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DESTRUCTION OF JOINT HOMOGRAFTS An Experimental Study ISADORE G . YABLON. KENNETH D. BRANDT. RONALD DELELLIS, and DAVID COVALL The host synovium undergoes a striking transformation at about 26 weeks after joint homografting. Histologically the synovium invaded and destroyed the graft with which it came in contact, becoming markedly hypercellular; the infiltrate consisted mainly of plasma cells and lymphocytes. The synovium at this stage closely resembled a rheumatoid pannus. The cartilage in contact with this invasive synovium lost its staining qualities, failed to take up S-35, and was gradually destroyed. The mechanism causing this transformation remains unclear. It is postulated that these changes could be due to an inflammatory or immune response.

The fate of osteochondral joint homografts has generally been that of degeneration, resorption, and collapse. Survival and function have variously been reported from 4 weeks ( I ) to 12 months (2,3). The reasons Isadore G . Yablon, M.D.: Orthopaedic Research Laboratories, Boston University Medical Center, University Hospital. 75 East Newton Street, Boston, Massachusetts; Kenneth D. Brandt, M.D.: Department of Rheumatology, University of Indiana, Indianapolis, Indiana; Ronald DeLellis, M.D.: Tufts University School of Medicine, Boston, Massachusetts: David Covall: Orthopedic Research Laboratories, Boston University Medical Center. Supported by Grant AM-17730-01, National Institute of Health, Bethesda, Maryland. Address reprint requests to lsadore G. Yablon, M.D.. Orthopaedic Surgery, Boston University Medical Center, University Hospital. 75 East Newton Street, Boston, Massachusetts 021 18. Submitted for publication March 14. 1977; revision accepted May 19. 1977. Arthritis and Rheumatism, Vol. 20, No. 8 (November-December 1977)

for homograft failure have been poorly understood despite new concepts concerning cartilage metabolism and structure, and more refined investigative techniques. The host synovium has received little attention regarding its role in graft survival, although recent work has strongly implicated this tissue as being closely associated with rejection of the homograft. Chesterman (4) demonstrated that chondrocytes survived when homografted into a defect in the iliac crest, a site devoid of synovium. but were resorbed when transplanted into a defect in the humeral head. Sengupta (9,reporting on the fate of articular cartilage transplants in rabbits, showed that when slivers of homograft cartilage were placed free within the recipient joint its cells survived until the slivers became adherent to the host synovium following which they were destroyed. When these slivers were sutured to the recipient synovium there was an intense cellular reaction with early destruction to the graft. This study was undertaken to demonstrate that the host synovium is responsible for the destruction of osteochondral joint homografts.

MATERIALS AND METHODS Surgical Technique Eighteen a d u l t m a l e mongrel d o g s w h o s e epiphyses were closed according t o radiological assessment were quara n t i n e d a n d given distemper prophylaxis. T h e y were divided

Fig 1. Photomicrograph of homograft articular cartilage ar 12 weeks. Normal appearance (Safianin-0: X 100).

Fig 2. Autoradiograph ofjoint homograft at I2 weeks. There is diffuse uptake of S-35

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( X 100).

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into 4 groups with 4 dogs in each group. The opposite knee of each dog served as a control. Under intravenous Nembutal anesthesia the right knee was exposed using a long median parapatellar incision (6). The origin of the extensor hallucis longus was divided from the lateral femoral condyle. The knee was hyperflexed and the distal articular portion of the femur was removed with an oscillating air-driven saw with about 5 mm of subchondral bone. Acute hyperflexion of the knee allowed the plane of section to fall anterior to the collateral and cruciate ligaments, thereby preserving its stability. The dogs were operated on in pairs and the grafts were exchanged between pairs and fixed securely with a compression screw countersunk beneath the articular surface to avoid damaging the opposite tibia1 cartilage. The 2 control dogs underwent autotransplantation whereby the graft was removed and immediately re-inserted into its original site. The synovium and skin were closed in the usual manner and the animals were permitted to mobilize their joints without any external immobilization. They were fed a standard laboratory diet and weighed weekly. Prophylactic Bicillin was administered for a period of 10 days following surgery.

