Abstract
Objective
To evaluate the microtensile bond strengths (μTBS) and nanoleakage (NL) of three universal or multi-mode adhesives, applied with increasing solvent evaporation times.
Methods
One-hundred and forty caries-free extracted third molars were divided into 20 groups for bond strength testing, according to three factors: (1) Adhesive – All-Bond Universal (ABU, Bisco, Inc.), Prime&Bond Elect (PBE, Dentsply), and Scotchbond Universal Adhesive (SBU, 3 M ESPE); (2) Bonding strategy – self-etch (SE) or etch-and-rinse (ER); and (3) Adhesive solvent evaporation time – 5 s, 15 s, and 25 s. Two extra groups were prepared with ABU because the respective manufacturer recommends a solvent evaporation time of 10 s. After restorations were constructed, specimens were stored in water (37 °C/24 h). Resin–dentin beams (0.8 mm 2 ) were tested at 0.5 mm/min (μTBS). For NL, forty extracted molars were randomly assigned to each of the 20 groups. Dentin disks were restored, immersed in ammoniacal silver nitrate, sectioned and processed for evaluation under a FESEM in backscattered mode. Data from μTBS were analyzed using two-way ANOVA (adhesive vs. drying time) for each strategy, and Tukey’s test ( α = 0.05). NL data were computed with non-parametric tests (Kruskal–Wallis and Mann–Whitney tests, α = 0.05).
Results
Increasing solvent evaporation time from 5 s to 25 s resulted in statistically higher mean μTBS for all adhesives when used in ER mode. Regarding NL, ER resulted in greater NL than SE for each of the evaporation times regardless of the adhesive used. A solvent evaporation time of 25 s resulted in the lowest NL for SBU-ER.
Significance
Residual water and/or solvent may compromise the performance of universal adhesives, which may be improved with extended evaporation times.
1
Introduction
New multi-mode or universal one-bottle adhesives have been recently introduced for use as either self-etch or as etch-and-rinse adhesives . The respective manufacturers suggest that multi-mode adhesives may also be used with separate etching of enamel margins, or selective enamel etching.
Due to the intrinsic wetness of the dentin substrate, hydrophilic monomers have been used in the composition of dentin bonding systems for years . Hydrophilic resins result in high dentin bond strengths. However, several studies have demonstrated that degradation of the resin–dentin interface occurs over time . It has been questioned whether current monomers have become too hydrophilic . In fact, all self-etch adhesives, including the newest universal adhesives, contain water, which is required for ionization of the hydrophilic acidic monomers .
Their hydrophilicity makes one-step self-etch adhesives behave as semi-permeable membranes, allowing fluid transudation across the resin–dentin interface . Some etch-and-rinse adhesives also contain water and hydrophilic monomers, which makes them behave as permeable membranes as well, allowing exudation of dentin fluid . The presence of residual water may accelerate the degradation of the bonding interface .
Commercial dental adhesives include organic solvents, such as ethanol or acetone, to facilitate monomer infiltration into the humid dentin substrate. Although water and organic solvents are essential components of one-step adhesives, solvents should be completely removed during clinical application of the adhesive. If solvents are not evaporated, residual water and organic solvents may inhibit the polymerization of monomers in current dentin adhesives .
Solvent evaporation is usually accomplished by agitating the adhesive on dentin/enamel surfaces followed by solvent evaporation with compressed air . An extended solvent evaporation time has been used to successfully improve the degree of conversion and mechanical properties of 1-step self-etch and 2-step etch-and-rinse adhesives . There is no consensus, however, regarding the proper solvent evaporation time for 1-step self-etch , or for 2-step etch-and-rinse adhesives .
Taking into account that new universal adhesives contain both water and, at least, one organic solvent (ethanol or acetone), the aim of this study was to compare the immediate microtensile bond strengths (μTBS) and nanoleakage (NL) of three universal or multi-mode adhesives, applied with increasing solvent evaporation times. The null hypotheses tested were that extended solvent evaporation time would not improve: (1) the immediate bond strengths of universal adhesives and; (2) the sealing ability of resin–dentin interfaces formed with universal adhesives.
2
Material and methods
2.1
Tooth selection and preparation
One hundred and forty extracted, caries-free human third molars were used. The teeth were collected after obtaining the patient’s informed consent under a protocol approved by the local Ethics Committee Review Board. The teeth were disinfected in 0.5% chloramine, stored in distilled water and used within six months after extraction.
