Pulp capping materials modulate the balance between inflammation and regeneration

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Thomas Giraud, Charlotte Jeanneau, Charlotte Rombouts, Hengameh

Bakhtiar, Patrick Laurent, Imad About

To cite this version:

Thomas Giraud, Charlotte Jeanneau, Charlotte Rombouts, Hengameh Bakhtiar, Patrick Laurent, et

al.. Pulp capping materials modulate the balance between inflammation and regeneration. Dental

Materials, Elsevier, 2019, 35 (1), pp.24-35. �10.1016/j.dental.2018.09.008�. �hal-02185271�

Thomas

Giraud

_,

_Charlotte

_Jeanneau

_,

_Charlotte

_Rombouts

_,

Hengameh

Bakhtiar

_,

_Patrick

_Laurent

_,

_Imad

_About

b_{APHM,HôpitalTimone,Serviced’Odontologie,Marseille,13005,France}

c_{DentalMaterialResearchCenter,TehranDentalBranch,IslamicAzadUniversity,Tehran,Iran}

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t

i

c

l

e

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f

o

Articlehistory:

Received20July2018 Receivedinrevisedform 14September2018

Accepted16September2018

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Theinterrelationsbetweeninflammationandregenerationareofparticularsignificance withinthedentalpulptissueinextensibleenvironment.Recentdatahavedemonstrated thepulpcapacitytorespondtoinsultsbyinitiatinganinflammatoryreactionanddentin pulpregeneration.Differentstudymodelshavebeendevelopedinvitroandinvivoto inves-tigatetheinitialstepsofpulpinflammationandregeneration.Theseincludeendothelial cellinteractionwithinflammatorycells,stemcellinteractionwithpulpfibroblasts, migra-tionchamberstostudycellrecruitmentandentirehumantoothculturemodel.Usingthese models,thepulphasbeenshowntopossessaninherentanti-inflammatorypotentialanda highregenerationcapacityinallteethandatallages.Thesamemodelswereusedto inves-tigatetheeffectsoftricalciumsilicate-basedpulpcappingmaterials,whichwerefound tomodulatethepulpanti-inflammatorypotentialandregenerationcapacity.Amongthese, resin-containingmaterialssuchasTheraCal®

shiftthepulpresponsetowardsthe inflamma-toryreactionwhilealteringtheregenerationprocess.Ontheopposite,resin-freematerials suchasBiodentineTM_{haveananti-inflammatorypotentialandinducethepulpregeneration}

capacity.Thisknowledgecontradictsthenewtendencyofdevelopingresin-basedcalcium silicatehybridmaterialsfordirectpulpcapping.Additionally,itwouldallowinvestigating themodulatoryeffectsofnewlyreleasedpulpcappingmaterialsonthebalancebetween tis-sueinflammationandregeneration.Itwouldalsosetthebasisfordevelopingfuturecapping materialstargetingtheseprocesses.

©2018TheAcademyofDentalMaterials.PublishedbyElsevierInc.Allrightsreserved.

∗ _{Correspondingauthorat:InstitutdesSciencesduMouvement(ISM),UMR7287CNRS&Universitéd’Aix-Marseille,Facultéd’Odontologie,}

27BdJeanMoulin,MARSEILLECedex513385,France.

E-mail addresses: thomas.giraud@univ-amu.fr (T. Giraud), charlotte.jeanneau@univ-amu.fr (C. Jeanneau),

romboutscharlotte@gmail.com(C.Rombouts),hengamehbakhtiar@yahoo.com(H.Bakhtiar),patrick.laurent@univ-amu.fr(P.Laurent),

imad.about@univ-amu.fr(I.About).

https://doi.org/10.1016/j.dental.2018.09.008

Contents

1. Introduction...25

2. Tricalciumsilicatesasdirectpulpcappingmaterials...26

3. Calciumsilicatecementsbyproductspromotemineralization...28

4. Impactofdirectpulpcappingontheinitialstepsofinflammation...28

5. Pulpcappingmaterialsmodulatethepulpregenerationpotential...29

6. Pulpcappingmaterialsoutcomeinvivo...32

7. Conclusions...33 8. Perspectives...33 ConflictofInterest ... 33 Acknowledgments...33 References...33

1.

