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

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.

The peripheral nervous system has an intrinsic capability to regenerate, crucially related to the ability of Schwann cells (SC) to create a permissive environment, for example, through production of regeneration-promoting neurotrophic factors. Survival, proliferation, migration and differentiation of SC into a myelinating phenotype during development and after injury is regulated by different Neuregulin1 (NRG1) isoforms. This study investigates the expression of different NRG1 isoforms and of their ErbB receptors in distal rat median nerve samples under regenerating conditions after a mild (crush) and more severe (end-to-end repair) injury and under degenerating condition. The expression of the NRG1/ErbB system was evaluated at mRNA and protein level, and demonstrated to be specific for distinct and consecutive phases following nerve injury and regeneration or the progress in degeneration. For the first time a detailed analysis of expression profiles not only of soluble and transmembrane NRG1 isoforms, but also of alpha and beta as well as type a, b and c isoforms is presented. The results of mRNA and protein expression pattern analyses were related to nerve ultrastructure changes evaluated by electron microscopy. In particular, transmembrane NRG1 isoforms are differentially regulated and proteolytically processed under regeneration and degeneration conditions. Soluble NRG1 isoforms alpha and beta, as well as type a and b, are strongly upregulated during axonal regrowth, while type c NRG1 isoform is downregulated. This is accompanied by an upregulation of ErbB receptors. This accurate regulation suggests that each molecule plays a specific role that could be clinically exploited to improve nerve regeneration.

Peripheral nerve injury treatment is a relevant problem because of nerve lesion high incidence and because of unsatisfactory regeneration after severe injuries, thus resulting in a reduced patient's life quality. To repair severe nerve injuries characterized by substance loss and to improve the regeneration outcome at both motor and sensory level, different strategies have been investigated. Although autograft remains the gold standard technique, a growing number of research articles concerning nerve conduit use has been reported in the last years. Nerve conduits aim to overcome autograft disadvantages, but they must satisfy some requirements to be suitable for nerve repair. A universal ideal conduit does not exist, since conduit properties have to be evaluated case by case; nevertheless, because of their high biocompatibility and biodegradability, natural-based biomaterials have great potentiality to be used to produce nerve guides. Although they share many characteristics with synthetic biomaterials, natural-based biomaterials should also be preferable because of their extraction sources; indeed, these biomaterials are obtained from different renewable sources or food waste, thus reducing environmental impact and enhancing sustainability in comparison to synthetic ones. This review reports the strengths and weaknesses of natural-based biomaterials used for manufacturing peripheral nerve conduits, analyzing the interactions between natural-based biomaterials and biological environment. Particular attention was paid to the description of the preclinical outcome of nerve regeneration in injury repaired with the different natural-based conduits.

OBJECTIVE Multiple factors may affect functional recovery after peripheral nerve injury, among them the lesion site and the interval between the injury and the surgical repair. When the nerve segment distal to the lesion site undergoes chronic degeneration, the ensuing regeneration (when allowed) is often poor. The aims of the current study were as follows: 1) to examine the expression changes of the neuregulin 1/ErbB system during long-term nerve degeneration; and 2) to investigate whether a chronically denervated distal nerve stump can sustain nerve regeneration of freshly axotomized axons. METHODS This study used a rat surgical model of delayed nerve repair consisting of a cross suture between the chronically degenerated median nerve distal stump and the freshly axotomized ulnar proximal stump. Before the suture, a segment of long-term degenerated median nerve stump was harvested for analysis. Functional, morphological, morphometric, and biomolecular analyses were performed. RESULTS The results showed that neuregulin 1 is highly downregulated after chronic degeneration, as well as some Schwann cell markers, demonstrating that these cells undergo atrophy, which was also confirmed by ultrastructural analysis. After delayed nerve repair, it was observed that chronic degeneration of the distal nerve stump compromises nerve regeneration in terms of functional recovery, as well as the number and size of regenerated myelinated fibers. Moreover, neuregulin 1 is still downregulated after delayed regeneration. CONCLUSIONS The poor outcome after delayed nerve regeneration might be explained by Schwann cell impairment and the consequent ineffective support for nerve regeneration. Understanding the molecular and biological changes occurring both in the chronically degenerating nerve and in the delayed nerve repair may be useful to the development of new strategies to promote nerve regeneration. The results suggest that neuregulin 1 has an important role in Schwann cell activity after denervation, indicating that its manipulation might be a good strategy for improving outcome after delayed nerve repair.