Preparation of Specimens Fig 3. Radiograph ofjoint homograft at 12 weeks. The graji has united to the hosr bone and is held securely with a compression screw countersunh beneaih rhe articular carrilage.

The animals were sacrificed with an overdose of Nembutal at 6, 12, 26, and 52 weeks. The controls were sacrificed at 26 and 52 weeks. Cultures of the operated knees were obtained at this time in order t o ensure that the joint was free of infection. Radiographs of the knees were obtained prior to and

Fig 4. Phoromicrograph ofjoint homograft at 26 weeks. The synooium is markedly hypercellular and has invaded the periphery 35 acticity is seen in the cartilage in contact with the synooium ( H & E: X 100).

OJ

the graJi. No S-

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Fig 5. Autoradiograph of transplant at 26 weeks. There is uptake ofs-35 in the areas of the grafi not invaded by the synovium ( H & E; original magnification X 100).

Fig 6. Photomicrograph of joint transplant at 26 weeks. The cartilage stains normally (Safranin-0; original magnijkation X 250).

after surgery, every 3 months, and prior to sacrifice. Twentyfour hours prior to sacrifice 100 pCi of S-35 (N.E. Nuclear Corp.. Boston, Massachusetts; specific activity 10-1000, pCi/pM) and 1 pCi per kg body weight of H3-thymidine (N.E. Nuclear Corp., Boston, Massachusetts; specific activity 40600, pCi/pM) were injected into each knee joint of the control and experimental animals. The right and left distal femora were removed and the graft was divided sagitally. One half was decalcified in Decal (Omega Chemical Corporation, Cold Springs, New York) and stained with H & E, Alcian Blue, and Safranin-0 counterstained with Fast Green. The remaining half was used to obtain autoradiographs by dipping unstained sections in Kodak NTB-3 (Kodak Company, Rochester, New York) nuclear track emulsion following which they were stored for 2 I days in a light-tight container at 4'C. They were processed using Kodak D-19 developer and subsequently stained with H & E.

graphs at this time were normal and the animals were walking and running normally (Figure 3). At 26 weeks the synovium was markedly hypercellular and began to invade the periphery of the graft (Figure 4). The homograft cartilage immediately adjacent to the invasive synovium showed loss of staining. S35 uptake was almost completely absent in this area, but it was present in areas further removed from the invading synovium (Figure 5 ) . This portion of the cartilage stained normally (Figure 6). There was marked activity of H3-thymidine in the synovium. Radiographs were normal (Figure 7). At 52 weeks tongues of synovial tissue had advanced and covered about 50% of the graft. The cartilage at the periphery of the graft, where the synovium first invaded it, had been completely resorbed (Figure 8). N o S-35 uptake was seen in these areas. The portion of the graft not in contact with the advancing synovium stained normally, had a normal histological appearance, and showed S-35 uptake (Figure 9). There was marked H3-thymidine activity in the synovium (Figure 10). The synovium itself was very hypercellular and contained numerous plasma cells and large lymphocytes (Figure

RESULTS Histological sections at 6 and 12 weeks appeared normal. The articular cartilage was intact and stained normally with Alcian Blue and Safranin-0 (Figure I ) . Autoradiographs showed activity in the chondrocyte lacunae and in the matrix (Figure 2). Radio-

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11). Lymphoid follicles were present in some areas (Figure 12). A t the place where the synovium had come in contact with the graft numerous lymphocytes appeared to be destroying the graft cartilage (Figure 13). At this stage the synovium was almost indistinguishable from a rheumatoid pannus. Radiographs indicated beginning degenerative changes consisting of joint space narrowing and small osteophyte formation (Figure 14). The synovium in the control and autografted knees had a normal histological appearance and did not show H3thymidine activity (Figure 15). The grafts were normal on gross examination as were the radiographs and histological sections (Figures 16 to 18).

DISCUSSION

Fig7. Radiographs of the transplant ar 26 weeks. Thegrafi has a normal radiological appearance.

