Abstract
Objectives
To examine the effects of the combined use of chlorhexidine and ethanol on the durability of resin-dentin bonds.
Methods
Forty-eight flat dentin surfaces were etched (32% phosphoric acid), rinsed (15 s) and kept wet until bonding procedures. Dentin surfaces were blot-dried with absorbent paper and re-wetted with water (water, control), 1% chlorhexidine diacetate in water (CHD/water), 100% ethanol (ethanol), or 1% chlorhexidine diacetate in ethanol (CHD/ethanol) solutions for 30 s. They were then bonded with All Bond 3 (AB3, Bisco) or Excite (EX, Ivoclar-Vivadent) using a smooth, continuous rubbing application (10 s), followed by 15 s gentle air stream to evaporate solvents. The adhesives were light-cured (20 s) and resin composite build-ups constructed for the microtensile method. Bonded beams were obtained and tested after 24-h, 6-months and 15-months of water storage at 37 °C. Storage water was changed every month. Effects of treatment and testing periods were analyzed (ANOVA, Holm–Sidak, p < 0.05) for each adhesive.
Results
There were no interactions between factors for both etch-and-rinse adhesives. AB3 was significantly affected only by storage ( p = 0.003). Excite was significantly affected only by treatments ( p = 0.048). AB3 treated either with ethanol or CHD/ethanol resulted in reduced bond strengths after 15 months. The use of CHD/ethanol resulted in higher bond strengths values for Excite.
Conclusions
Combined use of ethanol/1% chlorhexidine diacetate did not stabilize bond strengths after 15 months.
1
Introduction
Resin-dentin bonds are currently accepted as being more complex than previously thought . A lack of bond stability has been observed in several in vitro and in vivo studies for periods as short as 3 months . The hydrophilicity of contemporary etch-and-rinse adhesives and subsequent hydrolysis in combination with host-derived enzymatic degradation of collagen fibrils have been regarded as the two major causes of degradation of resin-dentin bonds over time.
Simplified etch-and-rinse adhesives incorporate hydrophilic monomers and solvents to properly bond to dentin, a naturally wet substrate. However, the use of increasing concentrations of hydrophilic resins raises concern that such adhesives have become too hydrophilic . The incorporation of hydrophilic monomers results in increased water sorption that expedites hydrolysis and decreases mechanical properties . Bonding to wet dentin has also been shown to be challenging even with the use of hydrophilic adhesives. The surface moisture required for collagen expansion may also cause phase separation of some etch-and-rinse adhesive systems, thus resulting in poor resin infiltration to the deepest regions of the demineralized dentin . Conversely, air-drying dentin to eliminate water also results in poorly infiltrated hybrid layer . The exposed, uninfiltrated collagen fibrils are then susceptible to the enzymatic action of host metalloproteinases (MMPs) that ultimately results in deterioration of the bond over time .
Adhesive formulations for simplified etch-and-rinse systems incorporate either ethanol or acetone to solvate hydrophobic monomers. These solvents also function as water-chasers to displace entrapped water simultaneously to adhesive infiltration . Anhydrous solvents play an important role in collagen matrix shrinkage, expansion, stiffness and overall infiltration . The ethanol wet-bonding concept has been presented as an alternative technique to overcome problems associated with the collapse of the collagen matrix if water is removed from the surface . As ethanol has been shown to be able to expand and maintain collagen fibrils apart, it can be used to replace water, leaving demineralized dentin saturated with ethanol. This concept has been proved successful when used with experimental adhesive resins or commercial etch-and-rinse adhesives . Ideally, protection and preservation of collagen should be achieved by complete infiltration of hydrophobic resins. This can be accomplished with the use of the ethanol wet-bonding concept .
