Calcified Tissues 1965 Proceedings of the Third European Symposium on Calcified Tissues held at Davos Edited by H. Fleisch H.J. J. Blackwood M. Owen Springer-Verlag New York Inc. V ^^^ ^m CD ^5^ TOEO III Illlil ilillll lAI Calcified Tissues 1965 Calcified Tissues 1965 Proceedings of the Third European Symposium on Calcified Tissues held at Davos (Switzerland), April Uth— 16th, 1965 Sponsored by the Laboratorium fiir experimentelle Chirurgie, Schweizerisches Forschungsinstitut, Davos Edited by H.Fleisch, H.J.J. Blackwood and M.Owen with the assistance of M. P. Fleisch-Ronchetti SPRINGER-VERLAG ■ NEW YORK • INC 1966 All rights, especially that of translation into foreign languages, reserved. It is also forbidden to reproduce this book, either whole or in part, by photomechanical means (photostat, microfilm and, or microcard) or by other procedure without written permission from Springer-Verlag. (g) by Springer-Verlag Berlin • Heidelberg 1966 Library of Congress Catalog Card Number 65-27 964 Printed in Germany Titelnummer 1323 Preface The papers collected in this volume represent the formal proceedings of the Third European Symposium on Calcified Tissues which was held in Davos, Switzerland from 11th to 16th April 1965 under the sponsorship of the Laboratorium fiir experi- mentelle Chirurgie, Schwelzerisches Forschungsinstitut Davos. This Symposium fol- lowed the now established tradition of the previous Symposia held in Oxford in 1963 and in Liege in 1964. Participation was again strictly on a residential basis. This year the Schatzalp Hotel provided a scenic and secluded meeting place high on a mountain side overlooking Davos yet close to the Forschungsinstitut in which the opening session of the Symposium was held. The papers and communications published in the volume are arranged in order of presentation and are grouped under the five main themes selected for discussion by the Symposium, namely, "Cell function in the formation, maintenance and destruc- tion of osseous tissue", "Response of calcified tissues to mechanical factors", "Mecha- nisms of mineralization and diseases related to mineral deposition", "Hormones and bone" and "Fundamental structure of dental hard tissues". The programme consisted of a number of review lectures given by invited speakers and of short communications in relation to each of the above themes. No attempt was made to record the dis- cussions to the papers as, being a residential meeting, the more valuable and interest- ing interchanges took place informally in small discussion groups and not within the time schedule of the prearranged programme. The Committees wish to express their thanks to the staff of the Laboratory for Experimental Surgery, Davos for their most valuable help and to all of the following who made this meeting possible through their financial support: Zentralverband schweizerischer Milchproduzenten; Nestle Alimentana S.A., Cham et Vevey; Sandoz AG., Basel; Robapharm AG., Basel; Ciba AG., Basel; J. R. Geigy AG., Basel; F. Hoff- mann-La Roche & Co. AG., Basel; Dr. A. Wander AG., Bern; Emser Werke AG., Domat/Ems; Laboratorien Hausmann AG., St. Gallen; Davos-Parsenn-Bahnen AG., Davos; Kleiner Rat des Kantons Graubiinden; Landschaft Davos; Migros-Genossen- schafts-Bund, Zurich; Schweizerische Unfallversicherungs-Gesellschaft, Winterthur; Treupha AG., Baden; Siegfried AG., Zofingen; Verkehrsverein Davos. The European Symposium on Calcified Tissues is held annually and Professor P. Gaillard's invitation on behalf of the University of Leiden to meet in Holland in 1966 has been warmly accepted. The editorial committee: The organizing committee: H. J. J. Blackwood C. A. Baud H. G. Haas H. Fleisch M. J. Dallemagne L. Richelle M. Owen H. Fleisch R. Schenk Contents Session I Cell Function in the Format toil, Maintenance and Destruction of Osseous Tissue Chairmen: P. Gaillard, I. MacIntyre, H. J. Dulce, A. B. Borle L. F. Belanger, T. Semba, S. Tolnai, D. H. Copp, L. Krook and C. Gries The Two Faces of Resorption 1 W. A. DE VooGD van der Straaten Some Remarks and Questions on Metabolic Patterns in the Family of Bone Cells ... 10 Ch. M. Lapiere Remodelling of the Bone Matrix 20 M. E. HOLTROP The Origin of Bone Cells in Endochondral Ossification 32 M. Owen RNA Synthesis in Growing Bone 36 J. S. Greenspan and H. J. J. Blackwood Histochemical Studies of Chondrocyte Function in the Cartilage of the Mandibular Condyle of the Rat 40 A. B. Borle Correlation between Morphological, Biochemical, and Biophysical Effects of Para- thyroid Hormone on Cell Membranes 45 P. A. Thornton Vitamin D-ascorbic Acid Association in Bone Metabolism 48 C. B. Sledge Lysosomes and Cartilage Resorption in Organ Culture 52 G. Vaes Acid Hydrolases, Lysosomes and Bone Resorption Induced by Parathyroid Hormone . 56 A. Dhem Le forage des canaux de Havers 60 E. Rutishauser, W. Taillard and B. Friedli Experimental Studies of the Non-inflammatory Vascular Pannus 63 L. V. AviOLi and Ph. H. Henneman Urinary Pyrophosphate in Disorders of Bone Metabolism 67 J. JOWSEY Bone Formation and Resorption in Bone Disorders 69 J. C. Birkenhager, J. VAN DER Sluys Veer, R. O. van der Heul and D. Smeenk Studies of Iliac Crest Bone from Controls and Patients with Bone Disease by Means of Chemical Analysis, Tetracycline Labelling and Histology 73 VIII Contents Session II Response of Calcified Tissues to Mechanical Factors Chairman: R. Amprino C. A. L. Bassett Electro-mechanical Factors Regulating Bone Architecture 78 G. Marotti and F. Marotti Topographic-quantitative Study of Bone Tissue Formation and Reconstruction in Inert Bones 89 H. Wagner Structure and Healing of Bone as a Response to Continuous and Discontinuous Strain and Stress 93 E. D. Sedlin and L. Sonnerup Rheological Considerations in the Physical Properties of Bone 98 M. Brookes Haemodynamic Data on the Osseous Circulation 101 N. E. Shaw The Influence of Muscle Blood-flow on the Circulation in Bones 104 Session III Mechanisms of Mineralization and Diseases Related to Mineral Deposition Chairmen: S. M. Partridge, B. Engfeldt, W. D. Armstrong, J. P. Aubert and H. C. G. Bauer F. G. E. Pautard A Biomolecular Survey of Calcification 108 L. J. RiCHELLE, C. Onkelinx and J. -P. Aubert Bone Mineral Metabolism in the Rat 123 P. Lerch and C. Vuilleumier Physico-chemical Methods for the Identification of Microcrystalline Basic Calcium Phosphates Prepared in Vitro 132 H. Newesely Umwandlungsvorgange bei Calciumphosphaten 136 J. MacGregor Some Observations on the Nature of Bone Mineral 138 A. Ascenzi and E. Bonucci The Osteon Calcification as Revealed by the Electron Microscope 142 H. J. Hohling, H. Themann and J. Vahl Collagen and Apatite in Hard Tissues and Pathological Formations from a Crystal Chemical Point of View 146 H. J. Arnott Studies of Calcification in Plants 152 R. Lagier, C. a. Baud and M. Buchs Crystallographic Identification of Calcium Deposits as Regards to their Pathological Nature, with Special Reference to Chondrocalcinosis 158 A. PoLiCARD, C. A. Baud, A. Collet, H. Daniel-Moussard and J. C. Martin Infrastructural and Crystallographic Study of Experimental Calcifications 162 S. M. Krane and M. J. Glimcher Protein Phosphorus and Phosphokinases in Connective Tissue 168 Contents IX W. M. McKernan and S. D. Dailly The Relationship between Swelling of Hard Tissue Collagen in Acid and Alkali and the Presence of Phosphate Cross-links 171 R. Steendijk, J. JowsEY, A. VAN DEN HooFF and H. K. L. Nielsen Microradiographic and Histological Observations in Primary Vitamin D-resistant Rickets 175 D. B. Morgan, C. R. Paterson, C. G. Woods, C. N. Pulvertaft and P. Fourman Therapeutic Response and Effect on the Kidney of 100 Units Vitamin D Daily in Osteomalacia after Gastrectomy 178 T. G. Taylor, A. Williams and J. Kirkley Changes in the Activities of Plasma Acid and Alkaline Phosphatases during Egg Shell Calcification in the Domestic Fowl 182 J. C. Stoclet and Y. Cohen Calcium Exchanges in the Aorta of the Rat 186 G. D. McPherson Estimation of the 24 Hour Exchangeable Calcium Pool in Children Using ^''Ca . . . 