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j ou rn a l h o m epa ge :w w w . i n t l . e l s e v i e r h e a l t h . c o m / j o u r n a l s / d e m a
Effect
of
an
ethanol
cross-linker
on
universal
adhesive
Allegra
Comba
a,
Tatjana
Maravi´c
a,
Virginia
Villalta
a,
Serena
Tozzola
a,
Claudia
Mazzitelli
a,
Vittorio
Checchi
b,
Edoardo
Mancuso
a,
Nicola
Scotti
c,
Franklin
R.
Tay
d,
Lorenzo
Breschi
a,∗,
Annalisa
Mazzoni
aaDepartmentofBiomedicalandNeuromotorSciences,DIBINEM,UniversityofBologna,AlmaMaterStudiorum,Via
SanVitale59,40125Bologna,Italy
bDepartmentofSurgery,Medicine,DentistryandMorphologicalSciencesWithTransplantSurgery,Oncologyand
RegenerativeMedicineRelevanceUnitofDentistryandOral-Maxillo-FacialSurgery,UniversityofModena&Reggio
Emilia,Modena,Italy
cDepartmentofSurgicalSciences,UniversityofTurin,Turin,Italy
dDepartmentofEndodontics,TheDentalCollegeofGeorgia,AugustaUniversity,1430JohnWesleyGilbertDrive,
Augusta,GA30912,USA
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Accepted6October2020 Keywords: Cross-LinkersDentinbondingsystems Matrixmetalloproteinases N,N’-dicyclohexylcarbodiimide Universaladhesives
a
b
s
t
r
a
c
t
Objective.ToevaluatetheeffectsofN,N’-dicyclohexylcarbodiimide(DCC),anethanol-based
dentincross-linker,ontheimmediateandlong-termmicrotensilebondstrength(TBS)and nanoleakageexpressionofauniversaladhesiveemployedinself-etchmode(SE)or etch-and-rinsemode(ER).TheeffectofDCConthedentinalMMPactivitywasalsoinvestigated bymeansofin-situzymography.
Methods.Eighty freshlyextractedhuman molarsweresectionedto exposemid-coronal
dentinsurfaces.Theteethwereassignedtooneofthefollowinggroups,accordingtothe dentinsurfacepriming/adhesiveapproach:(G1):DCCpre-treatmentandScotchbond Uni-versal(SBU)inERmode;(G2):SBUinERmode;(G3):DCCpretreatmentandSBUinSEmode; (G4):SBUinSEmode.TBStestwasperformedimmediately(T0)orafter1-yearaging(T12)
inartificialsaliva.Tenadditionalteethpergroupwerepreparedfornanoleakageevaluation
(N=5)andforin-situzymography(N=5).
Results.Three-factoranalysisofvariancerevealedsignificantdifferenceforthevariables
DCCpretreatment,applicationmodeandaging(p<0.05)forbothmicrotensilebondstrength testingandin-situzymography.Nanoleakageanalysisrevealedreducedmarginalinfiltration ofDCCexperimentalgroupsbothatT0andT12.
Significance.Theuseofanethanol-basedprimercontainingDCCappearstobepromisingin
preservingthestabilityoftheadhesiveinterfaceofauniversaladhesive,especiallyinthe SEmode.
©2020TheAcademyofDentalMaterials.PublishedbyElsevierInc.Allrightsreserved.
∗ Correspondingauthor.
E-mailaddress:lorenzo.breschi@unibo.it(L.Breschi). https://doi.org/10.1016/j.dental.2020.10.004
1.
Introduction
Adhesion between resin composite restorations and den-tal substrate is achieved through the infiltration of resin monomers into the demineralized dentin collagen matrix afterpartialdissolutionofthe mineralinorganic phase[1]. Theneedforaretentivecavitybecamelesscriticalafterthe adventofadhesive dentistry, withdentinbondingsystems provide reasonable immediate bond strength to the den-talsubstrate[2,3].Despitenearlysixdecadesofrefinement ofadhesivematerialsandprotocols,theinterdiffusionarea betweendentinandtheadhesiveresin,knownasthehybrid layer, stillremains the weakest region ofadhesively-based restorations[4–8].
The potential reasons behind the degradation of resin-dentinbond over time had been examined byin vitro and
invivostudies.Hydrolyticorenzymaticbreakdownofthe poly-merizedresincompoundsandendogenousprotease-initiated degradationofthedemineralizeddentincollagenmatrixhave emergedasthemostlikelycontributorsofinterfacial degra-dation[9–12].
Thedentinsubstratecontainscollagenfibrilswithbound non-collagenousproteinssuchasgrowthfactorsand endoge-nous proteases such as matrix-metalloproteinases (MMPs) and cysteine-cathepsin [13–16]. Endogenous proteases play an important role during dentin maturation and become trappedandinactivatedaftermineralizationofthecollagen matrix[17].Theseenzymesareexposedandreactivated dur-ingdemineralizationofthemineralizeddentin,progressively degradingthecollagenfibrilsthatarenotprotectedby adhe-siveresinwithinthehybridlayer,eventuallyresultinginthe lossofretention ofthe adhesive restorations[4,18–20]. For this reason, muchefforts have been devoted to increasing theresistanceoftheresin-sparse,water-richcollagenfibrils withinthehybridlayeragainstMMPs,withtheintentionof increasingthelongevityofadhesiverestorations[21].
