Effects of induced osteoporosis on bone tissue of rats with experimental periodontitis

(1)

Effects

of

glucocorticoid-induced

osteoporosis

on

bone

tissue

of

rats

with

experimental

periodontitis

Luzia

Hermínia

Teixeira

Sousa

a

,

Eveline

Valeriano

Moura

a

,

Ana

Larissa

Queiroz

b

,

Danielle

Val

c

_,

_Hellíada

_Chaves

a,b

_,

_Mario

_Lisboa

d

_,

_Flávia

_Furlaneto

e

_,

_Gerly

_Anne

_Brito

d,f

_,

Paula

Goes

a,g,

*

a_{Post-graduation}_Program_of_Health_Science,_Medical_School,_Federal_University_of_Ceará

–Sobral,AvenidaComandanteMaurocelioRochaPontes,100

-Derby,Sobral,CE,62.042-280,Brazil

b_School_of_Dentistry,_Federal_University_of_Ceará

–Sobral,R.Cel.EstanislauFrota–Centro,Sobral,CE,62.010-560,Brazil

c_Post_Graduation_Program_RENORBIO,_Federal_University_of_Pernambuco,_Av._Professor_Morais_Rego,₁₂₃₅

–CidadeUniversitária,Recife,PE,50670-901,Brazil

d_{Post-graduation}_Program_of_{Morphological}_Science,_Department_of_Morphology,_Medical_School,_Federal_University_of_Ceará

–Fortaleza,RuaDelmirode

Farias,s/n–RodolfoTeóﬁlo,Fortaleza,CE,60.430-170,Brazil

e_Department_of_Oral_&_{Maxillofacial}_Surgery_and_{Periodontology,}_Ribeirão_Preto_School_of_Dentistry,_University_of_São_Paulo,_Av._do_Café,_s/n

–VilaAmelia,

RibeirãoPreto,SãoPaulo,14050-904,Brazil,Brazil

f_Department_of_Morphology,_Medical_School,_Federal_University_of_Ceará

–Fortaleza,RuaDelmirodeFarias,s/n–RodolfoTeóﬁlo,Fortaleza,CE,CEP

60.430-170,Brazil

g_Department_of_Pathology_and_Legal_Medicine,_Medical_School,_Federal_University_of_Ceará

–Fortaleza,RuaMonsenhorFurtado,S/N–RodolfoTeóﬁlo,

Fortaleza,CE,60.441-750,Brazil

ARTICLE INFO

Articlehistory:

Received31March2016

Receivedinrevisedform4January2017 Accepted17January2017

Keywords:

Periodontitis Osteoporosis Glucocorticoids Alveolarboneloss

ABSTRACT

Objective:Toevaluatetheeffectsofosteoporosisinducedbyglucocorticoid(GIOP)onbonetissueofrats withexperimentalperiodontitis(EP).

Design:48maleWistarratsdividedintogroups:Naïve,EP,GIOPandGIOP+EP.RatsofGIOPandGIOP+EP groupsreceived7mg/kgofdexamethasoneintramuscularlyonceaweekfor5weeks.Following,EPand GIOP+EP groups were subjected to ligature-induced periodontitis. Naïve group experienced no manipulation.After11days,theanimalswereeuthanizedandleftmaxillaecollectedformacroscopic, radiographic,micro-tomographicandmicroscopicanalysisofalveolarboneloss(ABL).Bloodsamples werecollected fordetermination ofbone-speciﬁc alkalinephosphatase (BALP)levelsand theright femurswereremovedforradiographicandbiomechanicalanalysis.

Results:EPcausedABLandreducedBALPlevels(p< 0,05),butitdidnotchangethearchitectureor biomechanicsoffemur,comparedtoNaïve.GIOPdidnotcauseABL,butitsignificantlydecreasedalveolar bonemineraldensity(ABMD),bonepercentageandtrabecularthickness(Tb.Th)andincreasedalveolar boneporosity(p< _0.05)_and_signi_fi_cantly_reduced_BALP_serum_levels,_as_well_as_radiographic_density_and Young’smoduleoffemur,comparedtoNaïve.TherewasagreaterABLingroupGIOP+EPwhencompared toEP(p< 0.05).GIOP+EPcausedagreaterdecreaseonABMD,Tb.Th,bonepercentageandincreased bone porosity (p< 0.05) and also presented a significant reduction in BALP levels (p< 0.05), in radiographicdensityandinYoung’smoduleoffemurcomparedtoEP(p< 0.05).

