- Email: [email protected]
A 2-year evaluation of moisture on microtensile bond strength and nanoleakage
d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 862–870
available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/dema
A 2-year evaluation of moisture on microtensile bond strength and nanoleakage A. Reis a , R.H.M. Grande b , G.M.S. Oliveira c , G.C. Lopes c , A.D. Loguercio a,∗ a b c
School of Dentistry, University of Oeste de Santa Catarina, Joac¸aba, SC, Brazil ˜ Paulo, SP, Brazil School of Dentistry, University of Sao School of Dentistry, Federal University of Santa Catarina, SC, Brazil
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objectives. This study evaluated the effect of moisture on the resin–dentin -bond strength
Received 23 August 2005
(BS) and silver nitrate uptake (SNU) of three adhesive systems (Single Bond, One-Step and
Accepted 24 May 2006
Syntac Single Component) soon after bonding (IM) and after 2 years of water storage (2Y). Methods. Dentin surfaces were bonded on a dry (D), moist (W) or over-wet surfaces (OW). After restorations were constructed, specimens were stored in water (37 ◦ C/24 h). Resin–dentin
Keywords:
sticks were prepared (0.8 mm2 ) and they were divided for immediate (IM) and 2-year stor-
Adhesive systems
age (2Y) testing. Half of the specimens from each period of time were tested in tension at
Moisture degree
0.5 mm/min and the other half was immersed in silver nitrate and examined by SEM–EDX.
Degradation
The data was analyzed by three-way repeated measures ANOVA and Tukey’s tests (˛ = 0.05).
Microtensile bond strength
Results. The overall BS (MPa) in the IM group under W condition was higher than in D and
EDX–SEM
OW groups. After 2Y, the BS in W was lower than in the IM group, however higher than in the D and OW for OS and SB. The overall silver nitrate deposition (%) in the IM group under D, W and OW were similar. In the 2Y groups, the nanoleakage was higher than IM groups, however the increase was less pronounced in the W condition. Significance. Higher BS and a significantly lower nitrate uptake were observed for IM groups, for OS and SB. Under W conditions, the BS reduction over time was less pronounced and less nitrate uptake occurred. © 2006 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
The immediate bond strength of etch&rinse adhesive systems is dependent on a proper combination of surface moisture and solvent type [1–3]. It is not a new issue that low immediate bond strengths values of etch&rinse adhesives are obtained when the demineralized dentin is subjected to overdry or overwet conditions prior to adhesive application [3,4]. In both circumstances, the permeability of demineralized dentin is reduced, restricting the diffusion of resin monomers within a shrunken overdried collagen matrix [5] or within an excessively water-filled dentin that occurs on overwet surfaces [6].
Several studies have demonstrated that resin–dentin bond strengths of adhesives decrease after water storage [7–11]. This degradation may result from water movement within the hybrid and adhesive layer. Although some authors claim that the hydrolysis of collagen fibrils, not encapsulated by resin monomers at the base of the hybrid layer is responsible for such reduction [12], there are much more evidence showing that the water movement within the adhesive–dentin interface may extract unconverted monomers from the hybrid layer, rendering the interface weak [13,14]. One could also credit this reduction to inadequate management of surface moisture, however a recent study comparing the immedi-
∗ Corresponding author at: Universidade do Oeste de Santa Catarina, Campus Joac¸aba, SC, Faculdade de Odontologia, Disciplina de Mate´ ´ riais Dentarios e Dent´ıstica, Rua Getulio Vargas, 2125, Bairro Flor da Serra, CEP 89600-000 Joac¸aba, SC, Brazil. Tel.: +55 49 5512041. E-mail address: [email protected] (A.D. Loguercio). 0109-5641/$ – see front matter © 2006 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dental.2006.05.005
863
d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 862–870
ate and 6-month bond strength values of three two-step etch&rinse adhesives under extreme and ideal moisture conditions demonstrated reductions on the bond strengths values, after 6 months only when the adhesives were applied under ideal moisture conditions [15]. One hypothesis for this surprising finding is that the presence of water, from wet bonding technique, can render the polymer weaker than the one formed in a free-water environment, due to a poorly and incomplete polymerization [16]. This sub-polymerized polymer is therefore, more prone to the plasticizing effects of water over time. However, we cannot rule out the fact that the water storage period could have been too short (6 months) to explain the effects of surface moisture on the durability of resin–dentin bonds. Besides that, the aforementioned study has not evaluated the adhesive interfaces ultra morphologically. The evaluation of silver uptake (i.e. nanoleakage evaluation) provides good spatial resolution of submicron defects in resin infiltration or inadequate polymerization [13,17–19]. So far, no study has attempted to address the effects of moisture degrees on the microstructure of resin–dentin adhesive interfaces, after 2 years of water storage. Therefore, the objective of this study was to determine the effects of different surface moisture degrees on the immediate and 2-year specimens by means of microtensile bond strength test and silver nitrate uptake of three different solvent-based adhesive systems to dentin. The null hypothesis was that there is no influence of the surface moisture degree on the bond strengths or silver nitrate uptake of the adhesives, regardless of the type of solvent in their composition and the period of evaluation.
