Yutaka Moriwaki

Sintered carbonate apatites as bioresorbable bone substitutes

The dissolution behavior of sintered carbonate apatite was investigated in a 10 mM/L acetic acid solution adjusted to pH 5.0 at 37 degrees C, and compared to that of sintered hydroxyapatite and bone apatite for the purpose of establishing some similarities between the physicochemical dissolution of apatite biomaterials in vitro and their ability to be resorbed by osteoclasts in vivo. Both the sintered carbonate apatite and the bone apatite dissolved to an appreciable extent. Their solution compositions changed in an almost identical manner until toward the end of the reaction. The solution compositions for sintered carbonate apatite at 30 s was comparable with that for sintered hydroxyapatite at 3.8 days with respect to the degree of supersaturation, indicating that the former specimen is much more soluble than the latter specimen. Osteoclasts which were obtained from the long bones of 1-day-old neonatal rabbits resorbed bone and sintered carbonate apatite, but not sintered hydroxyapatite. These findings suggest that sintered carbonate apatites, which have characteristics that can be favorably compared with those of bone, especially with respect to its reactivity to acid media, would be useful as bioresorbable bone substitutes.

Osteoclastic responses to various calcium phosphates in cell cultures.

Disks made of hydroxyapatite, beta-tricalcium phosphate, carbonate apatite, tetracalcium phosphate, alpha-tricalcium phosphate, dicalcium phosphate dihydrate, and octacalcium phosphate were incubated in osteoclastic cell cultures for 2 days. The first five salts were sintered and the last two were compressed before incubation. Osteoclasts resorbed only the sintered carbonate apatite disks. However, osteoclasts were able to resorb octacalcium phosphate disks that were preincubated for 1 day in medium without cells, indicating that surface conditioning was important for osteoclastic resorption of this calcium phosphate. Although resorption did not occur, medium calcium and phosphorus changed to an appreciable extent after a 2-day incubation of beta-tricalcium phosphate, tetracalcium phosphate, alpha-tricalcium phosphate, and dicalcium phosphate dihydrate. These changes in the medium calcium and phosphate concentrations could explain why osteoclasts appeared to have lost their activity on these calcium phosphate disks and were not capable of resorbing them. With hydroxyapatite disks no changes were observed in the medium calcium and phosphorus before and after incubation. Moreover, the osteoclasts appeared to be essentially the same as with the sintered carbonate apatite disks and with bone slices used as a control. Nevertheless, no pits or lacunae were observed on the hydroxyapatite disks, indicating that sintered carbonate apatite should be superior to sintered hydroxyapatite as a bioresorbable bone substitute.

Formation of apatite—collagen complexes

An apatite-collagen complex was prepared in calcium beta-glycerophosphate solutions at pH 9.0 and 37 degrees C with the purpose of developing new bone substitutes that more closely resemble bone than currently available materials. Reconstituted type I collagen as well as sheet collagen were crosslinked in the presence of alkaline phosphatase and egg-yolk phosvitin. The crosslinked collagens were immersed in daily-renewed calcium beta-glycerophosphate solutions for 2 and 4 weeks to induce the deposition of apatite on the collagen fibers. After 2 weeks of reaction, for example, apatites deposited approximately two times the crosslinked collagen in weight. With reconstituted collagen, the complex showed some elasticity but no apatite was visually observed to detach under deformation with fingers and forceps. The complex, moreover, did not disintegrate when immersed in saline or animal blood. Nevertheless, the complex resorbed with no evidence of cytotoxicity when implanted in muscle tissues. These findings suggest that the apatite-collagen complex prepared would be useful as bone substitutes, especially for periodontal osseous lesion repair and alveolar ridge augmentation.

Elongated Growth of Octacalcium Phosphate Crystals in Recombinant Amelogenin Gels under Controlled Ionic Flow

Amelogenin proteins constitute the primary structural entity of the extracellular protein framework of the developing enamel matrix. Recent data on the interactions of amelogenin with calcium phosphate crystals support the hypothesis that amelogenins control the oriented and elongated growth of enamel carbonate apatite crystals. To exploit further the molecular mechanisms involved in amelogenin-calcium phosphate mineral interactions, we conducted in vitro experiments to examine the effect of amelogenin on synthetic octacalcium phosphate (OCP) crystals. A 10% (wt/vol) recombinant murine amelogenin (rM179, rM166) gel was constructed with nanospheres of about 10- to 20-nm diameter, as observed by atomic force microscopy. The growth of OCP was modulated uniquely in 10% rM179 and rM166 amelogenin gels, regardless of the presence of the hydrophilic C-terminal residues. Fibrous crystals grew with large length-to-width ratio and small width-to-thickness ratio. Both rM179 and rM166 enhanced the growth of elongated OCP crystals, suggesting a relationship to the initial elongated growth of enamel crystals.

