The regulatory effect of phosphates on bone metabolism in vitro

One of the most important indicators in vitro of the bone-cell phenotype is the synthesis of mineralized bone-like tissue. This has been achieved by supplementing isolated bone-cell and tissue cultures with organic phosphates, in particular, beta-glycerophosphate. To analyze the effects of beta-glycerophosphate on bone-cell metabolism and osteogenesis in vitro, both biochemical analyses and computer-assisted morphometry were used. Simultaneous autoradiographic and histochemical analyses of proliferating and alkaline phosphatase-positive cells were used to measure osteogenic events at the cellular level. Morphometric data showed that beta-glycerophosphate-treated cultures mineralized, but exhibited significantly less bone matrix (P less than 0.05) than non-mineralizing controls. Cultures treated with inorganic phosphate failed to mineralize. Cellular proliferation was unaffected by beta-glycerophosphate; however, there was a decrease in the amount of 3H-thymidine incorporation into the DNA of beta-glycerophosphate-treated cells as detected by autoradiography. The percentage of alkaline phosphatase-positive cells was identical in beta-glycerophosphate-treated or control cultures. In agreement with previous biochemical results, there was a decrease in the amount of alkaline phosphatase enzyme activity per cell. The kinetics of alkaline phosphatase enzymes were measured on individual cells by microdensitometry. beta-Glycerophosphate-treated cultures exhibited more rapid reaction rates than control cultures (p less than 0.05). Taken together, the results suggest that beta-glycerophosphate has global effects on bone-cell metabolism in vitro including its importance in mineralization.