Effectiveness of immediate bonding of etch-and-rinse adhesives to simplified ethanol-saturated dentin

This study examined the immediate bond strength of etchand-rinse adhesives to demineralized dentin saturated with either water or absolute ethanol. The research hypothesis was that there would be no difference in bond strength to dentin between water or ethanol wetbonding techniques. The medium dentin of 20 third molars was exposed (n = 5). The dentin surface was then acid-etched, left moist and randomly assigned to be saturated via either water wet-bonding (WBT) or absolute ethanol wet-bonding (EBT). The specimens were then treated with one of the following etch-and-rinse adhesive systems: a 3-step, water-based system (Adper Scotchbond Multipurpose, or SBMP) or a 2-step, ethanol / water-based system (Adper Single Bond 2, or SB). Resin composite build-ups were then incrementally constructed. After water storage for 24 h at 37°C, the tensile strength of the specimens was tested in a universal testing machine (0.5 mm/min). Data were analyzed by two-way ANOVA and Tukey’s test (α = 5%). The failure modes were verified using a stereomicroscope (40×). For both adhesives, no significant difference in bond strength was observed between WBT and EBT (p > 0.05). The highest bond strength was observed for SB, regardless of the bonding technique (p < 0.05). No significant interaction between adhesives and bonding techniques was noticed (p = 0.597). There was a predominance of adhesive failures for all tested groups. The EBT and WBT displayed similar immediate bond strength means for both adhesives. The SB adhesive exhibited higher means for all conditions tested. Further investigations are needed to evaluate long-term bonding to dentin mediated by commercial etch-and-rinse adhesives using the EBT approach. Descriptors: Dentin; Tensile Strength; Dentin-Bonding Agents; Dental Restoration, Permanent. Introduction The wet-bonding technique has been regarded as being the main method for bonding etch-and-rinse adhesives to dentin.1 In this bonding approach, the organic solvents added to hydrophilic monomers, such as acetone and ethanol, displace the water molecules from the demineralized collagen matrix.2 As a consequence, depending on the adhesive composition, the solvent evaporation will facilitate the diffusion of the monomers throughout the demineralized dentin. This favors the correct adhesive polymerization to form an interfacial hybrid layer and also imDeclaration of Interests: The authors certify that they have no commercial or associative interest that represents a conflict of interest in connection with the manuscript. Effectiveness of immediate bonding of etch-and-rinse adhesives to simplified ethanol-saturated dentin 178 Braz Oral Res. 2012 Mar-Apr;26(2):177-82 proves the bond strength to dentin.3 However, several studies have demonstrated that the monomer infiltration is considered mostly incomplete,4 mainly due to the hydrophilicity of the adhesive monomers. This favors the creation of a porous hybrid layer that is more prone to water sorption and solubility.5 These characteristics help the leachable monomers to reach the pulp tissue through intertubular and/ or dentin tubules and, consequently, increasing the potential toxicity and compromise the restoration longevity.6 Studies have shown that adding hydrophobic monomers to the adhesives allows for higher stiffness and increased stability in an aqueous environment, thus improving the durability of the bonded interface compared to that observed when hydrophilic-rich adhesives are applied.7 Recently, a study proposed to replace the residual water in a demineralized dentin matrix prior to the adhesive application by applying absolute ethanol to coax hydrophobic monomers into the ethanol-saturated dentin.8 The ultimate goal of this technique is to displace the residual water with ethanol, leaving the unsupported, uncollapsed demineralized collagen network suspended in ethanol-moist collagen fibrils. The closer the solubility parameter for hydrogen bonding forces (dh) of the mixture monomer/solvent is to that of the dentin, the better the compatibility and ability of the solvents to wet the dentin substrate.9 The EBT allows the relatively hydrophobic monomers to permeate the dentin substrate.8 Presumably, the more hydrophobic the resins, the lower the water absorption, the smaller the plasticization effect, and the more durable the bonding to dentin.5 The EBT is completed in two different ways: • a series of increased ethanol