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Collagen type III is consistently detected by immunofluorescence in samples of normal and osteoarthritic human articular cartilage (Aigner et al., 1993; Wotton and Duance, 1994; Fig. 3). By transmission electron microscopy, type III collagen was found co localized with type II collagen in the same banded fibrils and retaining its N-propeptide domain (Young et al., 2000). In another study of osteoarthritic cartilage, collagen III tended to be concentrated in the superficial and upper middle zones and to be synthesized by the chondrocytes in the absence of collagen I expression (Aigner et al., 1993).
Using an antibody that specifically recognizes the Ctelopeptide domain of type II collagen and Western blot analysis (Fig. 4), we could show that the pool of type III collagen extractable by pepsin from adult human and bovine articular cartilage included molecules that had been covalently linked to type II collagen in the tissue. The pool from 5-yr bovine articular cartilage had to be enriched by molecular sieve chromatography in order to be seen on SDS-PAGE by Coomassie Blue staining (Fig. 4, left panel). By isolating cross-linked peptides and determining their structure by sequence analysis and mass spectrometry, we established that type III collagen is copolymerized and linked to collagen II in adult human articular cartilage in significant amounts (Fig. 5) (Wu et al., 1994). It is tempting to speculate that collagen III is made by chondrocytes in addition to collagen II in response to matrix damage akin to the wound-healing role of collagen III in type I collagenbased tissues.
After skeletal growth has ceased, the rate of type II collagen synthesis by articular chondrocytes drops dramatically as assessed by proline labelling in vivo. In the adult tissue, however, some synthesis continues, and this can be accelerated up to 10-fold within 2 weeks after joint injury, for example after anterior cruciate ligament section in the mature dog (Eyre et al., 1980). Little is known of synthetic rates of the other collagen types in adult articular cartilage. Observations based on the synthetic rate of hydroxyproline indicate very little turnover of the collagenous component of the matrix as a whole, with an estimated turnover time of 400 years for human femoral head cartilage (Maroudas, 1979). This still leaves the possibility that a sub fraction of the collagenous matrix (e.g., fibril surface molecules and the pericellular domain) is remodelled more rapidly by chondrocytes in response to mechanical and molecular signals. If the bulk of the collagen mass, which is embodied in the thicker, mature fibrils of the interterritorial matrix, persists in maturity without turnover, then the average turnover rate of the collagen as a whole would still be very slow. Indeed, the mean diameter of banded collagen fibrils in mature human articular cartilage increases with age (Lane and Weiss, 1975), consistent with this remodelling concept.
Mechanisms of collagen degradation
Tissue sites of proteolysis and denaturation of matrix type II collagen can be observed in normal and osteoarthritic joint surfaces (Poole et al., 1995) using specific antibodies. The classical concept of collagen fibril degradation is through an initial cleavage of the collagen molecule (type I, II or III) by collagenase into three-quarter and onequarter length fragments. Articular chondrocytes can express collagenases, including collagenase-3 (MMP13) (which is the most active in cleaving type II collagen), as demonstrated in culture under interleukin-1 stimulation or directly in tissue removed from arthritic joints (Murphy et al., 1999). This enzyme therefore is implicated in the breakdown of cartilage collagen in osteoarthritis. Of the growing number of matrix metalloproteinases that may contribute to matrix protein metabolism (Krane et al., 1996), the collagenases are perhaps the best understood in terms of the natural substrate. However, an essential role for collagenases in all forms of collagen breakdown and turnover is becoming less certain. For instance, in mice genetically engineered to express type I collagen lacking a functional cleavage sequence at the three-quarter site, no phenotype was evident at birth. Only later did mild skin thickening and uterine fibroses develop, implying that alternative degradation mechanisms not requiring the three-quarter cleavage can provide for essentially normal development, growth and remodelling of most collagen type I-based tissues (Eyre and Wu, 1987).
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