Unwrapping microcomputed tomographic images for measuring cortical osteolytic lesions in the 5T2 murine model of myeloma treated by bisphosphonate

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Micron

jo u r n al h om ep 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

Unwrapping microcomputed tomographic images for measuring cortical osteolytic lesions in the 5T2 murine model of myeloma treated by bisphosphonate

Mambaye N’Diaye

, Hélène Libouban

, Eric Aguado

, Béatrice Bouvard

, Maurice Audran

, Daniel Chappard

aGEROMGroupeEtudesRemodelageOsseuxetbioMatériaux,LUNAMUniversité,IRIS-IBSInstitutdeBiologieenSanté,CHUd‘Angers,49933AngersCedex, France

bUPSP“BiologieetBiomatériauxduTissusOsseux–ChirurgieExpérimentale”,ONIRIS–NationalVeterinarySchoolofNantes,LUNAMUniversité,Routede Gâchet,BP44706,F44307Nantescedex03,France

cServicedeRhumatologie,CHUd‘Angers,49933AngersCedex,France

a r t i c l e i n f o

Articlehistory:

Received8May2014 Receivedinrevisedform 15September2014 Accepted2October2014 Availableonline13October2014

Keywords:

Myeloma Bisphosphonate Corticalbone Osteolysis MicroCT

a b s t r a c t

Multiplemyelomaisduetotheproliferationofmalignantplasmacellswhichincreasethenumberof osteoclastsleadingtotrabecularandcorticalboneosteolysis.The5T2MMmurinemodelreproducesthe humandiseaseandmicrocomputedtomographyisaprecisetooltoinvestigateboneloss.Bisphospho- nates(zoledronicacidorpamidronate)areusedinpreventingosteolysis.However,lossofcorticalbone innotpossibletoquantifybyhistomorphometryonhistologicalsectionsormicroCTimages.

Osteolysiswasstudiedinmicegraftedwiththe5THLsublinetoseeifonedrugwasmoreactiveafter 10weeks.Miceweredistributedinto4groups:control,untreated,treatedwithpamidronateorwith zoledronicacid.Theleftfemurswereembeddedundecalcifiedandsectionedat7␮m.Therighttibias andfemurswereanalyzedbymicroCTandtrabecularmorphometricparameterswereobtained.Cortical boneosteolysiswasanalyzedbydevelopinganewalgorithmtounwrapmicroCTsectionsofthecortices, allowingmeasurementofthenumberofperforations,porosityandmeanperforationarea.

Thebisphosphonateshadnosignificanteffectonthetumorgrowthasevidencebytheabsenceofeffect ontheM-proteinlevel.Corticalperforationswereevidencedonhistologicalsectionsandtheirnumber seemedtobereducedbybothbisphosphonates.MicroCTwasusedtoquantifythetrabecularbone:a bonelosswasevidencedintheuntreatedmyelomagroupandbothbisphosphonatesappearedequalto preservetrabecularmass.However,thenumberandsizeofcorticalperforationscannotbedetermined on3Dmodels.UnwrappingmicroCTimagesprovidedflatimagesallowingaprecisedeterminationof corticalperforations.Pamidronatedidnotreducethenumberandsizeofcorticalperforationsbutsig- nificantlyreducedporosity.Zoledronicacidappearedsignificantlysuperiorandconsiderablyreducedall parameters.

UnwrappingmicroCTimageisanewmethodallowingthemeasurementofcorticalperforationsin bonemalignancies,aparameterthatcannotbemeasuredcorrectlyon2Dhistologicalsections.

©2014ElsevierLtd.Allrightsreserved.

Abbreviations: 2D,twodimensions;3D,threedimensions;BM,bonemarrow;BP,bisphosphonates;BV/TV,trabecularbonevolume;Ec.Po,endostealporosity;FPP, farnesylpyrophosphate;IL-,interleukine;MicroCT,microcomputedtomography;MM,multiplemyeloma;N.Po,numberofpores;Pam,pamidronate;PamMM,micetreated withpamidronate;PBS,phosphatebufferedsaline;Po.Ar,meanareaofacorticalperforation;Ps.Po,periostealporosity;RANKL,receptoractivatorofNF␬Bligand;Tb.N, trabecularnumber;Tb.Sp,trabecularseparation;Tb.Th,trabecularthickness;UMM,micewithuntreatedmyeloma;VOI,volumeofinterest;Zol,zoledronicacid;ZolMM, micetreatedwithzoledronicacid.

∗ Correspondingauthorat:GEROM–LHEA,IRISIBSInstitutdeBiologieenSanté,LUNAMUniversitéNantesAngersLeMans,CHUd‘Angers,49933AngersCedex,France.

