dentin bonded interfaces formed by different adhesive strategies and exposed to NaOCl challenge

International Journal of Adhesion & Adhesives 59 (2015) 21–26

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Evaluation of resin/dentin bonded interfaces formed by different adhesive strategies and exposed to NaOCl challenge Fabianni Magalhaes Apolonio a, Lidiane Costa de Souza a, Francisco Claudio Fernandes Alves e Silva a, Mônica Yamauti b, Lorenzo Breschi c, Vicente de Paulo Aragão Saboia a,n a

Department of Restorative Dentistry, Federal University of Ceará, Monsenhor Furtado Street, Fortaleza, Ceará 60430-355, Brazil Graduate Program of Dentistry, Federal University of Ceará, Monsenhor Furtado Street, Fortaleza, Ceará 60430-355, Brazil c Department of Biomedical and Neuromotor Sciences, DIBINEM, University of Bologna – Alma Mater Studiorum & IGM-CNR, Unit of Bologna c/o IOR, Via San Vitale, 59, 40125 Bologna, Italy b

art ic l e i nf o

a b s t r a c t

Article history: Accepted 13 January 2015 Available online 2 February 2015

This study evaluated the importance of encapsulated collagen on resin/dentin interface created by different adhesive strategies. Composite build-ups were bonded to dentin using one of the following adhesive systems: Scotchbond Multi-Purpose (SBMP), Adper Scotchbond 2 (SB2), Clearfil SE (CSE) and Scotchbond SE Plus (SBSE), and cut into non-trimmed dentin–composite beams. Half of those beams were deproteinized using 10% NaOCl for 1 h and the other half was stored in water. Beams were pulled to failure and data were statistically analyzed by a two-way ANOVA and Tukey for multi-comparison test (α ¼0.05). Additional dentin disks were stained with Masson's trichrome acid and processed with light microscopy in order to identify the exposed collagen zones. All groups showed a significant reduction on bond strength after proteolytic challenge (po 0.05). Adhesive systems were ranked in the following order: SBMP4SB2 ¼CSE 4SBSE (po 0.05) for control and treated groups. Microscopy analysis showed different collagen exposed zones in relation with the adhesive strategy used. It can be concluded that collagen encapsulation affects the quality of bond interface, which is related to the adhesive system used. & 2015 Elsevier Ltd. All rights reserved.

Keywords: Dentin Hybrid layer Enzymatic degradation Collagen degradation

1. Introduction In etch-and-rinse adhesives, resin monomers are applied on a demineralized dentin substrate, thus infiltration occurs through the water-filled spaces between adjacent collagen fibrils producing the hybrid layer [1]. However, in these adhesive systems an incomplete resin infiltration occurs and a demineralized unprotected dentin collagen layer remains beneath the hybridized layer [2–4]. On the other hand, self-etch adhesives use acidic functional monomers that demineralize and infiltrate in the same step [1]. Theoretically, this procedure ensures that the whole demineralized dentin depth could be impregnated by the resin monomer, thus the number of exposed

n Correspondence to: Department of Restorative Dentistry, Federal University of Ceará, Gilberto Studart Street, 770 – AP. 901, Fortaleza, Ceará 60.190-750, Brazil. Tel.: þ 55 8588074623; fax: þ55 8533668232. E-mail addresses: [email protected] (F.M. Apolonio), [email protected] (L.C. de Souza), [email protected] (F.C.F. Alves e Silva), [email protected] (M. Yamauti), [email protected] (L. Breschi), [email protected] (V.d.P.A. Saboia).

http://dx.doi.org/10.1016/j.ijadhadh.2015.01.011 0143-7496/& 2015 Elsevier Ltd. All rights reserved.

