Bioelectromagnetics 31:277^285 (2010)

EMFActs on Rat Bone MarrowMesenchymalStem Cells to Promote Differentiation toOsteoblasts and to Inhibit Differentiation

to Adipocytes

YongYang,1 ChaoxiongTao,1 Dongming Zhao,1 Feng Li,1 Wenchun Zhao,2 and HuaWu1*1Department of Orthopedics,Tongji Hospital, Medical College,HuazhongUniversity of Science andTechnology,Wuhan, China

2NavyUniversity of Engineering,Wuhan, China

The use of electromagnetic fields (EMFs) to treat nonunion fractures developed from observations inthe mid-1900s. Whether EMF directly regulates the bone marrow mesenchymal stem cells (MSCs),differentiating into osteoblasts or adipocytes, remains unknown. In the present study, we investigatedthe roles of sinusoidal EMF of 15 Hz, 1 mT in differentiation along these separate lineages using ratbone marrow MSCs. Our results showed that EMF promoted osteogenic differentiation of the stem cellsand concurrently inhibited adipocyte formation. EMF increased alkaline phosphatase (ALP) activityand mineralized nodule formation, and stimulated osteoblast-specific mRNA expression of RUNX2,ALP, BMP2, DLX5, and BSP. In contrast, EMF decreased adipogenesis and inhibited adipocyte-specific mRNA expression of adipsin, AP-2, and PPARg2, and also inhibited protein expression ofPPARg2. These observations suggest that commitment of MSCs into osteogenic or adipogenic lineagesis influenced by EMF. Bioelectromagnetics 31:277–285, 2010. � 2009 Wiley-Liss, Inc.

Key words: electromagnetic fields (EMF); pulsed electromagnetic fields (PEMF); bonemarrow mesenchymal stem cells (MSCs); osteogenesis; adipogenesis

INTRODUCTION

Osteoporosis is a widespread condition, oftenunrecognized in clinical practice, which may havedevastating health consequences through its associationwith fragility fractures. Current osteoporosis therapiesare based mostly on antiresorptive agents the efficacy ofwhich is limited, in part because their effects mainlytarget bone resorption. Therefore, our ability to treatand augment osteoporosis remains limited.

It is well documented that electromagnetic fields(EMFs) play a role in the repair of human tissues.Electrical stimulation by means of capacitive coupling,direct current, combined magnetic fields, and pulsedEMFs has been used for over 30 years to augmenthealing [Bassett et al., 1981; Luna Gonzalez et al.,2005; Sutbeyaz et al., 2006; Taylor et al., 2006]. Bassettet al. [1974] demonstrated the therapeutic effects ofpulsed EMFs, which have led to clinical trials,commercial production, and widespread availabilityof devices to promote healing of bony nonunions. In1983–1984 a study was conducted on bone densitychanges in osteoporosis-prone women exposed topulsating EMFs (PEMF) [Bilotta et al., 1994]. The

study determined the effect of a 72 Hz PEMF of 3 or7 mT on bone density of the radii of post-menopausal(osteoporosis-prone) women, during and after 10 h ofdaily treatment for 12 weeks. Bone mineral densities ofthe treated radii increased significantly in the immedi-ate area of the field during the exposure period [Bilottaet al., 1994]. Thereafter, many experiments confirmedthat EMF exposure clearly stopped bone loss [Brightonet al., 1988; Rubin et al., 1989; Akca et al., 2007]. Morerecent reports continue to confirm the positive effects ofcapacitively coupled fields on bone density [Bilotta

72009Wiley-Liss, Inc.

——————Grant sponsor: National Natural Sciences Foundation of China(50477043).

