Rev. bras. ortop. vol.51 número3

SOCIEDADE BRASILEIRA DE ORTOPEDIA E TRAUMATOLOGIA

w w w . r b o . o r g . b r

Original

Article

Adverse

effect

of

beta-tricalcium

phosphate

with

zeta

potential

control

in

repairing

critical

defects

in

rats’

calvaria

夽

Daniel

Falbo

Martins

de

Souza,

Luciana

Correa,

Daniel

Isaac

Sendyk,

Rafael

Augusto

Burim,

Maria

da

Grac¸a

Naclério-Homem,

Maria

Cristina

Zindel

Deboni

FacultyofDentistry,UniversidadedeSãoPaulo,SãoPaulo,SP,Brazil

a

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t

i

c

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e

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n

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o

Articlehistory:

Received22June2015 Accepted7July2015 Availableonline26April2016

Keywords:

Boneregeneration Wistarrats

Biocompatiblematerials Zetapotential

a

b

s

t

r

a

c

t

Objective:Toevaluatewhetheranewbiphasiccementcomposedofcalciumsulfateandbeta tricalciumphosphatewithzetapotentialcontrolcouldinduceorleadtoboneneoformation incriticaldefects.

Methods:Acriticaldefectofdiameter8mmwasmadeinthecalvariaoffortymaleWistar rats.IntheTestGroup(n=20),thedefectswerefilledwithcement.IntheControlGroup (n=20),thedefectwasnotfilledandonlycoagulumwaspresent.Theanimalswere sac-rificed7, 14,21 and42 daysafterthe operation.Calvariaspecimensweresubjectedto microtomographyandwerethenpreparedforhistologicalanalysis.Theanalysesincluded morphologicalassessmentonthehistopathologyoftherepair;comparativemorphometric evaluationoftheareaofformationofbonetrabeculae betweenthegroups;and histo-chemicalstainingbymeansoftartrate-resistantphosphatase(TRAP)inordertoidentify osteoclasts.

Results:Microtomographicimagesofthedefectsfilledbythecement didnot showany decreaseinareaoverthecourseofpostoperativeevolution.IntheTestGroup,thematerial continuedtopresentaforeign-bodyresponseuntilthelastobservationalperiods. Histomor-phologicalanalysisshowedthatthereweremoresignificantgroupingsofgiantcellsinthe TestGroupandgreatermaturityofneoformedboneintheControlGroup.Exogenous mate-rialwasalsopresent.HistomorphometricanalysisshowedthatintheControlGroup,the totalareaofboneneoformationwassignificantlygreater(p=0.009)andgrewprogressively. ThegiantcellspresentedapositivereactiontoTRAPbutnoosteoclastswereobserved.

夽

StudycarriedoutattheDepartmentofSurgery,ProsthesisandOralandMaxillofacialTraumatology,FaculdadedeOdontologia, UniversidadedeSãoPaulo(USP),SãoPaulo,SP,Brazil.

∗ _{Correspondingauthor.}

E-mail:mczdebon@usp.br(M.C.Z.Deboni).

http://dx.doi.org/10.1016/j.rboe.2015.07.010

Conclusion: Theceramiccementdidnotinduceorleadtoboneneoformationfromthe microtomographicorhistologicalpointofview.

©2015SociedadeBrasileiradeOrtopediaeTraumatologia.PublishedbyElsevierEditora Ltda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

creativecommons.org/licenses/by-nc-nd/4.0/).

Efeito

adverso

do

beta-fosfato

tricálcico

com

controle

de

potencial

zeta

no

reparo

de

defeitos

críticos

em

calvária

de

ratos

Palavras-chave:

Regenerac¸ãoóssea RatosWistar

Materiaisbiocompatíveis Potencialzeta

r

e

s

u

m

o

Objetivo: Avaliarseumnovocimentobifásicocompostoporsulfatodecálcioebetafosfato tricálcicocomcontroledepotencialzetapoderiainduzirouconduziraneoformac¸ãoóssea emdefeitoscríticos.