A flat occlusal dentin surface was exposed in all teeth after wet grinding the occlusal enamel with # 180 grit SiC paper. The exposed dentin surfaces were further polished with wet # 600-grit silicon-carbide paper for 60 s to standardize the smear layer.
2.2
Experimental design, restorative procedure and specimen preparation
Teeth were randomly assigned into 20 groups ( n = 7) according to the adhesive strategy and different solvent evaporation times of three universal adhesive systems: All-Bond Universal (ABU – Bisco Inc., Schaumburg, IL, USA); Prime&Bond Elect (PBE – Dentsply Caulk, Milford, DE, USA); and Scotchbond Universal Adhesive (SBU – 3 M ESPE, St. Paul, MN, USA).
Each adhesive was applied (1) as etch-and-rinse (ER) adhesive or as self-etch (SE) adhesive; and (2) with three adhesive solvent evaporation times (5 s, 15 s, and 25 s). Two extra groups were tested to include the recommended manufacturer’s solvent evaporation time of 10 s for ABU in both adhesive strategies. All details regarding the adhesive composition are displayed in Table 1 .
| Materials | Composition | Application mode | |||
|---|---|---|---|---|---|
| Self-etch | Etch-and-rinse | ||||
| Manufacturer recommendations | Experimental groups | Manufacturer recommendations | Experimental groups | ||
| All-Bond Universal – ABU (1200002722) | 1. Etchant: 35% Phosphoric acid, benzalkonium chloride (SELECT HV-Etch) 2. Adhesive: MDP, Bis-GMA, HEMA, ethanol, water, initiators |
1. Apply two separate coats of adhesive, scrubbing the preparation with a microbrush for 10–15 s per coat. Do not light cure between coats. 2. Evaporate excess solvent by thoroughly air-drying with an air syringe for at least 10 s, there should be no visible movement of the material. The surface should have a uniform glossy appearance 3. Light cure for 10 s |
1. Apply adhesive as recommended by the manufacturer 2. The only difference is the solvent evaporation time: 5, 15 and 25 s a |
1. Apply etchant for 15 s 2. Rinse thoroughly for 10 s 3. Remove excess water with air syringe for 5 s. 4. Apply adhesive as in the self-etch strategy |
1. Apply etchant for 15 s 2. Apply adhesive as recommended by the manufacturer The only difference is the solvent evaporation time: 5, 15 and 25 s a |
| Prime & Bond Elect – PBE (1102221) | 1. Etchant: 34% Tooth Conditioner Gel (34% phosphoric acid) 2. Adhesive: Mono-, di- and trimethacrylate resins; PENTA Diketone; Organic phosphine oxide; Stabilizers; Cetylamine hydrofluoride; Acetone; Water |
1. Apply generous amount of adhesive to thoroughly wet all tooth surfaces 2. Agitate for 20 s 3. Gently dry with clean air for at least 5 s. Surface should have a uniform, glossy appearance 4. Light-cure for 10 s |
1. Apply adhesive as recommended by the manufacturer 2. The only difference is the solvent evaporation time: 15 and 25 s |
1. Apply etchant for 15 s. 2. Rinse for 15 s. 3. Dry with air syringe for 5 s. 4. Apply adhesive as in the self-etch strategy |
1. Apply etchant for 15 s 2. Apply adhesive as recommended by the manufacturer 3. The only difference is the solvent evaporation time: 15 and 25 s |
| Scotchbond Universal Adhesive – SBU (448716) | 1. Etchant: 32% phosphoric acid, water, synthetic amorphous silica, polyethylene glycol, aluminum oxide (Scotchbond Universal Etchant) 2. Adhesive: MDP Phosphate monomer, dimethacrylate resins, HEMA, methacrylate-modified polyalkenoic acid copolymer, filler, ethanol, water, initiators, and silane |
1. Apply the adhesive to the entire preparation with a microbrush and rub it in for 20 s 2. Direct a gentle stream of air over the liquid for about 5 s until it no longer moves and the solvent is evaporated completely 3. Light-cure for 10 s |
1. Apply adhesive as recommended by the manufacturer 2. The only difference is the solvent evaporation time: 15 and 25 s |
1. Apply etchant for 15 s 2. Rinse for 10 s 3. Air dry 5 s 4. Apply adhesive as in the self-etch strategy |
1. Apply etchant for 15 s 2. Apply adhesive as recommended by the manufacturer 3. The only difference is the solvent evaporation time: 15 and 25 s |
a Bisco recommends 10 s of solvent evaporation time. However, to standardize different groups, we also tested this adhesive for 5, 15, and 25 s of solvent evaporation time.