Conclusions:GIOPcanpotentiatethedestructiveeffectsofEPonalveolarboneandalterthesystemic boneloss,bypromotingboneresorptionandreducingosteoblastactivity.

1.Introduction

Periodontitisisaninfectious-inﬂammatoryandhighly

preva-lentdisease,characterizedbydestructionofconnectivetissueand

alveolar bone loss(ABL),and itis consideredthesecondmajor

cause of tooth loss (Tatakis and Kumar, 2005). This disease is

mainlyinitiatedbyoralbioﬁlm,howeverthedevelopmentofan

* Corresponding author at: Federal University of Ceará – Department of Pathologyand LegalMedicine, Medical School, Rua Monsenhor Furtado,s/n 60441–750,RodolfoTeóﬁlo,Fortaleza/CE,Brazil.

E-mailaddress:paulagpinheiro@yahoo.com.br(P.Goes).

http://dx.doi.org/10.1016/j.archoralbio.2017.01.014

ContentslistsavailableatScienceDirect

of

Oral

Biology

(2)

alteredhostresponseplaysanimportantroleontissuebreakdown (Pihlstrom,Michalowicz,&Johnson,2005).

Osteoporosisis acommondiseasecharacterizedbysystemic

bone loss and impaired bone microarchitecture. It can be a

consequenceofhormonalimbalanceinpostmenopausalwoman

(Jilka,Hangoc,Girasole,Passeri,&Williams,1992),butitalsocan

presentasecondarycause,mainlyasaresultoftheuseofsome

drugs, such as Glucocorticoids (GCs). Glucocorticoid-induced

osteoporosis (GIOP) is the most common cause of secondary

osteoporosis,theﬁrstcausebefore50yearsofageand theﬁrst

iatrogenic cause of the disease (Kok & Sambrook, 2009). In

addition,consideringthecontinuousraiseontheprevalenceofGCs

useinthecommunitypopulation(Overman,Yeh,&Deal,2013),it

seems interesting to understand the biological mechanisms

underlyingGIOP.

Intherecentdecades,numerousstudieshavefocusedonthe

associationbetweenosteoporosisand periodontitisat thebone

level.The majorityof thestudieshasfocused ontheeffects of

postmenopausalosteoporosisonthelossof periodontal

attach-ment (Hernández-Vigueras et al., 2015; Juluri et al., 2015).

However,littleisknownabouttheeffectsofGIOPonperiodontal

tissues.OnestudyshowedthatGCscaninduceanalveolarbone

lossinlong-term(28days)treatedmice(Bouvard,Gallois,Legrand,

Audran,&Chappard,2013)andanotheronereportedareductionof

bonedensityintheregionofinteralveolarsepta,insigniﬁcantbone

tissuelossofthehorizontaltypeandpathologicalteethmobilityin

periodontal tissues of patients with systemic osteoporosis

(Dmitrieva, Atrushkevich, & Pikhlak, 2006). There is no study

evaluatingtheeffectofGIOPwhentheperiodontalinﬂammationis

present.Inthiscontext,weaimedtoevaluatetheeffectsofGIOPon

bonetissueofratswithexperimentalperiodontitis.

2.Materialandmethods

2.1.Animalsandstudydesign

Theexperimentswereperformedonforty-eightyoungadult

male(fromtwolitters)Wistarrats(Rattusnorvegicus)fromcentral

AnimalFacilityofFederalUniversityofCeará,weighing180–220g,

keptinappropriatecageswithsixanimalseach.Theanimalswere

housed in standard conditions (12h light-dark cycles and

temperature-controlledrooms)withfood andwater adlibitum.

Theprotocolsforexperimentalproceduresandanimaltreatment

wereapprovedbyAnimalEthicsCommittee(number78/2014)of

FederalUniversityofCeará,Brazil.

Apowercalculationwas performedtodeterminethesample

size.Theanimalwasconsideredthestudyunit.Thesamplesize

wasdeterminedtoprovide80%powertorecognizeasigniﬁcant

differenceof20%amonggroupsandthestandarddeviationof15%

witha95%conﬁdenceinterval(p=0.05),consideringthechangein

alveolar bone loss (ABL) as the primary outcome variable.

Therefore,asamplesizeof6ratspergroupwasrequired.

