The Effect Of Different Platelet - Rich Plasma Concentration On Proliferation And Differentiation Of Minipig Periodontal Ligament Cells In Vitro

Periodontitics is a chronic disease which is caused by bacteria and characterized by destruction of the supporting tissues. Meanwhile, a lot of studies have approved that periodontitics have close contact with General health and systemic disease. Periodontitics represents changes of the structure and integrality of periodontal ligaments, alveolar bones, cementums and gums, makes destruction of connective tissue and cells, then causes pockets, receding of alveolar bones and attachment loose, even the teeth get loose. Repair of soft and hard periodontal tissues is a difficult question which is in dire need of resolving and the hotspot of fundamental research. In recent years, the rapid development of tissue engineering which is formed by cells, growth factors and scaffold brings regeneration of periodontal tissue a new turning point. Searching for effective growth factors to promote repair and regeneration of periodontal tissue, enhancing the function of related cells, have been the effectual way to advance repair of the tissue.The related cells in wound healing of periodontal tissue are gingival fibroblasts, periodontal ligament cells, osteoblasts and cementoblasts. Only periodontal ligament cells can be used as stem cells. Through constant proliferation and differentiation, can them not only form the new periodontal fibres but also attend rebuilding of alveolar bones and cementums, so periodontal ligament cells are seemed as the idealest stem cells in regeneration of periodontal tissue.The oral anatomical structure, dental system, physiology, pathology and metabolization of minipig are tremendous similar to the ones of human. The characteristics of its dwarfism figure, small oral cavity and easy to operate make it to be used in many fields of medical research. The most important thing is the subgingival bacteria of minipig are similar to the human's, which can cause periodontitis spontaneously, therefore minipigs are the ideal animal model to study periodontitis. As reported, the PDLCs of minipigs have the same shape of fibroblasts, can express type I collagen, osteopontin (OPN) and alkaline phosphatase (ALP). It also have the potential of differentiation, as the human's PDLCs cultured in vitro. Therefore, it brings well foreground to choose minipig as the model in medical research and clinic studies of periodontitis.Platelet- rich plasma(PRP) is the extract got by offentering blood。A large number of tests and studies have showed that after activated by thrombin and CaCl2, theα-granule of platelet can release many growth factors and cytokines in high concentration, including platelet-derived growth factor(PDGF), insulin-like growth factor(IGF), transforming growth factor(TGF), vaseular endothelial growth factor(VEGF), osteocalcin and fibronectin to repair tissues and promote organization healing. The advantages of PRP are as followed, autologous, completely natural and safe, many kinds and high concentration of growth factors, easy and to do with it and maturation in technology, having no harm to the bodies. Recently, most of the studies of PRP in repair of periodontal tissues focus on in vitro studies, moreover the biological characteristics of PRP to PDLCs are so few. The effective concentration and time to promote PDLCs'proliferation and differentiation haven't reached an agreement. Furthermore, PRP and the cells are not from the same way.As mentioned above, our study choose minipig as experimental subject to investigate the biological effect of PRP to self PDLCs in different concentration and different time, provide theoretical foundation to repair and regeneration of periodontal tissue, and laying foundations for further research.Methods: To extract 4 incisor teeth and 4 cuspid teeth from the minipig of 6 moon's age, collect the periodontal ligaments from the middle 1/3 of the root, cultured by tissue explant method, then do the cell identification, paint the growth curvature and make sure the best cells concentration to culture. Making self PRP of minipig with two step centrifuging, collecting preparation rich in growth factors(PRGF) after activated. Using PRGF in different concentrations, such as 2.5%,5%,10%,20% and 40% to act on the PDLCs of the minipig, to text the proliferation of the cells after 1-6 days with MTT, and then to text the level of cell differentiation with coomassie brilliant blue and ALP kit in the 3^th,5^th and 7^th day, contrasting with PBS. Results: The success ratio of cultured PDLCs is 50%, the incisor teeth's is 100%. The growth curvature of the minipig is expressed in the shape of S. The best cells concentration to culture are 5×10³ cells per hole. The data show statistical significance or statistically significant when contrasting the promotion of cell proliferation in each concentration of PRP and the activity of ALP to controls( P<0.05,P<0.01). The cells'proliferation and the activity of ALP raising with the concentration and time when the concentration of PRP increase from 2% to 10%. When the concentration reach 20%, PRP can inhibit proliferation but promote differention. When the concentration reach 40%, it shows obvious inhibition in both proliferation and differention. Conclusions: PRP in low concentration can promote the proliferation of minipigs'PDLCs and activity of ALP in 1-7 days, moreover, when it in high concentration, it can inhibit all the things above with dose dependent in some degrees.