Power-frequency electromagnetic field promotes mRNA expressions of bone morphogenetic protein-2 and transforming growth factor-beta 1 in bone marrow mesenchymal stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND:Studies confirm that electromagnetic field (EMF) can promote the synthesis and secretion of many bone growth factors,and some growth factors can induce the osteoblastic directional differentiation of bone marrow mesenchymal stem cells (MSCs).OBJECTIVE: To investigate the effect of power-frequency EMF on mRNA expressions of bone morphogenetic protein-2 (BMP-2) and transforming growth factor-beta 1 (TGF-β1) in mouse bone marrow MSCs cultured in vitro.DESTGN: Single sample, block design, observation and controlled animal trial.SETTING: Department of Traumatic Surgery, Tongji Hospital Affiliated to Tongji Medical .College, Huazhong University of Science and Technology.MATERIALS: This trial was carried out in the laboratory of Department of Traumatic Surgery, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology during February to December 2005. ①Twenty Kunming mice of clean grade were selected for harvest of bone marrow MSCs. ②Magnetic field generator,which could generate EMF with 0 to 100 mT field strength and successive adjustable 50 Hz sinusoidal wave, was developed by Wuhan Naval University of Engineering. ③ Primer was all synthesized by Saibaisheng Bioengineering Co.,Ltd., Beijing.NETHODS: ① The involved mice were sacrificed by cervical dislocation. Bilateral femora and tibia were harvested. Bone marrow MSCs were isolated and cultured in vitro, and the second generation of cells were used for the trial. ②Different intensities of EMF stimulation tests: Negative control group, positive control group, EMF 0.4, 0.8 and 1.6 mT stimulation groups were set. Five bottles of cells of the second generation were chosen from each group for test. The cells in the negative control group and positive control group were not exposed to EMF. But medium containing osteogenic inductor(10-8 mol/L dexamethasone, 10 mmol/L β-sodium glycerophosphate and 50 mg/L Vitamin C included) was added in the positive control group at passage. After adhering to the wall, the cells in the EMF 0.4,0.8 and 1.6 mT stimulation groups were exposed to EMF of 0.4,0.8 and 1.6 mT field strength, respectively, one hour per day, five days later, they were detected.③ EMF stimulation tests in the same field strength and different time: Negative control group, EMF 1.6 mT stimulation 15,30 and 60 minutes groups were set.Five bottles of cells of the second generation were chosen from each group for test. The cells in the negative control group were exposed to EMF. The cells in the EMF 1.6 mT stimulation 15,30 and 60 minutes groups were respectively given 15,30 and 60 minutes of EMF stimulation at 1.6 mT successively.Five days later, they were detected.④ The mRNA expressions of BMP-2 and TGF-β1 were detected in each group by reverse-transcriptase polymerase chain reaction (RT-PCR) technique.MAIN OUTCOME MEASURES: ① The effect of different field strength of exposure of 50 Hz EMF on mRNA expressions of BMP-2 and TGF-β1. ② The effect of the same field strength and different time of exposure of 50 Hz EMF on mRNA expressions of BMP-2 and TGF-β1.RESULTS: ①Five days after EMF stimulation, the mRNA expressions of BMP-2 and TGF-β1 in the positive control group and EMF 0.4,0.8 and 1.6 mT stimulation groups were significantly enhanced as compared with negative control group (all P ＜ 0.01), and the mRNA expression of BMP-2 in the EMF 1.6 mT stimulation group reached the peak [(57.74±0.23)％]and mRNA expression of TGF-β1 in the EMF 0.4 mT stimulation group also reached the peak [(126.20±0.21 )％].② Five days after EMF stimulation, the mRNA expressions of BMP-2 and TGF-β1 in the EMF 1.6 mT stimulation 15,30 and 60minutes groups were significantly enhanced (all P ＜ 0.01) as compared with negative control group, and the mRNA expressions of two factors in the EMF 1.6 mT stimulation 60 minutes group reached peak separately [(28.06±0.11 )％ and (75.20±0.16)％].CONCLUSION:Proper intensity and action time of exposure of power-frequency EMF can obviously promote the mRNA expressions of BMP-2 and TGF-β1 in mouse bone marrow MSCs cultured in vitro.