collagen fibrils within the hybrid layer is reduced compared to etchand-rinse adhesives [5]. As reported by Breschi et al. irrespective of the bonding strategy, such as in simplified adhesives, the bonded interface lacks a nonsolvated hydrophobic resin coating [6]. This happens because when applying BisGMA/HEMA based adhesives on the wet demineralized dentin, the water-soluble monomer HEMA can easily dissolve in water on the dentin surface, and penetrate the collagen fibrils on the etched dentin surface. On the other hand, due to the hydrophobic nature of the BisGMA, a hydrophobic phase will not readily penetrate the wet demineralized dentin matrices. Camphorquinone based photoinitiator system is hydrophobic and most part of it will remain associated with the BisGMA phase leading a poor polymerization of the HEMA zone [7]. An easy way to challenge the durability of this unprotected collagen is to store bonded interfaces in 10% NaOCl. This solution has a non-specific proteolytic effect that effectively removes organic components from resin-bonded teeth that are not completely enveloped. NaOCl has been used as a substitute for proteolytic enzymes [8] to challenge the adhesive interface and expedite degradation processes related to exposed collagen [9]. Previous

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studies showed that reduction on bond strength and increase in nanoleakage interfacial expression after storage in deproteinization solution (NaOCl 10%) occurs [9–11]. An approach to assay the presence of exposed collagen is the use of a trichromic staining [2,12]. This dye has a high affinity for cationic elements of normally mineralized type I collagen, resulting in staining collagen blue. Etching of dentin with phosphoric acid removes these elements from peptidic chains of collagen, resulting in different colorations, generally red [2], that could represent the collagen exposed non-encapsulate zone. With this technique it is possible to identify collagen fibrils exposed and not encapsulated by resin that would be able to react with stain [12]. The aim of the present study was to evaluate the influence of collagen non-encapsulated fibrils on resin/dentin interfaces formed by different adhesive strategies and exposed to NaOCl challenge using bond strength test and interfacial morphology analysis. The null hypothesis tested was regardless of the adhesive strategy, greater extent of exposed collagen fibrils more prone to degradation when exposed to NaOCl challenge.

2. Materials and methods Twenty-eight freshly extracted caries-free human third molars were used in this study. They were stored in 0.01% thymol solution

at 4 1C after obtaining the patient's informed consent under a protocol approved by the Institution Ethics Committee. After being cleaned and pumiced, dentin was exposed using a low-speed diamond saw (Isomet 1000, Buehler Ltd. Lake Bluff, USA) under water irrigation and the smear layer was created on dentin with a 600-grit SiC paper. Teeth were equally and randomly assigned to four groups and treated with one of the following adhesive systems: Adper Scotchbond MultiPurpose (SBMP), Adper Scotchbond 2 (SB2), Clearfil SE Bond (CSE), and Adper Scotchbond SE Plus (SBSE). All adhesives were applied in accordance with manufacturer's instructions as described in Table 1. 5 mm-thick increments of resin composite Filtek Z250 (Table 1) were applied over the bonded surfaces in increments of 2 mm and light cured for 40 s with a LED dental curing unit with intensity of 500 mW/cm (Ultraled; Dabi Atlante – Ribeirão Preto/SP, Brazil). 2.1. Microtensile bond strength (mTBS) Five teeth from each group were longitudinally sectioned in both “x” and “y” directions across the bonded interface with a diamond saw in order to obtain sticks with cross-sectional areas of approximately 0.9 mm2 like in accordance with the non-trimming technique for microtensile test. After being stored for 24 h in distilled water, sticks from each tooth were divided equally into two subgroups: control (stored in water for 1 h) or immersed in 10% NaOCl for 1 h at room temperature

Table 1 Adhesive and resin composite systems and their application protocols. Adhesive

Composition

Primer: HEMA, polyalkenoic acid Adper Scothbond Multi-purpose (3M copolymer Adhesive: Bis-GMA, HEMA, initiator – ESPE, Seefeld, Germany)

Instruction for use

 Etching: apply phosphoric acid to enamel and dentin. Wait 15 s. Rinse for 15 s.   

Adper Scothbond 2 (3M – ESPE, Seefeld, Germany)

Clearfil SE Bond (Kuraray, Osaka, Japan) pH¼2.0a

Adper Scothbond SE Plus (3M – ESPE, Seefeld, Germany) pHo 1.0a

Composite resin Z250 (3M – ESPE, Seefeld, Germany)

Dry with absorbent paper. Priming: apply Scotchbond Multi-Purpose primer to etched enamel and dentin. Dry gently for 5 s. Adhesive application: apply Scotchbond Multi-Purpose adhesive to the primed enamel and dentin. Adhesive curing: light-cure for 10 s.

