. <= CU eee OL ve oe eee = sian ceeachoenets ete my, eae ey Sa eS aa "s 5 2 ~ ey g Cee pecans PeSx eee Tso x ee nonsonne nee , 2 “toce a e eS ee tals sca aes nance r % Cee Are wacech regs mom mah Tee, er eee Wane ree yee eee ese eae eter een Ptr ocala ee coevneee yy ese eee) wes Digitized by the Internet Archive in 2009 with funding from University of Toronto http://www.archive.org/details/contributionstoeO9carn ry * ? Care CONTRIBUTIONS TO EMBRYOLOGY VotumeE IX, Nos. 27 To 46 A MEMORIAL TO FRANKLIN PAINE MALL PUBLISHED BY THE CARNEGIE INSTITUTION OF WASHINGTON Wasuineton, 1920 Re OO ee ee i ok ee i - qv MA, . \s : : ) %, e a © \ X _ ven! || , x " a “eres CARNEGIE INSTITUTION OF WASHINGTON Pusuication No. 272 PRESS OF GIBSON BROTHERS, INC. WASHINGTON, D.C. - FOREWORD. The papers included in this volume have been contributed as a memorial by present and former members of the staff of the late Professor Franklin Paine Mall, in recognition of his inspiring leader- ship and in response to the strong feeling of affection with which they had come to regard him. A volume of this nature had been under consideration, to commemorate the approaching twenty-fifth anni- versary of his occupancy of the chair of anatomy in the Johns Hopkins University. His untimely death, however, just at the close of a quarter century of remarkable productivity, interfered with the project as originally planned and left it possible to offer only a be- lated tribute in the form of the present volume. BaLtimMoreE, August 1, 1919. III BroGRAPHICAL Accounts OF FRANKLIN Patne MAtu. Fiexner, 8. Dr. Franklin P. Mall: An appreciation. Science, 1918, n. s., xLvi, 249-254. Howext, W. H. Franklin Paine Mall. Johns Hopkins News-Letter, 1917, xxm, No. 9. Huser, G.C. Franklin Paine Mall: In memoriam. Anat. Rec., 1918, x1v, 1-17. Luu, F. R. Annual Report of Marine Biological Laboratory for the year 1918. Biol. Bull., 1919, xxxvi, 354-355. Meyer, A. W. Franklin Paine Mall: An appreciation. Jour. Amer. Med. Assn., 1918, Lxx, 121-123. Sanry, F. R. Franklin Paine Mall; a review of his scientific achievement. Science, 1918, xLv11, 254-261. . Franklin Paine Mall. The Scientific Monthly, 1918, v1, 283-284. . Sverio, Barsosa. Franklin Paine Mall. Archivo de Anat. e Anthropol, rv, 357-359. Woopwarp, R. 8. Report of the President, Year Book No. 17, Carnegie Inst. Wash., 1918, 18-19. Memorial services in honor of Franklin Paine Mall, professor of anatomy, Johns Hopkins Uni- versity, 1893-1917, Addresses by FJ. Goodnow, R. 8. Woodward, W. H. Welch, L. F. Barker, 8. Flexner, and F. R. Sabin. Bull. Johns Hopkins Hosp., 1918, xxrx, 1 Boston Med. and Surg. Jour., cLxxvu, p. 31. British Med. Jour., 1918", p. 39. Jour. Amer. Med. Aer 1917, Lxrx, p. 1899. Lancet, 19177, p. 841. Nature, 1917, c, p. 328. South Med. Jour., 1918, L, p. 39. IV LIST OF CONTRIBUTIONS. BarDEEN, C. R. The height-weight index of build in relation to linear and volumetric proportions and surface area of the body during post-natal development. (11 charts, 2 text-figures.) No. 46... 483-554 Bran, Rosert B. Notes on the post-natal growth of the heart, kidneys, liver, and spleen in man. (8 text-figures.) No. 37.......... 263-284 Cuark, Extor R., and Exeanor L. Crark. On the origin and early development of the lymphatic system of the chick. (7 plates, Page. Move stmpures:)|. ING: 45. oc a. s . cles voy osc cecnccc ceed 447-482 Corner, GrorcE W. On the widespread occurrence of reticular fibrils : produced by capillary epithelium. (7 plates.) No. 29........ 85-93 Duessere, J. Cytoplasmic structures in the seminal epithelium of the opossum. (2 plates, 5 text-figures.) No. 28................ 47-84 Ksstck, Cuartes R. Formation of macrophages by the cells lining the subarachnoid cavity in response to the stimulus of particulate monuuene (byvplane:): ING. 426 ees es ac en cde 377-388 JENKINS, GEoRGE B. A study of the superior olive. (2 plates, 1 text- HEME MM NO SA eM Mr AR ee dae tg .. 157-172 Lewis, Marcarer R. Muscular contraction in tissue cultures. (2 plates, , (5) JURE are ET a1 08 MM [ls 1 191-212 Lewis, WarreN H. The cartilaginous skull of a human embryo 21 millimeters in length. (5 plates.) No. 39.................. 299-324 Mack.in, CHarites C. The development and function of macrophages in the repair of experimental bone-wounds in rats vitally stained with trypan-blue. (4 plates.) No. 27...................... 1-46 Meyer, Artour W. Hydatiform degeneration in tubal and uterine prepnancy. . (Giplatess, No: 4022. 22... ..-- ul: .~ * ise) + A ; meas bar ledolL ai % a! | ' be ae. : . ia alt ide t eriglig AN) * ai? | boorva ea he Rarely fli al pete f wel Well bre eer we A ruren-te i] wigifty RA el ue a ¢, , Wy? eit) ur hk 7 eae Ce a villares oe I een «i a ti dis Tina yts “) ees és j v Sul lidafet Te Pe i: oul ° ty A Th oh oii ; }' en hw vnige ‘ avererks gia Srp iy Tiida ! rie fr ed eal t t : of, : om ) tr) 4 ety at +l) NCU aT 9 } athhin hates) cal ie lie A” eas ” ‘ i "wag ovine’ aia ‘ venta th Telus) 2 eke wil ty ' , at) at Ve) =” ‘ vane ae all od oe dina ee ‘rid dio oy. 5 nt af vet one j . oes ad it > Ani sma or gate oA a ~! bg ae > he oly ey Dare ral wi ap es o! yes Pave on CONTRIBUTIONS TO EMBRYOLOGY, No. 27. __ THE DEVELOPMENT AND FUNCTION OF MACROPHAGES IN THE REPAIR OF EXPERIMENTAL BONE-WOUNDS IN RATS | _ VITALLY STAINED WITH TRYPAN-BLUE. By Cuarites Ciirrorp MAcKtLin, Associate Professor of Anatomy, University of Pittsburgh. With four plates. Mobilization AGtivity a: 352 95....isp eee eee ee ae hee nen 4s SOS THE DEVELOPMENT AND FUNCTION OF MACROPHAGES IN THE REPAIR OF EXPERIMENTAL BONE-WOUNDS IN RATS VITALLY STAINED WITH TRYPAN-BLUE. By CHARLES CLIrrorD MACKLIN. INTRODUCTION. The vital-staining route as an approach to the problem of bone-repair came as a natural consequence of the recent work of Shipley and Macklin (1916'?) on osteo- genesis. By subjecting very young, growing animals to trypan-blue, one of the azo-dyes belonging to the benzidine series of colors, these investigators were able to show that the regions of active bone-growth took a more intense stain than the remainder of the bone; and, furthermore, that the heightened coloration was largely referable to the presence in these areas of innumerable phagocytic cells, within whose cytoplasm the dyestuff was stored in multitudinous tiny segregations known as “dye-granules.”’ These phagocytes were identified as the reticulo-endothelial cells of the young bone-marrow. Their reaction to the dyestuffs of the benzidine group is the same as that of the host of cells found throughout the body, which have been extensively studied by different authors, and to which various names have been given, such as “pyrrhol-cells’” (Goldmann, 1909), ‘“‘clasmatocytes’’ (Ranvier, 1899-1900), “resting-wandering cells” (Maximow, 1906), ete. Recently Evans (1915) has employed Metschnikoff’s term ‘‘macrophage”’ to cover this entire group of phago- eytic cells which are united by a uniform functional response to these colloidal dyestuffs, and it is now well recognized that the term “macrophage” is a physiological designation, including within its compass very diverse morphological elements. This similar staining reaction, indeed, is but an expression of the phagocytic potentiality which these cells hold in common (Evans and Schulemann, 1914) and which mani- fests itself during their every-day existence in the ingestion and storage of certain elements of the surrounding tissue-fluids. In consideration of the vigorous phagocytic properties attributed to these cells, and also of their being present in large numbers where temporary bone and cartilage were being absorbed, it seemed evident to Shipley and Macklin (19167) that they were a very important factor in active osseous development and that their peculiar réle under these circumstances was played in connection with the resorp- ~ tion of the provisional cartilage and bone. Now bone-resorption is an active process in the later stages of bone-repair, for it is well known that the excess of provisional callus which is built up following a bone-injury, such as a fracture, is gradually removed. Since the resorption of this provisional callus is quite similar to that of provisional new bone it was decided to investigate the vitally stained cells in the callus of healing bone-wounds and to compare the findings with those in young, growing bone. 3 H DEVELOPMENT AND FUNCTION OF MACROPHAGES IN BONE-REPAIR. In the earlier stages of the reparative process following bone-wounds, too, there is a great deal of débris to be eliminated, such as injured bone, blood-clot, damaged muscle, and other devitalized tissue, and it is plain from the writings of other workers that the potentialities of the macrophages eminently fit them for the performance