Effect of immunosuppression on survival and growth of cartilage produced by transplanted allogeneic epiphyseal chondrocytes

Strong short-term immunosuppression improved survival of cartilage formed by transplanted allogeneic epiphyseal chondrocytes in mice. The agents tested were cortisone acetate (CA), cyclophosphamide (CY), procarbazine (PCH), and antithymocyte serum (ATS). Their effect on syngeneic grafts was examined morphologically and histomorphometrically. In untreated recipients, chondrocytes formed cartilage nodules that underwent endochondral ossification. Except for high repetitive doses of CY, none of the other agents interfered with normal cartilage formation. However, all agents affected endochondral ossification. In the allogeneic system, the effect of immunosuppression was examined morphologically and by evaluation of specific humoral and cellular antigraft immunity. Allogeneic chondrocytes evoked a strong immune response in untreated mice, and cartilage was gradually destroyed by infiltrating cells. Endochondral ossification did not occur in this system. Neither agent given alone exerted a marked, long-lasting protective effect upon the graft. However, combined treatment with ATS and PCH inhibited immune response and completely prevented infiltrate formation and allowed endochondral ossification similar to that in the syngeneic control. Although some weak signs of antigraft immunity were seen after six weeks, it is possible that they were due to secondary exposure of antigen-bearing chondrocytes in the course of endochondral ossification.     

Immunosuppression and rejection of cartilage formed by allogeneic chondrocytes in rats

Rat syngeneic and allogeneic chondrocytes were transplanted intramuscularly or into defects prepared in articular cartilage (intracartilaginous transplants). Recipients of allogeneic transplants received cyclosporin A (CsA), cladribine (2-chlorodeoxyadenosine, 2-CdA), or both drugs in combination. Transplants were taken for examination after 5 weeks. Cartilage formed intramuscularly by syngeneic chondrocytes was ossified. Allogeneic cartilage was resorbed by infiltrating cells. CsA or 2-CdA partially suppressed, and both these agents in combination strongly suppressed, formation of infiltrations. Both syngeneic and allogeneic chondrocytes formed cartilage in joint surface defects but only allogeneic cartilage was attacked by infiltrating cells. CsA + 2-CdA treatment slightly decreased intensity of infiltrations but did not prevent cartilage resorption. Antichondrocyte response was studied by evaluation of spleen mononuclear cells (SMC) stimulation in mixed splenocyte-chondrocyte cultures and by detection of antichondrocyte cytotoxic antibodies. SMC stimulation index (SI) was calculated separately for syngeneic and allogeneic chondrocytes. Comparison of SMC SI for syngeneic and allogeneic chondrocytes indicated lack of stimulation of SMC from control or syngeneic transplant recipients and significant stimulation of SMC from recipients of allogeneic transplants. SMC from animals treated with CsA + 2-CdA were not stimulated. Additional experiments aiming at an explanation of the lack of stimulation of SMC from intact animals by syngeneic chondrocytes reported in this work and contrary to other findings disclosed that it was caused by the use of collagenase solution containing N alpha-p-tosyl-l-lysine chloromethyl ketone for chondrocyte isolation. Spontaneous antichondrocyte cytotoxic antibody activity was found in intact rats raised only in sera from recipients of allogeneic intramuscular transplants without immunosuppression. Thus, strong immunosuppressive treatment of rats with allogeneic chondrocyte transplants was more effective in relation to the general immunological response than to the local reaction.     

Osteogenesis by chondrocytes from growth cartilage of rat rib

Chondrocytes were isolated from growth cartilage and resting cartilage of rat rib and cultivatedin vitro. The cultivated chondrocytes were placed in Millipore diffusion chambers, which were then implanted into the abdominal cavities of rats for several weeks and prepared for histological analysis. The results indicate that growth cartilage cells have a remarkable osteogenic potential, even after cultivationin vitro, whereas resting cartilage cells show no osteogenic activity. However, growth cartilage cells alone do not form new bone but require the participation of certain host cells to initiate osteogenic differentiation.     

