Effect of Pulsed Electromagnetic Field on the
Proliferation and Differentiation Potential of
Human Bone Marrow Mesenchymal Stem Cells
Li-Yi Sun,1,2Dean-Kuo Hsieh,3Tzai-Chiu Yu,4Hsien-Tai Chiu,2Sheng-Fen Lu,1Geng-Hong Luo,1
Tom K. Kuo,5Oscar K. Lee,5,6** and Tzyy-Wen Chiou1*
1
Department of Life Science and Graduate Institute of Biotechnology, National Dong Hwa University, Hualien,Taiwan, Republic of China
2
Department of Biological Science and Technology, National Chiao Tung University, Hsinchu,Taiwan, Republic of China
3
Department of Applied Chemistry, Chaoyang University of Technology,Taichung, Taiwan, Republic of China
4
Department of Orthopaedics, Buddhist Tzu Chi General Hospital, Hualien, Taiwan, Republic of China
5
Department of Orthopaedics and Traumatology,Taipei Veterans General Hospital,Taipei, Taiwan, Republic of China
6
Institute of Clinical Medicine, National Yang-Ming University,Taipei, Taiwan, Republic of China
Pulsed electromagnetic fields (PEMFs) have been used clinically to slow down osteoporosis and accelerate the healing of bone fractures for many years. The aim of this study is to investigate the effect of PEMFs on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells (BMMSC). PEMF stimulus was administered to BMMSCs for 8 h per day during culture period. The PEMF applied consisted of 4.5 ms bursts repeating at 15 Hz, and each burst contained 20 pulses. Results showed that about 59% and 40% more viable BMMSC cells were obtained in the PEMF-exposed cultures at 24 h after plating for the seeding density of 1000 and 3000 cells/cm2, respectively. Although, based on the kinetic analysis, the growth rates of BMMSC during the exponential growth phase were not significantly affected, 20–60% higher cell densities were achieved during the exponentially expanding stage. Many newly divided cells appeared from 12 to 16 h after the PEMF treatment as revealed by the cell cycle analysis. These results suggest that PEMF exposure could enhance the BMMSC cell proliferation during the exponential phase and it possibly resulted from the shortening of the lag phase. In addition, according to the cytochemical and immunofluorescence analysis performed, the PEMF-exposed BMMSC showed multi-lineage differentiation potential similar to the control group. Bioelectromagnetics 30:251–260, 2009.
2009 Wiley-Liss, Inc.
Key words: pulsed electromagnetic fields; human bone marrow; mesenchymal stem cells; growth kinetics; cell cycle; differentiation
INTRODUCTION
Pulsed electromagnetic fields (PEMFs) have been applied clinically to promote bone healing for many years [Heckman et al., 1981]. The clinical effectiveness was initially thought to be due to the accelerated formation of the bone matrix by the weak electric current generated by the magnetic field [Friedenberg and Brighton, 1966]. Studies have indi-cated that electromagnetic fields may heal bone fractures and slow down bone matrix loss in animal experiments [de Haas et al., 1980; Brighton et al., 1985a; McLeod and Rubin, 1992; Grace et al., 1998].
Despite the clinical success, electromagnetic fields have been reported to have negative effects on
2009 Wiley-Liss, Inc.
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*Correspondence to: Tzyy-Wen Chiou, Department of Life Science and Graduate Institute of Biotechnology, National Dong Hwa University, Hualien, No. 1, Sec. 2, Da Hsueh Rd., Shou-Feng, Hualien, Taiwan, Republic of China.
E-mail: [email protected]
**Correspondence to: Oscar K. Lee, Institute of Clinical Medicine, National Yang-Ming University, Veterans General Hospital-Taipei, No. 201, Sec 2, Shi-Pai Road, Taipei 11221, Taiwan, Republic of China. E-mail: [email protected] Received for review 29 December 2007; Final revision received 14 September 2008
DOI 10.1002/bem.20472
Published online 9 February 2009 in Wiley InterScience (www.interscience.wiley.com).
the proliferation and differentiation of bone cells [Norton, 1982; Norton et al., 1988]. Under specific electromagnetic fields, it has been observed that the proliferation of osteoblasts is limited and that alkaline phosphatase (ALP) activity is elevated [McLeod et al., 1993]. Recent research on the MG-63, ROS 17/2.8, and MLO-Y4 cell lines has shown that PEMF treatment either causes no effect or has a negative effect on cell proliferation [Sollazzo et al., 1997; Lohmann et al., 2000, 2003]. Furthermore, it was hard to explain the finding that PEMF stimulation significantly increased the proliferation of mouse osteoblasts, while the ALP staining and the mineralization remained the same [Chang et al., 2004b]. In another study, it was shown that treatment with PEMF alone or together with bone morphogenetic protein 2 (BMP-2) increased the cell proliferation and the differentiation of rat primary osteoblastic cells [Selvamurugan et al., 2007].