concentrations or • by saturating the dentin with absolute ethanol.9 Both strategies have shown promising performance, but a simplified bonding technique is considered to be more acceptable to the practitioners. Despite the promising findings of the ethanol wet bonding technique in coronal10 and intraradicular dentin,11 the association of a simplified ethanol-wet bonding technique with a commercial total-etch system deserves evaluation. This study evaluated the influence of the simplified ethanol-wet bonding technique on the bond strength to mid-coronal dentin by using commercial ethanol and water-based etch-and-rinse adhesive systems. The purpose of this in vitro study was to evaluate the effectiveness of both a 3-step and a 2-step etch-and-rinse adhesive system, which were applied by using water or ethanol wet bonding. The research hypothesis tested was whether the ethanol bonding technique would produce similar bond strengths to dentin when compared to those observed by the water wet-bonding technique, regardless of the adhesive system. Methodology Twenty sound human third molars were selected and used in accordance with a protocol approved by the institutional Research Ethics Committee. Teeth were stored in a saline solution containing 0.1  % thymol at 4°C and used within 4 months of extraction. The cusps were abraded with a water-cooled rotating diamond wheel (Isomet 1000, Buehler; Lake Bluff, USA) to expose a flat surface free of enamel tissue in the mid-coronal dentin surface. Using the highest pulp horn as a reference, a remaining dentin thickness varying from 1.5 to 2.0  mm was used to standardize the mid-coronal dentin. Also, a magnifying lens was used to check whether remaining enamel areas were noted. The dentin surfaces were further polished with a wet #600-grit SiC paper for 60  s to standardize the smear layer. After that, each exposed surface was acid-etched for 15 s with a 37% phosphoric acid gel and water-rinsed for 15 s. The specimens were then randomly divided into 2 groups (n = 10) according to the bonding approach: 1. Water wet-bonding technique (WBT) Excess water was removed from the surface with absorbing paper and the dentin remained moist. 2. Ethanol wet-bonding technique (EBT) The ethanol wet-bonding substrate was achieved by applying absolute ethanol (100% ethanol) to the demineralized dentin surface. Absolute ethanol was applied using a microbrush for 30  s. The dentin surface was saturated with 100% ethanol to completely replace the saturated water in the demineralized dentin matrix. Special care was Guimarães LA, Almeida JCF, Wang L, D’Alpino PHP, Garcia FCP 179 Braz Oral Res. 2012 Mar-Apr;26(2):177-82 taken to ensure that the dentin surface was always visibly moist. Excess ethanol was removed using an absorbing paper. The adhesive systems selected were a 3-step, etchand-rinse, water-based adhesive system (ScotchBond Multipurpose Plus, 3M ESPE, St. Paul, USA) and a 2-step, etch-and-rinse, ethanol / water-based adhesive system (Adper Single Bond 2, 3M ESPE, St. Paul, USA). Table 1 describes the adhesives and their compositions. Both adhesives were applied to the dentin surface after WBT or EBT according to the manufacturer’s directions. After application, the adhesives were light cured using a halogen light curing unit (Demetron, Kerr, Danbury, USA) with a power density of 570 mW/cm2 for 10 s. Four layers of 1 mm-thick resin composite (Filtek 350XT, 3M ESPE, St. Paul, USA) were applied to obtain specimens with the same dentin / resin composite proportion. Each layer was light-cured for 40 s using the same light-curing unit. The bonded teeth were stored in distilled water at 37°C for 24 h before testing. The roots were then sectioned approximately 2 mm below the cemento-enamel junction, perpendicular to the long axis of the tooth, using a diamond-impregnated disk (Extec, Enfield, USA) in a specific cutting machine (Isomet 1000, Buehler, Lake Bluff, USA), under water-cooling at 300 rpm. The teeth were then longitudinally sectioned in both “x” and “y” axes across the bonded interface using the same water-cooling cutting device. The resulting bonded stick-shaped specimens with a cross-sectional area of 0.8 (± 0.2) mm2 were then cemented to the testing device using cyanoacrylate cement (Zapit, DVA Inc., Corona, USA). The specimens were attached to a universal testing machine (Instron Model 3342, Instron Corp., Canton, USA) and stressed in tension at a cross-speed of 0.5  mm/min until failure. After testing, the fractured specimens were carefully