Type II collagen levels correlate with mineralization by articular cartilage vesicles.

OBJECTIVE: Pathologic mineralization is common in osteoarthritic (OA) cartilage and may be mediated by extracellular organelles known as articular cartilage vesicles (ACVs). Paradoxically, ACVs isolated from OA human cartilage mineralize poorly in vitro compared with those isolated from normal porcine cartilage. We recently showed that collagens regulate ACV mineralization. We sought to determine differences between collagens and collagen receptors on human and porcine ACVs as a potential explanation of their different mineralization behaviors. METHODS: ACVs were enzymatically released from old and young human and porcine hyaline articular cartilage. Western blotting was used to determine the presence of types I, II, VI, and X collagen and various collagen receptors on ACVs. Type II collagen was quantified by enzyme-linked immunosorbent assay. Biomineralization was assessed by measuring the uptake of (45)Ca by isolated ACVs in agarose gels and by ACVs in situ in freeze-thawed cartilage. RESULTS: As previously shown, isolated human ACVs mineralized poorly in response to ATP compared with porcine ACVs, but human and porcine ACVs mineralized similarly in situ in freeze-thawed cartilage. Type II collagen levels were 100-fold higher in isolated human ACVs than in porcine ACVs. Type II collagen in human ACVs was of high molecular weight. Transglutaminase-crosslinked type II collagen showed increased resistance to collagenase, suggesting a possible explanation for residual collagen on human ACVs. Expression of other collagens and collagen receptors was similar on human and porcine ACVs. CONCLUSION: Higher levels of type II collagen in human ACV preparations, perhaps mediated by increased transglutaminase crosslinking, may contribute to the decreased mineralization observed in isolated human ACVs in vitro.

Promotion of articular cartilage matrix vesicle mineralization by type I collagen.

OBJECTIVE: Calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals occur in up to 60% of osteoarthritic joints and predict an increased severity of arthritis. Articular cartilage vesicles (ACVs) generate CPPD crystals in the presence of ATP and BCP crystals with added beta-glycerophosphate. While ACVs are present in normal articular cartilage, they mineralize primarily in cartilage from osteoarthritic joints. The aim of this study was to explore the hypothesis that ACV mineralization is regulated by components of the surrounding extracellular matrix. METHODS: Porcine ACVs were embedded in agarose gels containing type II and/or type I collagen and/or proteoglycans. Mineralization was measured as (45)Ca accumulation stimulated by ATP or beta-glycerophosphate and reflects both nucleation and growth. Synthetic CPPD and BCP crystals were embedded in similar gels to isolate the effect of matrix components on crystal growth. RESULTS: After establishing baseline responsiveness of ACVs to ATP and beta-glycerophosphate in agarose gels, we examined the ability of ATP and beta-glycerophosphate to stimulate mineral formation in gels containing various matrix components. Type II collagen suppressed the ability of ATP to stimulate mineralization, while a combination of type II plus type I collagen increased the effect of ATP and beta-glycerophosphate on mineralization. Type I collagen affected ACV mineralization in a dose-responsive manner. Neither type of collagen significantly affected crystal growth or levels of mineralization-regulating enzymes. Proteoglycans suppressed mineral formation by ACVs in gels containing both type I and type II collagen. CONCLUSION: Cartilage matrix changes that occur with osteoarthritis, such as increased quantities of type I collagen and reduced proteoglycan levels, may promote ACV mineralization.