Abstract
Objective
A recently-launched universal adhesive, G-Premio Bond, provides clinicians with the alternative to use the self-etch technique for bonding to dentine without waiting for the adhesive to interact with the bonding substrate (no-waiting self-etch; Japanese brochure), or after leaving the adhesive undisturbed for 10 s (10-s self-etch; international brochure). The present study was performed to examine in vitro performance of this new universal adhesive bonded to human coronal dentine using the two alternative self-etch modes.
Methods
One hundred and ten specimens were bonded using two self-etch application modes and examined with or without thermomechanical cycling (10,000 thermal cycles and 240,000 mechanical cycles) to simulate one year of intraoral functioning. The bonded specimens were sectioned for microtensile bond testing, ultrastructural and nanoleakage examination using transmission electron microscopy. Changes in the composition of mineralised dentine after adhesive application were examined using Fourier transform infrared spectroscopy.
Results
Both reduced application time and thermomechanical cycling resulted in significantly lower bond strengths, thinner hybrid layers, and significantly more extensive nanoleakage after thermomechanical cycling. Using the conventional 10-s application time improved bonding performance when compared with the no-waiting self-etch technique. Nevertheless, nanoleakage was generally extensive under all testing parameters employed for examining the adhesive.
Conclusion
Although sufficient bond strength to dentine may be achieved using the present universal adhesive in the no-waiting self-etch mode that does not require clinicians to wait prior to polymerisation of the adhesive, this self-etch concept requires further technological refinement before it can be recommended as a clinical technique.
Clinical significance
Although the surge for cutting application time to increase user friendliness remains the most frequently sought conduit for advancement of dentine bonding technology, the use of the present universal adhesive in the no-waiting self-etch mode may not represent the best use of the adhesive.
1
Introduction
Science and technology constitute a primary productive force that stimulates society reform . As early as the 1700s, technological developments that led to the introduction of steam power catalysed the Industrial Revolution and provided the backdrop for modernisation on a global scale. Technologies triggered by the concepts of packet switching and wide-area networking in the 1960s incited the emergence of the internet and metamorphosed nearly all facets of human activity − society, politics, culture and the economy. On a lighter note, the technology triggered by Doctor Michael Buonocore’s adaptation of phosphoric acid treatment of metal surfaces to obtain better adhesion of paint primers spurred the rise of the resin-bonding epoch in dentistry .
Beginning with the initial technology trigger, the platform associated with the development and coming-of-age of new technologies appears to follow a similar cycle, irrespective of the field of endeavour . In the context of dentine bonding, the enthusiasms associated with the advent of simultaneous etching of enamel and dentine , multi-step etch-and-rinse adhesives and the water-wet bonding etch-and-rinse technique produced a plethora of successful dental adhesion stories, reaching a peak of inflated expectations during the early 1990s . No sooner had clinicians been besotted with these excitements than reports of post-operative sensitivity and adversarial pulpal responses associated with the use of those adhesives began to surface, particularly when the adhesives were applied to deep, vital dentine . By etching and priming tooth substrates simultaneously, two-step self-etch adhesives were subsequently introduced to solve those problems by reducing dentine permeability through the incorporation of smear layer components into the hybrid layer . Paradoxically, the problem-solving strategy contributed to unforeseen problems. Increases in water sorption associated with incorporating increasingly hydrophilic adhesive resin monomers , adhesive coatings with semi-permeable membrane-like characteristics , as well as bond degradation over time became the “Achilles heel” of both etch-and-rinse and self-etch adhesives. Waxing and waning of the promises to deliver speedier, easier and more user-friendly adhesive versions precipitated a trough of disillusionment in adhesive dentistry. As more is known about the control of resin-dentine bond degradation via the use of the water-compatible catalytic systems , less hydrophilic adhesive resin monomers and inhibition of adhesive-activated endogenous proteases , clinicians begin to appreciate the benefits associated with step reduction for both the etch-and-rinse and self-etch adhesives . The simplified clinical manipulation and shortened application time associated with these new adhesive genres moved the industry to a slope of enlightenment. Universal adhesives emerged as the latest player in this arena . They provided dentists with the option in choosing adhesive strategies (i.e. etch-and-rinse, self-etch or selective etch), the versatility in bonding to zirconia and the purported advantage of bonding to silica-based ceramics without the use of additional primers . Nevertheless, universal adhesives have been perceived as old wines in new bottles because they inherit the limitations of previous adhesive categories . In the absence of further enlightenment, it appears that dentine adhesive development has entered the plateau stage of a classical technology development cycle. New insights are thus required to transcend technological development of dentine adhesives to new heights.