Introduction

Thedentalpulptissueislocatedwithinrigiddentinalwalls

[1].Thisuniquelocationinaterminalbloodcirculation ren-dersthistissuevulnerableunlessalocalregulationprovides protectivemechanismstothisparticulartissue.Inthisregard, severallinesofevidencesuggestthatthedentalpulphaslocal regulationmechanismsofitsinflammationandregenerative capacity.Inafirst-linedefence,odontoblaststhatlieunderthe dentinbarrierandpulpfibroblastsexpresspattern recogni-tionreceptors(PRRs).TheseincludeToll-likereceptors(TLRs)

[2,3]whichareabletodetectbacterialinvadersbyrecognizing commonmoleculesontheirsurface,thepathogen-associated molecularpatterns(PAMPs).Afterthisrecognition,thesecells initiateaninflammatorycascadebyactivatingtheNF-kB path-way, essential for the inflammatory response by initiating theproductionofpro-inflammatorycytokines[4].Thesewill establishachemotacticgradientforguidinginflammatorycell migrationtowardstheinflammationsite(Fig.1).Duringthis process,inflammatorycellsadhereonthe activated vascu-larendothelium,then migrate throughthe endothelialcell layer and reach the inflammatory site guided by the pro-inflammatorycytokines[5].Thentheywillbeactivatedinto macrophage-likecells atthe inflammatorysitewhere they eliminatepathogensandcelldebris(Fig.1).

Besides cytokines, the complement system is another importantactorof theinflammatory process.Complement isactivatedbytheclassical,alternative,ormannose-binding lectinpathway[6].Uponpulptissuedamageand/orinfection, the Complement provides the signals required for elimi-nating invading pathogens and altered host cells. Recent data have shown that pulp fibroblasts are the first non-immunecellscapableofproducingallcomponentsrequired forComplementactivation[7].Complementactivationbypulp fibroblastsleadstotheproductionofinflammatorymediators andrecruitmentofinflammatorycellsbyanaphylatoxinssuch asC5aandC3a[8–10].Theseanaphylatoxinsinducethe vas-cularmodificationsrequiredtoallowinflammatorycells to migratetowardstheinflammationsiteinordertoeliminate theinfectiousagents[11].Additionally,Complement activa-tionbypulpfibroblastsleadstotheformationofthecytolytic membraneattackcomplex(MAC)[12].Afterfixationon cario-genicbacteria,thiscomplexleadstotheirdirectdestruction

[13].Thus,Complementactivationappearstobeessentialin

initiatingtheinflammatoryreactionandincontrolling cario-genicbacteria.

Althoughinflammationisaprerequisiteforhealingand regeneration[1]itcanalsobedetrimentalifitpersistsgiven thefactthatthepulpisconfinedinarigidenvironment, leav-ingnoroomforswelling[2].Incaseofsevereinflammation, thismayleadtopulpdestruction.Additionally,ifthe infec-tionpersists,theresultingchronicinflammationwillhamper the regenerativeprocessesandwilleventuallyleadtopulp necrosis.

Besides controlling bacterialprogression and inflamma-tion, it is well established that the dental pulp of both primary and permanent teeth, and at all ages, is rich in stemcells[14–16].Incaseoftraumaticinjuriesand/orpulp infection,regenerationsignals,suchasgrowthfactors,induce theirproliferation,migrationanddifferentiationtoregenerate the dentin-pulp tissue [14] (Fig. 2). Indeed, after stimula-tion, these dental pulp stem cells (DPSC) migrate to the injury/inflammatorysiteanddifferentiateintoodontoblast-like

cells.Upondifferentiation, theyexpressspecificmarkersof odontoblastssuchasthe intermediatefilament Nestinand DentinSialoProtein(DSP)whichisknownforitsimplication inthemineralizationprocess.Indeed,cellsexpressingNestin andDSPwereseenincontactandwithinmineralizedfociin thedentalpulpclosetotheinjurysite[17,18].

