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 β-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 aga-rose gels containing type II and/or type I collagen and/or proteoglycans. Mineralization was measured as45Ca accumulation stimulated by ATP or β-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 β-glycerophosphate in agarose gels, we examined the ability of ATP and β-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 β -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.

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.Pathologic calcification commonly occurs in the extracellular matrix of articular cartilage affected by severe osteoarthritis (OA). Two types of calciumcontaining crystals dominate: calcium pyrophosphate dihydrate (CPPD) crystals, which are relatively specific to articular cartilage, and basic calcium phosphate (BCP) crystals, which are similar to the hydroxyapatite mineral of bone and other calcified tissues. While the mechanisms of BCP and CPPD crystal formation have not been fully elucidated, small extracellular organelles known as articular cartilage vesicles (ACVs) have been implicated in this process (1). These organelles can be enzymatically isolated from both porcine and human articular cartilage, and when provided with calcium and a source of phosphate or pyrophosphate, they generate BCP and CPPD crystals identical to those found in human OA joints (2,3).ACVs can be easily isolated from both normal and OA cartilage (1). When removed from their extracellular milieu, normal porcine ACVs mineralize more effectively than ACVs derived from human OA cartilage (1). Since ACVs rarely mineralize in normal cartilage, this observation suggests a potential role for the normal extracellular matrix in suppressing ACV mineraliza...

Background Matrix vesicles (MVs) are membrane-bound organelles that are physiologic components of the extracellular matrix of connective tissues. MVs from articular cartilage are well characterised and range in size from 50-100 nm. MVs participate in mineralisation by acting as foci of calcium crystal formation. Acute gouty arthritis preferentially occurs in osteoarthritic joints and can co-occur with calcium crystals, but the role of MVs in monosodium urate (MSU) crystal formation is unexplored. We hypothesised that degradation of articular structures in osteoarthritis results in release of MVs into the synovial fl uid and that synovial fl uid MVs may promote MSU crystal formation. Methods MVs were isolated from human synovial fl uid (SF) by serial centrifugation using protocols identical to those used to isolate MVs from tissue digests. Isolated MVs were assayed for protein content and levels of alkaline phosphatase, and NTPPPH enzyme activities were measured and compared to articular cartilage MVs. 144 ug/ml of MVs were added to a supersaturated urate solution. Controls included identical quantities of SF supernatant or bovine serum albumin added to a supersaturated urate solution. The resulting crystal pellet was isolated, washed and weighed. Crystal structure was confi rmed by Fourier transform infrared (FTIR) spectroscopy and polarising light microscopy. Results MVs were easily isolatable from 30 ml of human osteoarthritic SF. The MV fraction contained 0.144 ± 0.01 mg/ ml protein. NTPPPH specifi c activity was considerably lower than from articular cartilage MVs at 14.7 pmol/mg. Alkaline phosphatase activity was undetectable. MSU crystals formed in all conditions. MVs increased MSU crystal production by 3.33 ± 0.07-fold over albumin control and 1.67 ± 0.08-fold over SF supernatant control. FTIR and polarising light microscopy confi rmed the identity of the crystals as MSU and showed that MVs were incorporated into the crystal structure. Conclusions MVs can be isolated from SF. These MVs differ in biochemical profi le compared to articular cartilage MVs, and thus, their tissue source is unclear. MVs facilitate MSU crystal formation, perhaps as acting as scaffolding for formation of crystal or altering the microenvironment. The presence of synovial fl uid MVs in OA joints may contribute to the strong association between gouty arthritis and OA.