Matrixmetalloproteinases,particularlyMMP-2and MMP-9,aremostlyresponsibleforthedegradationofthedentinal organicmatrix[22].Forthisreason,mucheffortshavebeen devotedtoincreasingtheresistanceoftheresin-sparse, water-richcollagenfibrilswithinthehybridlayeragainstMMPs,with theintensionofincreasingthelongevityofadhesive restora-tions[4,11,12,23–27].
DifferentMMPinhibitorssuchasquaternaryammonium methacrylates[17,28],andbenzalkonium chloride[12]have been used experimentallyto increasethe durability ofthe resin-dentininterface[29]. Collagencross-linkers havealso been used to enhance the mechanical properties of the demineralized collagen network as well as bond durabil-ity incoronal [11,30–33]and radicular dentin[34]. Because cross-linkingofthecollagenmatrix isanaturally-occurring phenomenonindentin,scientistshaveresortedtothe use ofchemicalsubstanceswithcross-linkingpropertiesto ren-derthedentincollagenmatrixlesssusceptibletoproteolytic attack[4,35].
Among the cross-linking reagents, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC),hasdemonstrated promisingresultsinthepreservationoftheintegrityofhybrid layersover time. Thisfeature was attributedto the ability
ofEDCtocross-linkpeptideswithoutintroducingadditional linkage groups [11,32,36]. N,N’-dicyclohexylcarbodiimide (DCC)isacrosslinkerbelongingtothesamefamilyasEDC, but with a different solubility behavior. Whereas EDC is solubleinwater,DCC issolubleinorganicsolventssuchas ethanoloracetone[37].Itisenvisagedthattheethanol-based cross-linkeragentmayhelpcounteractthedeleteriouseffects ofwaterduringdentinbondingandpreservethehybridlayer. Accordingly, the objective of the present in vitro study wastoevaluatetheabilityofanethanolsolutionofDCCto improvethebondstrengthofauniversaladhesiveemployed eitherinself-etchoretch-and-rinsemode,andtostabilizethe adhesiveinterfaces overtime.TheDDCwasappliedbefore adhesiveprocedurestocross-linkdentinalcollagen.Theeffect ofDCC ondentinMMPactivity wasfurtherinvestigatedby
in-situzymography.Thenullhypothesestestedwerethat
pre-conditioningofdentinwithDCCpriortoadhesiveapplication 1)doesnotbenefittheimmediatebondingperformanceofa universaladhesivetodentin,2)doesnotpreventinterfacial degradation over time,and 3) doesnotinhibit endogenous dentinMMPactivity.
2.
Materials
and
methods
Freshly-extractedsoundhumanthirdmolars(N=120)were obtainedfromanonymousindividualsfollowingtheirsigned consentundertheprotocolASLBON◦ 0013852,approvedon 02/01/2019bytheEthicsCommittee.
2.1. Microtensilebondstrengthtest(TBS)
Eighty teeth were selected to conduct TBS testing. The occlusal surface ofeach tooth was cuttransversely to the long axisto expose mid-coronal dentinusing a low-speed diamondsaw(Micromet,Remet,Bologna,Italy)withcopious watercooling.Astandardizedsmearlayerwascreatedwith 600-gritsilicon-carbidepaperoneachtoothsurface.The pol-ishedteethwererandomlyassignedtooneofthefollowing groupsaccordingtothedentinsurfacetreatmentand adhe-siveapproachperformed(N=20;Table1):
Group1(G1):DCCpre-treatmentandScotchbondUniversal adhesive(SBU;3MESPE,St.Paul,MN,USAusedinthe etch-and-rinsemode(ER).Thedentinsurfacewasetchedwith32% H3PO4(ScotchbondUniversalEtchant,3MESPE)for15s,
pre-treatedwithanethanolsolutionof0.5MDCCfor1min, air-driedandbondedwithSBUaccordingtothemanufacturer’s instructions(Table1).
Group 2 (G2):SBU inERmode. NoDCC was appliedon dentin;SBUapplicationwasthesameasG1.
Group3(G3):DCCpre-treatmentandSBUinself-etchmode (SE).Thedentinsurfacewaspre-treatedasinG1;insteadof etchingwith32%H3PO4,SBUwasapplieddirectlytothesmear
layer-covereddentinandagitatedfor20s.Theadhesivewas air-dried.Withoutlight-curing,DCCwasappliedfor1min.The secondlayerofuniversaladhesivewassubsequentlyapplied, air-driedandlight-cured(Table1).
Group 4 (G4): SBUinSE mode. NoDCC was appliedon dentin.SBUapplicationwasthesameasG3.
Table1–Adhesivesystem,compositecompositionandapplicationmode.
