Improving Nerve Regeneration of Acellular Nerve Allografts Seeded with SCs Bridging the Sciatic Nerve Defects of Rat

The objective of the paper is to evaluate the effect of acellular nerve allografts (ANA) seeded with Schwann cells to promote nerve regeneration after bridging the sciatic nerve defects of rats and to discuss its acting mechanisms. Schwann cells were isolated from neonatal Wistar rats. In vitro Schwann cells were microinjected into acellular nerve allografts and co-cultured. Twenty-four Wistar rats weighing 180–220 g were randomly divided into three groups with eight rats in each group: ANA seeded with Schwann cells (ANA + SCs), ANA group and autografts group. All the grafts were, respectively, served for bridging a 10-mm long surgically created sciatic nerve gap. Examinations of regeneration nerve were performed after 12 weeks by transmission electron microscope (TEM), scanning electron microscope (SEM), and electrophysiological methods, and then analyzed statistically. The results obtained indicated that in vitro Schwann cells displayed the feature of bipolar morphology with oval nuclei. Compared with ANA group, the conduction velocity of ANA + SCs group and autograft group was faster after 12 weeks, latent period was shorter, and wave amplitude was higher (P < 0.05). The difference between ANA + SCs group and autograft group is not significant (P > 0.05). Regeneration nerve myelinated fiber number, myelin sheath thickness, and myelinated fibers/total nerves (%) in both ANA + SCs group and autograft group are higher than that in ANA group; the difference is significant (P < 0.05). The difference between the former two is not significant (P > 0.05). In conclusion, ANA seeded with SCs could improve nerve regeneration and functional recovery after bridging the sciatic nerve gap of rats, which offers a novel approach for the repair peripheral nerve defect.     

Experimental Study of Low Dose Ultrashortwave Promoting Nerve Regeneration after Acellular Nerve Allografts Repairing the Sciatic Nerve Gap of Rats

Objectives To observe the effect of ultrashortwave (USW) therapy on nerve regeneration after acellular nerve allografts(ANA) repairing the sciatic nerve gap of rats and discuss its acting mechanisms. Methods Sixteen Wistar rats weighing 180–220 g were randomly divided into four groups with four rats in each group: normal control group; acellular group (ANA, treated by hypotonic-chemical detergent, was applied for bridging a 10 mm-long sciatic nerve defect); USW group (After 24 h of ANA repairing the sciatic nerve gap, low dose USW was administrated for 7 min, once a day, 20 times a course of treatment, three courses of treatment in all); and autografts group. 12 weeks after operation, a series of examinations was performed, including electrophysiological methods, the restoring rate of tibialis anterior muscle wet weight, histopathological observation (myelinated nerve number, myelin sheath thickness, and axon diameter), vascular endothelial growth factor (VEGF) mRNA expression of spinal cord, and muscle at injury site, and analyzed statistically. Results Compared to acellular nerve allografts alone, USW therapy can increase nerve conductive velocity, the restoring rate of tibialis anterior muscle wet weight, myelinated nerve number, axon diameter, VEGF mRNA expression of spinal cord, and muscle at injury site, the difference is significant. There were no differences between USW group and autografts group except myelin sheath thickness. Conclusions USW therapy can promote nerve axon regeneration and Schwann cells proliferation after ANA repairing the sciatic nerve gap of rats, the upregulation of VEGF mRNA expression of spinal cord and muscle may play an important role.     

Tissue engineering of peripheral nerves: A comparison of venous and acellular muscle grafts with cultured Schwann cells

