PulsedElectromagnetic Fields (PEMF)PromoteEarlyWoundHealingand

Myofibroblast ProliferationinDiabeticRats

Gladys Lai-YingCheing,1* Xiaohui Li,1,2 LinHuang,3

Rachel Lai-ChuKwan,1andKwok-KuenCheung11Department ofRehabilitationSciences,TheHongKongPolytechnicUniversity,

HongKongSpecialAdministrativeRegion, HongKong, China2Department of Endocrinology, First AffiliatedHospitalofXi’anJiaotongUniversity

College ofMedicine, Xi’an, China3Department of Surgery, Divisionof Plastic, Reconstructive andAesthetic Surgery,

TheChineseUniversity ofHongKong, Prince ofWalesHospital,HongKongSpecialAdministrativeRegion, HongKong, China

Reduced collagen deposition possibly leads to slow recovery of tensile strength in the healingprocess of diabetic cutaneous wounds. Myofibroblasts are transiently present during wound healingand play a key role in wound closure and collagen synthesis. Pulsed electromagnetic fields (PEMF)have been shown to enhance the tensile strength of diabetic wounds. In this study, we examined theeffect of PEMF on wound closure and the presence of myofibroblasts in Sprague–Dawley rats afterdiabetic induction using streptozotocin. A full-thickness square-shaped dermal wound (2 cm� 2 cm)was excised aseptically on the shaved dorsum. The rats were randomly divided into PEMF-treated(5mT, 25Hz, 1 h daily) and control groups. The results indicated that there were no significantdifferences between the groups in blood glucose level and body weight. However, PEMF treatmentsignificantly enhanced wound closure (days 10 and 14 post-wounding) and re-epithelialization (day10 post-wounding), although these improvements were no longer observed at later stages of thewound healing process. Using immunohistochemistry against a-smooth muscle actin (a-SMA), wedemonstrated that significantly more myofibroblasts were detected on days 7 and 10 post-woundingin the PEMF group when compared to the control group. We hypothesized that PEMF wouldincrease the myofibroblast population, contributing to wound closure during diabetic wound healing.Bioelectromagnetics 35:161–169, 2014. © 2014 Wiley Periodicals, Inc.

Key words: collagen; epithelialization; wound closure; diabetes; tensile strength; pulsedelectromagnetic fields; PEMF

INTRODUCTION

Wound healing involves complex processes con-sisting of distinct, but overlapping phases, such ashemostasis, inflammation, proliferation, and remodel-ing [Enoch et al., 2006]. Myofibroblast plays a keyrole in promoting wound remodeling [Hinz andGabbiani, 2003; Baum and Arpey, 2005] by enhancingtissue contraction to reduce the size of the wound.This produces new extracellular matrix components[Gabbiani, 2003; Desmouliere et al., 2005] andcontributes to the development of the vascular network[Mayrand et al., 2012]. The myofibroblasts of woundtissue are assumed to originate from the local recruit-ment of fibroblasts in the surrounding dermis andsubcutaneous tissue. Pericytes, vascular smooth mus-cle cells around vessels, and fibrocytes are alsoconsidered to be possible sources of myofibroblast[Desmouliere et al., 2005]. As the primary contractile

cells, myofibroblast exerts strong cell traction stressesin wound repair [Wipff and Hinz, 2009]. Upon theclosure of the wound after epithelialization, myofibro-blast will disappear by apoptosis and a scar is formed[Gabbiani, 2003; Hinz et al., 2007].

Grant sponsor: The Research Grants Council of the Hong KongSpecial Administrative Region Government; grant number: PolyU5600/11M.

*Correspondence to: Gladys Lai-Ying Cheing, Department ofRehabilitation Sciences, The Hong Kong Polytechnic University,Hung Hom, Kowloon, Hong Kong, China.E-mail: rsgladys@polyu.edu.hk

Received for review 16 April 2013; Accepted 8 November 2013

DOI: 10.1002/bem.21832Published online 3 January 2014 in Wiley Online Library(wileyonlinelibrary.com).