Tel.:+33244688349;fax:+33244688351.

E-mailaddress:daniel.chappard@univ-angers.fr(D.Chappard).

http://dx.doi.org/10.1016/j.micron.2014.10.001 0968-4328/©2014ElsevierLtd.Allrightsreserved.

108 M.N’Diayeetal./Micron68(2015)107–114 1. Introduction

Multiplemyeloma(MM)isaneoplastichematologicaldisease characterizedby the production of a monoclonal gammopathy (M-protein)and plasma Bcell infiltrationof thebone marrow.

Osteolyticlesionswilldevelopinabout80–90%ofMMpatients;

Theyareduetoanincreasedosteoclasticactivityresultingfrom interactionofplasmacellswiththebonemarrowenvironmentin associationwithadecreaseoftheosteoblasticfunction(Bataille etal.,1989,1991).Severalcytokineshavebeenshowntoberespon- sibleforanincreaseinosteoclastnumberandactivityincluding upregulationofRANKL(receptoractivatorofNF␬Bligand),repres- sionofosteoprotegerin,overproductionofIL-6andmacrophage inflammatoryproteinMIP-1␣bytheplasmacellswithinthebone marrowmicroenvironment(Lentzsch etal., 2003; Pearseet al., 2001;Roodman,2009).Inparallel,thereisanincreasedexpression ofDickkopf-1protein,IL-3 andsecretedfrizzled-relatedprotein 2which provokeaninhibition oftheosteoblastic activity(Tian et al., 2003). This complex alteration in the cytokine network withinbonemarrow invadedbyplasma cellsinduces a vicious circle betweenbone cells and themalignant cells. This is also responsible for the very specific aspect of bone lesions on X- rayimageswhichappearas multiple“punched-out”holes (also coinedcorticalperforations)withoutreactivesignofreconstruc- tionofthesurroundingbone(Dimopoulosetal.,2009).However theselesionsareonlyapparentwhen30–50%ofthebonemin- eraldensityhasbeenlost(Dimopoulosetal.,2009;Dinteretal., 2009).This radiologicalfindingcorresponds toa complete per- forationof thebone corticedeveloped in contact withnodules of plasma cellpresent in thebone marrow. Histologicalstudy, computed tomography or magnetic resonance imaging clearly illustrate that a perforation develops from the endosteal side andprogressestotheperiostealsurfaceofthecortices(Bauerle etal.,2009;Healyetal.,2011).InanimalmodelsofMMsuchas the5T2MMmodel reportedin theC57BL/KaLwRijmouse, sim- ilar cortical perforations (corresponding toosteolytic foci with disappearanceof the wholecortical thicknessfrom the endos- teumtotheperiosteum)havebeenreported(Radletal.,1985).

These modelsare useful tounderstand thepathophysiology of thediseaseand toevaluatenewpre-clinicaltherapeuticstrate- giesand pathophysiologicalhypotheses(Benameuret al.,2013;

Vanderkerkenetal.,2003,2005).Aspotentinhibitorsofosteo- clasts,bisphosphonates(BPs)have beenproposedveryearlyas bone-sparingagents inMM patients(Belch etal., 1991;Terpos etal.,2011).However,severalgenerationsofBPshavebeendevel- oped withincreasinganti-osteoclastic properties.Among them, amino BPs are the most active. Numerous clinical trials have showntheinterestoftheaminoBPspamidronate(Pam)andzole- dronic acid (Zol) in the prevention of osteolytic bone lesions andhypercalcemiain MM patients. Althoughthesecompounds are active in preventing trabecular bone loss, their activityon thecorticalbonelossappearslesspronounced(Chappardetal., 1991;Liboubanetal.,2003).Asingleanimalstudyconductedin osteosarcoma-inducedosteolysisintherathasraisedthepossibil- itythatZolcouldbeactiveoncorticallesionsbutnoquantitative evaluation of cortical perforations was done (Labrinidis et al., 2010).

Theaimofthepresentstudywastodevelopanewmethodto quantify,in3D,theefficiencyoftwoaminoBPscurrentlyusedin thetreatmentofMMpatients(PamandZol)inthe5T2MMmodelin themouse.Thetrabecularandcorticalbonelossweredetermined bymicrocomputedtomography(microCT).Becausenoalgorithm existstoquantifytheamountofcorticalperforations,wedevel- oped a methodology ofunwrapping imagesof corticalbone to evaluatethenumber,sizeandsurfaceofthesecorticalosteolytic defects.