Bioengineering is considered to be the laboratory-based alternative to human autografts and allografts. It ought to provide "custom-made organs" cultured from patient's material. Venous grafts and acellular muscle grafts support axonal regeneration only to a certain extent because of the lack of viable Schwann cells in the graft. We created a biologic nerve graft in the rat sciatic nerve model by implanting cultured Schwann cells into veins and acellular gracilis muscles, respectively. Autologous nerve grafts and veins and acellular muscle grafts without Schwann cells served as controls. After 6 and 12 weeks, regeneration was assessed clinically, histologically, and morphometrically. The polymerase chain reaction analvsis showed that the implanted Schwann cells remained within all the grafts. The best regeneration was seen in the control; after 12 weeks the number of axons was increased significantly compared with the other grafts. A good regeneration was noted in the muscle-Schwann cell group, whereas regeneration in both of the venous grafts and the muscle grafts without Schwann cells was impaired. The muscle-Schwann cell graft showed a systematic and organized regeneration including a proper orientation of regenerated fibers. The venous grafts with Schwann cells showed less fibrous tissue and disorganization than the veins without Schwann cells, but failed to show an excellent regeneration. This might be attributed to the lack of endoneural-tube-like components serving as scaffold for the sprouting axon. Although the conventional nerve graft remains the gold standard, the implantation of Schwann cells into an acellular muscle provides a biologic graft with basal lamina tubes as pathways for regenerating axons and the positive effects of Schwann cells producing neurotrophic and neurotropic factors, and thus, supporting axonal regeneration.     

The expression patterns of β1,4 galactosyltransferase I and V mRNAs, and Galβ1-4GlcNAc group in rat gastrocnemius muscles post sciatic nerve injury

Glycosylation is one of the most important post-translational modifications. It is clear that the single step of β1,4-galactosylation is performed by a family of β1,4-galactosyltransferases (β1,4-GalTs), and that each member of this family may play a distinct role in different tissues and cells. β1,4-GalT I and V are involved in the biosynthesis of N-linked oligosaccharides and play roles in sciatic nerve regeneration after sciatic nerve injury. In the present study, the expression of β1,4-galactosyltransferase (β1,4-GalT) I, V mRNAs and Galβ1-4GlcNAc group were examined in rat gastrocnemius muscles after sciatic nerve crush and transection. Real time PCR revealed that β1,4-GalT I and V mRNAs expressed at a high level in normal gastrocnemius muscles and decreased gradually from 6 h, reached the lowest level at 2 weeks, then restored gradually to relatively normal level at 4 weeks after sciatic nerve crush. In contrast, in sciatic nerve transection model, β1,4-GalT I and V mRNAs decreased gradually from 6 h, and remained on a low level at 4 weeks in gastrocnemius muscles after sciatic nerve transection. In situ hybridization indicated that β1,4-GalT I and V mRNAs localized in numerous myocytes and muscle satellite cells under normal conditions and at 4 weeks after sciatic nerve crush, and in a few muscle satellite cells at 4 weeks after sciatic nerve transection. Furthermore, lectin blotting showed that the expression level of the Galβ1–4GlcNAc group decreased from 6 h, reached the lowest level at 2 weeks, and restored to relatively normal level at 4 weeks after sciatic nerve crush. RCA-I lectin histochemistry demonstrated that Galβ1–4GlcNAc group localized in numerous membranes of myocytes and muscle satellite cells in normal and at 4 weeks after sciatic nerve crush, and in a few muscle satellite cells at 2 and 4 weeks after sciatic nerve transection. These results indicated that the expressions of β1,4-GalT I, V mRNAs and Galβ1–4GlcNAc group were involved in the process of denervation and reinnervation, which suggests that β1,4-GalT I, V mRNAs and Galβ1-4GlcNAc group may play an important role in the muscle regeneration.     

Allotransplanted neurons used to repair peripheral nerve injury do not elicit overt immunogenicity

A major problem hindering the development of autograft alternatives for repairing peripheral nerve injuries is immunogenicity. We have previously shown successful regeneration in transected rat sciatic nerves using conduits filled with allogeneic dorsal root ganglion (DRG) cells without any immunosuppression. In this study, we re-examined the immunogenicity of our DRG neuron implanted conduits as a potential strategy to overcome transplant rejection. A biodegradable NeuraGen® tube was infused with pure DRG neurons or Schwann cells cultured from a rat strain differing from the host rats and used to repair 8 mm gaps in the sciatic nerve. We observed enhanced regeneration with allogeneic cells compared to empty conduits 16 weeks post-surgery, but morphological analyses suggest recovery comparable to the healthy nerves was not achieved. The degree of regeneration was indistinguishable between DRG and Schwann cell allografts although immunogenicity assessments revealed substantially increased presence of Interferon gamma (IFN-γ) in Schwann cell allografts compared to the DRG allografts by two weeks post-surgery. Macrophage infiltration of the regenerated nerve graft in the DRG group 16 weeks post-surgery was below the level of the empty conduit (0.56 fold change from NG; p<0.05) while the Schwann cell group revealed significantly higher counts (1.29 fold change from NG; p<0.001). Major histocompatibility complex I (MHC I) molecules were present in significantly increased levels in the DRG and Schwann cell allograft groups compared to the hollow NG conduit and the Sham healthy nerve. Our results confirmed previous studies that have reported Schwann cells as being immunogenic, likely due to MHC I expression. Nerve gap injuries are difficult to repair; our data suggest that DRG neurons are superior medium to implant inside conduit tubes due to reduced immunogenicity and represent a potential treatment strategy that could be preferable to the current gold standard of autologous nerve transplant.     

