Taishi Yokoi

A bone substitute with high affinity for vitamin D-binding protein―relationship with niche of osteoclasts

The biological activity of osteoblasts and osteoclasts is regulated not only by hormones but also by local growth factors, which are expressed in neighbouring cells or included in bone matrix. Previously, we developed hydroxyapatite (HA) composed of rod‐shaped particles using applied hydrothermal methods (HHA), and it revealed mild biodegradability and potent osteoclast homing activity. Here, we compared serum proteins adsorbed to HHA with those adsorbed to conventional HA composed of globular‐shaped particles (CHA). The two ceramics adsorbed serum albumin and γ‐globulin to similar extents, but affinity for γ‐globulin was much greater than that to serum albumin. The chemotactic activity for macrophages of serum proteins adsorbed to HHA was significantly higher than that of serum proteins adsorbed to CHA. Quantitative proteomic analysis of adsorbed serum proteins revealed preferential binding of vitamin D‐binding protein (DBP) and complements C3 and C4B with HHA. When implanted with the femur of 8‐week‐old rats, HHA contained significantly larger amount of DBP than CHA. The biological activity of DBP was analysed and it was found that the chemotactic activity for macrophages was weak. However, DBP‐macrophage activating factor, which is generated by the digestion of sugar chains of DBP, stimulated osteoclastogenesis. These results confirm that the microstructure of hydroxyapatite largely affects the affinity for serum proteins, and suggest that DBP preferentially adsorbed to HA composed of rod‐shaped particles influences its potent osteoclast homing activity and local bone metabolism.

Biomimetic mineralization of calcium phosphates in polymeric hydrogels containing carboxyl groups

Abstract Biomimetic processing is an attractive method for the fabrication of inorganic crystalline materials with designed morphology under ambient conditions. Precipitation of inorganic solids in a hydrogel matrix could be regarded as mimicking the process of biomineralization and be called gel-mediated processing. The importance of the functional groups in reaction media and templates has been pointed out for such gel-mediated processing. In the present study, the role of carboxyl groups in hydrogel matrices on precipitation of calcium phosphate was investigated. Carboxyl groups in the hydrogel matrices provided pH buffering action, chelate formation with calcium ions and decreased the diffusion rate of calcium ions. The crystalline phase of the calcium phosphate changed from octacalcium phosphate to hydroxyapatite with increasing carboxyl group concentration in the hydrogels. The crystalline phase change is attributed to a decrease in the activation energy for calcium phosphate formation via a chelate structure involving calcium ions and carboxyl groups.

Synthesis of layered double hydroxide coatings with an oriented structure and controllable thickness on aluminium substrates

Layered double hydroxides (LDHs) are applied in environmental purification and biomedicine because of their anion intercalation and exchange properties. This study investigates the formation of LDH layers on aluminium substrates, through the reaction of substrates in solution containing magnesium ions at 30–90 °C. The resulting LDH layer was constructed from many plate-shaped crystals. The LDH crystal size increased with increasing reaction temperature. The LDH layer was preferentially oriented perpendicular to the substrate, thus the LDH crystal orientation was out-of-plane. The oriented structure was formed by crystal-growth-controlled geometrical selection. The LDH layer growth rate also increased with increasing reaction temperature. A high reaction temperature (90 °C) resulted in rapid formation of an approximately 10 μm-thick LDH layer. Milder temperatures (30 and 60 °C) resulted in a moderate LDH layer growth rate, which allowed the LDH layer thickness to be precisely controlled. The LDH layer removed toxic fluoride anions from aqueous solution without damaging the layer. These findings promote the development of oriented LDH coatings with precisely controlled microstructures and thickness.

