The enamel and dentin bond durability of the two-step adhesive systems in different etching modes were determined under different degradation conditions. The etching mode was either SE, or ER mode. Enamel and dentin bond durability were separately determined by measuring shear bond strength (SBS) after three different numbers of thermal cycles (TC): 0, 10,000, 20,000 or 30,000; and different periods of long-term water storage (WS): 24 h, 6-months, 1-year, or 2-year. The TC 0 (no cycling) group and WS 24 h group was set as a baseline group and were merged as a single group. Thus, there were a total of 42 treatment groups (3 adhesives × 2 etching modes × 7 degradation conditions (baseline group, 3 thermal cycling groups, and 3 water storage groups) in enamel and dentin. A total of 1092 bovine teeth were used in this study: 1008 teeth were used for enamel and dentin bond strength tests, 72 for the measurement of the surface free energy (SFE) of the cured bonding agent, and 12 for the scanning electron microscopy (SEM) observations. The necessary sample sizes for the enamel and dentin SBS test were calculated using Sigma Plot version 13 (Systat Software, Chicago, IL, USA). Based on our previous study , we assumed that the minimum important difference in the mean SBS values was 10 MPa, the expected standard deviation of residuals was 4.0, and the number of groups was 42 for shear bond strength. Power = 0.9 and α = 0.05 were set for SBS test. The calculations indicated that 12 specimens were required to effectively measure the shear bond strength in each group.
For SFE measurement, a statistical power analysis (G Power calculator) indicated that 10.7 specimens were required to measure the SFE effectively. The parameters used were as follows: α = 0.05 and power = 0.9. Thus, the experiments were conducted using 12 specimens for SFE tests.
Table 1 shows the materials used in this study. The two-step adhesive system using a universal adhesive-derived primer, G2-Bond Universal (GU; GC, Tokyo, Japan), was used. Clearfil SE Bond 2 (CS; Kuraray Noritake Dental, Tokyo, Japan) and OptiBond XTR (OX; Kerr, Orange, CA, USA) were the two traditional two-step SE adhesive systems used for comparison. Clearfil AP-X (Kuraray Noritake Dental) was used for bonding to the tooth substrate after bonding procedures. A light-emitting diode curing unit (Valo; Ultradent Products, South Jordan, UT, USA) was used with a 10 mm internal tip diameter, and a light irradiance of over 1,000 mW/cm2 (standard mode) was checked during the experiment.
Bovine teeth were used as a substitute for human teeth. Approximately two-thirds of each tooth’s apical root was removed using a diamond-impregnated disk in a precision sectioning saw (IsoMet 1000, Buehler, Lake Bluff, IL, USA). The labial surfaces were subjected to mechanical grinding/polishing (Ecomet 4, Buehler) with a wet 180-grit silicon carbide (SiC) paper (Fuji Star Type DDC, Sankyo Rikagaku, Saitama, Japan) to create a flat enamel and dentin surface. The prepared tooth was then mounted in self-curing acrylic resin (Tray Resin II, Shofu, Kyoto, Japan) to expose the flattened enamel and dentin surfaces. The adherent bonding surfaces were polished using 240-grit SiC paper followed by 320-grit SiC paper (Fuji Star Type DDC) under running water.
Adhesive application protocol and storage
Table 2 shows the adhesives' bonding procedures. Twelve specimens were prepared for each test group to measure the shear bond strength (SBS) to the enamel and dentin in the SE mode (without phosphoric acid pre-etching) and the ER mode (phosphoric acid pre-etching for 15 s). For the SE mode, the adhesives were applied to the adherent surface following the manufacturers' instructions. In contrast, for the ER mode, 35% phosphoric acid (Ultra-Etch, Ultradent Products, South Jordan, UT, USA) was applied to the enamel and dentin surfaces for 15 s, followed by rinsing with a water spray using a three-way dental syringe for 15 s. Bonded assemblies were fabricated by clamping plastic molds (height, 2.0 mm; internal diameter, 2.38 mm; Ultradent Products) to a fixture against the adherent surfaces. The resin composite was condensed into the mold and irradiated for 30 s. The bonded specimens were subjected to thermal cycling (TC groups) or underwent water storage (WS group) in distilled water (37°C). In the TC groups, the bonded assemblies were stored in distilled water at 37°C for 24 h and then subjected to 5,000, 10,000, 20,000, or 30,000 TCs (between 5°C and 55°C), with a dwell time of 30 s. The bonded assemblies in the WS groups were stored in distilled water (37°C) for 6-months, 1-year, or 2-year before the SBS tests (long-term storage). The storage water was changed every week during the experimental period. Baseline specimens were stored in distilled water at 37°C for 24 h before the SBS tests (baseline subgroups).
