10
 Inuence of chlorhexidine digluconate concentration and application time on resin–dentin bond strength durability Alessandro D. Loguercio 1 , Rodrigo Stanislawczuk 1 , Luceli G. Polli 1 , Jully A. Costa 1 , Milton D. Michel 2 , Alessandra Reis 1 1 Department of Restorative Dentistry, School of Dentistry, Universidade Estadual de Ponta Grossa, PR, Brazil;  2 Department of Mechanical Engineering, School of Mechanical Engineering, University Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil Rec ent st udies have pointed out that dent in bond- strength values measured immediately after formation of the bond do not always correlate with long-term bond st abili ty (1), as degradat ion throughout the dent in bonding interface occurs rapidly (i.e. within 6 months) (2, 3). Bon din g is cre ate d by impreg nat ing the dentin substrate with blends of resin monomers, and the sta- bility of the bonded interface relies on the creation of a compac t and homoge nou s hyb rid layer. In the etc h- and-r inse strategy, after preliminary etching to demin- eralize the sub strate, res in mon ome rs impreg nate the porous etched substrate (4, 5) and thus stable bonds may be achieved if the etched substrate is fully inltrated by the adhesive (6). However, when incompletel y inlt rated zones occur along the bottom of hybrid layers within the acid-etched dentin, a decreasing gradient of resin monomer diusion, wit h den ude d col lagen br ils (7– 9), is likely to occur. This region is susce ptible to hydr olytic degrada tion in the long term, leading to red uct ions in bon d str ength (10). Moreover, the elution of unpolymerized monomers from the hybrid layer in cont act wi th oral uids is respo nsibl e for the acceler ated process of bond interfac e degradation. This leaves more collagen brils within the hybrid layer exposed and susceptible to the degradation process (11). The combined degradation by hydrolysis of resin and/or collagen weakens the physical properties of the resin–dentin bond interface (11). Studies have revealed the contribution of host-derived prote inases , responsible for the break down of collag en matrices, in the pathogenesis of dentin caries (12, 13), periodontal disease (14), and in the degradation of dentin bon din g (15 ). It has bee n rep ort ed tha t mineralize d dentin contains collagenolytic and gelatinolytic enzyme act ivit ies der ived fro m the act ion of mat rix met all o- proteinases (MMPs) (16–18). Both  in vitro  and  in vivo studi es have shown that 2% chlor hexidi ne digluc onate (CHX), applied for 60 s on demineralized dentin, post- pon es the res in–den tin degradation of adhesiv e int er- faces, when compared with interfaces to which no CHX is applied (19–22). Despite these advantages, the use of 2% CHX for 60 s demands more chair-time during the adhesive procedure and this contrasts with the clinicians needs for simplication (23). It is also worthwhile mentioning that CHX solutions with concentrations (close to 0.01%) are highly cytotoxic to cultured cells (24, 25). Obviously, when CHX solution is applied as a cavity cleans er the re is eno ugh denti n structure to act as a barrier and protect the pulp cells against the cytotoxic eects of dental materials or rinsing Loguercio AD, Stanislawczuk R, Polli LG, Costa JA, Michel MD, Reis A. Inuence of chlor hexid ine diglu conat e concent ration and appli catio n time on resin –dent in bond strength durability. Eur J Oral Sci 2009; 117: 587–596.  2009 The Authors. Journal compilation  2009 Eur J Oral Sci Although it is known that chlorhexidine application may preserve resin–dentin bonds from degradation, the lowest optimal concentration and application time have yet to be established. This study evaluated the eects of dierent concentrations of chlor- hexid ine digluconat e and dierent applicatio n times on the preservati on of resin  dentin bonds formed using two etch-and-rinse adhesives. In experiment 1, after acid etchin g, the occlu sal demineral ized dentin was rewett ed either with water or with 0.002, 0.02, 0.2, 2, or 4% chlorhexidine for 60 s. In experiment 2, the surfaces were rewetted with water, or with 0.002% or 2% chlorhexidine for 15 or 60 s. After this, both adh esi ves and compos ite res in were app lied and light-cured. Bonded stic ks (0.8 mm 2 ) were tested under ten sion (0.5 mm min )1 ) immediatel y or after 6 month s of storage in water. Two bonded sticks from each tooth were immersed in silver nitrate and analyzed quantitatively using scanning electron microscopy. Reductions in mi- croten sile bond stren gths and higher silver nitrate uptake were obser ved for both adhesives when the rewetting procedure was performed with water. Stable bonds were mainta ined for up to 6 month s unde r all chlor hexid ine conditions tested, irrespec tive of the chlorhexidine concentration and application time. The use of 0.002% chlorh- exidine for 15 s seems to be sucient to preserve resin–dentin interfaces over a 6- month period. Alessandro D. Loguercio, Universidade Estadual de Ponta Grossa – Mestrado em Odontologia, Rua Carlos Cavalcanti, 4748, Bloco M, Sala 64A – Uvaranas, Ponta Grossa, Paran 84030-900, Brazil Telefax: +55–42– 32203741 E-mail: aloguerci [email protected] Key words: adhesive system; application time; chlorhexi dine concentrati on; dentin; longevity Accepted for publication May 2009 Eur J Oral Sci 2009; 117: 587–596 Printed in Singapore. All rights reserved  2009 The Authors.  Journal compilation   2009 Eur J Oral Sci European Journal of  Oral Sciences

Loguercio Et Al-2009-European Journal of Oral Sciences

Embed Size (px)

DESCRIPTION

,

Citation preview

  • Influence of chlorhexidine digluconateconcentration and application time onresindentin bond strength durability

    Alessandro D. Loguercio1, RodrigoStanislawczuk1, Luceli G. Polli1,Jully A. Costa1, Milton D. Michel2,Alessandra Reis1

    1Department of Restorative Dentistry, School ofDentistry, Universidade Estadual de PontaGrossa, PR, Brazil; 2Department of MechanicalEngineering, School of MechanicalEngineering, University Estadual de PontaGrossa, Ponta Grossa, PR, Brazil

