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d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 885–892

avai lab le at www.sc iencedi rec t .com

journa l homepage: www. int l .e lsev ierhea l th .com/ journa ls /dema

urability of resin–dentin bonds: Effects ofirect/indirect exposure and storage media

anuel Toledanoa,∗, Raquel Osorioa, Estrella Osorioa, Fatima S. Aguileraa,onica Yamautib, David H. Pashleyc, Franklin Tayc

Department of Dental Materials, School of Dentistry, University of Granada, Granada, SpainCariology and Operative Dentistry, Department of Restorative Sciences, Graduate School,okyo Medical and Dental University, Tokyo, JapanDepartment of Oral Biology & Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, GA, USA

r t i c l e i n f o

rticle history:

eceived 10 January 2006

ccepted 6 June 2006

eywords:

esin

entin

dhesive

ongevity

elf-etching

otal-etch

egradation

a b s t r a c t

Objectives. To evaluate the longevity of resin–dentin bonds of three adhesives using different

storage media and specimen size.

Methods. Flat dentin surfaces from extracted human third molars were bonded with: a two-

step etch-and-rinse self-priming adhesive (Single Bond), a two-step self-etching adhesive

(Clearfil SE Bond), and a one-step self-etching adhesive (One-Up Bond F). Composite build-

ups were constructed. The bonded teeth were stored under three conditions: dry, distilled

water, or mineral oil. Half of the specimens were stored as intact bonded teeth (Indirect

Exposure/IE). The other half were first sectioned into beams and stored under same condi-

tions (Direct Exposure/DE). After storage periods of 24 h, 3 months or 1 year, the intact teeth

(IE) were sectioned into beams and both subgroups (DE and IE) were tested for microtensile

bond strengths. Results were analyzed with multiple ANOVA and Student–Newman–Keuls

tests. Fractographic analysis was performed by SEM.

Results. After 24 h, Single Bond and Clearfil SE Bond performed equally and were superior

to One-Up Bond F. After 3 months of DE to water storage, decreases in bond strengths were

observed for Single Bond and One-Up Bond F, this decrease occurred for Clearfil SE Bond after

12 months of water storage. Bonded specimens aged in dry did not alter bond strengths over

time. Bond strength increased when Single Bond was stored in mineral oil after 3 and 12

months. Micromorphological alterations were evident after water storage.

Significance. Although dentin bond strength of all the adhesives fell over time in DE, SE Bond

fells the least.

emy

dentin with phosphoric acid to remove the smear layer and to

© 2006 Acad

. Introduction

onds created by adhesives to dentin are not as durable as they

ere previously conjectured, due to mechanical and chemi-

al degradation [1–3]. Thus, immediate and long-term bondtrength evaluations are necessary for product evaluation [4].

∗ Corresponding author at: Avda. de las Fuerzas Armadas 1, 1B, 18014 GE-mail address: toledano@ugr.es (M. Toledano).

109-5641/$ – see front matter © 2006 Academy of Dental Materials. Puoi:10.1016/j.dental.2006.06.030

of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Dentin bonding is usually achieved via two alternativestrategies. Etch-and-rinse adhesives are used pretreating

ranada, Spain. Tel.: +34 958 243788; fax: +34 958 240908.

demineralise the underlying 5–8 �m of dentin. This is followedby the application of a primer and an adhesive in two differentsteps or in a single step. With such a technique, incomplete

blished by Elsevier Ltd. All rights reserved.

l s 2

886 d e n t a l m a t e r i a

expansion of collapsed collagen matrix after air-drying mayimpair resin infiltration and compromise bonding [5]. In theself-etching approach, the acid and the primer are combinedinto one solution to form an acidic primer mixture [3] priorto the application of a subsequent solvent-free adhesive. Self-etch one-step adhesives contain all components in either atwo-bottle set or in a single bottle. With the use of self-etchingsystems, it is generally accepted that there is less discrepancybetween the depth of demineralisation and the depth of resininfiltration [3].