Solvent evaporation was accomplished with an oil-free air-water syringe. The air pressure was adjusted to 1 bar using a pressure regulator, and the air nozzle was held at 45° to the dentin surface at a distance of 1.5 cm. The adhesive systems were applied as per the respective manufacturer’s instructions, except for the different experimental solvent evaporation times. Please refer to Table 1 for more details.
After the bonding procedures, a nanofilled composite restoration (Filtek Z350, 3M ESPE, St. Paul, MN, USA) was built in two increments of 2 mm. Each increment was light polymerized for 40 s using a LED light-curing unit set at 1200 mW/cm 2 (Radii-cal, SDI Limited, Bayswater, Victoria, Australia).
2.3
Microtensile bond strength (μTBS)
After storage in distilled water for 24 h at 37 °C, one-hundred restored teeth ( n = 5 for each experimental group) were sectioned longitudinally in a mesio-distal and buccal-lingual directions across the bonded interface with a low-speed diamond saw (Isomet, Buehler Ltd, Lake Bluff, IL, USA) with water irrigation to obtain resin–dentin beams with a cross sectional area of approximately 0.8 mm 2 measured with a digital caliper (Digimatic Caliper, Mitutoyo, Tokyo, Japan).
Resin–dentin bonded beams were attached to a Geraldeli jig (Odeme Biotechnology, Joaçaba, SC, Brazil) with cyanoacrylate adhesive and tested under tension (Model 5565, Instron, Norwood, MA, USA) at 0.5 mm/min until failure. The μTBS values (MPa) were calculated by dividing the load at failure by the cross-sectional bonding area.
The failure mode was classified as cohesive ([C] failure exclusively within dentin or resin composite), adhesive ([A] failure at the resin/dentin interface), or mixed ([M] failure at the resin/dentin interface that included cohesive failure of the neighboring substrates). The failure mode analysis was performed under a stereomicroscope at 100× magnification (Olympus SZ40, Olympus Corporation, Tokyo, Japan). Specimens with premature failures (PF) were included in the tooth mean. We have attributed them the average value between zero and the lowest bond strength value obtained in all experiment. In this specific study, the value of 4.7 MPa was attributed when PF were recorded.
2.4
Nanoleakage (NL) evaluation
Forty restored teeth ( n = 2 for each experimental group) were used for nanoleakage evaluation assigned to each experimental group described in Table 1 . Two 1 mm-thick dentin disks were sectioned from each tooth parallel to the occlusal surface, using a slow-speed diamond saw (Isomet 1000, Buehler Ltd., Lake Bluff, IL, USA) under water-cooling. After ensuring that no enamel remains were present, a standard smear layer was created in the bonding surfaces of each disk (pulpal surface of the occlusal side disk and the occlusal surface of the pulpal side disk) with 600-grit SiC paper under water for 60 s to standardize the smear layer. The adhesives were then applied to each of the four bonding surfaces as described above. A 0.3 mm-thick layer of flowable composite (Filtek Bulk Fill, 3M ESPE, St. Paul, MN, USA) was then applied and cured for 40 s. The restored dentin disks were sectioned bucco-lingually in two halves using a slow-speed diamond saw (Isomet 1000, Buehler Ltd., Lake Bluff, IL, USA) under water-cooling to exposed the resin–dentin interface. All surfaces were coated with two layers of nail polish except for the bonding interface.
After the nail polish dried, the specimens were immersed in an aqueous solution of 50 wt% ammoniacal silver nitrate (pH 9.5) for 24 h at 37 °C, followed by 8 h in a photo-developing solution in order to permit reduction of the diammine silver ions to metallic silver grains . The specimens were washed in water for 1 min and the nail vanish removed with a periodontal scaler. Fixation was carried out with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.4 for 12 h at 4 °C. After fixation, the specimens were 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 for 1 min. The specimens were dehydrated in ascending grades of ethanol: 25% for 20 min, 50% for 20 min, 75% for 20 min, 95% for 30 min, and 100% for 30 min .
