Effects Of Low Intensity Pulsed Ultrasound On Expression Of Inflammatory Factors And Osteogenic Differentiation In Human Periodontal Ligament Cells

Periodontitis is a chronic infective disease and leads to tooth losing eventually by causing the destruction of the connective tissue matrix and the resorption of alveolar bone.Conventional periodontal treatment mainly eliminates local irritation factors by surgical or non-surgical methods,prevents periodontitis from occurring and developing,and creates favorable periodontal microenvironment to regenerate periodontal tissue.Low intensity pulsed ultrasound(LIPUS)has played a role in different aspects of the process of bone regeneration.It can act on the biologics components of the regeneration process via promotion of cell proliferation,cells pre-conditioning to orient their differentiation during culture,or cells transfection.LIPUS can modulate the micro-environment by triggering delivery of growth factors or gene expression in engineered cells;or by modulating the physical environment by heat deposition or mechanical stimulation.LIPUS can also be useful in tissue engineering approaches by acting on the scaffolds for improvements of scaffold integration,characterization and control of the rate of scaffold degradation.Within this arsenal,the LIPUS techniques aim at modulating the physical environment of the cells,in particular by mechanical stimulation.Previous animal experiments have demonstrated that LIPUS was able to enhance new bone formation when it stimulated acute periodontal defects,meanwhile,others' researches suggested that LIPUS could inhibit Lipopolysaccharide(LPS)inducing osteoblast's inflammation.As the most important pathogenic factor of periodontitis and a recognized inflammatory initiation factor,LPS can be used to simulate the microenvironment of periodontitis in vitro.Under LPS background,hPDLCs was irradiated with LIPUS to investigate the application of LIPUS in the microenvironment of periodontitis.This study was divided into the following five parts:Part 1 Culture and biological identification of human periodontal ligament cellsObjective: Culture human periodontal ligament cells and identify their biological characteristics for further research.Method: Enzyme digestion combined with tissue culture were used to harvest human periodontal ligament cells from human premolars and conduct cell passage.Immunofluorescence tested vimentin and CK14 to identify cell sources.Flow cytometry were used to identify cell surface markers.The multi-potent differentiation potential were tested.Results: purified periodontal ligament cells at passage 3 were all uniform shape as fibroblast like cells.Cells were vimentin positive and CK14 negative.Flow cytometry test showed hPLDCs expressed mesenchymal stem cell markers(CD73:99.88%,CD146:71.6%,Strol-1:1.21%)and no hematopoietic cells markers(CD34:0.62%).After 21 days' differentiation,the alizarin red staining,alcian blue staining and oil red O staining were all positive.Conclusion: Human periodontal ligament cells were successfully separated and cultured with high proliferation capacity and multiple potential differentiation.Part 2 Inhibitory effect of LIPUS irradiation on expression of IL-6 and IL-8 in hPDLCs under inflammatory microenvironmentObjective:Explore the inhibition effect of LIPUS stimulation on LPS induced hPDLCs through detecting the expression of inflammatory factors.Screen out the optimal LIPUS stimulation parameter and explore the safety of the optimal LIPUS parameter.Methods: LPS stimulated hPDLCs for 24 h to mimic periodontal inflammation.10?30?60?90 mW/cm2 LIPUS were applied to stimulate LPS stimulated hPDLCs for 2h to screen out the optimal parameter.Specific detections includes: ELISA tested the protein secretion level of IL-6 and IL-8;qPCR tested the gene expression level of IL-6 and IL-8;CCK-8 tested cell proliferation activity;.Results: ELISA and qPCR results showed that 30?60?90 mW/cm2 LIPUS inhibited the protein secretion level and the gene expression level of IL-6 and IL-8 both(p<0.05).And,90mW/cm2 LIPUSshowed the best inhibition effect to LPS induced inflammatory environment.CCK-8 suggested that 90mW/cm2 LIPUS didn't cause cell death and to some degree,it promoted cell proliferation(p<0.05).Flow cytometry tested cell apoptosis showed LIPUS could inhibit cell apoptosis(p<0.05).Conclusion: LIPUS can effectively inhibit the secretion and gene expression of IL-6 and IL-8.90mW/cm2 LIPUS shows the optimal inhibition effect on LPS induced inflammation.LIPUS has no harmful effect on cells,and it can enhance cell proliferation,inhibit cell apoptosis.Hence,LIPUS stimulation can be a safe and effective method applied to inhibit periodontal inflammation.Part 3 Effects of LIPUS irradiation