Abstract
Objectives
To evaluate the effect of intrinsic water permeation on the microtensile bond strengths of different adhesive systems to dentine and the quality of resin-dentine interfaces.
Methods
Ninety-six non-carious human third molars were divided into 4 groups: Clearfil S 3 Bond Plus (CSBP; Kuraray); Clearfil S 3 Bond (C3S; Kuraray); iBond Self-Etch (IB; Heraeus-Kulzer) and Prime&Bond NT (PB, control etch-and-rinse adhesive, Dentply-Sirona). For each adhesive, specimens from one subgroup (N = 10) were bonded using zero pulpal pressure, while specimens from the other subgroup (N = 10) were bonded using 15 cm water pressure (PP). Each bonded tooth was sectioned into 1 × 1 mm sticks and stressed to failure. Data were analysed using two-way ANOVA and Holm-Sidak pairwise comparisons to examine the effects of “adhesive”, “pulpal pressure” and their interaction on bond strength (α = 0.05). Representative fractured sticks were examined by SEM. The remaining tooth slabs in each subgroup were used for TEM and CLSM.
Results
Microtensile bond strengths (mean ± SD; in MPa) were: 33.4 ± 6.9 (CSBP), 33.2 ± 4.7 (CSBP-PP), 35.0 ± 8.6 (C3S), 25.5 ± 7.3 (C3S-PP), 18.4 ± 4.0 (IB), 16.5 ± 6.9 (IB-PP), 28.2 ± 5.5 (PB), 20.5 ± 7.2 (PB-PP). “Adhesive-type” (P < 0.001), “pulpal-pressure” (P < 0.001) and their interactions (P < 0.001) significantly affected bond strength results. No difference between no-PP and PP subgroups was found for CSBP and IB (P > 0.05). Water droplets were identified along the resin-dentine interface for IB, IB-PP and C3S-PP.
Conclusion
IB exhibits water sensitivity when bonding is performed with/without pulpal pressure. C3S exhibits water sensitivity when bonding is performed with pulpal pressure. CSBP does not exhibit water sensitivity when bonding is performed with/without pulpal pressure.
Clinical significance: Intrinsic water permeation during bonding procedures significantly affects bond strength results and the resin-dentine interface of contemporary single-bottle self-etch dentine adhesive systems.
1
Introduction
Water is the most common substance that exists in nature . It has been coined the “universal solvent” because water is capable of dissolving more substances than any other liquid . Because of this property, water is responsible for infiltration and enlargement of cracks in concrete blocks , decomposition of wood and initiating oxidation of iron-containing substances . Water is also the major component of the dentinal fluid . Free and bound water constitutes approximately 10% of the total weight of mineralised dentine . When dentine is demineralised with 32–37% phosphoric acid, apatite crystallites are completely dissolved along the top 5–10 μm of the mineralised dentine, with the spaces replaced by free water. In demineralised dentine, unbound (free water) constitutes 75–79% of the total water, while the remaining 21–25% is bound water . Although it is necessary to replace the unbound water with adhesive components during dentine bonding, replenishment of the intrinsic moisture lost by evaporation by pulp pressure often results in water transudation along the dentine-adhesive interface .
Water plays an antagonistic role in dentine bonding. In dentine demineralised by strong acids, water keeps the collagen matrix suspended and fully expanded by preventing the formation of interpeptide hydrogen bonds between adjacent collagen fibrils . Water is an intrinsic component of self-etch adhesives and keeps acidic resin monomers in their ionised states for demineralisation of the smear layer and the underlying intact dentine . Although water is initially necessary for dentine bonding, excess water that is not removed during the polymerisation of an adhesive compromises the integrity of the resin-dentine interface . When polar solvents are evaporated from adhesives without incorporating 2-hydroxylethyl methacrylate as a co-solvent, water-insoluble dimethacrylate resin monomers undergo phase separate into hydrophobic-rich domains when they come into contact with residual water; this results in an adhesive layer with non-uniform swelling/water-sorption characteristics and mechanical properties . Excessive moisture adversely affects solvent evaporation and adhesive resin polymerisation . Water provides a medium for enzymes to function. Hydrolysis of the resinous and collagenous components of the hybrid layer results in the degradation of resin-dentine bonds , which, in turn, compromises the clinical longevity of resin composite restorations . With the advent of newer single-bottle self-etch adhesive systems, it is important for clinicians to appreciate whether bonded resin-dentine interfaces created by these new systems are less susceptible to intrinsic water permeation.
Accordingly, the objective of the present study was to identify whether intrinsic water permeation affects the microtensile bond strengths and the quality of resin-dentine interfaces using recently commericialized single-bottle one-step self-etching and compared with the results achieved with a time-tested etchand-rinse adhesive. The null hypothesis tested was that bonding in the absence or presence of simulated pulpal pressure has no effect on the bond strength and the quality of the resin-dentine interface for the adhesives systems examined.