Métodos: Foifeitoumdefeitocríticode8mmdediâmetronacalváriade40ratosWistar machos. Nogrupo teste(n=20)os defeitosforampreenchidospelo cimento.Nogrupo controle(n=20)osdefeitosnãoforampreenchidos,permaneceuapenasocoágulo.Os ani-maissofrerameutanásiaem7,14,21e42diasdopós-operatório.Espécimesdacalvária forammicrotomografadoseposteriormentepreparadosparaanálisehistológica.Asanálises incluíramaavaliac¸ãomorfológicadahistopatologiadoreparoeaavaliac¸ãomorfométrica daáreadeformac¸ãodastrabéculasósseascomparativamenteentreosgruposecolorac¸ão histoquímicapormeiodafosfatasetartrato-resistente(TRAP)paraidentificac¸ãode osteo-clastos.

Resultados:Asimagensmicrotomográficasdosdefeitospreenchidospelocimentonão apre-sentaramdiminuic¸ãodaáreadeacordocomaprogressãodosperíodospós-operatórios.No grupotestehouvepermanênciadomaterialerespostacorpoestranhoatéosúltimos perío-dosdeobservac¸ão.Ahistomorfologiamostrouagrupamentosmaisexpressivosdecélulas gigantesnogrupotesteeossoneoformadomaismaduronogrupocontroleecomprovoua presenc¸adematerialexógeno.Nahistomorfometria,aáreatotaldeneoformac¸ãoósseafoi significativamentemaior(p=0,009)ecrescentenogrupocontrole.Ascélulasgigantes apre-sentaramexpressãohistoquímicapositivaparaTRAPenãoforamobservadososteoclatos.

Conclusão: Ocimentocerâmiconãoinduziuouconduziuaneoformac¸ãoósseasoboponto devistamicrotomográficoehistológico.

©2015SociedadeBrasileiradeOrtopediaeTraumatologia.PublicadoporElsevier EditoraLtda.Este ´eumartigoOpenAccesssobumalicenc¸aCCBY-NC-ND(http://

creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Autogenous graft is still the material of choice for the reconstructionofbonetissuelossinorthopedicand maxillo-facialsurgery.However,increaseinoperationtime,surgical traumaandpossiblecomplicationsinherenttothedonorarea approachdoes notalwaysmakeitfeasible.New biomateri-alsandsubstancesthatcanmimicthecharacteristicsofthe autogenousbonetissuehavebeenaconstantpursuitof bio-engineering.

Amongthealloplasticmaterialsmostoftenusednowadays arebioceramics,mainlyhydroxyapatiteandbeta-tricalcium phosphate(␤-TCP).Thelattershowsamorerapid biodegrada-tionthanhydroxyapatiteandinsomesituationsthismaybe amoreadvantageouscharacteristicforabiomaterial,mainly whenthere is noneed formechanical strength. Moreover, beta-tricalciumphosphatehasbeenwidelyusedasacarrier orscaffoldintissueengineering.

Recently,abiphasicceramicmaterialconsistingofcalcium sulfateand beta-tricalciumphosphate withanegative sur-facecharge,calledzetapotentialcontrol,waslaunchedinthe

international market with the proposal of making beta-tricalciumphosphateaninductivebonesubstituteandthus, promoteboneregeneration.1

According to the manufacturer,2 _{this ceramic is fully} synthetic and has what they called “intelligent porosity”, whichfacilitatescellgrowthandnutrientdistributioninthe extracellularmatrixinternallyinthemacroporositiesofthis compound. Someauthors havedemonstratedintensebone regenerationcapacityinvertebraldefectsinsheep.1

Theosteogenicpotentialofthiscompound,however,has beenquestioned.Someauthorshaveshownconcernaboutthe biologicalsafetyoftheproduct.3_{Otherresearchers}4 discontin-uedearlyclinicaltrialsduetotheappearanceofunexpected adverse effects, suchas aseptic inflammationand delayed repair.Theliteratureisscarceontheanalysisofthe biolog-icalbehaviorofthisnewbiomaterialinbonerepairininvivo

studies.

