Fate of Osteocytes in Adult Mouse Whole Bone Isografts and Homografts' KARL K. NISHIMURA: JAMES A. YAEGER3 AND TAWFIK Y. SABET Department of Histology, University of Illinois at the Medical Center and Department o f Orthopedic Surgery, Presbyterian-St. Luke's Hospital, Chicago, Illinois It is well established that fresh bone autografts or isografts are more successful than homografts (Herndon and Chase, '54; Curtiss, Chase and Herndon, '56; Anderson, Dingwall, Schmidt, Le Cocq and Clawson, '61; Sabet, Hidvegi and Ray, '61b). Autografts and isografts vascularize more completely, show continued growth and differentiation of young or embryonic bone with generous new bone formation, and are rapidly and more completely incorporated within the host (Ray, Degge, Gloyd and Mooney, '52; Chase and Herndon, '55; Siffert, '55; Felts, '57; Heslop, Zeiss and Nisbet, '60). In most of these studies a great proportion of the cellular elements was found to survive autotransplantation for a long period of time. Fresh bone homografts, on the other hand, evoke an immune host response (Campbell, Brower, MacFadden, Payne and Doherty, '53; Bonfiglio, Jeter and Smith, '55; Chalmers, '59; Sabet et al., '61b). This reaction is generally characterized by inferior vascularization of the recipient site, prolonged cellular reaction with round cell infiltration, fibrous encapsulation, sporadic and scanty osteogenesis, and progressive degeneration with final absorption or rejection of the transplant (Chalmers, '59; Heslop et al., '60; Anderson, '61; Sabet et al., '61b, '62). Since the failure of most osteocytes to survive homotransplantation was constantly associated with the inferior results obtained, one may conclude that there is a relation between cell survival and the homograft reaction. One previous comprehensive quantitation of osteocyte survival in bone transplantation has been reported. Although this work differed from the present study in the type of animal used, size and preparation of the bone transplant, age, and method of transplantation, it represents the only comparable study of which we are aware. Heslop et al. ('60) reported on osteocyte survival, osteogenesis, and the host reaction in a study comparing autologous and homologous bone transplants. Tibial segments were transplanted subcutaneously into young adult rats. The surviving osteocytes in a portion of the tubular cortex were counted and tabulated as a percentage of lacunae counted. They noted a band of osteocytes near the outer surface of the tubular cortex surviving up to 250 days in both experimental groups. The autografts showed a higher proportion of surviving cells than the homografts, with a significant difference being established by 35 days. The purpose of the present investigation was to determine the proportion of osteocytes persisting in the diaphyses of adult whole bone isografts and homografts. Differences in the survival levels of the two experimental groups might provide a quantitative index of the effects of the host-transplant reaction. Also, the study was designed to investigate the fate of osteocytes in different locations within the diaphysis to determine if the reaction was uniform throughout. MATERIALS AND METHOD The third metatarsals of inbred Strong A mice, six to eight months old, of both sexes, were used as grafts. They were 1 This investigation was supported by P.H.S. Research Grants A-3220 and A-1339 from. the National Institutes of Health. From a dissertatlon submitted by Karl K. Nishimura in partial fulfillment of the requirements for the M.S. Degree III the Graduate College, The University of Ilhnois at the Medical Center. 2 Supported by a Fund for Dental Education Fellow- shin. ----< 3 U.S.P.H.S. Research Career Development Award Recipient. 85 86 KARL K. NISHIMURA, JAMES A. YAEGER AND T A W F I K Y. SABET transplanted subcutaneously into transparent chambers (Sabet, Hidvegi and Ray, '61a) previously placed in six to eight month old inbred male mice of Strong A (as isografts) and CBA (as homografts) strains. Host animals were sacrificed at intervals of 10,20, 30,45, and 60 days post-grafting, and the grafts dissected out. A total of 114 specimens were available for this study. The grafts were ~ e indneutral formalin, decalcified with EDTA at pH 5.4 and imbedded in celloidin. Longitudinal serial sections were cut, and stained with hematoxylin and eosin. From each specimen, two to six sections were obtained. Generally, only one sec- I I I I PROXIY4L I tion extending the entire length of the diaphysis and representing the mid-longitudinal axis was selected for