Current studies on stem cells have revealed that the regeneration of human body tissues and the supplement of mature functioning cells are due to the proliferation and differentiation of stem cells [Korbling and Estrov, 2003]. It was suggested that the proli-feration and differentiation of human bone marrow mesenchymal stem cells (BMMSC) may lead to the replenishment of bone cells [Beresford, 1989; Pittenger et al., 1999; Halleux et al., 2001]. The mechanism of action underlying how PEMF promotes the formation of bone in an in vitro environment remains elusive. Therefore, we aim to investigate the influence of PEMF on the proliferation and differentiation potential of human BMMSC.
MATERIALS ANDMETHODS
Antibodies
Antibodies against human CD105 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against human CD13, CD14, CD29, CD34, CD44, CD45, HLA-ABC, and tau were purchased from Dako (Carpinteria, CA). Antibodies against human antigens CD73, CD90, HLA-DR, and microtubule-associated protein-2 (MAP2) were purchased from Becton Dickinson (Franklin Lakes, NJ). Antibodies against human antigens SH-2, SH-3, and SH-4 were purified from SH-2, SH-3, and SH-4 hybridoma cell lines (American Type Culture Collection, Rockville, MD). Secondary goat mouse or rabbit anti-bodies were from Chemicon (Temecula, CA).
BMMSC Isolation and Culture
The steps used for isolating human BMMSCs in this study were previously reported [Lee et al., 2004].
To maintain and expand the BMMSCs, the cells were cultured in expansion medium. Expansion medium consisted of Iscove’s modified Dulbecco’s medium (IMDM, Gibco, Grand Island, NY) and 10% fetal bovine serum (FBS, Hyclone, Logan, UT) supple-mented with 10 ng/ml basic fibroblast growth factor (FGF-2, R&D Systems, Minneapolis, MN), 100 units penicillin, 1000 units streptomycin, and 2 mM
L-glutamine. Once the cells reached 50–60%
con-fluence, they were washed twice with phosphate-buffered saline (PBS), detached by 0.25% trypsin-EDTA (Gibco BRL) and then replated at a ratio of 1:4 under the same culture conditions. BMMSCs at passages 10–15 were used for the experiments in this study.
PEMF Exposure
The PEMF device (EBI, L. P., Parsippany, NJ) was
placed in a CO2 incubator (5% CO2, 37 8C). For the
PEMF treatment experiments, the BMMSCs were seeded at different densities in tissue culture flasks or six-well plates using expansion medium. Cultures were placed in the PEMF device (as shown in Fig. 1) and were exposed to PEMF 8 h per day during culture period. The applied field consisted of 4.5 ms bursts of 20 pulses repeating at 15 Hz [Bassett et al., 1974]. During each pulse, the magnetic field increased from 0 to 1.8 mT in 200 ms and then decayed back to 0 in 25 ms. Other signal parameters used in this study were described in detail by Guerkov et al. [2001] and
Fig. 1. Schematic representation of the pulsed electromagnetic fields device.The pulsed electromagnetic field device provided by EBI, L. P. was placed in a 5% CO2and 378C incubator. BMMSCs seeded in tissue culture flasks or six-well plates were placed on the base of the device for PEMF stimulation 8 h per day.
Lohmann et al. [2000, 2003]. The control experiments were performed on BMMSCs seeded in tissue culture
flasks or six-well plates in a separate CO2incubator that
had not been equipped with the PEMF device. The magnetic field inside the incubators did not differ from that present in the normal terrestrial field. The incubators were alternated for the treatment and control experiments.
