2011 Fiscal Year Final Research Report
Exper imenta I study of reconstruct ion of peripheral nerve defect using tissue-engineered bioabsorbable nerve conduit with iPS cell induced neurosphere cell
| Project/Area Number |
21591904
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Orthopaedic surgery
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| Research Institution | Osaka City University |
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Principal Investigator |
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| Project Period (FY) |
2009 – 2011
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| Keywords | 再生医療 / 末梢神経 / 再生 / 生体吸収性材料 / iPS細胞 / 人工神経 / 組織工学 / マウス |
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| Research Abstract |
【Background】in spite ofextensirosearcliof induced pluripotent stem(iPS) cells, the therapeutic potential of iPS cells for peripheral nerve injury is largely unknown. This is the study to prove the feasibility of the combination of iPS cell-derived neurospheres and bioabsorbable nerve conduit.
【Purpose】<Aiml> The first purpose of this study was to examine the adhesion of the iPS cell induced neurospheres to the bioabsorbable nerve conduits and to examine their cellular characteristics.
<Aim2> The second purpose of this study wasto test tissue-engineered hioabsorbable nerve conduits coated with a three-dimensional(3D) culture of WS cell-derived neurospheres in peripheral nerve repair in vivo.
【Methods】<Aiml> The nerve conduit has 2 layers, The outer layer is composed of PLLA mesh and the inner layer is composedofPLLAand PCL porous sponge. We generated primary and secondary neurospheres from iPS cells by a published protocol[Editor1]. The primary and secondary neurospheres were suspended in
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each conduit. After suspension, the conduits were placed in a cell incubator for 7 or 14 days. After the conduits were seeded with iPs cells, they were subjected to immunohistological analysis by using antibodies specific to the glial marker(GFAP), Schwann cell marker(S-100) and neuronal marker.
<Aim2> The secondary neurospheres derived from mouse iPS cells were suspended in each conduit.(4.0×10^6 cells per miduit) and cultured in 3D-cultu fir 14 days. We then implanted them in the mouse sciatic nerve gap(5 mm)(iPs group ; n=10). The nerve conduit. alone was implanted in the control group(n=10). Motor and sensory function recovery was assessed. At 4, 8, 12 weeks, nerve regeneration in the nerve conduit was evaluated by histological analysis.
【Results】<Aiml> All the primary and secondary neurospheres that had differentiated for 7 or 14 days were found to have adhered to the inner surface of the conduits and migrated into the inner porous sponge. All neurospheres were positive for S-100 and GFAP but were negative for neurofilament protein. The cell adhesion and the immunostaining characteristics between the 7-and 14-day-differentiated neurospheres were not different.
<Aim2> Motor and sensory function recovery was significantly faster in the iPS group at weeks 4, 8, and 12.At 12 weeks, all the nerve conduits remained structurally stable without any collapse. Histological analysis indicated axonal regeneration in the nerve conduits of both groups. However, the iPS group showed more vigorous axonal regeneration.
【Conclusion】The bioabsorbable nerve conduits created by 3D culture of iPS cell-derived neurospheres promoted regeneration of peripheral nerves and functional recovery in vivo. The combination of iPS cell technology and bioabsorbable nerve conduits could represent a future tool for the treatment of peripheral nerve defects. Less
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