Aftertwoweeksofacclimationtothelaboratoryenvironment,

theratsweredivided,inablindmanner,intofourgroups(n=6):

Naïve, Experimental Periodontitis (EP), Glucocorticoid-induced

osteoporosis(GIOP)and GIOP+EP.Initially, rats fromGIOPand

GIOP+EPgroupsreceivedinjectionsof7mg/kgofdexamethasone

(Decadron,Aché1

–Guarulhos,SP,Brazil)intramuscularly,oncea

weekfor5weeks(Lucindaetal.,2012),andtheonesfromEPgroup

received0.5ml of0.9%salinesolution.Following,theratswere

anesthetized with 100mg/ml ketamine (Cetamin – Syntec1

–

SantanadeParaíba,SP,Brazil)and 20mg/mlxylazine(Xilazin–

Syntec1

–SantanadeParaíba,SP,Brazil)onthedoseof1ml/kg

intramuscularly.In ordertoinduceperiodontitis ratsof EPand

GIOP+EP groups received a sterile nylon thread ligature (3-0;

polysutureNP45330–SãoPaulo, SP,Brazil)aroundthecervical

areaoftheirmaxillaryleftsecondmolars (Bezerraetal.,2000;

Samejima, Ebisu, & Okada, 1990). After 11 days, all rats were

euthanizedwith20mg/kgthiopental(0.5gThiopentax;Cristália,

SãoPaulo,SP,Brazil).RatsofNaïveandGIOPgroupsdidnotreceive

theligature.RatsfromNaïvegroupexperiencednomanipulation.

Itwasperformed2studieswithparallelexperimentalgroups,one

formacroscopicandbiochemicalanalysesandtheotheronefor

microscopicevaluation.

2.2.Macroscopic,radiographicandmicro-computedtomographic(CT) analysesofalveolarbone

Formacroscopicanalysis,theexcisedmaxillaewereﬁxedin10%

neutralformalinfor24h.Hemi-maxillaewerethendeﬂeshedand

stainedwith1%aqueousmethyleneblueinordertodifferentiate

bonefromteeth.Thentheywereplacedonmicroscopeslidesand

photographed.Theareaofalveolarboneloss(ABL)wasmeasuredby

atrainedandblindedobserver(LHTS),usinganimagingsoftware

(ImageJ1_National_Institutes_of_Health,_Washington,_DC,_USA),_as

previouslydescribed(Goes,Lima,Melo,Rego,&Lima,2010).

Thesamespecimensusedformacroscopicstudywere

radio-graphedusingadigitalsystem(DigoraSoredexDigitalSystem1_,

Portslade-EastSussex, UK).Thespecimens wereposedoverthe

sensorandtheradiographicimageswereacquiredusing63kVp,

8mA,exposuretimeof0.06sandfocaldistanceof30mm.Bone

mineral density of the maxillae were evaluated using Image J

software,aspreviouslydescribed(Goesetal.,2010).

Thesamenon-demineralizedspecimenswerethenscannedby

a cone beam micro-CTsystem (Skyscan 1172, Bruker, Kontich,

Belgium). The x-ray generator was operated at an accelerated

potentialof50kVwithabeamcurrentof200

m

Aandanexposure

timeof560msperprojection.Imageswereproducedwithavoxel

size of 666

m

m. Using an appropriated software (Data

Viewer1_,_version_1.5.0,_Bruker,_Kontich,_Belgium),_the_generated

3 dimensionalmodelswererotatedintoa standard positionas

describedpreviously(Lisboaetal.,2015).Linearmeasurementsof

ABL were performedat 3 differentsites: buccal,furcation and

interproximal. For the interproximal site, coronal dataset was

analyzed using appropriated software (CT-Analyser1_, _version

1.13.5.1+,Bruker,Kontich,Belgium)(Lisboaetal.,2015).Volumetric

analyseswereperformedusingthesamesoftwareappliedforthe

analysisoftheinterproximalsite,aspreviouslydescribed(Lisboa

etal.,2015).Bonemineraldensity(BMD),bonepercentage,bone

porosity, and trabecular thickness (Tb.Th) were assessed. All

micro-CTanalyseswereperformedbyoneblindedandcalibrated

examiner(MRPL).