Cell Growth Kinetics Analysis
As mentioned above, six-well plates seeded with BMMSCs in expansion medium with two different
seeding cell densities (1000 and 3000 cells/cm2) were
placed in a CO2incubator in the presence or absence of
PEMF stimulation. To follow the growth kinetics, cells were detached by trypsin-EDTA and a cell scraper (Becton Dickinson) and then counted by a hemocy-tometer under a microscope. The growth rate, which is the reciprocal of generation time, is defined as the number of cell doublings per unit of time during the specified time interval. The calculation of growth rate is
as follows, m¼ (3.32[log10Nt log10N0])/t, where m,
Nt, and N0 are the growth rates, final and initial cell
densities, respectively. In this study, the calculated maximal growth rate represents the growth rate of the exponential phase during the culture process. These experiments were performed in triplicate and repeated in batches. Statistical analyses were performed using Student’s t-test.
Cell Cycle Analysis
For cell cycle analysis, 75 cm2 tissue culture
flasks (Becton Dickinson) seeded with BMMSCs in expansion medium with an initial density of 1000 cells/
cm2were placed in a CO2incubator in the presence of
PEMF stimulation as previously described. Every 6 h,
flasks (n¼ 3) were taken out of the incubator to detach
and harvest the cells for cell cycle analysis. The harvested cells were washed in PBS and then fixed in cold 70% ethanol for 30 min at 4 8C. Fixed cells were washed twice in PBS, and spun at 2000 rpm. The resulting cell pellet was treated with 50 ml Ribonuclease A (10 mg/ml, Sigma, St. Louis, MO) and 1 ml propidium iodide staining solution (50 mg/ml, Sigma) for 3 h at 4 8C. Samples were stored at 4 8C until analyzed by flow cytometry (Becton Dickinson). In the control experiments, the BMMSCs cultured under the same conditions but without PEMF exposure were used. Statistical analyses were performed using Student’s t-test.
Cell Surface Antigen Analysis
For the characterization of cell surface antigen
phenotype, the BMMSCs were cultured in 75 cm2tissue
culture flasks at an initial density of 1000 cells/cm2with
expansion medium under regular PEMF treatment (i.e.,
8 h a day) in a CO2 incubator. After 3 or 8 days,
cells were detached by trypsinization, stained with fluorescein isothiocyanate (FITC) or R-phycoerythrin (RPE) coupled antibodies, and then analyzed by flow cytometry (FACSCalibur, Becton Dickinson). In the control experiments, the BMMSCs were cultured under the same conditions but without PEMF treatment. In Vitro Differentiation, Cytochemical and Immunofluorescence Analysis
BMMSCs at an initial density of 1000 cells/cm2
were cultured under regular PEMF (8 h exposure each day) for 3 days and then redistributed into either
35 mm dishes or 25 cm2flasks at specified densities for
the following in vitro differentiation experiments. BMMSCs cultured without PEMF were used in the control experiments. The following in vitro differ-entiations were performed with no PEMF exposure.
Osteogenic differentiation. BMMSCs after 3 days’
PEMF treatment were seeded at a density of 5000 cells/
cm2 to study osteogenic differentiation. Cells were
treated with osteogenic medium for 3 weeks; the medium was changed twice a week. Osteogenic medium consisted of high-glucose DMEM (Gibco BRL) and 10% FBS supplemented with 0.1 mM dexamethasone (Sigma), 10 mM b-glycerol phosphate (Sigma), 0.2 mM ascorbic acid (Sigma), 100 U
penicillin, 1000 U streptomycin, and 2 mM
L-glutamine. At weekly intervals, osteogenesis was
evaluated by von Kossa staining and the activity of ALP.
von Kossa staining. The mineralized matrix of cells
was assessed using 5% silver nitrate (Sigma) under ultra-violet light for 60 min, followed by 3% sodium thiosulphate (Sigma) for 5 min, and then counterstained with van Gieson (Sigma) for 5 min. The mineral was stained to black and osteoid was stained to red by this method.
Alkaline phosphatase staining. The ALP activity of
cells was evaluated by an alkaline phosphatase histochemistry kit (Sigma #85). The reaction was performed for 60 min at 25 8C as recommended by the manufacturer. During incubation, culture dishes were protected from drying and light. Dishes were rinsed with deionized water, and air-dried prior to viewing.
Adipogenic differentiation. After 3 days’ PEMF
treatment, BMMSCs were seeded at a density of
Cells were treated with adipogenic medium for 3 weeks. Adipogenic medium consisted of high-glucose DMEM supplemented with 0.5 mM 3-isobutyl-1-methylxan-thine (IBMX, Sigma), 10 mg/ml insulin (Sigma), 1 mM dexamethasone, 0.1 mM indomethacin (Sigma), 100 U
penicillin, 1000 U streptomycin, 2 mM L-glutamine,
and 10% FBS. Medium changes were carried out twice a week and adipogenesis was assessed by oil red O staining at weekly intervals.
