Cold plasmaonfull-thicknesscutaneouswoundaccelerateshealing

throughpromotinginflammation, re-epithelialization and woundcontraction

Nasruddin a,b, YukariNakajima a, KanaeMukai a, HeniSetyowatiEstiRahayu b,
Muhammad Nur c, TatsuoIshijima d, HiroshiEnomoto d,e, YoshihikoUesugi d,f,
Junko Sugama g, ToshioNakatani g,n a
GraduateCourseofNursingScience,GraduateSchoolofMedical,PharmaceuticalandH
ealthSciences,KanazawaUniversity,Kanazawa-shi,Japan b Faculty
ofHealthScience,MuhammadiyahUniversityofMagelang,56172,CentralJava,Indone
sia c Department ofPhysics,DiponegoroUniversity,Semarang,Indonesia d
ResearchCenterforSustainableEnergyandTechnology,DivisionofEnergyandEnviron
mentalTechnology,KanazawaUniversity,Kanazawa-shi,Japan e Faculty
ofMechanicalEngineering,InstituteofScienceandEngineering,KanazawaUniversity,
Kanazawa-shi,Japan f Faculty
ofElectricalandComputerEngineering,InstituteofScienceandEngineering,Kanazawa
University,Kanazawa-shi,Japan g Division
ofNursing,FacultyofHealthSciences,InstituteofMedical,PharmaceuticalandHealthS
ciences,KanazawaUniversity,Kanazawa-shi,Japan a rticleinfo Article history:
Received1October2013 Receivedinrevisedform 11December2013
Accepted8January2014 Availableonline28January2014 Keywords: Cold plasma
Full-thicknesswound Woundhealing Inflammation Woundcontraction
Myofibroblast a b s t r a c t
Weinvestigatedcoldplasmaeffectsonacutewoundsofmice.Themicewereclassified
intoexperimental and
controlgroups.Intheformer,woundsweretreatedusingcoldplasmaoncedailyfor1min
,andthen
coveredwithhydrocolloiddressing;woundsinthecontrolwerelefttohealunderhydroc
olloiddressing. Daily evaluationwasconductedfor15days.Generalandspecific
stainingwasappliedtoevaluate re-epithelialization,
neutrophil,macrophage,myofibroblast andtransforminggrowthfactorbeta.Itwas
found
thatcoldplasmaacceleratedwoundhealingby1day.Plasmamaypromotethelatephas
eof inflammation, acceleratere-epithelializationandincreasewoundcontraction. &
2014ElsevierGmbH.Allrightsreserved. 1. Introduction Cutaneous
woundhealingisacomplexphysiologicalprocess consisting
oforchestratedeventscommunicatedbycollaborative factors [1].
Theutilizationofvariousexogenousagentsfrom
naturalproductslikeIndonesianhoney [2] and oleicorlinoleic acid [3] to
physicaltoolslikelight [4] and laser [5] has beenshown to
enhancetheoverlappinghealingphases,includinginflamma-tion,
proliferationandremodeling [1]. Amongthese,wound
therapybasedoncoldplasma,thatis,non-equilibriumplasma (with
anelectrontemperaturemuchhigherthanthegastem-
perature),withalowtemperatureofionizedgas [6], hasopened the
possibilityofaparadigmshiftinbiomedicaltherapy [7]; ithas
drawnsubstantialattentionfrombothplasmaandwoundcare scientists
sinceitsfeasibilitytoworkthroughlivingtissue [8,9] and
itspotencyforresolvingproblemsincontemporarywound care weredemonstrated
[10]. Asthefourthstateofmatter [6], plasma
hastheabilitytoproducecontrollablereactivespecies,like nitric
oxide(NO)andhydroxylradicals(OH),uponcontactingthe open air [11],
aswellasOHradicalsandhydrogenperoxide(H2O2) upon
contactinganaqueoussolution [12]. Althoughtheclinical efficacy
ofcarefullycontrolledtreatmentwithcoldplasmafor killing
bacteriacolonizingchronicwounds [13,14] and improving woundhealing [15] has
beendemonstrated,therehavebeenfew studies
abouttheeffectsofcoldplasmaanditsmechanismof action
onacutewoundsinmousemodels. Re-
epithelializationandwoundcontractionaretwokeyevents in thehealingoffull-
thicknesswounds.Theformeriscentralto
woundclosure,whichiscloselyconnectedtogranulationtissue formation
inaspatiotemporalmanner [16], andthelattermay account
foruptoa40%decreaseinwoundsize,correlatedwith the
expressionofmyofibroblasts [17]. Itiswellestablishedthat Contents listsavailableat
ScienceDirect journal homepage: www.elsevier.com/locate/cpme Clinical
PlasmaMedicine 2212-8166/$-seefrontmatter &
2014ElsevierGmbH.Allrightsreserved.
