data obtained from these 
field examinations is incomplete. 

Significance to Dental Research : 

The relationship between the various clinical signs of periodontal 
diseases has not been adequately described. Limitations associated 
with the clinical and cross-sectional epidemiological study of 
periodontal diseases, a chronic and progressive process, are well 
recognized. Consequently, the need for a long term study is not 
only indicated but appears to be the only method by which this 
information can be obtained. 

Proposed Course of Prp-ject ; 

The second series of field examinations are to commence in 
September 1968. In the interim, statistical analysis of the data 
obtained from the initial (cross sectional) examination will be 
completed. 

Part B not included 



3^k 



Serial No. NIDR-87 (c) (67) 
1. Oral Medicine and Surgery 
3. Bethesda, Maryland 



PHS -NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Post-surgical Tissue Healing 

Previous Serial Number: NIDR-67 (c) 

Principal Investigator: Dr. P.M. Lightbody 

Other Investigators: None 

Cooperating Units: None 

Man Years : 

Total: 3/4 
Professional: 1/2 
Other: 1/4 

Project Description: 

Objectives : 

To correlate age of patient, type of impacted tooth, systemic 
condition of patient and surgical trauma with post-operative 
localized osteitis. To study the effect of topical antibiotic 
administration on post-extraction healing. 

Methods Employed : 

Patients with bilateral impacted mandibular third molars are used. 
Age of patient, type of impaction, surgical procedure and medication 
in extraction wound are recorded. On one side no medication is 
used, and on the opposite side, neosporin powder is placed in the 
wound. Patients are observed until asymptomatic and healing of 
each side is compared. 

Major Findings: 

Thus far, the incidence of localized osteitis has not diminished 
on the side where medication is used as compared with the control 
side. The study is too young to report any findings. 

365 



Serial No. NIDR-87 (c) (67) 
Significance to Dental Research ; 

To try to eliminate localized osteitis, the most frequently 
encountered complication of third molar surgery. 

Proposed Course of Project ; 

A minimum of 500 impacted teeth are to be removed and results 
compared. Approximately 75 have been done. Approximately 200 
new cases of mandibular impacted third molars are completed. 

Part B not included 



366 



Serial No. NIDR-88 (c) (67) 
1. Oral Medicine and Surgery 
3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Sectional Roentgenographic Study of the Temporomandibular 
Joint Following Bilateral Osteotomy of the Ramus of the 
Mandible 

Previous Serial Number: NIDR-68 (c) 

Principal Investigator: Dr. P. M. Lightbody 

Other Investigators: None 

Cooperating Units: None 

Man Years: 

Total: 1/4 
Professional: 1/4 
Other: 

Project Description: 

Objectives ; 

Long term follow-up of patients subjected to bilateral osteotomy of 
the mandible is being done to evaluate the following: 

1) Any recurrence or relapse of the newly established occlusion. 

2) Any subjective or objective changes in the T.M.J. 

3) Any x-ray changes such as morphology change of condylar 
head, change in position of condylar head in glenoid fossa in 
open and closed positions. 

4) Any correlation between type of malocclusion and facial 
deformity correction and surgical procedure used. 

5) Any correlation between timing of auxiliary procedures, 
such as orthodontics, prosthetics and operative dentistry and 
surgical procedure. 



-3 b 



Serial No. NIDR-88 (c) (67) 



Methods Employed : 



Patients who have malocclusions or facial deformities that can be 
corrected by surgery are admitted to the Clinical Center and the 
operative procedure selected by the oral surgeon is carried out. 
Preoperative records include articulated models, pre- and post- 
operative Panorex x-ray, lateral and posterior-anterior head plates 
in open and closed position and lateral tomograms of the T.M.J, in 
open and closed position. Postoperative lateral and posterior- 
anterior head plates are then taken at 6 months, 1 year and each 
successive year for a minimum of 5 years. Lateral tomograms are 
taken at a 1 year postoperative. Clinical examination and 
evaluation is carried out each time the patient reports for x-rays. 

Major Findings : 

Approximately 15 patients are over 3 years postoperative. There are 
some minimal x-ray changes as seen in tomographic examination, 
however, these are not correlated to clinical findings. There have 
been no relapses or recurrences. 

Significance to Dental Research ; 

There is some question of what happens in the temporomandibular 

joint following bilateral osteotomy of the mandible. There is a 

need for a longitudinal study of this, both from a clinical 
evaluation and x-ray examination. 

There are no longitudinal studies reported that deal with follow- 
up of patients who have had bilateral osteotomies of the mandible. 
Possibly different surgical approaches should be used for varying 
types of malocclusions. 

Proposed Course of Project ; 

It is intended to have at least 50 of these cases with a documented 
follow-up. 

Part B not included 



368 



Serial No. NIDR-89 (c) (67) 
1. Oral Medicine and Surgery 
3. Be the s da, Maryland 



PHS -NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Evaluation of Premedication in Conjunction with. Local 
Anesthesia in Oral Surgical Procedures 

Previous Serial Number: NIDR-69 (c) 

Principal Investigator: Dr. P. M. Lightbody 

Other Investigator: None 

Cooperating Units: None 

Man Years : 



Total: 


1/2 


Professional: 


1/4 


Other: 


1/4 


Project Description 




Obiectives: 





To evaluate which premedicating drugs have the most potential for 
use on oral surgical patients being treated under local anesthesia. 

Methods Employed : 

Each patient has at least four separate oral surgical procedures 
done. Each procedure is done with a different premedication. The 
drug is given intravenously and is titrated for each patient. 
Vital signs are monitored prior to, during, and after the procedure 
and compared. Subjective evaluation of each drug is also evaluated. 

Major Findings : 

The study is too young to report any findings. 

Significance to Dental Research : 

To give the oral surgeon another tool for effective pain control 
and to evaluate which drug is best suited for this purpose. ' 

369 



Serial No. NIDR-89 (c) (57) 



There are many patients who require oral surgical procedures who 
do not have the benefit of general anesthesia. In many geographic 
locations in the country general anesthesia is not practiced on 
ambulatory patients. Hospitals today are too overcrowded to admit 
patients just for the benefit of general anesthesia; therefore, 
different types of premedication drugs are being employed. 

Proposed Course of Project : 

A minimum of 150 patients are to have at least four surgical 
procedures and results of each drug tabulated. 

Part B not included 



370 



Serial No. NIDR-90 (c) (68) 

1. Oral Medicine and Surgerv 

2. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Effects of Oral Fluids on the Dental Pulp 
Previous Serial Number: None 
Principal Investigator: Dr. T. Lundy 
Other Investigators: Dr. H. R. Stanley 
Cooperating Units: None 



Man 


Years: 








Total: 


2- 


■1/4 




Professional: 




1/4 




Other: 


2 




Pro, 


ject Description; 






Obi( 


actives: 







1. To determine the effects of saliva on the dental pulp over 
a three-month period after a Class V cavity preparation was 
prepared and unrestored. 

2. To establish a relationship between the clinical signs and 
symptoms and the microscopic pulpal changes. 

3. To evaluate the rate of movement of bacteria and the depth of 
bacterial penetration in the dental tubules. 

Methods Employed : 

Class V cavities are prepared in human teeth, intact, restored, or 
slightly carious, and left open to saliva. 

Although these specimens are slated for extraction for prosthetic 
or periodontal reasons, all are considered to be suitable for this 
study. None of the teeth show any clinical signs or symptoms prior 
to the procedure. Patients are accepted at all ages. The cavities 



371 



Serial No. NIDR-90 (c) (68) 

are prepared at the cemento-enamel junction or either the buccal 
or lingual intact surface with a Borden high-speed handpiece 
utilizing air-water spray and #2 or #4 bur. The preparation extends 
as deep into the dentin as possible and laterally to the mesial- 
distal borders. No preparations are left completely exposed to 
saliva without using any restorative material. Before the cavities 
are prepared and also at the time of extration, several diagnostic 
tests are performed to establish the level of response of 
sensitivity of the teeth, utilizing heat or cold stimuli, electrical 
pulp test, a dental probe tactile sensation test and a blast of air. 
Heat is applied utilizing gutta percha which is heated until it 
bends under its own weight. If a "no response" is obtained, a 
heated burnisher is then applied, because the burnisher maintains 
the heat for a longer period of time. If a "no response" is still 
obtained, then it is felt that the response is accurate and not a 
false negative. Cold is applied by two techniques: either 
utilizing a cotton pludget saturated with ethyl chloride or a cone 
of ice of standard size. In both the heat and cold tests various 
response times are recorded. 

First, the time interval necessary for the patient to respond to 
either stimuli; second, the time interval necessary for the 
sensation to disappear. Stopwatches are used to record these 
intervals. Electrical pulp tests utilize the Burton vitalometer. 
The teeth are extracted under local anesthesia between one and 120 
days postoperatively and routinely processed for microscopic 
interpretation. 

Ma.ior Findin_gs : 

1. Heightened tooth sensitivity decreased as the time period 
increased and it was associated with an acute state of pulpal 

inflammation. 

2. A delayed response to cold occurred in a group of teeth whose 
pulps had undergone the severest histopathological change. 

3. Saliva can be detrimental to the pulp as evidenced by the 
formation of intrapulpal abscesses. 

4. Bacterial penetration after 4 months averaged only 0.33 mm. 
from the cavity floor. Bacteria were found in the pulp only in the 
one specimen with a mechanical exposure. 



372 ( 



Serial No. NIDR-90 (c) (68) 

Significance to Dental Research ; 

Previous studies by other investigators have been unsuccessful in 
attempting to correlate clinical signs and symptoms with pulpal 
pathology. None of the previous investigators had intentionally 
produced pulpal irritation and measured patient responses. 

If a correlation could be established, the clinician's diagnostic 
ability would be greatly increased", and improved treatment offered. 
Knowing better the state of health of the pulp, the dentist could 
determine whether conservative or endodontic treatment would be 
recommended or necessary. ■ ' 

Few have investigated the effects of oral fluids on the dental pulp. 
Several investigators have mentioned that pulpal changes that have 
occurred under restorations are not due to the restorations them- 
selves but are actually due to leakage at the interface. If 
saliva proves to be detrimental to the pulp, then the clinicians 
and dental material manufacturers would take an even greater effort 
in manufacturing and utilizing a material that more closely adapts 
to the tooth surface. 

Proposed Course of the Project ; 

If a standarized pulpal response can be corrobrated into clinical 
signs and symptoms then various medicaments will be applied to 
counteract these changes. For instance, a corticosteriod 
preparation could be utilized to determine whether the steriod 
component can enhance healing of an irritated pulp, limit the extent 
of the reaction or have no effect. 

Part B not included 



ZIB 



Serial No. NIDR-91 (c) (67) 
1. Oral Medicine and Surgery 
3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Histopathology of the Periodontal Ligament and Alveolar 
Bone Following Endodontic Treatment 

Previous Serial Number: NIDR-71 

Principal Investigator: Dr. T. Lundy 

Other Investigators: Dr. H.R. Stanley 

Cooperating Units: None 

Man Years : 

Total: 3/4 
Professional: 3/4 
Other: 

Project Description: 

Objectives : 

To study the response of the periodontal ligament and alveolar bone 
following perforation of a previously intact root by an endodontic 
instrument or a bur. 

