Enzymatically-modified melt-extruded guides for peripheral nerve repair

In this work, melt-extruded guides for peripheral nerve repair were produced, based on blends between poly-(epsilon-caprolactone) and gelatin with a low gelatin amount (10 wt. %), with the aim of combining the good melt process ability of the synthetic polymer with the optimal biocompatibility of the natural polymer. In one case, a blend was produced between poly- (epsilon-caprolactone) and gelatin previously crosslinked by transglutaminase, using a solution mixing technique. The blend was then melt-extruded obtaining nerve guidance channels. In another case, a blend between poly- (epsilon-caprolactone) and uncrosslinked gelatin was first produced, then melt-extruded into tube-shaped manufacts. Finally, poly-L-lysine was grafted on the gelatin domains exposed on the inner tube surface using transglutaminase catalysis, with the aim to confer to the channel guide a specific signaling for nerve cells attachment, proliferation and migration. Scanning electron microscopy (SEM) and Fourier transform infrared-attenuated total reflectance spectroscopy (FTIR-ATR) coupled with Chemical Imaging analysis showed that the obtained binary blends were poor compatible, however, appropriate mixing techniques might lead to an homogeneous distribution of gelatin domains within the poly- (epsilon-caprolactone) matrix. Confocal microscopy (CM) was applied as a powerful tool to study the accessibility of mTGase towards gelatin substrates, using suitable model lysine-rich peptides (FITC-labeled KKKKGY). Confocal microscopy also gave a confirmation of poly-L-lysine enzymatic grafting on the exposed gelatin domains on the inner blend tube surface. In vitro cell tests using S5Y5 neuroblastoma cells showed that the produced nerve guides were biocompatible, however, gelatin was confirmed to be a non-specific protein for the attachment and proliferation of nerve cells. On the other hand, poly-L-lysine functionalization of the inner tube surface greatly improved the in-vitro cell response.