Oil red O staining. Adipogenic differentiation was
demonstrated by the accumulation of lipid vesicles. Cells were fixed with 10% formalin, stained with oil red O (Sigma) for 10 min, and then counterstained with Mayer’s hematoxylin (Sigma) for 1 min. The lipid vesicles were stained to red by this method.
Neurogenic differentiation. To induce neurogenic
differentiation, a multi-step protocol was used. At a
density of 10000 cells/cm2, BMMSCs were seeded in
35 mm tissue culture dishes coated with 10 ng/ml fibronectin (Sigma). The cells were cultured with Step1 medium for 3 days, Step2 medium for 3 days, followed by Step3 medium for 1 day. Step1 medium consisted of IMDM, 20 ng/ml FGF-2, 20 ng/ml EGF (R&D), 10 ng/ ml PDGF-BB (R&D), 0.5 mM retinoic acid (Sigma), 1 mM b-mercaptoethanol (Sigma), 100 U penicillin,
1000 U streptomycin, 2 mM L-glutamine, and ITSþ
(Gibco BRL). Step2 medium consisted of IMDM supplemented with 20 ng/ml FGF-2, 20 ng/ml EGF, 10 ng/ml PDGF-BB, 0.5 mM RA, 100 U penicillin, 1000
U streptomycin, 2 mML-glutamine, and ITSþ. Step3
medium comprised NEUROBASAL Medium (Gibco BRL) supplemented with 10 ng/ml BDGF(R&D), 1 mM cAMP (Sigma), 0.5 mM IBMX, 200 mM BHA (Sigma), 100 mM ascorbic acid, N2 supplement (Gibco BRL), 100 U penicillin, 1000 U streptomycin,
and 2 mML-glutamine. Neurogenesis was assessed by
immunofluorescence on day 7.
Immunofluorescence. For staining of intracellular
proteins, cells on culture dishes were fixed for 10 min with 10% formalin at 25 8C, and permeabilized with 0.1% Triton X-100 (Sigma) for 5 min on ice. They were incubated with mouse primary antibodies against human MAP-2 (1:1000), and human tau (1:1000) for 1 h, followed by fluorescein-coupled goat anti-mouse or goat anti-rabbit IgG secondary antibody for 1 h. Between incubations, dishes were washed with PBS-T (0.1% tween-20, Sigma). Undifferentiated BMMSC cells were used as the negative control of immuno-fluorescence.
RESULTS
The Effect of PEMF Exposure on Growth Kinetics and Cell Cycle of BMMSCs
The growth kinetics of BMMSCs in the control and PEMF treatment experiments were monitored at two different cell seeding densities (1000 and
3000 cells/cm2) as listed in Table 1. As examples, the
semi-log plots of the growth curves for one batch of experimental results for each specific seeding density are shown in Figure 2A,B. The ratios of cell density of PEMF-exposed to non-exposed control during the first 4 days of the cell culture batch experiments for each seeding density are shown in Figure 3.
As indicated in Figure 2 and Table 1, the cell density of 24 h after plating showed significant difference between the control and PEMF groups. In
the case of seeding density of 1000 cells/cm2, the cell
density increased from 1.00 103 cells/cm2to 1.18–
1.23 103in the control groups at day 1 after plating;
however, it increased to 1.75–1.84 103cell/cm2in the
PEMF-treated groups at the same time, that is, about 59% more viable cells were obtained in the PEMF exposed culture at 24 h after plating (shown in Fig. 3A). Likewise, about 40% more viable cells were obtained in the PEMF-treated groups at 24 h after plating in the case
of seeding density of 3000 cells/cm2(shown in Fig. 3B).
It seems that the proliferation of the PEMF-treated BMMSC took off earlier than the untreated control group.
Moreover, as shown in Figure 2, the slopes during the exponential phase for the two seeding densities of the PEMF-treatment group and the non-treatment control group were quite similar. The calculated maximal growth rates in the exponential phase (as
listed in Table 1) ranged from 5.59 to 5.88 102h1
and 5.39 to 5.81 102h1for the batches at seeding
density 1000 and 3000 cells/cm2, respectively. No
significant differences between the PEMF and control groups were shown among the batches for either seeding density. Although the growth rates in the exponential phase were comparable between the treat-ment and control groups, 20–60% higher cell densities were achieved in the PEMF group during the exponen-tially cell expanding stage, as shown in Figure 3. It is therefore suggested that the enhancement of cell proliferation by PEMF exposure might result from the shortening of lag phase.