counting. Where it was not possible to obtain a well oriented section, two to three slides of the same specimen were used for complete coverage of the diaphysis. The number of specimens counted is indicated in table 1. The relative number of persisting osteocytes was determined by counting under high power (X430) the areas of the diaphysis illustrated in figures l-a and l-b. Micrometer and Howard discs were used to demarcate the circumferential strata in each high power field. Corresponding CLYTR4L I I I DISTAL I I I A Diaphyssal Divisions PROXIMAL ......................P E R I O S T L A L ....................................................................... MID -CORTI C A L ....... ....................................................................................................... MCOULLARY MARROW CAVITY ..................................................................................................................... ..................................................................................................................... B Circumferential Strata Figure 1 a Outline drawing of a longitudinal section of a metatarsal indicating the longitudinal divisions. b Square from proximal division of figure 1-a enlarged, indicating circumferential strata. 87 OSTEOCYTES IN BONE TRANSPLANTS areas on either side of the marrow cavity were alternated for counting, several fields being required. The distribution of cells was not uniform throughout, and in many instances three to five fields were necessary to fulfill the quota for the periosteal and medullary strata. Counting was restricted to the lacunae of the orginal transplant and excluded those of any new bone formed after transplantation. Seventy-five lacunae were counted in each longitudinal division (proximal, central, and distal), 25 for each circumferential stratum (periosteal, mid-cortical, and medullary) in a division. The counts were also tabulated to indicate totals of 75 lacunae for each complete circumferential stratum. Thus, a grand total of 225 cells was counted per specimen. The data were expressed as a ratio of persisting osteocytes to the total number of lacunae counted (75 or 225). 0 I I I 10 20 The identification of persisting osteocytes was based on the presence of a purple-staining, intact, plump nuclear mass within the lacunae. Empty lacunae and those containing fragmented or obviously shrunken nuclei were counted as dead osteocytes. The great majority of persisting osteocytes exhibited normal morphology with purple-stained intact nuclei and distinct nuclear granules. However, it was difficult to distinguish between a normal nucleus and one that had reached the early stage of pycnosis. Accordingly, moderately pycnotic nuclei were probably tabulated as persisting in this study (fig. 5 ) . RESULTS The detailed description of the histological and vascular behavior of the grafts will be the subject of a later publication. In summary, the onset and pattern of vascularization appeared to be similar for both groups. Initial vascularization ap- I 30 I I I 40 50 60 Doys Fig. 2 Osteocytes persisting in whole isografts and homografts. Mean f 1 Standard Deviation. 88 KARL K. NISHIMURA, JAMES A. YAEGER AND TAWFIK Y. SABET TABLE 1 Osteocytes p e d s t i n g in entire diaphyses Ftsf Number of specimens counted grafting Experimental group 10 Isograft Homograft 7 20 Isograft Homograft 4 30 Isograft Homograf t 4 45 Isograft Homograf t 4 60 Isograft Homograft 2 0 Control Persisting osteocytes per 225 lacunae mean f 1 std. dev. 168 &16.2 2 161 -C 10.6 0.10 1202 11.3 0.05 98? 19.2 0.02 9 8 2 4.2 0.10 151 k19.9 3 129 k 6.3 5 122 219.6 2 115.5-C 2.1 3 4 peared as early as ten days and continued to become firmly established in isografts, but less well established in homografts. Following an initial inflammatory response, most of the homografts were encapsulated by fibrous tissue and were only loosely attached to the connective tissue of the host. Scanty periosteal and endosteal bone apposition was noted on the grafts of both series as early as ten days post-grafting. New bone overlaid cellular as well as acellular areas of some transplant. In both isografts and homografts the original whole bone transplant remained intact with little change in gross morphology. In general, the host immune response was mild (Sabet et al., ’62). 