Papers by Author: Masanori Kikuchi

Abstract: The combination of tetracalcium phosphate (TTCP; Ca₄(PO₄)₂O) and dicalcium phosphate anhydrous (DCPA; CaHPO₄) which are known as one system of apatite cements already used in the medical and dental application. In spite of several advantages of apatite cements, such as self-setting ability and biocompatibility, their mechanical strengths are still low. The aim of this study is to improve the mechanical strength of the TTCP-DCPA apatite cement using the hydroxyapatite/collagen nanocomposite (HAp/Col). The apatite cement powder was prepared using an equimolar TTCP and DCPA with addition of 10% and 20% of the HAp/Col. That without the HAp/Col was used as a control group. Each group was mixed with 1 mol/L Na_1.8H_1.2PO₄ aqueous solution at powder/liquid ratio of 0.5 and hardened at 37°C and 100 % of relative humidity for 24 hours. A setting time of the cement was evaluated using Vicat needle according to ISO 1566 for dental zinc phosphate cements. Morphology of the cements set were observed by the scanning electron microscopy (SEM), and crystalline phases were identified by the powder X-Ray diffractometry (XRD). The mechanical strength of the cement set was evaluated by the diametral tensile strength (DTS). The setting times of cements were the shortest for the cement with HAp/Col and the longest for the control. XRD patterns of the cement at 24 hours after mixing revealed that all cements changed into apatite from the mixture of TTCP and DCPA. The DTSs of cements were the highest for the cement with 20% HAp/Col and the lowest for the control with significant differences between the cement with 20 % HAp/Col and respective other two cements. The scanning electron micrographs of the surface and fracture surface of the cements suggested that the cement with HAp/Col showed denser structure in comparison to the control and the HAp/Col fibers and/or sheets covered the fracture surface. The HAp/Col would act as reinforcement fibers as well as an adhesive of apatite granules formed by the reaction between TTCP and DCPA. The setting time and mechanical strength of apatite cement was statistically significant improved by adding 20% HAp/Col.

Abstract: Calcium phosphate (CaP) coating is an effective technique for surface functionalization of biomaterials. The objective of our research is to prepare calcium phosphate (CaP) coatings on a hydroxyapatite/collagen (HAp/Col) nanocomposite and subsequently provide it with gene delivery function through the immobilization of DNA in the coating. We have specifically selected the HAp/Col nanocomposite since it has the high potential as bone substitutes due to its similar composition, nanostructure, and biological properties to those of human bone. CaP coatings consisting of different sized particles were prepared on the HAp/Col nanocomposite membrane by immersing it in supersaturaterd CaP solutions (so-called RKM solutions) with the varied Ca and P concentration levels. We immobilized DNA in the CaP coatings together with lipid and fibronectin by supplementing DNA, lipid, and fibronectin to the RKM solutions (DLF-RKM solutions). Gene transfer capability of the resulting HAp/Col nanocomposite membrane was improved with decreasing concentration level of the DLF-RKM solution. It was confirmed that the present CaP coating technique was effective in providing the HAp/Col nanocomposite membrane with gene transfer capability and that the Ca and P concentration level of the DLF-RKM solution was a controlling factor affecting the gene transfer efficiency.

Abstract: Hydroxyapatite/collagen (HAp/Col) nanocomposites with bone-like self-organized nanostructure show excellent bioactivity in vivo. However, they show quite high absorbability for cationic ions and lower culture medium ionic concentrations which adversely affects bone cell proliferation and osteogenic differentiation in in vitro cell culture condition. To address this limitation, in this study we have supplemented Ca²⁺ and Mg²⁺ ions to the HAp/Col nanocomposite membrane sample prior to cell culture to improve it’s in vitro biological properties. The HAp/Col nanocomposite membrane samples were fabricated by the simultaneous titration method using Ca(OH)₂, type-I atelocollagen and H₃PO₄ as starting precursor materials. Prior to in vitro cell culture experiments, the HAp/Col samples were pretreated with Ca²⁺ and/or Mg²⁺ ions by immersing in 10 ml of 20 mM CaCl₂ solution, 20 mM MgCl₂ solution, or a solution containing 20 mM CaCl₂ and 20 mM MgCl₂ for 7 days. In vitro bone cell-material interactions on the pretreated and untreated HAp/Col samples were studied by culturing MC3T3-E1 cells up to 7 days. Enhanced bone cell proliferation was found on all the pretreated HAp/col samples as confirmed by the CCK-8 assay. Interestingly, the HAp/Col samples pretreated with both Ca²⁺ and Mg²⁺ ions showed the maximum viable bone cell density.

Abstract: International standard for test method on cell migration into a scaffold is one of the important things to evaluate the scaffold. The "cell migration" ability can divide into two parts. One is infiltration of cell suspension before in vitro cell culture on the scaffold. Another is migration of adherent cells from the edge of scaffold. The latter one could be closely related to cell/tissue migration into the scaffold when it is implanted into bone. Thus, in the present study, the cell migration ability was evaluated toward standardization of in vitro evaluation method for in vivo cell/tissue migration ability using several bioactive ceramics and composites including commercially available materials. The specimen 5 mm in diameter was placed on confluent MG63 cell layer. After 3 days incubation, the specimen was harvested, fixed and divided into two parts. Inside and outside of the scaffold were stained by Giemsa and observed by optical microscopy. In addition, the same specimen was critical point dried and observed with scanning electron microscope (SEM). From microscopic observation, MG63 cells migrated to pore walls of the specimen as well as a sidewall. Maximum migration distances were different among specimens and seemed to depend on pore structure and size as well as porosity. Similar behaviors were observed with SEM.Even relations between this test method and in vivo cell/tissue migration have not been evaluated, this test method is potentially a good method for testing cell migration ability of porous bioactive ceramics as well as other porous scaffold materials.

