Experimental Study Of Schwann Cell-derived Exosomes Combined With Platelet Rich Plasma-derived Exosomes In Repairing Spinal Cord Injury

?Background?Spinal cord injury(SCI)is a common disabling injury of the central nervous system,which brings a heavy economic burden to society.Traditional treatments include surgery,medicine,and rehabilitation,while advanced therapeutic strategies are stem cell transplantation and tissue engineering repair.With the progress of tissue repair technology,new therapeutic strategies include stem cell transplantation and tissue engineering repair,the therapeutic effect has been improved,but a satisfactory repair effect still cannot be achieved.To date,there is no effective treatment for SCI.Therefore,studying safe,efficient,and biocompatible drugs for SCI repair is very important.Exosomes are about 40-160 nm diameter vesicles secreted by cells into the extracellular environment,containing nucleic acids,proteins,lipids,and metabolites.Exosomes have the following advantages compared with parental cells:(1)they are more stable,(2)they contain higher concentrations of growth factors,(3)they are biocompatible,(4)they reduce the safety risks inherent in using living cells,and(5)they can easily cross the blood-brain barrier.Exosomes with their biological advantages have gradually become the focus of medical tissue regeneration,and have been used to treat cancer and degenerative neurological diseases.After spinal cord damage,the spinal cord can produce a variety of pathophysiological changes,such as capillaries rupture,ischemia,axonal degeneration,nerve inflammation,demyelination changes,glial activation lipid peroxidation,glial scar formation,cell apoptosis,and ionic imbalance;these complex changes will affect the spinal cord in the local environment,thus increasing the difficulty of the SCI treatment.Cardiovascular growth and angiogenesis are two important goals in SCI therapy.Schwann cells(SCs)can promote axon growth and myelin sheath formation,while platelet-rich plasma(PRP)can promote angiogenesis.Schwann cell-derived exosomes(SCs-exos)and platelet-rich plasma-derived exosomes(PRP-exos),as the second-generation derivative of SCs and PRP,is likely to have stronger tissue repair ability than SCs and PRP,respectively,and their superior functions are different.However,SCs-exos combined with PRP-exos promote SCI repair,and related mechanisms remain unclear.We hypothesized that the combined application of SCs-exos and PRP-EXOS may play a synergistic repair effect and better effect of SCI treatment.Therefore,this research took SCs-exos and PRP-exos as the research object to study the combined application of SCs-exos and PRP-exos in promoting nerve and vascular regeneration after SCI and elucidate the related mechanisms.This study will provide a theoretical basis for exploring new therapeutic drugs for SCI repair and has a good clinical transformation prospect.?Objective?1.To observe the effects of combined SCs-exos and PRP-exos on the proliferation,migration,and differentiation of neural stem cells and vascular endothelial cells in vitro;2.The effects of SCs-exos combined with PRP-exos on nerve function recovery and nerve and vascular tissue repair in rats after SCI were observed in vivo.3.To elucidate the related mechanisms of the application of combined SCs-exos and PRP-exos to promote SCI repair.?Method?1.SCs-exos and PRP-exos were extracted from rat Schwann cell RSC96 cell line and rat PRP by supercentrifugation,respectively.The two exosomes were identified by transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA),and western blot(WB).2.After co-culture of SCs-exos + PRP-exos with neural stem cells or RAOEC rat vascular endothelial cells,the distribution of the two exosomes around neural stem cells and RAOEC rat vascular endothelial cells was observed.Subsequently,the experiment was divided into Control group(equal amount of medium),SCs-exos group,PRP-exos group,and SCs-exos + PRP-exos group.The four groups were co-cultured with neural stem cells or RAOEC rat vascular endothelial cells,respectively.Subsequently,the effects of SCs-exos + PRP-exos on the growth of neural stem cells and RAOEC rat vascular endothelial cells were observed.3.SCI model was established by clamp method,and the rats were locally given normal saline,SCs-exos,PRP-exos,and SCs-exos + PRP-exos.After 28 days,the neurological recovery of each group was evaluated,including BBB score,hind limb movement footprint analysis,and spinal cord electrophysiological monitoring.The tissue repair and regeneration of rats after SCI were also observed.The evaluation methods included HE staining,Masson staining,FLB staining,and tissue immunofluorescence staining(Ch AT,TH,?