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Tlfll JOUIlNAL o r Til!: AMERICAN DENTAL ASSOCIATlON The role of dentin moisture in the degradationof resin-dentin interfaces under clinical andlaboratory conditionsAlesssandra Reis, Ana C. Chibinski, RodrigoStanislawczuk, Denise S. Wambier, Rosa HelenaM. Grande and Alessandro D. LoguercioJADA 2012;143(7):e29-e36

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The role of dentin moisture in thedegradation of resin-dentin interfacesunder clinical and laboratory conditionsAlesssandra Reis. DDS. PhD: Ana C. Chibinski. DDS. MS: Rodrigo Stanislawczuk. DDS. MS:Denise S. Wambier. DDS. MS. PhD: Rosa Helena M. Grande. DDS. MS. PhD:Alessandro D. Loguercio. DDS. MS. PhD

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or almost 20 years,the wet bondingtechnique has beenused for dentin

bonding. As long as de-mineralized dentin is keptfully hydrated, the dentinmatrix will not collapse,and free space will beavailable for resin infiltra-tion.P The results of pre-vious studies have shownthat bonding to air-drieddemineralized dentin canlead to improper adhesiveinfiltration, with resininfiltration not exceedingone-half of the demineral-ized dentin zone.t"

However, water andsolvents need to beremoved completelybefore adhesive lightpolymerization takesplace'" and this removalcannot be achieved evenwhen using solvent evapo-ration times that are 10 to12 times longer thanthose recommended bythe manufacturers." Inhi-bition ofhydroxyethyl-methacrylate polymeriza-tion has been detectedwith intrinsic water atconcentrations of greaterthan 5 percent by volume,

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Dr. Reis is an adjunct professor, Department of Restorative Dentistry, School of Dentistry, Rua Garlos Gavalcanti, 4748, Universidade Estadual de PontaGrossa, Ponta Grossa, Paraná, Brazil, 84030-900, e-mail reis_ale@hotmail.com. Address reprint requests to Dr. Reis.Dr. Ghibinski is a professor and a doctoral student, Department of Pediatric Dentistry, School of Dentistry, Universidade Estadual de Ponta Grossa, Paraná,Brazil.Dr. Stanislawczuk is a doctoral student, Department of Restorative Dentistry, School of Dentistry, Universidade Estadual de Ponta Grossa, Paraná, Brazil,Dr. Wambier is an associate professor of pediatric dentistry, Department of Restorative Dentistry, School of Dentístry. Universidade Estadual de PontaGrossa, Paraná, Brazil,Dr. Grande ís an assocíate professor, Department of Dental Materials, University of São Paulo.Dr. Loguercio ís an adjunct professor, Department of Pediatric Dentistry, School of Dentistry, Universidade Estadual de Ponta Grossa, Paraná. Brazil.

JADA 143(7) http://jada.ada.org July 2012 eZ9Copyright © 2012 American Dental Association. Ali rights reserved.

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even with a 10-fold increase in photoinitiators."Trapped water also prevents the formation of apolymer with a high density of cross-linkingwithin the collagen fiber network.l'v" probablydue to phase separation ofhydrophobic andhydrophilic moieties.P'!'

Wet bonding is a technique-sensitive pro-cedure. Optimum bonding with current commer-cial adhesives occurs within a narrow range ofmoisture levels'<" and depends on the interpre-tation of manufacturers' instructions, dryingtime, tooth-air syringe distance" and operator'sskills." Under clinical conditions, it is unlikelythat uniform wetness on all of the walls of thetooth preparation can be achieved.

