Impact of Adhesive Application to Wet and Dry Dentin on Long-term Resin-dentin Bond Strengths
This study compared the effects of moisture and rubbing action on the immediate and one-year microtensile bond strength (BS) of an ethanol/water-based adhesive system (Single Bond [SB]) and an acetone-based system (One Step [OS]) to dentin. A flat superficial dentin surface on 60 human molars was exposed by wet abrasion. Two coats of the adhesives were applied on either a dry (D) or rewetted surface (W) with no (NRA), slight (SRA) or vigorous rubbing action (VRA). After light curing (600mW/cm2/10 seconds), composite buildups were constructed incrementally and the specimens were stored in water (37°C/24 hours). They were longitudinally sectioned in the “x” and “y” directions to obtain bonded sticks (0.8 mm2) to be tested in tension at 0.5 mm/minute. The sticks from each tooth were then divided, stored in water at 37°C and tested immediately and after 12 months (12M) at 0.5 mm/minute. The bond strength values of sticks from the same hemi-tooth were averaged for statistical purposes. The prematurely debonded specimens were included in the hemi-tooth mean. The data from each adhesive was analyzed by three-way ANOVA and Tukey's multiple comparison tests (α=0.05). In the dry groups, high bond strength values were obtained under VRA. When the dentin was kept moist, both SRA and VRA provided high resin–dentin bond strength values. Reductions in bond strength values after one year of water storage were not observed for the SB adhesive or were less pronounced for the OS adhesive when it was vigorously rubbed onto the dentin surface.SUMMARY
INTRODUCTION
As reported by Spencer and Wang,1 the two primary critical factors for achieving an adequate resin-dentin bonding are wetting of the dentin by components of the adhesive and micromechanical interlocking via resin penetration and the entanglement of exposed collagen fibrils in demineralized dentin.2–3
Exposure of the collagen fibrils depends on previous demineralization of the dentin substrate via acid-etching with mineral acids. Following rinsing, the mineral phase of the superficial 1–10 μm3 of dentin is completely removed, leaving the collagen fibrils literally suspended in water. If the demineralized dentin matrix is air-dried, the collagen fibrils are brought closer together, resulting in a demineralized zone with reduced permeability to resin monomers.4–5 Recent studies have shown that bonding to such air-dried demineralized dentin results in an improper adhesive infiltration—as much as one half of the zone of demineralized dentin.6–7 This less than ideal adhesive infiltration has been shown to produce lower immediate bond strengths with current etch&rinse adhesive systems.8–9
A common and widespread way to reverse such an undesirable condition is by maintaining a state of hydration for the demineralized dentin before adhesive application. This technique has been referred to as wet bonding and has been used for more than 10 years. Moist demineralized dentin provides a more porous collagen network and thus greater infiltration of adhesive monomers.410 However, several advantages can be listed for the wet bonding technique. A recent investigation has confirmed in a quantitative manner that etch&rinse adhesive systems achieve optimal bond strengths at different moisture degrees, which is dependent on the solvent present in each system.11 Unfortunately, in the manufacturer's directions of commercial adhesive systems, discrimination of the ideal moisture degree is not specified.
Also, as the adhesive is applied, the water within the collagen fibrils must evaporate in order to provide space for the formation of a highly cross-linked polymer entangled with the collagen fibrils. HEMA, which is a primary component in many simplified etch&rinse commercial adhesives, can dramatically reduce the evaporation of such water.12 The addition of HEMA reduces the mole fraction of water, affecting its partial pressure. As the molar fraction of water drops, it becomes more difficult to remove residual water from the demineralized dentin. This has two primary consequences. The first is that other hydrophobic monomers, such as Bis-GMA, would resist diffusing deeper into dentin, with phase separation likely to occur.1 This inhibits the formation of an integrated collagen/polymer network suppressing adhesive infiltration throughout the demineralized dentin matrix. Second, the entrapment of water within demineralized dentin precludes the formation of a highly cross-linked polymer,13–14 making the adhesive interface more prone to the degrading effects of water over time.15–20
As a result, any attempt to allow for an increased rate of water and solvent evaporation, along with deeper monomer infiltration as increased application times,21 multiple adhesive coatings,22 delayed polymerization23 and adhesives' rubbing1325 can improve the strength of the polymer formed within the collagen fibrils and allow for the achievement of high bond strength values. A recent investigation has demonstrated that high immediate resin-dentin bond strength values can be obtained for ethanol/water and acetone-based systems when they are vigorously agitated on a demineralized dentin surface. Interestingly, even when demineralized dentin was kept dry, high bond strength values were achieved under vigorous rubbing.25 This finding should be further investigated, since the maintenance of demineralized dentin in a dry state is easily accomplished by clinicians and minimizes the negative effects of water on hybrid layer formation.13–14
While it is clear that the immediate bond strength of simplified etch&rinse adhesives can be improved by rubbing the adhesive onto the dry or wet demineralized dentin surface,25 there is no information that documents this having any effect on long-term resin-dentin bond strength. Therefore, this study compared the effects of the degree of moisture and rubbing action on the immediate and one-year microtensile bond strength of two simplified etch&rinse adhesives with different solvent compositions.
