Abstract
Objective
To compare resin–dentin bond strengths and the micropermeability of hydrophobic vs. hydrophilic resins bonded to acid-etched or EDTA-treated dentin, using the ethanol wet-bonding technique.
Methods
Flat dentin surfaces from extracted human third molars were conditioned before bonding with: 37% H 3 PO 4 (15 s) or 0.1 M EDTA (60 s). Five experimental resin blends of different hydrophilicities and one commercial adhesive (SBMP: Scotchbond Multi-Purpose) were applied to ethanol wet-dentin (1 min) and light-cured (20 s). The solvated resins were used as primers (50% ethanol/50% comonomers) and their respective neat resins were used as the adhesive. The resin-bonded teeth were stored in distilled water (24 h) and sectioned in beams for microtensile bond strength testing. Modes of failure were examined by stereoscopic light microscopy and SEM. Confocal tandem scanning microscopy (TSM) interfacial characterization and micropermeability were also performed after filling the pulp chamber with 1 wt% aqueous rhodamine-B.
Results
The most hydrophobic resin 1 gave the lowest bond strength values to acid-etched dentin and all beams failed prematurely when the resin was applied to EDTA-treated dentin. Resins 2 and 3 gave intermediate bond strengths to both conditioned substrates. Resin 4, an acidic hydrophilic resin, gave the highest bond strengths to both EDTA-treated and acid-etched dentin. Resin 5 was the only hydrophilic resin showing poor resin infiltration when applied on acid-etched dentin.
Significance
The ethanol wet-bonding technique may improve the infiltration of most of the adhesives used in this study into dentin, especially when applied to EDTA-treated dentin. The chemical composition of the resin blends was a determining factor influencing the ability of adhesives to bond to EDTA-treated or 37% H 3 PO 4 acid-etched dentin, when using the ethanol wet-bonding technique in a clinically relevant time period.
1
Introduction
Etch-and-rinse adhesives require application of acid-conditioners to dentin, as the first step of the bonding procedure. The most commonly used acid etchant is ortho-phosphoric that modifies the smear layer and exposes the collagen fibril network . The 50 vol% of dentin previously occupied by minerals, is replaced by water after rinsing . This water is necessary to prevent the collapse of the collagen fibrils and allow the adhesive monomers to diffuse into the demineralized collagen network. The adhesive system should displace as much water as possible from the demineralized dentin in order to create a hypothetical perfect encapsulation of all the collagen fibrils with the intention of obtaining optimal and durable bonds .
Water sorption and plasticization of resins are determined by their hydrophilicity . The more hydrophilic the resin, the more the water sorption and plasticization . Nishitani et al. showed that adhesive bond strengths to acid-etched dentin matrices are directly related to the hydrophilicity of the resins. It has been hypothesized that if dentin could be bonded with more hydrophobic resins, a major decrease in water sorption would result in increased longevity of the resin–dentin interfaces. It is possible to coax hydrophobic monomers into a hydrophilic matrix by saturating the matrix with ethanol without any sign of phase change and/or micropermeability at the resin–dentin interface . Even though this technique leads to better infiltration of hydrophilic and/or hydrophobic dimethacrylates into ethanol-saturated matrices , concerns have been raised that the ethanol saturation of demineralized dentin requires 5 min, which may not be suitable for clinical practice.
Because EDTA creates a thinner demineralized layer compared to 37% phosphoric acid, it was thought that resins could infiltrate the thinner hybrid layers faster than is possible with thicker demineralized zones. The purpose of this study was to compare resin–dentin bond strengths of hydrophobic vs. hydrophilic resins bonded to acid-etched or EDTA-treated dentin created with a modified ethanol wet-bonding technique using a shorter period of ethanol saturation (1 min). Confocal tandem scanning microscopy (TSM) was used for interfacial characterization and micropermeability evaluation, with the intention of evaluating the quality and morphology of the resin–dentin bonded interfaces. The test null hypotheses were that there is no difference in bond strength following EDTA vs. phosphoric acid pretreatment using hydrophobic vs. hydrophilic resins and there is no difference in the micropermeability of the hybrid layers produced by those same procedures.
