Effect of alendronate on bonespecific alkaline phosphatase on periodontal bone loss in rats

Effect

of

alendronate

on

bone-specific

alkaline

phosphatase

on

periodontal

bone

loss

in

rats

Paula

Goes

,

Iracema

M.

Melo

,

Caio

S.

Dutra

,

Ana

Patrı´cia

S.

Lima

,

Vilma

Lima

*

b_{DepartmentofPhysiologyandPharmacology,FacultyofMedicine,FederalUniversityofCeara´,Fortaleza,Ceara´,Brazil}

1.

Introduction

Periodontitisisachronicinflammatorydiseaseofdestructive andnon-reversibleaction,that,ifnottreated,cancausetooth mobilityleadingtosubsequenttooth loss.1_Various

mecha-nisms are related to aetiopathogenesis of periodontitis; however, factors associated to immunoinflammatory host responseaffect mainlydevelopment anduse ofdrugs that mightpromoteamodulationforthisresponse.1

The mechanism of bone resorption in periodontitis is mediatedbyosteoclasts.Thesecellsareoriginatedbyblood precursorsfrombonemarrow,andareactivatedbyvarious mediators, especially cytokines, such as tumour necrosis factor(TNF)andinterleukin(IL)-1,whichinduceanincreaseof receptoractivatorofnuclearfactork-Bligand(RANKL)onthe osteoblast surface,2_{favouring RANK–RANKL linkage, which}

resultsinosteoclastactivationandosteoclastogenesis. On the resorption site, osteoclasts attach to the bone matrixthroughavb1integrin,formingasealingzone.3_Later,

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Accepted14July2012

Keywords: Alendronate

Bone-specificalkalinephosphatase Alveolarboneloss

Inflammation

a

b

s

t

r

a

c

t

Objective: The studyaimsto evaluate theeffect of alendronate(ALD) onbone-specific alkalinephosphatase(BALP)serumlevelsonperiodontalbonelossinWistarrats.

Design: Periodontitiswasinducedbyligaturearoundtheuppersecondmolarin36male Wistarrats(200g).Groupsofsixanimalsreceived0.9%saline(SAL)orALD(0.01;0.05; 0.25mgkg 1_{,s.c.),over11days;thentheyweresacrificedandtheirmaxillaewereremoved} tobedefleshedandstainedformacroscopicorhistopathologicalanalysis.Bloodsamples were collected for BALP, transaminases and total alkaline phosphatase (TALP) serum dosage,andhaematologicstudy.Ratswereweigheddaily.

Results: PeriodontitisinductioncausedreductionofBALP,intensealveolarboneloss(ABL), cementumandperiodontalligamentdestructionsandintenseleucocyteinfiltrationseen microscopically. Systemically,periodontitisinduced leucocytosis,weightloss andTALP reduction.ALD(0.25mgkg 1_{)preventedBALPreduction(19.17}

1.36Ul 1_{)whencompared} to SAL (13.61.5), as well as prevented ABL, by 57.2%, when compared to SAL (4.80+0.18mm2_{),whichwascorroboratedbyhistologicalfindings(ALD0.25mgkg} 1_=1.5 (1–2)andSAL=3(2–3))(p<0.05).ALDdidnotaltertransaminasesbutreducedTALPlevels (p<0.05).ALD0.25mgkg 1reduced6th-hneutrophilia(2.500.22cell103mm 3)and 7th-(12.290.66)and11th-daylymphomonocytosis(15.740.52)whencomparedtoSAL (5.200.28;18.241.05;and23.211.48,respectively).ALDdidnotaltertheweightloss.

Conclusion: ALDpreventedBALPreductionandABLandreducedinflammatoryinfiltrate, withoutcausingsystemicalterations.

#2012ElsevierLtd.Allrightsreserved.

*Correspondingauthorat:FederalUniversityofCeara´,DepartmentofPhysiologyandPharmacology,RuaCoronelNunesdeMelo,1127 -RodolfoTeo´filo,Fortaleza,Ceara´,Brazil.Tel.:+558599892199;fax:+558533668232.

E-mailaddresses:villima@yahoo.com.br,vilma@ufc.br(V.Lima).

Available

online

at

www.sciencedirect.com

journalhomepage:http://www.elsevier.com/locate/aob

0003–9969/$–seefrontmatter#2012ElsevierLtd.Allrightsreserved.

theyorganisetheircytoskeleton,andthen exhibitaruffled bordercalledtheresorptiveorgan.Bythen,agreatamountof acidvesiclesarereleasedon theresorptionsite,which are associatedtoaprotonpumpinordertostarthydroxyapatite crystaldissolution.3

Thenitrogen-containingbisphosphonates(nBPs)are phar-macologicalagentsthatpossessachemicalstructuresimilar topyrophosphate,whichprovidesastrongaffinitytocalcium. This structure promotes chelation to circulating calcium, binding it to the bone mineral surface.4 _Amongst

bispho-sphonates,sodiumalendronate(ALD)standsout duetoits highaffinitytobonetissue.

