ダブルネットワークゲルが生体内で誘導する関節軟骨自然再生現象の分子機序の解明

2009 Fiscal Year Annual Research Report

• Project/Area Number: 20240045
• Research Institution: Hokkaido University
• Principal Investigator: 安田 和則 Hokkaido University, 大学院・医学研究科, 教授 (20166507)
• Co-Investigator(Kenkyū-buntansha): 近江谷 克裕 産業技術総合研究所, ゲノムファクトリー研究部門, 主幹研究員 (20223951) ; 北村 信人 北海道大学, 大学院・医学研究科, 講師 (80447044) ; 小野寺 伸 北海道大学, 大学院・医学研究科, 特任講師 (00359481) ; 権 赫準 北海道大学, 大学院・医学研究科, 特任助教 (20455629) ; 黒川 孝幸 北海道大学, 大学院・理学研究院, 特任助教 (40451439)
• Keywords: 関節軟骨欠損 / 自然再生 / 硝子軟骨 / 分子機序 / 高機能生体材料 / ダブルネットワークゲル / 再生誘導

Research Abstract

(1)前年度に作製したDNAアレイを用いて、PAMPS/PDMAAm DNゲルで誘導した家兎自然再生軟骨(2および4週)に発現している遺伝子を網羅的に解析し、同様に解析した成熟家兎正常軟骨および幼弱家兎正常軟骨のそれと比較した。前者との比較で5倍以上の発現差を有する遺伝子は全遺伝子の約5%、後者との比較で3-4%であった。その中で発現差トップ30の遺伝子の中から軟骨分化マーカー候補(Col2,Col5,Col10,Agc1,Crtl1,Dcn,Comp,Prg4,Cilp,Ctgf,Pthr1)を探した。
(2)PDMAAmゲル、PAMPSゲル、PAMPS/PDMAAm DNゲルのin vivo軟骨自然再生誘導効果を、組織学的scoreおよび分子マーカーmRNAの発現に関して比較した。効果は後2者のゲルに認められ、DNゲルで最大であった。その結果、負電荷を持つPAMPSの材料化学的特性、およびDNゲル化したことによる物理学的特性が重要な役割を果たすことが解った。
(3)PAMPSゲルとPDMAAmゲルからなる種々のcopolymerゲル上でATDC5細胞をインシュリン添加および無添加培地で培養し、軟骨マーカーの発現を比較した。その結果、インシュリン非存在下でPAMPSゲルに強力な軟骨への分化誘導能があることが解った。またインシュリン存在下ではPDMAAmゲルに分化促進能があることが解った。
(4)前年度に作製した多色発光プローブを用いて、時計遺伝子Bmal1、Per2、Hes1および軟骨マーカー遺伝子Col2、SOX9の発現をリアルタイムイメージングによって観察した。その結果、軟骨分化が始まると、これらの遺伝子にUltradianリズムが発現し、それらは軟骨のパターン形成に関与していることが解った。そこでもっと上流でこれらのUltradianリズムを規定する因子の存在が推定され、新しい多色発光プローブの開発を進めている。

Research Products

• [Journal Article] In vitro differentiation of chondrogenic ATDC5 cells is enhanced by culturing on synthetic hydrogels with various charge densities. (2010)
  • Author(s): Kwon HJ, Yasuda K, Ohmiya Y, Honma KI, Chen YM, Gong JP
  • Journal Title: Acta Biomater 6 Pages: 494-501
  • Peer Reviewed
• [Journal Article] Implantation of autogenous meniscal fragments wrapped with a fascia sheath enhances fibrocartilage regeneration in vivo in a large harvest site defect (2010)
  • Author(s): Kobayashi Y, Yasuda K, Kondo E, Katsura T, Tanabe Y, Kimura M, Tohyama. H
  • Journal Title: Am J Sports Med 5(Epub ahead of print)
  • Peer Reviewed
• [Journal Article] Applications of Iuciferin biosynthesis : Bioluminescence assays for l-cysteine and luciferase (2010)
  • Author(s): Niwa K, Nakajima Y, Ohmiya Y
  • Journal Title: Anal Biochem 396 Pages: 316-318
  • Peer Reviewed
• [Journal Article] Formation of a strong hydrogel-porous solid interface utilizing the double network principle (2010)
  • Author(s): Takayuki Kurokawa, Hidemitsu Furukawa, Wei Wang, Yoshimi Tanaka, Jian Ping Gong
  • Journal Title: Acta Biomaterialia 6 Pages: 1353-1359
  • Peer Reviewed
• [Journal Article] A novel double-network hydrogel induces spontaneous articular cartilage regeneration in vivo in a large osteochondral defect. (2009)
  • Author(s): Yasuda K, Kitamura N, Gong JP, Arakaki K, Kwon HJ, Onodera S, Chen YM, Kurokawa T, Kanaya F, Ohmiya Y, Osada Y
  • Journal Title: Macromol Biosc 9 Pages: 307-316
  • Peer Reviewed
• [Journal Article] A Soluble factor(EMMPRIN)in exudate influences knee motion after total arthroplasty. (2009)
