Experimental model Antimicrobial therapy photodynamic combined to periodontaltreatment: Photodiagnosis and Photodynamic Therapy

PhotodiagnosisandPhotodynamicTherapy18(2017)275–278

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Photodiagnosis and Photodynamic Therapy

j o ur na l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / p d p d t

Antimicrobial photodynamic therapy combined to periodontal treatment: Experimental model

Estéfani L. Belinello-Souza

, Letícia H. Alvarenga

, Cintia Lima-Leal

,

Patrícia Almeida

, Carolina Guimarães Leite

, Tairine R. Lima

, Bianca Godoy-Miranda

, Jhosepher Previati-Oliveira

, Lucas de Pretto

, Anderson Zanardi de Freitas

,

Adjaci U. Fernandes

, Rodrigo Labat Marcos

, Renato A. Prates

aDep.ofBiophotonics,UniversidadeNovedeJulho(UNINOVE),SãoPaulo,Brazil

bSchoolofDentistry,UniversidadeNovedeJulho(UNINOVE),SãoPaulo,Brazil

cCenterforLasersandApplications−IPEN-CNEN/SP,SãoPaulo,Brazil

dUniversidadeAnhembiMorumbi,SãoPaulo,Brazil

a r t i c l e i n f o

Articlehistory:

Received1December2016

Receivedinrevisedform3March2017 Accepted13March2017

Availableonline19March2017

Keywords:

Antimicrobialphotodynamictherapy Periodontitis

Photosensitizer Porphyrin LED

a b s t r a c t

Background:Antimicrobialphotodynamictherapy(aPDT)hasbeenusedasanadjuvanttreatmentfor periodontitis.Itcombinesaphotosensitizerwithalightsourcetoinducereactiveoxygenspeciesandkill microbialcells.PpNetNIisaprotoporphyrinderivative,andithasachemicalbindingsiteatbiofilmand greataffinitytomicrobialcells.TheaimofthisstudywastoinvestigatetheeffectsofaPDTasanadjuvant treatmentforperiodontitis.

Methods:ThirtyhealthymaleratsWistar(Rattusnorvegicus)wereusedinthisstudy(ApprovedbyUNI- NOVEEthicalcommitteeAN0029/2015).Periodontitiswasinducedbyplacingacottonligaturearound thefirstmandibularmolarinasubgengivalposition.Thecontralateralmandibularfirstmolarreceived neitheraligaturenoranytreatment,andwasusedasacontrol.After7days,theligaturewasremoved andallanimalsreceivedscalingandrootplaning(SRP)andweredividedaccordingtothefollowingtreat- ments:SRPgroup(receivedSRPandirrigationwithPpNetNI,10␮M)andaPDTgroup(PpNetNI10␮M followedbyLEDirradiation).aPDTwasperformedwithaLED(630nm)withanoutputpowerof400mW (fluence-rate200mW/cm²;fluence18J/cm²).Ratswereeuthanizedat24h,48hand7dayspostopera- tively.TheareaofbonelossinvestibularregionofthefirstmolarwasevaluatedbyOpticalCoherence Tomography(OCT,THORLABSLTD.,Ely,UK).Datawereanalyzedstatistically(ANOVAandTukeytests, p<0.05).

Results:TheanimalstreatedbyaPDTshowedbonegainofapproximately30%comparedtotheSRPgroup following7daysfromthetreatment.

Conclusion:aPDTpromotedbonerecovery7daysafterperiodontalintervention.

©2017ElsevierB.V.Allrightsreserved.

1. Introduction

Periodontitisis aninflammatory disease of supportiveperi- odontal tissue characterized by gingival inflammation, loss of connectivetissueattachment,andreabsorption ofalveolarbone [1,2]. This disease is caused by microorganisms organized in biofilms that colonize tooth surface; the biofilm is a complex

∗Correspondingauthorsat:BiophotonicsProgram,NovedeJulhoUniversity(UNI- NOVE),RuaVergueiro235/249,Liberdade,SãoPauloZIP:01504−001,Brazil.

E-mailaddresses:leticiahalvarenga@uni9.pro.br(L.H.Alvarenga), pratesra@uni9.pro.br(R.A.Prates).

structure composedbymicrocolonies ofbacteriaimbibed inan extra-cellularmatrixthatpromotescellprotectionandadherence tothetooth[3,4].Theperiodontaltherapyisbasedonprofessional removalofthebiofilmandcalculus byscalingandrootplaning (SRP)inconjunctionwithpersonalplaquecontrol[5].However, themechanicaldebridementmayfailtoremovesomepathogenic organismsbecauseoftheirlocationinsubgengivaltissue,likefur- cationsordeeppocketsandinsomesituationstheuseofsystemic antibioticsmayalsobeindicated[6,7].