A recently-launched universal adhesive (G-Premio Bond, GC Corporation, Tokyo, Japan) provides the clinician with the alternative to use the self-etch technique for bonding to dentine without waiting for the adhesive to interact with the bonding substrate (no-waiting self-etch; Japanese version of manufacturer’s instructions), or after leaving the adhesive undisturbed for 10 s (10-s self-etch; international version of manufacturer’s instructions) . Introduction of the no-waiting concept, while appealing to clinicians, should not compromise adhesive bonding effectiveness and the durability of resin-dentine bonds. Hence, the present study was performed to examine in vitro performance of the single-component, universal adhesive bonded to human coronal dentine using the two aforementioned alternative self-etch modes. Accordingly, two null hypotheses were tested: 1) there is no difference between the use of no-waiting self-etch and 10-s self-etch on the microtensile bond strength of the universal adhesive to dentine; and 2) thermomechanical cycling has no effect on the microtensile bond strength to dentine when the universal adhesive is used in either the no-waiting or the 10-s self-etch mode.
2
Materials and methods
2.1
Dentine disc preparation
One hundred and ten freshly extracted intact human third molars were collected according to a protocol approved by the Human Assurance Committee of the Augusta University, with informed consent obtained from the donating subjects with respect to the use of human tissues. The extracted teeth were stored in 0.9% (w/v) NaCl containing 0.02% sodium azide at 4 °C for no longer than one month. The roots of the teeth were removed using a water-cooled low-speed saw (Isomet, Buehler Ltd, Evanston, IL, USA). For each tooth, a flat bonding surface was prepared by removing the occlusal third of each tooth crown to expose the mid-coronal dentine. The dentine surfaces were polished with 320-grit silicon carbide paper under water irrigation for 1 min.
2.2
Bonding procedures
The teeth were randomly assigned to two groups (50 teeth per group). The composition of G-Premio Bond (GP) is listed in Table 1 . Self-etch bonding procedures were performed according to the Japanese version of the manufacturer’s instructions (no-waiting self-etch mode) or the international version of the manufacturer’s instructions (10-s self-etch mode). For the no-waiting self-etch mode, the adhesive was applied on the dentine surface and immediately air-dried without waiting (see below). For 10-s self-etch mode, the adhesive was applied on the dentine surface and left undisturbed for 10 s. For both modes, the adhesive was dried for 5 s using oil-free air from the triple syringe of a clinical operatory unit under maximum air pressure. After light-curing of the adhesive, resin composite build-ups were constructed in four 1-mm thick increments using G-ænial Sculpt (GC Corporation). Each increment was light-cured for 20 s using a light-emitting diode (440–480 nm range) curing unit with an output intensity of 1200 mW/cm 2 . To facilitate sectioning of transmission electron microscopy (TEM) specimens, the bonded specimens were coupled with a 2 mm thick layer of Protect Liner F flowable resin composite (Kuraray Noritake Dental Inc., Tokyo, Japan) and light-cured for 20 s.
| Adhesive | pH | Composition | Manufacturer’s instructions |
|---|---|---|---|
| G-Premio Bond | 1.5 | 10-MDP, 4-META, 10-methacryoyloxydecyl dihydrogen thiophosphate, methacrylate acid ester, distilled water, acetone, phot initiators, silica | No-waiting self-etching mode
10-s self-etching mode
|
Because the manufacturer emphasised the mandatory use of maximum air pressure for drying the adhesive in the bonding instructions, a pilot study was also performed to understand the consequence of not adhering to this instruction. In the pilot study, additional dentine discs were bonded using the two aforementioned self-etch protocols (N = 2), but with the adhesive dried with 5 s of gentle air flow. After coupling with the flowable resin composite, the bonded specimens were aged for 24 h and prepared for nanoleakage examination of the resin-dentine interfaces (methods described in the TEM section).
