Anna Hyc

Differences in Cartilage Formed Intramuscularly or in Joint Surface Defects by Syngeneic Rat Chondrocytes Isolated from the Articular-Epiphyseal Cartilage Complex

Syngeneic rat chondrocytes isolated from the articular-epiphyseal cartilage complex were suspended in hyaluronic acid and transplanted intramuscularly or into joint surface defects. Transplants were fixed in ruthenium hexammonium trichloride and embedded in glycol methacrylate. In cartilage nodules produced intramuscularly, chondrocyte hypertrophy and matrix calcification were observed after 2 wk. Partial ossification occurred after 4 wk and the cartilage was almost completely replaced by an ossicle after 8 wk. Only small, dispersed groups of chondrocytes remained within the ossicle. In cartilage formed in joint surface defects a superficial and a deep zone were distinguished. Chondrocytes in the superficial zone did not hypertrophy and cartilage remained unossified. In the deep zone matrix calcification and bone formation occurred. These processes were, however, retarded in comparison with intramuscular transplants. Thus, either intraarticular environment exerted an inhibitory effect on chondrocyte hypertrophy and matrix calcification or articular chondrocytes present among transplanted cells accumulated close to the joint lumen and reconstructed normal articular cartilage.

Influence of LPS, TNF, TGF-ß1 and IL-4 on the expression of MMPs, TIMPs and selected cytokines in rat synovial membranes incubated in vitro

Synovial membranes are formed by four main types of cells, i.e. fibroblasts, macrophages, epitheliocytes and adipocytes. To study the combined effect of various factors on these cell populations, synovial membranes dissected from rat knee joints were incubated in control medium or medium with lipopolysaccharide (LPS), TNF, TGF-ß1 or IL-4 for 12 h. LPS stimulated TNF secretion and both agents stimulated secretion of IL-6. TGF-ß1 slightly increased IL-6 secretion. LPS increased the mRNA levels of IL-6, IL-1ß, TGF-ß1, MMP1a, MMP1b, MMP3, MMP9, MMP13, MMP14, TIMP1 and TIMP3 while the mRNA levels of MMP2, TIMP2 and TIMP4 were significantly decreased. Expression of IL-1ß, MMP1a, MMP1b, MMP3, MMP9, MMP13 and TIMP1 increased after TNF treatment, while mRNA levels of MMP2, MMP14, TIMP2, TIMP3 and TIMP4 were decreased. TGF-ß1 decreased the mRNA levels of IL-1ß, all MMPs, TIMP1, TIMP2, TIMP4 and increased mRNA levels of itself and TIMP3. IL-4 decreased mRNA levels of IL-1ß, TGF-ß1, MMP2, MMP9, MMP13 and all TIMPs. Only LPS decreased the amount and activity of MMP2. The effect of LPS and cytokines on most of the MMPs and TIMPs produced by whole synovial membrane was in good agreement with previous studies on their action on similar types of cells as those present in synovial membranes, but originating from other tissues. All tested agents decreased MMP2 mRNA expression levels and in the case of LPS also the protein level and its activity determined by zymography, contrary to previous observations on isolated cell populations. This indicates that the response of the organized tissue is an interplay of all components and cannot be deduced from the individual reactions.

Transplants of rat chondrocytes evoke strong humoral response against chondrocyte-associated antigen in rabbits.

