A Novel Scaffold for the Repair of Peripheral Nerve Injury

Abstract

INTRODUCTION: TPeripheral nerve injuries can result in lifelong disability. Primary nerve repair is used for short nerve defects. Though autologous nerve can bridge longer defects, the harvesting procedure creates donor site morbidity. Nerve conduits lack an aligned internal scaffold to support and guide axonal regeneration. E2 peptide amphiphiles (PA) can self-assemble into aligned nanofibers, and can potentially mimic the native internal architecture of peripheral nerve. Bioactive epitopes IKVAV (Ile-Lys-Val-Ala-Val) and RGDS (Arg-Gly-Asp-Ser) can be incorporated into E2PA nanofibers and have been shown to promote neuronal cell adhesion, growth, and migration. There are no studies to date that examine the ability of E2PA nanofibers to support the proliferation of Schwann cells, key components in peripheral nerve healing. In this preliminary study, we investigate the proliferation of rat Schwann cells after incorporation into aligned E2PA gels combined with bioactive epitopes. METHODS: E2PA was synthesized by Stupp et al, aqueously dissolved, and combined with Schwann cells (cell line RT4-D6P2T). Gels were then formed by pipetting E2PA/cell suspensions into CaCl2 solution. WST-1 proliferation assay was then performed on these gels and compared to collagen-cell gels and 2-dimensional cell culture. WST-1 cell proliferation assays were then performed at days 3,7,14, and 21. Following determination of cell viability in E2PA gels, E2PA and bioactive epitopes (also synthesized by Stupp et al) were then aqueously dissolved, divided into 5 groups (E2 only, E2+IKVAV, E2+RGDS, E2+VVIAK control, and E2+RSDG control) , incorporated with rat Schwann cells at the same cell density, and made into aligned gels. WST-1 assays were performed at days 1 and 7. Immunocytochemistry (ICC) was performed on Schwann cells cultured in aligned E2PA and E2PA combined with RGDS compared to collagen controls, staining for actin and cell-adhesion-molecules. RESULTS: Schwann cells demonstrated increasing proliferation in E2PA gels for at least 21 days. Schwann cells also demonstrated proliferation in aligned gels, which was increased by IKVAV and RGDS compared to E2 alone. ICC revealed attachment and alignment of Schwann cells to aligned E2 gels. CONCLUSION: Schwann cells embedded in aligned E2PA gels combined with epitopes RGDS and RSDG exhibit increasing proliferation compared with E2PA alone. Schwann cells demonstrated successful attachment and alignment to aligned E2. Currently we are investigating the effect that E2PA and epitope composition have on rat dorsal root ganglia neuronal migration, and will use these results for future in-vivo studies.