The Effect Of Infrasound On The Growth Of Bone Marrow Stromal Stem Cells

BackgroundsThe reseach of stem cells applies a new approach and hope for a renewable repair of major diseases. At present, the separation and identification, culture and amplification, ice save and recovery of stem cells have got the attention of some research institutions and individuals. BMSCs are very popular for it’s adequate source,and autologous transplantation.Infrasound is a mechanical vibration wave in the frequency range of0.0001～20Hz. It may have mechanical effect,warm effect, and chemical effect. It has effect on the most system of human body. It can cause the vivo cells change, and also have effects on the vitro cells. Infrasound has certain effect on the nervous system diseases, such as ischemia-reperfusion injury. It can also affect the profication of progenitor nervous cells. And BMSCs also has been widely used in the diseases of nervous system. So it is very necessary to reseach the effect of infrasound on the biological effect of BMSCs. At present,the chinese scholars have some researches about the effect of infrasound on the ultrastructure and proliferation of osteoblasts, progenitor nerve cells,microglia, corneal cells, and cancer cells, but few of BMSCs. A study found that low intensity ultrasound can promote BMSCs proliferation and cartilage directional differentiation. As a mechanical wave, infrasound may have the effect in common. The foreign scholars have very little reseaches of infrasound on the culture of BMSCs, But some people think mechanical factors have effect on it.The biological effect of infrasound is mainly decided by the parameters,such as frequency,time and strength. This reseach mainly observe the effect of different time on the biological effects of BMSCs.ObjectiveTo investigate the effect of infrasound on the growth of bone marrow stromal stem cells (BMSCs) and explore the reasonable parametre of using infrasound.MethodsThe culture of bone marrow stromal stem cells:The femurs and tibiae were removed from Sprague-Dawley rat after killing them with broken cervical spine. The BM plugs were flushed with medium (DMEM/F12). The liquid was centrifuged at1000r.p.m. For5min at room temperature. All cells were cultured with complete medium (DMEM/F12supplemented with10%fetal bovine serum) at37℃in humid air with5%CO2. Change the complete medium after2days, and then once every2days.At confluence, the cells were harvested for passage with0.25%trypsin containing0.02%EDTA and phosphate-buffered saline (PBS) solution (The ratio of concentration of trypsin and PBS was1:1). All experiments were performed using cells from the third passage. The expressions of CD29, CD90and CD45in MSC were analyzed by flow cytometer.The cells viability would be showed by trypan blue staining.The method of the trial:The cells of passage3were devided into trial groups with treating for10min,30min and60min by infrasound and control groups with treating for the same time without infrasound output. The cells were moved to the37℃incubated tank with5%CO2to culture. The proliferation of cells was measured respectively using CCK8method which were cultivated consecutively for3days after treatment. The cell apoptosis and cell cycle were analysed with flow cytometry(FACS) cultivated3days after treatment. The scanning electron microscopy(SEM) and transmission electron microscopy(TEM) were used to test the ultrastructure of the cells.The proliferation of the cells would be measured in96well plate,and there were6plates for each group having one.The number of the trial cells was adjusted to4000/100ul/well. The cells were divided into12wells in every plate, and carried to the incubator for3hours to make them adherent growth. Every day the cells of each group were exposed to infrasound or air at the same time point for3days. After treating, the96well plates were immediately carried to the incubator. The cck8test was carried out in4wells with cells growth after48hours for the first time,and it was done3times in all for3days.The cells were transfered to a5ml cryogenic freezing container to receive the exposure of infrasound or air after digesting and resuspensing. After treating, the cells were divided into the25cm2cell culture bottle to incubate3days for the experiment of apoptosis and cell cycle analysis.The cells used to observe the ultrastructure were treated as same as the experiment of apoptosis and cell cycle analysis. After treating, one part of the cells were transfered to the sterile microscopic glass in the culture dish with complete medium for SEM observation after1night. And the other part of the cells was transfered to the25cm2cell culture bottle with complete medium for TEM observation after4days.The date are expressed as Mean±SD.statistical analyses were done with SPSS, version13.0. P<0.05was considered significant.The differences between the means were determined by factorial analysis or T test.Results 1. The result of trypan blue assay showed that the rates of survival BMSCs were above95%.2.The result of the surface marker of BMSCs by flow cytometry:CD29and CD90were postive, but CD45was negative.3.The result of the CCK8kit:Factorial analysis found that after culturing for48h,72h and96h, the OD value of BMSCs treating for10min,30min and60min with infrasound was (0.929±0.042;1.094±0.013;1.410±0.016),(1.480±0.001;1.348±0.027;1.493±0.017) and (1.774±0.127;1.731±0.062;1.833±0.054) respectively, while the OD value of BMSCs of the control groups was (1.148±0.088;1.147±0.030;1.112±0.051),(1.479±0.051;1.267±0.006;1.227±0.126) and (1.567±0.032;1.563±0.043;1.632±0.071) respectively. It was visible that the OD value of cells of infrasound groups are larger than the control groups after culturing for72h.With the extension of time of treating, the OD value of BMSCs will increase or increase after decreasing, and the OD value of BMSCs treating with infrasound for60min was the highest. Single effect analysis was done with T-test which found the difference between the infrasound group and the control group was significant (P<0.05)4.The results of FACS indicated that there was no effect of infrasound on apoptosis of BMSCs when the treating time was30min, but the cell apoptosis can be inhibited when the treating time was10min or60min. The early phase apoptosis rate was1.07%±0.12%and0.97%±0.21%in the trial group treating forlOmin and60min respectively, and1.43%±0.06%and3.33%±0.15%in the control group respectively (P<0.01)5. The results of cell cycle analysis was that infrasound can disturb the cell division of BMSCs when the treating time was within30min, but there was no effect when the time is60min(P>0.05).When the treating time was10min of infrasound, the cells in a quiescent state were more than the cells in control group (P<0.05). However, the result was adverse when the treating time was30min (P<0.01)6.SEM:The BMSCs treated by infrasound exposure for60min and then cultivated for1day showed that the prominent, micro-floss of the membrane and the figure were very different from the cells of control group. The cell of control group showed that the prominent and micro-floss of the membrane become shorten or decreased.The figure of cells in infrasound group was common strip,but it in the control group was little with most roundness, which represented the figure of the dead cell increased.7.TEM:The cells in the infrasound group were in a good condition, with normal mitochondria and cell nuclear.However compared with the cells in the infrasound group,the condition of the cells in the control group was bad, with broken cell nuclear and some lysosomes. It revealed that the dead cells were more than those in the infrasound group.Conclusions1.Infrasound can improve the cell proliferation,disturb the cell division, but not cause cell apoptosis of BMSCs. Infrasound can change the ultrastructure of BMSCs, improving the cell viability.2. When the treating time is60min.the cell proliferation was improved steadily, the cell apoptosis rate was decreased, and the cell cycle was not changed.The ultrastructure of BMSCs revealed the growth condition were well after treating60min with infrasound. So the60min is the most reasonable treating time of infrasound to BMSCs.