Ethanol-wet bonding technique: Clinical versuslaboratory findings

Highlights

  • Ethanol and water-wet bonding were evaluate under clinical and laboratory conditions.

  • It is one of the first studies that evaluated the ethanol wet-bonding in an in vivo condition.

  • Results in the laboratory setting was not confirmed in clinical conditions.

  • Based on nanoleakage results, ethanol wet-bonding is a promise technique.

Abstract

Objectives

This study evaluated the microtensile bond strength (μTBS) and nanoleakage (NL) of dentin bonded interfaces produced with ethanol-wet and water-wet bonding protocols under clinical and laboratory conditions.

Methods

The sample was composed of forty primary second molars in advanced exfoliation process. Occlusal cavities were prepared leaving a flat dentin surface on the pulpal floor. In half of the teeth, the water-wet protocol was followed using a three-step etch-and-rinse adhesive. In the other half, dentin was dehydrated with ascending ethanol solutions (50%, 70%, 80%, 95% and 3 × 100%), 15 s each for the ethanol-bonding protocol. An experimental hydrophobic primer was used, followed by the neat adhesive application. Resin build-ups were prepared, stored for 24 h, sectioned into sticks and tested in tensile mode (0.5 mm/min). NL was performed for all groups. The μTBS and NL data were submitted to two-way ANOVA and Kruskall–Wallis tests, respectively ( α = 0.05).

Results

Under clinical conditions, the highest μTBS was observed for the water-wet bonding while under the laboratory setting, the highest μTBS was obtained for the ethanol-wet bonding. Increased NL was observed in the water-wet bonding groups irrespective of the bonding condition.

Significance

The immediate benefits of the ethanol-bonding observed in the laboratory setting was not confirmed when the same protocol was performed in vivo . However, as reduced nanoleakage was seen in adhesive interfaces produced with the ethanol-wet bonding technique, suggests that the hybrid layer may be more resistant to degradation.

Introduction

Early generations of dental adhesives were relatively hydrophobic, and dry substrates were required for bonding. However, drying acid-etched dentin causes collapse of the collagen meshwork that prevented resin infiltration. As a consequence, the resulting dentin bond strengths were very low . Modifications of adhesive formulations, by the inclusion of more hydrophilic monomers and acidic resin monomers, made adhesive solutions more compatible with moist dentin, which, in turn, yielded significant improvements in the immediate bonding effectiveness of most adhesive systems .

The potential problems associated with incorporation of hydrophilic formulations are well known . Increased water sorption rapid deterioration of mechanical properties of the adhesive layer as well as increased permeability of the adhesive interface that jeopardize the longevity of resin–dentin bonds after short- and long-term periods under in vivo and in vitro investigations .

In an attempt to solve these problems, some studies attempted to make acid-etched dentin more hydrophobic . The strategy involved the replacement of water within the demineralized collagen matrix with ascending concentrations of ethanol, allowing the latter to penetrate the collagen matrix without causing additional shrinkage of the interfibrillar spaces, in the so-called ethanol-wet bonding technique .

Although dentin bonding with hydrophobic resins using the ethanol-wet bonding technique has shown encouraging results in terms of resin–dentin bond stability the protocol is time consuming and technique-sensitive . Additionally, a complete replacement of water by ethanol may not be feasible under clinical conditions due to the constant presence of an outward physiological dentinal fluid coming up from the dental pulp. Although the ethanol-wet bonding technique was conceived to be a bonding philosophy rather than a bonding technique, due to its clinical difficulties, the understanding of the behavior of this technique in a clinical setting may complement what has been reported in laboratory studies and it may be a major contribution to the adhesive dentistry. So far, to the extent of the authors’ knowledge no study has attempted to investigate the ethanol-wet bonding protocol in an in vivo setting. Therefore, the aim of the present study was to compare the resin–dentin bond strength and nanoleakage of dentin-bonded interfaces produced with ethanol- and water-wet bonding protocols under clinical and laboratory conditions. The following null hypotheses were tested: (1) there are no differences in the resin–dentin bond strength and nanoleakage produced by an etch-and-rinse adhesive system bonded with the ethanol-wet and water-wet protocols; (2) there are no differences in the resin–dentin bond strength and nanoleakage produced by an etch-and-rinse adhesive system bonded under clinical or laboratory conditions.

