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Acousto-mechanical behaviour of ex-vivo skin: Nonlinear and viscoelastic properties

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HAL Id: hal-02009152

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Acousto-mechanical behaviour of ex-vivo skin:

Nonlinear and viscoelastic properties

Halima Ghorbel-Feki, Ali Masood, Michael Caliez, Michel Gratton, Jean Christophe Pittet, Martin Lints, Serge dos Santos

To cite this version:

Halima Ghorbel-Feki, Ali Masood, Michael Caliez, Michel Gratton, Jean Christophe Pittet, et al..

Acousto-mechanical behaviour of ex-vivo skin: Nonlinear and viscoelastic properties. Comptes Rendus Mécanique, Elsevier, 2019, 347 (3), pp.218-227. �10.1016/j.crme.2018.12.005�. �hal-02009152�

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Contents lists available atScienceDirect

Comptes Rendus Mecanique

www.sciencedirect.com

Acousto-mechanical behaviour of ex-vivo skin: Nonlinear and viscoelastic properties

Halima Ghorbel-Fekia,b,c,Ali Masooda,b,c,Michael Calieza,b, Michael Grattona,b,Jean Christophe Pittetc,Martin Lintsa,b, Serge Dos Santosa,b

aINSACentreValdeLoire,3,ruedelaChocolaterie,CS23410,41034Bloiscedex,France bUMR1253,ImagingandBrain:iBrain,UniversitédeTours,INSERM,Tours,France cOrionConcept,113,ruedesBordiers,37100Tours,France

a rt i c l e i n f o a b s t ra c t

Articlehistory:

Received15October2018 Accepted20December2018 Availableonline1February2019

Keywords:

Skin

Strengthtensiletest TR-NEWSmeasurements Hysteresis

Nonlinearity Viscoelasticity

Themechanicalbehaviourofskinissignificantforsomeapplicationsincludingdermato- logy,surgery, and impact biomechanicsscience. Inthis work,we have investigated the studyof the acousto-mechanical viscoelastic properties of skin. For that, both tensile- relaxation and ultrasonic tests were conducted on porcine tissue samples in fibre directions.Tounderstandthecomplexskinagingphenomena,weusedstrengthtensiletest correlatedwiththeNonlinearTimeReversalsignalprocessingtoolextension“TR-NEWS”.

Uniaxialtensiletestswerecarriedoutatastrain rateof5·103mm s1 onskinusing aload-relaxation-discharge loadpathwithincreasingamplitudeand offset.Thiswork is alsounderway toextend thefrequency rangeofultrasoundsto50 MHz.Digital Image Correlationwas used for 2D strain measurementofthe dermis. Fromthisanalysis, we concludethatfreshporcineskinshouldbemodelledasanonlinearviscoelasticmaterial withstrain-ratedependence.Theobtainedhysteresisloopshallbetakenassignificantskin damage.

©2019Académiedessciences.PublishedbyElsevierMassonSAS.Thisisanopenaccess articleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Skinisa livingcomplextissue whichcontainsheterogeneouslayer:epidermis, dermis,andhypodermis.Theepidermis consistsofcells andcellulardebris, thedermis consistsofmostly networksoffibrous proteincollagen, withinterspersed elastin, andreticulin [1], andthe hypodermisisprincipally madeup ofconnective tissue andfat lobules. Collagen fibers accountfor75%ofthedryweightofthedermaltissue[2].Thedamagetothesefibersisresponsibleforskinaging.Previous studies suggested that the deformation characteristics of skin are very complex [3,4]. The skin tissue is an anisotropic, nonlinear,viscoelasticmaterialunderlittleincompressibledeformation[5].

Skinanisotropicity wasrecognized by Langer,whomapped thenaturallinesoftensionthat occurwithin theskin[6].

TheselinesareknownastheLangerlines.Atensiletestshowedthatthemechanicaldeformationofskinisdependentupon thespecimen’sorientationalongLangerlines[7].Mostrecentstudieshaveusedopticalcoherenceinvivoimagestoindicate a large differencebetweenthe Youngmoduli ofskinalong theparallel andorthogonaldirections oftheLanger lines[8].