Material

and

methods

Experimentalprocedure

ThisresearchprojectwasapprovedbytheInstitutionalEthics Committee on Animal Use (CEUA) under protocol number 006/2014 and in accordance with the ethical principles of animalexperimentationadoptedbytheBrazilianSocietyfor LaboratoryAnimalScience(SBCAL).

Forty (40) male Wistar rats (Rattus norvegicus albinus), weighingbetween 200gand 250g and agedapproximately 45 days, were operated on under general anesthesia by intramuscular injection in the right rear paw of each ani-malof ketaminehydrochloride (Dopalen®_{, Vetbrands) at a}

doseof0.8mg/kgassociatedwithmusclerelaxantxylazine (Rompum®_{, Bayer) at a dose of 0.3mg/kg. All animals}

receivedantibioticprophylaxisthroughintramuscular injec-tion of benzathine benzylpenicillin (Roche®_{) at a dose of}

150,000IU/kg.

After trichotomy, followed by skin antisepsis with 2% chlorhexidine digluconate,anaccess wasmade tothe cal-varia througha 2cm-rectilinear incision in the skinofthe medialregionof theskull, extendingfrom the nasofrontal area tothe occipitalprotuberance. Theskin, subcutaneous tissue,temporal muscle and theperiosteum were divulsed laterally.

A bone defect was created in the central region ofthe animalcalvariausingan8mm-diametersteeltrephinedrill (Sistemas de Implantes Nacionais – SIN®_{), adapted to a}

counter-angleimplant motor (Driller® _{– Carapicuíba – São}

Paulo) under low speed and constant irrigation with 0.9% salinesolution.

Theanimalswererandomlydividedintotwogroups:Test Group(n=20),whichreceivedcementconsistingofbeta tri-calciumphosphate(␤-TCP)andcalciumsulphateataratioof 1:1(Genex–Biocomposites®_{–Staffordshire–England)tofill}

thecriticaldefectinthecalvariaattheamountof25mm3_per

defect,andtheControlGroup(n=20),whichremainedwith thecriticaldefectfilledonlybyaclot.

Synthesisoftheskinwasperformedwith3/0silkthread (Ethicon®_{).Throughout thestudy period, theanimals were}

keptinventilated polypropylenecages,coveredwith steril-izedwoodshavings,withday/nightcyclesof12/12h,andwere fedrodentchow(LabinaforRodents,Purina®_)andwaterad

libitum.

Fiveanimalsfromeach groupwereeuthanizedinaCO2

chamberafter 7, 14, 21 and 42 days postoperatively. After euthanasia,theskullsweredissectedandrepresentative frag-mentsofthecalvariawiththedefectareawerefixedin10% bufferedformalin.

Priortotheprocessingandhistologicalanalysis,a speci-menfromaratintheTestGroupandfromaratintheControl Group,ineachofthestudyperiods,weresubmittedto micro-tomographyina100kVto100␮ASkyScanmicrotomographer intheDepartmentofAnimalPhysiologyoftheBiology Insti-tuteofUniversidadedeSãoPaulo.

Thesampleswereplacedatandfixedtotheequipmentcell, whichwassetforaresolutionof2000×2000pixelsand16␮m sections.Thetimeofimageacquisitionwasapproximately2h

persample.Imageswereanalyzedandreconstructedintwo andthreedimensions,usingtheInVesaliussofware(Division ofThree-DimensionalTechnologiesofCentrodeTecnologia daInformac¸ãodeCampinas–SãoPaulo).

Thebiomaterialvolumewasquantifiedincubic millime-ters(mm3_{).Inthetwo-dimensionalimages,thesagittaland}

axialdistancesofthebonedefectandthree-dimensionalaxial distanceweremeasuredinmillimeters(mm).

