Papers by Author: Takafumi Yoshikawa

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Authors: Toshimasa Uemura, J. Dong, Takafumi Yoshikawa, Y. Hakamatsuka, H. Irie, Hisahiro Inoue, Tetsuya Tateishi
Authors: Jin Iida, Takafumi Yoshikawa, Y. Ueda, Hajime Ohgushi, Toshimasa Uemura, Y. Enomoto, Kunio Ichijima, Yoshinori Takakura, Tetsuya Tateishi
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.
Authors: Takafumi Yoshikawa, Tetsuji Ohmura, Yasunori Sen, Jin Iida, Yoshinori Takakura, Iehisa Nonaka, Kunio Ichijima
Authors: Takafumi Yoshikawa, Hajime Ohgushi, Toshimasa Uemura, Y. Ueda, Hideo Nakajima, Y. Enomoto, Kunio Ichijima, Yoshinori Takakura, Tetsuya Tateishi
Authors: Hajime Ohgushi, Y. Dohi, Toshiaki Noshi, M. Ikeuchi, Takafumi Yoshikawa, M. Okumura, Hideo Nakajima, Yoshinori Takakura
Authors: N. Satoh, Takafumi Yoshikawa, Kazuhide Miyazaki, Hideki Shigematsu, Y. Ueda, M. Koizumi, Yoshinori Takakura
Abstract: Availability, storage and transportation of engineered bone tissue fabricated in vitro are major practical problems associated with adequate use of bone replacement grafts for the treatment of bone diseases. The ability to maintain viable engineered bone tissue would facilitate future clinical applications. In the present study, we investigated time required for transportation of engineered bone removed from cool storage, from the culture room to the operating room; and examined effects of cool storage on survival of engineered bone tissue. Bone marrowcells were obtained from the iliac bone of a 60-year-old male affected with lumbar spondylosis, and then incubated in standard medium. After two weeks in primary culture, cultured cells were trypsinized, and a concentrated cell suspension was incubated with a porous beta-TCP block. After 3 weeks of subculture with the osteogenic medium containing dexamethasone etc., engineered bone tissue was collected, stored for 0, 6, 12, 24 hours at 4 °C, and was subcutaneously implanted into the back of nude mice. Six weeks after implantation, implants were harvested. Before and after implantation, significant activity could be detected in all animals. In in vitro and in vivo situations, osteogenic activity of engineered bone tissue could be maintained even after 24 hours. These results provided information on appropriate storage conditions for engineered bone tissue.
Authors: Takafumi Yoshikawa, Y. Ueda, Takahito Ohmura, Yasunori Sen, Jin Iida, M. Koizumi, K. Kawate, Yoshinori Takakura, Akitaka Nonomura
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