HEMA, bis-GMA, DMA's methacrylate functional copolymer of polyacrylic and polyitaconic acids, water, ethanol, nanofiller, photo-initiator

 Etching: apply phosphoric acid to enamel and dentin. Wait 15 s. Rinse for 15 s.

Primer: 10-MDP, HEMA, hydrophilic DMA, tertiary amine, water, photoinitiator Bond: 10-MDP, HEMA, bis-GMA, hydrophilic DMA, tertiary amine, silanated colloidal silica, photo-initiator

 Thoroughly wet brush tip with primer. Apply Primer to tooth surface and leave

Liquid A: water, HEMA, surfactante, Pink colorant Liquid B: UDMA, TEGDMA, TMPTMA (hydrophobic trimethacrylate) HEMA phosphates, MHP (methacrylated phosphates), bonded zirconia nanofiller, initiator system based on camphorquinone

 Wet brush tip with Liquid A. Apply to the entire bonding area so that a

Bis-GMA, UDMA, Bis-EMA, zircônia/ silica filler, aluminum oxide

 Place 3M Filtek Z250 restorative in increments less than 2.5 mm.  Light cure each increment for 20 s (increments less than 2.0 mm of B0.5, C4 and

 

 



 

Dry with absorbent paper Apply two consecutive coats of adhesive for 15 s with gently agitation. Gently air thin for five seconds to evaporate the solvent. Adhesive curing: light cure for 10 s.

in place for 20 s. Dry with a mild air stream to evaporate the volatile ingredients. Dispense the necessary amount of bond into the second mix well and apply bond to the tooth surface. Dry with a gently air steam to create a uniform film. Light cure for 10 s.

continuous red-colored layer is obtained on the surface. Wet second brush tip with Liquid B, and scrub into the entire wetted surface of the bonding area. The red color will disappear quickly, indicating that the etching components have been activated. Continue scrubbing with moderate finger pressure for 20 s. Air dry thoroughly for 10 s to evaporate water. Re-coat brush with Liquid B, and apply second coat to the entire bonding surface. Lightly air thin adhesive layer to adjust film thickness/consistency. Light cure for 10 s.

Type Three step etch-andrinse

Two step etch-andrinse

Two-step self-etch

Two-step self-etch

UD are cured 30 s).

Abbreviations: 10-MDP, 10-methacryloyoxydecyl dihydrogen phosphate; Bis-EMA, Bis-GMA: bisphenol A diglycidyl ether dimethecrylate; DMA, dimethacrylate; HEMA, 2-hydroxyethylmethacrylate; MHP, methacrylated phosphates; TEGDMA, triethylene glycol dimethacrylate; TMPTMA, trimethylopropane trimethacrylate; UDMA, urethane dimethacrylate. a

Information as received from manufacturer.

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Table 2 Microtensile bond strength values, number of sticks and failure mode. Adhesive system

lTBS Control groups

SBMP SB2 CSE SBSE

G1(49) 39.9 7 12.7a G2 (52) 26.6 7 8.3b G3 (52) 27.0 76.8b G4 (45) 13.2 73.1c

Failure mode (%) Aged in NaOCl groups

G5 (47) 26.4 7 9.9b G6 (42) 11.7 7 4.2c G7 (57) 16.17 3.7c G8 (44) 4.9 7 2.4d

Control groups

Aged in NaOCl groups

A

M

CD

CC

A

M

CD

CC

79.6%

4.1%

6.1%

10.2%

87.2%

8.5%

4.3%

0%

82.7%

9.6%

5.8%

1.9%

90.8%

4.6%

2.3%

2.3%

81.0%

2.3%

13.0%

3.7%

87.7%

1.8%

10.5%

0%

100%

0%

0%

0%

100%

0%

0%

0%

Mean 7 standard deviation (number of sticks) of mTBS results (expressed in MPa). A, adhesive failure; M, mixed failure; CD, cohesive failure in dentin; CC, cohesive failure in resin composite. Groups identified by different subscript letters are significantly different (p o 0.05).