http://dx.doi.org/10.1016/j.cpme.2014.01.001 n Correspondence
to:DivisionofNursing,FacultyofHealthSciences,Instituteof Medical,
PharmaceuticalandHealthSciences,KanazawaUniversity,5-11-80
Kodatsuno,Kanazawa-shi,Ishikawa-ken920-0942,Japan.Tel.: þ81 762652542; fax:
þ81 762344363. E-mail address: nakatosi@staff.kanazawa-u.ac.jp (T.Nakatani).
Clinical PlasmaMedicine2(2014)28–35
these processesareinfluenced bythepresenceofgrowthfactors like
epidermalgrowthfactor(EGF),keratinocytegrowthfactor
(KGF)andtransforminggrowthfactor(TGF) [15], whicharelikely mediated
byreactiveoxygenspecies(ROS)andNO [18–20]. Although
themechanismoftheinteractionbetweencold plasma
andcellsorlivingtissueisstillunclear [21], severalstudies
havereportedtheeffectsofcoldplasmaonkeywound-related cells orsub-
cells,includedpromotingtheproliferationof fibro-blasts [22] and endothelialcells
[23], aswellasthegrowthof epithelial cells [24], inhibitingthemigrationof fibroblasts
[25] and their surfaceexpression [26], andactivatingintegrinof fibroblasts and
epithelialcells [27]. Interestingly,someoftheseeffectsare likely
tobesimilartotheactivitiesofnaturalROSand/orNO during
woundhealing,particularlycoldplasma'seffectsonthe proliferationofboth fibroblasts
andendothelialcells [19]. There- fore,
theaimofthisstudywastoassesstheeffectsofcoldplasma on
acutecutaneouswoundhealinginaninvivoscenariowitha focus onre-
epithelializationandwoundcontraction. 2. Materialsandmethods
2.1.Coldplasmajetcharacterizationandmousewoundpositioning The
coldatmosphericpressureplasmajetsystemthatweused here
issimilartothedevicedevelopedbyTeschkeetal. [28]. Two metal
ringelectrodeswereusedaroundthequartztubeforthe cold
atmosphericpressureplasmajetsystemprovidedbythe
DivisionofElectricalEngineeringandComputerScience,Kana-
zawaUniversity,Kanazawa,Japan.Ithadaquartztubewitha
1.6mminnerdiameter.Alow-frequency(_20 kHz)AChigh voltage,withapeak-to-
peakvoltageof25kV,wasappliedtothe two
ringelectrodeswhencommercialargongas(99.995%purity) at a flow
rateof5slmwasinjectedfromoneendofthequartz tube.
Thedischargevoltageanddischargecurrentweremeasured with ahigh-
voltageprobe(P6015A;Tektronix,Inc.,Tokyo,Japan) and
acurrentprobe(8585C;PearsonElectronics,PaloAlto,CA, USA).
Theaveragepowerdensityattheelectrodewas85W/cm2 in this study. During
itstreatment,amousewoundwaspositionedabout
15mmunderthenozzleoftheplasmareactor,andwasnot touched
byitsjet.Opticalemissionspectroscopy(OES)measure- ment
atabout10mmunderthenozzleshowedtheemissionsof the OH(A-
Xtransition)transitionnear309nm,N2 (C-B transition) (band
headmaximumat337nm) [29] and ArI(maximum
763nm).Thisobservationrevealedthepresenceofbothhydroxyl
radical(OH)andnitrogen-basedreactivespeciesinthegasphase during
itsgeneration(Fig. 1). In ordertoevaluateitsthermaleffectonlivingtissue,acold plasma
jetwastestedonnormalskinofanesthetizedmouseprior to
itsapplicationonwound.Onthebasisofmeasurementwitha non-contact infra-
reddigitalcamera(F30S;NECAvioInfrared
Technologies,Tokyo,Japan)atroomtemperature(24 1C), during cold
plasmatreatment,thetemperatureoftheinfluenced skin,of non-influenced
skinandoftheendoftheplasmanozzlewere 32.3 1C, 28.8 1C and36.6 1C,
respectively.Afterthesession,injury wasnotobservedontreatedskin. 2.2.
