Abstract
Dentin bonding performed with hydrophobic resins using ethanol-wet bonding should be less susceptible to degradation but this hypothesis has never been validated.
Objectives
This in vitro study evaluated stability of resin–dentin bonds created with an experimental three-step BisGMA/TEGDMA hydrophobic adhesive or a three-step hydrophilic adhesive after one year of accelerated aging in artificial saliva.
Methods
Flat surfaces in mid-coronal dentin were obtained from 45 sound human molars and randomly divided into three groups ( n = 15): an experimental three-step BisGMA/TEGDMA hydrophobic adhesive applied to ethanol (ethanol-wet bonding—GI) or water-saturated dentin (water-wet bonding—GII) and Adper Scotchbond Multi-Purpose [MP—GIII] applied, according to manufacturer instructions, to water-saturated dentin. Resin composite crowns were incrementally formed and light-cured to approximately 5 mm in height. Bonded specimens were stored in artificial saliva at 37 °C for 24 h and sectioned into sticks. They were subjected to microtensile bond test and TEM analysis immediately and after one year. Data were analyzed with two-way ANOVA and Tukey tests.
Results
MP exhibited significant reduction in microtensile bond strength after aging (24 h: 40.6 ± 2.5 a ; one year: 27.5 ± 3.3 b ; in MPa). Hybrid layer degradation was evident in all specimens examined by TEM. The hydrophobic adhesive with ethanol-wet bonding preserved bond strength (24 h: 43.7 ± 7.4 a ; one year: 39.8 ± 2.7 a ) and hybrid layer integrity, with the latter demonstrating intact collagen fibrils and wide interfibrillar spaces.
Significance
Coaxing hydrophobic resins into acid-etched dentin using ethanol-wet bonding preserves resin–dentin bond integrity without the adjunctive use of MMPs inhibitors and warrants further biocompatibility and patient safety’s studies and clinical testing.
1
Introduction
Despite important technical advances that have enhanced resin–dentin bond performance , degradation of dentin-adhesive interfaces and bond strength reductions over time are continuously reported . This probably accounts for the reduced longevity of clinically applied adhesive restorations .
Previous works have correlated resin–dentin bond instability with increased hydrophilic resin monomer content in dentin adhesives , which expedites water sorption and decreases adhesive mechanical properties . Resin–dentin bonds created by contemporary hydrophilic adhesives are also susceptible to fluid permeation .
It is possible to coax comparatively hydrophobic monomers to acid-etched dentin with an ethanol-wet bonding protocol . The rationale behind this technique is that ethanol dehydration renders acid-etched dentin less hydrophilic, allowing the use of relatively hydrophobic monomers for infiltrating shrunken but non-collapsed demineralized collagen network that is suspended in ethanol . Theoretically, this would improve resin–dentin bond durability by minimizing water sorption through polymerized hydrophobic adhesive . To-date, the longevity of hydrophobic resin–dentin bonds created by ethanol-wet bonding has never been assessed. In vitro longevity evaluation should be performed to ensure that this new bonding concept confers real advantages over contemporary hydrophilic etch-and-rinse adhesives before clinical evaluation may be recommended. Thus, the objectives of this study were (1) to compare 24-h and one-year microtensile bond strengths of composite-dentin beams created with an experimental three-step hydrophobic adhesive using ethanol-wet bonding, vs those obtained with a three-step hydrophilic etch-and-rinse adhesive using water-wet bonding; and (2) to assess structural integrity of aged resin–dentin bonds. The null hypothesis tested was that there are no differences in bond strength and ultrastructural integrity of human coronal dentin bonded with the ethanol-wet bonded hydrophobic adhesive or the water-wet bonded hydrophilic adhesive after one year of accelerated aging.
2
Materials and methods
2.1
Experimental adhesive
A comonomer resin blend comprising 70 mass% BisGMA (Esstech, Essington, PA), 28.75 mass% TEGDMA (Esstech), 0.25 mass% camphorquinone (Sigma–Aldrich, St. Louis) and 1 mass% ethyl N,N-dimethyl-4-aminobenzoate (Sigma–Aldrich) was used to formulate the experimental three-step etch-and-rinse hydrophobic adhesive. The neat comonomer resin blend was employed as the adhesive component. The primer solution was prepared by diluting it with 50 mass% absolute ethanol .
2.2
Bonding procedures
Forty-five human molars were collected after patients’ informed consent was obtained under a protocol reviewed and approved by the Ethical Research Committee, School of Dentistry, University of São Paulo, Brazil. The teeth were stored in 0.02% sodium azide at 4 °C to prevent bacteria growth and were used within 2 months after extraction. Flat surfaces in mid-coronal dentin were obtained with an Isomet saw (Buehler Ltd., Lake Bluff, IL) under water-cooling and polished with 180-grit silicon carbide papers under running water for 20 s. Each surface was etched with 35% phosphoric acid gel (3 M ESPE, St. Paul, MN) for 15 s, rinsed with water and left moist prior to bonding procedures.
