| ObjectivePromoting nerve tissue regeneration is the key to the treatment of spinal cord injury(SCI).In recent years,researchers have implanted various biomaterials with stem cells and growth factors into the spinal cord injury area to promote the repair of spinal cord injury.In addition to biocompatibility,biomaterials also need to mimic the three-dimensional structure and microenvironment of spinal cord to promote cell proliferation and differentiation.Therefore,in order to simulate the three-dimensional niche microenvironment of spinal cord and realize the continuous release of growth factors in the area of spinal cord injury,this study used porcine spinal cord decellularized matrix scaffold(SC-DM)as a biological material to carry human umbilical cord mesenchymal stem cells(HUCMSCs)which overexpressing basic fibroblast growth factor(b FGF)to treat spinal cord completely transection in rats.To study the therapeutic effect of SC-DM loading HUCMSCs(b FGF-HUCMSCs)that can continuously release b FGF factor in spinal cord injury.Methods and Contents1.SC-DM biological scaffolds were obtained by decellularized technology,and SC-DM biological characteristics were measured.2.Preparation of HUCMSC and b FGF-HUCMSC,and HUCMSCs,b FGF-HUCMSCs and DRGs were culture on SC-DM.3.Preparation rats’ SCI model and implantation composite biomaterials.4.Open-field locomotion assessment after SCI.5.HE staining was used to detect the repair effect of SCI rats after 4 weeks of operation.6.Pathology related staining for axon regeneration,glial cells and the proliferation and migration of neural stem cells detection.Results1.SC-DM biomaterials were obtained from porcine spinal cord after decellularized.Scanning electron microscope(SEM)showed SC-DM had a porous structure,which provided a three-dimensional structural basis for cell adhesion and material metabolism.At the same time,HE staining suggested there was no cell residue on the prepared SC-DM scaffold,which reduced the immunogenicity of the biomaterial.Collagen Ⅰ immunohistochemistry showed that SC-DM material was rich in collagen Ⅰprotein,which provided adhesion sites for cells.2.The good biocompatibility of SC-DM was also confirmed by the culture of HUCMSCs/ b FGF-HUCMSCs and axon formation of DRGs on SC-DM.The expression of b FGF was detected by western blotting and the results showed the level of b FGF was significantly higher in b FGF-HUCMSCs group compared to HUCMSCs group.3.After spinal cord injury,the motor function of hind limbs in each group gradually recovered with time,and the scores of each group were lower than 2 points at day 7after operation.At 2,3 and 4 weeks after operation,the BBB score of SC-DM / b FGF HUCMSCs group was significantly higher than that of other groups.In addition,SC-DM/HUCMSCs group was also higher than SC-DM group and Control group.There was no significant difference between SC-DM group and Control group.4.HE staining was used for histological evaluation after SCI 4 weeks.In Control group,many inflammatory cells gathered and infiltrated into injury center.The density of linear fibrous tissues and cell infiltration were significantly increased in and around the SC-DM/b FGF-HUCMSCs group compared to Control and SC-DM groups.5.Tuj1 and NF200 immunofluorescence staining showed that the fluorescence intensity of SC-DM/b FGF-HUCMSCs group increased significantly in injured site.GFAP immunofluorescence staining showed that the number of astrocytes increased in SC-DM/HUCMSCs group and SC-DM/b FGF-HUCMSCs group.Nestin immunofluorescence staining significantly increased the number of positive cells in SC-DM/HUCMSCs group and SCDM/b FGF-HUCMSCs group.ConclusionsIn conclusion,SC-DM carrying HUCMSCs overexpressing b FGF can provide an effective three-dimensional microenvironment to enhance the ability of neuronal axon regeneration.And promote axon regeneration through the continuously releasing b FGF growth factor,so as to promote the repair of spinal cord injury tissue in rats. |
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