    Recent studies have pointed out that dentin bond-strength values measured immediately after formation ofthe bond do not always correlate with long-term bondstability (1), as degradation throughout the dentinbonding interface occurs rapidly (i.e. within 6 months)(2, 3). Bonding is created by impregnating the dentinsubstrate with blends of resin monomers, and the sta-bility of the bonded interface relies on the creation of acompact and homogenous hybrid layer. In the etch-and-rinse strategy, after preliminary etching to demin-eralize the substrate, resin monomers impregnate theporous etched substrate (4, 5) and thus stable bonds maybe achieved if the etched substrate is fully inltrated bythe adhesive (6).However, when incompletely inltrated zones occur

    along the bottom of hybrid layers within the acid-etcheddentin, a decreasing gradient of resin monomer diusion,with denuded collagen brils (79), is likely to occur.This region is susceptible to hydrolytic degradation inthe long term, leading to reductions in bond strength(10). Moreover, the elution of unpolymerized monomersfrom the hybrid layer in contact with oral uids isresponsible for the accelerated process of bond interfacedegradation. This leaves more collagen brils within thehybrid layer exposed and susceptible to the degradation

    process (11). The combined degradation by hydrolysis ofresin and/or collagen weakens the physical properties ofthe resindentin bond interface (11).Studies have revealed the contribution of host-derived

    proteinases, responsible for the breakdown of collagenmatrices, in the pathogenesis of dentin caries (12, 13),periodontal disease (14), and in the degradation of dentinbonding (15). It has been reported that mineralizeddentin contains collagenolytic and gelatinolytic enzymeactivities derived from the action of matrix metallo-proteinases (MMPs) (1618). Both in vitro and in vivostudies have shown that 2% chlorhexidine digluconate(CHX), applied for 60 s on demineralized dentin, post-pones the resindentin degradation of adhesive inter-faces, when compared with interfaces to which no CHXis applied (1922). Despite these advantages, the use of2% CHX for 60 s demands more chair-time during theadhesive procedure and this contrasts with the cliniciansneeds for simplication (23).It is also worthwhile mentioning that CHX solutions

    with concentrations (close to 0.01%) are highly cytotoxicto cultured cells (24, 25). Obviously, when CHX solutionis applied as a cavity cleanser there is enough dentinstructure to act as a barrier and protect the pulp cellsagainst the cytotoxic eects of dental materials or rinsing

    Loguercio AD, Stanislawczuk R, Polli LG, Costa JA, Michel MD, Reis A. Inuence ofchlorhexidine digluconate concentration and application time on resindentin bondstrength durability. Eur J Oral Sci 2009; 117: 587596. 2009 The Authors. Journalcompilation 2009 Eur J Oral Sci

    Although it is known that chlorhexidine application may preserve resindentin bondsfrom degradation, the lowest optimal concentration and application time have yet tobe established. This study evaluated the eects of dierent concentrations of chlor-hexidine digluconate and dierent application times on the preservation of resindentin bonds formed using two etch-and-rinse adhesives. In experiment 1, after acidetching, the occlusal demineralized dentin was rewetted either with water or with0.002, 0.02, 0.2, 2, or 4% chlorhexidine for 60 s. In experiment 2, the surfaces wererewetted with water, or with 0.002% or 2% chlorhexidine for 15 or 60 s. After this,both adhesives and composite resin were applied and light-cured. Bonded sticks(0.8 mm2) were tested under tension (0.5 mm min)1) immediately or after 6 months ofstorage in water. Two bonded sticks from each tooth were immersed in silver nitrateand analyzed quantitatively using scanning electron microscopy. Reductions in mi-crotensile bond strengths and higher silver nitrate uptake were observed for bothadhesives when the rewetting procedure was performed with water. Stable bonds weremaintained for up to 6 months under all chlorhexidine conditions tested, irrespectiveof the chlorhexidine concentration and application time. The use of 0.002% chlorh-exidine for 15 s seems to be sucient to preserve resindentin interfaces over a 6-month period.

    Alessandro D. Loguercio, UniversidadeEstadual de Ponta Grossa Mestrado emOdontologia, Rua Carlos Cavalcanti, 4748,Bloco M, Sala 64A Uvaranas, Ponta Grossa,Paran 84030-900, Brazil

    Telefax: +554232203741E-mail: [email protected]

    Key words: adhesive system; application time;chlorhexidine concentration; dentin; longevity

    Accepted for publication May 2009

    Eur J Oral Sci 2009; 117: 587596Printed in Singapore. All rights reserved

    2009 The Authors.Journal compilation 2009 Eur J Oral Sci

    European Journal ofOral Sciences

  • solutions. However, no data have so far been publishedconcerning the capacity of CHX molecules to diusethrough dentin tubules and reach the pulpal cells.The use of very low concentrations of CHX has been

    shown to strongly inhibit the collagenolytic activity ofpure MMPs (26) and dentin matrix-bound MMPs (27).Specic host-derived MMPs, responsible for the break-down of the collagen matrices (MMP-2, MMP-8, andMMP-9), can be inhibited by low concentrations ofCHX (0.00010.02%) (26). Fortunately, no cytotoxiceect on odontoblast-like cells has been reported forCHX at these concentrations (25). However, to the bestof the authors knowledge, no study has so far evaluatedthe eects of CHX concentrations on the durability ofresindentin bonding.The application time of MMP inhibitors is still a

    matter to be addressed. In a recent investigation,Stanislawczuk et al. (28) showed that when 2% CHXcontaining phosphoric acid was applied for 15 s thedurability of the resindentin bonds was preserved. Thisseems to indicate that even a short period of CHX incontact with the demineralized dentin appears to besucient to inhibit the action of specic host-derivedproteinases. Therefore, the ideal situation would be toapply CHX at a low concentration, and also for a shortperiod of time, as part of the strategy to simplify thebonding protocol.Therefore, the aim of this study was to evaluate the

    eect of dierent concentrations of CHX and dierentapplication times on the durability of the dentin bonds.To do so, the study was divided into two experiments.The objective of the rst experiment was to evaluate theearly and 6-month resindentin bond strength and silvernitrate uptake of two-step etch-and-rinse adhesivesrewetted with dierent CHX concentrations (0.002, 0.02,0.2, 2, and 4%) applied for 60 s. The second experimentexamined the early and 6-month resindentin bondstrength and the silver nitrate uptake (SNU) pattern oftwo-step etch-and-rinse adhesives after rewetting thedemineralized dentin with two CHX concentrations(0.002 and 2%) applied for two dierent applicationtimes (15 and 60 s). It was hypothesized that the lowestconcentration of CHX applied for only 15 s would be

    sucient to prevent reductions in resindentin bondstrength and deposition of SNU within adhesive andhybrid layers after 6 months of water storage.

