2022/01/27 更新

写真a

カワセ トモユキ
川瀬 知之
KAWASE Tomoyuki
所属
教育研究院 医歯学系 歯学系列 准教授
医歯学総合研究科 分子細胞医学専攻 准教授
歯学部 歯学科 准教授
職名
准教授
外部リンク

学位

  • 歯学博士 ( 1990年6月   新潟大学 )

研究キーワード

  • 血小板濃縮材料

  • 歯周組織の再生

  • Bone metabolism

  • Regeneration of periodontal tissue

  • 生体材料

研究分野

  • ライフサイエンス / 補綴系歯学

  • ライフサイエンス / 口腔再生医学、歯科医用工学

  • ライフサイエンス / 保存治療系歯学

経歴(researchmap)

  • -date Associate Professor, Graduate School of Medical

    2002年

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  • -date 新潟大学大学院 准教授

    2002年

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  • Associate Professor, School of Dentistry,

    1993年 - 2002年

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  • 新潟大学歯学部 助教授   Faculty of Dentistry

    1993年 - 2002年

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  • 新潟大学歯学部 講師   Faculty of Dentistry

    1992年 - 1993年

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  • Assistant Professor, School of Dentistry,

    1992年 - 1993年

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  • Research Associate, School of Dentistry,

    1986年 - 1992年

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  • 新潟大学歯学部 助手   Faculty of Dentistry

    1986年 - 1992年

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  • Niigata Univ.

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  • Niigata Univ.

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  • Niigata Univ.

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  • and Dental Sciences, Niigata Univ.

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▶ 全件表示

経歴

  • 新潟大学   医歯学総合研究科 分子細胞医学専攻   准教授

    2004年4月 - 現在

  • 新潟大学   歯学部 歯学科   准教授

    2004年4月 - 現在

  • 新潟大学   歯学部   講師

    1992年1月 - 1994年1月

  • 新潟大学   歯学部   助手

    1986年1月 - 1992年9月

学歴

  • 新潟大学

    - 1985年

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  • 新潟大学   歯学部

    - 1985年

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    国名: 日本国

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所属学協会

▶ 全件表示

委員歴

  • 歯科基礎医学会   評議員  

    1998年 - 現在   

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    団体区分:学協会

    歯科基礎医学会

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  • 日本薬理学会   学術評議員  

    1994年 - 現在   

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    団体区分:学協会

    日本薬理学会

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  • 日本歯周病学会   評議員  

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    団体区分:学協会

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  • 日本再生医療学会   代議員  

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    団体区分:学協会

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論文

  • Osteoclastogenic Potential of Tissue-Engineered Periosteal Sheet: Effects of Culture Media on the Ability to Recruit Osteoclast Precursors. 国際誌

    Kohya Uematsu, Takashi Ushiki, Hajime Ishiguro, Riuko Ohashi, Suguru Tamura, Mari Watanabe, Yoko Fujimoto, Masaki Nagata, Yoichi Ajioka, Tomoyuki Kawase

    International journal of molecular sciences   22 ( 4 )   2021年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Cell culture media influence the characteristics of human osteogenic periosteal sheets. We have previously found that a stem cell medium facilitates growth and collagen matrix formation in vitro and osteogenesis in vivo. However, it has not yet been demonstrated which culture medium is superior for osteoclastogenesis, a prerequisite for reconstruction of normal bone metabolic basis. To address this question, we compared chemotaxis and osteoclastogenesis in tissue-engineered periosteal sheets (TPSs) prepared with two types of culture media. Periosteal tissues obtained from adult volunteers were expanded with the conventional Medium 199 or with the stem cell medium, MesenPRO. Hematopoietic enhanced-green-fluorescent-protein (EGFP)-nude mice were prepared by γ-irradiation of Balb/c nu/nu mice and subsequent transplantation of bone marrow cells from CAG-EGFP C57BL/6 mice. TPSs were implanted subcutaneously into the chimeric mice and retrieved after intervals for immunohistopathological examination. EGFP+ cells were similarly recruited to the implantation site in both the TPSs prepared, whereas the distribution of CD11b+ cells was significantly lower in the TPS prepared with the stem cell medium. Instead, osteoclastogenesis was higher in the TPS prepared with the stem cell medium than in the one prepared with the conventional medium. These findings suggest that the stem cell medium is preferable for the preparation of more functional TPSs.

    DOI: 10.3390/ijms22042169

    PubMed

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  • Platelet adhesion on commercially pure titanium plates in vitro III: effects of calcium phosphate-blasting on titanium plate biocompatibility

    Masayuki Nakamura, Hachidai Aizawa, Hideo Kawabata, Atsushi Sato, Taisuke Watanabe, Kazushige Isobe, Yutaka Kitamura, Takaaki Tanaka, Tomoyuki Kawase

    International Journal of Implant Dentistry   6 ( 1 )   2020年12月

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:Springer Science and Business Media LLC  

    <title>Abstract</title><sec>
    <title>Background</title>
    Platelet-rich plasma (PRP) is often used to improve surface biocompatibility. We previously found that platelets rapidly adhere to plain commercially pure titanium (cp-Ti) plates in the absence, but not in the presence, of plasma proteins. To further expand on these findings, in the present study, we switched titanium plates from a plain surface to a rough surface that is blasted with calcium phosphate (CaP) powder and then examined platelet adhesion and activation.


    </sec><sec>
    <title>Methods</title>
    Elemental distribution in CaP-blasted cp-Ti plates was analyzed using energy-dispersive X-ray spectroscopy. PRP samples prepared from anticoagulated blood samples of six healthy, non-smoking adult male donors were loaded on CaP-blasted cp-Ti plates for 1 h and fixed for examination of platelet morphology and visualization of PDGF-B and platelet surface markers (CD62P, CD63) using scanning electron microscopy and fluorescence microscopy. Plain SUS316L stainless steel plates used in injection needles were also examined for comparison.


    </sec><sec>
    <title>Results</title>
    Significant amounts of calcium and phosphate were detected on the CaP-blasted cp-Ti surface. Platelets rapidly adhered to this surface, leading to higher activation. Platelets also adhered to the plain stainless surface; however, the levels of adhesion and activation were much lower than those observed on the CaP-blasted cp-Ti plate.


    </sec><sec>
    <title>Conclusions</title>
    The CaP-blasted cp-Ti surface efficiently entraps and activates platelets. Biomolecules released from the activated platelets could be retained by the fibrin matrix on the surface to facilitate regeneration of the surrounding tissues. Thus, PRP immersion could not only eliminate surface air bubbles but also improve the biocompatibility of the implant surface.


    </sec>

    DOI: 10.1186/s40729-020-00270-2

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    その他リンク: http://link.springer.com/article/10.1186/s40729-020-00270-2/fulltext.html

  • The Platelet Concentrates Therapy: From the Biased Past to the Anticipated Future 招待 査読

    Kawase T, Suliman Mubarak, Carlos Fernando Mourao

    Bioengineering   7 ( 3 )   82   2020年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Concentrated Growth Factor Matrices Prepared Using Silica-Coated Plastic Tubes Are Distinguishable From Those Prepared Using Glass Tubes in Platelet Distribution: Application of a Novel Near-Infrared Imaging-Based, Quantitative Technique 査読

    Sadahiro Yamaguchi, Hachidai Aizawa, Atsushi Sato, Tetsuhiro Tsujino, Kazushige Isobe, Yutaka Kitamura, Taisuke Watanabe, Hajime Okudera, Carlos Fernando Mourão, Tomoyuki Kawase

    Frontiers in Bioengineering and Biotechnology   8   600   2020年6月

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    担当区分:責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Frontiers Media SA  

    DOI: 10.3389/fbioe.2020.00600

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  • Use of platelet-rich fibrin for the treatment of gingival recessions: a systematic review and meta-analysis 査読

    Miron RJ, Moraschini V, Del Fabbro M, Piattelli A, Fujioka-Kobayashi M, Zhang Y, Saulacic N, Schaller B, Kawase T, Cosgarea R, Jepsen S, Tuttle D, Bishara M, Canullo L, Eliezer M, Stavropoulos A, Shirakata Y, Stahli A, Gruber R, Lucaciu O, Aroca S, Deppe H, Wang HL, Sculean A

    Clin Oral Invest   2020年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Quantitative Near-Infrared Imaging of Platelets in Platelet-Rich Fibrin (PRF) Matrices: Comparative Analysis of Bio-PRF, Leukocyte-Rich PRF, Advanced-PRF and Concentrated Growth Factors 査読

    Hachidai Aizawa, Tetsuhiro Tsujino, Taisuke Watanabe, Kazushige Isobe, Yutaka Kitamura, Atsushi Sato, Sadahiro Yamaguchi, Hajime Okudera, Kazuhiro Okuda, Tomoyuki Kawase

    International Journal of Molecular Sciences   21 ( 12 )   4426   2020年6月

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    担当区分:責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • A Comparative Study of The Effects of Anticoagulants on Pure Platelet-Rich Plasma Quality and Potency. 査読 国際誌

    Hachidai Aizawa, Hideo Kawabata, Atsushi Sato, Hideo Masuki, Taisuke Watanabe, Tetsuhiro Tsujino, Kazushige Isobe, Masayuki Nakamura, Koh Nakata, Tomoyuki Kawase

    Biomedicines   8 ( 3 )   2020年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    It is generally accepted that citrate or the A-form of acid-citrate-dextrose (ACD-A) are suitable for preparing platelet-rich plasma (PRP) for regenerative therapy. However, this is based on evidence from blood transfusions and not from regenerative medicine. Thus, we examined the effects of anticoagulants, such as ACD-A, ethylenediaminetetraacetic acid (EDTA), and heparin, on the regenerative quality of PRP to address this gap. The blood samples were collected in the presence of anticoagulants and were processed to prepare pure-PRP. Platelet size, activation status, and intra-platelet free Ca2+ concentration were determined while using a hematology analyzer and flow cytometer. Platelet-derived growth factor-BB (PDGF-BB) was quantified while using an ELISA. In pure-PRP samples, EDTA caused platelet swelling and activation, but yielded the highest number of platelets. Heparin aggregated platelets and disturbed the overall counting of blood cells. However, no significant differences in PDGF-BB levels were observed among the anticoagulants tested. Moreover, when considering the easy preparation of platelet suspensions, without the need for high-level pipetting skills, these findings suggest the comparable potency of EDTA-derived pure-PRP in tissue regeneration and support the use of EDTA in the preparation of pure-PRP. Further in vivo studies are required in animal models to exclude the possible negative effects of including EDTA in pure-PRP preparations.

    DOI: 10.3390/biomedicines8030042

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  • Acute cytotoxic effects of silica microparticles used for coating of plastic blood-collection tubes on human periosteal cells. 査読

    Hideo Masuki, Kazushige Isobe, Hideo Kawabata, Tetsuhiro Tsujino, Sadahiro Yamaguchi, Taisuke Watanabe, Atsushi Sato, Hachidai Aizawa, Carlos Fernando Mourão, Tomoyuki Kawase

    Odontology   2020年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Because of its simple operation, platelet-rich fibrin (PRF) is becoming more popular than the original form, platelet-rich plasma (PRP), in regenerative dentistry. PRF preparation requires plain glass blood-collection tubes, but not either anticoagulants or coagulation factors. However, such glass tubes designed for laboratory testing are no longer commercially available. Although several glass tubes specifically designed for PRF preparation are available, many clinicians prefer to obtain stably supplied substitutes, such as silica-coated plastic tubes produced by major medical device companies. The quality of PRF prepared by silica-coated tubes has not been assessed and we previously reported significant contamination of silica microparticles in the resulting PRF matrix and alerted clinicians against the use for PRF preparation. To further assess the biosafety of the silica microparticles, we presently examined their effects on human normal periosteal cells derived from alveolar bone. The periosteal cells were obtained from explant cultures of small periosteal tissues obtained from healthy donors. Silica microparticles were obtained from silica-coated tubes and added to cell cultures. Cellular responses were monitored using a tetrazolium assay, phase-contract inverted microscopy, an immunofluorescence method, and scanning electron microscopy. Silica microparticles adsorbed onto the cell surface with seemingly high affinity and induced apoptosis, resulting in significant reduction of cell proliferation and viability. These findings suggest that silica microparticles contained in plastic tubes for the purpose of blood coagulation are hazardous for various cell types around sites where silica-contaminated PRF matrices are implanted.

    DOI: 10.1007/s10266-020-00486-z

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  • Platelet Adhesion on Commercially Pure Titanium Plates in Vitro II. Immunofluorescence Visualization of PDGF-B, TGFβ1, and PPARγ Released from Activated Adherent Platelets. 査読

    Tsujino T, Takahashi A, Watanabe T, Isobe K, Kitamura Y, Okuda K, Nakata K, Kawase T

    Dentistry journal   7 ( 4 )   2019年11月

  • Distribution of platelets, TGFβ1, PDGF-BB, VEGF, MMP9 and fibronectin in advanced platelet-rich fibrin (A-PRF) and concentrated growth factors (CGF) matrices 査読

    Takahashi A, Tsujino T, Yamaguchi S, Isobe K, Watanabe T, Kitamura Y, Okuda K, Nakata K, Kawase T

    J Invest Clin Dent   10 ( 4 )   e12458   2019年11月

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    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Striking differences in platelet distribution between advanced-platelet-rich fibrin and concentrated growth factors: effects of silica-containing plastic tubes 査読

    Tsujino T, Masuki H, Nakamura M, Isobe K, Kawabata H, Aizawa H, Watanabe T, Kitamura Y, Okudera H, Okuda K, Nakata K, Kawase T

    J Funct Biomater   10 ( 3 )   43   2019年9月

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    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Evidence for contamination of silica microparticles in advanced platelet-rich fibrin matrix prepared using silica-coated plastic tubes 査読

    Tsujino T, Takahashi A, Yamagushi S, Watanabe T, Isobe K, Kitamura Y, Tanaka T, Nakata K, Kawase T

    Biomedicines   7 ( 2 )   45   2019年6月

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    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Spectrophotometric determination of the aggregation activity of platelets in platelet-rich plasma for better quality control. 査読

    Tsujino T, Isobe K, Kawabata H, Aizawa H, Yamaguchi S, Kitamura Y, Masuki H, Watanabe T, Okudera H, Nakata K, Kawase T

    Dent J   7 ( 2 )   61   2019年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Proposal for point-of-care testing of PRP quality. 招待

    T. Kawase, A. Takahashi, T. Watanabe, T. Tsujino

    Int J Growth Factors Stem Cells Dent   2 ( 1 )   13 - 17   2019年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Platelet adhesion on commercially pure titanium plates in vitro I. Effects of plasma components and involvement of the von Willebrand factor and fibronectin. 査読

    A. Takahashi, S. Takahashi, T. Tsujino, K. Isobe, T. Watanabe, Y. Kitamura, T. Watanabe, K. Nakata, Tomoyuki Kawase

    Int J Implant Dent   5   5   2019年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Platelet-rich fibrin extract: a promising fetal bovine serum alternative in explant cultures of human periosteal sheets for regenerative therapy. 査読

    Kawase T, Nagata M, Okuda K, Ushiki T, Fujimoto Y, Watanabe M, Ito A, Nakata K

    Int J Mol Sci   20 ( 5 )   1053   2019年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • 第5節 歯周組織再生医療の現状と細胞治療製品の開発 第2章 臓器・器官,疾病ごとの治療・製品ニーズの把握と製品開発 招待

    川瀬知之, 永田昌毅, 奧田一博, 中田 光, 伊藤 彰

    再生医療の開発戦略と最新研究事例集   81 - 89   2019年2月

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    担当区分:筆頭著者, 責任著者   記述言語:日本語  

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  • Imaging of Metastatic Cancer Cells in Sentinel Lymph Nodes using Affibody Probes and a Potential Theranostic Approach in vivo 査読

    Makoto Tsuchimochi, Haruka Yamaguchi, Kazuhide Hayama, Yasuo Okada, Tomoyuki Kawase, Takamasa Suzuki, Norio Tsubokawa, Noriaki Wada, Atsushi Ochiai, Satoshi Fujii, Hirofumi Fujii

    International Journal of Molecular Sciences   20 ( 2 )   427   2019年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.3390/ijms20020427

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  • An on-site preparable, novel bone-grafting complex consisting of human platelet-rich fibrin and porous particles made of a recombinant collagen-like protein. 査読

    Tsukioka T, Hiratsuka T, Nakamura M, Watanabe T, Kitamura Y, Isobe K, Okudera T, Okudera H, Azuma A, Uematsu K, Nakata K, Kawase T

    J Biomed Mater Res B Applied Biomaterials   2018年10月

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  • Spectrophotometric determination of platelet counts in platelet-rich plasma. 査読

    Kitamura Y, Suzuki M, Tsukioka T, Isobe K, Tsujino T, Watanabe T, Watanabe T, Okudera H, Nakata K, Tanaka T, Kawase T

    Int J Implant Dent   4   29   2018年10月

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  • Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma. 査読

    Toyoda H, Isobe K, Tsujino T, Koyata Y, Ohyagi F, Watanabe T, Nakamura M, Kitamura Y, Okudera H, Nakata K, Kawase T

    Int J Implant Dent   4   23   2018年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Quantitative evaluation by digital holographic microscopy of morphological changes of in activated platelets in vitro using digital holographic microscopy. 査読

    Kiatamura Y, Isobe K, Kawabata H, Tsujino T, Watanabe T, Nakamura M, Toyoda T, Okudera H, Okuda K, Nakata K, Kawase T

    Micron   113   1 - 9   2018年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Comprehensive quality control of the regenerative therapy using platelet concentrates: The current situation and prospects in Japan. 査読

    Kawase T, Okuda K

    BioMed Res Int   6389157   2018年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • HER2-targeted multifunctional silica nanoparticles specifically enhance the radiosensitivity of HER2-overexpressing breast cancer cells. 査読

    Yamaguchi H, Hayama K, Sasagawa I, Okada Y, Kawase T, Tsubokawa N, Tsuchimochi M

    Int J Molec Sci   19   908   2018年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Platelet counts in insoluble platelet-rich fibrin clots: a direct method for accurate determination. 査読

    Kitamura Y, Watanabe T, Nakamura M, Isobe K, Kawabata H, Uematsu K, Okuda K, Nakata K, Tanaka T, Kawase T

    Front Bioeng Biotechnol   6   4   2018年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • An updated proposal for terminology and classification of platelet-rich fibrin. 査読

    Kawase T, Tanaka T

    Regen Ther   7   80 - 81   2017年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Quality assessment of platelet-rich fibrin-like matrix prepared from extendedly stored whole blood samples. 査読

    Kawabata H, Isobe K, Watanabe T, Okudera T, Nakamura M, Suzuki M, Ryu J, Kitamura Y, Okudera H, Okuda K, Nakata K, Kawase T

    Biomedicines   5   57   2017年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Synergistic effects of the combined use of human cultured periosteal sheets and platelet-rich fibrin on bone regeneration: an animal study. 査読

    Horimizu M, Kubota T, Kawase T, Nagata M, Kobayashi M, Okuda K, Nakata K, Yoshie H

    Clin Exp Dent Res   3 ( 4 )   134 - 141   2017年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • 多血小板血漿とそこから派生した血小板濃縮材料: 再生医療に関与する歯科医が押さえておきたいポイント. 査読

    川瀬知之, 渡辺泰典, 奥田一博

    日本歯周病学会誌   59 ( 2 )   68 - 76   2017年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.2329/perio.59.68

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  • Mechanical and degradation properties of advanced platelet-rich fibrin (A-PRF), concentrated growth factors (CGF) and platelet-poor plasma-derived fibrin (PPTF). 査読

    Isobe M, Watanabe T, Kawabata H, Kitamura Y, Okudera T, Okudera H, Uematsu K, Okuda K, Nakata K, Tanaka T, Kawase T

    Int J Implant Dent   3   17   2017年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Platelet-rich fibrin prepared from stored whole-blood samples. 査読

    Isobe M, Suzuki M, Watanabe T, Kitamura Y, Suzuki T, Kawabata H, Nakamura M, Okudera T, Okudera H, Uematsu K, Nakata K, Tanaka T, Kawase T

    Int J Implant Dent   3   6   2017年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • An evaluation of the accuracy of the subtraction method used for determining platelet counts in advanced platelet-rich fibrin and concentrated growth factor preparations. 査読

    Watanabe T, Isobe K, Suzuki T, Kawabata H, Nakamura M, Tsukioka T, Okudera T, Okudera H, Uematsu K, Okuda K, Nakata K, Kawase T

    Dent J   5   7   2017年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Preparation of a poly(L-lactic acid) Membrane scaffold with open finger-Like pores prepared by a nonsolvent-induced phase separation method with the aid of a surfactant. 査読

    Minbu H, Kawase T, Ochiai A, Taniguchi M, Tanaka T

    膜   41 ( 6 )   304 - 310   2016年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.5360/membrane.41.304

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  • Growth factor and pro-inflammatory cytokine contents in PRP, plasma rich in growth factors (PRGF), advanced-platelet-rich fibrin (A-PRF) and concentrated growth factors (CGF). 査読

    Masuki H, Okudera T, Watanabe T, Suzuki M, Nishiyama K, Okudera H, Nakata K, Uematsu K, Su CY, Kawase T

    Int J Implant Dent   2   19   2016年8月

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  • High-Resolution Three-Dimensional Computed Tomography Analysis of the Clinical Efficacy of Cultured Autogenous Periosteal Cells in Sinus Lift Bone Grafting 査読

    Shin Ogawa, Hideyuki Hoshina, Koh Nakata, Kazuho Yamada, Kohya Uematsu, Tomoyuki Kawase, Ritsuo Takagi, Masaki Nagata

    CLINICAL IMPLANT DENTISTRY AND RELATED RESEARCH   18 ( 4 )   707 - 716   2016年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    Background and Purpose: Sinus lift (SL) using cultured autogenous periosteal cells (CAPCs) combined with autogenous bone and platelet-rich plasma (PRP) was performed to evaluate the effect of cell administration on bone regeneration, by using high-resolution three-dimensional computed tomography (CT).
    Materials and Methods: SL with autogenous bone and PRP plus CAPC [CAPC(+)SL] was performed in 23 patients. A piece of periosteum taken from the mandible was cultured in M199 medium with 10% fetal bovine serum (FBS) for 6 weeks. As control, 16 patients received SL with autogenous bone and PRP [CAPC(-)SL]. Three-dimensional CT imaging was performed before and 4 months and 1 year after SL, and stratification was performed based on CT numbers (HUs) corresponding to soft tissue and cancellous or cortical bone.
    Results: The augmented bone in CAPC(+)SL revealed an increase in HUs corresponding to cancellous bone as well as a decrease in HUs corresponding to grafted cortical bone. In addition, HUs corresponding to cancellous bone in the graft bed were increased in CAPC(+)SL but were decreased in CAPC(-)SL. Insertion torque during implant placement was significantly higher in CAPC(+)SL.
    Conclusion: By promoting bone anabolic activity both in augmented bone and graft bed, CAPCs are expected to aid primary fixation and osseointegration of implants in clinical applications.

