Exper imenta I study of reconstruct ion of peripheral nerve defect using tissue-engineered bioabsorbable nerve conduit with iPS cell induced neurosphere cell

[背景]尽管诱导性多能干细胞（iPS）的研究已经取得了很大进展，但iPS细胞在周围神经损伤治疗中的应用前景仍不清楚。本研究旨在证明iPS细胞源性神经球与生物可吸收神经导管结合的可行性。[目的]<Aiml>本研究的第一个目的是检测iPS细胞诱导的神经球与生物可吸收神经导管的粘附并检测其细胞特性。<Aim2>本研究的第二个目的是测试组织工程生物可吸收神经导管与三维（3D）培养的WS细胞衍生的神经球涂层在体内周围神经修复。[方法]<Aiml>神经导管分为2层，外层为PLLA网片，内层为PLLA和PCL多孔海绵。我们通过已发表的方案从iPS细胞产生了初级和次级神经球[编辑1]。初级和次级神经球悬浮在 关于我们 每一个管道悬浮后，将导管置于细胞培养箱中7或14天。在导管用iPs细胞接种后，通过使用对神经胶质标志物（GFAP）、许旺细胞标志物（S-100）和神经元标志物特异性的抗体对其进行免疫组织学分析。<Aim2>将源自小鼠iPS细胞的次级神经球悬浮在每个导管中。(4.0×10^6个细胞/培养基）培养14天。然后将它们植入小鼠坐骨神经间隙（5 mm）（iPs组; n=10）。神经导管。对照组10只。评估运动和感觉功能恢复情况。于术后4、8、12周行组织学观察。[结果]<Aiml>分化7 ~ 14 d的原代和次级神经球均粘附于导管内表面，并迁移至海绵内。所有神经球均为S-100和GFAP阳性，但神经丝蛋白阴性。分化7天和14天的神经球的细胞粘附和免疫染色特征没有差异。<Aim2>术后4、8、12周，iPS组运动和感觉功能恢复明显快于对照组。术后12周，所有神经导管结构稳定，无塌陷。组织学分析表明，两组的神经导管轴突再生。然而，iPS组表现出更旺盛的轴突再生。[结论] iPS细胞源性神经球三维培养构建的可吸收神经导管可促进周围神经的再生和功能恢复。iPS细胞技术和生物可吸收神经导管的结合可能代表了治疗周围神经缺损的未来工具。少

【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 … More 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