189 L. Miravet et D. Hioco Transfert du calcium intestinal chez I'homme dans Ics maladies demineralisantes de I'os 193 L. LUT^AK Absorption of Calcium in Man: Effect of Disease, Hormones and Vitamin D .... 198 J.-F. Dymling Therapeutic Results in Renal Tubular Osteomalacia with Special Reference to Calcium Kinetics 202 G. MiLHAUD et J. BOURICHON Etude du metabolisme du calcium chez I'homme a I'aide de calcium 45: I'osteopetrose et I'osteopsathyrose 207 D. A. Smith and C. J. Mackenzie Calcium Clearance and Re-absorption in Patients with Osteoporosis, Renal Stone and Primary Hyperparathyroidism 211 Session IV Hormones and Bone Chairmen: P. Fourman and P. H. Henneman G. Nichols, jr. Bony Targets of Non-"skeletal" Hormones 215 B. E. C. Nordin Hormones and Calcium Metabolism 226 H. A. Soliman, C. J. Robinson, G. V. Foster and L MacIntyre Mode of Action of Calcitonin 242 E. Uehlinger Of the Influence of Thyroxine, Thiouracil, Cortisone, Estrogen and Testosterone on Endo- chondral Ossification Utilizing Autoradiography 243 J. A. Fernandez de Valderrama and L. M. Munuera The Effect of Cortisone and Anabolic Agents on Bone 245 W. M. Rigal and W. M. Hunter Sites and Mode of Action of Growth Hormone 250 G. F. Mazzuoli and L. Terrenato Intestinal Absorption and Skeletal Dynamic of Calcium in Acromegaly ..... 254 X Contents Session V Fundamental Structure of Hard Dental Tissues Chairman: H. J. J. Blackwood R. M. Frank Ultrastructure of Human Dentin 259 E. P. Katz, G. Mechanic and M. J. Glimcher Preliminary Studies of the Ultracentrifugal and Free Zone Electrophoresis Charac- teristics of Neutral Soluble Proteins of Bovine Embryo Enamel 272 A. BOYDE The Development of Enamel Structure in Mammals 276 List of participants Allgowcr, M., Kantonsspital, Chur, Schwciz. Amprino, R., Istituto di Anatomia Umana, Universita, Bari, Italia. Armstrong, W. D., Department of Physiological Chemistry, University ot Minnesota, Min- neapolis, Minn., USA. Arnott, H. J., Cell Biology Institute, University of Texas, Austin, Texas, USA. Ascenzi, A., Istituto di Anatomia Patologica, Universita, Pisa, Italia. Aubert, J. -P., Institut Pasteur, Paris, France. Avioli, L. v., Seton Hall College, Division of Endrocrinology, Jersey City, N. J., USA. Bachra, B. N., Laboratory of Physiological Chemistry, Leiden, Holland. Bassett, C. A. L., Department of Research Orthopaedics, Columbia University, College of Physicians and Surgeons, New York, N. Y., USA. Baud, C. A., Institut de Morphologie, Ecole de Mcdecine, Geneve, Suisse. Bauer, G. C. H., Hospital for Special Surgery, New York, N. Y., USA. Belanger, L. P., Department of Histology, University of Ottawa, Ottawa, Canada. Bengtsson, A., Department of Pathology, University, Uppsala, Sweden. Bersin, T., Laboratorium Hausmann AG., St. Gallen, Schweiz. BIrkenhager, J. C, Department for Diseases of Metabolism, University Hospital, Leiden, Nederland. Birkenhager-Frenkel, D. H., Antoni van Leeuwenhoekhuis, Amsterdam, Nederland. Bisaz, S., Laboratorium fiir experimentelle Chirurgie, Schwelzerisches Forschungsinstitut, Davos, Schweiz. Blackwood, H. J. J., Royal Dental Hospital, School of Dental Surgery, London, England. Bohr, H., Orthopaedic Hospital, Copenhagen, Denmark. Bolognani, L., Istituto di Chimica Biologica, Universita, Pavia, Italia. Bonucci, E., Istituto di Anatomia Patologica, Universita, Pisa, Italia. Bordier, P., Centre du Metabolisme Phosphocalcique, Hopital Lariboisiere, Paris, France. Borle, A. B., Department of Physiology, University of Pittsburgh, Pittsburgh, Pa., USA. Boyde, A., Department of Anatomy, London Hospital Medical College, London, England. Brookes, M., Department of Anatomy, Guy's Hospital Medical School, London, England. Chalmers, J., Orthopaedic Department, Royal Infirmary, Edinburgh, Scotland. Cimasoni, G., Institut Dentaire, Geneve, Suisse. Collet, A., Cerchar B. P. 27, Creil (Olse), France. Coutelier, L., Laboratoire de Chirurgie Orthopcdique et Traumatologic, Louvain, Belgique. Czitober, H., I. Medizinische Universitatsklinik, Wien, Osterreich. Dallemagne, M. J., Institut de Therapeutique Experlmentale, Liege, Belgique. Dhem, A., Laboratoire de Chirurgie Orthopedique et Traumatologic, Louvain, Belgique. Dingle, J. T., Strangeways Research Laboratory, Cambridge, England. Dulce, H. J., Institut fiir angewandte physiologische Chemie und klinische Chemie, Berlin, Deutschland. XII List t)f participants Dupont, D., Institut de Morphologic, Ecole de Medecine, Geneve, Suisse. Dymling, J. F., Malmo General Hospital, Malmo, Sweden. Engfeldt, B., Department of Pathology II, University, Uppsala, Sweden. Ezra-Cohn, H., Nuffield Orthopaedic Centre, Oxford, England. Fitton Jackson, S., Strangeways Research Laboratory, Cambridge, England. Fleisch, H., Laboratorium fiir experimentelle Chirurgie, Schweizerisches Forschungsinstitut, Davos, Schweiz. Foster, G. V., Postgraduate Medical School, London, England. Fourman, P., The School of Medicine, Leeds, England. Franfois, P., Institut de Therapeutique Experimentale, Liege, Belgique. Frank, R. M., Institut Dentaire, Faculte de Medecine, Strasbourg, France. Gaillard, P., Laboratorium voor Cytologic en Experimentelc Histologic, Univcrsiteit, Leiden, Nederland. Greenspan, J. S., Royal Dental Hospital, School of Dental Surgery, London, England. Haas, H. G., Medizinische Universltatsklinik, Burgerspital, Basel, Schweiz. Hartles, R. L., School of Dental Surgery, University, Liverpool, England. Hattyasy, D., Leninkorut 66, Szeged, Hungary. Hekkelman, J. W., Laboratorium voor Cytologic en Experimentelc Histologic, Univcrsiteit, Leiden, Nederland. Henneman, Ph. H., Seton Hall College, Division of Endocrinology, Jersey City, N. J., USA. Hess, R., Ciba AG., Basel, Schweiz. Heuck, F., Katharinenhospital, Stuttgart, Deutschland. Hioco, D., Centre du Metabolisme Phosphocalcique, Hopital Lariboisicre, Paris, France. Hohling, H. J., Institut fiir medizinische Physik, Miinster, Deutschland. Holtrop, M. E., Laboratorium voor Cytologic en Experimentelc Histologic, Univcrsiteit, Leiden, Nederland. Horn, H. D., Urologisch-Balneologischc Forschungsstelle, Bad Wildungen, Deutschland. HooflF, A. van den, Jan Swammerdan Instituut, Amsterdam, Nederland. Huggler, A., Kantonsspital, Chur, Schweiz. Jasinski, B., Robapharm AG., Basel, Schweiz. Jasinski, W. K., Institute of Oncology, Warsaw, Poland. Jeffrce, G. M., Pathology Research Laboratory, University, Bristol, England. Johnson, N. W., Dental Research Unit, Bristol, England. Jowsey, J., Mayo Clinic, Rochester, Minn., USA. Knese, K. H., Institut fiir Biologic, Landw. Hochschule Hohcnhcim, Stuttgart-Hohenheim, Deutschland. Koskinen, E. V. S., Clinic for Orthopaedics and Traumatology, University, Helsinki, Finland. Kranc, S. M., Massachusetts General Hospital, Boston, Mass., USA. Kshirsagar, S. G., M.R.C. Bone-seeking Isotopes Research Unit, Churchill Hospital, Oxford, England. Kuhlencordt, F., Stoffwechsellaboratorium, I. Medizinische Univcrsitatsklinik, Hamburg- Eppendorf, Deutschland. Lagier, R., Institut Universitaire de Pathologic, Hopital Cantonal, Geneve, Suisse. Lapierc, Ch. M., Institut de Medecine, Dcpartement de Clinique ct dc Pathologic Mcdicales, Univcrsite, Liege, Belgique. Lemaire, R., Clinique Chirurgicale, Univcrsite, Liege, Belgique. Lentner, C, J. R. Geigy AG., Basel, Schweiz. Lerch, P., Institut