Effect Of Pulsed Electromagnetic Fields On Accelerating Human Mesenchymal Stem Cells Proliferation And Osteogenic Differentiation By Different Frequency

Objective: Bone tissue engineering can repair the defect of bone caused by different reasons which reveals good application prospects clinically.The human mesenchymal stem cells (hMSCs )in bone marrow are believed to be one of the best choices of seedling cells of bone tissue engineering for their strong potential and high activity of osteogenesis. How can we harvest enough seedling cells with superior quality and induce them to differentiate into osteoblasts as quickly as possible?Our research has been verified that the Pulsed Electromagnetic Fields (PEMFs) with specific frequencies and fields intensities could promote the proliferation and differentiation of hMSCs. If the frequency of PEMFs changed,the effects of PEMFs on accelerating hMSCs proliferation and osteogenesis differentiation would changed or not? Which frequency we should choose? The hMSCs in vitro are multitudinous, gap junctional intercellular communication(GJIC) play a important role in proliferation, differentiation and stabilization of environment. How can PEMFs cause the effects on GJIC of hMSCs? Theses questions are still unclear. This research observed the process of PEMFs' acting on hMSCs' multiplication and the competence of PEMF's induction on the hMSCs to be osteoblasts by different frequency in vitro. Method: The third generation human mesenchymal stem cells were irradiated by different frequency PEMF of 5,25,50,75,100,150Hz, 1.1mT, 30min/d, continue 21 days. The control group cultured with same environment but without PEMFs. 1. Morphological changes of the hMSCs were observed under phase contrast microscope. 2. The method of MTT was employed to evaluate the level of proliferation. and drawed the growth curves of the cells. 3. The parameter regarding the variation of the cell cycle were detected with the flow cytometry(FCM). 4. The activity of Alkaline phosphatase(ALP) was detected by enzyme test Kit. 5. The expressing of osteocalcin(OC) were detected by enzymology and radiate immunity methods to evaluate hMSCs differentiation. 6. The staining of chinalizarin were used to deteded the calcium nod. 7. The transmission electron microscopy(TEM) were used to reseach the structure of gap junction.of hMSCs. 8. The method of Fluorescence redistribution after photobleaching(FRAP) was used to evaluate the functions of GJIC through a laser scanning confocal mircroscope. Results: 1,The subcultured hMSCs after about 3 day's process by PEMFs changed their morphology. They aggregated into many calcification nodes something like a multi-storey structure after 3 weeks. 2,The growth curves of hMSCs irradiated by PEMFs antedisplacement compared with the controlled group.The 50Hz group was the most significant 3. The hMSCs cycle were changed irradiated by PEMFs , percentage of cells at the S phase was rised. In all groups , the 50Hz group was the most significant, which(28.89±0.25) was increased 107% compared with that of the control group(13.57±0.55). (P<0.05) 4,The activity of Alkaline phosphatase(ALP) were increased a lot with the time elongation, the concentration of ALP increased strikingly at the group irradiated by 50 Hz PEMFs and reached its peak on the 12th day, which much high than the controlled group(P<0.05). 5,The expressing of osteocalcin(OC) were little in the first week, and increased a lot in the third week. In all groups , the 50Hz group was the most significant, which(14.8±0.25ng/ml) was much more than that of the control group(0.99±0.13ng/ml) . (P<0.05) 6,Under the TEM we found that the cells were maturer comparatively, the ratio of N/P became lesser and the Endoplasmic reticulum a little dilatated. The structure of gap junction were observed between two hMSCs. 7,The mean fluorescence recovery rates of the hMSCs irradiated by PEMF(64.12±0.83)% was significantly higher than that of the control group cells(35.26±0.76)% (P<0.05). Results: Frequency was one of the most inportant factors on accelerating hMSCs proliferation and differentiation in vitro. Differnet frequency of PEMFs could cause the different effets. 50Hz maybe the suitable frequency. PEMFs can accelerate the function of GJIC of hMSCs .