Hua Jia

Adipose-derived stem cells promote peripheral nerve repair.

Introduction Recent evidence suggests that the implantation of bone marrow-derived mesenchymal stem cells improves peripheral nerve regeneration. In this study we aimed to investigate whether adipose-derived stem cells (ADSCs) can be used for peripheral nerve repair. Material and methods In a rat model, nerve regeneration was evaluated across a 15 mm lesion in the sciatic nerve by using an acellular nerve injected with allogenic ADSCs. The walking behaviour of rats was measured by footprint analysis, and electrophysiological analysis and histological examination were performed to evaluate the efficacy of nerve regeneration. Results Cultured ADSCs became morphologically homogeneous with a bipolar, spindle-like shape after ex vivo expansion. Implantation of ADSCs into the rat models led to (i) improved walking behaviour as measured by footprint analysis, (ii) increased conservation of muscle-mass ratio of gastrocnemius and soleus muscles, (iii) increased nerve conduction velocity, and (iv) increased number of myelinated fibres within the graft. Conclusions Adipose-derived stem cells could promote peripheral nerve repair in a rat model. Although the detailed mechanism by which ADSCs promote peripheral nerve regeneration is being investigated in our lab, our results suggest that ADSCs transplantation represents a powerful therapeutic approach for peripheral nerve injury.

Transplantation of adipose-derived stem cells for peripheral nerve repair

Mesenchymal stem cells have become a very attractive source of cell implantation for neural tissue engineering. The ideal stem cells for transplantation should be easily obtained, and should rapidly proliferate in vitro and have low immunogenicity. The purpose of this study was to investigate the regenerative potential of adipose-derived stem cells (ADSC) on peripheral nerve repair. ADSCs were isolated from rat adipose tissue and cultured until adherent cells became morphologically homogeneous with a fibroblast-like shape, and transplanted with acellular nerve allografts (ANAs) into rat models with a 10 mm gap of transected sciatic nerve defect. After cell transplantation, we found that ADSC implantation improved functional recovery of exercise behavior and increased wet weight ratio of the anterior tibial muscle. In the electrophysiological testing, we found that the percentage of activated fibers was higher in the ADSC-implanted animals as evidenced by the increase of nerve conduction velocity and amplitude. Histological examination revealed that the number of nerve fibers, axonal diameter and myelin thickness were significantly higher in the ADSC-implanted animals compared to the control. In addition, we demonstrated that the progression of the regenerative process after ADSC implantation was accompanied by elevated expression of neurotrophic factors at both the early and later phase. Taken together, these results suggest that ADSCs can promote the repair of peripheral nerve injury, and the combination of ADSC and ANA transplantation is a new therapeutic method for long distant peripheral nerve defects. Our data also provide evidence indicating the strong association of neurotrophic factor production to the regenerative potential of implanted ADSCs.

Sciatic nerve repair by acellular nerve xenografts implanted with BMSCs in rats xenograft combined with BMSCs.

Acellular nerves possess the structural and biochemical features similar to those of naive endoneurial tubes, and have been proved bioactive for allogeneil graft in nerve tissue engineering. However, the source of allogenic donators is restricted in clinical treatment. To explore sufficient substitutes for acellular nerve allografts (ANA), we investigated the effectiveness of acellular nerve xenografts (ANX) combined with bone marrow stromal cells (BMSCs) on repairing peripheral nerve injuries. The acellular nerves derived from Sprague‐Dawley rats and New Zealand rabbits were prepared, respectively, and BMSCs were implanted into the nerve scaffolds and cultured in vitro. All the grafts were employed to bridge 1 cm rat sciatic nerve gaps. Fifty Wistar rats were randomly divided into five groups (n = 10 per group): ANA group, ANX group, BMSCs‐laden ANA group, BMSCs‐laden ANX group, and autologous nerve graft group. At 8 weeks post‐transplantation, electrophysiological study was performed and the regenerated nerves were assayed morphologically. Besides, growth‐promoting factors in the regenerated tissues following the BMSCs integration were detected. The results indicated that compared with the acellular nerve control groups, nerve regeneration and functional rehabilitation for the xenogenic nervetransplantation integrated with BMSCs were advanced significantly, and the rehabilitation efficacy was comparable with that of the autografting. The expression of neurotrophic factors in the regenerated nerves, together with that of brain‐derived neurotrophic factor (BDNF) in the spinal cord and muscles were elevated largely. In conclusion, ANX implanted with BMSCs could replace allografts to promote nerve regeneration effectively, which offers a reliable approach for repairing peripheral nerve defects. Synapse, 2012.