dc Radiophysique Appliquee, Lausanne, Suisse. List of participants XIII Lindholm, B., Sahlgren's Hospital, Metabolic Laboratory, Medical Clinic II, Goteborg, Sweden. Little, K., Nuffield Orthopaedic Centre, Oxford, England. Lutwak, L., Cornell University, Sage Hospital, Ithaca, N. Y., USA. MacGregor, J., Bioengineering Unit, University of Strathclyde, Glasgow, Scotland. Maclntyre, I., Postgraduate Medical School, London, England. Marotti, G., Istituto di Anatomia Umana, Universita, Bari, Italia. Matrajt, H., Centre du Metabolisme Phosphocalcique, Hopital Lariboisiere, Paris, France. Matturri, L., Istituto di Anatomia e Istologia Patologica, Universita, Milano, Italia. Mazzuoli, G., Istituto di Patologia Medica, Universita, Policlinico Umberto I, Roma, Italia. McKernan, W. M., British Glues and Chemicals Ltd., Gelatine Division, London, England. McPherson, D., Hospital for Special Surgery, New York, N. Y., USA. Mechanic, G., Orthopaedic Research Laboratories, Massachusetts General Hospital, Boston, Mass., USA. Milhaud, G., Institut Pasteur, Paris, France. Miravet, L., Centre du Metabolsime Phosphocalcique, Hopital Lariboisiere, Paris, France. Morgan, D. B., Department of Chemical Pathology, The Medical School, Leeds, England. Morscher, E., Orthopadische Klinik, Universitlit, Basel, Schweiz. Moukhtar, M. S., Institut Pasteur, Paris, France. Miiller, M., Kantonsspital, St. Gallen, Schweiz. Newesely, H., Forschungsgruppe fiir Mikromorphologie, Fritz-Haber-Institut, Berlin, Deutsch- land. Nichols, G., Department of Medicine, Harvard Medical School, Boston, Mass., USA. Nordin, B. E. C, The General Infirmary, Leeds, England. O'Dell, D., Strangeways Research Laboratory, Cambridge, England. Onkelinx, C, Institut de Therapeutique Experimentale, Liege, Belgique. Owen, M., M.R.C. Bone-seeking Isotopes Research Unit, Churchill Hospital, Oxford, Eng- land. Partridge, S. M., Low Temperature Station, Cambridge, England. Pautard, F. G. E., The Astbury Department of Biophysics, University, Leeds, England. Perren, S., Laboratorium fiir experimentelle Chirurgie, Schweizerisches Forschungsinstitut, Davos, Schweiz. Price, C, Pathology Research Laboratory, University, Bristol, England. Probst, J.-Y., Sandoz AG., Basel, Schweiz Richelle, L., Institut de Therapeutique Experimentale, Liege, Belgique. Rose, G. A., Department of Medicine, General Infirmary, Leeds, England. Rowles, S. L., Birmingham Dental Hospital, Birmingham, England. Rutishauser, E., Institut de Pathologic Generale et Anatomic Pathologique, Universite, Geneve, Suisse. Schenk, R., Anatomisches Institut, Basel, Schweiz. Schibler, D., Laboratorium fiir experimentelle Chirurgie, Schweizerisches Forschungsinstitut, Davos, Schweiz. Sedlin, E. D., Department of Orthopaedic Surgery, University, Goteborg, Sweden. Shaw, N. E., Royal National Orthopaedic Hospital, Institute of Orthopaedics, Stanmore, Mddx., England. Sledge, C. B., Strangeways Research Laboratory, Cambridge, England. XIV List of participants Sluys Veer, J. van der. Department of Endocrinology and Metabolism, University Hospital, Leiden, Holland. Smeenk, D., Department of Endocrinology and Metabolism, University Hospital, Leiden, Holland. Smith, D. A., Department of Medicine, Gardiner Institute, Western Infirmary, Glasgow, Scot- land. Soliman, H., Postgraduate Medical School, London, England. Steendijk, R., Kinderkliniek, Binnengasthuis, Amsterdam, Nederland. Stoclet, J. C., Faculte de Pharmacia, Laboratoire de Pharmacodynamie, Paris, France. Strandh, J., Department of Pathology II, University, Uppsala, Sweden. Straumann, P., Institut Straumann, Waldenburg, Schweiz. Taylor, T. G., Department of Physiological Chemistry, University, Reading, England. Terrenato, L., Istituto di Patologia Medica, Universita, Policlinico Umberto I, Roma, Italia. Thornton, P. A., VA Hospital, University of Kentucky, Lexington, Ky., USA. Trautz, O. R., New York University, College of Dentistry, New York, USA. Uehlinger, E., Pathologisches Institut der Universitat, Zurich, Schweiz. Vaes, G., Laboratoire de Chlmie Physiologlque, Univcrsitc, Louvain, Belgique. Valderrama, J. P. de, Zurbano 39, Madrid, Espana. Vaughan, J., M.R.C. Bone-seeking Isotopes Research Unit, Churchill Hospital, Oxford, Eng- land. Vittali, P., Chirurglsche Unlversitatsklinik, Koln-Merheim, Deutschland. Voogd van der Straaten, W. A. de, Laboratorium voor Cytologic en Experimentele Histologic, Universiteit, Leiden, Nederland. Vuilleumier, C, Institut flir anorganische und physikalische Chemie, Universitat, Bern, Schweiz. Wagner, H., Orthopiidische Universltiitsklinik, Miinster, Deutschland. Wasserman, R. H., Institute of Biological Chemistry, University, Copenhagen, Denmark. Welgel, W., Robapharm AG., Basel, Schweiz. Wernly, M., 1 Sonnenbergstrafie, Bern, Schweiz. Zinn, M., Bad Ragaz, Schweiz. The Two Faces of Resorption L. F. Belanger, T. Semba ''', S. Tolnai Department of Histology and Embryology, Faculty of Medicine, University of Ottawa, Ottawa, Canada D. H. Copp Department of Physiology, Faculty of Medicine, University of British Columbia, Vancouver, B. C, Canada L. Krook, C. Gries Department of Pathology and Bacteriology, New York State Veterinary College, Ithaca, N. Y., U. S. A. 1. Development of the concept of osteolysis Recent reviews and textbooks state that "the osteoclast is the agent of bone destruction" (Lacroix, 1961), that this cell is "actively involved in some way in the resorption of bone" (Hancox and Boothroyd, 1963) or else that there is "much interesting controversy about the origin, the nature and the function of these cells" (Ham and Leeson, 1961) and that "indeed, the process of bone resorption is not understood as thoroughly as we might wish" (Ham and Leeson, 1961). "On the other hand, resorption may take place in the absence of osteoclasts for instance in the so-called creeping replacement in bone transplants" (Copenhaver, 1964). The concept of non-osteoclastic resorption has been itself, "creeping" through the years, towards the home of the bone investigator; perhaps this meeting will be the occasion when it will find its way inside . . . The idea of "remodeling absorption" from within, proposed by John Hunter towards the end of the 18th century (Home, 1800) might have been an early step in this direction. After cauterization of portions of bone. Hunter reported that "the earthy part of the living bone in contact with the dead portion, was first absorbed". Living cells within the bone were apparently needed for this process. Much later several investigators were attracted by changes in the staining proper- ties of the organic matrix in relation with interstitial loss of substance. These were manifested by basophilia (Zawisch-Ossenitz, 1927; Kind, 1951; Ruth, 1954, 1961) or by intensified staining for mucopolysaccharides (Heller-Steinberg, 1951; Engel, 1952;Gaillard, 1955 a, b). Changes in the appearance of the osteocytes and also in the size and shape of lacunae and canaliculi were observed by von Recklinghausen (1910) (oncosis), * Post-Doctorate Fellow of the Medical Research Council of Canada. 