| Objectives:Periodontitis is one of the most common diseases,which leads to irreversible loss of connective tissue and bone tissue,and the main cause of adult tooth loss.At present,the clinical treatment methods for periodontal disease mainly include supragingival scaling,subgingival curettage,flap surgery.But the periodontal tissue regeneration has not been truly realized.Exosomes,as new carriers of intercellular communication,show promising potential in promoting tissue repair and provide a new idea for tissue regeneration.At present,there are relatively few studies on exosomes in periodontal tissues.Dental follicle stem cells(DFSCs),as progenitor cells of various periodontal supporting tissues,have the potential to differentiate into alveolar bone,cementum and periodontal ligament.In our previous study,canine periodontal tissue like structures were successfully reconstructed using DFSCs sheets.However,the underlying mechanism was still elusive.Exosomes have been shown to promote tissue repair and regeneration by promoting cell proliferation and migration,inhibiting cell apoptosis,and regulating inflammation and immune response.Therefore,we hypothesized that exosomes are the key to promoting periodontal regeneration of DFSCs.This study will take DFSCs-exosomes as the main research object,explore the effects of DFSCs-exosomes on the proliferation,migration,osteogenic differentiation of PDLSCs in vitro,and verify the possibility of periodontal tissue regeneration by DFSCs-exosomes in vivo,so as to provide a theoretical basis for exploring the cell-free transplantation technology for periodontal tissue regeneration.Methods:1.DFSCs and PDLSCs were cultured by modified tissue culture technique,and the cells were characterized by multidirectional differentiation induction,clone formation,flow cytometry and immunofluorescence;2.Isolate DFSCs-exosomes from the supernatant of DFSCs,identify the morphology,particle size and marker molecules of DFSCs-exosomes,and observe the uptake of DFSCs-exosomes by PDLSCs by immunofluorescence;3.The effects of DFSCs-exosomes on the proliferation ability of PDLSCs were detected by EdU assay,CCK-8 and cell cycle assay,the effect of DFSCs-exosomes on the migration ability of PDLSCs was detected by wound healing assay,and the effect of DFSCs-exosomes on the mineralization ability of PDLSCs was detected by alizarin red staining,the effects of DFSCs-exosomes on the expression level of osteogenic related genes and proteins(COL1,ALP,RUNX2,BSP)in PDLSCs were detected by qRT-PCR and Western blot;4.The effect of DFSCs-exosomes on PDLSCs was studied by transcriptome sequencing,and the activation of MAPK pathway and its effects on the proliferation of PDLSCs were verified by Western blot,EdU assay and CCK-8;5.The periodontal defect model of SD rats was established,and the effects of repairing periodontal defect with DFSCs-exosomes were evaluated by micro-CT and HE staining.Results:1.DFSCs and PDLSCs cultured in this experiment showed typical fibroblast-like morphology,and expressed mesenchymal stem cell specific markers Nestin,Vimentin and CD 146,cells have the abilities of clone formation and osteogenic/adipogenic differentiation,flow cytometry showed that the cells expressed CD29,CD44,CD90 and CD105 positively,and CD34 and CD45 negatively,indicating that the cultured DFSCs and PDLSCs had the characteristics of mesenchymal stem cells;2.DFSCs-exosomes showed typical saucer-like morphology with a peak particle size of about 132.7nm and positively expressed exosomes marker molecules CD81,TSG101 and HSP90,immunofluorescence analysis showed that DFSCs-exosomes could be gradually internalized by PDLSCs;3.EdU assay and CCK-8 showed that 10μg/mL DFSCs-exosomes significantly promoted PDLSCs proliferation,cell cycle assay showed that DFSCs-exosomes increased the proportion of S and G2 phase in PDLSCs,suggesting that the proliferation activity of PDLSCs was enhanced,wound healing assay showed that 10μg/mL DFSCs-exosomes could significantly promote PDLSCs migration,a series of experiments on osteogenic induction showed that DFSCs-exosomes promoted the formation of mineralized nodules in PDLSCs and up-regulated the expression of osteogenic related genes and proteins in PDLSCs;4.Transcriptome sequencing results show that DFSCs-exosomes can up-regulate 234 mRNA and down-regulate 134 mRNA in PDLSCs,and the differentially expressed genes are mainly related to MAPK,mTRO,cMAP pathways,further verification shows that DFSCs-exosomes can activate p38 MAPK pathway in PDLSCs,thus promoting the proliferation of PDLSCs;5.DFSCs-exosomes can be internalized by host cells around the defect site after transplantation,micro-CT results show that DFSCs-exosomes can promote the formation of new bone in defective periodontal tissues of rats,and HE staining results show that DFSCs-exosomes can promote the formation of new periodontal ligament-like structures in defective periodontal tissues of rats.Conclusions:1.DFSCs and PDLSCs were successfully cultivated by modified tissue culture technique;2.DFSCs-exosomes could be internalized by PDLSCs;3.10μg/mL DFSCs-exosomes can promote the proliferation,migration and osteogenic differentiation of PDLSCs;4.DFSCs-exosomes promoted the proliferation of PDLSCs at least in part by activating the p38 MAPK pathway;5.Under the conditions of this study,DFSCs-exosomes can effectively repair the defective periodontal tissue. |
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