Experimental Study On The Repair Of Spinal Cord Injury By Light Curing GelMA Hydrogel With Metformin Loaded With IKVAV Polypeptide PLGA

BackgroundSpinal cord injury(SCI)is a devastating nervous system disease that includes a series of destructive events such as ischemia,inflammation,oxidative stress,and sensory motor disorders,which easily leads to disability or death of patients and brings a huge economic burden to the family and society.The commonly used therapeutic measures include decompression surgery and extensive hormone shock therapy.Due to the severity of the injury and the complexity of the mechanism,the therapeutic effect is still limited,more effective therapies need to be developed to deal with motor dysfunction and disruption of neural pathways after spinal cord injury.In recent years,with the rapid development of tissue engineering,it has shown unique advantages in the treatment of spinal cord injury.Tissue engineering materials are rich in types and functions,with good biocompatibility and biodegradability.They can perform adjustable mechanical matching according to tissue characteristics,fill in spinal cord defects,act as extracellular matrix,and carry nutrients or drugs to play the role of targeted or in situ therapy.The inflammatory microenvironment,neuronal death and glial scar formation after spinal cord injury are key factors that limit the therapeutic effect.Although it has been reported that tissue-engineered materials loaded with anti-inflammatory drugs or pro-differentiation substances can play a therapeutic role,few literatures have focused on the synergistic effect of anti-inflammatory and differentiation,as well as specific treatment for different injury periods.Therefore,on the basis of Gelatin methacryloyl hydrogel(GelMA),PLGA microspheres containing IKVAV polypeptide and Metformin(MET)were introduced in this study.It is expected that the inflammatory microenvironment after spinal cord injury can be improved through the anti-inflammatory effect of metformin in the acute stage,and the endogenous neural stem cells can be promoted to differentiate into neurons through sustained release of IKVAV polypeptides in the subacute and chronic stages,so as to repair the spinal cord injury with specific treatment according to the pathological process at different stages.ObjectiveTo explore whether metformin GelMA hydrogel loaded with IKVAV polypeptide PLGA microspheres can repair spinal cord injury and promote the recovery of motor function through anti-inflammatory and pro-differentiation effects at different time stages.MethodsThe microspheres of GelMA hydrogels and GelMA hydrogels containing PLGA were characterized to observe their pore size,elemental composition and mechanical strength.Afterwards,lipopolysaccharide(LPS)-induced inflammation model of mouse microglial cell line(BV2)was used.GelMA hydrogel loaded with metformin and BV2 cells were co-cultured through transwell chambers and set as the control,lipopolysaccharide,and metformin groups,respectively.The effect of metformin loaded GelMA hydrogel on levels of inflammatory cytokines and microglial polarization status was assessed by Western Blot,qRT-PCR,and immunofluorescence.GelMA hydrogel loaded with IKVAV peptide and neural stem cells were co-cultured through Transwell chamber and set as control group,GelMA hydrogel group,and GelMA+IKVAV peptide group,respectively.The effect of GelMA hydrogel containing IKVAV polypeptide on differentiation direction was evaluated by Western Blot,RT-qPCR and immunofluorescence.After that,in vivo experiments were carried out,and mice spinal cord injury model was used,which was divided into SCI group,GelMA group,GelMA+MET group,GelMA+MET+IKVAV polypeptide group.The recovery of motor function in different groups of mice was evaluated by electrophysiological test and motor function experiment,and the differentiation of neurons and regeneration of axons were evaluated by immunofluorescence staining experiment.ResultsThe characterization results showed that the addition of PLGA microspheres had no significant effect on the properties of GelMA hydrogels.Both GelMA hydrogels and GelMA hydrogels containing PLGA microspheres had suitable uniform pore size,matched the elastic modulus of spinal cord strength and good degradability.In vitro LPS-induced inflammation showed that compared with the Control group,the Metformin group decreased the expression of pro-inflammatory factors,increased the level of anti-inflammatory factors and promoted the polarization of microglia into M2 type.In vitro IKVAV polypeptide differentiation experiment showed that compared with the control group,the IKVAV polypeptide group increased the expression of Tuj-1 and decreased the expression of GFAP,which promoted the differentiation of neural stem cells into neurons.In animal experiments,compared with other groups,BMS score and electrophysiological potential amplitude of mice in GelMA+MET+IKVAV polypeptide group were significantly improved,which promoted the recovery of motor function.In addition,the number of NeuN,NF200 and Syn positive cells in the lesion area of GelMA+MET+IKVAV polypeptide group was significantly higher than that in other groups,indicating that the material promoted the differentiation and survival of neurons,and the regeneration of axons and synapses.ConclusionWith good biocompatibility and porosity,metformin GelMA hydrogel containing IKVAV polypeptide PLGA is a qualified tissue engineering material for the treatment of spinal cord injury.It can reduce inflammatory response and improve inhibitory microenvironment by releasing metformin.It also regulates the differentiation of neural stem cells by slowing the release of IKVAV polypeptide,reduces glial scar,promotes axon regeneration and improves motor function.