Animalsandexperimentalprotocol
FortyBALB/cCrSlcmalemiceaged8weeks(SankyoLabService Corporation,
Inc.,Toyama,Japan)andweighing21.3–26.0 gwere used.
Theywerecagedindividuallyinanair-conditionedroomat 25.072.0 1C
withlightfrom08:45to20:45h.Waterandlabora- tory
chowweregivenfreely.Theexperimentalprotocoland animal
carewereinaccordancewiththeGuidelinesfortheCare and
UseofLaboratoryAnimalsofKanazawaUniversity,Japan (AP: 112243). 2.3.
Woundhealingmodelandplasmatreatment After
beingcompletelyanesthetizedbytheinjectionofpento- barbital
sodium(0.5mg/10gweight)intotheperitonealcavity,we held
theskinofthedorsumincludingthesubcutaneoustissue betweenthumband
finger,foldeditalongtheapexofthemedian line onthedorsuminaU-
shape,putbothsidesoftheskin
together,madetwoholesthroughtheskinwithasteriledispo- sable
2mmbiopsypunch(KaiIndustriesCo.Ltd.,Gifu,Japan)and
finallymadetwocircular(2mmindiameter)full-thicknessskin
woundsincludingthepanniculuscarnosusmuscleandapartof the
subcutaneoustissueonbothsidesofthedorsumofthemouse.
Subsequently,themicewererandomlyclassified intotwogroups: (1)
experimentalgroup,withwoundstreatedoncedailybyacold plasma
jetduring1mininonespotonthewound,andthen
coveredbyhydrocolloiddressing(Tegaderm;3MHealthCare,
Tokyo,Japan)tomaintainitsmoistenvironment;and(2)control group,
withwoundsonlyallowedtohealunderhydrocolloid dressing. 2.4.
Macroscopicevaluation The daywhenwoundsweremadewasdesignatedasday0,and
the processofwoundhealingwasobserveddailyfromdays0to15
afterwounding.Beforeobservation,thesurroundingenvironment of
woundswascleanedwithsalinesolution.Woundededgeswere
tracedonpolypropylenesheetsandphotographsweretakenevery
day.Thetracesonthesheetswerecapturedwithascannerontoa personal
computerusingAdobePhotoshopElements7.0(Adobe
SystemInc.,Tokyo,Japan),andtheareasofwoundswerecalcu- lated
usingimageanalysissoftwareScionImageBeta4.02(Scion
Corporation,Frederick,Maryland,USA). 2.5. Calculationofhealingday The
dayofwoundhealingwascalculatedbasedonagraphof the
ratiosofareastooriginalareas.Initially,theoveralltrendof such
agraphwasevaluated.Woundhealingdaywasplottedon the y-axis
whenthetrendofreductionofwoundsizestartedto become flat,
whichwasat0.15.012345200300400500600700800900Intensity (arb.
units)Wavelength (nm)OH (A-X)N2(C-B)ArOH 309 nmN2 337 nmAr 763 nm
Fig. 1. Optical emissionspectroscopy(OES)measurementofcoldplasmajetnear the
woundsurface(about10mmunderthenozzleofthecoldplasmareactor) during
treatment.OHandnitrogen-basedreactivespecieswereidentified. Nasruddin
etal./ClinicalPlasmaMedicine2(2014)28–35 29
2.6. Histology The micewereeuthanizedbyamassivepentobarbitalsodium IP
injectionondays3,7,11or15post-wounding.Thewoundsand the
surroundingnormalskinwereexcisedforanareaofabout 10_10 mm2,
stapledontopolypropylenesheetstopreventover- contraction ofthesamplesand
fixedinneutralbuffered10% formalin
solutionin0.01Mphosphatebuffer,pH7.4,forabout
15h.Thesampleswerethenrinsedin0.01Mphosphate-buffered saline
(PBS)forabout8h.Subsequently,theyweredehydratedin an
alcoholseries,cleanedinxyleneandembeddedinparaffin to prepareserial5-mm
sections.Next,thesectionswereseparated into
twogroups:forgeneralstainingusinghematoxylin-eosin (H&E) forre-
epithelializationobservation,andforspecific staining using
immunohistochemicalstaining. 2.7. Immunohistochemistry(IHC)
Immunohistochemicalstainingformyofibroblastswascon-
ductedforthesectionsfromalldaysofmouseharvesting(days3,
7,11and15),whilethatformacrophages,neutrophilsandtrans-
forminggrowthfactorbeta(TGF-β) wasonlyconductedforthe
sectionsfromdays3and7ofmouseharvestingbecauseofa
limitationinthenumberofsections.Inbrief,theimmunohisto-
chemicalstainingwasperformedasfollows.Afterdeparaffinization
andrehydration,antigenunmaskingwasaccomplishedbyheating
slidesinawaterbathfollowedbyincubationinsodiumcitrate
buffer(10mMsodiumcitrate,0.05%Tween20,pH6.0)for20min at approximately100
1C. Then,theslideswerewashedwithPBS,pH
7.4,coveredwith0.03%hydrogenperoxidetoblockendogenous
peroxidasefor5minatroomtemperature,rinsedwithdistilled
water,coveredwithprotein-freenormalserumfor10minatroom
temperatureandthenrinsedwithPBS.Subsequently,sectionswere
incubatedwithprimaryandsecondaryantibodiesasfollows: a.
