Effect Of Conductive Hydrogel System Loaded With Neural Stem Cells And Donepezil In Repairing Spinal Cord Injury

Research purposes:This study hopes to prepare an injectable conductive hydrogel with multiple excellent properties and perform related characterization and determination,and verify through in vitro experiments that the combination of the hydrogel and DPL is beneficial to the proliferation and differentiation of NSCs.Then use the conductive water Gel-loaded NSCs and DPL were implanted in vivo on a rat model of total excision to treat spinal cord injury to achieve nerve regeneration and repair of damaged spinal cord and recovery of motor function.This will also provide a new theory for nerve tissue engineering to treat spinal cord injury basis.Research method:1.Synthesis and characterization of injectable conductive hydrogelBy modifying the biopolymers HA-ALD and NH2-Gelatin-PANI through the imine bond formed by the aldehyde group and the amino group as the crosslinking point,a new injectable conductive hydrogel NGP was successfully prepared.The related properties of the hydrogel were characterized and determined by the vial inversion method,infrared spectroscopy,scanning electron microscopy,dynamic rheology,electrochemical station,and drug release experiment.Evaluate the degradation properties of materials through in vitro and in vivo degradation experiments.The biocompatibility of the hydrogel was identified by subcutaneous injection and HE staining.2.Biological effects of donepezil on NSC in hydrogel environmentIn the experiments in this chapter,we first use the primary isolation of NSCs from the fetal rat cerebral cortex of SD rats at 13-15 days of pregnancy,and then use the general suspension culture method to cultivate and expand them,and use the characteristic Nestin antibody to immunize Fluorescence staining was used to identify NSCs.Then,1,3,and 5 days of living and dead cell staining experiments were used to detect the cytotoxicity of NGP hydrogels respectively.Finally,our purpose is to explore the proliferation and differentiation of NSCs when NGP hydrogel and DPL are used alone and in combination.The DPL concentration in the group is 8ng/μl.We used 1,4,and 7-day CCK-8 experiments to detect the effect of DPL on the proliferation of NSC in the hydrogel environment,and the immunofluorescence staining experiment and RT-PCR experiment of cells after 7 days of culture to qualitatively and quantitatively explore the NGP conductive hydrogel The influence of DPL on the differentiation-promoting effect of NSC in the environment.3.Injectable conductive hydrogel equipped with NSCs and DPL to treat spinal cord injuryWe first established the SD rat T9 total spinal cord injury model,and used the injectable conductive hydrogel NGP to carry NSCs and DPL to the injury site for orthotopic transplantation.The postoperative rats were observed for 6 weeks,and the BBB score table The motor function recovery of rats is evaluated,and the recovery of the conduction function of rat nerve electrophysiological signals is evaluated by the electromyography instrument.The experimental rats in each group were perfused with spinal cord tissue,and the area of cystic cavity at the center of the injury,remyelination,new neurons,glial scars,and axons were observed by HE staining,fast blue staining,and immunofluorescence staining.And analysis,the overall evaluation of the motor function recovery and nerve regeneration and repair of each group of experimental rats,especially the NGP+DPL+NSCs group.Research result:In this research topic,we first successfully and innovatively prepared a new type of injectable conductive hydrogel NGP(HA-ALD:NH2-Gelatin-PAN),and then characterized its various properties and found that it has excellent mechanical strength,Injectability,self-healing,electrical conductivity,biocompatibility,drug release performance,and degradability matching the transplantation time after spinal cord injury.Next,we successfully extracted,cultured and identified neural stem cells,and then in vitro live-death staining experiments proved that NGP conductive hydrogel is non-cytotoxic and has good adhesion to cells.CCK-8 experiment results show that NGP hydrogel has a certain degree of promotion effect on the proliferation of NSCs,while the proliferation effect of DPL is not obvious.The results of cell immunofluorescence staining experiments and RT-PCR experiments show that the drug DPL can significantly promote the differentiation of NSCs into newborn neurons and oligodendrocytes.Quantitative analysis of neurite length shows that NGP hydrogel,but not DPL,promotes the growth and extension of neurites of newborn neurons.In addition,the combination of the two inhibits the differentiation of astrocytes the most obvious.Finally,we used the injectable conductive hydrogel NGP to carry NSCs and DPL in the SD rat T9 total spinal cord injury model for orthotopic transplantation at the injury site.We found that in the recovery of motor function and nerve conduction function,NGP+DPL The +NSCs combination is very effective.At the same time,this treatment strategy also significantly increased the number of new neurons and the area of axons,and also minimized the hindrance of glial scars.The research process also confirmed the repair effect of NGP conductive hydrogel and NSCs on spinal cord injury.Research conclusion:The injectable conductive hydrogel NGP prepared by us meets the performance requirements of transplanted biomaterials after spinal cord injury,and is suitable as a common transplant carrier for DPL+NSCs.The NGP hydrogel environment can promote the proliferation of NSCs and the extension of new neurons and neurites,but DPL,a directional differentiation inducer of NSCs,is required to significantly enhance the differentiation of neurons and oligodendrocytes and inhibit astrocytes.Cell differentiation can prevent massive glial scar formation.In in vivo animal experiments,the treatment group of NGP+DPL+NSCs showed certain improvement in the recovery of motor function and nerve conduction function.At the same time,the formation of myelin sheath was significantly increased in the injury center of the rats in this group,the regeneration of new neurons and the growth and connection of new axons,and the obstruction of cystic cavity area and glial scars were also minimized.The new treatment combination of NGP conductive hydrogel + DPL drugs + NSCs also provides new ideas for nerve tissue engineering to repair spinal cord injury from both neuroprotection and nerve regeneration.