2. Materialsandmethods 2.1. Mice

C57BL/KaLwRijfemalemice6–8-weeks-oldwereusedforthe study(Harlan,Gannat,France).Theywereacclimatedforoneweek tothelocalvivarium conditions (24^◦C and 12h/12hlightdark cycle)wheretheyweregivenstandardlaboratoryfood(UAR,Ville- moisonsurOrge,France)andwateradlibitum.TheAnimalCareand UsecommitteeattheUniversityofAngersapprovedallprocedures (experimentation#49028).

2.2. Culturecellline

We used the 5THL cell subline as previously characterized (Croese et al., 1987; Libouban et al., 2004). 5THLcells can be propagated into youngsyngeneic mice by intravenous transfer ofthediseasedbonemarrow(BM).Progressionofthediseasein sevenrecipient micewasassessed bymeasuringtheserumM- proteinlevelusingagarelectrophoresis(HydragelProtein,SEBIA, Issyles Moulineaux, France). Around6 weeks post-injection of 5THL,micehada detectableserumM-proteinandwereeutha- nizedafter10–12weeksbycervicaldislocation.Femursandtibias weredissected,cleanedofsurroundingtissuesandBMwasflushed inDulbecco’smodifiedessentialmedium(DMEM.mod,GIBCO,Life Technologies,France)supplementedwithpenicillin-streptomycin, amphotericin-fungizoneandpyruvate.BMcellswerewashedonce inDMEM.mod.MononuclearcellswereisolatedbyaLympholyte- Mcentrifugationgradient(Cedarlane,Hornby,Ontario,Canada)at 1250gfor20min.Mononuclearcellswerethenwashedtwicein DMEM.modandcounted.

2.3. Experimentaldesign

Asrecommendedinallpapersconcernedwiththe5TMMmodel, mice of 6–8 weeks old were used (Vanderkerken et al., 2003, 2005).Sixtyfivemicewereinjectedwith1.5×10⁶ 5THLcellsin thetailveinand8additionalmicewereusedascontrolanddid notreceivethemalignantcells(Controlgroup).Theinjectedmice weredivided in 3 groups accordingto thetreatmentreceived:

25micereceivedaweeklyinjectionofphosphatebufferedsaline (PBS)andconstitutedtheuntreatedMMgroup(UMM)andwere usedtomeasuretheimportanceofthelossoftrabecularandcor- ticalbone.Twentymiceweretreatedbysubcutaneousinjection ofpamidronate(0.4mg/Kg/day,5daysperweek)andconstituted thePamMMgroup.Twenty miceweretreatedbysubcutaneous injectionofzoledronicacid(120␮g/Kg,2daysperweek)andcon- stitutedtheZolMMgroup(Croucheretal.,2003;Patakietal.,1997).

PamandZol(Novartis,Basel,Switzerland)wereobtainedfromthe hospitalpharmacy.ThesedosagesforPamandZolweresimilarto thoseusedforthetreatmentofhumansaccordingtothesupplier ofthedrugs(Green,J.,NovartisPharma,personalcommunication).

Thesuccessand progressionof thegraftwasascertainedby quantitativeelectrophoreticasaboveat6,8and10weeks.When electrophoresisdidnotevidence theM-proteinat 6weeks,the graftwasfoundunsuccessfulandthemousewasexcluded.Atthe endstageofthedisease(10–12weeks),whenosteolysiscanbe evidencedonX-rayimages(Fig.1),micewereeuthanizedforeth- icalreasonsbeforetheoccurrence of fractures.Theywerebled beforebeingsacrificedbycervicaldislocationandthetibiasand femursweredissectedandfixedinformalin.

2.4. Histologicalanalysis

Theleftfemurswereembeddedundecalcifiedinpoly(methyl- methacrylate). They were cut-dry (7␮m in thickness) on a

Fig.1.Radiographicaspectofcorticalperforationsonthetibiacrestandonthe femurofmouseoftheuntreatedgroupat10weekspostinjectionof5THLcells.

Notethemultiple“punched-out”holeswhichareidenticaltoimagesseeninhuman myeloma.

heavy-duty microtome equipped with tungsten carbide knives (LeicaPolycutS-RueilMalmaison,France).Sectionswerestained witha modifiedGoldner’strichrome.Allhistologicaltechniques havebeenextensivelydescribedelsewhere(Chappard,2009).

2.5. X-rayandmicrocomputedtomography

-RadiographsofmiceweredoneexvivousingaFaxitronX-ray systemwitha 12cm×12cm CCD camera (Edimex,Le Plessis Grammoire,France)everytwoweekstofollowtheappearance ofosteolyticlesions(Liboubanetal.,2003,2004).TheSkyscan 1172X-raycomputed microtomograph(Bruker MicroCT,Kon- tich,Belgium)wasusedintheconebeamacquisitionmode.The bones(righttibiaandfemur),stillinthefixativetopreventdesic- cation,werescannedaccordingtopreviouslypublishedprotocol at69kV,100␮Aand a0.5mmaluminumfilter.Thepixelsize wasfixedat3.75␮manda0.25^◦ rotationanglewasappliedat eachstep.Foreachbone,astackof2D-sectionswasobtained.