Effect of of ATP on Neurons of the Rat Intact Nodose Ganglion

Under intracellular recording, we studied the effect of ATP on nerve cells of the rat intact nodose ganglion. The resting membrane potential of the examined neurons was, on average, –60.3 ± 1.4 mV (n = 84); among such units, 88% were classified as C cells. Local application of 2 mM ATP to the surface of the ganglion using a modified laminar flow system led to depolarization of neurons by 7.1 ± 0.9 mV, on average (n = 19). A blocker of P2X receptors, PPADS (100 μM), suppressed these depolarization responses, decreasing their amplitude, on average, to 16 ± 3% (n = 3) of the initial value. The obtained data indicate that an overwhelming majority of neurons of the intact nodose ganglion possess functional P2X receptors on their membranes. The absence of the corresponding responses in a considerable part of neurons of intact spinal ganglia [13-15] was, apparently, determined by the fact that P2X receptors in the course of the described experiments had enough time to desensitize before ATP reached the effective concentration.     

Polyglycolic acid filaments guide Schwann cell migration in vitro and in vivo

Nerve conduits filled with longitudinal aligned filaments have demonstrated a better regenerative outcome for bridging large peripheral nerve gaps than hollow nerve conduits. In the present study, we investigated the in vitro and in vitro cellular behavior of Schwann cells on polyglycolic acid (PGA) filaments by immunocyto/histochemistry and light/electron microscopy. After 1-3-week culture of rat dorsal root ganglia (DRGs) onto PGA filaments, Schwann cells from rat DRGs adhered to and migrated along PGA filaments. Twenty-four rats received implantation of chitosan conduits inserted with PGA filaments to bridge 10-mm-long sciatic nerve gaps. At 1, 2, 3 and 4 weeks post-implantation (n = 6, each time point), Schwann cells were found to migrate along PGA filaments and form cell columns resembling bands of Büngner. These results suggest that PGA filaments may play a contact guidance role in Schwann cell migration and thus serve as a promising conduit-filling material to facilitate peripheral nerve repair.     

Preparation of cross-linked carboxymethyl chitosan for repairing sciatic nerve injury in rats

A successful nerve regeneration process was achieved with nerve repair tubes made up of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) cross-linked carboxymethyl chitosan (CM-chitosan) with improved biodegradability. Chitosan has a very slow degradation rate, while the EDC cross-linked CM-chitosan tubes degraded to 30% of original weight during 8 weeks of incubation in lysozyme solution. In vitro cell culture indicated that the CM-chitosan films presented no cytotoxicity to Schwann cells. From in vivo studies using a 10 mm rat sciatic nerve defect model investigated by histomorphometry analysis, the average diameter of the fibers and the average thickness of myelin sheath in the CM-chitosan tubes were 3.7 ± 0.33 and 0.33 ± 0.04 μm, respectively, which demonstrated equivalence to nerve autografts (the current “gold” standard); furthermore, the average fiber density in the CM-chitosan tubes was 20.5 × 10³/mm², which was similar to that of autografts (21 × 10³/mm²) and significantly higher than that of common chitosan tubes (15.3 × 10³/mm²).     

