Papers by Author: Hajime Ohgushi

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Authors: Yasuhito Tanaka, Yoshinori Takakura, K. Kadono, Akira Taniguchi, K. Hayashi, Jin Iida, Koh Ichi Sugimoto, Yasuaki Tohma, Hajime Ohgushi
805
Authors: Jin Iida, Takafumi Yoshikawa, Y. Ueda, Hajime Ohgushi, Toshimasa Uemura, Y. Enomoto, Kunio Ichijima, Yoshinori Takakura, Tetsuya Tateishi
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Authors: Kazuhide Miyazaki, Takafumi Yoshikawa, Jin Iida, Y. Ueda, M. Koizumi, N. Satoh, Hideki Shigematsu, Y. Dohi, Hajime Ohgushi, Yoshinori Takakura
Abstract: Introduction: Marrow mesenchymal cells contain stem cells and can regenerate tissues. We previously reported the clinical application of autologous cultured bone to regeneration therapy. However, in cases with low numbers of active cells, culture is often unsatisfactory. If frozen marrow cells retain their osteogenic potential, we could clinically use them in regeneration therapy as alternatives to high active cells obtained from youngsters. Here, we examined osteogenic potential of frozen human mesenchymal stem cells in combination with recombinant human bone morphogenetic protein (rhBMP) using biochemical and histological analyses. Method: Marrow fluid was aspirated from the human iliac bone of a 46-year-old man with lumbar canal stenosis during surgery. Two weeks after primary culture in standard medium, bone marrow mesenchymal stem cells (BMSCs) were trypsinized for the preparation of a cell suspension, and cells were concentrated to 106 cells/ml by centrifugation. Cells were kept at – 80 °C until use. To impregnate porous hydroxyapatite (HA) with rhBMP, 1 3g rhBMP/20 3l 0.1 % trifluoroacetic acid was applied on HA, and then desiccated under vacuum. In the present study, we used 4 subgroups: BMSC/rhBMP/HA, BMSC/HA, rhBMP/HA, and HA only. HA constructs from the 4 subgroups were implanted at subcutaneous sites on the back of 5-week-old nude mice (BALB/cA Jcl-nu). Eight weeks after implantation, implanted HA constructs were harvested, and biochemical and histological analyses were performed. Alkaline phosphatase activity (ALP) and human osteocalcin (hOs) levels were measured. Results and Discussion: ALP activity and hOs in the BMSC/BMP/HA subgroup were 2 or 3 times that in the BMSC/HA subgroup. Histological analysis showed that significant bone formation was observed in these two subgroups, and supported biochemical data. However, in the BMP/HA and HA only subgroups, significant bone formation could not be detected histologically nor biochemically. These results indicated that a combination of rhBMP and BMSCs, and only with a minimal amount of 1 3g rhBMP, allowed successful generation of human bone. In the human body, rhBMP in the order of milligrams is necessary for bone formation. However, by combining BMSCs, HA and rhBMP, only a small amount of rhBMP was needed to dramatically enhance osteogenic potential. As we reported here, cryopreserved BMSCs also showed high osteoblastic activity. In conclusion, this study provided histological and biochemical evidence that combination of cryopreserved BMSCs, BMP, and porous HA could enhance osteogenic potential.
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Authors: Takafumi Yoshikawa, Hajime Ohgushi, Toshimasa Uemura, Y. Ueda, Hideo Nakajima, Y. Enomoto, Kunio Ichijima, Yoshinori Takakura, Tetsuya Tateishi
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Authors: Hajime Ohgushi, Y. Dohi, Toshiaki Noshi, M. Ikeuchi, Takafumi Yoshikawa, M. Okumura, Hideo Nakajima, Yoshinori Takakura
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Authors: Asako Matsushima, Noriko Kotobuki, Mika Tadokoro, Hajime Ohgushi
Abstract: Hydroxyapatite (HA) ceramics together with various kinds of osteogenic cells have been used in bone tissue engineering. It is well known that the ceramics structure and composition affect cell proliferation / differentiation. In this study, three different types of HA ceramics were used to investigate initial cell attachment followed by osteoblastic differentiation of human mesenchymal stromal cells (MSCs). The results indicated that micro-pore affected the cell attachment and porosity (pore diameter and inter-pore connection) was the key to allow spacious distribution of the viable cells in the ceramics. This study also confirmed that surface pore areas of HA ceramics support the differentiation of human MSCs and thus the ceramics have the capability to regenerate damaged bone tissue.