Effects of F- on Apatite-Octacalcium Phosphate Intergrowth and Crystal Morphology in a Model System of Tooth Enamel Formation

SummaryIn order to study the effect of F- on tooth enamel-like apatite formation, crystal growth experiments were carried out in the presence of 0.1}2 ppm F- at 37°C and at pH 6.5 in a model system of enamel formation where octacalcium phosphate (OCP) was stable. Morphology changed from long and thin ribbons to small needle-like plates, and the product changed from OCP to apatite with an increase in F- concentration. In the presence of 0.1–1 ppm F-, apatite-OCP intergrowth took place, and crystals composed of apatite and OCP lamellas were formed. These crystals showed long and thin plate-like morphology and embedded an OCP lamella in the center of the crystal. The OCP lamella and its (100) planes were parallel to the (100) planes of apatite. The thickness of OCP decreased and that of apatite increased with an increase in F- concentration. Some apatite crystals obtained at 1 ppm F- embedded a central plane instead of the distinct OCP lamella. The result indicates that initially formed, thin, plate-like OCP acted as a template for the subsequent epitaxial overgrowth of apatite and, moreover, F- played an important role in regulating the apatite-OCP intergrowth.

Growth and structure of lamellar mixed crystals of octacalcium phosphate and apatite in a model system of enamel formation

Lamellar mixed crystals of octacalcium phosphate (OCP) and apatite were synthesized in a model system of enamel formation in the presence of 1 ppm F − at 37°C and at pH 6.5. The crystal has long and thin plate-like morphology and contained a distinct OCP lamella in the center of the apatite matrix. The thickness of the OCP lamella in the a -axis direction is one to several unit cells. Some apatite crystals embed a central layer instead of the distinct OCP lamella. The OCP lamella and the central layer are parallel to the (100) plane of the apatite, while the c -axis of the OCP is parallel to the c -axis of the apatite. Analysis suggests that (1) F − causes the growth of apatite on OCP and regulates the formation of the lamellar mixed crystals of OCP and apatite, (2) the OCP lamella acts as a template for the subsequent epitaxial growth of apatite, and (3) the lamellar mixed crystals grow mainly in the c-axis direction of both the OCP and apatite. These results strongly support the idea that enamel crystals take a thin and long ribbon-like morphology when the initially formed OCP acts as a template for the subsequent growth of apatite in the enamel formation.

Osteonectin inhibiting de novo formation of apatite in the presence of collagen.

SummaryThe effect of bone matrix protein of osteonectin onde novo formation of apatite was studied in a wide range of calcium phosphate solutions in the presence of collagen. In every solution, from which amorphous calcium phosphate, octacalcium phosphate, or apatite precipitated as a possible initial phase, osteonectin at concentrations less than 1 μM retarded the precipitation, subsequent transformation to apatite, and ripening crystal growth of apatite. Collagen present as either reconstituted or denatured form had no effect on the osteonectin-associated reactions as well as osteonectin-free reactions, and no structural correlation was observed between collagen fibrils and any of the calcium phosphates that appeared in our system. Direct measurement of free calcium levels in the solutions suggested that the reduction in calcium activity due to complexing with osteonectin hardly explained the inhibitory activity of osteonectin in retarding the formation of apatite. Instead, our transmission electron microscopic (TEM) observation strongly suggested that the primary mechanism for osteonectin to inhibit the formation of apatite is to block growth sites of calcium phosphates nucleated. The apatite thus formed in the presence of osteonectin showed less resolved X-ray diffraction patterns, partly because of smaller crystallites as suggested by TEM.

Influence of Carbonate on Sintering of Apatites

Sintering of carbonate apatite, prepared at 100°C and pH 9.0 for 3 days, was studied by thermal analysis, x-ray diffraction, and infrared spectroscopy. The sintering temperature, at which the linear thermal shrinkage of isostatically compacted specimens increased sharply, decreased in proportion to the amount of carbonate initially present in the apatite. For example, specimens with over 8 wt% carbonate could be sintered at a temperature (650°C) which was nearly 400°C lower than that needed for sintering a specimen with no carbonate. Amounts of carbonate lost at the end of sintering, estimated chemically and by infra-red specroscopy, were approximately equal to sample weight losses estimated thermogravimetrically

Transition of octacalcium phosphate to hydroxyapatite in solution at pH 7.4 and 37°C

Transition of octacalcium phosphate (OCP) to apatite was studied under the condition where Ca2+ ions were continuously supplied to the reacting solution at pH 7.4 and at 37°C. OCP crystals were grown and subsequently converted into apatite by discontinuing of Ca addition. The rectangular (100) blades of OCP crystals developed notches on their short edges in the early stage of transition. The depth of the notches increased along the c-axis direction with the progress of the reaction, giving rise to a slit-like texture. The direction of the slit formation seemed to relate to the spatial configuration of H2O molecules in the OCP lattice, which are released during the transition from OCP to apatite.

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.