ThemechanismofactionofnBPisbasedontheinhibition ofthe enzymefarnesyldiphosphate synthase(FPPS).5_FPPS

stimulatesthe isoprenylation ofsmall guanosine-50 -tripho-sphatases(GTPases),whichsignalisetoproteinsthat,when activated, regulate alterations on osteoclast morphology, cytoskeletonarrangement,vesicletraffic5_{andruffledborder.}

Whenthevesiculartrafficandruffledborderareinhibited,the activitiesthatelicitboneresorptionarealsoreduced.Finally, whenFPPSconcentrationreaches100mM,osteoclast apopto-sisinduction begins. Thus, nBPs areindicatedas excellent boneresorptioninhibitors.5

Theenzymealkalinephosphatasehasbeenknownformany years.6_{Alkalinephosphataseisametalloenzymeanchoredto}

thecellmembrane,anditisdistributedparticularlyintheliver, bowel,placentaandbone.6_{Bone-specificalkalinephosphatase}

(BALP), an isoenzyme of alkaline phosphatase, has been implicatedintheprocessesofboneformation6_anditisthe

majorenzymeinvolvedinremovinginorganicpyrophosphate, aninhibitorofbonemineralisation.6

BecauseBALPisanexoenzymethatfacestheextracellular compartment,itisconceivablethatitsactivityandfunction canbemodulated byenvironmentalconditions.6_Therefore,

weaimedtoevaluatetheeffectofALDonBALPonperiodontal bonelossinWistarrats.

2.

Materials

and

methods

2.1. Animalselection

Thirty-sixmaleWistarrats(Rattusnorvegicus)weighing180– 220g,fromourownanimalfacilities,wereusedinthisstudy. Theanimalswereacclimatisedforatleast1weekbeforethe beginningoftheexperimentandwerehousedundernormal laboratory conditions with laboratory chow and water available ad libitum. Experimental protocols were executed following ethicalprinciples for laboratory animal use,and wereapprovedbyinstitutionalEthicalCommitteeofAnimal Research and European Convention for the Protection of VertebrateAnimalsusedforExperimentalandOtherScientific Purposes (Protocol no. 101/2009). All efforts were made to reduceanimalnumber,theirpain,sufferingandstress.

2.2. Modelofexperimentalperiodontitis

Theratsweredividedintofourgroups,withsixanimalseach. Themodelofligature-inducedperiodontitisusedconsistedof insertionofnylonligaturearound thecervixofsecondleft

upper molar of rats anaesthetised with chloral hydrate (Vetec1_{, Duque de Caxias, RJ, Brazil).}7,8 _{The ligature was}

placedthroughtheproximalspaceoftherespectivetooth,and was knotted onthebuccal sideofthetooth,resultingina subgingivalpositionpalatinallyandinasupragingival posi-tionbuccallyoftheligature.Thecontralateralrightsidewas usedastheunligatedcontrol.Animalswereobserveduntilthe 11thday,theperiodofthemostintensealveolarboneloss, when they were then sacrificed. All ligature-induced peri-odontitiswasmaderandomly.

2.3. Experimentalgroups

2.3.1. Salinegroup

Thiscontrolgroupwasconstitutedbysixratssubmittedto periodontitis.Theanimalsreceived0.5mlof0.9%sterilesaline solution subcutaneously (s.c.), 30min before ligature and, afterthat,daily,foran11-dayperiod,whentheywerethen sacrificed.

2.3.2. ALDgroups

Theanimalsweresubdividedinthreegroupsofsixanimals each,whichreceivedALDsubcutaneously(Fosamax1_,Merck,

Sa˜oPaulo-SP,Brazil)dissolvedin0.9%sterilesalinesolutionin thedosesof0.01,0.05and0.25mgkg 1_{,respectively,30min} beforeligature,anddailyuntilthe11thday.

2.4. Morphometricstudyofbonetissue

On the 11thday,afterperiodontitisinduction,the animals weresacrificedandtheirmaxillaewereremovedandfixedin 10% neutralbufferedformalin(Reagen1_{,Riode Janeiro,RJ,}

Brazil),for24h.Followingthat,themaxillaewereseparatedin half,dissectedandstainedwith1%aqueousmethyleneblue (Vetec1_{,DuquedeCaxias,RJ,Brazil)andplacedonmicroscope}

slides.8,9 _{Then, they followed to photographic registration}

usingadigitalcamera,Nikon1_{(D40,Melville,NY,USA).The}

measurementoftheresorptionareawasmadebyadelimited region,involvingtheocclusalborderofthevestibularsideof the hemimaxilla until bone border. These areas were evaluated by ImageJ1 _{software (Software ImageJ 1.32j,}

