Synergistic Enhancement of Peripheral Nerve Defect Repair using Peptide Functionalized Aligned Nanofiber Conduits

摘要 周围神经再生经历了不同的阶段。在过去的几十年里，新的细节 关于周围神经再生的过程已经阐明。当轴突再生时 过程已经被研究很久了，最近注意到再生和修复是与 雪旺细胞(SC)渗入损伤的周围神经缺损处。SC招聘和定向迁移 一直是我们实验室感兴趣的话题，重点是使用地形和 模拟ECM的多肽。我们的体外初步数据显示明显诱导了SC的定向迁移 使用转化生长因子β多肽和YIGSR多肽的锚定浓度梯度。我们在体内的初步数据 进一步证明合成纳米纤维支持SC的渗透和成熟。总而言之，这些数据 为我们提供了大量的动力来进一步研究模仿 通过招募SC进行神经再生的过程。为了追求这些目标，我们开发了 功能性、可降解聚合物和多功能触摸纺丝制造策略，以产生空间- 定义的，生物活性的，定向的纳米纤维管道，我们建议使用这个平台来改进再生 受损周围神经的能力。我们认为，无细胞材料解决方案可以增强内源性 修复流程与翻译相关，并将为这些功能的翻译提供最佳选择 在短期内为诊所提供管道。我们假设，基于多肽的生物活性因子在不同的 浓度分布与地形线索相结合，将增加Sc的入渗，因此， 神经再生，跨越临界大小的缺口。我们将带着三个独立的目标来追求这一假说。 具体目标1：锚定的层粘连蛋白多肽梯度，以促进神经细胞迁移和SC渗透。我们会 研究浓度梯度的层粘连蛋白多肽如何单独增强轴突和SC的反应 在外植体(多细胞)模型中。该目标的结果将产生最佳的纳米纤维(直径， 层粘连蛋白-多肽梯度)，以推进我们在目标3中提议的体内研究。特定目标2：拴系的转化生长因子-β 促进神经细胞迁移和SC渗透的多肽梯度。我们将调查如何集中精力 梯度化转化生长因子-β肽基生长因子联合RGD增强轴突和SC 反应，单个和在外植体(多细胞)模型中。这一目标的结果将产生最佳的纳米纤维 (直径，转化生长因子-β多肽梯度)以推进我们建议的体内特定目标研究3.特定目标 3：结合层粘连蛋白多肽梯度和转化生长因子-β改善体内神经再生结果 渐变。我们将使用目标1和目标2中独立确定的最好的纳米纤维支架来研究 层粘连蛋白多肽和转化生长因子-β多肽浓度梯度联合应用是否具有协同作用 增强体内神经再生的初始过程和长期功能恢复- 建立大鼠坐骨神经损伤模型。重点放在内源性修复的早期步骤，以及 长期恢复指标，这项工作将为SC在神经中所起的作用提供基础证据 再生过程。这一知识将把我们对神经修复的关注从轴突转移到 已知支持再生过程，以促进恢复。

Abstract Peripheral nerve regeneration has moved through a variety of stages. Over the past few decades, new details regarding the process of peripheral nerve regeneration have been elucidated. While the axonal regrowth process has long been studied, it was noted recently that the regrowth and repair proceeds in tandem with Schwann cell (SC) infiltration into the injured peripheral nerve defect. SC recruitment and directed migration has been a topic of interest in our laboratories, with a focus on biased SC migration using topographical and ECM-mimicking peptides. Our in vitro preliminary data shows a clear induction of directional SC migration using tethered concentration gradients of both TGF-β peptide and YIGSR-peptide. Our in vivo preliminary data further demonstrates that synthetic nanofibers support SC infiltration and maturation. Together, these data have provided us with substantial motivation to further investigate mechanisms that mimic the neuroregenerative process through the recruitment of SC. To pursue these goals, we have developed functional, degradable polymers and versatile touch-spinning fabrication strategies to generate spatially- defined, bioactive, aligned nanofiber conduits and we propose to use this platform to improve the regenerative capacity of injured peripheral nerves. We believe that cell-free material solutions that enhance the endogenous repair process are translationally-relevant and will provide the best options for translation of these functional conduits to the clinic in the near term. We hypothesize that tethered, peptide-based bioactive factors in distinct concentration profiles, in combination with topographical cues, will increase SC infiltration, and therefore, neuroregeneration, across critical-sized gaps. We will pursue this hypothesis with three independent aims. Specific Aim 1: Tethered laminin peptide gradients to enhance neural cell migration and SC infiltration. We will investigate how concentration gradients of tethered laminin peptide enhance neurite and SC response, singly and in an explant (multicellular) model. The outcome of this Aim will yield an optimal nanofiber (diameter, laminin-peptide gradient) to advance to our proposed in vivo studies in Aim 3. Specific Aim 2: Tethered TGF-β peptide gradients to enhance neural cell migration and SC infiltration. We will investigate how concentration gradients of tethered TGF-β peptide-based growth factor in combination with RGD enhance neurite and SC response, singly and in an explant (multicellular) model. The outcome of this Aim will yield an optimal nanofiber (diameter, TGF-β peptide gradient) to advance to our proposed in vivo studies in Specific Aim 3. Specific Aim 3: In vivo neural regeneration outcomes improve with combinations of laminin peptide gradients and TGF-β gradients. We will use the best nanofiber scaffolds independently identified in Aims 1 and 2 to investigate whether combinations of laminin peptide and TGF-β peptide concentration gradients will synergistically enhance the initial process of neural regeneration and long- term functional recovery in vivo in a well- established rat sciatic nerve defect model. With a focus on the early steps in endogenous repair, along with a long-term recovery metric, this work will provide foundational evidence in the role that SC play in the nerve regeneration processes. This knowledge will shift our focus in nerve repair from the axon to cells that are known to support the regeneration process to enhance recovery.