Xueyu Hu

Electrical regulation of Schwann cells using conductive polypyrrole/chitosan polymers

Electrical stimulation (ES) can dramatically enhance neurite outgrowth through conductive polymers and accelerate peripheral nerve regeneration in animal models of nerve injury. Therefore, conductive tissue engineering graft in combination with ES is a potential treatment for neural injuries. Conductive tissue engineering graft can be obtained by seeding Schwann cells on conductive scaffold. However, when ES is applied through the conductive scaffold, the impact of ES on Schwann cells has never been investigated. In this study, a biodegradable conductive composite made of conductive polypyrrole (PPy, 2.5%) and biodegradable chitosan (97.5%) was prepared in order to electrically stimulate Schwann cells. The tolerance of Schwann cells to ES was examined by a cell apoptosis assay. The growth of the cells was characterized using DAPI staining and a MTT assay. mRNA and protein levels of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in Schwann cells were assayed by RT-PCR and Western blotting, and the amount of NGF and BDNF secreted was determined by an ELISA assay. The results showed that the PPy/chitosan membranes supported cell adhesion, spreading, and proliferation with or without ES. Interestingly, ES applied through the PPy/chitosan composite dramatically enhanced the expression and secretion of NGF and BDNF when compared with control cells without ES. These findings highlight for the first time the possibility of enhancing nerve regeneration in conductive scaffolds through ES-increased neurotrophin secretion.

Electrical stimulation induces calcium-dependent release of NGF from cultured Schwann cells

Production of nerve growth factor (NGF) from Schwann cells (SCs) progressively declines in the distal stump, if axonal regeneration is staggered across the suture site after peripheral nerve injuries. This may be an important factor limiting the outcome of nerve injury repair. Thus far, extensive efforts are devoted to modulating NGF production in cultured SCs, but little has been achieved. In the present in vitro study, electrical stimulation (ES) was attempted to stimulate cultured SCs to release NGF. Our data showed that ES was capable of enhancing NGF release from cultured SCs. An electrical field (1 Hz, 5 V/cm) caused a 4.1‐fold increase in NGF release from cultured SCs. The ES‐induced NGF release is calcium dependent. Depletion of extracellular or/and intracellular calcium partially/ completely abolished the ES‐induced NGF release. Further pharmacological interventions showed that ES induces calcium influx through T‐type voltage‐gated calcium channels and mobilizes calcium from 1, 4, 5‐trisphosphate‐sensitive stores and caffeine/ryanodine‐sensitive stores, both of which contributed to the enhanced NGF release induced by ES. In addition, a calcium‐triggered exocytosis mechanism was involved in the ES‐induced NGF release from cultured SCs. These findings show the feasibility of using ES in stimulating SCs to release NGF, which holds great potential in promoting nerve regeneration by enhancing survival and outgrowth of damaged nerves, and is of great significance in nerve injury repair and neuronal tissue engineering.

Electrical Stimulation to Conductive Scaffold Promotes Axonal Regeneration and Remyelination in a Rat Model of Large Nerve Defect

Background Electrical stimulation (ES) has been shown to promote nerve regeneration when it was applied to the proximal nerve stump. However, the possible beneficial effect of establishing a local electrical environment between a large nerve defect on nerve regeneration has not been reported in previous studies. The present study attempted to establish a local electrical environment between a large nerve defect, and examined its effect on nerve regeneration and functional recovery. Methodology/Findings In the present study, a conductive scaffold was constructed and used to bridge a 15 mm sciatic nerve defect in rats, and intermittent ES (3 V, 20 Hz) was applied to the conductive scaffold to establish an electrical environment at the site of nerve defect. Nerve regeneration and functional recovery were examined after nerve injury repair and ES. We found that axonal regeneration and remyelination of the regenerated axons were significantly enhanced by ES which was applied to conductive scaffold. In addition, both motor and sensory functional recovery was significantly improved and muscle atrophy was partially reversed by ES localized at the conductive scaffold. Further investigations showed that the expression of S-100, BDNF (brain-derived neurotrophic factor), P0 and Par-3 was significantly up-regulated by ES at the conductive scaffold. Conclusions/Significance Establishing an electrical environment with ES localized at the conductive scaffold is capable of accelerating nerve regeneration and promoting functional recovery in a 15 mm nerve defect in rats. The findings provide new directions for exploring regenerative approaches to achieve better functional recovery in the treatment of large nerve defect.

