The Effects And Mechanism Of Skeletal Muscle-derived Cells On Peripheral Nerve Regeneration

BackgroudsPeripheral nerve injury is a large problem that can lead to great morbidity in those afflicted,ranging from sensory loss,motor loss,chronic pain,or a combination of deficits,and seriously affect the daily life of patients.Autologous peripheral nerve transplantation is currently the golden standard for the treatment of peripheral nerve defects.However,due to shortcomings of this treatment such as with morbidity of the donor site and limited donor area,the reasonable and effective alternative method is needed.Seeding cells within the transplanted grafts is one of the important options in peripheral nerve tissue engineering.The mechanism for transplanted cells involved in peripheral nerve repair involves participating in neural construction as a cellular component,or secreting growth factors,or transmitting information for nerve breaks.Skeletal muscle,as an autologous tissue,has been demonstrated the effectiveness on peripheral nerve regeneration for several decades.Our previous studies have also confirmed that the use of mouse external oblique muscle-fabricated nerve conduits(EMC)can effectively promote peripheral nerve regeneration.The skeletal muscle-derived cells(MDCs)can secret a great number of growth factors,which include brain-derived neurotrophic factor(BDNF)and anti-inflammatory factor IL-6.Meanwhile,it can also increase the expression of neuregulin 1(NRG1)under the certain microenvironments.NRG1 binds to the ErbB2/3 receptor of Schwann cells(SCs)and activates focal adhesion kinase(FAK),which can regulate the migration of SCs.In addition,skeletal muscle-derived stem cells(MDSCs)of MDCs can differentiate into Schwann cell-like cells.Although MDCs have been applied to tissue-engineered muscles and tendons,MDCs has not yet been used to repair peripheral nerve defects.Objectives1.To evaluate the effectiveness of MDCs on peripheral nerve regeneration.2.To investigate the mechanisms of MDCs participated in the regeneration of peripheral nerve on the early stage and the expression of related signaling pathway.3.To investigate the impact of MDCs on the migration of SCs and related mechanisms.Methods and Results1.The evaluation of MDCs on repairing sciatic nerve defects in miceMethods:An animal model of 5 mm peripheral nerve defect of the right sciatic nerve in mice was used in this experiment.MDCs were injected into the 7 mm-long polyurethane(PUR)catheter to bridge the bilateral nerve stumps(MDC group),and the PUR catheter group(PUR group)was used as the negative control group.While the external oblique muscle-fabricated nerve conduit group(EMC group)and the sham-operation group(Sham group)were used as positive control groups.The effectiveness was evaluated by immunofluorescence,electron microscopy,gait experiment,and masson staining.Results:Implanted MDCs facilitated uniform distribution of myelin and axons,formation of new myelin and growth of nerve fibers and restored the sciatic functional index and function of the atrophied muscle.These results were comparable to those of the EMC-treated group and were superior to those of the PUR group.MDCs could accelerate peripheral nerve regeneration.2.The mechanisms of MDCs participated in the regeneration of peripheral nerve on the early stage and the expressions of ErbB2 and FAKMethods:An animal model of 5 mm nerve defect of the right sciatic nerve in mice was performed.MDCs with enhanced green fluorescent protein(EGFP)were injected into the PUR catheter to bridge the bilateral nerve stumps(MDC group),compared with the PUR group.The operative peripheral nerves were harvested and immunofluorescence staining of tissue sections was performed at 1 week and 2 weeks after operation to explore the mechanism of MDCs in the early stage of peripheral nerve regeneration.Otherwise,ErbB2 and FAK genes and activated proteins were respectively detected by quantitative real-time PCR(qPCR)and immunofluorescence.Results:MDCs could facilitate SCs migration.Meanwhile,MDCs could be involved in the reconstruction of regenerative peripheral nerves as cellular components of peripheral nerves in the early stage of nerve regeneration.MDCs promoted the expression of p-ErbB2 and p-FAK proteins in myelin sheath of peripheral nerve.3.Effects of MDCs on the cell migration and the expression of ErbB2 and FAK of SCs in vitroMethods:MDCs were cultured and serum-free MDCs conditioned medium(MDC-CM)was collected to detect the concentration of NRG1.Primary SCs was cultured with MDC-CM(Co group),which were compared with SCs cultured with serum-free DMEM(S group).The effects of MDC-CM on the cell migration of SCs and the expression of ErbB2 and FAK were measured by adherence experiment,transwell migration experiment,qPCR and western blot.The inhibitor of ErbB2 was used to verify the role of NRG1/ErbB2/3 signaling pathway on the effectiveness of MDC-CM.Results:MDCs could secret NRG 1 and activate ErbB2 that was positively correlated with FAK phosphorylation in vitro.MDC-CM promoted SC adherence,spreading,and migration in vitro study,which was depended on the NRG1/ErbB2/3 signaling pathway.Conclusions1.MDCs could promote peripheral nerve regeneration by uniform distribution of myelin and axons,formation of new myelin and growth of nerve fibers and restoration of sciatic functional index and atrophied muscle.2.MDCs could be used as cellular components involved in the reconstruction of peripheral nerve and promote SC migration of SCs in vivo.3.MDCs could express NRG land activate ErbB2,which was positively correlated with FAK phosphorylation in vivo.4.MDCs could secret NRG1,which facilitated SC migration and the expression of FAK via ErbB2/3-FAK signaling pathway.5.MDCs could be a potential cell source for the nerve tissue engineering research.