    Material and methods

    Preparation of teeth

    One-hundred and twenty extracted, caries-free humanthird molars were used. The teeth were collected afterobtaining the patients informed consent. The PontaGrossa State University Review Board approved thisstudy. Teeth were disinfected in 0.5% chloramine, storedin distilled water, and used within 6 months of extraction.A at and supercial dentin surface was exposed on eachtooth after wet grinding the occlusal enamel on #180-gritsilicon carbide (SiC) paper. The enamel-free, exposeddentin surfaces were further polished on wet #600-grit SiCpaper for 60 s to standardize the smear layer.

    Microtensile bond strength (lTBS) test Two dierentsolvent-based, etch-and-rinse adhesive systems were tes-ted: Adper Single Bond (SB; 3M ESPE, St Paul, MN,USA), an ethanol/water-based system; and Prime &Bond 2.1 (PB; Dentsply De Trey, Konztanz, Germany),an acetone-based system (Table 1).For the rst experiment, 60 teeth were divided into 12

    groups (n = 5) according to the combination of themain factors adhesive (two levels) and rewettingsolution (six levels). The exposed dentin surfaces wereconditioned with the respective phosphoric acids of eachadhesive system for 15 s, rinsed o (15 s), air-dried(Table 1), and rewetted either with water (control group)or with aqueous solutions of 0.002, 0.02, 0.2, 2, or 4%CHX (Fleming drugstore, Ponta Grossa, PR, Brazil). Allrewetting procedures were performed for 60 s. After-wards, the adhesive systems were applied in accordancewith the manufacturers directions (Table 1). The aim ofthis rst experiment was to select two extreme CHXconcentrations that had benecial eects on the preser-vation of the resindentin bonds over a period of6 months.

    Table 1

    Adhesive systems: composition, groups, and application mode

    Adhesive systems Composition Application mode*

    Prime & Bond 2.1 (PB)(Dentsply De Trey,Konstanz, Germany)

    1. Caulk Tooth Conditioner Gel 34% phosphoric acid2. Adhesive UDMA, Bis-GMA, PENTA,

    butylated hydroxitoluene, 4-ethyl dimethyl aminobenzoate,cetilamine hydrouoride, initiator, and acetone

    a, b, c, d, e1, f, g

    Adper Single Bond (SB)(3M ESPE, St Paul, MN, USA)

    1. Scotchbond Etchant: 37% phosphoric acid2. Adhesive Bis-GMA, HEMA, dimethacrylates,

    polyalkenoic acid copolymer, initiators, water, and ethanol

    a, b, c, d, e2, f, g

    *a, acid-etch (15 s); b, rinse (15 s); c, air-dry (30 s); d, dentin rewetted with water or chlorhexidine digluconate (CHX) solution for 15or 60 s; e1, two coats of adhesive were applied for 20 s; e2, one coat of adhesive was applied for 10 s; f, air-dry for 10 s at 20 cm; g,light-cure (10 s; 600 mW cm)2).Bis-GMA, bisphenol A diglycidyl methacrylate; HEMA, 2-hydroxyethyl methacrylate; PENTA, dipentaerythritol pentacrylatemonophosphate; UDMA, urethane dimethacrylate.

    588 Loguercio et al.

  • For the second experiment, 60 teeth were divided into12 groups (n = 5) according to the combination of themain factors adhesive (two levels), rewetting solution(three levels), and application time (two levels). Thedentin surfaces were acid etched with phosphoric acid,rinsed o, air-dried, and rewetted either with water(control group) or with aqueous solutions of 0.002 or 2%CHX (Fleming drugstore). In this case, all rewettingprocedures were performed for 15 or 60 s. Afterwards,thee adhesive systems were applied according to themanufacturers instructions.For both experiments, the light-curing procedure was

    performed by means of a quartz-tungsten-halogen light(VIP; Bisco, Schaumburg, IL, USA; 600 mW cm)2) forthe recommended time (10 s). Resin-composite build-ups(Opallis; FGM, Joinville, SC, Brazil) were placed on thebonded surfaces (three increments of approximately1.5 mm each) and individually light activated for 40 seach. All bonding procedures were carried out by a singleoperator, and at 24C and 50% relative humidity.After the bonded teeth had been stored in distilled

    water at 37C for 24 h, they were longitudinally sec-tioned in both x and y directions across the bondedinterface using a diamond saw in a Labcut 1010 machine(Extec, Eneld, CT, USA), under water cooling at300 r.p.m., to obtain bonded sticks with a cross-sectionalarea of approximately 0.8 mm2. The number of prema-turely debonded sticks (D) per tooth during specimenpreparation was recorded. The remaining dentin thick-ness (RDT) was measured with a caliper and recorded(Absolute Digimatic, Mitutoyo, Tokyo, Japan). Thecross-sectional area of each stick was measured with thedigital caliper to the nearest 0.01 mm and recorded forsubsequent calculation of the lTBS. The bonded sticksthat originated from the same teeth were randomlydivided and assigned to be tested immediately, or after6 months of storage in distilled water at 37C. Thestorage solution was not changed and its pH wasmonitored monthly.Each bonded stick was attached to a jig in the uni-

    versal testing machine (Emic, Sao Jose dos Pinhais, PR,Brazil) with cyanoacrylate resin (Zapit; Dental Venturesof North America, Corona, CA, USA) and subjected toa tensile force of 0.5 mm min)1. The failure modes wereevaluated at 400magnication (microhardness HMV-2;Shimadzu, Tokyo, Japan) and classied as cohesive(failure exclusively within the dentin or composite; C),adhesive (failure at resin/dentin interface; A), oradhesive/mixed (failure at the resindentin interface thatincluded cohesive failure of the neighboring substrates;A/M).

    SNU measurement under scanning electron microscopy For both experiments, the amount of SNU into thehybrid layer and the adhesive layer was measured. Twobonded sticks from each tooth for each storage periodwere coated with two layers of nail varnish applied up towithin 1 mm of the bonded interfaces. The specimenswere rehydrated in distilled water for 10 min beforeimmersion in the tracer solution for 24 h. Ammoniacalsilver nitrate was prepared according to the protocol

    previously described by Tay et al. (29). The sticks wereplaced in ammoniacal silver nitrate in the dark for 24 h,rinsed thoroughly in distilled water, and immersed inphoto developing solution for 8 h under uorescent lightto reduce silver ions into metallic silver grains withinvoids along the bonded interface.All sticks were wet-polished with 600 SiC paper to