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  • Dual-Labeled Near-Infrared/Tc-99m Imaging Probes Using PAMAM-Coated Silica Nanoparticles for the Imaging of HER2-Expressing Cancer Cells 査読

    Haruka Yamaguchi, Makoto Tsuchimochi, Kazuhide Hayama, Tomoyuki Kawase, Norio Tsubokawa

    INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES   17 ( 7 )   7   2016年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:MDPI AG  

    We sought to develop dual-modality imaging probes using functionalized silica nanoparticles to target human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer cells and achieve efficient target imaging of HER2-expressing tumors. Polyamidoamine-based functionalized silica nanoparticles (PCSNs) for multimodal imaging were synthesized with near-infrared (NIR) fluorescence (indocyanine green (ICG)) and technetium-99m (Tc-99m) radioactivity. Anti-HER2 antibodies were bound to the labeled PCSNs. These dual-imaging probes were tested to image HER2-overexpressing breast carcinoma cells. In vivo imaging was also examined in breast tumor xenograft models in mice. SK-BR3 (HER2 positive) cells were imaged with stronger NIR fluorescent signals than that in MDA-MB231 (HER2 negative) cells. The increased radioactivity of the SK-BR3 cells was also confirmed by phosphor imaging. NIR images showed strong fluorescent signals in the SK-BR3 tumor model compared to muscle tissues and the MDA-MB231 tumor model. Automatic well counting results showed increased radioactivity in the SK-BR3 xenograft tumors. We developed functionalized silica nanoparticles loaded with Tc-99m and ICG for the targeting and imaging of HER2-expressing cells. The dual-imaging probes efficiently imaged HER2-overexpressing cells. Although further studies are needed to produce efficient isotope labeling, the results suggest that the multifunctional silica nanoparticles are a promising vehicle for imaging specific components of the cell membrane in a dual-modality manner.

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  • Evaluating the Safety of Somatic Periosteal Cells by Flow-Cytometric Analysis Monitoring the History of DNA Damage 査読

    Tomoyuki Kawase, Kazuhide Hayama, Makoto Tsuchimochi, Masaki Nagata, Kazuhiro Okuda, Hiromasa Yoshie, Douglas M. Burns, Koh Nakata

    BIOPRESERVATION AND BIOBANKING   14 ( 2 )   129 - 137   2016年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:MARY ANN LIEBERT, INC  

    In preparing cell-based products for regenerative therapy, cell quality should be strictly controlled. Methodologies for evaluating cell viability, identity, and purity are established and used routinely, whereas current methodologies for evaluating cell safety, particularly genetic integrity or tumorigenicity, are time-consuming and relatively insensitive. As part of developing a more practical screening system, the authors previously demonstrated that gamma-H2AX and p53 were useful markers for evaluating the history of DNA damage. To validate these markers further and develop a more quantitative methodology, single cell-based expression of these markers and two additional candidates have now been examined using flow cytometry (FCM). FCM analysis and immunofluorescent staining demonstrated that gamma-ray-irradiation suppressed proliferation, enlarged cells, and cell nuclei, and immediately upregulated gamma-H2AX and p21(waf1) in large numbers of cells for up to 12 days. Gamma-H2AX foci were formed in the nuclei of many affected cells. An initial sharp increase in p53 expression declined slowly over 12 days, while Rb expression increased linearly. The present findings suggest that this high-throughput, cell-based, combinational evaluation of protein markers and cell size enables a small number of cells with a history of DNA damage to be detected quickly and routinely from within a very large cell population. Using this screening methodology will improve the ability to verify the quality of cell-based products used in regenerative therapy.

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  • Basic characteristics of plasma rich in growth factors (PRGF): blood cell components and biological effects. 査読

    Nishiyama K, Okudera T, Watanabe T, Isobe K, Suzuki M, Masuki H, Okudera H, Uematsu K, Nakata K, Kawase T

    Clin Exp Dent Res   2 ( 2 )   96 - 103   2016年3月

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  • Non-invasive, quantitative assessment of the morphology of γ-irradiated human mesenchymal stem cells and periosteal cells using digital holographic microscopy. 査読

    Kawase T, Okuda K, Nagata M, Tsuchimochi M, Yoshie H, Nakata K

    Int J Radiat Biol   92 ( 12 )   796 - 805   2016年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • In vitro immunological and biological evaluations of the angiogenic potential of platelet-rich fibrin preparations: a standardized comparison with PRP preparations. 査読

    Kobayashi M, Kawase T, Okuda K, Wolff LF, Yoshie H

    Int J Implant Dent   1 ( 1 )   31 - 41   2015年11月

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  • The heat-compression technique for the conversion of platelet-rich fibrin preparation to a barrier membrane with a reduced rate of biodegradation 査読

    Tomoyuki Kawase, Mana Kamiya, Mito Kobayashi, Takaaki Tanaka, Kazuhiro Okuda, Larry F. Wolff, Hiromasa Yoshie

    Journal of Biomedical Materials Research - Part B Applied Biomaterials   103 ( 4 )   825 - 831   2015年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:John Wiley and Sons Inc.  

    Platelet-rich fibrin (PRF) was developed as an advanced form of platelet-rich plasma to eliminate xenofactors, such as bovine thrombin, and it is mainly used as a source of growth factor for tissue regeneration. Furthermore, although a minor application, PRF in a compressed membrane-like form has also been used as a substitute for commercially available barrier membranes in guided-tissue regeneration (GTR) treatment. However, the PRF membrane is resorbed within 2 weeks or less at implantation sites
    therefore, it can barely maintain sufficient space for bone regeneration. In this study, we developed and optimized a heat-compression technique and tested the feasibility of the resulting PRF membrane. Freshly prepared human PRF was first compressed with dry gauze and subsequently with a hot iron. Biodegradability was microscopically examined in vitro by treatment with plasmin at 37C or in vivo by subcutaneous implantation in nude mice. Compared with the control gauze-compressed PRF, the heat-compressed PRF appeared plasmin-resistant and remained stable for longer than 10 days in vitro. Additionally, in animal implantation studies, the heat-compressed PRF was observed at least for 3 weeks postimplantation in vivo whereas the control PRF was completely resorbed within 2 weeks. Therefore, these findings suggest that the heat-compression technique reduces the rate of biodegradation of the PRF membrane without sacrificing its biocompatibility and that the heat-compressed PRF membrane easily could be prepared at chair-side and applied as a barrier membrane in the GTR treatment.

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  • Platelet-rich plasma and its derivatives as promising bioactive materials for regenerative medicine: basic principles and concepts underlying recent advances. 招待 査読

    Tomoyuki Kawase

    Odontology   103 ( 2 )   126 - 35   2015年5月

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    担当区分:筆頭著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Over the past decade, platelet-rich plasma (PRP), a platelet-concentrated plasma fraction, has been widely investigated and applied to regenerative medicine. The clinical utility of PRP is supported by evidence that PRP contains high concentrations of platelet-related growth factors and normal concentrations of plasma-derived fibrinogen, both of which contribute synergistically to the regenerative process. Additionally, its superior cost-efficacy versus conventional therapies is attractive to many clinicians. However, current disadvantages of PRP include a relatively complicated preparation procedure and variable operator-dependent efficacy. An additional disadvantage is the use of bovine thrombin, an animal-derived biological, as a coagulant. Many of these disadvantages are overcome by recent advances in preparation procedures and devices; for example, Joseph Choukroun simplified the platelet-rich fibrin preparation procedure and improved handling efficiency without the aid of animal-derived factors. With advancements in cell processing technology, there has been a general shift in cell therapy from autologous to allogeneic treatment; however, autologous PRP therapy will not easily be replaced by allogeneic treatment in the near future. Therefore, to provide more predictable regenerative therapy outcomes using autologous PRP, further investigations should address developing a standardized procedure for PRP preparation to augment its efficacy and potency, independent of donor variability. We would then propose that operators and clinicians prepare PRP according to the standardized protocol and to carefully evaluate the clinical scenario (i.e., recipient factors comprising skeletal defects) to determine which factor(s) should be added to PRP preparations. This careful approach will lead to improved clinical outcomes for patients.

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  • Preparation of poly(L-lactic acid) microfiltration membranes by a nonsolvent-induced phase separation method with the aid of surfactants 査読

    Hiromi Minbu, Akihito Ochiai, Tomoyuki Kawase, Masayuki Taniguchi, Douglas R. Lloyd, Takaaki Tanaka

    JOURNAL OF MEMBRANE SCIENCE   479   85 - 94   2015年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    Microfiltration membranes of poly(L-lactic acid) (PLEA) have been prepared by a nonsolvent-induced phase separation method with the aid of surfactants. Surfactants with hydrophilic-lipophilic balance (HLB) values of 14.9-15.6 were found to be useful in reducing the shrinkage in thickness of the PLEA membrane. Among the surfactants examined, Tween 80 was the best for preparing microfiltration membranes. The surfactant allowed instantaneous phase separation and seemed to enhance the diffusion of water in the PLEA solution during structure formation. The membrane had asymmetric finger-like structures and showed low membrane resistance and high bacterial cell retention when the membrane was prepared from a 10 wt% PLEA solution in 1,4-dioxane containing 10 wt.% Tween 80. Bovine serum albumin molecules passed through the membrane suggesting that the membrane functions as a microfiltration membrane. The membrane was stable at 25 degrees C but degradable at 60 degrees C in wet conditions. The membrane can be applied as a compostable microfiltration membrane in food and biochemical industries. (C) 2015 Elsevier B.V. All rights reserved.

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  • X-ray and ultraviolet C irradiation-induced γ-H2AX and p53 formation in normal human periosteal cells in vitro: markers for quality control in cell therapy. 査読

    Kawase T, Kamiya M, Hayama K, Nagata M, Okuda K, Yoshie H, Burns DM, Tsuchimochi M, Nakata K

    Cytotherapy   17 ( 1 )   112 - 123   2015年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • X-ray-induced damage to the submandibular salivary glands in mice: An analysis of strain-specific responses 査読

    Mana Kamiya, Tomoyuki Kawase, Kazuhide Hayama, Makoto Tsuchimochi, Kazuhiro Okuda, Hiromasa Yoshie

    BioResearch Open Access   4 ( 1 )   307 - 318   2015年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Mary Ann Liebert Inc.  

    Radiation therapy for head and neck cancers often causes xerostomia (dry mouth) by acutely damaging the salivary glands through the induction of severe acute inflammation. By contrast, the mechanism underlying the X-ray-induced delayed salivary dysfunction is unknown and has attracted increasing attention. To identify and develop a mouse model that distinguishes the delayed from the acute effects, we examined three different mouse strains (C57BL/6, ICR, and ICR-nu/nu) that showed distinct T-cell activities to comparatively analyze their responses to X-ray irradiation. Three strains were irradiated with X-rays (25 Gy), and functional changes of the submandibular glands were examined by determining pilocarpine-induced saliva secretion. Structural changes were evaluated using histopathological and immunohistochemical examinations of CD3, cleaved poly (ADP-ribose) polymerase (PARP), and Bcl-xL. In C57BL/6 mice, the X-ray irradiation induced acute inflammation accompanied by severe inflammatory cell infiltration at 4 days postirradiation, causing substantial destruction and significant dysfunction at 2 weeks. Fibrotic repair was observed at 16 weeks. In ICR-nu/nu mice, the inflammation and organ destruction were much milder than in the other mice strains, but increased apoptotic cells and a significant reduction in salivary secretion were observed at 4 and 8 weeks and beyond, respectively. These results suggest that in C57BL/6 mice, X-ray-induced functional and structural damage to the salivary glands is caused mainly by acute inflammation. By contrast, although neither acute inflammation nor organ destruction was observed in ICR-nu/nu mice, apoptotic cell death preceded the dysfunction in salivary secretion in the later phase. These data suggest that the X-ray-irradiated ICR-nu/nu mouse may be a useful animal model for developing more specific therapeutic methods for the delayed dysfunction of salivary glands.

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  • Quantitative single-cell motility analysis of platelet-rich plasma-treated endothelial cells in vitro 査読

    Tomoyuki Kawase, Takaaki Tanaka, Kazuhiro Okuda, Makoto Tsuchimochi, Masafumi Oda, Toshiaki Hara

    Cytoskeleton   72 ( 5 )   246 - 255   2015年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:John Wiley and Sons Inc.  

    Platelet-rich plasma (PRP) has been widely applied in regenerative therapy due to its high concentration of growth factors. Previous in vitro and in vivo studies have provided evidence supporting the angiogenic activity of PRP. To more directly demonstrate how PRP acts on endothelial cells, we examined the PRP-induced changes in the motility of human umbilical vein endothelial cells by examining the involvement of VEGF. Time-lapse quantitative imaging demonstrated that in the initial phase (~2 h) of treatment, PRP substantially stimulated cell migration in a wound-healing assay. However, this effect of PRP was not sustained at significant levels beyond the initial phase. The average net distance of cell migration at 10 h was 0.45±0.16 mm and 0.82±0.23 mm in control and PRP-stimulated cells, respectively. This effect was also demonstrated with recombinant human VEGF and was significantly attenuated by a neutralizing anti-VEGF antibody. Immunofluorescent examination of paxillin and actin fibers demonstrated that PRP concomitantly up-regulated focal adhesion and cytoskeletal formation. Western blotting analysis of phosphorylated VEGFR2 demonstrated that PRP mainly stimulated the phosphorylation of immature VEGFR2 in a dose- and time-dependent manner, an action that was completely blocked by the neutralizing antibody. Taken together, these data suggest that PRP acts directly on endothelial cells via the activation of VEGFR2 to transiently up-regulate their motility. Thus, the possibility that PRP desensitizes target endothelial cells for a relatively long period of time after short-term activation should be considered when the controlled release system of PRP components is designed.

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  • 再生医療による難治性疾患の幕開け 2 培養骨膜シート移植による歯周病治療. 招待

    奧田一博, 川瀬知之, 中田 光, 吉江弘正

    新潟医学会雑誌   128 ( 11 )   568 - 580   2014年11月

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    記述言語:日本語   掲載種別:研究論文(学術雑誌)  

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  • An atmospheric-pressure plasma-treated titanium surface potentially supports initial cell adhesion, growth, and differentiation of cultured human prenatal-derived osteoblastic cells 査読

    Tomoyuki Kawase, Takaaki Tanaka, Hiromi Minbu, Mana Kamiya, Masafumi Oda, Toshiaki Hara

    JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B-APPLIED BIOMATERIALS   102 ( 6 )   1289 - 1296   2014年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    An atmospheric-pressure plasma (APP) treatment was recently reported to render titanium (Ti) surfaces more suitable for osteoblastic cell proliferation and osteogenesis. However, the mechanism of action remains to be clearly demonstrated. In this study, we focused on cell adhesion and examined the effects of the APP treatment on the initial responses of human prenatal-derived osteoblastic cells incubated on chemically polished commercially pure Ti (CP-cpTi) plates. In the medium containing 1% fetal bovine serum, the initial cell adhesion and the actin polymerization were evaluated by scanning electron microscopy and fluorescence microscopy. The expression of cell adhesion-related molecules and osteoblast markers at the messenger RNA level was assessed by real-time quantitative polymerase chain reaction. Although the cells on the APP-treated CP-cpTi surface developed fewer cytoskeletal actin fibers, they attached with higher affinity and consequently proliferated more actively (1.46-fold over control at 72 h). However, most of the cell adhesion molecule genes were significantly downregulated (from 40 to 85% of control) in the cells incubated on the APP-treated CP-cpTi surface at 24 h. Similarly, the osteoblast marker genes were significantly downregulated (from 49 to 63% of control) at 72 h. However, the osteoblast marker genes were drastically upregulated (from 197 to 296% of control) in these cells by dexamethasone and beta-glycerophosphate treatment. These findings suggest that the APP treatment improves the ability of the CP-cpTi surface to support osteoblastic proliferation by enhancing the initial cell adhesion and supports osteoblastic differentiation when immature osteoblasts begin the differentiation process. (C) 2014 Wiley Periodicals, Inc.

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  • Real-time quantitative polymerase chain reaction and flow cytometric analyses of cell adhesion molecules expressed in human cell-multilayered periosteal sheets in vitro 査読

    Tomoyuki Kawase, Kohya Uematsu, Mana Kamiya, Masaki Nagata, Kazuhiro Okuda, Douglas M. Burns, Koh Nakata, Hiromasa Yoshie

    Cytotherapy   16 ( 5 )   653 - 661   2014年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Informa Healthcare  

    Background aims: Cultured human periosteal sheets more effectively function as an osteogenic grafting material at implantation sites than do dispersed periosteal cells. Because adherent cell growth and differentiation are regulated by cell-cell and cell-extracellular matrix contacts, we hypothesized that this advantage is a result of the unique cell adhesion pattern formed by their multiple cell layers and abundant extracellular matrix. To test this hypothesis, we prepared three distinct forms ofperiosteal cell cultures: three-dimensional cell-multilayered periosteal sheets, two-dimensional dispersed cell cultures, andthree-dimensional hybrid mock-ups of cells dispersed onto collagen sponges. Methods: Periosteal cells were obtained from human alveolar bone. Cell adhesion and extracellular matrix molecules were quantitatively determined at the messenger RNA and protein levels by means of real-time quantitative polymerase chain reaction and flow cytometry, respectively. Results: Real-time quantitative polymerase chain reaction analysis demonstrated that regardless of culture media α1 integrin, vascular cell adhesion molecule-1, fibronectin and collagen type 1 were substantially upregulated, whereas CD44 was strongly downregulated in periosteal sheets compared with dispersed cell monolayers. With increased thickness, stem cell medium upregulated several integrins (β1, α1 and α4), CD146, vascular cell adhesion molecule-1, fibronectin and collagen type 1 in the periosteal sheets. Flow cytometric analysis revealed that the active configuration of β1 integrin was substantially downregulated in the stem cell medium-expanded cell cultures. The cell adhesion pattern found in the mock-up cultures was almost identical to that of genuine periosteal sheets. Conclusions: Integrin α1β1 and CD44 function as the main cell adhesion molecule in highly cell-multilayered periosteal sheets and dispersed cells, respectively. This difference may account for the more potent osteogenic activity shown by the thicker periosteal sheets. © 2014 International Society for Cellular Therapy.

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  • ITGA3 and ITGB4 expression biomarkers estimate the risks of locoregional and hematogenous dissemination of oral squamous cell carcinoma 査読

    Masaki Nagata, Arhab A. Noman, Kenji Suzuki, Hiroshi Kurita, Makoto Ohnishi, Tokio Ohyama, Nobutaka Kitamura, Takanori Kobayashi, Kohya Uematsu, Katsu Takahashi, Naoki Kodama, Tomoyuki Kawase, Hideyuki Hoshina, Nobuyuki Ikeda, Susumu Shingaki, Ritsuo Takagi

    BMC Cancer   13   2013年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Background: Molecular biomarkers are essential for monitoring treatment effects, predicting prognosis, and improving survival rate in oral squamous cell carcinoma. This study sought to verify the effectiveness of two integrin gene expression ratios as biomarkers.Methods: Gene expression analyses of integrin α3 (ITGA3), integrin β4 (ITGB4), CD9 antigen (CD9), and plakoglobin (JUP) by quantitative real-time PCR were conducted on total RNA from 270 OSCC cases. The logrank test, Cox proportional hazards model, and Kaplan-Meier estimates were performed on the gene expression ratios of ITGA3/CD9 and ITGB4/JUP and on the clinicopathological parameters for major clinical events.Results: A high rate (around 80%) of lymph node metastasis was found in cases with a high ITGA3/CD9 ratio (high-ITGA3/CD9) and invasive histopathology (YK4). Primary site recurrence (PSR) was associated with high-ITGA3/CD9, T3-4 (TNM class), and positive margin, indicating that PSR is synergistically influenced by treatment failure and biological malignancy. A high ITGB4/JUP ratio (high-ITGB4/JUP) was revealed to be a primary contributor to distant metastasis without the involvement of clinicopathological factors, suggesting intervention of a critical step dependent on the function of the integrin β4 subunit. Kaplan-Meier curves revealed positive margin as a lethal treatment consequence in high-ITGA3/CD9 and YK4 double-positive cases.Conclusion: Two types of metastatic trait were found in OSCC: locoregional dissemination, which was reflected by high-ITGA3/CD9, and distant metastasis through hematogenous dissemination, uniquely distinguished by high-ITGB4/JUP. The clinical significance of the integrin biomarkers implies that biological mechanisms such as cancer cell motility and anchorage-independent survival are vital for OSCC recurrence and metastasis. © 2013 Nagata et al.
    licensee BioMed Central Ltd.

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  • An improved freeze-dried PRP-coated biodegradable material suitable for connective tissue regenerative therapy 査読

    Makoto Horimizu, Tomoyuki Kawase, Yu Nakajima, Kazuhiro Okuda, Masaki Nagata, Larry F. Wolff, Hiromasa Yoshie

    Cryobiology   66 ( 3 )   223 - 232   2013年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    We previously published an investigation indicating freeze-dried platelet-rich plasma (PRP)-coated polyglactin mesh was a promising wound-dressing material. However, one of its disadvantages was the inflammatory nature due to degradation of the polyglactin. Therefore, in this study, we investigated the use of a collagen sponge as the carrier for PRP. When implanted subcutaneously in nude mice, the PRP-coated sponge alone rapidly induced angiogenesis and infiltration of surrounding connective tissue without inducing appreciable inflammation. Moreover, addition of periosteal fibroblastic cells substantially augmented the angiogenic response. With in vitro studies, the PRP-coated sponge provided various major growth factors at high levels to stimulate the proliferation of cells cultured on plastic dishes, but did not stimulate the proliferation of cells inoculated into the PRP-coated sponge. Cells were embedded in the fibrin mesh and maintained their spherical shape without stretching. The atomic force microscopic analysis demonstrated that the fibrin gel formed on the PRP-coated sponge was much softer (approx. 22. kPa) than the cross-linked collagen that formed the sponge base (appox. 1.9. MPa). Because insoluble matrices have recently and increasingly been considered important regulatory factors of cellular behavior, as are soluble growth factors, it is suggested that this soft fibrin mesh possibly suppresses cell survival. Overall, our investigation has successfully demonstrated improved wound-healing and regenerative potential of the PRP-coated mesh by combining it with the collagen sponge. In the clinical setting, this PRP-coated collagen sponge is a promising material for connective tissue regenerative therapy, such as periodontal therapy, burn victim treatment and in cosmetic or plastic surgery. © 2013 Elsevier Inc.

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  • Biomechanical evaluation by AFM of cultured human cell-multilayered periosteal sheets 査読

    Makoto Horimizu, Tomoyuki Kawase, Takaaki Tanaka, Kazuhiro Okuda, Masaki Nagata, Douglas M. Burns, Hiromasa Yoshie

    Micron   48   1 - 10   2013年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    We previously demonstrated that thicker periosteal sheets with enhanced cell layering maintain their component cells at relatively immature stages of differentiation but express a high in vivo osteogenic potential. As it has been recently proposed that stiff scaffolds provide a mechanical cue to various cell types that promotes differentiation, we postulated that the maintenance of immature cells in our periosteal sheets is due to the mechanical stiffness of the multilayered-cell architecture. To demonstrate the biomechanical characteristics of our periosteal sheets, we have determined their stiffnesses with atomic force microscopy (AFM) and evaluated the expression of extracellular matrix (ECM) components specifically by both immunocytochemistry and a complementary DNA microarray technology. Compared to osteoblastic Saos2 cells, the cytoskeletal fibers were developed more in the periosteal cells, but the periosteal cells in monolayer culture developed before either the cells in the peripheral or central regions of the periosteal sheets developed. However, the nanoindentation by AFM distinguished the central region from the peripheral region. The peak stiffness values of cells were ordered as follows: tissue culture polystyrene (1.66. GPa). ⋙. dispersed (9.99. kPa). &gt
    . central region (5.20. kPa). &gt
    . peripheral regions (3.67. kPa). Similarly, the degree of development of α-smooth muscle actin (αSMA) filaments within cells was dispersed. &gt
    . central region. &gt
    . peripheral region. In conjunction with the abundantly deposited ECM in the periosteal sheets, these findings suggest that the order of cell stiffness may depend on the integration of the stiffness of individual ECM components and the extent of cytoskeletal fiber formation. Because recently published data have demonstrated that the optimal stiffness for osteogenic differentiation is 25-40. kPa, it is plausible that the periosteal cells residing in the less-stiff multilayer regions could be maintained at relatively immature stages under the control of the stem-cell medium in vitro but start differentiating when exposed to the proper stiffness upon release from the culture conditions at the implantation site. © 2013 Elsevier Ltd.