removed from the apparatus and the crosssectional area was measured with a digital caliper at the site of failure. The results were recorded, and the debonded stress values were converted into MPa. Pre-test failures were not noted. The distribution of the failure mode of remaining composite and dentin fragments was also evaluated at 40× magnification using a dissecting microscope (Stereozoom, Bausch & Lomb, Rochester, USA). Failure mode patterns were classified as follows: • A  =  adhesive between dentin and adhesive system; • M = mixed and • C = cohesive in resin composite or dentin. Data was statistically analyzed (2-way ANOVA, Tukey’s test [α = 0.05]). Results The bond strength means (in MPa) of the experiAdhesive systems Composition Lot # Adper Scotchbond Multipurpose Plus (SBMP) Primer: aqueous solution of HEMA and a polyalkenoic acid copolymer Adhesive: BisGMA and HEMA resins 7KP Adper Single Bond 2 (SB) BisGMA, GDMA, UDMA, HEMA, nanofillers, water, ethanol, methacrylate fun


Introduction
The wet-bonding technique has been regarded as being the main method for bonding etch-and-rinse adhesives to dentin. 1 In this bonding approach, the organic solvents added to hydrophilic monomers, such as acetone and ethanol, displace the water molecules from the demineralized collagen matrix. 2 As a consequence, depending on the adhesive composition, the solvent evaporation will facilitate the diffusion of the monomers throughout the demineralized dentin. This favors the correct adhesive polymerization to form an interfacial hybrid layer and also im-Declaration of Interests: The authors certify that they have no commercial or associative interest that represents a conflict of interest in connection with the manuscript.
proves the bond strength to dentin. 3 However, several studies have demonstrated that the monomer infiltration is considered mostly incomplete, 4 mainly due to the hydrophilicity of the adhesive monomers. This favors the creation of a porous hybrid layer that is more prone to water sorption and solubility. 5 These characteristics help the leachable monomers to reach the pulp tissue through intertubular and/ or dentin tubules and, consequently, increasing the potential toxicity and compromise the restoration longevity. 6 Studies have shown that adding hydrophobic monomers to the adhesives allows for higher stiffness and increased stability in an aqueous environment, thus improving the durability of the bonded interface compared to that observed when hydrophilic-rich adhesives are applied. 7 Recently, a study proposed to replace the residual water in a demineralized dentin matrix prior to the adhesive application by applying absolute ethanol to coax hydrophobic monomers into the ethanol-saturated dentin. 8 The ultimate goal of this technique is to displace the residual water with ethanol, leaving the unsupported, uncollapsed demineralized collagen network suspended in ethanol-moist collagen fibrils. The closer the solubility parameter for hydrogen bonding forces (d h ) of the mixture monomer/solvent is to that of the dentin, the better the compatibility and ability of the solvents to wet the dentin substrate. 9 The EBT allows the relatively hydrophobic monomers to permeate the dentin substrate. 8 Presumably, the more hydrophobic the resins, the lower the water absorption, the smaller the plasticization effect, and the more durable the bonding to dentin. 5 The EBT is completed in two different ways: • a series of increased ethanol concentrations or • by saturating the dentin with absolute ethanol. 9 Both strategies have shown promising performance, but a simplified bonding technique is considered to be more acceptable to the practitioners. Despite the promising findings of the ethanol wet bonding technique in coronal 10 and intraradicular dentin, 11 the association of a simplified ethanol-wet bonding technique with a commercial total-etch system deserves evaluation.
This study evaluated the influence of the sim-plified ethanol-wet bonding technique on the bond strength to mid-coronal dentin by using commercial ethanol and water-based etch-and-rinse adhesive systems. The purpose of this in vitro study was to evaluate the effectiveness of both a 3-step and a 2-step etch-and-rinse adhesive system, which were applied by using water or ethanol wet bonding. The research hypothesis tested was whether the ethanol bonding technique would produce similar bond strengths to dentin when compared to those observed by the water wet-bonding technique, regardless of the adhesive system.

