The Research Of The Effect Of Tensile Force On Periodontal Ligament Fibroblast And Metabolism Of Extracellular Matrix

The basic procedure of orthodontic treatment is tooth movement in a certain direction resulted from periodontal tissue biological responses and remodeling induced by exogenous mechanical forces. So. the mechanical force is the premise and the key to promote periodontal ligament fibroblast(PDLF) physiologic reaction and to result in orthodontic tooth movement. PDLF and extracellular matrix(ECM) are the main components of periodontal ligament. Proliferation and functional activity of PDLF affect periodontal tissue health in normal environment and periodontal tissue remodeling in orthodontic treatment. Degradation and synthesis of periodontal ligament ECM manifest in active remodeling of periodontal tissue induced by mechanical force. Previous studies indicates that matrix metalloproteinases / tissue inhibitor of metalloproteinases (MMPs/TIMPs) system directly controls ECM metabolism. MMP-1 is a kind of proteolytic enzyme closely associated with degradation of periodontal ECM and plays an important role in orthodontic periodontal tissue remodeling. Its main substrates include I, II, III collagen, Fibronectin (FN), etc. But there are few studies on biological changes of periodontal ligament fibroblast and the mechanism of the regulation of ECM metabolism under mechanicalstress is not clear.In the present study, we loaded tension stress to human periodontal ligament fibroblast cultured in vitro by muti-passage cell strain loading device designed by ourselves and observed the effects of different mechanical strain on human PDLF proliferation and the expressions of III collagen, FN, proteoglycan (PG) and MMP-1/ TIMP-1, trying to find out the effect and the molecular mechanism of mechanical stress on PDLF in orthodontic periodontal tissue remodeling. The research works are as follows: 1. Manufacture of multi-passage cell stress loading systemObjective: To manufacture multi-passage cell stress loading system to research the biological behavior of cells in vitro under stretch stress. Methods: The multi-passage cell stress loading device was designed and developed, which was controlled by singlechip. The negative pressure that changed periodically was achieved by manipulating the vac-pup connected with vacuo chamber, the changed negative pressure caused the elastic membrane on the culture plate to deform. The deformation of elastic membrane exerted stretch stress on the cells cultured on the membrane, so the periodical stretch stress on cells was obtained. Results: The steering system, easy to carry, with deformation rate (1%~21%) and frequency (0-0.5Hz) that could be adjusted correctly, could compare deformation rate of three passages at the same time. Conclusion: The system run stably and completely achieved design demand, providing a method to stretch basement membrane of tissue cell in vitro. 2. Effects of different tension force on the proliferation of HPDLFObjective: To investigate the effects of the different mechanical strain on the proliferation of HPDLF. Methods: HPDLF weresubjected to 6%, 12%, 18% elongation by stretching frequency 0.1 Hz, or 12% elongation by stretching frequency 0.1 Hz, 0.2Hz and sustained stretching force in this experiment. MTT was performed to observe the proliferation of HPDLF. Results: The frequency O.lHz stretching force could promote HPDLF proliferation at 6%, 12% elongation and the 12% elongation is more powerful. Stretching force could restrain HPDLF proliferation at 18% elongation by frequency 0.1 Hz. At 12% elongation, the best stretching frequency for HPDLF proliferation was 0.2Hz and the least was sustained stretching force. Conclusion: Different stretching force had different effects on the proliferation of HPDLF and the stretching force of 12% elongation by frequency 0.2 Hz was the best.3. Effects of cyclic-tension force on the expression of coIlagen-HI,proteoglycan and fibronectin in HPDLFObjective: To investigate the effects of the mechanical strain on the expression of collagen-III, proteoglycan and fibronectin in human periodontal ligament cells (HPDLF). Methods: HPDLF were subjected to 12% elongation by stretching frequency 0.2Hz in this experiment. The expression of proteoglycan by saffron stain and collagen-III was determined by immunofluorescence, collagen-III and fibronectin by sandwich-in ELISA. Results: The expression of proteoglycan in HPDLF was negative in control group while positive in experiment group, indicating that HPDLF began to secret proteoglycan after loading cyclic-tension force. The expression of collagen-III in HPDLF was weak positive in control group, while significantly increased after loading cyclic-tension force in experiment group, indicating that the synthesization and secretion of collagen-III increased. The expression of fibronectin wassignificantly increased after loading cyclic-tension force compared with the controls. Conclusion: The cyclic-tension force may promote the expression of some ECM such as proteoglycan, collagen-III and fibronectin.4. Effects of cyclic-tension force on the expression of MMP-1 mRNA in HPDLFObjective: To investigate the effects of the mechanical strain on the expression of MMP-1 mRNA in HPDLF. Methods: HPDLF were subjected to 12% elongation by stretching frequency 0.2Hz in this experiment. The expression of MMP-1 mRNA was determined at 12, 24, 48h by in situ hybridization(ISH) and RT-PCR. Results: The expression of MMP-1 was positive in HPDLF cultured in vitro and increased in a time-dependent manner after loading cyclic-tension force. Conclusion: The cyclic-tension force promoted the expression of MMP-1 in HPDLF, suggesting possibly important role of MMP-1 in the metabolism of ECM.5. Experimental investigations of collagen tape I and III of periodontal tissues during tooth movementObjective: To observe the variation of collagen tape I and III in the periodontal tissues of first maxillary molar of rats which applied to tension-stress. Methods: Ni-Ti extension spring was placed between the maxillary left first molar and central incisors of adult SD rat. Grouped according to tooth movement days of 0, 1, 3, 5, 7, 10 and 14days, collagen tape I and III expression was examined with Picrosirius Red Staining and image analyses at 0, 1, 3, 5, 7, 10 and 14days after tooth movement. Results: The normal periodontal tissue mainly was composed of Collagen-1 .When the tooth was stressed, the content of collagen III rose in the side of tension-stress.