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  • Tissue culture of human alveolar periosteal sheets using a stem-cell culture medium (MesenPRO-RS™): In vitro expansion of CD146-positive cells and concomitant upregulation of osteogenic potential in vivo. 査読

    Uematsu K, Kawase T, Nagata M, Suzuki K, Okuda K, Yoshie H, Burns DM, Takagi R

    Stem cell research   10 ( 1 )   1 - 19   2013年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.scr.2012.08.006

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  • Microporous membranes of PLLA/PCL blends for periosteal tissue scaffold 査読

    Tomoaki Kouya, Shin-Ichiro Tada, Hiromi Minbu, Yu Nakajima, Makoto Horimizu, Tomoyuki Kawase, Douglas R. Lloyd, Takaaki Tanaka

    Materials Letters   95   103 - 106   2013年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Flexible microporous scaffold membranes of poly(l-lactic acid) (PLLA)/poly(ε-caprolactone) (PCL) blend were developed for bone regeneration. The new membranes overcome the fragility problems of PLLA membranes. When the PCL content in the blend was increased to 50%, the mechanical property for elongation was significantly improved without sacrificing the pores on the membrane surface that help tissue segments adhere the membrane. However, further increases in PCL content reduced the surface pores. In addition, spontaneous cell invasion and formation of cell-multilayers were observed in the membrane of PLLA/PCL blend (50:50). These results indicate that the porous membrane of PLLA/PCL blend (50:50) is useful for the preparation of periosteal sheets in tissue engineering. © 2012 Elsevier B.V.

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  • Tissue-engineered cultured periosteum sheet application to treat infrabony defects: Case series and 5-year results 査読

    Kazuhiro Okuda, Tomoyuki Kawase, Masaki Nagata, Kanoko Yamamiya, Koh Nakata, Larry F. Wolff, Hiromasa Yoshie

    International Journal of Periodontics and Restorative Dentistry   33 ( 3 )   281 - 287   2013年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    One-year data after autologous grafting of infrabony periodontal defects with human cultured periosteum sheets in combination with platelet-rich plasma and hydroxyapatite granules have shown favorable clinical and radiographic results. A 5-year follow-up evaluation of 22 selected patients indicated that treated infrabony defects remained stable. Radiographically, there was an increase in osseous radiopacity and bone trabeculation suggesting further bone maturation. This novel tissue-engineered periodontal treatment approach has resulted in significant clinical improvement, and defects remained stable after 5 years. © 2013 by Quintessence Publishing Co Inc.

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  • Application of stem-cell media to explant culture of human periosteum: An optimal approach for preparing osteogenic cell material 査読

    Kohya Uematsu, Masaki Nagata, Tomoyuki Kawase, Kenji Suzuki, Ritsuo Takagi

    Journal of Tissue Engineering   4 ( 1 )   1 - 12   2013年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SAGE Publications Ltd  

    As part of our clinical tests on bone regeneration using cultured periosteal sheets, here, we prepared cultured periosteal sheets in two types of stem-cell culture media, STK1 and STK3. Human periosteum was expanded either in 1% human serum-supplemented STK1 for 28 days, in 1% human serum-supplemented STK1 for 14 days followed by 1% human serum-supplemented STK3 for 14 days (1% human serum-supplemented STK1+3), or in 10% fetal bovine serum-supplemented Medium 199 for 28 days (control). Cultured periosteal sheet diameter and DNA content were significantly higher, and the multilayer structure was prominent in 1% human serum-supplemented STK1 and 1% human serum-supplemented STK1+3. The messenger RNA of osteoblastic markers was significantly upregulated in 1% human serum-supplemented STK1+3. Osteopontin-immunopositive staining and mineralization were evident across a wide area of the cultured periosteal sheet in 1% human serum-supplemented STK1+3. Subcutaneous implantation in nude mice following expansion in 1% human serum-supplemented STK1+3 produced the highest cultured periosteal sheet osteogenic activity. Expansion in 1% human serum-supplemented STK1+3 successfully induced cultured periosteal sheet growth while retaining osteogenic potential, and subsequent osteoblastic induction promoted the production of homogeneous cell material. © The Author(s) 2013.

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  • Bioactivity of freeze-dried platelet-rich plasma in an adsorbed form on a biodegradable polymer material 査読

    Yu Nakajima, Tomoyuki Kawase, Mito Kobayashi, Kazuhiro Okuda, Larry F. Wolff, Hiromasa Yoshie

    Platelets   23 ( 8 )   594 - 603   2012年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Owing to the necessity for the immediate preparation from patients' blood, autologous platelet-rich plasma (PRP) limits its clinical applicability. To address this concern and respond to emergency care and other unpredictable uses, we have developed a freeze-dried PRP in an adsorbed form on a biodegradable polymer material (Polyglactin 910). On the polymer filaments of PRP mesh, which was prepared by coating the polymer mesh with human fresh PRP and subsequent freeze-drying, platelets were incorporated, and related growth factors were preserved at high levels. This new PRP mesh preparation significantly and reproducibly stimulated the proliferation of human periodontal ligament cells in vitro and neovascularization in a chorioallantoic membrane assay. A full-thickness skin defect model in a diabetic mouse demonstrated the PRP mesh, although prepared from human blood, substantially facilitated angiogenesis, granulation tissue formation, and re-epithelialization without inducing severe inflammation in vivo. These data demonstrate that our new PRP mesh preparation functions as a bioactive material to facilitate tissue repair/regeneration. Therefore, we suggest that this bioactive material, composed of allogeneic PRP, could be clinically used as a promising alternative in emergency care or at times when autologous PRP is not prepared immediately before application. Copyright © 2012 Informa UK Ltd.

    DOI: 10.3109/09537104.2011.645923

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  • A proposed protocol for the standardized preparation of PRF membranes for clinical use 査読

    Mito Kobayashi, Tomoyuki Kawase, Makoto Horimizu, Kazuhiro Okuda, Larry F. Wolff, Hiromasa Yoshie

    Biologicals   40 ( 5 )   323 - 329   2012年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Upon clinical application, thick platelet-rich fibrin (PRF) is usually compressed to fit the implantation site. However, it is speculated that the preservation of platelets and plasma content depends on the compression methods used. To accurately evaluate the clinical outcome of PRF, the preparation protocol should be standardized. Freshly prepared PRF clots were compressed into a thin membrane by our novel PRF compression device. The localization of platelets was examined by SEM and immunostaining. Growth factor levels were evaluated by bioassays and cytokine-antibody array techniques. The angiogenic activity was examined by the chick chorioallantoic membrane assay and the scratch assay using HUVEC cultures. Platelets were concentrated on the surface of the region adjacent to the red thrombus and this region was subjected to the experiments. Compared to the PRF membrane compressed by dry gauze (G-PRF), the preservation of the plasma content, 3D-fibrin meshwork, and platelets was more intact in the compressor-prepared PRF membrane (C-PRF). Among the growth factors tested, C-PRF contained PDGF isoforms at higher levels, and significantly stimulated cell proliferation and neovascularization. C-PRF may be useful for grafting while minimizing the loss of bioactive factors. This C-PRF preparation protocol is proposed as a standardized protocol for PRF membrane preparation. © 2012 The International Alliance for Biological Standardization.

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  • Irsogladine maleate ameliorates inflammation and fibrosis in mice with chronic colitis induced by dextran sulfate sodium 査読

    Hana Yamaguchi, Kenji Suzuki, Masaki Nagata, Tomoyuki Kawase, Vijayakumar Sukumaran, Rajarajan A. Thandavarayan, Yusuke Kawauchi, Junji Yokoyama, Masayuki Tomita, Hiroshi Kawachi, Kenichi Watanabe, Hiroyuki Yoneyama, Hitoshi Asakura, Ritsuo Takagi

    MEDICAL MOLECULAR MORPHOLOGY   45 ( 3 )   140 - 151   2012年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER JAPAN KK  

    Intestinal fibrosis is a common and severe complication of inflammatory bowel disease (IBD), especially Crohn's disease (CD). To investigate the therapeutic approach to intestinal fibrosis, we have developed a mouse model of intestinal fibrosis by administering dextran sulfate sodium (DSS) and examining the effects of irsogladine maleate (IM) [2,4-diamino-6-(2,5-dichlorophenyl)-s-triazine maleate], which has been widely used as an antiulcer drug for gastric mucosa in Japan, on DDS-induced chronic colitis. In this experimental colitis lesion, several pathognomonic changes were found: increased deposition of collagen, increased number of profibrogenic mesenchymal cells such as fibroblasts (vimentin(+), alpha-SMA(-)) and myofibroblasts (vimentin(+), alpha-SMA(+)) in both mucosa and submucosa of the colon with infiltrating inflammatory cells, and increased mRNA expressions of collagen type I, transforming growth factor (TGF)-beta, matrix metalloproteinase (MMP)-2, and tissue inhibitor of matrix metalloproteinase (TIMP)-1. When IM was administered intrarectally to this colitis, all these pathological changes were significantly decreased or suppressed, suggesting a potential adjunctive therapy for intestinal fibrosis. IM could consequently reduce fibrosis in DSS colitis by direct or indirect effect on profibrogenic factors or fibroblasts. Therefore, the precise effect of IM on intestinal fibrosis should be investigated further.

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  • A short-term preservation of human cultured periosteal sheets, osteogenic grafting materials, using a commercial preservation solution containing epigallocatechin-3-gallate (Theliokeep(®)) under hypothermic conditions. 査読

    Kamiya M, Kawase T, Kobayashi M, Sekine Y, Okuda K, Nagata M, Fuse I, Nakata K, Wolff LF, Yoshie H

    Biopreservation and biobanking   10 ( 3 )   245 - 252   2012年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1089/bio.2011.0051

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  • A clinical study of alveolar bone tissue engineering with cultured autogenous periosteal cells: Coordinated activation of bone formation and resorption 査読

    Masaki Nagata, Hideyuki Hoshina, Minqi Li, Megumi Arasawa, Kohya Uematsu, Shin Ogawa, Kazuho Yamada, Tomoyuki Kawase, Kenji Suzuki, Akira Ogose, Ichiro Fuse, Kazuhiro Okuda, Katsumi Uoshima, Koh Nakata, Hiromasa Yoshie, Ritsuo Takagi

    Bone   50 ( 5 )   1123 - 1129   2012年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    In ongoing clinical research into the use of cultured autogenous periosteal cells (CAPCs) in alveolar bone regeneration, CAPCs were grafted into 33 sites (15 for alveolar ridge augmentation and 18 for maxillary sinus lift) in 25 cases. CAPCs were cultured for 6. weeks, mixed with particulate autogenous bone and platelet-rich plasma, and then grafted into the sites. Clinical outcomes were determined from high-resolution three-dimensional computed tomography (3D-CT) images and histological findings. No serious adverse events were attributable to the use of grafted CAPCs. Bone regeneration was satisfactory even in cases of advanced atrophy of the alveolar process. Bone biopsy after bone grafting with CAPCs revealed prominent recruitment of osteoblasts and osteoclasts accompanied by angiogenesis around the regenerated bone. 3D-CT imaging suggested that remodeling of the grafted autogenous cortical bone particles was faster in bone grafting with CAPCs than in conventional bone grafting. The use of CAPCs offers cell-based bone regeneration therapy, affording complex bone regeneration across a wide area, and thus expanding the indications for dental implants. Also, it enables the content of particulate autogenous bone in the graft material to be reduced to as low as 40%, making the procedure less invasive, or enabling larger amounts of graft materials to be prepared. It may also be possible to dispense with the use of autogenous bone altogether in the future. The results suggest that CAPC grafting induces bone remodeling, thereby enhancing osseointegration and consequently reducing postoperative waiting time after dental implant placement. © 2012 Elsevier Inc.

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  • 歯周病の再生治療材料:ヒト自家骨膜シートの特性 招待 査読

    川瀬知之, 奥田一博, 吉江弘正

    顕微鏡   47 ( 4 )   216 - 222   2012年4月

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    担当区分:筆頭著者   記述言語:日本語   掲載種別:研究論文(学術雑誌)  

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  • An osteogenic grafting complex combining human periosteal sheets with a porous poly(l-lactic acid) membrane scaffold: Biocompatibility, biodegradability, and cell fate in vivo 査読

    Tomoyuki Kawase, Takaaki Tanaka, Takayuki Nishimoto, Kazuhiro Okuda, Masaki Nagata, Douglas M Burns, Hiromasa Yoshie

    Journal of Bioactive and Compatible Polymers   27 ( 2 )   107 - 121   2012年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    In this in vitro study, novel porous poly(l-lactic acid) membranes were developed to improve periosteal sheets by promoting initial adhesion of periosteal tissue segments and stimulating the formation of a viable multilayered cellular sheet. The biocompatibility, biodegradability, and osteogenicity were evaluated using human periosteal tissue segments cultured on porous poly(l-lactic acid) membranes
    the periosteal sheets were osteogen induced and were then implanted in the dorsal subcutaneous tissue of nude mice. In vivo, the membrane degraded into clusters of membrane particles separated by wide cracks
    fibroblastic cells invaded along with small blood vessels from the surrounding mouse connective tissue. In osteoinduced periosteal sheets, the membrane clusters were surrounded by numerous capillaries and a number of tartrate-resistant acid phosphatase-positive, multinucleated cells. Neither severe inflammation nor fibrous encapsulation was observed throughout the implantation (∼12 weeks). These porous poly(l-lactic acid) membranes were highly biocompatible and functioned well as biodegradable scaffolds that could enhance the use of osteogenic periosteal sheets in therapy. © The Author(s) 2012.

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  • In-vivo near-infrared optical imaging of growing osteosarcoma cell lesions xenografted in mice: dual-channel quantitative evaluation of volume and mineralization 査読

    Hitoshi Nakayama, Tomoyuki Kawase, Kazuhiro Okuda, Larry F. Wolff, Hiromasa Yoshie

    ACTA RADIOLOGICA   52 ( 9 )   978 - 988   2011年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ROYAL SOC MEDICINE PRESS LTD  

    Background: In a previous study using a rodent osteosarcoma-grafted rat model, in which cell-dependent mineralization was previously demonstrated to proportionally increase with growth, we performed a quantitative analysis of mineral deposit formation using (99m)Tc-HMDP and found some weaknesses, such as longer acquisition time and narrower dynamic ranges (i.e. images easily saturated). The recently developed near-infrared (NIR) optical imaging technique is expected to non-invasively evaluate changes in living small animals in a quantitative manner.
    Purpose: To test the feasibility of NIR imaging with a dual-channel system as a better alternative for bone scintigraphy by quantitatively evaluating mineralization along with the growth of osteosarcoma lesions in a mouse-xenograft model.
    Material and Methods: The gross volume and mineralization of osteosarcoma lesions were evaluated in living mice simultaneously with dual-channels by NIR dye-labeled probes, 2-deoxyglucose (DG) and pamidronate (OS), respectively. To verify these quantitative data, retrieved osteosarcoma lesions were then subjected to ex-vivo imaging, weighing under wet conditions, microfocus-computed tomography (mu CT) analysis, and histopathological examination.
    Results: Because of less scattering and no anatomical overlapping, as generally shown, specific fluorescence signals targeted to the osteosarcoma lesions could be determined clearly by ex-vivo imaging. These data were well positively correlated with the in-vivo imaging data (r &gt; 0.8, P &lt; 0.02). Other good to excellent correlations (r &gt; 0.8, P &lt; 0.02) were observed between DG accumulation and tumor gross volume and between OS accumulation and mineralization volume.
    Conclusion: This in-vivo NIR imaging technique using DG and OS is sensitive to the level to simultaneously detect and quantitatively evaluate the growth and mineralization occuring in this type of osteosarcoma lesions of living mice without either invasion or sacrifice. By possible mutual complementation, this dual imaging system might be useful for accurate diagnosis even in the presence of overlapping tissues.

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  • Improved adhesion of human cultured periosteal sheets to a porous poly(L-lactic acid) membrane scaffold without the aid of exogenous adhesion biomolecules 査読

    Tomoyuki Kawase, Takaaki Tanaka, Takayuki Nishimoto, Kazuhiro Okuda, Masaki Nagata, Douglas M. Burns, Hiromasa Yoshie

    JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A   98A ( 1 )   100 - 113   2011年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    Human cultured periosteal sheets, which are developed from small excised periosteum tissue segments (PTSs) in culture dishes by simple expansion culture, have been applied as a promising autologous osteogenic grafting material for periodontal regenerative therapy. However, the weak initial adhesion of PTSs to dish surfaces often hampers cellular outgrowth and limits the number of preparations. To correct this weakness and still avoid the use of animal-derived adhesion biomolecules, we have developed a novel, biodegradable, porous poly(L-lactic acid) (pPLLA) membrane. Freshly excised PTSs bound well to the highly porous pPLLA membrane, possibly due to the presence of semihemispheric 20-30 mu m diameter openings on the upper surface. Global gene expression analysis demonstrated that periosteal sheets cultured on pPLLA membranes upregulated expression of many adhesion molecules. Osteogenic induction stimulated the production of proteoglycans by these cells and concomitantly enhanced their expansion and penetration into the deep pore regions of the membrane in parallel with the progression of in vitro mineralization. These findings suggest that our pPLLA membranes not only facilitate initial adhesion, primarily mediated by adsorbed proteins, but also enhance biological adhesion by inducing endogenous adhesion molecules in periosteal sheet cultures. Therefore, the efficacy of periosteal sheets in therapy should be greatly enhanced by using this new pPLLA membrane. (C) 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 98A: 100-113, 2011.

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  • Nondestructive Microstructural Analysis of Porous Bioceramics by Microfocus X-ray Computed Tomography (mu CT): A Proposed Protocol for Standardized Evaluation of Porosity and Interconnectivity Between Macro-pores 査読

    Hitoshi Nakayama, Douglas M. Burns, Tomoyuki Kawase

    JOURNAL OF NONDESTRUCTIVE EVALUATION   30 ( 2 )   71 - 80   2011年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER/PLENUM PUBLISHERS  

    Microfocus X-ray computed tomography (mu CT) has now become widely available for the nondestructive evaluation of porous bioceramics suitable for use as a bone substitute in orthopedic surgery. As part of an official Japanese working committee, we recently participated in the preparation of a proposed standard protocol for the quantitative mu CT analysis of porous bioceramics sent to the International Organization for Standardization (ISO). In this protocol, the recommended basic conditions for analysis were [field of view (XY plane): 3.0 mm, spatial resolution: 6 mu m/pixel (or the closest minimal values available for both parameters on a particular mu CT system), matrix size: 512 pixels], and we have now further determined the optimal values for more detailed parameters (e.g., threshold determination). To validate the utility of the complete protocol, three different types of ceramic sample [a ceramic of beta-tricalcium phosphate (beta-TCP) and two types of hydroxyapatite (HAp) with different porosities] were evaluated with three different types of cone-beam mu CT scanner (the Shimadzu SMX-100CT, Shimadzu inspeXio-90CT, and Skyscan-1174 scanners). Acquired images were quantified using 3D-reconstruction software, VGStudio MAX (version 1.2). After comparing data obtained from these three mu CT scanners, we have found that determinations of both porosity and pore-interconnectivity were very similar from one system to the other although the total number of measured pores did vary between scanners. The present data indicate that our protocol for mu CT analysis is reliable enough to quantify the porosity and interconnectivity of porous bioceramics and would therefore facilitate both large-scale screening and quality control of porous bioceramic samples.

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  • Manual cryopreservation of human alveolar periosteal tissue segments: Effects of pre-culture on recovery rate 査読

    Tomoyuki Kawase, Hiroyuki Kogami, Masaki Nagata, Kohya Uematsu, Kazuhiro Okuda, Douglas M. Burns, Hiromasa Yoshie

    CRYOBIOLOGY   62 ( 3 )   202 - 209   2011年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    Cultured human periosteal sheets constitute a promising grafting material for periodontal tissue regenerative therapy. However, preparation of these sheets usually requires six weeks or longer, and this lengthy commitment and delay limits both clinical applicability and availability. The aim of this study is to develop an efficient, practical, cost-effective cryopreservation method for periosteal tissue segments (PTSs). Human PTSs were aseptically excised from alveolar bone and pre-cultured in Medium 199 + 10% fetal bovine serum (FBS) for the indicated number of days before they were slowly frozen down to -75 degrees C in a commercial freezing vessel using medium containing 10% dimethyl sulfoxide (Me(2)SO) and various concentrations of FBS. After fast-thawing at 37 degrees C, PTSs were again cultured, and their growth and responses to standard osteogenic induction were evaluated (vs. freshly excised PTSs). Proliferating cells were obtained at the highest levels from cryopreserved FTSs that were pre-cultured for 14 days before freezing. When a concentration of 50% or more FBS was included in the cryopreservation solution, cells migrated out more actively and grew faster. Importantly, osteoinduction up-regulated alkaline phosphatase (ALP) activity and osteoblastic marker mRNAs in cryopreserved PTS-derived sheets just as effectively as it did in native PTS-derived ones. These data suggest that pre-conditioned PTSs can be efficiently cryopreserved in a freezing solution containing high FBS by traditional manual cryopreservation methods without aid of a program freezer or more elaborate equipment. (C) 2011 Elsevier Inc. All rights reserved.

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  • The primary site of the acrocephalic feature in Apert syndrome is a dwarf cranial base with accelerated chondrocytic differentiation due to aberrant activation of the FGFR2 signaling 査読

    Masaki Nagata, Glen H. Nuckolls, Xibin Wang, Lillian Shum, Yukie Seki, Tomoyuki Kawase, Katsu Takahashi, Kazuaki Nonaka, Ichiro Takahashi, Arhab A. Noman, Kenji Suzuki, Harold C. Slavkin

    BONE   48 ( 4 )   847 - 856   2011年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE INC  

    Activation of osteoblastic bone anabolism in the calvarial sutures is considered to be the essential pathologic condition underlying mutant FGFR2-related craniofacial dysostosis. However, early clinical investigations indicated that abnormal cartilage development in the cranial base was rather a primary site of abnormal feature in Apert Syndrome (AS). To examine the significance of cartilaginous growth of the cranial base in AS, we generated a transgenic mouse bearing AS-type mutant Fgfr2IIIc under the control of the Col2a1 promoter-enhancer (Fgfr2IIIc(P253R) mouse). Despite the lacking expression of Fgfr2IIIc(P253R) in osteoblasts, exclusive disruption of chondrocytic differentiation and growth reproduced AS-like acrocephaly accompanied by short anterior cranial base with fusion of the cranial base synchondroses, maxillary hypoplasia and synostosis of the calvarial sutures with no significant abnormalities in the trunk and extremities. Gene expression analyses demonstrated upregulation of p21, Ihh and Mmp-13 accompanied by modest increase in expression of Sox9 and Runx2, indicating acceleration of chondrocytic maturation and hypertrophy in the cranial base of the Fgfr2IIIc(P253R) mice. Furthermore, an acquired affinity and specificity of mutant FGFR2IIIc(P253R) receptor with FGF2 and FGF10 is suggested as a mechanism of activation of FGFR2 signaling selectively in the cranial base. In this report, we strongly suggest that the acrocephalic feature of AS is not alone a result of the coronal suture synostosis, but is a result of the primary disturbance in growth of the cranial base with precocious endochondral ossification. (C) 2010 Elsevier Inc. All rights reserved.

