Effect And Mechanism Of Proanthocyanidins On Osteogenic Differentiation Of Human Periodontal Ligament Fibroblasts In TNF-? Environment

Background and purposesPeriodontitis which initiated by oral plaque biofilm is a chronic inflammatory disease with multiple factors involved.Inflammation and corresponding host immune response play important roles in the sustained destruction of periodontal tissue.Periodontal ligament fibroblasts(PDLFs)are the most abundant cells in periodontal ligament and have multidirectional differentiation potential,so that they become main origin of functional cells for periodontal tissue regeneration.In the process of periodontitis,lots of inflammatory cytokines are released,of which TNF-? plays important role in process of periodontitis,not only contributing to osteoclastic differentiation,but also inhibiting osteogenic differentiation.Thus,we need to look for some strategies which can inhibit inflammation and promote osteogenic differentiation to provide new idea for therapy of periodontitisProanthocyanidins(PA),belonging to the flavonoid group compounds,has been proved to have anti-inflammatory effect.There are also studies indicating that PA can promote osteogenesis in rheumatoid arthritis and experimental periodontitis rats.However,the effect of PA on osteogenic differentiation of PDLFs,especially under inflammatory microenvironment,has not been explored.The present study was conducted to observe the effect of PA on osteogenic differentiation of human PDLFs(hPDLFs)with or without TNF-? stimulation and explore underlying mechanism.Materials and methods(1)Isolation,culture and stemness identification of hPDLFs Healthy premolars were obtained for orthodontic reason with the consent of the 20 Patients(age 14-20 years),then we got primary hPDLFs via the periodontal ligament tissue block method.Colony-Forming Unit Assay was used to evaluate proliferation ability of hPDLFs.We made use of Flow Cytometry Analysis to detect the surface makers of hPDLFs.Alizarin red staining and oil red O staining were used to detect the multiple differentiation potential.(2)The effect of PA on osteogenic differentiation of hPDLFs hPDLFs were treated with different concentration PA for 3 and 7days.qRT-PCR and Western blot were used to detect osteogenic related makers ALP,Runx2,and OPN gene/protein.We also applied Alkaline phosphatase(ALP)assay kit to test ALP activity.(3)Effect of PA on TNF-?-inhibited osteogenic differentiation of hPDLFs.hPDLFs were cultured with PA,TNF-?,or their combination for 3,7,and 14 days.qRT-PAR and Western blot were used to detect ALP,Runx2,and OPN gene/protein expression.Alkaline phosphatase(ALP)assay kit was used to detect ALP activity Alizarin Red S and analysis of amount of calcium deposition were used to evaluate the degree of mineralization.(4)Mechanism of PA on reversing TNF-?-inhibited osteogenic differentiation of hPDLFs.Western blot was used to analyze whether NF-?B signal pathway is involved in reversing TNF-?-inhibited osteogenic differentiation by PA.Results(1)hPDLFs were got successfully via the tissue block method.Colony-Forming Unit Assay indicated hPDLFs had good proliferation ability.hPDLFs presented high expression of mesenchymal stem cell markers CD-44,CD-90,and CD-105/low expression of hematopoietic cell marker CD-34 and CD-45.The result of alizarin red staining and oil red O staining showed that hPDLFs had ability of multi-lineage differentiation potential(2)The result of CCK8 showed that there was no significant difference in cell viability between PA treatment groups within 50 ?g/ml and the control group.ALP activity assay revealed that 0.1-10 ?g/ml PA had positive effect on ALP activity.qRT-PCR and Western blot showed the same results for ALP,Runx2,and OPN gene/protein expression.(3)Compared with the control group,10 ng/ml TNF-? significantly decreased ALP,Runx2 and OPN gene/protein expression,but 1?g/ml PA reversed these results.ALP activity assay and Alizarin Red S had same results.(4)TNF-? significantly enhanced the phosphorylation of P65 and I?B? and nuclear translocation of P65 compared with the control,while these protein expression was significantly lower in the PA+TNF-? group than that in TNF-? group.ConclusionWe find that low concentration PA may can up-regulate osteogenic differentiation of hPDLFs.PA can reverse inhibition of osteogenic differentiation stimulated by TNF-?via inhibiting the phosphorylation and degradation of I?B?,and phosphorylation of P65 and nuclear transition of P65.