After bonding, the specimens from each experimental group (i.e. those not related to the pilot study) were divided into two batches. One batch was stored in deionised water at 37 °C for 24 h; the other batch was subjected to thermomechanical challenge, using 10,000 thermal cycles (10 °C for one min, 25 °C for one min and 55 °C for one min) and 240,000 mechanical cycles, corresponding to one year of intraoral functioning . The resin-dentine specimens were sectioned in both x and y directions across the adhesive interface to obtain beams with cross-sectional areas of approximately 0.9 mm × 0.9 mm using the ‘non-trimming’ version of the microtensile bond testing procedures . Simulated ageing was performed in a thermomechanical wear system (Model ER-37000; Erios, São Paulo, SP, Brazil). The four longest beams were obtained from the two central slabs of each bonded tooth for bond strength testing and one beam each was obtained from the two central slab of each bonded tooth for TEM. Hence, for each of the four subgroups, 80 beams from 20 teeth were used for bond strength testing and 10 beams from 5 teeth were used for TEM examination of the resin-dentine interface.
2.3
Microtensile bond strength
Each beam from a subgroup (N = 20) was secured with cyanoacrylate glue (Zapit; Dental Ventures of America, Corona, CA, USA) to a testing jig and stressed to failure under tension in a universal testing machine (Vitrodyne V1000; Liveco Inc., Burlington, VT, USA) at a cross-head speed of 1 mm/min. The tensile load at failure was recorded and divided by the measured cross-sectioned area of each beam to yield the tensile bond strength in megaPascals (MPa). The mean bond strength of the 4 beams derived from one tooth was used to represent the tensile bond strength of that tooth, yielding 20 values per subgroup. Data were analysed with parametric statistical methods after validating the normality (Shapiro-Wilk test) and homogeneity of variance (modified Levene test) assumptions of the data sets. A two-factor analysis of variance was used to examine the effects of adhesive application time (i.e. no-waiting or 10-s self-etch) and simulated ageing (i.e. without or with thermomechanical cycling), and the interaction of those two factors on the bond strength results. Post-hoc pairwise comparisons were performed using the Tukey statistic. Statistical significance was set at α = 0.05.
After bond strength testing, the two ends of a fractured stick were retrieved and examined with 10× magnification using a stereoscopical microscope to determine the mode of failure. Failure modes were classified as adhesive failure (failure along the adhesive interface), mixed failure (failure within the adhesive joint with failure within the resin composite or dentine), or cohesive failure (failure within the resin composite or dentine). For statistical analysis, the number of specimens exhibiting mixed and cohesive failures were combined into the non-adhesive category. Adhesive and non-adhesive failure modes in the 4 subgroups (zero-second or 10-s etch; with or without thermomechanical cycling) were arranged into a 2 × 4 contingency table and analysed with the Fisher-Freeman-Halton statistic . A 95% confidence level was used to determine if an association existed between the method in which the specimens were bonded and tested, and the category of failure (adhesive vs non-adhesive).
2.4
Transmission electron microscopy
Five of the 10 beams designated for TEM were used for nanoleakage examination. Each beam was coated with two layers of nail varnish applied to within 1 mm of the bonded interface. After drying, the varnish-coated beams were immersed in 50% ammoniacal silver nitrate solution for 48 hours. The silver-impregnated specimens were thoroughly rinsed with deionised water and placed in photo-developing solution for 8 h, under a fluorescent light, to facilitate reduction of the diaminesilver ions into metallic silver grains . The silver-impregnated specimens were polished, cleaned ultrasonically, dehydrated in an ascending ethanol series (50–100%), immersed in propylene oxide as transition medium, and embedded in epoxy resin. Ninety nanometre-thick sections were prepared using an ultramicrotome and examined without staining, using a JEM-1230 TEM (JEOL, Tokyo, Japan) at 110 kV.