3'''^ Europ. Symp. on Cal. Tissues \ 2 L. F. Belanger, T. Semba, S. Tolnai, D. H. Copp, L. Krook, C. Gries Jaffe (1933), Kind (1951) and Lipp (1954, 1956), with the light microscope and more recently by Baud (1962) with the electron microscope. RuTiSHAusER and Majno (1951) made the interesting observation that during its hypertrophic or oncotic phase, the osteocyte produced alkaline phosphatase. The sub- sequent death of the osteocyte, leaving empty lacunae which oftentimes fill with calcium salt (Sherman and Selakovitch, 1957; Sissons, 1964) has been related by DuFOUR (1952) and by Urist et al. (1963) to osteoporosis and other rarefying diseases of the skeleton. Empty lacunae have also been recognized as a conspicuous feature of old age (Frost, 1960; Sissons, 1964). Recently, Lipp (1959) has detected the presence of aminopeptidase in some of the osteocytes. On the other hand, Belanger and Migicovsky (1963 a) have shown that these cells contained an enzyme capable of digesting gelatin. Furthermore, Belanger et al. (1963 b) have been able to associate the same areas with toluidine blue meta- chromasia and also with organic matrix and salt depletion as revealed by alpharadio- graphy (Belanger and Belanger, 1959) and X ray microradiography (Belanger Fig ed by -diaphysis of an 2 days-old chick embryo. The cell is apparently closely idoplasmic reticulum, ribosomes and mitochondria are prominent features of the cytoplasma et al., 1963 a). To designate specifically this intimate form of resorption related to osteocytic activity, the abused term osteolysis has been proposed (Belanger et al., 1963 b; Belanger, in press). The Two Faces of Resorption 3 JowsEY (1963) and Jowsey et al. (1964) have reported on the basis of micro- radiographic studies of a variety of bone depleting conditions, "that the osteocytes may be capable of controlling the amount of mineral and also perhaps, the amount of matrix, deposited in the tissue adjacent to them". At the Second Parathyroid conference held at Noordwijk in August 1964, Talmage (in press) who had already demonstrated that the removal of hydroxyproline from bone followed a pattern quite similar to that of the mineral components (Talmage, 1962), reported that numerous enlarged osteocytes were observed early in bone fragments cultured with parathormone. 2. Electron microscopy of bone cells of the chick Further aspects of this osteocytic activity have been uncovered by electron micro- scopy during the past year. Very intense osteolysis has already been observed in the bones of young chicks (Belanger et oil., 1963 b). This type of material being not too hard, can be processed for electron microscopy with a minimum of pretreatment. ^|H-.iiMM, I ^ above. The laci una IS WKk- ,1 lui apparently sm.oph.lu n latenal (l)sosomc s>) ai c promi iient features ol cytoplasma In collaboration with Teruhiko Semba and Susan Tolnai, small fragments of the mid-diaphysis of the tibia from II days chick embryos were fixed in Palade's isotonic osmic mixture at 5 °C and then partly demineralized in an aqueous isotonic solution 4 L. F. Belanger, T. Semba, S. Tolnai, D. H. Copp, L. Krook, C. Gries of the disodium salt of ethylene-diamine tetra-acetic acid (EDTA) at pH 7.4. Sec- tions of ca. 800 A were cut in epoxy resin and submitted to electron microscopy. The low power pictures obtained so far have revealed that the ovoid cells (osteo- blasts) located at the immediate outer border of the peripheral trabeculae contained mitochondriae and an extensive endoplasmic reticulum network, as already described elsewhere in mammalian tissue (Dudley and Spiro, 1961; Cameron et al., 1963). Young osteocytes located immediately inside the bone matrix (Fig. 1) revealed similar features of the cytoplasma. These cells generally filled the lacunae in which they were contained. Osteocytes located further away from the border, inside larger lacunae (Fig. 2) showed apart from the above features, some osmiophilic vesicles of different sizes, which might well be lysosomes. This observation is of interest in view of the previous histochemical demonstration of protease activity over the more mature osteocytes (Belanger and Migicovsky, 1963 a). 3. The effects of acute parathyroid stimulation Parathyroid stimulation by perfusion of dogs and sheep with EDTA, over periods of a few hours only, such as performed by D. H. Copp at the University of British Columbia (Copp, 1963) has produced effects in cancellous bone particularly visible in alpharadiographs (Belanger, in press). The number of enlarged osteocytes was increased in the parietal bone after 4 hrs. Moreover, the surrounding matrix displayed already at this early stage, a remarkable loss of density. 4. The effects of prolonged parathyroid stimulation We were highly privileged during the years 1964 — 65 to be associated with Drs. Lennart Krook and Christian Cries from the Veterinary College of Cornell University in studies involving horses which were fed a diet containing an optimum Hi ••P ^i^- ^ • '^ ; ' h -^ ' ..^ "■■■ ■ *J^;j-V.W -•: ,; ■ Figs. 3, 4, 5, 6 represent alpharadiographs (X75) of lamina dura dentis from horses on high P diet: Fig. 3, 4 weeks, Fig. 4, 7 weeks; Fig. 5, 12 weeks; Fig. 6, 30 weeks. Osteolysis at first intense (Fig. 3); newly-formed tissue decreasing (Fig. 4, larger canals); compact bone replaced by spongy bone (Fig. 5); becoming more and more immature and abnormal (Fig. 6) The Two Faces of Resorption ^'m^^ amount of calcium (about 15 gm/day/horse) and an excessive amount of phosphorus (about, 3V2 times as much). The animals were killed after periods of 4, 7, 10, 12 and 30 weeks of abnormal feeding. The clinical observations have been similar to those already published by Krook and Lowe (1964). After 4 weeks, very minor signs of osteitis fibrosa were present but the parathyroids were enlarged about 3 times. After 8 weeks, extremely severe osteitis fibrosa was recognized and in the colourful language of the senior veterinary pathologist (K), "the parathyroids were almost as big as the testicles of a dachshund". The bone response was uneven throughout the body. On the other hand, the horses which were the youngest at the onset of the experiment were the most severely affected. Changes in the mandibles and maxillae were observed sooner and progressed at a more rapid rate than those in the metacarpi (Krook and Lowe, 1964) and long bones. Increased radiolucency was particularly evident in the bones of the skull and face. Complete resorption of the lamina dura dentis was evident after 30 weeks. Comparative alpharadiographs of the periodontal portion of the mandible and maxilla examined in the light of histochemical staining and with the knowledge of the mechanism of bone growth pro- vided by radioautography (Belan- GER and MiGicovsKY, 1963 b) have been very instructive. Short term studies have demon- strated that only mesenchymal cells, preosteoblasts or "osteopro- genitors" (Tonna, 1965; Belanger and MiGicovsKY, 1963 b; Young 1963) are originally labeled. The presence of radioactive osteoblasts and osteocytes occurring later is an indication of growth movements and also of tissue replacement in areas where the adult condition has already been achieved. In 3 weeks-old chicks, the re- placement rate of metaphyseal trabeculae of the tibia has been established at 4 days (Belanger and MiGicovsKY, 1963 b). In the lamina dura of the horse jaw, this time is unknown. However, a con- stant cell movement must be simi- larly taking plase from the border of the osteonic canal, towards the areas of lower tissue density where osteolysis occurs and where the cells die. If osteo- lysis and peripheral accretion are balanced, a static histological, histochemical and radiological picture is maintained. On the other hand, if osteolytic stimuli such as hyperparathyroidism are present, resorption Is presumably greater than accretion. L. F. Belanger, T. Semba, S. Tolnai, D. H. Copp, L. Krook, C. Gries The result should be a progressive loss of bone manifested by an enlargement of the osteonic canal and a gradual loss of bone substance. Alpharadiographs of demineralized sections of normal bone have revealed that the areas where osteolysis is occurring are characterized by the presence of enlarged Fig. 5 and oftentimes confluent lacunae surrounded by low-density matrix (Belanger et al., 1963 b). Newly-formed portions of the tissue, at the border of osteonic canals consist of denser matrix containing small lacunae (Belanger et al., 1963 b). The lower- density matrix of the osteolytic sites stains more intensely with the periodic acid- Schiff and exhibits toluidine blue and azur metachromasia, indicative of a concentra- tion of mucopolysaccharides in these areas (Belanger et al., 1963 b). In the present series, the periodontal bone (lamina dura) was still fairly compact after 4 weeks (Fig. 3). The canals were small but there was already a considerable Intensification of osteolysis. After 7 weeks (Fig. 4), the areas of osteolysis were more extensive and character- ized by lower density of matrix corresponding to more widespread p.a.