2.
Materials and methods
2.1. Selection of teeth, adhesive systems, experimental design and teeth preparation Three solvent-based, etch&rinse adhesive systems were tested: Single Bond (SB—3M ESPE, St. Paul, MN, USA),
an ethanol/water-based, One-Step (OS—Bisco, Schaumburg, IL, USA), an acetone-based, and Syntac Single Component (SC—Vivadent, Schaan, Liechtenstein, Germany), a waterbased system. The composition, application mode and batch number are described in Table 1. Forty-five extracted, caries-free human third molars were used. The teeth were collected after obtaining the patient’s informed consent under a protocol approved by the Univer˜ Paulo Institutional Review Board. The teeth were sity of Sao disinfected in 0.5% chloramine, stored in distilled water [20] and used within 6 months after extraction. A flat dentin surface was exposed after wet grinding the occlusal enamel on a # 180 grit SiC paper. The exposed dentin surfaces were further polished on wet # 600-grit silicon-carbide paper for 60 s to standardize the smear layer. The bonding area was demarcated by placing a piece of masking tape with an 8.45 mm punched hole in diameter on the center of the surface, yielding a standard area for bonding (52 mm2 ).
2.2.
Bonding procedures
After acid etching with the respective etchants, the surfaces were rinsed with distilled water for 20 s and extensively airdried for 30 s with oil-free compressed air. The adhesives were applied on the surface that was either kept dried (0 l of water) or rewetted for 10 s (Fig. 1) with different amounts of distilled water (2.5 or 4.0 l) pipetted on the surface (Micropipet, Pipetman, Gilson, NY, USA). The adhesives were light cured for the respective recommended time using a VIP light unit set at 600 mW/cm2 (Bisco, Schaumburg, IL, USA). The time elapsed between rewetting and adhesive application was the time required to open the bottles, pour the adhesive on the microbrush and take it to the tooth surface. Resin composite build-ups (Z250, 3M ESPE, St. Paul, MN, USA) were constructed on the bonded surfaces in increments of 1 mm that were individually light cured for 30 s with the same light intensity. All the bonding procedures were carried out by a single operator at a room temperature of 24 ◦ C and 75% relative humidity [21]. Five teeth were used for each combination of adhesive system and surface moisture.
Table 1 – Adhesive systems: composition, application mode and batch number Adhesive systems Single Bond (3M ESPE)
One Step (Bisco)
Syntac Single Component (Vivadent)
Composition Scotchbond—37% phosphoric acid Adhesive—bis-GMA, HEMA, dimethacrylates, polyalquenoic acid copolymer, initiators, water and ethanol Uni-Etch—32% phosphoric acid Adhesive—bis-GMA, BPDM, HEMA, initiator and acetone Total Etch—37% phosphoric acid Maleic acid, HEMA, polyacrylic acid modified by methacrylates, initiators and water
Application mode
Batch number
a, b, c, da , e1, f1, g1
9CX
a, b, c, da , e1, f1, g1
CE0459
a, b, c, da , e2, f2, g2, e2, f2, g2
D56201
a—acid-etch (15 s); b—rinse (15 s); c—air-dry (30 s); d—dentin kept dry or rewetted with different amounts of water; e1—two coats of adhesive systems, brushed for 10 s each; e2—one adhesive coat kept for 20 s, brushed for 10 s; f1—air-dry for 10 s at 20 cm; f2—air-dry for 20 s at 5 cm; g1—light-cure (10 s − 600 mW/cm2 ); g2—light-cure (20 s − 600 mW/cm2 ). a
According to each experimental condition (as shown in Fig. 1).
