Enzymatically‐Modified Melt‐Extruded Guides for Peripheral Nerve Repair

Abstract

In this work, melt-extruded guides for peripheral nerve repair were produced, based on blends between poly-(e-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-(e-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-(e-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-(e-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.

Melt-extruded guides for peripheral nerve regeneration. Part I: Poly(ε-caprolactone)

“…Modifications of PCL by blending with gelatin (Chiono et al 2008) or collagen (Cultispheres) (Bender et al 2004;Waddell et al 2003) have also been performed to improve its cell compatibility. Our group has recently studied PCL/gelatin melt-extruded guides (Chiono et al 2008), which, in some cases, have been modified by grafting poly(L-lysine) on the gelatin domains exposed on the inner tube surface using transglutaminase as an enzyme catalyst. In such a case, the aim of the study has been that to propose an enzymatic technique allowing surface modification of synthetic guides to obtain tubules with biomimetic characteristics (Chiono et al 2008).…”

“…Several studies on PCL have been performed to evaluate the ability of this polymer to be shaped into suitable tubular guides (Bender et al 2004;Chiono et al 2008) for peripheral nerve repair or its in vitro affinity towards the attachment and proliferation of PC12 cells (Waddell et al 2003), Schwann cells (Bender et al 2004) and rat cortical neurons (Bender et al 2004). Modifications of PCL by blending with gelatin (Chiono et al 2008) or collagen (Cultispheres) (Bender et al 2004;Waddell et al 2003) have also been performed to improve its cell compatibility.…”

Biomimetic Materials for Medical Application Through Enzymatic Modification

“…The thering of the two peptides (PPFLMLLKGSTREQQQIVM and PPFLMLLKGSTRKKKKG) using mTGAse enhanced cell attachment, spreading and actin cytoskeleton organization [25]. Chiono et al [21] successfully functionalized melt-extruded guides based on polycaprolactone and gelatin with poly(L-lysine) by mTGase catalysis. A blend between poly(e-caprolactone) and uncrosslinked gelatin was produced for the first time and was then melt-extruded into tube-shaped scaffolds.…”

“…Finally, poly(L-lysine) grafted on the gelatin domains exposed on the inner tube surface using transglutaminase catalysis was synthesized with the aim of conferring guide-specific signaling for nerve cell attachment, proliferation and migration. Confocal microscopy was successfully used to study the accessibility of mTGase towards gelatin substrates using suitable model lysine-rich peptides (FITC-labeled KKKKGY) [21]. Similarly, electrospun gelatin fiber mats were produced from water/acetic acid solutions (10% w/v concentration applied voltage 50 kV; distance needle-collector: 10 cm; collector: wire netting) and then crosslinked by dehydrothermal treatment (Fig.…”

Enzymatic Polymer Modification

“…Besides crosslinking, transglutaminases have been employed for grafting/coating of wool fabrics with silk sericin or keratin leading to increased bursting strength and softness and reduced felting shrinkage [17,18] . In biomedical applications, transglutaminases have been used for tissue engineering [19,20] or for the production of melt -extruded guides for peripheral nerve repair [21] .…”