-Schiff staining. After 12 weeks (Fig. 5), the canalicular arrangement of the lamina dura had in great part disappeared and the newly-formed bone was of the trabecular type. The central portion of the trabeculae showed the characteristic manifestations of osteo- lysis. At this stage consequently, bone formationhad regressed to a more primitive form, but this younger type of bone could still be considered as normal bone. At this stage also, some less dedifferentiated areas, showed the disappearance of the p.a.- Schiff positive areas characteristic of osteolytic resorption. Numerous, irregular cementing lines separated the areas of new growth. The newly-formed bone con- tained only osteocytes of small size and many lacunae were empty. After 30 weeks, the trabeculae were thin and either uniformly dense of fibrillar (Fig. 6). The tissue surrounding this abnormal bone was also de differentiated and inhabited mostly by mesenchymal cells. The Two Faces of Resorption 7 Osteoclasts which had been sparsely observed at 4 and 7 weeks, were more numerous at 12 weeks. They attained almost tumoral frequency at 30 weeks and appeared most active along the abnormal new trabeculae. 5. The role of osteoclasts These observations bring us to consider the role of osteoclasts and the factors which are responsible for the occurrence of these cells. In an excellent review, Hancox (1956) lists a series of generalized and localized factors as responsible for the appearance of osteoclasts. In the first group, hyperpara- thyroidism, hypervitaminosis A, the administration of glucose or lead and a diet low in calcium have been reported. "Remodeling", bone fractures, ectopic bones or teeth, degenerated deciduous roots, circulatory disorders, infections, introduction of inert material in the root canals of teeth, have been recognized as so many local factors. The introduction of Plutonium (Arnold and Jee, 1957) or Yttrium (Neuman et ai, 1960; Jowsey, 1963) into the organism, has produced beautiful demonstrations of surface resorption in the presence of osteoclasts. Grafts such as performed by Barnicot (1948) and also in vitro transplantation and culture of bone specimens (Gaillard, 1955 a, b; Goldhaber, 1962) have been followed by stimulation of osteoclastic activity. Irving and Handelman (1963) have recently published interesting results with devitalized autologous bone grafts. A multinucleated giant cell reaction was initiated from mesenchymal cells whether the grafted bones were mineralized or demineralized. The host response did not seem to be affected by parathormone. All these events and also the results of our current observations on horses seem to have something in common: The presence of abnormal skeletal tissues either bone, cartilage or the components of the teeth. 8 L. F. Belanger, T. Semba, S. Tolnai, D. H. Copp, L. Krook, C. Gries 6. Conclusions At this point we would like to propose the following conclusions: (1) Normal adult bone is a constantly renewed tissue in which resorption is equally matched by accretion. (2) Intimate resorption (osteolysis), taking place away from the bone surfaces seems to occur. The mature osteocytes are probably responsible for this phenomenon. One of the factors involved is the production by these cells, of a protease possibly linked to lysosome activity. (3) The mature osteocytes seem to respond readily (within a few hours) to endo- genous parathyroid stimulation. The role of these cells in the maintenance of calcium homeostasis, is perhaps more important than previously realized. (4) Osteoclasia appears as a specialized response to the presence of abnormal skeletal material, either of general or local origin. The osteoclasts which probably originate from primitive cells, may be akin to the giant multinucleated cells of connective tissue. Like those, their major role may be to take part in the maintenance of the integrity of the body. Acknowledgements The authors are indebted to Mrs. L. F. Belanger for skilled technique; also to the Medical Research Council of Canada, to the Canada Department of Agriculture and to the U.S. Atomic Energy Commission (Contract No. AT(30-l)-2779) for financial assistance. Para-Thormone was graciously supplied by Eli Lilly and Co. (Canada) Ltd., Toronto. References Arnold, J. S., and W. S. S. Jee: Bone growth and osteoclastic activity as indicated by radioautographic distribution of Pu-''". Amer. J. Anat. 101, 367 (1957). Barnicot, N. a.: The local action of the parathyroid and other tissues on bone in intra- cerebral grafts. J. Anat. 82, 233 (1948). Baud, C. A.: Morphologic et structure inframicroscopique des osteocytes. Acta anat. 51, 209 (1962). Belanger, L. F.: Osteolysis: An outlook on its mechanism and etiology. In Parathyroid Hor- mone, Ultrastructure, Secretions and Functions. Gailiard, P. G., and R. V. Talmage (eds.). Chicago: University of Chicago Press (in press). — , and C. Belanger: Alpharadiography: A simple method for determination of mass con- centration in cells and tissues. J. biophys. biochem. Cytol. 6, 197 (1959). — , and B. B. Migicovsky: Histochemical evidence of proteolysis in bone: The influence of Parathormone. J. Histochem. Cytochem. 11, 734 (1963 a). — — Bone cell formation and survival in H-'-thymidinc-labeled chicks under various con- ditions. Anat. Rec. 145, 385 (1963 b). — , J. RoBiCHON, and C. Belanger: Mounting hard tissues for microradiography. Experientia 19, 163 (1963 a). , B. B. Migicovsky, D. H. Copp, and J. Vincent: Resorption without osteoclasts (Osteolysis). In Mechanisms of Hard Tissue Destruction. Sognnaes, R. F. (ed.). Washing- ton: Amer. Ass. Advanc. Sci. 1963 b, p. 531. Cameron, D. A., H. A. Paschall, and R. A. Robinson: The ultrastructure of bone cells. In Bone Biodynamics. Frost, H. M. (ed.). Boston: Little, Brown 1963, p. 91. CoPENHAVER, W. M.: Bailcy's Textbook of Histology (15th ed.). Baltimore: Williams and Wilkins 1964. Copp, D. H.: The hormones of the parathyroid glands and calcium homeostasis. In Bone Biodynamics. Frost, H. M. (ed.). Boston: Little, Brown 1963, p. 441. The Two Faces of Resorption 9 Dudley, H. R., and D. Spiro: The fine structure of bone cells. J. biophys. biochem. Cytol. 11, 627 (1961). DuFOUR, J. J.: Etat pre-fractural du col femoral. Helv. chir. Acta 19, 15 (1952). Engel, M. B.: Mobilization of mucoprotein by parathyroid extract. A. M. A. Arch. Path. 53, 339 (1952). Frost, H. M.: In vivo osteocyte death. J. Bone Jt Surg. 42 A, 138 (1960). Gaillard, p. J.: Parathyroid gland tissue and bone in vitro. I. J. exp. Cell Res. Suppl. 