Kazuo Onuma

Zinc-releasing calcium phosphate for stimulating bone formation ☆

Zinc-containing tricalcium phosphate (ZnTCP) is biocompatible and bioactive, and functions as an effective zinc carrier. ZnTCP contains a maximum of 12 mol% of zinc. ZnTCP ceramics and composite ceramics of ZnTCP and hydroxyapatite (ZnTCP/HAP) with a (Ca+Zn)/P molar ratio of 1.60 were prepared by sintering at 1100 °C. ZnTCP/HAP continued to release zinc for more than 50 days in 0.9 wt.% sodium chloride solution. ZnTCP/HAP with a zinc content of 1.2 wt.% significantly increased osteoblastic MC3T3-E1 cell proliferation and alkaline phosphatase activity of rat stromal cells in vitro. ZnTCP/HAP with a zinc content of 0.316 wt.% increased bone formation by 51% (n=6; p=0.0509) 4 weeks after implantation in rabbit compared to the control without zinc. A zinc concentration within a noncytotoxic level of a solution does not completely block the crystal growth of apatite in the solution. When ZnTCP is added to calcium phosphate cement, the ZnTCP showed neither inhibitory nor promoting effect on the setting ability of the cement. All these findings suggest that the zinc-containing tricalcium phosphate is a biomaterial that has a pharmaceutical effect of promoting bone formation.

Direct transformation from amorphous to crystalline calcium phosphate facilitated by motif-programmed artificial proteins

An animals hard tissue is mainly composed of crystalline calcium phosphate. In vitro, small changes in the reaction conditions affect the species of calcium phosphate formed, whereas, in vivo, distinct types of crystalline calcium phosphate are formed in a well-controlled spatiotemporal-dependent manner. A variety of proteins are involved in hard-tissue formation; however, the mechanisms by which they regulate crystal growth are not yet fully understood. Clarification of these mechanisms will not only lead to the development of new therapeutic regimens but will also provide guidance for the application of biomineralization in bionanotechnology. Here, we focused on the peptide motifs present in dentin matrix protein 1 (DMP1), which was previously shown to enhance hydroxylapatite (HAP) formation when immobilized on a glass substrate. We synthesized a set of artificial proteins composed of combinatorial arrangements of these motifs and successfully obtained clones that accelerated formation of HAP without immobilization. Time-resolved static light-scattering analyses revealed that, in the presence of the protein, amorphous calcium phosphate (ACP) particles increased their fractal dimension and molecular mass without increasing their gyration radii during a short period before precipitation. The protein thus facilitated reorganization of the internal structure of amorphous particles into ordered crystalline states, i.e., the direct transformation of ACP to HAP, thereby acting as a nucleus for precipitation of crystalline calcium phosphate. Without the protein, the fractal dimension, molecular mass, and gyration radii of ACP particles increased concurrently, indicating heterogeneous growth transformation.

In vitro bioactivity of starch thermoplastic/hydroxyapatite composite biomaterials: an in situ study using atomic force microscopy

The in vitro bioactivity of a composite composed by a biodegradable starch-based polymeric matrix and hydroxyapatite fillers was investigated, in situ, as a function of immersion time in a simulated body fluid (SBF) using atomic force microscopy (AFM). The surface roughness of the composite started to increase after the initial 8h because of both the degradation of the polymer matrix and the nucleation of calcium phosphate. After 24h of immersion the surface of the composite was fully covered with calcium phosphate nuclei with diameters around 126 nm. As the immersion time increased, the nuclei increased both in number and size, and coalesced leading to the formation of a dense and uniform calcium phosphate layer on the surface of the composite only after 126 h of SBF immersion. The results of in situ AFM observation agreed with those of standard in vitro bioactivity tests in combination with scanning electron microscopy observations. Thin-film X-ray diffraction demonstrated that the ratio of apatite to the polymer matrix was higher within the surface layer (40 microm deep from the surface) than that in the bulk after the immersion for 7 days. The water-uptake capability of the polymer contributes to the nucleation and growth of the calcium phosphate layer. These results suggest the great potential of the composite for a range of temporary applications in which bone-bonding ability is a desired property.

In situ study of surface phenomena by real time phase shift interferometry

Abstract Real time phase shift interferometry is applied to the observation of a growing crystal surface. Detailed profiles of a growth hillock, such as local vibrations of slopes and height differences of growth steps, are clearly distinguished on a barium nitrate crystal, together with measurements of growth rate at each location. It was found that the height difference of 0.92 nm was directly detectable in this interferometry, indicating that the present method is a few ten times more sensitive than conventional two-beam interferometry, applicable to in situ study, such as Michelson interferometry. It was found that: (1) Step spacing gradually increases as a step approaches the edge of a crystal, which is inferred from the non-uniformity of surface supersaturation. (2) The observation of fluctuation of the growth rate under a constant condition was analyzed as due to the unequal separation of neighboring dislocations which produce the pertubation of steps.