E-mailaddress:ghorbel.halima@gmail.com(H. Ghorbel-Feki).

https://doi.org/10.1016/j.crme.2018.12.005

1631-0721/©2019Académiedessciences.PublishedbyElsevierMassonSAS.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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J.BenítezandF.J.Montáns haveestablishedan anisotropicmodeltodescribe themechanicalpropertiesofhumanskinin uniaxialtensionalongloadaxesinthe0,45,and90directions[9].Grovesetal.[10] developedexvivotestsoncircular samplesofbothmurineandhumanskin.Thesetestsareconsideredassignificantbecausetheywereachievedindifferent directionsforthesamespecimentostudytheanisotropicbehaviour ofskinandwerenumericallyanalyzedin[11].

Astress–strain skincharacteristicofsome previous works displays nonlinearbehaviour anddescribesthe response of skintodeformation[12–14].Accordingtothesestudies,intheloadingphase,skinisverycompliantandlargedeformations occurforrelativelylowappliedloads.Atthisstage,thefibersarelargelyunaligned.Afterward,skinstiffnessprogressively increasesasthe fibers alignthemselves underthe loadapplied. The stiffness ofskinincreases continuously,rapidly,and linearlyuntilthecollagenfibersbecomemostlyaligned.Atthisstage,theoverallmechanicalresponsebecomesdependent onthemechanicalpropertiesofthecollagenfibers,whicharestifferthanthoseofelastin.Aswasreportedbysomeauthors [15,16],thedirectionalbiasofcollagendistributionisresponsiblefortheanisotropicbehaviour ofskin.TheYoungmodulus of collagenis about 1.0–10 GPa. Furthermore,the modulus of elastin isabout 100kPa, i.e.several orders ofmagnitude lessthanthatofcollagen[17].Elastinaccountsfor4%ofthedermisvolumeandispresentedasthinstrandattachedtothe collagenfibers[15].Elastinandcollagenarecross-linkedtohyaluronicacidembeddedinthegroundsubstance’samorphous material,averyviscous,thixotropicsemi-fluid[18].

Severalworkswerefocusedonthedeterminationoftheviscousmechanicalbehaviour ofskin.Therheologicalproperties are physiologicallybased,primarily,onthepreviously mentioned viscousnature ofthematrixandalsoon itsinteraction withthefibers,aswellasontheinter-fiberextensions[19].

Theviscoelasticityofskinisdependentonstrainlevel,rate,andtemperature[20].Astrain-leveldependenceofrelaxation functionsisfound insome biologicaltissuessuch asskin[18].Anisotropyalsoplaysan interestingrole intheviscoelastic propertiesofskin[21].Viscoelastic behaviourinskinatsmalldeformation levelsisfrequentlymeasured throughdynamic methodslikewavepropagation,permittingalsoananisotropy analysis[22].Aswassuggestedbypreviousauthors[23,24], themaincontributorstotheviscoelasticbehaviour ofskinarethecollagenfibersandtheinteractionbetweencollagenand matrix.

Inthisstudy,we providenewexperimentaldataaboutex-vitroporcineskin,focusing,inparticular,ontheviscoelastic andanisotropicpropertiesofskin.Wealsocorrelatedthetypicaluniaxialtensiletestswithclassicalmedicalultrasonicimag- ingorNon-DestructiveTesting(NDT)toquantifytheskindeformation degreesandtounderstandthecomplexmechanical propertiesofsofttissues.

Althoughsomerecentresearches onthemechanicalpropertiesofskinreport invitroandinvivoexperiments[25,26], thereareonlyvery limitedworksconcerningthedeformation cycleallowing onetoevaluatetheviscoelasticitybehaviour ofskinusingsynchronizationwithanovelsetupformechanicalloadingandultrasonicmeasurements.

Inthiswork,wehavelimitedourexperimentstoinvitrouniaxialtensiletests.Itwasdecidedtoperforminvitrotensile tests withporcine tissues for two important reasons.Primarily, thistest provides simple stress–strainrelationships that canbe easily modelledandquantified withboundaryconditionsthat arewell defined.Secondly,inthiswork thefurther dynamic responseuntilfatigue andfailure istested. Manyinvitrostudiesusehumanskinsubstitutes suchassiliconeor polyurethane[27,28] andinvitrotestsonnaturalsoftskintissuearemainlylimited.Thisstudyaimstoprovidenewma- terialdataforporcineskinwhichcanbe appliedtoconstitutivemodelsinanumberoftechnicalareassuch ascosmetics, surgicalsimulation,forensicpathology,andimpactbiomechanics.Simpletensiontestsremainimportantbecausetheyserve toevaluatethelevelofanisotropy.However,uniaxialtensiletestsalonearenotenoughtodeterminemulti-dimensionalma- terialmodelsforsofttissues.Tounderstandthiscomplexmechanicalpropertyandthememoryeffectsthatareresponsible forthematerials’aging,classicalmedicalultrasonicimagingorNon-DestructiveTesting(NDT)wasinvestigated.