Forneutrophilidentification: Sectionswereincubatedwithanti-
neutrophilantibody(AbcamJapan,Tokyo,Japan)atadilutionof 1:100inPBSat4 1C
overnight,andthenwithsecondaryantibody polyclonalrabbitanti-
ratimmunoglobulins/HRP(Dako,North
AmericaInc.,CA)þmouseserum(DakoNorthAmericaInc.,CA)
(1:100inPBS)atroomtemperaturefor30min. b. Formacrophageidentification:
Sectionswereincubatedwithanti- macrophage-
3antibody(AbcamJapan,Tokyo,Japan)atadilution of 1:100inPBSat4 1C
overnightandthenwithsecondary antibodypolyclonalrabbitanti-
ratimmunoglobulins/HRP(Dako,
NorthAmericaInc.,CA)þmouseserum(DakoNorthAmericaInc., CA)
(1:100inPBS)atroomtemperaturefor30min. c. Formyofibroblast identification:
Sectionswereincubatedwith anti-α-smooth muscleactin(anti-α-SMA)
(AbcamJapan,Tokyo, Japan) (1:100inTween-PBS)at4 1C overnight,andthen with
secondaryantibodyDakoEnVisionþSystem-HRPlabeled polymeranti-
mouse(DakoNorthAmericaInc.,CA)atroom temperaturefor30min. d. ForTGF-β
identification: Sectionswereincubatedwithanti-TGF- β (1:100inPBS)at4 1C
overnight,andthenwithsecondary antibody DakoEnVisionþSystem-
HRPlabeledpolymeranti- mouse
(DakoNorthAmericaInc.,CA)atroomtemperaturefor 30 min.
Aftercompletionoftheincubationwiththesecondaryantibody, the
sectionswerereactedwith3,30-diaminobenzidinesubstrate (Dako
ENVISIONKit/HRP(DAB),DakoJapan,Kyoto,Japan)forstaining for about2–
5minatroomtemperature.Finally,counterstainingwas
conductedusinghematoxylin.Asnegativecontrols,sampleswere prepared
usingthesameprocedurewithPBSinsteadofthe first antibody. 2.8.
Microscopicobservations On thebasisoftheresultsofhematoxylin-eosinstaining, the
percentageofre-epithelializationwascalculatedasfollows: 100%_(length
ofnewepithelium/lengthofwoundbetween
woundedges).Inaddition,onthebasisoftheresultsofimmuno-
histochemicalstaining,thenumbersofneutrophils,macrophages,
myofibroblastsandcellsstainedwithTGF-β through observation using
anOlympusBX50lightmicroscope(Olympus,Tokyo,Japan) at magnification 400_
werealsocounted.Imageswerecaptured with
anOlympusDP72digitalcameraandOlympusDP2-BSW
software.Threesquareswereselectedateachwoundmarginand the
centerofthewound,onfourserialsectionsperwound.The data
arepresentedasthemeannumberofstainedcellscounted in
the12squares;fourserialwoundsectionsperwoundwere analyzed. 2.9.
Statisticalanalysis Data weresubjectedtostatisticalanalysesusingSPSS16.0.