Abstract: Injectable hydroxyapatite/collagen nanocomposite (HAp/Col) artificial bone was prepared utilizing gelation of sodium alginate (Na-Alg). Mass ratio of the HAp/Col powder, with or without Ca adsorption treatment and Na-Alg (80-120, 300-400, 500-600 cP in viscosity at 10 g/dm³) was fixed at 90/10. Injectable HAp/Col was prepared by mixing the HAp/Col powder with Na-Alg solution at several powder (HAp/Col)/liquid (Na-Alg solution) ratios (P/L ratio, g/cm³). The result of consistency measurement suggested that the operability of injectable HAp/Col paste could be controlled by both the P/L ratio and the viscosity of Na-Alg solution. According to the consistency measurement and practical feelings during mixing, P/L=1/1.67 (80-120 cP) and 1/1.89 (300-400, 500-600 cP) were considered to be the highest P/L ratio allowed to mix the HAp/Col paste with a spatula. At the P/L=1/2.33 (80-120 cP), the paste prepared with the non-treated HAp/Col powder, placed in an incubator (37 °C,relative humidity 100%) for 24h, demonstrated gel-like property, while the paste prepared with Ca-treated HAp/Col powder did putty-like property. The difference in their property might be caused by the initial bonding behavior between Alg and Ca²⁺ after mixing. The setting time measurement with Gillmore needle was impossible because they were toosoft for this method. Even though, their operability and coalescence/settingproperty could be used as the injectable bone filler.

Abstract: Osteoclast differentiation of bone marrow cells on hydroxyapatite/collagen self-organized bone-like nanocomposite (HAp/Col) disk in vitro was evaluated by coculture of mouse bone marrow cells with mouse osteoblasts with or without addition of osteoclastic inducers, 1,25-Dihydroxyvitamin D3 and 1 µM prostaglandin E2. Dentine slice and tissue culture polystyrene were used as controls. Good osteoclastic differentiation at day 7 were observed among the bone marrow cells cultured on the HAp /Col disk and controls with osteoclastic inducers. On the contrary, osteoclastic differentiation was observed only for marrow cells cultured on the HAp/Col disk. Nano- and micro- structures as well as chemical and mechanical properties have a potential to control cell differentiation.

Abstract: Effect of osteogenic activities of MG63 on the HAp/Col membrane was examined at day 10 and 14 by reverse-transcript and real-time polymerase chain reactions. Osteogenic activities of MG63 were upregulated by culture them on the HAp/Col membrane in comparison to those on tissue culture polystyrene. The novel three-dimensional HAp/Col scaffold was prepared from the HAp/Col wavy membrane. The cylindrical HAp/Col scaffold was successfully prepared and indicated at least 2.5-times higher compressive strength and Young's modulus compared to the previous HAp/Col composites. The novel scaffold could be useful for regenerative medicine.

Abstract: Biocompatible and water-soluble fibers (sodium carboxymethyl cellulose (CMC)) were fabricated via a wet-spinning method. The CMC fibers/ polymethyl methacrylate (PMMA) mixtures and CMC fibers/Poly (L-lactide-co-glycolide-co- -caprolactone) (PLGC) mixtures were prepared by a heat-kneading method. For CMC fibers/PMMA, after removal of the CMC fibers from the mixtures, the interconnected porous scaffolds with porosity from 27.79 % to 60.98 % (volume percent) were obtained. For CMC fibers/PLGC, the interconnected porous scaffolds with porosity 38.55 % and 76.83 % (volume percent) were prepared. The solid CMC fibers/PLGC mixtures had the higher ultimate tensile strength and Young, s modulus than those of porous PLGC scaffolds.

Abstract: The bone-like self-organized hydroxyapatite/collagen (HAp/Col) nanocomposite sponge was prepared from HAp/Col nanocomposite fibers. We analyzed osteogenic activity of human osteoblastic MG63 cells in the HAp/Col sponge under pressure/perfusion culture. Collagen (Col) sponge was used as a control. The MG63 cells were attached well and showed good proliferation in the HAp/Col sponge. The total DNA content in the HAp/Col sponge was approximately 1.8 times greater than that in the Col sponge at 21 days. The MG63 cells showed good osteogenic gene expression in the HAp/Col sponge by reverse transcription-polymerase chain reaction analysis. These result suggested that HAp/Col sponge can be useful for bone tissue engineering scaffold materials.

Abstract: Biomaterials Center is composed of five groups and collaborate each other to examine interdisciplinary fields of biomaterials. In the ceramics-based biomaterials research, we have been developing three novel bone regeneration materials, i.e., high-porosity hydroxyapatite (HAp) ceramics with high-strength, guided bone regeneration (GBR) membranes and bone-like nanocomposite composed of HAp and collagen. The GBR membrane composed of β-tricalcium phosphate and biodegradable copolymer of lactide, glycolide and ε-caprolactone has thermoplastic, pH auto-adjustment and enough mechanical property to protect an invasion of surrounding tissues. With the membrane, bone defect up to 20 × 10 × 10 mm3 in length in mandibles and segmental bone defect up to 20 mm in length in tibiae of beagles are regenerated without any additional bone fillers or cell transplantations. The bone-like nanocomposite is synthesized by a co-precipitation of HAp and collagen via their self-organization. The dense composite has a half to quarter mechanical strength (40 MPa) to cortical bone and the porous one demonstrates sponge-like viscoelasticity. The composites implanted into bone are incorporated into bone remodeling metabolism like as autogenous bone graft, i.e., they are resorbed by osteolasts followed by osteogenesis by osteoblasts.