Additionally, the incorporation of MMP inhibitors into the bonding procedure is desirable. In vivo and in vitro studies have shown that the application of aqueous solutions of 2% chlorhexidine digluconate plays an important role in preservation of resin-dentin bonds by inhibiting the collagenolytic activity of host-derived enzymes . Several studies have proposed chlorhexidine diacetate (CHD) as a potential bio-active antibacterial agent to be incorporated to resin composites, glass ionomers, adhesives and provisional cements . Chlorhexidine diacetate was selected in this study because it is available as a powder and is soluble in ethanol. It has been demonstrated that chlorhexidine digluconate concentrations in the range of 0.002–0.2% applied for shorter periods of time (15–30 s) are also capable to postpone the resin–dentin degradation of adhesive interfaces . Thus, the combined use of chlorhexidine and ethanol could represent a promising bonding technique. Although the effects of ethanol bonding and chlorhexidine on the durability of bond strengths to dentin have been extensively investigated in separate, the combination of both approaches in a clinically feasible protocol still requires further investigation.
This study investigated the hypothesis that the combined use of ethanol wet-bonding with 1% chlorhexidine diacetate in a short, and clinically feasible application time, would result in more stable resin-dentin bonds over time. The null hypothesis tested was that there is no effect of 1% chlorhexidine diacetate, ethanol or the combination of both on the stability of resin-dentin bond strength.
2
Materials and methods
2.1
Tooth preparation
Forty-eight extracted human caries-free third molars stored in saline containing 0.1% thymol at 4 °C for no longer than 6 months were used in this study. The study was approved by the Institutional Review Board of the university (# 164/07). A flat surface was prepared with a slow-speed Isomet saw (Isomet 1000 Precision Saw, Buehler Ltd., Lake Bluff, IL, USA) by transversally sectioning the crowns under water cooling to expose mid-coronal dentin. The dentin surface was polished (Ecomet 3000, Buehler Ltd., Lakebluff, IL, USA) with 320 and 600-grit SiC paper at 250 rpm to create a standard smear layer. The crown segments were randomly allocated to 8 groups of 6 teeth each. There were 4 solutions for dentin treatment (1% chlorhexidine diacetate in water, w/w) [CHD/W], 1% chlorhexidine diacetate in ethanol, w/w [CHD/E], distilled water [W, control] and 100% ethanol [E]); and 2 adhesive systems ( Table 1 ), All Bond 3 (Bisco Inc.) and Excite (Ivoclar Vivadent), comprising 8 test groups. The adhesives were selected as representatives of commercial, water-free, ethanol-based, simplified etch-and-rinse systems. This was relevant to prevent confounding effect when using the ethanol-wet bonding approach.
| Material | Composition | Manufacturer and bath numbers |
|---|---|---|
| Uni-etch 32% BAC | 32% phosphoric acid with benzalkonium chloride (BAC) | Bisco Inc. 0700006350 |
| All Bond 3 | Part A: ethanol, MgNTG-GMA (magnesium nitro-tri-glycyl glycidyl methacrylate) | Bisco Inc. 0700005251 |
| Part B: Bis-GMA (bisphenol A–diglycidyl, ester dimethacrylate); BPDM (bisphenyl, dimethacrylate); HEMA (2-hydroxyethyl methacrylate); photoiniciator, stabilizer | Bisco Inc. 0700005255 |
|
| Excite | Ethanol, HEMA (2-hydroxyethyl methacrylate); phosphonic acid acrylate, dimethacrylate, silica, fillers, photoiniciator, stabilizer | Ivoclar Vivadent J25791 |
| Aelite All Purpose Body | Bis-GMAE (bisphenol A ethoxylate–diglycidyl, esther dimethacrylate); TEGDMA: tri-ethilenoglycol, dimethacrylate | Bisco Inc. 0700005779 0700005705 0700005135 0800001576 |
2.2
Preparation of chlorhexidine diacetate solutions and bonding procedures
Chlorhexidine diacetate hydrate (Acros Organics, Fisher Scientific, Catalog number AC: 21498-0050) was used to prepare the experimental solutions. Solutions were prepared by gradually adding 1% by weight (Mettler Toledo, XP504 Delta Range) of chlorhexidine diacetate monohydrate to stirred 100% ethanol or water in a glass beaker. One single batch of the solutions was prepared, kept in the refrigerator and used for all bonding procedures where appropriate. No chlorhexidine diacetate precipitation was observed after the solutions were prepared or during the course of the experiment. The pH of the solutions was determined (Mettler Toledo, SevenMulti, pH mV/ORP, Schwerzenbach, Switzerland) as being 7.5 and 9.1 for water and ethanol solutions, respectively. They were not adjusted to a neutral pH.