Rat chondrocytes transplanted intramuscularly in rabbits produced cartilage. In 1-day-old transplants chondrocytes remained viable. After 1 week peripheral chondrocytes of the transplant were dead and the cartilage was surrounded and resorbed by macrophages. In 2-week-old transplants cartilage deteriorated and was invaded by fibroblast-like cells and macrophages. Sera of rabbits that received two or three consecutive transplants of rat chondrocytes with 2-week intervals contained high titer of antichondrocyte cytotoxic antibodies. A part of the cytotoxic activity could be removed by absorption with rat splenocytes. Western blot analysis of lysates from fresh or 24-h cultured chondrocytes with absorbed sera detected antigen with Mr of ~74 and ~23 kDa. Only the latter remained after reduction in 2-mercaptoethanol. In lysates of fibroblasts and endotheliocytes the 23-kDa antigen was not found but the serum reacted with Mr 39-kDa antigen. In lysates of thymocytes a weak band corresponding to Mr of 35 kDa was present. Serum from rabbits receiving transplants of living chondrocytes followed by chondrocytes suspended in complete Freunds adjuvant contained antibodies directed against components of crude collagenase used for cell isolation. Such antibodies could not be detected in sera of rabbits receiving transplants of living chondrocytes only. Molecular weight of detected antigen differs from that of collagen type II, core of aggrecan, link proteins, and several other macromolecules of cartilage matrix. It could represent either a component of chondrocyte membrane or a membrane-bound substance resistant to enzymes used for isolation. Availability of antibodies against presumably chondrocyte-specific antigen produced during transplant rejection may help to characterize it more precisely and to ascertain whether its presence may influence results of autogenous chondrocyte transplants in humans.

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 splenocyte-chondrocyte 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.

Effect of immunosuppression on rejection of cartilage formed by transplanted allogeneic rib chondrocytes in mice.

The effect of short-term immunosuppressive treatment with antithymocyte serum-procarbazine (ATS-PCH) and cyclosporin-A (Cy-A) on survival of allogeneic rib chondrocyte grafts was examined morphologically and by evaluation of specific humoral and cellular antigraft immunity. The latter were evaluated by means of leukoagglutination and the indirect migration inhibition assay, respectively. Untreated recipients of syngeneic rib chondrocytes and untreated recipients of whole syngeneic and allogeneic rib cartilage served as controls. Transplanted syngenic rib chondrocytes formed cartilaginous nodules similar to rib cartilage in situ. These nodules contained hypertrophied chondrocytes, but neither physiologic resorption by vascularized connective tissue nor bone formation occurred after an observation period of longer than six weeks. Transplantation of allogeneic chondrocytes resulted in development of humoral and cellular antigraft immunity, and the formed cartilage was destroyed by infiltrating immune cells. Immunosuppression by ATS-PCH resulted in inhibition of graft destruction and a marked decrease of specific humoral antigraft immunity. Cellular antigraft immunity did not occur. Moreover, neither the histologic appearance of the cartilaginous nodules nor the results of immunologic response evaluations in the ATS-PCH-treated group differed from those in untreated whole allogeneic cartilage recipients. Treatment with Cy-A did not significantly improve survival cartilage formed by allogeneic chondrocytes.

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 Nα-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.

Chondrocyte-associated antigen and matrix components in a 2- and 3-dimensional culture of rat chondrocytes

The goal of this study was to compare expression of chondrocyte-associated antigen (CAA) and cartilage matrix molecules in 2-D (monolayer) and 3-D (Matrigel) culture. Chondrocytes isolated from the cartilage of 3-day-old rats were expanded in monolayer culture for 28 days. CAA expression gradually decreased and was not detected beyond the 96th hour of monolayer culture. Collagen type II and aggrecan mRNA levels decreased during culture. The collagen type I mRNA level increased during the first week and remained high. The increase in the versican mRNA level was less pronounced during the first week and declined slightly after further cultivation. Freshly isolated chondrocytes introduced into Matrigel still expressed CAA after 7 days. Moreover, CAA expression returned in chondrocytes re-cultured in Matrigel after 7 days in monolayer. Similarly, the increase in the mRNA levels of collagen type II and aggrecan in Matrigel was limited to freshly isolated chondrocytes and to those that remained in monolayer for 1 week. Collagen type I mRNA in monolayer and Matrigel cultures of freshly isolated chondrocytes was at a similar level. The introduction of freshly isolated or 7-day monolayer-cultured chondrocytes into Matrigel caused a decrease in the versican mRNA level in comparison with 7- and 14-day 2-D cultures, respectively. On the other hand, chondrocytes seeded in Matrigel after 14 days of monolayer culture did not express CAA, showed decreased levels of collagen type II and aggrecan mRNA and an increase in versican mRNA. In conclusion, it appears that changes in the expression of CAA in 2- and 3-D cultures occur in parallel to changes in typical cartilage matrix molecule expression.