Material Composition ERmode DCC+ERmode SEmode DCC+SEmode
Scotchbond Universal(SBU;3M ESPE)
1.Etchant:32%phosphoricacid,water, syntheticamorphoussilica,polyethylene glycol,aluminumoxide(Scotchbond UniversalEtchant)2.Adhesive: methacryloyloxydecyldihydrogen phosphate(MDP)phosphatemonomer, dimethacrylateresins,2-hydroxyethyl methacrylate(HEMA),
methacrylate-modifiedpolyalkenoicacid copolymer,filler,ethanol,water, initiators,andsilane
1.Applyetchant for15s2.Rinse for10s3.Airdry 5s4.Applythe adhesivetothe entire
preparationwith amicrobrush andrubitinfor 20s5.Directa gentlestreamof airoverthe liquidfor5s untilitnolonger movesandthe solventis evaporated completely6. Repeatsteps4 and57. Light-curefor20 s 1.Apply0.5M DCC ethanol-based primerand brushitfor1min 2.Directagentle streamofair overtheliquid for5s3.Apply adhesiveasfor theERmode 1.Applyadhesive totheentire preparationwith amicrobrush andrubitinfor 20s2.Directa gentlestreamof airoverthe liquidfor5s untilitnolonger movesandthe solventis evaporated completely3. Repeatsteps2 and34. Light-curefor20 s 1.Applyfirstcoat ofadhesive2. Apply0.5MDCC ethanol-based primerand brushitfor1min 3.Directagentle streamofair overtheliquid for5s3.Apply adhesiveasfor theSEmode
FiltekZ250(3MESPE) Triethyleneglycoldimetacrylate (TEGDMA)<1–5%; Bisphenol-A-glycidylmethacrylate (Bis-GMA)<1–5%;Bisphenol-A polyethylenglycol dietherdimethacrylate(Bis-EMA) 5–10%;Urethanedimethacrylate (UDMA)5–10%Fillers:
Zirconia/silica;60vol%inorganic fillers(particlesize0.01–3.5m)
Eachbondedspecimen waslight-curedfor 20s using a light-emitting diodecuring light (DemiTM Plus, Kerr Corp., Brea,CA,USA)aftersolventevaporation.Four1-mmthick lay-ersofamicro-hybridresincomposite(FiltekZ250;3MESPE) wereincrementallyplacedoverthebondeddentinand indi-viduallypolymerized for20s each toobtain a4-mm thick composite build up for TBS testing. Each specimen was serially-sectionedtoobtainapproximately1-mmthicksticks, each containing resin composite and dentin and with the adhesiveinterfaceinbetween,inaccordancewiththe non-trimmingtechniqueoftheTBStest.Thedimensionofeach stick(0.9 mm× 0.9mm± 0.01mm)wasrecorded using a pairofdigitalcalipers.Thebondedareawascalculated for subsequentconversionofmicrotensilestrength valuesinto unitsofstress(MPa).Sticksfromeachtoothwererandomly assignedtotwostoragegroups:24h(T0)or1year(T12)of stor-ageinartificialsalivaat37◦C.Theartificialsalivaconsistedof CaCl2(0.7mmoles/L),MgCl26H2O(0.2mmoles/L),KH2PO4(4.0 mmoles/L),KCl(30mmoles/L),NaN3(0.3mmoles/L)inHEPES buffer[38].
Eachstick wasstressed tofailureundertensionusing a simplifieduniversaltestingmachine(Bisco,Inc.,Schaumburg, IL,USA)atacrossheadspeedof1mm/min.Thenumberof prematurely-debondedsticksineachgroupwasrecorded,but thosenullvalueswerenotincludedinthestatistical analy-sis.Thisisbecauseallprematurefailuresoccurredduringthe cuttingprocedureand thosefailuresdidnotexceedthe3% ofthetotalnumberoftestedspecimensandweresimilarly
distributed withinthe groups. A singleobserver evaluated the failuremodesunderastereomicroscope (Stemi2000-C; CarlZeissGmbH,Jena,Germany)at30×magnification.Failure modeswereclassifiedsadhesivefailure(A),cohesivefailurein dentin(CD),cohesivefailureincomposite(CC)ormixedfailure (M).
Statistical analysis was performed using the tooth as the statistical unit. Bond strength data from each tooth were averaged to obtain the mean bond strength for that tooth.Theacquireddatawereevaluatedforcompliancewith the normalityassumptionusing Shapiro-Wilktest,and the homoscedasticityassumptionusingthemodifiedLevenetest priortotheuseofparametricanalyticalmethods.Athree-way analysisofvariance(ANOVA)wasperformedtoidentifythe effectsofthreevariables,DCCpre-treatment(with/without), adhesiveapplicationmode(ER/SE)andaging(T0/T12)andtheir
interactions on bond strength.Post-hoc comparisonswere conductedusingTukeytest.Additionally,one-wayANOVAwas conductedtoevaluatedifferenceswithineachvariable.Forall tests,statisticalsignificancewaspre-setat˛=0.05. Statisti-calanalyseswereperformedusingStata12.0softwareforMac (StataCorp,CollegeStation,TX,USA).