Background-Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease caused by deficient ␤-glucuronidase (GUSB) activity resulting in defective catabolism of glycosaminoglycans (GAGs). Cardiac disease is a major cause of death in MPS VII because of accumulation of GAGs in cardiovascular cells. Manifestations include cardiomyopathy, mitral and aortic valve thickening, and aortic root dilation and may cause death in the early months of life or may be compatible with a fairly normal lifespan. We previously reported that neonatal administration of a retroviral vector (RV) resulted in transduction of hepatocytes, which secreted GUSB into the blood and could be taken up by cells throughout the body. The goal of this study was to evaluate the effect on cardiac disease. Methods and Results-Six MPS VII dogs were treated intravenously with an RV-expressing canine GUSB. Echocardiographic parameters, cardiovascular lesions, and biochemical parameters of these dogs were compared with those of normal and untreated MPS VII dogs. Conclusions-RV-treated dogs were markedly improved compared with untreated MPS VII dogs. Most RV-treated MPS VII dogs had mild or moderate mitral regurgitation at 4 to 5 months after birth, which improved or disappeared when evaluated at 9 to 11 and at 24 months. Similarly, mitral valve thickening present early in some animals disappeared over time, whereas aortic dilation and aortic valve thickening were absent at all times. Both myocardium and aorta had significant levels of GUSB and reduction in GAGs. Key Words: cardiovascular diseases Ⅲ gene therapy Ⅲ lysosomes Ⅲ mucopolysaccharidosis T he mucopolysaccharidoses (MPS) are a family of lysosomal storage diseases resulting from defective catabolism of glycosaminoglycans (GAGs) by 1 of 11 enzymes. 1,2 In humans, the most common cardiovascular lesion, regardless of MPS type, is thickening of the mitral valve with regurgitation or stenosis.3 Aortic valve thickening and hypertrophic cardiomyopathy are the next most common lesions, with endocardial thickening and dilated cardiomyopathy also recognized. 3 GAGs accumulate in the valve leaflets, with secondary fibrosis and nodular deformation. Primary myocardial involvement and infiltration of the coronary arteries with GAGs can also occur. 4 Cardiac involvement is present in most patients with MPS, and the lesions are progressive, with risk of death as a result of congestive heart failure. 3 A colony of MPS VII (␤-glucuronidase [GUSB]-deficient) dogs 5 have a mutation resulting in a single amino acid substitution, and cardiac abnormalities in affected dogs have been reported previously. 6,7 Enzyme replacement therapy, the intravenous injection of normal enzyme, 8 has been successful in MPS VI mice, MPS I dogs 9 and cats, 10 and human MPS I patients 11 ; however, cardiac function was not specifically evaluated. 12 Bone marrow transplantation (BMT) has been effective in MPS VII dogs 6 and also appeared to be beneficial in some MPS I, IV, and VI children. 13,14 Cardiac lysosomal storage was reduced in t...

Muscle-in-vein conduit is successfully employed for repairing nerve injuries: the vein prevents muscle fiber dispersion, while the muscle prevents the vein collapse and creates a favorable environment for Schwann cell migration and axon regrowth. However, it requires microsurgical skills. In this study we show a simple strategy to improve the performance of a chitosan hollow tube by the introduction of fresh skeletal muscle fibers. The hypothesis is to overcome the technical issue of the muscle-in-vein preparation and to take advantage of fiber muscle properties to create an easy and effective conduit for nerve regeneration. Rat median nerve gaps were repaired with chitosan tubes filled with skeletal muscle fibers (muscle-in-tube graft), hollow chitosan tubes, or autologous nerve grafts. Our results demonstrate that the fresh skeletal muscle inside the conduit is an endogenous source of soluble Neuregulin 1, a key factor for Schwann cell survival and dedifferentiation, absent in the hollow tube during the early phase of regeneration. However, nerve regeneration assessed at late time point was similar to that obtained with the hollow tube. To conclude, the musclein-tube graft is surgically easy to perform and we suggest that it might be a promising strategy to repair longer nerve gap or for secondary nerve repair, situations in which Schwann cell atrophy is a limiting factor for recovery.

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.