Fig. 1. Light micrographs of Adper Scotchbond Multi-Purpose adhesive interface. (a) Interface of control group shows no red zones indicating that collagen fibrils were protected by the adhesive system. (b) After 10% NaOCl storage, the dentin/resin interface showed no red zones, as in control group (400  ). Composite resin (C), dentin (D).

for removing of the exposed and non-encapsulated collagen. All specimens were extensively rinsed under tap water, individually measured with a digital caliper (Absolute Digimatic, Mitutoyo, Tokyo, Japan) and finally stressed until failure with a tensile force in a universal testing machine (Model 3345, Instron Corp., Canton, MA, USA) at crosshead speed of 1 mm/min. Microtensile bond strength data were analyzed using SPSS 17.0 (SPSS, Chicago, IL, USA). Data were analyzed using ANOVA twoway on ranks and Tukey multiple-comparison test with statistical significance set at α ¼0.05. The bond failures were evaluated with a stereomicroscope at 40  magnification (Leica Zoom 2000 – Leica Microsystems GmbH – Wetzlar, Germany) and classified as adhesive, cohesive in dentin, cohesive in resin or mixed (failure at resin/dentin interface or mixed with cohesive failure of the neighboring substrates). 2.2. Light microscopy—Masson trichrome After 24 h storage in distilled water, two teeth from each group were sectioned in only one direction to obtain 1 mm thick dentin– resin slabs. Resin–dentin slabs from each tooth were divided equally into two subgroups: control or immersed in 10% NaOCl for 1 h. Slabs were fixed in a glass holder with cianoacrilate glue (SuperBonder flex gel – Henkel Ltda, Düsseldorf, Germany) and ground with SiC papers on increasing fine grits (800, 1000, 1200 and 2500) in a polisher under running water (Buehler, Lake Bluff, IL, USA). Specimens were treated with Masson's trichromic acid staining technique. The trichrome is applied by immersion of the sample into: Heidenhain's hematoxylin (1 min), wash in water (3 min), picric alcohol (5 min), wash in water (10 min), xylidine ponceau (10 min),

briefly in acetic water and wash in water. Differentiate in phosphomolybdic acid in 45–56 1C for 10 min and immerse briefly in acetic water in 45–56 1C. Immerse in aniline blue for 3 min and wash copiously. Slices were examined in an optical microscope (Leica DM 1000 – Leica Microsystems GmbH – Wetzlar, Germany) at 400  magnification.

3. Results 3.1. Microtensile bond strength Means and standard deviations of microtensile bond strength, number of specimens and failure mode are shown in Table 2. Statistically differences were found within the experimental groups and the interaction between the statistical factors was not significant (p40.05). SBMP showed the highest mTBS values (po0.05), followed by SB2 and CSE values, then SBSE showed the lowest values under both control and deproteinized conditions (po0.05). Deproteinization with 10% NaOCl reduced the bond strength for all adhesives tested (p o0.05). Fracture mode analysis indicated that in control specimens, almost all fractures were adhesive failures. In deproteinized specimens the number of adhesive failures increased numerically for SBMP, SB2 and CSE while SBSE showed no differences. 3.2. Light microscope – Masson trichrome Representative images of Masson's trichrome staining sections of the resin–dentin interfaces are presented in Figs. 1–4.

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Fig. 2. Light micrographs of Adper Scotchbond 2 adhesive interface. (a) In the control group, an extensive and homogeneous red zone of exposed collagen is present. (b) The red zone is no longer visible after NaOCl storage, substituted by a light zone, due to collagen removal (400  ). Composite resin (C), dentin (D). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. Light micrographs of Clearfil SE Bond adhesive interface. (a) Control group: no red zones were present indicating that the collagen fibrils exposed by acid monomers were encapsulated by the adhesive system. (b) After 10% NaOCl storage no red zones were observed, as in control group (400  ). Composite resin (C), dentin (D).