Bioelectromagnetics 35:161^169 (2014)

� 2014 Wiley Periodicals, Inc.

Normal wound healing proceeds at a rapid ratewhen there are no complications. However, delayedhealing frequently occurs with diabetic wounds[King, 2001], and deficiencies of various physiologicfactors among individuals with diabetes can contributeto this delay. There is a marked decrease in collagensynthesis in people with diabetes, due to dysfunctionin the production of collagen peptides and to the post-translational modification of collagen degradation[Dinh and Veves, 2005]. The delayed onset andalterated spatial organization of myofibroblasts hasbeen shown to contribute to delayed healing and toinefficient contraction in diabetic wounds [Darbyet al., 1997]. A failure to re-epithelialize may result inan unhealed wound or even in further breakdown inthe diabetic wound [Harker and Moore, 2004].

For decades, investigators have attempted to lookfor an effective intervention to enhance the healing ofdiabetic ulcers. Pulsed electromagnetic fields (PEMF)have been adopted as a non-invasive intervention forpromoting tissue healing. Several clinical studies havedemonstrated that PEMF promote nerve regeneration[Musaev et al., 2003] and improve microcirculation[Webb et al., 2003] in people with diabetes. Someclinical studies have also found that PEMF acceleratethe recovery of chronic wounds [Kenkre et al., 1996]and increase the tensile strength of scar tissue [Bou-zarjomehri et al., 2000]. The application of PEMF canenhance the binding of calcium ions in the growthfactor cascades involved in tissue healing, leading to amarked increase in tensile strength at the repair site inrats [Strauch et al., 2006]. Furthermore, histologicalexaminations using animal studies have shown thatPEMF can promote angiogenesis [Callaghan et al.,2008] and re-epithelialization [Athanasiou et al.,2007], which are crucial factors in the healing ofdiabetic wounds. However, the effects of PEMF on themyofibroblast population in diabetic wounds have notbeen investigated.

We hypothesized that PEMF can promotewound healing by enhancing the myofibroblast popu-lation in diabetic rats. Therefore, the present studyexamined the effects of PEMF on enhancing therelease of myofibroblasts and promoting the healingof dermal wounds in streptozotocin-induced diabeticrat models.

MATERIALS AND METHODS

Diabetic Rat Model

Male Sprague–Dawley (SD) rats (8–10 weeks,280–320 g) were bred and supplied by the CentralizedAnimal Facilities of The Hong Kong PolytechnicUniversity (Hong Kong Special Administrative Re-

gion, China). Throughout the entire study period, allanimals were housed in a temperature- and humidity-controlled animal holding room with a 12 h dark/lightcycle. All procedures were performed in accordancewith the Animal Subjects Ethics Subcommittee of theuniversity. The study protocol complied with theguidelines of The Hong Kong Polytechnic University(Hong Kong Special Administrative Region, China)and all animals received humane care. Each rat wasgiven a single intraperitoneal injection of streptozoto-cin (STZ) (Sigma-Aldrich, St. Louis, MO, 50mg/kg incitrate buffer) after a 7-day acclimatization period.Those rats with a blood glucose level of more than300mg/dl (16.7mmol/L) 72 h after the STZ injectionwere defined as diabetic.

Wound Induction

Seven days after the diabetes induction, anes-thesia was administered for all animals via anintraperitoneal injection of a mixture of ketamine andxylazine (100mg/kg and 10mg/kg body weight,respectively; Alfasan International, Woerden, Hol-land). A full-thickness square shaped wound (2 cm� 2 cm) was excised aseptically on the shaveddorsum [Athanasiou et al., 2007], as shown inFigure 1A. Post-surgically, the wounds were disin-fected using a betadine solution and temporarilycovered with sterile gauze to prevent infection. Therats were housed individually thereafter.