864
d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 862–870
Fig. 1 – Experimental design.
2.3.
Specimen’s preparation and storage
After storage of the restored teeth in distilled water at 37 ◦ C for 24 h, they were longitudinally sectioned in both “x” and “y” directions across the bonded interface with a diamond saw in a Labcut 1010 machine (Extec Corp., Enfield, CT, USA) to obtain 30 bonded sticks per tooth, each with a crosssectional area of approximately 0.8 mm2 . The number of premature debonded sticks (D) per tooth during specimen preparation was recorded. Specimens originated from the areas immediately above the pulp chamber had their remaining dentin thickness (RDT) measured with a digital caliper and recorded (Absolute Digimatic, Mitutoyo, Tokyo, Japan). The cross-sectional area of each stick was measured with the digital caliper to the nearest 0.01 mm and recorded for posterior calculation of the bond strength (BS). The bonded sticks originated from the same teeth were randomly divided and assigned to be tested immediately or after 24 months of storage in distilled water containing 0.4% sodium azide [22] at 37 ◦ C. The storage solution was not changed [23] and its pH was monitored monthly. In each period of time, half of the specimens were divided to be tested by microtensile bond strength, while the other half was ultra morphologically examined by SEM. The experimental design of this study is shown in Fig. 1.
2.4.
Resin–dentin microtensile bond strength (BS)
At each storage time period, individual bonded sticks were attached to a modified device for microtensile testing with cyanoacrylate resin (Zapit, Dental Ventures of North America, Corona, CA, USA) and subjected to a tensile force in a ˜ Jose´ dos Pinhais, PR, universal testing machine (EMIC, Sao Brazil) at a crosshead speed of 0.5 mm/min. The failure modes were evaluated at 400× (HMV-2, Shimadzu, Tokyo, Japan) and classified as cohesive (failure exclusive within dentin or resin 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).
2.5.
Ultramorphological analysis by SEM/EDX
Bonded sticks were coated with two layers of nail varnish applied up to within 1 mm of the bonded interfaces. The specimens were re-hydrated in distilled water for 10 min prior to immersion in the tracer solution for 24 h. Ammoniacal silver nitrate was prepared according to the protocol previously described by Tay et al. [24]. The sticks were placed in the ammoniacal silver nitrate in darkness for 24 h, rinsed thoroughly in distilled water, and immersed in photo developing solution for 8 h under a fluorescent light to reduce silver ions
865
d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 862–870
for every testing group was expressed as the average of the five teeth used per group and expressed in MPa. The amount of the silver penetration (%) was determined for every testing group was expressed as the average of the five teeth. A three-way repeated measurement ANOVA (material versus moisture degree versus time) and Tukey’s multiple comparisons test was used to analyze the data at ˛ = 0.05 obtained in both methods. The correlation between the bond strength measurements and silver nitrate uptake of each experimental condition was analyzed by simple linear regression analysis. The strength of the association between these two variables (bond strength measurements versus silver nitrate uptake) was estimated with the Pearson product–moment correlation statistics (˛ = 0.05).
Fig. 2 – Schematic drawing of a resin–dentin interface, showing the areas where the silver nitrate uptake was measured by SEM–EDX.
into metallic silver grains within voids along the bonded interface. All sticks were wet-polished with 600 SiC paper to remove the nail varnish. Then, the specimens were placed inside an acrylic ring, which was attached to a double-sided adhesive tape, and embedded in epoxy resin. After the epoxy resin had set, the thickness of the embedded specimens was reduced to approximately half by grinding with silicon-carbide papers under running water. Specimens were polished with a 1000grit SiC paper and 6, 3, 1 and 0.25 m diamond paste (Buehler Ltd., Lake Bluff, IL, USA) using a polish cloth. They were ultrasonically cleaned, air-dried, mounted on stubs, and carbon coated (MED 010, Balzers Union, Balzers, Liechtenstein). Resin–dentin interfaces were analyzed in a field-emission scanning electron microscope operated in the backscattered electron mode and using energy dispersive X-ray spectrometry (EDX) (LEO 435 VP, LEO Electron Microscopy Ltd., Cambridge, UK). The amount of silver nitrate within the adhesive layer, hybrid layer and resin tags, in each stick, was measured with EDX in three regions (5 m × 5 m) of the bonded stick (left, center and right). The total length of the hybrid layer scanned for silver nitrate uptake measurement was approximately 75 m2 . The silver nitrate uptake was expressed as a percentage of the total area evaluated (Fig. 2).