As a standard procedure for all groups, tooth surfaces were acid-etched with 32% H 3 PO 4 gel for 15 s (Uni-etch BAC 32%, Bisco Inc., Schaumburg, IL, USA), rinsed with water for 15 s and kept wet until bonded. The surface was blot-dried with tissue paper (Kimwipes, Kimtech Science) before further treatment according to groups. Dentin surfaces remained slightly moist, but no excess water was present. One of the 4 solutions was applied and kept in the surface for 30 s. The solutions were re-applied in the event of evaporation before 30 s, never allowing the ethanol-saturated dentin to evaporate to dryness. At the end of the 30 s, excess solution was blot dried with tissue paper. The respective adhesive was immediately applied with smooth rubbing action for approximately 10 s, gently air-dried for 15 s. For All Bond 3, it was necessary perform a mixture of primers A and B before being applied. The third-step (adhesive resin layer) was omitted as permitted by the instructions. The rationale was to evaluate both ethanol-based adhesive systems under the same condition (as simplified etch-and-rinse adhesives). An additional, relatively hydrophobic layer could compromise the interpretation of the results because it is related to more stable bond strengths to dentin over time . All groups were light-cured at 500 mW/cm 2 (OptiLux 501, SDS KERR, Middleton, WI, USA) for 20 s. Immediately after bonding, the entire dentin surface received four layers of Aelite All Purpose Body resin composite (Bisco Inc., Schaumburg, IL, USA) ( Table 1 ), to build an approximate 4 mm crown. Each 1 mm increment was light-cured for 40 s (OptiLux 501, SDS KERR, Middleton, WI, USA).
2.3
Preparation of specimens for microtensile test and storage
The bonded teeth were stored in water at 37 °C for 24 h, and then sectioned perpendicular to the adhesive-dentin interface using an Isomet diamond saw (Isomet 1000 Precision Saw, Buehler Ltd., Lake Bluff, IL, USA) to obtain rectangular beams of approximately 0.8 mm 2 cross-sectional area. One-third of the beams obtained from each tooth were randomly selected and tested immediately after sectioning, while the remaining two-thirds were kept in a clear vial containing neutral (pH 7) distilled water at 37 °C. Storage water was renewed monthly to expedite storage effects . Preservatives and/or antimicrobial agents were not used in the storage water in this study.
2.4
Microtensile testing
Beams were tested immediately after sectioning as described above and then after 6 and 15 months of storage. Bonded beams were mounted on a microtensile testing jig with cyanoacrylate glue and pulled to failure at 1.0 mm/min in a Microtensile Tester machine (Bisco Inc., Schaumburg, IL, USA). The load (Kgf) at failure was divided by the cross-sectional area of the interface (mm 2 ) measured with a digital caliper to the nearest 0.01 mm (Fisher Scientific, Chicago, IL, USA) to calculate the microtensile bond strength that was expressed in MPa. The bond failure modes were evaluated under 40× using light microscope (Olympus, Tokyo, Japan).
Fractures were classified as cohesive, adhesive or mixed. When it occurred exclusively in either dentin or composite, as cohesive in dentin (CD) or cohesive in composite (CC). As adhesive (A) when occurred at dentin/resin bonded interface, and mixed (M) when two modes of failures, adhesive and cohesive, occurred simultaneously.
