Porphyromonas Gingivalis Stimulates Periodontal Ligament Fibroblasts In Vitro

Objective Periodontal ligament fibroblasts (PDLFs) were seprated and cultured. The viable Porphyromonas gingivalis (P. gingivalis) suspensions were adjusted to 109,108, 107,106,105 CFU/ml with the aid of visible UV-Vis spectrophotometer. PDLFs were challenged by viable P. gingivalis for 6 hours to study the cell viability and gene expression of inflammatory cytokines and bone metabolism.Methods Human PDLFs were obtained by tissue block method, then typsinizated and cultured. Experiments were performed with cells from passages 3-4. Viable P. gingivalis were centrifuged at 4,000 r/min for 5 min at 4℃. Then the medium was discarded and P. gingivalis were washed twice with phosphate-buffered saline (PBS). The resulted viable P. gingivalis were resuspended in PBS until reaching an optical density of 0.8 at 660nm, corresponding to 109 CFU/ml. This concentrated suspension was resuspended by antibiotic-free DMEM and then diluted into 108,107,106 and 105 CFU/ml. PDLFs were challenged in vitro by viable P. gingivalis at concentrations of 109,108,107,106 and 105 CFU/ml for 6 hours and then the cell morphology was observed by inverted phase contrast microscope and their viability was monitored via Trypan blue staining. The expression of cytokines interleukin 6 (IL-6), IL-8, IL-1β, tumor necrosis factor-α (TNF-a), RANKL and osteoprotegerin (OPG) were analyzed by real-time fluorescence quantitative polymerase chain reaction (RT-PCR).Results When challenged by viable P. gingivalis for 6 hours, all PDLF samples were fusiform or polygonal when observed by inverted phase contrast microscope. When the concentration of viable P. gingivalis was 109 CFU/ml, the medium was appreciably turbid and the cell morphology was anomalous, the cell membranes were incomplete and the nuclei were not distinct. Cell viability declined with the increasing concentration of viable P. gingivalis but there was no statistically significant difference compared with the control group. The expression of proinflammatory cytokines such as IL-6, I1-1β, TNF-a and RANKL were more strongly induced when the concentration of viable P. gingivalis gradually increased from 105 CFU/ml to 109 CFU/ml. The expression of IL-6 was strongly induced when the concentration of viable P. gingivalis was 108 CFU/ml. The expression of IL-1β and TNF-a also increased significantly when the concentration of viable P. gingivalis was 109 CFU/ml. The expression of chemokine such as IL-8 was strongly induced when the concentration of viable P. gingivalis was 107 CFU/ml. In additon, the gene expression of RANKL was also induced with the increasing concentration of viable P. gingivalis, and reach the highest amount when the concentration of viable P. gingivalis was 108 CFU/ml, but difference was not statistically significant. The expression of OPG was suppressed significantly by P. gingivalis in PDLFs.Conclusion No obvious difference in cell viability was observed when PDLFs were challenged by viable P. gingivalis for 6 hours. When challenged by P. gingivalis, PDLFs would participate in the regeneration and remodeling of periodontal tissue by producing cytokines.