For the histologicalanalysis, the specimens(n=5) from each group and preoperative observation period were immersed in10% EDTAsolution (pH7.4) for sixweeksfor bone tissue decalcification. The defect area was sectioned inhalf, exactlyatthe mediansagittalplaneofthe rat cal-varia, which resulted in two parts of the wound: the left sideand theright side(S1and S2).Eachsideofthe defect wasembeddedinparaffinandsubsequently,four4.5cm-thick parasagittalsectionswereobtainedandstainedwithMasson trichrome.Histologicalsectionswereassessedusinga conven-tionallightmicroscope(Olimpus®_{CH2,OlympusOpticalCo.}

Ltd.,Japan)inablindedfashionbyanexperienced patholo-gist.Thehistomorphologicalaspectsconsideredthepresence ofthefollowingrepaircharacteristics:edema,inflammatory infiltrate,granulationtissue,bleeding/bloodclot,bone neofor-mation,foreignbodyreactionandthepresenceofexogenous material. Theparameters were weighed usingan arbitrary scalebythefollowingscores:0=absent;1=mild;2=mildto moderate;3=moderatetointenseand4=intense.

Thehistomorphometricanalysiswasusedtoquantifythe neoformedbonetissueareainrandomlyselectedsectionson bothhalves(leftandright)inthreeregionsofthewound,the anteriorregion(closetothenasalbone),thecentralregionand theposteriorregion(closetotheoccipitalbone).Histological imagesofeachregionwereobtainedwitha40×magnification usingaCCDcamera(Sony®_{)coupledtoamicroscope(Jeol}®₎

andanimagecaptureboard(Captivator®_).

Then,theseimagesweretransferredtoadigital morphom-etry softwareprogram(ImageJ,NIH– NationalInstitutesof Health). The formation ofneoformed trabecular bone was identifiedanddelimitedusingahandfreesoftwaretool. Mea-surements were performed in four semi-serial histological sectionsofeachslide.

Thetotalareaofboneneoformationwasobtainedateach fieldandaddedtothevalueobtainedontheoppositesideof thewound.Attheend,meanwascalculatedforthetotalarea ofneoformedbonetissueforeachgroupandperiod.

Inordertoidentifyosteoclasticcells,tworandomizedcuts oneachsideofthewoundfromtwoanimalsfromeachgroup wererandomlychosenandsubmittedtohistochemical stain-ingtodemonstratetartarphosphatase-resistantacid(TRAP). Histologicalsectionswereincubatedfor3hatroom tempera-ture,insolutioncontainingsodiumtartrate,tartaricacidand disodiumsalt(SigmaTM_).

Table1–Volumeofremainingmaterialandmicrotomographictwo-dimensional,three-dimensionalmeasurementsof bonedefectsaccordingtotheperiodsofpostoperativeobservationintheTestandControlGroups.

Period Group n _{Volumeofbiomaterial(mm}3_{) Two-dimensionaldistances Tridimensionalaxialdistance(mm)}

Axial Sagittal

7days Control 1 8.97 8.95 8.96

Test 1 25.11 8.32 8.16 9.39

14days Control 1 9.46 9.41 9.50

Test 1 10.96 7.72 10.81 7.34

21days Control 1 8.41 8.58 8.15

Test 1 7.00 7.77 9.55 9.51

42days Control 1 8.75 8.56 9.06

Test 1 11.64 9.88 11.51 11.10

Histologicaldatawereanalyzedbynonparametric

inferen-tialstatisticsusingtheMann–WhitneytestwiththeBiostat

5.0softwarewithasignificancelevelof5%.

Results

Theresultsofmicrotomographicimageanalysisareshownin

Table1andFig.1.Theintensityingraylevelsofthe

bioma-terialwasclosetothatofbone,butthebiomaterialshowed granularity,allowingittobedifferentiatedfrombone.

Histologicalexaminationregardingrepairmorphology is showninTable 2andmainhistologicalaspectsare shown inFig. 2. Fig.3 shows the resultofstainingfor TRAP (tar-trate-resistantacidphosphatase)intheTestGroup42days postoperatively.

Resultsofthehistomorphometricanalysisofbone neofor-mationareaareshowninFig.4.