Thirty teeth were used for the experimental adhesive. In half of them (GI), a chemical dehydration protocol was used for ethanol-wet bonding. Briefly, acid-etched dentin surfaces were treated with a series of increasing ethanol concentrations (50%, 70%, 80%, 95% and 3 × 100%, 30 s each; i.e. 3 min 30 s in toto ). During this procedure, dentin surfaces were kept immersed in the liquid phase prior to the application of the more concentrated ethanol solution. Two consecutive coats of the experimental hydrophobic primer were then applied to ethanol-saturated dentin. Excess ethanol solvent was evaporated with a gentle air stream for 10 s. A layer of the neat BisGMA/TEGDMA adhesive was then applied, spread thin with moisture-free air and light-cured for 20 s using a halogen light-curing unit (VIP Junior, Bisco Inc., Schaumburg, IL) with an irradiance of 600 mW/cm 2 . The experimental hydrophobic adhesive was also used as the negative control (GII) for bonding to acid-etched dentin using a conventional wet bonding protocol in which the demineralized dentin was saturated with 100% deionized water. All bonding procedures were repeated as previously described, except that the selected hydrophobic primer was directly applied to visibly moist, water-saturated demineralized dentin. For the 15 teeth in the positive control group (GIII), Adper Scotchbond Multi-Purpose (3 M ESPE) was applied to water-saturated, visibly moist demineralized dentin according to manufacturers’ instructions. The primer contains HEMA, polyalkenoic acid copolymer and water, and the bonding resin contains BisGMA and HEMA. The presence of HEMA in both primer and bonding resin was used as a criterion for designating the commercial adhesive as hydrophilic, as opposed to the experimental system. Composite build-ups were constructed in all groups with a light-cured resin composite (EPIC-TMPT, Parkell Inc., Farmingdale, NY) in five 1-mm thick increments.
2.3
Tensile testing
After storage in artificial saliva at 37 °C for 24 h, each tooth was vertically sectioned into 0.9-mm thick serial slabs with the Isomet saw under water-cooling. The central slab of each tooth was used for morphologic examination. Adjacent slabs were sectioned into 0.9 mm × 0.9 mm beams, according to the microtensile “non-trimming” technique. The number of premature failures was extremely high for the negative control group (GII) (74, or 87%), while it was small for groups I and III (6, or 3%, for the experimental group with the ethanol-wet bonding technique and 8, or 4%, for Adper Scotchbond MP). Intact beams from each tooth from groups I and III were then randomly divided into two similarly sized subgroups. One subgroup was used for 24-h bond strength testing. The remaining specimens were stored in artificial saliva (CaCl 2 (0.7), MgCl 2 ·6H 2 O (0.2), KH 2 PO 4 (4.0), KCl (30), NaN 3 (0.3), HEPES buffer (20), in mM/L) at 37 °C for one year before testing. The storage medium was changed weekly. Each beam was stressed to failure under tension using the Geraldeli’s testing device mounted on a universal testing machine (model 5565, Instron Corp., Canton, MA) at a crosshead speed of 0.5 mm/min. To improve the robustness of the statistical testing with parametric methods, results from the negative control group (GII) that were much lower than those of the experimental and positive control groups and included many premature failures, were excluded from the statistical analysis. And since premature failures were evenly distributed for groups I and III, they were excluded from the statistical analysis. As bond strength data from the four subgroups were normally distributed (Kolmogorow–Smirnoff test) and homoscedastic (Levene test), they were statistically analyzed with a two-way ANOVA design (adhesive vs testing time). Post hoc multiple comparisons were performed with Tukey test, with statistical significance set at α = 0.05.
2.4
Transmission electron microscopy (TEM)
Four central slabs from each of the four subgroups statistically analyzed were used for TEM examination. These slabs were also sectioned into composite-dentin beams to facilitate accelerated aging in artificial saliva. For each subgroup, half of the beams were completely demineralized in 17% ethylenediaminetetra-acetic acid. The rest of the mineralized beams was immersed in aqueous ammoniacal silver nitrate for 48 h according to the tracer protocol for nanoleakage examination . Both demineralized and undemineralized, epoxy-resin-embedded, 90-nm thick sections were prepared using a standardized TEM protocol . Demineralized sections were stained with methanolic uranyl acetate and Reynolds’ lead citrate for examination of the characteristics of resin–dentin interfaces ( n = 5). Undemineralized sections were examined unstained for identification of silver tracer particles within resin–dentin interfaces ( n = 5). The sections were examined with a TEM (JEM-1230, JEOL, Tokyo, Japan) operating at 80 kV.
2
Materials and methods
2.1
Experimental adhesive
A comonomer resin blend comprising 70 mass% BisGMA (Esstech, Essington, PA), 28.75 mass% TEGDMA (Esstech), 0.25 mass% camphorquinone (Sigma–Aldrich, St. Louis) and 1 mass% ethyl N,N-dimethyl-4-aminobenzoate (Sigma–Aldrich) was used to formulate the experimental three-step etch-and-rinse hydrophobic adhesive. The neat comonomer resin blend was employed as the adhesive component. The primer solution was prepared by diluting it with 50 mass% absolute ethanol .