Calcium phosphate clusters

The potential energy surfaces associated with [Ca3(PO4)2n clusters are analyzed in detail using ab initio calculations for n ranging from one to four. Considering separated clusters, energy criteria favor the so-called Posners cluster Ca9(PO4)6, which is the core of the actual structural model of amorphous calcium phosphate. This is rationalized through the existence of a distinct CaO bonding pattern in this cluster. Considering aggregated clusters as a possible model for amorphous calcium phosphate, the aggregation of Ca3(PO4)2 clusters appears as an alternative to Posners hypothesis.

Growth kinetics of hydroxyapatite crystal revealed by atomic force microscopy

Abstract The growth process of the {1010} face of synthesized hydroxyapatite single crystal under physiological conditions were first investigated by in situ atomic force microscopy (AFM). It was confirmed that the growth proceeded by both simple flow of steps which appeared on the wall of an etch pit and a two-dimensional nucleus. Step velocities were in the order of 10 −3 nm/s and independent of the step heights. This means that the bulk and/or surface diffusion of a growth unit is not the rate-determining process. Microscopic Raman spectroscopy of grown crystals showed that the newly grown layer has a poorer crystallinity than the seed crystal.

Denaturation and aggregation of hen egg lysozyme in aqueous ethanol solution studied by dynamic light scattering.

We applied dynamic light scattering technique on the model system of hen egg lysozyme in salt-free aqueous ethanol solution to study the mechanism of denaturation and aggregation of protein. At low ethanol concentration [0-63% (v/v)], the fast relaxation mode was observed, which was caused by lysozyme molecules in the solution interacting with each other with strong repulsive electrostatic force. At 45 and 63% (v/v) ethanol, the slow relaxation mode was also observed, which showed translational diffusive nature, similar to that observed in salt-free polyelectrolyte solution. At 72 or 81% (v/v) ethanol, the slow mode disappeared, leaving only the fast mode. However, the mutual diffusion coefficients obtained from the fast mode at 72 and 81% (v/v) ethanol decreased by about one order of magnitude compared with those from the fast mode at 0-63% (v/v). The reported alcohol-induced conformational transformation of lysozyme molecules at >60% (v/v) ethanol from their native structure to an alpha-helix-rich structure might cause such drastic decrease in the mutual diffusion coefficients. At the highest ethanol concentration of 90% (v/v), the slow mode reappeared, and its relaxation rate was decreasing with elapsed time, which is possibly due to the growth of aggregates of lysozyme molecules. X-ray diffraction results suggested that the intermolecular beta-sheet formation caused the aggregation. Thus, our results indicated that the change in molecular structure of lysozyme closely relates to the diffusion of molecules and their aggregation.

Transformation of α-glycine to γ-glycine

Transformation in a cell of the a form of glycine crystals to the γ form under high humidity was observed by an in situ observation method. The transformation of α-glycine to γ-glycine was found to be solvent-mediated phase transformation. The kinetic equation was obtained, and the calculated values were in good agreement with experimental values.

Mesoporous bioactive glass coatings on stainless steel for enhanced cell activity, cytoskeletal organization and AsMg immobilization

Mesoporous bioactive glass (MBG) coatings with SiO2:CaO:P2O5 mol ratio of 100:0:0, 80:15:5 and 70:25:5 and a tunable pore size and pore structure were prepared on a stainless steel plate by spin-coating sol solutions containing a triblock copolymer and the inorganic precursors. The calcium content in the MBG coatings affected the mesoporous structure. With the increase in calcium content, the crystallinity of the MBG coatings increased and thus the Brunauer–Emmett–Teller (BET) surface area and pore volume decreased. The MBG coatings were evaluated on the basis of protein adsorption, cell attachment, cell proliferation, cell differentiation, cytoskeletal organization and L-ascorbic acid phosphate magnesium salt n-hydrate (AsMg) immobilization for their potential in improving implant-bone integration. The results showed that the osteoblast MC3T3-E1 cells were stimulated by the mesoporous structure and chemical composition of the MBG coatings, with enhanced cell attachment, proliferation, differentiation and better developed cytoskeleton. Moreover, AsMg was successfully immobilized on the MBG coatings by using an AsMg-containing supersaturated calcium phosphate solution. The AsMg immobilized on the MBG coatings was not denatured and showed high activity enhancing the fibroblast NIH3T3 proliferation in vitro. An appropriate range of pore size and a preferred alignment of the mesochannels of the MBG coatings on stainless steel are promising to improve the implant-bone integration.

Investigation of a growth unit of hydroxyapatite crystal from the measurements of step kinetics

Abstract Step velocities on a hydroxyapatite [lcub]101¯0[rcub] face growing under physiological conditions were measured by in situ atomic force microscopy with functions of step height, step spacing and supersaturations. It was shown that the rate determining process of growth was neither diffusion nor dehydration but incorporation of a growth unit at the step front. The evaluated step kinetic coefficient was in the similar order as that of macromolecular virus and protein crystals, being a few orders smaller than that of soluble inorganic crystals. It was suggested from these that the growth unit of hydroxyapatite crystal is in a form of cluster, and not in ionic entities.