For each beam, 5 images taken at 10,000× magnification were analysed with the Scion Image software (Scion Corp., Frederick, MD) for the percentage area within the resin-dentine interface (i.e. the adhesive layer and hybrid layer) occupied by the silver tracer. The mean of the percentage of interface containing silver deposits from the 5 images was used to represent the overall nanoleakage of a subgroup. Because the homogeneity of variance assumption of the data sets derived from the 4 subgroups was violated, the interval data (in percentages) were logarithmically transformed to satisfy both normality and equal variance assumptions. The transformed data were analysed with two-factor analysis of variance to examine the effects of adhesive application time and simulated ageing, and the interaction of those two factors on the nanoleakage results. Post-hoc pairwise comparisons were performed using the Tukey statistic. Statistical significance was set at α = 0.05.
The other five beams from each subgroup were completely demineralised in 0.1 M formic acid/sodium formate (pH 2.5). The end point of demineralisation was determined by drop-wise addition of a 10% potassium oxalate solution to the demineralisation medium, which formed a white calcium oxalate precipitate when calcium ions were present. Completely demineralised beams containing the resin-dentine interface were fixed with Karnovsky’s fixative (2.5 wt% glutaraldehyde and 2% paraformaldehyde in 0.1 mol/L cacodylate buffer; pH, 7.3) for 8 h, and post-fixed in 1% osmium tetroxide for 1 h. The fixed specimens were dehydrated in an ascending ethanol series (30–100%), immersed in propylene oxide as a transition medium and ultimately embedded in pure epoxy resin. Ninety-nanometre thick sections were prepared, stained with 2% aqueous uranyl acetate and Reynold’s lead citrate, and examined using the JEM-1230 TEM at 110 kV.
2.5
Attenuated total reflection-Fourier transform infrared spectroscopy
A Nicolet 6700 spectrophotometer (Thermo Scientific Inc., Waltham, MA, USA) with an attenuated total reflection setup was used to collect infrared spectra from dentine discs before self-etching (control), and after etching the mineralised dentine without waiting or for 10 s (N = 2 dentine discs). The universal adhesive was applied respectively to each disc without light-curing. The etched dentine surface was rinsed with absolute ethanol to completely remove the uncured adhesive prior to scanning. The rationale of adhesive dissolution was to prevent interference of the adhesive vibrational peaks with the peaks characteristic of mineralised dentine. Spectra were collected between 4000 and 500 cm −1 at 4 cm −1 resolution using 32 scans. The spectra were superimposed after correction of their baseline shifts, so that the intensities of the carbonated apatite-associated peaks could be compared.
2
Materials and methods
2.1
Dentine disc preparation
One hundred and ten freshly extracted intact human third molars were collected according to a protocol approved by the Human Assurance Committee of the Augusta University, with informed consent obtained from the donating subjects with respect to the use of human tissues. The extracted teeth were stored in 0.9% (w/v) NaCl containing 0.02% sodium azide at 4 °C for no longer than one month. The roots of the teeth were removed using a water-cooled low-speed saw (Isomet, Buehler Ltd, Evanston, IL, USA). For each tooth, a flat bonding surface was prepared by removing the occlusal third of each tooth crown to expose the mid-coronal dentine. The dentine surfaces were polished with 320-grit silicon carbide paper under water irrigation for 1 min.