Multi-modalbasedimagingapproaches havethepotential to imagesuch nonlinear informationasalreadyestablished inNDT.Thenonlinearsignature ofaginghasbeenmeasuredrecentlyinseveralexperimentsandconfigurations[29].Time Reversal (TR)-based NEWS (Nonlinear Elastic Wave Spectroscopy) methods have the potential to become powerful and promising toolsforthe NDT industry [30].The systemic approach ofthe TR-NEWSultrasoniccomplexity is unclear gen- eratedinnovationformedicalapplicationswithextended experimentalresultsdevelopedforecho-dentographyonhuman teeth[31] ortolocateanddestroyakidneystoneinthebodyduringlithotripsytreatment[32–34].Somestudiesincludea nonlinearanalysisofhystereticbehaviour.TheassociationbetweenTR-NEWSandMTSloadingset-upsenablestomeasure the nonlinearity of porcine skin. Digital Image Correlation (DIC) is used to evaluate the deformation field of the longi- tudinal andtransversal strain directions.The repeatable measurement resultspresented inthis work show a progressive deteriorationofthemechanicalproperties,confirmedbytheincreasing numberofhysteresiscycles,whicharefollowedby relaxation.

Theobjectiveaimofthispaperistoprovidean acousto-mechanicalmethodformeasuringtheelasticity,viscoelasticity, andrelaxationparametersofaninvitroskinsample.

2. Materialsandmethods 2.1. Specimenpreparation

Theusedporcineskinisobtainedfromtheabdomenareaofthesamedomesticpig.Fiveskinsampleswereexcisedfrom samesubject.withascalpelandcutintoarectangularshapeinthedirectionoftheskinfibers.

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Fig. 1.Posterior porcine skin in uniaxial tension along a loading axis at 0with respect to Langer’s natural lines.

The adipose tissue of each specimenwas then carefully removed usinga scalpel.The thickness ofskin afterremoval of adipose tissue was measured to be between3 and4 ± 0.2 mm.The dimensions of thetest posterior sampleswere measuredbeforeexcision.Allsamplesare150×40×3 mminsize.Atotalofthreespecimensweretestedsuccessfully.In accordancewithEshelandLanir’sstudy[35],theusedskinswerethereforenotpre-conditionedpriortotesting.

Fig. 1gives theorientation details of theloading axesused during the tensiletests, commonforposterior specimens fromporcineskin.

2.2. Tensiletests

The tensile tests, performedat 0 withrespect to Langer’s naturallines,were achievedusing a universal tensiletest machine“AnInstron8800”.Thesampleswereclampedusingspeciallydesignedanti-slipclampstocounteractthetendency ofsamplesto slipin ordinarygrips.The velocityofthecrossheadwas 0.5 mm/s.A500-Nloadcellwas usedtomeasure displacement andtensileload. Exceptforthefirstcycle, theloading starts wheneverazero forcelevel isreachedduring unloading.Itisnotedthatallthetestsperformedonthevarioussampleswerecompletedwithin3 htoensurethefreshness oftheskintissue.Theloadwassetto0beforestartingthetest.Thestrainratewasappliedfortheloadingandunloading steps.Theloadingstepsareperformeduntilaconstantmaximumimposedstrainrate(10%)isreached.Thestrainratesand thedisplacementsaredirectlyimposedthroughthemachine’scrosshead.Thesampleswereclampedusingspecialanti-slip clamps.Thevelocityofthecrossheadwas0.5mm/s.Theresultsareobtainedintermsofforce–displacement.Thenominal stress P andtheengineeringuniaxialstrain εaregivenbytherelations

P=F/S0(1)

ε=(D D0)/D0 (1)

where F istheappliedforce andS0 istheinitialarea ofthecrosssection oftheskinsamples, D and D0 arethecurrent andinitiallengthsofthesample.

Eachtensiletestwasrecordedwithadigitalvideocameratorecordanyirregularbehaviourduringthetensiletestand forthefollowinguseofDIC.Thegaugelengthandwidthwerebothmeasuredoptically.