NationalInstitutesofHealth;EUA)inaccordanceto method-ologydescribedbyGoesetal.8

2.5. Histologicalanalysisofalveolarbone

Extra groups of six animals with periodontitis that had received saline or ALD (0.25mgkg 1_{) were sacrificed as} described aboveand hadtheirmaxillaeexcised.The speci-menswerefixedin10%neutralbufferedformalinandwere demineralised in 10% ethylene diamine tetraacetic acid (EDTA) (DinaˆmicaQuı´mica Contemporaˆnea1_{, Diadema, SP,}

from0to3,basedontheintensityoffindings,asfollows:Score 0: absence of or only discrete cellular infiltration, few osteoclasts,preservedalveolarprocessandcementum;Score 1:moderatecellularinfiltration,presenceofsomeosteoclasts, some but minor alveolar process resorption and intact cementum; Score 2: accentuated cellular infiltration, large number of osteoclasts, accentuated degradation of the alveolarprocess and partial destruction ofcementum;and Score3:accentuatedcellularinfiltrateandtotaldestructionof alveolarprocessandcementum.9

2.6. SerumdosageofBALP

Blood samples were collected from the orbital plexus of anaesthetisedanimals(salineandALD)beforetheexperiment and on the 11th day. The BALP was evaluated using the thermoactivationmethod,byheatingthesampleat568Cfor 10min,10 _{since BALP is a thermosensible isoform of total}

alkalinephosphatase(TALP).BALPserumlevelswereobtained bythesubtractionofheatedalkalinephosphatasefromTALP serumlevels.Themethodologyusedtoevaluatetheenzymes’ serum levels followed the manufacturers’ directions (Labt-est1_{,LagoaSanta-MG,Brazil).}

2.7. Serumdosageoftransaminases(ASTandALT)and TALP

On the baseline and on the 11th day of the assay, blood sampleswerecollected fromtheorbitalplexuses of anaes-thetised animals (saline and ALD). Liver function was evaluatedthroughserumdosageoftransaminases:aspartate aminotransferase(AST)andalanineaminotransferase(ALT). TALP serum levels werealso evaluated. Specific kits were used,andmethodologyfollowedthemanufacturer’s instruc-tions(Labtest1_{,LagoaSanta-MG,Brazil).}

2.8. Haematologicstudy

Themethodusedtoanalysewhitebloodcellcounts,aswellas itssubpopulation(neutrophilandmononuclearcells),wasas follows:20mlofblood,takenfromtherattail,wasaddedto 380mlofTurksolution. Totalwhite blood cell countswere performed using a Neubauer chamber and the differential countsweremadeusingsmearsstainedbyrapidInstantProv Stain Set (Newprov Produtos para Laborato´rio; Pinhais-PR, Brazil).Aleucogramofthegroupsofanimals(salineandALD) wasperformedbeforeperiodontitisinduction,atthe6thhour and2nd,7thand11thdaysaftertheligature.

2.9. Corporalmassvariation

Animalsfrom salineand ALDgroupshad their body mass measuredbeforeperiodontitisinductionandafterthat,daily until the 11th day. Values were expressed as body mass variation(g)comparedtotheinitialbodymass.

2.10. Statisticalanalysis

Thedataarepresentedasmeanstandarderrorofthemean (SEM)ormedian(andrange),whereappropriate.Analysisof

variance(ANOVA),followedbyBonferroni’stestorStudent’st -test,wereusedtocompare means,andKruskal–Wallisand Dunntestswereusedtocomparemedians.Ap<0.05value was considered as indicating significant differences. All calculationswereperformedusingGraphPadPrism5software (GraphPad,Inc.,SanDiego,CA,USA).

3.

Results

3.1. Morphometricstudyofbonetissue

Themacroscopicanalysisofalveolarboneshowedthat11day ligature-inducedperiodontitiscausedintenseboneresorption

(Table1),associatedwithrootexpositionandfurcationlesion

(Fig. 1(d)). ALD, at the lowest dose (0.01mgkg 1_{), did not} protectalveolarbone(p>0.05)whencomparedtosaline.ALD athigherdoses(0.05and0.25mgkg 1_{)wasabletosignificantly} inhibit bone loss by 33.5% and 57.2%, respectively, when comparedtosaline(p<0.05).Although theanimalstreated with ALD (0.25mgkg 1_{) had not presented alveolar bone} preservation similar to normal hemimaxilla (Fig. 1(a)), the periodontalaspectwasdifferentfromsaline(Fig.1(g)).

3.2. Histologicalanalysisofalveolarbone

Forthehistological analysis,anotherassay wasperformed, and then thehemimaxillae wereprocessedforhistological analysis (Table 1). It was observed that alveolar bone and cementum resorptions were associated to an important inflammatory infiltrate (p<0.05) on animals submitted to periodontitis (Table 1; Fig. 1(e) and (f)), when comparedto normalperiodontium(Table1;Fig.1(b)and(c))(p<0.05).ALD (0.25mgkg 1_{)treatmentsignificantlyattenuatedthe} inflam-matory infiltrate and preserved periodontal ligament, root cementumandalveolarbone(Table1;Fig.1(h)and(i)),when comparedtosaline(p<0.05).