Adhesion behaviors of Escherichia coli on hydroxyapatite

Optimum design of support materials for microorganisms is required for the construction of bioreactors. However, the effects of support materials on microorganisms are still unclear. In this study, we investigated the adhesion behavior of Escherichia coli (E. coli) on hydroxyapatite (HA), polyurethane (PU), poly(vinyl chloride) (PVC), and carbon (Carbon) to obtain basic knowledge for the design of support materials. The total metabolic activity and number of E. coli adhering on the samples followed the order of HA ≈ Carbon>PVC>PU. On the other hand, the water contact angle of the pellet surfaces followed the order of HA<Carbon<PVC<PU. The high hydrophilicity of the HA surface might be one of the suitable characters for the adhesion of E. coli. The results implied that HA has a potential as a support material for microorganisms used in bioreactors.

Rapid and topotactic transformation from octacalcium phosphate to hydroxyapatite (HAP): a new approach to self-organization of free-standing thin-film HAP-based nanohybrids

Biomineralization-inspired processing is attractive for the preparation of functionalized inorganic/organic polymer hybrid materials because the materials are obtained under mild conditions and by using organic templates. As for the formation processes of ordered nanocrystalline hydroxyapatite (HAP), the preparation of self-standing hybrid films based on HAP has not yet been established. In the present study, self-standing thin-film hybrids composed of HAP and poly(vinyl alcohol) (PVA) are obtained by rapid and topotactic transformation of thin films based on octacalcium phosphate (OCP) as a precursor in the organic polymer matrix. Bioinspired crystallization of calcium phosphate on the PVA matrix in the presence of poly(acrylic acid) leads to the formation of nanocomposite structures with oriented OCP nanorod crystals 2–4 nm in width and 10–30 nm in length. The nanostructures allow the composites to transform rapidly into a HAP/PVA hybrid thin film in water. The transformation proceeds without a change in the original OCP/PVA nanostructures, resulting in the formation of a HAP/PVA hybrid thin film with oriented HAP nanorod crystals 5–6 nm in width and 20–50 nm in length. The HAP/PVA hybrids have been obtained as self-standing films with submicrometer scale thickness. The ratio of organic to inorganic components in the self-standing hybrid thin films is similar to that in bones of vertebrates.

Formation of octacalcium phosphates with co-incorporated succinate and suberate ions

Octacalcium phosphates (OCPs) co-incorporated with various molar ratios of succinate and suberate ions were synthesized by wet processing. The interplanar spacings of the (100) planes (d(100)) of OCPs formed in the presence of succinic acid (Suc) or suberic acid (Sub) were larger than those of OCPs formed without addition of a dicarboxylic acid to the reaction solvent. The increases in the interplanar spacings of the (100) planes were caused by substitution of HPO(4)(2-) by dicarboxylate ions. The OCPs with co-incorporated succinate and suberate ions, i.e. solid solutions of OCP with incorporated Suc and Sub, were formed by reactions in the presence of Suc and Sub. When the Suc/(Suc + Sub) values in the starting compositions were in the range 0.45-1.0, Suc was preferentially incorporated into the OCP. In contrast, when the Sub/(Suc + Sub) values in the starting compositions were in the range 0.60-1.0, Sub was preferentially incorporated into the OCP crystals.

Morphological control of layered double hydroxide through a biomimetic approach using carboxylic and sulfonic acids

Abstract Layered double hydroxides (LDHs) have intercalation properties and are used in various applications. The performances of the LDH materials can be improved by controlling crystal morphology. Morphology of inorganic crystals is controlled by organic molecules in biomineralization. Inspired by biomineralization, we investigated the effect of the addition of mono, di and triacids as morphological control agents on crystal morphology of LDH synthesized by the homogeneous precipitation method. Morphology of LDH was changed from hexagonal plate to stacked disc by addition of monoacids, namely acetic acid and methanesulfonic acid, in the reaction solution. Flower-shaped LDH crystals were formed in the presence of diacids and a triacid, namely succinic acid, 1,2-ethanedisulfonic acid and 1,2,3-propanetricarboxylic acid. We found that the morphology of the LDH crystals was controlled by the number of functional group on the morphological control agent rather than the type of functional group. These findings can contribute for the development of novel and functional LDH materials with precisely controlled morphology.

