Effects of photodynamic therapy mediated by nanoemulsion containing chloro-aluminum phthalocyanine: a histologic and immunohistochemical study in human gingiva

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ContentslistsavailableatScienceDirect

Photodiagnosis

and

Photodynamic

Therapy

j o ur na l ho me p ag 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

Effects

of

photodynamic

therapy

mediated

by

nanoemulsion

containing

chloro-aluminum

phthalocyanine:

a

histologic

and

immunohistochemical

study

in

human

gingiva

Maiara

de

Moraes

a,∗

_,

_Roseane

_Carvalho

_Vasconcelos

b

_,

_João

_Paulo

_Figueiró

_Longo

c

_,

Luis

Alexandre

Muehlmann

c

_,

_Ricardo

_Bentes

_de

_Azevedo

c

_,

Telma

Maria

Araújo

Moura

Lemos

d

_,

_Antonio

_de

_Lisboa

_Lopes

_Costa

e

a_DDS,_PhD,_{Post-Graduate}_Program,_Oral_Pathology,_Federal_University_of_Rio_Grande_do_Norte,_Natal,_RN,_Brazil b_DDS,_MSc,_{Post-Graduate}_Program,_Oral_Pathology,_Federal_University_of_Rio_Grande_do_Norte,_Natal,_RN,_Brazil c_Department_of_Genetics_and_Morphology,_Institute_of_Biological_Sciences,_University_of_Brasília,_{Brasília/DF,}_Brazil

d_Department_of_Clinical_and_{Toxicological}_Analysis,_Health_Sciences_Center,_Federal_University_of_Rio_Grande_do_Norte,_Natal,_RN,_Brazil e_DDS,_PhD,_Professor_of_the_{Post-Graduate}_Program,_Oral_Pathology,_Federal_University_of_Rio_Grande_do_Norte,_Natal,_RN,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received3August2015

Receivedinrevisedform15October2015 Accepted22October2015

Availableonline28October2015 Keywords:

Photodynamictherapy Photochemotherapy Photosensitizingagents

VascularendothelialgrowthFactorA

a

b

s

t

r

a

c

t

Background:Photodynamictherapy(PDT)usesphotosensitizingagents,whicharedeliveredintarget cells,followedbylocalapplicationofvisiblelightinspeciﬁcwavelengths.Thisreactionproducereactive oxygenspeciesabletoinducecelldeathbyapoptosisornecrosis,injuredtothelocalvasculature,and exertimportanteffectsontheimmunesystem.

Objective:Thepresentworkevaluatedtheclinicalﬁndings,histomorphologicalalterationsand immun-odetectionofVEGF afterPDTusingchloro-aluminumphthalocyanine(AlClPc)entrappedin alipid nanoemulsioninasplit-mouthclinicaltrial.

Materialandmethods:Eighthealthyvolunteerswithclinicalindicationforextractionwereincludedin thestudy.Sevendaysbeforetheextraction40ulofnanoemulsionAlClPc5␮Mwasinjectedinto gin-givaltissuefollowedbyirradiationwithdiodelaser,thecontralateralsidewasusedascontrol.Tissue specimenswereremovedsevendaysafterthePDTanddividedintotwogroups(testandcontrolgroups) forhistologicalandimmunohistochemicalanalysis.Patientsweremonitoredatdays,0,7,14and30to assessadverseeffectsofthetherapy.

Results:Thetherapywaswelltoleratedbyallpatients.Adverseeffectswereshort-timeandcompletely reversible.Areasofedema,vascularcongestion,andintensevascularizationwereviewedingingival samplesthatreceivedPDT.Additionally,dystrophiccalciﬁcationwasobservedinsubepithelialregion. VEGFshowedmoderatetostrongimmunostaininginspecimenssubjectedtoPDT.

Conclusions:Takentogether,theresultsshowed thatthe protocolused in thisstudymediated by nanoemulsioncontainingAlClPcissafeforclinicalapplicationingingivaltissueandsuggeststhatVEGF isincreasedafterPDT.