METHODS AND MATERIALS
Sixty extracted, caries-free human third molars were used. The teeth were collected after obtaining the patient's informed consent. The University of Oeste de Santa Catarina Institutional Review Board approved this study. The molars were disinfected in 0.5% chloramine, stored in distilled water and used within six months of extraction.
A flat and superficial dentin surface was exposed on each tooth after wet grinding the οcclusal enamel on #180-grit SiC paper. The enamel-free, exposed dentin surfaces were further polished on wet #600-grit silicon-carbide paper for 60 seconds to standardize the smear layer. An adhesive tape with a hole in its center (radius [r] = 4.1mm; area [πr2] = 52 mm2) was bonded onto the dentin surface before adhesive application.
Two different solvent-based etch&rinse adhesive systems were tested: Single Bond (SB, 3M ESPE, St Paul, MN, USA), an ethanol/water-based system and OneStep (OS, BISCO, Schaumburg, IL, USA). Both are presented in Table 1. The acid etching was performed with the respective acids of the different adhesives (Table 1). Contrary to the manufacturer's instructions, the surfaces were rinsed with distilled water for 15 seconds and air-dried for 30 seconds using oil-free compressed air to collapse the collagen fibers. The adhesives were applied onto the surface, which was either kept dry or was rewetted for 10 seconds, using different amounts of distilled water (1.5 or 3.5 μ1, for SB and OS, respectively) measured with a micropipette (Pipetman, Gilson, NY, USA).11 The differences in the amount of water used for rewetting the dentin are due to differences in the vapor pressure and Hansen's solubility parameters from solvents of each adhesive system.11 The adhesives were applied onto the dentin as follows:
No rubbing action (NRA): In this group, the adhesive was only spread over the entire surface for approximately three seconds and left undisturbed for seven seconds. Then, an air stream was applied for 10 seconds at a distance of 20 cm.
Slight rubbing action (SRA): The adhesive was slightly spread onto the surface for approximately 10 seconds; no intentional manual pressure was exerted on the microbrush. An air stream was applied for 10 seconds at a distance of 20 cm. Before performing the adhesive application and in order to determine the equivalent manual pressure that would be placed on the surface of the demineralized dentin, the operator was trained on the surface of an analytical balance (Mettler, type H6; Columbus, OH, USA). In this group, the pressure was equivalent to approximately 4.0±1.0g. After the operator determined the calibrated manual balance pressure, they repeated the balance procedure seven times and a mean ± standard deviation was calculated. This procedure was repeated at the beginning of every laboratory setting in order to ensure the operator's calibration.
Vigorous rubbing action (VRA): The adhesive was rigorously agitated on the entire dentin surface for approximately 10 seconds. The microbrush was scrubbed on the dentin surface under manual pressure (equivalent to approximately 34.5±6.9g). An air stream was applied for 10 seconds at a distance of 20 cm.
In all three groups, a second coat of adhesive layer was applied in the same manner as the first layer. The time lapse between starting the adhesive application and light curing (VIP, BISCO, Schaumburg, IL, USA; 600mW/cm2) was approximately 40 seconds. The light curing was performed for the respective recommended time (10 seconds). Resin composite build-ups (Z250, 3M ESPE) were placed on the bonded surfaces (1 mm increments), which were individually light activated for 30 seconds each. All bonding procedures were carried out by a single operator at 24°C at 50% relative humidity. Five teeth were used for each combination of adhesive system and surface moisture.
