KINETICS OF BETA-GLYCEROPHOSPHATE-INDUCED ENDOCHONDRAL MINERALIZATION INVITRO - CALCIUM ACCUMULATION, ALKALINE-PHOSPHATASE ACTIVITY, AND EFFECTS OF LEVAMISOLE

Isolated mesenchymal limb bud cells from day-12 mouse embryos grown at high density in organoid culture at the medium/air interphase differentiate into chondrocytes and form cartilage nodules. Upon addition of beta-glycerophosphate (beta-GP), cartilage undergoes endochondral mineralization. This beta-GP-induced mineralization was investigated by measuring the calcium content in the cultures and the activity of alkaline phosphatase (AP) in the cell mass and the medium. Calcium incorporation depended on the amount of beta-GP added. After continuous treatment, mineralization began on day 8 of the culture period and increased linearly until day 15. In long-term cultures, periodical treatment for 6 days caused an increase in mineralization the older the cultures were, but the slope of increase was proportionately less steep. Treatment at the latest period on days 19-24 resulted in a markedly reduced mineralization. After short-term treatment (48 hours), mineralization increased also the older the cultures were and proceeded during further cultivation in beta-GP-free medium. This kinetic behavior indicates a dependency of mineralization on cartilage maturation in this in vitro system. AP activity increased enormously and nearly logarithmically in the cell mass in beta-GP-free medium, whereas beta-GP treatment inhibited this drastic increase. In the medium, considerable activities of AP were also measurable from day 10 onward. It increased in beta-GP-free medium up to day 14, but was diminished after mineralization had been induced. Levamisole inhibited AP activity dose dependently when added directly to the enzyme-containing medium (100% inhibition at 10(-3) M). Added to the cultures from day 7 to 14, it partially inhibited AP activity and mineralization at 5 x 10(-5) M; mineralization was totally inhibited at 10(-3) M, but AP activity was still present. This high concentration was cytotoxic, as revealed ultrastructurally and by GAG estimation. This in vitro system comprises cartilage development and maturation, beta-GP-inducible endochondral mineralization, and final degenerative changes; it may be an appropriate model for investigations on endochondral mineralization.