Acceleration of fracture healing by growth factor fused with tandem collagen-binding domains from Clostridium histolyticum collagenase

• Project/Area Number: 15K20015
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Orthopaedic surgery
• Research Institution: Kitasato University
• Principal Investigator: Uchida Kentaro 北里大学, 医学部, 講師 (50547578)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000); Fiscal Year 2017: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2016: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2015: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
• Keywords: 骨折治癒促進 / コラーゲン結合型成長因子 / コラーゲン結合ドメイン / 難治性骨折 / 塩基性線維芽細胞増殖因子 / 骨形成促進 / 骨折治癒 / 人工コラーゲン

Outline of Final Research Achievements

We examined the binding affinity of four collagen anchors derived fromthe two clostridial collagenases to H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly)12-NH2, a collagenous peptide, by surface plasmon resonance and found that tandem CBDs (s3a-s3b) have the highest affinity for the collagenous peptide.We also constructed four fusion proteins consisting of bFGF and s3 (bFGF-s3), s2b-s3b (bFGF-s2b-s3), s3b (bFGF-s3b), and s3a-s3b (bFGF-s3a-s3b) and compared their biological activities to those of a previous fusion construct (bFGF-s2b-s3) using a cell proliferation assay in vitro and a mouse femoral fracture model in vivo. Among these CB-bFGFs, bFGF-s3a-s3b showed the highest capacity to induce mesenchymal cell proliferation and callus formation in the mice fracture model. The poly(Pro-Hyp-Gly)10/bFGF-s3a-s3b
construct may therefore have the potential to promote bone formation in clinical settings.

Research Products

• [Int'l Joint Research] University of Arkansas(米国)
• [Journal Article] Basic Fibroblast Growth Factor Fused with Tandem Collagen-Binding Domains from Clostridium histolyticum Collagenase ColG Increases Bone Formation (2018)
  • Author(s): Sekiguchi H, Uchida K, Matsushita O, Inoue G, Nishi N, Masuda R, Hamamoto N, Koide T, Shoji S, Takaso M
  • Journal Title: Biomed Res Int Volume: 8393194 Pages: 1-8
  • DOI: 10.1155/2018/8393194
  • Peer Reviewed / Open Access
• [Journal Article] Basic Fibroblast Growth Factor-Anchored Multilayered Mesenchymal Cell Sheets Accelerate Periosteal Bone Formation (2017)
  • Author(s): Uchida K, Inoue G, Matsushita O, Horikawa K, Sekiguchi H, Saito W, Takano S, Fujimaki H, Miyagi M, Takaso M.
  • Journal Title: Biomed Res Int Volume: 4371460 Pages: 1-8
  • DOI: 10.1155/2017/4371460
  • Peer Reviewed / Open Access
• [Journal Article] Effect of Freeze-Dried Allograft Bone with Human Basic Fibroblast Growth Factor Containing a Collagen-Binding Domain From Clostridium Histolyticum Collagenase on Bone Formation After Lumbar Posterolateral Fusion Surgery in Rats (2017)
  • Author(s): Inoue G, Uchida K, Matsushita O, Fujimaki H, Saito W, Miyagi M, Sekiguchi H, Nishi N, Ohtori S, Yogoro M, Takaso M.
  • Journal Title: Spine Volume: 印刷中 Issue: 17 Pages: 995-1001
  • DOI: 10.1097/brs.0000000000002074
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Adrenomedullin Regulates IL-1β Gene Expression in F4/80+ Macrophages during Synovial Inflammation. (2017)
  • Author(s): Takano S, Uchida K, Miyagi M, Inoue G, Aikawa J, Iwabuchi K, Takaso M.
  • Journal Title: J Immunol Res. Volume: - Pages: 1-10
  • DOI: 10.1155/2017/9832430
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Oriented collagen tubes combined with basic fibroblast growth factor promote peripheral nerve regeneration in a 15 mm sciatic nerve defect rat model (2017)
  • Author(s): Fujimaki H, Uchida K, Inoue G, Miyagi M, Nemoto N, Saku T, Isobe Y, Inage K, Matsushita O, Yagishita S, Sato J, Takano S, Sakuma Y, Ohtori S, Takahashi K, Takaso M.
  • Journal Title: J Biomed Mater Res A Volume: 105 Issue: 1 Pages: 8-14
  • DOI: 10.1002/jbm.a.35866
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Nerve growth factor regulation by TNF-alpha; and IL-1β in synovial macrophages and fibroblasts in osteoarthritic mice. (2016)
  • Author(s): Takano S, Uchida K, Miyagi M, Inoue G, Fujimaki H, Aikawa J, Iwase D, Minatani A, Iwabuchi K, Takaso M.
  • Journal Title: J Immunol Res Volume: - Pages: 1-8
  • DOI: 10.1155/2016/5706359
  • Peer Reviewed / Open Access
• [Journal Article] Acceleration of bone formation during fracture healing by poly(pro-hyp-gly)10 and basic fibroblast growth factor containing polycystic kidney disease and collagen-binding domains from Clostridium histolyticum collagenase (2016)
  • Author(s): Sekiguchi H, Uchida K, Inoue G, Matsushita O, Saito W, Aikawa J, Tanaka K, Fujimaki H, Miyagi M, Takaso M.
  • Journal Title: J Biomed Mater Res A Volume: 104 Issue: 6 Pages: 1372-1378
  • DOI: 10.1002/jbm.a.35670
  • Peer Reviewed / Acknowledgement Compliant
• [Presentation] Acceleration Of Bone Repair During Fracture Healing By Poly(pro-hyp-gly)10 And Basic Fibroblast Growth Factor Containing Collagen-binding Domains From Clostridium Histolyticum Collagenase (2017)
  • Author(s): Sekiguchi H, Uchida K, Inoue G, Saito W, Aikawa J, Fujimaki H, Nagura N, Takano S, Miyagi M Takaso M
  • Organizer: ORS 2017 Annual Meeting
  • Place of Presentation: San Diego(USA)
  • Year and Date: 2017-03-19
  • Int'l Joint Research
• [Presentation] 難治性骨折に対する新規コラーゲン結合型塩基性線維芽細胞増殖因子/人工コラーゲン複合体の有用性の検討 (2017)
  • Author(s): 関口裕之、内田健太郎、井上玄、宮城正行、相川淳、高野昇太郎、名倉直重、中脇充章、庄司真太郎、大貫裕子、高相昌士
  • Organizer: 第32回日本整形外科学会基礎学術集会
• [Presentation] 新規コラーゲン結合型線維芽細胞増殖因子による骨形成法の開発 (2017)
  • Author(s): 関口裕之、内田健太郎、井上 玄、高相晶士.
  • Organizer: 第36回日本運動器移植・再生医学研究会
• [Presentation] 人工コラーゲンゲルとコラーゲン結合型線維芽細胞増殖因子による骨折後の骨形成促進効果の検討 (2015)
  • Author(s): 関口裕之、内田健太郎、井上 玄、相川 淳、齋藤 亘、宮城正行、藤巻寿子、大貫裕子、高相晶士
  • Organizer: 第30回日本整形外科学会基礎学術集会
  • Place of Presentation: 富山国際会議場（富山県富山市）
  • Year and Date: 2015-10-22
• [Patent(Industrial Property Rights)] COLLAGEN-BINDING AGENT COMPOSITIONS AND METHODS OF USING THE SAME (2017)
  • Inventor(s): Skaon J, Uchida K et al.
  • Industrial Property Rights Holder: Skaon J, Uchida K et al.
  • Industrial Property Rights Type: 特許
  • Filing Date: 2017-02-10
  • Overseas