Experiment Of Exosome Derived From Dental Follicle Stem Cells In Bone Defect Repair

Objectives: Exosome is an extracellular vesicle with a double-layer membrane structure,which can transmit information between cells and plays a key role in cell communication.Therefore,exosome has become an emerging hot spot in the field of bone defect repair and treatment,which has received extensive attention and research by the medical community.Dental follicle stem cells(DFSCs)are odontogenic stem cells derived from mesenchymal tissues.Previous experiments conducted by our research group have demonstrated that the exocrine secretion of dental follicle stem cells can promote the proliferation,migration and osteogenic differentiation of periodontal ligament stem cells,and has also proved that Treated dentin matrix particles(TDMP)can repair rat skull defect tissues.Therefore,we assume whether exosomes can also play an important role in the repair of bone defects.Therefore,in this study,DFSCs-exosomes are taken as the object.In an in vitro experiment,DFSCs-exosomes are expressed in bone marrow mesenchymal stem cells,BMSCs)proliferation,migration,osteogenic differentiation and other processes,through the in vivo experiment to explore the potential of DFSCs-exosomes combined with dentin matrix particles to repair bone tissue,to further improve the theoretical basis of bone defect repair.Methods: 1.Isolating and cultivating DFSCs and BMSCs in vitro with the modified tissue mass technique and whole bone marrow method,colony-forming assay was used to assess their self-replication capability.The expression of related markers of mesenchymal stem cells between DFSCs and BMSCs was detected by cellular immunofluorescence,and the adipogenic and osteogenic differentiation abilities of DFSCs and BMSCs were detected by multi-directional induction and differentiation.2.Under the transmission electron microscope,the morphology of the extracted exosomes was observed;the Zeta View granulometer detected their particle size,and western blot detected their markers..Immunofluorescence assay revealed BMSCs’uptake of DFSCs-exosomes.3.Based on cell cycle experiments,Ed U experiments,and CCK-8 experiments,explore the impact of DFSCs exosomes on the proliferation ability of BMSCs,use scratch experiments to detect the impact of DFSCs exosomes on the migration ability of BMSCs,and use alizarin red staining to detect the impact of DFSCs exosomes on the osteogenic ability of BMSCs,Based on the results of q RT-PCR and Western blot,the effect of DFSCs exosomes on the expression level of osteogenic related genes and proteins(COL1,ALP,RUNX2,BSP)in BMSCs was analyzed;4.The model of skull defect in SD rats was constructed by surgical method and divided into three groups(blank control group,TDM group,TDM+Exosomes group).The results of micro-CT and HE staining were analyzed,and the specific effects of DFSCs-exosomes loaded dentin matrix particles in bone defect repair were summarized.Results:1.The cultured DFSCs and BMSCs have fibroblast-specific long spindle shapes.Both cells can positively express mesenchymal stem cell specific markers Nestin and Vimentin,and have good self-replication ability and osteogenic and adipogenic differentiation potential.DFSCs and BMSCs express mesenchymal stem cell surface markers CD44 and CD90,and negatively express non-mesenchymal stem cell surface markers CD34,CD45 and CD31;.2.The separated DFSCs-exosomes had a typical double-layer membrane structure,took the shape of a teacup,and the particle size peak was about 145.2nm.The positive expressions of CD81,TSG101 and CD63 in DFSCs-exosomes were detected by western blot,and immunofluorescence experiment showed that BMSCs could absorb DFSCs-exosomes into cells.3.Cell proliferation experiments(CCK-8 and EDU’s methods)showed that the 10μg/m L and 50μg/m L exosome groups could significantly promote the proliferation of BMSCs.Cell cycle experiments showed that the two exosome concentration groups could enhance their proliferation activities.Scratch experiments showed that the 10μg/m L and 50μg/m L exosome groups could significantly promote the migration of BMSCs.A series of osteogenic induction experiments showed that DFSCs-exosomes promoted the formation of mineralized nodules of BMSCs and up-regulated the expression of osteogenic related genes and proteins in BMSCs at different levels.4.micro-CT results showed that the TDM particles loaded with DFSCs-exosomes played an important role in the new bone formation in the defect area,and HE staining results also showed that it could promote the new bone formation in the defect area.Conclusions: 1.DFSCs and BMSCs were successfully cultured by improved tissue mass method and whole bone marrow method;2.BMSCs can ingest DFSCs exosomes;3.10μg/m L and 50μg/m L DFSCs exosomes can accelerate the proliferation,migration,and osteogenic differentiation of BMSCs;4.Dentin matrix particles loaded with DFSCs exosomes can effectively repair defective skull tissue.