of this duty. That they play a part in the healing of wounds of soft tissues, such as skin, kidney, and liver, may be inferred from the work of Gold- mann (1912), who demonstrated by the aid of vital-dyes that they were increased in the regions where repair was proceeding. Indeed, the evidence of numerous investigators points to the macrophages being concerned in all inflammatory con- ditions. Maximow (1902, 1909?), notably, has made a special study of these cellsin inflammatory areas, where he finds them increased in number and size, and speaks of them as “polyblasts”; and Tschaschin (1913) has recorded similar findings. The problem of the healing of wounds of bone, therefore, seemed to offer a particularly favorable field for the application of the vital-staining method, for it was expected that in the early stages of bone-repair, where damaged soft parts must be cleared away, as well as in the later stages, where provisional bone has to be eroded, the trypanophil cells—i. e., the macrophages—in the pursuance of — their physiological vocation as phagocytes, would become locally very numerous and would show hypertrophy and intensified phagocytic power. These expecta- — tions, as will be seen, were realized, and the following pages are devoted to the discussion of the gross and microscopic appearances presented in the progressiv stages of healing of fractures and trephine wounds in rats in whose tissues macrophages were made visible by the introduction of the dyestuff (trypan-blue) into the circulating fluids shortly before death. . =" MATERIAL AND METHOD. For the most part, the experiments were carried out on the albino rat, thou the black and crossed breeds were also used. The material was collected at admirable rat colony of the Wistar Institute of Anatomy and Biology, Phil phia,* and this insures that the rats were all perfectly healthy and were kept u the most favorable conditions during the time of experimentation. A comp) series of stages was secured, covering the entire period of repair. ] The operations were conducted as follows: The animal having been anesthet- ized with ether, the top of the head was carefully shaved and sterilized; a median incision was then made and the skin reflected over the parietal areas. The parie bone having been laid bare, a small trephine of 5 mm. diameter was used to forate it. In some cases the piece was removed altogether; in others it was replaced, sometimes upside down; and in still other cases a piece of living or dead bone from — another rat, or even dead bone from an animal of a different species, was inserted. As a rule two areas were trephined, one in each parietal bone. The wounds were *! winh to thank Dra. Greenman and Donaldson, of the Wistar Institute, for laboratory facilities and access to the rat ny; also Dr. McClung, of the University of Pennsylvania, for placing his laboratory at my disposal for the operations. Finally, I wish to -s ress to Miss Madge DeG. Thurlow my most cordial thanks for the invaluable assistance rendered by her carrying on this investigation. DEVELOPMENT AND FUNCTION OF MACROPHAGES IN BONE-REPAIR. 5 carefully sutured with sterile silk, and the most rigid asepsis was observed through- out. In the same animals, before recovery from the anesthetic, the tibia and femur of the left hind leg and a few of the ribs on the left side were fractured. No attempt was made to splint the bones, so that healing took place under the same conditions as in natural life. There were slight variations from this general type experiment; in some animals only trephining was done and in others only fracturing. In all, 20 animals were used, which furnished 16 trephined skulls, 15 fractured tibiew, 15 fractured femora, and 12 sets of fractured ribs. In table 1 a full description of the material is given. TasBLe 1.—Material. - | Specimens examined. Long bones. Dawak Animal | 48° t Skull. Rib. repair. No death Tibia Femur [ : (months). s : Cleared Sec- Cleared Sec- Cleared Sec- Cleared Sec- gross. tions. gross. tions. gross. tions. gross. tions. OAM Sm eteac § 18-1 3 CM Eten [A oom (ee se se ge) > en see Bohan Aa ae S$ 11-1 24 se se ce Bee See se se se se ths ORE ae $ 12-1 See | ecaeers se SELL Waste siere se se se se Giana t... seen a oimat| |) Reape eC dso range Seer ef = [a ee - 0 | Macrophages....... 9 (1 oye a] [PPM cee +44 +++ ]+4++4] (+4) Nn WES yea she (+) 0 Sections, long bones: | Petrie. fh5 28 Jette oe wae naka tae cyl i a tL ara =F =— Were - - — Macrophages....... Soap igre Sepa isc crater sess eonlocedsee mreig arf (att jean o. (EE) Cae | Sections, skull: 151 erm Ie oy 2 (a +++] +++ ae eee: Se Oot sae Sa PRS |-)-15--05 | Macrophages........|..... ++++ ++ Sa ak] ESE eee ++ 0} ‘eats — \ is we eee The plus signs indicate roughly the relative amounts of débris or macrophagic tissue, as the case may be. Thus + would denote a discernible increase in macrophages as compared with the normal, or the presence of an appreciable amount of débris; + + + + would mean an exceptional amount of macrophagic tissue or débris, ete. The sign — indicates an absence of débris or macrophages, as the case may be. The sign 0 means that no evidence is presented by the specimen. The bracketed signs indicate macrophages which have persisted in regions where formerly tissue was being absorted. They are comparatively small and weakly staining. The sections of the long bones show even more strikingly the same remarkable coexistence of débris and abundant macrophagic tissue over a period from the second to the tenth day, with macrophages remaining in the field in reduced amount as far as the thirtieth day. In the sections of the skull the same parallelism of débris and macrophages is found, extending (in the specimens examined) from the third to the tenth day. From this summary, the results of which are graphically set out in table 2, it is evident that the occurrence of excess of macrophage-tissue is strikingly syn- chronous with the presence of waste material. Following rapidly upon the injury, macrophages become excessive in numbers in and around the damaged tissue; these cells are not only larger in size than the usual resting macrophages, but are of DEVELOPMENT AND FUNCTION OF MACROPHAGES IN BONE-REPAIR. 31 heightened phagocytic power, as revealed by their increased dye-content. It may be inferred, therefore, that this waste material, the result of the trauma. is con- cerned in some way with the mobilization of the phagocytes and with their accel- erated activities; that, in fact, the increase in macrophagic tissue, in volume and in functional efficiency, is a response to the presence of dead or dying tissue. From table 2 it is also plain that as the dead tissue vanishes, as shown bv the tinctorial and histological evidence, the macrophages become less and less con- spicuous, their numbers being reduced and their staining weaker. This points to some connection of the phagocytes with the absorption of the débris. More will be said upon this point later. Another evidence that tissue resorption has been proceeding coincidently with the presence of macrophagic tissue in excess is the thinning and rounding of the ends of the bones surrounded by the phagocytes. This, for instance, is seen at the sixth day (S 11-2) in the cleared rib. As table 2 shows, some of the phagocytes are found in numbers somewhat above normal after demonstrable débris has disappeared. These are to be looked upon as cells which have persisted in the field after their work was done. They undergo gradual diminution in number and in reaction to the vital dye and are probably to be looked upon as resting rather than as actively functioning. To anyone familiar with the literature it will be quite obvious that the behavior of these phagocytes, in the reaction following bone injuries, is quite like that which obtains in the repair of any damaged tissue, and thus the problems involved are those common to inflammation. These problems have been investigated by various writers, as Maximow (1902, 1906, 1909?) and Goldmann (1912); the latter studied the tissues of the vitally stained animal following the application of turpentine and infection with the tubercle bacillus. Tschaschin (1913), too, investigated the reac- tion of the vitally stained cells in the neighborhood of foreign bodies in the loose connective tissue and in cauterized areas of the liver, spleen, and mesenteric lymph- nodes. Thorough discussions of the various aspects of macrophage behavior under these conditions are to be found in the literature, so that it is here sufficient to refer only to some of the more outstanding points and to emphasize the special application of macrophage function to the repair of bone-wounds. We have seen that in the rise and fall of