-tubulin,CD31,CD68).4.The concentrations of various growth factors in SCs-exos and PRP-exos with the same total protein amount were compared by ELISA to clarify the mechanism of SCs-exos + PRP-exos promoting functional recovery and tissue regeneration after SCI.?Results?1.SCs-exos and PRP-exos could be successfully extracted from rat Schwann cell RSC96 cell line and rat PRP by supercentrifugation method.TEM results showed that SCs-exos and PRP-exos were disc-shaped with diameters of about 60 nm and110 nm,respectively.NTA results showed that the particle sizes of SCs-exos and PRP-exos were 53.33 ± 23.83 nm and 96.46 ± 56.98 nm,respectively.WB results showed that SCs-exos and PRP-exos were highly expressed on marker proteins CD9,CD63 and CD81.The above identification results showed that the extracted nanovesicles were exosomes.2.SCs-exos and PRP-exos can be distributed around neural stem cells and RAOEC rat vascular endothelial cells,thus affecting the biological functions of the two cells.SCs-exos + PRP-exos group was better than other groups in promoting the proliferation and differentiation of neural stem cells into neurons(P < 0.05).SCs-exos + PRP-exos group had better effects on promoting vascular endothelial cell proliferation,migration,and angiogenic differentiation in RAOEC rats than other groups(P < 0.05).3.The clipping method can successfully prepare the rat SCI model.Compared with the control group,treatment groups including SCs-exos,PRP-exos,and SCs-exos+ PRP-exos groups have better improvement of lower limb function,and SCs-exos+ PRP-exos has the best improvement effect.In HE staining,the SCs-exos +PRP-exos group has the smallest area of the spinal cavity.The SCs-exos +PRP-exos group has the lowest collagen deposition in Masson staining.In FLB staining,the SCs-exos + PRP-exos group has the most myelin regeneration.Tuj-1,Ch AT,and TH immunofluorescence assay showed that the number of neurons,motor neurons,and sensory neurons in the SCs-exos + PRP-exos group was more than that in other groups(P < 0.05).CD34 immunofluorescence assay showed that the SCs-exos + PRP-exos group had a better angiogenesis-promoting effect than others(p<0.05).CD68 immunofluorescence assay results showed that the number of CD68(+)microglia/macrophages in the SCs-exos + PRP-exos group was lower than that in other groups(P < 0.05).4.Under the same total protein concentration,SCs-exos is rich in BDNF,NGF-?,b FGF,CNTF,NT-3,NT-4,NT-5,and PRP-exos is rich in VEGF,EGF,TGF-?1,PDGF-AA,PDGF-AB,and PDGF-BB.?Conclusions?1.In vitro experiments,SCs-exos and PRP-exos can be successfully extracted from RSC96 cells and PRP by ultra-centrifugation method,which has the advantage of simple operation.SCs-exos + PRP-exos could promote the proliferation,differentiate into neurons and anti-apoptosis of neural stem cells.Moreover,SCs-exos + PRP-exos can also promote proliferation,migration,and angiogenic differentiation of vascular endothelial cells.2.In vivo experiments,SCs-exos + PRP-exos can improve the recovery of nerve function after SCI in rats,promoting nerve regeneration,angiogenesis,and inhibiting local scar formation.3.SCs-exos contains a large number of growth factors promoting nerve regeneration,including NT-3/4/5,NGF-?,BDNF,and a certain amount of growth factors promoting angiogenesis,such as VEGF.Besides,PRP-exos contains a large number of angiogenesis promoting growth factors(PDGF-AB,PDGF-BB),a large number of cell differentiation and repair promoting growth factors(IGF-1,TGF-?),and a small number of nerve regeneration promoting growth factors(NT-3/4/5,CNTF).The difference in the types and concentrations of growth factors rich in SCs-exos and PRP-exos is the main reason for their different functions in the treatment of SCI,and also an important mechanism for the sequential interactive synergistic repair effect to maximize the therapeutic effect when combined application.