Study Of Overexpression Of Satb2 Promoting Neurogenesis During Bone Tissue Regeneration

Background and ObjectivesRecently,the rapid development of theory and technology of tissue engineering provides new possibilities for the remodeling of bone structure and functional recovery.However,maxillofacial tissue engineering bone has not yet achieved simultaneously vascularization and neuralization.This fails to achieve mechanical strength and plasticity physiologically,making it difficult for tissue engineering to transform from basic research to clinical application.Specific AT-rich sequence binding protein 2(SATB2)is a tissue-specifically expressed nuclear matrix-binding protein.It acts as a molecular node in maxillofacial development and bone modeling,regulating the differentiation and maturation of osteoblasts and promoting periodontal tissue regeneration.Moreover,SATB2 is also involved in the formation of projection neurons in corpus callosum of cerebral cortex.Although current researches suggest that SATB2 plays an important role in bone tissue regeneration,few studies have demonstrated the role and mechanism of SATB2 on neurogenesis during bone defect repair.If there is a breakthrough on the topic,it will lay a foundation for the possible clinical application of gene therapy of Satb2 and bring new hope and possibility for the formation of functional bone.We have demonstrated that overexpression of Satb2 can induce differentiation of rat bone marrow mesenchymal stem cells into neuron-like cells in vitro,with promoting the expression of NSE and NGF.Therefore,this study intends to further verify whether Satb2 plays a role in nerve regeneration while repairing bone defects in vivo,so as to provide new ideas and therapeutic targets for the application of Satb2 in bone regeneration.Methods1.Isolation,Culture and Identification of Rat Bone Marrow Mesenchymal Stem cellsRat bone marrow mesenchymal stem cells(rBMSCs)were isolated and cultured by the method of whole bone marrow adherent screening.Monoclonal cells were obtained by clonal formation assay and the proliferation ability was evaluated.Flow cytometry was used to detect the surface markers of rBMSCs.The multiple differentiation potential was determined by alkaline phosphatase,alizarin red staining after osteogenic induction and oil red O staining after lipogenic induction.2.Construction of rat bone mesenchymal stem cells with overexpressing Satb2 gene293T cells were infected by the lentivirus plasmid,and then the supernatant was collected at 72h.After filtration,a high titer virus solution was obtained,which was stored at-80 C for use.The target cells were infected by lentivirus and then screened by puromycin.Then efficiency was evaluated by qRT-PCR and Western blot.3.The effect of overexpression of Satb2 on nerve regeneration during the repair of critical-size calvarial bone defect1)The acellular dermal matrix membrane(ADM)was combined with cells,and the histocompatibility was evaluated by hematoxylin and eosin staining(HE staining),cell proliferation assay(CCK-8)and scanning electron microscopy(SEM).2)The cell-membrane material complex was applied to the bone defect of skull in nude mice.The experiment was divided into four groups:ADM group,rBMSCs/ADM group,Puro/rBMSCs/ADM group,Satb2/rBMSCs/ADM group.The formation of new bone was evaluated histomorphology by HE and Masson staining on 3,7 and 14 days,3)On 3,7,14 days after operation,the expression of NSE and NGF was detected by qRT-PCR.Immunohistochemical staining(IHC staining)was used to detected the level of Nestin,a neural stem/precursor cell marker.Results1.RBMSCs in good condition were isolated and cultured successfully.The colony formation rate showed that the cells had good proliferative potentiality.Flow cytometry analysis showed that rBMSCs high expressed the surface markers of stem cells':CD29(98.4%),CD44(98%)and CD90(98.5%),while the low expression of hematopoietic cell surface markers:CD45(0.24%)and CD11b(0.42%).After induction of osteogenic,adipogenic,it was found that the experimental group was positive compared with the control group,which proved that the cells have strong multiple differentiation potential.2.The stable overexpression of Satb2 rBMSCs cells were successfully constructed.The results of qRT-PCR and Western blot confirmed that the expression levels of Satb2 mRNA and protein in the experimental group were significantly higher than those in the control group(P<0.05).There was no significant difference between the control group and the empty vector group(P>0.05).3.The results of HE staining,CCK-8 and SEM showed that the cells adhered to the surface and inside of the membrane and maintained high proliferation activity,indicating that the material had good biocompatibility.4.HE and Masson staining showed that a small amount of fibrous callus and osteoid tissue were formed in the defect area near the dura mater in each group 14 days after operation.The quality and quantity of new hone in the group with overexpression of Satb2 was significantly higher than that in the other groups(P<0.05).On 3 and 7 days,there was no obvious osteogenesis in all groups.5.The expressions of NSE and NGF mRNA in four groups were detected.The results showed that overexpression of Satb2 could promote the expression of NSE and NGF.The expression of Nestin in Satb2/rBMSCs/ADM group was also significantly higher than that in the control group.We also found that the expression of NSE,NGF and Nestin began to rise from 3 to 7 days,and reached the peak on 7 days,and then descended in all groups,but the levels in the experimental group ware still higher than than in the control groups.ConclusionsOverexpression of Satb2 can promote nerve regeneration during the repairation of bone defects.The discovery of this topic provides a theoretical basis for a comprehensive understanding of the role of Satb2 on osteogenesis and neurogenesis.