The Explored Study For The Effect Of Constant Intensity Pulsed Electromagnetic Fields On Rat Bone Marrow Mesenchymal Stem Cells Proliferation And Osteogenic Differentiation

Objective:Pulsed Electromagnetic Fields (PEMFs) are used for treating the delay-union, non-union after bone fracture and osteoporosis as an noninvasive treatment clinically. Bone tissue engineering can repair the defect of bone caused by different reasons which reveals good application prospects clinically.The mesenchymal stem cells(MSCs)in bone marrow are believed to be one of the best choices for seeding cells of bone tissue engineering for their strong potential and high activity of ostengenesis.The problem is how can we harvest enough seeding cells with superior quality and induce them to differentiate into osteoblasts quickly? At an earlier stage our research has been verified that the high-frequency pulsed electromagnetic felds(HF-PEMFs) could inhibit the proliferation and differentiation of BMSCs in some extent.But if the frequency of PEMFs changed, how do the effects of PEMFs changed, accelerating BMSCs proliferation and osteogenesis differentiation or not? Which frequency we should choose in bone tissue engineering and clinic? The BMSCs in vitro are multitudinous, which signaling pathway play an important role in proliferation and osteogenesis differentiation? these questions are still unclear.This research observed the process of PEMFs’ acting on BMSCs’multiplication and the competence of PEMFs1induction on the BMSCs to be osteoblasts by1KHz frequency in vitro, aiming to provide the evidences for the mechanism of the PEMFs’intervention and the further research.Methods:Rat bone marrow mesenchymal stem cells were separated and cultured to the third generation while irradiated by frequency PEMF of1KHz,0.05mT,30min twice a day, continue15days.The control group cultured with same environment but without PEMFs.1.Morphological changes of the BMSCs were observed under phase contrast microscope.2.The method of MTT was used to evaluate the level of proliferation and draw the growth curves of the cells.3.Expression of CD29、CD31、CD44、CD45、CD105were detected in the control group. The proliferation rate and cell cycle of passage3cells were detected at day3、6、9、12with the flow cytometry method(FCM).4.The activity of alkaline phosphatase(ALP)was detected by enzyme test Kit at different time stages.5.The calcium nodules were stained by enzymology and immunity methods(Vonkossa) to evaluate BMSCs differentiation.6.Performing mRNAs microarray in BMSCs at day15of the stage of inducing osteogenesis differentiation. Screening the candidated mRNAs for regulating osteogenesis differentiation of BMSCs, employing bioinformatics to predict their trend and pathway, selecting the interested target genes and validating them by real time reverse transcriptase polymerase chain reaction.Results:1.The subcultured BMSCs after about3day’s process by PEMFs changed their morphology.They aggregated into many calcification nodes something like a multi-storey structure as the time passed.2.The growth curves of BMSCs irradiated by PEMFS showed antedisplacement compared with the controlled group.The day5group was the most significant3.CD29, CD44, CD105in the3rd passage non-stimulated BMSCs was positively expressed and the CD31, CD45was negatively expressed. The survival rate of passage3BMSCs and percentage of S phase following intervention of PEMFs were greater than those in the control group (P<0.05).4.The activity of alkaline phosphatase(ALP) was increased a lot with the time elongation, the concentration of ALP irradiated by PEMFs reached its peak on the day12, which was much higher than the control group(P<0.05).5.The calcium nodules were stained not obviously at the first week in all group.The stained calcium nodules in the PEMFs group increased significantly comparing with the control group from the second week.6.Our microarray results revealed that325mRNAs were significantly up-regulated which mainly involved in the bone mineralization and neuro differentiation, while636mRNAs were down-regulated which mainly involved in the T/B leukomonocyte differentiation. Interestingly, after using bioinformatics approaches, we selected four mRNAs BMP-2、Id2、ALP、Decorin correlated with the process of osteogenesis, validating them by qRT-PCR. Furthermore, the qRT-PCR results showed that the up-regulated (or down-regulated) expression of mRNAs were significantly(P<0.05). The PEMFs possibly promoted the proliferation and osteogenesis differentiation through TGF-Beta signaling pathway.Conclusion:Rat bone marrow mesenchymal stem cells were separated and cultured successfully. Frequency and irradiated time was the most important factors on accelerating BMSCs proliferation and differentiation in vitro.Different frequency of PEMFs could cause the different effets.1kHz maybe the suitable frequency for promoting the BMSCs’ proliferation and osteogenesis differentiation.PEMFs may paly its important role through TGF-Beta signaling pathway, in which the BMP-2and Decorin could accelerate the osteogenesis differentiation.But which genes are of promotion to the proliferation is still need a further research.