Application And Mechanism Of PHBV/PLA/Col-Based Scaffolds In The Treatment Of Spinal Cord Injury

[Backgrounds and Objectives]Because axonal regeneration in the adult spinal cord is rare,spinal cord injury(SCI)often results in incurable neurological dysfunction.Currently,one of the most important challenges in SCI regeneration is the creation of an artificial scaffold that can mimic the extracellular matrix(ECM)and support nervous system regeneration.Nanofibrous scaffolds are artificial ECMs that provide a natural environment for tissue regeneration,which is more efficient than other forms of scaffolds due to the high surface-to-volume ratio.Electrospun nanofibers can mimic the temporal cellular environment and might also provide signaling cues to the cells with which they are in direct contact.These types of interactions could guide cellular activities such as cell adhesion,migration,and differentiation.As nanofibers may alter the intracellular signaling pathways associated with the control of astrocyte behavior,and also suppress glial fibrillary acidic protein(GFAP)expression,we postulated that the micropeg of PHBV/PLA-based nanofibrous scaffolds may regulate the activation of astrocytes and produce more effective neural regeneration following SCI.The present study evaluated the biocompatibility of electrospun PHBV/PLA and PHBV/PLA/Col nanofibers in vitro with the application of astrocytes.Additionally,the potential of PHBV/PLA-based nanofibers for nerve tissue regeneration and functional recovery in vivo was investigated.[Methods]Experiment 1:The morphological and chemical properties of the electrospun scaffolds were investigated.The growth and proliferation of astrocytes on the scaffolds were assessed by MTT and LDH assay.The differentiation and gene expression of astrocytes on the scaffolds were measured by immunofluorescence and quantitative real-time polymerase chain reaction(q-PCR)assays.In a rat spinal cord hemisection model with 3-mm defects,80 Sprague-Dawley rats were randomly divided into five groups:Sham group,SCI group,SCI+PHBV/PLA group,SCI+PHBV/PLA/Col(70:30)group,and SCI+PHBV/PLA/Col(50:50)group.The Basso-Beattie-Bresnahan(BBB)scores were evaluated every week postsurgery,and(immuno)histological and protein analyses were performed on specimens at 8 weeks.Experiment 2:P-P-C 1.5 and P-P-C 4.5 nanofiber membranes were constructed.The fiber diameter were measured by SEM.The proliferation and toxicity test of micro/nano membrane on VSC 4.1 was determined by CCK-8 and LDH.Implantation experiments were carried out to determine the degradation cycle and inflammatory response.In a rat thoracic spinal cord injury model(Allen's method),72 SD rats were randomly divided into 6 groups:sham group,control group(injury without treatment),allograft membrane group,P-P membrane group,the P-P-C 1.5 membrane group,the P-P-C 4.5 membrane group.The hindlimb motor function was scored every weekly using the BBB score.After 3 days and 8 weeks postoperatively,the samples were obtained and detected by HE,LFB staining and western blot.[Results]Experiment 1:PHBV/PLA/Col scaffolds strongly inhibited the activation of astrocytes without decreasing their proliferation.qPCR assays revealed significant increases in the expression of brain lipid-binding protein(BLBP),glutamate transporter 1(GLT-1)and S100 calcium-binding protein B(S100-?),but decreases in the expression of glial fibrillary acidic protein(GFAP),chondroitin sulphate sulfate proteoglycan(CSPG),neurocan,and phosphacan in the PHBV/PLA/Col scaffold group.In a series of in vivo experiments,PHBV/PLA/Col scaffold-treated SCI groups showed significant reductions in the numbers of CD68-and GFAP-immunopositive astrocytes within the interface of the remodeled tissue layer,but increased expression of NF-200 in residual neurons with better locomotor functional recovery.However,there were no significant differences between the PHBV/PLA/Col(70:30)and PHBV/PLA/Col(50:50)groups,except in BBB scores.Experiment 2:The P-P-C micro/nano membranes were substantially non-toxic to VSC 4.1 cells and promoted its proliferation,and SEM showed that VSC 4.1 cells interacted well with the nanofiber.The inflammatory response of P-P-C 1.5 and P-P-C 4.5 nanofiber membranes were significantly lower than that of P-P group after subcutaneous implantation,with a decreased expression of CD68.After spinal cord contusion and dural decompression,the expression of NLRP3 was reduced in the allograft membrane group and micro/nano-membrane groups,and accompanied by a decreased expression of IL-1?,TNF-? and CD68,especially in P-P-C 4.5 group.At 8 weeks postsurgery,the expression of GFAP in the injured area of the micro/nano-membrane group was lower than that of control group and the allograft membrane group,but there was no significant difference among the micro/nano-membrane groups.However,there was no significant difference in GAP43 protein expression among all these groups.[Conclusion]PHBV/PLA/Col nanofibrous scaffolds were biocompatible and significantly promoted astrocyte differentiation but decreased astrocyte activation.The topographic structures of the PHBV/PLA/Col(70:30 and 50:50)nanofibers were favorable for neural regeneration due to a decrease in astrogliosis in SCI rats.The nanofibrous membrane of P-P-C 1.5 and P-P-C 4.5 was able to promote the adhesion and proliferation of VSC 4.1.After spinal cord contusion and early surgical decompression,PHBV/PLA/Col membranes were used for duraplasty,which reduced inflammation and decreased the expression of GFAP in the injury areas.