∗ Correspondingauthorat:MaiaradeMoraesUniversidadeFederaldoRioGrande doNorte—DepartamentodeOdontologia,Av.SenadorSalgadoFilho,1787–Lagoa Nova,Natal,RNCEP59056-000,Brazil.Fax:+8432154138.

E-mailaddresses:maiaramoraes@gmail.com(M.deMoraes),

r.roseane@hotmail.com(R.C.Vasconcelos),jplongo82@gmail.com(J.P.F.Longo), razevedo@unb.br(R.B.deAzevedo),telmaml@yahoo.com.br(T.M.A.M.Lemos), antoniolisboa@uol.com.br(A.d.L.L.Costa).

1. Introduction

Photodynamictherapy(PDT)isapromisingtherapeutic modal-itythathasbeenusedinresearchandclinicalinvestigationsfor various diseases suchas malignant and non-malignanttumors, periodontitis, potentially malignant disorders and other oral lesions.Thisnon-surgicaltreatmenthasbeenusedtobeminimally invasiveandeasytoperformbesidespresenting goodresultsin clinicalstudies[1].Theprincipleofthistherapyisthedeliveryof aphotosensitizingagentincellsandtissuesfollowedbythe appli-cationoflaserirradiation(lowlevellaser).Absorptionofphotons

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M.deMoraesetal./PhotodiagnosisandPhotodynamicTherapy12(2015)592–597 593

bythephotosensitizingdrugpromotesitstransformationintoan extremelyunstablemoleculethatreactswiththemolecular oxy-genandformionsperoxides,superoxideandhydroxylradicals,and singletoxygenwhoareextremelycytotoxic[2–5].

Inrecentyears, researchesinvolvingthemechanismsofPDT havebeenwidelyexploitedmainlyinregardtosignaltransduction pathways,transcriptionandcellcycleregulatorsfactors, inﬂamma-tion,andapoptosis.Mechanismsassociatedwiththedestruction of cells and tissues with theuse of this therapy can be medi-atedbymultiplesignalingpathwaysthatoftenoverlap.Celldeath, changesinlocalvascularization,andimmunomodulatoryeffects areinvolvedinphotosensitizationandmaycontributetoits ther-apeuticactionintissues[3,5,6].

Severalphotosensitizershavebeenusedinthephoto-activation ofbiologicalsystems.Developmentofnewphotosensitizingagents iscomplexandrequiresexperimentalpre-clinicaltestinginseveral stepslatertoenteronstageofclinicalresearch.Studieswith chloro-aluminumphthalocyanine(AlClPc)hasshownthatthisdrugkeeps allitsphotophysicalandphotochemicalpropertieswhen adminis-teredassociatedwithnano-carrieir,suchasnanoemulsionsinboth invitroandinvivostudies[7,8].Analysisofmitochondrialactivity andcellorganelles[9,10];analysisofcellviability,cytotoxicityand genotoxicpotentialincellsculture[11,12];vasculardamage,tissue necrosis[13]andreductionofmetastasistocervicallymphnodes

[14]wereinvestigatedinpreclinicalstudies.Resultsofthestudies describedabovedemonstratetheAlClPcasaneffectivedrugwith antitumoractivity.

ItiswellknownthatPDTapplicationproducesaseriesoftissue effectsthatincludeinﬂammationandvascularshutdowninregions closetothetargetarea.Ingeneral,thesevasculareffectsare fol-lowedbyanimportanttissuehypoxiathatpromotestherelease ofaseriesofmolecularsignalstorecoverythenormalconditions ofthetissues.Oneofthecentralmolecularpathwaysinvolvedin thisprocessismediatedbytheVascularEndothelialGrowthFactor (VEGF),anessentialmediatorduringtheprocessofangiogenesis, whichcanbeproducedbydifferentcelltypesinhypoxiaconditions. Numerousauthorsdescribeanup-regulationofVEGFafterPDTin tumorcells[15–18],andthismediatorisrelatedtotissuerecovery post-treatment.Thus,theobjectivesofthisstudyistoinvestigate thetissueandclinicaleffects,aswellasthepresenceofVEGF,of normalgingivaltissueafterapplicationofPDTmediatebyAlClPC entrappedinananoemulsioninasplit-mouthexperimentalclinical trialinvolvinghumanvolunteers.

