INTRODUCTION Peripheral nerve injuries often result in painful neuropathies owing to reduction in motor function and sensory perception. When large nerve gaps exist (20mm or longer in humans), sensory nerve autografts are conventionally used to treat neural defects. The main issues related to autografts are shortage of donor nerves, a mismatch of donor nerve size with the recipient site, and occurrences of neuroma formation. Recent advances in nanotechnology and tissue engineering have been found to cover a broad range of applications in regenerative medicine and offer the most effective strategy to repair neural defects. Prior work in this area has shown the utility of collagen-based scaffolds for the regeneration of nerve tissue. This work focuses on the fabrication of collagen scaffolds with two different pore sizes, with the aim of evaluating the effects of pore size on the migration of Schwann cell lines. EXPERIMENTAL METHODS Scaffold fabrication and crosslinking Porous cylindrical scaffolds (diameter=2mm, length=10mm) with aligned channels were fabricated by freeze-drying a 2wt% collagen suspension along a one-dimensional temperature gradient (along the length of the cylindrical scaffold). Scaffolds with two different pore sizes were fabricated by freezing the collagen suspension at two different final freezing temperatures (-20°C and -60°C). The scaffolds were then subjected to dehydrothermal (DHT) cross-linking, followed by a carbodiimide based chemical crosslinking. Qualitative characterization of the pore structure was performed by means of scanning electron microscopy (SEM). Cell culture and cytocompatibility A rat Schwann cell line, RSC96, was expanded in monolayer culture in a 96-well plate. The plate was then incubated at 37°C and 5% CO2 for 24 hours. After 24 hours, sterilized scaffolds were placed vertically to the wells of the 96-well plate and incubated again at 37°C and 5% CO2. At 1, 3, 7, and 10 days, the cell-seeded scaffolds were fixed in 10% formalin and processed for paraffin embedding. Schwann cells were quantified by embedding the cell-seeded scaffolds in paraffin blocks, sectioning, staining them with hematoxylin & eosin stain (H&E stain) and visualizing under a microscope. MTT assay was also performed at 1, 3, 7 and 10 days to evaluate the cell viability. RESULTS AND DISCUSSION SEM demonstrated that both freezing temperature and rate of freezing affect significantly the pore size. As shown in Fig.1, lower temperatures (-60°C) resulted in smaller pore sizes (~85µm), while higher temperatures (-20°C) resulted in much larger pores (~120µm). The longitudinal sections of the samples showed that the pores were in axial orientation disregard of the freezing temperature. MTT assay revealed that cell viability on the two different types of scaffolds increased gradually from first to the tenth day after seeding. Although there was not much difference between the two porous scaffolds on day 1, 3 and 7, on day 10 there was a slight increase in the cell number in the scaffolds with a larger pore size (-20°C). In spite of the different pore dimensions under investigation, the cell migration studies revealed that Schwann cells could migrate through the entire length of both types of scaffolds, by day 7. CONCLUSION Both types of scaffolds were found to support Schwann cell growth and migration, which is the key factor required for the regeneration of nerve tissue. Further studies are proposed regarding the addition of laminin and the evaluation of its effects on the cell growth and migration. REFERENCES 1. W. Daly et al., J. R. Soc. Interface 9:202-221, 2012.
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