Effects Of Lipopolysaccharides Extracted From Porphyromomanus Endodontalis On Osteoblast Cell Proliferation And Differentiation

ObjectiveAs one of the most important pathogenic bacteria, Porphyromonas endodontalis (P.e) could extracted kinds of virulence factor. Lipopolysaccharides (LPS) is the particularly important one of the virulence factors. The biological behaviour of osteoblasts is very critical in the rebuilding of bone tissue, which is not only possible for forming bone tissue but also producing inflammatory factors to accelerate bone resorption of osteoclasts. To observe the effect of lipopolysaccharides extracted from Porphyromomanus endodontalis on osteoblast cell proliferation and the activity of alkaline phosphatase, to quantify the IL-6 secretion induced by P.e-LPS. Try to investigate the action of P.e-LPS on osteoblast cell proliferation and differentiation.Methods1.Cultures of Porphyromonas endodontalisPorphyromonas endodontalis was cultured in solid Brain heart infusion(BHI) medium at 37℃in anaerobic atmosphere(80%N2,10%CO,10%H2). The bacteria that were multiplied in fluid BHI medium were collected and then stored at-20℃.2.Extraction of LPSLPS was extracted from Porphyromonas endodontalis by hot-phenol-water method. The tachypleus amebocyte lysate(TAL) was used for qualitative analysis of LPS. 3.Cultures of cellsMC3T3-E1 cells was cultured in MEM-a containing 10% fetal boving serum (FBS), 100u/ml penicillin and 100u/ml streptomycin at 37℃in a humidified atmosphere containing 5% CO2 in air. Cells of the third or fourth generation growing well were used for experiments.4.Cell proliferation rate was detected by methyl thiazolyl tetrazolium (MTT)MC3T3-E1 treated with different concentrations of P.e-LPS (10μg/ml,25μg/ml,50μg/ml) for different hours (12h,24h,48h,72h). The Cell proliferation rate was detected by MTT kit at 490nm.5. The activity of alkaline phosphatase was detected by enzyme kinetics assayMC3T3-E1 treated with different concentrations of P.e-LPS (10μg/ml,25μg/ml,50μg/ml) for different hours (6h,12h,24h,48h,72h). The activity of alkaline phosphatase was detected by enzyme kinetics assay at 520nm.6. IL-6 was detected by Enzyme-linked Immunosorbent Assay (ELISA)MC3T3-E1 cells were incubated for 48h with different concentrations of P.e-LPS (10μg/ml,25μg/ml,50μg/ml) in the presence of non-serum. Cells were incubated for 6, 12,24 and 48h with 25μg/ml P.e-LPS in the presence of non-serum. The secretion of IL-6 was detected by ELISA at 450nm.7.Statistical analysisOne-way analysis of variance (ANOVA) and Dunnett-t test was performed in SPSS 11.0 software for statistical analysis. Significance level:P<0.05 is considered statistically significant, P<0.01 is considered highly statistically significant. Results1. When MC3T3-E1 cell treated with P.e-LPS more than 72hrs, the Relative growth rate (RGR) of 25μg/ml group is 87.464%(P＜0.05),and the RGR of 50μg/ml group is 71.117% (P＜0.01)2. After MC3T3-E1 cells treated with P.e-LPS at more than 50μg/ml and for more than 12 hours, the ALP activitie of was inhibited (P＜0.05). After MC3T3-E1 cells treated with different concentrations (10μg/ml,25μg/ml,50μg/ml) P.e-LPS and for more than 24 hours, the ALP activities of were inhibited (P<0.05).3. MC3T3-E1 treated with different concentrations of P.e-LPS (10μg/ml,25μg/ml,50μg/ml) for 48h, the level of IL-6 production increased significantly (P＜0.01). MC3T3-E1 treated with 25μg/ml P.e-LPS, the level of IL-6 production increased significantly at 6h (P＜0.05),and the level of IL-6 production increased significantly for more than 12h (P<0.01).Conclusions1. The level of IL-6 production increased significantly after MC3T3-E1 treated with lipopolyseccharides extracted from Porphyromomanus endodontalis. Then the ALP activities of MC3T3-E1 decreased, the differentiation of osteoblast cell was inhibited.2. With the long-time toxicity action of P.e-LPS and the stimulation of IL-6, the proliferation rate of MC3T3-E1 also markedly decreased.