2. Materialandmethods

2.1. Subjectsandstudydesign

Experimental protocol was reviewed and approved by the Institution’sHuman Research Committeeand the protocolwas approvedon April20, 2012(protocol0156.0.051.000-11, Natal, FederalUniversityofRioGrandedoNorte,Brazil)andthe exper-iments were undertaken with the understanding and written consentofeachsubject.Eightpatients(5females,3males,aged 23–63)presentingtwoteethwithaclinicalindicationforextraction wereselectedforthestudy.Absenceofgingivalbleedingon peri-odontalprobingwastheinclusioncriteriaforbothteethincluded insamples.Additionally,thepatientshadneitherotheroralor sys-temicdiseases,noranyovertimmunologicalabnormalitiesanddid nottakeanypreoperatorymedication.

Thestudywasperformedusingthesplit-mouthdesignina non-randomizedmanner.Atotalof8pairsofcontralateralmaxillaryor mandibularyteethwereincluded.Ineachcontralateralpair,one toothwasassignedascontrolwhereastheothertoothwastreated withphotodynamictherapy (PDT).Allpatientsweretreatedby

thesameoperatorandtheextractionsofbothteeth(controland treated)were performed7days aftertreatmentby PDT. Gingi-valtissuesamples,whichotherwisewouldhavebeendiscarded, wereobtainedduringsurgery underlocalanaesthesia,avoiding local anaestheticinﬁltrationinto the biopsysite, and deforma-tionorcompressionofthesamples.Gingivalsamplesofcontrol andtreatedteethwereobtainedfromthemesialanddistalbuccal marginalgingiva.

2.2. Photosensitizersolutionandlaser

Aluminum-chloride-phthalocyanine(AlClPc)wassynthesized, puriﬁed, andprovided byAldrichChemicalCompany (St.Louis, MO, USA). Incorporation of aluminum-chloride-phthalocyanine (AlClPc) intothenanoparticleswasperformedaccording tothe methoddescribedbyMuehlmannetal.[12].Acontinuousredlaser (660nm,BioWaveDualLLLTKondortech,SãoCarlos,SãoPaulo, Brazil)wasusedinthisstudy.FortheclinicalPDTapplication,4min oflaserirradiationwith30mWofpotencywasappliedingingival tissue.Theequipmenthadaspotlaserof0.03cm2_,_which_confers

anenergyﬂuenceof7J/cm2_and_a_total_irradiance_of₃₀_mW/cm2_.

2.3. Treatment

Seven days prior to the extraction procedure, drug appli-cation was performed. Treatment consisted of injecting of 40␮lnanoemulsioncontainingchloro-aluminumphthalocyanine in the interdental papilla region (mesial or distal marginal gingiva—approximatelengthofatoothface),withoutlocal aneste-sia(withasterileinsulinsyringeandaminimumtime5minofdrug infusion).Fifteenminuteslater,diodelaser irradiationfor4min (7J/cm2_)._Analgesics _{(acetaminophen,} ₇₅₀_mg/tablet, _one _tablet

threeorfourtimesaday) wereprescribedtothepatientsafter PDT.

2.4. Clinicalassessment

Atbaseline,1week,2weekand1monthaftertreatment,the adverseeffectsofthetherapywereevaluated.Patientand inves-tigator painassessmentswererecordedona4-pointpainscale (0=no,1=slight,2=mild, 3=moderate,and 4=severe).Adverse effectsincludingstinging,burning,erythema,purpura,blistering, orcrustingwererecordedonpatientandinvestigatorassessment scales(0–4,asabove)[19].Clinicalphotographsweretakenateach patient’svisittoevaluatetheclinicaloutcomeofPDT.