The reasons for homograft failure have never been adequately explained. Because antibodies could not be demonstrated within the transplanted cartilage it was thought that cartilage was not antigenic (7). Alternately it was suggested that because the cartilage matrix is a gel it either prevents the release of antigen or the binding of antibody (8). A review of the history of the ultimate fate of osteochondral grafts shows that autogenous grafts survive indefinitely without showing radiological, histological, or histochemical changes of degeneration (3). Homografts on the other hand are eventually destroyed (2,9-11). The pertinent question is why grafts transplanted in a similar manner to similar

Fig 8. Phoromicrograph of rransplant at 52 weeks. The cartilage ar the periphery of rhe graft has been complere1.v resorbed and the synooium is now in contact wirh subchondral bone. The graft carrilage not in conlact with the s.pnovium appears normal. Occasional grains indicarive of's-35 actioiry are presenr ( H & E; original tnagnificarion X 1001.

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Fig 9. Autoradiograph ofjoini homografi ai 52 weeks. A portion ofthe grafied cartilage not in contact wiih ihe synovium shows a normal S-35 uptake ( H & E: original tnagniJicaiion X 100).

Fig 10. Hosi synouium ai 52 weeks. The graft cartilage has been completely resorbed. Marked H3-thymidine activity is present ( H & E: X 250 I .

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Fig 11. Oil immersion photomicrograph ofhosi synovium ai 52 weeks. Numerous plasma cells and lymphocytes areseen ( H & E;

Fig 12. Photomicrograph of host synovium at 52 weeks. The synovium is markedly hypercellular and shows villous hypertrophy resembling a lymphoid follicle ( H & E; original magnification X 250).

X

IOOO).

DESTRUCTION OF JOINT HOMOGRAFTS

Fig 13. Phototiiiiwgraph oJ'host synor;ium at 52 weeks. There is marked lwiphoid hyperplasia. A portion of the graft cartilage in the lower portion OJ'rhephorogruph is apparen~lj,being destroyed 6). the lymphoid W//A f H & E ; oripiiiu/ rrrup~rficationX 4(?f)1.

anatomical regions display such a discrepancy in their duration of survival. A major point to consider is the one of joint instability. which is an important cause of degenerative arthritis. The method used to anchor the graft in this study preserved the major ligaments of the knee. Thus, instability due to ligamentous laxity was avoided because all knees were stable when examined postoperatively. In addition. because the graft was secured with a compression screw, postoperative immobilization was not necessary and joint stiffness and loss of proteoglycan and glycosaminoglycan from the cartilage graft were thereby obviated. This result was confirmed by histological examination of the graft. sections of which showed S-35 activity and stained with Safranin-0 up to 12 weeks after surgery. I t is possible that the grafts degenerated because the surgical procedure interfered with joint proprioception and thereby promoted a Charcot-type arthropathy. Histologically one of the hallmarks of a Charcot

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Fig 14. Radiograph oJ'transplant a1 52 weeks. The joint surfaces irregulur and osteoph.~tc~ jortrratiun is ecidenr.

ure

joint is the appearance of loose cartilagenous fragments within the joint. This finding was never verified on any of the histological sections and no cartilage fragments were observed within the synovium. I n addition, as the joints began to deteriorate the animals displayed a limp and began to favor the affected extremity indicating that protective sensation was present. However, the most important factor regarding joint stability concerns the adequacy of graft fixation, the lack of which contributed greatly to joint failure in previous work (2,3,9,10). Because all animals were examined radiographically at frequent intervals, any slipping or loosening of the graft would be easily detected, as films obtained in the postoperative period were always compared with those films obtained at the termination of the surgical procedure. In order to ensure a congruous fit of the grafts, the animals were operated upon in pairs of approximately equal size and weight. Any residual incongruity was easily corrected by shaping the distal end of the recipient femur so that the graft

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Fig 15. Photomicrograph oJautotransplant at 52 weeks. The synovium and cartilage are normal. No H3-thymidine uptake is seen ( H & E: X 250).