Methods Employed : 

Teeth scheduled for extraction for prosthetic reasons were used for 
this study. Routine endodontic treatment was performed utilizing the 
rubber dam in order to maintain aseptic conditions. The root canal 
was properly debrided, and prepared for obturation. 

Before filling, a perforation through the previously intact root was 
made using an endodontic instrument or bur. Then the main canal was 
filled utilizing the laterally-condensed gutta percha technique. 
Therefore, only the area or the channel, which was created by the 
perforation procedure, was not filled. After various observation 
time periods the teeth were extracted with that portion of the invol- 
ved labial bone left intact. The hard and soft tissues were fixed in 



21^ 



Serial No. NIDR-91 (c) (67) 

10 % formalin, and processed for microscopic evaluation. The spa 
cimen were serially sectioned in a horizontal plane. In this way, 
one could examine the response of the periodontal ligament, alveolar 
bone, and the dentin and cementum in the region of the perforation. 

Major Findings ; 

The study is in process. 

Significance to Dental Research ; 

Perforation of a previously intact root can occur from the follow- 
ing situations: 

1, During endo- treatment , an endodontic instrument can penetrate 
the dentin and cementum of the root and involve the surrounding 
ligament and alveolar bone. 

2. Perforation of the root can occur when placing a post into the 
root canal in order to construct a crown. Previously the perfora- 
tion of a root surface would have resulted in the extraction of the 
tooth . 

At present time there is a trend toward a more conservative approach. 
If a perforation ensues the dentist will flap the area, locate the 
perforation and seal it off with amalgam. Prognosis of these teeth 
subjected to an amalgam repair has not been determined, although it 
is considered to be quite favorable. Presently, the surgical app- 
roach is the only way of salvaging such teeth. There are several 
undesirable aspects to repairing the perforation surgically; 

a. Post-operative discomfort. 

b. The necessity of removing the labial plate of bone in the region 
of the perforation to gain access to the lesion for amalgam sealing. 
Unfortunately, in the removal of the alveolar bone, one might affect 
the stability of the tooth already involved by bone loss. 

c. Unforeseen surgical problems may arise when, in throwing back 

a flap, one cannot always find the perforation, especially if it is 
located lingually or on a mesial or distal lingual aspect. In these - 
cases some clinicians would extract the teeth while others will re- 
sort to periodic observations. This study will determine what events 
occur in the region adjacent to the perforation. Does the periodon- 
tal ligament and alveolar bone regenerate, or does the initial in- 
jury cause a subsequent degeneration and expansive destruction in 



375 



Serial No. NIDR-91 (c) (67) 

i!i=; ares? If the study shows that the area can heal following a 
perforation then the clinician can resort to a non-surgical proce- 
dure in siffiilar situations. The only procedure that the clinician 
would have to do is to fill the main canal, thereby not subjecting 
the patient to surgery. 

Proposed Course of Project ; 

1. Even though the main canal is obturated, utilizing gutta percha 
with a root canal sealer, a small quantity of sealer will be ex- 
truded through the perforation into the injured alveolar bone and 
periodontal ligament. A thorough investigation of the toxicity of 
the root canal sealer on alveolar bone and periodontal ligament 
will be conducted in order to determine whether the cement impaired 
healing o 

2. The extent of the initial injury to the supporting structures 
could have a decisive role in whether healing can be achieved. The 
siae of the perforation will be correlated with the degree of heal- 
ing. 

Part B not included 



376 



Serial No. NIDR-92 (c) (57) 
1. Oral Medicine and Surgery 
3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Histopathology of the Human Dental Pulp 

Previous Serial Number: NIDR-72 (c) 

Principal Investigator: Dr. H.R. Stanley 

Other Investigators: Dr. H. Swerdlow, Dr. T. Lundy and Miss S. Knadle 

Cooperating Units: School of Dentistry, Indiana University, Indianapolis, 

Indiana; U.S.P.H.S. Dental Health Center, San Fran- 
cisco, California, Tufts College Dental School 

Man Years : 

Total: 2-1/4 
Professional: 1/4 
Other: 2 , 

Project Description 

Objectives ; 

1. To determine the range of pulpal responses to various operative 
and restorative procedures. 

2. To determine the healing potential of normal and diseased pulp. 

3. To determine the interval for odontoblastic regeneration and the 
rate of reparative dentin formation. 

4. To determine the effects of steroid medication on the pulpal 
tissues and response. 

5. To correlate clinical symptoms with microscopic pathology. 

6. To determine the part saliva plays in intensifying the pulpal 
response. 

7. To determine the mechanism of pulp stone formation. 

8. To establish criteria for human pulp studies that will be 



377 



Serial No. NIDR-92 (c) (57) 

acceptable for forthcoming international standards. 

9. To gain understanding concerning the cracked-tooth sjmdrome. 

Methods Employed ; 

Patients are selected with non-carious, non-restored teeth to be 
extracted for prosthetic or periodontal reasons. Except for full 
crown procedures, most of the studies require Class V cavity prep- 
arations cut on the labial or buccal surface. The teeth are treated, 
restored and extracted at varying postoperative intervals to meet 
the requirements of a specific protocol. After fixation in formalin, 
the teeth are decalcified in 57= formic acid, embedded in paraffin, 
serially sectioned, and routinely stained with hematoxylin and eosin. 
Masson's trichrome. Wilder 's reticulum stain, Feulgen's reaction, 
periodic acid-Schiff, toluidine blue, and other special stains are 
also utilized. Every attempt is made to standardize the categories 
to be compared in respect to age of patients, tooth size, postopera- 
tive extraction interval, and remaining dentin thickness. The pul- 
pal reactions are compared by recording in incidence and intensity 
of the inflammatory response, cellular displacement and reparative 
dentin formation. 

At present one collaborative research contract ($21,600) is underway 
at the University of Indiana School of Dentistry. Investigators 
there are evaluating the effectiveness of various types of liners 
beneath experimental epoxy resin restorative materials. At Tufts 
Dental College another collaborative research contract ($8,000) is 
nearing completion concerned with the effects of full crown procedures, 

Major Findings : 

1, When a prepared cavity was washed with a steroid formula contain- 
ing 1% prednisolone in a vehicle of parachlorophenol, cresatin and 
gum camphor, before restoration with zinc oxide and eugenol, the 
average pulpal response was minimized from one-third to one-half. 
When the prednisolone was used without the vehicle, the inflammatory 
responses returned after 12 days. The long-term effect was sustained 
only in the presence of the vehicle. When the inflammatory responses 
resulting from cavity preparation were permitted to become fully 
established, the minimizing or modifying effect of the steroid fona- 
ula was not observed until 48 hours after application. The steroid 
formula has definite efficacy for reversing an established focal 
pulpitis and shortening the resolution period. 

2, The rather misleading statements in the literature concerning 
the rate of reparative dentin formation indicated a necessity for 
establishing some guidelines for this phenomenon. Little evidence 
of reparative dentin formation is apparent prior to the thirtieth 

375 



Serial No. NIDR-92 (c) (57) 

postoperative day. The rate of formation was highest initially 

in the 27-48 day interval (3.5^ /day); decreased markedly after 

the forty-eighth day of the experimental period to 0.7^>t /day; 

and decreased further to 0.23,^* in the 72-132 day period. One should 

not expect more than 0.2 mm of reparative dentin to be produced 

within 100 postoperative days. 

3. Calcium hydroxide, when used as a cavity liner and not in direct 
contact with the pulpal tissues, did not increase the rate of repar- 
ative dentin formation. Only when actually controlling the pulp 
tissue was a stimulus apparent. 

4. The condensation of gold foil even with the newer mechanical 
malleting instruments produces a considerable pulpal response when 
the gold foil is applied directly to freshly cut primary dentin not 
lined by reparative dentin. A Copalite lining plus a cement base 
will reduce the pulpal response by 50%. When gold foil is condensed 
against dentin lined by reparative dentin little or no pulpal response 
en sues . 

5 . Before we can determine the pulp responses of carious teeth to 
various experimental technics, we must determine how to predict the 
quantity of pulpal pathology to be found in the carious teeth prior 
to experimentation. 

Ita a study based on the histological examination of non-operated 
carious human teeth, measurements were made between the most advanced 
point of bacterial penetration in the dentinal tubules and the pul- 
pal tissues. As long as the cariogenic organisms were more than 
1.0 mm from the pulp, tte pulpal pathology was insignificant. When 
the bacteria reached within 0.75 mm of the pulp, a definite increase 
in pulpal pathology occurred. But not until the reparative dentin 
Itself was invaded by the cariogenic organisms was significant path- 
ology (abscess formations and chemically inflamed granulation tissue) 
found . 

6. In the field of experimental epoxy resins, we have found them 
to be equal to silicate cements in terms of pulpal initiation. At 
this time they can be recommended for use only in conjunction with 
an Impermeable liner. The collaborative research contract results 
are not yet complete. However, the liners presently being marketed 
with certain epoxy resins and considered acceptable by the profession 
are not providing adequate pulpal protection. 

7. In a rat study utilizing trltlated thymidine, it was found that 
only one peak of mitotic activity occurred after operative Injury. 
Ninety percent of the mitotic divisions occurred between 72 and 96 
hours and differentiation of the daughter cells into odontoblasts 
was apparent. 

379 



Serial No, NIDR-92 (c) (57) 



Significance to Dental Research : 



1. Contradictory findings in the dental literature leave the pro- 
fession in a quandary concerning the use of certain operative tech- 
nics and filling materials. Much of this confusion apparently has 
emanated from the lack of appreciation of various investigators for 
the response of the pulp to operative trauma per se . Pulpal response 
can vary in incidence and intensity according to the speed and pres- 
sure employed in instrumentation, the thickness of remaining dentin, 
the postoperative extraction period, the type of coolant, the size 

of the cutting tool, the amount of salivery contamination. All of 
these factors must be considered before evaluating accurately the 
additional irritating properties of permanent and temporary filling 
materials, cavity liners and sterilizing agents, 

2. Specific biologic problems related to dental procedures are oc- 
curing at an ever-increasing rate and require a stepped-up program as 
described in this report to keep pace with the needs of the dental 
profession. 

Proposed Course of Project ; 

1. With the high speed technics, the traiimatic effect of cutting 
tooth structure is minimal enough that the incidence of reparative 
dentin is greatly reduced. Consequently, virgin tubules opened by 
operative technics remain open and permit the filtration of toxic 
products of cement and silicate to reach the pulp tissues. Measures 
must be found to either increase the incidence of reparative dentin 
formation and/or seal these tubules adequately against permeating 
substances. 

2. There is a need for describing the significant events within the 
entire panorama of odontoblastic regeneration. Although much of this 
information can be derived from fixed material, studies are in pro- 
gress utilizing tritiated thymidine on rats and primates » 

3. In order to cope with the increasing number of biologic problems 
in this field contract arrangements will be continued with various 
dental schools and hospitals to secure additional specimens for our 
studies. 



Part B 



Honors and Awards : 

1. Elected to President of the American Academy of Oral Pathology, 
April 5, 1967. 



380 



( 



Serial No. NIDR-92 (c) (57) 

2, Formal consultant to U.S. Naval Dental School, National Naval 
Medical Center, Bethesda, Maryland. 