TheCTAnSoftware(Skyscan,release1.10.1.0)wasusedforthe

Fig.2.(A)3DmodelofafemurfromamouseoftheUMMgroup.Notethenumberof perforationsvisibleontheperiostealsurface.Thesectioningplaneindicatesthelevel ofsection(B);(B)the2Dsectionselectedin(A)showingperforationsofthecortical ringandtrabeculaeinsidethecancellousspace;(C)Samesectionafterremovalof thetrabeculae;(D)binarizedimageofsection(C);(E)unwrappedimageofsection D;(F)3Dmodelofallunwrappedimagesviewedfromtheendostealside;(G)3D modelofallunwrappedimagesviewedfromtheperiostealside.

reconstructionofthewhole3Dmodel(Fig.2A)andtomeasure thebonemassandarchitectureatthesecondaryspongiosaof thetibia.Thefirstimageselectedforanalysiswasjustunderthe growthplate(excludingthethinprimaryspongiosa),andthen 6682Dsectionswereselectedforthefemurand800sectionsfor thetibia.Thevolumeofinterest(VOI)wasdesignedbydrawing polygonsonthe2Dsections.Onlyafewnumberofpolygonsneed tobedrawn(e.g.onthefirstsection,severalatthemiddle,andon thefinalsection)sincearoutinefacilitycalculatesalltheinterme- diarymasksbyinterpolation.TheVOIcomprisedonlytrabecular boneandmarrowcavity.Thefollowingparametersweremea- suredintheVOI(TV,inmm³)accordingtotherecommendations oftheAmericanSocietyforBoneandMineralResearch(Dempster etal.,2013).

-Trabecularbonevolume(BV/TV,in%)representsthepercentage ofthecancellousspaceoccupiedbytrabecularboneintheVOI.

-Trabecularthickness(Tb.Th,in␮m),trabecularseparation(Tb.Sp, in ␮m), and trabecular number (Tb.N, in/mm) provide a full descriptionofbonemicroarchitecture.

Then,trabecularbonewasdiscardedbyusingaroutinefacility ofthesoftwareandanewstackofbinaryimagescontainingonly thesectionofcorticalbonewasobtained(Fig.2B–D).

110 M.N’Diayeetal./Micron68(2015)107–114

Fig.3.Histologicanalysisofundecalcifiedfemur:(A)untreatedmousefromtheUMMgroup;(B)mousetreatedwithpamidronatefromthePamMMgroup;(C)mouse treatedwithzoledronicacidintheZolMMgroup.Arrowsidentifycorticalperforations.Goldner’strichrome;thebarstandfor500␮m.

2.6. Algorithmforunwrapping2Dimagesofbonesections obtainedbymicroCT

Themethodemployedhereisbased ona technique usedin vision technology to unwrap omnidirectional images acquired throughtheuseofacatadioptricsensor.Webasedonthechange ofreferencespacefollowedbyatranslationtounwrapastackof microCTimages.UnwrappingtheimageswasdoneusingaMat- lablab-madesoftware(MathWorks,Natick,MA,release7.10).In theory,weconsiderthatpixelsofeach2Dsectionofcorticalbone werepositionedinanorthogonalreferencewhosecentercoincides withthecenteroftheimage2D(Fig.2D).Thechangeofreference consistedtoexpressthecoordinatesofeachpixeloftheoriginal image2Dintheformofpolarcoordinates.Twocoordinates,andı wereobtainedforeachpixelofimage.Then,thepixelswerecopied inanewmatrixbyputtinginrowandıincolumn(ElKadmiri andMasmoudi,2011;Kepuckaetal.,2012).

Inpracticethealgorithmcanbedescribedasfollows:

Inthefirststep,theunwrappedmatrixwasinitializedandits sizedetermined.Toknowthesizeoftheunwrappedimagematrix, theradiusRandtheperimeterPofthe2Dwrappedimagewere determined;theyrepresentrespectivelythewidthandlength.Each pointofthe2Dwrappedimageisidentifiedbyitsx,ycoordinatesin thematrix.So,themasscenter(xc,yc)ofeach2Dsectionofthestack inaCartesianspacewasdetermined.Then,themasscenterwas translatedtotheoriginoftheCartesiansystem.Aftertranslation, thenewcoordinatesofeachpointwerelinkedtotheformersby therelation:x1=x−xcandy1=y−yc.Inthisway,themeanRand Pcanbeexpressedasfollows:

Rmoy= 1 N

1

(x_i−xc)²+(y_i−yc)²

P=2..Rmoy

In the second step, for each pixel of the unwrapped image matrix,thepolarcoordinates(,)wereobtained.