excess macrophagic tissue in the areas surrounding bone-wounds a curve is traced. From this three successive segments may be taken to block out periods of macrophage history in which the most out- standing features of the phagocytic tissue are consecutively development; activity, and decline; but it must be recognized that these periods grade insensibly into one another, so that if arbitrary limits be assigned to them there will be of necessity some overlapping. They serve, however, to separate the discussion into convenient subdivisions. DEVELOPMENT. Of special interest in connection with the development of the macrophage- tissue is the question of origin of these phagocytes of the soft parts. Among the possible sources there must be considered the macrophages found normally in the 32 DEVELOPMENT AND FUNCTION OF MACROPHAGES IN BONE-REPAIR. marrow-cavity and periosteum of bone and in the tissue of the immediate vicinity. When an injury occurs, such as a fracture, it is quite possible to think of these “resting-wandering”’ cells as undergoing extensive rapid multiplication until the aggregation of phagocytes which is so marked a feature of the early stages of the repair process is produced. But to accomplish this result the proliferation would have to be enormous, and it is noteworthy that but few mitotic figures were to be found among the macrophages. Again, it is known that endothelial cells can, under the stress of inflammation, take on phagocytic properties, as shown by their reaction to colloidal dyestuffs (MacCurdy and Evans, Tschaschin, and others), and hence it may be assumed that some of the macrophages are recruited from these elements. The reticular cells of bone-marrow, too, are known to be phagocytic, and these may contribute their quota to the sum total of the ‘“‘polyblasts.” It would be difficult, however, to conclude that any or all of these sources could account for the tremendous local increase in trypanophilic cells, as seen, for instance, on the third day, even providing for the immigration of considerable numbers from adjacent tissues. Again, such an hypothesis would have no place for transi- tional cells of the type seen in figure 8, and of these there are all grades, from the finished polyblast—large, filled with enormous blue granules, and with a relatively small nucleus—all the way down to what appears to be the parent cell. This is a small mononuclear element resembling a lymphocyte. The parent cells contain no dye-granules, but they soon gain phagocytic ability, as shown by their rapid increase in size and the larger and larger amount of dye which they take up (fig. 8). The transitional cells are particularly abundant in the early stages, and most of all on the third day. The idea that some at least of the macrophages are really metamorphosed lymphocytes has been steadily gaining in the literature. Even in normal connective tissue, transitional forms may be found linking together the small round amoeboid lymphocytes with the clasmatocytes (Ranvier, Tschaschin) and suggesting the deri- vation of the latter from the former. The origin of the macrophages of the ‘“taches laiteuses” in the rabbit is indicated by finding transitional cells connecting them with a small lymphoid element (Tschaschin). Maximow (1907, 1909") has estab- lished the relationship of these wandering cells by embryological researches. The same author (1902, 1909°) has long maintained that the hypertrophied mononuclear phagocytes or polyblasts of areas of inflammation are largely derived from lympho- cytes attracted thither from the tissue-spaces and from the blood-stream, and Tschaschin brings forward evidence to confirm this view—the lymphocytes, it is assumed, rapidly undergoing a metamorphosis, their powers of phagocytosis becoming intensified, as shown by their progressive increase in ability to ingest vital dyes. Tschaschin has illustrated a series of transitional forms in his figure 11, Taf. vir, which strikingly resembles that shown in figure 8. Maximow (1916), according to Downey (1917), has even been able to bring about the development of typical vitally staining polyblasts from lymphocytes in tissue-cultures of lymph-nodes of young DEVELOPMENT AND FUNCTION OF MACROPHAGES IN BONE-REPAIR. 33 and adult rabbits by the addition of tissue-extracts. Similar views as to the lvm- phogenic origin of the macrophages are expressed by other authors. In addition to the lymphocytoid cells, most workers recognize the participation, in the forma- tion of the wandering mononuclear phagocytes of inflammation, of the resting- wandering cells of the tissue-spaces, the endothelial cells, and, in the blood-forming organs, the cells of the reticulum, ete. It is probable, therefore, that while some of the large macrophages of the inflammatory region at the fracture-site come from the reticulum of the marrow and from endothelial cells, and that even more are mustered from the ranks of the resting-wandering cells of the surrounding tissues, yet the vast proportion develop from the small lymphocyte-like cells. Though the lymphocytes of the tissues (histiogenous lymphocytes) doubtless supply some of these, by far the greater proportion probably arises from the lymphocytes of the blood-stream, which have wandered from the vessels into the inflammatory zone (hematogenous lymphocytes). We may summarize the discussion of the source of the cells by saying that at the beginning of the inflammatory process cells of the types above mentioned are already present in the wound-area, and although these function actively, and even increase in effectiveness, they soon become inadequate to the demands made upon them. Cells from the surrounding tissues, too, may be presumed to wander to the inflammatory region; in these we may recognize representatives of the resting- wandering-cell type and also the closely related “histiogenous” lymphocyte. But even these reinforcements are insufficient for the performance of the work, and the vast bulk of the phagocytes, as we have seen, come by way of the blood-stream. Thus the macrophages of the inflamed tissue in the vicinity of wounded bone, though derived from cells of diverse morphological type, are yet united by the possession of a common physiological potentiality which manifests itself in a uni- form response to a common call te arms. This response consists in the metamor- phosis of these cells into enormous and rapacious phagocytes and in the assump- tion by the latter of an important service in the treatment of the waste products occasioned by tissue-injury. This phagocytic response is graphically demonstrated by the trypanophil reaction. We must postulate a progressive adaptation on the part of the cytoplasm of the mobilized cells until there is produced a mechanism of the highest efficiency in the function of phagocytosis. It is a striking fact that the working units of this phagocytic tissue, although derived from different sources, resemble one another so closely that the riper forms are indistinguishable. Indeed, this fact has been commented upon by Tschas- chin (1913), who, speaking of the resemblance of the ‘‘polyblasts’’ derived from resting-wandering cells to those from the lymphocytes, states (p. 388): “gegen Ende des zweiten Tages der Entziindung die Polyblasten nach der Quelle ihrer Entstehung nicht mehr unterschieden werden kénnen.” MOBILIZATION. Why, it may be asked, do the macrophages congregate at the fracture-site? What is the influence which causes a cell situated near the area of inflammation to 34 DEVELOPMENT AND FUNCTION OF MACROPHAGES IN BONE-REPAIR. move toward it, or which brings about the dislodgment of a lymphocyte from its ordinary depot into the blood-stream (for we can hardly regard the blood-stream as normally containing sufficient numbers of these cells to supply the requirements without replenishment)? The specific stimulant is undoubtedly the product of tissue breakdown. It may operate through its chemical properties, in which case its action would be described as a chemiotaxis, or its effect may be due to its pecu- liar physical condition and its influence be more accurately designated as a physi- cotaxis. Be that as it may, it is certain that the cells themselves are in some way specialized to react to this form of stimulation, not only by moving toward the source of the attraction—in the soft parts of the vicinity by amoeboid movement through the tissues, in the blood by movement of lymphocytes from their resting- places into the blood-stream and from this into the inflammatory area—but by developing prodigious phagocytic abilities. In connection with the hematogenous lymphocytes the attraction must be thought of as acting to get the cells from their depots into the blood-stream, where the attracting influence may be assumed to be circulating. But the latter would not control their course in the blood-current, for in this they float passively until they happen to reach the region of inflammation. Here the circulatory conditions favor their arrest, the blood-current in the dilated capillaries being very slow. Diapedesis ensues, and the embryo phagocytes are thus assembled on the field of their opera- tions. The inflammation-area, as it were, “screens out’’ the lymphocytes from the blood, just as, in certain types of septic inflammation, the leucocytes which are brought in the blood to the focus of inflammation are sifted out and retained there by the mechanical and other conditions which they encounter. The interesting fact is noted by Tschaschin and others that the local lym- phoid elements of adenoid-tissue do not normally stain with vital dyes and even in inflammation they are very slow in developing phagocytic power. It appears, however, that if these cells gain entrance into the blood-stream they soon become sensitive and react promptly to the stimulus of inflammation by wandering from the vessels and becoming metamorphosed into typical ‘“‘polyblasts.”’ ACTIVITY. The method of action of the macrophages has long been the subject of much study on the part of eytologists. There is nothing to suggest that these phagocytes actually break down the tissue. It may be that they secrete some enzyme which assists in this process, but no facts were discovered in support of this view. Their function is concerned with the clearing away of the waste products rather than with tissue solution. In the section describing the observations it has been noted that the cells are not typically in direct contact with the tissue being absorbed. More- over, they do not seem to operate merely by engulfing fragments of the moribund tissue, although their ability to act in this way on occasion is not questioned; rather, they are concerned with the imbibition of a colloidal solution of the tissue. In this solution they lie, their outer walls bathed with it. The way in which they absorb the material may be inferred from the way in which they are known to take up col- DEVELOPMENT AND FUNCTION OF MACROPHAGES IN BONE-REPAIR. 30D loidal dyestuffs (Evans and Schulemann, 1914). The ultramicrons of such dyestuffs in some way permeate the cell-membrane and are combined in aggregations which soon become visible by the aid of the higher powers of magnification as multi- tudinous, isolated granules. It appears, too, that often an aggregation of dye- molecules is situated within a vacuole in the cytoplasm. This fact has led some workers to suppose that the cell was attempting to subject the material so housed to a form of digestion in which it would be made useful, or at least harmless, and that its treatment of this colloidal dyestuff was an example of its behavior toward any material in the same physical condition. The same reasoning may be applied to the interpretation of the behavior of the macrophages in fractures, and we may postulate that the colloidal waste products resulting from the breakdown of the tissue are similarly acted upon within the cell economy. This may be a productive mechanism. It is a well-known fact that certain of the products of protein-splitting, if generally scattered throughout the circulation, will bring about great harm. Now it is quite possible that some at least of the products of proteolysis in fractures and other wounds are of this noxious character, and it may well be that the macrophages are called out to form a barrier against the escape of these materials into the general circulation. There is a stasis of fluids in these regions, and thus the conditions are most favorable for phago- cytosis (Downey, 1917). Indeed, in emergencies such as the healing of wounds and clearing away of damaged tissue resulting from gross insults, the macrophages may be considered as expressing in an exaggerated form the same function they express every day under normal routine conditions of metabolism—as in the breaking-down of red blood-cells, or it may be of the protoplasm of muscle-fibers. Here it is note- worthy that Goldmann (1909) finds these cells especially numerous in the heart—a hard-worked muscular organ. Not all tissues, however, are to be looked upon as producing materials which are dealt with in this way; for instance, nerve-tissue contains few or none of these cells (Goldmann, 1909), and hence the functional waste- products of this tissue may be considered as being treated in some other manner. DECLINE. As to the fate of the macrophages of the inflammatory area, it has been noted that, shortly after the disappearance of the tissue-waste, the macrophages, in all the specimens, become less and less evident, finally dwindling to their normal numbers (table 2). The examination of the sections throws some light on the final end of the individual cells. As early as the third day we have noted that certain of the phagocytes had a degenerate appearance, and these were more numerous on the fifth day. It seems evident that some of the cells start to degenerate quite early, even at the stage where new macrophages are developing and where the macrophagic tissue is, on the whole, increasing. On the sixth day the degenerate cells were present in enormous numbers, especially in the young scar- tissue, and it is at this time that most of the phagocytes undergo dissolution. After the first week they disappear more slowly, for but few degenerate forms are noted; indeed none was seen after the twelfth day. 36 DEVELOPMENT AND FUNCTION OF MACROPHAGES IN BONE-REPAIR. The morphology of these degenerate cells has been described in the text (sixth-day stage). It appears that many (probably most) of the phagocytes fall to pieces, their liquefied content of ingested material (which is assumed to be changed in character) being returned to the lymph-spaces, from which it is passed on to the blood-stream, to be excreted or utilized; and any fragments which remain are probably devoured by the tissue-phagocytes. A few macrophages, however, as has been noted, persist on the field after absorbable material has disappeared. In the fractures of the long bones these were observed in numbers more or less above normal as far as the thirtieth day. They haunt the surviving muscle-fibers and new scar-tissue. Their size and staining ability gradually diminish, and they are quite inferior in these respects to the active phagocytes; their numbers soon fall away to normal. It is difficult to say, from the evidence, whether or not these cells ever become transformed into fibroblasts, as Goldmann (1912), Maximow (1902, 1906), and Tschaschin (1913) suggest. The typical fibroblasts plainly are quite different from the typical macrophages, and it seems probable that most, if not all, of the scar-tissue arises independently of the macrophages. In the fifth-day stage, for instance, when there is no perceptible diminution in the number of macrophages, there is much new fibrous material. They, however, give place to scar-tissue, and, in a sense, may be said to prepare the way for the scar by assisting in the removal of waste material (Maximow, 1902). INTRAOSSEOUS MACROPHAGES. The second outstanding fact brought to light by the study of vitally stained, healing bone-wounds is that the reticulo-endothelial cells of the callus-spaces develop marked phagocytic power coincidently with the appearance of the erosive processes concerned in the enlargement of these spaces; the intensity of this power, too, seems to be roughly proportional to the amount of the callus breakdown. There is, indeed, a most obvious parallelism in the curves tracing the degree of activity of callus destruction, on the one hand, and the degree of phagocytic efficiency of the macrophagie reticulum-tissue on the other, which is consistently maintained throughout the entire history of the callus. From a review of the findings in the callus up to the sixtieth day it is possible to divide the life of its macrophagie tissue, like that in the degenerate soft parts, into the three phases: development, activity, and decline. Although these phases merge gradually into one another, yet arbitrary limits may be set for them, that of development covering the first nine days, that of activity, roughly, the period from the tenth to the twentieth day inclusive, and that of decline the remaining time. Having made this division, it is a simple matter to recount the most important features of each phase. DEVELOPMENT, Upon referring to the records it will be noted that the callus rapidly develops, following its first indication on the second day, and by the sixth day is quite well marked. In this callus, spaces filled with cells (which are derived apparently from ———- =. DEVELOPMENT AND FUNCTION OF MACROPHAGES IN BONE-REPAIR. 