*Correspondence to: Hua Wu, Department of Orthopedics, TongjiHospital, Medical College, Huazhong University of Science andTechnology, Wuhan, China. E-mail: wuhua360@yahoo.com.cn

Received for review 22 August 2008; Final revision received7 August 2009

DOI 10.1002/bem.20560Published online 29 December 2009 in Wiley InterScience(www.interscience.wiley.com).

et al., 1994; Akca et al., 2007]. Despite clinical evidencedemonstrating EMFs to be an effective treatmentmodality in bone healing and osteoporosis, its mech-anism of action is unknown. EMFs are able toupregulate several cytokines that are important inpromoting osteoblast differentiation during fracturerepair, including bone morphogenetic proteins 2 and 4,and transforming growth factor-b [Bodamyali et al.,1998; Jasti et al., 2001]. However, more directbarometers of osteoblast function such as collagensynthesis, proliferation, alkaline phosphatase (ALP)activity, and prostaglandin E2 production are notsignificantly altered in the presence of EMFs [Pattersonet al., 2006; Sakai et al., 2006; Martino et al., 2008].Thus, it seems unlikely that the clinical success ofEMFs is entirely attributable to an effect on osteoblastsalone.

The bone marrow contains mesenchymal stemcells (MSCs) that exhibit the ability to differentiate intoadipocytes and osteoblasts. A reciprocal relationshipexists between bone marrow MSCs adipogenesis andosteogenesis. The balance between MSCs osteoblastand adipocyte differentiation is disrupted in varioushuman diseases. For example, decreased bone forma-tion accompanied by an increase in bone marrowadipogenesis occurs with aging and immobility orosteoporosis [Meunier et al., 1971; Burkhardt et al.,1987; Kajkenova et al., 1997; Nuttall and Gimble, 2000;Verma et al., 2002], whereas increased bone formationis observed in patients with progressive osseous hyper-plasia who form heterotopic bone within their adiposetissue [Kaplan and Shore, 2000].

These observations prompted us to evaluate thepossible mechanism of EMFs in prevention or treat-ment of osteoporosis. We hypothesized that EMFs maypositively regulate bone formation by directly affectingdifferentiation of multipotential bone marrow MSCs.Our results showed that sinusoidal EMF of 15 Hz, 1 mTstimulated the differentiation of progenitor cells intoosteoblasts and concurrently inhibited adipogenesisformation.

MATERIALS AND METHODS

MSC Culture

Rat MSCs were isolated with a modified proce-dure as described previously [Jiang et al., 2002]. Briefly,ten 8-week-old Sprague–Dawley rats (male andfemale, 80–120 g) were sacrificed by neck dislocationaccording to the Guideline’s of Animal Care and UseCommittee for Teaching and Research of HuazhongUniversity of Science and Technology. Bone marrowwas collected from the femurae and tibiae of the animalwith a 18-gauge needle under sterile conditions,

suspended in a 15-ml test tube containing Dulbecco’smodified Eagle’s medium (DMEM, Invitrogen, GrandIsland, NY), and centrifuged at 2000 rpm for 5 min. Themarrow pellet was washed in Hanks’ balanced saltsolution (Boster Biological Technology, Wuhan,China), centrifuged at 1000 rpm for 10 min, and thenresuspended in DMEM. Nucleated cells were isolatedwith a Percoll density gradient (Invitrogen) by cen-trifuging at 14000 rpm for 12 min at 8 8C. The top 60%of the gradient was collected, and then washedwith the complete culture medium containing 10%fetal bovine serum (FBS, Invitrogen), penicillin(100 U/ml) (Sigma–Aldrich, St. Louis, MO), strepto-mycin (100mg/ml; Sigma–Aldrich), and amphotericinB (0.25 mg/ml; Sigma–Aldrich). The cells were thenseeded to a 25 cm2 tissue culture flask and incubated inthe medium as described above with 10 ng/ml leukemiainhibitory factor (Invitrogen) at 37 8C in a humidifiedatmosphere of 5% CO2 and 95% air. Nonadherentcells were removed by changing the medium after 24 h.The culture medium was changed twice a week there-after. For subculture, cells were detached with0.25% trypsin (Amresco, Cleveland, OH) and 1 mMethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetra-acetic acid (EGTA) (Sigma–Aldrich) and passaged ata ratio of 1:3 plates when cells grew to 80–90%confluence.