57 k 18.6 218 P value of unpaired “t” test isograft vs homograft * 5.8 0.05 - tion of the “t” test (unpaired) showed a significant difference at this time interval ( P = 0.05). In the succeeding periods both groups showed a progressive diminution in the number of persisting osteocytes, isografts decreasing less rapidly and remaining relatively constant after 30 days. By 60 days the percentage of remaining osteocytes in the isograft series was approximately twice that of the homograft (51.3% and 25.3%). A comparison of the means for time intervals of the two series showed a significant difference ( P = 0.02) of the overall reaction (table 4). In no instance was an isograft or homograft specimen found to be entirely devoid of lacunae with stained, intact nuclei. Persistence of osteocytes in the Persistence of osteocytes in the entire diaphysis longitudinal divisions Counts of non-transplanted control meCounts of persisting osteocytes showed tatarsals showed that an average of 96.8% no significant differences between the three of the lacunae contained normal osteo- longitudinal divisions in either the isocytes. The cells were evenly distributed graft or homograft series (table 2, figs. throughout the diaphysis. 3-a, 3-b). At ten days the proportion of In both experimental series, the total osteocytes remaining in all divisions was number of persisting osteocytes dropped similar for both experimental groups, but rapidly during the first ten days, both thereafter all the homograft divisions asshowing a loss of approximately 25% Figure 3 (table I, fig. 2 ) . By the end of 20 days, a Osteocytes persisting in isograft longituhowever, the isograft series showed a dinal divisions. higher proportion of persisting osteocytes b Osteocytes persisting in homograft longithan did the homograft series. Applica- tudinal divisions. 89 OSTEOCYTES IN BONE TRANSPLANTS 10 r 9( - 8( I a 2. Y 7c 0 v) 0 sc m . c ..-* 5c v) a 0 40 L 0 30 -= 0 0 20 - - Proximal ______ Y L Central e 10 Distal A 0 - I I I I I I 10 20 30 Days 40 50 so I so so Distal B I I I I 10 20 30 Days 40 Figure 3 I 90 KARL K. NISHIMURA, JAMES A. YAEGER AND TAWFIK Y . SABET TABLE 2 Osteocytes persisting in longitudinal divisions Persisting osteocytes per 75 lacunae. Mean -I- 1 std. dev. Days Homograft Isograft Proximal Proximal Distal Central Central Distal 10 57 2 7.5 57 2 7.3 55 i. 8.2 5 4 2 4.2 54 2 7 . 1 53 2 7 . 7 20 47 211.3 57 2 2.3 47 42rt11.2 44 34 2 2.9 2a* f 11.4 39.52~ 2.8 k5.6 26.4 30 46.5)- 6.8 43 35 k a . 0 35 28.9 45 41 f 1 3 42 k 6.3 39 5.7 2 9 5 5.6 34.5*9.2 34.5kQ.7 60 40 212.7 39.5*10.5 36 f 4.2 2 4 k 5.8 13 2 5 . 2 20 k 8 . 5 Control 73 2 2.3 73 2 2.6 72 -t- 1.5 -C sumed a lower level, reflecting the overall homograft-isograft difference described above. 4.5 grafts had only 6% and the isografts 30% of their original osteocytes persisting in the medullary stratum. The proportion of osteocytes persisting in the periosteal stratum of both series lay between the mid-cortical and medullary strata, and this relationship was maintained throughout the observation period. CeIl dissolution appeared to be most rapid in the medullary stratum, moderate in the periosteal, and least in the midcortical. In this respect the three strata behaved similarly in isografts and homografts with the latter assuming a lower level, again reflecting the overall difference. A comparison of the means for the time intervals of the three isograft and homograft circumferential strata showed highly significant differences between the periosteal and medullary strata but not between the mid-cortical strata. A summary of the results including tests of significance on the related behavior of these three circumferential strata is presented in tables 3 and 4. Persistence of osteocytes in the circumferential strata An unexpected observation was the high proportion of nucleated lacunae in the midcortical portion of the diaphysis. The persisting osteocytes seemed to predominate in this deep stratum in both homografts and isografts by a significant margin over the periosteal and medullary strata (tables 3 and 4, figs. 4-a, 4-b). The width of this layer of osteocytes approximated one-third of the cortex but varied slightly from specimen to specimen and also in different parts of the same transplant. In the isografts the proportion of osteocytes in this mid-cortical stratum decreased gradually. At the end of 60 days, 79% of the osteocytes still remained. The homograft midcortical stratum, as in the isograft, showed the highest percentage of remaining osteocytes. The decrease was gradual for the first 20 days, but thereafter cell dissoluDISCUSSION tion occurred at a more rapid rate dropThe results of this study do not agree ping to 52% by 60 days. with previous reports that all cellular eleA second interesting observation was ments are destroyed in cortical bone