Fig. 4. Light micrographs of Adper Scotchbond SE Plus adhesive interface. (a) Control group: red zone was present in all resin/dentin interface indicating exposed and nonencapsulated collagen fibrils. (b) After 10% NaOCl storage, no red zones were present indicating that all exposed collagen fibrils were degraded (400x). Composite resin (C), dentin (D). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Control specimens of the two simplified adhesive systems (SB2 and SBSE) showed a distinct red zone, representing non-encapsulated/exposed collagen, able to react with the stain (Figs. 2 and 4a, respectively). The three-step etch-and-rinse SBMP and two-step self-etch CSE showed no red zones in resin/dentin interface (Figs. 1 and 3a, respectively). After deproteinization with 10% NaOCl specimens showed no red stained zones within the hybrid layer, depicting that the exposed collagen fibrils were fully degraded (Figs. 1–4b).

4. Discussion The results of this study showed that dentin bonding systems with greater extent of exposed collagen fibrils are more prone to degradation when exposed to NaOCl challenge which allows us to accept the null hypothesis tested. For total-etch adhesives the three-step adhesive system (SBMP) showed the highest bond strength result (39.9 7 12.7 MPa) with lower reduction in μTBS after NaOCl deproteinization (33.8% of

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reduction). The simplified system (SB2) showed a lower result than SBMP (26.6 78.3 MPa) with a 56% bond strength reduction after deproteinization. These findings are in accordance with other results that showed the three-step system as the gold-standard etch-and-rinse adhesives with better results than two-step etchand-rinse systems [13]. In addition, morphological data showed unprotected collagen in SB2 interface showing that the resins monomers of adhesive did not infiltrate in all demineralized dentin, while SBMP showed no red zones indicating a more effective collagen encapsulation. This may occur because simplified adhesives put together hydrophilic and hydrophobic component, in the same bottle impairing proper encapsulation and protection of the collagen fibrils. Resin monomers (hydrophobic component), particularly those with high molecular weight, have limited diffusion into the wet demineralized dentin [14]. Therefore to increase the infiltration in wet substrate, the simplified systems contain more hydrophilic components than the multi-bottle leading to hydrolytic degradation [6], suboptimal polymerization [7], and phase separation [15], which have been reported to be important factors for degradation of the simplified adhesive interface over time. In particular, because of suboptimal polymerization, the interface created by simplified adhesives may result in a semipermeable adhesive layer [16]. A study using micro-Raman [17] spectroscopy calculated the extent of adhesive penetration into the adhesive interface and showed that at the first micrometer of the resin–dentin interface, only 68% of the concentration of adhesive penetrated the demineralized dentin. The discrepancy between dentin demineralization and monomer infiltration results in incompletely infiltrated zones along the bottom of the hybrid layer, which contain denuded collagen fibrils even immediately after bonding [17]. According to the self-etching approach, substrate infiltration by resin occurs simultaneously with the etching process [6], so nonencapsulated collagen fibers should not exist and no bond strength reduction is expected to occur after deproteinization with NaOCl. However, a reduction of mTBS values was observed for both SE systems tested. The CSE, a two-step system, showed higher mTBS control values (27.076.8 MPa) in comparison with the results of SBSE, a simplified system (13.273.1 MPa; po0.05), and lower reduction after NaOCl storage (40.37% and 62.87% respectively). In the morphological analysis, CSE (Fig. 3a) showed no red zones while the simplified SBSE system depicted a dentin demineralized red zone under resin infiltrated layer, corresponding to collagen fibrils exposed and non-encapsulated by the adhesive resin (Fig. 4a). This difference in results of self-etch adhesives may be explained by three reasons: 1) the application mode of the SBSE system that mix acid primer and bonding resin simultaneously in the cavity, acting like a simplified one-step system; 2) the difference in pH, since SBSE is classified as an aggressive self-etch system (pHo1.0) and CSE as a mild system (pH¼ 2.0); and 3) CSE incorporate the functional monomer methacryloyl-oxy-decyl dihydrogen phosphate (MDP) that is known for its primary chemical interaction with hydroxyapatite enhancing enhance long-term stability [18]. Some self-etch systems, especially the aggressive ones, produce a continuous demineralization of dentin even after adhesive polymerization, creating an etched non resin-infiltrated layer on the underlying dentin [19]. We hypothesize also that the higher percentage of fracture mode in dentin of the self-etch system sticks compared to etch-and-rinse ones represents the result of creation of a weak area with porous and no mineralized dentin. The potent proteolytic agent NaOCl degrades unprotected collagen due to the presence of superoxide radicals in aqueous solution acting as an indicator of complete infiltration into the hybrid layer [20]. NaOCl solution can affect the resin–dentin bond structures following two pathways: (1) the etched and non-infiltrated layer and (2) the collagen that was not properly resin-infiltrated and later