    remove the nail varnish. After this, the specimens wereplaced inside an acrylic ring, which was attached to adouble-sided adhesive tape, and embedded in epoxyresin. After the epoxy resin had set, the thickness of theembedded specimens was reduced to approximately halfby grinding with SiC papers under running water.Specimens were polished with a 1,000-grit SiC paper and1 and 0.25 lm diamond paste (Buehler, Lake Blu, IL,USA) using a polishing cloth. Specimens were ultra-sonically cleaned and demineralized in a 50% phosphoricacid solution for 3 s followed by immersion in 1%NaOCl for 10 min. Next, specimens were air-dried for24 h, mounted on aluminum stubs, and sputtered withgold (Sputter Coater IC 50; Shimadzu, Tokyo, Japan).Resindentin interfaces were analyzed using a scanningelectron microscope (SSX-550; Shimadzu) operated inthe back-scattering electron model. The working distancewas 10 mm and the accelerating voltage (ACCV) was15 Kv.Three pictures were taken of each specimen. The rst

    picture was taken in the center of the stick. The othertwo pictures were taken 0.3 mm to the left and right ofthe rst picture. As two sticks per tooth were evaluatedand a total of ve teeth were used for each experimentalcondition, 30 images were evaluated for each group.They were all taken by a technician who was blinded tothe experimental conditions under evaluation. The rela-tive percentage of SNU within the adhesive and hybridlayer areas was measured in all pictures using theuthscsa ImageTool 3.0 software (Department of DentalDiagnostic Science at The University of Texas HealthScience Center, San Antonio, TX, USA) by an authorblind to the test and control samples. First of all, thetotal area of the adhesive layer plus the hybrid layer wasrecorded. Then the area occupied by the silver nitratedeposits was delimitated by a software tool, summed,and the relative ratio between the total area vs. theimpregnated areas was calculated to give the percentageof SNU within each specic bonding interface.The experimental unit in this study was the tooth half,

    because half of the sample was tested immediately andthe other half was tested after 6 months. The lTBSvalues of all sticks from the same tooth half were aver-aged for statistical purposes. The prematurely debondedspecimens were included in the tooth half mean. Theaverage value attributed to specimens that failedprematurely during preparation was arbitrary andcorresponded to approximately half of the minimumlTBS that could be measured in this study (ca. 5.0 MPa).For SNU (%), the mean SNU of all pictures originatedfrom sticks from the same tooth half was averaged forstatistical purposes. The SNU of every test group wasexpressed as the mean of the ve tooth halves used pergroup and is expressed in per cent.

    Chlorhexidine on dentin adhesion 589

  • Before submitting the data for analysis using theappropriate statistical test, the KolmogorovSmirnovtest was performed to assess whether the data followed anormal distribution, and the Barletts test for equality ofvariances was performed to determine if the assumptionof equal variances was valid (30). After observing thenormality of the data distribution and the equality of thevariances, the lTBS (MPa) and SNU (%) data weresubjected to appropriate statistical analysis.In the rst experiment, the lTBS data and SNU were

    subjected to a two-way repeated-measures analysis ofvariance (chlorhexidine concentration vs. storage time)for each adhesive. The repeated measure was the storagetime. The post hoc test (Tukeys test at a = 0.05) wasused for pairwise comparisons. In the second experiment,the lTBS and SNU data were subjected to a three-wayrepeated-measures analysis of variance (chlorhexidineconcentration vs. application time vs. storage time) foreach adhesive. The repeated measure was the storagetime. A post hoc test (Tukeys test at a = 0.05) was usedfor pairwise comparisons.

    Results

    The mean cross-sectional area ranged from 0.78 to1.02 mm2 and no signicant dierence among groupswas detected (P > 0.05). The RDT for all specimensranged from 2.7 to 3.3, indicating that the interfaces werelocated in mid-dentin (31).

    lTBS

    For the rst experiment, the cross-product interactionCHX concentration vs. storage time for each adhesivesystem was statistically signicant (P < 0.05). Signi-cant reductions in the lTBS were observed in the controlgroup (i.e. when water was used instead of CHX as therewetting agent) (Table 2). The two etch-and-rinseadhesives tested had approximately the same overallfailure mode rates under similar experimental conditions(Table 3).For the second experiment, only the cross-product

    CHX concentration vs. storage time for both adhesives

    was statistically signicant (P < 0.05). Signicantreductions in resindentin bond-strength values wereobserved for both adhesives when the demineralizeddentin was rewetted with water, irrespective of theapplication time. By contrast, rewetting with CHX in thedierent concentrations and application times yieldedstable resindentin bonds after 6 months of water stor-age (Table 4). The two etch-and-rinse adhesives testedhad approximately the same overall failure mode ratesunder similar experimental conditions (Table 5).

    SNU

    In experiment 1, the cross-product interaction CHXconcentration vs. storage time was statistically signicantfor both adhesive systems (P < 0.05). For both adhe-sives, a lower percentage of SNU immediately aftertreatment was observed for the interfaces treated withCHX, irrespective of its concentration, when comparedwith the control group immediately after treatment(P = 0.01) (Table 6). After 6 months of water storage, asignicantly higher SNU was observed for both adhe-sives under the experimental conditions; however, thisuptake was much less pronounced for the CHX groupswhen compared with the controls (P = 0.001) (Table 6).Observe in Table 6 that among the dierent CHX con-centrations used, 4% CHX showed the highest SNU,which diered statistically from that of the lower CHXconcentrations (P = 0.01).In experiment 2, only the cross-product CHX con-

    centration vs. storage time for each adhesive system wasstatistically signicant (P < 0.05). For both adhesives, alower percentage of SNU immediately after treatmentwas observed for the interfaces treated with CHX, irre-spective of its concentration, when compared with thecontrol group immediately after treatment (P = 0.01)(Table 7). After 6 months of water storage, signicantlyhigher SNU was observed for both adhesives under theexperimental conditions; however, this uptake was muchless pronounced for the CHX-treated groups than for thecontrols (P = 0.001) (Table 7).Representative back-scattering scanning electron

    microscopy images captured at the resindentin inter-faces from experiment 2 are depicted in Figs 14.

    Table 2

    Overall microtensile bond strength means (in MPa) and the respective standard deviations obtained in each experimental condition inexperiment 1*

    Prime & Bond 2.1 Adper Single Bond

    Conditions Immediate 6 months Immediate 6 months

    Control 32.0 3.2 A 21.3 2.4 B 34.1 4.6 a 24.2 5.4 b0.002% CHX 28.2 3.4 A 25.1 3.2 A,B 35.2 6.2 a 31.1 5.3 a0.02% CHX 29.9 5.3 A 30.1 4.2 A 30.2 4.3 a 27.3 5.1 a,b0.2% CHX 30.9 4.7 A 27.4 4.6 A 34.2 4.1 a 36.2 4.0 a2% CHX 34.2 5.1 A 31.3 4.1 A 32.4 6.1 a 28.3 3.5 a4% CHX 26.7 4.7 A,B 21.1 3.5 B 26.3 4.2 a,b 24.3 4.2 b

    *Comparisons are only valid within each adhesive system. For each adhesive, means with the same capital letter or lower case letterare not signicantly dierent (P > 0.05).CHX, chlorhexidine digluconate.