Patientswerefollowedupat7,14and30days.Wereassessed: adverseeffects,reportsofcomplications,post-PDTpain;the effec-tivenessofthedrugforthepurposeofanalgesiaprotocol;wellas reportsofthepatienttowardthetherapyused.

2.5. Histologyandimmunohistochemistry

SevendaysafterthetreatmentbyPDT,foreach patient,the teethwereremovedandgingivaltissueswereimmediatelyfixedin 4%phosphatebufferedformalin,pH7.4andprocessedbyroutine laboratorytechniquesfor paraffin embeddingTissuespecimens dividedintotwogroups(testandcontrolgroups).Serialsections (5␮mthick)wereobtainedfromallspecimens.Forallspecimens wasperformedstainingwithhematoxylin-eosinandslidesofall sampleswerereviewedtoevaluatethemagnitudeof inflamma-tionatthehistologiclevelaccordingtoadaptationofTsaietal.

[20].Eachspecimenwasgradedat400×magnificationas:grade 1(slight),inflammatory cellslessthan 1/3;grade2(moderate), inflammatory cells between 1/3and 2/3; and grade3 (severe), inflammatorycellshigherthan2/3.Gradationofeachspecimenwas

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Fig.1.Clinicalaspectofregionsafterphotodynamictherapy.(A,C)Ondayofdeliveredofthedrugandirradiation;(B,C)14daysafterPDT.

basedontheinﬂammatoryconditionstartingfromthe epithelial-conective tissue border and proceeding gradually deeper into laminapropria.Forimmunohistochemistry,apoliclonalantibodie anti-VEGF-A(C1,dilution 1:600,SantaCruz Biotechnology)was usedinavidin–biotin–immunoperoxidase(LSAB—Labeled Strepta-vidinBiotin),techniqueaspreviouslydescribed intheliterature

[21].

2.6. Evaluationmethod

AnalysisofVEGFimmunostainingwasperformedaccordingto (i)thepresence and (ii)theintensity ofthestaining (1=weak, 2=moderate,3=strong), asperformedby Moriyamaetal. [22]. IntensityofVEGFstainingwasevaluatedaccordingtothecolorof diaminobenzidine(DAB)underlightmicroscopy.Whenthe inten-sityofstainingobservedwasidenticaltotheendothelialcellsof thespecimenwasassigned agrade 2. For themarking weaker orstronger than the endothelialcells in thesample was given ascoreof1or3,respectively.Slideshowwithout immunostain-ingwere marked with0 (no immunostaining). Immunostained slideswerescannedusingthePannoramic3DHISTECHMIDI equip-ment(3DHISTECHLtd.,Hungary).Thereafter,theywereanalyzed, quantiﬁedandphotographedwiththevirtualmicroscopysoftware PannoramicViewer,version1.15.2(©Copyright20133DHISTECH Ltd.,Hungary).

3. Results

3.1. ClinicalapplicationofPDT

DuringtheinfusionofAlClPcwasobservedthatthesensitizer iseasytoapplyandhandling.Thedrugwasappliedwithno com-plications,noclinicalsignsofdeteriorationofthemucosa;and2 in8patients(n=2,25%)complainedofpainscoredas1(slight).In 1patient,edemawasobservedatthetimeofdruginfusion(n=1, 12.5%).Duringexposuretolightsource,2patientscomplainedofa

mildburning.Fig.1illustratestheregionondayapplicationofPDT and14daysafterapplication.