2.6.
Data treatment
The average BS of each tooth was calculated and expressed as an index that assumes the relative contribution of the different failure modes. The value attributed to specimens that failed prematurely during preparation and could not be tested was arbitrary and corresponded to approximately half of the minimum bond strength value that could be measured in this study [3]. The formula assumed the cohesive strength of the resin composite (CR ) as the average value of all specimens (from a single tooth) that failed in the resin composite. Similarly, the cohesive strength of dentin (CD ) was calculated. The BS index
3.
Results
The remaining dentin thickness (RDT) for all specimens ranged from 2.3 to 2.7 mm. Regression analysis between RDT and BS revealed no influence of RDT in bond strengths. The mean cross-sectional area ranged from 0.78 to 0.82 mm2 and no differences among the treatment groups were detected (p > 0.05).
3.1.
Resin–dentin microtensile bond strength
The overall bond strength values for the experimental groups are expressed in Table 2. No stick, from the SC adhesive, survived the 24-month water storage and this material was not included in the statistical model. The interaction adhesive × moisture degree × time was statistically significant (p < 0.05). While SB achieved higher BS at 0 and 2.5 l of water, OS bond strength was higher at 2.5 and 4.0 l of water in the immediate time (Table 2). After 24 months of water storage, significant reductions in BS were observed for SB and OS when
Table 2 – Means and standard deviations (MPa) of bond strength indexes for each moisture degree at each time period Adhesive systems/ moisture (l)
Time Immediate
24 months
SB 0 2.5 4.0
26.1 (1.1) a,b 32.6 (1.5) a 21.3 (1.2) a,b
5.3 (4.9) c 21.8 (7.2) a,b 7.5 (6.5) c
OS 0 2.5 4.0
18.0 (1.9) b 32.0 (2.1) a 31.4 (6.2) a
5.8 (3.9) c 19.8 (7.6) a,b 13.5 (5.3) b,c
SCa 0 2.5 4.0
24.0 (3.6) 24.3 (0.7) 12.5 (2.6)
0.0 0.0 0.0
Groups with the same letter are not significantly different using Tukey’s test (p > 0.05). a
The bond strength values of this material were not included in the ANOVA model.
866
d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 862–870
Table 3 – Means and standard deviations (%) of silver penetration for each adhesive system in each moisture degree at each time period
Table 4 – Means, standard deviations (%) and statistical significance of silver penetration for each adhesive system at each moisture degree
Adhesive systems/ moisture (l)
Moisture (l)
Time Immediate 5.1 (1.8) 7.8 (2.5) 3.0 (2.2)
46.9 (4.7) 35.1 (2.6) 50.9 (5.8)
OS 0 2.5 4.0
11.1 (6.7) 2.0 (3.4) 9.3 (6.4)
57.5 (10.1) 33.5 (8.9) 49.6 (6.3)
SCa 0 2.5 4.0 a
13.4 (9.6) 9.9 (6.7) 10.8 (9.0)
SB
24 months
SB 0 2.5 4.0
0.0 0.0 0.0
The silver penetration values of this material were not included in the ANOVA model.
the moisture degree was set at the extreme conditions of moisture (i.e. 0 and 4.0 l) (p < 0.05) (Table 2). No significant reduction in BS was observed when the moisture degree was set at 2.5 l, regardless of the adhesive system employed (Table 2).
Adhesive systems
0 2.5 4.0
26.0 (22.5) a,b 21.4 (15.4) a 27.0 (25.2) a,b
OS 34.3 (24.8) b,c 18.0 (20.6) a 29.5 (23.4) b,c
Groups with the same letter are not significantly different using Tukey’s test (p > 0.05).