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  • Analysis of intestinal fibrosis in chronic colitis in mice induced by dextran sulfate sodium 査読

    Kenji Suzuki, Xiaomei Sun, Masaki Nagata, Tomoyuki Kawase, Hana Yamaguchi, Vijayakumar Sukumaran, Yusuke Kawauchi, Hiroshi Kawachi, Takayoshi Nishino, Kenichi Watanabe, Hiroyuki Yoneyama, Hitoshi Asakura

    PATHOLOGY INTERNATIONAL   61 ( 4 )   228 - 238   2011年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    Fibrogenic mesenchymal cells including fibroblasts and myofibroblasts play a key role in intestinal fibrosis, however, their precise role is largely unknown. To investigate their role in intestinal fibrosis, we analyzed the lesions of chronic colitis in C57BL/6 (B6) mice induced by dextran sulfate sodium (DSS). B6 mice exposed to single cycle administration of DSS for 5 days developed acute colitis that progressed to severe chronic inflammation with dense infiltrates of mononuclear cells, irregular epithelial structure, thickening of colonic wall, and persistent deposits of collagen. Increased mRNA expressions of proinflammatory cytokines are correlated with extensive cellular infiltration, and the mRNA expressions of collagen 1, transforming growth factor (TGF)-beta, and matrix metalloproteinases were also enhanced in the colon. In the colon of chronic DSS colitis, fibroblasts (vimentin+, alpha-smooth muscle actin (alpha-SMA)-) were increased in both mucosal and submucosal layers, while myofibroblasts (vimentin+, alpha-SMA+) were increased in mucosal but not in submucosal layers. Primary mouse subcutaneous fibroblast cultures experiments revealed that exogenously added TGF-beta 1 substantially augmented the expressions of both vimentin and alpha-SMA proteins with increased production of collagen. In conclusion, profibrogenic mesenchymal cells play an important role in the development of intestinal fibrosis in this chronic DSS-induced colitis model.

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  • Collagen-coated poly(L-lactide-co-ε-caprolactone) film: A promising scaffold for cultured periosteal sheets. 査読

    Kawase T, Yamanaka K, Suda Y, Kaneko T, Okuda K, Kogami H, Nakayama H, Nagata M, Wolff LF, Yoshie H

    J Periodontol   81 ( 11 )   1653 - 1662   2010年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Evaluation by Bone Scintigraphy of Osteogenic Activity of Commercial Bioceramics (Porous beta-TCP and HAp Particles) Subcutaneously Implanted in Rats 査読

    Hitoshi Nakayama, Tomoyuki Kawase, Hiroyuki Kogami, Kazuhiro Okuda, Hikaru Inoue, Takaaki Oda, Kazuhide Hayama, Makoto Tsuchimochi, Larry F. Wolff

    JOURNAL OF BIOMATERIALS APPLICATIONS   24 ( 8 )   751 - 768   2010年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SAGE PUBLICATIONS LTD  

    Osteogenic potential of biomaterials used in bone regenerative therapy has been mainly examined in an animal-implantation study. We have here evaluated the applicability of bone scintigraphy in imaging ectopic bone formation, especially its initial phase, by beta-tricalcium phosphate (beta-TCP) particles that were implanted in rat dorsal subcutaneous tissues. In implanted osteogenic osteosarcoma cells used as a positive control, osteoid formation was found by histological examination and bone scintigraphy using (99m)Tc- hydroxymethyl diphosphonate (HMDP) at 2 and 3 weeks post-implantation, respectively, while the microfocus-computed tomography (mu CT) system required further mineralization, which occurred at 4 weeks. Implantation of beta-TCP particles alone induced only faint biomineralization inside the particles, which could be microscopically detected by calcein chelation at 2 weeks post-implantation, but not by other histological examinations (e. g., HE staining) or mu CT. However, the bone scintigraphy successfully detected this microscopic change at 1 week. Implanted hydroxyapatite (HAp) particles alone used as a negative control did not induce mineralization at microscopic levels, and therefore nothing was detected by either calcein chelation or bone scintigraphy. In conclusion, the bone scintigraphic methodology, although exhibiting less quantitation and resolution, would be applicable as a non-invasive, highly sensitive methodology in detecting the initial, microscopic changes associated with mineralization.

    DOI: 10.1177/0885328209341845

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  • Human Periosteum-Derived Cells Combined With Superporous Hydroxyapatite Blocks Used as an Osteogenic Bone Substitute for Periodontal Regenerative Therapy: An Animal Implantation Study Using Nude Mice 査読

    Tomoyuki Kawase, Kazuhiro Okuda, Hiroyuki Kogami, Hitoshi Nakayama, Masaki Nagata, Tomokazu Sato, Larry F. Wolff, Hiromasa Yoshie

    JOURNAL OF PERIODONTOLOGY   81 ( 3 )   420 - 427   2010年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER ACAD PERIODONTOLOGY  

    Background: A superporous (85%) hydroxyapatite (HA) block was recently developed to improve osteoconductivity, but it was often not clinically successful when used to treat periodontal osseous defects. The primary purpose of this study is to develop a clinically applicable tissue-engineered bone substitute using this HA block and human alveolar periosteum-derived cells.
    Methods: Commercially available superporous HA blocks were acid treated and subjected to a three-dimensional (3D) culture for periosteal cell cultivation. Cells in the pore regions of the treated HA block were observed on the fracture surface by scanning electron microscopy. After osteogenic induction, the cell HA complexes were implanted subcutaneously in nude mice. Osteoid formation was histologically evaluated.
    Results: Acid treatment enlarged the interconnections among pores, resulting in the deep penetration of periosteal cells. Under these conditions, cells were maintained for &gt;2 weeks without appreciable cell death in the deep pore regions of the HA block. The cell HA complexes that received in vitro osteogenic induction formed osteoids in pore regions of the treated HA blocks in vivo. In contrast, most pore regions in the non-pretreated, cell-free HA blocks that were evaluated in vivo remained cell free.
    Conclusions: Our findings suggest that an acid-treated HA block could function as a better scaffold for the 3D high-density culture of human periosteal cells in vitro, and this cell HA complex had significant osteogenic potential at the site of implantation in vivo. Compared with the cell-free HA block, our cell HA complex using periosteal cells, which are the most accessible for clinical periodontists, showed promising results as a bone substitute in periodontal regenerative therapy. J Periodontal 2010;81:420-427.

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  • Osteogenic activity of human periosteal sheets cultured on salmon collagen-coated ePTFE meshes 査読

    Tomoyuki Kawase, Kazuhiro Okuda, Hiroyuki Kogami, Hitoshi Nakayama, Masaki Nagata, Hiromasa Yoshie

    JOURNAL OF MATERIALS SCIENCE-MATERIALS IN MEDICINE   21 ( 2 )   731 - 739   2010年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER  

    Our animal implantation studies have demonstrated that, after osteogenic processing, cultured human periosteal sheets form osteoid tissue ectopically without the aid of conventional scaffolding materials. To improve the osteogenic activity of these periosteal sheets, we have tested the effects of including a scaffold made of salmon collagen-coated ePTFE mesh. Periosteal sheets were produced with minimal manipulation without enzymatic digestion. Outgrown cells penetrated into the coated mesh fiber networks to form complex multicellular layers and increased expression of alkaline phosphatase activity in response to the osteoinduction. In vitro mineralization was notably enhanced in the original tissue segment regions, but numerous micro-mineral deposits were also formed on the coated-fiber networks. When implanted subcutaneously into nude mice, periosteal sheets efficiently form osteoid around the mineral deposits. These findings suggest that the intricate three-dimensional mesh composed of collagen-coated fibers substantially augmented the osteogenic activity of human periosteal sheets both in vitro and in vivo.

    DOI: 10.1007/s10856-009-3896-9

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  • 歯周組織再生を促す生理活性物質と細胞療法に関するトランスレーショナルリサーチ 招待 査読

    川瀬 知之

    日本歯周病学会誌   52 ( 1 )   3 - 11   2010年

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    記述言語:日本語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.2329/perio.52.3

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  • Characterization of human cultured periosteal sheets expressing bone-forming potential: In vitro and in vivo animal studies 査読

    Tomoyuki Kawase, Kazuhiro Okuda, Hiroyuki Kogami, Hitoshi Nakayama, Masaki Nagata, Koh Nakata, Hiromasa Yoshie

    Journal of Tissue Engineering and Regenerative Medicine   3 ( 3 )   218 - 229   2009年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Our recent clinical studies have demonstrated that autologous implantation of human cultured periosteal (hCP) sheets in combination with porous hydroxylapatite (HAp) particles at the site of periodontal bone defects strikingly facilitates tissue regeneration. To better understand how the hCP sheet functions at the implantation site, we have now examined its biochemical and morphological characteristics in vitro and its ectopic osteoinductivity in nude mice. Cultured human periosteal tissue segments produced periosteal cells that migrated out from the central region within 4-8 days and grew more rapidly with longer culture times. Alkaline phosphatase activity increased in parallel with actual osteoblastic induction. Cytokine array assays demonstrated that osteoblastic induction downregulated IL-6 and thrombopoietin, but upregulated IL-8, IL-13, IGF-I and IGFBP-2 in hCP sheets. When differentiated hCP sheets were implanted alone, areas of osteoid and mineralized tissue were formed within 2 weeks, but non-induced, immature hCP sheets did not produce much mineralization. These findings suggest that mature hCP sheets potentially function not only as seeds of ectopic bone formation without the need of synthetic tissue scaffolds, but also as living drugdelivery systems, to influence cells near implantation sites by producing several important cytokines. These two major characteristics indicate that a mature hCP sheet is a promising osteoinductive biomaterial, even without conventional scaffolds for periodontal regenerative therapy. Copyright © 2009 John Wiley &amp
    Sons, Ltd.

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  • X線マイクロCTによる生体活性セラミックス 多孔体の微小構造解析 査読

    中山 均, 川瀬知之

    歯科放射線   49 ( 3 )   33 - 40   2009年

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    記述言語:日本語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.11242/dentalradiology.49.33

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  • Treatment of human infrabony periodontal defects by grafting human cultured periosteum sheets combined with platelet-rich plasma and porous hydroxyapatite granules: case series. 査読

    Okuda K, Yamamiya K, Kawase T, Mizuno H, Ueda M, Yoshie H

    J Int Acad Periodontol   11 ( 3 )   206 - 213   2009年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • 歯肉細胞シート・骨膜シートを用いた歯周再生治療 招待 査読

    吉江弘正, 奥田一博, 川瀬知之

    日本口腔外科学会雑誌   55 ( 9 )   432 - 439   2009年

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    記述言語:日本語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.5794/jjoms.55.432

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  • Tissue-engineered cultured periosteum used with platelet-rich plasma and hydroxyapatite in treating human osseous defects 査読

    Kancko Yamamiya, Kazuhiro Okuda, Tomoyuki Kawase, Ken-ichiro Hata, Larry F. Wolff, Hiromasa Yoshie

    JOURNAL OF PERIODONTOLOGY   79 ( 5 )   811 - 818   2008年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER ACAD PERIODONTOLOGY  

    Background: The aim of the present controlled clinical study was to compare the clinical response of human cultured periosteum (HCP) sheets in combination with platelet-rich plasma (PRP) and porous hydroxyapatite (HA) granules to a mixture of PRP and HA in the treatment of human infrabony periodontal defects.
    Methods: Thirty interproximal infrabony osseous defects in 30 healthy, non-smoking subjects diagnosed with chronic periodontitis were included in this study. The subjects were randomly assigned to the test group (HCP sheets combined with PRP and HA) or the control group (PRP with HA). Clinical and radiographic measurements were made at baseline and the 12-month post-surgical evaluation.
    Results: Compared to baseline, the 12-month results indicated that both treatment modalities resulted in statistically significant changes (P&lt;0.01) in the gingival index, bleeding on probing, probing depth, clinical attachment level, and radiographic infrabony defect depth. Compared to the control group, the test group exhibited a statistically significantly more favorable change in clinical attachment gain (3.9 +/- 1.6 mm versus 2.7 +/- 1.3 mm; P&lt;0.05), vertical relative attachment gain (83.5% +/- 31.7% versus 55.0% +/- 21.9%; P &lt;0.05), and radiographic infrabony defect fill (4.9 +/- 1.2 mm versus 3.2 +/- 1.1 mm; P&lt;0.01).
    Conclusions: Compared to PRP with HA, treatment with a combination of HCP sheets, PRP, and HA led to a significantly more favorable clinical improvement in infrabony periodontal defects. A factor likely contributing to these favorable clinical results is the presence of osteogenic cells in the HCP sheets, which provided greater regeneration potential.

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  • Extracellular ATP and ATP gamma S suppress the proliferation of human periodontal ligament cells by different mechanisms 査読

    Tomoyuki Kawase, Kazuhiro Okuda, Hiromasa Yoshie

    JOURNAL OF PERIODONTOLOGY   78 ( 4 )   748 - 756   2007年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER ACAD PERIODONTOLOGY  

    Background: Periodontitis, similar to many other known inflammatory diseases, is thought to increase adenosine triphosphate (ATP) levels in extracellular spaces. Extracellular ATP acts on specific receptors to modulate the proliferation of various cell types. Platelet-rich plasma (PRP) contains high levels of ectonucleotidase activity capable of degrading ATP. The aim of this study was to investigate the effects of ATP on the proliferation of human periodontal ligament (PDL) cells and how these effects are altered by ectonucleotidases.
    Methods: PDL cells were derived from healthy young volunteers. ATP content and DNA systhesis were quantified by a bioluminescence and an enzyme-linked immunosorbent assay, respectively. CD39 and p21(WAF1/cip1) expression was analyzed by Western blot. Apoptosis was evaluated by caspase-3/7 activity, terminal deoxynucleotidyl transferase (TST)-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) activity, annexin-V-binding, and DNA fragmentation.
    Results: CD38 and ectonucleotidase-like activity were found in PDL cells and serum, respectively. Because less CD39 is expressed in freshly plated cells, both exongenous ATP and ATP gamma S, a slowly hydrolysable analog, inhibited cell proliferation under low serum condition. ATP upregulated p21(WAF1/cip1), an inhibitor of cell-cycle progression, whereas ATp gamma S induced capase-dependent apoptosis. Either upregulated of CD39 or added serum rescued cells from the cytostatic actions of exogenous ATP.
    Conclusions: In PDL cells expressing low CD39 levels, both ATP and ATP gamma S inhibited proliferation but by different mechanisms. ATP-induced growth arrest suggests that periodontal tissue regeneration is often supressed at the site of injury. Futhermore, added ectonucleotidases protected PDL cells from ATP's cytostatic actions, suggesting that ectonucleotidase-rich PRP augments the regenerative actions of its constituent growth factors by protecting against exogenous ATP at clinical sites.

    DOI: 10.1902/jop.2007.060283

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  • A hepatocyte growth factor (HGF)/receptor autocrine loop regulates constitutive self-renewal of human periodontal ligament cells but reduces sensitivity to exogenous HGF 査読

    Tomoyuki Kawase, Kazuhiro Okuda, Hiromasa Yoshie

    JOURNAL OF PERIODONTOLOGY   77 ( 10 )   1723 - 1730   2006年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER ACAD PERIODONTOLOGY  

    Background: In addition to its prominent role in liver regeneration, hepatocyte growth factor (HGF) is now generally thought to be produced by mesenchymal cells to promote the regeneration of epithelial tissue by a paracrine mechanism. However, it is not known how or if HGF could be involved in the regeneration of periodontal tissues. The purpose of this study was to characterize the ability of normal human periodontal ligament (PDL) cells to produce or respond to HGF.
    Methods: PDL cells derived from healthy young volunteers were used from passages four through 10. HGF receptors were detected both by immunocytochemical staining and Western-blotting analysis. Both DNA synthesis (by bromo-deoxyuridine [BrdU]-incorporation) and secreted HGF were quantified by enzyme-linked immunosorbent assays. Mitogen -activated protein kinase (MAPK) phosphorylation was also analyzed by Western blot.
    Results: Despite the immunocytochemical demonstration of HGF receptor protein in the cytoplasm and on the plasma membrane of PDL cells, exogenous recombinant human HGF did not exert the mitogenic effects expected. As reported for other mesenchymal cells, PDL cells were found to secrete HGF. Treatments with neutralizing anti-HGF antibody significantly suppressed constitutive PDL cell proliferation and sustained the receptor protein at higher levels than in non-treated cells. Under these conditions, exogenous HGF rapidly phosphorylated extracellular signal-regulated kinase (ERK), an action linked to the cell proliferation and downregulation of cell-surface receptors.
    Conclusions: Unlike other known mesenchymal or epithelial cells, these findings suggest that normal PDL cells from young donors possess a constitutive HGF/receptor autocrine loop that normally regulates their replacement self-proliferation but reduces sensitivity to exogenously applied HGF by acute receptor downregulation.

    DOI: 10.1902/jop.2006.060031

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  • Platelet-rich plasma combined with a porous hydroxyapatite graft for the treatment of intrabony periodontal defects in humans: A comparative controlled clinical study 査読

    K Okuda, H Tai, K Tanabe, H Suzuki, T Sato, T Kawase, Y Saito, LF Wolff, H Yoshie

    JOURNAL OF PERIODONTOLOGY   76 ( 6 )   890 - 898   2005年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER ACAD PERIODONTOLOGY  

    Background: The aim of the present controlled clinical study was to compare platelet-rich plasma (PRP) combined with a biodegradable ceramic, porous hydroxyapatite (HA) with a mixture of HA and saline in the treatment of human intrabony defects.
    Methods: Seventy interproximal intrabony osseous defects in 70 healthy, non-smoking subjects diagnosed with chronic periodontitis were included in this study. Thirty-five subjects each were randomly assigned to either the test group (PRP and HA) or control group (HA with saline). Clinical and radiographic measurements were determined at baseline and the 12-month evaluation.
    Results: When compared to baseline, the 12-month results indicated that, while both treatment modalities resulted in significant changes in all clinical parameters (gingival index, bleeding on probing, probing depth, clinical attachment level, and intrabony defect fill; P &lt; 0.001), the test group exhibited statistically significant changes compared to the control sites in probing depth reduction: 4.7 +/- 1.6 mm versus 3.7 +/- 2.0 mm (P &lt; 0.05); clinical attachment gain: 3.4 +/- 1.7 mm versus 2.0 +/- 1.2 mm (P &lt; 0.001); and vertical relative attachment gain: 70.3% +/- 23.4% versus 45.5% +/- 29.4% (P &lt; 0.001).
    Conclusion: Treatment with a combination of PRP and HA compared to HA with saline led to a significantly more favorable clinical improvement in intrabony periodontal defects.

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  • Platelet-rich plasma provides nucleus for mineralization in cultures of partially differentiated periodontal ligament cells 査読

    T Kawase, K Okuda, Y Saito, N Amizuka, H Suzuki, M Yoshie

    IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL   41 ( 5-6 )   171 - 176   2005年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOC IN VITRO BIOLOGY  

    Platelet-rich plasma (PRP) has been used to promote periodontal regeneration following the premise that constituent transforming growth factor beta 1 (TGF-beta 1) and platelet-derived growth factor-AB will stimulate cell proliferation at the site of application. In previous Studies, we demonstrated that PRP mimics TGF-beta 1 to modulate proliferation in a cell type-specific manner, that fibrin clot formation by PRP upregulates type 1 collagen, and that an unidentified factor(s) in PRP increases alkaline phosphatase (ALP) activity in human periodontal ligament (PDL) cell cultures. We have now examined the effects of PRP on in vitro mineralization. Platelet-rich plasma and PDL cells were prepared from human adult Volunteers or rats. After 20 d of continuous treatment with PRP in dexamethazone (Dex)-containing osteogenic medium, PRP time dependently promoted mineralization by rat PDL cells IRA failed to fully induce the osteoblastic phenotype. Furthermore, when human PDL cells were induced to increase ALP activity in osteogenic medium that lacked Dex, a condition that should delay (or suppress) osteoblastic differentiation, transmission electron microscopy revealed that mineralized spicules were initially deposited onto PRP-derived platelet aggregates. Taken together with our previous data, these findings suggest that PRP provides platelet aggregates as nuclei to initiate mineralization while stimulating PDL cell proliferation, differentiation, and collagen production. The combination of these effects may effectively mediate PRP's ability to promote regeneration of periodontal tissue, including skeletal tissue, at the site of injury.

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  • Platelet-rich plasma contains high levels of platelet-derived growth factor and transforming growth factor-beta and modulates the proliferation of periodontally related cells in vitro 査読

    K Okuda, T Kawase, M Momose, M Murata, Y Saito, H Suzuki, LF Wolff, H Yoshie

    JOURNAL OF PERIODONTOLOGY   74 ( 6 )   849 - 857   2003年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER ACAD PERIODONTOLOGY  

    Background: Platelet-rich plasma (PRP) is a fraction of plasma, in which platelet-derived growth factor (PDGF) and transforming growth factor-P (TGF-beta) are thought to be concentrated. It is plausible that topically-applied PRP up-regulates cellular activity and subsequently promotes periodontal regeneration in vivo. However, the concentrations of these growth factors in PRP have not been specifically determined and the biological effects of PRP at the cellular and molecular levels have not been determined.
    Methods: PRP obtained from 20 healthy subjects was prepared from plasma by centrifugation. These PRP preparations were immediately subjected to an evaluation for PDGF-AB and TGF-beta1 using enzyme-linked immunosorbent assay (ELISA) kits. The biological effects of the PRP preparations were evaluated on osteoblastic, epithelial, fibroblastic, and periodontal ligament cells. Cellular mitogenic activity was evaluated by counting cell numbers or evaluating 5 - bromodeoxyuridine (BrdU) incorporation. Expression of alkaline phosphatase (ALP) was immunocytochemically evaluated.
    Results: In the PRP preparations, platelets were concentrated up to 70.9 x 10(4) cells/mul (283.4% of the unconcentrated plasma). The levels of PDGF-AB and TGF-beta1 were also concentrated up to 182.0 ng/ml (440.6%) and 140.9 ng/ml (346.6%), respectively. Scatter plots revealed significant correlations between platelet counts and levels of these growth factors. PRP stimulated osteoblastic DNA synthesis and cell division (138% of control), with simultaneous down-regulation of ALP, but suppressed epithelial cell division (80% of control). PRP also stimulated DNA synthesis in gingival fibroblasts and periodontal ligament cells.
    Conclusions: These data demonstrated that both PDGF-AB and TGF-beta1 were highly concentrated in the PRP preparations. It is suggested PRP modulates cell proliferation in a cell type-specific manner similar to what has been observed with TGF-beta1. Since synchronized behavior of related cell types is thought to be required for successful periodontal regeneration, it is further suggested these cell type-specific actions may be beneficial for periodontal regenerative therapy.

