Contrast enhancement with uranyl acetate allows quantitative analysis of the articular cartilage by microCT: Application to mandibular condyles in the BTX rat model of disuse

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Micron

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / m i c r o n

Contrast enhancement with uranyl acetate allows quantitative analysis of the articular cartilage by microCT: Application to mandibular condyles in the BTX rat model of disuse

Jean-Daniel Kün-Darbois

^a,b

, Florence Manero

^c

, Louis Rony

^a,d

, Daniel Chappard

^a,c,∗

aGEROMGrouped’EtudeRemodelageOsseuxetbioMatériaux,IRIS-IBSInstitutdeBiologieenSanté,Universitéd’Angers,CHUd’Angers,4rueLarrey,49933 ANGERSCedex,France

bServicedechirurgiemaxillo-facialeetstomatologie,CHUd’Angers,4rueLarrey,49933ANGERSCedex,France

cSCIAM,ServiceCommund’ImagerieetAnalysesMicroscopiques,IRIS-IBSInstitutdeBiologieenSanté,Universitéd’Angers,CHUd’Angers,4rueLarrey, 49933ANGERSCedex,France

dServicedechirurgieorthopédiqueettraumatologique,CHUd’Angers,4rueLarrey,49933ANGERSCedex,France

a r t i c l e i n f o

Articlehistory:

Received17January2017

Receivedinrevisedform15March2017 Accepted15March2017

Keywords:

Botulinumtoxin Uranylacetate Softtissueimaging MicroCT Cartilage

Contrastenhancement

a b s t r a c t

Microcomputedtomography(microCT)iswelladaptedtoquantitativeanalysisofcalciﬁedtissuesbut softtissues(suchascartilage)areradiolucentandneedacontrastenhancementprocedureformicroCT.

Wedevelopeda“staining”methodallowingmicroCTimagingofarticularcartilageusinguranylacetate (UA).Themethodwasusedtoseewhetheradultratswithabotulinumtoxin(BTX)injectioninmastica- torymusclespresentachangeatthecondylarcartilageofthemandibleinassociationwithalocalized trabecularboneloss.

HumanfemoralheadsampleswereusedtodeveloptheenhancedcontrastmethodusingUAorlan- thanides(recentlyproposedasasubstituteforUA).Themethodwasthenappliedtothecondylarcartilage ofratmandibles.Maturemalerats(n=11)wererandomizedinto2groups:control(CTRL;n=4)andBTX group(n=7).RatsoftheBTXgroupreceivedasingleinjectionofBTXintotherightM.MasseterandM.

Temporalis.RatsoftheCTRLgroupweresimilarlyinjectedwithsaline.Ratsweresacriﬁced4weeksafter injection.Condyleswereharvested,ﬁxedinformalinandimmersedinUA.MicroCTwasperformedfor boneandcartilagemeasurements.

AfterUAimpregnation,articularcartilageofhumanfemoralheadsampleswasclearlyseenonitsfull thicknesswhereaslanthanidesproducedamuchlesspronouncedcontrast,withafaintlabelingatthe upperlayer.InBTXrats,microCTanalysisshowedasigniﬁcantbonelossattherightcondyles.After UA,thewholethicknessofarticularcartilagewasclearlyevidenced.Cartilagethicknessmeasurement showednodifferencewhencomparingtherightwiththeleftsidesoftheBTXgroupnorbetweenthe twosidesoftheCTRLgroup.

ContrastenhancementwithUAisasimpletechniqueallowingquantitativeanalysisofcartilageby microCT.290words.

1. Introduction

Articularcartilageisahighlyspecializedconnectivetissueof synovialjoints,itissupportedbysubchondralbone.Itsmainfunc- tionistoprovideasmootharticularsurfaceinordertofacilitate thetransmissionofloadswithalowfrictionalcoefﬁcient(Sophia Foxetal.,2009).Itismadeofadenseextracellularmatrixcom- posedmainlyofwater, typeIIcollagenand proteoglycanswith

∗Correspondingauthorat:GEROMLHEA,IRISIBS,CHUd’Angers,49933ANGERS Cedex,France.

