The fate of cancellous and cortical bone after transplantation of fresh and frozen tissue-antigen-matched and mismatched osteochondral allografts in dogs

After implantation, a massive osteochondral allograft cannot be completely protected from the stresses that are produced by weight-bearing, and it is susceptible to collapse during incorporation, revascularization, and substitution. How these processes are affected by disparities between the tissue antigens of the host and the graft remain unclear. To clarify the role of histocompatibility antigen-matching in the incorporation of cancellous and cortical bone, we orthotopically implanted both fresh and cryopreserved dog leukocyte-antigen-matched and mismatched proximal osteochondral radial allografts in beagles. Four groups of beagle dogs were used; they received (1) a dog leukocyte-antigen-mismatched frozen allograft, (2) a dog leukocyte-antigen-mismatched fresh allograft, (3) a dog leukocyte-antigen-matched fresh allograft, or (4) a dog leukocyte-antigen-matched frozen allograft. In twelve dogs, a sham operation was done in the contralateral limb (the first living donor had a sham operation), and in the remaining ten dogs, the proximal part of the contralateral radius was removed and then replaced as an autogenous (control) graft. The animals were given fluorochromes periodically, and they were killed eleven months after the operation. The osseous portion of the grafts was evaluated radiographically, biomechanically, and histomorphometrically. No dog had grossly obvious clinical abnormalities, all host-graft interfaces healed, and no joints dislocated. Radiographic examination of the allografts frequently showed deformation of the radial head and variable peripheral resorption. No significant difference in the modulus of elasticity at the host-graft interface was found among the groups. The repair process of the cortical bone was similar for all grafted segments. New periosteal and endosteal bone formed, and the cortical bone became porotic as vessels penetrated it. The uptake of fluorochrome was the most active in the autogenous grafts and the least active in the fresh antigen-mismatched grafts. The volume of cancellous bone was significantly greater and the trabeculae were thicker in all allografts compared with the bones on which a sham operation had been done and compared with the autogenous grafts. The volume of intertrabecular fibrous connective tissue was directly proportional to the immunogenicity of the allografts, and the percentage of the surface on which bone was forming tended to be inversely proportional to the immunogenicity of the allografts. The grafts were revascularized by the ingrowth of vessels into the intertrabecular spaces; necrotic trabeculae were not penetrated by vessels. This pattern was particularly pronounced in the antigen-mismatched grafts, regardless of whether they were fresh or frozen.(ABSTRACT TRUNCATED AT 400 WORDS)     

The viability of articular cartilage in fresh osteochondral allografts after clinical transplantation

The articular cartilage of four fresh osteochondral allografts was biopsied after transplantation, and its viability was studied by autoradiography. The biopsy specimens were labeled with both 3H-cytidine, for newly synthesized ribonucleic acid, and 35S-sulphate, for newly synthesized proteoglycans. The cartilage of a lateral humeral condylar graft at twelve months had 96 to 99 per cent labeled chondrocytes, the articular cartilage of a medial femoral condylar graft at twenty-four months showed 69 to 78 per cent labeled chondrocytes, and the cartilage of a medial tibial-plateau graft at forty-one months had 90 per cent labeled cells. At six years, a lateral tibial-plateau graft had 37 per cent labeled chondrocytes.     

Chondrocyte survival in cryopreserved osteochondral articular cartilage

Fluorescent staining techniques were used in combination with confocal and conventional fluorescent microscopy to determine the location of viable chondrocytes in frozen and thawed osteochondral articular cartilage. The results showed that cell survival was confined to the superficial layer of the cartilage matrix. The addition of a cryopreservative agent (dimethyl sulfoxide) increased chondrocyte survival but only in the same layer. There was no evidence of cell survival in the middle or deep layers of the cartilage with or without the use of dimethyl sulfoxide. Under the conditions employed in this study, chondrocyte survival in a cryopreserved osteochondral allograft appears to be limited to the superficial layer of the articula cartilage.     