2.2. Nanoleakageexpression
Twentyteeth(N=5)wereusedforexaminationof nanoleak-agewithintheresin-dentininterface.Mid-coronaldentinwere bondedinthesamemannerdescribedforTBStesting.Each
Fig.1–Schematicoftoothpreparationforin-situ
zymography.Adentindisk(1-mmthick)wasdividedinto fourquadrants,enablingbondingproceduresofthefour controlandexperimentalgroupstobeperformedonthe samedentinsubstrate.
specimenwascutverticallyinto1-mm-thickslabstoexpose theresin-dentininterface.Afterstorageinartificialsalivaat 37◦C for24h(T0)or12months(T12),thespecimenswere
immersedin50wt.%ammoniacalAgNO3solutionfor24hin
thedark,followingtheprotocoldescribedbyTayetal.[39].The specimenswerethenthoroughlyrinsedindistilledwaterand immersedinaphoto-developingsolutionfor8hundera flu-orescentlighttoreducesilverionsintometallicsilvergrains withinvoidsalongthebondedinterfaces.
For light microscopy, the specimens were fixed, dehy-drated,embeddedinepoxyresin(LRWhiteresin, Millipore-Sigma, Burlington, MA, USA), fixed on glass slides using cyanoacrylateglue,flattenedonagrindingdevice(LS2;Remet, Bologna, Italy) under water irrigation and polished with a gradedseriesofsiliconcarbideabrasivepapersofincreasing fineness(180-,600-,1200-,2400-,and4000-grit).Thepresence ofthe silver tracer was examined along the bonded inter-faceusinglightmicroscopy(E800;Nikon,Tokyo,Japan),at20× magnification.Interfacialnanoleakageexpressionwasscored bytwotrainedinvestigatorsbasedonthepercentageof adhe-sivesurfaceshowingAgNO3deposition,followingthemethod
ofSaboiaetal.[40].Ascale0–4wasusedforevaluation:(0)no nanoleakage;(1)<25%surfacewithnanoleakage;(2)25–50% surfacewithnanoleakage;(3)50–75%surfacewith nanoleak-age;and(4)>75%surfacewithnanoleakage.Intra-examiner reliabilitywasevaluatedusingtheCohen’skappa()statistic. Statisticaldifferencesamongnanoleakagescoreswere ana-lysedwiththechi-squarestatistic.Statisticalsignificancewas pre-setat˛=0.05.
2.3. In-situzymography
Twentyfreshly-extracted humanthirdmolars(N = 5) were
used for in-situ zymography. One mm-thick slabs of
mid-dle/deepdentinwereprepared.Eachslabwasfurtherdivided intofourpartstotestthe4controllandexperimentalgroups onthesamesubstrate(Fig.1).Siliconcarbidepaper(600-grit)
wasusedtocreateastandardizedsmearlayeroneachdentin surface.Onesurfaceofeachquarterofaslabwastreatedwith theadhesivesystemsasdescribedforTBStesting.
Theprocedurewasperformedusingthemethodpreviously reportedbyMazzonietal.[24,41].Afteragingforthe desig-nated period(24h or 1year), each bondedslabwas glued to aglass slideand polished to producean approximately 40-m thick section. To produce the substrate, 1.0mg/mL of a stock solution containing self-quenched fluorescein-conjugated gelatin (E-12055;Molecular Probes,Eugene, OR, USA)waspreparedbyadding1.0mLdeionizedwatertothe vial containing the lyophilized gelatin. The substrate was stored at−20 ◦C until use. Thegelatin stock solution was
diluted10timeswithdilutionbuffer(NaCl150mm,CaCl25
mm,Tris–HCl50mm,pH8.0),followedbytheadditionofan anti-fading agent(Vectashieldmountingmediumwith4 ,6-diamidino-2-phenylindole; Vector Laboratories, Burlingame, CA, USA). Then, 50 L of the fluorescent gelatin mixture wasplacedontopofeachpolisheddentinsectionand pro-tected with a cover slip. The glass slide assemblies were light-protectedandincubatedinahumidifiedchamberat37
◦Cfor48h.
Detection of endogenous gelatinolytic enzyme activity within the hybrid layer was based on hydrolysis of the quenchedfluorescein-conjugatedgelatinsubstrate.The pro-cess was evaluated by examining the glass slides with a multi-photonconfocallaserscanningmicroscope(LSM5Pa; CarlZeiss),usinganexcitationwavelengthof495nmandan emissionwavelengthof515nm.Sampleswereimagedusinga HCXPLAPO40×/1.25NAoilimmersionobjective.Seriesof x-y-zimages(0.145*0.145*1m3voxelsize)werecollected.Laser poweranddetectorgainweresetatthebeginningofthe exper-imentandkeptthesameforallspecimensinordertohavethe possibilitytocomparedifferentgroups.Sixteento20optical sectionswereacquiredforeachspecimen.Thestackedimages wereanalyzed,quantified,andprocessedwithZEN2009 soft-ware(CarlZeiss).Thefluorescenceintensityemittedbythe hydrolyzed fluorescein-conjugatedgelatin wasisolated and quantifiedusingImageJ(ImageJ;NationalInstituteofHealth, Bethesda,MD,USA).Theamountofgelatinolyticactivitywas expressedasapercentageofthegreenfluorescencewithinthe hybridlayer.