2
Materials and methods
2.1
Specimen preparation
Ninety-six human molars (age 20–40), extracted for periodontal reasons under a protocol approved by the institutional review board of Guy’s Hospital were used in this study for the microtensile bond strength and Confocal microscopy tests. The teeth were stored in 4 °C water for no more than one month. The specimens were sectioned below the dentin–enamel junction (Accutom-50, Struers, Copenhagem, Denmark) using a water-cooled diamond saw (330-CA RS-70300, Struers, Copenhagen, Denmark). The occlusal surfaces were groundd flat (LaboPol-4, Struers, Copenhagem, Denmark) using an abrasive paper (180- and 500-grit) under constant water irrigation to provide standardized smear-layer covered dentin surfaces.
2.2
Experimental resins
The five comonomer blends used in this study as dentin bonding agents (DBAs) were formulated based on known concentrations of all ingredients, including 50 wt% ethanol-solvated resin mixtures used as primers ( Table 1 ). All experimental neat resins contained 0.25 wt% camphoquinone and 1.0 wt% ethyl-dimethyl-4-aminobenzoate.
| Neat resin (pH: ∼7) | Solvated primer (pH: ∼7) | Total conc. (mole/L) of non-volatile ingredients a |
|---|---|---|
| Resin 1 (most hydrophobic) | ||
| 70 wt% E-BisADM | 34.4 wt% E-BisADM | 2.55 |
| 28.75% TEGDMA | 14.35% TEGDMA | |
| – | 50 wt% ethanol | |
| Resin 2 (second most hydrophobic) | ||
| 70 wt% BisGMA | 34.4 wt% BisGMA | 2.37 |
| 28.75% TEGDMA | 14.35% TEGDMA | |
| – | 50 wt% ethanol | |
| Resin 3 (mildly hydrophilic) | ||
| 70 wt% BisGMA | 34.4 wt% BisGMA | 3.58 |
| 28.75% HEMA | 14.35 wt% HEMA | |
| – | 50 wt% ethanol | |
| Neat resin (pH: ∼7) | Solvated primer (pH: ∼4) | |
|---|---|---|
| Resin 4 (more hydrophilic) | ||
| 40 wt% BisGMA | 20 wt% BisGMA | 3.57 |
| 30 wt% TCDM | 14.4 wt% TCDM | |
| 28.75% HEMA | 14.35% HEMA | |
| – | 50 wt% ethanol | |
| Neat resin (pH: ∼4) | Solvated primer (pH: ∼3) | |
|---|---|---|
| Resin 5 (most hydrophilic) | ||
| 40 wt% BisGMA | 20 wt% BisGMA | 3.92 |
| 30 wt% BisMP | 14.4 wt% BisMP | |
| 28.75% HEMA | 14.35% HEMA | |
| – | 50 wt% ethanol | |
| Neat adhesive (pH: ∼3) | Solvated adhesive (pH: ∼3) | |
|---|---|---|
| Scotchbond Multi-Purpose b (adhesive-bottle) (3M ESPE, St. Paul, MN, USA) | ||
| BisGMA | BisGMA | Unknown |
| HEMA | HEMA | |
| Polyalkenoic acid polymer | Polyalkenoic acid polymer | |
| Tertiary amines | Tertiary amines | |
| Photo-initiator | Photo-initiator | |
| – | 50 wt% ethanol | |
| Adhesive (pH: 8.2) | Primer (pH: 4.3) | |
|---|---|---|
| Scotchbond Multi-Purpose b (primer and adhesive) (3M ESPE, St. Paul, MN, USA) | ||
| BisGMA | HEMA | Unknown |
| Polyalkenoic acid polymer | Polyalkenoic acid polymer | |
| HEMA | Water | |
| Tertiary amines | – | |
| Photo-initiator | – | |
a Comonomer concentrations of neat resin blends were calculated by summing the molar concentrations of the non-volatile constituents .
b Percentage of the constituents of the primer and adhesive of the commercial Adper Scotchbond Multi-Purpose are not given by the 3M ESPE, St. Paul, MN, USA.