37 the tissue of the Haversian canals of the old bone) early make their appearance. First seen on the third day, they soon expand, the largest spaces naturally occupying the oldest part of the callus, situated, of course, in the vicinity of the original bone. With the hollowing-out of these spaces numbers of the reticulum cells con- tained in them gradually acquire the power to ingest and store colloidal dyestuffs. Faintly stained cells were found in the spaces in the third-day stage. This mani- festation of phagocytic activity on the part of the cells of the reticulum is intimately associated with the breaking down of tissue consequent upon the excavation of the callus spaces. It is noteworthy that the brightest staining occurs in the cells of the largest spaces, and especially in those at the edge of the old bone—an indication that the greatest phagocytic activity is resident in areas of greatest tissue destruction. As new spaces open out, trypanophilic cells, at first very weakly stained, appear in them, so that the number of these cells gradually increases, keeping pace with the growing volume of the callus recesses. Cell multiplication is by mitosis, as is proved by finding karyokinetic figures among them, even in dye-containing cells. Also, with advance in age of the reticulum cells, there is usually a slight concomitant progressive increase in their phagocytic potentiality which goes hand in hand with the gradually accelerated callus erosion; for, in the fifth and sixth- day stages, the cells in the older and larger spaces are somewhat more brightly stained than corresponding cells of earlier periods. No noteworthy increase in size of these phagocytesisso far apparent. Thin-walled blood-sinuses of large size appear in the callus-spaces of this period, and are a marked feature. A favorite position of the macrophages is between the walls of these vessels and the osseous plates. In no area of the callus during this period is the staining phenomenon of the reticulum cells at all comparable with that of the extraosseous macrophages; indeed, the reticulum cells are quite small and inconspicuously stained as compared with the deeply dyed “polyblasts” of degenerating soft tissue, so that it seems more rational to look upon them as cells whose powers are as yet potential rather than actively functioning—as elements, indeed, capable of developing very efficient phagocytic ability on short notice. Though their service in phagocytizing the products of tissue breakdown during this early stage is recognized, it is felt that the amount of colloidal waste resulting from callus erosion can not be very great, since the total callus destruction is as yet small. The main feature of the callus during this phase is construction (rather than destruction) of osseous tissue. ~ ACTIVITY. The history of the callus during the period from the tenth to the twentieth day inclusive is of the greatest interest. The first part of this interval is charac- terized by both development and destruction of bone, for as the callus expands in its more outlying regions the older parts are worn away, as shown by the progressively widening spaces. Gradually peripheral growth declines, it being practically non- existent by the twentieth day, but very active destruction of the trabecule is maintained throughout. During the first few days the area of most active bone destruction is in the older portions of the callus around the original bone, but as 3s DEVELOPMENT AND FUNCTION OF MACROPHAGES IN BONE-REPAIR. the tissue here is eroded this active zone shifts outward and at the fifteenth day it occupies the interior of the mass. From here the zone advances, in turn, to the periphery, and the redundant material is cleared away in that region. Certain areas of bone, necessary for the stability of the callus, are conserved, and these are thoroughly reinforced through the application of layer upon layer of bone by the activities of the osteoblasts. During this period the intraosseous phagocytes, as demonstrated by vital- staining, are exceedingly striking. In regions where obviously bone erosion is proceeding most vigorously they now devour the dyestuff much more greedily and store it in the form of larger and more numerous granules. In their more’ intense staining they form a striking contrast to the cells of the developmental phase. Thus, in the earlier stages of this period, at the tenth and twelfth days, the largest macrophages are found crowded in the spaces near the original bone; on the fifteenth day they have shifted to the interior of the callus, and on the twentieth day (keeping pace with the outward movement of the zone of most active bone destruction) they have again shifted their ranks to the peripheral regions of the callus, the more central areas containing relatively few of them. This localization of the demolition-zones is not absolute, for (especially in the later stages) detachments of hypertrophied macrophages may be found in nooks and corners throughout the callus wherever bone is being actively resorbed. Again, in some of the ribs (perhaps because there was less movement of the fragments) the callus was less and its removal was accomplished apparently by a process of paring down from the periphery, the bone becoming more and more slender, as the figures of cleared ribs indicate. It is quite obvious that concentration and hyper- trophy of macrophagic tissue are inseparably linked with active bone erosion. As time goes on, the macrophages of the reticulum undergo, in the areas of active bone destruction, a certain amount of progressive enlargement, the largest cells having an average long diameter of 7.6 u, 9.15 uw, 9.95 uw, and 12.6 uw on the tenth, twelfth, fifteenth, and twentieth day respectively. With this hypertrophy there is some increase in the amount of the dyestuff stored, indicating an exalta- tion of phagocytic power. Mitotic figures in reticulum cells, many of which contained dye, were found throughout this period of activity. A few smaller and less brilliantly stained reticulum cells are found in all parts of the callus tissue. The manner of action of the reticulum macrophages presents some features of interest. There is nothing to support the idea that they carry on, contribute to, or even initiate the actual process of callus destruction. Their position in the reticulum—never in actual contact with the bony strueture—does not, to say the least, lend support to any such hypothesis; nor is there any evidence pointing to the elaboration, by these cells, of a seeretion—such as an acid or a proteolytic enzyme—which would act in the liquefaction of the callus. On the other hand, there is positive evidence of the most convincing kind—the avidity with which these cells ingest colloidal dyestuffs—that they play the réle of phagocytes; like the polyblasts of degenerating soft tissues, or the reticulo-endothelial cells of developing bone, they ingest the products of tissue breakdown. ——— eC DEVELOPMENT AND FUNCTION OF MACROPHAGES IN BONE-REPAIR. 39 The particular tissue-destruction with which the reticulum macrophages have to do is that of callus. In this destructive process we recognize two aspects: (1) the removal of the bone-salts; (2) the removal of the matrix with its contained cells. It is becoming more and more clear that the process of removal of the bone- salts from the matrix is simply the reverse of that of their deposition in ossification: that we have here to do with the reverse phase of a chemico-physical reaction whose direction is determined solely by the conditions of the immediate environment. Under certain circumstances, apparently centering around definite and well-ordered changes in the local blood-vascular system, there are precipitated from the circu- lating fluids into a special matrix (elaborated by the activities of definite specialized cells, the osteoblasts) certain insoluble building-materials, the bone-salts. These consist mainly of calcium phosphate and calcium carbonate, their quantitative relations being determined by their relative solubilities in the blood-plasma. There is here simply a cell-controlled calcification (Wells, 1911; Macklin, 1917). In deossification, or more accurately decalcification, the reverse process is encountered. Changes, particularly in the circulating fluids, cause the bone-salts to be released from the matrix and again taken up into the blood; the matrix and bone-cells remain; the former is liquefied, by means at present obscure. As to the latter, there is ground for the view that they are sometimes left in heaps like drift- wood; that they even coalesce to form