In-vitro controlled mechanical testing of tendons from living donors

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In-vitro controlled mechanical testing of tendons from living donors

Matthieu Ollivier, Jaafar Sbihi, A. Sbihi, Martine Pithioux, Sebastien Parratte, Jean-Noël Argenson

To cite this version:

Matthieu Ollivier, Jaafar Sbihi, A. Sbihi, Martine Pithioux, Sebastien Parratte, et al.. In-vitro con-

trolled mechanical testing of tendons from living donors. Orthopaedics and Traumatology - Surgery

and Research, Elsevier, In press, 103 (7), pp.1027-1030. �10.1016/j.otsr.2017.05.024�. �hal-01593116�

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Original article

Ropivacaine alters the mechanical properties of hamstring tendons:

In vitro controlled mechanical testing of tendons from living donors

M. Ollivier

, J. Sbihi

, A. Sbihi

, M. Pithioux

, S. Parratte

, J.-N. Argenson

aInstituteformovementandlocomotion,orthopedicsurgery,boulevardSainte-Marguerite,13009Marseille,France

bICOS13,institutdechirurgieorthopédiqueetsportivedeMarseille,13008Marseille,France

cInstitutdessciencesdumouvementUMR7287,Aix-Marseilleuniversité,CNRS,13288Marseille,France

a r t i c l e i n f o

Articlehistory:

Received16January2017 Accepted2May2017

Keywords:

Ropivacaine Hamstringtendons Mechanicaltesting Mechanicalproperties

a b s t r a c t

Objective:Intraarticularorperiarticularinjectionofropivacaine(RI)isanelementofcurrentkneesurgery practices.ThegoalofthisstudywastodeterminetheeffectsofRIonthemechanicalpropertiesofham- stringtendons.WehypothesizedthatRIwouldhaveadetrimentaleffectonthemechanicalproperties ofperiarticularsofttissues

Methods:Atensiletesttofailurewasperformedon120hamstringtendonsegmentsharvestedduring ACLreconstructionsurgeryin120patients.Twosetsoftensiletestsweredone.Thefirstevaluatedthe effectofRIitselfonthemechanicalpropertiesoftendons:30samplesweresoakedfor1hourina2%RI solutionandcomparedto30samplessoakedinasalinesolution(controlgroup).Thesecondevaluatedthe effectofRIconcentrationonthemechanicalpropertiesofhamstringtendons:30samplesweresoaked for1hourina2%RIsolutionand30samplesweresoakedina7.5%RIsolution.

Results: Inthefirsttest,29samplesfromeachgroupwereanalyzedastwosamples(onein each group)failedatthegripinterface.Thespecimensexposedto2%RIhadlowerultimatetensilestrength (=4.4MPa,P=0.001),strainenergy(=13MPa,P=0.001)andYoung’smodulus(=1.6MPa,P=0.02) thanthespecimensinthecontrolgroup.Therewasnosignificantdifferenceinthestrainatfailure betweengroups(=5%,P=0.3).Inthesecondtest,onespecimenfromthe7.5%RIgroupfaileddur- ingthepreloadingandwasexcluded.Therewasnosignificantdifferenceintermsoftheloadatfailure andultimatetensilestress(=0.45MPa,P=0.6)andstrainenergy(=0.49MPa,P=0.49)betweenthe twogroups.Thereweresignificantdifferencesintermsofelongationatfailure(=28%,P=0.0003)and Young’smodulus(=2.6MPa,P=0.005),withthespecimensexposedto7.5%RIundergoinggreater deformationandhavingalowerYoung’smodulus.

Discussion:WhilelocalRIinjectionsarewidelyperformedinclinicalpractice,theresultsofthisinvitro studypointtoshort-termalterationsofthemechanicalpropertiesofhamstringtendons.Iftheseresults holdinvivo,thiscouldleadtoweaknessofthesofttissuesexposedtothisproduct,particularlythe tendonsandligamentsaroundtheinjectionarea.

Levelofevidence:Experimentalstudy.Level1

©2017ElsevierMassonSAS.Allrightsreserved.