1067
Authors: Norimasa Tsuji, Masataka Yoshikawa, Tadao Toda, Hiroko Machida, Hajime Ohgushi
Abstract: The purpose of this study was to estimate hard tissue formation in two types of porous columnar hydroxyapatite (HA) in order to use as a scaffold for regeneration of dentine-pulp complex. Hard tissue formation in the columnar HA scaffold with a hollow center was compared to that in the columnar HA scaffold without a hollow center. The scaffolds were immersed in hyaluronic acid sodium salt solution and were soaked in bone marrow cell suspension. They were respectively implanted into dorsal subcutis of rats for 4 weeks. Serially sectioned paraffin specimens were made and observed histologically. The scaffolds with a hollow center showed new hard tissue formation in many pores between the superficies and the wall of hollow. On the other hand, in the scaffolds without a hollow center, hard tissue formation was observed in only a few pores in the area near the external superficies. The results of this study suggested that the supply of nutrition and bioactive substance from the surrounding tissue were indispensable for differentiation of bone marrow cells and formation of new hard tissue in scaffold. A large contact area of a scaffold to the surrounding tissue may contribute to nutrition supply into the pores.
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Authors: H. Shimaoka, Y. Dohi, K. Narikawa, Hajime Ohgushi, M. Ikeuchi, M. Okamoto, T. Kirita, K. Yonemasu
Abstract: Various recombinant growth factors have been used for promoting osteoblastic differentiation cascade. To compare the growth/differentiation factor-5 (GDF-5) and bone morphogenetic protein-2 (BMP-2) in the in vivo osteogenic potential of bone marrow mesenchymal stem cells (MSCs), the bone formation was assessed by rat subcutaneous implantation of 5 kinds of hydroxyapatite (HA) implants; namely GDF/HA composites, BMP/HA composites, MSCs/HA composites and the MSCs/HA composites supplemented with recombinant mouse GDF-5 (GDF/MSCs/HA) or recombinant human BMP-2 (BMP/MSCs/HA). Neither the GDF/HA nor the BMP/HA composites exhibited any bone formation at any time after implantation. At both 2 and 4 weeks after implantation, obvious de novo bone formation together with active osteoblasts was seen histologically in many pores of the GDF/MSCs/HA and BMP/MSCs/HA composites. The GDF/MSCs/HA and BMP/MSCs/HA composites also showed high alkaline phosphatase (ALP) and osteocalcin expression determined at both the protein and gene levels. Compared with GDF/MSCs/HA, the BMP/MSCs/HA composites exhibited excellent osteogenesis with relatively early osteoblastic phenotype expression. These findings indicate that the two growth factors synergistically enhance de novo bone formation capability of MSCs/HA composites and the importance of ceramic surface to retain and to deliver the molecules of growth factors for the cell differentiation and maturation.
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Authors: Hajime Ohgushi, Shigeyuki Kitamura, Noriko Kotobuki, Motohiro Hirose, Hiroko Machida, Akira Oshima, Yasuhito Tanaka, Yoshinori Takakura
Abstract: Alumina ceramics have excellent mechanical and biocompatible properties, but are bioinert and hence have no bone-bonding properties. We took a tissue engineering approach in an attempt to modify the ceramic surface and so provide an osteogenic/osteoconductive milieu. We used fresh human bone marrow cells obtained from the iliac crest by needle aspiration for culture expansion of mesenchymal stem cells (MSC) followed by in vitro osteogenic differentiation on both tissue culture polystyrene (TCPS) and alumina ceramics. We have succeeded in expanding the number of MSC from all 35 cases and compared the differentiation capability of selected MSC on alumina ceramics to that on TCPS. The cells on both substrata showed extensive alkaline phosphatase staining and mineralization as evidenced by calcein uptake. Biochemical analyses revealed high levels of alkaline phosphatase activity, osteocalcin expression, and calcium content. These data indicate that an alumina ceramic surface can support a differentiation cascade of MSC resulting in osteoblastic phenotype expression of the cells. Based on these results, we have performed clinical applications of tissue engineered total joint replacements for osteoarthritic patients.
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