3, 154 (1955 a). — Parathyroid gland tissue and bone in vitro. III. Proc. kon. ned. Akad. Wet. C 58, 286 (1955 b). GoLDHHABER, P.: SoHie Current concepts of bone physiology. New Engl. J. Med. 266, 870 (1962). Ham, a. W., and T. S. Leeson: Histology (4th ed.). Philadelphia: J. B. Lippincott 1961. Hancox, N.: The osteoclast. In The Biochemistry and Physiology of Bone. Bourne, G. H. (ed.). New York: Academic Press 1956, p. 213. — , and B. Boothroyd: Structure-function relationships in the osteoclast. In Mechanisms of Hard Tissue Destruction. Sognnaes, R. F. (ed.). Washington: Amer. Ass. Advanc. Sci. 1963, p. 497. Heller-Steinberg, M.: Ground substance, bone salts and cellular activity in bone formation and destruction. Amer. J. Anat. 89, 347 (1951). Home, E.: Experiments and observations on the growth of bones; from the papers of the late Mr. Hunter. Trans, med. chir. Knowledge 2, 277 (1800). Irving, J. T., and C. S. Handelman: Bone destruction by multinucleated giant cells. In Mechanisms of Hard Tissue Destruction. Sognnaes, R. F. (ed.). Washington: Amer. Ass. Advanc. Sci. 1963, p. 515. Jaffe, H. L.: Hyperparathyroidism (Recklinghausen's disease of bone). Arch. Path. 16, 63 (1933). JowsEY, J.: Microradiography of bone resorption. In Mechanisms of Hard Tissue Destruc- tion. Sognnaes, R. F. (ed.). Washington: Amer. Ass. Advanc. Sci. 1963, p. 447. — , B. L. Riggs, and P. J. Kelly: Mineral metabolism in osteocytes. Proc. Mayo Clinic 39, 480 (1964). Kind, H.: Studien zur Frage der Osteolyse. Beitr. path. Anat. Ill, 283 (1951). Krook, L., and J. E. Lowe: Nutritional secondary hyperparathyroidism in the horse, with a description of the normal equine parathyroid gland. Path. vet. suppl. 1 (1964). Lacroix, p.: Bone and cartilage. In The Cell, Vol. V. Bracket, J., and A. E. Mirsky (eds.). New York: Academic Press 1961, p. 219. Lipp, W.: Neuuntersuchungen des Knochengewebes. II. Histologlsch erfaBbare Lebensaulkrun- gcn der Knochenzellen. Acta anat. 22, 151 (1954). — Neuuntersuchungen des Knochengewebes. III. Histologisch erfalsbare Lebensaufierungen der Osteozyten in embryonalen Knochen des Menschcn. Anat. Anz. 102, 361 (1956). — Aminopeptidase in bone cells. J. Histochem. Cytochem. 7, 205 (1959). Neuman, W. F., B. J. Mulryan, and G. R. Martin: A chemical view of osteoclasis based on studies with yttrium. Clin. Orthop. 17, 124 (1960). Recklinghausen, F. von: Ober Rachitis und Osteomalacic. Jena 1910. Ruth, E. B.: Further observations on histological evidence of osseous tissue resorption. Anat. Rec. 118, 347 (1954). — Basophilic islands in osseous tissue and their relation to resorption. Anat. Rec. 140, 307 (1961). Rutishauser, E., and G. Majno: Physiopathology of bone tissue: The osteocytes and fun- damental substance. Bull. Hosp. Jt Dis. 12, 468 (1951). Sherman, M. S., and W. G. Selakovitch: Bone changes in chronic circulatory insufficiency. J. Bone Jt Surg. 39 A, 892 (1957). Sissons, H. a.: Histological studies of normal and osteoporotic bone. In L'osteoporose. Hioco, D. J. (ed.). Paris: Masson 1964. Talmage, R. v.: Parathyroid function: A calcium replacement mechanism. Amer. Zoologist 2, 353 (1962). 10 W. A. DE VOOGD VAN DER StRAATEN Talmage, R. v.: Cytological and biochemical changes resulting from fluctuations in endo- genous parathyroid hormone levels. In Parathyroid Hormone, Ultrastructure, Secretions and Functions. Gaillard, P. G., and R. V. Talmage (eds.). Chicago: University of Chicago Press (in press). ToNNA, E. A.: Osteoclasts and the aging skeleton: A cytological, cytochemical and auto- radiographic study. Anat. Rec. 137, 251 (1960). Urist, M. R., N. S. MacDonald, M. J. Moss, and W. A. Skoog: Rarefying disease of the skeleton: Observations dealing with aged and dead bone in patients with osteoporosis. In Mechanisms of Hard Tissue Destruction. Sognnaes, R. F. (ed.). Washington: Amer. Ass. Advanc. Sci. 1963, p. 385. Young, R. W.: Nucleic acids, protein synthesis and bone. Clin. Orthop. 26, 147 (1963). Zawisch-Ossenitz, C: Uber Inseln von basophiler Substanz in den Diaphysen langer Rohren- knochen. Z. mikr.-anat. Forsch. 10, 472 (1927). Some Remarks and Questions on Metabolic Patterns in the Family of Bone Cells W. A. DE VoOGD VAN DER StRAATEN Laboratorium voor Celbiologie en Histologie, Rijksuniversiteit Leiden, Leiden, Nederland In order to speak sensibly about metabolic patterns in the family of bone cells at least three general points have to be clarified. The first point concerns the way in which the word family is used; the second point concerns the concept of metabolic pattern in general; and the last point concerns the Interconnection between some pure scientific and methodological questions in our special field. 1. By definition the members of a family are relatives, and they remain so even if they are miles apart. Now the different types of cells found in bone tissue, the bone cells, are relatives but they are not miles apart; on the contrary the members of the family of bone cells live closely together and form a functional and structural unit. In what I am going to say about bone cells, however, I will not look upon them as relatives but as different types of labourers in a "family factory". 2. It is common knowledge that the morphologically comparable cellular elements of a tissue e. g. osteocytes, are not by necessity all in the same functional state at the same time. "Without discussing the background of this phenomenon, I think that none of us would attribute these variations to differences in metabolic pattern. What then is covered by this term and what is the special meaning of the word pattern? Or to restate this problem in a positive way, why do we in all probability suppose different metabolic patterns to be existent in osteoblasts and osteoclasts even if we have no pertinent biochemical data at hand? To my mind a list of biochemical differences would not necessarily imply different metabolic patterns. Such a list would deserve our full attention and would be a challenge, stimulating our Ingenuity In making connections between these biochemical differences and the essential differences in function, the cells are known to have in the tissue. It Is only from the moment that we have succeeded in making reasonable connections that we are allowed to speak of different metabolic patterns. To press this point a little further, it is clearly a matter of convention how to define the term metabolic pattern. In fact I prefer an opera- Some Remarks and Questions on Metabolic Patterns in the Family of Bone Cells 1 1 tional definition linking up the specific biochemical organization and the specific function of the cell. However we will soon find out that our definition is a little pedantic as our present day knowledge hardly allows for even one single good example. 3. The final preliminary point will carry us forward to the heart of our subject. The number of publications concerning the metabolism of bone cells is increasing, and it is already apparent that there is a wide difference in scope and method among the various approaches. Concerning differences in scope there are two extremes. At one end we find investigations disclosing the activity of the cellular enzymatic apparatus. At the other end investigations dealing with the fate of certain substrates. Figuratively speaking one could say that the first group deals with the metabolic highway system of the cells, the other group with the real traffic on that highway system. This picture will make clear to us that the conditions of the highways and the intensity of the traffic do not necessarily run parallel. Concerning differences in method, we can more or less arbitrarily differentiate into methods that carry no considerable risk of changing the metabolic parameters under investigation and methods that do. To my mind histochemical and enzymo- logical investigations performed on previously intact bone tissue fall within the group of low risk. However metabolic investigations using slices or otherwise wounded pieces of bone tissue ask for a more critical