Despite the better immediate bond strengthsproduced with the wet bonding protocol.P-" theuse of this technique does not guarantee stableand durable bonds" across time, as dentalmateriaIs are not inert in an oral environ-ment.21,22 Degradation of the bonded interface isreported consistently in the literature.ê=" Inves-tigators have reported that as long as etch-and-rinse adhesives are rubbed vigorously''':" ondentin surfaces, strong immediate" and one-year" resin-dentin bonds can be achieved on drydemineralized dentin. Technique simplificationand the lower residual water content of drybonding bring additional advantages; nonethe-less, investigators in only two clinical studieshave attempted to study this issue in noncar-ious cervical lesions.êv"

Although in vitro laboratory studies are rela-tively simple and may provide faster results andcloser control ofthe variables involved in thebonding protocol than clinical studies, they donot take into account the pulpal pressure andthe presence of dentinal tubule fluid underphysiological conditions.P-" as well as otherenvironmental-related variables.ê=" which mayadversely affect dentin bonding.

Although the investigators of several studieshave attempted to validate the laboratory dataclinícally,":" few compared the microtensileresin-dentin bond strengths produced underboth clinical and laboratory conditions.v" Weconducted an investigation to evaluate the influ-ence of dentin moisture on the degradation ofresin-dentin bond strengths to primary toothdentin under both clinical and laboratory condi-tions. The null hypotheses tested were thatresin-dentin bond strengths would not beaffected by the bonding conditions (clinical andlaboratory) and the moisture of the dentin andthat no significant reductions in bond strengthwould be observed after six months of aging theteeth.

METHODSSpecimen preparation for the clínicalexperimento The ethics committee from theState University ofPonta Grossa, Paraná,Brazil, approved our study. After clinical andradiographic examination of approximately 200patients ranging from 10 to 12 years of age, weselected 20 patients for the study. These pa-tients were required to have a caries-free orincipient carious lesion in their primary secondmolar (extending by as much as the upper one-third into the dentin as seen in an interprox-imal radiograph) and be in need of restorativetreatment in the same hemiarch, so that theywould need to receive local anesthetic andwould require isolation of the tooth by means ofa rubber damo

Among these 20 patients, one-halfhad secondmolars that met the inclusion criteria and wereat an advanced stage of physiological root re-sorption and mobility, which indicated that thephysiological exfoliation process was occurring.Another 10 patients met the inclusion criteria,but had teeth that would be retained for at leastsix months to await the natural exfoliationprocesso The fact that exfoliated teeth can beused as source of stem cells is evidence that theteeth we used in the study had pulp vitality.5o,51

Restorative procedure. After we adminis-tered local anesthetic to patients and isolatedthe teeth with a rubber dam, we prepared ClassI restorations by using a cylindrical diamondbur with water cooling. We used each diamondbur for only three tooth preparations. We pre-pared the teeth to achieve the largest possibledimensions, completely flat pulpal floor dentinand complete enamel cavosurface margins. Wedivided the specimens randomly into twogroups, according to the adhesive procedures:wet (five teeth) and dry (five teeth) bondingtechniques.

The restorative procedures involved etchingthe prepared surface with 37 percent phosphoricacid gel for 15 seconds followed by water rinsingfor 15 seconds; bonding with a two-step etch-and-rinse adhesive system (XP Bond, batch0804002271, Dentsply DeTrey, Konstanz, Ger-many) and restoration with a microhybrid resin-based composite resin in three increments.

The bonding procedures differed between thestudy groups. For the wet bonding technique, we

ABBREVIATION KEY. AL: Adhesive layer. Co: Com-posite. De: Dentin. EDX: Energy-dispersive x-rayspectroscopy. HL: Hybrid layer. J.l.TBS:Microtensilebond strength. SEM: Scanning electron microscopy.SNU: Silver nitrate uptake.

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rinsed the teeth with water for 20 seconds andcompletely air dried them after we acid etchedthem. We then carefully rewet the tooth prepa-ration with a microbrush saturated with waterbefore applying the adhesive. The surface wasleft slightly moist without water pooling. For thedry bonding technique, we kept the dentin sur-faces dry before we applied the adhesive.