1. Introduction

Postoperativepainafterjointsurgery,whetherperformedby arthroscopyorasanopenprocedure,isamajorconcernforpatients andsurgeons[1–3].Whiletherearenostudiesspecificallyrecom- mendingintraarticularinjectionoflocalanesthetics,ropivacaine (RI)isofteninjectedinsideoraroundthejoint,oradministered bytheperineuralroutetomanagepain[3–5].RIisanamidelocal

∗ Correspondingauthor.

E-mailaddresses:ollivier.matthieu@yahoo.fr,ollivier.mt@gmail.com (M.Ollivier).

anesthetic(LA).Recentpublicationshavedescribednegativeeffects onchondrocytesfollowinginvitro jointinjection[6–9].Studies havealsoshowntoxiceffectsonthemesenchymalandfibroblas- tictissuesinvolvedinhealingandpostoperativerecovery[10–13].

Haastersetal.[11]showedthatRIhasaharmfuleffectontendon cells,withanelevatedapoptosisrateafter6hoursofincubation [11,12].

WehypothesizedthatRIwouldhaveadetrimentaleffecton themechanicalpropertiesofperiarticularsofttissues.Wehada dualprimaryobjective.First,toassessthetoxicityofRIbycompar- ingthemechanicalpropertiesoffreshhumanhamstringtendons soakedfor1hourina2%RIsolutiontotendonssoakedinasaline solution.Second,toassesstheRIconcentrationeffectbycomparing http://dx.doi.org/10.1016/j.otsr.2017.05.024

1877-0568/©2017ElsevierMassonSAS.Allrightsreserved.

1028 M.Ollivieretal./Orthopaedics&Traumatology:Surgery&Research103(2017)1027–1030

Table1

Demographicsofthepatientsinthevariousgroups.

Demographicdata Controlgroup(n=30) Ropivacaine2%A(n=30) P Ropivacaine2%B(n=30) Ropivacaine7.5%(n=30) P

Age(years) 30±8(18–44) 29±7(18–42) 0.7 31±8(18–43) 29±7(18–42) 0.7

Gender(F/M) 43/17 43/17 1 41/19 41/19 1

Sportslevel(UCLA) 9±1(8–10) 9±1(8–10) 1 9±1(8–10) 9±1(8–10) 1

Professionalathletes 2 2 NA 2 2 NA

Specimens(mm) 8±5(6–12) 7±4(5–10) 0.5 8±4(5–11) 8±5(5–12) 0.6

Dataareexpressedasmean±standarddeviation(minimum–maximum);Pwassignificantwhen<0.005.

60 specimens used to evaluate RI toxicity effect

60 specimens used to evaluate RI concentration effect

30specimens:

7.5% ropivacaine

Mechanical testing

29 RI specimens included in

29 control specimens analysis

120 specimens harvested

29 RI 7.5%

specimens analysis 30 RI 2%

specimens analysis Statistical analysis

30 specimens:

saline solution (control) 30 specimens:

2% ropivacaine Immersion 1 hr. 30 specimens:

2% ropivacaine

Fig.1.Flowchartforthestudyprocedures.

themechanicalpropertiesoffreshhumantendonssoakedina2%

versusa7.5%RIsolution.

2. Methods

Biomechanicaltestingwascarriedoutwith120hamstringten- donsegmentsharvestedduringACLreconstructionsurgeryin120 patients.Themeanpatientagewas29.7±8.8years;71%weremen and29%werewomen(Table1).

Digitalcalipers(AbsoluteDigimatic^®,Mitutoyo^TM,Kanagawa, Japan)withanaccuracyofU=±0.03(k=2mm)wereusedtocre- ateidentical,3cmlongsamples;thediameterwasbasedonthe widthoftheproximalgracilistendon.Twosetsoftensiletestswere done(Fig.1).ThefirstevaluatedtheeffectofRIonthemechani- calpropertiesoftendons:30samplesweresoakedfor1hourina 2%RIsolutionandcomparedto30samplessoakedinasalinesolu- tion(theRIdiluent).The1hoursoakingperiodcorrespondedtothe

“toxicperiod”ofspecimensafterACLsurgery,duringwhichmore than50%oftheproductisactive[14].Thehalf-lifeofintraarticu- larRIhasnotbeenreported,howevertheintravenoushalf-lifeis 1to2hours.ThesecondsetoftestsevaluatedtheeffectofRIcon- centrationonthemechanicalpropertiesofhamstringtendons:30 samplesweresoakedfor1hourina2%RIsolutionand30samples weresoakedina7.5%RIsolution.

