Calcium crystals in osteoarthritic (OA) joints promote enzymatic degradation of articular tissues. Matrix vesicles provide a nidus for calcium crystal formation in chick epiphyseal and mature porcine articular cartilage. In order to examine a potential role for matrix vesicles from OA cartilage in generating pathologic crystals, we sought to determine whether vesicles derived from human OA cartilage (OAMV) could mineralize; and we characterized the resultant mineral species. OAMV were isolated and examined for alkaline phosphatase (AP) and nucleoside triphosphate pyrophosphohydrolase (NTPPPH) activity. OAMV ATP-dependent and independent mineralization were measured in a radiometric biomineralization assay, and newly formed OAMV crystals were examined using Fourier transform infrared spectroscopy (FTIR) and compensated polarized light microscopy. The mean specific activity of OAMV AP was approximately 6 times higher and NTPPPH activity 11 times lower than that of previously characterized, mature, porcine, articular cartilage vesicles. OAMV progressively precipitated 45 Ca over time both in the presence and absence of ATP. The FTIR spectra of mineral formed in ATP-dependent assays most closely resembled the standard spectrum for calcium pyrophosphate dihydrate (CPPD). The FTIR spectra of OAMV mineral formed in the absence of ATP closely resembled apatite. These data support the hypothesis that OAMV may form mineral phases of two key crystals found in degenerating cartilage and provide further evidence for the role of matrix vesicles in pathologic articular cartilage biomineralization.

OBJECTIVE:We previously identified a unique fraction of porcine articular cartilage vesicles, sedimentable at 8 x 10(6) g/min, which generate calcium pyrophosphate dihydrate crystals (CPPD) in vitro. We sought to identify and characterize other fractions of articular cartilage digest, sedimentable at lower g forces, which may also contain mineralizing vesicles.METHODS:Electron microscopy and alkaline phosphatase and nucleoside triphosphate pyrophosphohydrolase (NTPPPH) assays were used to analyze each fraction. Radiometric mineralization assays, Fourier transform infrared (FTIR) spectroscopy, and compensated polarized light microscopy were used to analyze crystals formed by these fractions.RESULTS:Vesicles of varying sizes identical to epiphyseal cartilage matrix vesicles were seen in all sedimentable fractions examined, but were the exclusive component of fractions sedimentable at 3 x 10(6) g/min, termed the heavy vesicle fraction (HVF), and at 8 x 10(6) g/min, now termed the light vesicle fraction (LVF). All vesicle containing fractions supported ATP dependent calcium pyrophosphate precipitation. The HVF and LVF precipitated 30 x more calcium than vesicle poor supernatant (p < 0.01) and 1.5-4 x more than cell-free unfractionated digest (p < 0.01). HVF differed from LVF in that it contained 3-4 x higher NTPPPH specific activity (p < 0.05). HVF resembled LVF in that both precipitated crystals consistent with CPPD by FTIR spectroscopy and compensated polarized light microscopy.CONCLUSION:These data expand our previous estimate of the total number of vesicles available for biologic mineralization and demonstrate heterogeneity of vesicle fractions. They support a key role for vesicles in CPPD crystal formation.