Nobukazu Wakamatsu

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.

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.

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.

Effects of CO2-3 ion on the formation of octacalcium phosphate at pH 7.4 and 37°C

The effects of carbonate (CO2-3) ion on the formation of octacalcium phosphate (OCP) were studied at pH 7.4 and 37°C. The reaction was carried out in solutions with various amounts of KHCO3 (0–20mM) and with an initial solution Ca/P ratio of 0.075–0.25. CO2-3 ion changes the OCP/apatite ratio of the product, depending upon the Ca/P ratio of the solution, that is, with the increase in CO3 concentration, the amount of OCP increases and then decreases, while the amount of apatite decreases and then increases: when the Ca/P of the solution is high, a smaller amount of CO2-3 ion disturbs the crystal growth of OCP. The morphology of the products is plate-like under various amounts of CO3, but the thickness decreases and the crystal size becomes small with increasing CO3 concentration. These results indicate that CO2-3 ion significantly affects the product and morphology of final crystals. Possible roles of CO2-3 ion in the biological crystallization are discussed.

Sintering mechanism of hydroxyapatite by addition of lithium phosphate

The sintering mechanism of hydroxyapatite (HAp) by addition of lithium phosphate (Li3PO4) has been investigated. Using the X-ray diffraction method, HAp was confirmed to decompose into β-Ca3(PO4)2 (β-TCP) by addition of Li3PO4. The measurement of shrinkage rate by the isothermal firing made it clear that the densification process at the initial stage of sintering took place in the presence of liquid phase. Furthermore, the examination of the phase diagram on the binary system β-TCP-Li3PO4 revealed that there was an eutectic point at 1010°C in the composition of 60 wt% Li3PO4. From these evidences, we concluded that β-TCP produced by the decomposition of a part of HAp has formed the liquid phase by reacting with Li3PO4 above 1010°C, and that this liquid phase has largely promoted the densification by the rearrangement of HAp particles at the initial stage of sintering.

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.

Effects of adding silver nanoparticles on the toughening of dental porcelain

STATEMENT OF PROBLEM Dental porcelains currently used for ceramic restorations are brittle, and it is sometimes necessary to replace fractured or chipped restorations. Porcelain is fragile and exhibits elastic deformation rather than plastic deformation, leading to fracture or chipping of restorations. PURPOSE The purpose of this study was to investigate the toughening of porcelain through the addition of silver nanoparticles. MATERIAL AND METHODS Noritake Super (NS) Porcelain AAA modified with the addition of silver nanoparticles was used. The concentration of silver in the solution was adjusted to 100, 200, 500, and 1000 ppm (Ag100, Ag200, Ag500, and Ag1000). The Vickers hardness (Hv) and median crack length extending from the corner of each indent were measured. The fracture toughness (KIC) was calculated by the indentation method. Optical reflectance spectra were recorded by using a spectrometer in the wavelength range of 200 to 700 nm. X-ray diffraction spectroscopy, color measurement, Fourier transform infrared spectroscopy, and electron probe microanalysis were also performed. The observed values of Hv, 2a, E, and KIC were compared and evaluated with a 1-way ANOVA, followed by the Bonferroni method (α=.05). RESULTS The addition of silver nanoparticles significantly increased the Hv of all specimens with the exception of Ag100. The median crack length was significantly smaller in Ag500 (104.5 μm, SD: 11.9) and Ag1000 (100.0 µm, SD: 5.5). Significantly higher toughness values were observed for Ag500 (1.54 MPa·m(1/2), SD: 0.05) and Ag1000 (1.51 MPa·m(1/2), SD: 0.08) than for the control (1.36 MPa·m(1/2), SD: 0.03). In terms of color difference, Ag500 (5.08, SD:1.32) and Ag1000 (5.47, SD:1.05) had values significantly greater than ΔE*=2.69. CONCLUSIONS The addition of silver nanoparticles significantly increased the fracture toughness and Vickers hardness of the NS porcelain. A residual compressive stress was generated due to the ion exchange reaction and differential thermal expansion of the silver metal nanoparticles. However, the addition of Ag500 and Ag1000 nanoparticles led to a color change.

Thermal decomposition ofLingula shell apatite

SummaryLingula shell is composed of apatite with a preferred orientation. The shell apatites ofLingula unguis(Lu) andLingula shantoungensis(Ls) were characterized and compared with apatite of human tooth enamel. Insight into theLingula apatite was studied by following the change of lattice parameter, transformation to β-tricalcium phosphate (β-TCP), and the loss and change of CO3, OH, and H2O after heating up to 1,000°C in air and N2 for 24 hours. The OH stretching band was not observed in unheated apatites and in apatites heated in dried N2.Lu andLs apatite produced 26 and 17 wt% of β-TCP at 700°C, respectively. Fifty to 60% of H2O was lost at 200°C, being accompanied by a drastic contraction of the a- and c-axis and a drastic decrease in the crystallinity. These results indicate that (1)Lu andLs shell apatite is CO3 containing F+Cl-apatite, and (2) the structural H2O of theLingula apatite is loosely bounded such that they are lost at lower temperature than tooth enamel.

The influx of serum albumin to enamel matrix in rat incisors after trauma.

The most frequent results of trauma to tooth germs are enamel hypoplasia and enamel hypocalcification. These differing results may be due to the stage of amelogenesis at which trauma occurs. The cellular and biomolecullar events involved in the genesis of these defects are poorly understood. We hypothesized that one factor involved is the possibility that relatively high levels of serum albumin enter the enamel matrix through the damaged enamel organ, and impair mineralization of the matrix. The present study was undertaken to immunohistochemically and autoradiographically localize serum albumin in the enamel organs of rat incisors after trauma was inflicted to the mandibular incisor region of 4-day-old rats. Hemorrhage was seen surrounding the enamel organ and between the detached secretory-stage ameloblasts. One day after trauma, the most intense immunohistochemical (IHC) staining for albumin was localized in the outer layer of the enamel matrix adjacent to the detached secretory-stage ameloblasts. Albumin was also detected autoradiographically in the secretory-stage ameloblasts layer and enamel matrix. These findings indicate that serum albumin can leak between the detached ameloblasts and penetrate the enamel matrix after trauma. Leaked albumin was still present in the matrix during the maturation stage. Leaked albumin in the developing enamel could inhibit crystal growth and result in hypocalcification.