    590 Loguercio et al.

  • Discussion

    The results of this study conrm previously publishedndings that resindentin interfaces bonded with sim-plied etch-and-rinse adhesives can degrade after shortperiods of time (2, 3). One mechanism of degradationproposed in the literature is the incomplete impregnationof resin into the collagen network and hybrid layer itself(1, 11).

    It has recently been speculated that the autodegrada-tion of dentin collagen brils is responsible for suchndings (15). This mechanism is initiated from beneaththe bonded interface, with the breakdown of acid-demineralized collagen matrices by host-derived MMPs.The low, but persistent, endogenous collagenolyticactivity can be completely inhibited by the use of pro-tease inhibitors (15). Chlorhexidine digluconate has thepotential to minimize the reductions in the resindentin

    Table 5

    Number and percentage of specimens (%) according to fracture pattern mode and the premature debonded specimens of eachexperimental condition from experiment 2*

    Prime & Bond 2.1 Adper Single Bond

    Immediate 6 months Immediate 6 months

    Pattern fracture A/M C Debonded A/M C Debonded A/M C Debonded A/M C Debonded

    Control (15 s) 27 (69.2) 02 (5.1) 10 (25.7) 38 (84.5) 2 (4.4) 5 (11.1) 26 (74.3) 2 (5.7) 7 (20.0) 35 (85.4) 4 (9.7) 2 (4.9)Control (60 s) 35 (85.4) 2 (4.9) 4 (9.7) 37 (84.1) 3 (6.8) 4 (9.1) 33 (76.7) 7 (16.3) 3 (7.0) 36 (87.8) 2 (4.9) 3 (7.3)0.002% CHX (15 s) 42 (75.0) 0 (0) 14 (25.0) 39 (79.6) 3 (6.1) 7 (14.3) 32 (80.0) 6 (15.0) 2 (5.0) 40 (95.2) 1 (2.4) 1 (2.4)0.002% CHX (60 s) 38 (82.6) 1 (2.2) 7 (15.2) 33 (82.5) 3 (7.5) 4 (10.0) 39 (83.0) 2 (4.3) 6 (12.7) 36 (92.3) 0 (0) 3 (7.7)2% CHX (15 s) 41 (87.2) 2 (4.3) 4 (8.5) 39 (88.6) 4 (9.1) 1 (2.3) 35 (85.4) 1 (2.4) 5 (12.2) 42 (85.7) 3 (6.1) 4 (8.2)2% CHX (60 s) 36 (94.7) 2 (5.3) 0 (0) 40 (88.9) 0 (0) 5 (11.1) 38 (90.5) 3 (7.1) 1 (2.4) 37 (90.2) 4 (9.8) 0 (0)

    *A/M, adhesive/mixed fracture mode; C, cohesive fracture mode; CHX, chlorhexidine digluconate; Debonded, premature debondedspecimens.

    Table 3

    Number and percentage of specimens (%) according to fracture pattern mode and the premature debonded specimens from eachexperimental condition from experiment 1*

    Patternfracture

    Prime & Bond 2.1 Adper Single Bond

    Immediate 6 months Immediate 6 months

    A/M C Debonded A/M C Debonded A/M C Debonded A/M C Debonded

    Control 24 (75.0) 0 (0) 8 (25.0) 34 (80.9) 0 (0) 8 (19.1) 26 (81.2) 0 (0) 6 (18.8) 29 (69) 3 (7.2) 10 (23.8)0.002% CHX 38 (77.6) 9 (18.4) 2 (4.0) 33 (71.7) 5 (10.9) 8 (17.4) 36 (73.5) 7 (14.3) 6 (12.2) 35 (76.1) 5 (10.9) 6 (13)0.02% CHX 38 (67.9) 0 (0) 18 (32.1) 36 (75.0) 11 (22.9) 1 (2.1) 26 (46.4) 0 (0) 30 (53.6) 36 (75) 7 (14.6) 5 (10.4)0.2% CHX 35 (87.5) 0 (0) 5 (12.5) 32 (94.1) 0 (0) 2 (5.9) 32 (80) 3 (7.5) 5 (12.5) 30 (88.2) 2 (5.9) 2 (5.9)2% CHX 39 (75) 1 (2.5) 9 (22.5) 36 (94.4) 0 (0) 2 (5.6) 37 (84.1) 2 (4.5) 5 (11.4) 34 (97.1) 0 (0) 1 (2.9)4% CHX 30 (88.2) 0 (0) 4 (11.8) 25 (76) 0 (0) 6 (24) 38 (77.6) 4 (8.2) 7 (14.2) 33 (70.2) 7 (14.9) 7 (14.9)

    *A/M, adhesive/mixed fracture mode; C, cohesive fracture mode; CHX, chlorhexidine digluconate; Debonded, premature debondedspecimens.

    Table 4

    Overall microtensile bond strength means (in MPa) and the respective standard deviations obtained in each experimental condition fromexperiment 2*

    Prime & Bond 2.1 Adper Single Bond

    Immediate 6 months Immediate 6 months

    Applicationtime 15 s 60 s 15 s 60 s 15 s 60 s 15 s 60 s

    Control 28.3 4.3 A 32.4 5.4 A 20.1 4.2 B 21.2 3.8 B 39.2 5.4 a 41.5 6.4 a 27.9 6.2 b 25.4 4.1 b0.002% CHX 25.7 2.4 A,B 29.2 3.4 A 23.2 4.1 A,B 27.0 3.6 A,B 41.4 4.8 a 43.2 6.1 a 37.2 6.1 a 40.1 3.7 a2% CHX 33.1 6.5 A 31.3 5.1 A 27.3 4.2 A,B 28.1 4.4 A 43.5 4.1 a 41.2 4.2 a 40.1 5.7 a 37.6 3.3 a

    *Comparisons are only valid within each adhesive system. For each adhesive, means with the same capital letter or lower case letterare not signicantly dierent (P > 0.05).CHX, chlorhexidine digluconate.

