Wnt Signaling Pathway Regulates The Osteogenic Differentiation Of Human Periodontal Ligament Stem Cells And Its Mechanism

BackgroundPeriodontitis is a chronic infectious disease of the periodontal supporting tissue and is caused by plaque bacteria.It is the main cause of tooth loss in adults.Human periodontal ligament stem cells(hPDLSCs),as one of the most promising "seed cells" for periodontal regeneration tissue engineering,are often used to study periodontal bone regeneration.A large number of studies have shown that Wnt signaling pathway is intimately connected to the osteogenesis process,but the relationship between the canonical and non-canonical Wnt signaling pathways and the osteogenic differentiation of human periodontal ligament stem cells,and the molecular mechanism of its role in the process of osteogenesis is not yet clear.AimThis study aimed to investigate the impact of Wnt signaling pathway on the osteogenic differentiation of hPDLSCs,to explore the effect of using hPDLSCs and regulating the activation of Wnt signaling pathway on periodontal tissue regeneration and the corresponding molecular mechanisms,and to explore the possibility of periodontal tissue regeneration by modifying the Wnt signaling pathway.Methods1.The hPDLSCs were isolated and cultured by tissue enzymatic digestion method,and the source of the cells was determined by immunofluorescence.The multidirectional differentiation ability of hPDLSCs was tested via the induction of osteogenesis,adipogenesis and chondrogenesis.2.Add the canonical Wnt agonist LiCl,determine the concentration and duration of LiCl by CCK-8 and Real-time PCR firstly,then verify the activation of LiCl on the canonical Wnt signaling pathway through immunofluorescence,nucleoplasmic separation combined with western blot and dual-luciferase reporter gene system assay.LiCl was added to hPDLSCs to induce osteogenic differentiation,with the same concentration of Na Cl as the control group.Alkaline phosphatase(ALP)staining and ALP activity assay were performed on the 7th day of induction,and alizarin red staining and semi-quantitative analysis were performed on the 21 st day of induction.In addition,RNA was extracted from the control group and the experimental group that were induced for 7 days and 14 days,then analyze their differences in expression of bone formation-related genes and Wnt-related genes by Real-time PCR.3.Wnt 3a,4,5a,7a,7b and control conditioned medium were obtained by plasmid transfection,the above conditioned medium were added to hPDLSCs,then test cell proliferation by CCK-8.The effects of Wnt conditioned medium on the canonical Wnt signaling pathway were observed by immunofluorescence and dual-luciferase reporter gene system assay.Wnt conditioned medium was used to induce osteogenic differentiation of hPDLSCs,ALP staining and ALP activity assay were performed on the 7th day of induction,and alizarin red staining and semi-quantitative analysis were performed on the 14 th day of induction;on the 7th day of induction,extract the RNA of the experimental group and the control group,and analyze the expression of bone formation-related genes through Real-time PCR,the above experiment was used to detect the effect of adding Wnt conditioned medium on the osteogenic differentiation of hPDLSCs.Results1.The hPDLSCs were successfully isolated and cultured with spindle shape.Immunofluorescence showed that hPDLSCs express vimentin but not keratin.They are originating from mesenchymal and have shown multidirectional differentiation potential.2.Immunofluorescence,nucleoplasmic separation combined with western blot and dual-luciferase reporter gene system assay confirmed that 8 m M LiCl(doesn’t affect the proliferation of hPDLSCs)activated the canonical Wnt signaling pathway.After adding 8 m M LiCl to hPDLSCs,compared with the control group,the ALP staining of the experimental group was lighter and the ALP activity was lower after 7days of induction;the deposited calcium salt stained by alizarin red in the experimental group was significantly reduced after 21 days of induction;Real-time PCR results showed that the expression of bone formation-related genes decreased in the experimental group,indicating that the osteogenic differentiation ability of hPDLSCs was lower than that in the control group.3.Fluorescence microscope and Western Blot results showed that Wnt 3a,4,5a,7a,7b conditioned medium was successfully prepared.Select 10% Wnt conditioned medium that does not affect the proliferation of hPDLSCs for the experiment.Immunofluorescence and dual-luciferase reporter gene system assay showed that 10%Wnt 7a conditioned medium increased the expression of β-catenin in the nucleus,but did not affect downstream TCF transcription.After adding 10% control and Wnt conditioned medium,the results of osteogenic experiments showed that Wnt 3a,5a,7a,7b conditioned medium increased the expression of ALP during the osteogenic differentiation of hPDLSCs,and it has no significant effect on the amount of mineralized nodules formed by osteogenic differentiation of hPDLSCs.Conclusions1.After adding LiCl to activate the canonical Wnt signaling pathway,the osteogenic differentiation ability of hPDLSCs decreases.2.Wnt 7a conditioned medium increased the expression of β-catenin in the hPDLSCs nucleus.Wnt 3a,5a,7a and 7b conditioned medium increased the expression of ALP during the osteogenic differentiation of hPDLSCs,but the effect of Wnt conditioned medium on the bone mineralization ability of hPDLSCs is still inconclusive.