Conditioning effect on dentin, resin tags and hybrid layer of different acidity self-etch adhesives applied to thick and thin smear layer

journal of dentistry xxx (2006) xxx xxx available at www.sciencedirect.com journal homepage: www.intl.elsevierhealth.com/journals/jden Conditioning effect on dentin, resin tags and hybrid layer of different acidity self-etch adhesives applied to thick and thin smear layer Silvia Kenshima a, *, Carlos Francci a, Alessandra Reis b, Alessandro Dourado Loguercio b, Leonardo Eloy Rodrigues Filho a a University of São Paulo, FOUSP, Dental Materials Department, Av. Professor Lineu Prestes, 2227, Cidade Universitária, CEP 05508-000, São Paulo, SP, Brazil b University of Oeste de Santa Catarina, Campus Joaçaba, R. Getúlio Vargas, 2125, Bairro Flor da Serra, CEP 89600-000, Joaçaba, SC, Brazil article info Article history: Received 25 February 2006 Received in revised form 25 February 2006 Accepted 6 March 2006 Keywords: Dentin Self-etch adhesive Resin tags Smear layer Scanning electron microscopy abstract Objectives: to assess the conditioning effect (CE) of self-etch adhesives of different acidity applied to thick and thin smear layer (SL), the corresponding resin tags (RT) and hybrid layer (HL). Methods: Twenty-seven molars had their occlusal dentin exposed and were sectioned into two halves. Each of them was ground with a 60 or 600-grit SiC paper, respectively for thick and thin SL production. Three self-etch adhesives: a mild (Clearfil SE Bond), an intermediary (Optibond Solo SE and Solo Plus) and a strong (Tyrian Self Priming Etchant + One Step Plus) and an etch-&-rinse system (Scotchbond Multi Purpose Plus) were used. For CE evaluation, the selfetch primers were applied and rinsed off with acetone and alcohol prior to SEM preparation. For RT and HL specimens self-etch adhesives were applied and restored with Z-250. For RT specimens, dentin was removed with HCl (6N) and NaClO (1%) baths. The HL specimens were fixed, dehydrated, dried with HMDS, embedded, polished and slightly demineralized (6N HCl) and deproteinized (1% NaClO). After gold sputtering they were observed by SEM. Results: The thick smear layer was clearly not totally removed by the mild self-etch primer. RT varied in density and shape among the self-etch adhesives. Thicker HL was observed for the strong self-etch and etch-&-rinse adhesives. Conclusions: The etch-&-rinse adhesive presented the thickest HL and was the only adhesive to produce RT in high density and uniform distribution along the whole dentin surface, independently of the SL thickness. # 2006 Elsevier Ltd. All rights reserved. 1. Introduction The efficiency of bonding to dentin depends mostly on micromechanical retention promoted by resin infiltration in partially demineralized dentin with consequent hybrid layer and tags formation. 1,2 To meet these requirements, there are two strategies: the etch-&-rinse and self-etch approaches. The etch-&-rinse approach requires conditioning, rinsing and priming steps that promotes the most complete removal of the smear layer. The self-etch partially incorporates the * Corresponding author. Tel.: +55 11 30917842x229; fax: +55 11 30917842. E-mail address: silvia.k@uol.com.br (S. Kenshima). 0300-5712/$ see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2006.03.001 JJOD-994; No of Pages 9

2 journal of dentistry xxx (2006) xxx xxx smear layer providing simultaneous demineralization and infiltration during the application of the acidic primer. 3,4 Therefore, the demineralizing potential of these materials is an important feature and depends on the type of acidic monomer, the pk a, the applied concentration, the application duration, the osmolarity, the wettability, the viscosity, the water concentration and its ph. 5 According to the latter they can be classified as mild, intermediary and strong. 6 Another factor that might interfere with the demineralizing potential of a self-etch adhesive is the type of smear layer. Some studies reported low resin dentin bond strengths over thick dentin smear layers, 7,8 while others reported no influence. 