Differencesbetweentheexperimentalandcontrolgroupsforthe
ratioofwoundareaaveragetooriginalwoundareaandthe calculation
ofhealingdaywereevaluatedbytwo-tailedunpaired t-testsand P-values o0.05
wereconsideredsignificant. Differ- ences
betweentheexperimentalandcontrolgroupsfortheresults of
histologicalstainingwereevaluatedbyANOVAfollowedbythe Tukeytestand P-values
o0.05 wereconsideredsignificant. 3. Results 3.1.Macroscopicevaluation
Immediatelyaftercoldplasmairradiation,thewoundsurfaces in
theexperimentalgroupseemeddrierthanthoseinthecontrol group.
Woundswereobserveddaily(Fig. 2). Therewereno particular
differencesregardingtheappearanceofthewound surfaces
betweentheexperimentalandcontrolgroupsduringthis
observation.Apparentdifferencesjustinthewoundsizewere found
forseveraldaysofthisobservationperiod.
Woundsinboththeexperimentalandthecontrolgroups
experiencedslightexpansion(edema)onday1andthen
decreasedgraduallyuntiltheendoftheobservationperiod.On days1–3,
therewasexudateonthesurfaceofthewoundsinthe
twogroups.Thewoundareaoftheexperimentalgroupwas smaller
thanthatofthecontrolfromdays4to15.Fromdays 5
to15,thesurfacesofthewoundsinallgroupsweremostlyfresh with noexudate. 3.2.
Woundareaevaluationanddayofwoundhealing The
ratiosofwoundareastooriginalareasfromday0both until
day15anduntilthedayofwoundhealingweredetermined (Fig. 3a).
Ondays3,4,5,6and8,theratiosofareastotheoriginal2 mm 2 mm Control group
Experimental group Day 7Day 11 Day 15 Day 3 Day 0
Fig. 2. Macroscopicobservationofwoundhealing. Nasruddin
etal./ClinicalPlasmaMedicine2(2014)28–3530
Fig. 3. (a)
Ratioofareastooriginalareasduringwoundhealing.Ondays3,4,5,6and8,thetreatedwou
ndwassignificantly smallerthanthatofthecontrol.(b)Dayof wound
healing.Experimentalwoundshealedsignificantly
faster,byoneday,thanthoseofthecontrol.Control (3d)Experiment (3d)Control
(3d)Experiment (3d)
Fig. 4. (a) Percentageofre-epithelializationduringwoundhealing.
(b)Newepithelialimageonday3ofwoundhealing:
(1and3)HEstainingoftheexperimental group. (2
and4)HEstainingofthecontrol.AB,CD,EFandGHshowthelengthsofnewepithelium.IJ
andKLalsorevealthelengthsofnewepitheliumatmagnification 200_. Re-
epithelializationincreasedmorerapidlyintheexperimentalgroupthaninthecontrol.
Nasruddin etal./ClinicalPlasmaMedicine2(2014)28–35 31
areafortheexperimentalgroupweresignificantly lowerthan those
forthecontrolgroup(Po0.05). Ontheotherhand,ondays 9–
15,theratiosofareastotheoriginalareafortheexperimental group
weremostlythesameasthoseforthecontrolgroup (P40.05).
Daysofwoundhealingfortheexperimentalandcontrol groups were8.070.6
and9.071.3,respectively(Fig. 3b). These two meansweresignificantly
different(P¼0.04). 3.3. Coldplasmaacceleratedre-epithelialization Re-
epithelializationduringwoundhealingwasobserved(Fig. 4). On
day3,thepercentagesofre-epithelializationweresignificantly
differentbetweentheexperimentalgroupandthecontrolgroup
(Po0.001),withtheformerbeingmorethan25%greaterthanthe
latter.Thepercentagesofre-epithelializationofbothincreased
dramaticallyfromdays3to7(controlgroup: Po0.001;experimen- tal group:
Po0.001).Onday7,thepercentagesofre-epithelialization of
thetwogroupsweresimilar(P¼0.999).Ondays11and15,all
woundsinallgroupswerecoveredbynewepithelium. 3.4. Myofibroblastcount
Myofibroblast numberonday3wasevaluated.Onthisday,a few myofibroblasts
wereobservedintheexperimentalgroup.On the
otherhand,nomyofibroblastswereobservedinthecontrol. Myofibroblasts
werecountedondays7,11and15(Fig. 5). The number ofmyofibroblasts permm2 in
theexperimentalgroup peaked onday7andthendecreasedgraduallyuntilday15, while
thatinthecontrolwasstableondays7and11andthen
decreasedonday15.Onday7,thenumberofmyofibroblasts in the
experimentalgroupwashigherthanthatinthecontrolgroup, but
thetwomeanswerenotsignificantly different(P¼0.557). On
theotherhand,ondays11and15,thenumbersofmyofibro- blasts
intheexperimentalgroupwereslightlylowerthanthosein the
control,butthetwomeanswerenotsignificantly different (11days: P¼0.990, 15days:
P¼0.994). Onday15,thenumbersof
myofibroblastsintheexperimentalgroupandthecontrolwere
lowerthanthoseonday7.Thetwomeansintheformergroup
weresignificantlydifferent(Po0.001),butthoseinthelatterwere not (P¼0.148). 3.5.
