Effects Of Porphyromonas Gingivalis Lipopolysaccharide On Healing Of Impaired Periodontal Ligament Cells

Lipopolysaccharide(LPS) of Porphyromonas gingivlix(Pg),one of the main etiological factors of periodontitis,can lead to the loss of attachment and resorption of alveolar bone in periodontal tissue.Periodontal ligament cells(PDLCs) play an important role in periodontal tissue repairment and regeneration.The concentration of LPS may influence the progression of periodontal disease.Platelet-derived growth factor-BB(PDGF-BB) is one of factors on PDLCs mitosis,playing important roles in PDLCs regeneration biology.ObjectiveTo evaluate the influence of different concentrations of Pg-LPS alone or with PDGF-BB on PDLCs via an in vitro model of wound healing,and to observe its effects on attachment and proliferation of PDLCs on the slices of cementum,thus to elucidate the effects of LPS in PDLCs regeneration biology.Methods1.PDLCs were havested from the periodontal ligaments of the teeth extracted for orthodontic treatment from healthy humans.Immunocytochemical staining was utilized to detect expression of vimentin in PDLCs.Cells of the 4^th transfer in culture were cultured in 12-well plates,and then grown to confluence,and in vitro wound models were mechanically established by removing cells in an 8mm diameter area. Then the wounded models were incubated in the presence of LPS of a concentration of 0,0.001,0.01,0.1,1 and 10μg/ml for 1d,3d,6d,9d and 12d,then the cell quantities of each group and areas of the wound were evaluated.Levels of IL-1β,IL-6 and TNF-αon 1d,3d and 6d were detected by ELISA.2.0,1 or 10μg/ml LPS was incubated with 10ng/ml PDGF-BB in 10%DMEM for 1d,3d,6d and 9d to estimate the healing speed in the PDLCs wound model.3.PDLCs were seeded on the cementum surface of the slice in the presence of LPS(0,1 or 10μg/ml),Attachment and proliferation assays were detected by MTT assay on 1d and 3d.Apoptosis of the PDLCs were evaluated using AO/EB Doubly Staining.Results1.Cultured cells showed fibroblast-like morphology with a positive vimentin expression.The growth inhibitory effects of LPS were stastistically significant in 10μg/ml Pg-LPS group(P＜0.05) with increased secretion of IL-1β,IL-6 and TNF-α(P＜0.05).In contrast,treatment with low concentration of LPS were no stastistically significant in 0.001 to 1μg/ml groups(P＞0.05).2.The healing of PDLCs responses of PDGF was stastistically reduced in the presence of 10μg/ml Pg-LPS group(P＞0.05),however,0.001 to 1μg/ml LPS were no stastistically significant to control groups(P＞0.05).3.The number of cells attached and proliferated on the slices of cementum incubated with 10μg/ml Pg-LPS were deceased in contrast to control groups(P＜0.05). but low concentration of LPS were no stastistically difference to control groups(P＞0.05).Apoptotic cells could be observed using AO/EB Doubly Staining in 10μg/ml Pg-LPS group.Conclusions1.The inhibitory effects of PDLCs on growth and proliferation are related to the concentration of LPS.2.LPS with a concentration of lower than 1μg/ml did not affect the attachment and proliferation of PDLCs on slice of cementum,suggesting massive scraping of the cementum of the root slices may be not necessary for the therapy of periodontitis.