2.3.Microscopicanalysisofalveolarbone

Anothersetofexperimentwasperformedforhistopathological

analysis. After the euthanasia of the rats, the maxillae were

removed and ﬁxed in 10% neutral buffered formalin and

demineralized in 10% EDTA buffered solution. After complete

decalciﬁcation,thespecimensweredehydratedandembeddedin

parafﬁn. Serial sections,4

m

mthick,wereobtainedina

mesio-distaldirection.Thesectionswerestainedwithhematoxylinand

eosin.Sectionsrepresentingtheareabetweentheﬁrstandsecond

molarswereevaluatedbylightmicroscopy(40magniﬁcation).

TheparametersanalyzedwerebasedonthestudybyLeitãoetal.

(2005),using scoresvaryingfrom0 to3.The histopathological

analysiswasperformedbyacertiﬁedhistologist(GACB).

2.4.Serumlevelsofbone-speciﬁcalkalinephosphatase(BALP)

Bloodsampleswerecollectedfromtheorbitalplexus atthe

11thdayaftertheinductionofperiodontitis.Bone-speciﬁcalkaline

(3)

phosphatase (BALP), a thermosensible isoform of total alkaline

phosphatase, was evaluated using a thermoactivation method

(Whitby & Moss,1975). The samples wereheated to 56_C _for

10min. Serumlevelsof BALPwerecalculatedby thedifference

betweenheated alkalinephosphatase fromtotal alkaline

phos-phataseinserum(Labtest1

–LagoaSanta,MG,Brazil)(Goes,Melo,

Dutra,Lima,&Lima,2012;Goesetal.,2014).

2.5.Biomechanicaltestingandradiographicdensityoffemur

Therats’rightfemurswerecollectedaftereuthanasiaandﬁxed

in10%formaldehyde(ReagenProdutosparaLaboratóriosLtda1

–

Rio de Janeiro, RJ, Brazil) for 24h. The specimens were

radio-graphedusingDigoraSoredexSystem1_(Dental_Imaging_Company

Ltd,Portslade-EastSussex,UK)withthesameparametersusedfor

themaxillae.TheseimageswereevaluatedbyImageJ1_software._A

regionofinterest(ROI)wascreatedmeasuring0.50.5mm,inthe

formof a square, with473229pixels, and was posed in the

proximalfemurdiaphysis,and thesameevaluatordrewallROI

panels. Grey tone differences were considered the value of

radiographicdensity.

The same non-demineralized femurs were analyzed in a

materialstestingmachine(InstronE10000,InstronCorporation–

Norwood, MA, USA), using a three-point bending procedure.

Femurswerecompresseduntilfailureata displacementrateof

3mm/min (span=20mm)withthe anteriorside down,and all

forceanddisplacementdatawererecordeduntilthespecimenwas

broken.Young’smodulus(MPa) werecalculated,assumingthe

cross-sectional geometryof the femurs wereelipses (Xu etal.,

2014).

2.6.Statisticalanalysis

Thedataarepresentedasmeansstandarderrorsofthemeans

orasmedian(range),whenappropriate.Normalityand

homosce-dasticity of the data were veriﬁed. ANOVA followed by the

Bonferronitestwereusedtocomparethemeans,and Kruskal–

WallisandDunn testswereusedtocomparethemedians.The

signiﬁcancelevelwassetat5%inalltests.Allcalculationswere

performedusingPrism5(GraphPadSoftwareInc.,SanDiego,CA,

USA).Allprotocolsandanalyseswereperformedbyblindedand

calibratedexaminers.

3.Results

3.1.Macroscopic,radiographicandmicro-computedtomographic(CT) analysesofalveolarbone

Onthemacroscopicanalysisofhemimaxillae(Fig.1A),Naïve

group(Fig.1C)presentedABLclosetozero,i.e.Ligaturesforeleven

days caused a signiﬁcant (p<0.05) ABL in group EP, which

presentedfurcationlesionsandrootexposure,whencomparedto

groupNaïve(Fig.1E).TheresponseoftheratstoGIOP(Fig.1G)was

similar to that of the rats of the Naïve group. However, rats

subjectedtoGIOP+EP(Fig.1I)showedgreater(p<0.05)ABLwhen

comparedtogroupEP.

The results of the radiographic density (RD) analysis of

hemimaxillae are depicted on Fig. 1B. Radiographs of EP rats

showedreductionofRDby19%(Fig.1F)(p<0.05)whencompared

toNaïvegroup(Fig.1D).Therewerenosigniﬁcantdifferencesin

theRDofhemimaxillaebetweengroupsGIOP(Fig.1H)andNaïve.

However, hemimaxillae of the rats subjected to GIOP+EP had

lowerRD,by12%(Fig.1J)whencomparedtoEPgroup(p<0.05).