The overall silver penetration percentages for the experimental groups are expressed in Table 3. No stick, from the SC adhesive, survived the 24-month water storage and this material was not included in the statistical model. A significant effect of moisture degree on the silver nitrate penetration was observed among the materials (p = 0.01). The lowest percentages of silver nitrate occurred in the wet condition, for SB and OS (Table 4). A significant interaction between the moisture degree and the storage periods was also detected (p = 0.0001). The silver nitrate penetration in the immediate period was similar for all moisture conditions (Figs. 3 and 4), regardless the adhesive employed. After 24 months the silver nitrate pen-
Fig. 3 – Backscattered SEM images of the interface bonded with Single Bond to dentin.
d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 862–870
867
Fig. 4 – Backscattered SEM images of the interface bonded with One Step to dentin.
Table 5 – Means, standard deviations (%) and statistical significance of silver penetration for each moisture degree at each time period Time
Moisture (l) 0
Immediate 24 months
8.1 (4.3) a 52.2 (7.4) c
2.5 4.9 (3.0) a 34.3 (5.8) b
component and the moisture degree on the dentin surface, which lead us to reject the null hypothesis of the study. Although not statistically significant in the immediate time, SB and SC showed high bond strength means when the moisture degree was set at 0 or 2.5 l of water. Other previous
4.0 6.2 (4.3) a 50.3 (6.1) c
Groups with the same letter are not significantly different using Tukey’s test (p > 0.05).
etration increased when compared to baseline measurements (immediate period) (Figs. 3 and 4), however this increase was less pronounced when the moisture degree was set at 2.5 l (Table 5). Regression analysis revealed a linear and negative relationship between the mean bond strength and the mean silver nitrate uptake (Fig. 5) (R2 = 0.87, p = 0.001).
4.
Discussion
The results of this study suggests that the bond strengths of etch&rinse adhesive systems is dependent on the solvent
Fig. 5 – Regression analysis between the mean microtensile bond strength values (MPa) and the mean silver nitrate penetration (%).
868
d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 862–870
investigations have confirmed that water or water/ethanol based systems have higher immediate bond strength when applied in a rather dried surface [3,15]. It is likely that the number of variables involved in this study and the substantial reduction on the bond strengths values after 24 months could explain the lack of statistical significance observed in this study. The water component, in water-based adhesive systems, is able to break the undesirable interpeptide bonds formed between the collagen fibrils when in an overdry condition [25]. Water has a higher solubility parameter for hydrogen bonding (37.3 (J/cm3 )1/2 ) than that formed between the collagen fibrils (19.0 (J/cm3 )1/2 ) [25]. Consequently, the shrunken and stiffened air-dried demineralized dentin [26] can be re-expanded allowing the penetration of resin monomers. Acetone and resin monomers are not capable of re-expanding air-dried demineralized dentin [25,27]. Under overdry conditions, the infiltration ratio of the bonding resin within the hybrid layer for acetone-based systems was reduced by 50% when applied to dry instead of wet dentin [28], which must explain the inferior performance of OS, an acetone-based system. On the other hand, the OS system showed high bond strengths values when the moisture degree was set 4.0 l of water, contrary to what was observed for SB and SC. Presumably the high vapor pressure of acetone (ca. 200 mmHg) in the OS system allows removal of residual water in a higher extent than the other solvents like water (ca 47.1 mmHg) [29] or mixture of water and ethanol presented in the SC and SB adhesives. The more residual water entrapped in the hybrid and adhesive layer, the lower the mechanical properties of the polymer formed reducing the bond strength values. It was already demonstrated that the conversion degree of adhesives is substantially reduced in the presence of water [16,30]. A previous study, that evaluated the management of surface moisture on the durability of resin–dentin bonds, concluded that the specimens bonded to adverse moisture conditions were not more prone to degradation over time [15]. This finding was not corroborated in the present investigation. The differences between these two studies could be due to the differences in the period of evaluation. While the cited study evaluated the durability of resin–dentin bonds after 6 months of water storage [15], the present investigation stored the specimens for 24 months. Actually the present investigation observed reductions on bond strengths for all experimental groups albeit only those bonded to adverse conditions of moisture was statistically different from the immediate values, which explains why no experimental group from the present investigation showed interfaces free of silver nitrate uptake. This situation was even worse for extreme moisture conditions. It was already demonstrated that the infiltration ratio of resin monomers into collapsed demineralized dentin [5,28] and into overwet dentin [5,6] is substantially reduced compared to ideal conditions. For instance, recent studies have shown that collagen collapse means that adhesive does not infiltrate as much as one half of the zone of demineralized dentin [31,32]. Under overwet conditions, phase separation of the adhesive occurs and leads to a very porous hybrid layer characterized by hydrophobic bis-GMA-rich particles distributed in a hydrophilic HEMA-rich matrix [33].