Allspecimenswerecarefullydriedafterthesoakingstepand frozenat−20^◦Cuntilthemechanicaltesting,accordingtoapre- viouslyvalidatedprotocol[15].Tominimizebias,thespecimens wereassignedtoeachgroupafterbeingstratifiedonpatientage (±3years),genderandsportslevel(UCLAscore).Allpatientswere high-levelathletes(UCLAscore>8);theeightspecimensfrompro- fessionalathleteswereseparatedintotwopergroup.Allpatients

providedwrittenconsent,andthestudywasapprovedbyourhos- pital’sethicscommittee(No.2012-015724-11;26January2013).

2.1. Mechanicaltesting

Mechanical testing was performed on a universal testing machine(Instron5566-A, Instron^®,NorwoodMA,USA) (Fig.2).

Afterthespecimenswerethawedfor1hour,theendsofeachten- donwereplacedintwogrips(1cmfromeachend).Themechanical propertiesweredeterminedonthetissuebetweenthetwogrips (1cm)usingapreviouslyvalidatedmethod[16].Tendonswerepre- conditionedwith10cyclesof1mmextension.Theinterfaceofeach tendon/gripwasmarkedwithChinainktomonitorpotentialten- donsliding.Alltendonsweresubjectedtoatensiletesttofailure [17].Theinstanceandmethodoffailurewererecordedforeach specimen.Themaximumloadandstiffnessweredeterminedfrom theload–elongationcurvesusingBluehill3acquisitionsoftware (Instron^®,Norwood,MA,USA).Theultimatetensilestrength(UTS) wascalculatedbyassumingthespecimenshadacylindricalshape [18].Thefollowingparameterswerecalculated:UTS,elongationat failure,Young’smodulusandstrainenergy.

Aspecimenwasexcludedfromthefinalanalysisifitfailedatthe gripinterfaceorinsidethegrip,slippedduringthetest,orfailed duringpreconditioningorbefore150Noftensileloadhadbeen applied.

2.2. Statisticalanalysis

TheKolmogoroff-Smirnofftestwasusedtodeterminethenor- mality of the data distribution. Parametric tests were used to compare normallydistributed variables (demographic data and

Fig.2. Materialstestingsystemusedforthetensileloadtofailuretests.

mechanicalproperties)betweenthegroups.Non-parametrictests wereusedtocomparetheelongationatfailure,sincethisdatawas notnormallydistributed.Two-tailedtestswerecarriedoutusing PASWStatisticsversion20(SPSS,IBMInc.,Chicago,Illinois).The significancethresholdwassetatP<0.05.Sincewecouldnotfind anypublisheddataonthemechanicalpropertiesof“fresh”human hamstringtendons,samplesizecalculationswerebasedonthetest resultsofthefirst20specimens.Thesecalculationsshowedthat atleast28 tendonswereneededineach grouptoshowaclin- icallysignificant70Ndifferenceinthefailureloadbetweenthe groups(␣=0.05;␤=0.8SDforfailureloadof80N).Consequently, weincluded30patientspergroup,whichallowed2specimensto beexcludedifnecessary.

3. Results

3.1. RIversuscontrol

Inthefirstseries,29samplesfromeachgroupwereanalyzed astwosamples(oneineachgroup)failedatthegripinterface.The specimensexposedto2%RIhadlowerultimatetensilestrength (=4.4MPa,P=0.001),strainenergy(=13MPa,P=0.001)and Young’smodulus(=1.6MPa,P=0.02)thanthespecimensinthe controlgroup(Table2).Therewasnosignificantdifferenceinthe elongationatfailurebetweengroups(=5%,P=0.3).

3.2. RIconcentration

Inthesecondseries,onespecimenfromthe7.5%RIgroupfailed duringthepreloadingandwasexcluded.Therewasnosignificant differenceintermsofultimatetensilestress(=0.45MPa,P=0.6) andstrainenergy(=0.49MPa,P=0.49)betweenthetwogroups.

Thereweresignificantdifferencesintermsofelongationatfailure (=28%,P=0.0003)andYoung’smodulus(=2.6MPa,P=0.005),

withthespecimensexposedto7.5%RIundergoinggreaterdefor- mationandhavingalowerYoung’smodulus.