  • Author(s): Onodera J, Onodera S, Kondo E, Betsuyaku T, Yasuda K
  • Journal Title: Knee Surg Sports Traumatol Arthrosc. 7 Pages: 298-304
  • Peer Reviewed
• [Journal Article] In vivo biological responses and bioresorption of tilapia scale collagen as a potential biomaterial. (2009)
  • Author(s): Sugiura H, Yunoki S, Kondo E, Ikoma T, Tanaka J, Yasuda K
  • Journal Title: J Biomater Sci Polym Ed 20 Pages: 1353-1368
  • Peer Reviewed
• [Journal Article] Effects of stress shielding and subsequent restressing on mechanical properties of regenerated and residual tissues in rabbit patellar tendon after resection of its ce ntral one-third (2009)
  • Author(s): Maeda E, Asanuma H, Noguchi H, Tohyama H, Yasuda K, Hayashi K
  • Journal Title: J Biomech 42 Pages: 1592-7
  • Peer Reviewed
• [Journal Article] Atelocollagen-associated autologous chondrocyte implantation for the repair of chondral defects of the knee : a prospective multicenter clinical trial in Japan. (2009)
  • Author(s): Tohyama H, Yasuda K, Minami A, Majima T, Iwasaki N, Muneta T, Sekiya I, Yagishita K, Takahashi S, Kurokouchi K, Uchio Y, Iwasa J, Deie M, Adachi N, Sugawara K, Ochi M
  • Journal Title: J Orthop Sci 14 Pages: 579-88
  • Peer Reviewed
• [Journal Article] Stress deprivation from the patellar tendon induces apoptosis of fibroblasts in vivo with activation of mitogen-activated protein kinases. (2009)
  • Author(s): Kawabata H, Katsura T, Kondo E, Kitamura N, Miyatake S, Tanabe Y, Setoguchi T, Komiya S, Yasuda K
  • Journal Title: J Biomech 42 Pages: 2611-2615
  • Peer Reviewed
• [Journal Article] In vivo bioluminescence imaging of bone marrow-derived cells in brain inflammation. (2009)
  • Author(s): Akimoto H, Kwon HJ, Ozaki M, Yasuda K, Honma K, Ohmiya Y
  • Journal Title: Biochemical and Biophysical Research Communications 380 Pages: 844-849
  • Peer Reviewed
• [Journal Article] The in vivo effect of artificial cartilage made from a novel double-network hydrogel on the microstructure of the normal cartilage located in the opposite joint surface. (2009)
  • Author(s): Arakaki K, Kitamura N, Fujiki H, Kurokawa T, Iwamoto M, Ueno M, Kanaya F, Osada Y, Gong JP, Yasuda K
  • Journal Title: J Biomed Mater Res A 18(Epub ahead of print)
  • Peer Reviewed
• [Journal Article] A bioluminescent enzyme immunoassay for prostaglandin E2 using Cypridina luciferase. (2009)
  • Author(s): Wu C, Irie S, Yamamoto S, Ohmiya Y
  • Journal Title: Luminescence 24 Pages: 131-3
  • Peer Reviewed
• [Journal Article] In vivo far-red luminescence imaging of a biomarker based on BRET from Cypridina bioluminescence to an organic dye. (2009)
  • Author(s): Wu C, Mino K, Akimoto H, Kawab ata M, Nakamura K, Ozaki M, Ohmiya Y
  • Journal Title: Proc Natl Acad Sci USA 106 Pages: 15599-603
  • Peer Reviewed
• [Journal Article] Antifouling activity of synthetic polymer gels against cyprids of the barnacle(Amphibalanus amphitrite)in vitro (2009)
  • Author(s): Takayuki Murosaki, Takaya Noguchi, Akira Kakugo, Ananda Putra, Taka yuki Kurokawa, Hidemitsu Furukawa Yoshihito Osada, Jian Ping Gong, Yasuyuki Nogata, Kiyotaka Matsumura, Erina Yoshimura, Nobuhiro Fusetani