Negative controlsections were similarlyincubated,with the exception that 250 mLethylenediaminetetraacetic acid (EDTA)or2mM1,10-phenanthrolinewasdissolvedinthe mix-tureofquenchedfluorescein-conjugatedgelatin.The EDTA-and1,10-phenanthroline-containinggelatinwereusedas neg-ative controls.Inaddition,standardnon-fluorescentgelatin wasusedasthethirdnegativecontrol.
Becausethein-situzymographydatacomplied with nor-mality and homoscedasticity assumptionsafter non-linear transformation,athree-wayANOVAwasusedtoidentifythe effects ofthe threevariables,DCC pre-treatment, adhesive applicationmodeandaging, onthe densityoffluorescence signals.Additionalone-wayANOVAwasconductedto eval-uatedifferenceswithineachvariable.Statisticalsignificance waspre-setat˛=0.05.
Table2–ResultsofTBStestatT0andT12.SBU(ER):ScotchbondUniversalusedintheetch-and-rinsemode;SBU(SE):
ScotchbondUniversalusedintheself-etchmode.G1:0.5MDCCSBU(ER);G2:SBU(ER),control;G3:0.5MDCC+SBU(SE); G4:SBU(SE),control.T0:Dataobtainedafter24hofstorageat37◦C.T12:Dataobtainedafter1yearofaginginartificial
salivaat37◦C.
Applicationmode SBU(ER)a SBU(SE)a
Pre-treatment G1 G2 G3 G4
T0 46.0±15.3A,a 37.1±12.5A,b 39.4±11.1A,a,b 26.,3±11.4A,c
T12 33.5B±13.9B,a 31.0±11.0B,a 35.3±13.9A,a 13.4±9.1B,b
Differentsuperscriptupper-caselettersindicatedifferences(p<0.05)withinthecolumns. Differentsuperscriptlowercaselettersindicatedifferences(p<0.05)withintherows.
a Valuesaremeans±standarddeviations(inMPa).
Table3–Percentagesoffailuresmodeamongthedifferentgroups.
Applicationmode SBU(ER) SBU(SE)
Pre-treatment G1: G2: G3: G4:
T0 68%A32%M 35%A8%CD57%M 81%A1%CD18%M 74%A36%M
T12 45%A55%M 58%A42%M 53%A47%M 60%A40%M
3.
Results
3.1. Microtensilebondstrength
Microtensile bonds strengths of the four groups tested at T0andT12 aresummarizedinTable2.Three-factorANOVA revealed significant difference for the variables DCC pre-treatment,adhesive applicationmodeand aging(p <0.05), as well as forthe interaction between DCC pre-treatment and adhesive application mode (p < 0.05). Post-hoc com-parisons showed that the use of a 0.5 M DCC-containing ethanolsolutionbeforeadhesiveapplicationimprovedbond strength ofSBU (G1and G3)vs controlgroups(G2 andG4) (p<0.05),irrespectiveoftheadhesiveapplicationmodeand aging.Additionally,SBUgeneratedhigherbondstrengthwhen employedintheERmodevs theSEmode(p <0.05).Aging significantlyreducedTBSamongall theadhesive applica-tionmode/dentinpre-treatmentcombinations,exceptforG3 (p < 0.05). One-way ANOVA indicatedthat atT0, DCC
pre-treatmentsignificantlyimprovedTBSinbothexperimental groupscomparedtothecontrolgroups(G146.0±15.3;G237.1 ±12.5;G339.4±11.1;G426.3±11.4).Afteraging,DCC pre-treatmentshowedapreservationofthebondstrengthwhen SBUwasappliedintheSEmode(T0=39.4±11.1andT12=
35.3±13.9)thus,shovingafinalMPavalueafteraging compa-rabletothatoftheSBUERgroups.ForallgroupsatT0orT12
thepredominantfailuremodesweretheadhesivefailureand themixedfailure.Atbaseline,forallgroups,exceptforG2the adhesivefailurewerearound70%ofthefailure.Afteraging, thenumberofmixedfailureincreasedbetween40%and55% inthedifferentgroups(Table3).
3.2. Nanoleakageexpression
Descriptivestatistics ofinterfacialleakagescoresare repre-sentedinFig.2.Statisticallysignificantdifferenceswerefound amongthefourgroupsintheextentofsilvernitrate penetra-tionalongtheadhesiveinterfaces(p<0.05).Specimensthat werepretreatedwithDCC-containingsolutionpriorto
adhe-siveapplicationshowedlowernanoleakageexpressioninboth the ERandSEmodes,comparedtocontrolgroups,atboth T0andT12(p<0.05).AtbaselineSBUERandSBUSEshowed
anhigherpercentageofmarginalinfiltrationcomparedtothe experimentalgroups.However,SBUSEperformedworstthan SBUER.Afteraginginartificialsaliva,DCCpretreatedgroups shovedasignificantlylowermarginalleakagethanthe con-trolgroups.Inaddition,DCCSBUgroupsshowedcomparable resultsamongthem.
