The present study was undertaken to examine how osteoarthritis affects the expression of type-X collagen, a hypertrophic chondrocyte-specific collagen in articular cartilage. A well characterized sheep polyclonal antiserum, as well as three mouse monoclonal antibodies against canine type-X collagen, was used to immunolocalize type-X collagen in human and canine joints. Its expression in osteoarthritic cartilage was altered in several locations. In the canine osteoarthritic joints, type-X collagen increased in and just above the zone of calcified cartilage and was present diffusely throughout the calcified matrix. In both the human and canine cartilage, type-X collagen was localized around cell clones in the transitional zone of cartilage. This is surprising, since that region of the cartilage does not calcify and one of the proposed roles of type-X collagen is in mineralization. Thus, the osteoarthritic process may damage the matrix in the superficial layer and induce changes leading to the expression of the hypertrophic chondrocyte phenotype.
Summary: Type X collagen was extracted from ends of canine growth plates by pepsin digestion after 4 M guanidine hydrochloride extraction, purified by stepwise salt precipitation (2.0 M NaCl in 0.5 M acetic acid), and chromatographed on a Bio-Gel A1.5 M column in 1.0 M CaCl,. Without reduction on sodium dodecyl sulfate (SDS) polyacrylamide gels, the preparation yielded a single, high-molecular-weight (mol wt) band; after reduction, a single band of relative mol wt 5.0 x lo4 was found. Polyclonal sera were raised against the purified collagen and used in the immunolocalization of canine type X collagen. As expected, indirect immunoperoxidase (IP) or indirect immunofluorescent staining with the polyclonal sera demonstrated that most of the immunoreactivity was localized in the zone of provisional calcification of the growth plate and in cartilage remnants in the metaphyseal region of the physis. A progressive decrease in staining toward the diaphysis of the fetal canine long bone was apparent as the trabecular structures were remodeled to bone. Unexpectedly, type X collagen was also detected in the zone of calcified, mature articular cartilage. It was concentrated in the pericellular matrix of the chondrocytes, appeared at or just above the tidemark, and was expressed immediately before mineralization. Identification of type X collagen in both the canine growth plate and the zone of calcified articular cartilage suggests that cells in the deep layer of cartilage and in the zone of calcified cartilage in the adult animal retain some characteristics of a growth plate and may be involved in regulation of mineralization at this critical interface. The expression of growth plate-like properties would allow the deep chondrocytes of mature articular cartilage to play a role in remodeling of the joint with age and in the pathogenesis of osteoarthritis. Key Words: Type X collagen-Zone of calcified cartilageImmunohistochemistry-Growth plate-Mineralization-Tidemark.Much of the knowledge concerning type X collagen has been derived from the chick model (7,11,12,21,23,(35)(36)(37)(38)(39). The structure, biosynthesis, and location of type X collagen in chick cartilage have been demonstrated (7,11,12,17,(21)(22)(23)(24)27, 3540 pertrophic chondrocytes, is synthesized a s a procollagen composed of three identical pro a ( x ) chains of molecular weight (mol wt) of 59,000. The chick type X molecule is 138 nm long and consists of a triple helical domain flanked by a globular peptide on the amino terminus and a short noncollagenous piece on the carboxyl terminus.Type X collagen has been localized to zones of hypertrophic chondrocytes in chick cartilage by both biochemical and immunohistochemical tech-
Purpose: We and others have previously shown that periostin expression is dramatically elevated in human OA cartilage and in three surgical models of OA (medial menisectomy and anterior crucial ligament, partial meniscectomy) in rodents. In vitro periostin promotes collagen and proteoglycan degradation in human chondrocytes via activation of MMP-13 and ADAMTS4 expression. Therefore, we hypothesized that periostin-deficient mice may be protected from surgically-induced posttraumatic OA. Periostin has been shown to control gene expression in bone cells by interacting with avb3 integrin. However, the nature of periostin receptor(s) in chondrocytes is unknown. DDR1, a receptor tyrosine kinase, is highly expressed in chondrocytes and controls MMP-13 expression during chondrogenesis. Therefore, we hypothesized that the effect of periostin on chondrocytes is mediated by DDR1. Methods: Periostin (Postn) knockout (PostnÀ/À) mice were purchased from Jackson Laboratory (B6;129-Postntm1Jmol/J Stock No: 009067). We subjected 3-months old littermates (Postnþ/þ, Postnþ/À and PostnÀ/À) to partial medial menisectomy (PMX) or sham surgery, and harvested the knee joints 8 week post-surgery for histological assessment of OA progression. Human OA cartilage explant cultures were incubated in the presence or absence of the DDR1 inhibitor DDR1-IN-1 dihydrochloridein (100e500 nM) for 2 h before addition of periostin (1 mg/ml) or control vehicle to the culture medium. MMP-13 levels were determined by ELISA-24 h post stimulation. Results: PostnÀ/À deficient mice showed reduced PMX-induced cartilage degeneration and osteophyte formation relative to Postnþ/þ mice. We also observed reduced subchondral bone thickening in both Postnþ/À and PostnÀ/À mice relative to Postnþ/þ controls. In ex vivo studies, pre-incubation of human cartilage explants with the DDR1 inhibitor, DDR1-IN-1 dihydrochloridein, inhibited both constitutive and periostin-induced MMP-13 expression in a dose-dependent manner ( Fig. 1). In contrast, neutralizing antibody to avb3 integrin had no effect on periostin-induced MMP-13 expression. Conclusions: PostnÀ/À mice are protected from surgically-induced post-traumatic OA showing that periostin promotes cartilage degeneration.. DDR1 mediates the stimulatory effect of periostin on MMP-13 expression. Further studies are in progress to investigate the potential of periostin as a druggable target for the treatment of OA.Purpose: The aim of this study was to identify the expression of Latexin of chondrocytes from the three zones of the cartilage and their possible role during the OA pathogenesis in an animal model. Methods: We conducted a proteomic study of osteoarthritic cartilage during the early stages of OA using an experimental rat model. Then, we identify and evaluated the presence of Latexin (Lxn) in the three zones of the cartilage during OA pathogenesis. Results: In this work, we performed a proteomic study of osteoarthritic cartilage during the early stages of an experimental OA rat model. Ten proteins were differentially e...
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