For both groups, we applied two coats of theadhesive under vigorous rubbing action." Wethen gently air dried the adhesive layer for fiveseconds, left it undisturbed for 20 seconds andthen light polymerized it by using a light-emitting diode unit for 10 seconds at 900 milli-watts per square meter. The prepared teeth wererestored with three incrementallayers of resin-based composite light activated individually for20 seconds with the same light-curing unit.

Within 20 minutes after completion of thebonding procedures, we extracted the teeth fromthe 10 patients with advanced physiological rootresorption, immersed the teeth in distilledwater (pH = 7) and kept them in a moist envi-ronment for 24 hours at 37°C before we pre-pared them for a microtensile bond strength testand micromorphological analysis. The teethfrom the other 10 patients were left in the pa-tients' mouths and used under normal circum-stances until the physiological exfoliationprocess occurred.

Specimen preparation for the laboratoryexperimento We repeated the same experimentas the one performed clinically but under labo-ratory conditions by using 20 recently exfoli-ated, caries-free primary second molars inwhich we prepared 20 Class I restorations. Westored the teeth in saline solution for up tothree months before we conducted the labora-tory experimento We made preparations similarto those we made for the vital teeth, and thesame dentist (A.C.C.) performed all oftherestorative procedures. We divided the speci-mens into four groups as described for the clin-ical experiment (wet, dry, immediate and aftersix months of aging). We prepared the restora-tions the same way we did for the clinical exper-iment. After we completed these procedures, weplaced the teeth in a moist environment for 24hours at 37°C (n = 10 teeth, five wet and fivedry) or stored them in distilled water at 37°C forsix months (10 teeth, five wet and five dry).

Microtensile bond strength (IlTBS).Intheir respective testing periods, we longitudi-nally sectioned bonded teeth from both experi-ments in both the mesiodistal and buccolingualdirections across the bonded interface by usinga diamond saw in a cutting machine, with water

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cooling at 600 rotations per minute to obtainbonded specimens with a cross-sectional area ofapproximately 0.8 square millimeters.

We measured the remaining dentin thicknessof the specimens immediately above the pulpchamber and the cross-sectional are a of eachspecimen by using a digital caliper to thenearest 0.01 mm to evaluate dentin depth andbond strength, respectively. We attached indi-vidual bonded specimens to a microtensiletesting device by using cyanoacrylate resin(Super Bonder, Loctite-Henkel, São Paulo), sothat tensile forces acted perpendicularly to thedentin-adhesive interface and were subjected toa tensile force in a universal testing machine at1.0 mm per minute. We evaluated the failuremodes of the specimens in which the bondingfailed after they underwent microtensile testingat x400 magnification and classified them ascohesive (failure exclusively within dentin orresin-based composite), adhesive (failure at theresin-dentin interface) or adhesive-mixed(failure at the resin-dentin interface, whichincluded cohesive failure of the neighboringsubstrates) .

Micromorphological analysis. We evalu-ated one representative specimen from eachtooth from each experimental condition bymeans of scanning electron microscopy (SEM).After storing the specimens in ammoniacalsilver nitrate for 24 hours," we rinsed thesilver-impregnated specimens thoroughly indistilled water and placed them in a photo-developing solution for eight hours under a fluo-rescent light. We polished the adhesive inter-faces with descending grits of silicon-carbidepapers (1,000; 1,200; 1,500; 2,000; and 2,500)and 1.00- and 0.25-micrometer diamond pasteby using a polishing cloth. We cleaned the speci-mens by means ofultrasonography and leftthem in a desiccator for 24 hours at room tem-perature. We then mounted the specimens onstubs and sputter-coated them with a 10-nanometer gold layer so we could analyze themby means of SEM by using backscattered elec-tron mode and energy-dispersive x-ray spec-troscopy (EDX).As the dehydration took placeslowly at room temperature and with specimensof reduced dimensions, the specimens rarelycracked.