Resins 1 and 2 are similar to non-solvated hydrophobic resins used in the final step of three-step, etch-and-rinse, and two-step, self-priming adhesives. Resin 3 represents the formulation of typical two-step, etch-and-rinse adhesives, while resins 4 and 5 correspond to very hydrophilic one-step, self-etching adhesives, containing carboxylic- or phosphate-substituted methacrylates, respectively.
2.3
Solubility of the resins in water vs. ethanol
Most methacrylate-based monomers have poor water solubility even though they are claimed to be hydrophilic ( Table 2 ). A standard quantity (2 mL) of each experimental comonomer mixture was mixed with 10 mL of water or ethanol. The solvent-saturated with resin was removed and transferred to a tared container and the increase in mass was gravimetrically evaluated after solvent evaporation. This provided quantitative data of the relative solubility of these resins. Note that among the experimental comonomers, the most hydrophobic blend, resin 1, was 64 times more soluble in ethanol than water ( Table 2 ).
| Water | Ethanol | Ethanol/water | |
|---|---|---|---|
| Resin 1 | 0.0027 | 0.1735 | 64.3 |
| Resin 2 | 0.0038 | 0.1632 | 42.8 |
| Resin 3 | 0.0063 | 0.1268 | 20.1 |
| Resin 4 | 0.0118 | 0.1296 | 11.0 |
| Resin 5 | 0.0422 | 0.1515 | 3.6 |
| HEMA | 0.1120 | 0.1451 | 1.3 |
| TEGDMA | 0.0095 | 0.1692 | 17.8 |
| BisGMA | – | 0.1688 | – |
| BisMP | 0.1282 | 0.1890 | 1.5 |
2.4
Bonding procedures
Dentin surfaces were acid-etched for 15 s with 37% phosphoric acid (PA) or treated with 0.1 M EDTA (pH 7.8) for 60 s and then bonded using the ethanol wet-bonding technique. The ethanol wet-bonding substrate was achieved by covering the conditioned, water-rinsed dentin surfaces with absolute ethyl alcohol for 1 min. The procedure was performed by keeping the dentin specimens visibly moist with ethanol prior to the application of the resin blends .
Two consecutive coats of the five 50% ethanol/50% experimental primers were then applied onto the ethanol wet-dentin. Excess solvent was gentle air-dried from the primer/dentin for 3 s. Subsequently, a layer of each respective neat comonomer adhesive was applied, spread thin with moisture-free air, and light-cured for 20 s (Translux EC halogen light-curing unit, Kulzer GmBh, Bereich Dental, Werheim, Germany). The output intensity was monitored with a Demetron Radiometer (Model 100, Demetron Research, Danbury, CT, USA). A minimal light output intensity of 600 mW/cm 2 was employed for the experiments. A commercial adhesive Scotchbond Multi-Purpose (SBMP) (3M ESPE, St. Paul, MN, USA) was also applied with the ethanol wet-bonding either as per manufactures’ instructions (i.e. application of the primer and adhesive layers) or applying of the 50% adhesive diluted with 50% ethanol, in two consecutive coats followed by one layer of neat adhesive. Composite build-ups (6 mm) were constructed with a light-cured flowable resin composite Tetric EvoFlow ® (Ivoclar, Vivadent, Schaan, Liechtenstein – batch number: L26398) in four 1-mm-thick increments. The resin-bonded specimens were stored in de-ionized water for 24 h at 37 °C.