Factors that may contribute to the degradation ofresin–dentin bonds include exposure to water [6], incompletehybridization [4] and the presence of residual solvent or water[7]. Preservation of the adjacent composite–enamel bond hasbeen reported to offer protection against the degradation ofcomposite–dentin bonds [2].

The objective of this study was to investigate the effects ofprolonged aging in a dry condition, in distilled water, or min-eral oil, on the bond strengths of different adhesives to dentin.The null hypothesis tested was that there are no differences indentin bond strength when resin–dentin interfaces were agedfor variable periods under differential storage conditions.

2. Material and methods

One hundred sixty-two caries-free extracted human thirdmolars that were stored in 0.5% chloramine T (Sigma–Aldrich

S.A., Madrid, Spain) at 4 ◦C for less than 1 month were used forthe study. The specimens were sectioned below the dentino-enamel junction and ground flat (180-grit) under runningwater to provide smear layer-covered dentin surfaces. Tested

Table 1 – Bonding agents used in the experimental groups

Product (manufacturercomponents)

Principle ingredients (according to m

Single BondAdhesive 2-Hydroxyethylmethacrylate, water, eth

Bis-GMA, dimethacrylates, amines,methacrylate-functional copolymer of pand polyitaconic acids

Clearfil SE BondPrimer 10-Methacryloyloxydecyl dihydrogen ph

2-hydroxyethyl methacrylate; hydrophildimethacrylate; di-camphorquinone;N,N-diethanol-p-touidine; water.

Bond 10-Methacryloxydecyl dihydrogen phospBis-GMA; 2-hydroxyethyl methacrylate;dimethacrylate; di-camphorquinone;N,N-diethanol-p-toluidine; silanated coll

One-Up Bond FBonding agent A Phosphoric monomer, MAC-10, multi-fu

methacrylic monomers, photo-initiator

Bonding agent B Fluoroaminosilicate glass filler, water,mono-functional monomers, dye-sensitderivate

Abbreviations: Bis-GMA: bis-phenol A diglycidylmethacrylate; MAC-10: me

3 ( 2 0 0 7 ) 885–892

adhesives were: Single Bond (SB) (3MESPE Dental Products, St.Paul, MN, USA), a two-step etch-and-rinse self-priming adhe-sive, Clearfil SE Bond (SEB) (Kuraray Co. Ltd., Osaka, Japan),a two-step self-etching primer adhesive system and One-UpBond F (OUB) (Tokuyama Europe GmbH, Dusseldorf, Germany),a single-step self-etching adhesive. Adhesives were appliedin accordance with the manufacturers’ instructions (Table 1).After the application of the adhesives to dentin, 6 mm highresin composite build-ups were constructed incrementally(1.5 mm) with Tetric Ceram (Vivadent, Schaan, Liechtenstein).Each layer of composite was light activated for 40 s with aTranslux EC halogen light-curing unit (Kulzer GmbH, Bere-ich Dental, Wehrheim, Germany). The output intensity wasmonitored with a Demetron Curing Radiometer (Model 100)(Demetron Research Corporation, Danbury, CT, USA). A min-imal output intensity of 600 mW/cm2 was employed for theexperiments.

After the preparation of resin-bonded specimens, half ofthe teeth, designated as “indirect exposure” (IE) were storedintact (without sectioning) at 37 ◦C in: (a) distilled watercontaining 0.02% sodium azide (Sigma–Aldrich S.A., Madrid,Spain) pH 7.0, 37 ◦C; (b) dry condition (stored in air, 40% relativehumidity, 37 ◦C); (c) mineral oil (Sigma–Aldrich S.A., Madrid,Spain), 37 ◦C. These teeth were aged for 24 h, 3 months and 1year prior to bond testing. The other half of the teeth, desig-nated as “direct exposure” (DE) were vertically sectioned intoserial slabs and further into beams with cross-sectioned areasof 1 mm2. These resin-bonded beams were aged in the same

three media and for the same three periods as described above.