E-mailaddress:[email protected](D.Chappard).

specialized cells called chondrocytes. Histology is a commonly used methodtoanalyzearticularcartilage in pathologic condi- tions.Themainlimitationofthetechniqueisthatitisdestructive and causes tissue alterations. Furthermore, sample positioning is difﬁculttoreproduce,leading toadifferent sectionobliquity foreachslide.Microcomputedtomography(microCT)allowshigh resolution3Dimagingmakingprecisequantitativemeasurement possible(Ruegsegger,1994;SasovandVanDyck,1998).Thistech- nique is challenging for soft tissue imaging, such as cartilage, becauseX-rayattenuationofanon-mineralizedsofttissueisvery low(Navehetal.,2014).Contrastenhancementusinga“staining”

procedureisthereforemandatorywiththeuseofvariousmetal salts(Metscher,2009;Pauwelset al.,2013).Severalcontrasting http://dx.doi.org/10.1016/j.micron.2017.03.008

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thickness.Thetechniquewasﬁrstelaboratedonthehumanhip articularcartilageandthenappliedinananimalstudy.Wehave searchedifchangesinathinarticularcartilage(e.g.,thecondylar articularcartilageof themandible)occurin apathologiccondi- tion.Wechosetostudythechangesinducedbybotulinumtoxin (BTX)injectedinmasticatorymuscles.Inanimalmodels,BTXinjec- tionscauseamuscleatrophyassociatedwithadisuseboneloss (Chappardetal.,2001).Whetherdisusealsoactsonthisarticular cartilageislargelyunknownandamicroCTmethodaftercontrast enhancementofthecartilagematrixisofgreatinterest.

2. Materialsandmethods 2.1. Humanfemoralheads

Femoralheadsfrompatientswitharecenttrans-cervicalhip fracturewereusedinthisstudytodevelopthecontrastenhance- menttechniqueforarticularcartilage.Thespecimenswerecutat thetopof thehead, in theareaofthe maincompressive bun- dle of trabeculae. On a first femoral head, four samples were harvestedandfixedin10%formalinfor48h(MerckKGaA,Darm- stadt,Germany)(CarsonandHladik,2009).Thefirstspecimenwas included undecalcified in poly(methylmethacrylate) for routine histology.ThesecondsamplewasimagedbymicroCTwhileinthe fixative.Thesecondsamplewasimmersedfor48hinuranylacetate (UA).UA(Merck)waspreparedasa 3%solutionin50^◦ ethanol;

thesolutionwasfilteredona0.2␮msyringefilterandstoredat 4^◦Cinthedark.The“stained”specimenwasrinsedduring1hin runningtapwater,transferredto10%formalinandscannedinthe fixative.Thefourthspecimenwasstainedwithalanthanidesolu- tion(LFGDistribution,Lyon,France).Itcomprised1%lanthanum acetate,1%gadoliniumacetateand1%samariumacetateandwas preparedinethanol50^◦.Thespecimenwasimmersedsimilarlydur- ing48hinthelanthanidesaltssolution,rinsedduringonehourin tapwater,placedinformalinandscannedasabove.Additionaltri- alsweredonewithIKI(1%iodine,2%potassiumiodide),PTA(1.5%

inwater)andIopamiron300^®(BraccoImagingFrance),acontrast- ingagentforcomputedtomographycontaining300mgiodine/ml.

Thesethreeadditionalreagentswereusedonpiecescomingfrom anotherfemoralhead.

2.2. Animalsandexperimentalprocedure

Animalcareandexperimentalprotocolswereapprovedbythe FrenchEthicalCommittee(protocolagreementnumber01732.01) andunderthesupervision ofauthorized investigators.Eighteen weeks-oldmaleSprague-Dawleyrats(n=11),weighing587±26g, wereusedforthestudy(Janvier-Labs,LeGenest-Saint-Isle,France).