A Combination of Schwann-Cell Grafts and Aerobic Exercise Enhances Sciatic Nerve Regeneration

Background

Despite the regenerative potential of the peripheral nervous system, severe nerve lesions lead to loss of target-organ innervation, making complete functional recovery a challenge. Few studies have given attention to combining different approaches in order to accelerate the regenerative process.

Objective

Test the effectiveness of combining Schwann-cells transplantation into a biodegradable conduit, with treadmill training as a therapeutic strategy to improve the outcome of repair after mouse nerve injury.

Methods

Sciatic nerve transection was performed in adult C57BL/6 mice; the proximal and distal stumps of the nerve were sutured into the conduit. Four groups were analyzed: acellular grafts (DMEM group), Schwann cell grafts (3×10⁵/2 µL; SC group), treadmill training (TMT group), and treadmill training and Schwann cell grafts (TMT + SC group). Locomotor function was assessed weekly by Sciatic Function Index and Global Mobility Test. Animals were anesthetized after eight weeks and dissected for morphological analysis.

Results

Combined therapies improved nerve regeneration, and increased the number of myelinated fibers and myelin area compared to the DMEM group. Motor recovery was accelerated in the TMT + SC group, which showed significantly better values in sciatic function index and in global mobility test than in the other groups. The TMT + SC group showed increased levels of trophic-factor expression compared to DMEM, contributing to the better functional outcome observed in the former group. The number of neurons in L4 segments was significantly higher in the SC and TMT + SC groups when compared to DMEM group. Counts of dorsal root ganglion sensory neurons revealed that TMT group had a significant increased number of neurons compared to DMEM group, while the SC and TMT + SC groups had a slight but not significant increase in the total number of motor neurons.

Conclusion

These data provide evidence that this combination of therapeutic strategies can significantly improve functional and morphological recovery after sciatic injury.     

Behavioral evaluation of regenerated rat sciatic nerve by a nanofibrous PHBV conduit filled with Schwann cells as artificial nerve graft

Abstract

The aim of this study is to develop a nanofibrous polymeric nerve conduit with Schwann cells (SCs) and to evaluate its efficiency on the promotion of functional and locomotive activities in rats. The conduits were implanted into a 30-mm gap in the sciatic nerves of the rats. Four months after surgery, the rats were monitored and evaluated by behavioral analyses such as toe out angle, toe spreading analysis, walking track analysis, extensor postural thrust, open-field analysis, swimming test and nociceptive function, four months post surgery. Four months post-operatively, the results from behavioral analyses demonstrated that in the grafted groups especially in the grafted group with SCs, the rat sciatic nerve trunk had been reconstructed with functional recovery such as walking, swimming and recovery of nociceptive function. This study proves the feasibility of artificial conduit with SCs for nerve regeneration by bridging a longer defect in the rat model.     

Cutaneous Magnetic Stimulation Reduces Rat Chronic Pain via Activation of the Supra-Spinal Descending Pathway

Recent studies have demonstrated that magnetic stimulation (MS) can induce cellular responses such as Ca²⁺ influx into the cultured neurons and glia, leading to increased intracellular phosphorylation. We have demonstrated previously that MS reduces rat neuropathic pain associated with the prevention of neuronal degeneration. Thus, we aimed to elucidate the actions of MS in relation to modulation of spinal neuron–glia and the descending inhibitory system in chronic pain. The male SD rats intrathecally implanted with catheters were subjected to sciatic nerve ligation (CCI). MS is a low power apparatus characterized by two different frequencies, 2 KHz and 83 MHz. Rats were given MS to the skin (injured sciatic nerve) for 10 min from the seventh day after CCI. The paw withdrawal latency (PWL) evoked by thermal stimuli was measured for 14 days after CCI. Immunohistochemistry for Iba-1 or GFAP was performed after 4% paraformaldehyde fixation (microscopic analysis). We employed microdialysis for measuring CSF 5-HIAA as a reflection of 5-HT release by MS stimulation. Following CCI, rats showed a decrease in PWL after CCI, and the decrease continued until the 14th day. With MS treatment, the decrease in PWL was reduced during the 10–14 day after CCI. Injection of JNK-1 inhibitors on the 14th day antagonized the analgesic effect of MS. MS also eliminated the CCI-induced decrease in GFAP immunoreactivity. Moreover, MS evoked spinal 5-HT release reflected by increase in spinal 5-HIAA level. Thus, we demonstrate that a novel magnetic stimulator used cutaneously can ameliorate chronic pain by not only preventing abnormal spinal neuron–glia interaction, but also through the activation of the supra-spinal descending inhibitory system.     