giant-cells (Arey, 1917). It is probable that many are disintegrated, to swell the volume of the liquefied waste-products. That the liquefied bone-salts are phagocytized by the macrophages seems doubtful. They are non-toxic and probably pass off into the circulating blood in a manner the reverse of their incoming. It does seem probable, however, that the phagocytes ingest some, at least, of the products resulting from liquefaction of the protein-content of the callus. Their function would thus be closely allied with that assumed for the wandering macrophages of the degenerating extraosseous tissue. Here, too, it may be postulated that their service is protective; that they guard the organism from the harmful effects of toxic, nitrogen-containing compounds resulting from proteolysis. It is quite possible that only certain of the compounds arising from tissue breakdown are poisonous, and that only these are ingested. As in the ease of the polyblasts, it may be assumed that the materials so phagocytized are digested and rendered innocuous—or even useful—within the cytoplasmic laboratory of the macrophages. It has been noted that the treatment by the macrophages of high-molecular dyestuffs, such as trypan-blue, is an expression of their general behavior toward any material in the same physical condition; and it has been inferred, therefore, by Shipley and Macklin (19167), that the material resulting from the erosion of pro- visional cartilage and bone, which is phagocytized by the macrophages, is in a finely dispersed state. Such an inference may also be made for the waste products resulting from the erosion of callus or from the breakdown of the soft tissues around the wounded bone. The position of the macrophages of the demolition zones in the loose reticulum of the spaces is favorable to the exercise of their function, for they, like the poly- 40 DEVELOPMENT AND FUNCTION OF MACROPHAGES IN BONE-REPAITR. blasts of moribund tissue, are bathed by fluid containing colloidal waste products. Especially in the perisinusoidal spaces (where, as has been many times observed, the phagocytes are often thickly crowded) are they well situated to gather in waste products; for here not only do they have access to the materials coming directly from the dissolving tissues, but, owing to the thinness of the vessel-walls and the slowness of the circulation, diffusion of katabolites from the sinuses into the spaces may easily occur, so that these macrophages are in a position to gain some of their pabulum from the blood-stream. In any event, toxic materials in the blood- sinuses are readily extracted by the phagocytic endothelial cells. The enormous thin-walled blood-sinuses of the callus, which have been repeat- edly referred to, are a conspicuous feature of areas of active bone-demolition. They arise, as has been shown, through coalescence of smaller vessels. So striking are they on account of their enormous size and great number, as well as because they are invariably present in areas of active callus destruction, that the conclusion is forced upon us that they must play an important part in the breaking-down and removal of the redundant osseous tissue. Certain it is that the rate of flow in these vessels is very slow and that, judging from the thinness and insecurity of their walls, the pressure is very low. It is probable, too, that the CO, tension is high. It is significant that a very voluminous blood-current of slow speed and low pressure, flowing through a thin-walled channel, enveloped thickly with phagocytes of high-grade efficiency, is so constant a feature of callus resorption, as it is also — of the resorption of young developing bone. It suggests that this is part, at least, of the mechanism of bone erosion. DECLINE. During the third phase, after the twentieth day, osseous resorption gradually ebbs and the destructive and constructive activities of the callus slowly subside. Reinforcement of the permanent trabeculz is the principal industry of the cells, but, especially in the earlier days, evidence of the work of the wrecking-gang is still seen in the clearing away of the few remaining provisional spicules of bone and the trimming of the rough corners. The result is a firm osseous structure formed of material almost indistinguishable from the bone of the original shaft, containing relatively few (but very large) spaces filled with vascular and marrow tissue. Thisbony formation occupies the fracture-site and thoroughly immobilizes the shaft of the bone. In keeping with this falling away of destructive processes the macrophages gradually decline in size, concentration, and staining activity, and revert to the character of the ordinary bone-marrow reticulum cells. This does not take place uniformly in all stages, however, for in the specimens of the sixtieth day there was still evidence of bone erosion as well as of bone building, whereas in those of the fifty-first and fifty-ninth days these processes had apparently completely ceased. Involution and degenerate forms, like those of the macrophages of the soft parts, were not found in the callus-spaces. Cartilage was found almost constantly in the callus of the long bones and seems to be associated with movement of the parts during repair, since they were not splinted. It was not found in any of the skulls, where movement was absent. In DEVELOPMENT AND FUNCTION OF MACROPHAGES IN BONE-REPAIR. 41 the older stages the cartilage underwent changes similar to those of ossification of cartilage in normal skeletal development. A case was found where the cartilage had even taken all the characters of a typical epiphysial plate. Associated with the process of destruction of the cartilage, and of the trabecul of bone built upon the calcified remnants, macrophages played the same part as in the normal endo- chondral ossification, as shown by Shipley and Macklin (19162). Giant-cells or “osteoclasts” were found not infrequently in the callus of some of the specimens, and rarely in others. Their numbers seemed to have no relation to the amount of bone destruction; thus none was observed on the tenth day, a few on the twelfth, they were fairly numerous on the fifteenth, but scarce on the twentieth day. In all of these stages there was undoubtedly a great deal of bone erosion progressing. Again, at the same stage but in different specimens, they were inconstant in number; thus in one of the specimens of the six-day stage there were very few, while in the other specimen a fair number was found. In no case was there a large enough number, nor were the cells sufficiently well distributed, to warrant regarding them as the agents of bone erosion. Careful search was made for dye-granules in them, but not a trace was to be found; hence they are not phago- cytes of the type of the macrophages. Indeed, no ingested material of any kind could be found in them. Thus the giant-cells of the callus and the underlying old bone in process of erosion are in these respects quite the same as the giant-cells of developing bone described by Shipley and Macklin (1916°). COMPARISON OF EXTRAOSSEOUS AND INTRAOSSEOUS MACROPHAGES. It is of interest to compare briefly the macrophagic cells of the callus with those of degenerating extraosseous tissue. They have many points of difference. The callus cells are fixed and belong to the reticulum tissue, whereas the cells of the soft parts are mostly wandering and are derived from the “‘resting-wandering cells” of the tissues and from certain lymphocyte-like cells brought in the blood-stream. It is probable, however, that cells originating from the reticulum and endothelium of the bone-marrow of the broken bone-edges contribute to the forces of the extra- osseous macrophages in their vicinity. The callus cells develop in situ, whereas the cells of the soft parts begin their development probably for the most part out- side the zone of their operations; however, both no doubt undergo their hypertrophy mainly at the site of tissue-breakdown. The callus-cells multiply in situ by mitosis (typical figures being found in dye-containing cells as early as the sixth day and as late as the twentieth), whereas most of the cells of the soft parts undergo multipli- cation at their source. The largest of the extraosseous macrophages are of greater size than the phagocytes of the callus, and usually contain more dyestuff, their granules, as a rule, exceeding in magnitude those of the callus-cells. It seems probable that the demands upon the macrophages of the soft parts are much heavier than those which the callus-phagocytes are called upon to meet, the waste material being much greater in the damaged soft parts than in the resoly- ing callus. Hence the cells of the callus never reach the physical