9,10 This can be partially explained by differences in the smear layer thickness (ranging from 0.9 to 2.6 mm 9 ), in surface roughness, density and degree of smear layer attachment to the underlying tooth structure, which is dependent on the way the smear layer is produced. 11 New adhesive systems are frequently launched onto the market within a short period of time. Prior to the commercialization, they are routinely tested for bond strength 6,9 and leakage 12 as those methods provide immediate results. But micromorphological studies may provide additional information to the adhesive performance, such as the hybridization effectiveness. In this sense, the self-etch acidity had been previously correlated to their respective hybrid layer. 3,4 But the literature lacks reports about the conditioning effect on dentin and resin tags. As the bond strength of a etch-&-rinse dentin adhesive was theoretically calculated as the sum of the strengths of resin tags, hybrid layer and surface adhesion, 2 these aspects seem likely to be correlated also for self-etch adhesives, though for the latter, additional chemical bonding might occur at varied extents depending on the monomer system employed. Therefore, the objective of this study was to qualitatively assess the conditioning effect, the resin tags and hybrid layer of different acidity self-etch systems and an etch-&-rinse adhesive applied to thick and thin smear layer. The null hypothesis tested were: (1) there is no correspondence between the hybrid layer thickness and the conditioning effect and corresponding resin tags for different acidity selfetch adhesives applied to thick and thin smear layer; (2) there is no difference, regarding conditioning effect, resin tags and hybrid layer, among self-etch adhesives of different acidity applied to thick and thin smear layer; (3) there is no difference between these systems and the etch-&-rinse adhesive. 2. Materials and methods 2.1. Teeth preparation Twenty-seven non-carious, third molars previously disinfected in 0.5% chloramine were used (protocol approved by the University of São Paulo Institutional Review). The occlusal third was removed (2 mm remaining dentin thickness) and the teeth were longitudinally sectioned with a Labcut 1010 cutting machine (Extec Corp., Enfield, CT, USA) in a buccallingual direction in two halves (54 experimental units n = 3). One-half was ground on wet 60-grit SiC paper for 60 s (thick smear layer), and the other with 600-grit SiC (thin smear layer). 3 2.2. Smear layer measurement Immediately after that, for smear layer thickness measurements the teeth halves had pre-cut grooves made on their pulp side to allow segmentation and were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at ph 7.4 for 12 h at 4 8C, rinsed with 20 ml of 0.2 M sodium cacodylate buffer at ph 7.4 for 1 h with three changes, followed by distilled water rinsing (1 min) and dehydration in ascending grades of ethanol: 25% (20 min); 50% (20 min); 75% (20 min); 95% (30 min); 100% (60 min). After that, the specimens were immersed in hexamethyldisilazane (HMDS) for 10 min, 13 segmented and gold-sputtered to be observed at SEM (Philips Electronics N.V., Balzers, Liechtenstein). Each segment was tilted 208 in each direction and the smear layer thickness was measured at 10 equally spaced points 11 using an image analysis software, UTHSCSA Image Tool, Version 2.0 (Universisty of Texas Health Science Center, San Antonio, Texas). 2.3. Restorative procedures for resin tags and hybrid layer Three self-etch adhesive systems with different acidity 6 were selected (Table 1): a mild, Clearfil SE Bond (ph ffi 2); an intermediary, Optibond Solo Plus Self-Etch Primer + Optibond Solo Plus (1 < ph < 2) and a strong, Tyrian Self Priming Etchant SPE + One-Step Plus (ph < 1). The three-step etch-&-rinse Scotchbond Multipurpose Plus was included for comparison purpose. Each adhesive was randomly applied, by a single operator to both halves from the same tooth as follows: Clearfil SE Bond active application (rubbing) of two coats of primer (20 s); air-drying (10 s), application of one coat of adhesive (15 s), air-drying and light-activation (10 s 600 mw/cm 2 ); Optibond Solo Self-Etch and Solo Plus active application of one coat of primer (15 s); air-drying (10 s), application of one coat of adhesive (15 s), air-drying (10 s), light-activation (20 s 600 mw/cm 2 ), application of another adhesive coat (15 s), air-drying (10 s) and light-activation (20 s 600 mw/ cm 2 ); Tyrian SPE and One Step Plus activation of the mixture of Tyrian SPE (A and B) and active application of two coats of primer (10 s), air-drying (10 s), application of two consecutive coats of adhesive (10 s each), air-drying (10 s) and lightactivation (10 s 600 mw/cm 2 ). ScotchBond Multi Purpose Plus etching with phosphoric acid (15 s), rinsing (15 s), air-drying (10 s), active application of one coat of the primer (10 s), air-drying (10 s) and excess removal, application of the adhesive (10 s), air-drying (10 s), light-activation (10 s 600 mw/cm 2 ). Special care was taken to ensure that the dentin surfaces had been adequately covered by the primer after evaporation of the solvents producing a shiny surface prior to light curing of the adhesives. For resin tags and hybrid layer observation, after the adhesive application, a resin composite buildup (Filtek Z250

journal of dentistry xxx (2006) xxx xxx 3 Table 1 Formulation of the adhesive systems according to information provided by the manufacturers Adhesive system Composition Batch number Clearfil SE Bond SE (Kuraray Medical Inc., Osaka, Japan) Optibond Solo SE and Solo Plus SO (Kerr Orange, CA, USA) Tyrian SPE and One Step Plus TY (Bisco, Schaumburg, IL, USA) ScotchBond Multi Purpose Plus SC (3M ESPE, St. Paul, MN, USA) (1) Primer water, MDP, HEMA, camphoroquinone, hydrophilic dimethacrylate; (2) adhesive MDP, Bis-GMA, HEMA, camphoroquinone, hydrophobic dimethacrylate, N,N-diethanol p-toluidine bond, silanated colloidal silica (1) Alkyl dimethacrylate resins, Barium aluminoborosilicate glass, fumed silica (silicon dioxide), sodium hexafluorosilicate and ethyl alcohol; (2) Alkyl dimethacrylate resins (25 28%), ethyl alcohol, water, stabilizers and activators (1) Self-etching Part A ethanol (20-30%); (2) self-etching Part B 2-acrylamido-2-methyl propanesulfonic acid (30 50%); bis (2-(methacryloyloxyethyl) phosphate (5 15%)GMA; ethanol (40 70%) (200002694); (3) adhesive Bis-GMA and BPDM (15 40%), HEMA (15-40%), Dental glass (1 10%) and acetone (40 70%) (1) Scotchbond 37% phosphoric acid; (2) primer aqueous solution of HEMA, polyalkenoic acid copolymer (Vitrebond); (3) adhesive Bis-GMA, HEMA, dimethacrylates and initiators 00176A; 001185A 205187; 203D20 200004295 3008; 7543 3M ESPE, St. Paul, MN, USA) was prepared. The resin tags specimens were immersed in HCl (6N) and in NaClO (1%) until dentin removal was completed, washed and dried for 24 h before gold sputtering and SEM observation. The hybrid layer specimens were processed for SEM as indicated in Fig. 1, embedded in epoxy resin and polished to high gloss with SiC papers and tissues with diamond suspensions (Buehler Ltd. Lake Bluff, IL, USA) of decreasing abrasiveness. After sonication in distilled water for 5 min, specimens were demineralized in 6 N HCl for 30 s and deproteinized in 1% NaOCl for 10 min to reveal the hybrid layer. All specimens were mounted on aluminum stubs and, after gold sputtering, observed by SEM. 2.4. Restorative procedures for conditioning effect For the conditioning effect evaluation, following the self-etch primers application, they were rinsed off with a sequence of acetone bath (5 min), deionized water (5 min), 96% alcohol bath (5 min) and deionized water (5 min). 11 After that, they were SEM processed as previously described (Fig. 1). 3. Results 3.1. Smear layer thickness The 60-grit SiC prepared surface presented an uneven and apparently rougher and undulating surface with wide scratches (Fig. 2C). The smear layer was not uniformly distributed forming thick deposits of granular particles, which seemed loosely attached to the underlying dentin (Fig. 2A and C). The mean smear layer thickness measured along the fractured lateral view was 3.58 1.79 mm(fig. 2A). In contrast, the 600-grit SiC prepared surface presented a smoother surface with narrow and evenly distributed scratches (Fig. 2D). The mean smear layer thickness was 1.37 0.78 (Fig. 2B). Fig. 1 Specimen preparation protocols.