This means that bonding performed under extreme moisture conditions (overdry or overwet conditions) can leave more voids at the base of the hybrid layer not fully penetrated by resin that can act as pathway for extrinsic and intrinsic water penetration over time. Although nanoleakage pathway may be located within the adhesive resin, within the hybrid layer and within partially- or fully-demineralized dentin [13], it seems that porosities in the partially demineralized dentin, not infiltrated by adhesive resin, is the easiest way for penetration of water, since the energy barrier that water molecules must overcome during water uptake is much lower under this circumstance than when water needs to break intermolecular forces established among polymer chains. It is worth mentioning that even when the dentin is adequately rewetted, a full resin infiltration does not occur along the decalcified dentin for etch&rinse adhesive systems, applied according to the time recommended by the manufacturers [28,32,34,35], which explains the silver nitrate uptake under ideal moisture conditions, over a 24-month storage period. As more water is absorbed into the polymer over time, swelling of the resin by hydrogen bonding of water on hydrophilic domains of polymer chains takes place. In a second stage, residual unreacted monomers is eluted from the hybrid layer [36,37] creating new channels for water penetration, through it water diffuses even more in an auto-accelerative manner. The elution of residual and low molecular weight polymers also cause an increase in the porosity within the hybrid layer [13] and an inevitably reduction of the cohesive strength of the bonding resin in the adhesive interface. The lower the cohesive strength of adhesive, the lower the resulting bond strength values [10,38,39], which renders the resin–dentin bond weak and less stable over time [13,15,22,40]. The findings from the silver nitrate uptake from this study add further evidence on this hypothesis. By reference to Table 5, one can observe that after 24 months of water storage, the silver nitrate uptake was higher when compared to the baseline measurements, mainly for extreme conditions of moisture, where a less than optimal hybridization and polymerization could have occurred. Interestingly are the results of the groups bonded under ideal moisture conditions. Although a numerical decrease in the resin–dentin bond strength values occurred after 2 years of water storage, this reduction was not statistically different from the immediate values. From the SEM/EDX evaluation, one could observe that a significant increase in the silver nitrate uptake has occurred for ideal moisture bonded groups, which means that these groups also underwent a degradation process; however, in a slightly low rate compared to extreme moisture conditions. As the use of an air stream does not effectively remove water left in the demineralized dentin, from HEMA/water mixtures [16,29], it is fair to suppose that remaining water and solvents led to sub-polymerization [16]. The lack of specimens from the SC adhesive after 2 years can also be explained by the aforementioned hypothesis. Syntac Single Component is a water-based system and has the lowest vapour pressure among the solvents used in adhesive formulations. Consequently the evaporation of water and solvents from the adhesive after its application on demineralized dentin is rather slow, leading to their entrapment into the adhesive layer and consequent sub-optimal polymerization
d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 862–870