EMF Exposure

A continuous sinusoidal EMF (Bm¼ 1 mT,f¼ 15 Hz) was used in this study. Culture dishes wereplaced in an incubator between a pair of 30-cm diameterHelmholtz coils, placed 30 cm apart, which weredesigned and manufactured by the Naval Universityof Engineering of China. From the coil center to theorigin, in the 7 cm of the spherical region, theuniformity of the EMF is approximately 90%. Coil isvertical with the horizontal plane. Sham-exposedcontrol samples were kept in the same conditionswithout using EMF. The CO2 concentration andhumidity of the sham-exposed control were similar.The temperatures inside the two incubators (for sham-exposed controls and EMF treatments) were within0.2–0.8 8C of each other. The EMF waveform charac-teristics are presented in Figure 1.

Alkaline Phosphatase Activity

The MSCs (from passage 3) were plated ata density of 1� 105 cells/well in six-well plates(Corning, Corning, NY) for EMF or sham exposurefor 8 h/day for 3 days. The cells were collected andrinsed twice with PBS and then digested with 0.25%trypsin. Cells were added into a solution of 10 mmol/LTris–HCl, 5 mmol/L MgSO4, 0.1% Triton X-100, 0.1%

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NaNO3 (Amresco) for a total of 80ml, and frozen(�80 8C) 3 times to disrupt cell membranes. MSCsuspensions were sonicated for 5 min in order to extractthe enzyme. ALP activity was measured on thesupernatant by an established technique using p-nitro-phenylphosphate (Sigma–Aldrich) as a substrate at37 8C. After 30 min, the action was stopped with1 mmol/L NaOH, and the absorbance value at 405 nmwas measured with a microplate reader. Cell lysateswere analyzed for protein content using a Micro BCAAssay kit (Lipulai, Beijing, China), and ALP activitywas normalized for total protein concentration.

Mineralized Bone Matrix Formation Assayand Quantification

The cells (from passage 3) were plated at a densityof 1� 105 cells/well in six-well plates in osteogenicinduction supplement medium (low glucose DMEMcontaining 10% FBS, 0.1mM dexamethasone (Deca-dron, Merck, Whitehouse Station, NY), and 10 mM b-glycerophosphate (Amresco)) and exposed to EMF orsham treatment for 15 or 21 days. Medium was changedevery 3 or 4 days. The mineralized matrix formationwas monitored using von Kossa stain and wasperformed as follows: the cell cultures were washedtwice with PBS, fixed in 4% neutral formalin for 5 min,washed with water, serially dehydrated in 70%, 95%,and 100% ethanol, twice each, and then air dried. Theplates were rehydrated from 100% to 95% to 80%ethanol before staining. The water was removed, a 5%silver nitrate (Tianjing, Guangcheng, China) solutionwas added, and the plates were exposed to ultravioletlight for 1 h and rinsed with water after. A 5% sodiumthiosulfate was added for 5 min and the plates wereagain rinsed with water. The plates were washed withwater, 2� 95% ethanol and 2� 100% ethanol, and thendried and photographed. Mineral deposits were stainedblack. The mineral deposit area of each well wascalculated from the positive stained areas measuredfrom 20 randomly selected microscopic fields(96467.83mm2/field) using Image-Pro1 Plus analysis

software (Media Cybernetics, Silver Spring, MD)[Chang et al., 2007].

Real-Time Quantitative RT-PCR

Total RNA of rat MSCs (from passage 3) wasisolated using TRIzol method (Invitrogen) and furthertreated with DNase I (Invitrogen). Reverse transcription(RT) was performed with the RT system protocol in a20-ml reaction mixture. RNA (1mg) was used in thereaction, and a combination of oligo(dT) and randomhexamer primers was used for the initiation of cDNAsynthesis. Following this RT procedure, the reactionmixture (cDNA) was used for real-time quantitativepolymerase chain reaction (PCR). The forwardand reverse PCR oligonucleotide primers weredesigned with the Primer premier 5.0 software (PremierBiosoft, Palo Alto, CA), and the sequences were blasted(http://www.ncbi.nlm.nih.gov/BLAST/). The primersequences are shown in Table 1. PCRs were performedaccording to the real-time PCR machine manufacturer’sinstructions (DNA Engine Opticon, MJ Research,Waltham, MA), which allows real-time quantitativedetection of the PCR product by measuring the increasein SYBR green fluorescence caused by binding ofSYBR green to double-stranded DNA. The SYBR kitfor PCRs was purchased from Toyobo (Tokyo, Japan).For sample analysis, the threshold was set based on theexponential phase of products, and the CT value forsamples was determined. The resulting data wereanalyzed with the comparative CT method for relativegene expression quantification against the housekeep-ing gene b-actin.