attitude. And in fact without any data at hand one can foresee that e. g. by damaging the delicate network of osteocytes serious harm is probably done. In this connection unpublished data from Hekkelman (personal communication) have illustrative value: He observed that pieces of diaphyseal rabbit bone (roughly 15X5X2= 150 mm-*) put in Hanks balanced salt solution lose a considerable amount Table 1. The total amount of nucleic acid obtained after 2 hours of incubation and expressed as ugjml incubation fluid from 'Is g of hone Hanks (ph 7.4) , 2.7 ± 0.77 , 6.6 ± 1.96 | < 0.002 (6 exp.) I (6 exp.) Hanks without glucose (ph 7.4) 3.2 ± 0.4 4.3 ±1.2 ! < 0.01 ! (6 exp.) (12 exp.) Pd insignificant < 0.01 Table 2. The activity of isocitric dehydrogenase obtained in the medium after 2 hours of incubation: mu moles/minig of bone Hanks (ph 7.4) 6.28 + 2.2 13.2 ± 5.6 0.02 > p > 0.01 (6 exp.) (6 exp.) Hanks without glucose (ph 7.4) 4.82 ±1.3 6.42 4- 4.41 insignificant (9 exp.) (l?cxp.) Pd I insignificant 0,02 > p > 0.01 of nucleic acid and enzymes as e. g. isocitric and lactic dehydrogenase. The output was found to depend on the temperature of incubation and the presence or absence of glucose; see Tables 1 and 2. 12 W. A. DE VOOGD VAN DER StRAATEN Moreover the yield was not far below that obtained after homogenization followed by extraction, and to take Hekkelman's own words, "the phenomenon suggests a pumping out of cellular material". In a recent interesting publication (Peck et ai, 1964) we find another aspect of the same problem. The authors describe the isolation of bone cells from rat calvaria in buffered collagenase. The cells obtained passed successfully viability tests and proved fit for cultivation. From cytochemical alkaline phosphatase reactions they argue that their harvest probably contained osteoblasts and osteocytes. Moreover radioassay of CO2 and lactate after confrontation with labelled glucose under aerobic conditions revealed metabolic traits reminiscent of "intact" bone tissue. However they observed no bone formation in tissue culture and were not able to prevent dedifferen- tation and/or overgrowth by "fibroblasts". The latter point is not surprising at all. Is the absence of bone formation surprising? Taking the acceptable view that bone formation is a complex process dependent on the interplay of very subtle intra and extra cellular conditions, the experiments clearly show that somewhere these condi- tions were not fulfilled. However taking the biochemical data really to mean that the collagenase isolated cells have preserved their original metabolic organization, we come to the conclusion that perhaps even full knowledge concerning the metabolism of the individual cells will always be fundamentally insufficient to explain the formation of bone as a tissue. There is still another example to be considered In the clarification of my third preliminary point. Starting with the assumption that destruction is in general easier to understand than construction, one may be tempted to look upon the process of osteoclastic bone resorption as a nut easy to crack. In fact it is not uncommon for the specialist in tissue culture to be consulted about a method for cultivating osteoclasts in pure strain for investigational purposes. The bare facts are that such a culture is non existent and probably even impossible. Gaillard (1961) has repeatedly observed in PTE treated cultivated radius rudiments that the numerous typical osteoclasts disappear from the moment the bony shaft is totally resorbed. A good explanation seems to be that the development or the preservation of typical osteoclasts depends among other things on the presence of bone and/or breakdown products of bone. In the light of work of Belanger we arrive at the same conclusion; however he adheres to the restricting view that the development of the osteoclasts demands the presence of bone. Returning to the observation of Gaillard and leaving undiscussed what, in this situation, the fate of the osteoclasts might be, we conclude that the student of osteoclastic function, working with isolated cells, would in all probability waste his effort on cells in transition. Now coming to my main point and starting from the metabolic schemes known from general biochemistry, what lines can be drawn in black and what lines have to be dotted or even omitted? This specially concerns carbohydrate metabolism, which is responsible for at least 3 diff'erent tasks in bone tissue: 1. Generation of energy; 2. Provision of the cells with precursors to be used in the synthesis of proteins, muco- polysaccharides and pentose containing compounds of biological importance such as the pyridine nucleotides and RNA and DNA; and 3. Production of organic acids supposed to be important in the solubilization of matrix mineral. It is from histochemical investigations that we have at least some data concerning the different types of cells (Balogh et al., 1961; Herrmann-Erlee, 1962). Balogh Some Remarks and Questions on Metabolic Patterns in the Family of Bone Cells 13 worked with long bones of 4-week-old mice; Herrmann-Erlee with 15 day-old mouse radius rudiments. In reproducing their semi-quantitative data I restrict myself to some data concerning the carbohydrate metabolism of the cells of the inner periosteum and the osteoblasts, the osteocytes and the osteoclasts. Taking together data from both authors and omitting a number of minor discrepancies they demon- strate from each of the main pathways a number of enzymes to be active. However it is quite remarkable that they observed no 6-p-G Dehydrogenase activity as this is the second enzyme in the pentose phosphate cycle of which the initial enzyme, the G-6-p Dehydrogenase, was found to be active. The data of Balogh show in fact dis- appointingly little difference between the different types of cells. However Herr- mann-Erlee finds Succinic dehydrogenase to be active only in the osteoclasts and osteoblasts at the ossification ring. In Balogh's data the osteoclasts do not hold a monopoly, but here this enzyme is more active than in the other types of cells. Moreover and in sharp contradistinction to Balogh, she finds no Lactic dehydro- genase in osteoclasts. Finally she was unable to demonstrate a-glycerophosphate dehydrogenase activity in the cell types mentioned, with the only exception again of the osteoblasts at the ossification ring. Summarizing, no convincing argument is found to doubt the completeness of the carbohydrate metabolizing machinery. Moreover the osteoclasts and the osteoblasts at the diaphyseal-metaphyseal junction seem to keep a special position. However the data are absolutely insufficient to construe different metabolic patterns in the sense I have defined. Concerning the special position of the osteoclasts I would like to add another point: Both authors found the osteoclasts deprived of Glutamic Dehydro- genase activity, and as you know this enzyme belongs to the domain of protein catabolism. Before passing on to some investigations concerning metabolic pathways in bone cells it will repay to discuss, whether essential cellular potencies must find by necessity some reflection in the conditions of the metabolic pathways. I will try to formulate an answer on the basis of a special example: In the Liege symposium we have heard Vaes's paper on acid Hydrolases and Lysosomes in bone cells (Vaes, 1965). Moreover in Leyden we have seen his successfull work on the PTE intensified release of Lyso- somal enzymes from cultivated mouse calvaria. I will take the position that in all probability his finding will prove to be of essential importance in the understanding of the process of bone resorption. Now