Effect of static compression on proteoglycan biosynthesis by chondrocytes transplanted to articular cartilage in vitro

Transplantation of chondrocytes by injection or within carrier matrices has shown promise for augmenting the repair of articular cartilage defects. In vivo, transplanted chondrocytes are exposed to mechanical forces. This in vitro study examined the effect of a step application of compressive load to chondrocytes after the cells had been seeded onto a cartilage surface. Bovine chondrocytes were transplanted onto bovine cartilage disks, allowed to attach for 1 hour or 4 days, and subjected to compression through overlying cartilage disks in a confined compression configuration. Before use, the disks were lyophilized to lyse the endogenous chondrocytes and thereby allow assessment of the metabolic activity of the transplanted cells. During a 16-hour application of compressive stress of 0-24-0.72 MPa, proteoglycan synthesis, assessed as [³⁵S]sulfate incorporation into macromolecules. was inhibited by approximately 68% after the 1-hour attachment and by approximately 45% after the 4-day attachment. Cell retention after the application of load was assessed by use of [³H]thymidine-tagged chondrocytes and quantitation of the displacement of radioactivity. After the 1-hour seeding period, loading induced a dose-dependent dislodgment of [³H]radioactivity (as much as 35%) from the tissue bilayer. In contrast, after the 4-day seeding period, there was no detectable effect of loading on chondrocyte dislodgment with an 8–12% release of radioactivity. The inhibitory effect of a 16-hour compression of 0.48 MPa applied after the 4-day seeding period was studied further. This protocol did not appear to have an irreversible effect on chondrocyte metabolism; at 2 days after the release of load, proteoglycan synthesis by the loaded cells was stimulated by 41% compared with transplanted cells that were not subjected to loading. These results suggest that the application of static compressive stress to chondrocytes at a cartilage surface may affect biosynthesis by these cells and thus subsequent integrative cartilage repair. Such an effect may have implications for optimization of the tightness of the press fit of a cell-laden cartilaginous construct into an articular defect.     

Mechanical barrier as a protection against rejection of allogeneic cartilage formed in joint surface defects in rats

Cartilage formed in transplants of allogeneic chondrocytes into joint cartilage defects in rats was infiltrated by immune cells migrating from the bone marrow while the surface on the side of the joint cavity remained free of infiltrations. This suggested that immunization occurred via bone marrow and not via joint cavity. Because articular cartilage is nourished exclusively by the synovial fluid, we have attempted to prevent cartilage rejection by protecting transplants from the contact with bone marrow. Defects in articular surface were filled with bone cement and chondrocytes were transplanted into a cavity prepared within the bone cement plug. Cartilage formed within the cement shell remained free of infiltrations and did not evoke systemic immunological response. However, distribution of glycosaminoglycans in the matrix of protected transplants was irregular. Cultures of chondrocytes growing in vitro on cement contained less glycosaminoglycans than the controls. This suggests that some factor(s) released from the cement unfavorably influenced chondrocytes and matrix production in protected transplants.     

Studies on bone formation by cartilage reconstructed by isolated epiphyseal chondrocytes, transplanted syngeneically or across known histocompatibility barriers in mice

Calcified cartilage transplants induce bone formation. This process may be inhibited if the recipient is immunized by the transplant. To study the relation between the degree of antigen incompatibility between the donor and recipient and bone formation in more detail, chondrocytes isolated from cartilaginous epiphyses of five-day-old mice were transplanted within a fully compatible syngeneic system and across weak (H-Y, non-H-2) and strong (H-2) histocompatibility barriers. Reconstruction of cartilage occurred in all cases. In these transplants, which did not evoke immunologic reaction (fully compatible system, transplants across H-Y barrier in nonrejector strain), reconstructed cartilage hypertrophied, calcified, yielded to resorption by mesenchyme, and finally, was replaced by bone. When (independently of the degree of antigenic difference) cartilage was surrounded by mononuclear infiltration, bone formation did not occur or was delayed. The presence of infiltrations around transplants led to the degeneration of chondrocytes as well as of matrix in peripheral regions of cartilage. Immunologic infiltration may prevent endochondral osteogenesis by inhibiting cartilage invasion by vascularized mesenchyme, and/or interfering with matrix mineralization. The function of the chondrocyte is not yet defined, but in endochondral ossification, it plays more than a passive role.     