In order to further investigate the effect of PEMF on cell growth, the cell cycle of propidium iodide stained BMMSCs in the control and experimental groups was analyzed by flow cytometry (Fig. 4A). Using total cell numbers as the basis, the proportion of the cells at various cell cycle stages was examined. As
shown in Figure 4B, it is apparent that at the 6th hour (during the first PEMF exposure) and 12th hour (4 h after the first PEMF treatment), PEMF treatment led to a higher percentage of cells in the G2/M phase (repre-senting DNA synthesis stage) by a difference of 3–4%. At the 18th hour (10 h after first PEMF treatment) and 24th hour (16 h after the first PEMF treatment), the percentage of cells at both the G2/M phase and S (DNA-synthesis) phase decreased by 8–12% and 3–4%, respectively; however, the proportion of cells at the G0/ G1 phase (representing the newly divided cells) increased by 13–16%. At the 48th hour (16 h after the second PEMF exposure), the differences between the control and PEMF experiments were still signifi-cant, but the variations at this time were not as considerable as those in the 24th hour. These results imply that the first PEMF exposure affected the cell cycle and resulted in more newly divided cells than the control. The results support the findings obtained from the growth kinetics studies.
The Effect of PEMF Exposure on the Morphology and Immunophenotype of BMMSCs
As shown in Figure 5A, after human FGF-2 had been added to medium, the BMMSCs exhibited spindle-shape morphology [Martin et al., 1997; Halleux et al., 2001; Sottile et al., 2002; Bianchi et al., 2003]. No significant change in the morphology of the BMMSCs was observed after 3 days of PEMF treatment.
Analysis of the immunophenotype by flow cytometry showed that PEMF treatment for 3 or 8 days did not lead to significant changes in the surface phenotype of typical mesenchymal stem cells (Table 2). Effect of PEMF Exposure on In Vitro
Differentiation of BMMSCs
In order to determine whether the BMMSCs still have differentiation potential after PEMF treatment, in vitro osteogenic, adipogenic, and neurogenic dif-ferentiation experiments were conducted for both PEMF-treated and non-treated BMMSCs.
For osteogenic differentiation, BMMSCs at a
density of 5000 cells/cm2 were induced under
osteo-genic conditions. The mineralized matrix formed by the osteoblasts was confirmed by von Kossa staining (Fig. 5B), and positive staining for ALP (Fig. 5C). Both the PEMF-treated and non-PEMF treated BMMSCs possessed the potential for osteogenic differentiation.
To evaluate the adipogenic potential, BMMSCs with or without PEMF treatment at a density of
T ABLE 1. The Effect of PEMF Exposur e o n the Gr owth Kinetics of BMMSCs at T w o Differ ent Seeding Densities Cell density (10 3 cells/cm 2 ) Day 1 Day 2 Day 3 Day 4 Maximal gro wth rate (10 2 h 1 ) Batch number Control PEMF Control PEMF Control PEMF Control PEMF Control PEMF Seeding density 1000 cell/cm 2 1 1.23 0.09 1.79 0.22 { 3.19 0.31 4.53 0.47 { 6.85 0.20 10.29 0.77 { 17.03 3.15 23.80 2.48* 5.73 0.14 5.58 0.11 2 1.20 0.14 1.75 0.07* 3.15 0.15 4.40 0.28* 6.80 0.14 10.25 0.78* 14.30 0.14 21.70 0.42 { 5.80 0.85 5.54 0.63 3 1.18 0.13 1.84 0.23* 2.99 0.14 4.52 0.18 { 7.25 0.49 10.09 0.57 { 14.75 0.50 20.43 1.38 { 5.59 0.42 5.39 0.12 4 1.21 0.14 1.80 0.12* 3.22 0.31 4.67 0.47 { 7.00 0.47 9.56 0.52* 15.18 0.69 21.88 1.81* 5.88 0.42 5.73 0.24 Seeding density 3000 cell/cm 2 1 3.22 0.62 4.34 0.32* 5.11 0.16 7.34 0.32 { 10.65 0.64 14.80 0.71* 23.78 5.34 31.99 1.73* 5.71 0.80 5.81 0.02 2 3.49 0.39 4.57 0.16* 5.75 0.40 7.55 0.56* 10.88 0.50 13.64 0.32 { 27.89 3.09 34.12 2.65* 5.66 0.91 5.51 0.23 3 3.18 0.56 4.54 0.39* 5.55 0.62 8.00 0.11* 10.33 0.32 13.33 0.42* 26.28 0.92 34.15 0.64 { 5.61 0.31 5.65 0.63 * P < 0.05. { P < 0.01. { P < 0.005.