2.5
Microtensile bond strength (μTBS) test
Sixty teeth were used for the μTBS test. Each principal group (i.e. EDTA and H 3 PO 4 ) was constituted by 30 teeth that were subsequently sub-divided in 5 teeth for each sub-group according to the resin adhesives used in this study. The resin–dentin specimens were sectioned with a diamond wafering blade (Accutom-50, Struers, Copenhagem, Denmark) using a hard tissue saw (330-CA RS-70300, Struers, Copenhagem, Denmark) in both x and y directions across the adhesive interface to obtain beams with cross-sectional areas of 0.9 mm 2 . By excluding peripheral beams showing the presence of residual enamel, only the central 10 beams were used from each tooth. Thus, there were 10 beams × 4 teeth = 40 beam specimens in each sub-group ( Table 3 ).
| Dentin conditioning | EDTA | H 3 PO 4 |
|---|---|---|
| Resin 1 | 0 (0) c | 14.1 (13.3) d |
| (40/0) | (18/22) | |
| [100/0/0] | [85/0/15] | |
| Resin 2 | 36.5 (14.4) b | 30.9 (12.7) c |
| (0/40) | (0/40) | |
| [11/19/70] | [14/25/61] | |
| Resin 3 | 41.8 (10.2) b | 49.2 (12.6) b |
| (0/40) | (0/40) | |
| [0/62/38] | [0/75/25] | |
| Resin 4 | 48.2 (8.3) a | 51.1 (7.8) a |
| (0/40) | (0/40) | |
| [0/80/20] | [0/63/33] | |
| Resin 5 | 42.4 (8.3) a,b | 17.6 (14.3) d |
| (0/40) | (11/29) | |
| [11/29/60] | [30/0/70] | |
| Scotchbond Multi-Purpose (primer + adhesive) | 41.1 (10.0) b | 59.8 (9.9) a |
| (0/40) | (0/40) | |
| [10/68/22] | [0/71/29] | |
| Primer made from 50% ethanol-solvated SBMP adhesive + neat adhesive | 40.1 (12.5) b | 43.5 (14.3) b |
| (0/40) | (0/40) | |
| [21/27/52] | [8/50/42] | |
Each beam was attached to a modified Bencor Multi-T testing apparatus (Danville Engineering Co., Danville, CA, USA) with cyanoacrylate adhesive (Zapit, Dental Ventures of America Inc., Corona, CA, USA) and stressed to failure in tension using a universal testing machine (Instron 4411, Instron Corporation, Canton, MA, USA) at a crosshead speed of 0.5 mm/min. Bond strength data were calculated in MPa. Premature failures were included in the statistical analysis as zero values. Two-way ANOVA including interactions and Student–Newman–Keuls multiple comparisons were used for the statistical analysis. Adhesive systems and dentin surface treatment were considered as independent variables and μTBS as the dependent variable. Statistical significance level was set in advance at α = 0.05.
Modes of failure were classified as adhesive (A), mixed (M), or cohesive (C) when the failed bonds were examined at 30× by stereoscopic light microscopy or by SEM. Ten representative fractured specimens from each group were critical-point dried and then mounted on aluminum stubs with carbon cement. They were sputter-coated with gold (SCD 004 Sputter Coater; Bal-Tec, Vaduz, Liechtenstein) and viewed using a scanning electron microscope (SEM) (S-3500; Hitachi, Wokingham, UK) with an accelerating voltage of 15 kV and a working distance of 25 mm at increasing magnifications from 60× to 5000×.
2.6
SEM observations of the failed bonds
Ten failed specimens from each sub-group were examined by SEM to classify the modes of failure into the percentage of the total surface area of failed dentin exhibiting pure adhesive, cohesive or mixed failure modes.
2.7
Confocal microscopy characterization
Three resin–dentin bonded specimens were prepared for each sub-group. The pulpal tissue was carefully removed from the exposed pulp chamber of each crown segment, without crushing the pulpal-dentin walls using thin tissue forceps. A pincer-type calliper was used for measuring the remaining dentin thickness (RDT) from the dentin surface to the highest pulpal horns (0.7–0.9 ± 0.1 mm). Each tooth section was attached to a Perspex™ (Perspex Distributions Ltd., London, UK) platform (2 cm × 2 cm × 0.5 cm) that was perforated by an 18-gauge stainless steel tube glued in place using cyanocrylate adhesive (Rocket Heavy, Dental Ventures of America, Corona, CA, USA). Each specimen was connected to a 60 cc syringe barrel filled with 1 wt% aqueous rhodamine-B solution that was connected to the pulp chamber via polyethylene tubing as described by Sauro et al. . The height of the fluid was 20 cm above the dentin surface. After 3 h, the specimens were disconnected from perfusion system, separated from the Perplex platform and finally sectioned into 1 mm slabs using a slow-speed water-cooled diamond saw (Labcut, Agar Scientific, Stansted, UK). The surface of the slabs was slightly polished using 1200-grit silicon carbide paper and ultra-sonicated for 2 min at each step .