不同生长阶段软骨细胞构建同种异体软骨的研究

目的　研究不同生长阶段山羊软骨细胞 ,形成同种异体组织工程化软骨组织的能力 ,为软骨组织工程寻找种子细胞来源。方法　取胎羊 (孕 10 0d)、新生羊 (10d龄 )及成年羊 (9个月龄 )各大关节软骨组织。酶消化法获取软骨细胞与生物材料Pluronic形成复合物 ,接种于异体羊 (9个月龄 )腹部皮下。按不同时间段取材。所取标本进行大体观察、称重 ,并作组织学检测 ,对新生的软骨组织进行评价。结果　各组接种处均有组织工程化软骨组织形成。胚胎组新生软骨的重量最大 (81.8± 17.9)mg及存留时间最长 (>12个月 )。结论　胚胎软骨细胞可以作为组织工程化软骨的种子细胞来源。     

Incidence of graft rejection in small bowel transplanted pigs after immunosuppression withdrawal

As intestinal grafts require heavy immunosuppression, there are no reports of immunosuppression withdrawal after clinical small bowel transplantation. In this large-animal study, we investigated the occurrence of graft rejection in intestinal-transplanted pigs after withdrawal. Large-White unrelated piglets were transplanted and divided in three groups: group 1 (n = 5), intestinal transplantation (ITx) with no immunosuppression; group 2 (n = 7), Itx and 60 days of treatment with tacrolimus and mycophenolate mofetil; group 3 (n = 5), Itx and donor bone marrow infusion (DBMi) and 60 days of treatment with tacrolimus and mycophenolate mofetil. Follow-up time after withdrawal was 120 days. Group 1 pigs died of graft acute cellular rejection (ACR) after a median of 11 days. In group 2, two pigs died of ACR-related infection and another two of ACR within 90 days. The remaining three animals (43%) were sacrificed at day 180, and their grafts showed no signs of ACR. In group 3, two pigs died of ACR-related infection and one of graft versus host disease within 80 days; at day 180 the two surviving animals showed signs of chronic rejection in the allograft. This study demonstrates that total withdrawal after ITx is followed by sudden and lethal ACR (or ACR-related infection) in more than 50% of the recipients. When a tolerance-inducing strategy as DBMi is applied, lethal graft versus host disease may also occur. In group 3, the intestinal allograft, to which the recipients were partially tolerant, developed chronic rejection that was probably associated with a decline with time of donor-leukocytes chimerism, as recently demonstrated in rats.     

Immunological response against allogeneic chondrocytes transplanted into joint surface defects in rats

Rat chondrocytes isolated from the articular-epiphyseal cartilage complex were transplanted into defects prepared in articular cartilage and subchondral bone. Transplants were taken for examination after 3 and 8 wk. Cartilage formed by syngeneic chondrocytes did not evoke formation of infiltrations. Contrary to that, in the vicinity of cartilage produced by allogeneic chondrocytes numerous infiltrating cells were present and cartilage resorption could be observed. Cyclosporine-A (CsA) treatment of recipients of allogeneic chondrocytes only partially suppressed accumulation of infiltrating cells and matrix resorption. Antichondrocyte immune response of chondrocyte graft recipients was studied by evaluation of spleen mononuclear cells (SMC) stimulation in mixed splenocytechondrocyte cultures and by evaluation of antichondrocyte cytotoxic antibodies. No difference in stimulation of SMC from intact rats by syngeneic and allogeneic chondrocytes was observed. Stimulation by allogeneic chondrocytes was slightly but significantly higher in recipients of syngeneic grafts. SMC of allogenic chondrocyte recipients were strongly stimulated by allogeneic chondrocytes. This response was absent in recipients treated with CsA. Spontaneous antichondrocyte cytotoxic antibody activity was detected in intact rats and in recipients of syngeneic grafts. In recipients of allogeneic chondrocytes the antibody response against allogeneic chondrocytes was raised but was statistically not significant owing to the considerable variation in the level of spontaneously occurring antichondrocyte antibodies.     

自体肋软骨隆鼻术的效果观察

目的 探讨应用自体肋软骨行隆鼻术的方法和效果.方法 自2010年3月至2014年2月,我们应用自体肋软骨对9例患者进行了隆鼻术.切取右侧第7肋软骨,长5～6 cm,雕刻成“柳叶”形.选择鼻小柱侧切口,鼻骨骨膜下钝性分离,形成相应的软骨植入腔隙;将雕刻好的“柳叶”形软骨植入腔隙,并将剩余软骨修剪成“盾牌”形,植于鼻尖点.结果 本组共9例患者.1例患者术后出现肋骨切取伤口脂肪液化,其余患者手术切口均Ⅰ期愈合;1例术后鼻背软骨轻度弯曲,其余患者鼻外形改善,效果满意.结论 自体肋软骨隆鼻效果良好,克服了假体隆鼻的缺点.     