2
Materials and methods
Ninety-six recently extracted, non-carious human third molars were collected after receiving the patients’ informed consent under a protocol approved by the Augusta University Human Assurance Committee. These teeth were stored at 4 °C in 0.9% sodium chloride solution supplemented with 0.02% sodium azide to prevent bacteria growth. The teeth were divided into four groups and randomly assigned to one of the four groups:
Group I: Clearfil S 3 Bond Plus (Kuraray Noritake Dental Inc., Tokyo, Japan), a single-bottle, one-step, self-etching adhesive;
Group II: Clearfil S 3 Bond (Kuraray Noritake Dental Inc.), a single-bottle, one-step, self-etching adhesive;
Group III: iBond Self Etch (Heraeus-Kulzer, Hanau, Germany); a single-bottle, one-step, self-etching adhesive; and
Group IV: Prime & Bond NT (Caulk Division, Dentsply Sirona, York, PA, USA), a two-step etch-and-rinse adhesive.
The composition of these adhesives and their application procedures are listed in Table 1 . Each group was subdivided into two equal subgroups of 10 teeth each. Specimens in first subgroup were bonded using zero pulpal pressure. Specimens in second subgroup were bonded using 15 cm water pressure. For each tooth, a flat coronal dentine surface was prepared perpendicular to the longitudinal axis of the tooth using a slow-speed diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) in the presence of water-cooling. The dentine surface was wet-polished with 320 grit silicon carbide paper for 1 min to create standardized smear layer. A second section was made 1 mm below the cementoenamel junction to create a crown segment containing the top of the pulp chamber. The distance between the pulp horn and dentin surface was measured to closest 0.01 mm, and specimens with dentin thickness less than 1 mm thick were discarded. The cut root surface of the crown segment was attached to a 14 × 14 × 5 mm piece of polymethyl methacrylate using a cyanoacrylate glue (Zapit, Dental Ventures of American, Corona, CA, USA). The polymethyl methacrylate plate was penetrated by an 18-gauge stainless steel tubing that ended flush with the upper surface of the plate.
| Adhesive | Classification | Composition | Application |
|---|---|---|---|
| Clearfil S 3 Bond Plus | single-bottle | MDP, HEMA, BisGMA, dimethacrylates, water, ethanol, new photoinitiator, NaF, CQ, SiO 2 | Apply for 10 s; dry with mild air pressure. Air-thin the adhesive surface with mild air pressure for more than 5 s until the liquid no longer moves. Light-cure for 10 s. |
| one-step | |||
| self-etching | |||
| adhesive | |||
| Clearil S 3 Bond | single-bottle | MDP, HEMA, BisGMA, water ethanol, photo-initiator, CQ, SiO 2 | Apply for 20 s; dry with strong air pressure; light-cure for 10 s. |
| one-step | |||
| self-etching | |||
| adhesive | |||
| iBond Self Etch | single-bottle | UDMA, 4-MET, glutaraldehyde, acetone, water, stabiliser, photoinitiator | Apply 3 consecutive times, rub for 30 s, air-dry until adhesive stops moving, then thoroughly air-dry for 5 s. Light-cure for 20 s. |
| one-step | |||
| self-etching | |||
| adhesive | |||
| Prime & Bond NT (PB) (Caulk/Dentsply, Milford, DE, USA) | two-step | Etchant: 34% phosphoric acid | Etch 15 s, rinse 10 s, leave dentine moist, apply adhesive for 20 s, evaporate solvent for 5 s, light-cure for 10 s. |
| etch-and-rinse | |||
| adhesive | Adhesive: PENTA, BisGMA, TEGDMA, acetone, SiO 2 , CQ |
After filling the pulp chamber with isotonic saline, a 40 cm length of polyethylene tubing was attached via the 18-gauge tubing to a syringe barrel containing isotonic saline. The height of the column of isotonic saline was adjusted to 15 cm above the exposed dentine surface. This arrangement enabled one to apply simulated physiological pulpal pressure to dentine during bonding ( Fig. 1 ). For the no pulpal pressure subgroup, the polyethylene tubing was clamped to deliver a simulated pulpal pressure of 0 cm water pressure. Each tooth was bonded with one of the four adhesives by following meticulously the instructions supplied by the respective manufacturer. After evaporation of the adhesive solvent, each adhesive was polymerised for 20 s using a quartz-tungsten-halogen light curing unit with an output intensity of 600 mW/cm 2 . This was followed by incremental placement of two 2-mm thick layers of a universal nano-hybrid resin composite (Clearfil Majesty Esthetic, Kuraray Noritake Dental Inc.) that were light-cured separately for 40 s each.
