The Experimental Study On The Efficacy Of Exosomes Combining Hydrogel On Neural Repair After Traumatic Brain Injury

Background and objective: Traumatic brain injury(TBI)is a major cause of death and long-term disability worldwide,which imposes a heavy burden on families,economy and society.Currently,there has not been an efficient therapy for neural repair in TBI patients.As a paracrine factor,stem-cell-derived exosomes can exert biological function of stem cells in vivo and avoid cell-related adverse reactions,such as immune rejection,teratogenesis and tumorigenesis.However,the treatment is faced with problems such as low yield,loss of transplantation,and maintenance of local concentration.This study aims to explore an appropriate hydrogel for exosomes-hydrogel complex transplantation,and to explore an effective therapy to promote neurorepair after TBI.Methods: 1.HMSC-bm were mass-cultured and expanded in vitro,induced differentiation and flow cytometry(CD44,CD45,CD90,CD105)were performed to verify the differentiation ability and stemness of HMSC-bm.2.Exosomes were extracted from the cell supernatant by hypervelocity centrifugation.Then.the exosome markers(CD63,Tsg101)were detected by Western Blot,the particle size was detected by q Nano nano-analyzer,and the morphology was observed by transmission electron microscopy.3.Exosomes were mixed with hydrogel,detecting the concentration of exosomes in suspension by q Nano nano-analyzer to understand the sustained-release effect.4.The model of TBI rat was built by free-fall blow method.Then transplanting exosome-hydrogel,and evaluating the modified neurological deficit score(m NSS).After 28 days,the rats were sacrificed,and the brain tissues were stained with HE and immunohistochemistry(CD68,GFAP).Results: 1.HMSC-bm was fusiform in shape and could be induced to osteoblasts,adipocytes and chondrocytes.Flow cytometry showed that the specific markers CD105,CD44 and CD90 were expressed in more than 90% of P8 cells,but decreased significantly from P9 cells.2.The exosome markers(CD63 and Tsg101)were positive by Western Blot.The size of exosomes was 30-180 nm in diameter.And transmission electron microscopy of exosomes showed a typical “cup-shaped” morphology.3.Exosomes can be retained for at least 3 days with exosomes-hydrogel,and the retention rate was 28.47% after 3 days.4.From the 7th day after surgery,the m NSS scores of exosomes group and exosomes-hydrogel group was significantly lower than the control group(P < 0.05).From the second week,the exosomes-hydrogel group showed better repair effect than the exosomes group(P < 0.05).HE staining showed that the brain tissue injured area of exosomes-hydrogel group was less than the TBI group.Immunohistochemical results showed that the numbers of GFAP+ astrocytes and CD68+ macrophages/microglia in the cerebral cortex and dentate gyrus were significantly decreased in the exosomes-hydrogel group and the exosomes group(P < 0.05).However,there was no statistically significant difference between the two groups(P > 0.05).Conclusion: Human bone marrow mesenchymal stem cell-derived exosomes combined with hydrogel have shown a certain sustained release effect in vitro.The complexes can significantly improve the recovery of neurological function in TBI rats,and exosomes can reduce the incidence of neuroinflammation.