After storage of the bonded teeth in distilled water at 37°C for 24 hours, the teeth were longitudinally sectioned in both the “x” and “y” directions across the bonded interface using a diamond saw in a Labcut 1010 machine (Extec Corp, Enfield, CT, USA) under water cooling at 300 rpm to obtain bonded sticks with a cross-sectional area of approximately 0.8 mm2. The number of premature debonded sticks (D) per tooth during specimen preparation was recorded. The cross-sectional area of each stick was measured with the digital caliper to the nearest 0.01 mm and recorded for subsequent calculation of the BS (Absolute Digimatic, Mitutoyo, Tokyo, Japan). The bonded sticks that originated from the same teeth were randomly divided and assigned to immediate testing or after one year of storage in distilled water containing 0.4% sodium azide26 at 37°C. The storage solution was not changed and its pH was monitored monthly.
At each storage time period, each bonded stick was attached to a modified device27 for microtensile testing with cyanoacrylate resin (Zapit, Dental Ventures of North America, Corona, CA, USA) and subjected to tensile force in a universal machine (Emic, São José dos Pinhais, PR, Brazil) at 0.5 mm/minute.28 The failure modes were evaluated at 400X (HMV-2, Shimadzu, Tokyo, Japan) and classified as cohesive (failure exclusive within dentin or composite, C), adhesive (failure at resin/dentin interface–A) or adhesive/mixed (failure at resin/dentin interface that included cohesive failure of the neighboring substrates, A/M).
The experimental unit in this study was the hemi-tooth, since half of the sample was tested immediately and the other half after one year. The mean bond strength of all sticks from the same hemi-tooth was averaged for statistical purposes. The prematurely debonded specimens were included in the hemi-tooth mean. The average value attributed to specimens that failed prematurely during preparation is arbitrary and corresponds to approximately half of the minimum bond strength value that could be measured in this study (7.6MPa).18 The BS mean for every testing group was expressed as the average of the five hemi-teeth used per group and expressed in MPa.
The microtensile bond strength data was subjected to three-way repeated measures analysis of variance (Moisture degree/Rubbing action/Storage time) and a post hoc test (Tukey's test at α=0.05) was used for pairwise comparisons.
RESULTS
The mean cross-sectional area ranged from 0.76 to 0.91 mm2 and there was no difference among the groups was detected (p>0.05). The percentage of specimens with premature debonding and the frequency of each fracture pattern mode are shown in Table 2. Regardless of the condition of the moisture, SB and OS showed a lower pre-testing failure rate when the adhesive was slightly or vigorously rubbed (Table 2). SB had a lower overall failure rate than OS. No cohesive failure in composite occurred. A low percentage of dentin cohesive failure occurred for both adhesives after one year. The specimens with cohesive failure were excluded from the data analysis.
The overall microtensile bond strength (BS) values of SB and OS are shown in Table 3. For SB, the interactions moisture degree/rubbing action/storage time (p=0.562) and moisture degree/storage time (p=0.084) were not significant. The interactions moisture degree/rubbing action and rubbing action/storage time were statistically significant (p<0.00001 and p=0.0033, respectively). The means and respective standard deviations of BS for the significant interactions mentioned are shown in Tables 4 and 5. One can observe (Table 4) that, in dry and wet groups, high BS values were obtained when SB was vigorously rubbed on dentin. When the dentin was kept moist, both slight and vigorous rubbing action provided high resin–dentin BS, although the mean BS obtained under vigorous agitation was statistically higher than that obtained after slight agitation. Table 5 demonstrates that both slight and vigorous rubbing action provided high immediate BS for the SB. However, after one year, high BS values were observed only when SB was vigorously agitated. Although the interaction moisture degree/storage time was not significant, its results are shown in Table 6 for comparison purposes. It can be seen that the immediate BS was higher than that reported after one year.
For OS, the interactions moisture degree/rubbing action/storage time (p=0.879) was not significant. The interactions moisture degree/rubbing action (p=0.032), rubbing action/storage time (p=0.0008) and moisture degree/storage time (p=0.0177) were statistically significant. The BS means and the respective standard deviations for the significant interactions mentioned are shown in Tables 7, 8 and 9. High BS values for the dry groups were observed (Table 7) when the OS was vigorously rubbed on dentin. When the dentin was kept moist, both slight and vigorous rubbing action provided high resin–dentin bond strength values. The results presented in Table 8 demonstrate that, for OS, both slight and vigorous rubbing action allowed for the achievement of high immediate bond strength values. Significant bond strength reductions occurred in all conditions of rubbing action after one year of water storage; however, the bond strength mean of the VRA condition after one year was statistically higher than the mean value obtained under NRA and SRA. The results presented in Table 9 show significant reductions in bond strength values after one year for both wet and dry conditions for OS adhesive.