    Chlorhexidine on dentin adhesion 591

  • Table 6

    Overall mean percentage of silver nitrate uptake (%) within hybrid layer and adhesive layer and the respective standard deviations(in MPa) obtained in each experimental condition from experiment 1*

    Conditions

    Prime & Bond 2.1 Adper Single Bond

    Immediate 6 months Immediate 6 months

    Control 26.3 4.6 B,C 42.3 5.6 D 24.5 3.6 b,c 38.1 6.5 d0.002% CHX 12.1 3.3 A 24.6 4.3 B 10.1 2.3 a 22.9 4.2 b0.02% CHX 12.2 4.2 A 25.4 4.1 B 11.1 3.5 a 21.4 2.4 b0.2% CHX 10.3 2.9 A 22.1 3.8 B 9.6 2.7 a 22.5 3.2 b2% CHX 9.8 3.2 A 27.8 3.9 B,C 12.4 4.3 a 24.5 5.4 b,c4% CHX 14.6 5.6 A 32.4 4.6 C 16.2 3.6 a 27.4 3.6 c

    *Comparisons are only valid within each adhesive system. For each adhesive, means with the same capital letter or lower case letterare not signicantly dierent (P > 0.05).CHX, chlorhexidine digluconate.

    Table 7

    Overall mean percentage of silver nitrate uptake within hybrid layer and adhesive layer and the respective standard deviations (MPa)obtained in each experimental condition from experiment 2*

    Prime & Bond 2.1 Adper Single Bond

    Immediate 6 months Immediate 6 months

    Applicationtime 15 s 60 s 15 s 60 s 15 s 60 s 15 s 60 s

    Control 28.7 3.4 B 26.3 4.2 B 39.5 6.3 C 40.2 5.4 C 18.3 3.7 b 22.4 4.2 b 45.3 6.4 c 42.4 3.6 c0.002% CHX 12.3 2.6 A 12.6 2.3 A 24.1 3.9 B 23.6 2.8 B 10.3 1.8 a 9.8 2.5 a 21.3 5.6 b 24.3 4.8 b2% CHX 11.2 2.5 A 10.2 5.2 A 25.1 3.5 B 26.5 3.8 B 10.4 2.3 a 10.2 3.2 a 26.7 4.3 b 22.1 3.8 b

    *Comparisons are only valid within each adhesive system. For each adhesive, means with the same capital letter or lower case letterare not signicantly dierent (P > 0.05).CHX, chlorhexidine digluconate.

    A B C

    D E F

    Adper Single Bond

    15 s

    Fig. 1. Representative back-scattered scanning electron microcopy images of the resindentin interfaces bonded with Adper SingleBond immediately after treatment (AC) or 6 months after treatment (DF), in which the rewetting time was 15 s. In the samplestested immediately after treatment, only a few areas of silver nitrate uptake were observed within the hybrid layer (AC), mainly inthe group in which dentin was rewetted with water (white stains indicated by the white pointer, panel A). After 6 months, silvernitrate uptake was higher in the hybrid layer only for the control group (water) (white pointer, panel D). In panels E and F, only asmall amount of silver nitrate was taken up in the hybrid layer (white stains indicated by the white pointer, panel E) (AL, adhesivelayer; Co, composite; De, dentin; and HL, hybrid layer).

    592 Loguercio et al.

  • bond strengths commonly observed for simplied con-ventional adhesives after long-term water storage andalso to preserve the morphological properties of hybridlayers by inhibiting host-derived proteases (1922, 28).Therefore, one might consider that preventing the

    degradation of these incompletely resin-inltrated colla-gen brils and the hybrid layer by MMPs in durabilitystudies is an important issue to investigate, because thiscould be the key to increasing the durability of restora-tions that involve dentin bonding. While some in vitro

    and in vivo studies have suggested that the application of2% CHX solution, a MMP inhibitor, to acid-etcheddentin for 60 s could minimize the degradation of thedentin bond over time, the brillar network of the hybridlayers formed without pretreatment with CHX solutiondepicts a signicant and progressive disintegration(1922, 28). This was conrmed to some extent in thepresent investigation. Signicant reductions in the lTBSand higher SNU were seen within the adhesive andhybrid layers after 6 months of water storage in the

    A B C

    D E F

    Adper Single Bond

    Fig. 2 Representative back-scattered scanning electron microcopy images of the resindentin interfaces bonded with Adper SingleBond immediately after treatment (AC) or 6 months after treatment (DF), in which the rewetting time was 60 s. In the samplestested immediately after treatment, only a few areas of silver nitrate uptake in the hybrid layer (panels AC) were observed (whitestains indicated by the white pointer, panel A). After 6 months, silver nitrate uptake was highest in the hybrid layer and across theentire thickness of the adhesive layer (white stains indicated by the white pointer, panel D). After 6 months, in the 0.002% and 2%chlorhexidine digluconate (CHX) groups (panels E and F, respectively) only a small amount of silver nitrate was taken up (whitestains indicated by white pointer, panel E). (AL, adhesive layer; Co, composite; De, dentin; and HL, hybrid layer).

    Prime & Bond 2.1

    A B C

    D E F

    Fig. 3 Representative back-scattered scanning electron microcopy images of the resindentin interfaces bonded with Prime & Bond2.1 immediately after treatment (AC) or 6 months after treatment (DF), after rewetting for 15 s. In the samples tested immediatelyafter treatment, a considerable amount of silver nitrate uptake within the hybrid layer was observed in the control group (water)(white stains indicated by the white pointer, panel A), compared with the groups rewetted with 0.002% and 2% chlorhexidinedigluconate (CHX) (panels B and C, respectively). In panel A, signs of phase separation can be observed (black star). After 6 months,the silver nitrate uptake was higher in the hybrid layer only for the control group (water) (white stains indicated by white pointer,panel D). In panels E and F, only a small amount of silver nitrate uptake was seen in the hybrid layer (white stains indicated by thewhite pointer, panel F) (AL, adhesive layer; Co, composite; De, dentin; and HL, hybrid layer).