4. Discussion

Themain findingof this studyis thatropivacainealters the mechanicalpropertiesofhumanhamstringtendonsinvitro.Intra- orperiarticularinjectionofalocalanestheticisregularlyperformed toimprovepostoperativeanalgesiaafterkneesurgery.Itsthera- peuticbenefitsforcontrollingpainhavebeenclearlydemonstrated [2,3].However,severalwarningshavebeenissuedrelatedtothe cytotoxiceffectsoflocalanestheticsonchondrocytes,mesenchy- malcellsandfibroblasts[6–13].

Ourobjective wastodeterminewhetherRI,whichis known tobecytotoxic,affectsthemacroscopicpropertiesofsofttissues.

Our hypothesis was confirmed–RIaffects themechanical prop- erties of humanhamstring tendons.Although RIdid not affect tendonelongationrelativetoasalinesolution.Incomparisonto publishedresultsdescribing theload atfailure oftendons har- vestedfromcadavers[5,19],ourvaluesappearlow;however,our resultscorrespondtothemechanicalpropertiesofisolatedtendons testedinasimilarconfiguration(3cmlength)withoutreinforce- ment(i.e.,suture).Inaddition,weusedfreshtendonsfromliving donors,whilemosttestsweredonewithanimalorcadaverspeci- mens[17–20].Mechanicalpropertiesofligamentsandtendonsare relatedtovariousmorphologicalparameters[19].Potentialcon- foundingexternalfactorsinourstudywerecontrolledbypairing ourpatientsbasedonactivitylevel(UCLAscore,professionalath- letes),sexandage(±3years)beforethegroupswerecreated.

OurresultsontheeffectsofRIonthemechanicalproperties oftendontissuetendtosupportinvitrofindingsofcelltoxicity [5,7,8,11,13].PiperandKim[8]werethefirsttodescribethetoxic effectsoflocalanesthetics(bupivacine)onhumanchondrocytes invitro;however,RIdidnotincrease cellapoptosisincompar- ison tothe controlsaline solution.Grishkoet al. [7] published theopposite results–theyshowedthat lidocaine,bupivacaine, andropivacaineledtomitochondrialdysfunctionand apoptosis ofhumanchondrocytesinvitro. Fedderetal.[12] exploredthe effectoflocalanestheticsonhumanfibroblasts;theyconcluded thatcontinuousintraarticularinjectionoflidocaine,bupivacaine andropivacaineleadstoseverecelldamage.Aninvitrostudyon theeffectoflocalanestheticsonstemcellsfromhamstringten- donsshowedthatbupivacine(0.5%)andropivacaine(0.75%)have considerablecytotoxic effects onhumantendonprogenitor/cell stems[11].Theauthorsconcludedtheviabilityofprogenitorcells harvestedfrominvitroligamentreconstructiongraftswascom- promisedwhenusingbupivacineandropivacaine.Recently,Lehner etal.[20]evaluatedtheeffectofbupivacineonmechanicalprop- ertiesofratAchillestendon.Theyfoundthatbupivacainehada dose-dependentandtime-dependentnegativeeffectontheviabil- ityoftendoncellsinvitro.Asingleinvivoperitendinousinjection ofbupivacainecantriggerapoptosisofendotenoncells,increase metalloproteaseactivityandalterthequalityoftendoncollagen afteronly6hours[20].Theseresultscanhelptoexplainourstudy’s findings.Thecollagenandextracellularmatrixthatgivesstructure tosoft tissues[21]immediatelyloses17%ofthenormaltendon architecturewhenbupivacaineisadministered[20].

Our secondobjective wastoevaluatethe potentialeffectof variousropivacaineconcentrationsonthemechanicalproperties of hamstringgrafts.Therewasnodifferencebetween7.5%and 2%RIontheloadatfailureandUTS.However,thestrainenergy andelongationatfailurewasgreaterinthespecimensexposedto 7.5%ropivacaine.Thisconcentrationeffectwasknownonacellular level,asFedderet al.[12]foundnodifferenceinfibroblastvia- bilitywhenexposedtothreedifferentropivacaineconcentrations

1030 M.Ollivieretal./Orthopaedics&Traumatology:Surgery&Research103(2017)1027–1030

Table2

Mechanicalpropertiesofspecimensinthevariousgroups.

