Effects And Mechanisms Of Electromagnetic Fields On Proliferation And Differentiation Of Bone Marrow Mesenchymal Stem Cells Modulated By Dexamethasone

Objective1. To investigate the time dependent manner of SEMF exposure on rat BMSC proliferation and osteogenic differentiation.2. To investigate the effect of SEMF on BMSC proliferation and osteogenic or adipogenic differentiation modulated by DEX.3. To explore the effect of MEK/ERK signaling pathway in SEMF exerting effects directly and modulated by DEXMethods1. Rat BMSCs were isolated and expanded, and cells of passage three were used. After exposing with15Hz,1mT SEMF for different time (1d,4d,7d,10d,14d), the proliferation was detected by CCK-8. The cells were treated with15Hz,1mT SEMF for different duration (1h/d,4h/d,8h/d), and the genes expression of RUNX2, BSP, OPN, the activity of ALP and the numbers of calcium nodule were detected after7days and14days treatment.2. The effect of different concentrations DEX treatment1d,3d,5d,7d,9d on BMSC proliferation was detected by CCK-8. After DEX of different concentrations treatment for different days (7d,14d), the genes and proteins expression of osteogenic indicator-RUNX2, ALP, OPN and adipogenic indicator-PPARy2, AP2, ADIPOQ were detected and the activity of ALP, the numbers of calcium nodule and fat vacuole were compared. After5days of treatment by15Hz,1mT SEMF and0.1μM DEX the proliferation of BMSC was investigated. The effect of SEMF on the osteogenic and adipogenic differentiation modulated by DEX was investigated after7days,14days and21days treatment. 3.15Hz1mT SEMF lh/d was used to treat BMSC, the proteins expression of ERK and p-ERK were detected and compared. The specific inhibitor of MEK/ERK signaling pathway-U0126was used, and after3days treatment, the proteins expression of ERK and p-ERK among control, U0126group, SEMF group and U0126+SEMF group were detected, after14days treatment, the proteins expression of osteogenic indicator-RUNX2, BSP, OPN were detected and compared. The effect of U0126, SEMF alone and together on BMSC proliferation was investigated after treating for different days. The effect of SEMF,0.1μM DEX alone and together on the expression of ERK and p-ERK was detected, and investigated the effect of U0126on the expression of RUNX2, ALP and PPARy2, AP2modulated by SEMF and DEX.Results1. The cultured rat BMSCs were elongated spindle-shaped or rhomboid, grew colonially and they had the potential of multi-lineage differentiation. SEMF exposure promoted the BMSC proliferation, and the obvious effect occurred at day7and day10. After treating with SEMF for different duration, the expression of RUNX2, BSP, OPN were boosted, the activity of ALP were promoted and the number of calcium nodule were increased. Moreover, after7days treatment, the obvious effect was seen at4h/d group, while after14days treatment, the effect of1h/d was more obvilously.2. Higher concentrations of DEX (0.1μM and1μM) inhibited proliferation of BMSCs, while lower concentrations (1nM and10nM) of DEX promoted it. In the early stage of differentiation, Higher concentrations DEX restrained expression of RUNX2and ALP, but amplified the expression of ALP and OPN in the late stage. DEX promoted expression of adipogenic-related genes and proteins, increasing the number of lipid droplets in a time and concentration dependent manner. SEMF treatment partially attenuated the inhibition effect of DEX on BMSC proliferation. SEMF treatment of BMSCs influenced by0.1μM DEX inhibited the high expression of adipogenic-related genes, decreased the number of lipid droplets; stimulated the expression of osteogenic-related genes; and increased the activity of ALP.3.15Hz1mT SEMF1h/d promoted the expression of p-ERK after1day and3days treatment, and the effect can be inhibited by U0126. Furthermore, U0126attenuated the effect of SEMF on promoting BMSC proliferation and osteogenic differentiation.15Hz1mT SEMF evoked the ERK phosphorylation after only30min treatment, while DEX inhibited it. And SEMF treatment1hour partly attenuated the suppression effect of DEX on ERK phosphorylation. Moreover, the effect of SEMF on the osteogenic and adipogenic differentiation of BMSC modulated by DEX was partially inhibited by U0126.Conclusion1. SEMF treatment promoted the proliferation of rat BMSCs and induced them osteogenic differentiation, and the effects were time dependent.2. The DEX concentration and time dependent affected the BMCS proliferation and osteogenic or adipogenic differentiation. And SEMF could also promote BMSC proliferation and induce osteogenic differentiation, inhibit adipogenic differentiation in DEX modulated condition.3. MEK/ERK signaling pathway played an important role in the effect of SEMF on boosting the BMSC proliferation and osteogenic differentiation directly and promoting BMSC osteogenic differentiation, inhibiting it adipogenic differentiation modulated by DEX.