A comprehensive proteomic analysis for the Identification of factors contributing to hyaline cartilage regeneration

• Project/Area Number: 16H05455
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Orthopaedic surgery
• Research Institution: Tokai University
• Principal Investigator: SATO Masato 東海大学, 医学部, 教授 (10056335)
• Co-Investigator(Kenkyū-buntansha): 豊田 恵利子 東海大学, 医学部, 特定研究員 (90749269)
• Project Period (FY): 2016-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥17,160,000 (Direct Cost: ¥13,200,000、Indirect Cost: ¥3,960,000); Fiscal Year 2018: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2017: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2016: ¥7,930,000 (Direct Cost: ¥6,100,000、Indirect Cost: ¥1,830,000)
• Keywords: タンパク質 / miRNA / マイクロアレイ解析 / 細胞シート / 再生医学 / マイクロアレイ / 蛋白質

Outline of Final Research Achievements

To identify the mechanisms and anabolic factors that contribute to the hyaline cartilage regenerative properties of chondrocyte sheets, we performed a proteomic analysis (SOMAscan) of factors that vary in correlation with the cartilage regenerative properties of chondrocyte sheets fabricated under different culture conditions and identified 26 such factors. Through the analysis of the change in levels of gene expression and secreted factors of chondrocyte sheets with varying cartilage regenerative properties, regulators of Wnt signaling, regulators of inflammation, and growth factors were found to correlate with the cartilage regenerative properties. Furthermore, through the microarray analysis of exosomal miRNAs in culture supernatants, miRNAs that correlate with the cartilage regenerative properties were identified. Validation through RT-PCR revealed 4 such miRNAs.

Academic Significance and Societal Importance of the Research Achievements

軟骨細胞シートは、損傷した軟骨からのプロテオグリカンの流出の阻止や、関節液中の軟骨分解因子からの軟骨の保護に働き、軟骨成長因子の持続的な供給や、軟骨下骨から誘導される骨髄由来幹細胞に対する軟骨分化促進イニシエーターとしての機能等を介して軟骨修復を促進していると考えられるが、これらの作用にかかわる因子やメカニズムについては不明な点も多い。
自然には修復しない関節軟骨の再生に有用な因子の一端を明らかにした本研究成果は、軟骨細胞シートが軟骨修復を促進するメカニズムの解明に繋がるものである。