After each aging period, the intact teeth (Group IE), in eachstorage medium, were sectioned according to the methoddescribed to produce beams for microtensile bond strength

anufacturers) Mode/steps of application

anol,

olyacrylic

Etch for 15 s. Rinse with water spray for 10 s,leaving tooth moist. Apply two consecutivecoats of the adhesive with a fully saturatedbrush tip. Dry gently for 2–5 s. Light cure for10 s.

osphate;ic

Apply primer for 20 s. Mild air stream. Applybond. Gentle air stream. Light cure for 10 s.

hate;hydrophobic

oidal silica

nctional Mix Bonding Agent A and Bonding Agent Buntil the mixed turns homogeneously pink.Apply the mixture.

izer, borateLeave the surface undisturbed for 20 s. Lightcure for 10 s. The pink color should turn to apale brown after light irradiation

thacryloyloxyalkyl acid phosphate.

2 3 ( 2 0 0 7 ) 885–892 887

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d e n t a l m a t e r i a l s

esting. The aged sectioned beams (Group DE) were retrievedrom the corresponding medium and tested in the same man-er. Each beam was attached to a modified Bencor multi-Testing apparatus (Danville Engineering Co., Danville, CA, USA)ith cyanoacrylate adhesive (Zapit, Dental Venture of America

nc., Corona, CA, USA) and stressed to failure in tension using aniversal testing machine (Instron 4411, Instron Corporation,anton, MA, USA) at a crosshead speed of 0.5 mm/min. The

ractured beams were removed from the testing apparatus andhe cross-sectional area at the site of failure was measuredo the nearest 0.01 mm with a pair of digital callipers (Syl-ae Ultra-Call, Li, USA). Bond strength values were expressedn MPa and analyzed with a multiple ANOVA to examine theontributions of adhesive type, storage medium, storage timend exposure method, and the interaction of these four fac-ors on microtensile bond strength. Post hoc multiple com-arisons were conducted using Student–Newman–Keuls testst ˛ = 0.05. The fractured specimens were examined with atereomicroscope (Olympus SZ-CTV, Olympus, Tokyo, Japan)t 40× magnification to determine the mode of failure. Failureodes were classified as adhesive, mixed or cohesive failure.

epresentative specimens from each subgroup were dessi-ated and gold-coated and observed with a scanning electronicroscope (SEM) (Zeiss DSM-950, Karl-Zeiss, Germany) at an

ccelerating voltage of 20 kV to examine the morphology ofhe debonded interfaces.

. Results

he adhesive type (F = 148.0; P < 0.0001), the storage mediumF = 340.0; P < 0.0001), the aging period (F = 291.7; P < 0.0001)nd the mode of exposure (F = 40.6; P < 0.0001) significantlyffected the microtensile bond strength to dentin. Interactionsetween factors were also significant. The mean bond strengthalues obtained for the different groups are shown in Table 2.

After 24 h of water storage, Single Bond and Clearfil SE Bondhowed higher bond strengths (P < 0.05) than One-Up Bond F inoth IE and DE groups regardless of storage media. Direct expo-ure of resin–dentin interface resulted in a significant decreasen microtensile bond strengths when Single Bond and One-Upond F were tested after 3 and 12 months of water immersion,nd when Clearfil SE Bond was tested after 12 months of watermmersion. In indirectly exposed specimens, the only signifi-ant changes in bond strengths occurred in specimens bondedith One-Up Bond F stored in water. Bond strengths from

pecimens that were maintained in a dry environment weretable over time and followed the trend: Single Bond = ClearfilE Bond > One-Up Bond F. Bond strengths increased when Sin-le Bond was stored in mineral oil after 3 and 12 months andlearfil SE Bond and One-Up Bond F showed no changes.

Table 3 summarizes the percentage failure modes of theebonded specimens according to the adhesive type, the stor-ge medium, the aging time and their mode of exposure.ixed fracture modes were frequently identified in all groups.o pure cohesive failures were observed in any group. Low

ond strengths (i.e. One-Up Bond F) were associated withigher percentages of adhesive failures. In general, the num-er of adhesive failures also increased with aging of directlyxposed specimen beams in water.

