Electromagnetic Fields Act On Bone Marrow Mesenchymal Stem Cells To Promote Osteogenesis And The Possible Mechanism

Obiective:(1) To Culture bone marrow mesenchymal stem cells in vitro and identify the characteristic of bone marrow mesenchymal stem cells. (2) To study the effect of osteogenic differentiation of bone marrow mesenchymal stem cells promoted by electromagnetic fields through detecting osteogenesis markers expression and the formation of calcium nodules. (3) To study the effect of adipogenic differentiation of bone marrow mesenchymal stem cells effected by electromagnetic fields. (4) To study the signaling pathway of bone marrow mesenchymal stem cells activated by electromagnetic fields and investigate the relationship between the activated signaling pathway and osteogenesis promoted by the electromagnetic fields.Methods:Density gradient centrifugation and adherence screening methods were used to isolation of bone marrow mesenchymal stem cells from rats. Cells at the fourth to sixth passage were selected for all the experiments.(1) To determine the cells are bone marrow mesenchymal stem cells, multi-differentiation induction and flow cytometry were used. The flow cytometry were used to detect surface markers of bone marrow mesenchymal stem cells.(2) In order to study the effect of osteogenesis of bone marrow mesenchymal stem cells promoted by electromagnetic fields, ALP activity were assayed by PNPP method, ALP,RUNX2,BMP2,BSP and DLX5 mRNA expression were detected by RT-PCR and Realtime PCR, type I collagen expression were measured by Immunohistochemistry, the formation of calcium nodules were detected by Von Kossa staining.(3) In order to study the effect of adipose differentiation of bone marrow mesenchymal stem cells effected by electromagnetic fields, the adipose differentiation markers were detect by RT-PCR and Realtime PCR, the expression of PPARy were detected by western blot, the formation of fat droplets were detected by Oil O staining.(4) To detect the effect of MAPK,AKT and cAMP-PKA signaling pathway of bone marrow mesenchymal stem cells activated by electromagnetic fields and find the activated points with western blot and ELISA methods.(5) To study the effect of signaling pathway of bone marrow mesenchymal stem cells in osteogenesis promoted by electromagnetic fields, the agent and excitomotor of signaling pathways were added, And the relationship were discussed between osteogenesis and signaling pathways promoted by electromagnetic fields.Result:(1) The obtained cells were observed under inverted microscope, with the ship of spindle or polygonal and with good refraction. The cells grow rapidly, clustered and colony-like. With the osteogenic induction supplement medium for 21 days, a lot of calcium nodules formed; with the adipogenic supplement induction medium for 14 days, the large fat droplets formed. The flow cytometry analysis result show that the expression rate of CD90+, CD45- and CD29+, CD45- are over 95%. The expression rate of CD45+ is less than 5% indicate that less hematopoietic cell lines were contained and the uniformity of cells.(2) Three days of electromagnetic fields exposed significantly promote the expression of alkaline phosphatase activity, and promote the expression of collagen typeâ… , a variety of osteogenic markers such as ALP, BMP2, DLX5, BSP and RUNX2. In the role of osteogenic induction supplement medium for 15 days and 21 days, the numbers of calcium nodule were significantly higher compare electromagnetic fields group with the control group.(3) RT-PCR and real-time PCR results showed that the PPAR-y2, adipsin and AP2 mRNA expression were reduced under the exposure of electromagnetic fields, the protein expression of PPAR-y2 and the formation of fat drops were also reduced.(4) The cells were exposed in electromagnetic fields for 15min,30min,60min and 120min and then detect the activity of cAMP, PKA, ERK1/2, P38, JNK and AKT. The results show that electromagnetic fields increase the expression of cAMP and the activity of PKA, promote the expression of the phosphorylation ERK1/2 and P38, the phosphorylation JNK and AKT were not detected. Add the PKA inhibitor H-89 and ERK1/2 inhibitor PD98059, the effect of osteogenesis of bone marrow mesenchymal stem cells promoted by electromagnetic fields decreased.Conclusions:(1) Through gradient centrifugation and repeated adherent methods we can get high purity bone marrow mesenchymal stem cells.(2) Electromagnetic fields can promote differentiation to osteoblast of bone marrow mesenchymal stem cells.(3) Electromagnetic fields inhibit differentiation to adipocytes of bone marrow mesenchymal stem cells.(4) Electromagnetic field activated cAMP-PKA and MAPK signals of bone marrow mesenchymal stem cells, and the signaling pathway promoted by electromagnetic fields were associated with the osteogenesis effected by electromagnetic fields.