Specimens were polished with waterproof silicon carbide papers of decreasing abrasiveness (600-grit, 800-grit and, 1200-grit), followed by soft tissue disks with increasingly fine suspensions of 1 μm and 0.3 μm for 1 min each. The specimens were ultra-sonicated in 95% ethanol for 10 min, thoroughly dried, and demineralized in 0.5% silica-free phosphoric acid for 1 min to remove polishing debris. Sections were mounted on Al stubs with carbon adhesive tape and graphite paint (Ted Pella, Inc., Redding, CA, USA). Then, specimens were evaporated with carbon under a DV502A Vacuum Evaporator (Denton Vacuum, Moorestown, NJ, USA) for 1 min and observed in backscattered mode under a Hitachi S-4700 FESEM (Hitachi High Technologies America, Inc., Dallas, TX, USA) with an Autrata-modified YAG detector at an accelerating voltage of 8.0 kV and working distance of 13.0–13.2 mm.
A series of 9–10 micrographs were obtained from each of the four interfaces to include the entire length of the interface in secondary and in backscattered mode simultaneously. Micrographs were assembled through Adobe Photoshop CS3 Extended Version 10.0.1 (Adobe Systems Incorporated, San Jose, CA, USA) to reproduce the entire interface. The interface total length (in mm) was measured using ImageJ 1.44o (NIH, Bethesda, MD, USA). Nanoleakage areas were identified as the areas of the interface that displayed silver ions. All the measurements were calibrated to the same pixel/mm ratio.
For each interface, the observed length of the interface with signs of silver infiltration was added up and the percentage calculated as: (length of interface with silver ions/total length of the interface) × 100. Representative areas of each interface were imaged at high magnification (×5000) to analyze the nanoleakage patterns.
2.5
Statistical analysis
Each tooth was considered as a statistical unit. As the p -value obtained with the Levene’s test was >0.05, data from μTBS were analyzed separately using two-way ANOVA (adhesive vs. solvent evaporation time) for the etch-and-rinse and self-etch strategies, followed by Tukey’s post hoc test ( α = 0.05). Nanoleakage data were analyzed with non-parametric tests (Kruskal–Wallis and Mann–Whitney pair-wise comparisons, α = 0.05) for the etch-and-rinse and self-etch strategies considering each tooth-interface as an independent statistical unit.
2
Material and methods
2.1
Tooth selection and preparation
One hundred and forty extracted, caries-free human third molars were used. The teeth were collected after obtaining the patient’s informed consent under a protocol approved by the local Ethics Committee Review Board. The teeth were disinfected in 0.5% chloramine, stored in distilled water and used within six months after extraction.
A flat occlusal dentin surface was exposed in all teeth after wet grinding the occlusal enamel with # 180 grit SiC paper. The exposed dentin surfaces were further polished with wet # 600-grit silicon-carbide paper for 60 s to standardize the smear layer.
2.2
Experimental design, restorative procedure and specimen preparation
Teeth were randomly assigned into 20 groups ( n = 7) according to the adhesive strategy and different solvent evaporation times of three universal adhesive systems: All-Bond Universal (ABU – Bisco Inc., Schaumburg, IL, USA); Prime&Bond Elect (PBE – Dentsply Caulk, Milford, DE, USA); and Scotchbond Universal Adhesive (SBU – 3 M ESPE, St. Paul, MN, USA).
Each adhesive was applied (1) as etch-and-rinse (ER) adhesive or as self-etch (SE) adhesive; and (2) with three adhesive solvent evaporation times (5 s, 15 s, and 25 s). Two extra groups were tested to include the recommended manufacturer’s solvent evaporation time of 10 s for ABU in both adhesive strategies. All details regarding the adhesive composition are displayed in Table 1 .