5000 cells/cm2were induced with adipogenic medium. After 3 days of induction, cells of both groups revealed adipogensis based on the apparent changes in cell morphology and the visible accumulation of neutral lipid vacuoles. At day 14, more than 80% of the cells showed neutral lipid vacuoles in the cells as confirmed by oil red O staining (Fig. 5D).
Using the multi-step neurogenic methods des-cribed earlier, both the PEMF-treated and non-treated BMMSCs showed neurite-like structures and convex-shaped cell bodies, indicating that they have the potential to differentiate into neuron-like cells (Fig. 4E). Immuno-fluoresence staining on the differentiated cells indicated the expression of neuron-related proteins, such as MAP2 (Fig. 5F) and tau (Fig. 5G).
DISCUSSION
PEMF therapy has been used successfully to cure a wide range of bone diseases for many years, including bone fractures [Grace et al., 1998] and osteoporosis [Brighton et al., 1985b,c; McLeod and Rubin, 1992]. However, the mechanism by which PEMF stimulation induces cellular proliferation and bone regeneration remains unclear. PEMF stimulation affects not only proliferation of osteoblasts, but also apoptosis of osteoclasts [Luben, 1991; McLeod et al., 1993; Chang et al., 2004a, 2006]. In addition, different characteristics of PEMF signals can reduce or enhance osteoclastogenesis of bone marrow cells [Chang et al., 2004a, 2005].
Fig. 2. The effect of PEMF exposure on the proliferation of BMMSCs at different seeding densities. A: Semi-log chart of cell growth at the seeding density of 1000 cells/cm2. B: Semi-log chart of cell growthat the seedingdensityof 3000 cells/cm2.The celldensitiesinthe PEMF-exposedgroupswere significantly higher than those in the control group, especially during the exponential phase of cell culture experiments for both seeding densities tested. For both seeding densities, the maximal growth rates in the exponential phase were not significantly affected by PEMF treatment, as shown by the slope of the kinetic curves in the semi-log plots. The data points represent the mean cell number standard deviation (n ¼ 3). *P < 0.05.{P< 0.01.{P< 0.005.
Fig. 3. Ratios of cell density of PEMF-exposed to non-exposed control during the first 4 days of the culture process. A: Batch results at seeding density of 1000 cells/cm2. B: Batch results at seeding density of 3000 cells/cm2.The results represent the mean cell number standard deviation (n ¼ 3). *P< 0.05.{P< 0.01.{P< 0.005.
However, bone regeneration requires the proli-feration and differentiation of new osteoblasts from stem cells. The human bone marrow stromal system is believed to be the reservoir of stem cells [Civin et al., 1984; Owen and Friedenstein, 1988]. In particular, the BMMSC can repair a number of damaged tissues by the stimulation of mobilizing signals such as hypoxia, platelet-derived growth factor-AB (PDGF-AB) and insulin-like growth factor 1 (IGF-1) [Rochefort et al., 2006; Ponte et al., 2007]. Research has demonstrated the in vivo osteogenic potential [Goshima et al., 1991; Gundle et al., 1995; Quarto et al., 1995] and in vitro osteogenic differentiation of BMMSC [Pittenger et al., 1999].
There are quite a few studies analyzing the effects of PEMF stimulation on cell lines and on non-human osteoblasts in vitro [Lohmann et al., 2000, 2003; Diniz et al., 2002; Sakai et al., 2006; Selvamurugan et al.,
2007]. Diniz et al. [2002] showed that PEMF treatment of osteoblasts in the early stages accelerated cellular proliferation, enhanced cellular differentiation, and increased bone tissue-like formation. Chang et al. [2004b] reported that PEMF stimulation significantly increased the proliferation of osteoblasts, but had no effect on the synthesis of extracellular matrix. In this study, we investigated the effect of PEMF on expanding BMMSCs in medium with human FGF-2. In the medium containing human FGF-2, the PEMF treatment led to the early take-off of cell proliferation and consequently resulted in higher cell densities during the exponentially growing phase (Fig. 2A,B). The enhancement of cell proliferation is shown in Figure 3. The achieved cell densities in PEMF group were 20– 60% higher than those in the control group during the exponential growth phase.