2.2
Bonding procedures
Forty-five human molars were collected after patients’ informed consent was obtained under a protocol reviewed and approved by the Ethical Research Committee, School of Dentistry, University of São Paulo, Brazil. The teeth were stored in 0.02% sodium azide at 4 °C to prevent bacteria growth and were used within 2 months after extraction. Flat surfaces in mid-coronal dentin were obtained with an Isomet saw (Buehler Ltd., Lake Bluff, IL) under water-cooling and polished with 180-grit silicon carbide papers under running water for 20 s. Each surface was etched with 35% phosphoric acid gel (3 M ESPE, St. Paul, MN) for 15 s, rinsed with water and left moist prior to bonding procedures.
Thirty teeth were used for the experimental adhesive. In half of them (GI), a chemical dehydration protocol was used for ethanol-wet bonding. Briefly, acid-etched dentin surfaces were treated with a series of increasing ethanol concentrations (50%, 70%, 80%, 95% and 3 × 100%, 30 s each; i.e. 3 min 30 s in toto ). During this procedure, dentin surfaces were kept immersed in the liquid phase prior to the application of the more concentrated ethanol solution. Two consecutive coats of the experimental hydrophobic primer were then applied to ethanol-saturated dentin. Excess ethanol solvent was evaporated with a gentle air stream for 10 s. A layer of the neat BisGMA/TEGDMA adhesive was then applied, spread thin with moisture-free air and light-cured for 20 s using a halogen light-curing unit (VIP Junior, Bisco Inc., Schaumburg, IL) with an irradiance of 600 mW/cm 2 . The experimental hydrophobic adhesive was also used as the negative control (GII) for bonding to acid-etched dentin using a conventional wet bonding protocol in which the demineralized dentin was saturated with 100% deionized water. All bonding procedures were repeated as previously described, except that the selected hydrophobic primer was directly applied to visibly moist, water-saturated demineralized dentin. For the 15 teeth in the positive control group (GIII), Adper Scotchbond Multi-Purpose (3 M ESPE) was applied to water-saturated, visibly moist demineralized dentin according to manufacturers’ instructions. The primer contains HEMA, polyalkenoic acid copolymer and water, and the bonding resin contains BisGMA and HEMA. The presence of HEMA in both primer and bonding resin was used as a criterion for designating the commercial adhesive as hydrophilic, as opposed to the experimental system. Composite build-ups were constructed in all groups with a light-cured resin composite (EPIC-TMPT, Parkell Inc., Farmingdale, NY) in five 1-mm thick increments.
2.3
Tensile testing
After storage in artificial saliva at 37 °C for 24 h, each tooth was vertically sectioned into 0.9-mm thick serial slabs with the Isomet saw under water-cooling. The central slab of each tooth was used for morphologic examination. Adjacent slabs were sectioned into 0.9 mm × 0.9 mm beams, according to the microtensile “non-trimming” technique. The number of premature failures was extremely high for the negative control group (GII) (74, or 87%), while it was small for groups I and III (6, or 3%, for the experimental group with the ethanol-wet bonding technique and 8, or 4%, for Adper Scotchbond MP). Intact beams from each tooth from groups I and III were then randomly divided into two similarly sized subgroups. One subgroup was used for 24-h bond strength testing. The remaining specimens were stored in artificial saliva (CaCl 2 (0.7), MgCl 2 ·6H 2 O (0.2), KH 2 PO 4 (4.0), KCl (30), NaN 3 (0.3), HEPES buffer (20), in mM/L) at 37 °C for one year before testing. The storage medium was changed weekly. Each beam was stressed to failure under tension using the Geraldeli’s testing device mounted on a universal testing machine (model 5565, Instron Corp., Canton, MA) at a crosshead speed of 0.5 mm/min. To improve the robustness of the statistical testing with parametric methods, results from the negative control group (GII) that were much lower than those of the experimental and positive control groups and included many premature failures, were excluded from the statistical analysis. And since premature failures were evenly distributed for groups I and III, they were excluded from the statistical analysis. As bond strength data from the four subgroups were normally distributed (Kolmogorow–Smirnoff test) and homoscedastic (Levene test), they were statistically analyzed with a two-way ANOVA design (adhesive vs testing time). Post hoc multiple comparisons were performed with Tukey test, with statistical significance set at α = 0.05.
2.4
Transmission electron microscopy (TEM)
Four central slabs from each of the four subgroups statistically analyzed were used for TEM examination. These slabs were also sectioned into composite-dentin beams to facilitate accelerated aging in artificial saliva. For each subgroup, half of the beams were completely demineralized in 17% ethylenediaminetetra-acetic acid. The rest of the mineralized beams was immersed in aqueous ammoniacal silver nitrate for 48 h according to the tracer protocol for nanoleakage examination . Both demineralized and undemineralized, epoxy-resin-embedded, 90-nm thick sections were prepared using a standardized TEM protocol . Demineralized sections were stained with methanolic uranyl acetate and Reynolds’ lead citrate for examination of the characteristics of resin–dentin interfaces ( n = 5). Undemineralized sections were examined unstained for identification of silver tracer particles within resin–dentin interfaces ( n = 5). The sections were examined with a TEM (JEM-1230, JEOL, Tokyo, Japan) operating at 80 kV.
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