Conductive Conduit Combined With Electric Stimulation For Long-range Peripheral Nerve Regeneration And The Associated Mechanism

OBJECTIVESLong-range peripheral nerve defects is a big challenge for clinical treatment at present and tissue engineering nerve conduit has long been explored as the potential alternative treatment.The ideal nerve conduit should not only bridge the defects and offer a physical channel for nerve regeneration,but also stimulate nerve regeneration and accelerate this process.Conductive polymer composites and electric stimulation(ES)are both promising methods to support neuron adhesion and promote nerve regeneration.In this study,we aimed to investigate the abilities of direct current ES through electrospinning conductive polymer composites composed of polypyrrole and Poly(l-lactic acid-co-?-caprolactone)(PPY/PLCL)in peripheral nerve regeneration,to reveal the best regeneration methods for long-range peripheral nerve defects and underlying its associated mechanism.METHODSPPY/PLCL composite conduits were synthesized by polymerizing pyrrole coated electrospun PLCL scaffolds.Morphologies and chemical compositions were characterized by scanning electron microscope and attenuated total reflection fourier transform infrared(ATR-FTIR)microscope.Rat pheochromocytoma 12(PC12)cells and dorsal root ganglia(DRG)cells cultured on PPY/PLCL scaffolds were stimulated with 100 mV/cm for 4 h per day.The median neurite length and cell viability were measured in PC-12 cells.The levels of brain-derived neurotrophic factor(BDNF),glial cell derived neurotrophic factor(GDNF)and neurotrophin-3(NT-3)were analyzed in DRG cells.In rats,15 mm gaps of sciatic nerves were bridged using an autograft,non-stimulated PPY/PLCL conduit and PPY/PLCL conduit stimulated with 100 mV potential,respectively.A 100 mV potential direct current ES was applied for 1 h per day at 1,3,5 and 7 days post-implantation.Electrophysiological assessment,histological and immunofluorescence analysis were used to evaluate the repair efficiency.RESULTSPPY/PLCL conduits have great mechanical properties,good conductivity and good biocompatibility,without obvious cytotoxicity and in vivo inflammatory response.Compared with the conductive PPY/PLCL film without ES,the PPY/PLCL film combined with ES significantly elevated the median neurite length in PC12 cells(p<0.05).The results of ELISA,Western-blot and RT-PCR of DRG cells show that the PPY/PLCL film combined with ES increased the concentration of GDNF,BDNF and NT-3,significantly higher than other two groups(p<0.05).The in vitro study results show that PPY/PLCL conductive conduit's combined use with ES seems to improve nerve regeneration and functional recovery for rat sciatic nerve 15 mm defects,similar performance compared with the autograft group,and significantly better than the non-stimulated PPY/PLCL conduit group.CONCLUSIONSThe conductive polymeric nanofibers nerve conduit PPY/PLCL combined use with ES(100mV/cm)has good biocompatibility,induce neuron differentiation,promote the secretion of neurotrophic factors,improve the repair efficiency for long-range sciatic nerve defects,which worth further study and has great potential for clinical application.