which caused, in turn, the premature failure of these specimens. As previously mentioned, nanoleakage pathways within resin–dentin interfaces is not only those areas of incomplete resin infiltration into demineralized dentin but also the microvoids within the sub-polymerized resin matrix. This is one of the reasons of why two-step etch&rinse adhesives function as permeable membranes [41,42] permitting the diffusion of water molecules from dentin across the adhesive layer. The retention of unbound water, both from residual water that is incompletely evaporated from the adhesive, or from the underlying dentin as a result of the high osmolarity of the hydrophilic adhesive mixture, creates water-filled channels within the adhesive that can be visualized after silver nitrate impregnation. The higher amount of silver nitrate uptake observed after storage in water for 24 months indicates that the permeability of the adhesive layer was increased due to the degradation process caused by water uptake [40,43]. Several studies have attempted to correlate the data from marginal sealing, microleakage and nanoleakage with bond strengths values [19,44–48]. However none of these studies has succeeded in obtaining a significant correlation between these two properties. In this study a negative and linear correlation was observed between these two properties. The main difference observed in this study and the aforementioned ones is that the bonded interfaces were evaluated in the short- and long-term. This allowed time for the degradation process takes place and to promote significant changes in the resin–dentin adhesive interface. One could speculate that the highly significant correlation between decreases in bond strength and increases in silver nitrate uptake reflect degradation at the resin–dentin interfaces. Okuda et al. [17] also observed an inverse relationship between bond strength and silver nitrate uptake for the Clearfil Liner Bond 2V adhesive system mainly when the 9 month’s specimens were evaluated. However, this matter still deserves further attention since another study that also evaluated the durability of bonded interfaces by means of bond strength measurements and silver nitrate uptake has not observed any correlation between these two properties [40]. Although the wet bonding has been advocated for etch&rinse adhesive systems, this technique does not avoid the degradation process that occurs to highly hydrophilic monomers included in the composition of simplified etch&rinse adhesives. New material’s formulations should focus on the use of adhesives with more hydrophobic features [49], which seems to be less prone to the damage caused by water sorption over time. Further studies should be conducted in order to evaluate the hypothesis arose in this study.
5.
Conclusions
Higher bond strength values and a significantly lower nitrate uptake were observed for the immediate specimens, for all adhesives tested. Under the ideal moisture condition, the bond strength reduction and the silver nitrate uptake was less pronounced than in the extreme moisture conditions, however it did not avoid the degradation process that adhesives interfaces are prone to.
869
Acknowledgments This study was supported, in part, by School of Dentistry, University of Oeste de Santa Catarina and by CNPq (302552/030, 551043/02-2, 474226/03-4 and 300481/95-0) and FAPESP (01/06140-1 and 02/06682-1).
references
¨ [1] Jacobsen T, Soderholm KJ. Effects of primer solvent, primer agitation, and dentin dryness on shear bond strength to dentin. Am J Dent 1998;11:225–8. [2] Van Meerbeek B, Yoshida Y, Lambrechts P, Vanherle G, Duke ES, Eick JD, et al. A TEM study of two water-based adhesive systems bonded to dry and wet dentin. J Dent Res 1998;77:50–9. [3] Reis A, Loguercio AD, Azevedo CLN, Carvalho RM, Singer JM, Grande RHM. Moisture spectrum of demineralized dentin for different solvent-based adhesive system. J Adhes Dent 2003;5:183–92. [4] Nakajima M, Kanemura N, Pereira PNR, Tagami J, Pashley DH. Comparative microtensile bond strength and SEM analysis of bonding to wet and dry dentin. Am J Dent 2000;13: 324–8. [5] Tay FR, Gwinnett JA, Wei SH. Relation between water content in acetone/alcohol-based primer and interfacial ultrastructure. J Dent 1998;26:147–56. [6] Tay FR, Gwinnett JA, Wei SH. The overwet phenomenon: a transmission electron microscopic study of surface moisture in the acid-conditioned, resin–dentin interface. Am J Dent 1996;9:161–6. [7] DeMunck J, Van Meerbeek B, Yoshida Y, Inoue S, Vargas M, Suzuki K, et al. Four-year water degradation of total-etch