Conductive nanocomposite and micro-grooved scaffolds based on oxidized polyvinyl alcohol and carbon nanotubes for peripheral nerve regeneration

The surgical approach to peripheral nerve injuries (PNI) with substance loss depends on injury severity. In case of severe lesions that cannot be managed by end-to-end suture, interposition of a nerve autograft represents the current gold standard; however, this approach is not free from limitations, boosting intense research over the development of comorbidities-free alternatives: “on-the-bench” resorbable nerve conduits (NCs). To date, the Food and Drug Administration (FDA) has approved 11 conduits for PNIs repair but the results they guarantee for are still not fully satisfactory. In this research project, the new synthetic and bioresorbable polymer oxidized polyvinyl alcohol (OxPVA) was considered for innovative NCs prototyping. To this purpose, OxPVA cryogels behaviour was modulated by a twofold strategy: (i) hybridization with multi-walled carbon nanotubes (MWCNTs), (ii) exerting precise control over scaffold superficial geometry. The new nanocomposite hydrogels were fabricated by physical cross-linking (freeze-thawing) after mechanical embedding of MWNTs (OxPVA+CNTs) (CNTs concentration of 0.1 wt%) into OxPVA polymer solution. Hence, scaffolds electroconductivity, mechanical behaviour, in vitro cytotoxicity (cytotoxicity extract test over SHSY-5Y cells) and in vivo biocompatibility (dorsal subcutaneous implant in BALB/c mice) were verified. The hybridization with CNTs conferred an enhanced superficial electroconductivity of 1.81 · 10^(-6) ± 2.00 · 10^(-7) Sm-1 to the material, but, as proved by uniaxial tensile test, it did not modify the mechanical properties. Potential CNT-related toxicity was excluded, in fact no cytotoxic response was detected over SH-SY5Y cells at both 24 and 72 h. After 14-days of subcutaneous implant, the OxPVA+CNTs scaffolds elicited only a mild immune reaction but no evident thick fibrotic capsule was observed. After this preliminary characterization of OxPVA+CNTs scaffolds, the hybrid polymer solution was used for NCs fabrication by cast-moulding technique. At first, the NCs were investigated for their mechanical behaviour, and then implanted in an animal model of disease (Sprague Dawley rat; sciatic nerve, gap: 5 mm) to verify their effectiveness in supporting nerve regeneration; a period of 6 weeks was considered. In accordance with experimental data referring to membranes, uniaxial tensile test did not highlight significant difference in OxPVA+CNTs NCs behaviour than OxPVA NCs. Focusing on the preclinical study evidences, at 6 weeks from surgery, the NCs were clearly identifiable without showing dislocations or neuroma formation; moreover, axonal regeneration triggered by OxPVA+CNTs NCs was confirmed by histology. To furtherly improve OxPVA bioactivity, ultrastructural modification was introduced. In particular, linear patterned grooves were adopted to enhance an adequate neuron directional growth. Patterned moulds were designed with a Computer Aided Design (CAD) software and printed with a Fused Filament Fabrication (FFF) 3D printer. Hence, OxPVA surface was impressed with the moulds and the bioactivity of the derived scaffolds was assessed over SHSH-5Y cells. The scaffolds with more pronounced grooves (groove (g): 300 um / depth (d): 500 um, g: 300 um / d: 700 um, g: 500 um / d: 700 um) proved to be the most effective in promoting cell adhesion and proliferation which was evaluated at 7 and 14 days from seeding. According to the results gathered within this study, OxPVA hybridization with CNTs seems to be a promising approach for the development of conductive NCs, triggering nerve regeneration; in addition, as highlighted by in vitro evidences, neuro-instructive geometries are also an interesting strategy to induce cell-scaffold interaction. Future direction of this study may focus on combining these two methods, obtaining an appealing complex device supporting regeneration of severely injured peripheral nerves.