3.3. SerumdosageofBALP

SerumdosagesofBALPwereanalysed(Fig.2).Salinepresenteda significant decrease by 45.6% on BALP serum levels (13.621.56Ul 1_{) when compared to its baseline} (25.041.43Ul 1_{). The treatment with ALD (0.01 and} 0.05mgkg 1_{)causedareductionofBALPserumlevels,although} notsignificant(p>0.05),by17.6%(19.922.97Ul 1)and19.5% (21.622.39Ul 1_{),respectively,whencomparedtoits} respec-tive baseline (ALD 0.01=24.191.62; ALD 0.05mgkg 1₌ 26.672.15Ul 1). The treatment with ALD (0.25mgkg 1)

inducedasignificantdecreaseby28.1%(19.171.36Ul 1_)for this enzyme after 11days of ligature-induced periodontitis whencomparedtoitsbaselinedata(26.672.15Ul 1_); howev-er,thetreatmentwiththehighestdoseofALDpreventedBALP reductionby17.5%,whencomparedtosalineafter11daysof periodontitis(p<0.05).

3.4. SerumdosageoftransaminasesandTALP

Onthe11thday,forASTandALT,therewasnostatistical difference in the saline group when compared to its respectivebaseline.However,asignificantdecreaseinTALP serumlevelswas observedintheanimalsfromthe saline groupafter11days,whencomparedtoitsbaselinedata.The treatment with ALD did not cause significant alteration (p>0.05) in AST and ALT serum levels, but it reduced

(p<0.05)TALPserumlevelswhencomparedtoitsrespective baselinedata.

3.5. Haematologicstudy

Regarding total leucocyte counts, it was observed that periodontitiscausedleucocytosisatthe6thhourafterligature Table1–Macroscopicandhistologicalanalysisofnormalhemimaxillaorsubmittedtoperiodontitisreceivingsalineor ALD.

Normal Saline ALD0.01mgkg 1 _ALD0.05mgkg 1 _ALD0.25mgkg 1

Macroscopicanalysis MeanSEM

– 4.80.2 4.10.4 3.20.5* _2.1

0.1*

Histologicalanalysis Scores

0(0–0) 3(2–3)# _{– – 1.5(1–2)}*

(–)Indicatesthattherewasnoanalysis.Formacroscopicanalysis,valuesrepresentthemeanfollowedbyS.E.Mofaminimumof6animalsper groupbyAnovaandBonferronitest.Forhistologicalanalysis,valuesrepresentthemediansfollowedbyscoresvariation(lower–higher)ofa minimumof6animalspergroupbyKruskal–WallisandDunntest.

* _{Statisticallysignificantdifferencewhencomparedtosaline.}

# _{Statisticallysignificantdifferencewhencomparedtonormalhemimaxillae(p<0.05).}

Fig.1–Macroscopicandmicroscopicaspectrespectivelyofnormalperiodontium(A)–(C)andperiodontiumofratsubmitted

toperiodontitisreceivingsaline,showingmacroscopicboneresorption(D)andalveolarboneandcementumresorption,

andinflammatorycellinfiltrationseeninhistopathology(E)and(F).(G)(H)and(I)illustratethereductionofinflammation andalveolarbonelossinperiodontiumofratstreatedwithALD(0.25mgkgS1_{)for11days(macroscopicoriginal}

(18.771.66 leucocytes103mm 3) (Fig. 3(a)), when

com-paredtoitsbaselinedata(11.560.31).Thisleucocytosiswas marked (p<0.05) by neutrophilia (5.200.28 neutrophil103_mm 3_{), when compared to its baseline} (1.370.08)(Fig.3(b)).Following,onthe2ndday,therewas adecreaseintotalleucocytecount;however,the basalcell countswerenotachieved.Newleucocytosiswasobservedon the 7th (21.730.87 leucocytes103_mm 3_{) and 11th} (25.841.23)days,withpredominationofmononuclearcells (7th day=18.241.05;11th day=23.211.48 mononuclear cells103_mm 3_{)whencomparedtoitsbaseline(10.19}

0.25) (Fig.3(c)).AlldosesofALDpreventedneutrophiliaatthe6th hour (ALD 0.01=4.000.42; ALD 0.05=2.980.21; ALD 0.25=2.500.22), when compared to saline (5.200.28) (p<0.05) (Fig. 3(b)).However, onlyALD (0.25mgkg 1) pre-ventedmononuclearcellpeaksonthe7th(12.290.66)and 11th(15.740.52)days(Fig.3(c)).

3.6. Corporalmassvariation

Periodontitiscausedbodyweightlossnotedonthe3rdday afterligatureplacementwhencomparedtonormalanimals. Afterthat,animalsshowedgainofweightandatendencyto follow the normal animal corporal mass curve. Animals

treatedwithALDshowedasimilarcorporalmasspatternto saline. ALD did not alter initial loss of weight, when compared to saline. After the 3rd day, gain of mass was observed accompanying animals from the saline group (Fig.4).

Fig.2–EffectofALDonbone-specificalkalinephosphatase.