DPSCrecruitmenttotheinjury/inflammationsiterequires the presence ofactivemolecules to directtheir migration. RecentworksonComplementactivationhavedemonstrated thatpulpfibroblastComplementactivation isalsoinvolved indental-pulpregenerationbyprovidingComplementactive fragmentssuchasC5aandC3a.Indeed,ithasbeenshown thatDPSCrecruitment,isselectivelyguidedbyaC5agradient

[19].Anotherfragment,C3a,alsopromotestheregenerative processes byincreasingDPSCand pulpfibroblast prolifera-tion,mobilizingDPSCandguidingpulpfibroblastmigration to theComplement activation site [20]. Thisindicates that in addition to its role in initiatingthe inflammatory reac-tion,Complementactivationbypulpfibroblastsproducethe regenerationsignalsrequiredfortheregenerationprocess par-ticularlyinguidingDPSCmigrationtotheinjurysite.Thus, Complement activation provides the missing linkbetween inflammationandregeneration[21].

Fig.1–Schematicrepresentationoftheinitialstepsofpulpinflammation.

Followingacariouslesion,theinflammatoryreactionimpliessecretionofpro-inflammatorycytokines(1)byresidentcells, suchaspulpfibroblasts.Circulatinginflammatorycellsadhereontheactivatedvascularendothelium(2),thenmigrateand reachtheinjuredsite(3)tobefinallyactivatedasmacrophage-likecells(4).

2.

Tricalcium

silicates

as

direct

pulp

capping

materials

Calcium-silicatebased cements(CSC) havebeen developed more than 20years ago with Mineral Trioxide Aggregate (MTA®)being the mostwell-known and most widely used formulation [22]. It is a Portland Cement (PC) based for-mulationcontainingmainlytricalcium(C3S) and dicalcium silicates (C2S) [23] which sets and develops its properties in the presence ofmoisture. CSC were initially developed asendodontic repair and root-endfilling materials[22–24]. Given their biocompatible properties, their clinical usage rapidlyexpandedtowardsdirectand indirectpulpcapping.

Nevertheless,therearesomedisadvantagesassociatedwith theseclassicMTAformulationsincludingalongsettingtime, difficulthandling,poormechanicalpropertiesandtooth dis-coloration [24–26]. Researchers have thus been working to improve CSC’sphysico–mechanicaland handlingproperties andmanynewproductshavebeenintroducedonthemarket, eachhavingtheirspecificsetofcomponents(setting modu-lators,radiopacifyingagentsanddrugs).However,besidesthe requiredphysico-mechanicalproperties,pulp-capping mate-rials should have suitable biological properties given their directcontactwithvitalpulptissue.

Takingintoconsiderationthehandlingandbiological prop-erties,twoCSCmaterialshavebeendevelopedwithenhanced mechanical properties: 1.) BiodentineTM _(Septodont,

Fig.2–Schematicrepresentationoftheinitialstepsofdentin-pulpregeneration.

Followingacariouslesion,dentalpulpcellssuchasfibroblastssecretegrowthfactors(1).Thesegrowthfactorscreatea gradientleadingtoperivascularstemcellproliferation(2)andtheirmigrationtotheinjuredsite(3).Finally,migratingstem cellsdifferentiateintoodontoblasts-likecellsandsecretemineralizedmatrixtoprotectthepulptissue(4).

Saint-Maur-des-Fossés, France) is resin-free and mainly composedofpuretricalciumsilicatesandcalciumchlorideas asettingaccelerator.Itispresentedaspowderandliquidtobe preparedbymixingbothcomponentswithanamalgamator. It sets in 12min which is much shorter than MTA® that setsafter2h45min[27].2.)TheraCal® (Bisco,Schaumburg, IL,USA), iscomposed ofPC and contains 43%of resins.It is presented as a ready-to-use material in a syringe and sets by photopolymerization (20s per 1mm increment) in a hydrophobic environment. The complete composition of thesematerialshasbeenreported[28].