proportions nor the great numbers of the cells of the soft parts. The effort of the extraosseous cells 12 DEVELOPMENT AND FUNCTION OF MACROPHAGES IN BONE-REPAIR. is sudden, vigorous, and of short duration, their period of maximum efficiency being from the third to the sixth days inclusive; whereas the effort of the intraosseous elements begins later, is more gradual in onset and less violent, and is of longer duration, the period of maximum efficiency of these cells ranging from the tenth to the twentieth days. Both types, however, are apparently stimulated to hyper- trophy and functional efficiency by the same type of material—waste resulting from proteolysis; but the call is apparently much more sudden, forceful, and per- emptory in the soft parts than in the callus, so that here an expeditionary force of potential macrophages from distant regions must be rushed in. The needs of the callus, on the other hand, in the matter of its resorption, are apparently served adequately by the exaltation of the powers of the resident phagocytes. Both macrophagie types are concerned in like manner in the treatment of dissolved tissue-waste, and they are thus physiologically similar. In both types phagocytic activity has been developed coincidently with the occurrence of tissue breakdown. Both have a reconstructive as well as a scavenger function, for they prepare the ground for scar-tissue or permanent callus, as the case may be. Most of the cells of the soft parts perish in situ, while the fate of the callus-cells is obscure; no involution forms, however, were found among them. Macrophages were found in healing wounds of membrane-bone, not only in the surrounding soft parts but in the spaces of the callus itself. Here the cells were of the reticulum type and were related to blood-sinuses as in the long bones. The callus of membrane-bone was rather slower in development and was relatively small in volume, so that the pictures presented by callus in the trephine-wounds of the skull were not so striking and instructive as those in the long bones. They were useful, however, in confirming the findings in the long bones. It is worthy of emphasis that the method of vital-staining, as applied to developing callus on the one hand and to developing bone on the other, demonstrates very forcibly that the same cellular elements are at work in the performance of a like task. Macrophages of identical morphological type are found in mass forma- tion in each case, intimately related to areas of bone or cartilage resorption, and for them a common physiological significance is claimed—that of phagocytizing the products of disintegration of provisional cartilage and bone. More than this, there was found absolutely no trace (in the osteoclasts of either callus or developing bone) of any phagocytic activity. Thus the conclusions of Shipley and Macklin (1916*) in regard to developing bone are upheld by the findings in eallus. From an examination of all the cleared and sectioned skulls it may be said that no difference can be discerned in the staining reactions consequent upon putting the insert back right side up, or upside down, or inserting living or dead bone from another rat, or even foreign dead bone. Thus the macrophages, as far as could be made out, behave alike toward all these types of insert. In no case was the insert removed, although the edges were trimmed and rounded off; and, although here and there in some of the older skulls there was some slight evidence of erosion in the inserts and in the surrounding bony edges, it is remarkable how little of the insert disappears. The absence of an increased macrophagic tissue, DEVELOPMENT AND FUNCTION OF MACROPHAGES IN BONE-REPAIR. 43 after the débris has been cleared up, is what would be expected, since so little of the insert is eroded. A note may here be made as to the condition of the healing areas of skin over the trephined bones. Several of these were cleared and some were sectioned. It was found that macrophages are increased in certain places, especially around the suture holes, in the earlier stages. This was noticed in the skulls of the following days: second (818-1), ninth (S 17-2), tenth (S 5-1), and twentieth (S 12-2). Thus the macrophages appear to have a function to perform in the solution of the sutures and probably in the repulsion of infection as well. In the actual scar-tissue the trypanophil phagocytes are few in number. It is of interest to observe that Gold- mann (1912, p. 80) noted a similar increase of macrophages in healing skin wounds of the rat. CONCLUSIONS. The following conclusions are based solely upon investigations with the rat. It seems probable, however, that they would hold good in general principle for the other mammals, and no doubt for many of the lower forms. In the healing of bone-wounds, macrophages, which stain brilliantly with trypan-blue, soon congregate at the site of the injury and become very numerous, hypertrophied, and of increased phagocytic power. They assist in dealing with the tissue-waste resulting from the trauma. These phagocytes are developed prin- cipally from the lymphocyte-like cells from the blood-stream, but also from local mononuclear cells with phagocytic potentialities. Most of them ultimately dis- integrate in situ. During the structural changes attending the transformation of provisional into permanent callus, trypanophilic macrophages develop in the callus-spaces from the reticulum cells and become numerous, large, and phagocytic. They function in the removal of redundant bony spicules, their particular réle being con- cerned with the absorption of the waste products from the breaking down of the matrix. When cartilage is present in the callus they also play a part in its removal. Their action here is thus the same as that of the macrophages of developing bone. The macrophages of soft parts and bone, though morphologically different, are physiologically similar. They phagocytize the products of proteolysis and segregate the material within their cytoplasm, where they probably subject it to a form of digestion. Limited numbers of polymorphonuclear leucocytes were encountered among trypanophilic macrophages in areas undergoing repair. No dye-granules were found in them. Physiologically they are distinct from the macrophages. The osteoclasts of the callus did not show dye-granules. Their numbers did not bear any relation to the apparent amount of bone-destruction which was going on. Osteoblasts, too, contained no dye-granules. BIBLIOGRAPHY. B., 1917. On the origin and fate of the osteo- clasts. Anat. Record, vol. 11, p. 319. Downey, H., 1917. Reactions of blood and tissue cells to acid colloidal dyes under experimental condi- tions. Anat. Record, vol. 12, p. 429. Evans, H. M., and W. Scuutemann, 1914. The action of vital stains belonging to the benzidine group. Science, n. s., vol. 39, p. 443. Evans, H. M., F. B. Bowman, and M. C. WInTERNITz, 1914. An experimental study of the histogenesis of the miliary tubercle in vitally stained rabbits. Jour. Exper. Med., vol. 19, p. 283. Evans, H. M., 1915. The macrophages of mammals. Amer. Jour. Physiol., vol. 37, p. 243. Gotpmann, E. E., 1909. Die fiussere und innere Sekre- tion des gesunden Organismus im Lichte der vitalen Farbung. Beitr. z. klin. Chir., Bd. 64, p. 192. Gotpmany, E. E., 1912. Neue Untersuchungen iiber die fiussere und innere Sekretion des gesunden und kranken Organismus im Lichte der vitalen Far- bung. Beitr. z. klin. Chir., Bd. 78, t. 2, p. 1. MacCorpy, J. T.,and H. M. Evans, 1912. Experi- mentelle Lisionen des Centralnervensystems, untersucht mit Hilfe der vitalen Farbung. Berl. klin. Wehnschr., vol. 49, p. 1695. Mackuin, C. C., 1917. Studies in calcification by the use of vital dyes. Jour. Med. Research, vol. 36, p. 493. Maxrwow, A., 1902. Experimentelle Untersuchungen iiber die entziindliche Neubildung von Binde- gewebe. Beitr. z. pathol. Anat. u. z. allg. Pathol., Bd. 5, Suppl. p. 1. ——— 1906. Uber die Zellformen des lockeren Binde- gewebes. Arch. f. mikr. Anat., Bd. 67, p. 680. Angy, L. 44 Maximow, A., 1907. Uber die Entwicklung der Blut. und Bindegewebszellen beim Siugetierembryo- Folia Haematol., Bd. 4, p. 611. ——— 1909'. Untersuchungen iiber Blut und Binde- gewebe. I. Die frithesten Entwicklungsstadien der Blut- und Bindegewebszellen beim Sauge- tierembryo bis zum Anfang der Blutbildung in der Leber. Arch. f. mikr. Anat., Bd. 73, p. 444. 1909*. Die Histiozenese der Entziindung. Ver- handl. d. 16. Intern. Med. Kongr. Budapest 1909, Sekt. 4b. (cited by Tschaschin, 1913). 