Prolonged storage effects on the articular cartilage of fresh human osteochondral allografts

BACKGROUND: Fresh osteochondral allograft transplantation is a well-established technique for the treatment of cartilage defects of the knee. It is believed that the basic paradigm of the technique is that the transplantation of viable chondrocytes maintains the articular cartilage matrix over time. Allograft tissue is typically transplanted up to forty-two days after the death of the donor, but it is unknown how the conditions and duration of storage affect the properties of fresh human osteochondral allografts. This study examined the quality of human allograft cartilage as a function of storage for a duration of one, seven, fourteen, and twenty-eight days. We hypothesized that chondrocyte viability, chondrocyte metabolic activity, and the biochemical and biomechanical properties of articular cartilage would remain unchanged after storage for twenty-eight days. METHODS: Sixty osteochondral plugs were harvested from ten fresh human femoral condyles within forty-eight hours after the death of the donor and were stored in culture medium at 4 degrees C. At one, seven, fourteen, and twenty-eight days after harvest, the osteochondral plugs were analyzed for (1) viability and viable cell density by confocal microscopy, (2) proteoglycan synthesis by quantification of (35)SO(4) incorporation, (3) glycosaminoglycan content, (4) indentation stiffness, (5) compressive modulus and hydraulic permeability by static and dynamic compression testing, and (6) tensile modulus by equilibrium tensile testing. RESULTS: Chondrocyte viability and viable cell density remained unchanged after storage for seven and fourteen days (p > 0.7) and then declined at twenty-eight days (p < 0.001). Proteoglycan synthesis remained unchanged at seven days (p > 0.1) and then declined at fourteen days (p < 0.01) and twenty-eight days (p < 0.001). No significant differences were detected in glycosaminoglycan content (p > 0.8), indentation stiffness (p > 0.4), compressive modulus (p > 0.05), permeability (p > 0.3), or equilibrium tensile modulus after storage for twenty-eight days (p > 0.9). CONCLUSIONS: These data demonstrate that fresh human osteochondral allograft tissue stored for more than fourteen days undergoes significant decreases in chondrocyte viability, viable cell density, and metabolic activity, with preservation of glycosaminoglycan content and biomechanical properties. The cartilage matrix is preserved during storage for twenty-eight days, but the chondrocytes necessary to maintain the matrix after transplantation decreased over that time-period.     

新鲜和深低温冷冻同种异体骨软骨移植长期转归实验研究

[目的]观察和比较新鲜和深低温冷冻同种异体骨软骨移植后的长期转归情况,为同种异体骨软骨移植治疗关节软骨缺损进一步提供理论基础。[方法]建立兔膝关节软骨缺损模型,行新鲜和深低温冷冻同种异体骨软骨移植。在术后12个月和18个月,骨软骨移植物取材进行关节软骨蛋白多糖、软骨细胞活性检测和软骨细胞超微结构观察。[结果]新鲜和冷冻骨软骨移植物中蛋白多糖阿尔新蓝染色强度和存活软骨细胞比率术后均下降,且在各时间点新鲜移植组的结果优于冷冻移植组,有统计学差异(P0.05)。超微结构显示软骨细胞退行性改变。[结论]新鲜和深低温冷冻同种异体骨软骨在移植术后较长时间均出现严重退变,冷冻骨软骨移植物退变较严重,其作为关节软骨缺损治疗方法目前是不可行的。     

同种异体半月板联合骨软骨移植的实验研究

目的:探讨新鲜同种异体半月板骨软骨联合移植治疗胫骨平台毁损伤后骨关节炎的疗效。方法:成年新西兰大白兔36只,随机分为A、B、C3组,各12只。A组行右膝内侧半月板连同胫骨平台骨软骨移植,克氏针交叉固定骨块。B组行右膝内侧半月板移植,左膝内侧半月板取出制备新鲜冷冻半月板。C组行左膝内侧新鲜冷冻半月板移植。术后4、8、12周分批取材行大体观察、组织学检查和胫骨平台软骨氨基己糖(GAG)测定。结果:12周时A组移植胫骨平台软骨与B、C组半月板移植术后的内侧胫骨平台软骨氨基己糖含量差异无统计学意义;A、B组移植的半月板纤维软骨细胞数差异无统计学意义;A组半月板移植的纤维软骨细胞数多于C组。结论:新鲜同种异体半月板骨软骨联合移植能修复胫骨平台毁损伤。     