The additional 16 teeth were used for transmission electron microscopy (TEM). The teeth in each subgroup (N = 2) were bonded in the manner described previously using the respective adhesive. Clearfil Protect Liner F (Kuraray Noritake Dental Inc.), a flowable resin composite with nanoscopical silica fillers, was used for buildup so that specimens could be sectioned without damaging the diamond knife by chunky inorganic fillers that are usually incorporated in hybrid resin composites.
2.1
Microtensile bond testing
After storage in deionised water at 37 °C for 24 h, each tooth was vertically sectioned into 1 mm-thick serial slabs using the Isomet saw with water cooling. The two central slabs were sectioned into 1 × 1 (±0.2) mm wide sticks, each containing the adhesive joint in the centre of the stick. The two longest sticks with an average dentin thickness of 2–2.5 mm from each slab were randomly selected for tensile testing, yielding 4 sticks per tooth. Thus, the specimens for microtensile testing in each subgroup comprised 4 sticks × 10 teeth = 40 sticks to enable valid statistical comparisons to be performed .
The “non-trimming” version of the microtensile technique was employed for bond strength evaluation . Each stick was secured via cyanoacrylate glue to a testing jig and stressed to failure under tension using a universal tester (Vitrodyne V1000, Liveco Inc., Burlington, VT, USA) at a cross-head speed of 1 mm/min. After bond testing, the two ends of the fractured stick were removed and examined under a surgical microscope to determine the mode of failure. Failure modes were classified as adhesive failure ( i.e. failure along the adhesive interface), mixed failure ( i.e. failure within the adhesive joint and with attached resin composite or dentine) or cohesive failure ( i.e. failure within the resin composite or dentine). Beams that failed prematurely during specimen preparation were not included in the statistical analysis.
The mean strength of the four beams in each tooth was used to compile the mean microtensile bond strength for that tooth. Analysis was performed using tooth number as the statistical unit (N = 10 teeth). Because the normality (Shapiro-Wilk test) and equal variance assumptions (modified Levene test) of the tensile strength data appeared to be violated, the data were logrithymically transformed to enable a two-factor analysis of variance to be performed. The analysis was used to determine the effects of “adhesive” and “pulpal pressure” and the effect of the interaction of those two factors on the microtensile bond strength results. All pairwise comparisons were performed using the Holm-Sidak procedure. Statistical significance was set at α = 0.05.
2.2
Scanning electron microscopy of fractured interfaces
The dentine side of four fracture beams from each subgroup with either adhesive or mixed failures and with bond strengths that are closest to the mean bond strength of the respective subgroup were selected for detailed fractographic analysis using scanning electron microscopy (SEM). The specimens were mounted on aluminum stubs, coated with gold/palladium and examined with a field emission scanning electron microscope (XL-30 FEG; Philips, Eindhoven, The Netherlands) operated at 10 KeV.
2.3
Transmission electron microscopy of resin-dentine interfaces
Two 1 mm-thick slabs derived from the bonded teeth designated for transmission electron microscopy were examined for each of the eight subgroups. Each slab was completely demineralised in formic acid-sodium formate (pH 2). After demineralisation of the bonded slabs for 7 days at 25 °C, the specimens were fixed in Karnovsky’s fixative (2.5 wt% glutaraldehyde and 2% paraformaldehyde in 0.1 mol/L cacodylate buffer, pH 7.3), rinsed in 0.1 mol/L sodium cacodylate buffer and post-fixed with 1% osmium tetroxide. The slabs were progressively dehydrated through an ascending ethanol series (50–100%). The absolute ethanol was replaced with propylene oxide as a transitional medium. Specimens were subsequently embedded in pure epoxy resin. Ninety nanometre-thick sections were prepared and collected with single-slot carbon- and formvar-coated copper grids, stained with 2% uranyl acetate and Reynold’s lead citrate and examined using a JEM-1230 TEM (JEOL, Tokyo, Japan) at 110 kV.
2.4
Confocal laser scanning microscopy of resin-dentine interfaces
Two remaining tooth slabs from each subgroup were used for confocal laser scanning microscopy (CLSM). Each non-demineralised tooth slab was ultrasonicated for 5 min and immersed in 0.1 wt% rhodamine B (MilliporeSigma, St. Louis, MO, USA) dissolved in phosphate-buffered saline (pH 7.4). After 24 h, the dye-infiltrated slabs were rinsed with deionised water and examined using a CLSM (LSM 510 META; Carl Zeiss, Thornwood, NY) that was coupled with a helium neon gas laser (80% of 543 nm excitation, 1.2 mW). Images were captured at 5 μm beneath the polished surface to avoid superficial specimen preparation artefacts.