Cartilage produced after transplantation of syngeneic chondrocytes is rejected in rats presensitized with allogeneic chondrocytes.

Cartilage produced in 2-week-old intramuscular transplants of syngeneic chondrocytes in rats did not display any signs of rejection. Cartilage produced by similar transplants in animals presensitized with intramuscular transplants of allogeneic chondrocytes was surrounded by infiltrations composed mainly of lymphocytes and was partially resorbed. Spleen mononuclear cells (SMC) from recipients of syngeneic transplants alone were not stimulated in mixed splenocyte–chondrocyte cultures by syngeneic or allogeneic chondrocytes. SMC from recipients of allogeneic and subsequent syngeneic transplants were strongly stimulated by both syngeneic and allogeneic chondrocytes, although stimulation by the latter was significantly more pronounced. Sera from naive rats usually contained cytotoxic antichondrocyte antibodies but their level varied considerably in various individuals. In rats chosen as transplant recipients on the basis of low antichondrocyte cytotoxicity of their sera, this toxicity was markedly raised after sensitization with allo- and syngeneic chondrocytes. Absorption with thymocytes or fibroblasts decreased but did not abrogate cytotoxicity. These observations support previous reports suggesting expression of tissue-specific antigen(s) by chondrocytes.

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.

Influence of cartilage interstitial fluid on the mRNA levels of matrix proteins, cytokines, metalloproteases and their inhibitors in synovial membrane

Articular cartilage and the synovial membrane both ensure the smooth action of synovial joints; however, the influence of chondrocytes on synovial metabolism remains unclear. The secretory activity of chondrocytes is usually studied in cell cultures and may differ from that in intact cartilage. According to McCutchens theory of weeping joint lubrication, loading of the articular cartilage during motion squeezes the fluid with lubricating properties from the cartilage. The purpose of the study was to obtain cartilage interstitial fluidxa0(CIF) from intact cartilage and to evaluate its influence on gene expression in the synovial membrane cells. CIF was rinsed out from the cartilage of newborn rats at a pressure of three bar. The chondrocytes survived rinsing and grew in culture. Cytokines in CIF were detected using the enzyme-linked immunosorbent assayxa0(ELISA). The influence of CIF and CIF-like cocktail (all cytokines found in CIF) on gene expression in the synovial membrane cells was studied after a 4xa0h-incubation, by real-time PCR. Data were analyzed using the Wilcoxon matched-pair test or by the Mann‑Whitney Uxa0test. CIF contained basic fibroblast growth factorxa0(bFGF), insulin-like growth factorxa0(IGF)‑1, transforming growth factorxa0β1xa0(TGFβ1), bone morphogenetic proteinxa07xa0(BMP7), macrophagexa0(M)-colony-stimulating factorxa0(CSF), granulocytexa0(G)-CSF and leukemia inhibitory factorxa0(LIF). CIF stimulated the expression of hyaluronan synthasexa0(HAS)1xa0andxa02, lubricin, collagenxa0I, versican, aggrecan, matrix metalloproteinasesxa0(MMPs)2xa0andxa03, tissue inhibitors of metalloproteinasesxa0(TIMPs)xa01-3, interleukinxa0(IL)-6 and TGFβ1, and decreased the expression of tumor necrosis factorxa0(TNF) and IL-1β. Incubation of the synovial membrane with CIF-like cocktail partially imitated the effects of CIF. Analysis of CIF composition may help to characterize the secretory activity of chondrocytes in their natural environment under various physiological and pathological conditions and to understand the interactions between articular cartilage and the synovial membrane.