| Materials | Composition | Application mode | |||
|---|---|---|---|---|---|
| Self-etch | Etch-and-rinse | ||||
| Manufacturer recommendations | Experimental groups | Manufacturer recommendations | Experimental groups | ||
| All-Bond Universal – ABU (1200002722) | 1. Etchant: 35% Phosphoric acid, benzalkonium chloride (SELECT HV-Etch) 2. Adhesive: MDP, Bis-GMA, HEMA, ethanol, water, initiators |
1. Apply two separate coats of adhesive, scrubbing the preparation with a microbrush for 10–15 s per coat. Do not light cure between coats. 2. Evaporate excess solvent by thoroughly air-drying with an air syringe for at least 10 s, there should be no visible movement of the material. The surface should have a uniform glossy appearance 3. Light cure for 10 s |
1. Apply adhesive as recommended by the manufacturer 2. The only difference is the solvent evaporation time: 5, 15 and 25 s a |
1. Apply etchant for 15 s 2. Rinse thoroughly for 10 s 3. Remove excess water with air syringe for 5 s. 4. Apply adhesive as in the self-etch strategy |
1. Apply etchant for 15 s 2. Apply adhesive as recommended by the manufacturer The only difference is the solvent evaporation time: 5, 15 and 25 s a |
| Prime & Bond Elect – PBE (1102221) | 1. Etchant: 34% Tooth Conditioner Gel (34% phosphoric acid) 2. Adhesive: Mono-, di- and trimethacrylate resins; PENTA Diketone; Organic phosphine oxide; Stabilizers; Cetylamine hydrofluoride; Acetone; Water |
1. Apply generous amount of adhesive to thoroughly wet all tooth surfaces 2. Agitate for 20 s 3. Gently dry with clean air for at least 5 s. Surface should have a uniform, glossy appearance 4. Light-cure for 10 s |
1. Apply adhesive as recommended by the manufacturer 2. The only difference is the solvent evaporation time: 15 and 25 s |
1. Apply etchant for 15 s. 2. Rinse for 15 s. 3. Dry with air syringe for 5 s. 4. Apply adhesive as in the self-etch strategy |
1. Apply etchant for 15 s 2. Apply adhesive as recommended by the manufacturer 3. The only difference is the solvent evaporation time: 15 and 25 s |
| Scotchbond Universal Adhesive – SBU (448716) | 1. Etchant: 32% phosphoric acid, water, synthetic amorphous silica, polyethylene glycol, aluminum oxide (Scotchbond Universal Etchant) 2. Adhesive: MDP Phosphate monomer, dimethacrylate resins, HEMA, methacrylate-modified polyalkenoic acid copolymer, filler, ethanol, water, initiators, and silane |
1. Apply the adhesive to the entire preparation with a microbrush and rub it in for 20 s 2. Direct a gentle stream of air over the liquid for about 5 s until it no longer moves and the solvent is evaporated completely 3. Light-cure for 10 s |
1. Apply adhesive as recommended by the manufacturer 2. The only difference is the solvent evaporation time: 15 and 25 s |
1. Apply etchant for 15 s 2. Rinse for 10 s 3. Air dry 5 s 4. Apply adhesive as in the self-etch strategy |
1. Apply etchant for 15 s 2. Apply adhesive as recommended by the manufacturer 3. The only difference is the solvent evaporation time: 15 and 25 s |
a Bisco recommends 10 s of solvent evaporation time. However, to standardize different groups, we also tested this adhesive for 5, 15, and 25 s of solvent evaporation time.
Solvent evaporation was accomplished with an oil-free air-water syringe. The air pressure was adjusted to 1 bar using a pressure regulator, and the air nozzle was held at 45° to the dentin surface at a distance of 1.5 cm. The adhesive systems were applied as per the respective manufacturer’s instructions, except for the different experimental solvent evaporation times. Please refer to Table 1 for more details.
After the bonding procedures, a nanofilled composite restoration (Filtek Z350, 3M ESPE, St. Paul, MN, USA) was built in two increments of 2 mm. Each increment was light polymerized for 40 s using a LED light-curing unit set at 1200 mW/cm 2 (Radii-cal, SDI Limited, Bayswater, Victoria, Australia).
2.3
Microtensile bond strength (μTBS)
After storage in distilled water for 24 h at 37 °C, one-hundred restored teeth ( n = 5 for each experimental group) were sectioned longitudinally in a mesio-distal and buccal-lingual directions across the bonded interface with a low-speed diamond saw (Isomet, Buehler Ltd, Lake Bluff, IL, USA) with water irrigation to obtain resin–dentin beams with a cross sectional area of approximately 0.8 mm 2 measured with a digital caliper (Digimatic Caliper, Mitutoyo, Tokyo, Japan).
Resin–dentin bonded beams were attached to a Geraldeli jig (Odeme Biotechnology, Joaçaba, SC, Brazil) with cyanoacrylate adhesive and tested under tension (Model 5565, Instron, Norwood, MA, USA) at 0.5 mm/min until failure. The μTBS values (MPa) were calculated by dividing the load at failure by the cross-sectional bonding area.