1916. The cultivation of connective tissue of adult mammals in vitro. Arch. Russes d’Anat., d’Histol. et d’Embryol., t. 1, fase. 1 (cited by Downey, 1917). Ranvirr, L. A., 1899-1900. Des clasmatocytes. Arch. d’Anat. mier., t. 3, p. 122. Spavrenoiz, W., 1914. Uber das Durchsichtigmachen von menschlichen und tierischen Priparaten. Leipzig. Surrey, P. G., and C. C. Mackin, 1916". The demon- stration of centers of osteoblastic activity by use of vital dyes of the benzidine series. Anat. Record, vol. 10, p. 597. Sarpcey, P. G., and C. C. Mack.in, 19162. Some features of osteogenesis in the light of vital staining- Amer. Jour. Physiol., vol. 42, p. 117. Tscuascam, S., 1913. Uber die “ruhenden Wander. zellen”’ und ihre Bezeihungen zu den anderen Zellformen des Bindegewebes und zu den Lym- phozyten. Folia Haematol., Bd. 17, p. 317. Wetts, H. G., 1911. Caleification and ossification Arch. Int. Med., vol. 7, p. 721; also Harvey Lectures, 1910-1911. DESCRIPTION OF FIGURES. B, original bone. M, macrophage. N, nerve. BS, blood-sinus. MC, marrow-cavity. P, periosteum. C, callus. Mus., muscle. S, sear-tissue. Puate 1. Fic. 1. Portion of normal rib from rat vitally stained with trypan-blue. The black dots represent macrophages Fic. Fia. Fia. Fic. Fic. Fic. Fic. Fic. Fic. Fia. Fic. normally present in the marrow-cavity, periosteum, and tissue around the bone. % 27. This and the next five figures are free-hand drawings from gross cleared preparations under the binocular microscope. The macrophages are represented somewhat larger than they actually are, for the sake of plainness. 2. Fractured rib of rat on second day of repair. Note the débris around the ends of bone and the slight increase in macrophages as compared with figure 1. 27. 3. Fractured rib of rat on third day of repair. Great increase in macrophages is seen, with swelling due to young callus. Diffusely stained débris present. > 27. 4. eS rib of rat on ninth day of repair. Macrophages somewhat less numerous. Callus much more ense. X 27. 5. Fractured rib of rat on twentieth day of repair. The contour of bone is almost normal, the medullary cavity is being restored, and macrophages are but little in excess over the normal. X 27. PLATE 2. 6. Trephined skull of rat on ninth day of repair. A bone-disk has been inserted on the left side, while the right has been left open. Note the crowds of macrophages in the open spaces and around the bone edges. Here and there are areas almost free from phagocytes; these are patches of scar tissue. Early callus spicules line the bone edges. X 19. 7. Photomicrograph of area near broken end of long bone from rat on third day of repair. The edge of Lone (B) and the fragments of dead tissue are stained blue. Myriads of macrophages (M) are found through- out the figure. x 40. 8. A few cells selected from specimen from which figure 7 was made, showing the development of the large macrophages from the small lymphocyte-like cells. As a rule, more and more dyestuff is taken up with increase in size of the cell. Besides the dye the cells also contain the tissue-waste which they have phagocytized. Free-hand drawing. X 1,000. PLATE 3. 9. Photomicrographs from a cleared and uncounterstained section of the sixth-day stage in fracture-repair. Exactly the same field is seen in the two pictures, but that on the left received a much longer exposure, so that practically the only objects seen in it are the macrophages. These are larger and more thickly distributed in the central and lower right regions. Here the greatest amount of diffusely stained tissue- waste is present, as is seen by the denser staining in this region in the right-hand picture. The field is from an area of degenerating muscle. Above and to the right is scar-tissue (S), while below and to the left the muscle is beginning to deteriorate. In the central and lower right areas the muscle is degen- erate, and here the macrophages (M) are largest and most numerous. X 50. 10. High-power drawing (camera lucida) of area of degenerating muscle from a region similar to that shown in figure 9 (M). Fragmented muscle-fibers are seen, together with young fibroblasts, polymorphonu- clear leucocytes, and macrophages. Three small lymphocytoid cells appear. Some of the macro- phages contain more blue dye than others; the latter are usually stuffed full of phagocytized material. One small macrophage has engulfed a polymorph. > 1,000. 11. A few degenerate macrophages from an area of scar-tissue, similar to that shown in figure 9 (S). They are of different sizes. Some appear vacuolate and ragged; others are mere fragments. They contain comparatively little dye. Some young fibroblasts are shown. X 1,000. 12. A portion of a callus-space from the sixth-day stage. A thin-walled blood-sinus (BS) is conspicuous, and between its walls and the bone (C) are situated reticulum cells, some of which contain dyestuff (7) and are thus phagocytic. X 1,000. . 13. Three reticulum phagocytes from the callus of the sixth-day stage, showing mitosis in dye-containing cells. There are two metaphases and an anaphase. > 1,000. PuaTe 4. . 14. Old bone (B) and callus from the tibia of the tenth day. Camera-lucida drawing, from cleared section. The spaces near the original bone are filled with the largest and brightest macrophages (M). Under high magnification these large phagocytes are shown in figure 15 a. Here, too, there are blood-sinuses. Farther out the macrophages are less conspicuous and are absent at the periphery. > 80. 45 Fic Fic Fic _15. The groups a, b, c, and d (see plates) were drawn with the aid of the camera lucida from typical large 16. A space in callus, showing developing macrophages of the reticulum (M). Camera-lucida drawing from _17. Drawing made from a tracing from a photomicrograph of the twentieth-day stage of repair. Section cl _ 18. A small field from the callus of figure 17, much enlarged. Drawing traced from a photomicrograph. Thro DESCRIPTION OF FIGURES. : b ra : Piate 4—Continued. =! Fy Saha gocytie reticulum cells selected from the callus spaces of the tenth, twelfth, fifteenth and twe1 days respectively. The sections were cleared without counterstaining and the dye-granule contour only is represented. In the interior of the cell the nucleus is indicated by the clear space. Only mat ; phagoeytes were selected in each stage. They resemble one another closely and show a certain amount of increase in size with age of callus. In each case the drawings were made from cells situated in areas where active osseous resorption was going on. XX 1,000. arr. peripheral region of callus at the twelfth day of repair in long bone. Some of the cells are clear, while others contain varying amounts of dyestuff. The granules are at first small, but gradually increase in size and number. Large intercellular spaces will be noticed. Blood vessels are not shown in this figure, but in the older spaces at this stage they were large and abundant. 1,000. he without counterstaining. In the lower left corner the old bone and marrow cavity is seen. Aro’ it the callus is arranged,and in many of its spaces there are crowds of black granules, represen macrophages of the reticulum. X 50. of macrophages (M), heavily loaded with dye-granules, present themselves between the osseous beculae (C). These cells are seen more highly magnified in figure 15d. X 190. * PLATE 1 MACKLIN B. E. Stocking feci ia MACKLIN a ‘* a MACKLIN ” ao M Macklin fecit ‘ ’ : : - Bi fe . 7 E : o ‘ = <° a he Ve ‘ MACKLIN 15 tin fecit Mack 17 CONTRIBUTIONS TO EMBRYOLOGY, No. 28. CYTOPLASMIC STRUCTURES IN THE SEMINAL EPITHELIUM : OF THE OPOSSUM. . By J. DursBere, Adjunct Professor of Anatomy, Faculty of Medicine, University of Liége, Belgium, Research Associate, Department of Embryology, Carnegie Institution of Washington. With two plates and five text-figures. 47 CONTENTS. PAGE Material. oic..:5.56. 6c, arcic.es ls che ered estat ra lees aE ACTS ACR tae ee 49 Technique. <..,...:0:: «six ss /ssaveisynyaos © so yals e olnne ls eee Clee leer Eee 50 Short survey of the process of spermatogenesis. ..................0.-- 51 First panied s goicsiys). caccrcpacc onap go sae ee WERE ln Serres ee eee 55 Second periods ini. < tho ks sloe dis file hae od ot ceca ace oma 56 Third period ...'2 Jissis a. cacanae Dae dee eee eee 59 Fourth period. $5. .5402a.85o: i tap tactile ocanio ar emieiete 60 Chondriosomes’ 5)005:5si54,0.59- S29 tee see ene ORR C Oe COC E Eee 62 Sertoliicells). <6 cicic ic diuk, cae acacien se ade roe CES ee eto 63 SPeLMAbOPONIA. 5... 5:o.4,:si5:0'e 5:10 ete seaseppy aves sist ciemsterede Csae ere aetna eae 63 Spermatocytes....< <5... sd Hees kee eres eee eiseieteashe a erere ate 63 Spermiogenesis.-.< «... ....-,-/-is0 ais: s aaless/cepaep eles tale Ree ieee eee 64 First Period... sc chess cree cies Bie terete tiene ee ae eee eet 64 Second period... ys xts.2. ASSIS. che SEIT, oe ee eee oe 65 Third period... :<,2:c:00'22' son ee Pe ER EO OEE eee 65 Fourth period ; « «.%,,