Papers by Author: Masaru Murata

Abstract: Commercial hydroxyapatite (HAp) porous granules were designed by the supersonic treatment at 120W and 38 kHz for 15 min in parenteral fluids (amino-acids (PF-A), electrolytes and carbohydrates (PF-EC), the mixtures (PF-M)) to form PF-A/HAp, PF-EC/HAp and PF-M/HAp. The modified HAp kept spherical shape and exhibited adhesive aggregates originated from parenteral fluid components on the surface layer. The BET specific surface areas decreased from 39 m²・g^-1 to 19-24 m²・g^-1. The granules dried at 293K were stirred at 309.5 K in 0.5-2.5 mg・cm^-3 CEZ saline solutions. Adsorption isotherms of CEZ for all the granules almost obeyed the Langmuir type-equation. The amounts of CEZ adsorbed on the modified HAp were larger than those on HAp. Regarding CEZ-release characteristics in a biomimetic environment, the CEZ-adsorbed granules were freeze-dried or dried at 293K and they were stirred at 309.5K and pH 7.40 in simulated body fluid (SBF). The CEZ-release efficiencies for granules freeze-dried into SBF were significantly higher than those for granules dried at 293K. The values of granules freeze-dried were 37-43% for PF-A/HAp, 75-83% for PF-EC/HAp, and 56-64% for PF-M/HAp, which were related to the interface function, such as hydrophilicity of surface and multilayer film thickness of water molecule.

Abstract: Bio-absorbable materials have been strongly needed in bone regenerative surgery. β-TCP ceramics have been widely used as bone tissue scaffold materials, due to their bio-compatibility and bio-degradation. The aims of this study are to estimate blood permeation into different porous β-TCP blocks (75% and 67% in porosity), and to evaluate the behaviors of the 75% porous β-TCP block in rat subcutaneous tissue and sheep iliac bone defect by histological observation and 3-dimensional (3D) imaging analysis by μ-CT. The 75% β-TCP block revealed better performance in blood permeation than the 67% β-TCP in a dish including 3ml of sheep blood at 2 and 10 minutes. Almost area of the 75% β-TCP block turned to red at 10 minutes. In rat subcutaneous tissue, the bulk region of the 75% β-TCP was stained with HE. TRAP-positive multinucleated giant cells appeared on the surface of bulk at 4 weeks. In sheep iliac bone defect (10×15×9 mm³) model, μ-CT showed bone ingrowth into almost pores of the 75% β-TCP block at 2 months, and the block was absorbed and replaced by new bone until 4 months. The block was reduced to one-third in horizontal length and from 10 mm to 4 mm in vertical length at 2 months by 3D images. Body fluid stained by HE was found in the bulk region. We believe the body fluid permeation inside the bulk of the 75% porous β-TCP should contribute to the initial cell adhesion, proliferation and differentiation, and its biodegradation. It was concluded that the super porous β-TCP block with hydrophilic property might be a biological scaffold, harmonized with bone remodeling.

Abstract: Microcracks and trabecular fractures can be observed in physiological bone. Biomimetic hydroxyapatite (HAp) scaffolds have been strongly needed in bone regenerative medicine. We have been developing the combination method of the partial dissolution-precipitation techniques involving the stirring-supersonic treatment in 1.7×10^-2 N HNO₃ solution containing Ca^2＋and PO₄^3- ions to improve the surface and the bulk of commercially available synthetic HAp block (82.5% in porosity, 50-300µm in macropore). The modified HAp was named as a partially dissolved and precipitated HAp (PDP-HAp). The aims of this study are to characterize the PDP-HAp and to observe cell response for the ceramics in rat scalp tissue. The PDP-HAp exhibited the macropore sizes of 50-200µm, the porosities of 85-90%, and the specific surface areas of 1.0-2.0 m²･g^-1, with many micro-cracks. Twenty rats were divided into 2 groups. At 9 months, bone induction occurred inside the many pores in the PDP-HAp group, while bone and cartilage were not found in the HAp group. We believe that osteoinduction by the PDP-HAp is different from the process of BMP-loaded HAp-induced bone formation. The PDP-HAp might be applied as potential ceramics with osteoinductive properity and excellent biocompatibility in difficult bone regenerative cases.