DISCUSSION
As previously reported in an in vitro study,25 both slight and vigorous rubbing of water/alcohol and acetone adhesives is essential to providing high immediate bond strengths to either dry or wet dentin. Good infiltration in conditioned, wet bonding specimens can be obtained if the adhesive resin replaces all the water within the demineralized matrix that was previously occupied by mineral, without the collagen matrix collapsing. Under a moist condition, the demineralized dentin preserves the nanospaces within collagen fibrils2 into which the adhesive monomers diffuse to envelope the collagen fibrils prior to polymerization. High molecular weight monomers from simplified etch&rinse adhesives have limited diffusion into wet demineralized dentin.29–31 The rubbing action can increase the moieties kinetics and allow for better monomer diffusion inward, while solvents are diffusing outward, which may explain the high bond strength values obtained under slight and vigorous agitation.
Surprising were the bond strength findings from dry demineralized dentin. For both adhesives, high immediate bond strength values were also obtained when the adhesives were vigorously rubbed onto the dry dentin. These results were not expected, since it is known that, when demineralized dentin is air-dried, the water within the collagen matrix is removed and the collagen fibrils are brought into close contact. They form weak interpeptide bonds that render the matrix shrunk, stiff532 and practically impermeable to resin adhesives.6–7 Studies have demonstrated that the infiltration ratio of the bonding resin within the hybrid layer for acetone-based systems is reduced approximately 50% when applied to dry instead of wet dentin.6–729 However, none of these studies reported that the adhesives were vigorously rubbed onto the dentin surface. These studies usually report that the adhesives were applied according to the manufacturers' instructions, which implies that they were, at best, rubbed slightly. This difference in application procedure may be the reason why previous studies were not able to achieve high bond strengths on dry demineralized dentin,8–10 as in the present investigation.
It is believed that the only way to revert to the reduction in permeability that occurs to air-dried demineralized dentin532 is to use the wet bonding technique. As water has a solubility parameter for hydrogen bonding (δh) that is higher than the interpeptide hydrogen bonds created by the close contact of collagen fibrils in the air-dried condition,33–34 the wet bonding technique can preserve the nanospaces within the collagen fibrils, which allow for resin infiltration.
However, the wet bonding technique is not easily accomplished by clinicians, which becomes a challenge in a clinical practice. Although it was demonstrated that the proper degree of moisture needed with such etch&rinse adhesives varies according to the solvent presented in the adhesives composition,1135 an examination of several in vitro and in vivo studies indicate that there is no consensus regarding the ideal degree of moisture. Laboratory investigators usually report that adhesives were applied “according to manufacturers' directions;” however, the moisture concept varies widely among the instructions of different adhesive systems and investigators. The adoption of a standard surface moisture may lead to less than optimal quality of bonding, depending on the type of adhesive used.1135 Although the proper ratio of water volume per surface area of dentin has already been established,11 from a clinical standpoint, it is practically impossible to precisely determine the ideal surface moisture in a clinical situation.
The aforementioned factors, along with the detrimental effects of water on the formation of a highly cross-linked polymer13–14 within the hybrid layer, make the wet bonding technique a difficult and unpredictable task. However, a recent in vitro investigation has observed that resin-dentin bonding performed under ideal moisture conditions is still prone to the degrading effects of long-term water storage.18
Contrary to several in vitro laboratory investigations,8–10 the current investigation demonstrated that it is possible to achieve high bond strength on air-dried demineralized dentin, depending on how the adhesives are applied. The mechanical pressure applied to the demineralized dentin surface during vigorous rubbing might compress the collapsed collagen network like a sponge. As the pressure is relieved, the compressed collagen expands and the adhesive solution may be drawn into the collapsed collagen mesh.24
With the exception of the NRA groups, for both adhesives, high immediate BS values were observed when the adhesives were slightly or vigorously rubbed on the dentin surface. After one-year water storage, no significant reductions in BS values were noted for NRA specimens, which maintained the low values reported for the immediate period. However, when comparing SRA and VRA, pronounced reductions in BS values, in the order of approximately 60%, were only observed when the adhesives were slightly rubbed.