3.3. In-situzymography
Representativemicrographicimagesofthedifferentgroups are shown in Fig. 3 for time T0 and Fig. 4 for time
T12. The percentages of hybrid layers exhibiting
hydroly-sis of the quenched fluorescein-conjugated gelatin at T0
and T12 are shown in Fig. 5. For all specimens, the
high-est enzymatic activity appeared to be concentrated in the hybridlayerandthedentinaltubulesunderneaththehybrid layer.
Statistical analysis of the in-situ zymography identified significant differencesforthe variables DCC pre-treatment, adhesiveapplicationmodeandaging(p<0.05),andforthe interaction betweenapplicationmode andaging (p< 0.05). Forthevariable“DCCpre-treatment”,post-hoccomparisons showedthatDCCpre-treatmentsignificantlyreduced fluores-cenceatthelevelofthehybridlayer,comparedtonon-treated groups(p<0.05),irrespectiveoftheadhesiveapplicationmode andtheagingperiod.Forthevariable“adhesiveapplication mode”,ERgroupsresultedinasignificantlyhigherenzymatic activity compared tothe SE groups (p < 0.05), irrespective ofDCCapplicationandagingperiod.Forthevariable“aging period”,especiallywhenassociatedwithERmode,significant increasesinfluorescenceatthelevelofthehybridlayerwas identifiedwiththeERmodeatbothT0andT12(p<0.05).
One-way ANOVA calculated across all groups indicated thatatT0,therewasreducedfluorescencewithinthehybrid
layers,irrespectiveoftheadhesiveapplicationmode,when 0.5MDCCsolutionwasappliedpriortoadhesiveapplication
Fig.2–Distributionofinterfacialnanoleakage(in%)intheresin-dentininterfacescreatedwiththeScotchbondUniversal (SBU)adhesiveintheetch-and-rinsemode(ER)ortheself-etchmode,withorwithoutDCCpre-treatmentofdentin.Testing wasperformedafter24h(T0)orafteroneyearofaginginartificialsaliva(T12).
Fig.3–Representativeexamplesofin-situzymographyoftheresin-dentininterfacesatT0.DentintreatedwithSBU
adhesiveintheSEmode(a,b);SBU(SE)+DCC0.5M(c,d);SBUintheERmode(e,f);SBU(ER)+DCC0.5M(g,h).D:dentin;HL: hybridLayer;R:resincomposite.
Fig.4–Representativeexamplesofin-situzymographyoftheresin-dentininterfacesatT12(agingfor12monthsinartificial
saliva).DentintreatedwithSBUadhesiveintheSEmode(a,b);SBU(SE)+DCC0.5M(c,d);SBUintheERmode(e,f);SBU(ER) +DCC0.5M(g,h).D:dentin;HL:hybridLayer;R:resincomposite.
(G1andG3).However,thedecreasewasnotstatistically sig-nificantfortheSEmode(Fig.5).AtT12,bothexperimental(G3
andG4)andcontrolgroups(G1andG2)showedcomparable endogenousenzymaticactivityfortheERgroup(Fig.5).When SBUwasappliedintheSEmode,0.5MDDCsolution signifi-cantlyreducedtheactivitywithinthehybridlayer(Fig.5).
No fluorescence was detected in two negative con-trol groups prepared with non-specific inhibitors (EDTA or
1,10-phenanthroline) or when non-fluorescent gelatin was employed(datanotshown).
4.
Discussion
Theresultsofthepresentstudyshowedthatbondstrength wasincreasedbyexposingdentinsurfacesto0.5MDCC treat-ment priorto bonding with SBU at T0, irrespective of the
Fig.5–Gelatinolyticactivity,expressedastheintensityof greenfluorescence(pixels/m2)withinthehybridlayers (HL)createdwithSBUinERmodeorSEmodeforthe experimental(DCCpre-treatment)andcontrol(noDCC pre-treatment)groupsatT0andT12.Valuesaremeansand standarddeviations.Forcomparisonofthefactor“adhesive applicationmode”,columnslabelledwiththesameupper caseletters(T0)orlowercaseletters(T12)arenot
significantlydifferent(p>0.05).Forcomparisonofthe factor“DCCpre-treatment”,columnslabelledwiththe samenumeralsarenotsignificantlydifferent(p>0.05)(For interpretationofthereferencestocolourinthisfigure legend,thereaderisreferredtothewebversionofthis article).
adhesiveapplicationmode.Furthermore,dentinspecimens pre-treatedwiththeethanol-basedDCC cross-linker exhib-itedlessinterfacialnanoleakage comparedtospecimensin thecontrolgroups.Thus,thefirstnullhypothesisthat “pre-conditioningofdentinwithDCCpriortoadhesiveapplication doesnotbenefitimmediatetensile bondstrengthofa uni-versaladhesivetodentin”hastoberejected.Likewise,the applicationofDCCbeforeSBUresultedinhigherbondstrength andlowernanoleakageattheadhesiveinterfaceinspecimens thathas been agedfor12 months.Hence, thesecond null hypothesisthat“pre-conditioningofdentinwithDCCpriorto adhesiveapplicationdoesnotpreventinterfacialdegradation overtime”alsohastoberejected.