Tabl

e2

–M

Spec

ime

Ind

irec

t-IE

2D

irec

t-D

E24

Ind

irec

t-IE

3D

irec

t-D

E3

Ind

irec

t-IE

1D

irec

t-D

E12

For

each

hor

dif

fere

nce

(P

888 d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 885–892

Table 3 – Percentage distribution of failure mode: A: adhesive, M: mixed

Time Watera Drya Oila Waterb Dryb Oilb Waterc Dryc Oilc

A M A M A M A M A M A M A M A M A M

24 h 22 78 28 72 27 73 25 75 22 78 25 75 48 52 14 86 74 263 months 55 45 19 81 9 91 24 76 17 83 22 78 78 22 10 90 83 1712 months 49 51 11 89 15 85 47 53 91 9 28 72 87 13 49 51 84 16

a Single Bond (SB).

b Clearfil SE Bond (SEB).c One-Up Bond F (OUB).

Fractographic analysis of the debonded dentin surfaces areshown in Figs. 1–4. After water immersion, Single Bond fre-quently failed at the top of the hybrid layer (Fig. 1) during theinitial period of testing. Resin was not observed on dentin sur-faces after 12 months of water immersion and the morphologyof the intertubular dentin was altered as a result of the lossof the incompletely infiltrated collagen fibrils (Fig. 1d). Whenthe self-etching adhesive systems were used, failures alongthe top, the bottom (Fig. 2) or within the hybrid layers wereobserved for Clearfil SE Bond (Fig. 3). The One-Up Bond F spec-imen exhibited mixed failures, frequently failing at the top of

the hybrid layer with evidence of a more aggressive etchingpattern compared to that of Clearfil SE Bond (Fig. 2). When thespecimens from the three adhesives were maintained in a dryenvironment, the predominant failure mode was mixed fail-

Fig. 1 – SEM observations of the fractured dentin surface of two safter 3 months of water immersion. Main fracture is at the top ofpreparation may be observed). (b) At a higher magnification, som(c) An adhesive failure, after 12 months of water immersion. (d) Asurface showed a lack of both collagen and resin.

ure, with frequent fracture within the resin composite (datanot shown). When the specimens were aged in mineral oil,most of the failures were mixed. An example of optimal resinintertubular infiltration is shown in Fig. 4.

4. Discussion

Microtensile bond strengths of the etch-and-rinse adhesive(SB) and the two-step self-etch adhesive (SE) were signifi-cantly higher than the one-step self-etch adhesive (OUB) after

24 h of storage in the different media (Table 2). OUB alsoexhibited the highest frequency of adhesive failures (Table 3).A general consensus exists regarding the poor performanceof one-step adhesive systems [2,8–10]. Previous studies have

pecimens bonded with Single Bond. (a) A mixed failure,the hybrid layer (remaining scratches from the surfacee residual resin is observed in the left side of the picture.t a higher magnification, a porous, intertubular dentin

d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 885–892 889

Fig. 2 – SEM observations of the fractured surface along the dentin side. (a) Specimen bonded with Clearfil SE Bond, after 3months of water immersion presented a mixed failure. The failure was located both at the top and bottom of the hybridcomplex. Peritubular dentin was observed and good resin infiltration of intertubular dentin was evident. Tubule diameter islimited and peritubular dentin is present. (b) A mixed failure of a One-Up Bond specimen, after 3 months of waterimmersion. At the left side of the picture, failure occurred at the top of the hybrid layer, showing that some tubule orificesare observed partially covered by hybridized smear layer. At the right and top side, the underlying dentin is exposedshowing an aggressive etching pattern.