In vitro study of isolated chondrocytes concerning the prognosis of transplanted cartilage]

In order to find out reliable criteria to conclude on the prognosis of transplanted cartilage the quality of isolated rabbit chondrocytes was assayed in vitro. Using a semisolid medium a culture system is introduced, which is able to detect colony forming units of chondrocytes. Furthermore their proliferative and differentiating capacity was investigated. Colony formation in vitro follows a strict dose response relationship. An inverse relationship was observed between the age of the donor and the colony incidence in vitro. Cells from neonatal cartilage could be induced to grow as fibroblastic colonies in the presence of conditioned medium from fibroblastic monolayer cultures. No effect, however, could be observed with chondrocyte derived conditioned medium.     

Paracrine effect of transplanted rib chondrocyte spheroids supports formation of secondary cartilage repair tissue

The study's objective was to investigate if transplanted chondrocyte or periosteal cell spheroids have influence on ingrowing bone marrow‐derived cells in a novel cartilage repair approach in miniature pigs. Autologous rib chondrocytes or periosteal cells were cultured as spheroids and press‐fitted into cavities that were milled into large, superficial chondral lesions of the patellar joint surface. Within the milled cavities, the subchondral bone plate was either penetrated or left intact (full‐thickness or partial‐thickness cavities). The transplantation of chondrocyte spheroids into full‐thickness cavities induced the formation of additional secondary repair cartilage that exceeded the original volume of the transplanted spheroids. The resulting continuous tissue was rich in proteoglycans and stained positive for type II collagen. Cell labeling revealed that secondarily invading repair cells did not originate from transplanted spheroids, but rather from arroded bone marrow. However, secondary invasion of repair cells was less pronounced following transplantation of periosteal cells and absent in partial‐thickness cavities. According to in vitro analyses, these observations could be ascribed to the ability of chondrocyte spheroids to secrete relevant amounts of bone morphogenetic protein‐2, which was not detected for periosteal cells. Transplanted chondrocyte spheroids exert a dual function: they provide cells for the repair tissue and have a stimulatory paracrine activity, which promotes ingrowth and chondrogenesis of bone marrow‐derived cells. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res     

抑制同种异体软骨细胞复合支架免疫反应研究进展

损伤或疾病造成的关节软骨缺损在临床上较常见,许多用于修复关节软骨损伤的方法各有优点,但均存在一定缺陷。组织工程学技术为关节软骨缺损的再生与修复提供了新思路,利用体外培养扩增的同种异体软骨细胞复合支架修复软骨缺损已有大量报道。该文就同种异体软骨细胞复合支架修复关节软骨损伤的免疫反应,在软骨细胞免疫原性、免疫反应发生途径、降低免疫反应机制及方法等方面作一综述。     

Immunologic pathways in a quantitative model of immunosuppression based on rejection of an allogeneic or xenogeneic tumor graft

BACKGROUND: A quantitative model of immunosuppression was previously developed based on the rejection of the allogeneic A/J murine tumor sarcoma 1 (Sa1) in immunocompetent mice. Here, the model is used to evaluate the immunologic mechanisms of graft rejection and to determine the potential of this model to detect synergistic effects of combined immunosuppressive therapies. METHODS: Wild-type, genetically-deficient, or drug-treated mice were used. Mice were challenged subcutaneously with the allogeneic murine tumor cell line, Sa1, or with the xenogenic human tumor, MDA435. Tumor growth was monitored with time, with increasing tumor size reflecting greater immunosuppression. In some cases, the mice were presensitized with either Sa1 or with A/J splenocytes. RESULTS: In na?ve recipient mice, studies in major histocompatibility complex (MHC)-I-deficient mice and with depleting anti-CD8 monoclonal antibody (mAb) demonstrate that CD8 T cells are important for Sa1 rejection. A modest role for perforin but not for Fas/Fas ligand could be demonstrated. Blockade of CD4 T cells was more effective with decreasing histocompatibility barriers. In contrast, CD4 T cells were critical in second-set rejections, but CD8 T cells were not. Rejection of xenogeneic tumors was also T-cell dependent as demonstrated by anti-CD3 mAb, dependent on both CD4 and CD8 T cells as demonstrated using MHC-I- and MHC-II-deficient mice, but was more vigorous as demonstrated by the lack of effectiveness of immunosuppressive drugs in this model. CONCLUSIONS: This model can be used to define dominant and partial effects of immunologic pathways as well as synergistic interactions of agents to develop immunosuppressive strategies.     