Themainfocusofthisworkisaninvestigationofthehyperplasticpropertiesofskin.Furthercreepandstressrelaxation tests,requiringadditionalskinsamples,wouldneedtobeapprovedtocharacterizetheviscoelasticityofskin.

2.3. Digitalimagecorrelation

ThemechanicalcharacterizationiscarriedoutduringtheexperimentsusingDIC.Thestretchratiowascalculatedviathe displacementcellattachedtothecross-headofthetensilemachineanditwasalsocalculatedviaDIC.Itisafull-fieldoptical strain measurement methodusing image registrationto measurethe 2D or3D material deformations. Thesecorrelations are basedonimagestakenwithanIDSvideocamera(1:2.8 50 mm30.5TAMRONlens).Blackspraypaintwasappliedto theskinsurfacetogeneratethedesiredrandomspeckledpatternnecessaryforthecorrelation.

2.4. TR-NEWSsignalprocessing

An advancedsignal processingtechnique basedon multi-scaleanalysisandmultimodalimagingwasinvestigated.The NonlinearTimeReversalmethodwasused,inthiswork,todetectthestructuraldamagesinthecomplexmedium.

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Fig. 2.The synchronization diagram of ultrasonic and mechanical measurements on the porcine skin using DIC.

The TR-NEWSsignal processing approach,which isused here, consistsof the followingsignal processing steps:chirp broadcasting,reception,correlation,time reversal,rebroadcasting,andreturnreception.Suchsignalprocessingallowsusto betterunderstandthephysicalmeaningofthecross-correlationfunctionofacomplexmaterial.Indeed,theTR-NEWSsignal approachcomingfromnonlineareffectswouldbemeasuredby thesymmetry’sfields.Thedevicesusedforimplementing the process are calibrated andrelated to pulseexcitations that allow us todetect the nonlinear break behaviour of the porcineskin.TheTR-NEWSDataAcquisition(DAQ) systemwas designedbyJuvitek (TRA-02“0.02–5 MHz”).The synchro- nizationandamplificationwereperformedusinganamplifierENImodelA150(55 dBat0.335 MHz)andapulsegenerator (GPG-8018G)asapulseextender.

Twosensortypeswere choseninthiswork:theshear wavetransducer(ABFP-0202-70 “2.25MHz”)forwaveemission and the longitudinal transducer (V155 “5 MHz”) for wave reception. An ultrasonic transducer is attached to each skin sample bya handclampaspresentedinFig. 2.Inaddition,elasticwavesguidedperiodicallyatspecifiable time intervals andemitted by an actuator orultrasonic transducerare detected by sensors or ultrasonictransducers andexamined for changesinthe emittedwaves.TheTR-NEWSmeasurement,whichiscountedaccordinglyby theloadframe, iscontrolled byspecificsoftware.

3. Resultsanddiscussion 3.1. Uniaxialtensiletests

Somebasicbiomechanicalpropertiesarefirstinvestigatedbyuniaxialtests,whichareveryusefulforunderstandingthe ratchetingbehaviour moredeeply.TheobtainedexperimentalresultsareshowninFig.3.

Foreachtensiletestperformed,astress–deformationcurveandastrain–timecurvewereobtained. Thenominalstress was then calculatedby dividingthe force by the undeformed cross-sectional area of thesamples. The stretch ratio was calculatedby dividingthecurrentlengthofthespecimenby itsinitial length.Inthisway,nominalstressvsstretchratio graphswereplottedforeachspecimen.Acharacteristicnumberofthesecurveswereidentifiedasdescriptiveparameters.

Fig. 3(a)illustrates thehyperplastic behaviour obtained inthe tension test.The stress–strain response ofporcine skinis extremelynonlinear,andthetensilestress–straincurve canbeseparatedintothreeparts:thefirstpartwithan increased tangentmodulus,thesecond partwithaconstanttangentmodulus,andthethirdpartwithadecreasedtangentmodulus.