3.2. AdverseeffectsofPDT

Therapywaswelltoleratedbymostpatients.Inallparticipants, 14 daysafterthePDTwasshown edema anderythema. It was observedalloftheseeventswereofshortdurationandcompletely reversible.Safetyassessmentsdemonstratedthatstinging/burning sensationafterAlClPcapplicationwasscoredas0(no)in6patients (75%)and1(slight)in2patients(25%)inday7.Therewasa sig-niﬁcantincreaseinerythemaafterlaserirradiationandextraction. After14days,erythemawasscoredas1in5patients(62,5%)and2 in3patients(37,5%).Nopurpura,crusting,orscarringoccurredin anypatient.Dysphagiaandallergicreactionswerenotobserved. 3.3. Histologicalﬁndings

Specimensofthetreatedteethshowedcollectionsof inflamma-torycells, predominantlymononuclear,and areasofedemaand vascular congestion(Fig.2A–C), markingan exudativereaction. NocorrelationswasfoundbetweentheVEGFimmunostainingand degreeofinflammationingroups(p>.05,datanotshown).Vascular modificationswereobservedinthegingivalsamplesthatreceived PDT.Intwocasesitcanbeobservedthepresenceofnumeroustiny bloodvesselsinthesubepithelialregion(Fig.2D),andhistological appearanceofnumerousvascularsprouts(Fig.2C)characteristicof theprocessofneoangiogenesis.Itisfurtherhighlightthepresence ofintenseextravasationofplasmaandinterstitialfluidprecipitated insidethebloodvessels.Inthreecaseswerevisualizedthe pres-enceofanamorphous,acellularmaterial,well-defined,basophilic (Fig.2E,andF)andinproximitytobloodvesselscompatiblewith dystrophiccalcificationprocess.Thesefindingswereinthe vicin-ityofareaswithintensehemorrhagicextravasationandvascular congestionandinsubepithelialregion.Inspecimensofthe con-trolgroupwasdetectedepithelialandconnectivetissuewithinthe normalpattern.

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Fig.2.Phtotomicrographyofclinicallyhealthygingivaintestgroup.(A)Thehighlightshowscollectionsofinflammatorycellsinfibrovasculartissue(PannoramicViewer,HE, 200␮m).(B)Detailofnumerouscongestedbloodvesselsinthesubepithelialregion(PannoramicViewer,HE,100␮m).(C)Myxomatousconnectivetissueexhibitingedema, mononuclearcellinfiltration,andnumerousbloodvesselswithsomevascularsprouts(PannoramicViewer,HE,100␮m).(D)Largenumberofbloodvesselsinsubepithelial regionproliferationcontributestochronicinflammation.(PannoramicViewer,H.E.,200␮m).(E)Basophilicdepositsinsub-epithelialregion,consistentwithdystrophic calcificationprocess(PannoramicViewer,HE,500␮m).Detailofcalcification.(F)Basophilicdepositsinsubepithelialregion,consistentwithdystrophiccalcificationprocess. Asteriskshighlightthevascularcalcifications(PannoramicViewer,HE,200␮m).

3.4. Immunohistochemicalﬁndings

VEGFshowedanuclearandcytoplasmicpatternof immunore-activity. Immunostaining was visualized in endothelial cells, epithelialcells,ﬁbroblasts andmononuclearinﬂammatory cells. Wasobserved a predominanceof nuclearstainingin basal and suprabasallayeroftheepithelium,whereasatconnectivetissue cytoplasmicstaining(Fig.3).Whenpresent,muscles and blood vesselsassociated,alsoshowedpositivestaining.Nostatistical dif-ferencewasobservedwhencompared VEGFintest andcontrol group.Somecasesoftestgroupshowedatendencytopresenta strongpatternofexpressionforVEGFcomparedtocontrolgroup. Nopositivestainingwasobservedwhenprimaryantibodieswere omitted.Positivecontrolsamplesshowedstrongreactivity.

