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

摘要 周围神经再生已经经历了不同的阶段。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 这个过程已经研究了很久，最近注意到再生和修复是同步进行的， 雪旺细胞（SC）浸润到损伤的周围神经缺损中。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 ECM模拟肽。我们在体外的初步数据显示，明确诱导定向SC迁移 使用TGF-β肽和YIGSR-肽两者的栓系浓度梯度。我们的体内初步数据 进一步证明了合成纳米纤维支持SC浸润和成熟。这些数据一起 为我们提供了大量的动力，以进一步研究模仿的机制， 神经再生过程通过招募SC。为了追求这些目标，我们已经开发了 功能性、可降解的聚合物和多功能触摸纺丝制造策略， 明确的，生物活性的，对齐的血管导管，我们建议使用这个平台来改善再生的 损伤的周围神经的能力。我们认为，无细胞材料解决方案，增强内源性 修复过程是实用相关的，并将为这些功能的翻译提供最佳选择 在短期内，诊所。我们假设，在不同的细胞中，以肽为基础的生物活性因子是不同的。 浓度分布，结合地形线索，将增加SC渗透，因此， 神经再生，跨越临界尺寸的间隙。我们将以三个独立的目标来追求这个假设。 具体目标1：栓系层粘连蛋白肽梯度增强神经细胞迁移和SC浸润。我们将 研究栓系层粘连蛋白肽浓度梯度如何单独增强神经突和SC反应 以及在外植体（多细胞）模型中。该目标的结果将产生最佳的直径， 层粘连蛋白-肽梯度）以推进我们在目标3中提出的体内研究。具体目标2：栓系TGF-β 肽梯度以增强神经细胞迁移和SC浸润。我们将研究浓度如何 基于TGF-β肽的生长因子梯度与RGD组合增强神经突和SC 反应，单独和外植体（多细胞）模型。这一目标的结果将产生一个最佳的收益率。 （直径，TGF-β肽梯度），以推进我们在特定目标3中提出的体内研究。具体目标 3：层粘连蛋白肽梯度和TGF-β的组合改善体内神经再生结果 梯度。我们将使用在目标1和2中独立鉴定的最佳生物支架来研究 层粘连蛋白肽和TGF-β肽浓度梯度的组合是否会协同作用 增强体内良好的神经再生和长期功能恢复的初始过程， 建立大鼠坐骨神经缺损模型。通过关注内源性修复的早期步骤，沿着 长期恢复指标，这项工作将提供基础证据的作用，SC在神经 再生过程。这些知识将使我们的神经修复重点从轴突转移到 已知支持再生过程以提高回收率。

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.