Abstract: As interface design of autotransportation materials and bioactive ceramics by supersonic treatment, human teeth-originated granules and commercial hydroxyapatite (HAp) were partially dissolved in different acid solutions, such as electrolyzed water and HNO₃ aqueous solution to control bio-absorption, adsorption-release of bone growth factor and anti-bacterial characteristics. Human teeth were pulverized with cooling, dissolved in strongly acidic electrolyzed water (pH 2.6-3.1) or 2.0% HNO₃ solution to obtain demineralized dentin matrix granules. For supersonic or stirring demineralization in the acidic electrolyzed water, dissolution efficiencies (DE) were 3-12% and dentinal tubules with 1-2μm were found, while for supersonic demineralization in the HNO₃ solution, DE was attained to the highest value of 86%. When HAp was dissolved with stirring in the acidic electrolyzed water, DE of porous ceramics was lower than that of spherical particles due to smaller specific surface area and good crystallinity. For supersonic dissolution of porous ceramics in the acidic electrolyzed water, DE was 30% and extension of grain boundary and micro-crack were observed. Concerning supersonic irradiation of the electolyzed water to parietal bone in rats by using ultrasonic scaler, enlargement and propagation of micro-crack were recognized on the hard tissues.

Abstract: Biomimetic composites of hydroxyapatite (HAp) and collagen with fast bio-absorption and good biocompatibility were designed utilizing salmon bone and skin at 283-293 K and pH 7.5-7.9 by a dissolution-precipitation method. Simultaneously, dissolved-precipitated HAp (dp-HA) was prepared at pH 9-10 using the calcined bone. The HAp/collagen composites (HA-C) were constituted by Ca²⁺-deficient HAp, I type-collagen and gelatin. At the synthetic temperature of 283 K, collagen fiber and HAp microcrystals were seen, while at 293 K, frock like-agglomerated particles or fiber like-oriented columnar ones were observed depending on the composition ratio (H/C) of HAp to collagen. Specific surface areas and total pore volumes for the HA-C synthesized at 293 K clearly increased with increasing the H/C, although there were micro-and-meso-pores in the pore diameters of 3-30 nm. Concerning water vapor-adsorption isotherms at 298 K for the HA-C powders, hysteresis-curves of the amounts of water vapor adsorbed (V) were recognized in the adsorption-desorption processes. The V values increased with increasing the H/C under the low relative pressures (P/P_S) of 0-0.3 that mean monolayer-adsorption of water molecule. However, for H/C=2.2, the V values were the highest under the high P/P_S of 0.70-0.90 that mean multilayer-adsorption and the biggest hysteresis-curve was found under the P/P_S of 0.45-0.60, suggesting that the HA-C (H/C=2.2) powder not only adsorb water molecule on the surfaces but also absorb one into the crystal structure. Accordingly, the biomimetic HA-C powders will be applied as water-absorbable adsorption materials for cosmetic products or bone-regeneration therapy.

Abstract: We previously reported functionally graded hydroxyapatite (fg-HAp) with the characteristics of blood permeability into the bulk and osteoinduction by adding low dose of bone morphogenetic protein-2 (BMP-2). In this study, we evaluated the bioactivity of the obtained dense HAp bodies in a simulated body fluid (SBF) and the osteoinductive activity with or without SBF treatment in vivo. The fg-HApSBF was prepared by immersing the fg-HAp in the SBF solution at 14 days and the surface structure was observed by SEM. The fg-HApSBF showed fine bone-like crystal on the surface of the HAp. Ectopic bone formation occurred in the fg-HAp/BMP-2 (1.0, 0.5μg) system at 3 weeks, while only in the fg-HApSBF/BMP-2 (1.0μg), bone induction was found. The histological finding showed body fluid permeation into the fg-HApSBF bulk and bone formation were obserbed.

Abstract: Our approach to the design of biological material scaffolds for bone regeneration is the creation of cell environments that mimic natural tissues. Recently, we confirmed hitologically that the material surfaces of conventional, nonabsorbable ceramics shed body fluid. For bone engineering, it is known that ideal scaffolds should be bioabsorbable, mimetic, and hydrophilic materials that allow for the permeation of liquid components, such as blood and/or extracellular fluid. In our previous study showed functionally graded hydroxyapatite (fg-HAp) absorbed body fluid including albumin. In this study, we investigated the behavior of human blood adsorption to the fg-HAp by using the scanning electron microscope (SEM). The adsorption of the platelets and the formation of the fibrinous network were observed in the fg-HAp group incubated 20 minutes.

