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 meas ured [H-3]-thymidine incorporation into cell DNA during the final four hr of a 24-hour incubation with phenytoin. Phenytoin at micromolar co ncentrations significantly stimulated the [H-3]thymidine incorporation in a dose-dependent, biphasic, manner with a maximal effect at from 1 0 to 50 mu M. We confirmed the proliferative effect of phenytoin by co unting cell number. To evaluate the effects of phenytoin on osteoblast ic differentiation, we determined alkaline-phosphatase specific activi ty and found that phenytoin at micromolar concentrations significantly increased that activity in a dose-dependent manner, with maximal stim ulation at approximately 1 mu M. To investigate the effects of phenyto in on mature osteoblastic activities, we measured de novo collagen syn thesis and osteocalcin secretion. Mitogenic doses of phenytoin signifi cantly increased collagen synthesis and osteocalcin secretion in a dos e-dependent, biphasic, manner, with the maximal stimulatory dose at fr om 5 to 10 mu M. In summary, phenytoin at micromolar ranges increased (a) [3H]-thymidine incorporation and cell number, (b) alkaline-phospha tase specific activity, (c) collagen synthesis, and (d) osteocalcin se cretion in monolayer cultures of normal human-mandible-derived bone ce lls. These observations are consistent with the premise that low doses of phenytoin act on human craniofacial bone cells to stimulate cell p roliferation, differentiation, and mature osteoblastic activities to s timulate bone formation.