Assessment of Cytoplasmic LipidDroplet Formation

The cells were plated at a density of 2� 104 cells/well in six-well plates in adipogenic induction supple-ment medium (high glucose DMEM containing 10%FBS, 10mg/ml insulin, Invitrogen), 0.5 mM isobutyl-methylxanthine, 1mM dexamethasone, and 100 mMindomethacin (Sigma–Aldrich) and exposed to EMF orsham treatment for 21 days. Cytoplasmic inclusions ofneutral lipids were assessed by Oil Red O staining(Sigma–Aldrich). Adipogenic cultures were fixed with10% paraformaldehyde for 10 min at room temperatureand rinsed in distilled water. Working solution of OilRed O was prepared from 1% (w/v) Oil Red O in 99%isopropanol and diluted to 0.3% (v/v) with distilledwater. Cultures were stained with Oil Red O workingsolution for 10 min by gentle rocking, rinsed withdistilled water, counterstained with hematoxylin for5 min, rinsed again with distilled water, stored under50% glycerol, and photographed. After staining, thecells were rinsed with distilled water, and 0.5 ml of

Fig. 1. EMF waveform characteristics. Electromagnetic fieldinduction Bm¼1mTand the net frequency f¼15 Hz of sinusoidalelectromagnetic field.

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isopropyl alcohol was added to the stained dish.Aliquots of the extracted Oil Red O were measured at540 nm with spectrophotometer (Ultrospec 3000,Pharmacia Biotech, Washington, DC) [Baksh et al.,2007].

Immunoblot Analysis

MSCs were cultured in adipogenic inductionmedium and stimulated with EMF or sham exposurefor 14 days. Then the cells were lysed with 50 mMTris, 40 mM b-glycerophosphate, 100 mM NaCl,10 mM EDTA, 10 mM p-nitrophenol phosphate,1 mM Na3VO4, 10 mM NaF, 1% Nonidet P-40, 0,1%SDS, 1 mM phenylmethylsulfonyl fluoride, 250 U/mlaprotinin, 40 mg/ml leupeptin, and pH 7.4. (All theseagents were purchased from Amresco.) The lysate wascentrifuged at 12000g for 10 min before some of thesupernatant was sequestered for a protein assay.Subsequently, the appropriate volume of the super-natant was removed, based upon the concentrationvalues provided by the protein standard assay, andresuspended in the appropriate volume of Laemmlisample buffer. Proteins were denatured at 100 8C for5 min, and the supernatant was dissolved in Laemmlisample buffer. Aliquots of the protein (30 mg) wereresolved on a SDS–polyacrylamide gel (12%) (BosterBiological) and transferred to nitrocellulose mem-branes (Boster Biological). The membranes wereincubated at 4 8C with blocking solution (5% nonfatdried milk in phosphate-buffered saline (PBS), 0.2%Tween-20 (PBST), Boster Biological) for 1 h, thenprobed with various primary antibodies (1:500)(Santa Cruz Biotechnology, Santa Cruz, CA) overnight.After three washes with PBST, membranes were

incubated with horseradish peroxidase (HRP)-conjugated secondary IgG (1:25000) (Boster Biolog-ical) for 2 h, followed by three more washes with PBST.Immunoreactive bands were detected using theSuper Signal Chemiluminescent reagent (ThermoScientific, Waltham, MA) and analyzed quantitativelyby normalizing band intensities to the controls onscanned films or by using a ChemiImager 4400 Gelimaging system (Alpha Innotech, San Leandro, CA) byAdobe Photoshop software.

Statistical Analysis

All values were expressed as means� SD. Theanalyses were conducted with the SPSS 13.0 software(SPSS, Chicago, IL). Statistical significance wasassessed by Student’s t-test. For PCR analysis, statisticalanalyses were conducted according to instructions for theGel-Pro Analyzer (Alpha Innotech). For all statisticaltests, P< 0.05 was considered significant.