Electrical Stimulation Accelerates Motor Functional Recovery in the Rat Model of 15-mm Sciatic Nerve Gap Bridged by Scaffolds With Longitudinally Oriented Microchannels

BACKGROUND Electrical stimulation (ES) can enhance the regenerative capacity of axotomized motor and sensory neurons. However, the impact of ES on axonal regeneration and functional recovery has not been investigated in an animal model of a lengthy peripheral nerve defect. OBJECTIVE To determine whether ES accelerates axonal regeneration and functional recovery of a 15-mm sciatic nerve defect in rats. METHODS A 15-mm excision of the sciatic nerve was bridged with a chitosan scaffold with longitudinally or randomly oriented pores or with autologous grafting of the segment. In half of the animals with chitosan grafts, the proximal nerve stump was electrically stimulated for 1 hour at 20 Hz immediately after the nerve repair with the scaffolds. Axonal regeneration was investigated by retrograde labeling and morphometric analysis. The rate of motor functional recovery was evaluated by electrical nerve stimulation, behavioral tests of stepping, and histological appearance of the target muscles. RESULTS Axonal regeneration and motor functional recovery were improved by ES in animals that received longitudinal pore grafts as compared with others. The maximal number of axons that regenerated across the longitudinal graft was achieved 2 to 4 weeks earlier in rats with ES. In addition, the latency of compound muscle action potentials (CMAPs), the peak amplitude of CMAPs, and nerve conduction velocity were improved by ES. Stepping indices were better, with less atrophy of target muscle in ES rats managed with longitudinal pores. CONCLUSION Brief ES may accelerate axonal regeneration and motor recovery after focal peripheral nerve transection when repaired with optimally tissue-engineered grafts.Background. Electrical stimulation (ES) can enhance the regenerative capacity of axotomized motor and sensory neurons. However, the impact of ES on axonal regeneration and functional recovery has not been investigated in an animal model of a lengthy peripheral nerve defect. Objective. To determine whether ES accelerates axonal regeneration and functional recovery of a 15-mm sciatic nerve defect in rats. Methods. A 15-mm excision of the sciatic nerve was bridged with a chitosan scaffold with longitudinally or randomly oriented pores or with autologous grafting of the segment. In half of the animals with chitosan grafts, the proximal nerve stump was electrically stimulated for 1 hour at 20 Hz immediately after the nerve repair with the scaffolds. Axonal regeneration was investigated by retrograde labeling and morphometric analysis. The rate of motor functional recovery was evaluated by electrical nerve stimulation, behavioral tests of stepping, and histological appearance of the target muscles. Results. Axonal regeneration and motor functional recovery were improved by ES in animals that received longitudinal pore grafts as compared with others. The maximal number of axons that regenerated across the longitudinal graft was achieved 2 to 4 weeks earlier in rats with ES. In addition, the latency of compound muscle action potentials (CMAPs), the peak amplitude of CMAPs, and nerve conduction velocity were improved by ES. Stepping indices were better, with less atrophy of target muscle in ES rats managed with longitudinal pores. Conclusion. Brief ES may accelerate axonal regeneration and motor recovery after focal peripheral nerve transection when repaired with optimally tissue-engineered grafts.