Theywereacclimatedfortwoweekstothelocalvivariumcondi- tions(24^◦Cand12h/12hlightdarkcycle)wheretheyweregiven standardlaboratoryfood(UAR,Villemoison-sur-Orge,France)and water adlibitum. Rats were randomizedinto 2 groups:control

cartilageisthinnerthanthehumanhipcartilage,eachsamplewas thenimmersedfor24hintheUAsolution,rinsedintapwaterfor onehourtoremoveunlabeledUA,transferredin10%formalinand scannedasabove.AftermicroCTanalysis,sampleswereembedded undecalciﬁedasaboveforhistologicalanalysis.

2.3. Microcomputedtomography

MicroCT of human femoral samples and rat mandibular condyles were performed using a Skyscan 1172 X-ray com- puterized microtomograph (Bruker microCT, Kontich, Belgium) equippedwithanX-raytubeworkingat70kV/100␮A.Boneswere placedinplastictubesfilledwithformalin.Thetubeswerefixed onabrassstubwithplasticine.Analysiswasdonewithapixelsize correspondingto13␮m(humanfemoralheads)and2.94␮m(rat condyles).Therotationstepwasfixedat0.20^◦witha0.5mmalu- minumfilter.Foreachsample,astackof2D-sectionswasobtained andreconstructedusingNReconsoftware(Bruker)andanalyzed withtheCTAn,DataViewerandCTvolsoftwares(Bruker).MicroCT acquisitionparameters,reconstructionandrenderingsettingswere strictly alike between all the concerned specimens and for all images.AftermicroCTanalysis,thecondyleswereembeddedin poly(methylmethacrylate)forhistologicalanalysis.

2.4. QuantitativeanalysisofcartilageandboneeffectsofBTX injections

Cartilagethickness(Cart.Th)measurementofthecondyleswas performedon2Dsagittalandfrontalsectionsobtainedbyusing thecuttingplanefacilityofthesoftware.Foreachcondyle,12mea- surementpointswereusedin3serialfrontalsectionsand1sagittal section (Fig. 1C). The measured width (in ␮m) was comprised betweenthetide-markand theexternal surfaceofthearticular cartilage.

Bonemorphometricparameterswereobtainedfromtheﬁrst scanimagesobtainedbeforeimmersioninUA.Theabsolutebone volume(C.BV/C.TV,expressedin%andrepresentingthepercentage of volumeoccupiedby corticaland trabecularbone)was mea- suredin3DusingtheCTAnsoftwareforeachcondyle.Trabecular bonevolumecouldonlybeaccuratelymeasuredin2D(B.Ar/T.Ar, expressedin%,representingthefractionalareaoccupiedbytra- becularbone)aspreviouslydescribedbecausethedelineationof theregionofinterestis difﬁcultonsucha smallbonesegment (Kün-Darboisetal.,2015).

2.5. Histologyonundecalciﬁedbones

Because we wanted to see if theUA location corresponded tospecifichistologicalareas,theboneswereprocessedundecal- cified.Samplesweredehydratedinamixtureofacetone/xylene andembeddedundecalcifiedinpoly(methylmethacrylate)aspre- viouslydescribed(Chappard,2014).Sections(7␮minthickness) werecutdryonaheavy-dutymicrotomeequippedwith50^◦tung-

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Fig.1.(A)Lateralviewofaratmandible.Arrowsindicatetherightandleftcondylarprocesses(=articularprocesses)thathavebeenanalyzedinthepresentstudy.Thescale barstandsfor1cm.(B)Lateralviewofa3DreconstructionaftermicroCTanalysisofarightcondylarprocesswithoutmetallic“staining”.(C)Upperviewofacondylarprocess showingthedistributionofthe12pointsusedforcartilagethicknessmeasurement.Inallimages(exceptimageA)thescalebarstandfor500␮m.

stencarbide(LeicaPolycutS,Rueil-Malmaison,France).Sections werestainedbyGoldner’strichromeforthedifferentiationofmin- eralizedandosteoidtissues.