Rat brain Na channel mRNAs in non-excitable Schwann cells

The expression of rat brain voltage-sensitive Na⁺ channel mRNAs in Schwann cells was examined using in situ hybridization cytochemistry and RT-PCR. The mRNAs of rat brain Na⁺ channel subtype II and III, but not subtype I, were detected in cultured Schwann cells from sciatic nerve and in intact sciatic nerve, which contains Schwann cells but not neuronal cell bodies. These results indicate that rat brain Na⁺ channel mRNAs, which have been considered as mainly neuronal-type messages, are also expressed in glial cells in vitro and in vivo.     

Uptake of Endoneurial Lipoprotein into Schwann Cells and Sensory Neurons Is Mediated by Low Density Lipoprotein Receptors and Stimulated After Axonal Injury

During Wallerian degeneration of rat sciatic nerve, the expression of apolipoprotein E increases and apolipoprotein E-containing endoneurial lipoproteins accumulate in the distal nerve segment. In established primary cultures dissociated from dorsal root ganglia, Schwann cells and sensory neurons internalized rhodamine-labeled lipoproteins isolated from crushed rat sciatic nerve as well as low density lipoprotein (LDL) from human serum. The uptake of endoneurial lipoproteins could be inhibited by an excess of LDL or at low temperature (4 degrees C). After transection of nerve fibers in dorsal root ganglia explant cultures, the uptake of lipoproteins was markedly stimulated in Schwann cells that were in close proximity to degenerating neurites. A specific monoclonal antibody directed to the bovine LDL receptor (clone C7) was shown to cross-react with LDL receptor preparations of rat endoneurial cells. LDL receptor immunoreactivity was expressed by cell bodies and processes of cultured Schwann cells, sensory neurons, and fibroblasts from dorsal root ganglia. Incubation of Schwann cells and neurons with the LDL receptor antibody strongly inhibited the uptake of endoneurial lipoproteins. Our results provide direct evidence for the important role of the LDL receptor-mediated pathway to internalize endoneurial lipoproteins into Schwann cells and peripheral neurons required for reuse of cholesterol and other lipids in myelin and plasma membrane biogenesis during nerve repair.     

Effects of Acrylamide on the Nervous Tissue Antioxidant System and Sciatic Nerve Electrophysiology in the Rat

To investigate the time-dependent effects of acrylamide (ACR) on the antioxidative status in rat nerve tissues, adult male Wistar rats were given ACR (40 mg/kg, i.p., 3 times/week) for 2, 4, 6 and 10 weeks, respectively. The time-dependent changes of the lipid peroxidation (malondialdehyde, MDA) and antioxidative status (glutathione, GSH; glutathione peroxidase, GSH-Px; glutathione reductase, GR; superoxide dismutase, SOD and anti-reactive oxygen species, anti-ROS) in nerve tissues were investigated. The electrophysiology indices (nerve conduction velocity, NCV; compound action potential duration, CAPD; compound action potential amplitude, CAPA; compound action potential latency, CAPL) in the sciatic nerve were determined using BL-420E Biologic Function Determining System. The results showed that MDA levels increased significantly (P < 0.05) in nerve tissues, while GSH levels markedly decreased (P < 0.05) in a time-dependent manner. SOD activity (in the spinal cord and sciatic nerve) and GR activity (in the sciatic nerve) increased significantly after 4 weeks ACR treatment (P < 0.01), but then decreased (P < 0.05). The anti-ROS activity in the sciatic nerve was markedly decreased at the end of week 6 and 10 (P < 0.01). The above indices changed most in the sciatic nerve. The levels of GSH, MDA and anti-ROS in rat sciatic nerve were in high correlation (P < 0.05, |r| > 0.80) with the electrophysiology indices according to the exposure time. Thus, ACR-induced neurotoxicity may be associated with the enhancement of lipid peroxidation and reduction of the antioxidative capacity. Depletion of neural GSH level might be one of the primary events in ACR-induced neuropathy. Ying-Jian Zhu and Tao Zeng––These authors contributed equally to this work.     

