Phenytoin at micromolar concentrations is an osteogenic agent for human-mandible-derived bone cells in vitro

The present study sought to test the hypothesis that phenytoin acts on normal human-mandible-derived bone cells to induce osteogenic effects. To test the effects of phenytoin on bone cell proliferation, we measured [3H]-thymidine incorporation into cell DNA during the final four hr of a 24-hour incubation with phenytoin. Phenytoin at micromolar concentrations significantly stimulated the [3H]-thymidine incorporation in a dose-dependent, biphasic, manner with a maximal effect at from 10 to 50 microM. We confirmed the proliferative effect of phenytoin by counting cell number. To evaluate the effects of phenytoin on osteoblastic differentiation, we determined alkaline-phosphatase specific activity and found that phenytoin at micromolar concentrations significantly increased that activity in a dose-dependent manner, with maximal stimulation at approximately 1 microM. To investigate the effects of phenytoin on mature osteoblastic activities, we measured de novo collagen synthesis and osteocalcin secretion. Mitogenic doses of phenytoin significantly increased collagen synthesis and osteocalcin secretion in a dose-dependent, biphasic, manner, with the maximal stimulatory dose at from 5 to 10 microM. In summary, phenytoin at micromolar ranges increased (a) [3H]-thymidine incorporation and cell number, (b) alkaline-phosphatase specific activity, (c) collagen synthesis, and (d) osteocalcin secretion in monolayer cultures of normal human-mandible-derived bone cells. These observations are consistent with the premise that low doses of phenytoin act on human craniofacial bone cells to stimulate cell proliferation, differentiation, and mature osteoblastic activities to stimulate bone formation.