RESULTS

Morphology of Rat MSCs in Culture

The cultures of primary rat MSCs underwent aninitial lagphase of about 5 days, during which thecolonies were seen to arise from single cells (Fig. 2A).The colonies contained both spindle-shaped cells andlarge flat cells. By day 12, the colonies were grown toconfluency (Fig. 2B).

Effects of EMF on Osteogenesis by Rat MSCs

To test the effect of EMF on osteogenic dif-ferentiation of rat MSCs, ALP activity was measured by

TABLE 1. Primers for Real-Time PCR

No. Gene Oligonucleotide Product length (bp)

NM_013059 ALP 5-TGACTGACCCTTCCCTCTCG-3 1005-TCAATCCTGCCTCCTTCCAC-3

NM_012943 Dlx5 5-TACTGCTCTCCTACCTCTGCTTCT-3 1955-CACTTCTTTCTCTGGCTGGCT-3

XM_001060656 RUNX2 5-GAGTCAGATTACAGGTGCTTC-3 1245-AGTGAAGGTGGCTGGATAG-3

NM_017178 BMP2 5-CAGCGAGTTTGAGTTGAGG-3 1025-CGGTACAGGTCGAGCATAT-3

DQ213013 BSP 5-AGAAAGAGCAGCACGGTTGAG-3 1615-TCATAGCCATGCCCCTTGTAG-3

AB019561 PPARg2 5-CCTTTACCACGGTTGATTTCTC-3 1395-GGCTCTACTTTGATCGCACTTT-3

NM_001077642 Adipsin 5-TGGTGGATGAGCAGTGGGT-3 1115-AGGGTTCAGGACTGGACAGG-3

NM_053365 AP2 5-GCGTAGAAGGGGACTTGGTC-3 1095-TTCCTGTCATCTGGGGTGATT-3

NM_031144 b-Actin 5-ACCTCTATGCCAACACAGTGC-3 1565-AGCCACCAATCCACACAGAG-3

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the method described above. Cultures were exposed for8 h/day for 3 days. Control treatments were maintainedin a different incubator. EMF exposure for 3 daysresulted in an increase in ALP activity over thenontreated control (n¼ 6/group; P< 0.01; Fig. 3).

Bone cell maturation is accompanied by extrac-ellular matrix mineralization. Therefore, we tested theeffect of EMF exposure on the ability of rat MSCsto mineralize the extracellular matrix that they produce.After 21 days of EMF exposure (8 h/day), no extrac-ellular matrix mineralization was observed based on thevon Kossa staining, which detects calcium phosphatedeposits. Because osteogenic inducing medium (OS)-treated rat MSCs undergo osteocyte maturation within3 weeks, we tested whether EMF treatments followedby the OS could facilitate this process. As demonstratedin Figure 4, at days 15 and 21 the treatments of rat MSCswith EMF and OS clearly enhanced the intensity of vonKossa staining compared with the control treatment.These results suggest that the EMF may play promotionroles not only in osteogenic specification of rat MSCsbut also in bone cell maturation.

EMF Exposure Reduces Adipocyte MasterGene Expression and Increases OsteoblastMaster Gene Expression of Rat MSCs

To characterize the molecular response of theeffect of EMF exposure on differentiation of rat MSCs,we detected the expression of adipocyte-specific genessuch as adipsin, AP-2, and PPARg2, at the expense ofosteogenic-specific genes such as ALP, BMP2, DLX5,RNUX2, and BSP, in rat MSCs incubated in standardgrowth medium in the presence of EMF exposure orcontrol. The expression of ALP was induced to highlevels in EMF-exposed rat MSCs for 8 h/day for 3 days.Concurrently, EMF inhibited these cells to enter the

Fig. 2. Rat MSCs were cultured from marrow and are shown 5days after plating (A) and 12 days after plating (B). (Magnifica-tion�100).

Fig. 3. EffectsofEMF-inducedALPactivityonratMSCsosteogen-esis.The cellswere cultured instandardgrowthmediumwith EMForshamexposurefor3days. (means�SEM;n¼ 6,p< 0.01). [Thecolor figure for thisarticle isavailable onlineat www.interscience.wiley.com.]