=i

= _P^j ×2whereiandjstandfortherawandcolumnnumber inthematrix.

Inthethirdstep,thepolarcoordinates(,^{)wereconverted} intoCartesiancoordinates(X,Y)asfollows:

[X,Y]=

cos(),sin()

Inthefourthstep,the(X,Y)coordinateswereexpressedinthe orthonormalreferencecenteredinthemiddleofthecenterofthe unwrappedimage.Becausethe0,0originofanimageinMatlab alwaysappearsonthetopleftoftheimage,(X,Y)wereconverted to(X,Y)expressedinthereferenceframecenteredas:

X=X+xc

Y=Y+yc

In thefifth step, each pixel values of thematrix (of the2D wrappedimagewithindex(X,Y))wascopiedinacelldefinedbyits i,jindexesintheunwrappedimagematrix.So,the2Dunwrapped matrixwasconvertedintoanewimagewiththe‘imshow’function ofMatlabandsavedinthe.bmpformat(Fig.2E).Thisprocedure wasrepeatedforall2D bonesectionsof thestack producedby themicroCT;in sucha way,anewstack ofunwrapped images wasobtained.The3Dreconstructionoftheunwrappedbonewas obtainedwiththeAntsoftwareandbothendosteal(Fig.2F)and periosteal(Fig.2G)sidescanbeimaged.

2.7. Measurementofboneperforationsontheunwrappedimages The3Dreconstructionoftheunwrappedbonewastransferred toMatlabsoftware.Twotypesofimageprocessingoperationswere appliedonthe3Dunwrappedimage.Inthefirststepasegmenta- tionwasperformedtodifferentiatebonefromperforations(bone inwhiteandperforations inblack).Inthesecondstep,labeling ofeach perforationwasdoneandtheirnumber N.Powascom- puted.Oneachofbothsurface(periostealandendosteal)ofbone, the total area of thebone unwrapped surface (resp. Ps.Ar and Ec.Arinmm²)andthetotalareaofperforations (Po.Ar,inmm²) were determined. So the periosteal porosity Ps.Po (in %) was derivedas100*Po.Ar/Ps.ArandendostealporosityEc.Po(in%)as 100*Po.Ar/Ec.Ar.

2.8. Statisticalanalysis

StatisticalanalysiswasperformedusingtheSystatstatistical softwarerelease13.0(SystatSoftwareInc.,SanJosé,CA,USA).All datawereexpressedasmean±standarderrorofthemean.Differ- encesbetweengroupswereanalyzedbyanon-parametricANOVA

Fig.4.3Dreconstructedmodelsoftheuppertibiaextremityinthedifferentgroupsofmice.(A)controlmouse;(B)untreatedmousefromtheUMMgroup;(C)mousetreated withpamidronatefromthePamMMgroup;(D)mousetreatedwithzoledronicacidintheZolMMgroup.

test(Kruskall–Wallis)withtheConover-Inmanpost-hoctest.Dif- ferenceswereconsideredsignificantwhenp<0.05.

3. Results

3.1. EvolutionoftheM-protein

EvolutionoftheM-proteinduringthediseaseisknowntoreflect thetumor growthand representsan indirectevaluation ofthe tumormassinthemouse.Thegraftof5THLcellswassuccessful inthe25casesoftheUMMgroup,17ofthePamMMgroupand14 oftheZolMMgroup.Asexpected,theM-proteinlevelsignificantly increasedineachgroupduringthetimecourseofthedisease.There wasnosignificantdifferencebetweenthegroupsatsix,eightorten weeks.

3.2. Evolutionoftrabecularandcorticalbone

Histologicalanalysisevidencedamassiveinfiltrationofthebone marrow of thefemur in each groupof animals (Fig. 3).Perfo- rationsof thecorticalbone wereeasilyshown;malignant cells

expansioninthesofttissuewasmostoftenlimitedbytheperios- teum.TheamountofperforationsseemedreducedinthePamand Zolgroups.However,due totheshapeof thebone andsection obliquity,nohistomorphometricevaluationofthenumberandsize oftheseperforationscouldbedone.

AnalysiswaseasieronbonesevaluatedbymicroCT.Thetypical aspectofthetibiametaphysisineachgroupappearsinFig.4.The amountoftrabeculaeislimitedinthisstrainofmouseasevidenced inFig.4A.Inthefemur,theaspectsweresimilar.Thetumorpro- vokedanetreductionofthetrabecularnetworkassociatedwith largeperforationsofthecorticalboneduetoincreasedresorption (Fig.4B).InthePamMMandZolMMgroups,trabecularbonewas preservedbutcorticalperforationswerestillnoticed(Fig.4C–D).