Micro-CTanalysisoflinearandvolumetricparameterscanbe

observedinFig.2.Consideringthelinearmeasurements,EPgroup

showedincreasedABLatbuccal(Fig.2A),furcation(Fig.2B)and

interproximal (Fig. 2C) sites, when compared to Naïve group

(p<0.05). GIOP did not affect the alveolar bone level when

comparedtoNaïve(p>0.05).Ontheotherhand,GIOP+EPgroup

presentedagreaterincreaseofABLwhencomparedtoEPinall

three sites analyzed (p<0.05). The assessment of volumetric

parameters revealed that EP reduced BMD (Fig. 2D), bone

percentage (Fig. 2E), trabecularthickness (Tb.Th) (Fig. 2F) and

increasedboneporosity(Fig.2G)inrelationtoNaïvegroup.No

differenceswereobservedbetweenGIOPandEPgroups(p>0.05).

However,groupGIOP+EPpresentedlowerBMD,bonepercentage

and trabecular thicknessand greaterincrease in bone porosity

whencomparedtogroupEP(p<0.05).

Fig.1.A)MacroscopicandB)Radiographicdensityanalysisofthealveolarboneofrats.BarsrepresentthemeansSEMof6animalspergroup.#_Signi_ﬁ_cant_difference

comparedtoNaïvegroup.*SigniﬁcantdifferencecomparedtoEPgroup(p< 0.05;ANOVAfollowedbyBonferroniTest).Macroscopicandradiographicimagesofhemimaxillae oftheanimalsfromNaïvegroup(CandD),EPgroup(EandF),GIOPgroup(GandH),andGIOP+EPgroup(IandJ).

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3.2.Microscopicanalysisofalveolarbone

Periodontalhistopathologicanalysisoftheregionbetweenﬁrst

andsecondmolars of naïveratsshows thenormalstructure of

gingiva,periodontalligament(PDL),alveolarboneandcementum

(Fig. 3A). The periodontium of rats subjected to experimental

periodontitis(EPgroup)demonstratedaccentuatedinﬂammatory

cell inﬁltration, breakdown of alveolar bone, collagen ﬁber

derangementwithintheperiodontalligament,andresorptionof

cementum,receiving a medianscoreof 3(range,2–3)(Fig.3B;

Table1).TheperiodontaltissueoftheratssubjectedtoGIOPdid

notdemonstrateanychangeonthearchitecturecomparedwith

thenaïverats(Fig.3C;Table1).However,theratssubjectedto

GIOP+EP showed worse bone loss and inﬂammatoryinﬁltrate

thangroupEP(Fig.3D;Table1).

3.3.SerumbiochemicalanalysisofBALP

Serum dosage of BALP was analyzed (Fig. 4). EP caused a

signiﬁcantdecrease,by27%,onBALPserumlevels(21.642.48U/

L)whencomparedtoNaïve(29.525.14U/L;p<0.05).GIOPalso

causedareductionby41%(17.42.65U/L)onBALPserumlevels

compared to Naive (p<0.05). GIOP+EP group presented a

reductionby55% (13.252.11U/L)onBALPserum levelswhen

comparedtoEP(p<0.05).

3.4.Biomechanicaltestingandradiographicdensityoffemur

AssessingRDoffemur(Fig.5E),thoseratsundergoingonlyEP

(Fig.5B)showednosigniﬁcantdifferencewhencomparedtothe

ratsofNaïvegroup(Fig.5A).GIOPcausedasigniﬁcantreductionof

RDoffemur(Fig.5C)whencomparedtoNaïveandEPgroups.In

thegroupofratssubmittedtoGIOP+EP,therewasareductionof

RDof femur(Fig.5D)when compared toNaïve and EPgroups

(p<0.05).

We further checked the effects of GIOP and experimental

periodontitisonfemurbiomechanical properties(Fig.5F).After

11daysofligature,groupEPdidnotshowdifferenceonYoung’s

moduluswhencomparedtothefemursofNaïvegroup(p>0.05).

TherewasasigniﬁcantdecreaseinYoung’smodulusinthegroup

subjectedtoGIOPwhencomparedtoNaïvegroup,aswellasinthe

groupGIOP+EPwhencomparedtoEPgroup(p<0.05).