transthe more rapid and higher mortality found plants (Phemister, '14; Pollock, McKenney in the medullary stratum than in the peri- and Blaisdell, '29; Reynolds and Oliver, osteal stratum of both series. The statis'50) nor do they suport the hypothesis tical signscance of this difference was that all cellular elements survive (Albee, marginal when individual values were compared within separate time intervals Figure 4 (table 3), but the T' values were much a Osteocytes persisting in isograft circumlower, denoting greater significance, when ferential strata. Mean f 1 Standard Deviation. mean values for time intervals were comb Osteocytes persisting i n homograft circumpared (table 4). By 60 days the homo- ferential strata. Mean -C 1 Standard Deviation. 91 OSTEOCYTES IN B O N E T R A N S P L A N T S 100 -) 5 2 0 1 Poriorteol - Mid- Cor l i c a I Medullary A 0 ------ _._.-.I I I I I0 9.0 I I 30 Days Figure 4 I I 40 I J I 1 80 a0 92 KARL K. NISHIMURA, JAMES A. YAEGER AND TAWFIK Y. SABET TABLE 3 Osteocytes persisting in circumferential strata P value of paired “t” test Days Experimental SOUP Persisting osteocytes per 75 lacunae Mean 1 std. dev. +- pen!:kal Midcortical Periosteal VS. VS. medul- medullary larv Periosteal Midcortical Medullary czhzH1 46 214.2 41 f 6.3 0.02 0.17 0.004 0.04 0.33 0.20 10 Isograft Homograft 54 flO.O 54 f 0.1 68 k 2.2 66 k 4.2 20 Isograft Homograft 49 2 5.3 34 & 8.3 67 7.2 63 2 2.0 35 8.6 22 2 3.2 0.002 0.02 0.001 0.002 0.02 0.15 30 Isograft Homograft 44.5212.0 34 214.6 62.5f 2.6 52 f 8.5 22 12 4.2 4.9 0.08 0.08 0.001 0.001 0.07 0.03 45 Isograft Homograft 42 2 3.3 28.52 3.5 56 2 8.3 56 f 0.1 24 f 9.0 13.52 0.7 0.02 0.06 0.002 0.01 0.01 0.09 60 Isograft Homograft 32 C 7.7 13 2 5.8 59 2 1.4 39 k10.6 24 2 4.2 5 3.6 * 0.17 0.02 0.04 0.02 0.37 0.13 Control 73 2 1.5 72 2 3.3 73 f 1.7 - - - 0 * * f * TABLE 4 Comparison o f combined means in circumferential strata P values f r o m paired “t” tests between means f o r time intervals HOMOGRAFT ISOGRAFT 0.0I M i d - Cortical 0.11 Mid-Cortical Total 0.02 Total ’44). They do however, confirm the findings of Chalmers (’59), Heslop et al. (’60) and Anderson (’61), that a proportion of osteocytes persist in both isografts and homografts. In the present study a significant difference between the isografts and homografts is apparent by 20 days. 0.004 The proportion of persisting osteocytes shows an apparent quantitative relationship to the adverse host response in the homografts. Although statistically significant, the difference may not be sufficiently great to provide an accurate index of a milder homograft reaction. Therefore, the OSTEOCYTES IN BONE TRANSPLANTS 93 94 KARL K. N I S H I M U R A , JAMES A. YAEGER AND TAWFIK Y. SABET usefulness of "relative osteocyte persistence" as a method for testing measures enhancing the acceptance of homografts is limited. The question of viability or non-viability of osteocytes persisting in cortical bone transplants has received attention in recent literature (Ray, La Violette, Buckley and Mosiman, '55; Kiehn and Gutentag, '57; Anderson et al., '61). An empty lacuna is an indication that the osteocyte has died and disappeared. However, the routine histological criterion for establishing viability, i.e., the presence of morphologically normal, purple staining nuclei, is now disputed as sufficient evidence of cell survival. Harris and Ham ('56), Anderson ('61) and Ham and Leeson ('61) asserted that cells deep in cortical bone were non-viable after transplantation regardless of the presence of stainable material. They reasoned that the canalicular mechanism of cortical bone was so inefficient that deeper cells could not possibly survive transplantation after being severed from their normal blood supply and environment. Osteocytes may appear to be living as long as they are surrounded by intact bony matrix which protects them from lysis by the host. Therefore, the presence of stained, intact nuclear material in a lacuna may not necessarily indicate viability of the osteocyte. Heslop et al. ('60) and Chalmers ('59) on the other hand, also observed histologically that osteocytes persisted in cortical bone transplants but suggested that these osteocytes occupied a protected position similar to the interior of a diffusion chamber. The cortical bone could be a barrier to host cells but permit fluid exchange. Abbot, Schottstaedt, Saunders and Bost ('47) reported that cells of a bone graft could survive up to 15 days, without a demonstrable blood supply, in the fluids of the host bed. By the intracellular uptake of a radioactive isotope (P39, Kiehn