Fig. 4. Effects of EMFon osteogenesis.The cellswere cultured inosteogenesis inducing medium and exposed to EMF or shamexposure.Von Kossa staining was performed at days 15 and 21.The mineral deposit area of each well was calculated from 20randomly selected microscopic fields. Each bar represents themean � S.E.M. of three replicated cultures. Data evaluated byone-way ANOVA. (A, C): EMF exposed (B, D):Control. (Magnifica-tion�100). (E):Quantificationof VonKossastaining.

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adipocytic lineage with low expressions of adipsin,AP-2, and PPARg2. The expressions of BMP2, DLX5,RUNX2, and BSP were also induced to high levels inEMF exposure. The mRNA expression was assessedusing real-time quantitative PCR, and b-actin mRNAexpression remained constant with time and treatmentin culture. Each gene expression was normalized tob-actin and reported as the fold over b-actin. We foundthat EMF exposure significantly increased geneexpression of BMP2, DLX5, RNUX2, and BSP. Also,EMF exposure reduced gene expression of adipsin,AP-2, and PPARg2 (Fig. 5).

EMF Downregulates the PPARg2Expression of MSCs

To further study whether expression of PPARg2protein is downregulatated by EMF, protein expressionanalysis was performed using Western blot and anti-PPARg2 antibodies (Santa Cruz Biotechnology). First,MSCs were cultured in adipogenic induction mediumwith EMF or sham exposure for 8 h/day for 14 days.Second, the cells were lysed and the immunoblotanalysis was detected as described in the Materials andMethods Section. As shown in Figure 6, PPARg2significantly decreased after 14 days of EMF stimula-tion, according to the real-time quantitative RT-PCRresult.

EMF Exposure of Effects on Adipogenesisby Rat MSCs

Under normal conditions the adipogenesis of ratMSCs is induced by the adipogenic induction medium.To further confirm the effect of EMF on MSCsadipogenesis, the cells were incubated in adipogenicinduction medium with EMF (8 h/day) or shamexposure for 14 days and then stained with Oil Red O.In Figure 7A,B we can see that the control has morelipid droplets than the EMF exposed. Similar resultswere also obtained in the quantification of the lipiddroplets, as shown in Figure 7C.

DISCUSSION AND CONCLUSION

Bone marrow-derived MSCs are multipotent, butthey do not differentiate spontaneously. When culturedunder appropriate in vitro conditions, MSCs arecapable of differentiating into at least three lineages(osteogenic, chondrogenic, and adipogenic) [Beresfordet al., 1992; Mackay et al., 1998; Pittenger et al., 1999].For example, cultured in the presence of dexametha-sone, ascorbic acid, and b-glycerophosphate, MSCsdifferentiate into the osteogenic lineage, producingbone-like nodules with a mineralized extracellularmatrix [Rickard et al., 1994].

Fig. 5. A^E:Effectsof PEMFonexpressionofosteogenic-specificgenesin culturesof ratMSCs; F-H:Effectsof PEMFonexpressionof adipose-specific genes in cultures of rat MSCs.The rat MSCswere cultured and total RNAwas isolated and analyzed by real-time PCR with the indicated primers. Real-time PCR resultsshowed a significant increase over vehicle control in ALP, BMP2,DLX5,RUNX2 and BSPmRNAafter 3 days of treatment with EMF.By contrast, a significant decrease in PPARg2, Adipsin and AP-2mRNAwas observed after 3 days of treatment with EMF. (n¼ 3,p< 0.01). [The color figure for this article is available online atwww.interscience.wiley.com.]

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Under normal conditions there is a balancebetween osteoblasts and adipocytes in bone marrow.However, this balance is lost in diseases such asosteoporosis where a decrease in bone volume isaccompanied by an increase in adipose tissue [Meunieret al., 1971; Gimble et al., 1996; Nuttall and Gimble,2000]. The increased adipose tissue volume anddecreased trabecular bone volume with aging in vivoare the result of alterations in cellular composition andcharacteristics of MSCs. Enhanced adipogenesis in

bone marrow with aging may result from compositionchanges of bone marrow microenvironment, age-related changes in levels of hormones [Bellows et al.,1994], and growth factors [Gimble et al., 1989] thatregulate adipogenesis.