同种异体软骨移植修复关节软骨缺损实验研究

目的 采用兔膝关节软骨标本经打孔梯度降温冻存后行同种异体移植,研究打孔梯度降温冻存对兔关节软骨的影响及其修复关节软骨缺损的效果.方法 自16只2月龄新西兰白兔膝关节股骨髌面取分别取3块骨软骨移植物,随机分为3组.Ⅰ组为实验组,在软骨上以3 mm×3 mm矩阵打孔,Ⅱ、Ⅲ组为对照组,不打孔.分别将软骨标本置于二甲基亚砜冷冻保护溶液中,并经梯度降温至-80℃(Ⅰ、Ⅱ组)或直接置于-80℃(Ⅲ组)保存1周,复温后移植到成年兔相应膝关节部位.术后分批处死动物,通过对移植物的大体形态学、组织学、免疫组化染色光镜观察,研究各组移植物保存效果的差异.结果 Ⅰ、Ⅱ组光镜观察结果明显优于Ⅲ组;Ⅰ组与Ⅱ组结果差异不明显,但Ⅰ组对中间层软骨组织的保护明显加强.结论 关节软骨的梯度降温冷冻保存效果明显优于快速降温冷冻保存,且关节软骨打孔冷冻保存对深层软骨细胞有一定的保护作用,可提高软骨细胞存活率,延缓移植软骨组织的退变过程.     

同种异体骨软骨移植修复关节软骨缺损研究现状

关节软骨缺损常因软骨再生能力低而难以自行修复.新鲜的同种异体骨软骨移植修复关节软骨缺损的疗效稳定,成功率逐渐提高.冷冻保存的同种异体骨软骨移植修复关节软骨缺损的成功率可与新鲜的同种异体骨软骨移植媲美,梯度降温法是目前保存软骨细胞存活率最好的冷冻方法.该文就同种异体骨软骨移植的实验和临床研究、免疫排斥问题及其相关研究动态作一综述.     

Histological and biomechanical assessment of articular cartilage from stored osteochondral shell allografts

Normal and stored articular cartilage from the medial tibial plateaus of mature canine knee joints were evaluated histologically and biomechanically. The medial plateaus from the right knee (control) were assessed fresh, while the left (stored) were preserved in culture media at 4 degrees C for 3, 7, 14, or 28 days and then evaluated. Biomechanically, confined compression tests were performed on all specimens to determine the aggregate modulus and apparent permeability of the articular cartilage. Histologically, Safranin O- and hematoxylin and eosin (H&E)-stained sections were evaluated. All stored cartilage specimens had an aggregate modulus on average lower than normal, but the differences were not significant (p greater than 0.10). The apparent permeability was on average higher than but also not significantly different from normal (p greater than 0.10). Time in storage (up to 28 days) did not have a significant effect on the biomechanical properties of stored cartilage normalized by control values (p greater than 0.50). Safranin O and H&E histological evaluation also showed no overall changes in cell appearance or staining of the stored cartilage when compared with control for the time periods studied.     

Time-sequential changes in biomechanical and morphological properties of articular cartilage in cryopreserved osteochondral allografting

This study examined time-sequential changes in the biomechanical and morphological properties of articular cartilage that had received cryopreserved osteochondral allografting. Osteochondral blocks obtained from the femurs of 18 rabbits were cryopreserved with dimethylsulfoxide (DMSO), using a two-step freezing method, and allografted to the femurs of another 18 rabbits. Specimens for biomechanical and morphological examinations were prepared at the second, fourth, and twelfth weeks after allografting (n = 18). In 12 allografted rabbits, biomechanical features were examined with an indentation test apparatus, and histological changes were studied with a light microscope (second week, n = 4; fourth week, n = 4; twelfth week, n = 4). In the other 6 allografted rabbits, cartilage surfaces were studied with a scanning electron microscope (second week, n = 2; fourth week, n = 2; twelfth week, n = 2). For controls, fresh, DMSO-treated, or DMSO-treated + cryopreserved specimens were examined biomechanically and morphologically. In the time-sequential examination of biomechanical features, both the parameter for elasticity (i.e., ratio of instant elastic strain to maximum strain) and the parameter for viscosity (i.e., average retardation time) significantly changed. Light microscopy showed chronological decreases in safranin-O staining intensity in the matrix, and progression of degeneration. On scan-ning electron microscopy, disruption of the cartilage surface was also recognized. Therefore, changes in biomechanical properties due to cryopreservation could cause irreversible changes in the cartilage in cryopreserved osteochondral allografting. Received: August 31, 2000 / Accepted: January 16, 2001     

Impact loading of articular cartilage during transplantation of osteochondral autograft