Pulsed Electromagnetic Fields (PEMF)Treatment

The diabetic rats were randomly allocated intoeither the PEMF group or the control group. ThePEMF treatment was given on post-wounding day 1.The rats in the PEMF group (n¼ 28) were fixed in aplastic cylindrical container placed under the applica-tor of the PEMF device (Fig. 1B, model XKC-600W;Magnetopulse International, Griffin, Australia), whichis an inverted U-shaped applicator with an internaldiameter of 12 cm and length of 30 cm. The woundwas placed at the central space area close to the wallof the applicators to ensure the delivery of a uniformmagnetic field. The magnetic field was generated bypassing through a pair of concentric 200-turn coils in arectangular shape of 15 cm� 25 cm positioned next toeach other and mounted to the shape of the applicator(Fig. 1C), to produce a uniform magnetic field in thelateral direction. The maximum density was distribut-ed along the applicator's central area where the woundwas positioned. The generator produces a train ofsinusoidal pulses with a width of 0.04ms, giving anoverall frequency of 25Hz. The maximum fieldapplied to the sample was 5mT and was measured by

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the hand-held Gauss/Tesla meter (Model 4048, F.W.Bell, Milwaukie, OR).

Based on our pilot study (data not shown), thewounds were exposed to active electromagnetic fields(5mT, 25Hz) for 60min on a daily basis until the ratswere sacrificed. The rats in the control group (n¼ 28)were fixed in a plastic cylindrical container withoutexposure to PEMF.

Wound Assessment

Body weight and blood glucose levels weremeasured on day 0 (the day that wound inductionwas performed) and on days 7, 10, 14, and 21 afterwounding. Images of the wound were taken using adigital camera (Nikon Coolpix P5100; Nikon, Tokyo,Japan) and were imported to the Verge VideometerMeasurement Documentation (VeV MD) system(Version 1.1.14, Vista Medical, Winnipeg, Canada) inorder to calculate the area of the wound. The percent-

age of wound closure (WC %) was calculated usingthe following formula: WC %¼ (WA0�WAx)/WA0,where WA0 represents the wound area on day 0whereas WAx represents the wound area on the date ofthe assessment.

After wound assessment, the rats from the PEMFgroup and control group were killed by asphyxiationwith a rising concentration of CO2 (between 0% and100%) at different time points, which were chosenaccording to previous studies [Patiño et al., 1996;Milgram et al., 2004; Matic et al., 2009].

Tissue Preparation and Histochemical Staining

After the animals were euthanized, wound tissueswere excised in full thickness with subcutaneous fat,including a margin of at least 5mm of healed skin allaround. The specimens were fixed in 4% paraformal-dehyde in phosphate buffered saline (PBS, pH 7.4)and were kept in 70% ethanol until the tissue was

Fig. 1. PEMF exposure of a diabetic wound. A: Representative image of a square shaped full-thickness wound (2 cm� 2 cm) excised on the dorsum of the back of a diabetic SD rat (a VeVorientationcard(3 cm� 3 cm)wasplacedalongsidetocalculatetheareaofthewound).B: Theani-malswereplacedunder theapplicator for PEMFexposure.C: Schematics of PEMFexposure in adiabeticSDrat.

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processed. Each piece of wound tissue was cut in halfbefore being processed and embedded in paraffin.Sections with a thickness of 5mm were cut for routinehematoxylin and eosin (H&E) staining in order tomeasure epidermal gaps.

For immunohistochemistry, the tissue sectionswere deparaffinized and rehydrated through gradedalcohol into water. After blocking with a 5% normalhorse serum (NHS) in PBS, the tissue sections wereincubated with mouse monoclonal primary antibodyagainst a-SMA (1:50000; Sigma-Aldrich) at 4 8Covernight for myofibroblasts labeling. The sectionswere subsequently incubated with Alexa Fluor 555-conjugated donkey anti-mouse secondary antibodies(Molecular Probes, Carlsbad, CA) at 1:300 dilutionsfor 1 h at room temperature. Immunostaining wasperformed in triplicate for each section of tissue.Images taken under 200 and 400 magnifications at therepresentative area of the slides were captured usingan epifluorescent microscope (Eclipse 80i, NikonInstruments, Melville, NY) and a SPOT Flex DigitalCamera (Diagnostic Instruments, Sterling Heights,MI).