Materials and methods

Specimen preparation for the clinical and laboratory experiments

The present investigation was approved by the local Ethics Committee under protocol number 24/2009. For the clinical experiment, clinical and radiographic examination of approximately 100 patients ranging from 10 to 12 years old were performed in order to find 20 patients to take part in this study. These patients were required to have an incipient caries lesion in the primary second molar (up to the upper 1/3 of the dentin in the interproximal radiograph) and be in need of restorative treatment in the same hemi-arch, so that patients would necessarily receive anesthesia and rubber dam isolation. All teeth selected were at an advanced stage of physiological root resorption and mobility, indicating advanced physiological exfoliation process. The fact that these teeth have been used as source of stem cells was evidence of their pulp vitality .

For the laboratory evaluation, 20 recently exfoliated primary second molars free of caries or having an incipient caries lesion were used. Teeth were stored in saline solution at 4 °C for up to 3 months before the in vitro experiment.

Tooth preparation and restorative procedures

In each tooth, one standardized occlusal cavity was prepared under local anesthesia without vasoconstrictor (3% mepivacaine solution, Mepisv, Nova DFL, Rio de Janeiro, RJ, Brazil) and rubber dam isolation, using a cylindrical diamond bur (#1092, KG Sorensen, São Paulo, Brazil) under water-cooling. Each diamond bur was used in four preparations and then discarded. The cavities were prepared in order to achieve: 1 – the largest possible dimensions, which averaged 7 mm wide, 6 mm long and 2 mm deep; 2 – completely flat cavity floor dentin; 3 – complete enamel cavo-surface margins. The specimens were randomly divided into two groups, according to the bonding technique: water-wet ( n = 10 teeth) and ethanol-wet bonding techniques ( n = 10 teeth). Due to the cavity dimensions, the bonding was performed in deep dentin.

For the water-wet bonding technique, preparations were total-etched with 35% phosphoric acid gel for 15 s, followed by water rinsing (15 s). Two coats of the Scotchbond Multi-Purpose primer (Adper Scotchbond Multi-Purpose Plus – 3M ESPE, St. Paul, MN, batch number # 0804002271; Table 1 ) were applied to visibly moist demineralized dentin according to the manufacturer’s directions. After briefly air-drying for 10 s, Scotchbond Multi-Purpose Adhesive was applied and light cured for 10 s.

Table 1
Composition and application mode of the adhesive system used in this study.
Adhesive system brand Original adhesive used for the water-wet bonding protocol Modified adhesive used for the ethanol-wet bonding
Adper Scotchbond MultiPurpose Plus Component 1: Etchant 35% phosphoric acid
Component 2: Scotchbond Multi-Purpose primer (HEMA, polyalkenoic acid polymer, water).
Component 3: Scotchbond Multi-Purpose adhesive (Bis-GMA, HEMA, tertiary amines, photo-initiator)
Component 1: Etchant 35% phosphoric acid
Component 2: Scotchbond Multi-Purpose adhesive diluted in 50 wt% ethanol (Bis-GMA, HEMA, tertiary amines, photo-initiator and ethanol)
Component 3: Scotchbond Multi-Purpose adhesive (Bis-GMA, HEMA, tertiary amines, photo-initiator)

After acid-etching, dentin in the ethanol-wet bonding group was treated with a series of increasing ethanol concentrations: 50%, 70%, 80%, 95% and three 100% ethanol applications for 15 s each following a chemical dehydration protocol . Dehydration procedures were meticulously performed to ensure that the dentin surface was always immersed in a liquid phase by keeping it visibly moist prior to the application of the subsequent solution.

Two consecutive coats of the experimental hydrophobic primer were applied to ethanol-saturated dentin. The experimental primer solution was prepared by diluting the Scotchbond Multi Purpose Adhesive ( Table 1 ) with 50 wt% absolute ethanol. This procedure was performed to produce a water-free bonding resin with similar composition of the hydrophilic adhesive employed in the water-wet protocol. Excess ethanol solvent was evaporated with a gentle air stream for 10 s. Then, a layer of the neat Scotchbond Multi-Purpose Adhesive was applied and spread over the primed surface and light cured for 10 s.