BarsrepresentmeanWSEMofBALP(U/l)ofaminimumof

6animalspergroup.(*)indicatesstatisticallysignificant differencewhencomparedtosaline11daydata.(§) indicatesstatisticallysignificantdifferencewhen

comparedtoitsrespectivebaselinedata[Two-wayAnova;

BonferronitestandStudent’st-test](p<0.05).

Fig.3–EffectofALDonleukocytecounts.Pointsrepresent meanWSEMoftotalleukocytes(A),neutrophils(B), mononuclearcells(C)T103_mmS3_{ofaminimumof6} animalspergroup.(*)indicatesstatisticallysignificant differencewhencomparedtosaline.(§_)indicates statisticallysignificantdifferencewhencomparedtoits baselinedata[AnovaandBonferronitest](p<0.05).

Table2–SerumdosageofASTandALTandTALPofanimalssubmittedtoperiodontitisandreceivingsalineorALD.

Days Groups

Saline ALD0.01mgkg 1 _ALD0.05mgkg 1 _ALD0.25mgkg 1

AST(U/l) 0 44.512.13 40.612.97 45.443.92 47.443.33

11 48.644.74 38.722.50 46.043.86 42.513.52

ALT(U/l) 0 18.443.89 19.193.81 17.363.27 19.324.18

11 22.033.44 19.911.30 21.572.72 16.021.99

TALP(U/l) 0 95.611.21 96.511.52 97.071.97 93.061.09

11 70.141.74§ _77.29

1.99§ _75.75

2.11§ _69.64

1.71§

ValuesrepresentmeanSEMof6animalspergroup.

§ _{Statisticallysignificantdifferencewhencomparedtoitsrespectivebaselinedata[Two-wayAnova;BonferronitestandStudent’st-test]}

4.

Discussion

In the present study, it was seen that ligature-induced periodontitis caused intense alveolar bone resorption and periodontalinflammation,asdemonstratedbymacroscopic andhistologicalanalyses.Inaddition,asignificantdecreasein BALPandTALPserumlevelswasobserved,andnochangein ASTandALTserumlevels.Periodontitiscausedleucocytosis markedbyneutrophiliaatthe6thhandmarkedby lympho-monocytosisonthe7thand11thdays.Inaddition,aninitial weightlossfollowedbytendencytoaccompanynormalrat corporal mass curve was observed. Treatment with ALD preventedalveolarboneresorptionofanimalssubmittedto ligature-inducedperiodontitis,confirmedinmacroscopicand histologicalanalyses,whencomparedtosaline.ALD,atthe higherdose, prevented thereduction ofBALPserum levels whencomparedtosaline,and didnotaltertransaminases’ serumlevels.Besides,ALDprevented6th-hneutrophilia,as wellas lymphomonocytosis observedon the 7th and 11th days.ALDdidnotpreventtheinitialweightloss,althoughthe animalshadshown gainofcorporalmasssimilartosaline corporalmasscurve.

IthasbeendescribedthatALDisrapidlyeliminatedfrom plasma,andmainlydistributedtothebone,whereabout60% ofthedoseislocalisedinbonetissueofrats.11_Accordingly,

Azumaet.al.12_{observedtheconcentrationof[}14 C]-alendro-nate in several bone tissues at various times after the 0.05mgkg 1 _{IV dose. The maximal concentration in long} boneandlumbarspineoccurredabout8and24h, respective-ly,aftertheIVdose,andtheconcentrationofALDinboneat 2hvariedinadose-dependentmanner,when0.05–15mgkg 1 of [14_{C]-alendronate was injected IV. Furthermore, reports} fromtheliteraturehaveshownthatnBPsnotonlyactedon osteoclastboneresorption, but alsoaffected the behaviour and metabolismofother bone-related cells,suchas osteo-blasts,osteocytesandmacrophages.13,14_{Therefore,weaimed}

toevaluateBALPserumlevelsaftertreatmentwithALD.BALP, an isoform of TALP, acts specifically as a bone formation marker. Its mechanism of action is based on inorganic pyrophosphatehydrolysis,removingthisosteogenicinhibitor, while it creates inorganic phosphate, required for the

generation and deposition of hydroxyapatite.15 _{BALP is}

secretedfromosteoblast membranetowardmatrixvesicles, allowing the mineralisationprocessto occur.15_{Itis known}

thatmammalian-tissueBALPisstronglyactivatedbydivalent cations such asMg2+_{and Zn}2+_{, and hasanactive siteand} contains twoZn2+ _{ionsthatstabiliseitstertiarystructure.}14