Analysis of the Staining

All sections were scored using a scale from 0 to4 for myofibroblasts; the scoring was completed bytwo blinded evaluators who are specialists in laborato-ry animal pathology and who did not know whichintervention the specimen had been given. The scoresmade by the two evaluators were then averaged foreach specimen. The criteria for scoring have beendescribed previously [He et al., 2012].

Statistics Analysis

Data are presented as mean� standard error ofthe mean (SEM) in the text and figures. An indepen-

dent Student's t-test was conducted to compare thedifference between the PEMF and the control groupsat each time point. A Mann–Whitney U-test wasapplied to test the score of the a-SMA staining of twogroups at different time points. Data analysis wasperformed using IBM SPSS Statistics 17.0 (IBM,Chicago, IL). The level of significance was set at 0.05for all measurements.

RESULTS

Throughout the entire study period, all of the 56rats stayed healthy and showed no signs of infectionat their wound sites until they were sacrificed. Nosignificant group difference in blood glucose level wasfound at any assessment time point (Fig. 2A).

The rats in both groups lost weight after thediabetes induction, and exhibited metabolic abnormali-ties characteristic of uncontrolled diabetes, includingfailure to gain weight and frequent urination duringthe course of the study. No significant difference inbody weight was observed between the two groupsthroughout the study (Fig. 2B).

The results obtained from the planimetric evalua-tion of the total wound area, including the crust, onday 7, 10, 14, and 21 post-wounding, are listed inTable 1. Our results revealed that the PEMF grouptended to experience a smaller wound area and agreater percentage of wound healing than did thecontrol group (Fig. 3A). By day 7 post-wounding,although wound healing appeared faster in the PEMFgroup than in the control group, the difference did notreach statistical significance (P¼ 0.25). By day 10, asignificant between-group difference was noticed withfaster wound healing observed in the PEMF group(Fig. 3B, P< 0.05). A highly significant difference inwound closure was observed on day 14 post wounding

Fig. 2. Graphssummarizingbasic signsof the rats in the PEMFand controlgroupsduring the ex-periment.A:Bloodglucoselevels.B:Bodyweight.

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TABLE 1. Wound Area Measured by VeV System

Wound area (cm2) Percentage of wound closure (%)

PEMF group(mean� SEM)

Control group(mean� SEM) P-Value

PEMF group(mean�SEM)

Control group(mean� SEM) P-Value

0 day 4.16� 0.07 4.24� 0.08 0.427 days 2.42� 0.30 2.72� 0.12 0.37 42.80� 6.37 34.17� 3.00 0.2510 days 0.96� 0.13 1.76� 0.30 0.03� 76.60� 3.10 57.32� 6.83 0.02�

14 days 0.13� 0.02 0.31� 0.03 0.0006� 96.73� 0.40 92.93� 0.57 0.0003�

21 days 0.04� 0.02 0.04� 0.01 0.81 98.98� 0.49 99.13� 0.25 0.80

�The P values considered statistical significant when P< 0.05.

Fig.3. Woundclosurein thePEMFandcontrolgroups.A:Representative imagesof woundstakenon days 0,7,10,14, and 21post-wounding.Graphs show the percentage of thewound closure (B)and epidermal gaps (C) in the PEMF and control groups recorded at various time points.�Representsasignificantbetween-groupdifference (P< 0.05).Scalebar¼ 5 mm.

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(P¼ 0.0003) even though the percentage ofclosure appeared very close between the PEMF(96.73� 0.40%) and control (92.93� 0.57%) groups.By day 21, the wound area and percentage of woundclosure between the two groups were very similar, asthe wounds had almost completely healed.

Similar findings were found in the measure-ments of epidermal gap in that a significant between-group difference was only found on day 10 post-wounding (Fig. 3C, P< 0.05). By day 21, the woundsof both the PEMF and control groups had epithelial-ized completely.

The representative photographs of the a-SMAimmunoreactive myofibroblasts in the PEMF groupand control group measured at each time point areshown in Figure 4A. Although a-SMA was alsointensely expressed in the smooth muscle layers of theblood vessels (and also in glands), the typical round ortubular morphology of blood vessels allows for easydifferentiation from myofibroblasts.