Malondialdehyde oxidation of cartilage collagen by chondrocytes

OBJECTIVE: The damage to cartilage collagen is a central event in the pathogenesis of cartilage aging and osteoarthritis (OA). We have previously developed an in vitro model of cartilage degradation which shows that chondrocyte-dependent lipid peroxidation mediates cartilage collagen degradation. The goal of our study was to investigate the role of vitamin C in this degradation model and to investigate effect of chondrocyte-dependent lipid peroxidation in the oxidation of cartilage collagen. METHODS: We studied primary articular chondrocytes. Effect of vitamin C was investigated in the previously described model. Serum-free stimulated and unstimulated chondrocyte-matrix extracts were subjected to SDS-PAGE and immunoblot analysis. Malondialdehyde (MDA)-protein oxidation of cartilage proteins was demonstrated by the reactivity of chondrocyte extracts to a monoclonal antibody, MDA2, which detects MDA-lysine adducts. RESULTS: Vitamin C treatment of chondrocyte cultures resulted in significant enhanced incorporation of 3H-proline label in cell-matrix. Cells treated with vitamin C, as compared to control untreated cells showed decreased spontaneous release of labeled matrix. Vitamin C treated or not treated chondrocytes responded comparably to stimulation with the agonist calcium ionophore A23187. The serum-free in vitro culture of chondrocytes resulted in MDA-protein oxidation. The treatment of chondrocytes with A23187 resulted in the enhancement of MDA-protein oxidation. The immunoblot reactivity pattern of extracts to MDA2 antibody and to polyclonal anti-type II collagen antibody was somewhat similar, which suggests that these two different types of antisera exhibit a crossreaction to chondrocyte proteins. Chondrocyte extracts were pretreated both with and without pure collagenase, and then subjected to immunoblot analysis. Only collagenase treated extracts showed a disappearance, or significant reduction, of larger than 60 kDa size MDA2 immunoreactive proteins. This suggests that the proteins that disappeared after the enzyme treatment were collagen proteins and which had also been modified by MDA oxidation. CONCLUSIONS: These observations suggest that collagen hydroxylation of matrix by vitamin C does not play a role in this model of chondrocyte-dependent collagen degradation. Also, this study demonstrates that chondrocyte-derived lipid peroxidation product MDA mediates oxidation of cartilage collagens. Oxidative modification of cartilage collagen in vivo could result in alteration of biochemical and biophysical properties of cartilage collagen fibrils, making them prone to degradation, thus initiating the changes observed in aging and OA.     

Effect of gravity on localization of chondrocytes implanted in cartilage defects.

The transplantation of autologous chondrocytes under a periosteal flap has been used to treat focal cartilage defects. Results have been promising but occasionally involve complications ranging from graft hypertrophy to detachment. The objective of this study was to determine if gravitational forces affect the uniformity of cell distribution within the defect. Using an ex vivo bovine model, the orientation relative to gravity of a repaired full-thickness articular cartilage defect was found to affect the initial distribution of transplanted chondrocytes, prelabeled with 3H-thymidine. After 4 hours, the injected cells (3H-radioactivity) were primarily at the base of the defect (79%) in samples oriented in the up position, primarily at the dependent semicylindrical half of the defect (83%) in samples oriented to the side, and primarily at the periosteal top of the defect (78%) in samples oriented upside-down. Subsequently, the cell distribution remained unchanged after reorientation into other positions. These results indicate that injected chondrocytes localize under the influence of gravity within the initial few hours after injection. Therefore, the defect orientation during this time can be an important factor in the uniformity of cell distribution in the autologous chondrocyte implantation procedure and may be an important determinant of the ultimate clinical outcome.