We measured the amount of silver nitrateuptake (SNU) inside the hybrid layer in eachspecimen by using EDX at three different sites(5 x 5 um: left, middle and right) of the bondingarea in each specimen. The totallength of thescanned hybrid layer used for silver nitratepenetration measurement was approximately

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TABLE 1

Laboratory

wet] versus testingperiod [immediate andsix months]) and Tukeytest (a = .05) for pair-wise comparisons.

Dry

RESULTS~TBS. The mean cross-sectional areas of thebonded specimensranged from 0.82 to 1.03mm", and we observedno significant differenceamong the groups (datanot shown; P > .05).Seven specimens fromthe clinical experimentexfoliated after sixmonths and three speci-mens from the clinicalexperiment exfoliatedafter eight months ofclinical use in the pa-tient's mouth. TheoveralI mean bondstrength values areshown in Table 1. Thefracture pattern mode ofthe bonded specimens isshown in Table 2. Mostof the failures wereadhesive-mixed, irre-spective of the experi-mental condition.

The results of thethree-way ANOVAshowed that the interaction ofdentin moisture and testing period (P < .001), aswell as of the main factors (bonding condition[P = .001] and testing period [P < .001]), werestatistically significant. The bond strengthvalues for the wet bonding technique were thehighest in the immediate period (Table 3).Aftersix months of aging, however, we observed sig-nificant degradation ofthe resin-dentin bondsonly for the wet bonding technique (overalIimmediate bond strength [standard deviation] =31.3 [4.5] MPa versus overalI six-month bondstrength = 21.3 [2.1] MPa; P < .05). We observedno degradation of the resin-dentin bonds whenbonding was performed for the dry bonding tech-nique. Irrespective ofbonding conditions, theoveralllaboratory bond strength values (SD)(28.5 [4.4] MPa) were significantly higher thanthe clinical values (25.2 [3.6] MPa) (P = .001).Despite the numerical difference in terms ofbond strength values, the results of both theclinical and laboratory experiments led to sim-

Wet

21.6 (2.3)

29.2 (3.1) 33.4 (3.0)

28.9 (3;0)

TABLE 2

Immecllate: .

Specimens with cohesivefailure. in resin-basedcomposite· .

Prematurely debonded specimens

Slx Months

751lm.30The SNU was expressed in percentageof the total area evaluated.

Statistical analysis. We averaged the IlTBSvalues of all specimens from the same tooth forstatistical purposes.P>' We included pretest fail-ures in the tooth mean. For SNU, we averagedthe mean SNU of all specimens originating fromthe same tooth for statistical purposes. TheSNU of every test group was expressed as themean of the five teeth used per group.

Before analyzing data by using the appro-priate statistical test, we performed theKolmogorov-Smirnov test to assess whether thedata folIowed a normal distribution. We per-formed a Bartlett test for equality of variancesto determine ifthe assumption of equal vari-ances was validoAfter observing the normalityof the data distribution and the equality of thevariances, we submitted the IlTBS (megapas-cals) and SNU data to a three-way analysis ofvariance (ANOVA)(bonding condition [clinicaland laboratory] versus dentin moisture [dry and

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ilar conclusions; that is,bonding to dentin by fol-lowing the dry bondingtechnique producedmore stable resin-dentinbond strength values.

SNU. The mean rela-tive percentages of SNUwithin the hybrid andadhesive layers areshown in Table 4. Onlythe main factor, testingperiod, was statisticallysignificant (P < .001).Higher SNU occurred inall groups after sixmonths of aging, irre-spective of the otherexperimental conditions.Representative SEMimages of the restorativeinterfaces can be seen inthe figure.

TABLE 3

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TEsTlNG PERIOD DENTIN MOlsTURE, MEAN MEGAPASCALs(sTANDARD DEVlATION)

Dry WetImmedlate 27.3 (3.4)bt 31.3 (4.5)'

slx Months 27.4 (2.9)b 21.3 (2.1)<

* The overall data from both clinical and laboratory conditions are reported for each group (n = 10 teethper experimental condition).

t Means identified with the same superscript letters are statistically similar (P < .05).