3. Appointed to Subcommittee of F.D.I, on Toxicity of Filling Ma- 
terials. 



Publications; 



1. Stanley, H.R., Swerdlow, H. and Buonocore, M.G. : Human pulpal 
response to experimental restorative resins. J.A.D. A. ; 75:132- 
141, July , 1967. 

2. Stanley, H.R.: Design for a human pulp study. Oral Surg.. Oral 
Med. . and Oral Pth. 

3. Stanley, H.R. and Weaver, K. : A technique for the preparation 
of human pulpal tissues. Accepted for publication in a workshop 
monograph entitled "Biology of the Dental Pulp Organ." 

4. Sundell, J.R., Stanley, H.R,: The relationship of coronal pulp 
stone formation to experimental operative procedures. Oral Surg. , 
Oral Med., and Oral Path. : April, 1968. 

5. Stanley, H.R. Dental Science Handbook, Chapter on "Oral Path- 
ology". To be published by the American Dental Association. 

6. Stanley, H.R. : Human pulpal response to new drugs and restorative 
materials. Mass. Dental Society Journal 16: 80-84, Spring, 1967. 

7. Stanley, H.R.: "The cracked tooth syndrome". To be published 
in the Journal of the American Academy of Gold Foil Operators . 



381 



Serial No. NIDR-93 (c) (65) 

1. Oral Medicine and Surgery 

2. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Autogenous Replantation of Human Teeth 

Previous Serial Number: NIDR-73 (c) 

Principal Investigator: Dr. H. R. Stanley 

Other Investigators: Drs. P. N. Baer, P. Lightb.ody, H. Swerdlow and 

T. Lundy 

Cooperating Units: None 

Man Years : 

Total: 2-1/4 
Professional: 1/4 
Other: 2 

Project Description: 

Objectives : 

1. To observe in time sequence the reaction of the periodontal 
tissues to autogenous reimplantation of human teeth. 

2. To establish the regenerative period for cementob lasts . 

3. To study the phenomena of ankylosis and resorption. 

4. To determine why a cemental surface that has been involved by a 
periodontal lesion is apparently incapable of offering a suitable 
surface for the deposition of new cementum. 

Methods Employed : 

On specially selected patients that have teeth to be extracted for 
prosthetic or periodontal reasons and who will eventually receive 
full dentures, one or two teeth were extracted, received endodontic 
therapy, cleansed and re-inserted into the same tooth socket. Before 
re-insertion, portions of the cemental surface were scored with a 
rotary cutting instrument to expose the primary dentin. Each re- 
implanted tooth was splinted until reattachment and stability occur- 



382 



Serial No. NIDR-93 (c) (66"^ 

red. At intervals of two weeks, one month, three months, and six 
months, the teeth were again removed with their attached tissues. 
The specimens were properly fixed and processed for microscopic 
evaluation. The specimens were serialed horizontally from the 
occlusal or incisal surface to the apex. Particular attention was 
given to the status of the cementum, whether that cementum inicially 
left had resorbed or been coated with new cementum and whether new 
cementum had been deposited on the exposed dentin. Characteristics 
of immune rejection represented by resorption and ankylosis were 
detailed. 

Major Findings ; 

1. Most of the reimplanted teeth are accepted and become firmly 
attached. A few teeth are exfoliated within several days. This is 
a problem in itself that needs clarification. 

2. New functioning cementoblasts have not been found earlier than 
29 days. Incidentally the regeneration of odontoblasts with produc- 
tion of reparative dentin takes 30-35 days. 

It is possible that the same interval of time is required for the 
differentiation of cementoblasts. 

3. It appears that resorption is generally accompanied by ankylosis. 
It must be determined whether resorption will occur in the complete 
absence of ankylosis. 

Significance to Dental Research ; 

Through the centuries tooth replantation has been attempted. Present- 
day methods are not much more successful than experienced 100 years 
ago. However, with progress in the science of immunology, the reasons 
for failure are now more understandable. Replanted teeth do not 
produce a typical rejection phenomenon but are eventually resorbed 
and exfoliated after about 18 to 24 months. To date no one has 
carried out a basic study of the initial and progressive tissue 
responses related to human replanted teeth in time sequence fashion. 
Instead, the teeth are usually permitted to remain in the mouth until 
exfoliated and then examined by a pathologist. 

For a long time, the periodontists have been stating that their best 
clinical results in regard to reattachment of the periodontal tissues 
occurred within an intrabony pocket. Reattachment otherwise has been 
generally unsuccessful, the clinician being satisfied to obtain 
shrinkage of gingival tissues with increased tissue tone. Recently, 

383 



Serial No. NIDR-93 (c) (66) 

however., long term clinical studies have shown, unfortunately, that 
intrabony pockets offer no particular advantage for reattachment. 

In other words, a cemental surface that has been involved by a 
periodontal lesion is apparently incapable of offering a suitable 
surface for the deposition of new cementum, a requirement for 
reattachment of periodontal collagen fibers. 

In other words, the problem of reattachment facing thousands of 
periodontists throughout the world, is the same problem thwarting 
the success of autogenous replantation of human teeth. Any knowledge 
that might be derived from the study of the autogenous replantation 
of human teeth could also be beneficial to the entire practice of 
periodontology. 

Proposed Course of Project : 

1. During the first 18 months of this program, nineteen patients 
have been included in the study. During the coming three-year 
period we hope to obtain a total of fifty cases. 

2. There is a new industrial technique for coating glass which makes 
it more acceptable for tissue culture growth. Our interest in this 
technique is to determine whether a thin coating of this same material 
on a duplicated acrylic tooth before replantation in the original 
socket would provide a surface suitable for the differentiation of 
cementob lasts and the deposition of cementum. 

3. Some recent studies have indicated that decalcified bone en- 
courages bone regeneration at an amazing rate. It is planned to 
subject the root surface of teeth to minimal decalcification in order 
to affect the outer surface but not enough to weaken the total struc- 
ture of the tooth. 

Part B not included. 



38t^ 



Serial No. NIDR-94 (c) (44) 
1. Oral Medicine and Surgery 
3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Studies on the Etiology and Control of Rampant Dental 
Caries: Clinical and Experimental Animal Studies on 
the Differentiation of Cariogenic and Non-car iogenic 
Foods 

Previous Serial Number: NIDR-74 (c) (44) 

Principle Investigators: Dr. R. M. Stephan 

Other Investigators: None 

Cooperating Units: None 

Man Years: 

Total: 2 
Professional: 1 
Other: 1 , 

Project Description: 

Objectives : 

Rampant dental caries is a very severe form of the disease in 
which practically all of the teeth are attacked by decay in a re- 
latively short period of time. It is found chiefly in young child- 
ren, but may develop in adults who previously had little or no 
caries experience. Under suitable experimental conditions, com- 
parable forms of rampant caries can be developed in laboratory 
animals such as rats and hamsters. From a research standpoint, 
rampant carias offers a most favorable opportunity to study the 
basic factors which activate or control the caries process because 
the usually prolonged time element in the development of carious 
lesions is reduced to a minimum, and the determination of caries 
activity can be much more certain than in caries of usual severity. 
The purpose of this project is to evaluate in clinical studies the 
many factors which may be important in different cases of rampant 
caries, and to study the more important of these factors in labor- 
atory and animal experiments with the goal of establishing more 
effective means for solution of the caries problem. 

3SS 



Serial No. NIDR-94 (c) (44) 
Methods Employed ; 

1, Clinical Studies 

In addition to the usual oral and medical examination and labora- 
tory tests, a detailed history is taken in regard tos (a) caries 
experience in the family; (b) diet and drinking water, illnesses 
and medication during the period of tooth formation; (c) food 
selection, eating habits and other factors affecting oral hygiene 
and food retention around the teeth during the period in which 
caries developed; and (d) a comparison with these factors in 
siblings and other patients with minimal caries. 

Direct stereomicroscopic observations are made on the growth of 
bacterial plaques and the retention of food material in carious 
tooth surfaces as compared with intact tooth surfaces, using the 
Zeiss "otoscope" at 6X to 40X magnification. pH measurements are 
also made in these areas and salivary flow rates are measured. 

A large number of plaque samples for the fluorescent antibody 
study of specific strains of alpha streptococci which had accumu- 
lated from previous years, have now been studied by Mrs. Shirley 
Gels and this part of the rampant caries study is reported by her 
in a separate project report. 

2. Laboratory Animal Experiments 

The clinical studies have indicated that the frequent eating of 
foods containing sucrose or other fermentable carbohydrates is a 
very important factor in the etiology of rampant caries. During 
the past year animal experiments using OM and pathogen free SD 
rats have been continued to find foods which may not be cariogenic 
even though they contain considerable amounts of fermentable car- 
bohydrates. These experiments have included the addition of grad- 
ed levels of sucrose or glucose to some of the world's basic foods 
which are relatively non-cariogenic, such as milk, whole wheat, 
meat and fish meal, or fish protein concentrates. The sucrose or 
glucose was given in several ways, either mixed with the food 
tested, given separately in a second food cup, or given as a solu- 
tion in the drinking water. In addition certain minerals, includ- 
ing calcium salts, phosphates, calcium phosphate, magnesium, sod- 
ium or calcium fluoride were added at graded levels to the sucrose 
or glucose to determine the relative extent to which these addit- 
ions would inhibit caries. 

Major Findings ; 

The study of new patients with rampant caries during the past year 
has further extended our knowledge of the conditions under which 



386 



Serial No. NIDR-94 (c) (44) 

rampant caries develop. The social and economic situations which 
lead people to substitute frequent between meal eating for regular 
meals was again emphasized. For example, a child had developed 
rampant caries six months after his mother hal started working 
and was no longer at home during the day to feed him. An adult 
patient developed rampant caries within a year after she had fM arc- 
ed sucking candy mints to replace her previous cigarette smoking 
habit. The pattern of eating sweets between meals has been a coir- 
mon finding in rampant caries patients. 

Previous animal experiments have shown that some foods commonly 
eaten by people, such as sucrose, glucose, aandies, cookies, some 
fruits, bread with jelly and similar foods containing fermentable 
carbohydrates were highly cariogenic to rats, whereas other foods 
such as milk, whole wheat, peanuts, popcorn, cheese, and similar 
materials were relatively non-cariogenic to rats. 

During this past year we have continued our rat experiments or. the 
effect of fish protein concentrate (f.P.C.) and fish meals to re- 
duce the cariogenic effects of sugar and other cariogenic foods 
when mixed with them; although F.P.C. contains from 100 to 250 ppm. 
fluoride, this fluoride probably accounts for only part of the 
anticariogenic effect since in other experiments in which compar- 
able amounts of fluoride were added to diets containing 
66% or 83% of sucrose, the inhibition of caries was not as great 
as with the fish products. 

Some of the anticariogenic effect is probably due to the relatively 
high level of calcium, phosphorus and basic amino acids in F.P.C, 
as well as to its effect in raising the nutritional value of high 
carbohydrate foods such as sugar and cereals. 

Significance to Dental Research : 

The chief significance of the clinical studies is to point up the 
problem of between meal eating of sweets and snacks as conducive 
to the high rate of dental caries in some members of one population, 
and to indicate the need for both «ore regular eating habits, and 
the selection of non-cariogenic foods for between meal eating. 
In addition, the need to develop more non-cariogenic snacks and to. 
make them available for people instead of the usual cariogenic 
•weets is obvious. In this regard the demonstration that fish pro- 
tein concentrates exert a great anticariogenic effect on sucrose 
when fed to laboratory rats suggests that this material and food 
supplements like it may not only be helpful in overcoming human 
malnutrition, but also in the control of caries. 