Neutrophilcount Numerousneutrophilswereobservedintheexperimentalgroup
andthecontrolgroupondays3and7afterwounding(Fig.6). On
day3,thenumbersofneutrophilspermm2 in theexperimental
andcontrolgroupswererelativelysimilarandwerenotsignifi-
cantlydifferent(P¼0.966).Onday7,thenumberofneutrophilsin
theexperimentalgroupwaslowerthanthatinthecontrolgroup,
butthetwomeanswerenotsignificantlydifferent(P¼0.762).TheFig. 5. (a)
Histogramofmyofibroblast numberondays7,11and15.Byday7,myofibroblast
numberintheexperimentalgroupwasgreaterthanthatinthecontrol,butby day
15,theformerwaslowerthanthelatter.(b)Alpha-
SMAstainingondays7and15.Blackarrowsshowmyofibroblasts
coloredbrown.Control ( 7 days) Experiment ( 7
days )20μm20μm20μm20μmExperiment (15 days)Control (15 days)
Experiment (7days)Experiment (3days)Control (7 days)Control (3 days)20μm20μm20μm20μm

Fig. 6. (a)
Histogramforneutrophilnumberondays3and7.Neutrophilnumberoftheexperimentalg
roupwassignificantly lowerthanthatofthecontrolatday7. (b)
Immunohistochemicalstainingforneutrophils.Blackarrowsshowneutrophilscoloredb
rown. Nasruddin etal./ClinicalPlasmaMedicine2(2014)28–3532
numberofneutrophilsinthetwogroupsdecreasedrapidlyfrom
days3to7(experimentalgroup: Po0.05;controlgroup: Po0.05). 3.6. Macrophagecount
Numerousmacrophagesintheexperimentalgroupandthe control
groupwereobservedondays3and7afterwounding (Fig. 7).
Onthesedifferentobservationdays,themacrophage number permm2 in
theexperimentalgroupwaslowerthanthat in
thecontrol,butthetwomeanswerenotsignificantly different (day 3: P¼0.203; day7:
P¼0.676).Macrophagenumbersinthe control groupdecreasedsignificantly
fromdays3to7(Po0.05). Macrophagenumberintheexperimentalgroupalsodecreased
from days3to7,butthetwomeanswerenotsignificantly different (P¼0.913). 3.7.
CountofcellsstainedwithTGF-beta
Numerouscellsstainedwithtransforminggrowthfactorbeta (TGF-β)
wereobservedintheexperimentalgroupandthecontrol group
ondays3and7afterwounding(Fig. 8). Onday3,the number ofcellswithTGF-β per
mm2 in theexperimentalgroup
waslowerthanthatinthecontrolgroup,butthetwomeanswere not significantly
different(P¼0.688).Incontrast,onday7,the number ofcellswithTGF-β in
theexperimentalgroupwasslightly higher
thanthatinthecontrolgroup,butthetwomeanswerenot significantly
different(P¼0.897).ThenumbersofcellswithTGF-β in
theexperimentalgroupweresimilarondays3and7(P¼0.991). On
theotherhand,thenumberofcellswithTGF-β in thecontrol group
decreasedfromdays3to7,butthetwomeanswerenot significantly different(P¼0.209).
4. Discussion This researchdesignseparatedbetweentreatedanduntreated
micebecauseitwasconsideredthatcoldplasmaproducednotonly
reactivespecieslikenitricoxide(NO)andhydrogenperoxide(H2O2)
thatinappropriatedosagemayhaveefficacyforwoundhealing [20],
butalsothattemperaturechangemayhavethesameeffect [30].
