The effects of fluoride on osteoblast progenitors in vitro

The number of discrete, three-dimensional bone nodules formed in vitro from a class of osteoprogenitor cells present in fetal rat calvaria cell populations (RC cells) is linearly related to the number of cells plated, implying that this system functions as a colony assay for the expression of osteoprogenitor cells. To determine the effect of fluoride on the expression of these cells, primary RC cells were grown for periods of up to 21 days in alpha-MEM (minimal essential medium) containing 5-15% heat-inactivated fetal bovine serum (FBS), 50 micrograms/ml ascorbic acid, 10 mM Na beta-glycerophosphate, and NaF at concentrations from 10 microM to 5 mM. The continuous presence of NaF resulted in an increase in the number of bone nodules with maximal response occurring at 500 microM (p less than 0.001). A similar response at 500 microM NaF was observed also with regard to alkaline phosphatase activity. NaF levels up to 500 microM did not affect the growth of the mixed RC cell population, however, higher concentrations (1 mM) significantly reduced cell numbers (p less than 0.001) suggestive of cytotoxicity. Plating efficiency tests for colony formation in the presence of 0.5 to 2 mM NaF showed that the decreases in nodule formation observed at concentrations above 500 microM correlated with cytotoxicity. NaC1 at 1 mM had no effect on nodule formation, alkaline phosphatase activity, or cell growth. The results show that NaF stimulates osteoprogenitor cell number in vitro and that the maximal effect occurs at concentrations close to toxic levels.