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  • NaF induces early differentiation of murine bone marrow cells along the granulocytic pathway but not the monocytic or preosteoclastic pathway in vitro 査読

    A Oguro, T Kawase, M Orikasa

    IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL   39 ( 5-6 )   243 - 248   2003年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOC IN VITRO BIOLOGY  

    The stimulatory effects of sodium fluoride (NaF) on bone formation have been explained solely by its activation of osteoblasts. However, whether and how NaF acts on the osteoclast lineage is poorly understood. We previously found that NaF differentiates HL-60 cells to granulocytic cells. To further test this action, we have employed here primary cultures of progenitor cells derived from murine bone marrow. NaF at subtoxic concentrations (&lt;0.5 mM) significantly up-regulated activities of several intracellular enzymes (lactate dehydrogenase, beta-glucuronidase, acid phosphatase), cellular reduction of nitroblue tetrazolium, and nitric oxide (NO) production; which are all accepted as general differentiation markers. NaF (&lt;0.5 mM) also up-regulated granulocyte-specific markers (chloroacetate esterase, cell surface antigens [Mac-1, Gr-1]) but not any of the monocyte-specific markers (nonspecific esterase, cell surface antigens [F4/80, MOMA-2]). Although other general differentiation markers (phagocytosis, adhesion, appearance, nuclear:cytoplasmic ratio) were not appreciably influenced by NaF. essentially in support of our previous data from HL-60 cells, the present findings suggest that NaF induces early differentiation of bone marrow hemopoietic progenitor cells along the granulocytic pathway but not the monocytic pathway that is linked to osteoclast formation. Therefore, in addition to its potent stimulatory effects on osteoblastic bone formation, NaF applied to patients with osteoporosis could be expected to indirectly reduce osteoclastic bone resorption.

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  • Anti-TGF-beta antibody blocks enamel matrix derivative-induced upregulation of p21(WAF1/CiP1) and prevents its inhibition of human oral epithelial cell proliferation 査読

    T Kawase, K Okuda, H Yoshie, DM Burns

    JOURNAL OF PERIODONTAL RESEARCH   37 ( 4 )   255 - 262   2002年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:BLACKWELL MUNKSGAARD  

    We have previously demonstrated that porcine enamel matrix derivative (EMD) contains TGF-beta1 (or a TGF-beta-like substance). and that EMD rapidly translocates smad2, which is an effector of the TGF-beta signaling pathway. into the nucleus and modulates the proliferation of both human gingival fibroblastic and oral epitheiial cells in a cell type-specific manner. To investigate the involvement of TGF-beta in the growth modulatory action of EMD, two approaches have been used in the present study: i) a neutralizing anti-TGF-beta antibody to block EMD action, and ii) authentic porcine TGF-beta1 to compare with EMD. Both in epithelial and fibroblastic cells, TGF-beta1 closely mimicked EMD in nuclear accumulation of smad2, phosphorylation of MAP kinase family members, and consequent cell type-specific growth modulation, Anti-TGF-beta antibody, at levels which completely blocked TGF-beta1-induced smad2 translocation. strongly blocked EMD-induced smad2 translocation. This antibody also blocked other actions of EMD in epithelial cells, i.e. p38-MAP kinase (p38-K) phosphorylation, p21(WAF1/cip1) expression. and inhibition of DNA synthesis. In support of our previous proposal, these data suggest that TGF-beta1 (or a TGF-beta-like substance), which is delivered as a principal bioactive factor in EMD, inhibits epithelial cell proliferation probably by a smad2- mediated, p21(WAF1/cip1)-dependent mechanism. However, the same neutralizing antibody failed to convincingly block EMD-induced fibroblastic proliferation, which suggests that EMD may contain additional unidentified mitogenic factor(s), which act in combination with TGF-beta to fully stimulate fibroblastic proliferation.

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  • Cytostatic action of enamel matrix derivative (EMDOGAIN (R)) on human oral squamous cell carcinoma-derived SCC25 epithelial cells 査読

    T Kawase, K Okuda, H Yoshie, DM Burns

    JOURNAL OF PERIODONTAL RESEARCH   35 ( 5 )   291 - 300   2000年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:MUNKSGAARD INT PUBL LTD  

    During surgical treatment of periodontal disease, enamel matrix derivative (EMD) is topically applied as a substitute for extracellular matrix in order to facilitate regeneration of damaged periodontal tissue. However, the mechanism for EMD action is poorly understood, We have now examined the effects of EMD on the proliferation of oral epithelial (SCC25) cells in vitro. After 3 days of treatments, EMD (25-100 mu g/ml) dose-dependently inhibited cell division and concomitantly arrested cell cycle at the G1 phase. Prior to this inhibition, EMD significantly up-regulated p21(WAF1/cip1), a cyclin-dependent kinase inhibitor, induced G1-arrest, and inhibited DNA synthesis. In addition, EMD down-regulated expression of cytokeratin-18 (CK18) protein, which was most due to decreased production, but less to increased degradation. However, EMD did not discernibly increase the number of apoptotic cells over 8 days of treatment. These findings indicate (I)that EMD acts as a cytostatic agent, rather than a cytotoxic agent, on epithelial cells, and (2) that this anti-proliferative action is probably due to p21(WAF1/cip1)- mediated G1-arrest. Furthermore, our in vitro cellular data clearly verify and provide an explanation for the clinical observation that EMD application suppresses the down-growth of junctional epithelium onto dental root surfaces, a process that frequently interferes with the formation of new connective tissue attachments.

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  • Calcitonin gene-related peptide acts as a mitogen for human Gin-1 gingival fibroblasts by activating the MAP kinase signalling pathway 査読

    T Kawase, K Okuda, CH Wu, H Yoshie, K Hara, DM Burns

    JOURNAL OF PERIODONTAL RESEARCH   34 ( 3 )   160 - 168   1999年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:MUNKSGAARD INT PUBL LTD  

    In many peripheral tissues. calcitonin gene-related peptide (CGRP) is released from peptidergic sensory nerve fibres and acts like a growth factor during tissue development and regeneration. However, the ability of CGRP to influence gingival tissue has not been studied. To address this question, we have now examined the effects of CGRP on the proliferation of human gingival fibroblasts (Gin-l) in vitro. Gin-1 cells have approximately 3100 specific CGRP-binding sites with a Kd of 38.6 pM on their surface. Treatment with CGRP (0.1-100 nM) significantly stimulated cell proliferation in a dose-dependent manner, with maximal effects at 1-10 nM CGRP after 2 d. As one early cellular response to CORP, p44-MAPK protein (also known as the extracellular signal response kinase [ERK]) was tyrosine- and threonine-phosphorylated within 2 min, and this phosphorylation was sustained for at least 1 h. The dose-response curve of MAPK activation was very similar to that observed for CGRP's stimulation of cell proliferation. In addition, CGRP's activation of MAPK stimulated its ability to phosphorylate the Elk-l transcription factor. When cells were pretreated with PD98059, a selective inhibitor of MAPK kinase (also known as MEK), CGRP not only failed to induce phosphorylation of MAPK but also failed to stimulate Gin-1 cell proliferation. Our present data indicate that CGRP rapidly activates the MAPK signalling pathway, an effect which consequently stimulates the proliferation of gingival fibroblasts. Our data demonstrate specific cellular responses to CGRP by gingival fibroblasts and support the possibility that CGRP acts as a targeted local factor in the regulation of development, generation and/or regeneration of gingival tissues.

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  • Possible regulation of epidermal growth factor-receptor tyrosine autophosphorylation by calcium and G proteins chemically permeabilized rat UMR106 cells 査読

    T Kawase, M Orikasa, A Oguro, DM Burns

    ARCHIVES OF ORAL BIOLOGY   44 ( 2 )   157 - 171   1999年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PERGAMON-ELSEVIER SCIENCE LTD  

    A model using chemically permeabilized cells was developed to examine mechanisms that regulate protein tyrosine phosphorylation in osteoblastic cells. Using either permeabilized UMR106 osteoblastic or A431 (reference) cells, epidermal growth factor (EGF)-induced cellular tyrosine phosphorylation, and whether there are previously unrecognized interactions between this transduction pathway and Ca-24- or G protein-dependent signalling pathways, were investigated. Both permeabilized cell types, when maintained in non-supplemented cytoplasmic substitution solution (basic CSS), responded to EGF (1-100 ng/ml) with dose-dependent increases in tyrosine phosphorylation. A complex and time-dependent pattern of phosphotyrosine-containing proteins resulted, but the profile of tyrosine phosphorylated proteins was appreciably less complex than in intact cells. Supplementation of basic CSS with MgATP restored the normal complexity of the profiles for EGF-induced tyrosine phosphorylation proteins in both permeabilized cell lines and produced a more sustained accumulation of phosphoprotein products in A431 cells. Adding Ca2+ (less than or equal to 10(-6)M), with or without exogenous MgATP, dose-dependently attenuated EGF-induced tyrosine phosphorylation of EGF receptors (EGFR) and other substrates in UMR106 cells, but was less effective in A431 cells. In both cell types, genistein, an inhibitor of tyrosine kinases, was more effective in attenuating EGF-induced receptor tyrosine phosphorylation in permeabilized cells. Similarly, orthovanadate, an inhibitor of protein tyrosine phosphatases, stimulated the accumulation of phosphoprotein products more effectively in permeabilized cells. Thus, the permeabilization preserves many features of intact cells while facilitating manipulation of intracellular conditions. NaF reproducibly produced a significant vanadate-like action in permeabilized cells that was somewhat stronger than its effect on intact cells. In contrast, the well-known inhibition of tyrosine phosphorylation by phorbol 12-myristate 13-acetate (PMA) was less effective in permeabilized cells than in intact cells; these actions of PMA were Ca2+-dependent. In addition, guanylyl-imidodiphosphate (Gpp(NH)p) attenuated tyrosine phosphorylation in UMR106 cells, and this effect was specifically blocked by guanosine 5'-O-(2-thiodiphosphate) (GDP beta s). These results strongly suggest that there is crosstalk between EGFR-activated tyrosine phosphorylation/dephosphorylation pathways and both Ca2+- and G protein-mediated pathways un UMR106 cells, revealing a previously unrecognized modulation of EGF signalling in osteoblast-like: cells that contrasts with the simpler regulatory mechanisms found in A431 cells. (C) 1999 Elsevier Science Ltd. All rights reserved.

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  • Calcitonin gene-related peptide stimulates potassium efflux through adenosine triphosphate-sensitive potassium channels and produces membrane hyperpolarization in osteoblastic UMR106 cells 査読

    T Kawase, DM Burns

    ENDOCRINOLOGY   139 ( 8 )   3492 - 3502   1998年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ENDOCRINE SOC  

    In previous studies, we have shown that calcitonin gene-related peptide (CGRP) acutely inhibits Ca-45(2+) uptake in osteoblastic UMR106 cells, and we have proposed that ATP-sensitive potassium (K-ATP) channels are involved in mediating this action of CGRP. To directly test this proposed mechanism, we have now examined the effects of CGRP on both membrane potential (Em) and K+ mobilization in UMR106 cells, using specific fluorometric dye assays. CGRP (0.01-100 nM) induced membrane hyperpolarization in a dose-dependent manner, with a half maximal effect (ED50) at approximately 0.2 nM and a maximal effect at 100 nM. Both pinacidil (Pina; a K-ATP channel opener) and forskolin (FSK) induced similar membrane hyperpolarization, but the actions of these three agents could be easily distinguished: both CGRP and Pina actions were well antagonized by glibenclamide (Glib; a selective K-ATP channel blocker), whereas FSK action was strongly attenuated only by tetraethylammonium (a K-Ca channel blocker) or compound H-89 (an inhibitor of cAMP-dependent protein kinases). Cells pretreated with Pina no longer responded to CGRP, but they could still respond to FSK; furthermore, pretreatment with FSK failed to block successive treatment with either CGRP or Pina. In parallel with observed changes in Em, CGRP (0.01-100 nM) decreased intracellular K+ concentrations ([K+](i)) in a dose-dependent manner, with an ED50 identical to that obtained for alterations in Em. This action of CGRP was sensitive to Glib and had only slight sensitivity to tetraethylammonium; this CGRP effect was mimicked by Pina but not by FSK. Interestingly, CGRP significantly elicited changes in cell shape by a Glib-sensitive mechanism that included notable decreases in cross-sectional cytoplasmic area. These observations strongly support our proposal that CGRP primarily stimulates K+ efflux via activation of K-ATP channels and thereby induces membrane hyperpolarization in UMR106 cells. Furthermore, our data also suggest that this cascade of initial cellular events may result in rapid changes in cell morphology and decreases in cellular area of the type that are thought to act as triggers for proliferation and/or differentiation in many cellular phenotypes.

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  • Characteristics of NaF-induced differentiation of HL-60 cells 査読

    T Kawase, A Oguro, M Orikasa, DM Burns

    JOURNAL OF BONE AND MINERAL RESEARCH   11 ( 11 )   1676 - 1687   1996年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:BLACKWELL SCIENCE INC  

    Sodium fluoride (NaF) is known to stimulate osteoblastic bone formation, but little attention has been given to the possibility that NaF also affects bone resorption and the differentiation of osteoclastic progenitor cells, Wen human promyelocytic HL-60 cells were treated with NaF (0.5 mM, 0-4 days), cell proliferation was inhibited, and the addition of 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) (10 nM, 0-4 days) augmented this antiproliferative effect. NaF increased cel)dar reduction of nitroblue tetrazolium (NET), and this effect was strongly augmented by 1,25(OH)(2)D-3. In addition, NaF produced marked changes in cellular morphology, increased cellular adhesion to plastic, reduced the nuclear/cytoplasmic ratio, and increased cellular expression of chloroacetate esterase, but failed to alter cellular nonspecific esterase activity. Furthermore, NaF increased expression of CD11b and CD66b, and this stimulation was enhanced by adding 1,25(OH)(2)D-3. The sum of these changes in classical promyelocytic cellular indices suggest: (1) that NaF stimulates the early stages of HL-60 differentiation toward a granulocyte-like cell and (2) that 1,25(OH)(2)D-3 promotes these actions of NaF. Additional experiments aimed at further understanding the NaF-induced conversion of HL-60 cells identified further changes. NaF also increased cellular production of prostaglandin E(2) (PGE(2)) and nitric oxide (NO) and induced expression of inducible nitric oxide synthase (iNOS); 1,25(OH)(2)D-3 once again augmented these NaF-induced effects. Similarly, NaF stimulated the production of interleukin 1 alpha (IL-1 alpha). IL-6, and tumor necrosis factor-alpha and 1,25(OH)(2)D-3 again strongly enhanced these effects. Indomethacin completely blocked stimulation of NET reduction, NO production, and iNOS expression induced by NaF plus 1,25(OH)(2)D-3; adding exogenous PGE(2) (0.1-10 ng/ml) to these indomethacin-blocked cultures dose-dependently restored NO production. These additional findings together with the observed slow onset (24-18 h) of NaF and 1,25(OH)(2)D-3 interaction strongly suggest that 1,25(OH)(2)D-3 acts as a cofactor with NaF primarily through interaction with an endogenous NaF-induced cyclo-oxygenase product(s), quite possibly PGE(2) itself. Such a mechanism for NaF and 125(OPI)(2)D-3 interaction would be strongly analogous to the interaction we have recently demonstrated between 1,25(OH)(2)D-3 and PGE(1) on the differentiation of HL-60 cells.

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  • 1,25-DIHYDROXYVITAMIN D-3 PROMOTES PROSTAGLANDIN E(1)-INDUCED DIFFERENTIATION OF HL-60 CELLS 査読

    T KAWASE, S OGATA, M ORIKASA, DM BURNS

    CALCIFIED TISSUE INTERNATIONAL   57 ( 5 )   359 - 366   1995年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER VERLAG  

    Human promyelocytic HL-60 cells can be induced by biochemical agents to differentiate in vitro towards divergent types of myelomonocytic cells. It has been reported that prostaglandin E(1) (PGE(1)) can induce granulocytic differentiation and that 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) can induce monocytic differentiation. We have now examined the effects of these compounds, both alone and in combination, on HL-60 cell differentiation. PGE(1) (1 mu g/ml) or 1,25(OH)(2)D-3 (10 nM) each inhibited cell proliferation over 48-96 hours of treatment, but combined treatment with both agents was necessary to produce a strong inhibition. The percentage of HL-60 cells that can reduce nitroblue tetrazolium (NBT) (a characteristic index of early monocytic or granulocytic differentiation) increased 13-fold within 72 hours of PGE(1) treatment, and 1,25(OH)(2)D-3 produced a fivefold stimulation. However, combined treatment (PGE(1) plus 1,25(OH)(2)D-3) produced a dramatic 35-fold increase. HL-60 cells did not produce significant levels of nitric oxide (NO) before 48 hours in culture, and treatment with PGE(1) or 1,25(OH)(2)D-3 did not significantly increase cellular NO elaboration over control levels. However, combined treatment produced a striking 12-fold increase over control levels. Similarly, combined treatment was necessary to obtain the maximal time-dependent stimulation of cellular lactate dehydrogenase (LDH) activity (a marker of granulocytic differentiation) as well as acid phosphatase (ACP) activity. During this same period of time, PGE(1), but not 1,25(OH)(2)D-3, markedly stimulated cellular elaboration of interleukin (IL)-1 alpha, IL-6, and tumor necrosis factor (TNF)-alpha, and 1,25(OH)(2)D-3 cotreatment strongly augmented these effects. Thus, combined treatment with 1,25(OH)(2)D-3 plus PGE(1) generally augmented the apparent conversion of these cells, producing synergistic (multiplicative) or additive effects. Furthermore, PGE(1) induced within 48 hours the more general phenotypic changes classically associated with the differentiation of these cells: increased expression of chloroacetate esterase (ChAE) (a granulocytic marker), decreases in the nuclear/cytoplasmic ratio (characteristic of development beyond the promyelocyte/myelocyte stage), and major alterations in morphology from floating spherical cells to loosely adherent, elliptical polygons. 1,25(OH)(2)D-3 had little effect itself on most of these parameters, but augmented the morphological changes induced by PGE, treatment. Within 48 hours, the ability of these cells to reduce the tetrazolium salt WST-1, a general measure of cellular metabolic activity, was increased by PGE(1), but not by 1,25(OH)(2)D-3; however, the combination of 1,25(OH)(2)D-3 and PGE(1) again produced the strongest stimulation. Similarly, only PGE(1) significantly reduced intracellular ATP levels, but combined treatments produced a more pronounced decrease. In summary, our findings suggest that PGE(1), not 1,25(OH)(2)D-3, is sufficient to promote rapid in vitro differentiation of HL-60 cells along the granulocytic pathway; however, the PGE(1)-induced conversion of these cells is markedly augmented by cotreatment with 1,25(OH)(2)D-3.
    In addition, these converted HL-60 cells preferentially utilize the glycolytic pathway, rather than the citric acid cycle, for production of ATP, a metabolic characteristic that resembles that described for mature granulocytes.

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  • PROTEIN-TYROSINE PHOSPHORYLATION-INDUCED BY EPIDERMAL GROWTH-FACTOR AND INSULIN-LIKE GROWTH-FACTOR-I IN A RAT CLONAL DENTAL PULP-CELL LINE 査読

    T KAWASE, M ORIKASA, S OGATA, DM BURNS

    ARCHIVES OF ORAL BIOLOGY   40 ( 10 )   921 - 929   1995年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PERGAMON-ELSEVIER SCIENCE LTD  

    Both epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) produce a dose-dependent stimulation in the rate of cell division in a rat clonal dental pulp-cell line (RDP 4-1). To elucidate the initial mitogen-induced cellular events that may mediate mitogenic action, the effects of EGF and IGF-I on cellular protein tyrosine phosphorylation were examined. In a dose-dependent manner, EGF (1-100 ng/ml) transiently stimulated tyrosine phosphorylation in four major proteins with apparent molecular weights of 220, 180, 140 and 120 kDa, and in five other more minor proteins (90, 80, 65, 55 and 44 kDa). IGF-I (1-100 ng/ml) dose-dependently stimulated the tyrosine phosphorylation of 160- and 140-kDa proteins, and had a smaller effect on the 80-, 65- and 44 kDa proteins. In contrast to the action of EGF, IGF-I-induced tyrosine phosphorylation was sustained for more than 60 min, particularly that of the 160-kDa phosphoprotein. From the results of specific immunoprecipitation/Western-blot analyses, the 180-kDa EGF-sensitive protein could be identified as the EGF receptor (EGF-R). Among the IGF-I-sensitive pulp cell proteins, the 160-kDa protein was identified as insulin-receptor substrate-1. Both mitogenic treatments stimulated the tyrosine phosphorylation of a weak, 44-kDa protein, which we have identified as the extracellular signal-regulated kinase-1. Despite the presence of phosphoproteins of the correct size, neither the IGF-I receptor (IGF-I-R) nor the phospholipase C gamma-isoform could be identified as tyrosine kinase substrates in either treatment. Pretreatment with the tyrosine kinase inhibitor genistein (20 mu g/ml) significantly inhibited EGF- and IGF-I-induced tyrosine phosphorylation in permeabilized RDP 4-1 cells, and the tyrosine phosphatase inhibitor orthovanadate (1 mM) significantly prolonged the duration of the mitogen-stimulated tyrosine phosphorylation in both intact or permeabilized cells. Phorbol 12-myristate 13-acetate (100 nM), which activates protein kinase C (PKC), inhibited the tyrosine phosphorylation induced by either growth factor. This action was blocked by pretreatment with staurosporine (200 nM, 15 min), a selective PKC inhibitor. However, neither removing external Ca2+ with EGTA (1 mM) nor inducing Ca2+ influx with A23187 ionophore (2 mu M) significantly altered EGF- or IGF-I-induced phosphorylation. These findings strongly suggest that authentic EGF-R and IGF-I-R on RDP 4-1 cells are coupled to complex, tyrosine kinase-mediated, intracellular signalling systems that are sensitive to a PKC-dependent mechanism. EGF- and IGF-I-induced tyrosine phosphorylation cascades may have important roles in vivo in the regulation of dental pulp-cell proliferation and ultimately may affect dentine formation.

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  • DIVERSE ACTIONS OF CALCITONIN-GENE-RELATED PEPTIDE ON INTRACELLULAR FREE CA2+ CONCENTRATIONS IN UMR-106 OSTEOBLASTIC CELLS 査読

    T KAWASE, GA HOWARD, BA ROOS, DM BURNS

    BONE   16 ( 4 )   S379 - S384   1995年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE PUBL CO INC  

    Calcitonin gene-related peptide (CGRP) was examined for its effects on intracellular free Ca2+ concentration ([Ca2+](i)) in UMR 106 osteoblast-like cells, Cells loaded with the Ca2+ dye FURA-2 dose-dependently responded to CGRP (1-100 nM) with transient two-fold increases in [Ca2+](i), An intracellular source for this Ca2+ transient was suggested by the failure of membrane depolarization with high extracellular K+ or acute depletion of extracellular Ca2+ ([Ca2+](e) with EGTA to attenuate this response, After cells were incubated for 45 min with 0.1 mM extracellular Ca2+ to deplete intracellular Ca2+ stores, CGRP produced a 25-30% decrease in [Ca2+](i) rather than a transient increase, This calcium decrease was mimicked by membrane depolarization or by pinacidil, a specific activator of adenosine triphosphate (ATP)-sensitive potassium (K-ATP channels, and blocked by glybenclamide, a specific blocker of K-ATP channels. Our data suggest that CGRP has diverse Ca2+ regulatory effects in UMR 106 cells, mobilizing Ca2+ from intracellular stores via classical signaling while possibly promoting cellular Ca2+ efflux or inhibiting uptake through voltage-dependent Ca2+ channels via K-ATP-mediated hyperpolarization.

