The Research On Bionic Conductive Artificial Nerve Scaffold

Objective:(1)To prepare multi-microchannel multi-wall carbon nanotube agarose conductive scaffold with conductive function.And then to characterize and verify the biocompatibility of the prepared scaffolds.The effect of electrical stimulation on the growth of RSC96 cells was studied by three-dimensional cell culture on scaffolds.(2)Using prepared PLLA fiber films with aligned nanofibers,to characterize and verify their biocompatibility,and to study the effect of orientation on RSC96 cell signaling proteins.(3)The bionic conductive artificial nerve scaffolds were prepared and implanted into the sciatic nerve defect of rats.Then the effect and mechanism of the bionic conductive artificial nerve scaffold on the sciatic nerve repair of rats were investigated.Method:(1)Gradient freezing and freeze-drying techniques were used to prepare a multi-microchannel multi-wall carbon nanotube agarose conductive scaffold.The prepared multi-channel multi-wall carbon nanotube agarose conductive scaffolds were characterized by microstructural,swelling and de swelling,conductivity,shape memory function,drug loading and drug release properties,mechanical properties and biocompatibility.Threedimensional culture of RSC96 cells were histologically evaluated to observe the cell growth behavior under different culture conditions.(2)PLLA fiber films with aligned nanofibers were prepared by electrostatic spinning technology.The prepared PLLA fiber films was characterized by SEM,mechanical properties,degradation and biocompatibility.Immunofluorescence was used to evaluate the effect of aligned fiber on ERK,P38 MAPK,MEK and LRP4 proteins in RSC96 cells.(3)The multi-microchannel conductive scaffolds were prepared by using customized molds.The high aligned PLLA fiber membrane prepared in the second part and the multi-microchannel conductive scaffolds were combined into bionic conductive artificial nerve scaffolds which were implanted into the sciatic nerve defect of rats.The experimental animals were divided into scaffold group(S),scaffold with electrical stimulation group(S&E)and autologous nerve transplantation group(AT).The effect of bionic conductive nerve scaffolds on peripheral nerve regeneration was analyzed by sciatic nerve function index,nerve electrophysiological test,muscle histology test and nerve regeneration histology test.Results:(1)The addition of MWCNT did not affect the pore composition ratio of and shape memory of agarose scaffolds,but 0.025% wt MWCNT in scaffolds improved the swelling ratio and water retention at the swelling equilibrium state.MWCNT changed the electrical conductivity,drug release ability and mechanical properties of agarose scaffold.MWCNTs in high concentration had slight effect on proliferation of RSC96 cells and PC12 cells.High concentration of MWCNTs had a small effect on the proliferation of RSC96 and PC12 cells.Under electron microscopy,PC12 cells grew well on the MWCNTS agarose scaffold.The addition of MWCNTS did not change the expression of S100 in RSCs.Under electrical stimulation,the RSC96 cell layers arranged well along the longitudinal axis of the scaffold,and the 0.025% MWCNT-agarose scaffold and 0.05% MWCNT-agarose scaffold showed better cellular adhesion than the others.(2)The prepared PLLA fiber film had a porous structure,and most of the fibers are aligned.The highly aligned fiber film has a higher fiber orientation,with smooth surface and uniform thickness.Threedimensional pores were formed among the fibers and distributed throughout the fiber film.The tensile strength of the aligned PLLA fiber membrane was(59.06±2.49)MPa,and the tensile strength of the aligned PLLA fiber membrane was(56.82±2.80)MPa.There was no statistical difference in the tensile strength of the two fiber films.The Young's modulus of the aligned PLLA fiber membrane was(1037.07±133.30)MPa,and that of the highly aligned PLLA fiber membrane was(971.45±112.39)MPa.There was no statistical difference between the two types of fiber membrane.The elongation at break of aligned PLLA fiber film was 364.48±53.79,and that of Highly aligned PLLA fiber membrane was356.89±40.21.There was no statistical difference between the two types of fiber membrane elongation at break.Compared with aligned PLLA fiber membrane,PLLA fiber membrane with higher orientation did not change the tensile strength,Young's modulus and elongation at break of the fiber membrane significantly.Changing the fiber orientation of the fiber film had no effect on the degradation rate of PLLA fiber membrane.Both aligned PLLA and high-aligned PLLA had no effect on the proliferation of RSC96 cells.RSC96 cells on the highly aligned PLLA fibers showed distinct filamentous pseudopodia,extending along the neatly arranged nanofibers,compared with the aligned fibers.Highly aligned PLLA fibers increased ERK,P38 MAPK,MEK and LRP4 protein expression in RSC96 cells(P<0.01).(3)The bionic conductive artificial nerve scaffolds were well matched with rat sciatic nerve.Foreign body reaction was not observed after the scaffolds were implanted in the nerve.The sciatic nerve function index,electrophysiological results of muscle histology,axon and myelin regeneration in group E were similar to those in group AT,but better than those in group S.12 weeks postoperation.Immunohistochemical results showed that(1)the expression levels of NF-H protein and S00-? protein in group S&E and group AT were higher than those in group S(P<0.01),while those in group S&E was close to those in AT;Compared with group S,the expression levels of LRP4 protein and p38 MAPK protein in group AT were higher(P<0.01),while there was no significant difference between group AT to group S&E and group S&E to group S;The expression levels of ERK protein and MEK protein in AT group was higher than those in S and S&E group(P<0.01),there was no statistically significant difference between S&E group and S group.Conclusion:(1)Multi-walled carbon nanotubes(MWNT)agarose scaffolds have high porosity,certain shape memory function,drug loading and drug release ability,electrical conductivity ability,and good biocompatibility.Under electrical stimulation,the conductive multi-walled carbon nanotubes(MWCNTS)agarose scaffold guided the growth of RSC96 cells(2)Compared with the aligned PLLA fiber membrane,the highly aligned PLLA fiber membrane did not change its mechanical properties,degradability and biocompatibility,but increased the expression levels of ERK,P38 MAPK,MEK and LRP4 proteins,which as beneficial to the growth and migration of RSC96 cells and the application in peripheral nerve regeneration.(3)Bionic conductive artificial nerve scaffolds combined with electrical stimulation can enhance peripheral nerve regeneration.Electrical stimulation may promote peripheral nerve regeneration through activation of MAPK signaling pathway,involving ERK,p38 MAPK,MEK and LRP4.