Synergistic Effects of Bone Mesenchymal Stem Cells and Chondroitinase ABC on Nerve Regeneration After Acellular Nerve Allograft in Rats

This study aimed to evaluate whether combination therapy of bone marrow stromal cells (BMSCs) transplantation and chondroitinase ABC (ChABC) treatment further enhances axonal regeneration and functional recovery after acellular nerve allograft repair of the sciatic nerve gap in rats. Eight Sprague–Dawley rats were used as nerve donors, and 32 Wistar rats were randomly divided into four groups: Group I: acellular rat sciatic nerve (ARSN) group; Group II: ChABC treatment; Group III: BMSCs transplantation; and Group IV: ChABC treatment and BMSCs transplantation. The results showed that compared with ARSN control group, BMSC transplantation promoted axonal regeneration, the secretion of neural trophic factors NGF, BDNF and axon angiogenesis in nerve graft. ChABC treatment degraded chondroitin sulfate proteoglycans in ARSN in vitro and in vivo and improved BMSCs survival in ARSN. The combination therapy caused much better beneficial effects evidenced by increasing sciatic function index, nerve conduction velocity, restoration rate of tibialis anterior wet muscle weight, and myelinated nerve number, but did not further boost the therapeutic effects on neurotrophic factor production, axon angiogenesis, and sensory functional recovery by BMSC transplantation. Taken together, for the first time, we demonstrate the synergistic effects of BMSC transplantation and BMSCs treatment on peripheral nerve regeneration, and our findings may help establish novel strategies for cell transplantation therapy for peripheral nerve injury.

Effects of 660-nm gallium–aluminum–arsenide low-energy laser on nerve regeneration after acellular nerve allograft in rats

Purpose: The purpose of this study was to explore and discuss the effects of 660‐nm gallium–aluminum–arsenide low‐energy laser (GaAlAs LEL) irradiation on neural regeneration after acellular nerve allograft repair of the sciatic nerve gap in rats. Methods: Eight male and female Sprague–Dawley rats were used as nerve donors, and 32 healthy Wistar rats were randomly divided into four groups: normal control group, acellular rat sciatic nerve (ARSN) group, laser group, and autograft group. Twelve weeks after surgery, nerve conduction velocity, restoration rate of tibialis anterior wet muscle weight, myelinated nerve number, and calcitonin gene‐related peptide (CGRP) protein and mRNA expression of the spinal cord and muscle at the injury site were quantified and statistically analyzed. Results: Compared with the ARSN group, laser therapy significantly increased nerve conduction velocity, restoration rate of tibialis anterior wet muscle weight, myelinated nerve number, and CGRP protein and mRNA expression of the L4 spinal cord at the injury site. Conclusions: These findings demonstrate that 660‐nm GaAlAs LEL therapy upregulates CGRP protein and mRNA expression of the L4 spinal cord at the injury site and increases the rate of regeneration and target reinnervation after acellular nerve allograft repair of the sciatic nerve gap in rats. Low‐energy laser irradiation may be a useful, noninvasive adjunct for promoting nerve regeneration in surgically induced defects repaired with ARSN. Synapse 64:152–160, 2010.

Synergistic effects of ultrashort wave and bone marrow stromal cells on nerve regeneration with acellular nerve allografts

Acellular nerve allografts (ANA) possess bioactivity and neurite promoting factors in nerve tissue engineering. Previously we reported that low dose ultrashort wave (USW) radiation could enhance the rate and quality of peripheral nerve regeneration with ANA repairing sciatic nerve defects. Meanwhile, ANA implanted with bone marrow stromal cells (BMSCs) exhibited a similar result. Thus, it is interesting to know whether it might yield a synergistic effect when USW radiation is combined with BMSCs‐laden ANA. Here we investigated the effectiveness of ANA seeded with BMSCs, combined with USW therapy on repairing peripheral nerve injuries. Adult male Wistar rats were randomly divided into four groups: Dulbeccos modified Eagles medium (DMEM) control group, BMSCs‐laden group, ultrashort wave (USW) group and BMSC + USW group. The regenerated nerves were assayed morphologically and functionally, and growth‐promoting factors in the regenerated tissues following USW administration or BMSCs integration were also detected. The results indicated that the combination therapy caused much better beneficial effects evidenced by increased myelinated nerve fiber number, myelin sheath thickness, axon diameter, sciatic function index, nerve conduction velocity, and restoration rate of tibialis anterior wet weight. Moreover, the mRNA levels of brain‐derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) in the spinal cord and muscles were elevated significantly. In conclusion, we found a synergistic effect of USW radiation and BMSCs treatment on peripheral nerve regeneration, which may help establish novel strategies for repairing peripheral nerve defects. Synapse 67:637–647, 2013.

Molecular examination of bone marrow stromal cells and chondroitinase ABC‑assisted acellular nerve allograft for peripheral nerve regeneration

