INITIATION AND PROGRESSION OF MINERALIZATION OF BONE NODULES FORMED INVITRO - THE ROLE OF ALKALINE-PHOSPHATASE AND ORGANIC PHOSPHATE

Osteoid nodules form but do not mineralize in fetal rat calvaria cell cultures grown in alpha-minimal essential medium with 10% fetal bovine serum in the absence of Na beta-glycerophosphate (beta-GP). To study factors involved in the initiation and progression of mineralization, cultures were treated with beta-GP and radiolabelled with 0.1-0.2-mu-Ci/ml Ca-45 after nodules had formed (17-19 days in medium without beta-GP). Concentrations of beta-GP from 1 to 14 mM induced a dose-dependent increase in Ca-45 uptake. Ca-45 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 beta-GP. Calcium uptake from medium was slow during the first 12 h of beta-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 beta-GP exposure) showed a linear calcium uptake into nodules (r = 0.990) over a 7 h period at a rate of 9.2-mu-g calcium/h/culture. Initiation of mineralization was prevented by 100-mu-M levamisole, but not by 100-mu-M dexamisole. When 100-mu-M levamisole was added 24 h after mineralization had been initiated by the addition of beta-GP, the progression of mineralization was unaffected. Similarly, after mineralization had been initiated for 24 h by 10 mM beta-GP, mineralization continued independent of the presence of beta-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.