Methodology
Twenty sound human third molars were selected and used in accordance with a protocol approved by the institutional Research Ethics Committee. Teeth were stored in a saline solution containing 0.1 % thymol at 4°C and used within 4 months of extraction. The cusps were abraded with a water-cooled rotating diamond wheel (Isomet 1000, Buehler; Lake Bluff, USA) to expose a flat surface free of enamel tissue in the mid-coronal dentin surface. Using the highest pulp horn as a reference, a remaining dentin thickness varying from 1.5 to 2.0 mm was used to standardize the mid-coronal dentin. Also, a magnifying lens was used to check whether remaining enamel areas were noted. The dentin surfaces were further polished with a wet #600-grit SiC paper for 60 s to standardize the smear layer. After that, each exposed surface was acid-etched for 15 s with a 37% phosphoric acid gel and water-rinsed for 15 s. The specimens were then randomly divided into 2 groups (n = 10) according to the bonding approach: 1. Water wet-bonding technique (WBT) -Excess water was removed from the surface with absorbing paper and the dentin remained moist.

Ethanol wet-bonding technique (EBT) -The
ethanol wet-bonding substrate was achieved by applying absolute ethanol (100% ethanol) to the demineralized dentin surface. Absolute ethanol was applied using a microbrush for 30 s. The dentin surface was saturated with 100% ethanol to completely replace the saturated water in the demineralized dentin matrix. Special care was taken to ensure that the dentin surface was always visibly moist. Excess ethanol was removed using an absorbing paper.
Both adhesives were applied to the dentin surface after WBT or EBT according to the manufacturer's directions. After application, the adhesives were light cured using a halogen light curing unit (Demetron, Kerr, Danbury, USA) with a power density of 570 mW/cm² for 10 s. Four layers of 1 mm-thick resin composite (Filtek 350XT, 3M ESPE, St. Paul, USA) were applied to obtain specimens with the same dentin / resin composite proportion. Each layer was light-cured for 40 s using the same light-curing unit. The bonded teeth were stored in distilled water at 37°C for 24 h before testing.
The roots were then sectioned approximately 2 mm below the cemento-enamel junction, perpendicular to the long axis of the tooth, using a diamond-impregnated disk (Extec, Enfield, USA) in a specific cutting machine (Isomet 1000, Buehler, Lake Bluff, USA), under water-cooling at 300 rpm. The teeth were then longitudinally sectioned in both "x" and "y" axes across the bonded interface using the same water-cooling cutting device. The resulting bonded stick-shaped specimens with a cross-sectional area of 0.8 (± 0.2) mm² were then cemented to the testing device using cyanoacrylate cement (Zapit, DVA Inc., Corona, USA). The specimens were attached to a universal testing machine (Instron Model 3342, Instron Corp., Canton, USA) and stressed in tension at a cross-speed of 0.5 mm/min until failure. After testing, the fractured specimens were carefully removed from the apparatus and the crosssectional area was measured with a digital caliper at the site of failure. The results were recorded, and the debonded stress values were converted into MPa. Pre-test failures were not noted. The distribution of the failure mode of remaining composite and dentin fragments was also evaluated at 40× magnification using a dissecting microscope (Stereozoom, Bausch & Lomb, Rochester, USA). Failure mode patterns were classified as follows: • A = adhesive between dentin and adhesive system; • M = mixed and • C = cohesive in resin composite or dentin.    mental groups are summarized in Table 2. Tukey's test showed no significant difference between the bond strength means for water and ethanol-wet bonding for both adhesive systems (p > 0.05). SB exhibited higher bond strength mean values regardless of the bonding approach (p < 0.05). ANOVA revealed that the interaction between adhesive and technique was not significant (p = 0.597). No premature failures were reported either during the cutting procedure or during the test. Failure modes of the tested interfaces demonstrated that the majority of the bonds failed in an adhesive mode.

Discussion
Adhesive systems are designed to provide dentin adhesion via the interaction of hydrophilic monomers in an organic solvent throughout a collagenrich humid tissue. 12 Depending on the solvents, replacing the water contained in the dentin matrix may cause shrinkage. 13 Water has not been shown to produce stiffening in the demineralized matrix. 14 Humid, water-saturated dentin matrix, d h = 40.4(J/ cm³) 1/2 , favors the breaking of interpeptide H-bonds, allowing the dentin matrix to maintain itself expanded. 15 As the collagen fibrils are intrinsically wet, a full impregnation of hydrophilic monomers such as HEMA would be expected. However, this may only occur under ideal conditions. The results of the present study warrant the acceptance of the research hypothesis that there is no difference in the bond strength means when water and ethanol bonding techniques were compared. A statistical equivalence was noticed when the EBT was used prior to the application of both adhesive systems (p > 0.05).