2.6. Statisticalanalysis

StatisticalanalysiswasperformedusingtheSystatstatistical softwarerelease13.0(SystatSoftwareInc.,SanJosé,CA).Alldata wereexpressedasmean±standarderrorofthemean(SEM).Dif- ferencesamonggroupswereanalyzedbyanon-parametricANOVA (Kruskal–Wallis)andbetweengroupsbytheMannandWhitney’sU test.Datafromrightandlefthemimandibleswerecomparedusinga pairedt-test.Differenceswereconsideredsigniﬁcantwhenp<0.05.

3. Results 3.1. Femoralheads

Theresultsofthe3typesofmicroCTanalysesappearinFig.2.

Routinehistology revealed nosigns of necrosis in the bone or marrowcompartments.Onthesampleprocessedwithoutmetallic impregnationformicroCT,thesubchondralbonewasevidencedas arathersmoothsurface.Thenetworkoftrabeculaewasanchored atitsendostealsurfaceandthemarrowcavitieswerewelldelin- eated.Thebonemarrowwasradiolucentasthearticularcartilage thatdidnotabsorbX-rays.AfterUAimpregnation,thearticular cartilagewasclearly seenonitsfullthickness.Therewasa line withanincreaseddensityabovethesubchondralbonewhichcould correspondtothetide-markseparating thecalcified/uncalcified layersofthearticularcartilage.UAwasalsofaintlydepositedin thebonematrixandinsidethemarrowspacesmakingthediffer- encebetweenboneandmarrowlessobvious.Whenlanthanides wereused,thearticularcartilagewaslessclearlyidentified,with afaintlabeling.However,therewasnolabelatthetide-marknor insidethemarrowcavities.Thecartilagethicknessforthispatient was2200–2400␮montheUAblockand 2100–2400␮monthe lanthanideblock.Trialswithothermetalsaltsreportedinthelit- eraturegave unsatisfactoryresults. PTAcouldlabelthearticular cartilagebutstainingwaslessintense(Fig.2E).IKIlabeledthecar- tilagebutthehematopoieticareaswereheavilystainedwhilethe fatcellsdidnottakeiodine(Fig.2E).Iopamironfaintlystainedthe upperlayersofthearticularcartilage(Fig.2G).

3.2. CondylarcartilageandboneinBTXinjectedrats

MicroCTmeasurementsaresummarizedinTable1.Asmaller condylarC.BV/C.TVwasobservedattherightvs.lefthemimandible in theBTXgroup(p<0.05).Therewasnosigniﬁcant difference

Table1

.QuantitativeanalysisonMicroCTimages.Bonemorphometricparametersobtained beforeUAimpregnation(absolutebonevolumeandtrabecularbonevolume);car- tilagethicknessdeterminedafterUAimpregnation.

CTRLleft CTRLright BTXleft BTXright C.BV/C.TV(%) 76.83±1.38 74.35±1.12 80.23±1.02 74.85±1.63^a B.Ar/T.Ar(%) 66.98±3.53 63.54±2.59 70.86±1.27 38.96±3.81^a,b Cart.Th(␮m) 164±25 161±25 168±12 169±12

aSigniﬁcantdifferencebetweentherightvs.theleftside.

bSigniﬁcantdifferencevs.CTRL.

betweentherightsideintheBTXgroupandthevaluesobtained intheCTRLgroup.Therewasnosigniﬁcantdifferencebetweenthe leftsideintheBTXgroupandbothsidesintheCTRLgroup.When trabecularbonewasaccuratelymeasuredon2Dsection,abone losswasclearlyseenonthecondylarboneattherightside(i.e.

paralyzed)intheBTXgroupwithareducedtrabeculardensityand widermarrowcavities.Nodifferencewasnotedwhencomparing bothsidesoftheCTRLgroup.TheBTXgrouphadasignificantly lowercondylarB.Ar/T.Ar(−32%)attherightsidecomparedtothe leftside(p<0.05).TheBTXgrouphadasignificantlylowercondy- larB.Ar/T.Ar(−25%)attherightsidecomparedtotherightside oftheCTRLgroup(p<0.05).Therewerenosignificantdifferences betweenleftsidesoftheBTXgroupandboth sidesoftheCTRL group.

