Acellular nerve allografts (ANGs) represent a promising alternative in nerve repair. Our aim is to improve the structural and biomechanical properties of biocompatible Sondell (SD) and Roosens (RS) based ANGs using genipin (GP) as a crosslinker agent ex vivo. The impact of two concentrations of GP (0.10% and 0.25%) on Wistar rat sciatic nerve-derived ANGs was assessed at the histological, biomechanical, and biocompatibility levels. Histology confirmed the differences between SD and RS procedures, but not remarkable changes were induced by GP, which helped to preserve the nerve histological pattern. Tensile test revealed that GP enhanced the biomechanical properties of SD and RS ANGs, being the crosslinked RS ANGs more comparable to the native nerves used as control. The evaluation of the ANGs biocompatibility conducted with adipose-derived mesenchymal stem cells cultured within the ANGs confirmed a high degree of biocompatibility in all ANGs, especially in RS and RS-GP 0.10% ANGs. Finally, this study demonstrates that the use of GP could be an efficient alternative to improve the biomechanical properties of ANGs with a slight impact on the biocompatibility and histological pattern. For these reasons, we hypothesize that our novel crosslinked ANGs could be a suitable alternative for future in vivo preclinical studies.

In critical nerve gap repair, decellularized nerve allografts are considered a promising tissue engineering strategy that can provide superior regeneration results compared to nerve conduits. Decellularized nerves offer a well-conserved extracellular matrix component that has proven to play an important role in supporting axonal guiding and peripheral nerve regeneration. Up to now, the known decellularized techniques are time and effort consuming. The present study, performed on rat sciatic nerves, aims at investigating a novel nerve decellularization protocol able to combine an effective decellularization in short time with a good preservation of the extracellular matrix component. To do this, a decellularization protocol proven to be efficient for tendons (DN-P1) was compared with a decellularization protocol specifically developed for nerves (DN-P2). The outcomes of both the decellularization protocols were assessed by a series of in vitro evaluations, including qualitative and quantitative histological and immunohistochemical analyses, DNA quantification, SEM and TEM ultrastructural analyses, mechanical testing, and viability assay. The overall results showed that DN-P1 could provide promising results if tested in vivo, as the in vitro characterization demonstrated that DN-P1 conserved a better ultrastructure and ECM components compared to DN-P2. Most importantly, DN-P1 was shown to be highly biocompatible, supporting a greater number of viable metabolically active cells.

Nerves are subjected to tensile forces in various paradigms such as injury and regeneration, joint movement, and rehabilitation treatments, as in the case of neurodynamic treatment (NDT). The NDT induces selective uniaxial repeated tension on the nerve and was described to be an effective treatment to reduce pain in patients. Nevertheless, the biological mechanisms activated by the NDT promoting the healing processes of the nerve are yet still unknown. Moreover, a dose–response analysis to define a standard protocol of treatment is unavailable. In this study, we aimed to define in vitro whether NDT protocols could induce selective biological effects on sensory and motor neurons, also investigating the possible involved molecular mechanisms taking a role behind this change. The obtained results demonstrate that NDT induced significant dose-dependent changes promoting cell differentiation, neurite outgrowth, and neuron survival, especially in nociceptive neurons. Notably, NDT significantly upregulated PIEZO1 gene expression. A gene that is coding for an ion channel that is expressed both in murine and human sensory neurons and is related to mechanical stimuli transduction and pain suppression. Other genes involved in mechanical allodynia related to neuroinflammation were not modified by NDT. The results of the present study contribute to increase the knowledge behind the biological mechanisms activated in response to NDT and to understand its efficacy in improving nerve regenerational physiological processes and pain reduction.

Peripheral nerves are highly susceptible to injuries induced from everyday activities such as falling or work and sport accidents as well as more severe incidents such as car and motorcycle accidents. Many efforts have been made to improve nerve regeneration, but a satisfactory outcome is still unachieved, highlighting the need for easy to apply supportive strategies for stimulating nerve growth and functional recovery. Recent focus has been made on the effect of the consumed diet and its relation to healthy and well-functioning body systems. Normally, a balanced, healthy daily diet should provide our body with all the needed nutritional elements for maintaining correct function. The health of the central and peripheral nervous system is largely dependent on balanced nutrients supply. While already addressed in many reviews with different focus, we comprehensively review here the possible role of different nutrients in maintaining a healthy peripheral nervous system and their possible role in supporting the process of peripheral nerve regeneration. In fact, many dietary supplements have already demonstrated an important role in peripheral nerve development and regeneration; thus, a tailored dietary plan supplied to a patient following nerve injury could play a non-negotiable role in accelerating and promoting the process of nerve regeneration.