The present study aimed to evaluate the molecular mechanisms underlying combinatorial bone marrow stromal cell (BMSC) transplantation and chondroitinase ABC (Ch-ABC) therapy in a model of acellular nerve allograft (ANA) repair of the sciatic nerve gap in rats. Sprague Dawley rats (n=24) were used as nerve donors and Wistar rats (n=48) were randomly divided into the following groups: Group I, Dulbeccos modified Eagles medium (DMEM) control group (ANA treated with DMEM only); Group II, Ch-ABC group (ANA treated with Ch-ABC only); Group III, BMSC group (ANA seeded with BMSCs only); Group IV, Ch-ABC + BMSCs group (Ch-ABC treated ANA then seeded with BMSCs). After 8 weeks, the expression of nerve growth factor, brain-derived neurotrophic factor and vascular endothelial growth factor in the regenerated tissues were detected by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. Axonal regeneration, motor neuron protection and functional recovery were examined by immunohistochemistry, horseradish peroxidase retrograde neural tracing and electrophysiological and tibialis anterior muscle recovery analyses. It was observed that combination therapy enhances the growth response of the donor nerve locally as well as distally, at the level of the spinal cord motoneuron and the target muscle organ. This phenomenon is likely due to the propagation of retrograde and anterograde transport of growth signals sourced from the graft site. Collectively, growth improvement on the donor nerve, target muscle and motoneuron ultimately contribute to efficacious axonal regeneration and functional recovery. Thorough investigation of molecular peripheral nerve injury combinatorial strategies are required for the optimization of efficacious therapy and full functional recovery following ANA.

Study on molecular mechanism for improving neural regeneration after repair of sciatic nerve defect in rat by acellular nerve allograft

Objective: Discuss the molecular mechanism for improving neural regeneration after repair of sciatic nerve defect in rat by acellular nerve allograft (ANA). Methods: Randomly divide 36 Wistar rats into six groups as normal control group, autografting group, and bridging groups of 2, 4, 8, 12 weeks, six rats for each group. Observe the expression of brain‐derived neurotrophic factor (BDNF) in L4 spinal cord and anterior tibial muscle at the injury site, calcitonin gene‐related peptide (CGRP) protein as well as mRNA, respectively. 12w after operation, histopathological observation was performed. Results: 2w after ANA bridging the sciatic nerve defect in rats, it was observed that the expression level of BDNF in spinal cord at the injury site and CGRP protein increased, reaching the peak level at 4w, lasting till 8w, then decreased but still significantly higher than that in normal control group at 12w, and was not significantly different compared with that in autografting group. However, the expression level of BDNF in anterior tibial muscle decreased gradually within the initial 4w, then increased progressively, reaching normal level at 12w, and was not significantly different compared with that in autografting group. The expression of BDNF mRNA and CGRPmRNA was essentially the same. 12w after operation, there was nerve regeneration in bridging group of 12w and autografting group. Conclusions: ANA possessed fine histocompatibility, and might substitute autograft to repair long‐segment defect of sciatic nerve in rats. This action might be related to upregulation of protein and mRNA expression for BDNF and CGRP in spinal cord. Synapse, 2012.

KLF7-transfected Schwann cell graft transplantation promotes sciatic nerve regeneration.

Our former study demonstrated that Krüppel-like Factor 7 (KLF7) is a transcription factor that stimulates axonal regeneration after peripheral nerve injury. Currently, we used a gene therapy approach to overexpress KLF7 in Schwann cells (SCs) and assessed whether KLF7-transfected SCs graft could promote sciatic nerve regeneration. SCs were transfected by adeno-associated virus 2 (AAV2)-KLF7 in vitro. Mice were allografted by an acellular nerve (ANA) with either an injection of DMEM (ANA group), SCs (ANA+SCs group) or AAV2-KLF7-transfected SCs (ANA+KLF7-SCs group) to assess repair of a sciatic nerve gap. The results indicate that KLF7 overexpression promoted the proliferation of both transfected SCs and native SCs. The neurite length of the dorsal root ganglia (DRG) explants was enhanced. Several beneficial effects were detected in the ANA+KLF7-SCs group including an increase in the compound action potential amplitude, sciatic function index score, enhanced expression of PKH26-labeling transplant SCs, peripheral myelin protein 0, neurofilaments, S-100, and myelinated regeneration nerve. Additionally, HRP-labeled motoneurons in the spinal cord, CTB-labeled sensory neurons in the DRG, motor endplate density and the weight ratios of target muscles were increased by the treatment while thermal hyperalgesia was diminished. Finally, expression of KLF7, NGF, GAP43, TrkA and TrkB were enhanced in the grafted SCs, which may indicate that several signal pathways may be involved in conferring the beneficial effects from KLF7 overexpression. We concluded that KLF7-overexpressing SCs promoted axonal regeneration of the peripheral nerve and enhanced myelination, which collectively proved KLF-SCs as a novel therapeutic strategy for injured nerves.

Biocompatibility of acellular nerves of different mammalian species for nerve tissue engineering.

Abstract: To explore the biocompatibility of acellular nerves of different mammalian species, for the acellular nerves derived from rats and rabbits, the morphology, immunocompatibility, and cytocompatibility with bone marrow stromal cells (BMSCs) were evaluated. The results indicated that the tridimensional architecture and main proteins of endoneurial tubes in both biomaterials were well retained. The nerve scaffolds did not show immunogenicity or cytotoxicity, but facilitated growth of BMSCs and secretion of neurotrophic factors in vitro. In conclusion, acellular nerves of different species possess favorable biocompatibility, and xenogenic acellular nerves combined with BMSCs have potential to replace allografts for peripheral nerve reconstruction.