Onaquantitativepointofview,thetumorinducedaninsignif- icant decrease in BV/TV (Fig. 5A) in both femur and tibia.

MorphometricresultsarefiguredinTable1.Quantitatively,trabe- cularbonelosswasduetoasignificantreductioninthenumberof trabeculaeassociatedwithaparallelincreaseinTb.Sp.Pamidronate provokedanincreasein BV/TVbothatthefemurand tibia;the number of trabeculaewaselevatedand Tb.Thwassignificantly increasedatthetibiabutnotatthefemur.IntheZolMMgroup,

112 M.N’Diayeetal./Micron68(2015)107–114

Table1

histomorphometricresultsatthefemurandtibiainthedifferentgroupsofmice.

Control UMM PamMM ZolMM

Femur

BV/TV(%) 3.54±0.4 2.51±0.37 8.27±1.18^a,b 5.65±0.29^a,b

Tb.Th(␮m) 46±2 51±2 61±1^a,b 53±1^a

Tb.Sp(␮m) 354±9 664±39^a 462±43^a,b 481±21^a,b

Tb.N(/mm) 0.76±0.06 0.49±0.06 1.35±0.19^a,b 1.078±0.05^a,b

N.Po 1.9±0.3 31.2±2.9^a 32.7±2.23^a 20.15±3.28^a,b,c

Po.Ar(␮m²) 12±0.02 79.34±12.3^a 88.99±12.9 32.35±9.0^a,b,c

Ps.Po(%) <0.01 10.18±1.43^a 6.85±0.98^a 1.89±0.48^a,b,c

Tibia

BV/TV(%) 4.28±0.68 3.93±0.55 7.12±0.81^a,b 8.58±0.49^a,b

Tb.Th(␮m) 49±2 51±2 55±2^a 62±7^a

Tb.Sp(␮m) 414±26 638±27^a 455±30^b 404±21^b

Tb.N(/mm) 0.86±0.11 0.74±0.1^a 1.28±0.14^a,b 1.53±0.13^a,b

N.Po 2.25±3.14 43.13±3.97^a 42.25±5.13^a 23.93±5.01^a,b,c

Ps.Po(%) 0.06±0.01 7.03±1.15^a 5.31±1.16^a 1.83±0.52^a,b,c

Po.Ar(␮m²) 12.7±0.3 93.75±14.0^a 109.0±24.2^a 35.52±12.25^a,b,c

aSignificantlydifferentfromcontrols.

b SignificantlydifferentfromUMM.

c significantlydifferentfromPanMM.

BV/TVwasalsosignificantlyincreasedvs.controlsbutdidnotdiffer fromvaluesofthePamMMgroup.However,Tb.Thwasmoderately increasedatthefemur;Tb.Nwasincreasedvs.controlinbothtypes ofbone.Thiseffectisduetothelackofremodelingoftheprimary spongiosainanimalsreceivingBPs.

Corticalboneappeared more stronglyaffectedbythetumor thantrabecularbone.Largeosteolyticlesionswereobservedinthe UMMgroup(Fig.5B).Morphometricanalysisoftheunwrapped imagesshowedthatbothBPshadreducedthesizeofperforations (Fig.4C–D)butthenumberofperforations(N.Po)wasreducedonly intheZolMMgroup(Fig.5C–DandTable1).Thesurfaceofeachcor- ticallesionwasreducedinthePamMMandZolMMgroups,sothe fractionsurfaceofthecorticalbonewithperforations(Ps.Poand Ec.Po)wasreducedwhencomparedtotheUMMgroup.Ps.Poand Ec.Powereexactlysimilarineachanimal,soonlyPs.Poappearsin Table1.Ps.PowassignificantlylowerintheZolMMgroupwhen comparedtothePamMMgroup(Fig.5C).N.Poandporositywere calculatedinthecontrolgroupandcorrespondedtothevascular channelswhichbringthevascularsupplytothecorticalboneand thebonemarrow.

4. Discussion

In thepresent study,unwrapping the3D imagesof a tubu- larbone was foundtobethe mostreliable methodtoanalyze cortical porosity. In a preliminary approach, filling the holes on2D microCTsections wasourfirst approach; we have tried a number of techniques classically used in image analysis to reconstructtheperiostealand/orendostealsurfaces:conditional opening/closing,x³ polynomialreconstruction afterray tracing, snake reconstruction... However, none of these methods was foundsatisfactorybecausetheyworkedwellonasinglemicroCT sectionandconsiderabledifferenceswereobtainedonthesuperior orinferiorsections.Theunwrappingmethoddescribedherecauses aminimumofartifactssuchasalittlealiasingeffectobservedon theflattenedimages.

