The Development Of Multi-passage Cellular Stretching System And The Proliferative Effects Of Stretch On ST2 Cells

Objective:Cells are under static or dynamic physiological stresses in vivo, which make physiological changes occur, these changes are the biological foundation of osteogenesis, orthodontic tooth movement etc. The most ideal research condition is to observe various reactions of cells to stress under physiological state, however, the mechanical environment of somatic cells is complex and diverse. Research of physiological or pathological phenomenon of cells is still dependent on cell culture in vitro. Development of cells plus experimental system is therefore the major way to study the mechanics and biology of cells. To study the biological behavior of cells under stretch in vitro, a multi-passage cyclic stretching loading system was developed.The ST2 cell line was chosen to complete the experiments in vitro, ST2 cell line is a stromal cell line, which is separated from the bone marrow of BC8 mice. ST2 cell line shares characteristics with bone marrow mesenchymal stem cells (Mesenchymal Stem Cells, MSCs). MSCs are multipotent differentiational stem cells, which are derived from the mesoderm. MSCs distribute in the connective tissues of different species of animals and the human body, mainly in the bone marrow. Mesenchymal stem cells-stretching stimulation model was built by exerting different stretches on cultured ST2 cells under certain conditions to obtain the best condition for cell proliferation, in order to provide foundation for further study in future. Materials and Methods:1. Microcontroller embedded software suite system based on C language was applied to develop the multi-channel cellular stretching loading system, which can control the vacuum pump connected with vacuum chamber in order that negative pressure in vacuum chamber can change cyclically, therefore, the basement membrane of the cell culture plate received deformation accordingly, in consequence, cyclic stretch was exerted to cells.2. ST2 cells were cultured by conventional DMEM culture method.The ST2 cells were seeded in 6-well BioFlex afterburner board with a concentration of 2105/ml, then cells were removed to incubator(37℃,5%CO2, humidified). The cell culture plate was connected to multi-channel stretching device after cell cycle synchronization in sterile condition, the morphological changes of ST2 cells under cyclic stretch were observed in vitro after 48 h.3. In experimental groups, the stretches exerted on ST2 cells were with frequency 0.125 Hz,0.25 Hz,0.5 Hz and size of 5%,10%,15%, while in control group, no stretch was exerted on ST2 cells. MTT assay was used to detect cell proliferation after 24 h. The experimental data were analyzed used SPSS 13.0 Statistical Package.Results:1. Stretch loading system can compare different elongations in three-passage, with precise adjustment and convenient operation. The range of 0-20% in deformation rate and 0-0.5 Hz in frequency was adjustable.2. ST2 cells reached confluence at day 2or 3. ST2 cells grew well in plate in 48 h after stretch exerted, adhering stably without stripping phenomenon and arraying at a direction gradually.3. The best proliferative effect was achieved by stretch of 15%,0.25 Hz, followed by stretches of 5% 0.125 Hz,0.5 Hz,10% 0.125 Hz,0.25 Hz,15% 0.5Hz,all of them showed significant difference compared with control group(P<0.01).The difference between stretch of 5%,0.25 Hz and control group was not significant. stretch of 10%, 0.5 Hz and 15%,0.125 Hz showed negative effect on cell proliferation. Conclusion:1. Loading device operated stably and reached design requirements, providing the experimental method of exerting cyclic stretch on adherent cells in vitro.2. The proliferative effects of different stretches is varied according to the measurement and frequency of stretch.15% of the deformation rate, frequency of 0.25 Hz periodic tension stress on the ST2 cell proliferation attained the best effect. Mesenchymal stem cells-stretch stimulation model was successfully constructed which can meet the needs of the experiment and the follow-up experiment to select the appropriate size and lay foundation for the ideal stretching load conditions in follow-up experiments.