4 journal of dentistry xxx (2006) xxx xxx Fig. 2 (A) lateral view of 60-grit SiC prepared surface with thick granular deposits (black arrowhead), fractured dentin with tubule lumen (white arrowhead); (B) lateral view of 600-grit SiC prepared surface with thin smear layer (between open arrows); (C) 60-grit SiC prepared surface with thick granular deposits (black arrowhead) and wide scratch (between open arrows); (D) 600-grit SiC prepared surface with narrow scratches (between open arrows) and more uniform surface. (original magnification: (A) and (B) 5000 and (C) and (D) 1000). 3.2. Conditioning effect The thick smear layer (60-grit) was partially removed by the mild self-etch primer and there was incomplete dissolution of the smear plugs (Fig. 3A). In contrast, when applied to thin smear layer (600-grit) its removal was evidently more effective (Fig. 3B). The intermediary self-etch primer showed an intermediary conditioning effect (Fig. 3C and D). And for the strongly acidic primer (Fig. 3E and F), similarly to the observed for the 35% phosphoric acid conditioning effect (Fig. 3G and H), there was no apparent difference in the conditioning effect between the thick and thin smear layer. 3.3. Resin tags For the self-etch adhesives, areas of apparently incomplete resin monomer infiltration could be identified at varied extensions depending on the primer acidity: the more acidic, the higher density of tags per unit of area and the fewer areas with incomplete tags formation. The mild self-etch system was clearly affected by the thick smear layer: characteristic areas with scarce and short needlelike resin tags were observed (Fig. 4A). This effect was not observed in any of the other experimental conditions. For both surfaces (60 and 600-grit SiC), tags of regular cross-section area and variable lengths were observed (Fig. 4B). No clear effect of the smear layer thickness could be observed for the intermediary self-etch adhesive. For both dentin surfaces, there were areas with higher resin tags concentration (Fig. 4D) and areas with probably incomplete resin monomer infiltration (Fig. 4C). It is interesting to observe the incidence of some voids along the tags length that were concentrated at their tips (Fig. 4D). For the most acidic self-etch system, regardless of the smear layer thickness, two distinct tags formation patterns were observed: (1) areas of incomplete resin monomer infiltration denoted by the irregular diameter and shape of the resin tags remnants (Fig. 4E); (2) areas that resembled the etch-&-rinse pattern with longer and more concentrated resin tags of regular cross-section area (Fig. 4F). The first pattern was restricted to spotted areas. Only the etch-&-rinse showed a regular and consistent pattern of resin tags on the whole dentin surface regardless of the smear layer thickness. A uniform and high concentration of long funnel-shaped solid resin tags (Fig. 4G) with several lateral branches could be observed (Fig. 4H). 3.4. Hybrid layer Hybrid layer thickness was apparently not affected by the smear layer thickness. Although not uniform throughout the whole extension, there was a clear distinction in the hybrid layer thickness between self-etch adhesives (0.7 1.5 mm) and the etch-&-rinse (4.2 mm). Among the self-etch adhesives, it was not possible to distinguish differences in hybrid layer thickness between the mild and the intermediary, but the strong adhesive presented markedly thicker hybrid layer (Fig. 5).