Fig. 3 – SEM observations of the fractured surface along the dentin side of two specimens bonded with Clearfil SE Bond. (a)After 3 months of water immersion, a mixed failure may be observed. (b) At a higher magnification, the hybrid complexseems fully hybridized. Tubules are occluded by resin tags. (c) After 12 months of water immersion, a mixed failure can beobserved. (d) At a higher magnification, the failure occurred at the bottom of the hybrid layer, exposing the underlyingdentin or within the hybrid layer. Few resin tags remain occluding the tubules.

890 d e n t a l m a t e r i a l s 2

Fig. 4 – SEM observations of the fractured surface along thedentin side of one specimen bonded with Single Bond,after 12 months of mineral oil storage. (a) A mixed failurecan be observed. Most of the surface is covered by anadhesive layer although some dentin is exposed. (b) At ahigher magnification of the exposed dentin, resin tags inthe tubule entrances may be observed. Infiltratedintertubular dentin is shown. Some dissociation between

peritubular and intertubular dentin can be determined.

shown that the aggressive versions of these simplified self-etch adhesives completely dissolve the smear layer and formrelatively thick hybridized complexes [8,10,11] that incorpo-rate the smear layer [11]. These aggressive self-etching sys-tems not only open up dentinal tubules but also enlarge theirdiameter (Fig. 2b), creating thick resin tags [8,10]. Several rea-sons have been advocated to account for the suboptimal bond-ing performance of these all-in-one adhesives: (1) the strongeretching process (Fig. 2b) may destabilize the collagen, lead-ing to a decrease in bond strength [13], (2) weaker cohesivestrength of the adhesive [12,14], (3) the combination of acidichydrophilic and hydrophobic monomers into a single step thatcompromises the polymerization of the adhesives [2], and

(4) a low degree of conversion of the resin monomer that iscaused by the effect of oxygen inhibition [7]. The increasedpermeability of resin–dentin interfaces created by one-stepself-etching adhesives [15,16] may also contribute to their

3 ( 2 0 0 7 ) 885–892

hydrolytic instability after aging in water. Wang and Spencer[17] demonstrated that the degree of polymerization droppedfrom 93% to 36% as water content of experimental adhesivesincreased from 20% to 60%. Even though water is an essentialcomponent for ionization of self-etching acid monomers [18],the entrapment of water within the resin [19] and the con-tinuous supply of water within the dentin/tubules [20] mightcause incomplete polymerization of the adhesive, i.e. by dilut-ing soluble monomers to an extent that there is proof notadequate free radicals propagation. Water is a major interfer-ing factor in polymerization, and, as result, unpolymerizedacidic and aggressive monomers may continue to etch thedentin [21], leading to lower bond strengths. When ethanolstarts to evaporate from water–ethanol mixtures, there is aphase separation of the sparingly water-soluble resin compo-nents. This results in the entrapment of water droplets withinthe adhesive layer [22]. Although a single-step adhesive isdesirable clinically, the combination of acidic, hydrophilic andhydrophobic monomers into a single solution may compro-mise the function of each one of these components.

Reducing the cross-sectioned area of the aged specimensreduces the diffusion distances for water diffusion throughthe exposed resin–dentin interface [23]. Direct exposure(DE) of the specimen beams to water storage for 3 monthsresulted in a significant decline in the microtensile bondstrength of SB and OUB to 50% of that obtained when theresin–dentin interface was surrounded by bonded enamel(IE groups, Table 2). Moreover, the percentage of adhesivefailures significantly increased with direct water exposure.A combination of the following factors could account for theobserved decreases in resin–dentin bond strengths duringwater storage: (1) the collagen fibrils may be made moresusceptible to hydrolytic degradation after they were treatedwith very low pH solutions [6,13]; (2) restriction of monomerdiffusion may have prevented optimal resin impregnationinto demineralised collagen matrices [8,17]; (3) exposed colla-gen may be degraded if matrix components are left exposedafter resin infiltration [4,24]; (4) water sorption may decreasethe mechanical properties of the hydrophilic adhesive resinsby lowering their glass transition temperatures [5,8,19,25]; (5)a continuing etching effect [21] may also expose unprotectedcollagen in the case of OUB.

