Effect of pyrophosphate and two diphosphonates on 45Ca and 32Pi uptake and mineralization by matrix vesicle-enriched fractions and by hydroxyapatite

Inorganic pyrophosphate (PPi) and two diphosphonates, ethane-1-hydroxy-1, 1-diphosphonate (EHDP) and dichloromethylene diphosphonate (Cl2MDP), were found to inhibit in vitro mineralization induced by matrix vesicle-enriched fractions from chicken epiphyseal cartilage. Inhibitor concentrations from 0.20 to 20 microM caused a dosage-dependent decrease in 45Ca and 32Pi uptake by the vesicle fraction. These inhibitors were also tested in a hydroxyapatite (HA)-seeded system to help distinguish between effects on the mineral vs nonmineral portions of the vesicle fraction. The order of inhibition of the HA-seeded system was EHDP greater than PPi greater than Cl2MDP, except for inhibitor concentrations of 0.20 microM where EHDP was the least inhibitory. This variation may be due to differences in the binding of the inhibitors to HA crystals. In general, inhibition of HA mineralization was greatest during later time periods, whereas vesicle ion uptake was affected more during early stages of incubation. The differential effects of the three inhibitors were most obvious at the 2.0 microM concentration. With PPi substantial inhibition of HA-seeded mineralization was observed even in late stages of the study; in contrast, with time the vesicle fraction overcame this inhibition. This suggests that alkaline phosphatase, an enzyme notably enriched in matrix vesicles, catalyzed the hydrolysis of PPi, reducing its concentration to a level where mineralization could proceed. Our findings show that matrix vesicle-induced mineralization differs significantly from apatite-induced mineralization. The data support the concept that vesicle alkaline phosphatase acts, at least in part, to remove physiological crystal growth inhibitors.