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

Abstract

Osteoid nodules form in cultures of fetal rat calvaria (RC) cells grown in medium containing 10% fetal bovine serum (FBS) and 50 microns/ml of ascorbic acid. When 10 mM beta-glycerophosphate (beta-GP) is added, the nodules mineralize in two phases: an initiation phase that is dependent upon alkaline phosphatase activity for cleavage of beta-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 (Al3+)on mineralization. When AlCl3 was added to culture medium at concentrations of 0, 3, 10, 30, 100, and 300 muM, the total concentrations of aluminum were 0.98, 6.07, 16.82, 40.19, 88.45, and 284.52 muM, respectively. The corresponding free Al3+ concentrations, assessed after ultrafiltration, were found to be 1.11, 1.75, 3.40, 6.22, 5.38, and 12.11 muM. In cultures in which osteoid was formed and mineralization initiated in the presence of added Al+ (3-300 muM), a dose-dependent inhibition of mineralization occurred. Osteoid formed in the presence of added Al3+ mineralized normally when Al3+ was removed from cultures at the time of initiation of mineralization with beta-GP. In osteoid nodules grown in the absence of Al3+, addition of Al3+ (3-300 muM) at the start of the initiation phase of mineralization resulted in a dose-dependent inhibition of mineralization. Addition of Al3+ to cultures after mineralization had been initiated in the absence of Al3+ inhibited progression of mineralization at added Al3+ concentrations of 10 muM and above. Al3+ did not decrease the conversion of beta-GP to P(i) and caused a small but significant increase in alkaline phosphatase activity at added concentrations of 100 muM or greater. The data show that Al3+ inhibits both the initiation and progression phases of mineralization starting at added concentrations of 3-10 muM (approximately 1.7-3.4 muM free Al3+) and that mineralization of osteoid formed in the presence of Al3+ is unaffected if Al3+ is removed prior to the initiation of mineralization.