The Role Of Cyclical Stretch In The Vascular Smooth Muscle Cells Proliferation And The Regulatory Mechanism

Background:In orthodontic treatment, the dynamic environment of periodontal tissue changes with the application of mechanical forces to the tooth crown, and at the same time, the stress and strain of periodontal membrane also changed. Underlying orthodontic force, the periodontal ligament cells mediate stress, and then the morphology and function of the cells changed which inducing the remodeling of periodontal tissue, and then the tooth moved gradually from one spot in the oral cavity to a more desirable one.Periodontal ligament microvasculature system is an important part of periodontal tissue, it plays an important role for the maintenance of physiology function. Vascular smooth muscle cells (VSMC) is a major component of vascular wall cells, in orthodontic treatment, it is affected by the blood flow generated shear stress and cyclical strain and other mechanical factors. In orthodontia process, if given appropriate cyclical stress, the VSMC would proliferate, migrate and differentiate, resulting in adaptive alterations. But it is not clearly about the specific mechanism of VSMC adaptive remolding.Mitogen-activated protein kinase (MAPK) is a kind of cell proliferation-related intracellular key enzyme discovered recently. It is a common channel of growth factors, cytokines, vasoactive peptides involved in the process of VSMC proliferation. P38 MAPK is the newest member of the MAPK family, involved in a variety of cells' intracellular signal transduction.P38 plays an important role in mediating inflammation, stress and other events. It is also involved in cell survival, differentiation and development process. It is one of the hottest in the field of signal transduction research in recent years.Objective:This study first constructs a vascular smooth muscle cells in vitro model of mechanical stimulation, in the field of cell mechanics to explain the effect of cyclical stretch in vascular smooth muscle cell proliferation. Then we imposed p38 MAPK specific inhibitors to clarify the role of the p38 MAPK playing in vascular smooth muscle cell proliferation, and it is can be known the relationships between cyclical stretch, p38 MAPK and vascular smooth muscle cell proliferation.It is also can be clearly the specific mechanisms for PDL adaptive remodeling underlying orthodontics force. It is hoped that clinicians can use different means and methods of regulation of vascular alterations, and provide a theoretical basis for clinical intervention. Materials and Methods:Cyclic stretch was applied on the VSMC respectively for 0h,6h,12h and 24h, The Flexcell Strain Unit FX-4000T was used to expose VSMC to an equiaxial cyclic stretch of 10% magnitude and a frequency of 1 Hz, a frequency of 6 cycles/min, each cycle including the 5-s stretch/5-s relaxation. MTT assay was used to observe the proliferation of the VSMC; flow cytometry analysis was used to examine the ratio of cells in proliferation period; RT-PCR was used to measure the expression of CDK2; western blotting was used to detect the activity of phospho-p38MAPK. This time we make the VSMC prior to impose p38 MAPK-specific inhibitor SB203580, and then imposed on the same tensile stress, and then we detected the changes of cell proliferation and the expression of phosphor-p38MAPK. The experimental data were analyzed used SPSS11.0 Statistical Package. Results:1. The results of MTT assay showed that the cyclical stretch of 10% magnitude and a frequency of 1 Hz applied in this experiment can induce vascular smooth muscle cell proliferation. In addition, the proliferation ability of vascular smooth muscle cell enhanced with the prolonged of cyclical stretch time. The highest result occurred in 12h group, and then the proliferation became lower, but it was higher than Oh group. 2. The results of flow cytometry analysis found that the ratio of vascular smooth muscle cell in S period was increased. With the extension of time exposed on cyclical stretch, the ratio of vascular smooth muscle cell in S period was also increased, the ratio of 12h group was highest compared with Oh group.3. The results of RT-PCR showed that the expression of CDK2 increased after exposed on cyclical stretch. It reached peck in 12h group and then decreased but it was higher than Oh group.4. The results of western blotting found that phosphor-p38 MAPK expressed in all groups, the longer of cyclical stretch time, the higher expression of phosphor-p38 MAPK. It was also reached peck after 12h exposing on cyclical stretch. Then, it decreased but still higher than Oh group.5. Cells imposed on SB203580 before imposing cyclical stretch, the results showed that phosphor-p38 MAPK expression decreased apparently. Moreover, MTT assay showed that the proliferation ability of vascular smooth muscle cell decreased compared with the group without using SB203580.The results of RT-PCR founded that the expression of CDK2 decreased too, but it was higher than Oh group.Conclusion:1. Cyclical stretch of 10%,1HZ might induce the proliferation of vascular smooth muscle cells.2. Vascular smooth muscle cell proliferation induced by cyclical stretch is related to cyclical stretch time. In our study, we found that the proliferation ability of vascular smooth muscle cells reached peck after 12 hours exposed on cyclical stretch.3. Underlying in the cyclical stretch the expression of phosphor-p38 MAPK may change with the proliferation of the vascular smooth muscle cell, otherwise, the proliferation decreased when we restrained the p38 MAPK pathway. The results suggest that p38MAPK is one of the main mechanisms of vascular smooth muscle cell proliferation induced by the cyclical tensile stretch.