Mechanical Strain Promote Human Periodontal Ligament Stem Cell Proliferation And Osteogenic Differentiation Via The Exosome MiR-181b-5p

Objectives:The oral cavity is the bacterium environment.Periodontal tissues are constantly challenged by various pathogenic microorganisms.The subsequent host immunoinflammatory reaction can greatly affect the normal physiological performance of tissue cells.In addition to the influence of microorganisms,periodontal tissue has been in the mechanical environment for a long time as one of the main structures that deliver the bite force.Mechanical stimulation has become an important factor affecting periodontal tissue remodeling.However,the mechanism of the effects of mechanical force and inflammation on periodontal tissue remodeling and periodontal diseases progression is still not very clear.Periodontal ligament stem cells have been shown to play an important role in maintaining periodontal tissue homeostasis,and can participate in immune response caused by microorganisms.However,their functions are greatly affected by the surrounding microenvironment,including cell-cell and/or cell-matrix interactions.As the most abundant cells in bone tissue,bone cells also have an important impact on the periodontal tissue reconstruction,but there are still few research on the exchange of information between bone cells and periodontal ligament cells.Therefore,this study applied uniaxial cyclic tensile strain of specific frequency to mouse bone-like cells(MLO-Y4)and purified the exosomes produced by them.Exosomes were applied to human periodontal ligment stem cells(hPDLSCs)cultured in vitro to explore the effects of uniaxial cyclic tension on the proliferation and osteogenic differentiation of hPDLSCs after exosomes produced by MLO-Y4 were stimulated.At the same time,Porphyromonas gingivalis lipopolysaccharides(P.g LPS)was used to establish an inflammation model to explore the role of mechanically induced exosomes in regulating cells in the inflammatory environment.It offers new ideas for further exploring the mechanism of normal bite force to maintain the metabolism and normal structure of periodontal tissues,and the relationship between abnormal bite force and the progression of periodontitis.Methods:1.Culture and identification of periodontal ligament stem cells.Human periodontal ligament stem cells were cultured by enzyme digestion.Cell morphology was observed under an inverted microscope.Flow cytometry was used to identify mesenchymal stem finely related surface antigens CD73,CD90,CD29,and CD45.2.Isolation and identification of MLO-Y4 exosomes induced by uniaxial cyclic tension.8%,0.1Hz uniaxial cyclic pulling force acts on MLO-Y4 cells.The exosomes secreted by the afterburner group and conventionally cultured MLO-Y4 were collected by ultracentrifugation and labeled Exosome-MS and Exosome respectively.Transmission Electron Microscope(TEM),Western blot,and Nanoparticle Tracking Analysis(NTA)were used to identify exosomes.3.The effects of exosomes and P.g LPS on hPDLSCs proliferation and osteogenic differentiation.Exosomes and P.g LPS stimulate or hPDLSCs separately and are divided into a blank control group(Control group),P.g LPS group(LPS group)plus exosomes group(Exosome-MS group),P.g LPS + Exosomes-MS group(LPS + Exosome-MS group),and P.g LPS + normal exosomes group(LPS + Exosome group).Cell proliferation was observed with CCK8 and Ed U staining;osteogenic differentiation ability of cells was observed with alkaline phosphatase and alizarin red staining.4.Potential mechanisms of exosomes-mediated mechanical force to promote the proliferation and osteogenic differentiation of periodontal ligament stem cells.Highthroughput sequencing of micro RNAs in exosomes produced by MLO-Y4 under stress and conventional culture,and prediction of signal pathways involved in target genes through databases such as Target Scan.Fluorescence in situ hybridization(FISH),luciferase report analysis,RT-q PCR and Western blot further confirmed that exosomes regulate hPDLSCs proliferation and osteogenic differentiation through the miR-181b-5p / PTEN / AKT pathway.Results:1.Human periodontal ligament stem cells were successfully cultured in vitro.Under an inverted microscope,the cells were arranged in a vortex,long spindle or star shape.Flow cytometry results showed that the cell surface antigens were CD45 negative,CD73,CD146,and CD90 positive,which proved to be mesenchymal stem cells.2.MLO-Y4 secreted exosomes increased under cyclic stress,and exosomes were successfully collected by ultracentrifugation.Western blot showed exosomal marker proteins CD63,CD81 and Alix were positive.TEM and NAT results showed that the diameter of the pellets obtained by ultracentrifugation was between 30-140 nm.3.The results of CCK8 and Ed U showed that P.g LPS inhibited cell proliferation and division activity,and exosomes produced by MLO-Y4 mechanically stimulated the proliferation of hPDLSCs,and could partially improve LPS inhibition,while the exosomes in the control group had no significant improvement.ALP and alizarin red staining results showed that the osteogenic differentiation ability of hPDLSCs in each group showed the same trend as the change in cell proliferation ability.4.MiRNA sequencing results show that the functions of exosomes miRNA are mainly concentrated in cell metabolism,information exchange between cells in the microenvironment,and immune response to microbial infection.RT-q PCR showed that miR-181b-5p expression was up-regulated in Exosome-MS,which was statistically different from the control group(P <0.01).FISH results showed that miR-181b-5p was mainly distributed in the cytoplasm;luciferase report experiments proved that miR-181b-5p and phosphatase and phosphate and tension homolog deleted on chromosome ten(PTEN)existed Binding site,miR-181b-5p is a negative regulator of PTEN.Overexpression or inhibition of miR-181b-5p can affect the expression levels of PTEN / PI3 K / AKT and BMP2 / Runx2 in cells,and then regulate cell proliferation and osteogenic differentiation.Conclusion:1.Appropriate mechanical force plays an important role in maintaining the normal structure of periodontal tissues and tissue repair after periodontal treatment.2.Uniaxial cyclic tensile stress induces upregulation of miR-181b-5p in exosomes secreted by osteocytes,binds and silences PTEN,and activates AKT signaling pathway in hPDLSCs.The miR-181b-5p / PTEN / AKT pathway is one of the potential mechanisms for mechanical force to promote the proliferation of hPDLSCs.3.Besides,miR-181b-5p can also up-regulate the expression of BMP2 and Runx2,and promote osteogenic differentiation of hPDLSCs.