Influx changes of calcium ion during the proliferation and differentiation of bone marrow mesenchymal stem cells stimulated by electromagnetic fields

Guohua YANG; Hua WU; Dongming ZHAO; Weijun LIU

ABSTRACT

BACKGROUND:

It has been proved that electromagnetic field can adjust and control proliferation and differentiation of bone marrow mesenchymal stem cells v/a cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signal transduction system. However, there are few relevant reports about Ca2+ as the second messenger in application.

OBJECTIVE:

To study the effects of verapamil on the proliferation and differentiation of bone marrow rnesenchymal stem cells stimulated by electromagnetic fields and to conclude influx changes of Ca2+.DESIGN, TIME AND

SETTING:

Electrostimulative cytological observation in vitro, which was performed in Laboratory of Orthopedic Surgery, Tongji Hospital between April and June 2005.MATERIALS Six 4-5-week SD rats of clean grade were selected in this study. Verapami| was provided by Sigma Company, USA, and Helmholtz coil-magnetic field producer was made in Department of Electric Machine, Navy Engineering University.

METHODS:

The bone marrow mesenchymal stem cells were isolated and cultured in vitro with adherence method and digested with trypsin. The fourth-passage cells were harvested, adjusted to 1 × 107 L-1 in density, and divided into A, B, C and D groups in 96-well plate with 200 μ I/well. Cells in the normal control group were not performed with any agent. On the second day of inoculation, cells in the magnetic field (EMF) group were cultured in Helmholtz-coil magnetic field (0.8 mT, 50 Hz) in 0.05% CO2 saturated humidity incubator at 37 ℃, 30 minutes for each, 12 hours for interval, six time in total. Cells in the verapamil group were cultured with 20 μ mol/L verapamil, and cells in the combination group were cultured with 20 μ mol/L verapamil and magnetic stimulation.MAIN OUTCOME

MEASURES:

Proliferative activity was tested with MTT method, content of alkaline phosphate differentiated to osteoblasts was measured, and cells were stained with modified Gomori Ca-Co staining.

RESULTS:

Proliferative activity was significantly increased in the EMF group as compared with that in the normal control group after 3-day magnetic stimulation (P < 0.01), but verapamil could inhibit promotive effect on proliferation. Content of alkaline phosphate in the normal control group was similar to that in the EMF group, while those two contents were significantly higher than those in the verapamil group and the combination group (P < 0.01); furthermore, content of alkaline phosphate in the combination group was significant higher than that in the EMF group (P < 0.01). Qualitative analysis of alkaline phosphate showed a coincident result as mentioned above.

CONCLUSION:

EMF of 50 Hz frequency and 0.8 mT intensity can change intracellular free calcium ion concentration of bone marrow mesenchymal stem cells, and the change play a key role in the cellular proliferation and play a partial role in the differentiation of bone marrow mesenchymal stem cells into osteoblasta.