Shear bonding strength test
To reduce ununiform distribution stress on the bonded specimen when using shear bond strength test, the notched-edge SBS test measured the SBS of the bonded specimens after undergoing the different degradation conditions according to ISO 29022 . The bonded specimens were fixed with the Ultradent shearing fixture and loaded until bonding failure occurred at 1.0 mm per min using a universal testing machine (Type 5500R, Instron, Canton, MA, USA). The SBS values (MPa) were obtained by dividing the peak load at failure by the bonded adherent area. The de-bonded sites on the tooth surfaces and resin composite rods were observed under an optical microscope (SZH-131; Olympus, Tokyo, Japan) at a magnification of ×10 to determine the bond failure mode. The failure modes were categorized as an adhesive failure, cohesive failure in resin, or cohesive failure in enamel or dentin when more than 80% of the adherent area comprised the adhesive, resin composite, or tooth substrate, respectively. Other failure patterns, such as partially adhesive and partially cohesive, were categorized as a mixed failure.
WCA and SFE measurements
Twelve enamel and dentin specimens in each adhesive were prepared as described for the SBS tests. The cured adhesive layer’s hydrophilicity is related to the bond hydrolysis; hence, the cured adhesive layer’s WCA and SFE were evaluated to determine the degree of hydrophilicity. An oxygen inhibited layer is normally present on the top surface of a cured adhesive layer. Therefore, SFE measurements of the cured adhesive layers were performed without the oxygen inhibited layer to measure the interior layer’s hydrophilicity. According to the manufacturers' instructions, each adhesive was applied to the enamel and dentin in the SE mode, followed by light irradiation for 10 s. The cured adhesive’s upper surface was removed with ethanol-impregnated cotton pellets to remove the oxygen inhibited layer. Subsequently, each group’s SFE values were obtained by measuring three test liquids' surface contact angles with known SFE parameters, 1-bromonaphthalene, di-iodomethane, and distilled water. The equilibrium contact angle (θ) of each test liquid on the specimens was then measured at room temperature (23°C ± 1°C) via the sessile-drop method using a contact angle meter (Drop Master DM500; Kyowa Interface Science, Saitama, Japan) with a built-in charge-coupled device camera. Sessile liquid drops (1.0 μL) were dispensed with a micropipette, and the surface characteristics were calculated based on the fundamental concepts of wetting, as described previously [4,11]. The components of the SFE (γs) arising from the dispersion force (γsd), the polar (permanent and induced) force (γSp), and the hydrogen-bonding force (γSh) were obtained using an add-on software (FAMAS, Kyowa Interface Science).
Scanning electron microscopy observations
Representative restorative/tooth interfaces were observed via scanning electron microscopy (SEM; ERA-8800FE, Elionix, Tokyo, Japan). Bonded specimens stored in 37ºC distilled water for 24 h were embedded in epoxy resin (Epon 812, Nisshin EM, Tokyo, Japan) and then longitudinally sectioned with a precision sectioning saw (IsoMet 1000 Precision Sectioning Saw). The resin/tooth interfaces' sectioned surfaces were polished to a high gloss using abrasive disks (Fuji Star Type DDC), followed by diamond pastes down to 0.25 μm in particle size (DP-Paste, Struers, Ballerup, Denmark); this was followed by ultrasonic cleaning for 30 min. The interface and treated surface specimens were dehydrated in ascending grades of tert-butyl alcohol (50% for 20 min, 75% for 20 min, 95% for 20 min, and 100% for 2 h) and transferred to a freeze dryer (Model ID-3, Elionix) for 30 min. The resin/tooth interface specimens were subjected to argon-ion beam etching (EIS-200ER, Elionix) for 45 s with the ion beam (accelerating voltage 1.0 kV, ion current density 0.4 mA/cm2) directed perpendicular to the polished surfaces. Finally, all the SEM specimens were coated with a thin film of gold in an automatic ion sputter (Quick Coater Type SC-701, Sanyu Electron, Tokyo, Japan). The observations were carried out using an SEM at an operating voltage of 10 kV.
The homogeneity of variance (Bartlett’s test) and distribution (Shapiro-Wilk test) for each test data set was confirmed before analysis of variance (ANOVA) was performed. In the SBS data in the TC and WS groups, the enamel and dentin data were analyzed separately, and three-way ANOVA followed by Tukey’s honest significant difference (HSD) test (α = 0.05) was used to analyze each data set. The factors evaluated were: the etching mode, storage period, and adhesive system. One-way ANOVA followed by Tukey’s HSD test (α = 0.05) was used for comparisons within subsets.
For the CA and total SFE data, two-way ANOVA followed by Tukey’s HSD test (α = 0.05) was used to analyze the full data set. The tooth substrate and adhesive system were evaluated. A one-way ANOVA followed by Tukey’s posthoc test (α = 0.05) was used to analyze the results for the components of SFE. All statistical analyses were performed using a statistical analysis software system (Sigma Plot 13; SPSS, Chicago, IL, USA).