Effects of Fluoride on Synaptic Membrane Fluidity and PSD-95 Expression Level in Rat Hippocampus

The objective of this study is to investigate the neurotoxicity of drinking water fluorosis on rat hippocampus. Just weaning male Sprague–Dawley rats were randomly divided into four groups and given 15, 30, and 60 mg/L NaF solution and distilled water, respectively, for 9 months. The fluidity of brain synaptic membrane and expression level of postsynaptic density 95 (PSD-95) were tested. Results showed that the fluidity of brain synaptic membrane decreased gradually with increasing of fluoride concentration, and it was significantly decreased (P < 0.05) in moderate-fluoride group compared with control group, and expression level of PSD-95 was significantly decreased (P < 0.01) in moderate-fluoride group when compared with that of control group. These results indicate that decrease of synaptic membrane fluidity and PSD-95 expression level may be the molecular basis of central nervous system damage caused by fluoride intoxication; PSD-95 in CA3 region of hippocampus is probably a target molecule for fluoride.     

A nanofibrous PHBV tube with Schwann cell as artificial nerve graft contributing to Rat sciatic nerve regeneration across a 30-mm defect bridge

Abstract

A nanofibrous PHBV nerve conduit has been used to evaluate its efficiency based on the promotion of nerve regeneration in rats. The designed conduits were investigated by physical, mechanical and microscopic analyses. The conduits were implanted into a 30-mm gap in the sciatic nerves of the rats. Four months after surgery, the regenerated nerves were evaluated by macroscopic assessments and histology. This polymeric conduit had sufficiently high mechanical properties to serve as a nerve guide. The results demonstrated that in the nanofibrous graft with cells, the sciatic nerve trunk had been reconstructed with restoration of nerve continuity and formatted nerve fibers with myelination. For the grafts especially the nanofibrous conduits with cells, muscle cells of gastrocnemius on the operated side were uniform in their size and structures. This study proves the feasibility of artificial conduit with Schwann cells for nerve regeneration by bridging a longer defect in a rat model.     

Lentiviral-mediated transfer of CDNF promotes nerve regeneration and functional recovery after sciatic nerve injury in adult rats

Peripheral nerve injury is often followed by incomplete and unsatisfactory functional recovery and may be associated with sensory and motor impairment of the affected limb. Therefore, a novel method is needed to improve the speed of recovery and the final functional outcome after peripheral nerve injuries. This report investigates the effect of lentiviral-mediated transfer of conserved dopamine neurotrophic factor (CDNF) on regeneration of the rat peripheral nerve in a transection model in vivo. We observed notable overexpression of CDNF protein in the distal sciatic nerve after recombinant CDNF lentiviral vector application. We evaluated sciatic nerve regeneration after surgery using light and electron microscopy and the functional recovery using the sciatic functional index and target muscle weight. HE staining revealed better ordered structured in the CDNF-treated group at 8 weeks post-surgery. Quantitative analysis of immunohistochemistry of NF200 and S-100 in the CDNF group revealed significant improvement of axonal and Schwann cell regeneration compared with the control groups at 4 weeks and 8 weeks after injury. The thickness of the myelination around the axons in the CDNF group was significantly higher than in the control groups at 8 weeks post-surgery. The CDNF group displayed higher muscle weights and significantly increased sciatic nerve index values. Our findings suggest that CDNF gene therapy could provide durable and stable CDNF protein concentration and has the potential to enhance peripheral nerve regeneration, morphological and functional recovery following nerve injury, which suggests a promising strategy for peripheral nerve repair.     