4.Discussion

Therehas beenan increase in the useof animal modelsin

studies of initiation and progression of bone diseases, such as

osteoporosis and periodontal disease. The ligature-induced

periodontitis model has been used extensively as a highly

Fig.2. Micro-CTanalysisoflinearandvolumetricparametersofthealveolarboneofrats.A)Buccalsite,B)Furcationsite,C)Interproximalsite,D)BoneMineralDensity (BMD),E)BonePercentage,F)TrabecularThickness(Tb.Th),G)BonePorosity.BarsrepresentthemeansSEMof6animalspergroup.#_Signi_ﬁ_cant_difference_compared_to

Naïvegroup.*SigniﬁcantdifferencecomparedtoEPgroup(p < 0.05;ANOVAfollowedbyBonferroniTest).

Fig.3. Microscopicanalysisoftheperiodontaltissuesintheregionbetweenthefirstandsecondmolarsofrats.A)Naïve,B)EP,C)GIOP,D)GIOP+EPgroups.Dentin(d); Cementum(c);AlveolarBone(ab);Gingiva(g);PeriodontalLigament(pdl).Inflammatoryinfiltrate(*)andboneresorption(!).Bars–500mm.Hematoxylin&eosin(H&E). (40magnification).

Table1

Histopathologicalanalysisoftheperiodontaltissuesofhemimaxillaeofrats.

Naïve EP GIOP GIOP+EP Histopathological

Analysis (Scores)

0(0–0)a ₃₍₂

–3) 0(0–0) 3(3–3)a

Dataarepresentedasmedians(extremevalue).

EP=experimentalperiodontitis;GIOP=glucocorticoid-inducedosteoporosis;

a _Signi_ﬁ_cant_compared_to_EP_(Kruskal_Wallis_followed_by_Dunn

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reproduciblemodelthatevaluatestheprogressionofperiodontitis (Bezerraetal.,2000;Goesetal.,2014;Leitãoetal.,2005).Aswith

humanperiodontitis,experimentallyinducedperiodontitisinrats

involves inﬂammatorycell recruitment with overproduction of

proinﬂammatory cytokinesand osteolytic enzymes,with

osteo-clast activation, and ultimately leads toalveolarbone and soft

tissueattachmentloss(Bezerraetal.,2000;Bezerra,Brito,Ribeiro,

&Rocha,2002;Goesetal.,2010;Goesetal.,2012,2014;Leitão etal.,2005;Limaetal.,2004;Lisboaetal.,2015;Messoraetal., 2016;Ozdemiretal.,2012;deLimaetal.,2000).Ontheotherhand,

consistentwithourﬁndings,thereisnoevidencethat

periodonti-tisbyitselfcanprovokesystemicboneloss(Xuetal.,2014).

Systemicriskfactorsmaydetermineaccelerationoftheonsetof

periodontaldiseases(Borrell&Papapanou,2005;Kornman,2008),

as well as increase in the rate of progression and severity of

periodontitis(Genco&Borgnakke,2013).Amongtheseriskfactors,

osteoporosisis consideredone ofthesix mainsystemic factors

(Genco & Borgnakke, 2013). Therefore, using reproducible

experimentalmodels,thepresentstudyinvestigatedtheinﬂuence

of glucocorticoid-induced osteoporosis on bone tissue of rats

subjectedtoexperimentalperiodontitisassessingdifferentaspects

ofthealveolarboneandfemuraswellastheosteoblasticactivity.

Osteoporosis is mainly caused by postmenopausal estrogen

deﬁciencyanditsassociationwithperiodontitisitwelldescribed

inliterature(Hernández-Viguerasetal.,2015;Julurietal.,2015).

However,osteoporosismayalsopresentasecondarycause,related

to the long-term use of glucocorticoids (GCs). Considering the

periodontaltissues,tothebestofourknowledge,thistheﬁrsttime

thattheeffectsofGIOPareevaluatedonexperimental

periodonti-tisinrats.

OurresultsshowedthatGIOPworsenstheABLinratswithEP.