Surgical reconstruction of articular surfaces by transplantation of osteochondral autografts has shown considerable promise in the treatment of focal articular lesions. During mosaicplasty, each cylindrical osteochondral graft is centred over the recipient hole and delivered by impacting the articular surface. Impact loading of articular cartilage has been associated with structural damage, loss of the viability of chondrocytes and subsequent degeneration of the articular cartilage. We have examined the relationship between single-impact loading and chondrocyte death for the specific confined-compression boundary conditions of mosaicplasty and the effect of repetitive impact loading which occurs during implantation of the graft on the resulting viability of the chondrocytes. Fresh bovine and porcine femoral condyles were used in this experiment. The percentage of chondrocyte death was found to vary logarithmically with single-impact energy and was predicted more strongly by the mean force of the impact rather than by the number of impacts required during placement of the graft. The significance of these results in regard to the surgical technique and design features of instruments for osteochondral transplantation is discussed.     

Chondrocyte viability in press-fit cryopreserved osteochondral allografts.

The viability of chondrocytes in press-fit glycerol-preserved osteochondral allografts was compared to that in fresh autografts, after transplantation into load-bearing and non-load-bearing sites in mature sheep stifle joints. We used macroscopic grading, tonometer pen indentation testing, histology, sulfate uptake and viability as determined by confocal-microscopy to assess cartilage condition. Despite there being no statistical differences between macroscopic appearance and tonometer testing of all grafts, confocal microscopy and histology demonstrated a positive effect of load-bearing placement on cryopreserved osteochondral allografts. Allografts transplanted into load-bearing sites demonstrated superior confocal microscopy-measured chondrocyte viability (77%+/-17%SD) than those transplanted into non-load-bearing sites (25%+/-2%). Load-bearing effect was not seen in autografts (78%+/-15%), and was comparable in adjacent cartilage (83%+/-9%). Similarly, load-bearing allografts demonstrated histological scoring closer to that of autografts and adjacent cartilage, all of which fared significantly better than non-load-bearing allografts. Load-bearing allografts had a greater amount of fibrocartilage than autografts or adjacent cartilage but less fibrocartilage than non-load-bearing allografts. Both autografts and allografts had non-significant increases in metabolism compared to adjacent cartilage as measured by sulfate-uptake. Load-bearing placement improved chondrocyte viability of glycerol cryopreserved osteochondral allograft following a press-fit implantation.     

The fate of meniscus cartilage after transplantation of cryopreserved nontissue-antigen-matched allograft. A case report.

A cryopreserved nontissue-antigen-matched meniscus was transplanted in the knee of a 48-year-old man with lateral compartment osteoarthrosis. After six months, diagnostic arthroscopy was performed. Clinical, histologic, and histochemical studies showed that meniscal chondrocytes survived cryopreservation and transplantation. One year after surgery, the outline of the donor meniscus on an arthrogram appeared normal.     

The immune response to osteochondral allografts in dogs

Dog leukocyte antigen (DLA)-matched and mismatched, fresh and frozen (cryopreserved) osteochondral allografts of the proximal part of the radius were implanted orthotopically in beagles. The systemic and local (intra-articular) immune responses were monitored for eleven months using a 51chromium release assay with donor peripheral-blood lymphocytes as target cells. DLA-mismatched fresh grafts markedly and persistently stimulated the formation of antibody directed against donor cell-surface antigens, while DLA-matched grafts did not elicit systemically detectable antibody. The same general pattern was noted when antibody-dependent cell-mediated cytotoxicity was assayed. There was measurable cell-mediated immunity to donor cells from six weeks after surgery onward, although no distinct pattern or differences between experimental groups were noted. Higher titers of anti-DLA antibody were found in synovial fluid than in serum; in fact, synovial antibody was detectable when systemic antibody was not. Joints that received DLA-mismatched grafts had the highest titer of antibody and the intra-articular response was significantly reduced when the graft had been frozen.     

Transplantation of cryopreserved osteochondral Dowel allografts for repair of focal articular defects in an ovine model.

