The Study On Spinal Cord Injury Repair By Thermosensitive Hydrogels Carrying Nafamostat

Objective: The aim of this study was to explore the effectiveness of the independently developed Nafamostat/PLGA-PEG-PLGA thermosensitive hydrogel with drug slow-release in repairing spinal cord injury(SCI).By measuring its properties,analyzing its biocompatibility,transplanting it to the SCI area of rats,the safety and application effect of the hydrogel drug-loaded system were evaluated,and providing a new idea for the repair of SCI.Methods: 1.Preparation and characterization of drug-loaded thermosensitive hydrogels: Prepared the drug-loaded thermosensitive hydrogel solution,observed and recorded its phase transition temperature to verify its rheological change with the change of temperature,and determine its sustained-release ability.2.In vitro biocompatibility test: 20% drug-loaded thermosensitive hydrogel solution(Nafamostat content 10mg/ml)was co-cultured with He La cells for 3 days.Observed the changes of cell morphology and proliferation under microscope and evaluated the effects of Nafamostat on cell activity by MTT cytotoxicity assay.3.Evaluation of the effectiveness of hydrogel drug delivery system in repairing SCI: The SCI model of rats was established by using Impactor model-II,injected drug-loaded thermosensitive hydrogel solution into the injured site,and evaluated BBB motor function in rats.After 8 weeks,we can observed the cavity area of SCI area by HE staining of spinal cord tissue section,inspected the astrocyte aggregation in injury center by GFAP immunohistochemical staining and observed the ability of nerve fibers to cross spinal cord injury region by BDA anterograde tracing.Result: 1.Properties of hydrogel drug delivery system: The hydrogels were observed to be spherical micelles with diameters ranging from 10 nm to 30 nm,and the drug particles were uniformly dispersed in the middle of the micelles or micelles.2.Biocompatibility evaluation: In cytotoxicity test,Nafamostat/PLGA-PEG-PLGA and Hela cells co-cultured for 3 days showed no abnormal cell morphology under microscope,and the cell proliferation was good.MTT research showed that 20% drug-loaded thermosensitive hydrogel solution had no significant effect on cell relative activity compared with other experimental groups(P>0.05).3.Motor function evaluation: There was no significant difference in BBB score between groups from 1 day to 1 week after SCI(P>0.05).Two weeks after operation,the scores of each group were statistically significant(P<0.05).The BBB score of Nafamostat loaded thermosensitive hydrogel group was 12.10±0.88 at the end of the experiment,that is,8 weeks after operation,which was significantly higher.4.Histomorphological observation: After 8 weeks of SCI,through observing the slices of spinal cord tissue,it was found that cavity area in the injured area of the control group was the largest,a large number of scar tissue was formed,and the area of injured area was significantly larger than other three groups(P<0.001).The damage area of Nafamostat-loaded thermosensitive hydrogel group was the smallest.5.Immunohistochemical staining: After 8 weeks of operation,the spinal cord tissue was cut longitudinally,performed GFAP immunohistochemical staining and observed the damage center,we can see that the GFAP positive area of Nafamostat-loaded thermosensitive hydrogel group was the smallest than that of other groups,and the results were statistically significant(P<0.01).In the Nafamostat-loaded thermosensitive hydrogel group,BDA staining of each group showed that the nerve fibers of rats could cross the spinal cord injury region,while the other groups had fewer nerve fibers across the injury area,and the control group had the fewest nerve fibers.Conclusion: Nafamostat-loaded thermosensitive hydrogel solution prepared by our team has good drug dispersibility,stable and slow drug release process and good biocompatibility.Compared with the single drug transient local action,injected the drug carrier gel into the spinal cord injury part of the rat can act on the injury part for a longer time by the way of drug diffusion and slow release.This will significantly improve the motor function of rats after spinal cord injury,reduce the cavity area of spinal cord injury area,reduce the formation of astrocytes and glial scar,promote the regeneration of neural synapses,regulate the microenvironment of spinal cord injury,and play a role in repairing spinal cord injury.This study provides a theoretical basis for improving the repair efficiency of spinal cord injury.