2.5
Data treatment
Bond strength values were averaged per tooth for each experimental condition and adhesive. The means calculated from 6 teeth were then considered for further analysis ( n = 6). There were no pre-test failures of beams.
A two-way ANOVA design with the general linear model was used to examine the effects of four treatments, and three storage times, on microtensile bond strength per adhesive (SigmaPlot 11, Systat Software Inc.). Least-square means (LSM) analysis was used and the variances were given in standard error of the mean (SEM) . Post hoc multiple comparison tests were performed with Holm–Sidak method. Significance level was pre-set to α = 5%.
2
Materials and methods
2.1
Tooth preparation
Forty-eight extracted human caries-free third molars stored in saline containing 0.1% thymol at 4 °C for no longer than 6 months were used in this study. The study was approved by the Institutional Review Board of the university (# 164/07). A flat surface was prepared with a slow-speed Isomet saw (Isomet 1000 Precision Saw, Buehler Ltd., Lake Bluff, IL, USA) by transversally sectioning the crowns under water cooling to expose mid-coronal dentin. The dentin surface was polished (Ecomet 3000, Buehler Ltd., Lakebluff, IL, USA) with 320 and 600-grit SiC paper at 250 rpm to create a standard smear layer. The crown segments were randomly allocated to 8 groups of 6 teeth each. There were 4 solutions for dentin treatment (1% chlorhexidine diacetate in water, w/w) [CHD/W], 1% chlorhexidine diacetate in ethanol, w/w [CHD/E], distilled water [W, control] and 100% ethanol [E]); and 2 adhesive systems ( Table 1 ), All Bond 3 (Bisco Inc.) and Excite (Ivoclar Vivadent), comprising 8 test groups. The adhesives were selected as representatives of commercial, water-free, ethanol-based, simplified etch-and-rinse systems. This was relevant to prevent confounding effect when using the ethanol-wet bonding approach.
| Material | Composition | Manufacturer and bath numbers |
|---|---|---|
| Uni-etch 32% BAC | 32% phosphoric acid with benzalkonium chloride (BAC) | Bisco Inc. 0700006350 |
| All Bond 3 | Part A: ethanol, MgNTG-GMA (magnesium nitro-tri-glycyl glycidyl methacrylate) | Bisco Inc. 0700005251 |
| Part B: Bis-GMA (bisphenol A–diglycidyl, ester dimethacrylate); BPDM (bisphenyl, dimethacrylate); HEMA (2-hydroxyethyl methacrylate); photoiniciator, stabilizer | Bisco Inc. 0700005255 |
|
| Excite | Ethanol, HEMA (2-hydroxyethyl methacrylate); phosphonic acid acrylate, dimethacrylate, silica, fillers, photoiniciator, stabilizer | Ivoclar Vivadent J25791 |
| Aelite All Purpose Body | Bis-GMAE (bisphenol A ethoxylate–diglycidyl, esther dimethacrylate); TEGDMA: tri-ethilenoglycol, dimethacrylate | Bisco Inc. 0700005779 0700005705 0700005135 0800001576 |
2.2
Preparation of chlorhexidine diacetate solutions and bonding procedures
Chlorhexidine diacetate hydrate (Acros Organics, Fisher Scientific, Catalog number AC: 21498-0050) was used to prepare the experimental solutions. Solutions were prepared by gradually adding 1% by weight (Mettler Toledo, XP504 Delta Range) of chlorhexidine diacetate monohydrate to stirred 100% ethanol or water in a glass beaker. One single batch of the solutions was prepared, kept in the refrigerator and used for all bonding procedures where appropriate. No chlorhexidine diacetate precipitation was observed after the solutions were prepared or during the course of the experiment. The pH of the solutions was determined (Mettler Toledo, SevenMulti, pH mV/ORP, Schwerzenbach, Switzerland) as being 7.5 and 9.1 for water and ethanol solutions, respectively. They were not adjusted to a neutral pH.
