Research On Neural Cell Differentiation And Spinal Cord Injury Repair Induced By 3D Electrospinning Controllable Nanofiber Catheter

Objective: Due to the poor regeneration ability of nerve fiber bundles and the complex microenvironment,spinal cord injury(SCI)has become a difficult problem in medical field today.Based on the anatomical characteristics of the directional arrangement of neurons in the spinal cord into nerve conduction bundles,the purpose of this study intends to construct multi-component directional neural tube to realize the directional growth of neurons,thus contributing to the treatment of spinal cord injury.This research not only provides new ideas for the research of 3D electrospinning,but also provides new ideas and methods for the clinical treatment of spinal cord injury.Methods:(1)3D electrospinning materials and process screening: Screen spinning properties and freeze-thaw properties of series polyvinyl alcohol(PVA)to determine the materials and proportions of 3D printing electrospinning,then screen the optimal process parameters of 3D electrospinning to prepare induced PVA fibers.(2)Investigation on the biocompatibility and cell growth of directional PVA fibers:Isolate ectodermal mesenchymal stem cells(EMSCs)from rat nasal mucosa,then investigate the biocompatibility of PVA electrospun fibers and their effects on cell growth direction.Morphological observation and immunofluorescence technology(IF)were used to screen the formula for inducing EMSCs differentiation into neural cells,and investigate effect of PVA electrospun fiber spacing on inducing cell growth.(3)Anti-inflammatory study of isoliquiritigenin(ISL)loaded red blood cell membrane nanoparticles(RBCM): Extraction and preparation of RBCM by hypotonic method.Characterize ISL@RBC NPs by FT-IR,SEM,TEM,and DLS particle analysis,and investigate the anti-inflammatory effect of ISL@RBC NPs on LPS polarized macrophages by protein expression and reactive oxygen species(ROS)changes.(4)Construction of neural tube: Prepare methacrylate gelatin(Gel MA)loaded with ISL@RBC NPs by 3D extrusion printing as the base film.Print directional structures on the base film by 3D electrospinning technology,and curling after inoculate EMSCs to form neural tube.Investigated the cell activity and the release of ISL@RBC NPs in neural tube.(5)Repair of spinal cord injury in rats: Implant the neural tube into the spinal cord injury SD rats and observe the repair effect.Initial evaluation of spinal cord injury repair based on BBB score,oblique plate tests and footprint analysis.The appearance of the spinal cord and HE staining were used to detect histopathological differences.The recovery of neurons and myelin sheath at the injured site was tested by Nissl and myelin staining.Immunofluorescence staining was used to detect the expression of neural related proteins(NF200,Tu J1,syn GAP,GFAP).Results:(1)Identification of the 3D electrospinning material and process:through material screening and printing conditions screening,determined the optimal3 D electrospinning material and the proportion of 5%PVA4688 + 5%PVA117;The optimal process parameters of electrostatic spinning were determined as follows: needle type 22 G,electric field voltage 6 k V,extrusion speed 0.05 μL/s,printing speed 20mm/s,needle height 0.5 mm.SEM observation showed that the diameter of PVA fibers was about 1μm,and multiple fibers were oriented and evenly spaced.(2)Directed PVA fibers showed excellent biocompatibility and successfully induced the directed growth and neuronal differentiation of EMSCs: EMSCs were successfully extracted from the nasal septum mucosa of SD rats,and the positive expression of mesenchymal stem cell markers were identified by immunofluorescence and flow cytometry.MTT showed no obvious cytotoxicity of PVA.Cytoskeleton staining showed that the directed arrangement of PVA fibers could effectively realize the directed arrangement of 3T6 and EMSCs cells.The results of Ed U and live/dead cell staining showed that PVA had no effect on cell proliferation and survival.The prescription was successfully screened to induced EMSCs differentiate into nerve cells and express neuronal markers.And induced neuronal growth along PVA.The PVA spinning interval was determined to be20 μm.(3)Successfully prepared ISL@RBC NPs with anti-inflammatory effect:RBCM was successfully extracted and the mean particle size of RBC NPs increased from 225.16±12.68 nm to 264.59±44.08 nm after ISL loading.TEM images showed that ISL@RBC NPs has obvious coating structure,ζ potential and FT-IR indicate the successful formation of ISL@RBC NPs.Encapsulation rate was 55.63%,drug loading was 38.9±2.99%;ISL@RBC NPs could promote the expression of CD206 and Arg-1,reduce the expression of i NOS and inhibit the production of reactive oxygen species in RAW264.7 cells induced by LPS,so as to effectively inhibit inflammation and protect damaged neurons.(4)Successfully constructed oriented neural tube containing multi-component drug release system: The Gel MA base film was successfully prepared by 3D extrusion printing and the PVA oriented fibers were spun on the film by 3D electrostatic spinning.In vitro release results showed that ISL could be released slowly in the neural tube.Cell experiment results showed that the survival rate of EMSCs cells and 3T6 cells in neural tube group was 88.25% ± 7.42 and 96.46% ± 2.86,respectively.In addition,EMSCs cells could be directed to grow and differentiate in the PVA/Gel MA neural tube.(5)Repair results of spinal cord injury in rats: SCI results showed that BBB score recovered to about 15 and Angle of oblique plate recovered to about 36° after 8 weeks of directional neural tube group,which was significantly higher than model group(P<0.01).Combined with the appearance of spinal cord and H&E results,the overall repair of the directional neural tube group was excellent.Nissl and myelin staining showed that Nissl bodies in the directional neural tube group were arranged in orderly manner,with no obvious demyelination or cavity.A large number of purple neurons and light blue myelin sheath could be seen in the injured area in directional neural tube group,indicating that the neural tube could connect the upper and lower nerve fibers,promote the regeneration of myelin sheath and improve the survival of neurons at the injured area.Immunofluorescence results showed that the expression of NF200,Tu J1 and syn GAP proteins at the injured site of spinal cord was up-regulated and the expression of GFAP was down-regulated in directional neural tube group.These results demonstrate the critical role of multi-component directional neural tube in spinal cord repair,including inhibiting inflammation and scar hyperplasia,promoting the growth of new neurons and nerve repair.Conclusion: The directed growth and neuronal differentiation of EMSCs were successfully induced by directed PVA fibers using 3D electrospinning technology.At the same time,oriented neural tube containing multi-component drug release system combined with ISL@ RBC nanoparticles,directional neural tube containing a multicomponent drug delivery system was successfully constructed to repair rat spinal cord injury.This provides new ideas and technologies for the construction of new drug release system,spinal cord injury repair,and the innovation and application of 3D printing technology.