Mechanicalproperties(atfailure) Controlgroup(n=30) Ropivacaine2%A(n=30) P Ropivacaine2%B(n=30) Ropivacaine7.5%(n=30) P

Ultimatetensilestrength(MPa) 13±7.1 8.2±3.8 0.001 8.3±4 8.8±3.7 0.6

Strain(%) 77±31 82±29 0.367 69±17 97±32 0.0003

Energy(MPa) 30±24 16.5±9.5 0.001 17.4±9.8 15.5±9.6 0.49

Young’smodulus(MPa) 5.9±3.7 4.3±2.9 0.019 4.2±2 3.24±3.3 0.04

Dataareexpressedasmean±standarddeviation;Pwassignificantwhen<0.005.

(0.31,0.62 and1.25mg/mL).Thiseffectisalsopresent inham- stringtendoncells,sinceHaasteretal.[11]concludedthat0.5%

RIledtomoderatelossofviablecells,while0.75%RItriggeredsig- nificantapoptosisandreductioninmetabolismoftheremaining cells.Lehnerandal.[20]alsodescribedaconcentration-dependent effectandtime-dependenteffectofbupivacaine,anotheramide localanesthetic.

Ourstudyhasseverallimitations.First,aninvitropreparation doesnotreproduceintraarticularorperiarticularconditions.The tendonsweresubmergeddirectlyintheRIsolutionorthecontrol salinesolutionatconcentrationsequaltostandardintraarticularor periarticularadministration.Sincethehalf-lifeofRItopicalinjec- tionisestimatedat1to2hours[14–22],welimitedthetendon immersiontimeto1hour.Anotherlimitationisthattheimpactof bloodorsynovialfluidwasnotconsidered;thismayhavecaused ustooverestimatethecytotoxiceffect.Ropivacainewasusedin thisstudybecausethisamidelocalanestheticisoftenusedincur- rentsurgicalpracticesbyanesthesiologistsandsurgeons.Also,RI issaidtobetheleasttoxicofalltheamide-typelocalanesthetics [8,11,13].Lastly,themechanicaltestusedinthisstudyhasnever beendescribedfor humantendons,but wasvalidated oncolon specimensbyMassalouetal.[16].Topreventbiasduringtheanal- ysis,alltestsweredonebyatrainedengineerfollowingaspecified protocol.Thetestingonmorethan100freshhumantendonsam- plesharvestedusingstrictinclusion/exclusioncriteriawasblinded andcontrolled.

Thisstudydemonstrates only theimmediate effect ofRIon hamstring tendons. Given that we did an in vitro analysis on non-vascularized specimens, we could not analyze short-term remodelingin the tissue and potentialadaptation of thecolla- genand extracellularmatrixtoRIexposure. Thisinformation is neededtounderstandtherelationshipbetweencelltoxicityand themechanicaleffectsofRI.WecanonlypresumethatRIdeeply modifiesthemicro-architectureoftendontissue,therebyaltering itsmechanicalproperties.Itisessentialtodeterminehowthese substancespenetrateandalterthegrafttissueandchangeitscon- figuration[20].

Itisalsoimportanttonotethatourspecimens(exvivoprox- imal gracilis tendon) only provide us with an approximation of the general effect of ropivacaine on other tendons. We are currentlydoingahistologicalandmechanicalstudyofothertis- sues.

Despitetheselimitations,ourstudyshowsthepotentialharmful effectsofRIadministrationonthemechanicalpropertiesoften- dons.Ourfindingsmustbeconfirmedbyhistologicalandclinical studiestounderstandthecauseoftheharmfulmechanicaleffects ofRIonperiarticulartissues,butalsotoanalyzetheinvivoconse- quencesofthesetissuemodifications.

Disclosureofinterest

Jean-Noël Argenson is an educational consultant for Zim- mer/Biomet.

SébastienParratte is aneducational consultant for Newclip, Zimmer/BiometandArthrex.

MatthieuOllivier,JaafarSbihi,AbderrahmaneSbihiandMartine Pithiouxdeclarethattheyhavenocompetinginterest.

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