BPsarehighlypotentanti-osteoclasticdrugswhichareincor- poratedintothemanagementofosteolyticlesionsinmetastatic bonedisease(Body,2003)andmultiplemyeloma(Bataille,1996;

Berenson, 1998;Mahindra et al.,2012).Pamidronate and zole- dronicacidarenowadaysthemostcommonlyusedBPsforthe preventionofMM-relatedosteolysis.Bothcompoundsbelongto thesubgroupofaminobisphosphonateswhicharesuperiortothe firstgenerationofBPssuchasclodronate,etidronate,tiludronate.

Atthetissue level, Zolis byfarmore potentthan Pamin pre- servingtrabecularbone in rat models(Patakiet al.,1997).The potencyofbisphosphonates,atthecellularlevel,isrelatedtotheir potencytoinhibitthetargetenzymefarnesylpyrophosphatesyn- thase(FPP-synthase), a key branchingpoint of the mevalonate pathway(Dunfordetal.,2001;Greenetal.,1994).

Inthepresent study,wefoundthatboth BPsweresimilarly effectivein preservingthetrabecularbone massin the5T2MM model. Pam and ibandronate were found to reduce osteolytic lesionsin the5T2MM model without anyeffect on thetumor burden and thetumormass becausebone marrowwasalmost completelyinvadedinthe3groupsofanimals(Dallasetal.,1999;

Liboubanetal.,2003;Radletal.,1985).PamandZolpreventedthe bonelossduetothehyper-resorptioninducedbythe5THLcells.

TheincreaseinBV/TVisnotduetoastimulationoftheosteoblas- ticactivity.Becauserodentshaveacontinuousgrowth(thegrowth platedoesnotdisappearinratsandmice),BPsinduceaconden- sationdue tothelack of remodelingofthe primaryspongiosa.

Thiseffectisoftenreferredasthe“Schenkeffect”(Schenketal., 1986).Asimilareffecthasbeendescribedinratmodelsofmetas- tasiswhereasingleZolinjectioncanconvertosteolyticmetastases intoosteoscleroticones(Nyangogaetal.,2010).

Iftrabecular morphometricanalysis iseasy to perform,it is moredifficulttoanalyzecorticalboneathistomorphometricand microCTlevels.Corticalperforationsareobservableduringrota- tionofthemodelinthe3Dspaceallowingadirectvisualization oftheselesionsbutnoquantitativeanalysisispossible.Wehave foundthatunwrappingimagesofthecorticescouldrepresenta suitablemethodtoquantifyboneresorptioninthisenvelope.The 3Dmodelscanbecomparedtoasceneanalyzedbyaunidirectional cameraandtheirrotationissimilartotheuseofmultidirectional camera.Multidirectionalcamerasystemsaremoreandmoreused byresearchersworkingincomputervision,becausetheycancap- turelargepartofasurroundingscenewithananglerangingfrom 0to360^◦.Manyapplicationssuchasrobotics,videosurveillance, andvideo-conference have founda great interestinusing such systems.Invariousapplications,theunwrappingprocessisnec- essaryto obtainpanoramicimageswhich are more adaptedto humanvisualsystemofconcentricannulardistortion.Themost usedmethodandeasiesttoimplemented,isthemethodofcon- vertingpolarcoordinatestoCartesiancoordinates,precededbya geometrictranslation,althoughthistechniqueaffectsthesizeand resolutionoftheoutputimage(Potúˇcek,2006;StraussandComby, 2005).

Nb of periosteal perforations (N.Po) 0 20 40 60 0

BV/TV (in %)

2.5 5 7.5 10 12.5

Porosity Ps.Po (in %)

0 2 4 6 8 10 12

tibia femur

tibia femur

tibia femur

* *

*

*

§

§

§

* *

* *

* *

§

#

#

*

*

§

§ #

#

*

*

*

*

control UMM

PamMM ZolMM

A

B

C

Fig.5. Mainmorphometricresultsobtainedintrabecularandcorticalboneby microCTatthefemurandtibia.(A)Trabecularbonevolume;(B)numberofperfora- tions;(C)Porosity.*:significantlydifferentfromthecontrolgroup;§:significantly differentfrom theuntreatedgroup(UMM);#: significantlydifferentfromthe pamidronatetreatedgroup(PamMM).

Becauseit is technicallydifficulttounwrapa 3D image,we chosetounwrapeach2Dsectionobtainedafterbinarizationand toproceedtoa reconstructionofa new3D modelof thestack of the unwrapped images. The method was developed under Matlabwhichwasalsousedtoprovidequantitativeanalysisofthe perforations.

