Effect of bound phosphoproteins and other organic phosphates on alkaline phosphatase-induced mineralization of collagenous matrices in vitro.

The aim of the present study was to determine to what extent the rate at which collagen mineralizes correlates with the amount and nature of bound phosphate groups. Sheets of collagen prepared from demineralized bovine dentin or cortical bone were complexed with various concentrations of phosphoserine [(P)Ser] or rat dentin phosphoproteins (PP; lowly or highly phosphorylated PP, LPP or HPP). Alternatively, phosphate groups were removed from the collagenous carrier material by treatment with phosphatases. Mineralization was achieved by incubation in culture medium supplemented with 45Ca, alkaline phosphatase and 10 mM beta-glycerophosphate. The sheets were monitored for uptake of 45Ca and lag times calculated and plotted against the amount of bound phosphate. It was observed that dephosphorylation of the carrier causes an increase in lag time and that rat PP decreases lag times in a concentration-dependent way. HPP were more effective than LPP. (P)Ser or other small organic P-containing molecules had hardly any influence on lag time. It is concluded that next to the amount of bound phosphate, the nature of phosphorylated substances has considerable influence on the rate of mineralization of a collagenous carrier.

Alkaline phosphatase induces the deposition of calcified layers in relation to dentin: an in vitro study to mimic the formation of afibrillar acellular cementum.

An attempt was made to test the hypothesis that alkaline phosphatase, an enzyme which is abundant in periodontal ligament, plays a role in the formation of acellular root cementum. Thin slices of bovine dentin were incubated in Iscove Modified Dulbecco's Medium supplemented with 10% normal rabbit serum and 10 mmol/L beta-glycerophosphate (beta-GP) or folded into pericardial explants. Intestinal bovine alkaline phosphatase (APase), covalently linked to agarose beads, was added to the cultures. In the presence of the enzyme, the dentin slices were covered with thin layers of mineralized material. Such layers were not observed in cultures not provided with APase-beads or beta-GP. They also did not form in relation to demineralized dentin. The layers of calcified material appeared to consist of crystallites embedded in a granular matrix of moderate electron density, which often exhibited the presence of incremental lines and resembled the matrix of afibrillar acellular cementum formed under in vivo conditions. When pericardial explants were interposed between the enzyme-containing beads and the dentin, mineral deposition in relation to the dentin was retarded. This finding lends support to the view that soft connective tissues interfere with the free diffusion of phosphate.

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

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.