Bassett et al. [1982] used a pair of Helmholtz coilsto produce a magnetic field across a fracture site andenhance osteogenesis. Since then several experimentalstudies have examined the influence of EMF onosteoporosis. The effect appears to vary with thewaveform of magnetic field used [Bassett et al., 1981;Brighton, 1984; Rubin et al., 1989; Skerry et al., 1991].The degree of effect has been variable, but in somecases, osteoporosis has been prevented or evenreversed. However, the exact mechanism by whichEMF stopped bone loss has not been defined. Here, ourfindings indicate that sinusoidal EMF of 15 Hz, 1 mTpromotes osteogenesis and inhibits adipogenesis asevidenced by a decrease in adipocyte formation and anincrease in ALP activity and mineralized nodulenumbers. These effects appear to be at the level ofcommitment rather than at the level of maturation. Asshown here, EMF enhanced the expression of Run-x2(cbfa1), BSP, DLX5, and BMP2, which are osteo-genesis markers of osteoblast differentiation [Glimcheret al., 2007; Holleville et al., 2007; Komaki et al., 2007].The primary effect of EMF appears to be the commit-ment to the osteoblast pathway through an earlyincrease in cbfa1 gene expression, rather than at thelevel of osteoblast maturation. Concomitantly to theenhanced expression of the osteogenic differentiationgene in rat MSCs, EMF decreased the expression ofthe adipogenic differentiation gene. Notably, PPARg2,adipsin, and AP-2, three transcription factorsinvolved in adipogenic differentiation [Noer et al.,2007; Hasegawa et al., 2008], were decreased markedlyin MSCs treated with EMF. The present finding thatEMF concomitantly corrected osteoblastogenesis andadipogenesis suggests that EMF may act directly on thecommon precursor cell to promote its commitment inthe osteoblastic lineage at the expense of the adipocytelineage. We demonstrated that EMF promoted osteo-genic differentiation of the stem cells and concurrentlyinhibited adipogenesis. This may be one of themechanisms by which EMF stops bone loss.

There are two limitations of this study. First, someresearch suggests that EMF can promote cell release ofBMP2, BMP4, and PGE2 [Li et al., 2007]; however, inour study these were not measured. Our study showsthat under EMF stimulation the mRNA expressionof BMP2 was promoted. BMP2 and BMP4 aretranscription factors which can promote MSCs toosteogenesis differentiation. Here our study revealedthat EMF acts on MSCs to promote differentiation to

Fig. 6. EMF downregulated PPARg2 protein expression. MSCscells were cultured in adipogenic induction medium with EMFor sham exposure for 14 days. The total protein was isolatedfor Immunoblot analysis.

Fig. 7. EMF effects on adipogenesis by rat MSCs. The rat MSCswere cultured in standard growth medium supplemented withEMForshamexposure.Theyweregrownunderadipogenic condi-tions, fixed with paraformaldehyde and stained with Oil Red O atday14 (magnification�100). (A):PEMF (B):Control (C):Quantifica-tionofOil RedO.

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osteoblasts; whether this effect is direct or indirect isstill an open question. A second important limitationis whether or not EMF promotes MSCs to osteogenesisdifferentiation in vivo and, therefore, more studies areneeded to confirm this effect.

Despite these limitations, our findings haveseveral implications for the biology and therapy ofhuman diseases associated with tissue loss. Osteopo-rosis is becoming more important as the elderlypopulation increases. Despite some successes withdrugs that inhibit bone resorption, there is a compellingneed for anabolic agents that will substantially enhancenew bone formation and restore bone in people whohave already suffered osteoporosis. Here, we confirmthat EMF play a vital role in balancing the differ-entiation of osteoblastic and adipogenic lineages from acommon progenitor (MSCs) in vivo, and in preventingor treating osteoporosis. In conclusion, we showed thatEMF directly regulates progenitor rat MSCs differ-entiating into more osteoblasts and less adipocytes. Theresults of this study might be relevant for the patho-genesis and treatment of osteoporosis.

ACKNOWLEDGMENTS

The authors thank Zhihua Zhao for technicalassistance and Wenchun Zhao for help in producing theEMF signal generator.

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