Carbonate-containing hydroxyapatite synthesized by the hydrothermal treatment of different calcium carbonates in a phosphate-containing solution

Abstract Carbonate-containing hydroxyapatite (CHA) particles were synthesized by the hydrothermal treatment of calcium carbonates in a phosphate-containing solution. Three types of calcium carbonates were synthesized: aragonite particles with rough surfaces, calcite particles with rough surfaces by heat treatment of aragonite particles with rough surfaces, and aragonite particles with smooth surfaces. The effects of the calcium carbonate crystal phase and morphology on the synthesized CHA were investigated and morphological changes in the formed CHA particles were observed. The reaction rates varied depending on the calcium carbonate crystal phase and morphology and this difference in reaction rates mainly affected the morphology of the synthesized CHA. On the other hand, the carbonate content and lattice constants of formed CHA were almost independent of the calcium carbonate crystal phase and morphology. This is because the equilibrium mainly governed the composition of the synthesized CHA. The characteristics of the starting material were highly important factors in controlling the morphology of the synthesized CHA.

Regulation and Biological Significance of Formation of Osteoclasts and Foreign Body Giant Cells in an Extraskeletal Implantation Model

The implantation of biomaterials induces a granulomatous reaction accompanied by foreign body giant cells (FBGCs). The characterization of multinucleated giant cells (MNGCs) around bone substitutes implanted in bone defects is more complicated because of healing with bone admixed with residual bone substitutes and their hybrid, and the appearance of two kinds of MNGCs, osteoclasts and FBGCs. Furthermore, the clinical significance of osteoclasts and FBGCs in the healing of implanted regions remains unclear. The aim of the present study was to characterize MNGCs around bone substitutes using an extraskeletal implantation model and evaluate the clinical significance of osteoclasts and FBGCs. Beta-tricalcium phosphate (β-TCP) granules were implanted into rat subcutaneous tissue with or without bone marrow mesenchymal cells (BMMCs), which include osteogenic progenitor cells. We also compared the biological significance of plasma and purified fibrin, which were used as binders for implants. Twelve weeks after implantation, osteogenesis was only detected in specimens implanted with BMMCs. The expression of two typical osteoclast markers, tartrate-resistant acid phosphatase (TRAP) and cathepsin-K (CTSK), was analyzed, and TRAP-positive and CTSK-positive osteoclasts were only detected beside bone. In contrast, most of the MNGCs in specimens without the implantation of BMMCs were FBGCs that were negative for TRAP, whereas the degradation of β-TCP was detected. In the region implanted with β-TCP granules with plasma, FBGCs tested positive for CTSK, and when β-TCP granules were implanted with purified fibrin, FBGCs tested negative for CTSK. These results showed that osteogenesis was essential to osteoclastogenesis, two kinds of FBGCs, CTSK-positive and CTSK-negative, were induced, and the expression of CTSK was plasma-dependent. In addition, the implantation of BMMCs was suggested to contribute to osteogenesis and the replacement of implanted β-TCP granules to bone.

Effects of Monocarboxylic Acid Addition on Crystallization of Calcium Phosphate in a Hydrogel Matrix

In biomineralization, it is thought that water-soluble organic substances control crystal growth of minerals in hard tissues. The roles of organic substances are not well understood, because the biomineralization process is established by complicated parameters. Crystal growth in hydrogel matrixes can be regarded as simplified model system of biomineralization. In the present study, we investigated the effects of propionic acid (Pro) on crystalline phases and crystal morphologies of calcium phosphate formed in polymeric hydrogel matrixes as the model system of biomineralization. Crystalline phase of the precipitates was octacalcium phosphate (OCP) with spherical shape regardless of Pro concentrations. The fibrous crystals formed under the condition without addition of Pro. The crystal morphologies composing spherical crystals were changed from fibrous to plate-like shape with increasing Pro concentrations. Generally, OCP crystal has plate-like shape exposing (100) face, which calcium ions exist on. Therefore, crystal growth rate of [100] direction of OCP was decreased by Pro adsorbed on (100) face. As a result, crystal morphology composing spherulite became plate-like shape with increasing Pro concentrations.