  • Journal Title: Biofouling The Journal of Bioadhesion and Biofilm Research 25 Pages: 313-320
  • Peer Reviewed
• [Journal Article] Friction between like-charged hydrogels-combined mechanisms of boundary, hydrated and elastohydrodynamic lubrication (2009)
  • Author(s): Shinsuke Oogaki, Go Kagata, Takayuki Kurokawa, Shinya Kuroda, Yoshihito Osada, Jian Ping Gong
  • Journal Title: Soft Matter 5 Pages: 1879-1887
  • Peer Reviewed
• [Journal Article] Direct Observation of Damage Zone around Crack Tips in Double-Network Gels (2009)
  • Author(s): Qiu Ming Yu, Yoshimi Tanaka, Hidemitsu Furukawa, Takayuki Kurokawa, Jian Ping Gong
  • Journal Title: Macromolecules 42 Pages: 3852-3855
  • Peer Reviewed
• [Journal Article] Antifouling of tough gels against barnacles in long-term marine environment (2009)
  • Author(s): Takayuki Murosaki, Takaya Noguchi, Kazuo Hashimoto, Akira Kakugo, Takayuki Kurokawa, Junji Saito, Yong Mei Chen, Hidemitsu Furukawa, Jian Ping Gong
  • Journal Title: Biofouling 25 Pages: 657-666
  • Peer Reviewed
• [Journal Article] First observation of stick-slip instability in tearing of poly(vinyl alcohol)gel sheets (2009)
  • Author(s): Yoshimi Tanaka, Hiroyuki Abe, Taka yuki Kurokawa, Hidemitsu Furukawa, Jian Ping Gong
  • Journal Title: Macromolecules 42 Pages: 5425-5426
  • Peer Reviewed
• [Journal Article] Ultrathin tough double network hydrogels showing adjustable muscle-like isometric force generation triggered by solvent (2009)
  • Author(s): I Songmiao Liang, Qiu Ming Yu, Haiyan Yin, Ziliang Wu, Takayuki Kurokawa, Jian Ping Gong
  • Journal Title: Chemical Communications 48 Pages: 7518-7520
  • Peer Reviewed
• [Journal Article] (2009)
  • Author(s): Kitamura N,Yasuda K,Gong JP, Osada Y
  • Journal Title: Molecular Imaging for Integrated Medical Therapy and Drug Development. Editors Tamaki N, Kuge Y(Springer) Pages: 116-125
• [Presentation] Joint Immobilization Inhibits The Spontaneous Hyaline Cartilage Regeneration Induced By PAMPS/PDMAAm Double-network Hydrogel (2010)
  • Author(s): Arakaki K, Kitamura N, Kurokawa T, Gong JP, Kanaya F, Yasuda K
  • Organizer: 56th Annual Meeting of the Orthopuedic Research Society
  • Place of Presentation: Morial Convention Center Hall H, New Orleans, Louisiana
  • Year and Date: 20100306-20100309
• [Presentation] Negatively Charged PAMPS Gel Component Plays A Significant Role in The In Vivo Spontaneous Cartilage Regeneration Induced by Implantation of PAMPS/PDMAAm Double-network Gel (2010)
  • Author(s): Ogawa M, Kitamura N, Arakaki K, Onodera S, Kurokawa T, Gong JP,Tanaka Y, Takakura Y, Yasuda K
  • Organizer: 56th Annual Meeting of the Orthopaedic Research Society
  • Place of Presentation: Morial Convention Center Hall H, New Orleans, Louisiana
  • Year and Date: 20100306-20100309
• [Presentation] Implantation of a PAMPS/PDMAAm Double-Network Gel Can Induce Spontaneous Hyaline Cartilage Regeneration In Vivo Within a Large Osteochondral Defect in a Sheep Model (2010)