AfterUA impregnation, thewhole thicknessof the articular cartilagewasclearlyseenwhereasitwasnotvisiblebeforeimpreg- nation(Fig.3AandB).Thetide-markwasclearlyseen.UAwasalso depositedinthebonematrixandinsidethemarrowspaces.Insome locations,bandswithanincreasedX-raydensitywerenoticedat thesurfaceoftrabecularandendostealbone.Nodifferencewas notedwhencomparingCart.Thbetweentherightandleftsidesin theBTXgroupandsimilarlyintheCTRLgroup(Table1).Nodif- ferencecouldbefoundbetweentherightsidesofCTRLandBTX rats.Theaveragecartilagethicknessforallratsofthestudywas 167±8␮m.

3.3. Histologicalanalysis

ThehumanfemoralheadappearsinFig.2A.Thetidemarkwas clearlyidentiﬁedandthelayerofcalciﬁedcartilageappearsvery thin.Inratmandibularcondyles,histologicalanalysisrevealedthe presenceofarticularcartilage(Fig.3C).Ithadthesameappearance asthecartilageseenin2DsectionsafterMicroCT.Thepresence ofosteoidseamsatthesurfaceoftrabecularbonearoundmarrow spaceswasnoted.

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Fig.2. (A)Histologicalaspectofafemoralheadembeddedundecalcified(Goldner’strichrome).C=articularcartilage,scb=subchondralbone,tn=trabecularnetwork.The arrowindicatesthetide-mark.(B)MicroCTanalysisofapieceofthesamefemoralheadwithoutmetallicimpregnation;thearticularcartilageisnotseen.(C)MicroCTanalysis ofapieceofthesamefemoralheadimpregnatedwithUA,thetide-markisheavilymarkedwiththemetallicsalt(arrow).(D)MicroCTanalysisoffragmentfromthesame femoralheadimpregnatedwiththesolutionoflanthanides;thearticularcartilageislessclearlyidentified.(E)MicroCTanalysisfromapieceofanotherfemoralheadstained withPTA.Thecartilageisfaintlystainedbutthetide-markisevidenced(arrow).(F)MicroCTanalysisfromapieceofanotherfemoralheadstainedwithIKI.Areasofmarrow cellsareheavilystained(arrowheads).(G)MicroCTanalysisfromapieceofanotherfemoralheadstainedwithIopamiron300.Theupperlayerofthecartilageisill-defined.

Inallimagesthescalebarstandsfor1mm.(Forinterpretationofthereferencestocolorinthislegend,thereaderisreferredtothewebversionofthearticle.)

4. Discussion

Asignificantadvanceinthebonefieldarosewiththedevel- opmentofmicroCTaroundthe2000s.Thismethodissuperiorto scanningelectronmicroscopy atlow magnificationsand allows direct3Dmeasurementsofthebonemass,porosityandmicroar- chitecture (Ruegsegger, 1994; Sasov and Van Dyck, 1998). The

methodisfaster thanmorphometric analysisdoneonhistolog- icalsections; moreovernew microCTs havea resolution inthe 2–3␮m/pixelrange.InadditionmicroCTisnondestructiveandthe samplescanbeprocessedforadditionalhistologicalstudies.Unfor- tunatelymicroCTisonlysuitablefor tissueswithhighinherent X-raycontrast,suchasbonewhichisamineralizedtissue.Because softtissues,suchasarticularcartilage,arenon-mineralized,they

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Fig.3.(A)2DfrontalsectionofarightcondylarprocessaftermicroCTanalysis,showingtheaspectwithoutUAimpregnation.Thearticularcartilageisnotvisible.(B)2D frontalsectionofarightcondylarprocessafterUAimpregnation.ArticularcartilageisstronglyvisibleTendosteum,sometrabeculaeandtheperiostealsurface(probably correspondingtoosteoidseams)isnoted(yellowarrows).(C)Histologicalaspectofacondylarprocessembeddedundecalciﬁed(Goldner’strichrome)showingarticular cartilage(greenarrow)andosteoidseamsattheendosteum(yellowarrow).Inallimagesthescalebarstandfor500␮m.(Forinterpretationofthereferencestocolorinthis legend,thereaderisreferredtothewebversionofthearticle.)

absorbX-raylessintensely,makingthemnotvisiblewithmicroCT.