Previously published works have already reported that resin-based materials cannot be recommended for direct

pulpcapping[29].However,therecentdevelopmentof resin-containing hybrid materials for direct and indirect pulp capping, stating their improved mechanical and handling properties,raisesquestionsabouttheirconsequencestothe pulphealingpotential.Forinstance,thebyproductformation fromcalciumsilicateinthesehybridmaterialsonsettingis differentfromthoseobservedinresin-freeCSCmaterials.In addition,thedevelopmentofthesematerialsrepresentsarisk tothe pulpvitality duetothe resin componentsand their potentialtoxicity.

In this review we willfocus on the effect ofthese two recently developed CSC, the resin-free BiodentineTM _and

Fig.3–Silicate-basedmaterialhydrationbyproductsonsettingandtheirbiologicaleffects.

Schematicrepresentationofthesettinghydrationreactionofsilicate-based(C2S:dicalciumsilicate,C3S:tricalciumsilicates) materials.Thisreactionleadstobyproductsformation:OH−,Ca2+_andSi4+_{.ThereleasedhydroxylionsincreasethepHin}

theunderlyingtissueleadingtoanti-microbialeffect.Calciumionsareinvolvedindentin-bridgeformationasthey stimulateDPSCdifferentiation.Siliconionsalsopromotemineralization.Calciumhydroxideinducesdentinbridge formation.

crucial biological processes which determine the suc-cess/failure of the clinical outcome: inflammation and regeneration.

3.

Calcium

silicate

cements

byproducts

promote

mineralization

Calciumsilicatesettingreactionishydration.Duringthis reac-tion,hydrationbyproductscanform/bereleased(Fig.3).Most CSC lead to calcium hydroxide formation, and leaching of hydroxylionsandcalciumionsasdemonstratedforMTA®and BiodentineTM_{,amongstothers[30–32].Thereleasedhydroxyl}

ionsuponhydrationwillincreasethepHintheunderlying tis-sueleadingtoathinnecroticlayerbetweentheremainingvital tissueandthepulpcappingagent[33,34].Thepresenceofthis necroticzoneprotectstheunderlyingvitalpulpcellsfromthe material’salkalinepH.Furthermore,itallowstheunderlying pulpcells tocarry out the healingand regeneration func-tions[35].ThealkalinepHalsoensuresanti-microbialactivity

[27].Subsequentcalcificationofthissuperficialnecroticlayer followed bytertiary dentinformation from stimulated and differentiateddentalpulpstemcellsgiverisetoaprotective dentin-bridge[36].Calciumionscontributetothisprotective dentin-bridgeformationastheystimulateDPSC differentia-tionandincreasetheformationofmineralizedmatrixnodules

[37,38]. Interestingly,TheraCal® has been shown torelease lesscalciumionscomparedtoBiodentineTM_andnocalcium

hydroxideformationwasseenwhenstudiedbyX-ray diffrac-tion analysis. Thismay bedue to the lackof moisture to allowproperhydrationofthetricalciumsilicateelementsin TheraCal®,whichexplainstheabsenceofcalciumhydroxide formation[32].

Besides the release of these ions involved in dentin-bridgeformation,a“bioactive”surfaceisformedduetothe nucleation of calcium phosphates and subsequent apatite formation, in a moist environment. This apatite layer is suggestedtostimulatecelldifferentiation,tissuerepair,

osteo-genesis and cementogenesis [22]. Silicon ions are another elementthatmayplayaroleindentin-bridgeformation.Their releasehasbeenknowntostimulateyoungboneformation by stimulatingosteoblasts [39]. In caseof directpulp cap-ping,it isbelieved thatthepresenceofsiliconionsinCSC, such as BiodentineTM_{, also promote mineralization. An ex}

vivotoothculturemodelshowedthatafterpulpcappingwith BiodentineTM_{,smallCSCparticleswereentrappedinthe}

min-eralized noduleswhich suggests that the materialitself is involvedinodontoblasticdifferentiationandmineralization

[17,18].

4.