Research Products

• [Journal Article] Combined surgery and chondrocyte cell-sheet transplantation improves clinical and structural outcomes in knee osteoarthritis (2019)
  • Author(s): Sato M, Yamato M, Mitani G, Takagaki T, Hamahashi K, Nakamura Y, Ishihara M, Matoba R, Kobayashi H, Okano T, Mochida J, Watanabe M
  • Journal Title: NPJ Regen Med Volume: 4 Issue: 1
  • DOI: 10.1038/s41536-019-0069-4
  • Peer Reviewed / Open Access
• [Journal Article] Rabbit xenogeneic transplantation model for evaluating human chondrocyte sheets used in articular cartilage repair (2018)
  • Author(s): Takahashi T, Sato M, Toyoda E, Maehara M, Takizawa D, Maruki H, Tominaga A, Okada E, Okazaki K, Watanabe M
  • Journal Title: J Tissue Eng Regen Med Volume: 12 Pages: 2067-2076
  • DOI: 10.1002/term.2741
  • Peer Reviewed / Open Access
• [Presentation] 多指症由来軟骨細胞シートの拡大培養と特性変化の解析 (2019)
  • Author(s): 豊田恵利子, 佐藤正人, 前原美樹, 高橋匠, 野中謙, 飯島寛, 的場亮, 川口佑加, 佐藤千香子, 高木岳彦, 阿久津英憲, 梅澤明弘, 赤松正, 渡辺雅彦
  • Organizer: 第32回日本軟骨代謝学会
• [Presentation] Analysis of micro-RNAs in exosomes secreted by human polydactyly-derived chondrocyte sheets (2018)
  • Author(s): Toyoda E, Sato M, Maehara M, Takahashi T, Takagi T, Nonaka K, Iijima H, Matoba R, Akutsu H, Umezawa A, Akamatsu T, Watanabe M
  • Organizer: The 2018 Osteoarthritis Research Society International (OARSI) World Congress
  • Int'l Joint Research
• [Presentation] Humoral factors produced by polydactyly-derived chondrocyte sheets in articular cartilage repair (2018)
  • Author(s): Takahashi T, Sato M, Toyoda E, Maehara M, Sogo Y, Tominaga A, Takagi T, Akamatsu T, Okazaki K, Watanabe M
  • Organizer: The 2018 Osteoarthritis Research Society International (OARSI) World Congress
  • Int'l Joint Research
• [Presentation] Identification of altered gene expression in the expansion polydactyly-derived chondrocytes (2018)
  • Author(s): Toyoda E, Sato M, Maehara M, Takahashi,T, Takagi T, Kotoku T, Sato C, Nonaka K, Matoba R, Akutsu H, Umezawa A, Akamatsu T, Watanabe M
  • Organizer: 5th TERMIS World Congress 2018
  • Int'l Joint Research
• [Presentation] Exploration of humoral factors related to cartilage regeneration produced by polydactyly-derived chondrocyte sheets and adult chondrocyte sheets (2018)
  • Author(s): Maehara M, Sato M, Toyoda E, Takahashi,T, Okada E, Takagi T, Akamatsu T, Akutsu H, Watanabe M
  • Organizer: 5th TERMIS World Congress 2018
  • Int'l Joint Research
• [Presentation] 多指症由来軟骨細胞シートと成人軟骨細胞シートの特性の比較研究 (2018)
  • Author(s): 鷹取直希, 佐藤正人, 三谷玄弥, 高垣智紀, 浜橋恒介, 芹ケ野健司, 十河泰之, 渡辺雅彦
  • Organizer: 第10回日本関節鏡・膝・スポーツ整形外科学会(JOSKAS)
• [Presentation] Evaluation of Humoral Factors Produced by Polydactyly-derived Chondrocyte Sheets using SOMAscanTM (2018)
  • Author(s): Takahashi T, Sato M, Toyoda E, Maehara M, Takizawa D, Sogo Y, Tominaga A, Okada E, Takagi T, Okazaki K, Akamatsu T, Watanabe M
  • Organizer: Orthopaedic Research Society 2018 Annual Meeting
  • Int'l Joint Research
• [Presentation] SOMAscanTMを用いた多指症由来同種軟骨細胞シートの液性因子解析：培養条件の影響 (2017)
  • Author(s): 高橋匠, 佐藤正人, 豊田恵利子, 前原美樹, 滝澤大智, 十河泰之, 冨永絢子, 岡田恵里, 渡辺雅彦
  • Organizer: 第32回日本整形外科学会基礎学術集会
• [Presentation] ヒト軟骨細胞シートが産生するタンパク質の網羅的解析. (2016)
  • Author(s): 豊田恵利子, 佐藤正人, 高橋匠, 前原美樹, 高垣智紀, 浜橋恒介, 幸得友美, 岡田恵里, 渡部綾子, 佐藤千香子, 滝澤大智, 的場亮, 阿久津英憲, 梅澤明弘, 持田讓治.
  • Organizer: 第31回日本整形外科学会基礎学術集会
  • Place of Presentation: 福岡国際会議場（福岡県福岡市）
  • Year and Date: 2016-10-13
• [Presentation] アプタマーアレイを用いた軟骨細胞シートが産生する因子の網羅的解析. (2016)
  • Author(s): 豊田恵利子, 佐藤正人, 高橋匠, 前原美樹, 高垣智紀, 濱橋恒介, 的場亮，阿久津英憲，梅澤明弘，渡辺雅彦.
  • Organizer: 第35回日本運動器移植・再生医学研究会
  • Place of Presentation: 広島県医師会館（広島県広島市）