  • Author(s): Yokota M, Kitamura N, Arakaki K, Kondo E, Onodera S, Kurokawa T, Gong JP, Yasuda K
  • Organizer: 56th Annual Meeting of the Orthopuedic Research Society
  • Place of Presentation: Morial Convention Center Hall H, New Orleans, Louisiana
  • Year and Date: 20100306-20100309
• [Presentation] Pattern of Peri-Prosthetic Osteolysis After Total Knee Arthroplasty in Patients With Rheumatoid Arthritis : Computer Tomography Study at Minimum 10-year Follow-up (2009)
  • Author(s): Kitamura N, Arakaki K, Susuda K, Kondo E, Yasuda K
  • Organizer: ISTA2009 The 22^<nd> Annual Congress of the International Society for Technology in Arthroplasty
  • Place of Presentation: Big Island, Hawaii
  • Year and Date: 20091222-20091224
• [Presentation] The In vivo Effect of Artificial Cartilage Made from a Novel Double-Network Hydrogel on the Microstructure of the Counterface Normal Cartilage (2009)
  • Author(s): Kitamura N, Arakaki K, Fujiki H, Kurokawa T, Iwamoto M, Ueno M, Kanaya F, Osada Y, Gong JP, Yasuda K
  • Organizer: ISTA2009 The 22^<nd> Annual Congress of the International Socicty for Technology in Arthroplasly
  • Place of Presentation: Big Island, Hawaii
  • Year and Date: 20091222-20091224
• [Presentation] An in vivo application of interleukin-1 receptor antagonist inhibits deterioration of mechanical properties of the stress-shielded patellar tendon without significant changes in the fascicle properties (2009)
  • Author(s): Miyatake S, Tohyama H, Kondo E, Katsura T, Onodera S, Yasuda K
  • Organizer: The 7^<th> Biennial ISAKOS Congress
  • Place of Presentation: The Osaka International Convention Center, Osaka, Japan
  • Year and Date: 20090405-20090409
• [Presentation] Implantation of comminuted autogenous meniscus tissue into a large meniscus defect enhances spontaneous in situ regeneration of the meniscus an experimental study (2009)
  • Author(s): Kobayashi Y, Yasuda K, Kondo E, Katsura T, Kumura M
  • Organizer: The 7^<th> Biennial ISAKOS Congress
  • Place of Presentation: The Osaka International Convention Center, Osaka, Japan
  • Year and Date: 20090405-20090409
• [Presentation] In vivo local administration of osteogenic protein-1 increases structural properties of the overstretched anterior cruciate ligament with partial midsubstance laceratio n : A biomechanical study (2009)
  • Author(s): Hayashi R, Kondo E, Miyatake S, T ohyama H, Saito T, Yasuda K
  • Organizer: The 7^<th> Biennial ISAKOS Congress
  • Place of Presentation: The Osaka International Convention Center, Osaka, Japan
  • Year and Date: 20090405-20090409
• [Presentation] Local administration of synovium-derived fibroblasts cultured in TGF-betal-supple mented medium inhibits natural reduction of the structural properties of the tendon autograft after ACL reconstruction : A sheep model study (2009)
  • Author(s): Kondo E, Hayashi R, Katsura T, Azuma C, Tohyama H, Yasuda K
  • Organizer: The 7^<th> Biennial ISAKOS Congress
  • Place of Presentation: The Osaka International Con vention Center, Osaka, Japan
  • Year and Date: 20090405-20090409