Impact

of

direct

pulp

capping

on

the

initial

steps

of

inflammation

Upon cariousand/orphysicalinjury ofthe dental-pulp,an inflammatoryreactionisinitiatedintheremaininghealthy pulp tissue [40]. While mild or moderate inflammation is required to stimulate the regenerative process, severe and/orchronicinflammationwillbedetrimentaltothepulp. Significant advances in investigating the initial steps of inflammationusingdifferentcellcultureandco-culture mod-elsinvitroclearlyestablishedthatpulpfibroblastsplayamajor roleintheinitialstepsofinflammatoryprocess.Secreted pro-inflammatorycytokinessuchasVascularEndothelialGrowth Factor (VEGF), Interleukine 6 (IL-6) and Complement frag-ments such asC3a and C5ainduce vascular modifications to initiate inflammatorycell recruitment to the inflamma-tionsite[41–44].Theseincludeadhesionofinflammatorycells toactivated vascularendothelium, theirmigrationthrough theendothelialcelllayerandsubsequentrecruitmenttothe inflammationsitewheretheyareactivated(Fig.1).

In order to evaluate in vitro inflammatory mediators secreted indamagedandinflamed pulptissue,pulp fibrob-lastshavebeeninjuredand/orstimulatedwithGrampositive lipoteichoicacid(LTA)and/orGramnegative lipopolysaccha-ride (LPS). Quantification ofthe pro-inflammatory released

Fig.4–Pulpcappingmaterialeffectsontheinitialstepsof inflammationinvitro.

(A)Effectsofmaterialsoncytokinesecretion.IL-6andVEGF secretionbypulpfibroblastssignificantlyincreasedwith bothBiodentineTM_andTheraCal®

conditionedmediaafter 48hbuttoalesserextentwithBiodentineTM_ascompared

tothecontrolasdescribed[46].(B)Effectsofmaterialson inflammatorycell(THP-1)recruitmentsequence.

BiodentineTM_{significantlydecreasesTHP-1adhesionto}

endothelialcells,migrationandactivation[46].(*) correspondstosignificantdifferenceascomparedtothe control,(**)representssignificantdifferencesbetweenthe twobiomaterials(p-value<0.05).

mediatorsshowedthat IL-6, IL-8, IL1␣ andTumor Necrosis Factor-␣(TNF-␣)secretionincreasedinLPS-stimulatedhuman DPSC[45].ApplicationofCSChasbeenshowntomodulate pro-inflammatorymediatorsecretion.Forinstance, applica-tionofCSCextracts(TheraCal®andBiodentineTM_)oninjured

and LTA-stimulatedpulp fibroblasts showed increasedIL-6 andVEGFsecretion(Fig.4A),whichwassignificantlyhigher forTheraCal®[46].IL-8secretionbypulpfibroblastswasalso significantlyhigherwithTheraCal®comparedtoBiodentineTM [18].ThismodulationofIL-8secretionbypulpcapping materi-alsisofinterestasIL-8isapotentchemokineandplaysarole incontrollingthedurationoftheinflammatoryprocess.MTA® hasalsobeenshowntoincreaseIL-8secretionbyhuman neu-trophils,thefirstinflammatorycellstoreachthedamagedsite

[47].

or BiodentineTM _{demonstrated thatpulpcappingmaterials}

modulate the inflammatory response. Inflammatory THP-1 cells adhesion to endothelial cells and their activation werereducedbyBiodentineTM_andTheraCal®

.However,their migrationdecreasedonlywithBiodentineTM[46](Fig.4B).

Given that severe pulp inflammation is detrimental to clinical outcome, pulp capping materials that reduce the inflammatoryprocessareofparticularinterest.Several stud-ies haveshown favourable clinicaloutcome withmaterials suchasMTA®andBiodentineTM_{,whichismostlikelyrelated}

toanattenuatedinflammatoryresponse.Forinstance,Kang etal.demonstratedthat,afterdirectpulpcappingfor8weeks, acalcifieddentinbarrierwasformedwithProRoot®MTAand Ortho® MTAwhereas dentin barrierformationwas incom-plete with Endocem® MTA. Thelatterwas associatedwith aninflammatoryreaction[48]. Anotherstudy alsosuggests thatmildinflammationisassociatedwiththickerandmore continuousdentinebridgeformation.Thiswasobservedafter 45daysforBiodentineTM_{asopposedtoDycal}®

[49].