387 



Serial No. NIDR-94 (c) (44) 

Pro posed Course of Project : 

It is planned to follow up the findings evaluating the cariogenic 
and anticariogenic properties of different food materials, partic- 
ularly the effects of fish protein concentrates, and to complete 
analysis of the extensive clinical data which have been developed 
on rampant dental caries and its control. 

Part B not included 



388 



Serial No. NIDR-95 (c) (66) 

1. Oral Medicine and Surgery 

2. Oral and Pharyngeal Development 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Studies of Oral and Pharyngeal Form and Function in 
Infants 

Previous Serial Number: NIDR-76 (c) - - ' - 

Principal Investigator: Dr. J. F. Bosma 

Other Investigators: None 

Cooperating Units: Dr. W. J. Logan, Stanford University; Dr. M, Silbiger, 

Division of Radiology, Clinical Center, NIH; Division 
of Photography, NIH 



Man Years : 




Total: 


1/4 


Professional: 





Other: 


1/4 


Project Description 




Obiectives : 





To describe development of functions of pharynx and mouth in the 
normal human infant and in infants impaired by malformation or by 
neurological disorder. 

Methods Employed : 

Cineradiographic, cinephotographic and acoustical methods of 
observation of suckle feeding and of respiratory functions, 
including cry. 

Major Findings : 

Studies have been obtained on 8 additional impaired infants 
including two examples of pharyngeal incoordination of the newborn, 
and one of familial dysautonomia. 

389 



Serial No. NIDR-95 (c) (66) 

A form of swallow recognized in the mature human as "pint swallow", 
of sustained opening of the pharynogoesophageal segment without 
elevation of the hyoid and larynx, was found for the first time 
in an infant. 

The classification of m.echanisms of infant pharyngeal disabilities, 
described in 1967 by Logan and Bosma, has been extended to include 
criteria of potential mobility of the pharynx, which may be limited 
in its displacements by muscle contractures. 

Significance to Dental Research : 

The disabilities of function and form in childhood, which are the 
usual concern of dentists, originate in earlier years. Definition 
of disabilities in infancy, and description of their evolution into 
the patterns of disordered function in childhood, should provide 
meaningful etiologic perspective to the dentistry of impaired 
children. 

Proposed Course of Project : 

Continuation of these studies. 

These observations are being accumulated into a portion of a general 
publication: Development of Form and Function of the Normal and 
Abnormal Pharynx. 

Part B not included 



390 



Serial No, NIDR-96 (c) (66) 

1. Oral Medicine and Surgery/ 

2. Oral and Pharyngeal Development 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Studies of Development of the Head Skeleton of the Rat 

Previous Serial Number: NIDR-75 (c) 

Principal Investigator: Dr. J. F. Bosma 

Other Investigators: None 

Cooperating Units: University of Michigan, School of Dentistry 

Dr. M. Baer and Miss E. Hirshfeld; Dr. J. Ackerman, 
National Library of Medicine - by a Special Publica- 
tion Grant; Colorfax Laboratories, Inc. 

Man Years: 

Total: 1 
Professional: 1/2 
Other: 1/2 

Project Description: 

Objectives : 

Demonstration of patterns of growth of the head skeleton of the rat, 
as a representative mammal. 

Methods Employed : 

Rats were alizarinated in different groups at ages selected to 
illustrate specific phenomena of development of individual head 
bones. 

The sites of successive red and blue deposition are demonstrated by 
thin sectioning of rat skulls. Unstained individual bones were also 
dissected at 15, 33, and 130. days and photographed in standard 
comparison orientations. A notable element of this project is the 
form of its publication in a major volume. An Atlas of the Postnatal 
Development of the Rat Skull, with atlas-style illustrations and 120 
matching color transparencies. The preparation and duplication of 
these slides for each of 1500 copies of the book is sponsored by 

351 



Serial No. NIDR-95 (c) (66) 

the National Library of Medicine as a publication demonstration 
project. In collaboration with Dr. Baer and Miss Hirshfeld, 
Colorfax Laboratories has developed special printing procedures 
for this project. 

Major Findings : 

By these methods, it has been possible to demonstrate the patterns 
of incremental growth and modulation of form of individual bones, 
both separately and in situ , as well as the resultant increments 
and form modulations of general areas of the skull. 

Significance to Dental Research ; 

This study demonstrates and elucidates the patterns and principles 
of skull growth in a mammal. These patterns and principles are 
applicable to mammalia, in general. 

These particular demonstrations, in the rat, afford baselines for 
evaluation of experimental deformations in the laboratory rat. 

Proposed Course of Project : 

The book, An Atlas of the Postnatal Development of the Rat Skull , 
under authorship of M. Baer, J. Bosma, and J. Ackerman, is scheduled 
for submission to the Government Printing Office in FY 1969. The 
projection transparencies and matching drawings will be completed 
in May, 1968. 

In this continuing project. Dr. Baer has alizarinated at selected 
ages a basic series of 24 pigs, as representatives of omnivora, and 
14 sheep, as representatives of herbivora. These animals were 
grown and sacrificed and the skulls are now ready for sectioning. 
The study will also be extended to rabbits. 



Part B 



Publications: 



1. Baer, M. J. , and Ackerman, J. L. : "A Longitudinal Vital Stain- 
ing Method for the Study of Apposition in Bone" in Studies on the 
Anatomy and Function of Bone and Joints . Springer-Verlag, Berlin, 1966. 

As an extension of this project interest. Dr. M. Baer has also 
arranged and conducted a Symposium on In Vivo Bone Markers, presented 
at the 1967 Meeting of the American Association of Physical Anthro- 
pologists. The contents of this Symposium will appear as a single 
issue (July, 1968) of the American Journal of Physical Anthropology. 

332 



Serial No. NIDR-97 (c) (62) 

1. Oral Medicine and Surgery 

2. Oral and Pharyngeal Development 

3. Bethesda, Maryland 



PHS-NIH 

Individual Project Report 

July 1, 1967 through June 30, 1968 



Part A 



Project Title: Studies of Sensory and Motor Functions in Subjects 
Impaired by Malformations of Neurological Disease 

Previous Serial Number: NIDR-77 (c) (62) 

Principal Investigator: Dr. J. F. Bosma 

Other Investigators: Dr. R. D. Christensen, Dr. R. L. Christiansen, 

Dr. L. Krames, and Dr. B. Weinberg 

Cooperating Units: Dr. D. Brodie, National Institute of General Medical 

Sciences; Dr. R. Henkin, National Heart Institute; 
Dr.. J. Kavanagh, National Institute of Child Health 
and Human Development; Dr. M. Silbiger, Clinical 
Center Division of Radiology and Division of Medical 
Illustrations and Photography; Cleft Palate Clinic 
of the Johns Hopkins Medical Center; University of 
Indiana Department of Neurology; Department of 
Speech, Catholic University of America. 

Man Years: 

Total: 11/2 
Professional: 1/2 
Other: 1 

Project Description: 

Objectives : 

Continuation of studies of sensory, perceptual and motor mechanisms 
in the mouth and pharynx. 

Methods Employed : 

The clientele continue to include a variety of subjects impaired by 
malformation and/or neurological impairment in the oral and pharyn- 
geal area. The basic study methods continue to include standard 
routines of cinephotography , cine and still radiography, etc. 



393 



Serial No. NIDR-97 (c) (62) 

Major Findings : 

The correlative studies of anatomical form and of sensory and 
motor function of an array of persons having primary abnormalities 
of function has afforded opportunities of recognition of abnormali- 
ties of form in the neurologically impaired and, reciprocally, of 
abnormalities of function in those malformed by antenatal anomaly. 

Particular studies have been performed in 3 subjects having severe 
hypoplasia of the tongue. Clearly articulated speech is achieved 
in two of these by distinctive motions of the lips, and in all 
three by distinctive motions of the dorsal portion of the anomalous 
tongue mass in relation to the soft palate and the walls of the 
mesopharynx. Our appreciation of the potential mechanisms of 
speech articulations are thus increased. Following identification 
of articulatory valving in the lateral portion of the lip, this lip 
manuever was discovered in corresponding /s/ articulation in an 
adult who had learned new speech articulation after surgical 
excision of a major part of his tongue, in therapy of cancer. 

This correlative study approach has also been applied to children 
having hypoplasia of the facial skeleton and submucous cleft palate. 
In 5 children having anatomically similar patterns of hypoplasia 
and similar abnormalities of speech and of feeding, R. Henkin found 
hypesthesia of the palate and similarly patterned abnormalities of 
taste and smell; their form of hyposmia (designated "type 2") was 
found also in their mothers. Analogous coincidences of abnormal- 
ities of facial form, of smell and of taste were found in subjects 
with Turner's syndrome of hypogonadism. 

Since different motor mechanisms are employed by the oral and 
pharyngeal area in feeding and in speech functions, these two 
categories of function were observed in comparison for the demon- 
stration of potentialities of motor performance. This comparison 
has been made in the upper pharyngeal function of cleft palate 
children. In most children with hypoplasia of the palate and in 
some with simple cleft palate who have had the usual form of 
surgical repair of the cleft, the upper pharynx closes adequately 
in the initial phase of swallow; nasal regurgitation of bolus does 
not occur. But the palatopharyngeal isthmus fails to close 
consistently or adequately in speech, and these subjects are 
correspondingly "hypernasal." This duality of performance mecha- 
nisms is also applicable to the analysis or study of neurological 
impairments. In the current report year, studies were brought to 
publication in two neurological syndromes which are manifested 
principally in impairments of the motor unit; amyotrophic lateral 
sclerosis and myotonic dystrophy. In each of these conditions, 
discrepancies were found between the actions of the mouth and 
pharynx in feeding actions, which were commonly adequate in function. 



39i| 



Serial No. NIDR~97 (c) (62) 

compared with the actions of speech and the respiratory function of 
maintenance of the pharyngeal airway, which were relatively more 
distorted and impaired. This approach of dual observation oi 
separate motor functions is applicable to other forms of neurologi- 
cal abnormality. A similar discrepancy between adequacy of feeding 
functions and failure of speech functions was reported previous. ly 
in two clinically similar subjects whose syndrome of abnormality 
was defined by deficiencies and impairments of oral sensation and 
perception (Bosma, J.F., Grossman, R. C, and Kavanagh, J. F. , 
A Sjmdrome of Impairment of Oral Perception, Chapter 18, in First 
Symposium on Oral Sensation and Perception , 1967). 

The tests of oral form perception described in the first Symposiam 
on Oral Sensation and Perception were further standardized by 
Dr. Weinberg, assisted by a graduate speech student from George 
Washington University and another student from Catholic University 
of America. Time parameters of the test were employed, and were 
found to afford separate additional criteria of test performance. 
A major effort has been devoted to the arrangement of a Second 
Symposium on Oral Sensation and Perception, and its transcription, 
editing and preparation for a volume publication. The contributions 
included 11 NIH Intramural and 18 Extramural persons. The publica- 
tion is of 29 titled presentations, of 180 edited discussion items 
and 4 final comments. Its estimated text is 660-680 pages. 
Included are original publications on mechanisms of taste sensation, 
on central representations of oral area afferents, on reflex 
functions of the mouth of the fetus and the neonate, on oral 
sensation and perception testing in normals and in subjects impaired 
by neurological disease. 

