Comparative calcification of native articular cartilage matrix vesicles and nitroprusside-generated vesicles

Objective: Articular cartilage matrix vesicles generate calcium pyrophosphate- and basic calcium phosphate-like mineral in vitro. We sought to determine the morphologic features and calcifying capacity of sodium nitroprusside (SNP)-induced vesicles for comparison to those of controls. Methods: Porcine articular cartilage was exposed to 1 mM SNP for 24 h and vesicles were isolated by enzymatic digestion and serial ultracentrifugation. Control vesicles were derived from an equal weight of untreated articular cartilage. Vesicles-containing fractions pelleted at 2 × 10⁵ · min (pellet I), 3 × 10⁶ g · min (pellet II, the heavy vesicle fraction) and 8 × 10⁶ g · min (pellet III, the light vesicle fraction) were analysed for Lowry protein content, nucleoside triphosphate pyrophosphohydrolase specific activity (NTPPPH) and ATP-dependent calcifying capacity. Results: Electron micrographs (EM) revealed two populations of vesicular structures in both SNP and control pellets. In most experiments, there were no significant differences between the protein contents or ATP-dependent calcium accumulation of SNP vesicles compared to control vesicles. SNP vesicles in pellets I and II had lower NTPPPH activities than their respective controls, P≤0.01. Conclusions: Our data confirmed that 24-hour treatment with the apoptosis-inducing agent did not increase matrix vesicle protein or alter the calcifying activity of vesicles compared to those from control cartilage. SNP did generate vesicles with lower NTPPPH specific activity in most experiments. SNP vesicles, although morphologically similar to controls, are not biochemically identical to them.