Initiation and progression of mineralization of bone nodules formed in vitro: the role of alkaline phosphatase and organic phosphate

Abstract

Osteoid nodules form but do not mineralize in fetal rat calvaria cell cultures grown in α-minimal essential medium with 10% fetal bovine serum in the absence of Na β-glycerophosphate (β-GP). To study factors involved in the initiation and progression of mineralization, cultures were treated with β-GP and radiolabelled with 0.1–0.2 μCi/ml 45Ca after nodules had formed (17–19 days in medium without β-GP). Concentrations of β-GP from 1 to 14 mM induced a dose-dependent increase in 45Ca uptake. 45Ca uptake was restricted to nodule-containing cultures and did not occur in cultures without nodules. Continuous labelling over 72 h compared with 2 h pulses over the same time period showed that little mineralization occurred over the first 8–12 h and that the rate of mineralization was maximal and constant after 24 h exposure to β-GP. Calcium uptake from medium was slow during the first 12 h of β-GP exposure but increased rapidly thereafter until the medium calcium concentration reached a steady state of between 0.5 and 0.6 mM. Measurement of calcium concentration in the medium after mineralization had been initiated (24 h after β-GP exposure) showed a linear calcium uptake into nodules ( r = 0.990) over a 7 h period at a rate of 9.2 μg calcium/h/culture. Initiation of mineralization was prevented by 100 μM levamisole, but not by 100μM dexamisole. When 100μM levamisole was added 24 h after mineralization had been initiated by the addition of β-GP, the progression of mineralization was unaffected. Similarly, after mineralization had been initiated for 24 h by 10 mM β-GP, mineralization continued independent of the presence of β-GP. The data show that the initiation and progression of mineralization are separate phenomena and that organic phosphate and alkaline phosphatase play a crucial role in the initiation of mineralization but are not required for the continuation of mineralization of bone nodules.