2.2
Bonding procedures
The teeth were randomly assigned to two groups (50 teeth per group). The composition of G-Premio Bond (GP) is listed in Table 1 . Self-etch bonding procedures were performed according to the Japanese version of the manufacturer’s instructions (no-waiting self-etch mode) or the international version of the manufacturer’s instructions (10-s self-etch mode). For the no-waiting self-etch mode, the adhesive was applied on the dentine surface and immediately air-dried without waiting (see below). For 10-s self-etch mode, the adhesive was applied on the dentine surface and left undisturbed for 10 s. For both modes, the adhesive was dried for 5 s using oil-free air from the triple syringe of a clinical operatory unit under maximum air pressure. After light-curing of the adhesive, resin composite build-ups were constructed in four 1-mm thick increments using G-ænial Sculpt (GC Corporation). Each increment was light-cured for 20 s using a light-emitting diode (440–480 nm range) curing unit with an output intensity of 1200 mW/cm 2 . To facilitate sectioning of transmission electron microscopy (TEM) specimens, the bonded specimens were coupled with a 2 mm thick layer of Protect Liner F flowable resin composite (Kuraray Noritake Dental Inc., Tokyo, Japan) and light-cured for 20 s.
| Adhesive | pH | Composition | Manufacturer’s instructions |
|---|---|---|---|
| G-Premio Bond | 1.5 | 10-MDP, 4-META, 10-methacryoyloxydecyl dihydrogen thiophosphate, methacrylate acid ester, distilled water, acetone, phot initiators, silica | No-waiting self-etching mode
10-s self-etching mode
|
Because the manufacturer emphasised the mandatory use of maximum air pressure for drying the adhesive in the bonding instructions, a pilot study was also performed to understand the consequence of not adhering to this instruction. In the pilot study, additional dentine discs were bonded using the two aforementioned self-etch protocols (N = 2), but with the adhesive dried with 5 s of gentle air flow. After coupling with the flowable resin composite, the bonded specimens were aged for 24 h and prepared for nanoleakage examination of the resin-dentine interfaces (methods described in the TEM section).
After bonding, the specimens from each experimental group (i.e. those not related to the pilot study) were divided into two batches. One batch was stored in deionised water at 37 °C for 24 h; the other batch was subjected to thermomechanical challenge, using 10,000 thermal cycles (10 °C for one min, 25 °C for one min and 55 °C for one min) and 240,000 mechanical cycles, corresponding to one year of intraoral functioning . The resin-dentine specimens were sectioned in both x and y directions across the adhesive interface to obtain beams with cross-sectional areas of approximately 0.9 mm × 0.9 mm using the ‘non-trimming’ version of the microtensile bond testing procedures . Simulated ageing was performed in a thermomechanical wear system (Model ER-37000; Erios, São Paulo, SP, Brazil). The four longest beams were obtained from the two central slabs of each bonded tooth for bond strength testing and one beam each was obtained from the two central slab of each bonded tooth for TEM. Hence, for each of the four subgroups, 80 beams from 20 teeth were used for bond strength testing and 10 beams from 5 teeth were used for TEM examination of the resin-dentine interface.
2.3
Microtensile bond strength
Each beam from a subgroup (N = 20) was secured with cyanoacrylate glue (Zapit; Dental Ventures of America, Corona, CA, USA) to a testing jig and stressed to failure under tension in a universal testing machine (Vitrodyne V1000; Liveco Inc., Burlington, VT, USA) at a cross-head speed of 1 mm/min. The tensile load at failure was recorded and divided by the measured cross-sectioned area of each beam to yield the tensile bond strength in megaPascals (MPa). The mean bond strength of the 4 beams derived from one tooth was used to represent the tensile bond strength of that tooth, yielding 20 values per subgroup. Data were analysed with parametric statistical methods after validating the normality (Shapiro-Wilk test) and homogeneity of variance (modified Levene test) assumptions of the data sets. A two-factor analysis of variance was used to examine the effects of adhesive application time (i.e. no-waiting or 10-s self-etch) and simulated ageing (i.e. without or with thermomechanical cycling), and the interaction of those two factors on the bond strength results. Post-hoc pairwise comparisons were performed using the Tukey statistic. Statistical significance was set at α = 0.05.