The hydrophilic nature of ionic and acidic methacrylate copolymers facilitates water sorption36–38 from the oral environment when exposed externally to salivary fluids and internally from the underlying hydrated dentin. Water sorption swells the polymer and reduces the frictional forces between the polymer chains, causing a decrease of their mechanical properties.39–40 However, the water sorption of resin depends on resin polarity and chain topology.41 Resin polarity influences the number of hydrogen bonding sites and the attraction between the polymer and water molecules, while chain topology determines the spatial configuration of the molecular segments and the availability of nanopores within the polymer structure.41
Interestingly, the degradation effects of water storage were not observed for SB adhesive and were less pronounced for OS when vigorously rubbed on the dentin surface. The nature of the adhesive (hydrophilicity) was not altered by varying the application mode of the adhesive. However, the chain topology could have been altered. The intrinsic fraction of nanopores inherent to a glassy polymer is typically one-fifth to three-fifths of the total volume fraction, with extremes occurring in low crosslink density (flexible) and high crosslink density (rigid) resins, respectively.41 Considering moisture absorption, a larger intrinsic hole volume leads to an increased moisture uptake within a series of resins with similar polarity. It is likely that the vigorous agitation improved the removal of residual water (in case the dentin was kept moist) and solvents, which increased the degree of conversion and cross-linking of the polymer and, consequently, the mechanical properties of the resin inside the hybrid layer.13–1442–43 A lower volume of nanopores is likely to be presented within a highly cross-linked polymer formed under vigorous rubbing action, which may have played a role in its lower susceptibility to the degradation effects of water storage.
The plasticizing effects of water on polymers have been extensively documented.3944–45 As water sorption occurs, the intermolecular interactions between the polymer chains are broken. A more pronounced effect may occur for poorly polymerized hydrophilic adhesive systems.1337 Although several studies have already reported the degradation of resin-dentin bonds applied on wet substrates, the long-term stability of resin-dentin bonds performed on air-dried demineralized dentin is yet to be studied.
For more than 10 years, the wet bonding technique has been recommended for dentin bonding.810 The rationale is that, as long as dentin is kept fully hydrated, the dentin matrix does not collapse and free space is available for resin infiltration.4 Based on such rationale and on the immediate higher bond strengths obtained under wet-bonding conditions, adhesive solutions with more hydrophilic monomers, along with a high concentration of solvents were included in the compositions of most adhesive systems currently on the market. Unfortunately, the maintenance of moist demineralized dentin is difficult to achieve in a daily clinical practice, since the ideal moisture degree varies, depending on the solvent presented in each adhesive system.11 This can lead to bonding failures and may also turn the adhesive interface into permeable membranes that are highly susceptible to the degrading effects of water.46 The results of this study show that bonding to demineralized and air-dried dentin can be an option in order to reduce the amount of water entrapped within the hybrid layer. Additionally, the wet bonding technique may not have practical clinical advantages over dry bonding in vivo.47
CONCLUSIONS
As long as adhesives are vigorously rubbed onto the dentin surface, high immediate and long-term bond strengths can be obtained to either air-dried or wet demineralized dentin.
Contributor Notes
Alessandra Reis, DDS, MS, PhD, professor, Department of Dental Materials and Operative Dentistry, University of Oeste de Santa Catarina, Joaçaba, SC, Brazil
Arlete Pellizzaro, DDS, Department of Dental Materials and Operative Dentistry, University of Oeste de Santa Catarina, Joaçaba, SC, Brazil
Karen Dal-Bianco, DDS, Department of Dental Materials and Operative Dentistry, University of Oeste de Santa Catarina, Joaçaba, SC, Brazil
Osnara Mongruel Gomes, DDS, MS, PhD, professor, Department of Restorative Dentistry, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil
Rafael Patzlaff, engineer, Department of Dental Materials and Operative Dentistry, University of Oeste de Santa Catarina, Joaçaba, SC, Brazil
Alessandro Dourado Loguercio, DDS, MS, PhD, professor, Department of Dental Materials and Operative Dentistry, University of Oeste de Santa Catarina, Joaçaba, SC, Brazil