As a standard procedure for all groups, tooth surfaces were acid-etched with 32% H 3 PO 4 gel for 15 s (Uni-etch BAC 32%, Bisco Inc., Schaumburg, IL, USA), rinsed with water for 15 s and kept wet until bonded. The surface was blot-dried with tissue paper (Kimwipes, Kimtech Science) before further treatment according to groups. Dentin surfaces remained slightly moist, but no excess water was present. One of the 4 solutions was applied and kept in the surface for 30 s. The solutions were re-applied in the event of evaporation before 30 s, never allowing the ethanol-saturated dentin to evaporate to dryness. At the end of the 30 s, excess solution was blot dried with tissue paper. The respective adhesive was immediately applied with smooth rubbing action for approximately 10 s, gently air-dried for 15 s. For All Bond 3, it was necessary perform a mixture of primers A and B before being applied. The third-step (adhesive resin layer) was omitted as permitted by the instructions. The rationale was to evaluate both ethanol-based adhesive systems under the same condition (as simplified etch-and-rinse adhesives). An additional, relatively hydrophobic layer could compromise the interpretation of the results because it is related to more stable bond strengths to dentin over time . All groups were light-cured at 500 mW/cm 2 (OptiLux 501, SDS KERR, Middleton, WI, USA) for 20 s. Immediately after bonding, the entire dentin surface received four layers of Aelite All Purpose Body resin composite (Bisco Inc., Schaumburg, IL, USA) ( Table 1 ), to build an approximate 4 mm crown. Each 1 mm increment was light-cured for 40 s (OptiLux 501, SDS KERR, Middleton, WI, USA).
2.3
Preparation of specimens for microtensile test and storage
The bonded teeth were stored in water at 37 °C for 24 h, and then sectioned perpendicular to the adhesive-dentin interface using an Isomet diamond saw (Isomet 1000 Precision Saw, Buehler Ltd., Lake Bluff, IL, USA) to obtain rectangular beams of approximately 0.8 mm 2 cross-sectional area. One-third of the beams obtained from each tooth were randomly selected and tested immediately after sectioning, while the remaining two-thirds were kept in a clear vial containing neutral (pH 7) distilled water at 37 °C. Storage water was renewed monthly to expedite storage effects . Preservatives and/or antimicrobial agents were not used in the storage water in this study.
2.4
Microtensile testing
Beams were tested immediately after sectioning as described above and then after 6 and 15 months of storage. Bonded beams were mounted on a microtensile testing jig with cyanoacrylate glue and pulled to failure at 1.0 mm/min in a Microtensile Tester machine (Bisco Inc., Schaumburg, IL, USA). The load (Kgf) at failure was divided by the cross-sectional area of the interface (mm 2 ) measured with a digital caliper to the nearest 0.01 mm (Fisher Scientific, Chicago, IL, USA) to calculate the microtensile bond strength that was expressed in MPa. The bond failure modes were evaluated under 40× using light microscope (Olympus, Tokyo, Japan).
Fractures were classified as cohesive, adhesive or mixed. When it occurred exclusively in either dentin or composite, as cohesive in dentin (CD) or cohesive in composite (CC). As adhesive (A) when occurred at dentin/resin bonded interface, and mixed (M) when two modes of failures, adhesive and cohesive, occurred simultaneously.
2.5
Data treatment
Bond strength values were averaged per tooth for each experimental condition and adhesive. The means calculated from 6 teeth were then considered for further analysis ( n = 6). There were no pre-test failures of beams.
A two-way ANOVA design with the general linear model was used to examine the effects of four treatments, and three storage times, on microtensile bond strength per adhesive (SigmaPlot 11, Systat Software Inc.). Least-square means (LSM) analysis was used and the variances were given in standard error of the mean (SEM) . Post hoc multiple comparison tests were performed with Holm–Sidak method. Significance level was pre-set to α = 5%.
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