    Chlorhexidine on dentin adhesion 593

  • control groups (rewetted with water) for both adhesivesystems tested.However, contrasting ndings were observed for

    CHX-treated groups. Very few silver nitrate depositswere seen within the hybrid layer after 6 months and nodegradation of the resindentin bond strengths wasdetected.Interestingly, the good performance of CHX (in terms

    of resindentin bond strengths) in the preservation of thedentin bonds over time was independent of its concen-tration. This is in agreement with the conclusion previ-ously reached by Gendron et al. (26), who found thatextremely low concentrations of CHX (around 0.01%)can inhibit protease activity. It has been demonstratedthat pure MMP-2, MMP-8, and MMP-9 can be inhibitedby CHX concentrations of 0.0001, 0.02, and 0.002%,respectively, through proteolytic action (26). Recently,Carrilho et al. (27) evaluated the collagenolytic activityof the dentin-bound MMPs and showed that the colla-genolytic activity of demineralized dentin was signi-cantly reduced by pretreatment with 2% CHX incomparison with non-treated specimens.Gendron et al. (26) proposed two dierent mecha-

    nisms of action involved in MMP inhibition: a chelatingmechanism for the inhibition of MMP-2 and MMP-9;and the interaction of CHX with the essential sulfhydrylgroups and/or cysteine present in MMP-active sites inthe case of MMP-8.Therefore, it seems that 0.002% CHX is sucient to

    prevent host-derived MMPs from degrading exposedcollagen brils, at least during the time-period evaluatedin the present investigation (i.e. 6 months). The use oflow CHX concentrations (0.002%) conveys a distinctadvantage in that it is not toxic to human periodontal

    cells (24) or odontoblast-like cells (25); therefore, this lowCHX concentration can be regarded as a potentialchemical agent for use as a cavity cleanser.The high substantivity of CHX may explain why the

    application time did not have a signicant eect foreither the 0.002 or 2% CHX solutions. The termsubstantivity has been much more correlated in the lit-erature to the residual antibacterial activity of CHX inhuman dentin (32, 33) than to the inhibitory eect ofCHX on MMP activity. Chlorhexidine digluconate isone of the most commonly used antimicrobial agentsbecause it retains a therapeutic eect for a prolongedperiod of time. The substantivity of CHX is related to therelease of positively charged molecules from CHX-treated surfaces and its ability to adsorb onto surfaces ofthe oral cavity (33, 34). Theoretically, this can also occurin the demineralized exposed collagen brils, and is theexplanation for the bonds being preserved after long-term water exposure.One may suggest that CHX is likely to bind to collagen

    brils at a very fast rate, and thus even short periods oftime, such as 15 s, seem to be sucient to guarantee suchbinding. This hypothesis was raised on the basis of theresults of a recent investigation (28). A 2% CHX solu-tion containing phosphoric acid was able to maintain thestability of the resindentin bond of two simplied etch-and-rinse adhesives after 6 months of water storage,suggesting that only a few seconds of contact with CHXmight be sucient to inhibit the MMP activity, corrob-orating the ndings of the present investigation.Several studies have so far evaluated the eect of CHX

    in the immediate and long-term durability of dentinbonds (1922), but among them only one has measuredthe amount of silver nitrate deposits within the hybrid

    Prime & Bond 2.1

    A B C

    D E F

    Fig. 4 Representative back-scattered scanning electron microcopy images of the resindentin interfaces bonded with Prime & Bond2.1 immediately after treatment (AC) or 6 months after treatment (DF), when the rewetting was applied for 60 s. In the samplestested immediately after treatment, no signs of silver nitrate uptake (panels B and C), in comparison with a small amount of silvernitrate uptake within the hybrid layer, was observed in the control group (water) (white stains indicated by the white pointer, panelA). After 6 months, the groups rewetted with chlorhexidine digluconate (CHX) (0.002 and 2%, respectively in panels E and F)showed preservation of the hybrid layer. Only a small amount of silver nitrate uptake was seen in panel E (white stains indicated bywhite pointer). The highest amount of silver penetration occurred in the hybrid layer in the group in which demineralized dentin wasrewetted with water (white stains indicated by a white pointer, panel D) (AL, adhesive layer; Co, composite; De, dentin; and HL,hybrid layer).

    594 Loguercio et al.

  • layer (28). It was previously hypothesized that CHXcould reduce the amount of SNU after 6 months but thatimprovements would not be expected immediately aftertreatment. Immediately after treatment with CHX, veryfew silver nitrate deposits were observed within thehybrid layer compared with the control, as shown byStanislawczuk et al. (28). As CHX has cationic prop-erties it can bind to sites containing collagen, ashypothesized by Carrilho et al. (22) and possibly tocalcium at the bottom at the hybrid layer, reducing thenanopores within the hybrid layer to which silver nitratecan deposit. However, this still deserves further investi-gation.One cannot rule out the fact that despite the advan-

    tages of using 2% CHX, its use for 60 s after acid etchingincludes another bonding step during the restorativeprocedure and this works against the clinicians need forsimplication (23). In this research, the application timeof CHX was reduced (15 s) and this reduction in appli-cation time did not jeopardize the benets of CHX in thepreservation of the dentin bonds.Therefore, the results of the present investigation

    suggest that a low concentration of CHX, applied for15 s, is sucient to preserve dentin bonds for at least6 months under the laboratory conditions of this study.However, further in vivo studies are needed to clarifywhether the use of a 0.002% CHX solution for 15 s isable to preserve resindentin bonds after long-termfunction.

    Acknowledgements This study was partially supported byCNPq grants 473101/2006-8 and 305870/2004-1. The authorswould like to thank FGM Dental Products for the donation ofthe composite resin used in this study.

    References1. De Munck J, Van Landuyt K, Peumans M, Poitevin A,

    Lambrechts P, BraemM, VanMeerbeek B. A critical reviewof the durability of adhesion to tooth tissue: methods andresults. J Dent Res 2005; 84: 118132.

    2. Reis A, Grandi V, Carlotto L, Bortoli G, Patzlaff R,Rodrigues AccorinteMLR, Loguercio AD. Effect of smearlayer thickness and acidity of self-etching solutions on early andlong-term bond strength to dentin. J Dent 2005; 33: 549559.

    3. Tay FR, Pashley DH, Suh BI, Hiraishi N, Yiu CK. Watertreeing in simplified dentin adhesives deja` vu? Oper Dent2005; 30: 561579.

    4. Nakabayashi N, Kojima K, Masuhara E. The promotion ofadhesion by the infiltration of monomers into tooth substrates.J Biomed Mater Res 1982; 16: 265273.

    5. VanMeerbeek B, DeMunck J, Yoshida Y, Inoue S, VargasM, Vijay P, Van Landuyt K, Lambrechts P, Vanherle G.Buonocore memorial lecture. Adhesion to enamel and dentin:current status and future challenges. Oper Dent 2003; 28:215235.

    6. Yoshida Y, Van Meerbeek B, Snauwaert J, Hellemans L,Lambrechts P, Vanherle G, Wasaka K, Pashley DH. Anovel approach to AFM characterization of adhesive tooth-biomaterial interfaces. J Biomed Mater Res 1999; 47: 8590.

