SilkBridge™: a novel biomimetic and biocompatible silk-based nerve conduit.

Silk fibroin (Bombyx mori) was used to manufacture a nerve conduit (SilkBridge™) characterized by a novel 3D architecture. The wall of the conduit consists of two electrospun layers (inner and outer) and one textile layer (middle), perfectly integrated at the structural and functional level. The manufacturing technology conferred high compression strength on the device, thus meeting clinical requirements for physiological and pathological compressive stresses. In vitro cell interaction studies were performed through direct contact assays with SilkBridge™ using the glial RT4-D6P2T cells, a schwannoma cell line, and a mouse motor neuron NSC-34 cell line. The results revealed that the material is capable of sustaining cell proliferation, that the glial RT4-D6P2T cells increased their density and organized themselves in a glial-like morphology, and that NSC-34 motor neurons exhibited a greater neuritic length with respect to the control substrate. In vivo pilot assays were performed on adult female Wistar rats. A 10 mm long gap in the median nerve was repaired with 12 mm SilkBridge™. At two weeks post-operation several cell types colonized the lumen. Cells and blood vessels were also visible between the different layers of the conduit wall. Moreover, the presence of regenerated myelinated fibers with a thin myelin sheath at the proximal level was observed. Taken together, all these results demonstrated that SilkBridge™ has an optimized balance of biomechanical and biological properties, being able to sustain a perfect cellular colonization of the conduit and the progressive growth of the regenerating nerve fibers.

Evaluation of Vascular Endothelial Growth Factor (VEGF) and Its Family Member Expression After Peripheral Nerve Regeneration and Denervation.

Vascular endothelial growth factor (VEGF) represents one of the main factors involved not only in angiogenesis and vasculogenesis but also in neuritogenesis. VEGF plays its function acting via different receptors: VEGF receptor1 (VEGFR-1), VEGF receptor2 (VEGFR-2), VEGF receptor3 (VEGFR-3), and co-receptors Neuropilin-1 (NRP1) and Neuropilin-2 (NRP2). This study reports on the first in vivo analysis of the expression of VEGF and VEGF family molecules in peripheral nerve degeneration and regeneration: for this purpose, different models of nerve lesion in rat were adopted, the median nerve crush injury and the median nerve transaction followed or not by end-to end microsurgical repair. Results obtained by real time polymerase chain reaction showed that VEGF and VEGF family molecules are differentially expressed under regenerating and degenerating condition, furthermore, in order to study the modulation and involvement of these factors in two different regenerative models, crush injury and end-to-end repair, protein expression analysis was evaluated. In addition, immunohistochemical analysis allowed to state a glial localization of VEGF and VEGFR-2 after peripheral nerve crush injury. Finally in vitro assay on primary Schwann cells culture show that VEGF165 stimulation increases Schwann cells migration, a major process in the promotion of neurite outgrowth. Anat Rec, 301:1646-1656, 2018. © 2018 Wiley Periodicals, Inc.

Chitosan crosslinked flat scaffolds for peripheral nerve regeneration.

Chitosan (CS) has been widely used in a variety of biomedical applications, including peripheral nerve repair, due to its excellent biocompatibility, biodegradability, readily availability and antibacterial activity. In this study, CS flat membranes, crosslinked with dibasic sodium phosphate (DSP) alone (CS/DSP) or in association with the γ-glycidoxypropyltrimethoxysilane (CS/GPTMS_DSP), were fabricated with a solvent casting technique. The constituent ratio of crosslinking agents and CS were previously selected to obtain a composite material having both adequate mechanical properties and high biocompatibility. In vitro cytotoxicity tests showed that both CS membranes allowed cell survival and proliferation. Moreover, CS/GPTMS_DSP membranes promoted cell adhesion, induced Schwann cell-like morphology and supported neurite outgrowth from dorsal root ganglia explants. Preliminary in vivo tests carried out on both types of nerve scaffolds (CS/DSP and CS/GPTMS_DSP membranes) demonstrated their potential for: (i) protecting, as a membrane, the site of nerve crush or repair by end-to-end surgery and avoiding post-operative nerve adhesion; (ii) bridging, as a conduit, the two nerve stumps after a severe peripheral nerve lesion with substance loss. A 1 cm gap on rat median nerve was repaired using CS/DSP and CS/GPTMS_DSP conduits to further investigate their ability to induce nerve regeneration in vivo. CS/GPTMS_DSP tubes resulted to be more fragile during suturing and, along a 12 week post-operative lapse of time, they detached from the distal nerve stump. On the contrary CS/DSP conduits promoted nerve fiber regeneration and functional recovery, leading to an outcome comparable to median nerve repaired by autograft.