Whiletheformermayworkinalocallyspecific manner [25], the
lattermayoperateataphysiologicallysystematiclevelundera hypothalamicregime
[31]. Inthisdesign,thereweretwowoundsina mouse,
ontheleftsideandtherightside.Whentheleftwoundwas
subjectedtoplasmatreatment,therightwoundmayalsohavebeen
influencedbyitswarmth.Therefore,itwasimportanttoensurethat
therewasnoinfluenceofplasmaagentsintheuntreatedmice.
Althoughthisapproachwasintendedtomimictheclinicalsettingin a
hospital,itdifferedfromtheworkofHeinlinetal. [15], whoplaced twointhesamepatient.
On thebasisofhistologicaldatafrom3daysafterwounding, we
showedthatcoldplasmaisefficacious fortheaccelerationof woundre-
epithelialization.This finding seemedtobeinlinewith
Nastutaetal.andHeinlinetal.Nastutaetal.reportedthathelium cold
plasmatreatmentacceleratedthere-epithelializationofburn woundtissue [32].
Furthermore,byaninvestigationofclinical
standardizedphotographsofwoundsonaskingraftdonorsite, Heinlin
etal.reportedthatargoncoldplasmatreatmenthada positiveeffect [15].
However,therewerenohistologicaldatain these tworeports.Thisisthe first
reportdescribingsuch findings on thistopicsupportedbythehistologicaldata. In
thepresentstudy,wefoundthatcoldplasmatreatment caused
accelerationofwoundhealingbyonedaycomparedwith that
ofuntreatedwounds.Suchreductionintheperiodrequired for
woundhealingmaybecorrelatedwiththeearlypresenceand the peakofmyofibroblasts
inthecoldplasma-treatedwoundsby
days3and7afterwoundcreation,asweobserved.Itiswell established
thattherearemultiplewaysbywhichmyofibroblasts originate,
oneofwhichisthroughthedifferentiationfrom Fig. 8.
Histogramforthenumberofcellsstainedwithanti-TGF-β antibody ondays 3
and7.Experiment (7 days)Experiment (3 days)Control (7 days)Control (3
days)20μm20μm20μm20μm

Fig. 7. (a)
Histogramformacrophagenumberondays3and7.Macrophagenumberoftheexperimen
talgroupwassignificantly lowerthanthatofthecontrolondays3and 7.
(b)Immunohistochemicalstainingformacrophages.Blackarrowsshowmacrophagesco
loredbrown. Nasruddin etal./ClinicalPlasmaMedicine2(2014)28–35 33
fibroblaststomyofibroblasts mediatedbyactivatedtransforming
growthfactorbeta(TGF-β) [33]. Coldplasmamayplaytwomain roles ininfluencing
thismechanism:(1)promotingtheprolifera- tion of fibroblasts
onthewoundsurface,inlinewithanother reportedstudy [21] in
whichthehigherthenumberof fibroblasts, the
possiblyhigherthenumberofmyofibroblasts; and(2)activat- ing “latentTGF-β”on
thewoundsurfacetobecome “activeTGF-β”, which mayincreasemyofibroblasts.
Thepossibilityofcoldplasma activatingTGF-β may
involveoneorbothoftwopossiblemechan- isms asfollows.The first oftheseisROS-
basedactivation,as described previously [34,35]. ROSinthiscontextaregeneratedby
cold plasma.Secondly,itcouldinvolveintegrin-basedactivation by
coldplasma,asalsoreportedelsewhere [26]. The activationofTGF-β may
bedetectedbyanincreaseofthe cells stainedwithanti-TGF-β
antibody,butinthisresearch,we found nosignificant
differenceinthenumberofcellswithTGF-β
betweentreatedanduntreatedwoundsondays3and7after
wounding.Thismaybeinlinewithareportmentionedpreviously that
statedthattheconventionalhistologicalmethod,asused in
thisresearch,couldnotclearlydifferentiatebetweenlatent TGF-β and activeTGF-β
[36]. Thus,itisdifficult todetermine whether immunohistochemicalstainingofTGF-β,
asinthis research,wasassociatedwithlatentTGF-β, activeTGF-β, orboth. In
thehealingofnormalwounds,inflammation isclassified into
earlyandlatephases.Neutrophil-richandmononuclear-cell- rich
infiltratesarerepresentativeoftheformerandthelatter, respectively [1].
Inthisresearch,theobservationthatby3days
afterwoundcreation,therewasthesamenumberofneutrophils in
theexperimentalandcontrolgroupssuggestedthattherewas no
differenceinthetimecourseoftheearlyphaseofinflammation
betweenthem.Itshowedthatcoldplasmahadnoeffectonthe earlyphaseofinflammation.