Variousinvitrostudieshaveprovidedinsightinthe under-lyingprocesses bywhichparticularpulpcappingmaterials shift thebalance from inflammationtowards regeneration. Forinstance,TNF-␣,animportantpro-inflammatorycytokine releasedduringpulpinflammation,hasbeenshowntoinduce Transient Receptor PotentialAnkyrin 1 (TRPA1)expression, which plays a role in nociception and neurogenic inflam-mation.Interestingly,BiodentineTM_{isabletoattenuatethis}

TNF-␣-inducedTRPA1expressionandtoreduceitsfunctional activity[50].

5.

Pulp

capping

materials

modulate

the

pulp

regeneration

potential

Inagreementwiththe aboveinflammation-reducingeffect, BiodentineTM _{isabletopromoteregenerativeprocesses.For}

instance,BiodentineTM_{increasesTransforminggrowthFactor}

␤1 (TGF-␤1) and Fibroblast Growth Factor 2 (FGF-2) secre-tion by injured pulp fibroblasts, as opposed to TheraCal® (Fig.5A)[17].TGF␤-1growthfactorhasbeenshownto stimu-lateodontoblasticdifferentiation[51]contributingassuchto dentin-bridgeformation.Recently,theinterestofthisgrowth factorrelease hasbeeninvestigatedbyencapsulatingthese growth factors in PLGA microspheres allowing their grad-ualrelease.Thisinvivostudydemonstratedthatthegradual releaseofFGF-2inducesfibroblastandstemcellproliferation whileTGF-␤1guidesDPSCrecruitment[52],odontoblastic dif-ferentiationandtertiarydentinformation[53].

Whenthepulpinjurywassimulatedinthescratchassay, an experimentalmodel which simulates a physical tissue injury,pulpfibroblastmigrationtocolonizetheinjurysitewas significantlyhigherwithBiodentineTM _{thanwithTheraCal}®

[46](Fig.5B).InvestigatingDPSCmigrationinBoyden cham-ber also demonstrated a significantly higher migration of thesecellsinthepresenceofBiodentineTM _ascomparedto

TheraCal®[54](Fig.5B).

Thesedataclearlyshowthatpulpcappingmaterialsalso modulatetheinitialstepsofpulpregeneration[46].Moreover, whilecellviabilitywasmaintainedwithBiodentineTM_,a

sig-Fig.5–Pulpcappingmaterialeffectsontheinitialstepsofpulpregenerationinvitro.

(A)Effectsofmaterialsongrowthfactorsecretion.PulpfibroblastssecretedsignificantlymoreTGF-␤1andFGF2after24hof incubationwithBiodentineTM_{thanwithTheraCal}®

asdescribed[46].(B)Effectsofmaterialsonstemcellmigrationand fibroblastcolonization.(a)Representativepicturesand(b)quantificationofpulpfibroblastsscratchwoundhealingassayand DPSCsBoydenchambermigrationassaywithBiodentineTM_andTheraCal®

asdescribed[46,54].BiodentineTM_{significantly}

inducedfibroblastcolonizationwhileTheraCal®significantlydecreasedDPSCsmigration.(*)correspondstosignificant differenceascomparedtothecontrol,(**)representssignificantdifferencesbetweenthetwobiomaterials(p-value<0.05). Scalebars:200␮mforscratchwoundhealingassaysand50␮mforBoydenchamberassays.

nificantdecreaseinpulpcellproliferationwasreportedwith TheraCal®(Fig.6A).Investigatingthepulpcelldifferentiation potentialwiththematerialsdemonstratedahigher expres-sionofodontoblasticmarkerssuchasNestinandDSPwith BiodentineTM _{than withTheraCal}®

(Fig. 6B). When applied

as direct pulp capping material in entire human tooth cultures,asignificantnumber ofmineralizedfoci wasseen underBiodentineTM_{inanintactpulptissuewhilea}

disorga-nizedpulptissuewasobservedunderTheraCal® withsmall andadispersedmineralization(Fig.6C)[18].Thisresultwas