Fig 16. Gross specimen of’auiotransplant at 52 weeks. The synoviutu was not adherent to the cartilage which appears normal.

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Fig 17. Radiograph of autotransplant at 52 weeks. Normal appearance.

Fig 18. Autoradiograph of cartilage autotransplant at 52 weeks. S-35 uptake i.t .seen in the lacunae and matrix ( H & E; X 2501.

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1536 assumed an anatomical position in its new site. Most important, however, if graft failure were due to mechanical instability, one would have expected the autografts to undergo the same fate as the homografts and this result was not observed. The other question to consider is whether graft failure was due to alterations in the nutrition of the cartilage. Because the synovium was abnormal, such a consideration is plausible. If, however, degeneration of the grafts were due to inadequate or altered nutrition, one should have seen a generalized diffuse change in the histological appearance of the grafts. That degeneration and destruction were seen only in areas where the synovium was in direct contact with the graft, and that portions of the cartilage not in contact with the synovium showed normal staining and S-35 uptake, would tend to mitigate against the hypothesis that nutritional factors were a cause of failure. It is also unlikely that the graft was destroyed by an ingrowth of granulation tissue from the subchondral area because such destruction was not observed histologically and because, if this were the case, there should have been an increased H3-thymidine uptake in the area of the advancing granulation tissue. However, no such increase was observed. Other possibilities include infection and a nonspecific inflammatory response of the synovium due to the release of proteoglycans and glycosaminoglycans from the transplanted cartilage. None of the joints reported on in this study showed any evidence of bacterial growth on culture. With regard to the release of proteoglycans and glycosaminoglycans, George and Chrisman (12) demonstrated that injection of chondroitin-sulfate A and C and keratosulfate into normal joints produced a marked inflammatory response within the synovium. Photographs of sections of synovium in their study appeared similar to the low-power photomicrographs shown here. However, the proteoglycan fractions used by these investigators were not injected in a totally pure form and the inflammatory response that they observed could easily have been due to impurities present in the injected fractions. Only the synovial changes were described and no high-power photomicrographs of the synovium are present. These facts are important because one would have to interpret a cellular infiltrate consisting predominantly of polymorphonuclear leukocytes and monocytes differently from an infiltrate consisting mainly of lymphocytes and plasma cells. The appearance of the articular cartilage is not described in detail and it is not clear whether the inflammed synovium invaded the articular cartilage and whether the cartilage not in contact with the synovium maintained a normal state of function. A careful search

of the literature failed to reveal any study in which it was demonstrated that glycosaminoglycan release was responsible for an invasive transformation of the synovium. Finally, one must consider whether the changes observed could be due to an immune response. The histological appearance of the synovium characterized by lymphoid follicles, lymphocyte and plasma cell hyperplasia, lymphocyte destruction of the graft, and its aggressive behavior is suggestive of such a response, which is occurring within that tissue. It has been demonstrated previously that an immune response can induce the changes in the synovium which were observed in this study and which are histologically almost identical to those seen in rheumatoid arthritis (13-18). There is also ample evidence that antigens are present in the cartilage matrix (8,19-24) and in bone (25). It has also been shown that an immune response occurs to histocompatibility antigens (9,26). Some doubts exist whether transplanted cartilage is immunogenic in nonsensitized animals. Heyner (27) demonstrated that transplanted cartilage survived for 30 days in nonpresensitized animals without showing evidence of an immune response. In her study the cartilage was transplanted to a heterotopic site devoid of synovium. I n addition, cartilage from endochondral embryonic bone was utilized, the nutrition of which differs from mature cartilage. Most important, the grafts were removed after only 30 days, whereas most studies have shown that it requires a longer time for the immune response to become manifest. Although. in an attempt to find the reasons for graft failure. the present study was not directed toward proving the existence of an immune response, the histological appearance and aggressive behavior of the synovium warranted the inclusion of an immune mechanism as a possible cause. The majority of the studies concerning joint homograft transplantation have demonstrated that the homografts were eventually destroyed, but the mode of this destruction was never clearly elucidated. The authors believe that the present study shows, for the first time, the manner in which the grafts were destroyed and focuses attention on the synovium as the prime tissue responsible for homograft failure.