4. Discussion

Aluminum-phthalocyanine chloride is a highly hydrophobic photosensitizerthatstronglyaggregateinaqueoussolutions, fea-ture that decreases their photodynamic effect, and impairs its usein aqueoussolutions alone.Onewellknowstrategyto pre-ventthisaggregationandkeepallthephotophysicalproprieties istheassociationofthephotosensitizerwithnano-carriers,such

asthenanoemulsionsusedinthisstudy.Thenanoemulsionused in thisstudywaspreviously developedshowedthat this nano-carrierkept,andsometimesimprovedtheAlClPcproprieties[23]

andseveralstudieshavedemonstratedeffectivepropertiesagainst tumors[11,13,14,24].Currentlyonlytwostudiesinhumans[8,18]

haveevaluatedtheeffectsofAlClPcinassociationwithlaser ther-apy.Nosideeffectswerereportedintheeightvolunteersincluded inthis studywhencomparedtootherPDTprotocols,especially thoseusingaminolevulinicacid(ALA)whichispainfulrequiring theuseoflocalanesthetics[25–27].Thelackofsideeffectscanbe anadvantageforusingAlClPc-PDT.However,therepeateduseofa nanoemulsionofAlClPcstillneedsfurtherinvestigationsincesingle doseshaveonlyshowedshorttermandreversibleadverseeffects (erythemaandedema).Erythemaandedema14daysafter ther-apycanbetheresultofthelocalinﬂammatoryresponsenormally associatedwiththesurgicalextractionprocedure.

Microscopicobservationsofprimaryvascularreactionsonthe specimenssubjectedtoPDT,includingvascularcongestion, throm-boseandedemawerealsoobservedbyotherauthors[13,28–30]. Althoughsomeauthors[11,13,24,28]haveshownareasofnecrosis intumorsaftertreatmentwithPDT,thisﬁndingwasnotobserved inthesamplestreatedwithPDT,theseresultsmaybeduetothe drug’shighafﬁnitytocancercells[11–13].Since,thisstudyutilized

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Fig.3.(A)PhotomicrographshowingmoderateimmunoreactivityforVEGFinbasalandsuprabasallayeroftheepitheliumandconnectivetissueofclinicallyhealthygingiva (controlgroup).(PannoramicViewer,LSAB,100␮m);(B)PhotomicrographshowingstrongstainingforVEGFinthebasallayerandsuprabasalepithelialandconnective tissueclinicallyhealthygingiva(testgroup).(PannoramicViewer,LSAB,200␮m);(C,D)PhotomicrographshowingmoderateimmunoreactivityofVEGFinendothelialand inﬂammatorycells(mononucleartype)ofclinicallyhealthygingiva(testgroup).(PannoramicViewer,LSAB,200␮m).

healthytissuesnotissueselectivitytothedrugoccurredwhich explainsthelackoftissuenecrosis.

Thehistologicalfindingofabasophilic,amorphousand acel-lularmaterialcompatiblewithdystrophiccalcificationwasnoted ontestgroupspecimens.Webelievethatoxidativestress,induced byPDT,mayhavecausedtheabnormaltissuecalcification.These calcificationsoftenoccurintissuesundergoingnecrosisor degen-eration[31].andshouldbecarefullyevaluatedsince,thedrugunder investigationhasanti-tumorproperties,amongstthemcelldeath bynecrosis[11,13,14,24].

Vascular calciﬁcation is a process of active mineralization whichoccurs inboth pathological andphysiological conditions. This process is cell-mediated and is a result of the imbalance betweenfactorsthat stimulate orinhibit mineralization.Under lightmicroscopy,samplesshowinitiallyacidophiliccharacteristics, followedbythepresenceofirregularbasophilicgranules (some-timesmistakenforbacteria)whichconvergeorgrowintolarger granules.Thesegranulesmay,sometimes,befragmentedduring histologicalprocessing,similartothatobservedinourspecimens. Dystrophicorlocalcalciﬁcationsdonotcausesignsorsymptoms, howevertheymayoccasionallyproduceedemaandtissue ulcer-ation[32–34].Thus,itcanbesuggestedthatPDTappliedtothe gingivapromotedinjurytothemucosa,reachingtheendothelial cellsinthesubepitheliallayer,whichmayexplainthealterations foundinourspecimens.