Fig.6–Effectsofsilicate-basedpulpcappingmaterialsonpulpcellproliferationandpulpmineralizationinvitroandinvivo. (A)EffectofBiodentineTM_andTheraCal®

onhumanpulpfibroblastproliferation[18].Asignificantdecreaseinpulp fibroblastproliferationwasobservedwithTheraCal®conditionedmediaforallincubationperiods.(*)correspondsto significantdifferenceascomparedtothecontrolmedium(p-value<0.05).(B)EffectofthecappingbiomaterialsonDSPand NestinexpressionbyDPSCsasdescribed[18].Anincreaseofbothmarkerswasobservedbyimmunofluorescencewhen DPSCswereculturedincontactwithBiodentineTM.Scalebars:50␮m.(C)Histologyresultsafterdirectpulpcappinginvitro usingthehumantoothculturemodel(15days)asdescribed[18]andinvivoaftertoothextraction(8weeks)asdescribed[55]

withBiodentineTM_andTheraCal®

.EntiretoothculturehistologyshowedahighermineralizationwithBiodentineTM_while

significanttissuedisorganizationwasobservedwithTheraCal®.Afterpartialpulpotomyinhumanteethacompletedentin bridgeformedwithBiodentineTM_{whileonlydispersedmineralizationsinadisorganizedpulptissuewereobservedwith}

Fig.7–Biologicaleffectsofsilicate-basedmaterialsonthedentalpulp.

confirmed on partialpulpotomies in humanthird molars. Indeed,whileacompletebridgeunderBiodentineTM_formed

inanintactpulpafter8weeks,onlyadispersedmineralization inadisorganizedpulpwasobservedunderTheraCal®(Fig.6C)

[55].

6.

Pulp

capping

materials

outcome

in

vivo

Clinical successofpulpcappingproceduresinvolves many aspectsand isdifficultto mimicin vitro. In vivoand clini-calstudies are thus an essential researchpart to evaluate not only dentin-pulp regeneration, but also inflammation and overall pulp response. CSC such as BiodentineTM _and

MTA®showedgoodclinicaloutcomeswithhighsuccessrates whereasTheraCal®waslesssuccessful.Forinstance,aclinical trialonpulpotomyinprimaryteethshowedahighrateof clin-icalandradiographicsuccessforMTA®andBiodentineTMafter 12months(92%and97%,respectively)[56].Anotherstudyin permanentteethalsoshowedfavourableoutcomesforpulp cappingwithBiodentineTM_andMTA®

.Theirhistological anal-ysisshowedthatthemajorityofteethhadcompletedentinal bridgeformationandnoinflammationafter6weekswithboth BiodentineTM_andMTA®

[57].Asimilarstudycomparedpulp cappingwithcalciumhydroxide,MTA®,BiodentineTM_and

Sin-gleBondUniversalinhumanteeth.After6weeks,thedentin bridges formedin BiodentineTM _{group showed the highest}

averagemineralizationandmaximumvolumeswhileSingle BondUniversalthelowest[58].Indogpartialpulpotomy,itwas observedthatTheraCal®cappingledtoextensive inflamma-tionandincompletecalcifiedbarrierformation.Ontheother hand,completedentin-bridgeformationwithno inflamma-tion was observed with ProRoot® MTA [59]. A clinical trial

in adults showed complete dentin-bridge formation in all BiodentineTM_{caseswhereasthisratewasonly11%and56%in}

TheraCal®andProRoot®MTAgroups.IntheTheraCal®group, twopatientshadseverepainanddiscomfortafteroneweek

[55].ThiscanbeexplainedbytheobservedTheraCal® toxi-cityininvitrostudies[18,60].Indeed,leachingofmonomers duetoincompletehydrationofTheraCal®leadstotoxicityof theunderlyingpulpcellsandinduceassuchaninflammatory response. Thisisparticularly disadvantageousinthe tooth confinedenvironmentwheresevereinflammationwillleadto painandsubsequentclinicalfailure.Ithasalsobeenshown thatnontoxicconcentrationsofthesemonomersinhibitthe secretionofdentinsialoproteinsandosteonectin,whichare involvedinthemineralizationprocess[61].