Significance to Dental Research : 

The increments of understanding of oral and pharyngeal function in 
normal and in impaired subjects are strategic to the development of 
additional techniques of study and therapy and of extension of 
dental therapy to additional categories of impaired persons. 

Proposed Course of Project : 

Continuation of current study routines, graduating generally to 
clientele of neurologically impaired subjects, and fewer subjects 
impaired by primary malformation in the facial area. 



395 



Serial No, NIDR-97 (c) (62) 



Part B 



Publications: 



1. Henkin, R.t Impairment of Oral Sensation and Perception and 
. Hyposmia in Association with Facial Hypoplasia and Growth 

Retardation, Chapter in publication in Second Symposium on Oral 
Sensation and Perception , in preparation. 

2. Weinberg, B., Bosma, J. F., Shanks, J, C, and DeMyer, W.: 
Myotonic Dystrophy Initially Manifested by Speech Disability, 
J. of Speech and Hearing Diseases , 33:51-58, 1968. 

3. Bosma, J, F. and Brodie, D.: Disabilities of the Pharynx in 
Amyotrophic Lateral Sclerosis, as Demonstrated by Cineradiogra- 
phy, submitted to Neurology . 

4. Bosma, J. F.: Editor, Second Symposium on Oral Sensation and 
Perception , in preparation. 

5. Weinberg, B., Lyons, M. J., and Liss, G. M.: Studies of Oral, 
Manual, and Visual Form Identification Skills in Children and 
Adults, in Second Symposium on Oral Sensation and Perception , 
in preparation. 

6. Weinberg, B., Liss, G. M. and Hillis, J.: A Comparative Study 
of Visual, Manual, and Oral Form Identification in Speech 
Impaired and Normal Speaking Children, in Second Symposium on 
Oral Sensation and Perception , in preparation. 

7. Henkin, R. I., Christiansen, R. L. , and Bosma, J. F.: Impair- 
ment of Oral Sensation and Perception and Hyposmia in Associa- 
tion with Facial Hypoplasia and Growth Retardation, in Second 
Symposium on Oral Sensation and Perception , in preparation, 

8. Henkin, R. I.: Manual and Oral Stereognosis in Normal Volun- 
teers and in Patients with Various Abnormalities of Taste and 
Olfaction, in Second Symposium on Oral Sensation and Perception , 
in preparation. 

9. Henkin, R. I.: The Neuro -Endocrine Control of Sensation and 
Perception, in Second Symposium on Oral Sensation and Percep- 
tion , in preparation. 



39S 



Serial No. NIDR-98 (c) (66) 

1. Oral Medicine and Surgery 

2. Oral and Pharyngeal Development 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Serial Extraction Study on Preadolescent Children 
Having Crowded Class I Occlusion 

Previous Serial Number: NIDR-51 

Principal Investigator: Dr. R. D. Christensen 

Other Investigators: Dr. L. F. Mills, and Dr. R. L. Christiansen 

Cooperating Units: Carole Highland Elementary School; Holly Park 

Elementary School; 0. W. Phair Elementary School; 
and Hollywood Elementary School, Prince Georges 
County, Maryland 

Man Years: 

Total: 11/4 
Professional: 3/4 
Other: 1/2 ' 

Project Description: 

Objectives : 

1. Compare the changes which occur within the dentition of 
subjects who undergo serial extraction, and subjects with 

a similar diagnosis but do not receive extractions. Special 
notice will be made of arch width, length, crowding of teeth, 
over jet, overbite, root length and formation, and status of 
supporting tissues. 

2. Compare the degree and direction of development of the facial 
bones of these two groups. Special interest will be taken in 
observing the size of maxilla and mandible achieved within 
these two groups.. 



39? 



Serial No. NIDR-98 (c) (66) 
Methods Employed ; 

Intraoral mirror examinations on approximately 1200 first, second, 
and third grade children in four elementary schools have been 
performed. Initial identification of sample subjects was based on 
clinical evidence of: 

1. moderate to severe crowding of permanent anterior teeth. 

2. harmonious size, form, and relationship of maxilla and 

mandible 

3. good oral hygiene and minimal decay of teeth 

4. a mixture of deciduous and permanent teeth present in the 

mouth 

5. medically healthy and normal and oral and facial muscle 

function 

Subjects found to meet these criteria will undergo more through 
examination including collection and analysis of the following 
records: (a) medical and dental histories; (b) detailed mouth 
examination; (c) five cephalometric radiographs; (d) radiographs 
of individual teeth; (e) orthodontic study models; (f) facial 
and intraoral photographs; and (g) medical examination. 

Those subjects found after thorough examination to meet the most 
rigid criteria for serial extraction procedures will be re-examined 
every six months until the permanent dentition is complete. 

Major Findings: 

The preliminary examinations of 1200 children have revealed: 

1. a wide range of variation in permanent tooth eruption 

time and sequence 

2. an apparently closer correlation between time of tooth 

eruption and other physical growth parameters than 
exists between eruption time and chronologic age 

3. a lower incidence of subjects meeting the criteria for 

the long term study than was anticipated. 

Significance to Dental Research : 

Serial extraction procedures are used frequently in dentistry to 
help guide the eruption of permanent teeth into more favorable 
positions in the dental arches. The procedure was designed to 
anticipate and hopefully prevent the development of a fully matured 
deformity in the permanent dentition. It is applied by extraction, 
in proper sequence, of a predetermined series of deciduous and 
permanent teeth. Such procedures are presented in graduate and 
undergraduate orthodontic texts as a method of interceptive 

338 



Serial No. NIDR-98 (c) (66) 

orthodontics, following a thorough diagnosis, which will not 
necessarily result in an ideal occlusion but will produce a more 
desirable alignment of teeth. There are, however, questions about 
serial extraction which still remain unanswered including 
documentation of the concomitant changes occurring dentally anri 
skeletally and their frequency of occurrence. 

Proposed Course of Project : 

The findings of the preliminary screening examinations have 
suggested a need for revision of subject selection criteria 
before continuing the long term study proposed. The findings 
related to variation in tooth eruption are being more thoroughly 
analyzed in anticipation of publication. 

Part B not included*) . , 



399 



Serial No. NIDR-99 (c) (67) 

1. Oral Medicine and Surgery 

2. Oral and Pharyngeal Development 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Comparative Skeletal and Soft Tissue Cephalometric 
Analysis of Acromegalic and Normal Human Adults 

Previous Serial Number: None 

Principal Investigator: Dr. R. D. Christensen 

Other Investigator: None 

Cooperating Units: Dr. J. Roth and Dr. P. Gorden, Clinical Endocri- 
nology Branch, National Institute of Arthritis and 
Metabolic Diseases 

Man Years: 

Total: 1/2 
Professional: 1/4 
Other: 1/4 

Project Description: 

Objectives : 

1. Describe the skeletal and soft-tissue changes in the cranio- 
facial complex of individuals with acromegaly. 

2. Assess changes, if any, in cranio-facial skeletal and soft 
tissue features of the acromegalic following irradiation of 
the pituitary tumor. 

Methods Employed : 

Lateral cephalometric headfilms on 2 7 male and 15 female humans 
with acromegaly have been analyzed. Similar measurements have 
been made on headfilms of 20 male and 20 female humans with no 
evidence of acromegaly. Comparative statistical analyses have 
been employed to determine areas of significant difference between 
the acromegalic and non-acromegalic samples. 



^00 



Serial No. NIDR-99 (c) (67) 

Additional headfilms have been taken at annual intervals following 
irradiation of the pituitary ttimors of approximately 20 of the 
acromegalic patients. These are analyzed and compared with the 
pre-irradiation analyses. 

Major Findings : 

1. The greatest differences between groups was (a) increased 
thickness of soft tissue at the midsagittal plane in the region 
of lower lip and chin in the acromegalics, (b) increased 
mandibular length, thickness, and density in the acromegalics, 
(c) increased area (measured with a planimeter) between mid- 
sagittal soft tissue profile and a plane from nasion through 
pogonion in the acromegalics. 

2. No significant change in skeletal or soft tissue measures in 
the acromegalics treated by irradiation. 

3. Greater soft tissue profile thickness in the acromegalics than 
in a group of 13 young adults with prognathic mandibles but no 
evidence of elevated growth hormone levels. 

Significance to Dental Research : 

Many questions regarding mechanisms of growth of the facial complex 
are unanswered. The role of growth hormone in normal facial growth 
is not understood. The description of facial changes in the 
presence of elevated growth hormone provides information necessary 
for a better understanding of facial growth processes. 

Proposed Course of Project : 

The patients who have had radiation therapy should continue to have 
periodic radiographic evaluations. All new patients with acromegaly 
entering the Clinical Center should be included in this study. 

Part B not included. 



i^Ol 



Serial No. NIDR-100 (c) (66) 

1. Oral Medicine and Surgery 

2. Oral and Pharyngeal Development 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Study of Taste Thresholds, Tastebud Distribution, and 
Associated Dentofacial Form 

Previous Serial Number: NIDR-78 (c) 

Principal Investigator: Dr. R. L. Christiansen 

Other Investigator: Dr. J. F. Bosma 

Cooperating Units: Dr. R. I, Henkin, National Heart Institute 

Man Years: 

Total: 1/2 
Professional: 1/2 
Other: 

Project Description: 

Objectives : 

To define the specific area or areas of the oral cavity responsible 
for normal detection and recognition of the four basic taste 
modalities in normal subjects and in edentulous subjects habitually 
wearing dentures . 

Methods Employed : 

Data on baseline taste and smell thresholds and associated oral and 
facial forms have been secured on ten young normal subjects over 21 
years of age. The taste and smell examinations involved forced 
choice among three solutions. Taste is tested under four 
conditions: no oral anesthesia, palatal anesthesia only, lingual 
anesthesia only, and both palatal and lingual anesthesia. 
Infiltration of Lidocaine is performed to produce anesthesia of 
the hard and soft palate and the tongue. 



^02 



Serial No. NIDR-100 (c) (66) 

Ma.jor Findings ; 

The taste receptors on the palate are concentrated at the midline 
near the junction of hard and soft palate. P.eception on the tongue 
is primarily at the tip, lateral borders, and occasionally on the 
dorsum, near the tongue base. When one surface is anesthetize- the 
physical contact of tongue against palate still greatly facilitates 
taste perception regardless of which surface has been injected. 
Under anesthesia of the palate and tongue, little oral taste 
perception persists. In this condition it is possible to detennine 
thresholds of taste in the pharynx after swallowing the test 
solution. 

Significance to Dental Research ; 

Recent observations by Drs. Bosma, Henkin, and Christiansen on five 
children with deficiencies of taste and olfaction, and orofacial 
development suggest a possible relationship between the development 
of these special senses and malocclusions resulting from skeletal 
discrepancies. 

Proposed Course of Project ; 

Continue study of the senses of taste and smell in normal subjects, 
followed by studies on patients with specific forms of malocclusion. 



Part B 



Publications: 



1. Henkin, R. I. and Christiansen, R. L.: Taste Localization on 
Tongue, Palate and Pharynx of Normal Man, J. Appl. Physiol. 
February, 1967. 

