Differential effects of fluoride during initiation and progression of mineralization of osteoid nodules formed in vitro

Osteoid nodules form in cultures of fetal rat calvarial (RC) cells grown in medium containing 10% FBS and 50 μg/ml of ascorbic acid. When 10 mM β‐glycerophosphate (β‐GP) is added, osteoid nodules mineralize in two phases: an initiation phase, which is dependent upon alkaline phosphatase activity for conversion of β‐GP to P i , and a progression phase that proceeds independently of alkaline phosphatase activity and does not require exogenous phosphate. We have now used this system to investigate the effects of fluoride (F − ) on mineralization. In cultures in which osteoid was formed and mineralization initiated in the presence of F − , a dose‐dependent inhibition of the initiation of mineralization occurred over a concentration range of 25–500 μM F − ( p < 0.001 in all cases). The initiation of mineralization was not inhibited if F − was removed from the cultures at the time when mineralization was initiated with β‐GP. In osteoid nodules grown in the absence of F − , addition of F − resulted in a dose‐dependent inhibition of the initiation of mineralization, with significant decreases in 45 Ca uptake occurring at F − concentrations of 3 μM ( p < 0.01) and higher. However, if F − was added to cultures after mineralization was initiated in the absence of F − , a stimulation of 45 Ca uptake was observed at F − concentrations of 250 μM and above ( p < 0.001). F − (1–1000 μM) did not affect the conversion of β‐GP to P i or alkaline phosphatase activity in the cultures. The data show that mineralization of osteoid formed in the presence of F − is unaffected when F − is removed before initiation, that F − inhibits the initiation phase of mineralization at concentrations as low as 3 μM, and that F − stimulates mineralization during the progression phase. These data agree with clinical observations of hypermineralization of existing bone and hypomineralization of newly formed osteoid in association with F − administration.