The dentin/adhesive interfaces were examined using a confocal tandem scanning microscope (TSM: Noran Instruments, Middleton, Wisconsin, USA) in both the fluorescence and reflection modes. Reflection and fluorescence images were recorded using a Andor iXon EM , EMCCD camera (Andor Instruments, Belfast, Northern Ireland), digitized and image processed or reconstructed with suitable computer hardware and software (iQ, Andor iXon EM , Andor Instruments).
To measure the micropermeability of the bonded interface, all the specimens were examined using a X100/1.4 NA oil immersion objective with a 10× ocular and phototube in the fluorescence mode with 546 nm excitation and 600 nm long-pass filters, and in the reflection mode. Since peripheral dentin located close to the dentin–enamel junction has a low dentin permeability due to its reduced density of dentinal tubules , only the resin-bonded dentin surfaces located at least 1 mm from the enamel were analyzed in order to avoid underestimating micropermeability . Three representative images from resin-bonded dentin surfaces were recorded 1 mm from the dentin–enamel junction. One image was taken from the centre of the bonded interface and two additional images in proximity of the pulpal horns were obtained from each site after a complete investigation of the entire resin–dentin interface . These images were intended to be representative of the most common features observed in each specimen. Two additional specimens for each group were bonded with the resin adhesives previously mixed with 0.1 wt% rhodamine-B in order to image monomer diffusion of the tested resin adhesives into differently pretreated dentin . In those specimens, the pulp chamber, polyethylene tubing and syringe barrel were full of water without the presence of fluorochrome. That is, no micropermeability was performed in these specimens.
2
Materials and methods
2.1
Specimen preparation
Ninety-six human molars (age 20–40), extracted for periodontal reasons under a protocol approved by the institutional review board of Guy’s Hospital were used in this study for the microtensile bond strength and Confocal microscopy tests. The teeth were stored in 4 °C water for no more than one month. The specimens were sectioned below the dentin–enamel junction (Accutom-50, Struers, Copenhagem, Denmark) using a water-cooled diamond saw (330-CA RS-70300, Struers, Copenhagen, Denmark). The occlusal surfaces were groundd flat (LaboPol-4, Struers, Copenhagem, Denmark) using an abrasive paper (180- and 500-grit) under constant water irrigation to provide standardized smear-layer covered dentin surfaces.
2.2
Experimental resins
The five comonomer blends used in this study as dentin bonding agents (DBAs) were formulated based on known concentrations of all ingredients, including 50 wt% ethanol-solvated resin mixtures used as primers ( Table 1 ). All experimental neat resins contained 0.25 wt% camphoquinone and 1.0 wt% ethyl-dimethyl-4-aminobenzoate.
| Neat resin (pH: ∼7) | Solvated primer (pH: ∼7) | Total conc. (mole/L) of non-volatile ingredients a |
|---|---|---|
| Resin 1 (most hydrophobic) | ||
| 70 wt% E-BisADM | 34.4 wt% E-BisADM | 2.55 |
| 28.75% TEGDMA | 14.35% TEGDMA | |
| – | 50 wt% ethanol | |
| Resin 2 (second most hydrophobic) | ||
| 70 wt% BisGMA | 34.4 wt% BisGMA | 2.37 |
| 28.75% TEGDMA | 14.35% TEGDMA | |
| – | 50 wt% ethanol | |
| Resin 3 (mildly hydrophilic) | ||
| 70 wt% BisGMA | 34.4 wt% BisGMA | 3.58 |
| 28.75% HEMA | 14.35 wt% HEMA | |
| – | 50 wt% ethanol | |
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