journal of dentistry xxx (2006) xxx xxx 5 Fig. 3 From top to bottom: the conditioning effect on dentin of Clearfil SE Bond (A and B), Optibond Solo SE (C and D), Tyrian SPE (E and F) and 35% phosphoric acid (G and H). The images on the left correspond to 60-grit SiC surfaces and the ones on the right to the 600-grit SiC. Original Magnification: 5000.

6 journal of dentistry xxx (2006) xxx xxx Fig. 4 From top to bottom: resin tags of Clearfil SE Bond (A and B), Optibond Solo SE and Solo Plus (C and D), Tyrian SPE + One Step Plus (E and F) and Scotchbond Multi Purpose Plus (G and H). Figure A corresponds to resin tags of Clearfil SE Bond applied to 60-grit prepared surface and B to 600-grit. Note in A, needle-like tags (white arrowhead), mark left by the 60-grit SiC paper (between open arrows) and in B, regular cross-section area of resin tags. Regular diameter remnants of fractured resin tags can be observed in C and a porous aspect of the resin tags in D. Two distinct patterns were observed for Tyrian SPE + One Step Plus: pattern 1 irregular shape and diameter of resin tags remnants (white arrowhead in E) and pattern 2 high concentration of long resin tags with regular cross-section area (F). Figures G and H correspond to Scotchbond Multi Purpose Plus, in which, a high density of funnel-shaped tags (white arrowhead in G) and lateral branches (white arrowhead in H) can be observed. Original magnification: 1500 for all figures except for D (3000). 4. Discussion Based on the findings of this study, the first null hypothesis was rejected as the thick smear layer removal was more effective with the strong self-etch system; likewise, the resin tags were more numerous and the corresponding hybrid layer followed this trend and with strong self-etch adhesive produced the thickest hybrid layer in accordance with previous studies. 3,4,6 In agreement with reported SEM and TEM (transmission electron microscope) observations, 3,4,11 there was incomplete dissolution of the smear plugs (Fig. 3A) and needle-like resin tags were formed (Fig. 4A) in some areas when the mild selfetch primer was applied to thick smear layer. Therefore, the

journal of dentistry xxx (2006) xxx xxx 7 Fig. 5 From top to bottom: Adhesive interface of Clearfil SE Bond (A and B), Optibond Solo SE and Solo Plus (C and D), Tyrian SPE + One Step Plus (E and F) and Scotchbond Multi Purpose Plus (G and H). The images on the left correspond to 60-grit SiC prepared surfaces and the ones on the right to the 600-grit. The letters RC, A and D correspond to resin composite, adhesive layer and dentin, respectively. The hybrid layer was indicated between arrows. Original magnification: 5000. second null hypothesis was also rejected as these patterns of conditioning effect and resin tag were exclusive of this experimental condition. In spite of this, the mild self-etch system, Clearfil SE Bond, have shown to be one of the most reliable adhesive systems and presented high bond strength values in numerous studies, 5,6,10 which is comparable to the three-step etch-&- rinse systems that remains the gold standard in bond durability. 14 In this sense, the high mechanical properties of this adhesive 15,16 and the chemical bond produced by the 10- methacryloxydecyl dihydrogen phosphate (MDP), 1,17 one of the main constituents of Clearfil SE Bond might explain the reported high bond strength results. Nevertheless, concerns on the complex reaction to stress had arisen for mild self-etch systems as hybrid layer thickness decreases with higher ph self-etch primers 4,6 and this thinner hybrid layer could limit its contribution in polymerization shrinkage stress absorption. 18 Indeed, regarding different ph self-etch systems, acidity was previously correlated to interfacial gap widths and the mild self-etch system, that presented the thinnest hybrid layer in the present study, presented larger mean gap widths. 5 Additionally, in another study, Optibond FL, a three-step etch- &-rinse adhesive, with thicker hybrid layer, comparable to the etch-&-rinse system observed in this study, was more resistant to fatigue than Clearfil SE Bond. 19 Regional differences in the smear layer denseness, 11 might have affected the resin monomer infiltration for the mild selfetch system and could explain the larger interfacial gap widths previously associated to thick smear layer. 5 Therefore, it is reasonable to suppose that more acidic self-etch systems

