Masanori Adachi

Fluoride Analysis of Apatite Crystals with a Central Planar OCP Inclusion: Concerning the Role of F− Ions on Apatite/OCP/Apatite Structure Formation

Abstract. To study the roles of F− ions in the formation of apatite crystals embedding octacalcium phosphate (OCP) lamella in the center of apatite (Ap), a range of the Ap/OCP/Ap lamellar-mixed crystals were synthesized under various concentrations of fluoride ion (F−) from 0.1–1.0 ppm at pH 6.5 and 37°C. The products were analyzed for the F− incorporation, F− distribution, and the amount of OCP and Ap by chemical analysis, X-ray diffraction (XRD), electron probe microanalysis (EPMA), and nuclear magnetic resonance (NMR) techniques. The F− content and the amount of apatite in the crystalline product increased with an increase in the F− concentration in solution, whereas the amount of OCP and the yield of total product decreased. EPMA indicated that F− ions are distributed in the crystals almost homogeneously. The combined analysis suggested that a low-substituted fluoridated hydroxyapatite (FHAp) grew on a small amount of F−-containing OCP or on a surface-reaction layer of OCP, which has accumulated a small amount of F−. The roles of F− ions were hypothesized as the reduction of the growth rate and/or the critical thickness in the a*-axis direction of OCP, the enhancement of hydrolysis of OCP, and the activation of the growth of FHAp, resulting in thinner OCP lamella and thicker apatite lamella in the a*-axis direction with an increase in F− concentration.

Effects of non-collagenous proteins on the formation of apatite in calcium β-glycerophosphate solutions

The effects of the non-collagenous proteins; osteonectin, bone Gla protein and dentine phosphoprotein, on the formation of apatite were studied in calcium beta-glycerophosphate solutions containing catalytic amounts of alkaline phosphatase under physiological conditions. In the system used, calcium phosphate precipitates de novo at levels of supersaturation precisely determined through the enzymatic hydrolysis of beta-glycerophosphate. At 1.7 mM of calcium beta-glycerophosphate, calcium phosphate precipitated when inorganic phosphate accumulated to about 1.4 mM. In the presence of the proteins, however, a greater accumulation of inorganic phosphate was needed for calcium phosphate to precipitate, suggesting that a higher degree of supersaturation, though still a slight undersaturation with respect to dicalcium phosphate dihydrate, is required for calcium phosphate to precipitate in the presence of the proteins. At the same protein (micrograms/ml) concentration, dentine phosphoprotein was approximately four times as effective as bone Gla protein, which was about twice as effective as osteonectin in delaying precipitation. The proteins also retarded subsequent crystal growth, with apatite formed in the presence of the more inhibitory proteins having the smallest crystals, especially in width.

Immobilized DPP and other proteins modify OCP formation

SummaryOsteonectin, γ-carboxyglutamic acid-containing (Gla) protein, and dentin phosphoprotein were covalently attached to sepharose beads and inoculated in solutions at two different degrees of supersaturation with respect to both octacalcium phosphate (OCP) and hydroxyapatite. In both solutions, the inhibitory activity towards de novo formation of calcium phosphate that these proteins display when freely dissolved in solution was completely eliminated when they were immobilized on the sepharose at concentrations of up to 5 μg/mg wet beads. In the solution that was more highly supersaturated with respect to OCP, the immobilized dentin phosphoprotein, moreover, was found to induce de novo formation of OCP in proportion to the concentration of the protein immobilized. For example, at 10 μg/ml of the immobilized dentin phosphoprotein, the induction period was reduced more than 50%. However, in the solution considerably less supersaturated with respect to OCP, none of the immobilized proteins were capable of inducing OCP or apatite deposition. These findings suggest that the immobilized dentin phosphoprotein could work as a nucleating substrate for the OCP phase in solutions where calcium and phosphate concentrations are sufficiently higher than equilibrium saturation levels for the OCP phase.