Abstract: In our previous clinical study, autogenous demineralized dentin matrices (DDM) prepared from the functional vital teeth (#38, #41) of thirty-five-year-old female were grafted on the bone defect, using newly developed mill, and then received to the host without troubles. In this study, we implanted the human tooth dentin adjusted previously and the dental implants into the regions of missing tooth simultaneously. Fifty-seven-year-old female presented with missing teeth (#35-#37, #45-#47). First, a non-functional vital tooth (#18) were extracted and cryopreserved immediately. 11 months after extraction, the tooth was crushed by newly developed auto-crash mill using ZrO2 vessel and ZrO2 blade for 1 minute. The crushed granules were demineralized completely in 2% HNO3 solution, rinsed in cold distilled water and lyophilized (granule size: 0.5-2.0mm). The bacteria-free of the DDM were confirmed by the bacteriological examination before use. Drilling of the prospective implant beds were then performed according to the manufacture’s protocol and a screw-type rough surface implants (Nobel Biocare® Mk III) were placed. The adjusted DDM granules were implanted into the bone defect (#45). There are no postoperative complications at 3 years after implantation. This case indicates that the preserved autogenous DDM can be used as collagenous biomaterials with osteoinductive potency.

Abstract: Bone and dentin consist of hydroxyapatite, collagen and body fluid. From biological points of view, we have been focusing on HAp and collagen materials for bone regeneration. The aim of this study is to estimate the appearance of multinuclear giant cells for non-organic (functionally graded HAp: fg-HAp) and organic materials (demineralized dentin matrix: DDM), histologically. The fg-HAp ceramic: Biomimetic fg-HAp was designed by using the partial dissolution-precipitation methods. The fg-HAp with micro-pores of 10-160 nm had larger specific surface areas (30-40 m²･g^-1) than the synthetic HAp. Acid- insoluble dentin matrix (DDM): Human teeth were crushed under the cooling, completely demineralized in 0.026N HNO₃ solution, and dried. The materials were implanted into the subcutaneous tissues (Wistar rats, 4 week-old, male), and removed at 1 and 4 weeks after the operations. Multinucleated giant cells were counted in the H-E sections. Giant cells predominantly appeared on the biodegradable micro-crystals at 1 week. The number of giant cells was more numerous in fg-HAp than in DDM. There was a significant difference in the cell number between fg-HAp and DDM. The absorption mechanism of fg-HAp should be predominantly cellular phagocytosis, while that of DDM might be predominantly enzymatic digestion. These data support the hypothesis that the biological HAp crystals may function as mineral signal in the recruitment and differentiation of multinucleated giant cells.

Abstract: Bio-absorbable and biomimetic composites (HA-C) of hydroxyapatite (HAp) and collagen were designed utilizing salmon bone and skin at 298K and pH 7.5-7.9 by a dissolution-precipitation method. The HA-C powders were consistuted by Ca²⁺-deficient HAp containing small amounts of Na⁺ and Mg²⁺ ions and I type-collagen. Microstructure and surface characteristics of the HA-C powders gradually changed depending on the composition ratio of HAp to collagen (H/C). The HA-C powder of H/C=3.5 had frock like-agglomerated particles consisted of nano-crystals, micro-pore, and meso-pore so that it exhibited high specific surface area (75m²¥g^-1) and large total pore volume (0.543 cm³¥g^-1). From adsorption isotherms of water vapor at 298K for the HA-C powders, hysteresis - curves of the amounts of water vapor adsorbed were obtained in the adsorption-desorption processes. The amounts of water vapor adsorbed for the H/C=3.5 powder were the highest values under the relative partial pressures of 0.73-0.93. At 2 weeks after implantation of the HA-C powders into the subcutaneous tissue of the back region in rats, collagen was completely bio-absorbed and body fluid permeated into large agglomerated particles, although bio-absorption by multi-giant cell- infiltration was recognized around the surface layers of HAp particles.