Fibrous substrates, functioning as a temporary extracellular matrix, can be easily prepared by 5 electrospinning which allows to obtain fibrous matrices suitable as internal filler for nerve guidance 6 channels. In this study, gelatin micro-or nano-fibres have been prepared by electrospinning 7 technique by tuning gelatin concentration and solution flow rate. The influence of gelatin fibre 8 diameter on cell adhesion and proliferation was tested in vitro using Schwann cells (SC) and dorsal 9 root ganglia (DRG) explant cultures. Cell adhesion was evaluated by quantifying cell spreading area, 10 actin cytoskeleton organization and focal adhesion complex formation. Nano-fibres showed to 11 promote cell spreading and actin cytoskeleton organization, resulting in higher cellular adhesion 12 and proliferation rate. Yet, cell migration and motility were quantified by transwell and time lapse 13 assays respectively and results showed that cells cultured on micro-fibres displayed higher motility 14 and migration rate. Finally, DRG axon outgrowth resulted to be higher on micro-fibres. These data 15 suggest that gelatin electrospun fibres topography can be adjusted in order to modulate SC and 16 axons organization and that both nano-and micro-fibres are promising fillers for the design of 17 devices for peripheral nerve repair. 18 19

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Electrospun fibrous substrates mimicking extracellular matrices can be prepared by electrospinning, yielding aligned fibrous matrices as internal fillers to manufacture artificial nerves. Gelatin aligned nano-fibers were prepared by electrospinning after tuning the collector rotation speed. The effect of alignment on cell adhesion and proliferation was tested in vitro using primary cultures, the Schwann cell line, RT4-D6P2T, and the sensory neuron-like cell line, 50B11. Cell adhesion and proliferation were assessed by quantifying at several time-points. Aligned nano-fibers reduced adhesion and proliferation rate compared with random fibers. Schwann cell morphology and organization were investigated by immunostaining of the cytoskeleton. Cells were elongated with their longitudinal body parallel to the aligned fibers. B5011 neuron-like cells were aligned and had parallel axon growth when cultured on the aligned gelatin fibers. The data show that the alignment of electrospun gelatin fibers can modulate Schwann cells and axon organization in vitro, suggesting that this substrate shows promise as an internal filler for the design of artificial nerves for peripheral nerve reconstruction.

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Hydrogels are promising materials in regenerative medicine applications due to their hydrophilicity, biocompatibility and capacity to release drugs and growth factors in a controlled manner. In this study, biocompatible and biodegradable hydrogels based on blends of natural polymers were used in in vitro and ex vivo experiments as a tool for VEGF controlled release to accelerate the nerve regeneration process. Among different candidates, the angiogenic factor VEGF was selected since angiogenesis has been long recognized as an important and necessary step during tissue repair. Recent studies pointed out that VEGF has a beneficial effect on motor neuron survival and Schwann cell vitality and proliferation. Moreover, VEGF administration can sustain and enhance the growth of regenerating peripheral nerve fibres. Hydrogel preparation process was optimized to allow VEGF functional incorporation, while preventing its degradation and denaturation. VEGF release was quantified through ELISA assay whereas released VEGF bioactivity was validated in Human Umbilical Vein Endothelial Cells (HUVEC) and in a Schwann cell line (RT4-D6P2T) by assessing VEGFR-2 and downstream effectors Akt and Erk1/2 phosphorylation. Moreover, dorsal root ganglia explants cultured on VEGF releasing hydrogels displayed increased neurite outgrowth proving confirmation that released VEGF maintained its effect, as also confirmed in tubulogenesis assay. In conclusion, a gelatin based hydrogel system for bioactive VEGF delivery was developed and characterized for its applicability in neural tissue engineering.

Various biomaterials have been proposed to build up scaffolds for promoting neural repair. Among them, chitosan, a derivative of chitin, has been raising more and more interestamongbasicandclinicalscientists.Anumberofstudieswithneuronalandglial cellcultures haveshownthat thisbiomaterial has biomimetic properties,which make it a good candidate for developing innovative devices for neural repair. Yet, in vivo experimental studies have shown that chitosan can be successfully used to create scaffolds that promote regeneration both in the central and in the peripheral nervous system. In this review, the relevant literature on the use of chitosan in the nervous tissue, either alone or in combination with other components, is overviewed. Altogether, the promising in vitro and in vivo experimental results make it possible to foresee that time for clinicaltrialswithchitosan-basednerveregenerationpromotingdevicesisapproaching quickly.

Neuregulin 1 (NRG1) is a multifunctional and versatile protein: its numerous isoforms can signal in a paracrine, autocrine or juxtacrine manner, playing a fundamental role during the development of the peripheral nervous system and during the process of nerve repair, suggesting that the treatment with NRG1 could improve functional outcome following injury. Accordingly, the use of NRG1 in vivo has already yielded encouraging results.The aim of this review is to focus on the role played by the different NRG1 isoforms during peripheral nerve regeneration and remyelination and to identify good candidates to be used for the development of tissue engineered medical devices delivering NRG1, with the final goal to promote better nerve repair.

Viral vector-mediated gene transfer of neurotrophic factors is an emerging and promising strategy to promote the regeneration of injured peripheral nerves. Unfortunately, the chronic exposure to neurotrophic factors results in local trapping of regenerating axons or other unwanted side effects. Therefore, tight control of therapeutic gene expression is required. The tetracycline/doxycycline-inducible system is considered to be one of the most promising systems for regulating heterologous gene expression. However, an immune response directed against the transactivator protein rtTA hampers further translational studies. Immunogenic proteins fused with the Gly-Ala repeat of the Epstein-Barr virus Nuclear Antigen-1 protein have been shown to successfully evade the immune system. In this article, we used this strategy to demonstrate that a chimeric transactivator, created by fusing the Gly-Ala repeat with rtTA and embedded in a lentiviral vector (i) retained its transactivator function in vitro, in muscle explants, and in vivo following injection into the rat peripheral nerve, (ii) exhibited a reduced leaky expression, and (iii) had an immune-evasive advantage over rtTA as shown in a novel bioassay for human antigen presentation. The current findings are an important step toward creating a clinically applicable potentially immune-evasive tetracycline-regulatable viral vector system.