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exposed because of the bonding resin dissolution by the NaOCl [21]. Over time, resin elution from hydrolytically unstable polymeric hydrogels within hybrid layers [22] may continue to occur through the nanoleakage channels during aging, producing continuous further collagen fibril exposure. Subsequent hydrolysis of the exposed collagen peptides could lead to degradation of the resin– dentin bond, resulting in decreased bond strength and increased interfacial microleakage and nanoleakage over time [23,24]. The morphological data of this study showed the efficacy of NaOCl as nonspecific deproteinizing agent on degradation of nonencapsulated collagen, especially for the two-step etch-and-rinse adhesive system (SB2), where no collagen fibrils were present after the use of NaOCl, and a light zone was noted in the place of the red staining (Fig. 2B). These results are in accordance with those obtained by Sauro et al. that, using confocal microscopy, demonstrated complete removal of non-encapsulated collagen from acid-etched and adhesive-infiltrated dentin after the immersion time in NaOCl between 10 and 45 min [10]. Taschner et al. either showed that immersion in NaOCl reduced bond strength and may act as a great challenge to the adhesive interface [25]. Whereas previous findings obtained with different adhesive systems showed bond strength reductions comparable to six-month storage in artificial saliva [9,26].

5. Conclusion Regardless of the bond strategy, the encapsulation of all exposed collagen fibrils by resin monomers is crucial for stabilizing the resin/dentin interface. It can be concluded that collagen encapsulation affects the quality of bond interface, which is related to the adhesive system used.

Acknowledgments The authors thank Prof. Dr. Pedro Marcos Gomes Soares and Profa. Dra. Gerly Anne Brito (LAFICA – Laboratory of Pharmacology of Inflammation and Cancer) for photographical assistance, and Prof. Luis Flávio Gaspar Herculano and Natanael Wagner Sales Morais (LACAM – Laboratory of Materials Characterization) for mechanical assistance. This research was supported by Grant from FUNCAP-Brazil (Grant no. 2430/07). References [1] Pashley DH, Tay FR, Breschi L, Tjäderhane L, Carvalho RM, Carrilho M, et al. State of the art etch-and-rinse adhesives. Dent Mater 2011;27:1–16. [2] Bolanõs-Carmona V, González-López S, Briones-Luján T, De Haro-Munõz C, Macorra JC. Effects of etching time of primary dentin on interface morphology and microtensile bond strength. Dent Mater 2006;22:1121–9. [3] Sano H, Takatsu T, Ciucchi B, Horner JA, Matthews WG, Pashley DH. Nanoleakage: leakage within the hybrid layer. Op Dent 1995;20:18–25. [4] Spencer P, Swafford JR. Unprotected protein at the dentin-adhesive interface. Quintessence Int 1999;30:501–7. [5] Van Meerbeek B, Yoshihara K, Yoshida Y, Mine A, De Munck J, Van Landuyt KL. State of the art of self-etch adhesives. Dent Mater 2011;27:17–28. [6] Breschi L, Mazzoni A, Ruggeri A, Cadenaro M, Di Lenarda R, Dorigo E. Dental aging review: aging and stability of the bonded interface. Dent Mater 2008;24:90–101. [7] Cadenaro M, Antoniolli F, Sauro S, Tay FR, Di Lenarda R, Prati C, et al. Degree of conversion and permeability of dental adhesives. Eur J Oral Sci 2005;113:525–30. [8] Saboia VP, Nato F, Mazzoni A, Orsini G, Putignano A, Giannini M, et al. Adhesion of a two-step etch-and-rinse adhesive on collagen-depleted dentin. J Adhes Dent 2008;10:419–22. [9] Saboia VP, Silva FC, Nato F, Mazzoni A, Cadenaro M, Mazzotti G, et al. Analysis of differential artificial ageing of the adhesive interface produced by a twostep etch-and-rinse adhesive. Eur J Oral Sci 2009;117:618–24. [10] Sauro S, Mannocci F, Tay FR, Pashley DH, Cook R, Carpenter GH, et al. Deproteinization effects of NaOCl on acid-etched dentin in clinically-relevant vs prolonged periods of application. A confocal and environmental scanning electron microscopy study. Op Dent 2009;34:166–73.

