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. 2009 Sep;27(9):1169-74.
doi: 10.1002/jor.20862.

Modulation of osteogenesis in human mesenchymal stem cells by specific pulsed electromagnetic field stimulation

Ming-Tzu Tsai  1 , Wan-Ju Li, Rocky S Tuan, Walter H Chang
Affiliations

Modulation of osteogenesis in human mesenchymal stem cells by specific pulsed electromagnetic field stimulation

Ming-Tzu Tsai et al. J Orthop Res. 2009 Sep.

Abstract

Human mesenchymal stem cells (hMSCs) are a promising candidate cell type for regenerative medicine and tissue engineering applications by virtue of their capacity for self-renewal and multipotent differentiation. Our intent was to characterize the effect of pulsed electromagnetic fields (PEMFs) on the proliferation and osteogenic differentiation of hMSCs in vitro. hMSCs isolated from the bone marrow of adult patients were cultured with osteogenic medium for up to 28 days and exposed to daily PEMF stimulation with single, narrow 300 micros quasi-rectangular pulses with a repetition rate of 7.5 Hz. Relatively greater cell numbers were observed at late stages of osteogenic culture with PEMF exposure. The production of alkaline phosphatase (ALP), an early marker of osteogenesis, was significantly enhanced at day 7 with PEMF treatment in both basal and osteogenic cultures as compared to untreated controls. Furthermore, the expressions of other early osteogenic genes, including Runx2/Cbfa1 and ALP, were also partially modulated by PEMF exposure, indicating that osteogenesis in hMSCs was associated with the specific PEMF stimulation. Based on ALP and alizarin red S staining, the accumulation of ALP protein produced by the hMSCs as well as calcium deposits reached their highest levels at day 28. Our results indicate that extremely low-frequency PEMF stimulation may play a modulating role in hMSC osteogenesis. Taken together, these findings provide insights on the development of PEMF as an effective technology for regenerative medicine.

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Figures

Figure 1
Figure 1
The PEMF stimulation system: (A) solenoid coils with porous plastic board placed horizontally in the center; (B) control units for regulating and monitoring the signals derived from solenoids placed inside an incubator; (C) induced electric field with single quasi-rectangular pulse; (D) a search coil for detecting induced electric fields; and (E) PEMF stimulator and solenoid coil.
Figure 2
Figure 2
DNA quantitation of hMSCs cultured in basal medium without the addition of osteogenic supplement for 7 days. hMSCs were cultured at seeding densities of 1,500 and 3,000 cells/cm2. The PEMF-treated cells were exposed for 2 h per day. Data are from two independent experiments, with two 4-well chamber slides per group in each (N=16 total) *, p<0.05.
Figure 3
Figure 3
Proliferation and ALP activity of hMSCs at seeding densities of 1,500 and 3,000 cells/cm2 during 14 days of osteogenic culture. (A) DNA amount and (C) ALP activity in low-density hMSCs cultures; and (B) DNA amount and (D) ALP activity in high-density hMSCs cultures. Data are from two independent experiments, with two 4-well chamber slides per group in each experiment (N=16 total). *, p<0.05; **, p < 0.01.
Figure 4
Figure 4
Early osteoblastic gene expression of Runx2/Cbfa1, ALP, and collagen type I during hMSC osteogenesis. (A) Electrophoretic RT-PCR analysis of RNA samples extracted at day 3, 7, and 10 (C, control; P, PEMF-treated). (B) Densitometric quantification of RT-PCR results. Data presented are from two independent experiments, with triplicate samples (6-cm Petri dish) per experiment (N=6 total). Electrophoresis was carried out twice for each sample for gel image analysis. *, p < 0.05.
Figure 5
Figure 5
Osteogenic differentiation and matrix mineralization of hMSCs under PEMF exposure during 28 days of osteogenic culture. Cells were stained for ALP and with alizarin red S at different time points for osteogenic differentiation and matrix mineralization, respectively. ALP activity increased in a time-dependent manner, reached the highest level at day 28 in both groups (e and f; E and F). Matrix mineralization accumulated over time, reaching its highest level at day 28 (g and h; G and H). Magnification, 50×.
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