Inthisstudyweevidencedthatthenumberofperforationswas decreasedinthePamMMgroupbuttheirmeansurfaceremained similartothoseoftheUMMgroup.Thiswaspreviouslynoticed byourgroupinMMmicetreatedwithPamatthesamedosage butwewerenotabletoquantifythisparameter(Liboubanetal., 2003).Zolcausedaconsiderablereductioninporositybyreducing thenumberandthemeansurfaceofperforations.

Significant differences were noted between ZolMM and PamMM,meaningthatZol,asa3^rdgenerationBP,ismorepotent toinhibitprogressionofosteolyticlesionsincorticalbonewhen administeredinclinicallyrelevantdosesthanPam.

Therearefewclinicalstudiesconcernedwiththecomparison ofPamandZol.BothBPsareconsideredtohaveasimilarpower forthetreatmentofPaget’sbonedisease(Merlottietal.,2007).

In alargemeta-analysis,comparingZol,Pamand clodronatein patientswithbonemetastases,it wasfoundthatZolwassupe- riortoPamand clodronatetoreducetheoccurrenceofskeletal relatedevents(Machadoetal.,2009).HowevernoneoftheBPswas associatedwithareducedmorbidityinthesepatients.Inanother meta-analysisconductedinMMpatientstreatedbyZol,etidronate orotherBPs,Zolwassuperiortoetidronateinimprovingsurvival (Mhaskaretal., 2012).All BPsappeared tohave similareffects inreducingthenumberofvertebralfractures,theotherskeletal relatedeventsandpainwithoutevidenceofsuperiorityofZolover Pam,ibandronate,etidronateorclodronate.However,theinfluence onperipheralfractureswasnot takeninto consideration.Kanis reviewed theeffect ofPam and clodronate in thetreatmentof MMpatients.BothBPswereactiveinpreventinghypercalcemia, painandtheskeletalrelatedeventswithoutmakingacleardis- tinctionbetweenvertebralfractures(vertebralbodiesbeingmainly composedoftrabecularbone)andlongbonefractures(mainlycom- posedofcorticalbone)(KanisandMcCloskey,2000).Ina study comparingcorticalandtrabecularboneinoncologicpatientswith metastasisandtreatedwithZol,noincreaseinbonedensityofcorti- calbone(measuredbyvolumetriccomputedtomographydensity) wasnotedinthenon-invadedbone(Quattrocchietal.,2012).How- ever,osteolyticmetastasescontainingbothtrabecularandcortical bonehadanincreasedbonedensity.

EquivalentresultswereobtainedinMMpatientstreatedbyper- fusionofPam(90mg)orZol(2or4mg)(Berensonetal.,2001).In thisstudy,ZolwassuperiortoPaminpreservingcorticalboneinthe 5T2modelofMMasevidencedbytheunwrappingofthemicroCT images.AllBPsinhibittheFPPsynthaseandtheirpotencydepends bothoftheirhalfmaximalinhibitoryconcentration(IC50)onthis enzyme(Kavanaghetal.,2006)andtheiraffinityforthehydroxy- apatiteofthebonematrix(Nancollasetal.,2006).BPaffinityalso affectsdrugdistributionincorticalandtrabecularbone(Tureketal., 2012).Zolhasthehighestaffinityforhydroxyapatiteand isthe mostpotentinhibitorofthetargetenzymeFPP-synthase,which mayexplainitssuperiorityoverPamwithregardtotheinhibition ofcorticalosteolysisinourmousemodelofMM.

5. Conclusion

Inthepresentstudy,weproposeanewsoftware-basedimage processing methodfor‘unwrapping’microCTimagesof cortical bones,thusallowingforthefirsttimetomeasureporosityandpore characteristicscausedbyosteolytictumorsinamousemyeloma model.ThismethodcouldbetransferredtoCT(orHighresolution peripheralquantitativeCT)scansobtainedinhumans,toevaluate theprotectiveeffectofdrugsoncorticalbonewhichforthelackof asuitablemethodhasreceivedmuchlessattentionthantrabecular bone.

Conflictofinterest

Theauthorsdisclosenopotentialconflictsofinterest.

Acknowledgments

This work was made possible by grants from Contrat Region Pays de la Loire: Bioregos2 program. Many thanks

114 M.N’Diayeetal./Micron68(2015)107–114

to Mrs. Lechat for secretarial assistance and to the SATT Ouest Valorisation for their help with the deposit of this software (Unwrappos) at the Agency for Programs Protection IDDN.FR.001.310043.000.S.P.2013.000.31230.

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