Rapid repair and regeneration of damaged rabbit sciatic nerves by tissue-engineered scaffold made from nano-silver and collagen type I

A tissue-engineered scaffold with nano-silver and collagen type I was constructed and investigated for its ability to adsorb laminin and the usefulness in the repair and regeneration of damaged peripheral nerves in animals. The nano-silver scaffold displayed ideal microtubule structure under electronic microscope; even distribution of the nano-silver particles was also seen with energy spectrometry. After immersion in a laminin solution, the laminin-attached scaffolds were implanted into rabbits to repair a 10-mm injury of the sciatic nerve. At 30 days post-implantation, regeneration of the damaged nerve was evaluated by transmission electron microscopy, electrophysiological examination and fluoro-gold (FG) retrograde labelling. Compared with the control collagen-scaffold without nano-silver, the nano-silver-containing scaffold showed a higher rate of laminin adsorption, regenerated a nerve with a thicker myelin sheath and improved the nerve conduction velocity and nerve potential amplitude. FG retrograde labelled the newly grown axons in the spinal cord cortex anterior horn and the dorsal root ganglion. These results demonstrate the superior functionality of the nano-silver-collagen scaffold in the adsorption to laminin and subsequent regeneration of damaged peripheral nerves.

Electrical regulation of olfactory ensheathing cells using conductive polypyrrole/chitosan polymers.

Electrical stimulation (ES) applied to a conductive nerve graft holds the great potential to improve nerve regeneration and functional recovery in the treatment of lengthy nerve defects. A conductive nerve graft can be obtained by a combination of conductive nerve scaffold and olfactory ensheathing cells (OECs), which are known to enhance axonal regeneration and to produce myelin after transplantation. However, when ES is applied through the conductive graft, the impact of ES on OECs has never been investigated. In this study, a biodegradable conductive composite made of conductive polypyrrole (PPy, 2.5%) and biodegradable chitosan (97.5%) was prepared in order to electrically stimulate OECs. The tolerance of OECs to ES was examined by a cell apoptosis assay. The growth of the cells was characterized using DAPI staining and a CCK-8 assay. The mRNA and protein levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neural cell adhesion molecule (N-CAM), vascular endothelial growth factor (VEGF) and neurite outgrowth inhibitor-A (NOGO-A) in OECs were assayed by RT-PCR and Western blotting, and the amount of BDNF, NGF, N-CAM, VEGF and NOGO-A secreted was determined by an ELISA assay. The results showed that the PPy/chitosan membranes supported cell adhesion, spreading, and proliferation with or without ES. Interestingly, ES applied through the PPy/chitosan composite dramatically enhanced the expression and secretion of BDNF, NGF, N-CAM and VEGF, but decreased the expression and secretion of NOGO-A when compared with control cells without ES. These findings highlight the possibility of enhancing nerve regeneration in conductive scaffolds through ES increased neurotrophin secretion in OECs.

Electrical stimulation accelerates nerve regeneration and functional recovery in delayed peripheral nerve injury in rats

The present study aims to investigate the potential of brief electrical stimulation (ES; 3 V, 20 Hz, 20 min) in improving functional recovery in delayed nerve injury repair (DNIR). The sciatic nerve of Sprague Dawley rats was transected, and the repair of nerve injury was delayed for different time durations (2, 4, 12 and 24 weeks). Brief depolarizing ES was applied to the proximal nerve stump when the transected nerve stumps were bridged with a hollow nerve conduit (5 mm in length) after delayed periods. We found that the diameter and number of regenerated axons, the thickness of myelin sheath, as well as the number of Fluoro‐Gold retrograde‐labeled motoneurons and sensory neurons were significantly increased by ES, suggesting that brief ES to proximal nerve stumps is capable of promoting nerve regeneration in DNIR with different delayed durations, with the longest duration of 24 weeks. In addition, the amplitude of compound muscle action potential (gastrocnemius muscle) and nerve conduction velocity were also enhanced, and gastrocnemius muscle atrophy was partially reversed by brief ES, indicating that brief ES to proximal nerve stump was able to improve functional recovery in DNIR. Furthermore, brief ES was capable of increasing brain‐derived neurotrophic factor (BDNF) expression in the spinal cord in DNIR, suggesting that BDNF‐mediated neurotrophin signaling might be one of the contributing factors to the beneficial effect of brief ES on DNIR. In conclusion, the present findings indicate the potential of using brief ES as a useful method to improve functional recovery for delayed repair of peripheral nerve lesions.