Administration of antibiotictreatments hasbeen decreasing over the years because its side effects, such as stomach pain and increaseofbacterialresistancetoantibiotic.In somecases, patientleavestheantibiotictreatmentbeforeitsend,anditgives http://dx.doi.org/10.1016/j.pdpdt.2017.03.008

1572-1000/©2017ElsevierB.V.Allrightsreserved.

276 E.L.Belinello-Souzaetal./PhotodiagnosisandPhotodynamicTherapy18(2017)275–278

a chance to select resistant bacteria [6,8]. Antimicrobial pho- todynamic therapy (aPDT) hasbeen proposed as adjunctive to periodontaltreatment[9–11].aPDTcombinesanon-toxicphoto- sensitizer(PS)andlowintensityvisiblelight,whichinthepresence ofoxygenproducecytotoxicreactiveoxygenspecies(ROS),suchas singletoxygen[12,13],thataretoxictobacterialcells.

TheeffectofaPDThasbeenevaluatedbyseveralratligature models,withinductionofexperimentalperiodontitisbyligature placement [14–16].The ligature leads tobiofilm accumulation, resultinginlossofattachmentandbonepredictablyin7days[17].

Consideringthattheresultsofclinicaltrialsevaluatingthebene- fitsofaPDTarestillinconclusive[18–20],thepurposeofthisstudy wastoevaluatetheefficacyofaPDTasanadjuvanttoperiodon- taltreatmentinratsusingproto-porphyrin PpNetNIasPS[21].

Porphyrins-PDThavedemonstratedgoodresultsintheinactiva- tionofbacteria[22–24].PpNetNIwasusedforthefirsttimeinthis animalmodelassociatedwithirradiationwithLED.Opticalcoher- encetomography(OCT)assessmentwasusedtoevaluatetheeffects providedbyaPDTinperiodontaltissue.

2. Materialsandmethods

TheexperimentswereapprovedbytheEthicsCommitteefor AnimalResearchofUNINOVE/SP(n.AN0029/2015).Allratswere housedunderthesameconditionsandmaintainedonfoodand wateradlibitum.

2.1. Studydesign

ThirtyhealthymaleWistarrats(Rattusnorvegicus)withabody massofapproximately250gwereusedforthisstudy.Periodon- titiswas inducedby placinga cottonligature around theright inferiorfirstmolarinasubgingivalposition.After7days,thelig- atureswere removedfrom theanimals and theywere divided intotwo groups.SRP group(n=15)receivedSRPand irrigation with10␮MPpNetNI,withoutirradiationandaPDTgroup(n=15) received10␮MPpNetNIfollowedbyLEDirradiationfor90s.Rats wereeuthanizedat24h,48hand7dayspostoperatively(n=5ani- malspergroup)bytheadministrationofalethaldoseofanesthetics (150mg/kgthiopental).

TheexperimentalscheduleisillustratedinFig.1.

2.2. Ligatureinductionofexperimentalperiodontitis

The rats were anesthetized with ketamine (80mg/kg) and xylazine(10mg/kg)byintraperitonealinjection.Acottonligature wasplacedaroundtherightfirstmolarofthejawinasubmarginal positiontoinduceperiodontalinflammationandthecontralateral wasusedascontrol,withoutperiodontitisinduction(Fig.2).

2.3. Photodynamictherapy

ThePS(10␮MPpNetNI)wasappliedaroundtherightinferior firstmolarandapreirradiationtimeof5minwasset.Thetested

Fig.1. Inductionofperiodontitisonday−7.Alltreatmentswereperformedatday 1.Theanimalswereeuthanizedfollowing24h,48h,and7daysfromthetreatment.

Fig.2.Cottonligaturearoundtherightinferiorfirstmolarinasubgingivalposition.

Table1

Irradiationparametersusedintheexperiment.Thebuccalareaoftherightfirst molarwasirradiatedonce.

Irradiationparameters

Wavelength 630nm

Radiantoutputpower 400mW

Irradiance 200mW/cm²

Radiantexposure 18J/cm²

sizereceivedLEDirradiationduring90sthroughthebuccalside withtheparametersdescribedaboveinTable1.

2.4. Opticalcoherencetomography

Themaxilla of theratswas removedand thesampleswere immediatelyfixedin 4%paraformaldehydein smallglassesand identifiedaccordingtherespectivegroups.

Theareaofbonelossinbuccalregionofthefirstmolarwas evaluatedbyOpticalCoherenceTomography(OCT,THORLABSLTD., Ely,UK).Theanalysiswasmadebymeasuringthedistanceofthe coronalmarginofthebonecresttothecementoenameljunction (CEJ).Eachsamplewasevaluatedanda percentagevalueofthe amountofbonelosswascalculatedbyusingthecontralateraltooth asparameter.

