Biological Effects Of Connective Tissue Growth Factor On Cultured Human Periodontal Ligament Fibroblasts In Vitro

The periodontal disease incidence is about10%～15%in the world. The latest statistics show that, in our country and Japan the age over30have periodontal disease are more than80%[1]. Periodontal disease as the main reason why people over35teeth missing, and has become a variety of chronic systemic disease (such as coronary heart disease, diabetes, etc.), the risk factors of the development of a serious threat to human health [2]. The ultimate goal of periodontal therapy is to rebuild the destruction of periodontal tissue by inflammatory process, restore its normal structure and function, the true realization of periodontal tissue regeneration. But in the restoration of periodontal tissue physiological structure and function is far did not achieve the desired goal. Over the years, people have been seeking can effectively increase the tooth week attached, promote periodontal tissue regeneration method, and carry out the guiding periodontal tissue regeneration, periodontal bone graft technology aspects of research, has made some progress, but in the restoration of periodontal tissue physiological structure and function is not achieve the desired goal. In recent years, tissue engineering, stem cells and its related technology development by leaps and bounds, the regeneration of tissue or organ of tissue engineering research also ascendant, it provides an opportunity for the research of periodontal tissue regeneration. Tissue engineering research focus on mainly in the following three aspects:(1) the source of seed cells;(2) biological carrier scaffold choice;(3) effective cytokines. So select effective cytokines is used tissue engineering technology to obtain the prerequisite for periodontal regeneration.Connective tissue growth factor (CTGF) is a class of trend in recent years to promote tissue repair cell growth factor, can promote mitosis and cell proliferation, chemotactic cells, inducing the synthesis of cell adhesion and extracellular matrix. In the eye, liver and kidney and other disciplines have carried on the thorough research, but rarely involved in the mouth cavity. Also contains a large amount of fibrous tissue in the periodontal support tissues, the periodontal ligament fibroblasts is the most important function of cells in periodontal tissue, and the relationship between the fibroblasts of CTGF and periodontal tissue study very little.This topic selection, double-pointed root in1/3of the periodontal membrane for in vitro culture, through the tissue pieces culture method to extract the periodontal ligament fibroblasts, after the success of the extract by connective tissue growth factor effect on fibroblast, to observe the influence of its biological activities, so as to provide new research direction for dental treatment of periodontal disease, offers a new growth factor for periodontal disease clinical drug research and development.Aims:The periodontal ligament fibroblasts in vitro people as the research object, observe the different concentrations of connective tissue growth factor (CTGF) on the periodontal ligament fibroblasts proliferation, differentiation, adhesion, migration, the influence of the synthesis of extracellular matrix, discuss different concentrations of connective tissue growth factor (CTGF) on the biological changes of the periodontal ligament fibroblasts and its significance, provide the experimental basis for the further research of periodontal tissue regeneration.Methods:1、In vitro using tissue explant culture method of the periodontal ligament fibroblasts, isolation and purification of inverted phase contrast microscope and wood grain-eosin staining observation of cell morphology, cell growth curve drawing, immune cell chemical identification of the cells.2、CCK-8method is applied to detect different concentrations of connective tissue growth factor (1、5、10、50、100ng/ml) to act on human periodontal ligament fibroblasts proliferation after24,48,72h activity.3、The application of alkaline phosphatase kits of different concentrations of connective tissue growth factor (1、5、10、50、100ng/ml) of the periodontal ligament fibroblasts of alkaline phosphatase activity.4、Application of hydroxyproline kits of different concentrations of connective tissue growth factor (1、5、10、50、100ng/ml) of human periodontal ligament fibroblasts synthesis of collagen.5、Application of alizarin red staining method to detect different concentrations of connective tissue growth factor (1、5、10、50、100ng/ml) of the periodontal ligament fibroblasts, the effects of formation of mineralized nodules.6、Application Transwell Chambers to detect different concentrations of connective tissue growth factor (1、5、10、50、100ng/ml) of the influence of the periodontal ligament fibroblast migration ability.7、The application of real-time quantitative PCR method to detect different concentrations of connective tissue growth factor (1、5、10、50、100ng/ml) of the periodontal ligament fibroblasts express I type collagen (Col-I), alkaline phosphatase (ALP), fiber link protein (FN), bone sialoprotein (IBSP), osteocalcin (OC), integrin beta1(ITGB1) mRNA level.All all use SPSS17.0statistical