2.5
Tracer-infused water-rich zones within the resin-dentine interfaces
Six additional non-demineralised tooth slabs from each adhesive subgroup were wet-polished with 2000-grit silicon carbide paper and coated with nail varnish applied 1 mm from the bonded interface. The slabs were immersed in 50 wt% ammoniacal silver nitrate solution (pH 9.5) for 24 h The tracer-infused slabs were immersed in a photodeveloping solution for 8 h under a fluorescent light to convert the tracer into metallic silver. Each slab was then gently wet-polished with 1200-grit silicon carbide papers to remove the silver-rich surface layer.
For SEM examination, three tooth slabs from each subgroup were air-dried, coated with gold-palladium and examined with the XL-30 SEM at 30 kV. Imaging was performed using a combination of 70% backscattered electron mode and 30% secondary electron mode. The other three slabs from each subgroup were processed using the TEM embedding protocol described in Section 2.3 . After section, the 90 nm thick sections were examined unstained using the JEM-1230 TEM at 110 kV.
2
Materials and methods
Ninety-six recently extracted, non-carious human third molars were collected after receiving the patients’ informed consent under a protocol approved by the Augusta University Human Assurance Committee. These teeth were stored at 4 °C in 0.9% sodium chloride solution supplemented with 0.02% sodium azide to prevent bacteria growth. The teeth were divided into four groups and randomly assigned to one of the four groups:
Group I: Clearfil S 3 Bond Plus (Kuraray Noritake Dental Inc., Tokyo, Japan), a single-bottle, one-step, self-etching adhesive;
Group II: Clearfil S 3 Bond (Kuraray Noritake Dental Inc.), a single-bottle, one-step, self-etching adhesive;
Group III: iBond Self Etch (Heraeus-Kulzer, Hanau, Germany); a single-bottle, one-step, self-etching adhesive; and
Group IV: Prime & Bond NT (Caulk Division, Dentsply Sirona, York, PA, USA), a two-step etch-and-rinse adhesive.
The composition of these adhesives and their application procedures are listed in Table 1 . Each group was subdivided into two equal subgroups of 10 teeth each. Specimens in first subgroup were bonded using zero pulpal pressure. Specimens in second subgroup were bonded using 15 cm water pressure. For each tooth, a flat coronal dentine surface was prepared perpendicular to the longitudinal axis of the tooth using a slow-speed diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) in the presence of water-cooling. The dentine surface was wet-polished with 320 grit silicon carbide paper for 1 min to create standardized smear layer. A second section was made 1 mm below the cementoenamel junction to create a crown segment containing the top of the pulp chamber. The distance between the pulp horn and dentin surface was measured to closest 0.01 mm, and specimens with dentin thickness less than 1 mm thick were discarded. The cut root surface of the crown segment was attached to a 14 × 14 × 5 mm piece of polymethyl methacrylate using a cyanoacrylate glue (Zapit, Dental Ventures of American, Corona, CA, USA). The polymethyl methacrylate plate was penetrated by an 18-gauge stainless steel tubing that ended flush with the upper surface of the plate.
| Adhesive | Classification | Composition | Application |
|---|---|---|---|
| Clearfil S 3 Bond Plus | single-bottle | MDP, HEMA, BisGMA, dimethacrylates, water, ethanol, new photoinitiator, NaF, CQ, SiO 2 | Apply for 10 s; dry with mild air pressure. Air-thin the adhesive surface with mild air pressure for more than 5 s until the liquid no longer moves. Light-cure for 10 s. |
| one-step | |||
| self-etching | |||
| adhesive | |||
| Clearil S 3 Bond | single-bottle | MDP, HEMA, BisGMA, water ethanol, photo-initiator, CQ, SiO 2 | Apply for 20 s; dry with strong air pressure; light-cure for 10 s. |
| one-step | |||
| self-etching | |||
| adhesive | |||
| iBond Self Etch | single-bottle | UDMA, 4-MET, glutaraldehyde, acetone, water, stabiliser, photoinitiator | Apply 3 consecutive times, rub for 30 s, air-dry until adhesive stops moving, then thoroughly air-dry for 5 s. Light-cure for 20 s. |
| one-step | |||
| self-etching | |||
| adhesive | |||
| Prime & Bond NT (PB) (Caulk/Dentsply, Milford, DE, USA) | two-step | Etchant: 34% phosphoric acid | Etch 15 s, rinse 10 s, leave dentine moist, apply adhesive for 20 s, evaporate solvent for 5 s, light-cure for 10 s. |
| etch-and-rinse | |||
| adhesive | Adhesive: PENTA, BisGMA, TEGDMA, acetone, SiO 2 , CQ |
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