    7. Armstrong SR, Vargas MA, Fang Q, Laffoon JE. Micro-tensile bond strength of a total-etch 3-step, total-etch 2-step,self-etch 2-step, and a self-etch 1-step dentin bonding systemthrough 15-month water storage. J Adhes Dent 2003; 5: 4756.

    8. Wang Y, Spencer P. Hybridization efficiency of the adhesive/dentin interface with wet bonding. J Dent Res 2003; 82:350354.

    9. Hashimoto M, Ohno H, Kaga M, Sano H, Endo K, OguchiH. The extent to which resin can infiltrate dentin by acetone-based adhesives. J Dent Res 2002; 81: 7478.

    10. BurrowMf, SatohM, Tagami J. Dentin bond durability afterthree years using a dentin bonding agent with and withoutpriming. Dent Mater 1996; 12: 302307.

    11. HashimotoM, OhnoH, SanoH,KagaM,OguchiH. In vitrodegradation of resindentin bonds analyzed by microtensilebond test, scanning and transmission electron microscopy.Biomaterials 2003; 24: 37953803.

    12. Tjaderhane L, Larjava H, Sorsa T, Uitto VJ, Larmas M,Salo T. The activation and function of host matrix metallo-proteinases in dentin matrix breakdown in caries lesions. J DentRes 1998; 77: 16221629.

    13. Sulkala M, Wahlgren J, Larmas M, Sorsa T, Teronen O,Salo T, Tjaderhane L. The effects of MMP inhibitors onhuman salivary MMP activity and caries progression in rats.J Dent Res 2001; 80: 15451549.

    14. Lee W, Aitken S, Sodek J, Mcculloch CA. Evidence of adirect relationship between neutrophil collagenase activity andperiodontal tissue destruction in vivo: role of active enzyme inhuman periodontitis. J Periodont Res 1995; 30: 2333.

    15. Pashley DH, Tay FR, Yiu C, Hashimoto M, Breschi L,Carvalho RM, Ito S. Collagen degradation by host-derivedenzymes during aging. J Dent Res 2004; 83: 216221.

    16. Martin-De Las Heras S, Valenzuela A, Overall CM. Thematrix metalloproteinase gelatinase A in human dentine. ArchOral Biol 2000; 45: 757765.

    17. Mazzoni A, Mannello F, Tay FR, Tonti GA, Papa S,Mazzotti G, Di Lenarda R, Pashley DH, Breschi L.Zymographic analysis and characterization of MMP-2 and -9forms in human sound dentin. J Dent Res 2007; 86: 436440.

    18. Sulkala M, Tervahartiala T, Sorsa T, Larmas M, Salo T,Tjaderhane L. Matrix metalloproteinase-8 (MMP-8) is themajor collagenase in human dentin. Arch Oral Biol 2007; 52:121127.

    19. Hebling J, Pashley DH, Tjaderhane L, Tay FR. Chlorhex-idine arrests subclinical degradation of dentin hybrid layers invivo. J Dent Res 2005; 84: 741746.

    20. Brackett WW, Tay FR, Brackett MG, Dib A, Sword RJ,Pashley DH. The effect of chlorhexidine on dentin hybridlayers in vivo. Oper Dent 2007; 32: 107111.

    21. CarrilhoMR, Carvalho RM, De GoesMF, Di Hipolito V,Geraldeli S, Tay FR, Pashley DH, Tjaderhane L. Chlor-hexidine preserves dentin bond in vitro. J Dent Res 2007; 86:9094.

    22. CarrilhoMR, Geraldeli S, Tay F, De GoesMF, CarvalhoRM, Tjaderhane L, Reis AF, Hebling J, Mazzoni A,Breschi L, Pashley D. In vivo preservation of the hybrid layerby chlorhexidine. J Dent Res 2007; 86: 529533.

    23. Tay FR, Pashley DH. Have dentin adhesives become toohydrophilic? J Can Dent Assoc 2003; 69: 726731.

    24. Chang YC, Huang FM, Tai KW, Chou MY. The effect ofsodium hypochlorite and chlorhexidine on cultured humanperiodontal ligament cells. Oral Surg Oral Med Oral PatholOral Radiol 2001; 92: 446450.

    25. Souza LB, De Aquino SG, De Souza PP, Hebling J, CostaCA. Cytotoxic effects of different concentrations of chlor-hexidine. Am J Dent 2007; 20: 400404.

    26. Gendron R, Greiner D, Sorsa T,Mayrand D. Inhibition ofthe activities of matrix metaloproteinases 2, 8 and 9 bychlorhexidine. Clin Diagn Lab Immunol 1999; 6: 437439.

    27. Carrilho MR, Tay FR, Donnelly AM, Agee KA,Tjaderhane L, Mazzoni A, Breschi L, Foulger S, PashleyDH. Host-derived loss of dentin matrix stiffness associated withsolubilization of collagen. J Biomed Mater Res B Appl Biomater2009; 90: 373380.

    28. Stanislawczuk R, Amaral RC, Zander-Grande C, GaglerD, Reis A, Loguercio AD. Chlorhexidine-containing acid

    Chlorhexidine on dentin adhesion 595

  • conditioner preserves longevity of resin-dentin bonds. OperDent 2009; 34: 483492.

    29. Tay FR, Pashley DH, Yoshiyama M. Two modes of nano-leakage expression in single-step adhesives. J Dent Res 2002; 81:472476.

    30. Montgomery DC. Design and analysis of experiments. 5th edn.New York: John Wiley & Sons, 1991.

    31. Yoshiyama M, Carvalho RM, Sano H, Horner J, BrewerPD, Pashley DH. Interfacial morphology and strength ofbonds made to superficial versus deep dentin. Am J Dent 1995;8: 297302.

    32. Basrani B, Santos JM, Tjaderhane L, Grad H, GorduysusO, Huang J, Lawrence HP, Friedman S. Substantive anti-microbial activity in chlorhexidine-treated human root dentin.Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002; 94:240245.

    33. Rosenthal S, Spangberg L, Safavi K. Chlorhexidine sub-stantivity in root canal dentin. Oral Surg Oral Med Oral PatholOral Radiol Endod 2004; 98: 488492.

    34. Gjermo P, Bonesvoll P, Rolla G. Relationship betweenplaque-inhibiting effect and retention of chlorhexidine in thehuman oral cavity. Arch Oral Biol 1974; 19: 10311034.

    596 Loguercio et al.