Discussion

Theliteraturedescribesthat calcium phosphatesand their derivatives, including beta-tricalcium phosphate, act more directlyonosteoblastsandhavebeenextensivelystudiedas bonereplacementformorethan twodecades,asthey pro-moteboneneoformationbyosteoconductionandhavegood biocompatibility.5–7

Itseffectsasinorganicscaffoldforbonetissueregeneration arealreadywelldocumented,includingdentallesions,8–11_in spiteofstillcontroversialfindings,rangingfrombone neo-formationthatissuperiortotheoneobservedinautogenous grafts12_{andanorganicbovinebonegraft,}13_{torobustevidence} ofinsufficientosteogenesis.14,15

Thebiomaterialusedinthisstudy (GeneX®_{)isrelatively}

newanditisaceramiccompound,whichhasnegative sur-face charge control. Investigations about its efficacy have

Table2–Valuesofthemedians[max–min]ofthescoreintensitiesforthehistologicalvariablesofthebonedefect.

Variables 7days 14days 21days 42days

Control Test p Control Test p Control Test p Control Test p

Edema 2[2-1] 1[2-1] 0.22 2[2-1] 1[1-0] 0.075 1[1-0] 1[1-0] 1 0[2-0] 0[1-0] 0.916 Inflammatoryinfiltrate 1[2-1] 4[4-4] 0.004 1[3-1] 4[4-3] 0.008 1[1-0] 4[4-4] 0.003 1[1-0] 4[4-3] 0.006 Granulationtissue 3[4-3] 3[3-2] 0.212 2[3-1] 4[4-3] 0.011 1[1-1] 2[3-2] 0.004 1[2-1] 1[1-1] 0.317 Hemorrhage/thrombus 3[3-1] 2[3-1] 0.496 2[3-2] 1[4-0] 0.193 3[3-1] 0[4-0] 0.106 2[3-0] 1[3-0] 0.335 Boneneoformation 1[2-1] 1[1-1] 0.317 2[2-1] 2[3-1] 0.605 2[3-1] 2[2-1] 0.339 2[3-1] 1[2-1] 0.418 Foreignbodyreaction 0[1-0] 4[4-4] 0.005 1[2-1] 4[4-4] 0.005 0[2-0] 4[4-3] 0.007 1[2-0] 4[4-3] 0.006 Exogenousmaterial 0[0-0] 4[4-3] 0.007 0[0-0] 4[4-4] 0.005 0[0-0] 2[3-2] 0.005 0[0-0] 3[4-2] 0.008

0,absent;1,mild;2,mildtomoderate;3,moderatetointense;4,intense. PvaluesforKruskall–Wallistest.Significantwhenp<0.05.

Mann–Whitneytestissignificantwhenp<0.05.

most oftenfocused on repair defects of the spine or long

bones.16_Yangetal.1_{demonstratedinaninvivostudythatthis}

ceramicmaterialpromotedsuperiorboneformationin verte-braldefectsineightweekswhencomparedwiththeuseofa polymercement(polymethylmethacrylate).However,intheir conclusions,theyindicatethatthe␤-TCPceramicneedstobe furtherassessed.

Asthemanufacturer’sproposalwastoaddasurface treat-mentontheceramicparticlesthatallowedosteoinduction,a criticalbonedefectmodelwouldprovideimportant informa-tioniftheboneneoformationoccurredwhereitwouldnotbe expected.

Thistypeofmodelisquiteoftendescribed inthe litera-tureanditis,inaway,acknowledgedforthistypeofinvivo

assay.17–24 _{Weusedthecriticaldefect modelinrat calvaria} becauseit iseasy tocreate,reproduce andthefactthatan 8mm-extensiondefect would allowusto verify the occur-rence ofosteoconductionor inductionofthe materialina standardizedandreliablemanner.

Contrary tothefavorableoutcome describedbyGerman researchers,25 _{our resultsdidnotshow boneneoformation} after insertionofthis ceramiccompoundin filling defects. Cement withzetapotentialcontrol didnotstimulatebone neoformation in critical defects created in rat calvaria or showedrapidabsorptionbythevolumeofexogenousmaterial observedinmicrotomographicimagesinallstudyperiods.