Therefore,contrastenhancementusinga“staining”procedureis aprerequisiteforcartilageimaging.Severaldifferentprocedures havebeendescribedintheliteratureto“stain”softtissuesusing ionicornon-ioniccontrastagents:ioxaglicacid,osmiumtetroxide, IKI,PTA,PMA,gadoliniumoriodinatedcontrastsagentsforcom- putedtomography(Cockmanetal.,2006;DasNevesBorgesetal., 2014;Kerckhofsetal.,2013;Metscher,2009;Nieminenetal.,2015;

Pauwelsetal.,2013;Rendersetal.,2014;Xieetal.,2009;Yooetal., 2011).Todatenoneofthemisfullysatisfactoryandasimple,repro- ducible,non-destructiveandhigh-standardtechniqueforcartilage contrastenhancement,allowing3Danalysisisstilllacking.Results fromourpre-studyshowthatnoneofthesecompoundsappeared satisfactoryonﬁxedarticularcartilage.

UAcanbe utilizedas acontrast enhancerfor soft tissuesin transmissionelectronmicroscopy,andhasbeenusedinthisman- nerfor decades.Theuranyl ionis knowntobindtophosphate groupsofnucleicacidsandtoproteins(especiallycollagen)(Hayat, 2000).Recentreportshaveraisedthatradioactivitygeneratedby UAcouldbedangerousandlanthanideshavebeenproposedasa surrogateforUA(Hosogietal.,2015).However,uraniumistoxic atahighconcentrationinhumananditsharmfuleffectsaremore relatedtodirecttoxicitythanradioactivity(Keithetal.,2007).UA isclassiﬁedashazardoussubstanceanddangerousgooddueto itsdirecttoxicity.Standardprecautionsmustthereforebetaken asineveryelectronmicroscopylaboratory(useofglovestohan- dlethesolutionunderalaboratoryfumecabinet,wastesolutions discardedinspeciﬁcvials).Inlivingbeings,uraniumingestionor inhalationcanundergodepositionandaccumulationinbone.Itwas recentlyfoundthaturaniumpreferentiallyaccumulatesinthecal- cifyingareasofbone(endosteal,periosteal)afterasingleinjection ofuranylnitrate(Bourgeoisetal.,2015).In ourpreliminarytri- als,weexperimented witha numberofmetallicsaltsthathave beenreportedtoenhancecartilagecontrastinmicroornanoCT.

Iodinecreatedaheavystainingofmarrowcells;shrinkageisalso describedintheliteratureforhighlyhydratedtissuesbutthiswas notinvestigateinthispre-study(Vickertonetal.,2013).PTAgavea palecontrastofthearticularcartilage.Gadolinium,proposedforMR imagingdidnotworkproperlyonﬁxedtissues(Bashiretal.,1997;

Cockmanetal.,2006).Iodinatedanioniccontrastagentsrecom- mendedforperipheralquantitatecomputedtomography(pQCT) (Silvast etal.,2009)werealsoineffectivetoprovideasufﬁcient contrastonﬁxedcartilage,especially ontheupperlayerofthe cartilage.Onthecontrary,UAwasfoundtoprovidethebestcon- trastinmicroCTforthehumanhipcartilage.Lanthanidesenhanced thehumanarticularcartilagefaintlybybindingtoglycosamino-

