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

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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.