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  • INDUCTION OF MACROPHAGIC AND GRANULOCYTIC DIFFERENTIATION OF MURINE BONE-MARROW PROGENITOR CELLS BY 1,25-DIHYDROXYVITAMIN-D(3) 査読

    M ORIKASA, T KAWASE, A SUZUKI

    CALCIFIED TISSUE INTERNATIONAL   53 ( 3 )   193 - 200   1993年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER VERLAG  

    1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) was recently shown to promote maturation of 5-fluorouracil (5FU)-treated bone marrow cells by up-regulating macrophage-colony stimulating factor (M-CSF) receptors in the presence of interleukin 1alpha (IL-1alpha). In order to reveal how 1,25(OH)2D3 interacts with colony-stimulating factors and regulates the differentiation of bone marrow progenitor cell populations, in the present study, natural bone marrow cells were isolated from untreated mice and used in alpha-minimum essential medium supplemented with 20% heat-inactivated horse serum without added appropriate cytokines. Under the conditions, cells spontaneously differentiated gradually with days of culture, as assessed by expression of macrophage differentiation antigens such as Mac-1 (CD11b) and F4/80. Both M-CSF and granulocyte macrophage-colony stimulating factor (GM-CSF) induced only Mac-1 antigen expression. Simultaneous treatment with M-CSF and 1,25(OH)2D3 enhanced the M-CSF's effect on expression of both antigens, although 1,25(OH)2D3 per se has no effect on the expression for up to 11 days. In addition, successive treatment with 1,25(OH)2D3 and M-CSF or GM-CSF dramatically enhanced expression of both antigens or Mac-1 antigen, respectively. Similarly, both simultaneous and successive treatment with 1,25(OH)2D3 and M-CSF significantly enhanced phagocytic activity and H2O2 production, whereas successive treatment with 1,25(OH)2D3 and GMCSF significantly enhanced only phagocytic activity. Enzyme-histochemical study demonstrated that cells treated simultaneously or successively with 1,25(OH)2D3 and M-CSF were strongly positive for nonspecific esterase (NSE), a macrophage-specific marker, and that simultaneous or successive treatment with 1,25(OH)2D3 and GM-CSF yielded cells strongly positive for NSE or for chloroacetate esterase (ChAE), a granulocyte-specific marker, respectively. These findings suggest that 1,25(OH)2D3 primes bone marrow progenitor cell populations not only to M-CSF but also to GM-CSF and thereby accelerates the CSFs-dependent differentiation of the cells to the macrophage or granulocyte.

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  • EFFECT OF BRADYKININ ON INTRACELLULAR SIGNALING SYSTEMS IN A RAT CLONAL DENTAL PULP-CELL LINE 査読

    T KAWASE, M ORIKASA, A SUZUKI

    ARCHIVES OF ORAL BIOLOGY   38 ( 1 )   43 - 48   1993年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PERGAMON-ELSEVIER SCIENCE LTD  

    The cloned pulp-cell line RDP4-1 increases cAMP production, hydrolyses phosphoinositide (PI) and mobilizes calcium in response to prostaglandin E2. (PGE2) and PGF2-alpha. The effect of bradykinin (BK) on intracellular signalling systems and DNA synthesis was studied in these cells. BK (10 muM) transiently increased cytoplasmic free calcium ([Ca2+]i) both in the presence and absence of external calcium. After stimulation with BK (10 muM), cells did not respond significantly to PGE2 (0.5 mug/ml). Pretreatment with indomethacin (30 muM) inhibited the [Ca2+]i increment by BK (10 muM), but not by the subsequent addition of PGE2 (0.5 mug/ml). Also, pretreatment with PGE2 (0.5 mug/ml) blocked the action of BK (10 muM). BK (0.1-100 muM) stimulated PI hydrolysis and cAMP production in a dose-dependent manner. Both the PI and the cAMP responses were inhibited by indomethacin (30 muM), as was the calcium response. BK (0.01-10 muM) also stimulated release of arachidonic acid and its metabolites dose-dependently. However, prolonged exposure to BK in serum-deficient medium did not exert any effect on DNA synthesis. RDP 4-1 cells, therefore, appear to respond to BK with increased cAMP production, PI hydrolysis and calcium mobilization. The inhibition of these effects of BK by indomethacin raises the possibility that cyclo-oxygenase product(s), especially PGE2 or PGE2-like compounds, may be responsible for evoking these effects. These results indicate that BK may stimulate or modulate cell metabolism in the dental pulp.

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  • PHORBOL ESTER-LIKE ACTION OF STAUROSPORINE ON THE CAMP RESPONSE TO PROSTAGLANDIN-E2 IN 2 MACROPHAGE-LIKE CELL-LINES AT DISTINCT DIFFERENTIATION STAGES 査読

    T KAWASE, M ORIKASA, A SUZUKI

    CELLULAR SIGNALLING   4 ( 5 )   479 - 485   1992年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PERGAMON-ELSEVIER SCIENCE LTD  

    The purpose of this study is to investigate the involvement of protein kinase C (PKC) in prostaglandin E2 (PGE2)-stimulated cAMP production of two macrophage-like cell lines (G3 and XC). XC cells are thought to be placed at a more differentiated stage than G3 cells [Orikasa et al. (1991) Cell Immunol. 132, 350-365]. In RPMI 1640 containing 10% (v/v) heat-inactivated foetal calf serum (FCS), in which the cAMP response of both cells to PGE, increased with duration of culture, XC cells showed greater response than G3 cells at 2 days of culture. In alpha-minimum essential medium (alpha-MEM) containing 20% (v/v) heat-inactivated horse serum (HS), the cAMP response of both cells was apparently greater than that in RPMI 1640 containing 10% (v/v) FCS. These cells increased cAMP production also in response to PGE, and PGF2alpha, and the order of potency in increase was PGE1 &gt; PGE2 much greater than PGF2alpha. Interestingly, a short-term (20 min) treatment with phorbol 12-myristate 13-acetate (PMA), a direct activator of PKC or staurosporine, a relatively specific inhibitor of PKC, augmented the PGE2-stimulated cAMP production in these cells cultured in alpha-MEM containing 20% (v/v) HS. However, a long-term (24 h) treatment with these compounds did not alter the cAMP response. In G3 cells, PMA appeared more potent than staurosporine in terms of augmentation, whereas in XC cells, the former appeared less potent than the latter. Although the mechanism of this augmentation seems complex and is still unclear, our findings suggest that PKC modulates the regulation of the PGE2-cAMP signalling system, and that the macrophage differentiation may decrease the involvement of PMA-sensitive PKC, but not that of staurosporine-sensitive PKC, in this cAMP system.

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  • EFFECT OF PHORBOL-MYRISTATE ACETATE ON RELEASE OF ARACHIDONIC-ACID AND ITS METABOLITES IN THE OSTEOBLASTIC MOB 3-4 CELL-LINE AND ITS SUBCLONE, MOB 3-4-F2 査読

    T KAWASE, M ORIKASA, A SUZUKI

    CELLULAR SIGNALLING   4 ( 1 )   51 - &   1992年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PERGAMON-ELSEVIER SCIENCE LTD  

    We examined the effect of phorbol 12-myristate 13-acetate (PMA) on release of arachidonic acid (AA) and its metabolites in osteoblastic cells in an attempt to study mechanism of the regulation of phospholipase A2 (PLA2) activity. In the MOB 3-4-F2 cell line, a subclone of the clonal osteoblastic MOB 3-4 cell line, PMA (0.1-100 nM) changed its appearance and increased AA release in a dose- and time-dependent manner, whereas 4-alpha-phorbol 12, 13-didecanoate (4-alpha-PDD) did not show a significant affect on the release. The addition of 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, greater-than-or-equal-to 1.5 mM), a Ca2+ chelator, almost completely inhibited the PMA-induced AA release without affecting the intrinsic AA release. Preincubation with staurosporine (5-20 nM), an inhibitor of protein kinase C (PKC), partially (approximately 60%) blocked the AA release. However, 30-min preincubation with H-7 (50-200-mu-M), an inhibitor of PKC, failed to block the AA release. PMA, thus, appeared to stimulate AA release partially by a staurosporine-sensitive mechanism, probably an activation of PKC, in an external Ca2+-dependent manner. On the other hand, MOB 3-4 cells responded to PMA with an increased AA release but not with a drastic change of its shape. Both staurosporine and BAPTA exerted similar inhibitory effects. Prolonged exposure (48 h) to PMA (0.1-10 nM) enhanced DNA synthesis of MOB 3-4-F2 cells, but not MOB 3-4 cells. Taken together with Ca2+-dependence of PLA2, these findings suggest that the PMA-induced release of AA and its metabolites in both MOB 3-4 and MOB 3-4-F2 cells is due to activation of PLA2 by both a staurosporine-sensitive mechanism, probably an activation of PKC, and a staurosporine-insensitive mechanism. In addition, the comparison between the MOB 3-4 cell line and its subclone, MOB 3-4-F2, may provide a useful system for studying the physiological or pathological role of PKC in bone-derived cells.

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  • ALUMINOFLUORIDE-STIMULATED AND EPIDERMAL GROWTH FACTOR-STIMULATED DNA-SYNTHESIS IN MOB 3-4-F2 CELLS 査読

    T KAWASE, M ORIKASA, A SUZUKI

    PHARMACOLOGY & TOXICOLOGY   69 ( 5 )   330 - 337   1991年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:MUNKSGAARD INT PUBL LTD  

    In attempt to study the mechanism of F--induced, osteoblast-mediated bone formation, we tried to show the characteristics of Al-F complex-induced mitogenesis in osteoblastic cells. The MOB 3-4-F2 cell line, an osteoblast-like cell line derived from neonatal mouse calvaria, responded to F- (1-2 mM) combined with Al3+ and epidermal growth factor (EGF, 0.01-100 ng/ml) with increased DNA synthesis. Of the several types of Al-F complexes, AlF4- is thought to act as a mitogenic factor. On the other hand, NaF at high concentrations (&gt; 2 mM) markedly decreased cell viability. The AlF4--stimulated DNA synthesis at least with a delay of 48 hr, while EGF stimulated DNA synthesis within a few hours (4-6 hr). Both 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and staurosporine, inhibitors of protein kinase C (PKC), further enhanced DNA synthesis in AlF4--treated cells, whereas 12-O-tetradecanoyl-13-acetate (TPA), an activator of PKC, decreased the DNA synthesis. In EGF-treated cells, staurosporine and TPA, but not H-7, decreased DNA synthesis. In addition, indomethacin, an inhibitor of cyclooxygenase, partly inhibited the EGF-induced mitogenesis, which, however, was restored by addition of PGE2. AlF4-, as well as EGF, stimulated the release of arachidonic acid and its metabolites. Indomethacin failed to inhibit the AlF4--induced mitogenesis. Thus, the mitogenic response of MOB 3-4-F2 cells to F- in the presence of Al3+ had the following characteristics: (1) it was effective over a narrow range, (2) it had a slow onset, (3) included a PKC-sensitive mechanism and (4) a PG(E2)-independent mechanism. In contrast, a wide range of EGF concentrations rapidly stimulated DNA synthesis by a PKC-sensitive, PG(E2)-dependent mechanism in these cells.

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  • PHORBOL ESTER (TPA) REDUCES PROSTAGLANDIN-E2-STIMULATED CAMP PRODUCTION BY DESENSITIZATION OF PROSTAGLANDIN-E2 RECEPTORS IN A CLONAL OSTEOBLAST-LIKE CELL-LINE, MOB 3-4 査読

    T KAWASE, M ORIKASA, A SUZUKI

    CALCIFIED TISSUE INTERNATIONAL   48 ( 3 )   167 - 175   1991年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER VERLAG  

    A clonal osteoblast-like cell line, MOB 3-4, increased cAMP production in response to prostaglandin E2 (PGE2) (5-500 ng/ml). The purpose of this study was to show the effects of tumorpromoting phorbol ester (e.g., 12-O-tetradecanoylphorbol 13-acetate, TPA) on basal and PGE2-stimulated cAMP production and the affinity of PGE2 receptors in the cells. Pretreatment with TPA (1 nM-10-mu-M) for 30 minutes increased basal cAMP production, whereas it markedly reduced the PGE2-stimulated cAMP production in the presence of 0.1 mM isobuthylmethyl xanthine. Both the TPA increase and reduction were dose- and time-dependent. However, TPA exerted no effect on forskolinor cholera toxin-stimulated cAMP production. Copretreatment with TPA and H-7, an inhibitor of protein kinase C (PKC), prevented the TPA-induced increase in basal cAMP production, whereas it did not prevent the reduction of the PGE2-stimulated cAMP production. On the other hand, TPA (0.1-10 mu-M) decreased H-3-PGE2 binding in a dose- and time-dependent manner. Scatchard analysis revealed that TPA decreased the apparent affinity of PGE2 receptors without effect on their apparent number. In addition, 1-oleoyl-2-acetylglycerol (12.6 mu-M), a synthetic diacylglycerol analog, did not mimic the TPA action on H-3-PGE2 binding. Thus, TPA at relatively high concentrations appeared to increase basal cAMP production by a PKC-mediated mechanism, and it appeared to directly act on and thereby reduce the PGE2-stimulated cAMP production in the clonal osteoblast-like MOB 3-4 cell line.

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  • EFFECTS OF PROSTAGLANDIN-E2 AND PROSTAGLANDIN-F2-ALPHA ON CYTOPLASMIC PH IN A CLONAL OSTEOBLAST-LIKE CELL-LINE, MOB 3-4 査読

    T KAWASE, M ORIKASA, A SUZUKI

    JOURNAL OF CELLULAR PHYSIOLOGY   146 ( 1 )   141 - 147   1991年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-LISS  

    Prostaglandin E2 (PGE2, 5 ng/ml to 5-mu-g/ml) induced a dose-dependent increase in cAMP accumulation, inositol phosphates (IPs) accumulation, and cytoplasmic free Ca2+ ([Ca2+]i) in a clonal osteoblast-like cell line, MOB 3-4. In contrast, prostaglandin F2-alpha (PGF2-alpha, 5 ng/ml to 5-mu-g/ml) stimulated increases in IPs accumulation and [Ca2+]i without stimulating an increase in cAMP accumulation. Both PGE2 (&gt; 0.5-mu-g/ml) and PGF2-alpha (greater-than-or-equal-to 0.5-mu-g/ml) increased cytoplasmic pH (pHi) from approximately 7.15 to 7.35 in BCECF-loaded cells. A tumor promotor, phorbol 12-myristate 13-acetate (PMA, 0.1-100 nM) also increased pHi without effect on phosphoinositide hydrolysis. Both PGE2-(5-mu-g/ml) and PMA- (100 nM) induced cytoplasmic alkalinization was inhibited by removal of extracellular Na+, or by pretreatment of the cells with amiloride (0.5 mM), an inhibitor of Na+/H+ exchange, or H-7 (100-mu-M), a nonspecific inhibitor of protein kinase C. Thus, MOB 3-4 cells appeared to possess PGE2 receptors and PGF2-alpha receptors: the former are coupled to adenylate cyclase and phospholipase C, and the latter are predominantly coupled to phospholipase C. Also the cells appeared to possess an amiloride-sensitive Na+/H+ exchange activity, which increases pHi in response to PGE2 and PGF2-alpha, as well as to PMA. Long-term (48 hr) exposure of the cells to PGE2 at a high concentration (5-mu-g/ml), but not to PGF2-alpha and PMA, decreased DNA synthesis in the serum-deficient medium. Thus, cytoplasmic alkalinization appeared insufficient for cell replication. At least in MOB 3-4 cells, the inhibitory effect of PGE2 on DNA synthesis may be due to the cAMP messenger system.

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  • STARVATION OF A CLONAL OSTEOBLAST-LIKE CELL-LINE, MOB 3-4-F2, DOWN-REGULATES PROSTAGLANDIN-E2 RECEPTORS BUT INCREASES CAMP RESPONSE TO PROSTAGLANDIN-E2 査読

    T KAWASE, M ORIKASA, A SUZUKI

    CELLULAR SIGNALLING   3 ( 2 )   153 - 158   1991年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PERGAMON-ELSEVIER SCIENCE LTD  

    Prostaglandin E2 (PGE2) stimulated cAMP production in the MOB 3-4-F2 cell line, a subclone of the osteoblast-like MOB 3-4 cell line. After being cultured in alpha-minimum essential medium supplemented with 10% heat-inactivated foetal calf serum (HIFCS), cells responded to PGE2 (greater-than-or-equal-to 50 ng/ml) with a small, but significant, increase in cAMP production. This response did not vary with duration of culture. In 2% HIFCS-containing medium, despite their lower basal cAMP level, cells responded to PGE2 (greater-than-or-equal-to 5 ng/ml) with strikingly increased cAMP production. In addition, prolonged culture in this serum-deficient medium enhanced this response. On the other hand, culture of cells in 2% HIFCS-containing medium decreased the apparent number of PGE2 receptors, which was also enhanced by prolonged culture, without effect on their apparent affinity. Their number in 10% HIFCS-containing medium, more than that in 2% HIFCS-containing medium, was almost constant, independent of the culture period. Starvation of MOB 3-4-F2 cells in serum-deficient medium, therefore, appeared to down-regulate PGE2 receptors but increase the cAMP response to PGE2. Moreover, prolonged starvation of cells appeared to facilitate these phenomena. Our findings suggest that cAMP response to PGE2 does not always reflect the number of available PGE2 receptors in the cells.

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  • A CLONAL PROSTAGLANDIN-RESPONSIVE CELL-LINE (RDP-4-1) DERIVED FROM RAT DENTAL-PULP 査読

    T KAWASE, M ORIKASA, A SUZUKI

    BONE AND MINERAL   11 ( 2 )   163 - 175   1990年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCI IRELAND LTD  

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  • Initial responses of a clonal osteoblast-like cell line, MOB 3-4, to phosphatidic acid in vitro 査読

    Tomoyuki Kawase, Akitoshi Suzuki

    Bone and Mineral   10 ( 1 )   61 - 70   1990年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    A number of studies have shown membrane phospholipid metabolism to have an important role in biological mineralization. We considered the effects of exogenously applied phosphatidic acid (PA), a minor component of membrane phospholipids, on an osteoblast-like cell line, MOB 3-4. Exogenous PA (10-40 μg/ml) raised the level of cytoplasmic free Ca2+ ([Ca2+]i), independent of the level of extracellular Ca2+, in a dose-dependent fashion, and this Ca2+ response to PA gradually fell on serial application of PA. In a dose-dependent manner, exogenous PA also increased inositol 1,4,5-trisphosphate (IP3) accumulation and cytoplasmic pH, but decreased basal cAMP level. This cytoplasmic alkalinization was inhibited by pretreatment with nonspecific protein kinase C (PKC) inhibitors, such as sphingosine or H-7. A long-term incubation with PA increased alkaline phosphatase (ALP) activity and cell proliferation. Exogenous PA thus appeared to increase IP3 accumulation by activating phospholipase C, raise [Ca2+] by releasing Ca2+ from intracellular stores, induce cytoplasmic alkalinization via a PKC-dependent mechanism, and simultaneously decrease basal cAMP level. We suggest that these initial responses may be responsible for the increase in ALP activity and the proliferation of PA-treated MOB 3-4 cells. © 1990.

    DOI: 10.1016/0169-6009(90)90049-L

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  • Fluoride‐Induced Cytoplasmic Acidification: Possible Role of Protein Kinase C in BCECF‐Loaded L929 Cells 査読

    Tomoyuki Kawase, Akitoshi Suzuki

    Pharmacology &amp; Toxicology   64 ( 5 )   426 - 428   1989年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Abstract: The effect of fluoride on cytoplasmic pH (pHi) in L929 cells was investigated by using a fluorescent pH indicator, BCECF. Fluoride decreased pHi in a dose‐dependent manner. This cytoplasmic acidification was composed of two phases: 1) a rapid decrease in pHi occurring within seconds, and 2) a slow decrease in pHi occurring 1–2 min. after stimulation with fluoride. The phase one decrease in pHi at external pH (pHe) 7.7 was more rapid than that at pHe 6.8, whereas the phase two decrease at pHe 7.7 was slower than that at pHe 6.8. In addition, both in the protein kinase C‐inhibited and depleted cells, the fluoride‐acidified pHi gradually returned to the resting pHi level in phase two, though the initial cytoplasmic acidification (i.e. phase one) was not affected. These results suggest that the fluoride‐induced cytoplasmic acidification is dependent upon pHe and is sustained by the protein kinase C‐dependent Na+/H+ exchange. 1989 Nordic Pharmacological Society

    DOI: 10.1111/j.1600-0773.1989.tb00680.x

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  • Studies on the transmembrane migration of fluoride and its effects on proliferation of L-929 fibroblasts (L cells) in vitro 査読

    T. Kawase, A. Suzuki

    Archives of Oral Biology   34 ( 2 )   103 - 107   1989年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    A decrease in the pH of the medium facilitated fluoride (F-) influx but depressed its efflux, which is consistent with the hypothesis that simple diffusion of hydrogen fluoride (HF) contributes to F- migration across the cell membrane. Long-term exposure to F- (&gt
    1 mM) induced F- accumulation and, as a result, inhibited cell proliferation. In contrast, short-term exposure to F- (&gt
    1 mM), followed by careful washing, did not inhibit cell proliferation but rather stimulated it. Moreover, this stimulatory effect was enhanced by 1 μM Al3+ and was inhibited by 1 μM H-7, a specific inhibitor of protein kinase C. Thus F- may stimulate cell proliferation by activating protein kinase C through GTP-binding protein. The stimulatory effect of F- on cell proliferation, which is usually hidden by its inhibitory effect, can be observed by preventing the accumulation of F- in the cytoplasm. © 1989.

    DOI: 10.1016/0003-9969(89)90133-7

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  • Phosphatidic acid-induced calcium mobilization in osteoblasts 査読

    Tomoyuki Kawase, Akitoshi Suzuki

    Journal of Biochemistry   103 ( 4 )   581 - 582   1988年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Oxford University Press  

    Phosphatidic acid (PA) evoked a transient increase in the cytosolic free Ca2+ concentration ([Ca2+]1) in osteoblasts isolated from neonatal mouse calvaria. This increase was observed in both low (below 150 μM) and high (1.26 mM) Ca2+-containing medium. In contrast, other phospholipids, such as phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol, failed to increase [Ca2+]1 in osteoblasts. In high Ca2+-containing medium, A23187 also increased [Ca2+]1 in the cells, but the mode of the change was different from that in the case of PA. These results suggest that PA may induce Ca2+-mediated cellular responses through Ca2+ release from intracellular stores in osteoblasts. © 1988 BY The Journal of Biochemistry.