software analysis on the experimental data, the data results are expressed as a mean±standard deviation (x±s), with P<0.05think have statistical significance.Results:1、In vitro tissue pieces culture method is used to successfully obtain the periodontal ligament fibroblasts. Normal cells form, stable shape, in good condition. Immunocytochemistry staining positive waveform silk protein, keratin negative, conforms to the characteristic of ectomesenchymal cells from mesoderm source, prove to be cultured cells are the periodontal ligament fibroblasts. Cell growth curve presented "S" shape.The most suitable for experimental study in the logarithmic phase of the periodontal ligament fibroblasts, active best, vigorous growth. This experiment take4-6cells for subsequent experiments.2、Compared with the control group,1、5、10ng/ml of CTGF in2、48、72h of the periodontal ligament fibroblasts have obvious effect on promoting proliferation, statistically significant difference (P<0.05), and50ng/ml CTGF compared with control group, there was no statistically significant difference (P>0.05), and100ng/ml of CTGF in the periodontal ligament fibroblasts have inhibition. Two comparison showed that different concentrations of CTGF stimulate the periodontal ligament fibroblasts proliferation between three different time points, difference was not statistically significant (P>0.05).The10ng/ml of CTGF is the best concentration of proliferation for promoting the periodontal ligament fibroblast.3、Compared with the control group,1、5、10、50ng/ml of CTGF in7、14、28d of the periodontal ligament fibroblasts were significantly enhanced the activity of ALP, statistically significant difference (P<0.05),100ng/ml CTGF on the periodontal ligament fibroblasts showed inhibitory effect on the ALP activity. Two comparison showed that different concentrations of CTGF stimulate the periodontal ligament fibroblasts proliferation between three different time points, difference was not statistically significant (P>0.05). The10ng/ml of CTGF promote periodontal ligament fibroblasts optimal concentration of alkaline phosphatase activity.4、Compared with the control group,1、5、10ng/ml of CTGF in24、48、72h of the periodontal ligament fibroblasts have significantly enhanced the role of collagen protein synthesis, statistically significant difference (P<0.05),50,100ng/ml of CTGF compared with control group, there was no statistically significant difference (P>0.05). The10ng/ml of CTGF promote human periodontal ligament fibroblasts synthesis and optimal concentration of collagen.5、Six mineralization rate comparison between the experimental group, the difference is statistically significant (P<0.05), compared with the control group, the mass concentration of10ng/ml, CTGF has the biggest cu mineralization (P<0.01).6、1～100ng/ml of connective tissue growth factor can significantly promote the periodontal ligament fibroblast migration, statistically significant difference (P<0.05). Cell migration ability as the connective tissue growth factor (CTGF) increased with increasing concentration, the concentration of the apparent dependence.7^RT-PCR, according to the results of cell ALP mRNA expression level, the concentration of CTGF for5、10、50ng/ml can promote ALP mRNA expression, when compared with control group, the difference is statistically significant (P<0.05), peaked in the10ng/ml. And1ng/ml and100ng/ml difference compared with the control group. Cell IBSP mRNA expression level in CTGF concentration of1、10、50、100ng/ml can promote IBSP mRNA expression, compared with control group, the difference is statistically significant (P<0.05), peaked in the10ng/ml. And5ng/ml differences compared with the control group. Cell OC mRNA expression level in CTGF concentration of1、10、50ng/ml, effect can promote OC mRNA expression, compared with control group, the difference is statistically significant (P<0.05), peaked in the10ng/ml. And5ng/ml and100ng/ml difference compared with the control group. Cell FN, COL-I mRNA expression level, when CTGF in certain concentration range (1to100ng/ml) can promote the expression of FN, COL-the mRN I, compared with control group, the difference is statistically significant (P <0.05), peaked in the10ng/ml. Cell ITGB1mRNA expression level in CTGF concentration of1、5、10、50ng/ml can promote ITGB1mRNA expression, compared with control group, the difference is statistically significant (P<0.05), peaked in the10ng/ml. The100ng/ml difference compared with the control group.Conclusion:1、 Using the tissue pieces culture method in vitro can successfully get the periodontal ligament fibroblasts. This method is simple, reliable results and can be used as a person the periodontal ligament fibroblasts in vitro model of experimental research.2、Connective tissue growth factor to act on human periodontal ligament fibroblasts, one can increase the activity of periodontal fibroblasts by increasing the expression of integrin beta subunit1to promote the adhesion, promote the periodontal ligament fibroblasts proliferation, migration and participate in the regulation of the extracellular matrix, encourage people to osteoblast differentiation of periodontal ligament fibroblasts, promote the regeneration of periodontal support tissues.