Oxidativestressisknownasoneofthemaincontributorstothe immuneresponseobservedinPDT[6].Releaseofpro-inﬂammatory cytokinesafterPDTactivatestranscriptionalfactorssuchasnuclear factorKappa-B[5],aswellasothergrowthfactorssuchasVEGF. Oxygenreactivespecies lead tooxidativestress,tissue hypoxia andactivationofhypoxiainduciblefactor(HIF-1),whichinturn inducesVEGFexpression[15,35,36].The dropinthecirculating levelsofoxygen,attheinitialstagesofPDT,actsasastimulusfor endothelialcellstoreleaseHIF-1whichinturninducesthe expres-sionofVEGF,apotentstimulatorofmitosisforendothelialcells

[15,35,36].Someauthors[15,16,35–39]haveshownupregulation ofVEGFafterPDT.Oursampleswereevaluated7daysafterPDTby

histologyandimmunohistochemistryandshowedanincreasein VEGFafterPDTwhichisinagreementwithotherstudies.However, thelengthoftimebetweentreatmentandspecimenharvestingwas notdisclosedinsomeofthesestudies.

StudiesindicatethatVEGFcanpromotecalciﬁcationeitherby neovascularizationorbydirectlyaffectingbonecells.Inbone for-mationand regeneration, angiogenesisandbone resorptionare twoessentialandintimatelyassociatedprocesses,whichsuggestsa commonmediatorforbothevents.Inthiscontext,VEGFappearsas apossiblestimulatorviasecretionofgrowthfactorsandcytokines byendothelialcells,whichcandirectmesenchymalcell differen-tiationtowardstheosteogenicpathwayandosteogenesis[36,40]. IncreasedVEGFlevelsintheareastreatedwithPDTmayfavora phenotypicchangeofendothelialcellsintoosteoblasticcells,thus promotingmineral depositionand calciﬁcation build-up. Previ-ousstudieshavesuggesteda possibleroleofoxidativestressin mediatingdifferentiationofvariouscellssuchasperipheralblood dendriticcells andosteoclasts[41,42].Modyet al.[33]showed thatendothelialcells,whenstimulatedbyoxidativeagents,suchas xanthineoxidase,producedalkalinephosphatase,andwiththese resultshavesuggestedthatoxidativestressfavorsosteoblastic dif-ferentiation.

Therelationshipbetweenthetherapeuticeffects of PDTand cytokineexpression,suchasVEGF,is stillcontroversial. Several PDTprotocolshavebeenstudied(differentphotosensitizingagents, concentrationsandlengthofapplication;lightsourceswith differ-entwavelengths,power,ﬂuencyandlengthofapplication)which makes it difﬁcult tocompare the results. Nevertheless, clinical studies have allowedfor a betterunderstanding of the role of tissuemicroenvironment,andhowthebodyrespondstothe treat-mentwithPDT.Thisstudyconsistedofclinicalobservationsonthe responseofgingivaltissuestotheapplicationoftheinvestigated drug,whichallowedforobservationsthatwouldnotbepossiblein aninvitrooranimalstudy.Whentakingintoconsiderationthe lim-itationsofthisstudy,theapplicationofthenanoemulsionofAlClPc inthecurrentprotocoldemonstratedtobesafeforuseinhumans. ItisalsosuggestedtoincreasetheexpressionofVEGFafterPDT.

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However,furtherclinicaltrialsarerequiredtovalidatetheuseof thisphotosensitizingagentandrecognizeitasatherapyofchoice withanti-tumor,angiogenicandosteogenicproperties.

Conﬂictofinterest

Noneoftheauthorshaveanyconﬂictsofinterestsregardingthis manuscripttodeclare.

Acknowledgments

We thank Hévio Freitas de Lucena for immunohistochem-istry expertise. This work has been supported by funds from theBrazilianagenciesMCT/CNPq,MCT/CAPES,FAP/DF,and INCT-Nanobiotechnology.

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