In vitro models for peripheral nerve regeneration.

The study of peripheral nerve repair and regeneration is particularly relevant in the light of the high clinical incidence of nerve lesions. However, the clinical outcome after nerve lesions is often far from satisfactory and the functional recovery is almost never complete. Therefore, a number of therapeutic approaches are being investigated, ranging from local delivery of trophic factors and other molecules to bioactive biomaterials and complex nerve prostheses. Translation of the new therapeutic approaches to the patient always requires a final pre-clinical step using in vivo animal models. The need to limit as much as possible animal use in biomedical research, however, makes the preliminary use of in vitro models mandatory from an ethical point of view. In this article, the different types of in vitro models available today for the study of peripheral nerve regeneration have been ranked by adopting a three-step stair model based on their increasing ethical impact: (i) cell line-based models, which raise no ethical concern; (ii) primary cell-based models, which have low ethical impact as animal use, although necessary, is limited; and (iii) organotypic ex vivo-based models, which raise moderate ethical concerns as the use of laboratory animals is required although with much lower impact on animal wellbeing in comparison to in vivo models of peripheral nerve regeneration. This article aims to help researchers in selecting the best experimental approach for their scientific goals driven by the 'Three Rs' (3Rs) rules (Replacement, Reduction or Refinement of animal use in research) for scientific research.

Effect of vascular endothelial growth factor gene therapy on post-traumatic peripheral nerve regeneration and denervation-related muscle atrophy.

Functional recovery after peripheral nerve injury depends on both improvement of nerve regeneration and prevention of denervation-related skeletal muscle atrophy. To reach these goals, in this study we overexpressed vascular endothelial growth factor (VEGF) by means of local gene transfer with adeno-associated virus (AAV). Local gene transfer in the regenerating peripheral nerve was obtained by reconstructing a 1-cm-long rat median nerve defect using a vein segment filled with skeletal muscle fibers that have been previously injected with either AAV2-VEGF or AAV2-LacZ, and the morphofunctional outcome of nerve regeneration was assessed 3 months after surgery. Surprisingly, results showed that overexpression of VEGF in the muscle-vein-combined guide led to a worse nerve regeneration in comparison with AAV-LacZ controls. Local gene transfer in the denervated muscle was obtained by direct injection of either AAV2-VEGF or AAV2-LacZ in the flexor digitorum sublimis muscle after median nerve transection and results showed a significantly lower progression of muscle atrophy in AAV2-VEGF-treated muscles in comparison with muscles treated with AAV2-LacZ. Altogether, our results suggest that local delivery of VEGF by AAV2-VEGF-injected transplanted muscle fibers do not represent a rational approach to promote axonal regeneration along a venous nerve guide. By contrast, AAV2-VEGF direct local injection in denervated skeletal muscle significantly attenuates denervation-related atrophy, thus representing a promising strategy for improving the outcome of post-traumatic neuromuscular recovery after nerve injury and repair.

Use of poly(DL-lactide-ε-caprolactone) membranes and mesenchymal stem cells from the Wharton's jelly of the umbilical cord for promoting nerve regeneration in axonotmesis: in vitro and in vivo analysis.

Cellular systems implanted into an injured nerve may produce growth factors or extracellular matrix molecules, modulate the inflammatory process and eventually improve nerve regeneration. In the present study, we evaluated the therapeutic value of human umbilical cord matrix MSCs (HMSCs) on rat sciatic nerve after axonotmesis injury associated to Vivosorb® membrane. During HMSCs expansion and differentiation in neuroglial-like cells, the culture medium was collected at 48, 72 and 96 h for nuclear magnetic resonance (NMR) analysis in order to evaluate the metabolic profile. To correlate the HMSCs ability to differentiate and survival capacity in the presence of the Vivosorb® membrane, the [Ca(2+)]i of undifferentiated HMSCs or neuroglial-differentiated HMSCs was determined by the epifluorescence technique using the Fura-2AM probe. The Vivosorb® membrane proved to be adequate and used as scaffold associated with undifferentiated HMSCs or neuroglial-differentiated HMSCs. In vivo testing was carried out in adult rats where a sciatic nerve axonotmesis injury was treated with undifferentiated HMSCs or neuroglial differentiated HMSCs with or without the Vivosorb® membrane. Motor and sensory functional recovery was evaluated throughout a healing period of 12 weeks using sciatic functional index (SFI), extensor postural thrust (EPT), and withdrawal reflex latency (WRL). Stereological analysis was carried out on regenerated nerve fibers. In vitro investigation showed the formation of typical neuroglial cells after differentiation, which were positively stained for the typical specific neuroglial markers such as the GFAP, the GAP-43 and NeuN. NMR showed clear evidence that HMSCs expansion is glycolysis-dependent but their differentiation requires the switch of the metabolic profile to oxidative metabolism. In vivo studies showed enhanced recovery of motor and sensory function in animals treated with transplanted undifferentiated and differentiated HMSCs that was accompanied by an increase in myelin sheath. Taken together, HMSC from the umbilical cord Wharton jelly might be useful for improving the clinical outcome after peripheral nerve lesion.