To our knowledge, this is the first study that utilized ethanolic DCC as dentincollagen cross-linker. This is dif-ferent from the water-based cross-linkers that had been usedin previous studies,such asEDC, grape seedextract, riboflavin, proanthocyanidin and chitosan [11,42–44]. N,N’-dicyclohexylcarbodiimide is an organic compound whose primaryuseistocoupleaminoacidsduringartificialpeptide synthesis.Understandardconditions,itexistsintheformof whitecrystalswithaheavy,sweetodor.Thiscross-linkeris commonlyusedasacondensationreagentinamidesynthesis oresterificationreactions[45].UnlikeEDC,DCChasthe dis-tinctadvantageofbeinginsolubleinwaterbutiswellsoluble inotherpolarorganicsolventssuchasethanolandacetone. ThisenablesDCCtobemorerealisticallyblendedwith exist-ingadhesivesystems,withouttheneedforrinsingoffpriorto theapplicationethanol-baseduniversaladhesive.
Eachcollagenmoleculehasaprimaryaminegroup( NH2) at the N-terminus, and a carboxyl group ( COOH) at the
C-terminusofthe polypeptidechain, bothofwhichare on thesurfaceoftheproteinstructure,makingthemaccessible fortheconjugationofproteins[46].EDCandDCCconjugate carboxylates toprimary amines directlyand cross-link the neighboring collagen molecules, without becoming part of thefinalcross-linkedtargetmolecules.Hence,theyare zero-length cross-linkingagents [47]. Similarlytoan EDC-based dentinprimer,[11,34],theresultsofthepresentstudyshowed thattheuseofanethanol-basedDCCprimerimprovedboth bondstrengthandresininfiltrationaswellaspreservedbond strengthandminimizednanoleakageafteroneyearofaging inartificialsaliva.
Inpreviousstudies,improvementinthebonddurabilityby EDCinbothcoronalandradiculardentinwasnotattributed onlytostrengtheningofthedemineralizedcollagenmatrix, but alsotoinactivationofthecatalyticsitesofendogenous MMPspresentindemineralizeddentin.Thelatterisachieved bymodifyingthethree-dimensionalconformationoftheMMP enzymes[48–51].InactivationofMMPsinducedbyacollagen cross-linkerisanon-specificmechanisminvolvingcovalent bondsthatareclaimedtobeverystableovertime[16,52].For thisreason,theeffectof0.5MDCConMMPactivitywas fur-ther investigatedusingin-situzymography.Atbaseline(T0),
Decreaseofendogenousenzymaticactivitywasidentifiedin thebaseline(T0)specimensaftertheuseofDCC,althoughthe
reductionwasstatisticallysignificant onlyintheERgroup. Afteraging(T12),onlytheDCC/SEgroupshowedsignificant
reduction in MMP activity. For this reason, the third null hypothesisthat “pre-conditioningofdentinwithDCCprior toadhesiveapplicationdoesnotinhibitendogenousdentin MMPactivity”canonlybepartially-rejected.
In-situzymographyshowedclearlydetectablegelatinolytic
activitywithinthehybridlayersoftheexperimentalgroups bothatT0andT12.Theseactivitiesweresignificantlylower
forthegroupsbondedintheSEmode,irrespectiveofthe stor-agetimeortheuseofDCC.Thegelatinolyticactivitywithin thehybridlayerswereeithermaintainedorincreasedafter1 yearofstorageinartificialsalivaforthecontrolgroups with-out DCCpre-treatment.TheeffectivenessofDCC usedasa conditioning primerwas evidentintheDCC/SE group(G3), withsignificantlylowergelatinolyticactivitywithinthehybrid layersafter1yearstorageinartificialsaliva.Itcouldbe spec-ulated that, theDCC primerappliedbetweentwolayersof adhesiveandnotaftertheetchingstep,asforthe experimen-talERgroup,couldleadtoalongerperiodofactivity ofthe molecule.Indeed,DCCcouldremainboundinsidethehybrid layerforalongerperiodoftimeandslowlyreleasedwith age-ing,similarlytootherexperimentalmoleculesblendedinside theadhesive[12].
