| Background and Objectives Periodontitis is a chronic infectious disease characterized by gingivitis and attachment loss.And the tissue destruction is mainly caused by the host’s immune response to infection.As the most important active cell in inflammatory response,macrophages are of great significance in the process of periodontitis occurrence and development.In this study,in order to provide an experimental basis for"cell-free therapy" of periodontitis,M2 macrophages derived exosomes(M2 exo)were taken as the main research object to explore their effects on osteogenic differentiation,proliferation and migration of periodontal ligament stem cells(PDLSCs)in vitro.For the vivo part,we use rat alveolar bone defect model to verify that M2 exo promotes the repair of bone defects.Material and Methods 1.PDLSCs were cultured by tissue culture method and identified by flow cytometry,multiple differentiation induction and clone formation.2.IL-4 and IL-10 were used to polarize RAW264.7 cells into M2 type,which was identified by flow cytometry.The exosomes were extracted by high-speed centrifugation technique and the morphology,particle size and surface markers were identified by TEM,NTA,and Western blot.The uptake of M2 exo by PDLSCs was observed by confonal microscope.3.The expression of osteogenesis related genes and proteins(ALP,Runx2,OPN)was detected by QT qPCR and Western blot,and the effects of M2 exo on the mineralization of PDLSCs was detected by ALP staining and alizarin red staining.4.The expression of Wnt pathway related proteins was detected by Western blot,osteogenesis was induced after using pathway inhibitors,and the expression of osteogenesis related genes and proteins was detected by QT qPCR and Western blot.5.The effects of M2 exo on adipogenic differentiation,migration and proliferation of PDLSCs were respectively detected by oil red o staining,wounding healing assay and cck-8 assay.6.The bone defect model of Wistar rats was established.The alveolars were taken at 4 and 8 weeks after operation,and the repairing effect was evaluated by micro CT scanning and immunohistochemical staining.Results 1.The cultured PDLSCs are long spindle shaped and have the ability of clone formation and multidirectional differentiation.The results of flow cytometry showed that the cultured cells have the characteristics of mesenchymal stem cells.2.Flow cytometry showed that polarized cells expressed CD206 positively and iNOS negatively,indicating that the polarized cells were M2 macrophages.The extracted M2 exo was concave hemispherical,with a peak particle size of 136.0 nm,and positively expressed CD9,CD63 and HSP70.The immunofluorescence results showed that M2 exo could be ingested by PDLSCs.3.RT qPCR and Western blot showed that M2 exo could promote the osteogenic differentiation of PDLSCs,which was consistent with the results of ALP staining and alizarin red staining.4.Western blot showed Wnt pathway related protein increased and the expression of osteogenic related protein decreased after adding pathway inhibitor,indicating that M2 exo promotes bone formation through Wnt pathway.5.M2 exo inhibits adipogenic differentiation of PDLSCs and promotes the proliferation and migration.6.The results of micro CT scanning and immunohistochemical staining showed that M2 exo could promote the repair of bone defect.Conclusions 1.PDLSCs was successfully isolated by tissue culture method;2.Macrophages were polarized into M2 type by IL-4 and IL-10.M2 exo was isolated by ultracentrifugation,and could be absorbed by PDLSCs;3.M2 exo can promote the osteogenic differentiation through Wnt pathway,promote the proliferation and migration of PDLSCs and inhibit their adipogenic differentiation;4.M2 exo can effectively repair the bone defect in rats... |