Theintestinalandplacentalisoenzymesarelessinfluencedby thesecations.16

Inthisstudy,wehaveshownthatthelowestdosesofALD (0.01and0.05mgkg 1_{)preventedthereductionofBALPserum} levels,whencomparedtoitsbaselinedata.Ontheotherhand, the highest dose of ALD (0.25mgkg 1_{) prevented BALP} reductionwhencomparedtosalineafter11daysof periodon-titis,but itwassignificantlydifferentonBALPserumlevels when compared toits baseline. Although slight, the lower levelofBALPaftertreatmentwithALDmayberelatedtotwo aspects:thechemicalstructure,whichiscloselylinkedtothe anti-resorptiveeffectofthisdrug,anditsconcentration.17,18

nBPs,likeALD,havetworadicalslinkedtothecarbonatom, one,calledR1thathasahydroxylgroup(–OH)andimproves mineralaffinity,andtheotherone,calledR2,whichincreases nBP potency to inhibit bone resorption.14 _{This chemical}

structureelicitsthedevelopmentofastructuralmotifcalled ‘bonehook’thatbindstothemineralbychelationofdivalent cations.18 _{Therefore, considering that BALPneeds divalent}

cations to become activated and that the ALD bone hook reducestheofferofthesecations,ourpresentobservations suggestthatthehighestdoseofALDinhibitedBALPactivity through divalent cation chelation within the bone hook structure.Thissuggestionisbasedonapreviousreportwhere BALPinhibitionwasreversedbyanexcessofZn2+_orMg2+_.13

However,itwasseenthatlowerdosesofALDprevented BALP reduction while the highest dose did not, when comparedtoitsrespectivebaseline;therefore,wecaninfer thatALDmayhaveadose-dependenteffectonBALPserum levels. In fact, reports from the literature had already confirmedourfinding.17,18_{ForStilletal.}17_atlower

concentra-tions (10 9 _{to 10} 7_{M), ALD increased the formation of} fibroblastic colonies,suggestinga mildanabolic effect. The treatmentwithhighconcentrations(10 4_{M)ofALDcauseda} total inhibitionofcolonyformation. Itwasalsofoundthat intermediate concentrations (10 6_{M) ofALDdecreased the} formationofcoloniesdisplayingosteoblastic characteristics suchasalkalinephosphataseexpression,collagen accumula-tionandcalcification.ItwasalsoobservedbyVaismanetal.18

that low doses of nBPs (10 10 _{to 10} 5_{M) stimulated BALP} activity, whereas high concentrations (10 4_{M) inhibited it.} Levelsof10 4_{MofALDareestimatedtobefoundinvivoat} resorptionlacunaeinexperimentalanimalmodels.Thus,our present observations are physiologically relevant in the context of alocal action ofnBPs used inthe treatment of differentbonediseases,suchasperiodontitis.

In order to corroborate BALP serum level results, we evaluatedthebone-sparingactionofALDonmorphometric andhistologicalanalyses.Asignificantboneprotectionwas observedwhenthehighestdoseofALDwasused.Thealveolar bone protection performed by ALD after ligature-induced periodontitishasbeendemonstratedinpreviousreports,in studiesusingthesimilarmethodology.19,20_This

anti-resorp-tiveeffectmaybeexplainedbytheattractionofALDtothe Fig.4–EffectofALDoncorporalmassvariation.Points

representmeanWSEMofaminimumof6animalsper

group.(*)indicatesstatisticallysignificantdifferencewhen

comparedtosaline[AnovaandBonferroni’stest]

boneand itsinterferenceonenzymeactivity.21,22_nBPs,like

ALDinhibitFPPS,amevalonatepathwayenzymeresponsible forisoprenylationofsmallGTPases,suchasRab,Rac,Rasand Rho.23_{ThesesmallGTPasesaresignallingproteinsthat,when}

activated,regulateseveralstructuralpropertiesimportantfor osteoclast function, including morphology, cytoskeletal ar-rangement,vesiculartraffickingandmembraneruffling.24,25

Bythetimethatvesiculartraffickingandmembraneruffling areinhibited bone resorptionis alsoreduced, due toFPPS inhibitionandconsequentGTPasesisoprenylationdecrease. Therefore,FPPS inhibitionseems to be responsible forthe pharmacologiceffectsofthenBPsattissuelevel.26

Themacroscopicaspectwascorroboratedbyhistological analysis,demonstratingpartialpreservationofalveolarbone, cementumandperiodontalligamentaswellasreductionof inflammatoryinfiltrateinanimalsreceivingALD.Beyondthe anti-resorptive action, ALD has shown anti-inflammatory activity,byinhibitionofpro-inflammatorycytokinesrelease, suchasIL-1,IL-6andTNF,andofnitricoxide(NO).27–29_This

anti-inflammatoryactivity may alsorebound on ALD anti-resorptive action, since IL-1 and TNF, mainly stimulate expressionofRANKL,aTNFfamilycytokine,whichisessential forosteoclastogenesisinduction.30

TreatmentwithALDseemedtobesafe.Animalstreated withALDshowedinitialweightloss,similartosaline,which mayhavebeencausedbyligatureplacement.7,9_Afterthat,it

wasseenthatALDtherapydidnotinduceadditionallossof weight, according to previous data.20 _{ALD therapy did not}

cause significant changes in AST and ALT serum levels, suggesting thatALDdoes notinterfere withliverfunction, whichwasexpected,sincethisdrugisnotmetabolisedinthe liver.31 _{Studies in patients who received liver transplant}

demonstrated that ALD has been well tolerated without deleteriouseffects on liverfunctiontests.32_{Patients taking}