The present study also showed that more endogenous MMPswereactivatedwhentheuniversaladhesivewasapplied inthe ERmode [53].When amulti-mode adhesiveisused in the ER mode, acid-etching with phosphoric acid com-pletelyremovesthemineralcomponentsofthedentinmatrix, exposesthe collagenfibrilscompletelywithincreased acti-vation of the endogenous enzymes. Similarly to a 2-step etch-and-rinseadhesive,theenzymaticactivity ishigh and localizedatthebottomofthehybridlayer.Thisisprobablydue totheinabilityoftheadhesiveresinmonomerstocompletely infiltratethedemineralizeddentinmatrix[24,54].Conversely,
whenSBUisusedintheSEmode,thehybridlayeris simulta-neouslydemineralizedandinfiltratedbytheacidicadhesive resinmonomers.Inaddition,thereislesscompletelydenuded collagenfibrilswithinthethinnerhybridlayers.Becausethe apatitecrystallites areonlypartially-dissolved, hereis pre-sumablylessactivationoftheendogenousMMPs[55].Ithas beenreportedthatmoreMMPsareactivatedwhenthepHof thedentinmatrixislower[56],asthedropinpHvalueturns onthecysteineswitchmechanismandcausestheMMP pro-formstobeactivatedintofully-functionalMMPs[57].
Because dentin MMP activity could be reduced by the application of DCC, the results of the present study sug-gest that the ethanolic collagen cross-linker stabilizes the adhesive interface by strengthening of the collagen and inactivatingendogenousdentinproteases[4].Severe, albeit non-fatal limitation ofthe present study isthat no exper-iment was performed to confirm that there was increase incross-linking of the collagen matrix afterpre-treatment with DCC. This should be performed in future studies to identify the type and quantity of cross-links involved and to determine if the cross-linking produced by the use of DCC is reversible or irreversible. It would also be interesting to compare the degree of cross-linking pro-duced by the water-based EDC versus the ethanol-based DCC.
Another important aspect related to the use of DCC is that ethanol is used as the solvent instead of water. The use of an ethanol-based cross-linking agent may result in betterinfiltration of the collagen matrix byadhesive resin monomers,comparedwiththeuse ofawater-based cross-liningagent[58].Afteracid-etching,thedentincollagenfibrils are in a wet environment saturated with residual water. Tosubstitutewaterwithwater-insolubleadhesive resins,a primeragentbasedonavolatilesolventsuchasethanolor acetone is required. Formation of an ideal hybrid layer in dentinrequiressubstitutionofallunboundwaterwithresin monomers.However,completeremovalofallresidualwater cannotbeachievedduetothepresenceofboundwaterthat intrinsicallywet thecollagen fibrils.Thisprocess,however, maybeimprovedbyfirstreplacingtheunboundwaterwitha non-water-containingpolarsolventwhichiscapableof solu-bilizingtheadhesiveresinmonomers[59].Previousstudieson the“ethanol-wetbonding”conceptdemonstratedthatbetter resininfiltrationoccurredwithahighercontentofethanolin theadhesiveprimer[60].Whendentinisfullysaturatedwith ethanol,ethanol-solublehydrophobicresin monomersmay beintroducedinto ademineralized collagenmatrix [61,62]. Infiltrationofhydrophobicmonomersdecreaseswater sorp-tion/solubilityandresinplasticization,butalso,ithasbeen suggestedthattheeliminationofresidualwatercouldreduce oreliminateenzyme-catalyzedhydrolyticcollagen degrada-tion[63].TheethanolintheDCCprimerusedinthepresent research probablycontributes to furtherdissolution ofthe adhesiveresinmonomers,reducingtheviscosityandthe wet-tabilityofthemixture,andfacilitatingitsdiffusionintothe dentin.Inaddition,apossibleinhibitoryeffectofethanolon MMPsactivitycouldnotbeexcluded,althoughtheapplication oftheethanol-basedDCCprimerwasnotperformedfollowing thetraditional“ethanol-wet”protocolthatimpliesincreasing concentrationsofethanol(50%, 70%,80%,95% and 3100%
ethanolapplicationsfor30seach)appliedondentinal sub-strate.
In lightofthe favorableresults obtainedwithDCC pre-treatment, it is envisaged that DCC may be incorporated intoethanol-basedadhesivesystemstoproduceself-priming adhesivesthatcross-linksthecollagenmatrixastheadhesive infiltratesthecompletely-orpartially-demineralizeddentin. This removesanadditionalstepand furthersimplifies the bonding procedures. However, further studies on different adhesivesystemsandprotocolsarerequiredtoclarifytherole ofDCConpreservingtheadhesivebondovertimeandto val-idatethepossibilitytoincorporateDCCintocurrentadhesive systemswithoutcompromisingtheiradhesiveproperties.
5.
Conclusions
Withinthelimitations ofthe presentstudy,it maybe con-cludedthattheuseoftheethanolicDCCcollagencross-linker improves bond strength and reduces nanoleakage when a universal adhesive is applied on coronal dentin in the etch-and-rinsemodeorself-etchmode.TheuseofDCC pre-treatmentpriortotheapplicationofauniversaladhesivedoes notadverselyaffectbaselineMMPactivity.Whentheadhesive isemployedintheself-etchmode,theuseof0.5MDCCfurther helpsinpreventingthedegradationofdentinorganicmatrix byinhibitingendogenousdentinmatrixmetalloproteinases.
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