    DOI: 10.1093/oxfordjournals.jbchem.a122309

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  • NaF-induced Ca2+ mobilization is dependent upon the culture density in a parathyroid hormone-responsive osteoblast-like cell line 査読

    Tomoyuki Kawase, Ichijiro Ishikawa, Akitoshi Suzuki

    Life Sciences   43 ( 26 )   2241 - 2247   1988年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The effect of NaF on cytosolic free Ca2+ concentrations ([Ca2+]i) was examined in a clonal osteoblast-like cell line (MOB 3-4) loaded with Fura 2. MOB 3-4 cells in a sparse culture, which exhibited neither alkaline phosphatase (ALP) activity nor the response to parathyroid hormone (PTH), responded to NaF (0.1-10 mM) to increase [Ca2+]i transiently. In contrast, the cells in a dense culture, which exhibited both ALP activity and the response to PTH, responded to NaF (above 4 mM) to increase [Ca2+]i slowly. [Ca2+]i in osteoblasts in primary culture slowly increased in response to both NaF (above 4 mM) and PTH (3 U/ml). Thus, the sensitivity and the response of MOB 3-4 cells to NaF and PTH varied with the culture density, and high culture density matured the cells like osteoblasts in primary culture. These NaF-induced Ca2+ mobilizations were not dependent upon external Ca2+ and were enhanced by Al3+ (1 μM), whereas the PTH-induced Ca2+ mobilizations were due to Ca2+ influx. These results suggest that the maturation of MOB 3-4 cells, dependent upon the culture density, modulates intracellular signal transduction pathways and thereby alters the NaF-induced Ca2+ mobilization, and that the culture density must be taken into consideration in studying Ca2+ mobilization in such an osteoblast-like cells line as MOB 3-4 cell line. © 1988.

    DOI: 10.1016/0024-3205(88)90417-1

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  • 骨膜シートの骨再生機序における骨髄由来細胞の役割

    上松晃也, 牛木隆志, 石黒創, 永田昌毅, 川瀬知之, 中田光

    日本再生医療学会総会(Web)   18th   ROMBUNNO.O‐11‐3 (WEB ONLY)   2019年

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  • ヒト下顎骨分散骨膜細胞のゼノフリー骨分化誘導試験

    伊藤祐子, 米山奈保, 牛木隆志, 川瀬知之, 中田光, 對比地久義, 伊藤彰, 中村孝人

    日本再生医療学会総会(Web)   17th   ROMBUNNO.P‐02‐076 (WEB ONLY)   2018年

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  • 骨膜シートは移植局所に骨髄由来造血細胞を動員する

    上松晃也, 石黒創, 牛木隆志, 永田昌毅, 星名秀行, 今井秀明, 中田光, 川瀬知之

    日本再生医療学会総会(Web)   17th   ROMBUNNO.O‐39‐6 (WEB ONLY)   2018年

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  • ヒトPlatelet‐rich fibrin抽出物を用いた異種血清を用いない間葉系幹細胞培養系の開発

    伊藤祐子, 米山奈保, 牛木隆志, 川瀬知之, 中田光, 對比地久義, 伊藤彰, 中村孝人

    再生医療   16   417   2017年2月

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  • 培養骨膜シートおよび培養骨膜細胞による歯周組織・顎骨の再生療法と今後の課題

    奥田一博, 川瀬知之, 永田昌毅, 高木律男, 中田光, 吉江弘正

    再生医療   16   185   2017年2月

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  • デジタルホログラフィック顕微鏡による非接触的細胞品質評価の試み

    川瀬知之, 奥田一博, 永田昌毅, 土持眞, 吉江弘正, 中田光

    再生医療   16   271   2017年2月

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  • ヒトPlatelet‐rich fibrin抽出物はウシ胎児血清(FBS)の代替となり得るか?

    伊藤祐子, 川瀬知之, 牛木隆志, 中田光, 對比地久義, 伊藤彰, 中村孝人

    再生医療   15   330   2016年2月

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  • Platelet‐rich fibrin(PRF)とヒト培養骨膜シートの複合化による相乗的骨再生促進効果

    堀水慎, 久保田健彦, 川瀬知之, 奥田一博, 吉江弘正

    日本歯周病学会会誌(Web)   57   119   2015年8月

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  • 歯科手術用血小板成形スプーン「PRFスタンパー」血小板と構造を破壊することなく、容易にかつ迅速にPRFを成形するデバイス 招待

    川瀬知之, 奧田一博

    Dental Diamond   ( 7 )   162 - 165   2015年7月

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    記述言語:日本語   掲載種別:記事・総説・解説・論説等(商業誌、新聞、ウェブメディア)  

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  • 酸化ストレス刺激ヒト骨膜細胞モデルにおけるDNA修復履歴を指標とした品質管理

    川瀬知之, 神谷真菜, 羽山和秀, 永田昌毅, 奥田一博, 吉江弘正, 土持眞, 中田光

    再生医療   14   286   2015年2月

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  • 骨膜シート調製における無血清培養に関する基礎的研究

    渡辺真理, 藤本陽子, 牛木隆志, 川瀬知之, 奥田一博, 永田昌毅, 伊藤彰, 吉江弘正, 中田光

    再生医療   14   308   2015年2月

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  • 培養骨膜シート移植による顎骨再生医療

    永田昌毅, 川瀬知之, 中田光, 高木律男

    新潟医学会雑誌   128 ( 11 )   566 - 568   2014年11月

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  • The unique structure-function relationship found in osteogenic periosteal sheets

    T. Kawase, K. Okuda, M. Nagata, D. Burns, K. Nakata, H. Yoshie

    JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE   8   408 - 408   2014年6月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:WILEY-BLACKWELL  

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  • 細胞重層化したヒト培養骨膜シートと単層骨膜細胞シートの細胞接着様式の比較

    川瀬知之, 上松晃也, 永田昌毅, 奥田一博, 中田光, 吉江弘正

    再生医療   13   194   2014年1月

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  • Platelet‐rich fibrin(PRF)との複合化によるヒト培養骨膜シートの骨再生能向上

    堀水慎, 川瀬知之, 久保田健彦, 永田昌毅, 奥田一博, 冨田尊志, 両角俊哉, 吉江弘正

    再生医療   13   193   2014年1月

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  • コラーゲンスポンジと複合化した凍結乾燥PRPの有用性

    堀水慎, 川瀬知之, 中島悠, 奥田一博, 永田昌毅, 吉江弘正

    新潟歯学会雑誌   43 ( 2 )   154   2013年12月

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  • Platelet‐rich fi brin(PRF)との複合化によるヒト培養骨膜シート骨形成活性の亢進

    堀水慎, 川瀬知之, 久保田健彦, 永田昌毅, 奥田一博, 冨田尊志, 両角俊哉, 吉江弘正

    新潟歯学会雑誌   43 ( 2 )   150   2013年12月

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  • 培養自家骨膜シートを用いた歯槽骨再生療法の臨床試験

    小川信, 永田昌毅, 星名秀行, 山田一穂, 上松晃也, 川瀬知之, 吉江弘政, 魚島勝美, 高木律男

    新潟歯学会雑誌   43 ( 2 )   162 - 163   2013年12月

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  • 自家培養骨膜シートの移植による歯周再生・顎堤形成治療 より高活性な移植材料をめざして

    川瀬知之, 奥田一博, 永田昌毅, 吉江弘正, 中田光

    今日の移植   26 ( 5 )   425 - 433   2013年10月

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  • Platelet‐rich fibrin‐ヒト培養骨膜シート複合体移植による骨再生能の向上

    堀水慎, 久保田健彦, 川瀬知之, 奥田一博, 冨田尊志, 両角俊哉, 吉江弘正

    日本歯科保存学会学術大会プログラムおよび講演抄録集(Web)   139th   P132 (WEB ONLY)   2013年10月

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  • 自己培養骨膜シートを用いた歯槽骨再生研究 : データをどのように収集してどのように評価するか (シンポジウム 新潟大学の臨床研究を活性化するためにはどのような仕組みが必要か?)

    奥田 一博, 吉江 弘正, 川瀬 知之, 中田 光, Okuda Kazuhiro, Yoshie Hiromasa, Kawase Tomoyuki, Nakata Koh

    新潟医学会雑誌   127 ( 7 )   349 - 354   2013年7月

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    記述言語:日本語   出版者・発行元:新潟医学会  

    To regenerate periodontal tissue destroyed by chronic periodontitis, autologous cultured periosteal sheets in combination with platelet-rich plasma (PRP) and hydroxyapatite (HA) granules were used as a clinical application. The periosteum specimens were dissected from each patient, and were incubated for approximately six weeks until the sheets were formed. Prior to placement of the cultured periosteal sheets, osseous defects were thoroughly debrided and filled with HA granules in combination with PRP. Standardized clinical and radiographic measurements were determined during follow-up examinations at one, two and three years. Clinical attachment level (CAL) and radiographic infrabony defect depth (IBD) were determined as primary end-point. As secondary end-point, inflammatory indices including plaque index (PI), gingival index (GI) and bleeding on probing (BOP) were determined. Results were averaged for the clinical and radiographic parameters. Taking into account the paired nature of the changes from baseline to one year, and three years in each group, the Wilcoxon signed-rank test was performed. The Mann-Whitney U test was applied to compare clinical and radiographic outcomes between test and control groups at baseline and 1 year post surgery. The null hypothesis was rejected when the risk percentage was < 5% (p<0.05). One year results of this treatment have shown favorable clinical results, and these effects were stable and maintained after three years, when compared to baseline. The cultured periosteal sheets function as a living drug delivery system to favorably influence cellular functions and serves as seeds for ectopic bone formation near the implantation site through production of important growth factors related to periodontal regeneration.

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    その他リンク: http://hdl.handle.net/10191/36051

  • Platelet‐rich fibrin(PRF)との複合体によるヒト培養骨膜シートの骨形成活性の亢進

    堀水慎, 久保田健彦, 川瀬知之, 永田昌毅, 奥田一博, 冨田尊志, 両角俊哉, 吉江弘正

    日本歯周病学会学術大会プログラムおよび講演抄録集   56th   97   2013年4月

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  • BIOLOGICAL AND BIOMECHANICAL CHARACTERIZATION OF HIGHLY SELF-MULTILAYERED HUMAN PERIOSTEAL SHEETS AS AN OSTEOGENIC GRAFTING MATERIAL

    T. Kawase, K. Uematsu, M. Nagata, K. Okuda, D. M. Bums, H. Yoshie

    CYTOTHERAPY   15 ( 4 )   S45 - S46   2013年4月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:INFORMA HEALTHCARE  

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  • Platelet‐rich fibrin(PRF)とヒト培養骨膜シートの複合化による骨形成活性の亢進

    堀水慎, 川瀬知之, 久保田健彦, 永田昌毅, 奥田一博, 冨田尊志, 両角俊哉, 吉江弘正

    再生医療   12   284   2013年2月

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  • 幹細胞用培地(MesenPRO)は骨膜シート中のCD146<sup>+</sup>細胞の増加と骨形成ポテンシャルの向上に貢献する

    上松晃也, 川瀬知之, 永田昌毅, 奥田一博, 吉江弘正, 高木律男

    再生医療   12   182   2013年2月

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  • 幹細胞用培地で重層化が促進されたヒト培養骨膜シートの機械的特性と分化抑制との関連性

    川瀬知之, 堀水慎, 田中孝明, 永田昌毅, 奥田一博, 吉江弘正

    再生医療   12   182   2013年2月

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  • 歯槽骨再生療法に用いる培養骨膜シートの質的向上を目的とした培地の最適化

    上松晃也, 永田昌毅, 川瀬知之, 星名秀行, 小川信, 池田順行, 高木律男

    新潟歯学会雑誌   42 ( 2 )   143   2012年12月

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  • 培養骨膜シートを用いた歯周組織再生療法の長期予後

    奥田一博, 川瀬知之, 山宮かの子, 永田昌毅, 関根優, 白山早紀, 藤本陽子, 布施一郎, 中田光, 吉江弘正

    日本歯科医師会雑誌   65 ( 5 )   642   2012年8月

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  • Platelet‐rich fibrinとの複合化によるヒト培養骨膜シートの機能向上

    堀水慎, 久保田健彦, 川瀬知之, 奥田一博, 冨田尊志, 両角俊哉, 吉江弘正

    日本歯科保存学会学術大会プログラムおよび講演抄録集(Web)   136th   P23 (WEB ONLY)   2012年5月

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  • 培養骨膜シートを用いた歯周組織再生療法の5年予後

    奥田一博, 川瀬知之, 山宮かの子, 永田昌毅, 関根優, 白山早起, 藤本陽子, 布施一郎, 中田光, 吉江弘正

    再生医療   11   252   2012年5月

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  • 培養自家骨膜細胞シートを用いた歯槽骨再生臨床試験~骨形成と骨吸収の協調的な活性化~

    永田昌毅, 星名秀行, 上松晃也, 小川信, 川瀬知之, 奥田一博, 魚島勝美, 中田光, 吉江弘正, 高木律男

    再生医療   11   179   2012年5月

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  • 生体骨膜により近似した培養骨膜シートの作成を目指した幹細胞用培地でのアプローチ

    上松晃也, 川瀬知之, 永田昌毅, 奥田一博, 吉江弘正, 高木律男

    再生医療   11   179   2012年5月

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  • ポリ乳酸製多孔質膜を用いた骨膜シートの培養

    田中孝明, 川瀬知之, 西本崇之, 奥田一博, 永田昌毅, BURNS Douglas M, 吉江弘正

    日本膜学会年会講演要旨集   34th   76   2012年4月

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  • 患者までとどいている再生医療―培養骨膜シート移植を応用した歯周組織再生法― 招待

    奥田一博, 川瀬知之, 中田 光, 吉江弘正

    再生医療   11 ( 1 )   51 - 56   2012年

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    記述言語:日本語   掲載種別:速報,短報,研究ノート等(学術雑誌)  

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  • 幹細胞用培地は骨膜シートのポテンシャル向上に貢献するか?

    上松 晃也, 川瀬 知之, 永田 昌毅, 奥田 一博, 吉江 弘正, 高木 律男

    日本歯周病学会会誌   53 ( 秋季特別 )   117 - 117   2011年9月

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    記述言語:日本語   出版者・発行元:(NPO)日本歯周病学会  

    DOI: 10.14833/amjsp.2011f.0.93.0

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  • Analysis of Intestinal Fibrosis in Chronic Colitis in Mice Induced by Dextran Sulfate Sodium

    Xiaomei Sun, Masaki Nagata, Kawase Tomoyuki, Hana Yamaguchi, Yusuke Kawauchi, Xiafen Tang, Xu Ren, Mitsuhiro Anzai, Takayoshi Nishino, Kenichi Watanabe, Hiroyuki Yoneyama, Hitoshi Asakura

    GASTROENTEROLOGY   140 ( 5 )   S520 - S520   2011年5月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:W B SAUNDERS CO-ELSEVIER INC  

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  • 歯科インプラントを目的とした培養自家骨膜併用による歯槽骨再生

    永田 昌毅, 高木 律男, 川瀬 知之, 星名 秀行, 荒澤 恵, 山田 一穂, 嵐山 貴徳, 中田 光

    日本形成外科学会会誌   30 ( 6 )   326 - 326   2010年6月

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    記述言語:日本語   出版者・発行元:(一社)日本形成外科学会  

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  • 歯科インプラント適応を目的とした培養自家骨膜併用による歯槽骨再生

    永田 昌毅, 川瀬 知之, 奥田 一博, 中田 光, 吉江 弘正, 高木 律男

    日本歯周病学会会誌   51 ( 秋季特別 )   105 - 105   2009年9月

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    記述言語:日本語   出版者・発行元:(NPO)日本歯周病学会  

    DOI: 10.14833/amjsp.2009f.0.88.0

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  • Immature osteoblastic MG63 cells possess two calcitonin gene-related peptide receptor subtypes that respond differently to [Cys(Acm)(2,7)] calcitonin gene-related peptide and CGRP(8-37)

    T Kawase, K Okuda, DM Burns

    AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY   289 ( 4 )   C811 - C818   2005年10月

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    記述言語:英語   出版者・発行元:AMER PHYSIOLOGICAL SOC  

    Immature osteoblastic MG63 cells possess two calcitonin gene-related peptide receptor subtypes that respond differently to [Cys(Acm)(2,7)] calcitonin gene-related peptide and CGRP8-37. Am J Physiol Cell Physiol 289:C811-C818, 2005. First published June 15, 2005; doi:10.1152/ajpcell.00504.2004. Calcitonin gene-related peptide ( CGRP) is clearly an anabolic factor in skeletal tissue, but the distribution of CGRP receptor (CGRPR) subtypes in osteoblastic cells is poorly understood. We previously demonstrated that the CGRPR expressed in osteoblastic MG63 cells does not match exactly the known characteristics of the classic subtype 1 receptor (CGRPR(1)). The aim of the present study was to further characterize the MG63 CGRPR using a selective agonist of the putative CGRPR(2), [Cys( Acm) 2,7] CGRP, and a relatively specific antagonist of CGRPR(1), CGRP8-37. [Cys( Acm) 2,7] CGRP acted as a significant agonist only upon ERK dephosphorylation, whereas this analog effectively antagonized CGRP-induced cAMP production and phosphorylation of cAMP response element-binding protein ( CREB) and p38 MAPK. Although it had no agonistic action when used alone, CGRP8-37 potently blocked CGRP actions on cAMP, CREB, and p38 MAPK but had less of an effect on ERK. Schild plot analysis of the latter data revealed that the apparent pA(2) value for ERK is clearly distinguishable from those of the other three plots as judged using the 95% confidence intervals. Additional assays using 3-isobutyl-1-methylxanthine or the PKA inhibitor N-(2-[p-bromocinnamylamino] ethyl)5-isoquinolinesulfonamide hydrochloride (H-89) indicated that the cAMP-dependent pathway was predominantly responsible for CREB phosphorylation, partially involved in ERK dephosphorylation, and not involved in p38 MAPK phosphorylation. Considering previous data from Scatchard analysis of [I-125] CGRP binding in connection with these results, these findings suggest that MG63 cells possess two functionally distinct CGRPR subtypes that show almost identical affinity for CGRP but different sensitivity to CGRP analogs: one is best characterized as a variation of CGRPR(1), and the second may be a novel variant of CGRPR(2).

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  • In vitro evidence that the of platelet-rich plasma on biological effects periodontal ligament cells is not mediated solely by constituent transforming-growth factor-beta or platelet-derived growth factor

    T Kawase, K Okuda, Y Saito, H Yoshie

    JOURNAL OF PERIODONTOLOGY   76 ( 5 )   760 - 767   2005年5月

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    記述言語:英語   出版者・発行元:AMER ACAD PERIODONTOLOGY  

    Background: The biological actions of platelet-rich plasma (PRP) are thought to be mediated primarily by constituent transforming-growth factor-beta 1 (TGF-beta 1) and platelet-derived growth factor-AB (PDGF-AB). However, we previously demonstrated that type I Collagen expression in periodontal ligament (PDL) cells is acutely stimulated through fibrin clot formation produced by the fibrinogen within PRP, rather than by the known growth factors. To investigate the possible involvement of other unidentified components in PRP action, we have now compared the effects of PRP with those of known recombinant growth factors on cell proliferation, alkaline phosphatase (ALP) activity, and collagen synthesis in human PDL cell cultures.
    Methods: PRP was prepared by an established two-step centrifugation protocol using blood obtained from adult human volunteers. Cells cultured in serum-reduced medium on native or collagen-coated plates were treated with PRP, TGF-beta 1, or PDGF-AB. Cellular DNA synthesis was evaluated by bromodeoxyuridine incorporation. ALP activity was assessed using p-nitrophenyl phosphate with formalin-fixed cells, and cellular DNA content was subsequently quantified using bis-benzimide. Collagen synthesis was evaluated using a specific dye-based assay kit.
    Results: 1) As did both TGF-beta 1 and PDGF-AB, PRP stimulated cell proliferation. 2) However, only the initial mitogenic action of PRP was attenuated in collagen-coated plates. 3) PRP, but neither growth factor, immediately induced fibrin clot formation and subsequently stimulated cellular adhesion and Collagen synthesis. 4) These effects were significantly augmented on collagen-coated plates. 5) PRP enhanced ALP activity, but neither TGF-beta 1 nor PDGF-AB replicated this effect.
    Conclusions: When evaluated versus the concentrations of growth factors known to be contained by our PRP preparations, these data support the concept that PRP-constituent TGF-beta 1 acts as a significant growth factor on PDL cells. However, our findings also strongly suggest that the PRP-induced increase in ALP activity is mediated by an as-yet-unidentified component(s). In conjunction with previously demonstrated fibrinogen-mediated actions, our data provide evidence that PRP produces a number of potent effects on PDL cells that does not solely reflect simple combination of its major known growth factors.

    DOI: 10.1902/jop.2005.76.5.760

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  • 扁平上皮癌細胞とヒト歯肉由来不死化ケラチノサイトの分化, 増殖に及ぼす副甲状腺ホルモン関連蛋白(PTHrP)の影響

    原田 美樹子, 土持 眞, 川瀬 知之

    日本口腔科学会雜誌   54 ( 1 )   12 - 21   2005年1月

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    記述言語:日本語   出版者・発行元:日本口腔科学会  

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    その他リンク: http://search.jamas.or.jp/link/ui/2005188797

  • Calcitonin gene-related peptide elevates calcium and polarizes membrane potential in MG-63 cells by both cAMP-independent and -dependent mechanisms

    DM Burns, L Stehno-Bittel, T Kawase

    AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY   287 ( 2 )   C457 - C467   2004年8月

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    記述言語:英語   出版者・発行元:AMER PHYSIOLOGICAL SOC  

    Published data suggest that the neuropeptide calcitonin gene-related peptide ( CGRP) can stimulate osteoblastic bone formation; however, interest has focused on activation of cAMP-dependent signaling pathways in osteogenic cells without full consideration of the importance of cAMP-independent signaling. We have now examined the effects of CGRP on intracellular Ca2+ concentration ([Ca2+](int)) and membrane potential (E-m) in preosteoblastic human MG-63 cells by single-cell fluorescent confocal analysis using fluo 4-AM-fura red-AM and bis(1,3-dibarbituric acid)- trimethine oxanol [DiBAC(4)(3)] bis-oxonol assays. CGRP produced a two-stage change in [Ca2+] (int): a rapid transient peak and a secondary sustained increase. Both responses were dose dependent with an EC50 of similar to0.30 nM, and the maximal effect (initially similar to3-fold over basal levels) was observed at 20 nM. The initial phase was sensitive to inhibition of Ca2+ mobilization with thapsigargin, whereas the secondary phase was eliminated only by blocking transmembrane Ca2+ influx with verapamil or inhibiting cAMP-dependent signaling with the Rp isomer of adenosine 3', 5'-cyclic monophosphorothioate (Rp-cAMPS). These data suggest that CGRP initially stimulates Ca2+ discharge from intracellular stores by a cAMP-independent mechanism and subsequently stimulates Ca2+ influx through L-type voltage-dependent Ca2+ channels by a cAMP-dependent mechanism. In addition, CGRP dose-dependently polarized cellular Em, with maximal effect at 20 nM and an EC50 of 0.30 nM. This effect was attenuated with charybdotoxin ( - 20%) or glyburide ( glibenclamide; - 80%), suggesting that Em hyperpolarization is induced by both Ca2+-activated and ATP-sensitive K+ channels. Thus CGRP signals strongly by both cAMP-dependent and cAMP-independent signaling pathways in preosteoblastic human MG-63 cells.

