Effects Of 50Hz Electromagnetic Fields And Transforming Growth Factor-beta3 On Proliferation And Differentiation Of Mouse Precartilaginous Stem Cells In Vitro

Objective To investigate the effects of 50Hz pulsed electromagnetic fields (PEMFs) of different intensity on proliferation and apoptosis of mouse precartilaginous stem cells (PSCs) in vitro; To study the effects of various doses of transforming growth factor-beta3 (TGF-β3) on the chondrogenic differentiation of mice precartilaginous stem cells (PSCs) in vitro.Methods The mouse PSCs were isolated and purified in vitro. The third passage PSCs were randomly divided into four experiment groups and a control group. Four experiment groups were radiated (twice daily,45min each time) by 50Hz PEMFs of different intensity (0.10mT,0.50mT,1.00mT and1.50mT), respectively. MTT was used to observet the proliferation of PSCs and flow cytometer was performed to detect apoptosis. The level of Bcl-2 expression was evaluated with Western blot. The third passage PSCs were randomly divided into five experiment groups and a control group. Five experiment groups were cultured in serum-free induced mediums containing different doses of TGF-β3, respectively. Control group cells were cultured in the medium without TGF-β3. The production of collagen II was detected by immunohistochemistry staining. The transcription level of cartilage-related gene (collagen II gene) was also evaluated by quantitative RT-PCR assay.Results 50Hz PEMFs of different intensity could significantly promote the proliferation of PSCs and PEMFs of 1.0mT intensity had the most obvious effect. There were no significant differences after radiation for 48 hours between the experiment groups and control group, but the proliferation was significantly promoted after PEMFs intervention for 96 hours (P<0.05). Meanwhile, apoptosis rates of all experiment groups were significantly decreased (P<0.05) and the expression of Bcl-2 protein was obviously increased. The immunohistochemistry staining showed the synthesis of collagen II in all experiment groups with TGF-β3, but the expression of collagen II in the control group was negative. The production of collagen II in 10μg/L group was significantly higher than other groups(P<0.05). The chondrogenic differentiation of PSCs was also proved by the expression of collagen II gene in mRNA level with RT-PCR.Conclusions 50Hz PEMFs could significantly promote the proliferation of mice PSCs in vitro and inhibit apoptosis by the up-regulation of Bcl-2 expression. The effects are related with intensity and time of PEMFs intervention. 1.0mT is the most appropriate intensity to promote PSCs proliferation. TGF-β3 can effectively induce chondrogenic differentiation of PSCs and increase cartilage extracellular matrix(ECM)in vitro and these effects are dose-dependent.