On day 7 post-wounding, the a-SMA immunore-active staining was detected in the granulation tissueof wounds in both groups, indicating that myofibro-blasts already existed in the first week post-wounding.

Fig.4. Myofibroblast population duringdiabeticwound healing.A: The population of a-SMA-im-munoreactivemyofibroblasts (arrows)measuredin the PEMF-treatedandcontrolgroupsat vari-ous time points. B: Graphs show the a-SMA scores of the PEMF and control wounds.�Representsasignificantbetween-groupdifference (P< 0.05).Scalebar¼ 50mm.

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While most of the wounds in the control groupexhibited scattered myofibroblasts, the wounds of thePEMF group showed significantly more myofibro-blasts in the granulation tissue. The wound tissues ofboth groups harvested on day 10 post-woundingshowed higher scores of a-SMA staining whencompared to those harvested on day 7 post-wounding(Fig. 4B), indicating that the myofibroblasts were stillin a proliferative phase. The discrepancy in themyofibroblast population between the two groupsbecame more significant on day 10 post-wounding,when myofibroblasts were observed across the entirearea of the wound in the PEMF group whereas thosein the control group appeared less abundant (Fig. 4A).Statistically significant differences in the population ofmyofibroblasts were detected between the two groupson both days 7 and 10 (P< 0.05). However, fromday 14 post-wounding onwards, no significant be-tween-group difference in the myofibroblast popula-tion were observed. On day 21 post-wounding, mostof the wounds in both groups showed no or fewmyofibroblasts. Although more myofibroblasts weredetected in the wounds of the PEMF group than in thecontrol group, the difference was not significant.

DISCUSSION

In the present study, we demonstrated that PEMFtreatment exerted no influence on the blood glucoselevel of the treated animals, but accelerated earlywound healing in STZ-induced diabetic rat modelsand increased (as shown for the first time) themyofibroblast population in the diabetic wound.

PEMF has previously been shown to producepositive effects on dermal wound healing in bothnormal and diabetic rats [Athanasiou et al., 2007;Strauch et al., 2007; Callaghan et al., 2008; Matic etal., 2009; Goudarzi et al., 2010]. Callaghan et al.[2008] and Matic et al. [2009] reported that healing inPEMF-treated wounds was accelerated as compared tocontrol wounds. However, Milgram et al. [2004] foundthat PEMF did not produce any beneficial effectson wound healing. The effects of PEMF on woundclosure varied among the studies, possibly due to thedifferent treatment protocols that were applied. Fieldintensities of 2 and 5mT were explored in our pilotexperiment with the frequency of 25Hz, yielded themost effective intensity to be 5mT for wound closure.Thus, the frequency of 25Hz and intensity of 5mTwas applied in the present study. Our study showedthat PEMF generally accelerated wound closure whencompared with the situation in the control group, andthat the difference between the groups reached signifi-cance on day 10 post-wounding. The wound area on

day 14 post-wounding showed even a higher statisticalsignificance between the groups, but it appeared thatthe significance was due mainly to the very smallSEM instead of an obvious difference in the actualwound area between the groups. However, no signifi-cant difference in wound closure was noticed onday 21. The results for the epidermal gaps were alsoconsistent with those for the wound closure data,indicating that the control group was able to catch upwith the PEMF group with regard to wound closure.These findings appear to indicate that PEMF onlytransiently improved wound closure and re-epitheliali-zation, which is consistent with the findings of earlierstudies [Athanasiou et al., 2007; Goudarzi et al.,2010]. Athanasiou et al. [2007] suggested that PEMFmight function to create a favorable environment inthe early stage of healing to facilitate tissue regenera-tion, for example by increasing the release of cyto-kines and reducing early inflammation. Here we haveshown that PEMF transiently upregulated the myofi-broblast population for healing to take place on days7–10 post-wounding, the period that partially over-lapped but preceded the time frame when an increasein wound closure after PEMF treatment was observed.Myofibroblasts are specialized types of fibroblasts thatare transiently present when tissue is damaged. Uponinjury, myofibroblasts activate and migrate into thedamaged wound site and synthesize extracellularmatrix, mainly collagen, to replenish lost tissue [Hinzet al., 2007]. An important feature differentiatingmyofibroblasts from fibroblasts is the possession ofa-smooth muscle actin in their stress fibers. Thesestress fibers confer myofibroblasts high in contractileactivity for enhancing wound closure as well asproducing tension for wound remodeling [Hinz et al.,2001; Hinz, 2007]. It is known that in rodents such asrats, the primary mechanism of wound healing iswound contraction [Davidson, 1998]. We believe thatthe PEMF treatment transiently increased the myofi-broblast population that were involved in woundcontraction, thus resulting in enhanced wound closurethrough the contractile activity of the myofibroblasts.Enhancing wound contraction would facilitate moreeffective cicatrization, although an acceleration ofwound closure at the end may not necessarily beobserved [Medrado et al., 2003].