26

F.M. Apolonio et al. / International Journal of Adhesion & Adhesives 59 (2015) 21–26

[11] Toledano M, Osorio R, Perdigão J, Rosales JI, Thompson JY, Cabrerizo-Vilchez MA. Effect of acid etching and collagen removal on dentin wettability and roughness. J Biomed Mater Res 1999;47:198–203. [12] Erhardt MCG, Toledano M, Osorio R, Pimenta LA. Histomorphologic characterization and bond strength evaluation of caries-affected dentin/resin interfaces: effects of long-term water exposure. Dent Mater 2008;24:786–98. [13] Yang H, Pei D, Chen Z, Lei J, Zhou L, Huang C. Effects of the application sequence of calcium-containing desensitising pastes during etch-and-rinse adhesive restoration. J Dent 2014;42:1115–23. [14] Argolo S, Mathias P, Aguiar T, Lima A, Santos S, Foxton R, et al. Effect of agitation and storage temperature on water sorption and solubility of adhesive systems. Dent Mater J 2014;34:1–6. [15] Cardoso MV, Almeida Neves A, Mine A, Coutinho E, Van Landuyt K, De Munck J, et al. Current aspects on bonding effectiveness and stability in adhesive dentistry. Aust Dent J 2011;56:31–44. [16] Tay FR, Frankenberger R, Krejci I, Bouillaget S, Pashley DH, Carvalho RM, et al. Single-bottle adhesives behave as permeable membranes after polymerization I. In vivo evidence. J Dent 2004;32:611–21. [17] Wang Y, Spencer P. Hybridization efficiency of the adhesive/dentin interface with wet bonding. J Dent Res 2003;82:141–5. [18] Erhardt MC, Pisani-Proença J, Osorio E, Aguilera FS, Toledano M, Osorio R. Influence of laboratory degradation methods and bonding application parameters on microtbs of self-etch adhesives to dentin. Am J Dent 2011;24:103–8.

[19] Carvalho RM, Chersoni S, Frankenberger R, Pashley DH, Prati C, Tay FR. A challenge to the conventional wisdom that simultaneous etching and resin infiltration always occurs in self-etch adhesives. Biomaterials 2005;26:1035–42. [20] Hashimoto M, Tay FR, Ohno H, Sano H, Kaga M, Yiu C, et al. SEM and TEM analysis of water degradation of human dentin collagen. J Biomed Mater Res 2003;66:287–98. [21] Toledano M, Osorio R, Albaladejo A, Aguilera FS, Osorio E. Differential effect of in vitro degradation on resin-dentin bonds produced by self-etch versus totaletch adhesives. J Biomed Mater Res 2006;77:128–35. [22] Ito S, Hashimoto M, Wadgaonkar B, Svizero N, Carvalho RM, Yiu C, et al. Effects of resin hydrophilicity on water sorption and changes in modulus of elasticity. Biomaterials 2005;26:6449–59. [23] Nagpal R, Tewari S, Gupta R. Effect of various surface treatments on Microtensile and ultrastructure of resin–tooth interface. Op Dent 2007;32:16–23. [24] Reis A, Carrilho M, Breschi L, Loguercio AD. Overview of clinical alternatives to minimize the degradation of the resin–dentin bonds. Op Dent 2013;38:E1–25. [25] Taschner M, Kümmerling M, Lohbauer U, Breschi L, Petschelt A, Frankenberger R. Effect of double-layer application on dentin bond durability of one-step self-etch adhesives. Op Dent 2014;39:416–26. [26] Taschner M, Nato F, Mazzoni A, Frankenberger R, Falconi M, Petschelt A, et al. Influence of preliminary etching on the stability of bonds created by one-step self-etch bonding systems. Eur J Oral Sci 2012;120:239–48.