Faculty Bibliography

Anchorin CII is a collagen binding protein of the annexin family associated with plasma membranes of chondrocytes, osteoblasts, and many other cells. As a major constituent of cartilage-derived matrix vesicles it has been shown to bind to native type II and X collagen. In accordance with this observation, here we show the localization of anchorin CII in the extracellular matrix of calcifying cartilage in the fetal human growth plate, and that it was restricted to the chondrocyte surface in proliferating and resting cartilage. Furthermore, we present evidence, using a slot blot assay, that anchorin CII not only binds to native type II and X collagen, but also to chondrocalcin, the carboxy-terminal extension of type II procollagen, in a calcium-independent manner. Pepsin digestion of type II collagen results in loss of anchorin CII binding, confirming our previous notion that the telopeptide region of type II collagen carries anchorin CII binding sites

The tissue localization was analysed of collagen X during human fetal and juvenile articular cartilage-bone metamorphosis. This unique collagen type was found in the hypertrophic cartilage zone peri- and extracellularly and in cartilage residues within bone trabeculae. In addition, occasionally a slight intracellular staining reaction was found in prehypertrophic proliferating chondrocytes and in chondrocytes surrounding vascular channels. A slight staining was also seen in the zone of periosteal ossification and occasionally at the transition zone of the perichondrium to resting cartilage. Our data provide evidence that the appearance of collagen X is mainly associated with cartilage hypertrophy, analogous to the reported tissue distribution of this collagen type in animals. In addition, we observed an increased and often 'spotty' distribution of collagen X with increasing cartilage 'degeneration' associated with the closure of the growth plate. In basal hypertrophic cartilage areas, a co-distribution of collagens II and X was found with very little and 'spotty' collagen III. In juvenile cartilage areas around single hypertrophic chondrocytes, co-localization of collagens X and I was also detected

Evidence from recent studies on type X collagen in hypertrophic chick cartilage suggests that it may be involved in cartilage calcification. Here we compare the distribution of type X collagen with that of calcium mineral deposition in fetal human growth plate cartilages of long bones and ribs. Using a specific antibody we demonstrate the presence of type X collagen in a narrow, sharply defined zone of hypertrophic chondrocytes. Type X collagen was also localized in the calcifying cartilage remaining within spongy bone trabecules. Calcium deposits were, however, detected by alizarine red S only in the lower hypertrophic zone and in bone, confirming the notion that type X collagen is deposited in the hypertrophic cartilage before mineral deposition. By immunofluorescence double staining we demonstrate codistribution of type II and X collagen in the hypertrophic zone, while type I collagen was absent from hypertrophic cartilage matrix; it was detected only in the perichondrium, in vascular cavities, and in osteoid and bone. From these observations we conclude that the sequence of events leading to cartilage mineralization begins with chondrocyte hypertrophy, followed by type X collagen synthesis and finally by deposition of calcium mineral

OBJECTIVE: To investigate the appearance of hypertrophic chondrocytes in osteoarthritic (OA) cartilage, using type X collagen as a specific marker. METHODS. The biosynthesis of type X collagen was examined by metabolic labeling of freshly isolated articular chondrocytes with 3H-proline, immunoprecipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the synthesized collagens. Extracellular deposition of types X and II collagen was analyzed immunohistochemically. RESULTS. Immunostaining revealed an irregular distribution of type X collagen, which was localized around chondrocyte clusters in fibrillated OA cartilage, but was absent from the noncalcified region of normal articular cartilage. Freshly isolated OA chondrocytes synthesized predominantly type X collagen, while control chondrocytes synthesized mostly type II collagen. CONCLUSION. Our findings indicate focal premature chondrocyte differentiation to hypertrophic cells in OA cartilage

Type X collagen is a developmentally regulated collagen that is only synthesized by chondrocytes of the hypertrophic and calcifying zone in fetal cartilage. There is evidence in the literature that type X collagen may be involved in cartilage calcification. Here we show that type X collagen synthesis precedes calcium deposition in nodules of fetal human chondrocytes forming in cell culture and present evidence that type X collagen binds calcium in a specific and dose dependent manner. In an assay using bovine type X collagen coupled to beads and 45Ca2+ we determined a total of about 15 binding sites per alpha 1(X) chain with a dissociation of 32 microM

Type X collagen was extracted with 1 M NaCl and 10 mM dithiothreitol at neutral pH from fetal human growth plate cartilage and purified to homogeneity by gel filtration and anion-exchange chromatography. The purified protein migrates in SDS/polyacrylamide gels with an apparent Mr of 66,000 under reducing conditions, and as a high-Mr oligomer under non-reducing conditions. Purified collagenase digests most of the molecule; pepsin digestion at 4 degrees C decreases the Mr of the monomer to 53,000. A rabbit antiserum was raised against purified human type X collagen; the IgG fraction was specific for this collagen by criteria of ELISA and immunoblotting after absorption with collagen types I, II, VI, IX and XI. Immunohistological studies localized type X collagen exclusively in the zone of hypertrophic and calcifying cartilage

Type X collagen was extracted with 1 M-NaCl and 10 mM-dithiothreitol at neutral pH from fetal-bovine growth cartilage and purified to homogeneity by using f.p.l.c. gel filtration on a Superose 12 column, followed by ion-exchange chromatography on a Mono Q column. The purified protein migrates in SDS/polyacrylamide gels with an apparent Mr of 58,000 under reducing conditions and as a high-Mr oligomer in its unreduced form. The amino acid composition is similar to the published composition of chick type X collagen. Pepsin digestion at 4 degrees C decreases the Mr of the monomer to 43,000; purified bacterial collagenase digests most of the molecule, leaving a non-collagenous domain of apparent Mr 15,000, which probably represents the C-terminal globular domain. The IgG fraction from a rabbit antiserum raised against purified bovine type X collagen was specific for this collagen by the criteria of e.l.i.s.a. and immunoblotting after immunoabsorption with collagen types I, II, IX and XI. Immunofluorescence localization of type X collagen in sections of fetal-bovine and human cartilage was possible after acetone fixation of sections and hyaluronidase treatment. Type X collagen was restricted to the zone of hypertrophic and calcified cartilage inside the bone spicules of the growth plate