Toassesstheeffectofthetreatments,One-WayANOVAtestwas usedfollowedbyTUKEYtest.Theresultswereconsideredstatisti- callysignificantwhen(p<0.05).Dataispresentedasthemeanand standarddeviationofeachgroup.

3. Results

OpticalCoherenceTomographywasabletodetectbonelossin thesamplesanditwasanondestructivemethodforthisexperi- mentalmodel(Fig.3).

After7daysfromthetreatment, theanimalstreatedbyPDT showedbonegainofapproximately30%comparedtotheSRPgroup (Fig.4).

Furthermore,therewasanincreaseinbonelossintheSRPgroup, whiletheaPDTgrouppresentedregressioninthesizeofboneloss after7days(Fig.5).

E.L.Belinello-Souzaetal./PhotodiagnosisandPhotodynamicTherapy18(2017)275–278 277

Fig.3. Samplesphotographs(left)andOCTimages(right)fromananimal24haftertreatmentfromaPDTgroup.Wecanobservethedistancebetweenthecementoenamel junction(CEJ)andthebonecrest(BC).NotetheFurcationLesion(FL)andthemagnitudeofboneloss.A−controlside;B−testside.

Fig.4.Samplesphotographs(left)andOCTimages(right)fromananimal7daysaftertreatmentfromaPDTgroup.Wecanobservethedistancebetweenthecementoenamel junction(CEJ)andthebonecrest(BC).A−controlside;B−testside.

Fig.5. QuantificationdataofbonelosscomparingaPDTandSRPgroup.Theanimalswereeuthanizedfollowing24h,48h,and7daysfromthetreatment.IntheYaxiswe canobservetherelationbetweenthecontrolandthetestside.Dataaremeansandstandarddeviation.Statisticalsignificantdifferenceisrepresentedbythesymbol(*).

4. Discussion

In the present study, we reproduced a previously reported modelofperiodontitisinratsinducedbythepresenceofaligature

aroundatooth[25].Ourstudydemonstratedthatcottonligatures aroundtheinferiorrightfirstmolarresultedinprogressivebone lossinthefurcationregionafter7days.

278 E.L.Belinello-Souzaetal./PhotodiagnosisandPhotodynamicTherapy18(2017)275–278

Themeasurementofbonelossareawasperformedbyoptical coherencetomography,whichdemonstratedtobeagoodappara- tusforthistypeofanalysis,sinceitdidnotcausedamagetothe samplesandprovidesbetterimagequalityand resolutionwhen comparedtothetraditionalimagingmethods.Thiswasthefirst timethatOCTwasusedinexperimentalmodelsofperiodontitisin rats.Previously,theanalysisofbonelosswasperformedwithradio- graphs[14,26]andhistometricanalysis[15,16,27,28].TheOCTdata demonstratedsignificantlyreducedbonelossintheaPDTgroup comparedtotheSRPgroupat7days.Severalanimalstudiesand clinicaltrialsthatinvestigatetheaPDTasadjuvantofperiodon- taltherapyusethePSmethyleneblue(MB)[9–11,14,16].Animal studieshaveshowngoodresultswiththiscombination[16,27],but inclinicaltrialstheefficacyofaPDTusingMBisstillinconclusive [18–20].InattempttoimprovetheresultsobtainedbyaPDT,we proposedtheuseofPpNetNIasphotosensitizer.ThePpNetNIwas usedforthefirsttimeinanimalmodelassociatedwithirradiation withLED.

Theresultsobtainedinthepresentstudyshowedthattheasso- ciationofPpNetNIwithLEDirradiationatthesettingsmentioned beforerevealedmoresatisfactoryresultsthanisolatedutilizationof SRP.SignificantbonegainwasachievedintheaPDTgroupfollow- ing7daysfromthetreatment,andthismightbeduetoeffectsof lowlevellasertherapyonosteogenicdifferentiationandprolifera- tion[29].Similarresultswerealsoverifiedinotheranimalstudies [16,27].TheresultssuggestedthataPDTreducedperiodontaltissue destructionandmaybeusefulasanadjuvanttoSRP.However,fur- therstudiesshouldbeconductedtoelucidatequestionsaboutthe efficacyofaPDTonperiodontaldiseaseandtoestablisheffective protocolsfortheuseofaPDTinclinicalprocedures.5.Conclusions

Inconclusion,withintheparametersusedinthisstudy,aPDT wasaneffectivealternativetoheldperiodontalhealthaftertreat- ment,anditwasabletoimproveregenerationandpreventfurther tissueloss.

Acknowledgement

TheauthorsthankstoUNINOVEtoresearchsupportandFAPESP (grantnumber2016/10269-5).TheauthorsalsothanksDanielle Vieiraforthetechnicalsupportwiththeexperimentalanimals.

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