Aluminum inhibits both initiation and progression of mineralization of osteoid nodules formed in differentiating rat calvaria cell cultures.

Osteoid nodules form in cultures of fetal rat calvaria (RC) cells grown in medium containing 10% fetal bovine serum (FBS) and 50 μg/ml of ascorbic acid. When 10 mM β-glycerophosphate (β-GP) is added, the nodules mineralize in two phases : an initiation phase that is dependent upon alkaline phosphatase activity for cleavage of β-GP to inorganic phosphate (P i ) and a progression phase that proceeds independently of the activity of alkaline phosphatase and does not require exogenous phosphate. We have used this system to investigate the effects of aluminum (Al 3+ ) on mineralization. When AlCl 3 was added to culture medium at concentrations of 0, 3, 10, 30, 100, and 300 μM, the total concentrations of aluminum were 0.98, 6.07, 16.82, 40.19, 88.45, and 284.52 μM, respectively. The corresponding free Al 3+ concentrations, assessed after ultrafiltration, were found to be 1.11, 1.75, 3.40, 6.22, 5.38, and 12.11 μM. In cultures in which osteoid was formed and mineralization initiated in the presence of added Al 3+ (3-300 μM), a dose-dependent inhibition of mineralization occurred. Osteoid formed in the presence of added Al 3+ mineralized normally when Al 3+ was removed from cultures at the time of initiation of mineralization with β-GP. In osteoid nodules grown in the absence of Al 3+ , addition of Al 3+ (3-300 μM) at the start of the initiation phase of mineralization resulted in a dose-dependent inhibition of mineralization. Addition of Al 3+ to cultures after mineralization had been initiated in the absence of Al 3+ inhibited progression of mineralization at added Al 3+ concentrations of 10 μM and above. Al 3+ did not decrease the conversion of β-GP to P i and caused a small but significant increase in alkaline phosphatase activity at added concentrations of 100 μM or greater. The data show that Al 3+ inhibits both the initiation and progression phases of mineralization starting at added concentrations of 3-10 μM (∼1.7-3.4 μM free Al 3+ ) and that mineralization of osteoid formed in the presence of Al 3+ is unaffected if Al 3+ is removed prior to the initiation of mineralization.