    DOI: 10.1152/ajpcell.00274.2003

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  • Granulocyte colony-stimulating factor synergistically augments 1,25-dihydroxyvitamin D3-induced monocytic differentiation in murine bone marrow cell cultures

    T. Kawase, A. Oguro

    Hormone and Metabolic Research   36 ( 7 )   445 - 452   2004年7月

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    記述言語:英語  

    In a series of studies, we have reported that 1,25-dihydroxyvitamin D 3, a known stimulator of monocytic differentiation, primes bone marrow progenitor cells or promyelocytic HL-60 cells to the actions of several factors involved in both monocytic and granulocytic differentiation. In the present study, we have further examined the combinational effects of 1,25-dihydroxyvitamin D3 and the other inducer of granulopoiesis, granulocyte colony-stimulating factor, on non-fractionated native murine bone-marrow cell culture. Over 6 days of treatment, human granulocyte colony-stimulating factor sustained cell viability, increased the size of small rounded non-adherent cells, and induced granulocytic differentiation, while 1,25-dihydroxyvitamin D3 decreased cell viability, promoted the development of large adherent flattened cells, and upregulated some monocytic differentiation markers. Combining these two factors over 6 days synergistically upregulated phagocyte activity, membrane-bound interleukin-1α, NAD(P)H oxidase, monocytic Mac-1, and non-specific esterase. Similar effects were observed in successive treatment with granulocyte colony-stimulating factor followed by 1,25-dihydroxyvitamin D3, but successive treatment in reverse order was somewhat less effective. No combinational treatment upregulated granulocytic lactate dehydrogenase, Gr-1, or chloroacetate esterase to as great an extent as was obtained with granulocyte colony-stimulating factor alone, indicating that granulocytic differentiation is attenuated by addition of 1,25-dihydroxyvitamin D3. Therefore, in contrast to our previous data, the present findings suggest that granulocyte colony-stimulating factor synergistically augments 1,25-dihydroxyvitamin D3-induced monocytic differentiation in our murine bone-marrow cell cultures. Considering previously published data, we also suggest that these synergistic effects may be mainly due to the combination of two distinct effects such as the primary proliferative effects of granulocyte colony-stimulating factor on multipotent stem cells and the subsequent differentiative effects of 1,25-dihydroxyvitamin D3 on proliferating cells.

    DOI: 10.1055/s-2004-825728

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  • Immature human osteoblastic MG63 cells predominantly express a subtype I-like CGRP receptor that inactivates extracellular signal response kinase by a cAMP-dependent mechanism (vol 470, pg 125, 2003)

    T Kawase, K Okuda, DM Burns

    EUROPEAN JOURNAL OF PHARMACOLOGY   485 ( 1-3 )   345 - 345   2004年2月

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    記述言語:英語   出版者・発行元:ELSEVIER SCIENCE BV  

    DOI: 10.1016/j.ejphar.2003.11.054

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  • Immature human osteoblastic MG63 cells predominantly express a subtype 1-like CGRP receptor that inactivates extracellular signal response kinase by a cAMP-dependent mechanism

    Tomoyuki Kawase, Kazuhiro Okuda, Douglas M. Burns

    European Journal of Pharmacology   470 ( 3 )   125 - 137   2003年6月

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    記述言語:英語   出版者・発行元:Elsevier  

    Although accumulated data suggest that calcitonin gene-related peptide (CGRP) produces anabolic effects in skeletal tissue by directly acting on osteogenic cells, neither the distribution of CGRP receptor subtypes nor the associated cellular signaling pathways are well understood. In this study, we have pharmacologically and biochemically characterized CGRP-binding sites in immature human osteoblastic MG63 cells. In a [125I]CGRP whole-cell-binding assay, nonlinear regression curve-fitting analysis demonstrated a single binding site (KD=405±29 pM
    13,100±223 sites per cell). Immunocytochemical and Western blot analyses demonstrated that 48-, 52-, and 120-kDa forms of the calcitonin receptor-like receptor (CRLR) and a 15-kDa form of the receptor-activity-modifying protein-1 (RAMP-1) was expressed on the plasma membrane. CGRP strongly stimulated cellular cAMP production and this effect was antagonized not only by an antagonist of the subtype-1 CGRP (CGRP1) receptor, CGRP-(8-37), but by an agonist of the putative subtype-2 CGRP (CGRP2) receptor, [Cys(Acm)2,7]-CGRP, that also itself acted as a weak agonist. In contrast to published data, CGRP dose- and time-dependently dephosphorylated and inactivated extracellular signal response kinase (ERK). This action was blocked by CGRP-(8-37), by an inhibitor of cAMP-dependent protein kinase (H-89), or by an inhibitor of protein phosphatases (vanadate). Prolonged CGRP treatments significantly suppressed DNA synthesis at 27 h, but up-regulated type I collagen. Both these actions were blocked by CGRP-(8-37) and mimicked by a specific inhibitor of ERK (PD98059). In summary, our data suggest that the CGRP receptors in MG63 cells meet many, but not all, of the classical criteria used to define CGRP1 receptors. These receptors that functioned in a pharmacologically distinct manner could inhibit cell proliferation, and were substantially more sensitive to a CGRP2 receptor agonist than are typical CGRP1 receptors. These receptor proteins were not exactly matched with the known components of a CGRP1 receptor that have been reported. Therefore, it is possible that the CGRP receptors expressed in immature osteoblastic human MG63 cells represent a variation of the known CGRP1 receptor.

    DOI: 10.1016/S0014-2999(03)01763-1

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  • Platelet-rich plasma-derived fibrin clot formation stimulates collagen synthesis in periodontal ligament and osteoblastic cells in vitro

    T Kawase, K Okuda, LF Wolff, H Yoshie

    JOURNAL OF PERIODONTOLOGY   74 ( 6 )   858 - 864   2003年6月

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    記述言語:英語   出版者・発行元:AMER ACAD PERIODONTOLOGY  

    Background: Platelet-rich plasma (PRP) contains several growth factors, including platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta), at high levels. We have demonstrated the PRP functions like TGF-beta to modulate cell proliferation in a cell-type specific manner. In addition, PRP forms gel-like materials in several, but not all, cell cultures tested. This study was designed to investigate PRP's action on extracellular matrix production in periodontal ligament (PDL) and osteoblastic MG63 cell cultures.
    Methods: PRP was prepared from the plasma obtained from autologous blood of healthy volunteers and stored at -20degreesC until used. Cells treated with PRP (0.5% to 2%) were immunocytochemically stained for type I collagen and fibrin and the viscosity of the culture media was visually evaluated. Fibrinogen in PRP was detected by immunodot-blotting while endogenous thrombin expression in cells was detected by a modified enzyme-linked immunosorbent assay.
    Results: Gel-like material rapidly (&lt;30 minutes) formed in cultures of either PDL or osteoblastic MG63 cell cultures after addition of PRP (greater than or equal to0.5%). PRP changed cell shape and up-regulated type I collagen at 24 hours. Fibrinogen was detected in the PRP preparations and insoluble fibrin networks were found in the newly formed gel-like material. PRP's action on collagen synthesis was mimicked by purified fibrinogen and blocked by thrombin inhibitor. Thrombin was expressed both in PDL and MG63 cells.
    Conclusions: These findings demonstrated that the gel-like material formed in cell cultures of either PDL or MG63 cells is fibrin clot that is capable of up-regulating collagen synthesis in the extracellular matrix. Our data suggest the possibility that fibrinogen, converted to fibrin, in combination with growth factors present in PRP might effectively promote wound healing at sites of injury in periodontal tissue.

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  • Platelet-rich plasma-derived fibrin clot formation stimulates collagen synthesis in periodontal ligament and osteoblastic cells in vitro

    T Kawase, K Okuda, LF Wolff, H Yoshie

    JOURNAL OF PERIODONTOLOGY   74 ( 6 )   858 - 864   2003年6月

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    記述言語:英語   出版者・発行元:AMER ACAD PERIODONTOLOGY  

    Background: Platelet-rich plasma (PRP) contains several growth factors, including platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta), at high levels. We have demonstrated the PRP functions like TGF-beta to modulate cell proliferation in a cell-type specific manner. In addition, PRP forms gel-like materials in several, but not all, cell cultures tested. This study was designed to investigate PRP's action on extracellular matrix production in periodontal ligament (PDL) and osteoblastic MG63 cell cultures.
    Methods: PRP was prepared from the plasma obtained from autologous blood of healthy volunteers and stored at -20degreesC until used. Cells treated with PRP (0.5% to 2%) were immunocytochemically stained for type I collagen and fibrin and the viscosity of the culture media was visually evaluated. Fibrinogen in PRP was detected by immunodot-blotting while endogenous thrombin expression in cells was detected by a modified enzyme-linked immunosorbent assay.
    Results: Gel-like material rapidly (&lt;30 minutes) formed in cultures of either PDL or osteoblastic MG63 cell cultures after addition of PRP (greater than or equal to0.5%). PRP changed cell shape and up-regulated type I collagen at 24 hours. Fibrinogen was detected in the PRP preparations and insoluble fibrin networks were found in the newly formed gel-like material. PRP's action on collagen synthesis was mimicked by purified fibrinogen and blocked by thrombin inhibitor. Thrombin was expressed both in PDL and MG63 cells.
    Conclusions: These findings demonstrated that the gel-like material formed in cell cultures of either PDL or MG63 cells is fibrin clot that is capable of up-regulating collagen synthesis in the extracellular matrix. Our data suggest the possibility that fibrinogen, converted to fibrin, in combination with growth factors present in PRP might effectively promote wound healing at sites of injury in periodontal tissue.

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  • Calcitonin gene-related peptide stimulates cAMP production, increases intracellular calcium ion, and hyperpolarizes Em in human MG63 cells.

    T Kawase, L Stehno-Bittel, DM Burns

    JOURNAL OF BONE AND MINERAL RESEARCH   17   S283 - S283   2002年9月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:AMER SOC BONE & MINERAL RES  

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  • Enamel matrix derivative (EMDOGAIN((R))) rapidly stimulates phosphorylation of the MAP kinase family and nuclear accumulation of smad2 in both oral epithelial and fibroblastic human cells

    T Kawase, K Okuda, M Momose, Y Kato, H Yoshie, DM Burns

    JOURNAL OF PERIODONTAL RESEARCH   36 ( 6 )   367 - 376   2001年12月

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    記述言語:英語   出版者・発行元:MUNKSGAARD INT PUBL LTD  

    In our previous study, we demonstrated that porcine enamel matrix derivative (EMD) induces p21(WAF1/cip1) within 8 hours and subsequently arrests the cell cycle of human oral epithelial cells in G1 phase. In contrast, EMD markedly stimulates the proliferation of gingival fibroblasts without inducing p21(WAF1/cip1). To investigate the mechanism of how EMD produces these differential effects, we have focused on the initial response of these two cell types to EMD. In epithelial cell cultures, EMD stimulated cytoskeletal actin polymerization within 30 min and promoted cell adhesion within 3 hours, but EMD had no substantial effects on fibroblastic cell adhesion in our experimental system. EMD failed to stimulate either intracellular Ca2+ mobilization or CAMP production in either cell type. In both epithelial and fibroblastic cells, EMD (25-100 mug/ml) rapidly produced dose-dependent phosphorylation of the mitogen-activated protein kinase (MAPK) family: extracellular signal response kinase (ERK), p38-MAPK (p38-K), and c-Jun-terminal kinase/stress-activated protein kinase (JNK). However, neither inhibitors of MEK (ERK kinase) nor p38-K could block EMD's anti-proliferative action on epithelial cells. On the other hand, EMD rapidly stimulated translocation of smad2 into the nucleus in both cell types. Spurred by this finding, we assayed for TGF-beta1, a ligand for one receptor associated with smad2 activation, and detected significant levels in EMD preparations. The sutra of these pharmacological findings indicates that EMD contains at least one bioactive factor, which is most probably TGF-beta1 (or TGF-beta -like substances). In conjunction with the similarities in the differential growth-modulating actions between EMD and what is known for TGF-beta, we suggest that TGF-beta might act as the principal growth regulating agent of oral fibroblastic and epithelial cell types in EMD despite being present in only low levels.

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  • Effects of N-G-monomethyl-L-arginine on Ca2+ current and nitric-oxide synthase in rat ventricular myocytes

    S Matsumoto, T Takahashi, M Ikeda, T Nishikawa, S Yoshida, T Kawase

    JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS   294 ( 1 )   216 - 223   2000年7月

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    記述言語:英語   出版者・発行元:AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS  

    The effects of N-G-monomethyl-L-arginine (L-NMMA), a nitric-oxide synthase (NOS) inhibitor, on the L-type Ca2+ current (ICa) and NO effects on NOS were determined in rat ventricular myocytes. L-NMMA (10 and 100 mu M) had no significant effect on basal ICa, but in a cAMP-stimulated condition due to forskolin (1 mu M) or milrinone (10 mu M), a cGMP-inhibited cAMP-phosphodiesterase (PDE), L-NMMA (10 and 100 mu M) concentration dependently augmented ICa. The enhancing effects of L-NMMA (10 and 100 mu M) on ICa were not seen in the presence of either a nonselective inhibitor of PDE, 3-isobutyl-1-methylxanthine (20 mu M), resulting in a stimulated ICa condition or a cGMP-dependent protein kinase activator, 8-bromo-cGMP (200 mu M). 8-Bromo-cGMP (200 mu M) inhibited 100 mu M L-NMMA-induced ICa increase in the simultaneous application of forskolin (1 mu M). Acetylcholine (ACh; 1 and 3 mu M) inhibited 1 mu M forskolin-stimulated ICa in a concentration-dependent manner, but this inhibitory action of ACh was significantly attenuated by the additional application of L-NMMA (100 mu M). In the continuing presence of both L-NMMA (100 mu M) and forskolin (1 mu M), ACh (6 mu M) had no inhibitory effect on ICa. In another series of experiments with isolated ventricular myocytes, we obtained both the positive staining of NADPH-diaphorase activity and the expression of the endothelial isoform of NOS. These data suggest that the effect of L-NMMA on ICa in a cAMP-stimulated condition with or without cholinergic inhibition is due to inhibition (acute effects) of a cGMP-stimulated cAMP-PDE via inhibition of the endothelial isoform of NOS.

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  • Specific calcitonin gene-related peptide receptors stimulate the proliferation and function of osteoblasts through stimulation of MAPK and membrane potassium channel activity.

    T Kawase, DM Burns

    JOURNAL OF BONE AND MINERAL RESEARCH   14   S319 - S319   1999年9月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:AMER SOC BONE & MINERAL RES  

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  • Up-regulation of inducible nitric oxide (NO) synthase and NO production in HL-60 cells stimulated to differentiate by phorbol 12-myristate 13-acetate plus 1,25-dihydroxyvitamin D-3 is not obtained with dimethylsulfoxide plus 1,25-dihydroxyvitamin D-3

    T Kawase, M Orikasa, A Oguro, DM Burns

    CALCIFIED TISSUE INTERNATIONAL   63 ( 1 )   27 - 35   1998年7月

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    記述言語:英語   出版者・発行元:SPRINGER VERLAG  

    In previous studies we found that the calciotropic hormone 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3] augments the action of either prostaglandin E-1 (PGE(1)) or NaF to induce differentiation of human promyelocytic HL-60 cells, a process that features increased generation of nitric oxide (NO) via up-regulation of inducible nitric oxide synthase (iNOS). We have now examined the short-term interaction of 1,25(OH)(2)D-3 with phorbol 12-myristate 13-acetate (PMA) and dimethylsulfoxide (DMSO) in these cells. PMA (100 nM) alone generally up-regulated several classical indices of macrophagic differentiation and stimulated cellular production of interleukin (IL)-1 alpha, IL-6, tumor-necrosis factor (TNF)-alpha, PGE(2), and NO. Increased generation of NO primarily resulted from increased expression of cellular iNOS. When 1,25(OH)(2)D-3 (10 nM) was added to PMA treatments, most PMA-induced changes, particularly its effects to up-regulate iNOS-dependent NO production and change cell morphology, were multiplicatively augmented. In contrast, DMSO (1.3%) alone, an inducer of granulocytic differentiation, increased cytokine production, but failed to stimulate NO production or induce iNOS. In contrast to its striking interaction with PMA, 1,25(OH)(2)D-3 could not augment DMSO's differentiative effects. Changes in cellular cytokine production were eliminated as the driving force in HL-60 differentiation when specific neutralizing antibodies failed to produce any attenuation of iNOS up-regulation or of the shifts in cell morphology. However, indomethacin (30 mu M) blocked the synergistic interaction between 1,25(OH)(2)D-3 + PMA to shift cell morphology and stimulate NO production. Subsequently adding PGE(2) (1 ng/ml) to indomethacin-treated cells restored the ability of 1,25(OH)(2)D-3 + PMA to interactively increase cellular NO production, but failed to fully replicate the strong shift in cell morphology typical of PMA + 1,25(OH)(2)D-3 treatments. Our findings suggest that interaction between 1,25(OH)(2)D-3 and PMA to induce macrophagic differentiation increases iNOS-dependent NO production by a mechanism involving a cyclooxygenase product(s), possibly PGE(2).

    DOI: 10.1007/s002239900485

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  • Calcitonin gene-related peptide, amylin, or parathyroid hormone stimulate in vitro biomineralization

    DM Burns, T Kawase

    JOURNAL OF BONE AND MINERAL RESEARCH   12   F392 - F392   1997年8月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:BLACKWELL SCIENCE INC  

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  • Parathyroid hormone, prostaglandin E(2), calcitonin gene-related peptide, or forskolin acutely inhibit Ca-45(2+) uptake by osteoblasts

    T Kawase, GA Howard, BA Roos, DM Burns

    JOURNAL OF BONE AND MINERAL RESEARCH   11   T349 - T349   1996年8月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:BLACKWELL SCIENCE INC  

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  • Characteristics of NaF-induced differentiation of HL-60 cells

    T Kawase, A Oguro, M Orikasa, DM Burns

    JOURNAL OF BONE AND MINERAL RESEARCH   11 ( 11 )   M411 - M411   1996年8月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:BLACKWELL SCIENCE INC  

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  • Calcitonin gene-related peptide rapidly inhibits calcium uptake in osteoblastic cell lines via activation of adenosine triphosphate-sensitive potassium channels

    T Kawase, GA Howard, BA Roos, DM Burns

    ENDOCRINOLOGY   137 ( 3 )   984 - 990   1996年3月

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    記述言語:英語   出版者・発行元:ENDOCRINE SOC  

    In certain neurons, alternative RNA processing generates calcitonin gene-related peptide (CGRP) from the same gene that encodes the hormone calcitonin. As CGRP-containing nerve fibers are prominent in skeleton, we evaluated the effects of CGRP on osteoblasts. Because the vasodilatory effect of neural CGRP in smooth muscle probably involves inhibition of unstimulated Ca2+ uptake, we examined the acute effects of CGRP on this parameter in rat osteoblastic cells. CGRP inhibits Ca-45(2+) uptake in both UMR 106 osteosarcoma and RCOB-3 osteoblastic cells. This inhibition is rapid (0.5 min), occurs with an EC(50) of 1 nM, and cannot be demonstrated in the presence of 0.1 mM diltiazem, a blocker of voltage-dependent Ca2+ channels. Depolarization of bone cells with high extracellular potassium (K+) also blocks the effect of CGRP on Ca-45(2+) uptake, suggesting a central role for K+ channels in mediating this action. In agreement with this hypothesis, the effect of CGRP is blocked by 1 mu M glybenclamide, a specific inhibitor of ATP-sensitive potassium (K-ATP) channels, or by pretreatment of cells with 1 mM iodoacetic acid to deplete intracellular ATP, Blocking Ca2+-activated potassium channels with 1 mM tetraethylammonium does not prevent CGRP's effect. Pinacidil, a specific activator of K-ATP channels, mimics CGRP's effect. Both CGRP and pinacidil also produce a small significant stimulation of cellular Ca2+ efflux in UMR 106 cells. These data suggest that inhibition of diltiazem-sensitive Ca2+ channels occurs secondary to the hyperpolarization engendered by CGRP activation of K-ATP channels in osteoblastic cells, an effect similar to that of CGRP on smooth muscle cells.

    DOI: 10.1210/en.137.3.984

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  • 1,25-DIHYDROXYVITAMIN-D3 PROMOTES PROSTAGLANDIN-E1 INDUCED HL-60 CELL-DIFFERENTIATION

    T KAWASE, S OGATA, M ORIKASA, DM BURNS

    JOURNAL OF BONE AND MINERAL RESEARCH   10   S495 - S495   1995年8月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:BLACKWELL SCIENCE PUBL INC CAMBRIDGE  

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  • CALCITONIN-GENE-RELATED PEPTIDE OPPOSES PARATHYROID-HORMONE EFFECTS ON OSTEOBLASTIC GENE-REGULATION VIA POTASSIUM CHANNEL ACTIVATION

    DM BURNS, T KAWASE, GA HOWARD, BA ROOS

    JOURNAL OF BONE AND MINERAL RESEARCH   8   S173 - S173   1993年8月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:BLACKWELL SCIENCE INC  

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  • NITRIC-OXIDE STIMULATION OF MINERALIZATION IN OSTEOBLASTIC CELL-CULTURES

    T KAWASE, GA HOWARD, BA ROOS, DM BURNS

    JOURNAL OF BONE AND MINERAL RESEARCH   8   S372 - S372   1993年8月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:BLACKWELL SCIENCE INC  

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  • CALCITONIN GENE-RELATED PEPTIDE AND PARATHYROID-HORMONE ACUTELY BLOCK CA-45(2+) UPTAKE IN OSTEOBLASTIC CELLS BY DIFFERENT MECHANISMS

    T KAWASE, GA HOWARD, BA ROOS, DM BURNS

    JOURNAL OF BONE AND MINERAL RESEARCH   7   S207 - S207   1992年8月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:BLACKWELL SCIENCE INC  

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  • ALUMINUM ENHANCES THE STIMULATORY EFFECT OF NAF ON PROSTAGLANDIN-E2 SYNTHESIS IN A CLONAL OSTEOBLAST-LIKE CELL-LINE, MOB 3-4, INVITRO

    T KAWASE, ISHIKAWA, I, M ORIKASA, A SUZUKI

    JOURNAL OF BIOCHEMISTRY   106 ( 1 )   8 - 10   1989年7月

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    記述言語:英語   掲載種別:記事・総説・解説・論説等(学術雑誌)   出版者・発行元:JAPANESE BIOCHEMICAL SOC  

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  • Calcitonin gene-related peptide stimulates potassium efflux through ATP-sensitive potassium channels, resulting hyperpolarization in osteoblastic UMR106 cells.

    Endocrinology   138 ( 8 )   3492 - 3502   1988年

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受賞

  • 歯科基礎医学会賞

    1993年  

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    受賞国:日本国

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共同研究・競争的資金等の研究

  • 歯周組織の再生

    その他の研究制度 

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    資金種別:競争的資金

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  • Regeneration of periodontal tissue.

    The Other Research Programs 

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    資金種別:競争的資金

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担当経験のある授業科目(researchmap)

  • 薬理学

    機関名:新潟大学

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担当経験のある授業科目

  • 歯科薬理学

    2018年
    -
    現在
    機関名:新潟大学

  • 骨組織再建学演習ⅡA

    2018年
    機関名:新潟大学

  • 歯科基礎移植・再生学演習

    2009年
    機関名:新潟大学

  • 組織工学実習

    2008年
    -
    2013年
    機関名:新潟大学

  • 薬理学

    2007年
    -
    2018年
    機関名:新潟大学

  • 疾病とその病態

    2007年
    -
    2009年
    機関名:新潟大学

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