Atalaterstage,theobservationin this researchthatby7daysafterinjury,therewerefewer
macrophagesandneutrophilsintheexperimentalgroupthanin the
controlsuggestedthattherewasanaccelerationofthe
inflammatoryphaseofrepairintheexperimentalgroup,sothat the
latephaseofinflammation endedrapidly.Sofar,ithasbeen
revealedthatcoldplasmatreatmentacceleratedsuchaprocess. It
hasalsobeenindicatedthattreatedwoundsstartedthe
proliferationphaseearlierthanuntreatedones.Thisphenomenon may
becorrelatedwiththeproliferativeeffectofcoldplasma,as reportedpreviously [22],
andtheearlypresenceofmyofibroblasts, as reportedinthisresearch. It
iswellknownthattheoutcomeofacuteinflammation is elimination
ofthenoxiousstimulus [37]. Inthiscontext,Shekhter et
al.discussedthepossibleinfluence ofplasma-basedNOonthe
phagocytosisofmacrophagestoimprovetheregenerationof wounds [38].
Theoretically,acuteinflammation hastwomajor components:
vascularchangesandcellularevents [37]. Regarding the
former,plasmamayplaytheimportantroleofinfluencing
vasculargrowth,vasculardilation,andmicrocirculationnormal- ization,
asdiscussedpreviously [38]. Regardingthelatter,the direct
effectofcoldplasmaontheeventsofinflammatorycellslike
macrophageshasbeenunclear,butaswewrotepreviously,itwas
reportedthatcoldplasmareducedthemigrationof fibroblasts [25] and
activatedtheirintegrin [27]. Migrationandintegrinactivation
arecrucialeventsforfamilymembersofleukocytesduringthe
inflammatoryphase.Iftheeffectsofcoldplasmaon fibroblasts and
thoseoninflammatorycellsarethesame,thiscouldbeused to
demonstratethemechanismofthehealingeffectofcoldplasma during
thelatestageoftheinflammatoryphase. Cold
plasmatreatmentischaracteristicallytopical,withthe maximum
penetrationdepthofitsreactiveoxygenspecies(ROS)/
reactivenitrogenspecies(RNS)beingatmostafewtensof micrometers [39].
Fromacellularstudy,itwasalsoreportedthat the effectsofcoldplasmawereconfined
totheareathatcanbe reachedbyROS/RNS [25]. Inthepresentresearch,itisunclear
whether reactivespeciesofacoldplasmajetcoulddirectlyreach cells
relatedtowoundcontraction,myofibroblasts and fibroblasts.
However,ifthiscouldnotbeachieved,thehypothesisofHeinlin et
al.shouldbeconsidered.Heinlinetal.hypothesizedthatthe effect
ofcoldplasmatreatmentwouldprobablyoccurnotonlyin the
treatmentarea,butalsointheadjacentwoundareathrough
microenvironmentalmodification [15]. In
thisresearch,wedidnotdetectreactivespeciesofcold plasma
atadistanceof15mmunderthenozzleusinganOES
spectrophotometer,butwedetectedslighttemperaturechangeon normal
skinofanesthetizedmiceatsuchadistanceusingan
infraredthermalcamera.Ofcourse,thedetectionofsuchreactive species
couldbeachievedbyothermethods,buttheslight
temperaturechangeshouldalsobeconsideredifamicroenviron- mental
perspectivewereapplied. In conclusion,itwasdeterminedthatcoldplasmaaccelerated
woundhealingbyonedaythroughthemodification ofre- epithelialization,
thelatestageofinflammation andwoundcon- traction.Coldplasmamayinfluence
thewoundhealingmechan- ism
atthemicroenvironmentallevelthroughnotonlyitsreactive species,
butalsoitswarmingeffect,simultaneously. Conflict ofinterest The
authorshavenoconflicts ofinteresttoreportinregardto this manuscript.
Acknowledgments Nasruddin wouldliketoacknowledgethehelpoftheDirecto-
rateGeneralofHigherEducation(DIKTI),Indonesia,whichsup- ported him
financiallyduringhisPh.D.studythroughtheJoint Scholarship
ProgramDIKTI,Indonesia-KanazawaUniversity,Japan. Part
ofthisworkwassupportedbyGrants-in-AidforScientific
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