Peripheral nerves are characterised by the ability to regenerate after injury. Schwann cell activity is fundamental for all steps of peripheral nerve regeneration: immediately after injury they de-differentiate, remove myelin debris, proliferate and repopulate the injured nerve. Soluble Neuregulin1 (NRG1) is a growth factor that is strongly up-regulated and released by Schwann cells immediately after nerve injury. To identify the genes regulated in Schwann cells by soluble NRG1, we performed deep RNA sequencing to generate a transcriptome database and identify all the genes regulated following 6 h stimulation of primary adult rat Schwann cells with soluble recombinant NRG1. Interestingly, the gene ontology analysis of the transcriptome reveals that NRG1 regulates genes belonging to categories that are regulated in the peripheral nerve immediately after an injury. In particular, NRG1 strongly inhibits the expression of genes involved in myelination and in glial cell differentiation, suggesting that NRG1 might be involved in the de-differentiation (or “trans-differentiation”) process of Schwann cells from a myelinating to a repair phenotype. Moreover, NRG1 inhibits genes involved in the apoptotic process, and up-regulates genes positively regulating the ribosomal RNA processing, thus suggesting that NRG1 might promote cell survival and stimulate new protein expression. This in vitro transcriptome analysis demonstrates that in Schwann cells NRG1 drives the expression of several genes which partially overlap with genes regulated in vivo after peripheral nerve injury, underlying the pivotal role of NRG1 in the first steps of the nerve regeneration process.

Conduits for the repair of peripheral nerve gaps are a good alternative to autografts as they provide a protected environment and a physical guide for axonal re-growth. Conduits require colonization by cells involved in nerve regeneration (Schwann cells, fibroblasts, endothelial cells, macrophages) while in the autograft many cells are resident and just need to be activated. Since it is known that soluble Neuregulin1 (sNRG1) is released after injury and plays an important role activating Schwann cell dedifferentiation, its expression level was investigated in early regeneration steps (7, 14, 28 days) inside a 10 mm chitosan conduit used to repair median nerve gaps in Wistar rats. In vivo data show that sNRG1, mainly the isoform α, is highly expressed in the conduit, together with a fibroblast marker, while Schwann cell markers, including NRG1 receptors, were not. Primary culture analysis shows that nerve fibroblasts, unlike Schwann cells, express high NRG1α levels, while both express NRG1β. These data suggest that sNRG1 might be mainly expressed by fibroblasts colonizing nerve conduit before Schwann cells. Immunohistochemistry analysis confirmed NRG1 and fibroblast marker co-localization. These results suggest that fibroblasts, releasing sNRG1, might promote Schwann cell dedifferentiation to a “repair” phenotype, contributing to peripheral nerve regeneration.

Schwann cell reprogramming and differentiation are crucial prerequisites for neuronal regeneration and re-myelination to occur following injury to peripheral nerves. The neurotrophin receptor p75 NTR has been identified as a positive modulator for Schwann cell myelination during development and implicated in promoting nerve regeneration after injury. However, most studies base this conclusion on results obtained from complete p75 NTR knockout mouse models and cannot dissect the specific role of p75 NTR expressed by Schwann cells. In this present study, a conditional knockout model selectively deleting p75 NTR expression in Schwann cells was generated, where p75 NTR expression is replaced with that of an mCherry reporter. Silencing of Schwann cell p75 NTR expression was confirmed in the sciatic nerve in vivo and in vitro , without altering axonal expression of p75 NTR . No difference in sciatic nerve myelination during development or following sciatic nerve crush injury was observed, as determined by quantification of both myelinated and unmyelinated nerve fiber densities, myelinated axonal diameter and myelin thickness. However, the absence of Schwann cell p75 NTR reduced motor nerve conduction velocity after crush injury. Our data indicate that the absence of Schwann cell p75 NTR expression in vivo is not critical for axonal regrowth or remyelination following sciatic nerve crush injury, but does play a key role in functional recovery. Overall, this represents the first step in redefining the role of p75 NTR in the peripheral nervous system, suggesting that the Schwann cell-axon unit functions as a syncytium, with the previous published involvement of p75 NTR in remyelination most likely depending on axonal/neuronal p75 NTR and/or mutual glial-axonal interactions.

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