Preparations from both groups were restored with a microhybrid composite resin (shade EA2; Opallis – FGM Dental Products, Joinville, SC, Brazil; batch number # 160708) in three increments. Each increment was light-cured for 20 s. All light-curing procedures were performed for 10 s using a Radii LED light-curing unit (SDI Limited, Bayswater, Victoria, Australia) with an output intensity of 800 mW/cm 2 .

Within 20 min after completion of the bonding procedures, teeth were extracted, immersed in distilled water (pH 7), and kept in a moist environment for 24 h at 37 °C before being prepared for the microtensile bond test and nanoleakage analysis. All operative and restorative procedures reported for the clinical experimental was repeated under laboratory conditions in the extracted teeth.

Microtensile bond strength test (μTBS)

Bonded teeth from both experiments were longitudinally sectioned in both “ x ” and “ y ” directions across the bonded interface with a diamond saw in an ISOMET 1000 machine (Buehler, Lake Bluff, IL, USA), under water cooling at 300 rpm to obtain bonded sticks with a cross-sectional area of approximately 0.8 mm 2 .

Individual bonded sticks were attached to a device (Odeme Biotechnology, Joaçaba, SC, Brazil) for microtensile testing, with cyanoacrylate resin (Super Bonder, Locitec, São Paulo, SP, Brazil), so that tensile forces acted perpendicularly to the dentin/adhesive interface. Specimens were subjected to a tensile force in a universal testing machine (Kratos, São Paulo, SP, Brazil) at a crosshead speed of 0.5 mm/min. The failure modes were evaluated at 400× magnification (HMV-2, Shimadzu, Tokyo, Japan) and classified as cohesive in dentin (CD) or composite resin (CR) (failure exclusively within dentin or resin composite), adhesive (A, adhesive failure, restricted to the resin–dentin interface without partial cohesive failure of the neighboring substrates) and mixed (M, adhesive failure along with partial cohesive failure of the neighboring substrates). The number of specimens that showed premature failure was also recorded.

Nanoleakage analysis

One resin–dentin bonded stick of each tooth, randomly selected and not tested under tensile forces, was prepared for nanoleakage evaluation. These bonded sticks were immersed in ammoniacal silver nitrate for 24 h and the silver impregnated specimens were rinsed thoroughly in distilled water and placed in a photo-developing solution for 8 h under a fluorescent light. The adhesive interfaces were polished with descending grits of SiC papers (1000; 1200; 1500; 2000 and 2500) and 1 and 0.25 μm diamond paste (Erios Prod. Odont. Ltda, São Paulo, SP, Brazil) using a polishing cloth. Specimens were ultrasonically cleaned and left in a desiccator for 24 h at room temperature. Specimens were then mounted on stubs and sputter-coated with a 10-nm gold layer to be analyzed by scanning electron microscopy (SEM) (JSM 6360LV, Jeol Ltd., Tokyo, Japan) using a backscattered detector.

A representative image per tooth were obtained at 600× magnification by a blinded operator, not aware of the experimental conditions under investigation, and the amount of silver nitrate impregnation along the adhesive interface was evaluated by scoring nanoleakage interfacial expression by two calibrated observers as reported in Table 2 . Disagreements between observers were resolved by consensus.

Table 2
Classification of nanoleakage scores according to Saboia et al. .
Score % of adhesive interface showing nanoleakage
0 No nanoleakage
1 ˂25% with nanoleakage
2 25 ≤ 50% with nanoleakage
3 50 ≤ 75% with nanoleakage
4 >75% with nanoleakage

Statistical analysis

We based our sample size calculation on a previous study . In order to detect a significant difference of 8 MPa between means, with an average standard deviation of 6 MPa, at a power of 80% and a level of significance of 5%, ten teeth were required per experimental condition.