Ionically cross-linked chitosan microspheres for controlled release of bioactive nerve growth factor

Controlled release of neurotrophic factors to target tissue via microsphere-based delivery systems is critical for the treatment strategies of diverse neurodegenerative disorders. The present study aims to investigate the feasibility of the controlled release of bioactive nerve growth factor (NGF) with ionically cross-linked chitosan microspheres (NGF-CMSs). The microspheres were prepared by the emulsion-ionic cross-linking method with sodium tripolyphosphate (STPP) as an ionic cross-linking agent. The size and distribution of the microspheres, SEM images, Fourier transform infra red spectroscopy (FT-IR), encapsulation efficiency, in vitro release tests and bioactivity assay were subsequently evaluated. We found that the microspheres had relatively rough surfaces with mean sizes between 20 and 31μm. FT-IR results provided evidence of ionic interaction between amino groups and phosphoric groups of chitosan and STPP. The NGF encapsulation efficiency ranged from 63% to 88% depending on the concentration of STPP. The in vitro release profiles of NGF from NGF-CMSs were influenced by the concentration of STPP. NGF-CMSs which were cross-linked with higher concentration of STPP showed slower but sustained release of NGF. In addition, the released NGF from NGF-CMSs was capable of maintaining the viability of PC12 cells, as well as promoting their differentiation. Taken together, our findings suggest that NGF-CMSs are capable of releasing bioactive NGF over 7 days, thus having potential application in nerve injury repair.

Rapid sciatic nerve regeneration of rats by a surface modified collagen–chitosan scaffold

In the previous study, we attempted to use a collagen-chitosan (CCH) scaffold to mimic the bio-functional peripheral nerve and to bridge sciatic nerve defects in rats. The results demonstrated that it could support and guide the nerve regeneration after three months. In the current study, a type of peptide which carried RGD sequences was connected to the CCH surface by a chemical method. After this process, the microtubule structure of the scaffold was not changed. Then the coated scaffolds were used to repair a 15mm sciatic nerve defect in rats. Four weeks after implantation, linear growth of axons in the longitudinal structure was observed, and the number of regenerated axons remarkably increased. Two months later, the scaffold was partly absorbed and replaced by large quantity of regenerated axons. Importantly, the functional examinations also support the morphological results. Compared with the CCH group, all of the achievements revealed the superior function of RGD-CCH in the rapid regeneration of injured sciatic nerve.

Incorporation of chitosan microspheres into collagen-chitosan scaffolds for the controlled release of nerve growth factor.

Background Artifical nerve scaffold can be used as a promising alternative to autologous nerve grafts to enhance the repair of peripheral nerve defects. However, current nerve scaffolds lack efficient microstructure and neurotrophic support. Methods Microsphere–Scaffold composite was developed by incorporating chitosan microspheres loaded with nerve growth factor (NGF–CMSs) into collagen-chitosan scaffolds (CCH) with longitudinally oriented microchannels (NGF–CMSs/CCH). The morphological characterizations, in vitro release kinetics study, neurite outgrowth assay, and bioactivity assay were evaluated. After that, a 15-mm-long sciatic nerve gap in rats was bridged by the NGF–CMSs/CCH, CCH physically absorbed NGF (NGF/CCH), CCH or nerve autograft. 16 weeks after implantation, electrophysiology, fluoro-gold retrograde tracing, and nerve morphometry were performed. Results The NGF–CMSs were evenly distributed throughout the longitudinally oriented microchannels of the scaffold. The NGF–CMSs/CCH was capable of sustained release of bioactive NGF within 28 days as compared with others in vitro. In vivo animal study demonstrated that the outcomes of NGF–CMSs/CCH were better than those of NGF/CCH or CCH. Conclusion Our findings suggest that incorporation of NGF–CMSs into the CCH may be a promising tool in the repair of peripheral nerve defects.