title: 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 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 microCi/ml 45Ca 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 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 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 micrograms calcium/h/culture. Initiation of mineralization was prevented by 100 microM levamisole, but not by 100 microM dexamisole. When 100 microM 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.
authors.affiliation: M.R.C. Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Ontario, Canada.
authors.forename: C G
authors.initials: CG
authors.lastname: Bellows
authors.forename: J E
authors.initials: JE
authors.lastname: Aubin
authors.forename: J N
authors.initials: JN
authors.lastname: Heersche
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D001842:Bone and Bones / Q000196:embryology*; D002113:Calcification, Physiologic / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D005994:Glycerophosphates / Q000494:pharmacology*; D007091:Image Processing, Computer-Assisted; D007978:Levamisole / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013773:Tetramisole / Q000494:pharmacology
chemical_list: D005994:Glycerophosphates; D007978:Levamisole; D003907:Dexamethasone; D013773:Tetramisole; D000469:Alkaline Phosphatase; D001205:Ascorbic Acid; D002118:Calcium; C028075:dexamisole; C031463:beta-glycerophosphoric acid