Use of hybrid chitosan membranes and N1E-115 cells for promoting nerve regeneration in an axonotmesis rat model.

Many studies have been dedicated to the development of scaffolds for improving post-traumatic nerve regeneration. The goal of this study was to develop and test hybrid chitosan membranes to use in peripheral nerve reconstruction, either alone or enriched with N1E-115 neural cells. Hybrid chitosan membranes were tested in vitro, to assess their ability in supporting N1E-115 cell survival and differentiation, and in vivo to assess biocompatibility as well as to evaluate their effects on nerve fiber regeneration and functional recovery after a standardized rat sciatic nerve crush injury. Functional recovery was evaluated using the sciatic functional index (SFI), the static sciatic index (SSI), the extensor postural thrust (EPT), the withdrawal reflex latency (WRL) and ankle kinematics. Nerve fiber regeneration was assessed by quantitative stereological analysis and electron microscopy. All chitosan membranes showed good biocompatibility and proved to be a suitable substrate for plating the N1E-115 cellular system. By contrast, in vivo nerve regeneration assessment after crush injury showed that the freeze-dried chitosan type III, without N1E-115 cell addition, was the only type of membrane that significantly improved posttraumatic axonal regrowth and functional recovery. It can be thus suggested that local enwrapping with this type of chitosan membrane may represent an effective approach for the improvement of the clinical outcome in patients receiving peripheral nerve surgery.

Neural cell transplantation effects on sciatic nerve regeneration after a standardized crush injury in the rat.

The goal of the present study was to assess whether in vitro-differentiated N1E-115 cells supported by a collagen membrane would enhance rat sciatic nerve regeneration after a crush injury. To set up an appropriate experimental model for investigating the effects of neural cell transplantation, we have recently described the sequence of functional and morphologic changes occurring after a standardized sciatic nerve crush injury with a nonserrated clamp. Functional recovery was evaluated using the sciatic functional index, the static sciatic index, the extensor postural thrust, the withdrawal reflex latency, and ankle kinematics. In addition, histomorphometric analysis was carried out on regenerated nerve fibers by means of the 2D-disector method. Based on the results of the EPT and of some of the ankle locomotor kinematic parameters analyzed, the hypothesis that N1E-115 cells may enhance nerve regeneration is partially supported although histomorphometry disclosed no significant difference in nerve fiber regeneration between the different experimental groups. Therefore, results suggest that enrichment of equine type III collagen membrane with the N1E-115 cellular system in the rat sciatic nerve crush model may support recovery, at least in terms of motor function. The discrepancy between functional and morphological results also suggests that the combined use of functional and morphological analysis should be recommended for an overall assessment of recovery in nerve regeneration studies.

Long-term functional and morphological assessment of a standardized rat sciatic nerve crush injury with a non-serrated clamp.

We have recently described the sequence of functional and morphologic changes occurring after a standardized sciatic nerve crush injury. An 8-week post-injury time was used because this end point is the far most used. Unexpectedly, both functional and morphological data revealed that animals had still not recovered to normal pre-injury levels. Therefore, the present study was designed in order to prolong the observation up to 12 weeks. Functional recovery was evaluated using sciatic functional index (SFI), static sciatic index (SSI), extensor postural thrust (EPT), withdrawal reflex latency (WRL) and ankle kinematics. In addition, quantitative morphology was carried out on regenerated nerve fibers. A full functional recovery was predicted by SFI/SSI, EPT and WRL but not all ankle kinematics parameters. Moreover, only two morphological parameters (myelin thickness/axon diameter ratio and fiber/axon diameter ratio) returned to normal values. Data presented in this paper provide a baseline for selecting the adequate end-point and methods of recovery assessment for a rat sciatic nerve crush study and suggest that the combined use of functional and morphological analysis should be recommended in this experimental model.