Re-epithelialization, apart from wound contrac-tion, also plays an important role in wound healing.Although we have observed a transient reduction inepidermal gaps in the PEMF group, which implies anincrease in re-epithelialization, the reduction in epider-mal gaps could well be a result of increased woundcontraction that pulled the epidermal gaps towardseach other, to say nothing of the fact that wound

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contraction is a predominant mechanism in rodents.In order to determine if PEMF also promoted re-epithelialization (a preferential healing mechanism inhuman wounds) independent of wound contraction,further studies on the effects of PEMF using splintingto minimize wound contraction should be examined[Chung et al., 2010].

Another feature of myofibroblasts is the synthe-sis and deposition of an extracellular matrix such ascollagen into the wound site [Hinz, 2007]. Diabeticwounds are known to exhibit reduced tensile strengthdue to a defect in fibroblast proliferation, thus leadingto a decrease in collagen deposition [Hehenbergeret al., 1998; Stadelmann et al., 1998]. Recently,Goudarzi et al. [2010] reported that PEMF enhancedthe tensile strength of the dermal wound in diabeticrat models. They suggested that an increase in thesynthesis and deposition of well-aligned collagen aswell as its maturation during wound modeling canaccount for the increase in tensile strength observedin PEMF-treated diabetic wounds [Goudarzi et al.,2010]. The increase in the myofibroblast populationobserved in our study is solid evidence of the PEMF-mediated increase in the tensile strength of diabeticwounds. In fact, recent studies have revealed thatPEMF upregulated transforming growth factor-b(TGF-b) in bone [Shen and Zhao, 2010; Ding et al.,2011]. It is highly likely that transforming TGF-b, awell-known inducer of myofibroblasts [Desmoulièreet al., 1993; Dabiri et al., 2008], should also beinvolved in the increase in myofibroblasts observed inPEMF-promoted wound healing.

The present study demonstrated that PEMF didnot exert any influence on the blood glucose level ofthe treated animals. A previous study working withdiabetic wound models also found that PEMF did notaffect blood glucose level [Goudarzi et al., 2010],which is consistent with our findings. On the contrary,previous studies performed with streptozotocin-induced diabetic rats reported that PEMF could partial-ly inhibit the glucose increase in blood [Mert et al.,2010; Jing et al., 2011]. However, these studies wereperformed with diabetic rats without wound induction.It seems that wound induction might influence theeffect of PEMF on diabetic animals. In addition,the PEMF parameters adopted in these studies wereentirely different from the present protocol. Thereforeit is difficult to compare the PEMF effect on bloodglucose level in the present study with those previous-ly reported.

In conclusion, we have demonstrated for the firsttime that PEMF treatment transiently increased themyofibroblast population in STZ-induced diabeticrats, and that this could account for the beneficial

effects observed during the early phase of woundhealing.

ACKNOWLEDGMENTS

The authors are grateful for the animal husbandrysupport received from the Centralised Animal Facili-ties of The Hong Kong Polytechnic University (HongKong Special Administrative Region, China). Thiswork was supported by the General Research Fundprovided by the Research Grants Council of the HongKong Special Administrative Region Government(PolyU 5600/11M).

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Bioelectromagnetics