TABLE 4

TEsTlNGPERIOD

RELATlVE PERCENTAGE, MEAN* (sTANDARD DEVIATION)

Cllnlcal

38.5 (4.9)<

Immediate

six Months

* Means identified with the same superscript letters are statistically similar (P < .05).

Laboratory

Dry Wet WetDry15.7 (3.6» 13.1 (5.6» 14.1 (6.3)' 12.5 (4.2)'

DISCUSSIONThe results of our investigation showed thathigher resin-dentin bond strengths wereachieved for the material we evaluated whenthe restorations were created under laboratoryconditions, which confirms the findings of pre-vious studies.47-49 This finding may be due to thefact that in vital teeth there are odontoblasticprocesses, fluid in the dentinal tubules andintrapulpal pressure.P-" ln addition, there issignificantly higher relative humidity and tem-perature intraoralIy than there is under labora-tory conditions,":" which can reduce the adhe-sive systems' solvent evaporation rates. Com-bined, these factors make the bonding proceduremore chalIenging in a clinical scenario, whichmay result in lower bond strength values.

Laboratory investigators have reported thatdemineralized dentin needs to be kept moist" tomaintain interfibrillar porosity for resin mono-mer infiltration and to allow for high immediatebond strength values.15,18,19 Under moist condi-tions, the demineralized dentin preserves thenanospaces within colIagen fibrils':" into whichthe adhesive monomers diffuse to envelop thecolIagen fibrils before polymerization. Theresults of studies have shown that for an ace-tone-based system, the infiltration rate of thebonding resin within the hybrid layer is reducedby approximately 50 percent when applied todry dentin instead ofwet dentín.ê-"

The higher infiltration rate of resin mono-mers into wet demineralized dentin might

26.6 (2.4)b 33.9 (4.4)< 37.4 (6.4)<

explain the higher immediate resin-dentin bondstrength. It does not, however, explain thehigher susceptibility of the wet bonding tech-nique to degradation. Although the exact mech-anism that causes the degradation of the hybridlayer is not yet completely understood, the firststage ofbiodegradation involves extraction ofthe resins that had infiltrated the dentin matrixvia water-filIed nanometer-sized voids withinthe hybrid layer.20,23

If solvent and water are retained within theadhesive resin, they can compromise the struc-tural integrity of the hybrid layer severely,reducing its mechanical properties.v? lncreasingthe concentration of acetone in two-step etch-and-rinse adhesives can cause a decrease intheir f.1TBSand morphological manifestations ofcracks and interfacial gaps along the bondedinterfaces.ê-" The remaining water also mayinterfere with polymerization of the resin mono-mers from the adhesive systems. The addition of0.2 milliliter per mL of comonomer mixture canreduce the degree of conversion of bonding resinfrom 53.5 to 25.0 percent."

On the basis of the above findings, it appearsthat light activation of the adhesive in a water-containing environment, such as the wet dem-ineralized dentin, may produce a loosely cross-linked polymer inside the hybrid layer" that ismore susceptible to water sorption and solu-bility. This means that the ingress of water isfacilitated when the adhesives are light-

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·Cliniçal

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Figure. Representative scanning electron microscopic images ofthe resin-dentin interfaces for laboratory and clinical specimenstested immediately or after six months of water storage. Only asmall amount of silve r nitrate uptake can be seen in the hybridlayers of specimens in the immediate groups (A and B) (whitearrows). After six months, the amount of silver nitrate penetra-tion within the hybrid layer was higher in ali groups (e and O)(white arrows). Co: Composite. AL: Adhesive layer. HL: Hybridlayer. De: Dentin.

