Calcification of dentinal collagen by cultured rabbit periosteum: the role of alkaline phosphatase.

ABSTRACT

Periostea were dissected from 1-2 weeks old rabbit calvaria and folded around decalcified and extracted bovine dentin matrix slices (DMS). The cocultures were grown in serum-containing medium supplemented with beta-glycerophosphate or other organic phosphate esters. [45Ca]-uptake measurements indicated that the DMS calcified. Initiation of the calcification process was associated with alkaline phosphatase activity and could be prevented by adding the inhibitor L-levamisole to the culture medium. Using [32P]-adenosine-monophosphate as a substrate for phosphatase activity it was demonstrated that very little, if any, phosphate was utilized for the phosphorylation of higher molecular weight substances. The results suggest that over 99% of the phosphate produced was laid down in inorganic form. Further, it was noted that mineral deposition in the DMS was accompanied by the simultaneous inclusion of methylene blue and PAS-positive substances whose nature, origin and function remain to be determined. The results lend support to the theory that alkaline phosphatase is involved in the initiation of calcification processes by raising the local concentration of phosphate ions.