8 journal of dentistry xxx (2006) xxx xxx might be able to minimize possible adverse effects of smear layer incorporation in the adhesive interface. 4 In this sense, micromorphological similarities with etch-&- rinse systems would be beneficial features for self-etch systems, since high bond strength and good interfacial adaptation 5,20 was previously reported for these systems, especially for the three-step systems that presented better long-term performance regarding bond strength than their simplified versions. 20 However, contrary to what would be expected of higher tags concentration, lower bond strength values had been reported for the strongly acidic self-etch systems. 5,6,9 Possibly, these findings might have relation with the adhesive approach as the third null hypothesis was reject because only the etch- &-rinse adhesive presented uniform conditioning effect and resin tags throughout the whole extent of observed samples. It is interesting to observe that, in spite of the closest resemblance among the self-etch adhesives with the etch-&- rinse system in hybrid layer thickness, the strong self-etch system studied presented areas with irregular tags remnants with varied size and shape. This suggests incomplete removal of the smear layer in spotted areas (Fig. 4E) and might indicate that even for strongly acidic self-etch systems there might have, though to very limited extent, an adverse effect of the smear layer. It was previously demonstrated that phosphoric acid denatures collagen and a gelatinized smear layer that cannot be rinsed off is formed. 21 If the most acidic primer had a similar effect, as no rinsing step is performed for a self-etch adhesive technique, it is possible that in denser smear layer areas its removal would be incomplete preventing full resin monomers infiltration. However, this issue should be directly addressed in a specific study. These observations suggest that either excessive acidity of the self-etch primer can adversely affect its bond strength or other factors have stronger effects on bond strength, such as solvent concentration, 12,22 the adhesive conversion, 22 cohesive strength of the adhesive system 15 and the hybridization efficiency. High solvent content at the primer composition, for instance, reduces the adhesive layer thickness, leaving it more susceptible to oxygen inhibition 23 and reduces its mechanical properties. 22 Considering the intermediary self-etch system, although the bond strength was not adversely affected, 5 extensive voids formation at the tags tips was observed (Fig. 4D), which is a possible indication of lack of homogeneity within the infiltrated adhesive. Incomplete solvent evaporation is a possible explanation for this finding as the adhesive contains ethanol in the composition and even adhesive systems with solvents of higher vapor pressure, such as acetone, presented incomplete solvent evaporation. 22 Another explanation could be related to phase separation. The differential diffusion of monomers within the hybrid layer may result in regional variations in composition, which may affect the curing performance and consequently decrease resin tags strength. 22 The absence of 2-hydroxyethyl methacrylate (HEMA) in the composition of the intermediary strong self-etch primer could have been a contributing factor to phase separation. HEMA is a transitional polymerizable solvent, since many of the acidic monomers are only mildly water soluble. 24 Therefore, the presence of this monomer can reduce the occurrence of solvent droplets and phase separation. 25 The bond strength performance of these adhesives cannot be predicted by the micromorphological findings of this study. However, the lack of uniformity in the conditioning effect, resin tags formation and hybrid layer thickness remains a source of concern as a discrepancy between the depth of demineralization and resin monomers infiltration of a mild self-etch system was reported 26 and there is evidence of continuing etching for a strongly acidic self-etch system. 27 Under the experimental conditions of this in vitro study it is possible to conclude that only the etch-&-rinse adhesive produced tags in high density and uniform distribution along the whole dentin surface, independently of the smear layer thickness. 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