Itisworthnotingthatrecentdatahavedemonstratedthat usingbioactivematerialssuchasBiodentineTM_andMTA®

in partialorfullpulpotomytotreatirreversiblepulpitisleadsto pulpfunctionrestorationanddentinbridgeformation.This hasbeenreportednotonlyinimmaturebut alsoinmature teeth[62–65].Theseresults,whichrepresentaparadigmshift inirreversiblepulpitistreatment,appeartobeduetomultiple factors:

1) Thelocalregulationofpulpinflammationandregeneration

[66]

2) Thepresence ofstem cells and the inherenthigh pulp regenerationcapacity[51,67]

3) Theanti-inflammatoryactivityofbioactivematerialssuch asBiodentineTM_[46,50]

4) Thematerialbyproductsonsettingwhichinducestemcell differentiationanddentinbridgeformation[28]

subsequentrelease offactors suchasFGF-2 andTGF-␤1 involvedinpulptissueregeneration[52]

7.

Conclusions

Overall,eveniftheinitialinflammationisapre-requisitefor healing,arapidresolutionofinflammationwouldfavourthe regenerativeprocesswhichiskeyforasuccessfulclinical out-come [68]. Choosingan appropriate pulpcapping material iscrucialgiventhat theycanmodulatethecourseofthese events.ItappearsclearlythatthepresenceofresinsinCSC pulpcappingmaterialssuchasTheraCal®shiftsthebalance towards inflammation. Their incomplete photopolymeriza-tionleadstofreemonomersrelease.Whenthesemonomers reachtheunderlyingpulp,theyexerttheirtoxicityas demon-stratedbydecreasedcellviability,releaseofpro-inflammatory cytokinesand recruitment ofinflammatory cells.Itcan be assumedthatthiscreatesaninflammatorystatewhich com-promisestheregenerativeprocess.

Inaddition,theincompleteTheraCal®hydrationduetothe presenceofahighpercentageofresin,leadstoareduced Cal-ciumionreleaseandtheabsenceofCa(OH)2,bothofwhich

are known for their positive impact on the mineralization process (Fig. 7). Thus,pulpcappingwithTheraCal® can be heldresponsibleforadisorganizedpulptissuewithoutdentin bridgeformation[18,55].Basedonthesescientificfindings,it canbeconcludedthatevencombinedwithCalciumsilicates, resin-containingmaterialsarenotcompatiblewiththespirit ofdirectpulpcapping.

On the opposite, CSC pulp capping materials such as BiodentineTM _{and MTA}®

shift the balance towards regen-eration. Indeed, recent investigations demonstrated that BiodentineTM_{hasananti-inflammatoryactivitybycontrolling}

pro-inflammatoryfactorssecretionanddecreasing inflamma-torycells recruitment [46]. Atthe same time, the material hydration is complete leading to the formation/release of byproductswhichshiftsthepulpresponsetowards regenera-tionasdemonstratedthroughincreasedexpressionoffactors involvedintheregenerationprocesssuchasFGF-2and TGF-␤1,andtheinductionofdentinbridgeformationwhilekeeping anintactpulp(Fig.7).

8.

Perspectives

Currentknowledgeofpulpanti-inflammatoryand regenera-tionpotentialwouldpavethewayfordevelopmentoffuture therapeuticagentsthatcantargetnotonlytheregeneration but atthe same timethe pulpinflammation.Indeed, both inflammationandregenerationarerequiredforasuccessful clinicaloutcomewithinthepulpinextensibleenvironment. Thefactthattissuelysisanddestructionmayresultfroma severeinflammationsuggeststhat,beyondthepulp,thiswill alsosetthebasisfordevelopmentofbioactivematerialsinthe treatmentofothertissueslocatedinaterminalcirculation.

Conflict

of

Interest

Pr.Imad ABOUTreportsfinancialsupportofresearchfrom SeptodontduringthedevelopmentofBiodentineTM.

Acknowledgments

Theoriginalworksreportedinthispaperweresupportedby Aix-MarseilleUniversityandCNRS.

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