polymerized in a water-containing environment.The role of water in plasticization of the

polymer network has been discussed in the liter-ature. Water softens the polymer by swellingthe polymer network and reducing the frictionalforces between the polymer chains.21,22 Unre-acted monomers trapped in the polymer net-work are released into the surrounding area,creating new channels for water penetrationthrough which even more water diffuses in self-perpetuating processoAs a consequence, the pre-viously resin-infiltrated collagen matrix be-comes exposed and vulnerable to attack by host-derived proteolytic enzymes," allowing for anenzymatic attack of the exposed collagen fíb-rils," which can lead to their degradation.F-"

The relatively stable resin-dentin bondstrength in the dry groups does not necessarilymean that these interfaces were resistant todegradation. AlI groups, irrespective of theirmoisture and bonding conditions, had increaseddeposition of silver nitrate along the adhesive

and hybrid layer after six months of aging.Although the deposition of silver nitrate did notaccount for the reduction in resin-dentin bondstrength of the dry groups under clinical andlaboratory conditions, it is evident that thesedry-bonded interfaces also underwent resinplasticization and elution. One may envisionthat a polymer with improved quality mighthave been formed in the dry groups, leading toreduced degradation rates not detected by thebond strength test in the aging periods of ourinvestigation.

Contrary to the results of in vitro laboratoryinvestígations.P:" the results of our investiga-tion showed that it is possible to achieve highbond strength with air-dried demineralizeddentin, as we noted in earlier studies.P-" Themechanical pressure applied to the demineral-ized dentin surface during vigorous rubbing"might compress the collapsed collagen network,in such a way that adhesive solution is drawninto the collapsed collagen mesh when the pres-sure is relieved." Moreover, this approach mightincrease the rate of exchange and allow forbetter monomer diffusion inward, while solventsare diffusing outward.

lnvestigators who evaluated the effect ofdentin moisture on the retention rates of com-posite restorations in noncarious cervicallesionsdid not detect significant differences in theretention rates of two simplified etch-and-rinseadhesives when demineralized dentin waseither kept moist or dry.34,35The dry bondingtechnique should be considered for use withbonding etch-and-rinse adhesives, as it is aneasy and more reproducible clinical technique.

Our investigation had limitations. For ethicalreasons, the teeth we selected for the clinicalexperiments had advanced root resorption. It isunlikely that these teeth would have beenrestored clinically even if they had deep cariouslesions. Therefore, the clinical specimens do notexactly resemble those that clinicians would seein their practices. Another source of concern isthe vitality of the primary teeth used in theimmediate group, because they had advancedroot resorption. Exfoliated primary teeth are aviable source of stem cells for dental pulp tis sueengineering.ê'v" which can be used as evidenceof their tooth vitality. In addition, potentialendodontic problems could result from improperair drying of dentin for bonding and these po-tential side effects need to be clarified. Finally,the relevance of bond strength testing on theoverall clinical performance of restorations hasyet to be addressed. Although the results of astudy show that bond strength values can pre-

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dict the retention of adhesive systems under aclinical scenario," the study still is the first andonly one that has evaluated this issue. There-fore, further investigations should be conducted.

The resin-dentin bond strengths producedunder laboratory conditions may be higher thanthose obtained under clinical circumstances,although both methods (clinical and laboratory)seemed to yield similar results. Bonding a sim-plified etch-and-rinse adhesive to dry deminer-alized primary tooth dentin produced resin-dentin interfaces that were more resistant todegradation than were those produced on wetdemineralized dentin.

CONCLUSIONSThe bonding of adhesives to dry demineralizeddentin produces adhesive interfaces that aremore resistant to degradation, regardless of thebonding condition .•

Disclosure. None ofthe authors reported any disclosures.

The study was supported in part by National Couneil for Seientificand Teehnological Development (CNPq), Brasilia, Federal Distriet,Brazil, grants 301937/2009-5 and 303933/2007-0.

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