The samephenomenon hasbeen obtainedandcommented by Fungetal.[36].One can alsosee that thebiomechanical response ofporcineskinis anisotropic inboth directions.Themean value oftheinstantaneous initial Young modulusin tension ofthe porcine skin E0 inthe parallel direction ofthe uniaxial tensile test is evaluated as E0=0.35±0.2 MPa, whereastheinitialYoungmodulus E0intheorthogonaldirectionisevaluatedasE0=2.1±0.4 MPa.Thus,we noticethat the instantaneousinitial Young modulus issignificantly higherfor afresh tissuein crossfiberdirections. Thisis intotal agreementwiththefindings ofsome previousstudies [8,37],wherealarge differencebetweentheYoung moduliofskin alongparallelandorthogonaldirectionstonaturallineshasbeenclearlydemonstrated.Indeed,ithasbeensuggestedthat thedeformationresponsesofskinaredependentuponthespecimen’sorientation.

Anillustrativeviscoelasticbehaviour throughtensiontestsisdisplayedinFig.3(b).Arelativedeformation–timecurveis examined,aswellastheinitialaxialstrainvalueproducedatthemomentwhentheprescribedpeakforceisreachedinthe test.Itappearsthatthedeformationinparallelofthealongnaturallinesoftheporcineskinincreaseswiththetestingtime, whilethedeformationinorthogonaldirectiondecreaseswithtime.Thesefindingsareconsistentwiththeincompressibility theoryofbiologicaltissues.ThePoissonratioisdeterminedinthex–y planeasshownasFig.1.ThemeanPoissonratiois evaluatedas0.51±0.12.

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Fig. 3.Typicalstress–deformationgraphfortheexperiments.Theinitialelasticmodulusisdefinedastheslopeofthelinearportionofthecurve(a).Typical axialstrain–timeandtransversestrain–timecurves(b)ofporcineskin.

3.2. Cyclictensiontests

Five-nominalforce-displacementcyclictests,usingloading–unloadingandrelaxationtime“25s”,wereperformedonthe porcine skinsamples inthe orthogonal direction. The obtainedexperimental results are showninFig. 4. It appearsthat therelativepeakandvalleystrainsincreaseprogressivelywithincreasing thenumberofcycles(Fig.4a).Thisresultseems similartothosefromsomepreviousresearchthatusedastress-controlledcyclicloadingmethod[38,39].

Theratchetingstrainincreaseswiththeincreasingmeanforceandtime,asshowninFig.4(b).Whentheloadinglevelis highandafterthefirstcycle,theratchetingax-strainratecontinuestoincreaseuntiltherelaxation-imposedtime(25 s),and thendecreases.Theexperimentalcycletensileresultsinfunctiontimeillustrativeviscoelasticbehaviouroftheporcineskin.

Theabove-mentionedexperimentalobservationshowsthatratchetingalsooccursfortheporcineskinatroomtemperature whentheskinsamplesaresubjectedtocyclictensionunloading,aswellastocyclicloading–unloadingtests.However,the

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Fig. 4.Cycle tensile stress–strain curves (a); axial (Ax) and transverse (Tr) strain–time curves (b) testing on porcine skin during five loading cycles.

porcineskintissuehasadifferentphysicalmechanism.Theratchetingoftheporcineskinismainlycausedbyitsremarkable viscosity,whichhasbeendemonstratedclearlybythetensiletestsatdifferentloadingratesbyKangetal.[40].

TheinitialYoungmodulusofeachcyclewasdeterminedfromtheelasticpartofthecyclicloading–unloadingcurves.The obtainedresultsarepresentedinFig.5.OnecanseethattheinitialYoungmodulusincreasesprogressivelywithincreasing thestressrates.Itmaybeexplainedthat thecollagenfibersarereorientedinthedirectionoftheappliednormaltension forceandregeneratethestructureandincreasethemechanicalstrength.Accordingtoseveralpapers[41],thedermis,which actsasasupporttissue,isresponsibleforskin’sresistance.Whenviewedfromitsultra-structure,thedermisisstructured asacomplex3D networkofcollagen, elastin,andreticulinfibers thatareimmersedina semi-liquidcalled“fundamental substance”.Mechanically,thefundamentalsubstanceisaviscousgel.Invitrotestshaveshownthatcollagenfibersarehighly resistant,whileelastinfibershavehighextensibility[24].Severalstudieshaveshownthatcollagenfibersareorientedinthe stress directionaccordingto thethreeuniaxial tractionphases. Kangandal.[40] basedtheir findings onthemicroscopic observationofthecollagenfiberbundlesandontheirvariationduringthemonotonictensiletest.Theyconcludedthatsuch viscoelasticperformanceissatisfiedonlywithinacertainappliedload.

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