The failure mode was classified as cohesive ([C] failure exclusively within dentin or resin composite), adhesive ([A] failure at the resin/dentin interface), or mixed ([M] failure at the resin/dentin interface that included cohesive failure of the neighboring substrates). The failure mode analysis was performed under a stereomicroscope at 100× magnification (Olympus SZ40, Olympus Corporation, Tokyo, Japan). Specimens with premature failures (PF) were included in the tooth mean. We have attributed them the average value between zero and the lowest bond strength value obtained in all experiment. In this specific study, the value of 4.7 MPa was attributed when PF were recorded.
2.4
Nanoleakage (NL) evaluation
Forty restored teeth ( n = 2 for each experimental group) were used for nanoleakage evaluation assigned to each experimental group described in Table 1 . Two 1 mm-thick dentin disks were sectioned from each tooth parallel to the occlusal surface, using a slow-speed diamond saw (Isomet 1000, Buehler Ltd., Lake Bluff, IL, USA) under water-cooling. After ensuring that no enamel remains were present, a standard smear layer was created in the bonding surfaces of each disk (pulpal surface of the occlusal side disk and the occlusal surface of the pulpal side disk) with 600-grit SiC paper under water for 60 s to standardize the smear layer. The adhesives were then applied to each of the four bonding surfaces as described above. A 0.3 mm-thick layer of flowable composite (Filtek Bulk Fill, 3M ESPE, St. Paul, MN, USA) was then applied and cured for 40 s. The restored dentin disks were sectioned bucco-lingually in two halves using a slow-speed diamond saw (Isomet 1000, Buehler Ltd., Lake Bluff, IL, USA) under water-cooling to exposed the resin–dentin interface. All surfaces were coated with two layers of nail polish except for the bonding interface.
After the nail polish dried, the specimens were immersed in an aqueous solution of 50 wt% ammoniacal silver nitrate (pH 9.5) for 24 h at 37 °C, followed by 8 h in a photo-developing solution in order to permit reduction of the diammine silver ions to metallic silver grains . The specimens were washed in water for 1 min and the nail vanish removed with a periodontal scaler. Fixation was carried out with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.4 for 12 h at 4 °C. After fixation, the specimens were 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 for 1 min. The specimens were dehydrated in ascending grades of ethanol: 25% for 20 min, 50% for 20 min, 75% for 20 min, 95% for 30 min, and 100% for 30 min .
Specimens were polished with waterproof silicon carbide papers of decreasing abrasiveness (600-grit, 800-grit and, 1200-grit), followed by soft tissue disks with increasingly fine suspensions of 1 μm and 0.3 μm for 1 min each. The specimens were ultra-sonicated in 95% ethanol for 10 min, thoroughly dried, and demineralized in 0.5% silica-free phosphoric acid for 1 min to remove polishing debris. Sections were mounted on Al stubs with carbon adhesive tape and graphite paint (Ted Pella, Inc., Redding, CA, USA). Then, specimens were evaporated with carbon under a DV502A Vacuum Evaporator (Denton Vacuum, Moorestown, NJ, USA) for 1 min and observed in backscattered mode under a Hitachi S-4700 FESEM (Hitachi High Technologies America, Inc., Dallas, TX, USA) with an Autrata-modified YAG detector at an accelerating voltage of 8.0 kV and working distance of 13.0–13.2 mm.
A series of 9–10 micrographs were obtained from each of the four interfaces to include the entire length of the interface in secondary and in backscattered mode simultaneously. Micrographs were assembled through Adobe Photoshop CS3 Extended Version 10.0.1 (Adobe Systems Incorporated, San Jose, CA, USA) to reproduce the entire interface. The interface total length (in mm) was measured using ImageJ 1.44o (NIH, Bethesda, MD, USA). Nanoleakage areas were identified as the areas of the interface that displayed silver ions. All the measurements were calibrated to the same pixel/mm ratio.
For each interface, the observed length of the interface with signs of silver infiltration was added up and the percentage calculated as: (length of interface with silver ions/total length of the interface) × 100. Representative areas of each interface were imaged at high magnification (×5000) to analyze the nanoleakage patterns.
2.5
Statistical analysis
Each tooth was considered as a statistical unit. As the p -value obtained with the Levene’s test was >0.05, data from μTBS were analyzed separately using two-way ANOVA (adhesive vs. solvent evaporation time) for the etch-and-rinse and self-etch strategies, followed by Tukey’s post hoc test ( α = 0.05). Nanoleakage data were analyzed with non-parametric tests (Kruskal–Wallis and Mann–Whitney pair-wise comparisons, α = 0.05) for the etch-and-rinse and self-etch strategies considering each tooth-interface as an independent statistical unit.
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