It has been claimed that, while the demineralized dentin matrix is dehydrated by the solvents, the interfibrillar spaces would be preserved if the d h values of these solvents were high. 14 That study demonstrated the need to examine and optimize a short, clinically acceptable application time compared to that currently used in dentin bonding procedures. In addition, the optimal infiltration of collagen fibrils by the adhesive monomers would occur when the polar surface-free energy components are similar. According to Nishitani et al., 15 the solubility param-eter for polar forces (d p ) for ethanol-saturated dentin is 12.5 (J/cm³) 1/2 , which is closer to that of the adhesives tested (data not shown). Adding ethanol as a solvent to adhesive systems has shown to favor the infiltration of collagen fibrils and makes the dentin matrix more hydrophobic and stable over time. 16 For this reason, manufacturers have blended hydrophobic monomers in the adhesive formulations to promote bonding impregnation in a clinically acceptable time. 17 It is important to consider that applying absolute ethanol causes the dentin matrix to shrink 15% 18 due to the formation of matrix interpeptide Hbonds. In this study, the demineralized dentin was treated with a copious amount of absolute ethanol for 30 s in order to create an ethanol-saturated dentin matrix. The saturation time varies extensively in the literature and has been previously reported as 15 s, 19 20 s 20 and 1 min, 17 and a series of increased ethanol concentrations 9 for 30 s has also been used. To test our hypothesis, we used 30 s to saturate the dentin matrix with ethanol in a clinically acceptable timeframe. The ideal clinical condition would be the application of adhesives in which solvent evaporation prevented further shrinkage during infiltration of the monomers. 14 Thus, it can be speculated that applying absolute ethanol for 30 s lessens the stiffening effect on the dentin matrix because the bonding to dentin was unaffected.
It has been demonstrated that in ethanol-saturated dentin, the diameter of the collagen fibrils is smaller than those in water-saturated dentin, leaving larger interfibrillar spaces available for monomer impregnation. 20 As higher bond strength is correlated with wider interfibrillar spaces, 21 the use of ethanol can increase bond strength. 15 Saturating the dentin matrix with ethanol creates favorable circumstances for methacrylates (as BisGMA) to diffuse into interfibrillar spaces forming a hybrid layer and producing a higher mechanical property. 18 A reasonable explanation for the equivalence of bond strength of both SB and SBMP to ethanol pretreated dentin could be related to both HEMA and BisGMA methacrylates present in the composition of both formulations. Despite their hydrophilicity (monomethacrylate HEMA) or hydrophobicity (di-methacrylate BisGMA), both monomers are soluble in ethanol. The more resin (both mono-and dimethacrylates) that infiltrates acid-etched matrices, the higher are the resin-dentin bond strengths. 15 Even though no improvement in the bond strength was noticed when using EBT, it is important to consider that recent studies have demonstrated that endogenous dentin matrix metalloproteinases (MMPs) are present within the dentin matrices and may be activated in the presence of water after the adhesive application. 22 Consequently, the use of ethanol to replace water may stabilize the bonding to dentin over time. 17 The results of the present study demonstrate that the bonding technique using absolute ethanol for 30 s had no influence on the immediate bond strength when associated with water and water/ethanol-based etch-and-rinse systems. Even though the EBT presents an additional clinical step, it may reduce technique sensitivity when both water/ethanol-based adhesives (SB and SBMP) are used. The EBT may successfully coax more hydrophobic monomers into the dentin matrix, thereby creating more hydrophobic and more stable hybrid layers that are less susceptible to hydrolysis over time. Recent studies have shown promising results for EBT when using dehydration protocols with an increased series of ethanol concentrations associated with hydrophobic monomers. Future studies are necessary to evaluate the possible effects, both direct and indirect, of using adhesives that contain water and hydrophilic monomers (such as HEMA) on the long-term dentin bonding durability when associated with simplified ethanol saturated dentin.

Conclusion
Within the limitations of the present study it can be concluded that ethanol wet-bonding presents equivalent bonding to dentin when compared to the results observed with the water wet-bonding technique, regardless of the adhesive system tested (hypothesis accepted).