After bond strength testing, the two ends of a fractured stick were retrieved and examined with 10× magnification using a stereoscopical microscope to determine the mode of failure. Failure modes were classified as adhesive failure (failure along the adhesive interface), mixed failure (failure within the adhesive joint with failure within the resin composite or dentine), or cohesive failure (failure within the resin composite or dentine). For statistical analysis, the number of specimens exhibiting mixed and cohesive failures were combined into the non-adhesive category. Adhesive and non-adhesive failure modes in the 4 subgroups (zero-second or 10-s etch; with or without thermomechanical cycling) were arranged into a 2 × 4 contingency table and analysed with the Fisher-Freeman-Halton statistic . A 95% confidence level was used to determine if an association existed between the method in which the specimens were bonded and tested, and the category of failure (adhesive vs non-adhesive).
2.4
Transmission electron microscopy
Five of the 10 beams designated for TEM were used for nanoleakage examination. Each beam was coated with two layers of nail varnish applied to within 1 mm of the bonded interface. After drying, the varnish-coated beams were immersed in 50% ammoniacal silver nitrate solution for 48 hours. The silver-impregnated specimens were thoroughly rinsed with deionised water and placed in photo-developing solution for 8 h, under a fluorescent light, to facilitate reduction of the diaminesilver ions into metallic silver grains . The silver-impregnated specimens were polished, cleaned ultrasonically, dehydrated in an ascending ethanol series (50–100%), immersed in propylene oxide as transition medium, and embedded in epoxy resin. Ninety nanometre-thick sections were prepared using an ultramicrotome and examined without staining, using a JEM-1230 TEM (JEOL, Tokyo, Japan) at 110 kV.
For each beam, 5 images taken at 10,000× magnification were analysed with the Scion Image software (Scion Corp., Frederick, MD) for the percentage area within the resin-dentine interface (i.e. the adhesive layer and hybrid layer) occupied by the silver tracer. The mean of the percentage of interface containing silver deposits from the 5 images was used to represent the overall nanoleakage of a subgroup. Because the homogeneity of variance assumption of the data sets derived from the 4 subgroups was violated, the interval data (in percentages) were logarithmically transformed to satisfy both normality and equal variance assumptions. The transformed data were analysed with two-factor analysis of variance to examine the effects of adhesive application time and simulated ageing, and the interaction of those two factors on the nanoleakage results. Post-hoc pairwise comparisons were performed using the Tukey statistic. Statistical significance was set at α = 0.05.
The other five beams from each subgroup were completely demineralised in 0.1 M formic acid/sodium formate (pH 2.5). The end point of demineralisation was determined by drop-wise addition of a 10% potassium oxalate solution to the demineralisation medium, which formed a white calcium oxalate precipitate when calcium ions were present. Completely demineralised beams containing the resin-dentine interface were fixed with Karnovsky’s fixative (2.5 wt% glutaraldehyde and 2% paraformaldehyde in 0.1 mol/L cacodylate buffer; pH, 7.3) for 8 h, and post-fixed in 1% osmium tetroxide for 1 h. The fixed specimens were dehydrated in an ascending ethanol series (30–100%), immersed in propylene oxide as a transition medium and ultimately embedded in pure epoxy resin. Ninety-nanometre thick sections were prepared, stained with 2% aqueous uranyl acetate and Reynold’s lead citrate, and examined using the JEM-1230 TEM at 110 kV.
2.5
Attenuated total reflection-Fourier transform infrared spectroscopy
A Nicolet 6700 spectrophotometer (Thermo Scientific Inc., Waltham, MA, USA) with an attenuated total reflection setup was used to collect infrared spectra from dentine discs before self-etching (control), and after etching the mineralised dentine without waiting or for 10 s (N = 2 dentine discs). The universal adhesive was applied respectively to each disc without light-curing. The etched dentine surface was rinsed with absolute ethanol to completely remove the uncured adhesive prior to scanning. The rationale of adhesive dissolution was to prevent interference of the adhesive vibrational peaks with the peaks characteristic of mineralised dentine. Spectra were collected between 4000 and 500 cm −1 at 4 cm −1 resolution using 32 scans. The spectra were superimposed after correction of their baseline shifts, so that the intensities of the carbonated apatite-associated peaks could be compared.
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