The μTBS values of all sticks from the same tooth were averaged for statistical purposes. The bonded sticks that showed cohesive failures either in dentin or composite resin were not included in the tooth mean. With regard to premature failures, two statistical analyses were performed, with and without the inclusion of premature failures in the tooth mean. We assigned a value of zero to the premature failures. The Kolmogorov–Smirnov test was performed to assess whether the data followed a normal distribution, and the Barlett’s test for equality of variances was performed to determine if the assumption of equal variances was valid. After observing the normality of the data distribution and the equality of the variances (normality; p = 0.821 and equal variance p = 0.817), the μTBS (MPa) were submitted to a two-way ANOVA and Tukey’s test at α = 0.05. The nanoleakage scores and the fracture modes were statistically evaluated by the Kruskall–Wallis test and pairwise comparisons were performed with the Mann–Whitney test at α = 0.05.

Materials and methods

Specimen preparation for the clinical and laboratory experiments

The present investigation was approved by the local Ethics Committee under protocol number 24/2009. For the clinical experiment, clinical and radiographic examination of approximately 100 patients ranging from 10 to 12 years old were performed in order to find 20 patients to take part in this study. These patients were required to have an incipient caries lesion in the primary second molar (up to the upper 1/3 of the dentin in the interproximal radiograph) and be in need of restorative treatment in the same hemi-arch, so that patients would necessarily receive anesthesia and rubber dam isolation. All teeth selected were at an advanced stage of physiological root resorption and mobility, indicating advanced physiological exfoliation process. The fact that these teeth have been used as source of stem cells was evidence of their pulp vitality .

For the laboratory evaluation, 20 recently exfoliated primary second molars free of caries or having an incipient caries lesion were used. Teeth were stored in saline solution at 4 °C for up to 3 months before the in vitro experiment.

Tooth preparation and restorative procedures

In each tooth, one standardized occlusal cavity was prepared under local anesthesia without vasoconstrictor (3% mepivacaine solution, Mepisv, Nova DFL, Rio de Janeiro, RJ, Brazil) and rubber dam isolation, using a cylindrical diamond bur (#1092, KG Sorensen, São Paulo, Brazil) under water-cooling. Each diamond bur was used in four preparations and then discarded. The cavities were prepared in order to achieve: 1 – the largest possible dimensions, which averaged 7 mm wide, 6 mm long and 2 mm deep; 2 – completely flat cavity floor dentin; 3 – complete enamel cavo-surface margins. The specimens were randomly divided into two groups, according to the bonding technique: water-wet ( n = 10 teeth) and ethanol-wet bonding techniques ( n = 10 teeth). Due to the cavity dimensions, the bonding was performed in deep dentin.

For the water-wet bonding technique, preparations were total-etched with 35% phosphoric acid gel for 15 s, followed by water rinsing (15 s). Two coats of the Scotchbond Multi-Purpose primer (Adper Scotchbond Multi-Purpose Plus – 3M ESPE, St. Paul, MN, batch number # 0804002271; Table 1 ) were applied to visibly moist demineralized dentin according to the manufacturer’s directions. After briefly air-drying for 10 s, Scotchbond Multi-Purpose Adhesive was applied and light cured for 10 s.

Table 1
Composition and application mode of the adhesive system used in this study.
Adhesive system brand Original adhesive used for the water-wet bonding protocol Modified adhesive used for the ethanol-wet bonding
Adper Scotchbond MultiPurpose Plus Component 1: Etchant 35% phosphoric acid
Component 2: Scotchbond Multi-Purpose primer (HEMA, polyalkenoic acid polymer, water).
Component 3: Scotchbond Multi-Purpose adhesive (Bis-GMA, HEMA, tertiary amines, photo-initiator)
Component 1: Etchant 35% phosphoric acid
Component 2: Scotchbond Multi-Purpose adhesive diluted in 50 wt% ethanol (Bis-GMA, HEMA, tertiary amines, photo-initiator and ethanol)
Component 3: Scotchbond Multi-Purpose adhesive (Bis-GMA, HEMA, tertiary amines, photo-initiator)

After acid-etching, dentin in the ethanol-wet bonding group was treated with a series of increasing ethanol concentrations: 50%, 70%, 80%, 95% and three 100% ethanol applications for 15 s each following a chemical dehydration protocol . Dehydration procedures were meticulously performed to ensure that the dentin surface was always immersed in a liquid phase by keeping it visibly moist prior to the application of the subsequent solution.