REFERENCES R, Tile M: Transplantation of epiphyseal plates. J Bone Joint Surg.47A:897-914, 1965 2. Campbell C, Ishida J. Takahashi M, et al: The transplantation of articular cartilage. J Bone Joint Surg 45A:1576-1592, 1963 1. Harris WR, Martin

DESTRUCTION OF JOINT HOMOGRAFTS 3. DePalma A, Tsaltus T , Mauler G: Viability of osteochondral grafts as determine by uptake of S-35. J Bone Joint Surg 45A:1565-1575, 1963 4. Chesterman PJ, Smith A: The homotransplantation of articular cartilage and isolated chondrocytes. J Bone Joint Surg 50B: 184-1 97, 1968 5. Sengupta S: The fate of transplants of articular cartilage in the rabbit. J Bone Joint Surg 56B:167-177, 1974 6. Yablon IG. Franzblau C, Leach R E Response of transplanted articular cartilage to growth hormone. J Bone Joint Surg 56A:322-327, 1974 7. Craigmyle MBL: Studies of cartilage autografts and homografts in the rabbit. Br J Plastic Surg 8:93-100, 1955 8. McKibbin B: Immature joint cartilage and the homograft reaction. J Bone Joint Surg 53B:123-135, 1971 9. Aichroth PM, Burwell RG. Lawrence M: Transplantation of synovial joint surfaces. J Bone Joint Surg 54B:747. 1972 10. Entin M, Daniel G, Kahn D: Transplantation of autogenous half joints. Arch Surg 96:359-368, 1968 Hjertquist SW: Microscopical and microchemical studies 11. of osteochondral articular defects. Calc Tiss Res: 107- 109. 1970 (suppl) 12. George RC, Chrisman OD: The role of cartilage polysaccharides in osteoarthritis. Clin Orthop 57:259-265, 1968 13. Dingle JT: The role of lysozomal enzymes in skeletal tissue. J Bone Joint Surg 55B:87-95, 1973 14. Fell HB, Weiss L: Effect of antiserum alone and with hydrocortisone on fetal mouse bone in culture. J Exp Med I2 I :55 1-560, 1965

1537 15. Hirschorn R, Kaplan JM. Goldberg AF, et al: Acid phosphataserich granules in human lymphocytes induced by phytohemagglutinin. Science 147:55-57, 1965 16. Norton W, Ziff M: Electron microscope observations on localization of antigen in tubercular reactions. Immunology 8:81-87, 1965 17. Quie PG, Hirsch JG: Antiserum to leucocyte lysozomes: its cytotoxic, granulocytic and hemolytic activities. J Exp Med 120149-160, 1964 18. Weissmann G: Lysozomes and joint disease. Arthritis Rhem 9:834-840, 1966 19. Barland P, Janis R, Sandson JJ: lmmunofluorescent studies of human articular cartilage. Ann Rheum Dis 25: 156-16 I , 1966 20. Loewi G: The antigenicity of chondromucoprotein. Arthritis Rheum 7:323, 1964 21. Sadjera S, Hascall V: Protein polysaccharide complex from bovine nasal cartilage. J Biol Chem 244:77-86. I969 22. Sanders AM, Matthews MB, Dorfman A: Antigenicity of chondroitin sulfate. Fed Proc 21:26-32, 1962 23. White D. Sandson J. Rosenberg L, et al: The role of the protein moiety in the antigenicity of chondromucoprotein. Arthritis Rheum 6:305, 1963 24. Rosenberg L: Personal communication. 1973 25. Burwell RG: Studies in the transplantation of bone. V111. J Bone Joint Surg 48B:532-536, 1966 26. Langer F, Gross AF: lmmunogenicity of allograft articular cartilage. J Bone Joint Surg 56A:297-304. 1974 27. Heyner S: The significance of intercellular matrix in the survival of cartilage allografts. Transplantation 8:666677, 1969