ALD and diagnosed with primary biliary cirrhosis did not present significant hepatic effects regarding biochemical parameters of liver disease.33 _{Our study also revealed}

significantinhibitionofTALP serumlevels after11days of periodontitisinanimalsreceivingeithersalineorALD.This inhibitionmaybeduetothereductionoftheboneisoform, sinceBALPrepresentsabout90%oftheTALP.16

Wealsoobservedthat ALDprevented neutrophiliaand lymphomonocytosis.Thesefindingsareinaccordancewitha previousreportinwhichALDtreatmentinducedasignificant decreaseintotalwhitebloodcell,neutrophilandlymphocyte counts,inpatientswithPaget’sdisease.34_{Thereductionin}

neutrophil count may effect neutrophil migration and activity,onceitwasseenthatALDdecreasedonneutrophil influxusing a carrageenan-induced peritonitis model and reducedmyeloperoxidaseactivityaswell.20_{Inaddition,the}

reductioninperipheralmononuclearcells,whichincludes monocytesandlymphocytes,wasalsoanimportantfinding considering that circulating monocytes can migrate and differentiate locally on osteoclasts,thereby exerting bone resorption activity.22 _{Thus, the reduction of mononuclear}

cellsmaycontributetothebone-sparingeffectofALDinthis model.

Insummary,ourresultsdemonstratedthatALDprevented BALPreductionandABL,andreducedinflammatoryinfiltrate, withoutcausingsystemicalterations.

Funding

This work was supported by Brazilian grants from the ConselhoNacionaldeDesenvolvimentoCientificoe Tecnolo´-gico(CNPq,Grants471407/2009-7),Coordenac¸a˜ode Aperfei-c¸oamentodePessoaldeNı´velSuperior(CAPES)andFundac¸a˜o CearensedeApoioaoDesenvolvimentoCientı´ficoe Tecnolo´-gico(FUNCAP,Grants247.01.00/09).

Competing

interests

Nonedeclared.

Ethical

approval

Theexperimentalprotocolswereexecutedfollowingethical principlesforlaboratoryanimaluse inaccordancewiththe EuropeanConventionfortheProtectionofVertebrateAnimals usedforExperimentalandOtherScientificPurposes,andthey wereapprovedbyInstitutionalEthicalCommitteeofAnimal Research(ProcessNo.101/2009).

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s

1. GiannobileWV.Host-responsetherapeuticsfor periodontaldiseases.JournalofPeriodontology

2008;79(Suppl.8):1592–600.

2. CochranDL.Inflammationandbonelossinperiodontal disease.JournalofPeriodontology2008;79_{(Suppl.8):1569–76.}

3. VaananenK.Mechanismofosteoclastmediatedbone resorption—rationaleforthedesignofnewtherapeutics.

AdvancedDrugDeliveryReviews2005;57_{(7):959–71.}

4. RussellRG.Bisphosphonates:modeofactionand pharmacology.Pediatrics2007;119_{(Suppl.2):150–62.}

5. KimmelDB.Mechanismofaction,pharmacokineticand pharmacodynamicprofile,andclinicalapplicationsof nitrogen-containingbisphosphonates.JournalofDental Research2007;86_{(11):1022–33.}

6. WhyteMP.Physiologicalroleofalkalinephosphatase exploredinhypophosphatasia.AnnalsoftheNewYork AcademyofSciences2010;1192:190–200.

7. LimaV,VidalFDP,RochaFAC,BritoGAC,RibeiroRA.Effects ofTNF-ainhibitorspentoxifyllineandthalidomideon alveolarbonelossinshort-termexperimentalperiodontal diseaseinrats.JournalofPeriodontology2004;75(1):162–8. 8. GoesP,LimaAPS,MeloIM,RegoRO,LimaV.Effectof

atorvastatinonligature-inducedperiodontitisinWistar rats:radiographicandmacroscopicanalysis.BrazilianDental Journal2010;21_(3):193–8.

9. LimaV,BezerraMM,AlencarVBM,VidalFD,daRochaFA,de CastroBritoGA,etal.Effectsofchlorpromazineonalveolar bonelossinexperimentalperiodontaldiseaseinrats.

EuropeanJournalofOralSciences2000;108_(2):123–9.

10.MossDW,WhitbyLG.Asimplifiedheat-inactivation methodforinvestigatingalkalinephosphataseisoenzymes inserum.ClinicaChimicaActa1975;61_(1):63–71.

11.LinJH,DugganDE,ChenIW,EllisworthRL.Physiological dispositionofalendronate,apotentantiosteolytic

12.AzumaY,SatoH,OueY,OkabeK,OhtaT,TsuchimotoM, etal.Alendronatedistributedonbonesurfacesinhibits osteoclasticboneresorptioninvitroandinexperimental hypercalcemiamodels.Bone1995;16_{(2):235–45.}

13.RussellRG.Bisphosphonates:thefirst40years.Bone

2011;49_(1):2–19.

14.RogersMJ,CrockettJC,CoxonFP,Mo¨nkko¨nenJ.Biochemical andmolecularmechanismsofactionofbisphosphonates.