The detrimental effects of hydrolytic degradation could bereadily appreciated when the specimens were stored in undernon-aqueous conditions. Under such conditions, hydrolyticdegradation could not occur, resulting in the retention of orig-inal bond strengths during specimen aging (Table 2). Paul etal. [26] demonstrated an increase of mechanical properties inspecimens that were not immersed in water subsequent tocuring. The negative effects of water storage on bond strengthsis further supported by the relatively constant bond strengthsobserved for the specimens that were stored in mineral oil.Indeed, bond strength increased significantly in the SB/24-h-DE, and both SB/3-month-DE and IE subgroups (Table 2) whenthese specimens were stored in mineral oil. These results weresimilar to those reported by Carrilho et al. [27]. Pashley et al.

[28] found that dentin matrix contains endogenous collage-nase activity thought to be due to matrix metalloproteinases(MMPs). These enzymes are hydrolases (EC 3.424.24). That is,the break peptide bonds by adding water. In the absence of

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ater (i.e. dry condition or incubation in oil), these enzymesannot act (Fig. 4). However, in the presence of water, decreasesn microtensile bond strength occur as a consequence ofydrolytic degradation of collagen (Fig. 1). In addition, waterorption may plasticize the polymer networks, lowering thelass transition temperature of the cured resin [25,29] in aanner that ultimately weakens resin–dentin bonds over-

ime. Such plasticiation cannot occur in the absence of water.For SEB, decreases in MTBS after water immersion was

elayed compared to SB or OUB (Table 2). De Munck et al.2] demonstrated that different adhesives responded differ-ntly when dentin was unprotected by enamel. Self-etchingystems have been found to create more stable bonds ofesin restorations than total-etch systems [1,30]. The high per-entage of camphorquinone [7], the absence of a discrepancyetween the depths of demineralisation and infiltration [9],nd the protective effect of both resin-coated collagen andhe calcium salts of MDP which hardly dissolves in water [31]Fig. 3a and b), may have contributed to these results. Whenonding dentin with SB or SEB, surrounding resin-infiltratednamel exerted a protection role on resin–dentin bonds (evenfter 1 year of aging) [32]. However, this protective effect wasot evidenced when OUB system was used as the adhesive,robably due to the increased water sorption and permeabil-

ty of this enamel–resin layer [15,16].The results require rejection of the null hypothesis that

here are no differences in dentin bond strength whenesin–dentin interfaces were aged for variable periods underifferent storage conditions. The three adhesives that wereelected for this study showed different dentin bond strengthshen they were aged in different storage media for different

ime periods and under different modes of exposure.

. Conclusions

. Water plays an important role in resin–dentin bonds degra-dation.

. The presence of surrounding resin-infiltrated enamelexerted a protective effect on resin–dentin bonds exceptfor the one-step adhesive (OUB).

. When the two-step self-etching primer adhesive system(SE) is used as adhesive system, resin–dentin bonds degra-dation is delayed.

cknowledgments

his research project was supported by grants CICYT/FEDERAT2004-06872-C03-02, RED CYTED VIII. J. And R01 DE014911,01 DE015306 from the National Institute of Dental and Cran-

ofacial Research, Bethesda, MD (D.H. Pashley).

e f e r e n c e s

[1] Sano H, Yoshikawa T, Pereira PNR, Kanemura N, MorigamiM, Tagami J, et al. Long-term durability of dentin bondsmade with a self-etching primer. J Dent Res 1999;78:906–11.

[2] De Munck J, Van Meerbeek B, Yoshida Y, Inoue S, Vargas M,Suzuki K, et al. Four-year water degradation of total-etch

( 2 0 0 7 ) 885–892 891

adhesives bonded to dentin. J Dent Res 2003;82:136–40.

[3] Nakabayashi N, Pashley DH. Acid conditioning andhybridization of substrates. In: Nakabayashi N, Pashley DH,editors. Hybridization of dental hard tissues. Tokyo:Quintessence Publishing Co. Ltd.; 1998. p. 37–56 (Chapter 3).

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