The Role Of Nicotine-α7nAChR Signaling Pathway In Nicotine Affecting The Osteogenic Potential Of HPDLSC

Periodontitis is a common seen disease in oral system which has already been considered as a main reason of losing teeth for adults and cigarette smoking is a well established risk factor in its etiology. The most important toxicant in cigarette is nicotine. Compared with normal control, smokers have a higher prevalence and a more severe condition in periodontitis suffering. Published reports indicated that smoking has something to do with the formation of periodontal pocket, loss of periodontal attachment and loss of bone volume and teeth.The effect of nicotine on periodontitis has two sides. For one thing it would increase the susceptibility for smokers and accelerate the disease progress; for the other, the prognosis of a smoker is unsatisfactory even if he/she receives systematic therapy and a worse regenerative of periodontal bony substance would be detected compared with non smokers.Periodontal ligament stem cells (PDLSC) exist in periodontal ligament tissues which possess cloning ability and basic characteristics of stem cell. PDLSC could differentiate into osseous tissues under osteogenic inducement microenvironment in vitro and if given proper condition, it could form periodontal/cementum-like complex tissues in vivo with sharpey fiber processed through the newly formed structure. As a result, PDLSC has a potential of regenerating periodontal new attachment and repairing periodontal defect.In vitro study was carried out in researching the effect of nicotine on the osteogenic ability of PDLSC and further experiment was performed to observe whether a7nAChR expressed in PDLSC and whether it took part in the down regulating process of nicotine on the osteogenic ability of the cell together with the downstream signaling pathway of the receptor. We wished to perfect the mechanisms for smoking related periodontitis and provide some theoretical basis for possible new therapy method in the near future.Study1:Isolating, Culturing and Identifying PDLSC in VitroClones for PDLSC were isolated by the method of limiting dilution and the surface antigen markers were detected by flow cytometer. Certain inducement microenvironments were given to PDLSC to further explore its differentiating potential. This experiment was designed to provide appropriate seed cells for further study. Results indicated that PDLSC positively expressed surface antigen makers of cells from mesenchymal tissues; under certain inducement conditions it could exhibit osteogenic and adipogenic differentiation characteristics.Study2:Effect of Nicotine on Morphology and Proliferation Ability of PDLSCIn this experiment PDLSCs were cultured in vitro and gradient concentrations of nicotine were made up to observe the morphological and proliferation ability changes of PDLSC after a certain time to find out a proper concentration of nicotine for further study. Results indicated that nicotine with different concentrations would make morphological changes for PDLSC to different extents and suppress the cell proliferation ability while the later effect had a concentration dependent characteristic.Study3:Effect of Nicotine on the Osteogenic Ability of PDLSCThis experiment was designed to explore the effect of nicotine on the osteogenic ability of PDLSC wishing to explain the reason why smokers had a worse therapeutic effect of periodontal regeneration and provide some theoretical basis for clinical treatment. Results indicated that nicotine with different concentrations could suppress the osteogenic potential of PDLSC in early and terminal stage with a concentration dependent characteristic. Nicotine could suppress the expression of transcriptional factors in the osteogenic process such as ALP, BSP, OCN and Runx2both in gene and protein levels.Study4:a7nAChR Expressed in PDLSC and Its Role in the Down Regulating Effect of Nicotine on the Osteogenic Potential of PDLSCThis experiment explored whether α7nAChR expressed in PDLSC and its role in the down regulating effect of nicotine on the osteogenic potential of PDLSC. Results indicated that there existed gene and protein expression of α7nAChR in PDLSC. If given a-BTX which was a competitive antagonist of nicotine in combing with the receptor, the suppressive effect of nicotine could be partially reversed as revealed by histological stainings. Compared with nicotine treated group, same tendency was observed both in gene and protein levels.Study5:Effect of Wnt Signaling Pathway in Nicotine Affecting the Osteogenic Potential of PDLSCThis experiment aimed to explore whether there existed Wnt signaling pathway in the downstream of the activated α7nAChR and whether the Wnt signaling pathway took part in the negative regulation of nicotine on the osteogenic potential of PDLSC. Results indicated that there existed protein changes for Wnt related transcriptional factors when α7nAChR was activated. If given Dkkl which was an inhibitor of Wnt signaling pathway, the suppressive effect of nicotine could be partially reversed as revealed by histological stainings. Compared with nicotine treated group, same tendency was observed both in gene and protein level s.