Synthetic scaffold coating with adeno-associated virus encoding BMP2 to promote endogenous bone repair

Biomaterial scaffolds functionalized to stimulate endogenous repair mechanisms via the incorporation of osteogenic cues offer a potential alternative to bone grafting for the treatment of large bone defects. We first quantified the ability of a self-complementary adeno-associated viral vector encoding bone morphogenetic protein 2 (scAAV2.5-BMP2) to enhance human stem cell osteogenic differentiation in vitro. In two-dimensional culture, scAAV2.5-BMP2-transduced human mesenchymal stem cells (hMSCs) displayed significant increases in BMP2 production and alkaline phosphatase activity compared with controls. hMSCs and human amniotic-fluid-derived stem cells (hAFS cells) seeded on scAAV2.5-BMP2-coated three-dimensional porous polymer Poly(ε-caprolactone) (PCL) scaffolds also displayed significant increases in BMP2 production compared with controls during 12 weeks of culture, although only hMSC-seeded scaffolds displayed significantly increased mineral formation. PCL scaffolds coated with scAAV2.5-BMP2 were implanted into critically sized immunocompromised rat femoral defects, both with or without pre-seeding of hMSCs, representing ex vivo and in vivo gene therapy treatments, respectively. After 12 weeks, defects treated with acellular scAAV2.5-BMP2-coated scaffolds displayed increased bony bridging and had significantly higher bone ingrowth and mechanical properties compared with controls, whereas defects treated with scAAV2.5-BMP2 scaffolds pre-seeded with hMSCs failed to display significant differences relative to controls. When pooled, defect treatment with scAAV2.5-BMP2-coated scaffolds, both with or without inclusion of pre-seeded hMSCs, led to significant increases in defect mineral formation at all time points and increased mechanical properties compared with controls. This study thus presents a novel acellular bone-graft-free endogenous repair therapy for orthotopic tissue-engineered bone regeneration.     

The effects of Weichangshu tablet on intracellular Ca<Superscript>2+</Superscript> concentration in cultured rat gastrointestinal smooth muscle cells

The objective of this study was to explore the effects of Weichangshu tablets on free Ca²⁺ concentration of the gastrointestinal smooth muscle cells of rats and the molecular mechanism of the Weichangshu tablets. Cultured SD rat gastrointestinal smooth muscle cells were divided into control group, Cisapride group, Weichangshu group, and control + ethylene glycol tetraacetic acid (EGTA) group, Cisapride + EGTA group, and Weichangshu + EGTA group. Laser scanning microscope and spectrophotometer detection were applied to detect the calcium concentration. The calcium concentrations in Weichangshu group and Cisapride group significantly increased vs. control, and those in Weichangshu group were higher than those in Cisapride group. Ca²⁺ concentration in Weichangshu + EGTA group, Cisapride + EGTA group vs. control + EGTA group were not significantly different. Weichangshu could increase gastrointestinal smooth muscle free Ca²⁺ concentration, and this role may be achieved through the promotion of cells within the flow of calcium ions.     

Lentiviral vectors enveloped with rabies virus glycoprotein can be used as a novel retrograde tracer to assess nerve recovery in rat sciatic nerve injury models

Retrograde labeling has become the new “gold standard” technique to evaluate the recovery of injured peripheral nerves. In this study, lentiviral vectors with rabies virus glycoprotein envelop (RABV-G-LV) and RFP genes are injected into gastrocnemius muscle to determine the location of RFP in sciatic nerves. We then examine RFP expression in the L4-S1 spinal cord and sensory dorsal root ganglia and in the rat sciatic nerve, isolated Schwann cells, viral dose to expression relationship and the use of RABV-G-LV as a retrograde tracer for regeneration in the injured rat sciatic nerve. VSV-G-LV was used as control for viral envelope specificity. Results showed that RFP were positive in the myelin sheath and lumbar spinal motorneurons of the RABV-G-LV group. RFP gene could be detected both in myelinated Schwann cells and lumbar spinal motor neurons in the RABV-G-LV group. Schwann cells isolated from the RABV-G-LV injected postnatal Sprague Dawley rats were also RFP-gene positive. All the results obtained in the VSV-G-LV group were negative. Distribution of RFP was unaltered and the level of RFP expression increasing with time progressing. RABV-G-LV could assess the amount of functional regenerating nerve fibers two months post-operation in the four models. This method offers an easy-operated and consistent standardized approach for retrograde labeling regenerating peripheral nerves, which may be a significant supplement for the previous RABV-G-LV-related retrograde labeling study.