glycanes.Becausethethickhumanhipcartilagewasmoreclearly identifiedbyUA,weselectedthisproceduretoenhancethecon- trast ofthethin cartilageofthemandibularcondylesinrat. As describedbyothers, UAtendstolocalizeinthebonematrixby asubstitutionmechanismwiththemineralandanadsorptionon somenon-collagenousproteins(Bourgeoisetal.,2015).Thiscould explainwhyaheavylabelingofthetide-markandsomeareasof theendosteumortrabeculae(probablycorrespondingtoosteoid seams)werenoted inadditiontoa faintmarrow fixation.Both PTAand UAareknowntobefixedbycollagen.Asimilarlabel- ingofthetide-markisthereforereportedwithPTAstaining(Das NevesBorgesetal.,2014;Nieminenetal.,2015).X-rayattenuation obtainedwithPTAandPMAstainingappearstodependoncollagen distribution,i.e.,cartilagedepth(Nieminenetal.,2015).Cartilage stainslessstronglywithPTAcomparedtoothertissues(Metscher, 2009).Inourhands,PTAprovidescartilage3Dimagingofpoorer qualitythanUA(DasNevesBorgesetal.,2014).

Histomorphometryisthestandardprocedureforarticularcar- tilagethicknessmeasurement.Nevertheless,thistechniqueseems unreliableforquantitativemeasurementsincetherearedifﬁculties inreproducingthesameorientationforeachsample,duetosection obliquity(HowardandReed,2004;Vesterbyetal.,1987).Morpho- metricanalysisofcartilagethicknesshasbeendoneaftercontrast enhancementwithHexabrixinmicroCTalthoughthecartilagewas lessclearlydelineated thaninthepresentstudy(Rendersetal., 2014;Xieetal.,2009).Ahistomorphometricanalysisofthecarti- lageofmandibularcondylesinyoungrabbitwasrecentlypresented (Matthysetal.,2015,2016).

Inthepresentstudybonelosswasfoundattherightcondyles afterBTXinjectionintherightM.MasseterandM.temporalisin adultrats.Theseresultsfitin wellwiththeliteratureand con- firmourpreviousfindings(Kün-Darboisetal.,2015;Matthysetal., 2015).MandibularbonelossisduetotheBTX-induceddisuseof thetemporomandibularjoint.Whetherdisusealsoaffectscondy- larcartilageisstillcontroversial(Matthysetal.,2016).Condylar cartilageassessment inthis modelofdisusehasbecomeasub- jectofinterest.Itisknownthatarticularcartilagecanbestrongly affectedbymechanicalstatusofthejoint(Matthysetal.,2015).In younggrowingratshavingreceivedBTXinjectioninM.Masseter, themandiblegrowthwasimpairedandthecartilagethicknesswas significantlyreduced(Kimetal.,2008).Thiswasassociatedwith anincreaseinthenumberofapoptoticcells(determinedbythe TUNELassay)intheproliferationandhypertrophiczones.Onthe contrary,nochangeincondylarcartilagethicknesswasfoundafter aunilateralBTXinjectioninM.Masseterinyoungrabbits(Matthys etal.,2015,2016).Itshouldbenotedthatbothpapershaveused

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contrastenhancement byUA usingmicroCTthat would permit quantitativeanalysisof articularcartilage thickness.It allows a simpleandnon-destructiveassessmentofarticularcartilagemor- phology,whereascartilage isnormallyradiolucent.Othermetal saltsreportedintheliteraturecouldalsolabelcartilagebutthe intensityofstainingwaslowerinmicroCTimages.UA“staining”

wasfoundeffectiveintheassessmentand quantiﬁcationofthe condylararticularcartilageofthemandibleintheBTXratmodel of disuse.Although a reduction ofbone masswas observed in thecondyle,nodifferencewasobservedincartilagethicknessone monthafteraBTXinjection.

Conﬂictofinterest

Theauthorsdeclarethattheyhavenoconﬂictsofinterestcon- cerningthisarticle.

Acknowledgments

ThisworkwasmadepossiblebygrantsfromtheFrenchMinistry of Research. Many thanks to Mrs. Lechat for secretarial assis- tance.AuthorsaregreatlyindebtedtotheSCAHU(Servicecommun d’animalerie hospitalo-universitaire) of Angers, especially to P.

LegrasandJ.Rouxfortheirhelpwiththeanimalcare.Theyalso thankMrs.NadineGaboritforherskillfulassistancewithmicroCT andhistotechnology.JDKDreceiveda scholarshipfromtheARS (Agencerégionaledesanté)«PaysdelaLoire».

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