on NF-?B inflammatory signal pathway in hPDLCsObjective: Explore the mechanism of LIPUS stimulation on inflammation by LPS induced hPDLCs.Method: LPS stimulate hPDLCs for 24 h to mimic periodontal inflammation.90mW/cm2 LIPUS continuous stimulate LPS induced hPDLCs for 2h.Western Blot tested changes of NF-?Bsignal pathway relative proteins:I?B? ? p-I?B? ? p65;immunofluorescence tested the location of p65.Result: WB showed that I?B? phosphorylation and p65 translocated into nucleus and phosphorylated,suggested that NF-?B signal pathway activated under LPS stimulation.After LIPUS stimulation,I?B? phosphorylation decrease and p65 translocated into cytoplasm,suggested that LIPUS could inhibit NF-?B signal pathway.Immunofluorescence tested the location of p65 showed that under LPS stimulation,p65 translocated into nucleus.LIPUS inhibited p65 translocated into nucleus.It demonstrated that LIPUS could inhibit NF-?B signal pathway.Conclusion: Under LPS stimulation,LIPUS could effectively inhibit inflammation through NF-?B signal pathway.Part 4 Effects of LIPUS irradiation on osteogenic differentiation of hPDLCs under inflammatory microenvironmentObjective: Osteogenic differentiation of hPDLCs decrease under inflammation.LPS stimulation mimic periodontal inflammation in vitro and then LIPUS stimulated hPDLCs with osteogenic medium to explore whether LIPUS stimulation could enhance hPDLCs' osteogenic differentiation ability under LPS induced inflammation.Method: LPS stimulated hPDLCs with osteogenic medium,and then LIPUS with 20min/day and intensity of 90mW/cm2 LIPUS continuously stimulated for 7days and conducted relative tests.Tests includes: q PCR detected IL-6 and IL-8 gene expression level and osteogenic differentiation RUNX2?OPN?OSX?OCN gene expression level.Alkaline phosphatase staining and quantitative analysis were applied to detect osteogenic differentiation.CCK-8 tested cell proliferation.Result:After 90mW/cm2 LIPUS continuous stimulating hPDLCs under inflammation for 7days,qPCR testing the gene expression of IL-6 and IL-8 showed 90mW/cm2 LIPUS continuous stimulation could effectively inhibit the gene expression of IL-6 and IL-8(p < 0.05),suggesting that 90mW/cm2 LIPUS continuous stimulation inhibited periodontal inflammation.q PCR testing the gene expression of RUNX2?OPN?OSX?OCN found 90mW/cm2 LIPUS continuous stimulation could effectively enhance osteogenic gene expression level(p<0.05).Also,Alkaline phosphatase staining and quantitative analysis showed 90mW/cm2 LIPUS continuous stimulation promote hPDLCs osteogenic differentiation(p<0.05).CCK-8 result showed that 90mW/cm2 LIPUS continuous stimulation had no harmful effect on cell proliferation.Conclusion: LIPUS could enhance osteogenic differentiation of hPDLCs as well as inhibit inflammation.Part 5 Effects of LIPUS irradiation on osteogenic differentiation of hPDLCs in inflammatory microenvironment and the relationship with NF-?B inflammatory signal pathwayObjective: Explore the mechanism of LIPUS enhance hPDLCs osteogenic differentiation under periodontal inflammation,and NF-?B signal pathway inhibitor was applied to confirm.Method: WB tested NF-?B signal pathway relative protein I?B??p-I?B??p65 changes under LPS stimulation and LIPUS continuous stimulation.BAY 11-7082,NF-?B signal pathway inhibitor,was applied to inhibit NF-?B signal pathway with LPS stimulation and LIPUS continuous stimulation.qPCR detected osteogenic differentiation RUNX2?OPN?OSX?OCN gene expression level.Alkaline phosphatase staining and quantitative analysis were applied to detect osteogenic differentiation.WB tested NF-?B signal pathway relative protein I?B??p-I?B??p65 changes.Result: After LIPUS stimulating hPDLCs with LPS stimulation in osteogenic medium,WB showed that LIPUS could inhibit NF-?B signal pathway.After BAY 11-7082 inhibiting NF-?B signal pathway,qPCR found the expression level of osteogenic gene RUNX2?OPN?OSX?OCN increase when BAY 11-7082 was applied or LIPUS stimulation or both combined(p<0.05).Alkaline phosphatase staining and quantitative analysis suggested BAY 11-7082 applied or LIPUS stimulation or combined both could enhance cell osteogenic differentiation(p<0.05.WB showed that LIPUS had similar effect with BAY 11-7082,which could inhibit NF-?B signal pathway.Conclusion: After BAY 11-7082 inhibited NF-?B signal pathway we found that inhibitng NF-?B signal pathway could enhance hPDLCs' osteogenic differentiation,which means NF-?B signal pathway was involved in hPDLCs' osteogenic differentiation under inflammation environment and inhibiting NF-?B signal pathway could enhance hPDLCs' osteogenic differentiation.Also,this part demonstrated that LIPUS could act as NF-?B inhibitor,inhibiting inflammation and enhance hPDLCs' osteogenic differentiation.