2. Henkin, R. I. and Christiansen, R. L.; Taste Thresholds in 
Patients with Artificial Dentures, J. Am. Dent. Assoc. 
75:118, 1967. 



Serial No. NIDR-101 (c) (65) 

1. Oral Medicine and Surgery 

2. Oral and Pharyngeal Development 

3. Bethesda, Maryland 



PHS-NIH 

Individual Project Report 

July 1, 1967 through June 30, 1968 



Part A 



Project Title: Study of Oral Area Motor Mechanisms by Use of 
Pressure Transducers 

Previous Serial Number: NIDR-79 (c) 

Principal Investigator: Dr. R. L. Christiansen 

Other Investigators: None 

Cooperating Units: Instrument Fabrication Division, NIH; Dr. W. R. 

Prof fit. University of Kentucky, Department of 
Orthodontics; Dr. R. E. McGlone, Department of 
Speech, State University of New York, Buffalo 

Man Years: 

Total: 1/2 
Professional: 1/2 
Other: 

Project Description: 

Objectives : 

1. To further improve performance and reliability of intra-oral 
pressure transducer designs. 

2. To obtain data on pressure- time integral for forces acting on 
the teeth. 

3. To obtain data regarding tongue positioning and activity 
during swallowing and articulation of selected consonant sounds. 

Methods Employed : 

In cooperation with NIH Instrument Fabrication Division, two types 
of pressure transducers have been constructed, dynamically 
calibrated and tested by this section. The cantilever-beam trans- 
ducers utilize resistance strain gauges and the miniature trans- 
ducers use foil or semiconductor strain gauges. 



Serial No. NIDR-101 (c) (65) 

Tongue activity during speech and swallowing was studied in ten 
normal control subjects. The test instrument consisted of a 
removable maxillary appliance containing three transducers, two 
mounted bilaterally, lingual to the first molars, and one located 
just palatal to the central incisors. 

Planimetric determination of the area under the pressure curves, 
the pressure-time integral, was used in data collection. 
Computerization of pressure-time integral measurement is being 
explored. 

Major Findings: , 

It appears that 10 cps is the maximum fundamental frequency of 
lingual pressure waves even during rapid speech. Dynamic 
calibrations revealed that the cantilever-beam transducer has 
frequency response characteristics linear beyond 150 cps while the 
diaphragm transducer has linear response to 25-30 cps. Therefore, 
both designs give an adequate safety factor for studies of tongue 
movements and pressures. The diaphragm design offers the improve- 
ments of reduced size with increased output characteristics. 

Lingual pressures were measured during speech of the ten normal 
control subjects for various consonant-vowel combinations (/di/, 
/id/, /ta/, /at/, /da/, /ad/, etc.). Results indicated that mean 
lingual pressures for the consonants were unaffected by changing 
the vowel within the syllable. However, differences in pressure 
values were found between each of the consonants when syllabic 
position and vowel were held constant. Pressure values for the 
consonant in the initial position were greater than the mean 
values for the same consonant in the final position. A further 
difference was found in the time relation of maximum pressure to 
the onsent of phonation for each of the consonants. Greater 
pressure values were obtained from rapidly produced syllables as 
compared with slow speech utterances. 

Results showed considerably greater integrated pressure associated 
with swallow pressure values than with any speech activity. The 
female subjects exhibited greater lingual pressure during swallow- 
ing than did the males. This relationship was not found during 
either speech activity involving rapid and normal rates. 

Signifiaance to Dental Research : 

Muscle pressures play an ill-defined but probably important role in 
the normal development of the dentition and in the etiology of mal- 
occlusion. This study is designed to better define that role. 

^♦05 



Serial No. NIDR-101 (c) (65) 

Proposed Course of Project : 

Continued instrumentation development and intensive analysis of 
intra-oral pressure patterns during speech and swallowing activity. 



Part B 



Publications: 



McGlone, R. E., Prof fit, W. R. , and Christiansen, R. L.: 
Lingual pressures associated with alveolar consonants, J. Speech 
and Hearing Research , 10:606, 1967. 

Proffit, W. R. , Fogle, J. L. , Heitlinger, L. W. , Christiansen, 
R.L. , and McGlone, R. E.: Dynamic calibration of lingual pressure 
transducers, J. Applied Physiology , 21:1417, 1966. 



i^OS 



Serial No. NIDR-102 (c) (68) 

1. Oral Medicine and Surgery 

2. Oral and Pharyngeal Development 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Restitution of Mandibular Form After Condylar Injury 

Previous Serial Number: None 

Principal Investigator: Dr. P.J. Coccaro 

Other Investigators: None 

Cooperating Units: None 

Man Years: 

Total: 2/3 
Professional: 2/3 
Other: 

Project Description: -= 

Objectives : 

1. To prove that facial asymmetry (due to trauma of mandibular 
condyle at age 2) becomes progressively more severe because 
of accompanying aberrant muscle function on the affected side. 

2. To demonstrate influence of stimulating muscular activity thus 
creating favorable growth changes in condyle, ramus and body 
of hypoplastic hemi mandible in a child with facial asjmmietry. 

3. Demonstrate annually with photographs and radiographs a 
diminution of facial asymmetry supported by favorable skeletal 
changes in condylar and mandibular growth on the side of 
mandible which previously exhibited growth arrest. 

Methods Employed ; 

An occlusal index was made in acrylic with patient deviating as far 
laterally to the unaffected side. The appliance was cemented over 
deciduous teeth and patient wore this and similar appliances for 3 
years. The rationale behind such therapy was clinical evidence that 
the patient had marked limitation of lateral and protrusive mandibu- 
lar movements originating from the affected side. The appliance 

^07 



Serial No. NIDR-102 (c) (68) 

compelled the child to more effectively utilize muscles (internal 
and external pterygoids) on the affected side. 

Major Findings : 

1. Clinical and radiographic evidence over a 7 year period 
demonstrate a dramatic diminution of facial asymmetry present 
when patient first came to clinic. 

2. Initial laminographs of temporo-mandibular joint show marked 
hypoplasia of condyle, shallow glenoid fossa, short ramus and 
body as well as ante-gonial notching, all present on the 
affected side of patient's mandible before treatment. Final 
laminographs of temporo-mandibular joint exhibited the impact 
of muscular activity through the use of an occlusal guide 
plane. Restitution of condylar form and a glenoid fossa that 
became less shallow and more concave. Ramus height and body 
length reflect demonstrable growth over the years of study. 

3. Opening, closing and protrusive motions are more in line with 
normal parameters since being treated. This plus improved 
facial appearance is in direct proportion to obvious skeletal 
changes noted on the affected side. 

4. Study indicates that abnormal function could very well tend to 
compound severity of facial asymmetry, after initial trauma, 
and if an effort is made early enough progressive facial 
asjmmetry could be reduced or eliminated. 

5. P. A. radiographs, over a seven year period, show the change 
in facial appearance (due to favorable growth which created an 
equalization of structure size on both sides that was non- 
existent before. 

Significance to Dental Research : 

Traumatic injuries to the mandible in preschool children can 
produce damage to the growth site in the mandibular condyle. 
Facial asymmetry usually ensues and reports in the literature have 
indicated that it becomes progressively worse with growth and 
development. Abnormal mandibular movements in opening and closing 
and protrusion have also been noted in these children. Such patients 
are usually relegated to "waiting and watching" periods and recom- 
mendations for surgery when they have reached maturity. Other have 
had many surgical procedures to correct the size disparity on one 
side with the other. 



i*08 



Serial No. NIDR-102 (c) (68) 

This study certainly indicates that improved muscular functional 
activity, when restored early, can contribute significantly to 
a diminution of facial asymmetry resulting from favorable growth 
on affected side. _ . 

Proposed Course of Project : 

More cases of this type should be followed and results documented 
to further substantiate the impact of muscular activity in 
reducing or minimizing the progressive facial asymmetry which 
is expected with growth and development. 

Part B not included 



it09 



Serial No. NIDR-103 (c) (68) 

1. Oral Medicine and Surgary 

2. Oral and Pharyngeal Development 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Clinical and Roentgenographic Analysis of Orthodontics 
(on a Continuing Basis) in Cleft Palate Habilitation 

Previous Serial Number: None 

Principal Investigator: Dr. P. J. Coccaro 

Other Investigators: None 

Cooperating Units: None 

Man Years: 

Total: 7/12 
Professional: 1/3 
Other: 1/4 

Project Description: 

Objectives : 

1. To study the impact of early orthodontics on dentition and 
palate in severe palato-dental abnormalities. 

2. To discern degree of growth of palatal and alveolar processes 
bordering the cleft after early orthodontic treatment. 

3. Evaluate permanency of results in palatal and dental arch form 
after early expansion procedures in the presence of removable 
retainers and bone grafts, 

4. Document character of bone graft, on a time basis and determine 
its contribution to palatal and dental habilitation in children 
with cleft lip and palate. 



^10 



Serial No. NlDR-103 (c) (68) 
Methods Employed : 

The data selected for this study were obtained on a group ox 
patients ranging in age from 3 years to 7 years of age with cleft 
lip and palate. All received orthodontic therapy for 5 years - 
some in deciduous dentition and others in mixed dentition, 
Cephalometric , panorex, intra-oral occlusal X-rays were taken 
annually along with intra- and extra-oral photographs. Plaster 
models were made of patients' palato-dental abnormalities. All 
records reflected original problem and corresponding changes 
associated with orthodontic therapy. 

Major Findings ; 

1. Early orthodontic treatment procedures proved effective in 
correcting palato-dental abnormalities - this was observed in 
mixed dentition. 

2. Impacted palatal segments were unlocked after which time 
discernible growth of palatal and alveolar processes bordering 
the cleft was noted. 

3. Palatal expansion to achieve acceptable palatal form was 
adequate and permitted bone grafting to be electively performed, 

4. Bone graft restored integrity of dental arch and remained to 
allow unerupted teeth to emerge through it. 

5. Maintenance of results were not complete after early ortho- 
dontics and retainers. Slight palatal collapse was recorded 
even in the presence of removable retainers and bone grafts. 

6. Advantages of early orthodontics are clinically evident and 
outweigh reasons for delay. Early correction of malformed and 
malposed palates along with irregular dental arches result in 
more normal anatomical relationship of parts. It also 
contributes significantly to favorable growth for impacted 
palatal and alveolar bony processes adjacent to the cleft. 

7. Bone grafting and retainers do not create permanency of results 
when utilized during early years of dynamic change inherent in 
palatal and dental structures. Their contribution is more 
critically challenged particularly in view of the fact that 
further orthodontics is needed to obtain the final desired 
palatal and dental form and position. 



^11 



Serial No. NIDR-103 (c) (68) 
Significance to Dental Research : 

Studies of this nature will produce some of the answers to 
questions of validity, and judiciousness in advancing early- 
orthodontic and bone grafting procedures in cleft lip and palate 
habilitation. It could very well help decide the merits of 
surgical bone grafting procedures done during the early years of 
oral-facial growth and development. 

Proposed Course of Project : 

Patients will continue to be observed and treated until all 
permanent teeth are erupted and in occlusion - this is essential 
to prove the need of orthodontics, as a continuing process, for 
children with cleft lip and palate. 

