Co-culture of mechanically injured cartilage with joint capsule tissue alters chondrocyte expression patterns and increases ADAMTS5 production.

We studied changes in chondrocyte gene expression, aggrecan degradation, and aggrecanase production and activity in normal and mechanically injured cartilage co-cultured with joint capsule tissue. Chondrocyte expression of 21 genes was measured at 1, 2, 4, 6, 12, and 24h after treatment; clustering analysis enabled identification of co-expression profiles. Aggrecan fragments retained in cartilage and released to medium and loss of cartilage sGAG were quantified. Increased expression of MMP-13 and ADAMTS4 clustered with effects of co-culture, while increased expression of ADAMTS5, MMP-3, TGF-beta, c-fos, c-jun clustered with cartilage injury. ADAMTS5 protein within cartilage (immunohistochemistry) increased following injury and with co-culture. Cartilage sGAG decreased over 16-days, most severely following injury plus co-culture. Cartilage aggrecan was cleaved at aggrecanase sites in the interglobular and C-terminal domains, resulting in loss of the G3 domain, especially after injury plus co-culture. Together, these results support the hypothesis that interactions between injured cartilage and other joint tissues are important in matrix catabolism after joint injury.

Structure of pericellular matrix around agarose-embedded chondrocytes.

OBJECTIVE: Determine whether the structure of the type VI collagen component of the chondrocyte pericellular matrix (PCM) generated by agarose-embedded chondrocytes in culture is similar to that found in native articular cartilage. METHODS: Confocal microscopy, quick-freeze deep-etch electron microscopy, and real-time polymerase chain reaction (PCR) were used to investigate temporal and spatial patterns of type VI collagen protein deposition and gene expression by bovine chondrocytes during 4 weeks of culture within a 2% agarose hydrogel. Similar analyses were performed on chondrocytes within samples of intact cartilage obtained from the same joint surfaces as those used for cell isolation for comparison. RESULTS: Type VI collagen accumulated uniformly around cells embedded in agarose, with the rate of deposition slowing after the second week. After 1 week, PCM fibrils were observed to be oriented perpendicular to the cell surface, in contrast with the primarily tangential fibrillar arrangement observed in native articular cartilage. Expression of col6 in agarose-embedded cells was initially much higher ( approximately 400%) than that in chondrocytes within cartilage. Expression of col6 in the cultured chondrocytes declined by approximately 60% after 1 week, and remained stable thereafter. CONCLUSIONS: PCM structure and composition around cells in a hydrogel scaffold may be different than that in native cartilage, with potential implications for mass transport, mechanotransduction, and ultimately, the success of tissue engineering approaches.

Integrative articular cartilage repair: dependence on developmental stage and collagen metabolism.

OBJECTIVES: The objectives of this research were to determine whether the integrative repair of bovine cartilage explants was dependent on developmental stage, and whether observed differences in integration with developmental stage were related to deposition of newly synthesized collagen and lysyl oxidase-mediated collagen cross-linking. METHODS: Pairs of fetal, newborn calf, and adult bovine cartilage blocks were cultured in partial apposition for 2 weeks in medium supplemented with serum, ascorbate, and [3H]proline. Following culture, mechanical integration between apposed cartilage blocks was assessed by measuring adhesive strength in a single-lap shear configuration. Formation and stabilization of newly synthesized protein and collagen was investigated by determination of [3H]proline and [3H]hydroxyproline in tissue digests and guanidine extracts. RESULTS: Calf cartilage exhibited a relatively high integrative repair phenotype, achieving an adhesive strength that was three--four-fold that of adult or fetal specimens. The low and high integrative repair phenotypes appeared related in part to different levels of collagen biosynthesis, which was approximately four--five-fold higher in calf cartilage samples than in the adult. However, fetal cartilage also exhibited a high level of biosynthesis. The different integrative repair phenotypes were not associated with marked differences in the kinetics of chemical stabilization of newly synthesized collagen, as the proportion of incorporated [3H]proline and newly-formed [3H]hydroxyproline that was resistant to extraction by 4M guanidine-HCl following culture was similar for cartilage from all developmental stages. Integration of calf cartilage appeared to depend on lysyl oxidase-mediated collagen cross-link formation, since inclusion of beta-aminopropionitrile (BAPN) in the culture medium completely eliminated development of adhesive strength. BAPN treatment also increased the percentage of newly synthesized protein in the guanidine extracts from 10% to 36% of the total, and that of newly synthesized collagen from 2% to 20%, while having only slight inhibitory effects on overall protein and collagen biosynthesis. CONCLUSION: The finding that cartilage exhibits enhanced integrative repair at a certain developmental stage suggests that it may ultimately be possible to enhance repair when needed in clinical situations.

Integrative cartilage repair: adhesive strength is correlated with collagen deposition.

Procedures to repair focal articular cartilage defects often result in poor integration between the host cartilage and the graft tissue, and this may be related to the lack of matrix deposition and the death of chondrocytes near a cut cartilage surface. The objective of this study was to determine if cartilage repair was related to deposition of newly synthesized collagen. The mechanical integration that occurred between two live adult bovine cartilage blocks cultured in partial apposition for two weeks was correlated with [3H]proline incorporation, a measure of protein synthesis, of which more than 66% was accounted for by collagen. A similar level of mechanical integration occurred in sample pairs consisting of a live and killed cartilage block, and this adhesive strength was also correlated with [3H]proline deposition into both the live and the killed blocks. In these samples, the [3H]proline deposited into the killed cartilage appeared to originate from chondrocytes in the live cartilage, since live cells were not detected in the killed cartilage block by either viability staining or [35S]sulfate incorporation. These results suggest a mechanism of integrative cartilage repair in which live chondrocytes within cartilage secrete matrix molecules that are components of a collagen network, and subsequent deposition of these molecules near the repair interface contributes to functional integration.