Bone2011;49_(1):34–41.

15.BalcerzakM,HamadeE,ZhangL,PikulaS,AzzarG,Radisson J,etal.Therolesofannexinsandalkalinephosphatasein mineralizationprocess.ActaBiochimicaPolonica

2003;50(4):1019–38.

16.AndersonRA,BosronWF,KennedyFS,ValleeBL.Roleof magnesiuminEscherichiacolialkalinephosphatase.

ProceedingsoftheNationalAcademyofSciencesoftheUnited StatesofAmerica1975;72_{(8):2989–93.}

17.StillK,PhippsRJ,ScuttA.Effectsofrisedronate,

alendronate,andetidronateontheviabilityandactivityof ratbonemarrowstromalcellsinvitro.CalcifiedTissue International2003;72_{(2):143–50.}

18.VaismanDN,McCarthyAD,CortizoAM.Bone-specific alkalinephosphataseactivityisinhibitedby

bisphosphonates:roleofdivalentcations.BiologicalTrace ElementResearch2005;104_{(2):131–40.}

19. DuartePM,deAssisDR,CasatiMZ,SallumAW,Sallum EA,NocitiJrFH.Alendronatemayprotectagainst increasedperiodontitis-relatedbonelossin estrogen-deficientrats.JournalofPeriodontology2004;75(9):1196–202. 20.MenezesAM,RochaFA,ChavesHV,CarvalhoCB,Ribeiro

RA,BritoGA.Effectofsodiumalendronateonalveolarbone resorptioninexperimentalperiodontitisinrats.Journalof Periodontology2005;76(11):1901–9.

21.PapapoulosSE.Bisphosphonateactions:physicalchemistry revisited.Bone2006;38_(5):613–6.

22.RussellRG,WattsNB,EbetinoFH,RogersMJ.Mechanismsof actionofbisphosphonates:similaritiesanddifferencesand theirpotentialinfluenceonclinicalefficacy.Osteoporosis International2008;19_{(6):733–59.}

23. LuckmanSP,HughesDE,CoxonFP,GrahamR, RussellG,RogersMJ.Nitrogen-containing

bisphosphonatesinhibitthemevalonatepathwayand preventpost-translationalprenylationofGTP-binding proteins,includingRas.JournalofBoneandMineralResearch

1998;13(4):581–9.

24.AlakangasA,SelanderK,MulariM,HalleenJ,LehenkariP, Mo¨nkko¨nenJ,etal.Alendronatedisturbsvesicular traffickinginosteoclasts.CalcifiedTissueInternational

2002;70_(1):40–7.

25.PavlosNJ,XuJ,RiedelD,YeohJS,TeitelbaumSL, PapadimitriouJM,etal.Rab3Dregulatesanovelvesicular traffickingpathwaythatisrequiredforosteoclasticbone resorption.MolecularandCellularBiology2005;25_{(12):5253–69.}

26.FisherJE,RogersMJ,HalasyJM,LuckmanSP,HughesDE, MasarachiaPJ,etal.Alendronatemechanismofaction: geranylgeraniol,anintermediateinthemevalonate pathway,preventsinhibitionofosteoclastformation,bone resorption,andkinaseactivationinvitro.Proceedingsofthe NationalAcademyofSciencesoftheUnitedStatesofAmerica

1999;96(1):133–8.

27.GiulianiN,PedrazzoniM,PasseriG,GirasoleG. BisphosphonatesinhibitIL-6productionbyhuman osteoblast-likecells.ScandinavianJournalofRheumatology

1998;27_(1):38–41.

28.MakkonenN,SalminenA,RogersMJ,FrithJC,UrttiA, AzhayevaE,etal.Contrastingeffectsofalendronateand clodronateonRAW264macrophages:theroleofa bisphosphonatemetabolite.EuropeanJournalof PharmaceuticalSciences1999;8_{(2):109–18.}

29.Mo¨nkko¨nenJ,Simila¨ J,RogersMJ.Effectsoftiludronateand ibandronateonthesecretionofproinflammatorycytokines andnitricoxidefrommacrophagesinvitro.LifeSciences

1998;62(8):PL95–102.

30.TakayanagiH.Inflammatorybonedestructionand osteoimmunology.JournalofPeriodontalResearch

2005;40(4):287–93.

31.LambrinoudakiI,ChristodoulakosG,BotsisD.

Bisphosphonates.AnnalsoftheNewYorkAcademyofSciences

2006;1092:397–402.

32.AtamazF,HepgulerS,AkyildizM,KarasuZ,KilicM.Effects ofalendronateonbonemineraldensityandbonemetabolic markersinpatientswithlivertransplantation.Osteoporosis International2006;17_(6):942–9.

33.ZeinCO,JorgensenRA,ClarkeB,WengerDE,KeachJC, AnguloP,etal.Alendronateimprovesbonemineraldensity inprimarybiliarycirrhosis:arandomized placebo-controlledtrial.Hepatology2005;42_{(4):762–71.}