The purpose of this study was to test whether successful cryopreservation of osteochondral tissue is possible and whether, with the appropriate surgical procedure, it can be used for the successful repair of focal articular defects within joints. Fresh (nonfrozen) and snap-frozen (plunged in liquid nitrogen and thawed in a water bath at 37 degrees C, repeated three times) autografts were used as positive and negative controls, respectively. Snap-frozen, frozen (fresh tissue placed in a freezer at -80 degrees C), and cryopreserved (immersed in 10% dimethyl sulfoxide for 30 minutes and then frozen at 1 degrees C/min to -80 degrees C) allografts were transplanted into the knees of adult sheep. Outcomes were evaluated 3, 6, and 12 months after transplantation. The morphological, histological, biochemical, and biomechanical behaviors and characteristics of the graft cartilage, the host cartilage adjacent to the grafts, and the opposing tibial cartilage were assessed. Freezing protocols that yielded poor chondrocyte recovery after thawing (frozen and snap-frozen) resulted in poor overall graft outcome. The cryopreservation protocol, however, resulted in intermediate recovery (50%) of chondrocytes and in intermediate overall graft outcome compared with fresh autografts. The membrane integrity of the allograft chondrocytes immediately following cryopreservation was identified as the most reliable predictor of long-term outcome of the graft. Further improvements in cryopreservation technique may lead to an effective method of banking osteochondral tissue for successful transplantation for the repair of focal defects and larger joint reconstructions.     

The effects of storage on fresh human osteochondral allografts

Historically, fresh human osteochondral allografts have been stored in lactated Ringer's solution at 4 degrees C and then transplanted as quickly as possible, generally within 2 to 5 days, to ensure delivery of a high level of viable chondrocytes. Recently, allograft distribution companies have begun to provide fresh osteochondral allografts that are stored in a proprietary culture medium usually for at least 2 weeks before delivery to the surgeon for implantation. The effects of such storage on human cartilage have not been well-defined. In the current study the effects of storage in lactated Ringer's solution and in culture media were assessed. After 7 days of storage in lactated Ringer's solution, a significant decline in chondrocyte viability and metabolic activity was seen. Culture media provided significantly better preservation of the cartilage with viability and metabolic activity remaining essentially unchanged from baseline for as many as 14 days. The biochemical and biomechanical properties of the extracellular matrix remained stable with storage in both solutions with time. These data suggest that osteochondral allografts stored under traditional conditions in lactated Ringer's solution should continue to be implanted as quickly as possible and certainly within 7 days of donor death. If kept in culture media, the storage duration may be extended to approximately 2 weeks.     

Mini-pig fresh osteochondral allografts deteriorate after 1 week of cold storage

As a well-defined animal transplantation model, the mini-pig potentially is well-suited for large animal studies of fresh osteochondral allograft transplantation. This study was done to determine the histologic characteristics and function of proteoglycan synthesis of mini-pig articular cartilage after refrigeration in basal media for as much as 6 weeks. Osteochondral sections of 10 mini-pig knees were refrigerated in various media at 4 degrees C for 1 to 42 days after slaughter. Four hundred twenty samples were evaluated by 35S uptake and 260 samples by histologic evaluations. Proteoglycan synthesis declined by 7 days to 21% of the level measured on Day 1 and was undetectable at 42 days. Histologic evaluation revealed progressive degeneration. Mankin scores rose from 3.69 +/- 0.27 on Day 1 to 6.40 +/- 0.18 on Day 7, and logarithmically increased to 10.83 +/- 0.07 on Day 42. These results indicate that the metabolic characteristics of porcine articular cartilage were not retained after refrigeration in basal media for 7 days. Optimum cold storage of porcine osteochondral allografts for cartilage transplantation research may be less than 7 days. Because osteochondral grafts for clinical use currently are stored for greater than 7 days, similar studies of the viability of human articular cartilage are needed.     

Immune response in patients receiving fresh osteochondral allografts

Immunologic response to cartilage-specific protein (CSP) antigens was tested in patients treated with fresh osteochondral allografts. Eight of 14 allograft recipients showed immune reactivity against CSP, whereas only 2 of 14 controls were reactive (P<.05). The molecular weights of the most common immunoreactive proteins were 220 kd and 95 kd. The precise identity of these proteins could not be determined using standard anticollagen antibodies. These data indicate the presence of antibodies to CSP--which supports the concept of immunologic response to the cartilage component of osteochondral allografts.     

同种异体骨软骨移植修复软骨损伤的研究进展

关节软骨损伤的治疗是骨科领域一个难题,同种异体骨软骨移植是修复全层软骨损伤的有效方法.文中综述了同种异体骨软骨移植修复软骨损伤的研究进展,包括该方法的适应证和禁忌证,目前在临床的应用情况及治疗效果,并进一步探讨了同种异体骨软骨移植中的3个热点话题,如疾病传播、免疫排斥和移植物的保存.这些文献综述显示同种异体骨软骨移植修复关节软骨损伤具有特定优势,但进一步推广该方法仍有一些需要解决的问题.