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Objectives: To evaluate: (i) the neuro-regenerative potential of chitosan membrane (CS-Me) on acutely axotomised autonomic neurones in vitro; (ii) to exclude the possibility that a pro-regenerative biomaterial could interfere with the proliferation activity of prostate cancer cell lines; (iii) to provide an in vivo proof of the biocompatibility and regeneration promoting effect of CS-Me in a standardised rat model of peripheral nerve injury and repair; (iv) finally, to evaluate the tissue reaction induced by the degrading material; as previous studies have shown promising effects of CS-Me for protection of the neurovascular bundles for potency recovery in patients that undergo nerve-sparing radical prostatectomy (RP)., Materials and Methods: Addressing aim (i), the neuro-regenerative potential, organotypic cultures derived from primary sympathetic ganglia were cultured on CS-Me over 3 days and neurite extension and axonal sprouting were evaluated. Addressing aim (ii), effects of CS on cancer cells, different human prostate cancer cell lines (PC3, DU-145, LN-Cap) were seeded on CS-coated plates or cultured in the presence of CS-Me dissolution products. Addressing aims (iii) and (iv), functional recovery of peripheral nerve fibres and tissue reaction with the biomaterial, CS-Me and CS nerve guides were used to repair a median nerve injury in the rat. Functional recovery was evaluated during the post-recovery time by the behavioural grasping test., Results: CS-Me significantly stimulated axon elongation from autonomic ganglia in comparison to control conditions in organotypic three-dimensional cultures. CS coating, as well as the dissolution products of CS-Me, led to a significantly lower proliferation rate of prostate cancer cell lines in vitro. Tissue reaction towards CS-Me and standard CS nerve guides was similar in the rat median nerve model, as was the outcome of nerve fibre regeneration and functional recovery., Conclusion: The results of this study provide the first experimental evidence in support of the clinical safety of CS-Me and of their postulated effectiveness for improving functional recovery after RP. The presented results are coherent in demonstrating that acutely axotomised autonomic neurones show increased neurite outgrowth on CS-Me substrate, whilst the same substrate reduces prostate cancer cell line proliferation in vitro. Furthermore, CS-Me do not demonstrate any disadvantage for peripheral nerve repair in a standard animal model., (© 2019 The Authors BJU International © 2019 BJU International Published by John Wiley & Sons Ltd.)

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 muscle-in-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.

Background: Delayed reconstruction of transection or laceration injuries of peripheral nerves is inflicted by a reduced regeneration capacity. Diabetic conditions, more frequently encountered in clinical practice, are known to further impair regeneration in peripheral nerves. Chitosan nerve guides (CNGs) have recently been introduced as a new generation of medical devices for immediate peripheral nerve reconstruction. Here, CNGs were used for 45 days delayed reconstruction of critical length 15 mm rat sciatic nerve defects in either healthy Wistar rats or diabetic Goto-Kakizaki rats; the latter resembling type 2 diabetes. In short and long-term investigations, we comprehensively analyzed the performance of one-chambered hollow CNGs (hCNGs) and two-chambered CNGs (CFeCNGs) in which a chitosan film has been longitudinally introduced. Additionally, we investigated in vitro the immunomodulatory effect provided by the chitosan film., Results: Both types of nerve guides, i.e. hCNGs and CFeCNGs, enabled moderate morphological and functional nerve regeneration after reconstruction that was delayed for 45 days. These positive findings were detectable in generally healthy as well as in diabetic Goto-Kakizaki rats (for the latter only in short-term studies). The regenerative outcome did not reach the degree as recently demonstrated after immediate reconstruction using hCNGs and CFeCNGs. CFeCNG-treatment, however, enabled tissue regrowth in all animals (hCNGs: only in 80% of animals). CFeCNGs did further support with an increased vascularization of the regenerated tissue and an enhanced regrowth of motor axons. One mechanism by which the CFeCNGs potentially support successful regeneration is an immunomodulatory effect induced by the chitosan film itself. Our in vitro results suggest that the pro-regenerative effect of chitosan is related to the differentiation of chitosan-adherent monocytes into pro-healing M2 macrophages., Conclusions: No considerable differences appear for the delayed nerve regeneration process related to healthy and diabetic conditions. Currently available chitosan nerve grafts do not support delayed nerve regeneration to the same extent as they do after immediate nerve reconstruction. The immunomodulatory characteristics of the biomaterial may, however, be crucial for their regeneration supportive effects.

Object: This study evaluated a chitosan tube for regeneration of the injured peripheral nerve in a rodent transected sciatic nerve model in comparison to autologous nerve graft repair., Methods: Chitosan hollow tube was used to bridge a 10-mm gap between the proximal and distal ends in 11 rats. In the control group, an end-to-end coaptation of 10-mm long autologous nerve graft was performed in 10 rats for nerve reconstruction., Results: SFI showed an insignificant advantage to the autologous group both at 30 days (P = 0.177) and at 90 days post procedure (P = 0.486). Somato-sensory evoked potentials (SSEP) and compound muscle action potentials (CMAP) tests showed similar results between chitosan tube (group 1) and autologous (group 2) groups with no statistically significant differences. Both groups presented the same pattern of recovery with 45% in group 1 and 44% in group 2 (P = 0.96) showing SSEP activity at 30 days. At 90 days most rats showed SSEP activity (91% vs.80% respectively, P = 0.46). The CMAP also demonstrated no statistically significant differences in latency (1.39 ms in group 1 vs. 1.63 ms in group 2; P = 0.48) and amplitude (6.28 mv vs. 6.43 mv respectively; P = 0.8). Ultrasonography demonstrated tissue growth inside the chitosan tube. Gastrocnemius muscle weight showed no statistically significant difference. Histomorphometry of the distal sciatic nerve, 90 days post reconstructive procedure, showed similar number of myelinated fibers and size parameters in both groups (P ≥ 0.05)., Conclusions: Chitosan hollow tube used for peripheral nerve reconstruction of rat sciatic nerve showed similar results in comparison to autologous nerve grafting. © 2015 Wiley Periodicals, Inc. Microsurgery 36:664-671, 2016., (© 2015 Wiley Periodicals, Inc.)

Biosynthetic nerve grafts are developed in order to complement or replace autologous nerve grafts for peripheral nerve reconstruction. Artificial nerve guides currently approved for clinical use are not widely applied in reconstructive surgery as they still have limitations especially when it comes to critical distance repair. Here we report a comprehensive analysis of fine-tuned chitosan nerve guides (CNGs) enhanced by introduction of a longitudinal chitosan film to reconstruct critical length 15 mm sciatic nerve defects in adult healthy Wistar or diabetic Goto-Kakizaki rats. Short and long term investigations demonstrated that the CNGs enhanced by the guiding structure of the introduced chitosan film significantly improved functional and morphological results of nerve regeneration in comparison to simple hollow CNGs. Importantly, this was detectable both in healthy and in diabetic rats (short term) and the regeneration outcome almost reached the outcome after autologous nerve grafting (long term). Hollow CNGs provide properties likely leading to a wider clinical acceptance than other artificial nerve guides and their performance can be increased by simple introduction of a chitosan film with the same advantageous properties. Therefore, the chitosan film enhanced CNGs represent a new generation medical device for peripheral nerve reconstruction., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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. [ABSTRACT FROM AUTHOR]