Part B not included. 



itl2 



( 



Serial No. NIDR-104 (c) (68) 

1. Oral Medicine and Surgery 

2. Oral &. Pharyngeal Development 

3. Bethesda, Maryland 



PHS-NIH 

Individual Project Report 

July 1, 1967 through June 30, 1968 



Part A 



Project Title: Prenatal Development of the Larynx; Human and 
Comparative Investigation 

Previous Serial Number: None 

Principal Investigator: Dr. L. A. Krames 

Other Investigators: None 

Cooperating Units: Carnegie Institute of Embryology, Baltimore, 

Maryland; Department of Anatomy and Embryology, 
University of Toronto Dental School, Toronto, Canada 

Man Years: 

Total: 1 
Professional: 1 
Other: . 

Project Description: 

Objectives : 

1. To review the literature and the Carnegie human slide material 
concerning the development of the larynx. 

2. To apply autoradiographic, his tochemical and fetal surgical 
techniques to many unanswered questions concerning the development 
of the larynx: e.g. Neural crest contribution to the laryngeal 
cartilages; Branchial arch contributions to laryngeal structures 
and epiglottis; Early laryngeal cartilage and muscle relationships; 
Epithelial fusion and separation of the Laryngotracheal groove. 

Methods Employed : 

1. Review of histologic material of normal laryngeal development 
in the human, rat, and chick. 

2. Chick neural crest transplantations using tritiated thymidine 
for autoradiographic mapping. 



^13 



Serial No. NIDR-104 (c) (68) 

3. Exteriorization of living rat fetuses into abdominal cavity 
of anaesthetized mothers for surgical access to branchial arch 
region. 

Major Findings : 

1. After comprehensive review of the literature it was evident that 
the material presented to date was incomplete, fragmented, and 
inconclusive. This was discussed with Dr. Ebert of the Carnegie 
Institute and it was his opinion that a comprehensive review article 
is indicated at this time. 

2. In collaboration with Dr. Mac Johnston of the University of 
Toronto, using autoradiographic techniques, it was found that the 
laryngeal cartilages and connective tissue in the chick are neural 
crest in origin, demonstrating separate mesenchymal origins of 
muscle and cartilage in the larynx. 

Significance to Dental Research : 

It is clearly evident that laryngeal function is closely related to 

general oral and pharyngeal function and that basic knowledge of 

larjmgeal development may shed light on the development of more 
cranial visceral arches. 

Proposed Course of Project : 

1. Further elaboration and documentation of neural crest contribu- 
tions to the chick and possibly the rat larynx. 

2. Autoradiographic mapping of 4th and 6th arch laryngeal struc- 
tures in the rat with the development of fetal rat surgical tech- 
niques applicable to the larynx. 

3. Anticipate publication of: a review article on the development 
of the Human Larynx; Neural crest contribution to the larynx in 
the chick; Application of fetal rat surgery to the development of 
the larynx. 

Part B not included 



^1^ { 



Annual Report of the Dental Services Branch 
National Institute of Dental Research 
Summary Statement 

As in previous years, the major objective or purpose of the Dental Services 
Branch is to render detailed service to both inpatients and outpatients of 
the National Institute of Dental Research. Dental Services Branch furnisbes 
the clinical facilities for many NIDR investigators and the collaboration 
of our staff has served to enhance the clinical dental research program. lu 
addition, our responsibility to provide optimum dental care for the researcli 
beneficiaries of the various categorical Institutes has been continued in an 
effective manner. Complete oral examination, evaluation, consultation and 
dental therapy is performed at the request of the patient's attending physician. 
The Dental Services Branch staff, composed of six professionals and twenty-three 
supporting personnel, is understandably limited in performing extensive dental 
care for all patients in the 500-bed complex; however, our staff is capable 
of performing all types of dental treatment which may be required. 

The Branch has actively collaborated with all of the categorical Institutes 
in the discharge of our responsibilities to the total National Institutes of 
Health research effort. The following examples may be cited: 

National Cancer Institute : 

During the past year an increasing number of complicated, time-consuming to 
produce, maxillofacial prostheses have been constructed for patients with 
cancer. Prostheses of very high quality, which often defy detection even 
to the critical observer, are produced in almost routine fashion. 

An efficient working relationship exists between the Dental and NCI surgery 
staffs. In one example of collaboration, a three-stage maxillofacial prosthesis 
is designed and fabricated for each patient undergoing surgical treatment for 
neoplasm of the paranasal sinus area. Surgical procedures for tumors of the 
maxilla or mandible usually leave the patient with a considerable defect 
resulting in the impairment of speech, mastication and deglutition. The 
objective in every case is to develop a prosthesis which will restore the 
anatomical defect, improve function and esthetics, thereby benefitting the 
general well-being of the patient. 

In the post- surgical management of laryngectomized patients, the Dental 
Services Branch has developed a new tracheal prosthesis. These are one-piece, 
case-hardened pyrex glass appliances which are esthetic, hygienic, non- 
irritating and capable of maintaining humidity while protecting the trachea 
from debris. Approximately 75 patients are being maintained on these improved 
tracheal tubes with excellent response from the medical staff and the patients. 

Participation in operations about the head and neck, including neck dissections, 
has been of mutual benefit to our staff and the maxillofacial surgeons. Sugges- 
tions and procedures by the dentist during surgery are of major importance to 
the ultimate success of the final prosthesis and the rehabilitation of the 
ora-facial region. 



^15 



i 



Dental Services Branch 
Katloaal Institute of Arthritis and Metabolic Diseases : 

The development and fabrication of custom submaxillary saliva collectors for 
cystic fibrosis and normal control patients has provided a means to study the 
immunology and various chemical parameters of submaxillary saliva. The fun- 
damental metabolic defect resulting in cystic fibrosis of the pancreas has 
not been determined. Study of the biochemistry and physiology of mucous and 
serous glands in all locations may provide the ultimate explanation for this 
disease. 

National Heart Institute : 

A greater number of dental treatments were performed on the patients of the 
National Heart Institute than for patients of the other Institutes. Cardiac ^ 
surgery patients with congenital heart defects, or those requiring prosthetic 
valve replacement, pose problems of dental management in both the pre- and 
postoperative surgical periods. In the absence of proper dental care and 
preparation a simple procedure such as oral prophylaxis can precipitate a 
fatal, acute bacterial endocarditis. This program demonstrates clearly the 
importance of preventive care for patients who are to undergo or have undergone 
cardiac surgery. It is, therefore, imperative that candidates for cardiac 
operations obtain a thorough dental examination and completion of all necessary 
dental procedures before heart surgery in order to eliminate any possible sites 
of focal infection in the oral cavity. fl 

Collaborative investigations were performed with the Cardiology Branch, National 
Heart Institute, in an effort to determine if there is an association between 
high arched palatal vaults and congenital heart disease. X-rays of the skull, 
teeth, and wrist and a cast of the upper arch were made on each study participant. 
The wrist films will be compared with accepted norms to determine bone age and 
to compare this with the chronologic age; the skull x-rays will be utilized for 
cranio-facial landmark measurements. Palatal vault measurements were taken of 
a group of patients with congenital heart disease and a normal control series 
of patients for comparison. All data obtained will be analyzed statistically 
for degree of correlation. 

Extensive renovation of the Clinic was completed during this fiscal year. 
Three operator ies were modernized to provide better physical facilities within 
the limited space available. The well-lighted easily maintained, functionally 
efficient suites reflect the thought and time given to their redesign, and to 
the seeking, developing and installing of the most advanced equipment available. 
These operatories now allow the dentist and dental assistant to be seated 
during four-handed dental procedures resulting in the performance of more 
dentistry for the chronically ill patient with less time and energy expended. 

The renovation which was originally scheduled to be completed in three and 
one-half months experienced many delays and eleven months were required for 
final completion. This resulted in a loss of efficiency within the Clinic; 
however, the staff managed to perform an even greater number of treatments 4 
than in the previous year. ^ 



Fiscal Year 


Fiscal Year 


Fiscal Year 


1966 


1967 


1968 (Estimated) 


4,195 


4,076 


4,023 


1,398 


1,510 


1,481 


15,109 


14,759 


12,768 


36,546 


31,259 


32,460 



Dental Services Branch 

The Dental Services Branch staff is prepared to perform and has rendered all 
types of dental treatment found necessary for the patient examined. 

The following table documents briefly the statistical, facts of importance: 



Admissions to 
Clinical Center 

Examinations 

Vis it s 

Treatment s 

Publications 

1. Stanley, H.R. , and Swerdlow, H. : Minimizing Pulpal Reactions With 
Prednisolone Therapy. The Effects of a Delayed Steroid Application 
To Cut Dentin. J. Oral Ther. & Pharm. 3; 1-8, July 1967. 

2. Stanley, H.R. , Swerdlow, H. , and Buonocore, M.G.: Pulp Reactions 
to Anterior Restorative Materials. JADA 73: 132-141, July 1967. 

3. Hamner, J.E., Lightbody, P.m., Ketcham, A.S., and Swerdlow, H. : 
Cemento-ossifying Fibroma of the Maxilla. J. Oral Surg , (in Press). 

4. Gugler, E. , Pallavicini, C.J., Swerdlow, H. , and di Sant'Agnese, P.A. : 
The Role of Calcium in Submaxillary Saliva of Patients With Cystic 
Fibrosis. J. of Pediatrics. 71 : 585-588, October 1967. 

5. Stanley, H.R. , Swerdlow, H. , : Prednisolone Therapy for Pulpitis. 
Dental World 22 : Page 189, 3rd quarter 1967. , 

6. Gugler, E. , Pallavicini, J.C., Swerdlow, H. , Zipkin, I., di Sant'Agnese, 
P.A., Immunological Studies of Submaxillary Saliva From Patients With 
Cystic Fibrosis and Normals, Journal of Pediatrics (in press). 

Published Abstracts 

1. Pallavicini, J.C., Handwerger, S., Wiesmann, U. , Swerdlow, H. , 

di Sant'Agnese, P.A. : Albumin and Iga in Relation to Flow Rate in 
Normal Human Submaxillary Saliva, Federation Proceedings 27: p. 253, 
1968, 52nd Annual Meeting', Atlantic City, New Jersey. 

2. Wiesmann, U. , Pallavicini, J.C., Swerdlow, H. , di Sant'Agnese, P.A. : 
Effect of Rate on Electrolytes and Carbohydrates in Normal Submaxillary 
Saliva, Federation Proceedings 22[^: p. 676, 1968, Atlantic City, New Jersey, 



^♦11 



Dental Services Branch 

3. Wiesman, U. , Pallavicini, J, G. , Handwerger, H. , Swerdlow, H. , 

di Sant'Agnese, P.A. : Effect of Flow Rate on Electrolytes, Carbo- 
hydrates and Proteins in Submaxillary Saliva of Normal Subjects and 
Patients with Cystic Fibrosis (CFP) , Cystic Fibrosis Round Table 
Conference, Atlantic City, 1968. 

k. Swerdlow, H. : Prosthetic Management of Maxillofacial Defects, 
Proceedings of the Third Joint Meeting of Clinical Society & 
Commissioned Officers Association of USPHS, San Francisco, California, 
March 25-29, 1968. 