Recently,Saadounetal.3_{andFriesenbichleretal.}4_showed significant complications ofthis materialon tissues when

Fig.2–Histologicalsectionsrepresentativeofcriticaldefectsineachgroupintheperiodsof21and42dayspostoperatively. In(A)ControlGroup–21days,showsdepositionofcollagenandosteoidmatrixadjacenttothedefectborder(arrow).In(B) TestGroup–21days,showsexogenousmaterialcluster(longarrow)encapsulatedbyintensedepositionoffibrous

Fig.3–Positiveexpressionoftartrate-resistantacid phosphatase(TRAP)ofmultinucleatedgiantcells(large arrows)adjacenttotheexogenousmaterialclusters(thin arrows)–TestGroupperiodof42days(TRAPstaining, magnification40×).

usedtofillbonedefectsinclinical trials,althoughLaycock andCooper26_{attributedtheseadverseeffectstothestill} inad-equateuseofthisbiomaterial.

Obviously,thepresenceofasignificantlyhigheramountof inflammatoryinfiltrateintheTestGroupisdirectlyrelatedto thegreaterresponsetothepresenceofaforeignbodywhen comparedtotheControlGroup.Thefactthattheforeignbody responseremainedfortheentiredurationoftheexperiment canleadtotheformationofmaterialclusters,whichshould beexpelledfromthewoundareaintheencapsulatedform. Thisfactmayhelptoexplainthedescriptionof“softtissue cysts”madebyFriesenbichleretal.4

Theclinicalrelevanceofthisstudyshouldbeemphasized fromthehistopathologicalpointofview,becausetherewere

7 days 14 days 21 days

Periods of observation

Kruskall-Wallis test – Significant when P<.05 Area of bone neoformation

42 days 1 600 000

P=.009

P=.009

Test group Control group

P=.009

P=.0283

µ

m 2

1 400 000

1 200 000

1 000 000

800 000

600 000

400 000

200 000

0

Fig.4–Histomorphometricanalysisforthearea(␮m2_)of boneneoformationrelatedtothegroupsandperiods. Means(±standarddeviation).

nostandardizedexperimentalpreclinicalstudiestodisclose the behaviorofthis materialontherepairofhealthybone tissueyet.

Accordingtothehistomorphometricfindings,theproposal ofamoresignificantboneneoformationintheTestGroupwas notdemonstratedeither.Theresultsshowthatbone forma-tionwasmoreintenseandsignificantlyhigherintheControl Groupanddeniedourhypothesisoftryingtodemonstratethe osteoinductivepotentialpromisedbythemanufacturer.

Resultsshowedthatthesuperiorityinboneformationin controlsled ustobelievethat this ceramiccomposite pre-vented the small bone formation that would occur in the defect.

Ourresultsarealsoinaccordancewiththosefromother authors,27,28_{accordingtowhomtheapplicationofothertypes} ofbeta-tricalciumphosphate forbonelossrepair,inminor defectsandcrossoverstudiesinthesameanimaldidnot influ-encetheamountofboneformation.

Toassesswhethertherewasincreasedosteoclastogenesis andincreasedboneresorption,ahistochemicalreactionwas carriedoutbyTRAPstaininginthewoundarea.However,the foreignbodyresponsegiantcellsweretheonesthatshowed positivityforthisbiochemicalmarker.Thismayleadtothe hypothesisthatthematerialinthetissuemightpossiblyhave inducedspecificbiochemicalsignals.

Thus, giant cells, probably in an attempt to absorb exogenous material, might alsohave resorbed bone. Stud-ies demonstratingthe expressionofother specificmarkers relatedtorepairandinductionofbonetissueformationcould becarriedouttobetterexplainthisfact.

Bythetimethisstudywasplanned,weexpectedtofind beneficialeffectsofGenex®_{inboneregeneration,especially}

consideringthatthismaterialhadfavorablereportsinclinical use.Finally,ourresultsarerelevant,astheyconfirmthatthis typeofceramicmustbereconsideredasabonesubstitute.

Conclusion

Within the limitations of this study, the biphasic ceramic cement consisting of calcium sulfate and beta-tricalcium phosphate andzetapotentialdidnotinduceorresultedin boneneoformationof8mmcriticalbonedefectscreatedinrat calvariafromthemicrotomographicandhistologicalpointsof view.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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