and Gutentag ('57), further demonstrated the viability and survival of osteocytes in homografted bone that was kept in a millipore diffusion chamber for four months. Evidence seems to support the possibility of viable osteocytes persisting in cortical bone under proper conditions. No essential differences in morphological or staining characteristics of persisting nuclei between isografts and homografts and normal bone were apparent in this study. Normally staining, intact nuclei containing granules were observed in the lacunae of 60 day homografts (fig. 5 ) and isografts. The supposition that an osteocyte dies without showing degenerative nuclear changes within 60 days does not seem plausible. It is doubtful whether viability or non-viability can be established with certainty using routine hematoxylineosin staining and light microscopy. However, this does not detract from accepting the morphologic evidence as indicating relative, rather than absolute, values of osteocyte survival. In the present study no significant differences were apparent in the proportion of osteocytes persisting between the diaphyseal divisions (figs. 3-a, 3-b). This is not surprising in view of the morphology of the mouse metatarsal. The tubular cortex is fairly uniform in thickness, diameter and structure throughout the diaphysis (fig. l-a) unlike other long bones such as the femur and tibia. The behavior of the three circumferential strata is of interest since the pattern is similar in both isografts and homografts. Osteocyte persistence is greatest in the mid-cortical stratum, moderate in the periosteal, and least in the medullary. The periosteal circumferential stratum comes into immediate and direct contact with the tissue fluids of the host bed. This surface presumably would be accessible to both host tissue fluids and inflammatory cells, favoring the death and dissolution of some osteocytes, but allowing the survival of those not destroyed by the host. The medullary circumferential stratum occupies a unique position because the transplants were whole bones, in which the marrow cavity is isolated from the host environment until vascularization occurs. Therefore, there is a lag period (ten days) before re-establishment of circulation to the marrow cavity. During this interval the osteocytes closest to the medullary surface must compete with the myeloid elements for materials in the stagnant fluids of the marrow cavity. Due to the limited OSTEOCYTES IN BONE TRANSPLANTS access of these fluids to the osteocytes, most die and eventually disappear. The mid-cortical circumferential stratum, on the other hand, is farthest from either periosteal or medullary environment (70-80 p for the deepest cells) and yet, apparently, it is the least affected. This portion of the cortical diaphysis has the highest proportion of nucleated lacunae in both isografts and homografts. The reason for the persistence of these osteocytes is unknown. Assuming they are living, they must depend on diffusion from either the periosteal or medullary surfaces for obtaining nutrients and removal of waste products, but they are protected from destruction by the barrier of matrix surrounding them. The fate of osteocytes i n freshly transplanted bone depends on a multiplicity of factors interacting with a cumulative effect to produce a favorable or unfavorable environment. Since there is no clear understanding of the physiology of transplanted bone, a n explanation of osteocyte persistence based on granulation tissue formation, circulatory, and immunological factors is necessarily inconclusive. However, the consistency of the present observations suggests a predictable pattern of osteocyte response to bone transplantation, the response of the cells depending on their positions within the bone and the nature of the transplant. SUMMARY Metatarsals from adult Strong A (isografts) and CBA (homografts) mice were transplanted into subcutaneous sites in Strong A hosts using a transparent chamber technique. After intervals of 10, 20, 30, 45 and 60 days the 114 transplants were dissected free and longitudinal sections prepared. The proportion of intact nuclei in 75 lacunae was counted in each of three longitudinal diaphyseal divisions (proximal, central and distal) and three circumferential strata (periosteal, midcortical and medullary). Nuclei persisted u p to 60 days in both isografts (51.3% of lacunae nucleated) and homografts 25.3% ). A significantly higher proportion of osteocytes was found to -persist in isografts than- homografts as 95 early as 20 days after transplantation (67.1% vs. 53.3%, P = 0.05). No significant differences were found among the longitudinal divisions in either isografts or homografts. As early as ten days after transplantation, osteocyte persistence in the mid-cortical circumferential stratum was greatest and that in the medullary stratum least in both isografts and homografts. These differences persisted to 60 days ( P s at 60 days, 0.01-0.001). Osteocyte persistence depended upon the position of the cells within the bone. Cells in the mid-cortical stratum were best protected from the host immune reaction, while cells in the medullary stratum apparently had a n inadequate tissue fluid exchange until the transplants were vascularized. ACKNOWLEDGMENT We are indebted to Dr. Robert D. Ray for his encouragement and for the courtesy of his laboratory facilities and staff i n the preparation of this material. LITERATURE CITED Abbot, L. C., E. R. Schottstaedt, J. B. deC. M. Saunders and F. C. Bost 1947 The evaluation of cortical and cancellous bone as grafting material. A clinical and experimental study. J. Bone Jt. Surg., 29A: 3 8 1 4 1 4 . Albee, F. H. 1944 Evolution of bone graft surgery. Am. J. Surg., 63: 421-436. Anderson, K. J. 1961 The behavior of autogenous and homogenous bone transplants in the anterior chamber of the rat’s eye. J. Bone Jt. Surg., 43A: 980-995. Anderson, K. J., J. A. Dingwall, J. Schmidt, J. F. LeCocq and D. K. Clawson 1961 The effect of particle size of the heterogenous bone transplant on the host tissue. 11. A histological study. Ibid., 43A: 996-1004. Bonfiglo, M., W. S . Jeter and C. L. Smith 1955 Immune reactions in tissue homotransplants. Ann N. Y. Acad. Sci., 59: 4 1 7 4 3 2 . Campbell, C. J., T. Brower, D. G. MacFadden, E. B. Payne and J. Doherty 1953 Experimental study of the fate of bone grafts. J. Bone Jt. Surg., 35A: 332-346. Chalmers, J. 1959 Transplantation immunity in bone homografting. Ibid., 41B: 160-179. Chase, S. W., and C. H. Herndon 1955 The fate of autogenous and homogenous bone grafts. A historical review. Ibid., 37A: 809-841. Curtiss, P. H., Jr., S. W. Chase and C. H. Herndon 1956 Immunological factors in homogenous bone transplantation. 11. Histological studies. Ibid., 38A: 324-330. Felts, W . J. L. 1957 A comparison of subcutaneous implants of isolog&s and homolo- 96 KARL K. NISHIMURA, JAMES A. YAEGER AND TAWFIK Y . SABET gous immature whole mouse bones. Transplant. Bull., 4: 131-135. Ham, A. W., and T. S. Leeson 1961 Histology, 4th Edition. J. B. Lippincott Co., Philadelphia, pp. 334-338. Harris, W. R., and A. W. Ham 1956 The mechanism of nutrition i n bone and how it affects its structure, repair and fate on transplantation - In Ciba Symposium on Bone Structure and Metabolism (Ed. by G. E. W. Wolstenholme). Little, Brown and Co., Boston, pp. 135-143. Herndon, C. H., and S. W. Chase 1954 The fate of massive autogenous and homogenous bone grafts including articular surfaces. Surg. Gyn. Obst., 98: 273-290. Heslop, B. F., I. M. Zeiss and N. W. Nisbet 1960 Studies on transference of bone. I. A comparison of autologous and homologous bone implants with reference to osteocyte survival, osteogenesis, and host reaction. Brit. J. Exper. Path., 41: 269-287. Kiehn, C. L., and J. Gutentag 1957 The uptake of radio phosphorus by bone homografts in diffusion chambers. Transactions of the International Society of Plastic Surgeons, First Congress. The Williams and Wilkins Co., Baltimore, pp. 510-512. Phemister, D. B. 1914 The fate of transplanted bone and regenerative power of its various constituents. Surg. Gyn. Obst., 19: 303-333. Pollock, W. E., P. W. McKenney and F. E. Blaisdell 1929 The viability of transplanted bone: a n experimental study. Arch. Surg., 18: 607623. Ray, R. D., J. Degge, P. Gloyd and G. Mooney 1952 Bone regeneration. A n experimental study of bone-grafting materials. J. Bone Jt. Surg., 34A: 638-647. Ray, R. D., D. La Violette, H. D. Buckley and R. S. Mosiman 1955 Studies of bone metabolism. I. A comparison of the metabolism of strontium 90 in living and dead bone. Ibid., 37A: 143-155. Reynolds, F. C., and D. R. Oliver 1950 Experimental evaluation of homogenous bone grafts. Ibid., 32A: 283-297. Sabet, T. Y., E. B. Hidvegi and R. D. Ray 1961a A chamber for In V i m observation of living organs. Transplant. Bull., 27: 105108. 1961b Bone immunology. 11. Comparison of embryonic mouse isografts and homografts. J. Bone Jt. Surg., 43A: 1007-1021. 1962 Bone immunology of embryonic and adult bone grafts. Presented before American Academy of Orthopaedic Surgeons, Chicago. Siffert, R. S. 1955 Experimental bone transplants. J. Bone Jt. Surg., 37A: 742-758.