Fig. 4. The cell cycle of BMMSCs with or without PEMF exposure seeded at an initial density of1000 cells/cm2. A: The cell cycle of BMMSCs analyzed by propidium iodide staining and flow cytometry from 0 to 48 h. B: The percentages of cells in each phase of the cell cycle. At the 6th and12th hours, PEMF treatment resulted in a 3 ^ 4% increase in the proportion of cells in the G2/M phase. At the 18th and 24th hours, the percentage of cells in the G2/M phase and S phase decreased by 8 ^12% and 3 ^ 4%, respectively; however, the proportion of cells in the G0/G1phase increased by 13 ^16%.Theresultsrepresent the mean percentage of cells standarddeviation (n ¼ 3). *P < 0.05.
Minguell et al. [2001] and Conget and Minguell [1999] reported that while a small number of BMMSCs were actively engaged in proliferation (approximately
10% at Sþ G2 þ M), the vast majority of cells were in
the quiescent state (G0). However, in the study of Nicolin et al. [2007], flow cytometric analysis revealed that PEMF treatment of chondrocytes led to
signifi-cantly increased cell numbers in the Sþ G2 þ M cell
cycle phase. Our findings also showed that PEMF stimulation led to a higher percentage of BMMSCs in the G2/M phase between the 6th and 12th hours, and a higher percentage of BMMSCs in the G0/G1 phase
between the 18th and 24th hours in comparison with the control group (Fig. 4B). The results manifested that the first PEMF exposure significantly affected the cell cycle and resulted in more newly divided cells.
The change of cell cycle progression of BMMSCs under PEMF treatment might be attributed to the alteration of cell membrane potential resulting from PEMF exposure. Previously, Panagopoulos et al. [2002] suggested that the possible mechanism of electro-magnetic fields on cells is the forced-vibration of all the free ions on the surface of a cell’s plasma membrane. Recently, Garner et al. [2007] reported that the
Fig. 5. PEMFhadno effect onthemorphologyordifferentiationpotentialof BMMSCs.A: morphology of BMMSCs at 3 days’ culture with or without PEMF exposure. Cells possessed fibroblast-like morphology. Bar¼1 mm. To verify whether the BMMSCs still have differentiation potential after PEMF treatment, the differentiation experiments were conducted for both PEMF-treated and non-treated BMMSCs. Osteogenic differentiation after 14 days of induction was corroborated by formation of mineralized matrix as shown by (B) von Kossa staining and (C) ALP staining. Bar¼1 mm. Adipogenic differentiation was corroborated by (D) oil red O staining. Bar ¼ 500 mm. Neurogenic differentiation was corroborated by (E) morphology after 7 days of induction, and by immunofluorescence assay for (F) MAP2 and (G) tau.Bar¼ 500 mm. [The color figure for thisarticle is available online at www.interscience.wiley. com.]
cytoplasm and nucleoplasm conductivities of Jurkat cells decreased dramatically following nanosecond duration pulsed electric fields. Moreover, Deng et al. [2007] demonstrated that voltage-gated delayed
rec-tifier Kþ current and Ca2þ-activated Kþ current
channels were changed during progress from G1 to
S phase, and functional expression of ion channels could regulate proliferation in undifferentiated rat mesenchymal stem cells. These findings led us to infer that PEMF exposure changed the expression of ion channels and induced membrane hyperpolarization of BMMSCs and therefore resulted in the alteration of the cell cycle progression.
Although the surface phenotype (Table 2), mor-phology and multi-lineage differentiation potential of the BMMSCs were not significantly changed by the PEMF (Fig. 4), growth kinetic data and the cell cycle analysis indicated an enhanced growth rate after the first PEMF treatment. In addition, the cell densities achieved by PEMF were significantly higher than those achieved in non-treated conditions. This phenomenon of accelerated growth of BMMSCs due to PEMF may provide more osteoblast progenitor cells, thereby contributing to the healing of bone fractures. This finding facilitates the understanding of the interaction of PEMFs with BMMSC and may partly explain the mechanism underlying the effect of PEMF on bone healing.
ACKNOWLEDGMENTS
We thank Dr. Bruce Simon of EBI for generously providing us the PEMF device for this study.
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