GCsadverselyaffectbonetissueinanumberofways(Compston,

2010).Notably,afterinitiationofGCstherapy,thereisanearlyand

transient increase in bone resorptionin patients withmultiple

sclerosis(Dovioetal.,2004;Teitelbaum,2015).GCsincreasethe

expressionofthemacrophagecolonystimulatingfactor(M-CSF)

and RANKL, and decrease the expression of its soluble decoy

receptor, osteoprotegerin, in stromal and osteoblastic cells

(Canalis, Mazziotti, Giustina, & Bilezikian, 2007; Compston,

2010). It leads toosteoclastogenesis and a prolongation of the

lifespanofosteoclasts(Compston,2010).Inaddition,ithasbeen

reportedthat,wheninﬂammatorydisorderssuchasperiodontitis

arepresent,GCsmaypotentiatetheresorptiveprocess.Patients

with rheumatoid arthritis showed increased bone degradation

Fig.4.SerumbiochemicalanalysisofBALP.BarsrepresentthemeansSEMof6animalspergroup.#_Signi_ﬁ_cant_difference_compared_to_Naïve_group._*Signi_ﬁ_cant_difference

comparedtoEPgroup(p< 0.05;ANOVAfollowedbyBonferroniTest).

Fig.5.Biomechanicaltestingandradiographicdensityanalysesofthefemurofrats.RadiographicimagesofthefemurofanimalsfromA)Naïvegroup,B)EPgroup,C)GIOP group,D)GIOP+EPgroup.E)RadiographicdensityoffemurandF)Young’smodulus(MPa)offemur.BarsrepresentthemeansSEMof6animalspergroup.#_Signi_ﬁ_cant

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duringtheﬁrstthreemonthsoftherapywithGCswhencompared

tohealthyindividuals(Hall,Spector,Grifﬁn,Jawad,Hall,&Doyle,

1993). This initial rapid and greater bone loss can reﬂect

persistenceof theprior effects ofinﬂammatory cytokines, such

as TNF-

a

and IL-1, as wellas the osteoclastic cytokine RANKL

(Teitelbaum, 2015). Therefore, these ﬁndings can explain the

greaterABLandtheworsemicroarchitectureofthealveolarbone

observedinGIOP+EPratsinthepresentstudy.

GIOP is characterized by increased apoptosisof osteoblasts.

Thereis evidence that GCs decreaseosteoblastogenesis, impair

osteoblastic differentiation and maturation and decrease the

number and function of osteoblasts (Canalis et al., 2007).GCs

alsofavorthedifferentiationofbonemarrowstromalcellstoward

cellsoftheadipocytelineageinsteadoftheosteoblasticlinageby

blockingWnt/

b

-cateninsignalingpathway(Canalisetal.,2007;

Compston,2010).Inaddition,whenassociatedwithinﬂammatory

bonedisorders,suchasperiodontitis,theosteoblasticactivitymay

alsobesuppressedinagreaterway(Algate,Haynes,Bartold,Crotti,

&Cantley,2016).TNF-

a

,IL-1,6and17canperturbtheWntand

bonemorphogeneticprotein(BMP)signalingpathways,leadingto

lowerosteoblastdifferentiationandactivity(Walsh&Gravallese,

2010). TNF-

a

can also induce osteoblasts apoptosis (Jilka,

Weinstein,Bellido,Parﬁtt,&Manolagas,1998;Wei,Kitaura,Zhou, Ross,&Teitelbaum, 2005).Thus, theseﬁndings can justify the

decreasein BALP serum levelsobserved inGIOP and GIOP+EP

groups,sinceBALPisahomodimericglycoproteinthatisanchored

tothemembraneofosteoblastsandconsequentlyitisconsidereda

general indicator of the bone formation rate in skeletal tissue

(Sardiwal,Magnusson,Goldsmith,&Lamb,2013).

5.Conclusion

Insummary,withinthelimitsofthepresentstudy,weconclude

thatGIOPcanpotentiatethedestructiveeffects ofexperimental

periodontitisonalveolarboneofratsandaltertheirsystemicbone

loss, by promoting bone resorption and reducing osteoblast

activity. Nevertheless, more studies are necessary in order to

betterunderstandthebiochemical mechanismsof GCsin bone

cellsduringinﬂammatoryconditions.

Conﬂictofinterests

Theauthorshavenoconﬂictofinteresttodisclose.

Fundings

ThisstudywassupportedbygrantsfromCNPqandFUNCAPand

bytheauthorsthemselves.

Ethicalapproval

Theexperimentalproceduresdescribedherewereapprovedby

theInstitutionalAnimalCareandUseCommittee(#78/2014)and

performed in accordance with the Animal Care Standards of

FederalUniversityofCeará.

Acknowledgments

The authors gratefully thank Socorro França Monte of the

DepartmentofMorphology,FacultyofMedicine,Federal

Universi-tyofCeará,Ceará,Brazil,fortechnicalassistance.

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