Beth A. Derfus

Human Osteoarthritic Cartilage Matrix Vesicles Generate Both Calcium Pyrophosphate Dihydrate and Apatite In Vitro

AbstractCalcium 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 45Ca 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.

Comparison of Matrix Vesicles Derived from Normal and Osteoarthritic Human Articular Cartilage

Articular cartilage matrix vesicles (MVs) from normal human adult articular cartilage were examined for protein and enzyme content and biomineralizing capacity for comparison to MVs derived from human osteoarthritic (OA) cartilage. Femoral condylar and tibial plateau cartilage from each of 9 healthy donors ages 17-37 y was enzymatically digested and serially ultracentrifuged to pellet MVs at 3 x 10(6) g-min. MV protein content, nucleoside triphosphate pyrophospho hydrolase (NTPPPH) specific activity (SA) and capacity for 45Ca precipitation were determined. MV precipitated mineral was examined using Fourier transform infrared spectroscopy (FTIR). Normal human cartilage yields 50% less MV protein/g cartilage than OA cartilage (p < .01). Normal human articular MVs possess 30-70x higher NTPPPH SA than cell-free digest. Mean NTPPPH SAs of MVs derived from normal human cartilage are 3x higher than that of OA MVs (p < .05) and normal MV NTPPPH SA appears to decrease with age (p < .01). Normal human MVs support significantly higher calcium precipitation/mg MV protein in both ATP-dependent (p < .01) and -independent (p = .05) systems. The FTIR spectrum of MV mineral generated in the presence of ATP strongly resembles the standard spectrum for calcium pyrophosphate dihydrate (CPPD). The FTIR spectrum of MV mineral generated without ATP resembles that of carbonate-substituted apatite (AP). The fact that isolated MVs from normal cartilage generate pathologically relevant crystal phases in vitro implies that matrix integrity and substrate availability may be crucial factors in the control of pathologic biomineralization.