Two consecutive coats of the experimental hydrophobic primer were applied to ethanol-saturated dentin. The experimental primer solution was prepared by diluting the Scotchbond Multi Purpose Adhesive ( Table 1 ) with 50 wt% absolute ethanol. This procedure was performed to produce a water-free bonding resin with similar composition of the hydrophilic adhesive employed in the water-wet protocol. Excess ethanol solvent was evaporated with a gentle air stream for 10 s. Then, a layer of the neat Scotchbond Multi-Purpose Adhesive was applied and spread over the primed surface and light cured for 10 s.

Preparations from both groups were restored with a microhybrid composite resin (shade EA2; Opallis – FGM Dental Products, Joinville, SC, Brazil; batch number # 160708) in three increments. Each increment was light-cured for 20 s. All light-curing procedures were performed for 10 s using a Radii LED light-curing unit (SDI Limited, Bayswater, Victoria, Australia) with an output intensity of 800 mW/cm 2 .

Within 20 min after completion of the bonding procedures, teeth were extracted, immersed in distilled water (pH 7), and kept in a moist environment for 24 h at 37 °C before being prepared for the microtensile bond test and nanoleakage analysis. All operative and restorative procedures reported for the clinical experimental was repeated under laboratory conditions in the extracted teeth.

Microtensile bond strength test (μTBS)

Bonded teeth from both experiments were longitudinally sectioned in both “ x ” and “ y ” directions across the bonded interface with a diamond saw in an ISOMET 1000 machine (Buehler, Lake Bluff, IL, USA), under water cooling at 300 rpm to obtain bonded sticks with a cross-sectional area of approximately 0.8 mm 2 .

Individual bonded sticks were attached to a device (Odeme Biotechnology, Joaçaba, SC, Brazil) for microtensile testing, with cyanoacrylate resin (Super Bonder, Locitec, São Paulo, SP, Brazil), so that tensile forces acted perpendicularly to the dentin/adhesive interface. Specimens were subjected to a tensile force in a universal testing machine (Kratos, São Paulo, SP, Brazil) at a crosshead speed of 0.5 mm/min. The failure modes were evaluated at 400× magnification (HMV-2, Shimadzu, Tokyo, Japan) and classified as cohesive in dentin (CD) or composite resin (CR) (failure exclusively within dentin or resin composite), adhesive (A, adhesive failure, restricted to the resin–dentin interface without partial cohesive failure of the neighboring substrates) and mixed (M, adhesive failure along with partial cohesive failure of the neighboring substrates). The number of specimens that showed premature failure was also recorded.

Nanoleakage analysis

One resin–dentin bonded stick of each tooth, randomly selected and not tested under tensile forces, was prepared for nanoleakage evaluation. These bonded sticks were immersed in ammoniacal silver nitrate for 24 h and the silver impregnated specimens were rinsed thoroughly in distilled water and placed in a photo-developing solution for 8 h under a fluorescent light. The adhesive interfaces were polished with descending grits of SiC papers (1000; 1200; 1500; 2000 and 2500) and 1 and 0.25 μm diamond paste (Erios Prod. Odont. Ltda, São Paulo, SP, Brazil) using a polishing cloth. Specimens were ultrasonically cleaned and left in a desiccator for 24 h at room temperature. Specimens were then mounted on stubs and sputter-coated with a 10-nm gold layer to be analyzed by scanning electron microscopy (SEM) (JSM 6360LV, Jeol Ltd., Tokyo, Japan) using a backscattered detector.

A representative image per tooth were obtained at 600× magnification by a blinded operator, not aware of the experimental conditions under investigation, and the amount of silver nitrate impregnation along the adhesive interface was evaluated by scoring nanoleakage interfacial expression by two calibrated observers as reported in Table 2 . Disagreements between observers were resolved by consensus.

Nov 23, 2017 | Posted by in Dental Materials | Comments Off on Ethanol-wet bonding technique: Clinical versuslaboratory findings
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