one cannot foresee that the intracellular presence of even a high number of Lysosomes can be read in some way or another from certain peculiarities in the metabolic pathways. However it is possible that the production of the acid hydrolases and of the organic acids, necessary to bring the released enzymes in their pH optimum, or even the mechanism of extrusion, do ask for certain provisions that indeed can be read from the pathways. My answer then is, that perhaps many peculiarities in the metabolic pathways of bone cells will become explainable, provided data from other origins become available. With only a few exceptions data concerning the main metabolic routes in bone tissue are not specific to the different types of cells; on the contrary they mostly reflect the combined contribution of different types. In his thesis "Bone metabolism and the action of Parathyroid extract" Hekkel- MAN gave us an excellent survey, describing the 1963 situation. Leaving out some im- portant controversial points in the literature, we meet a picture in which 1. the glyco- 14 W. A. DE VOOGD VAN DER StRAATEN lactate -^'^^ pyruvate " flavoprot red ftavoprot c oxalo^acetate citrate NADH aconitase 'r^oj^'^^o ,50 citrate fumarate \^nadp / C Deh. X lytic pathway, the pentose phosphate cycle and the Krebs cycle are operative. 2. However concerning the oxidative phosphorylation nearly nothing is known. 3. The activities of the pentose shunt dehydrogenases are suprisingly high in dia- physeal rabbit bone tissue as com- glucose /^ gtuco5e-6-P <~ poiysaccha- pared with e.g. kidney, liver and brain tissue. Moreover the strik- ingly high relative activity of pyri- dine nucleotide transhydrogenase — being as it is, involved in the reoxydation of NADPH — seems to underline the importance of the NADP dependent dehydrogenases in bone. 4. On the other hand the relatively low activity of Succinic Cytochrome C reductase suggests a less important role of the tri- carboxylic acid cycle. 5. Concern- ing the citrate metabolism, espe- cially under the influence of PTH, the problem about the balance be- tween citrate synthesis and oxida- tion is still open to some contro- versy. 6. Finally, in this picture we meet already some connections between carbohydrate metabolism and protein synthesis, especially collagen synthesis. These connec- tions concern carbohydrate iner- mediates being precursors of some amino acids and furthermore the ribose synthesis being prerequisite to RNA synthesis and so indirectly to protein synthesis; see Fig. 1. Now although the data concerning the action of PTFi on bone tissue belong to a subgroup of our universe of discourse, they are of direct relevance, because they reveal in all probability the key points of bone metabolism. Moreover, though not being essential, work on the main pathways is mainly done by students in the PTE field. And so an essential contribution to our picture is Hekkelman's hypothesis stating that PTE induces a decrease of the amount of NADP and/or NADPH available for catabolic processes in the bone cells (Hekkelman, 1963, 1965). His argument hinges on phenomena concerning the extractahility of Isocitric dehydrogenase from dia- physeal bone homogenates and more specially on the influence of NADP or NADPH — additional or naturally present — on this extractahility. In fact he did no direct determinations of the cofactors mentioned. It will be interesting now to compare this picture with a number of observations communicated at the 2nd Parathyroid symposium held in 1964 in the Netherlands: CO2 NADPH A-CO2 aketoglutarate ammo acids Simplified scheme of the mam metabolic pathways Fig. 1. A simplified scheme of the main metabolic pathways, according to J. W. Hekkelman: Bone metabolism and the action of parathyroid extract, 1963. Note: NAD(H) = DPN(H): NADP(H) = TPN(H) Some Remarks and Questions on Metabolic Patterns in the Family of Bone Cells A most interesting paper on the influence of PTE on bone metabolism and the levels of Nicotinamide nucleotides was given by Van Reen (1965). He determined the pyridine nucleotides in the epiphyseal — metaphyseal area of rabbit femora fluoro- metrically and found quite to his own surprise the NADP content in the tissue of PTE pretreated animals to be increased by a factor 2.25. The NAD content was not changed significantly, NADH and NADPH were decreased by a factor 0.7. His surprise does not primarily concern a possible controversy with Hekkelman's data but far more a seeming incompatibility of a PTE induced increase of NADP and the well established PTE induced increase of citrate concentration in bone. (At least this holds as long as one supposes the activity of the NADP dependent Isocitric dehydrogenase to be of importance in the balance of citrate production and oxi- dation.) In fact in Van Reen's opinion the "old" concept of a metabolic block at the Isocitric dehydrogenase level cannot be considered as the final answer in explaining the accumulation of citrate in bone. In trying to reflect a little upon this new situation it is good to keep in mind, that in most organs NADP is present mainly in the reduced state, the reverse being true for NAD. In bone tissue, according to van Reen, the ratio NADP/NADPH comes near to unity; under the influence of PTE there is a dramatic shift towards the oxidized state. Returning then to Hekkelman's hypothesis concerning the action of PTE, it has been my contribution to show that in Gaillard's system of cultivated mouse radius rudiments, additional NAD or NADP are capable of inhibiting the development of the morphological effect of the hormone. Moreover the same was found to apply for a number of NADP-ase inhibi- tors, suggesting that by arti- ficially keeping up the NADP level the hormone effect is ham- pered in its development (de VooGD van der Straaten, 1965). Finally I have been able to demonstrate that PTE in- duces in radius rudiments a decreased binding of i-*C-Nico- tinic Acid, a compound being preeminently the precursor of NAD and NADP; see Fig. 2. Summarizing, my observations seem to be in favour of Hekkel- man's hypothesis and rather % mo 80 '^■-■^ ^.^ 60 - "'^ 70 vo 20 I 6 12 18 hours 2i4 Period of confrontation witti PTE 0.5 I U/ml Fig. 2. The effect of PTE 0.5 lU/ml on the Nicotinic acid-Z-'-'C bind- ing capacity of mouse radius rudiments in vitro. The radii were taken from 15-day-old embryo's; the left hand radii were cultivated in standard medium, the corresponding right hand radii being confronted with PTE. In all experiments this period of cultivation was followed ,.^- .... by a 1 hour period of confrontation with labelled Nicotinic acid. The dlfhcult to reconcile with Van graph presents the relative activities of the washed bone shafts of Reen's data ^^^ experimental radii, the activities of the corresponding control shafts being t.tken as 100 Against this controversial background there seems to be some need for a "unifying concept". Looking again upon Van Reen's data we meet so to say an "internal discrepancy", possibly even pointing to a way out; see Fig. 3. This "discrepancy" is found in the tremendous rise in NADP without any accom- panying rise in the amount of the reduced form. And from the very fact that on the 16 W. A. DE VOOGD VAN DER StRAATEN contrary NADPH decreases under the influence of PTE one gets the feeling that the oxidized and the reduced state do not keep the simple relation to one another one should perhaps suppose to exist in an uncomplicated chain of oxido-reductions. In Biosynthesis NADP PTE eltect of oddiliono NAD and NADP NADP (12S* increased) ' Substr(H)\ + (nADp' (decreased 07x) ^ nad(h) r^ 1 Cytochrome I '\ y\ system 1 (t^ ) NAD -< X etc ♦ \