Assessment of the keratin effect on the angiogenic response of chitosan membranes envisioning peripheral nerve regeneration

Peripheral nerve injuries have severe consequences, as loss of mobility and sensitive function. In the last years, great efforts have been made to develop an advanced tissue engineering (TE) approach as an alternative to autologous nerve transplantation [1]. Chitosan has been widely used to guide peripheral nerve regeneration (PNR). Besides its appealing properties for TE applications, as biocompatibility, biodegradability and its similarity to natural glycosaminoglycans, chitosan has shown to have superior performance on proliferation and orientation of Schwan cells and on differentiation of neuronal cells [2]. Keratin has also been reported as a suitable biomaterial for PNR by its neuroinductive properties [3]. A keratin-enriched chitosan membrane has been developed by our group for PNR. Vascularization is a critical factor for nerve regeneration by supporting the viability of Schwan and associated neuronal cells and also by guiding the migration of Schwan cells due to the chemotactic effect of endothelial cells’ secreted molecules [4]. The angiogenic response of chitosan and chitosan/keratin membranes was explored by using an optimized version of a simple and widely used platform for angiogenesis research: the chick embryo chorioallantoic membrane assay [5]. The results were expressed as described by Silva-Correia et al. [5], by semiquantification of the number of blood vessels converging to implanted materials, after four days of implantation, and qualitatively evaluated by Hematoxylin & Eosin staining and immunohistochemical detection of chick-endothelial cells. Results suggest that keratin increments the angiogenic potential of chitosan membranes by demonstrating a statistically higher number of convergent blood vessels when compared to chitosan membranes. Histological characterization reinforces this evidence by showing that, although the membranes were impermeable to cellular infiltration, in chitosan/keratin implanted CAM sections it’s possible to notice endothelial cells’ migration and microvessels formation on the proximity of keratin-enriched membranes. The improvement of the angiogenic properties of chitosan by keratin blending has proven to be a promising strategy to increase vascularization of chitosan nerve-grafts, impacting simultaneously on neurogenesis and nerve regeneration.