5. Stanley, H. , Baer, P., Lightbody, P., Lundy, T. , and Swerdlow, H. : 
Autogenous Reimplantation of Human Teeth, Proceedings of the Third 
Joint Meeting of the Clinical Society & Commissioned Officers 
Association of USPHS, San Francisco, California, March 25-29, 1968. 

Presentation of Lectures, Papers, and Table Clinics Included : 

1. Swerdlow, H. ; Prosthetic Management of Maxillofacial Defects, 
Proceedings of the Third Joint Meeting of Clinical Society & 
Commissioned Officers Association of USPHS, San Francisco, 
California, March 2 5-29, 1968. 

2. Stanley, H. , Baer, P., Lightbody, P., Lundy, T. , and Swerdlow, H. : 
Autogenous Reimplantation of Human Teeth, Proceedings of the Third 
Joint Meeting of the Clinical Society & Commissioned Officers 
Association of USPHS, San Francisco, California, March 25-29, 1968. 

3. Swerdlow, H. : Newer Anterior Restorative Materials, 36th Annual 
Postgraduate Clinic of the District of Columbia Dental Society, 
Washington, D.C., March 10-13, 1968, 

4. Swerdlow, H. : Maxillofacial Prosthesis, Postgraduate Seminar, 
Dental Assistants Association, USPHS Hospital, Baltimore, Maryland, 
June 9, 1968. 

5. Hamill, M.R. : Fluid Resin Denture Fabrication, West Virginia State 
Dental Society, July 26, 1967. 

6. Hamill, M.R. : Fluid Resin Denture Fabrication, Maryland State Dental 
Association, Baltimore, Maryland, September 20, 1967. 



kl8 



Serial No. NlDR-105 (c) (65) 

1. Dental Services Branch 

2. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Reaction of the Human Dental Pulp to Cavity 
Preparations and Filling I-laterials 

Previous Serial Number: NIDR-85 (c) 

Principal Investigator: Dr. Herbert Swerdlow 

Other Investigators: Dr. Harold R. Stanley 

Cooperating Units: None 



Man Years: 






Total : 




1/4 


Professional: 


lA 


Other : 







Project Desc 


ription 


• 
• 


Objectives: 







1. To study the efficacy of cavity liners, base and restorative 
materials in maintaining and protecting the dental pulp. 

2. To discover better ways to control the inflammatory reactions 
elicited by routine operative procedures. 

3. To test new cement or restorative material capable of chemically 
and/or mechanically bonding to the tooth. 

Met hods Etaployed : 

1. Patients selected for study must have non-carious, non- infected, 
vital teeth to be extracted for periodontal, prosthetic and/or ortho- 
dontic reasons. 

2. Teeth are prepared with specific regard for the following: (1) rpms, 
(2) cutting tool, (3) coolant, (4) type and area of tooth to be used, 
(5) time intervals until extraction, (6) restorative material, and (7) 
cavity liner. 



^19 



Serial No. NIDR-105 (c) (65) 

3o Teeth are ejctraetad at varying intervals. After fixation, embedding, 
sectioning, and staining, a histologic evaluation is made and related to 
the clinical experience of the tooth. 

4. The following experimental medicaments and restorative materials have 
been studied under the typical experimental design we have utilized in 
our previous work. 

a. The toxic effects of newly developed adhesive materials (Addent 
35, 3M Co.) and (Eastman Experimental Material, Dakor, L. D. 
Caulk Co.) which have the potential of supplanting the commonly 
used anterior restorative materials such as silicate and acrylic 
resin. These new direct dental filling materials are based on 
acrylic resins derived from epoxy resins. More than 50 percent 
of the binder is composed of chemically treated glass filler. 

An organic liquid catalyst polymerizes the binder in 3-5 minutes. 
These materials reputedly provide advantages such as low shrink- 
age during cure, high adhesive strengths, toughness, abrasion 
resistance and color stability. 

b. Additional experimental work was completed to more clearly 
determine relative effects of cutting and grinding teeth "wet" 
or "dry". It has been suggested that the frictional heat 
developed in cavity preparation could be adequately controlled 
when using only air as coolant. 

c. The use of a Copal ite varnish under class V amalgam restorations 
is a continuation of the amalgam study published in 1962. In 
order to block the thermal, chemical and mechanical irritation 
of amalgam to the pulp tissue, two thin layers of varnish are 
applied to cavity walls. 

d. A group of teeth have been used to evaluate the biologic com- 
patability of a gallium-tin-palladium alloy developed at the 
National Bureau of Standards, Dental Research Section. The 
physical properties of gallium are reputedly superior to dental 
amalgam. The results are being published by Waterstrat at the 
Bureau of Standards. 

Q. A new temporary protective packing developed in Sweden was 
placed in twenty-five teeth using the experimental design 
described above. This product composed of calcium sulfate, 
calcium hydroxide and zinc oxide etc. has been reported to 
produce superior characteristics in sedating, sealing and 
insulating cavity preparations when compared to zinc-oxide 
and engeuol. This material (Pharmatec) was designed to 
function as a temporary filling material as well as a pro- 
visional adhesive agent for crowns and bridges. Presently, 
histopathologic analysis is underway. 



^20 



Serial No. NIIR-IOS (c) (65) 
Pat lent Mater lal ; 

This year 46 teeth in 11 patients were utilized on these studies. 
Major Findings : 

1. If the health of the pulp is to benefit from the minimal pulp 
reactions associated with the new cutting methods, some treatment of 
the freshly cut dentinal tubules not lined by reparative dentin is 
imperative prior to restoration. 

2. The application of the steroid formula inhibits the pulp inflammatory 
response to one of the more traumatic operative procedures. There is 

no evidence at this time to suggest that the application of this formula 
in this dosage is detrimental to the human dental pulp. 

3. Reparative dentin was found at approximately the same time interval 
and occurred at a similar rate as seen in control teeth. This indicates 
that healing was not interfered with in the experimental group. 

4. The pulpal response of 293 intact human teeth were evaluated to 
compare several new composite anterior restorative materials (with and 
without liners) to zinc oxide-eugenol and silicate cements. It was 
found that the initial pulpal reactions created by the Eastman product 
were similar to silicate restorations. Addent 35 without a liner was 
initially less irritating than silicate. The pulpal response intensity 
subsided with the Eastman product after extended postoperative intervals, 
whereas the lesions produced by the 3M product became more severe. The 
vinyl -copolymer liner recommended by the 3M Company was not adequate to 
protect the pulp tissues from the irritating properties of addent 35 
filling material. 

5. Those experimental teeth prepared with only air cooling developed 
significantly more severe pulp pathology than a comparable group of 
teeth prepared with a water-spray coolant. The air-cooled teeth demon- 
strated a high percentage of pulp lesions exhibiting burn characteristics, 
such as lesions extending beyond cut tubules, massive reactions leading 
to intra-pulpal abscesses and an increased rate and amount of reparative 
dentin. Therefore, if pulp damage is to be reduced to a minimum when a 
high-speed handpiece is used, adequate water spray should be used during 
cavity preparation. 

Significance to Dental Research ; 

1. The development and acceptance of a restorative material, cavity 
liner, or base material must not only be governed by adequate measures 
of evaluation for physical properties, but must also conform to rigid 
biological requirements. The maintenance and protection of the dental 
pulp and supporting structures is a fundamental principle in restorative 
dentistry. 



^21 



Serial No. NIDR-105 (c) (65) 

2. The baseline data accumulated provide the ability to evaluate 
properly any restorative material introduced for dental procedures. 

3. The evidence now available from numerous histopathological studies 
supports the following recommendations as guidelines for minimizing 
pulpal trauma in restorative dental procedures. 

a. Treat all teeth as potentially "sick". 

b. Keep the cavity shallow in dentin. 

c. Speeds above 50,000 rpm are more biologically compatible. 

d. Use a water coolant when cutting tooth structure. 

e. Exert light force to exposed dentin. 

f. Use smaller cutting tools at higher speeds. 

g. Keep irritating drugs away from exposed dentin. 

h. Use zinc oxide and eugenol on dentin when possible. ■ 
i. Delay final setting of restoration, when possible, for 

secondary dentin deposition, 
j. Sedate, seal and insulate all cavity preparations. 

Proposed Course of Project ; 

Investigations of the following: 

1. Rationale for the use of corticosteroids to arrest an established 
inflammatory reaction. 

2. Com.parative toxic effect of experimental adhesive restorative 
materials (Addent, Dakor, Experimental Epoxy) . 

3. Post-extraction cavity preparation for observation of displacement 
of odontoblasts. 

4. Cavity preparation response in teeth with incipient carious lesions. 

5. Silicate - an anterior restorative material. 

6. Full crown preparations vs. the vertical parallel pin preparation. 

7. Cementing procedures. 

8. Horizontal pin-lay. 

9. Gold foil vs. amalgam condensation. 

10. Evaluation of reduced temperature of the water coolants used in 
cavity preparation. 

11. The value of a varnish cavity liner under amalgam restorations to 
reduce the established trauma from amalgam insertion. 



422 



Serial No. NIDR-105 (c) (65) 

12. The value of new temporary filling materials improving, sedation, 
sealing and insulation. 



Part B 



Publications 

1. Stanley, H. R. , Swerdlow, H. , and Buonocore, M. G. : Pulp Reactions 
to Anterior Restorative Materials. J.A.D.A. 7 3 : 132-141 July 1967. 

2. Stanley, H. R. , Swerdlow, H. , : Prednisolone Therapy for Pulpitis. 
Dental World 22: Page 189, 3rd quarter 1967. 



k23 



Serial No. NIDR-106 (c) (68) 
X. Dental Services Branch 
2. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1967 through June 30, 1968 



Part A 



Project Title: Palatal Vault Measurements in Patients with 
Congenital Heart Disease 

Previous Serial Number: None 

Principal Investigator: Dr. Barry Goldman 

Other Investigators: None 

Cooperating Units: Dr. Lawrence S. Cohen, Cardiology Branch, NHI, NIH 

Man Years: 

Total : 1/4 
Professional: 1/4 

Other: 

Project Description: 

Objectives : 

Preliminary observations suggest that patients with congenital heart 
disease have a high incidence of high arched palates. This is a pilot 
study to determine if an unusually high arched palate is characteristic 
of patients with congenital heart disease. 

Methods Ehiployed : 

Patients with documented congenital heart disease from the Cardiology 
Branch of NHI are utilized. Dental radiographs, a growth and develop- 
ment chart, wrist radiographs, and an impression of the maxillary arch 

are obtained for each patient. 

Accurate palatal measurements are made on the stone cast to determine 
the Palate Height Index. This index will be compared to that of normal 
control patients and analyzed for statistical correlation. 

Major Findings: 

This study is presently in progress and findings have not been completed. 



^2^ 



Serial No. NIDR-106 (c) (68) 

Sig nificance to Dental Research 

Previously the association' between high arched palate and congenital 
heart defects has been based on subjective observations of the palate. 
This study will obtain valid palatal dimensions in congenital heart 
patients. A significant correlation may be a useful tool in the 
diagnosis of congenital heart disease. 

Further study may also demonstrate a relationship between congenital 
heart defects and other factors such as cranial growth and development 
or orthodontic abnormalities. 

Proposed Course of Project : 

Twenty-five to fifty patients will be included in this pilot study. 
To date, nineteen patients have been examined. 



^25 






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