adhesives bonded to dentin. J Dent Res 2003;82:136–40. [8] Koshiro K, Inoue S, Tanaka T, Koase K, Fujita M, Hashimoto M, et al. In vivo degradation of resin–dentin bonds produced by a self-etch vs. a total-etch adhesive system. Eur J Oral Sci 2004;112:368–75. ¨ [9] Frankenberger R, Strobel W, Lohbauer U, Kramer N, Petschelt A. The effect of six years of water storage on resin composite bonding to human dentin. J Biomed Mater Res Part B: Appl Biomater 2004;69B:25–32. [10] Reis A, Grandi V, Carlotto L, Bortoli G, Patzlaff R, Accorinte MLR, et al. Effect of smear layer thickness and acidity of self-etching solutions on early and long-term bond strength to dentin. J Dent 2005;33:549–59. [11] Loguercio AD, Uceda-Gomez N, de Oliveira Carrilho MR, Reis A. Influence of specimen size and regional variation on long-term resin–dentin bond strength. Dent Mater 2005;21:224–31. [12] Hashimoto M, Ohno H, Kaga M, Sano H, Endo K, Oguchi H. In vivo degradation of resin–dentin bonds in humans over 1 to 3 years. J Dent Res 2000;79:1385–91. [13] Sano H, Yoshikawa T, Pereira PNR, Kanemura N, Morigami M, Tagami J, et al. Long-term durability of dentin bonds made with a self-etching primer, in vivo. J Dent Res 1999;78:906–11. [14] Carrilho MROMR, Tay FR, Pashley DH, Tjaderhane L, Carvalho RM. Mechanical stability of resin–dentin bond components. Dent Mater 2005;21:232–41. [15] Reis A, Loguercio AD, Carvalho RM, Grande RHM. Durability of resin dentin interfaces: effects of surface moisture and adhesive solvent component. Dent Mater 2004;20:669–76. [16] Paul SJ, Leach M, Rueggeberg FA, Pashley DH. Effect of water content on the physical properties of model dentine primer and bonding resins. J Dent 1999;27:209–14.
870
d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 862–870
[17] Okuda M, Pereira PN, Nakajima M, Tagami J, Pashley DH. Long-term durability of resin dentin interface: nanoleakage vs. microtensile bond strength. Oper Dent 2002;27:289–96. [18] Li H, Burrow MF, Tyas MJ. The effect of load cycling on the nanoleakage of dentin bonding systems. Dent Mater 2002;18:111–9. [19] Hashimoto M, De Munck J, Ito S, Sano H, Kaga M, Oguchi H, et al. In vitro effect of nanoleakage expression on resin–dentin bond strengths analyzed by microtensile bond test, SEM/EDX and TEM. Biomaterials 2004;25:5565–74. [20] De Wald JP. The use of extracted teeth for in vitro bonding studies: a review of infection control considerations. Dent Mater 1997;13:74–81. [21] Asmussen E, Peutzfeldt A. The influence of relative humidity on the effect of dentin bonding systems. J Adhes Dent 2001;3:123–7. [22] Burrow MF, Sato HM, Tagami J. Dentin bonding durability after three years using a dentin bonding agent with and without priming. Dent Mater 1996;12:302–7. [23] Kitasako Y, Burrow MF, Nikaido T, Tagami J. The influence of storage on dentin bond durability of resin cement. Dent Mater 2000;16:1–16. [24] Tay FR, Pashley DH, Yoshiyama M. Two modes of nanoleakage expression in single-step adhesives. J Dent Res 2002;81:472–6. [25] Pashley DH, Agee KA, Nakajima M, Tay FR, Carvalho RM, Terada RS, et al. Solvent-induced dimensional changes in EDTA-demineralized dentin matrix. J Biomed Mater Res 2001;56:273–81. [26] Maciel KT, Carvalho RM, Ringle RD, Preston CD, Russel CM, Pashley DH. The effects of acetone, ethanol, HEMA, and air on the stiffness of human decalcified dentin matrix. J Dent Res 1996;11:1851–8. [27] Pashey DH, Carvalho RM, Tay FR, Agee KA, Lee KW. Solvation of dried dentin matrix by water and other polar solvents. Am J Dent 2002;15:97–102. [28] Hashimoto M, Ohno H, Kaga M, Sano H, Endo K, Oguchi H. Extent to which resin can infiltrate dentin by acetone-based adhesives. J Dent Res 2002;81:74–8. [29] Pashley EL, Zhang Y, Lockwood PE, Rueggeberg FA, Pashley DH. Effects of HEMA on water evaporation from water–HEMA mixtures. Dent Mater 1998;14:6–10. ¨ [30] Jacobsen T, Soderholm KJ. Some effects of water on dentin bonding. Dent Mater 1995;11:132–6. [31] Wieliczka DM, Kruger MB, Spencer P. Raman imaging of dental adhesive diffusion. Appl Spectrosc 1997;51: 1593–6. [32] Spencer P, Swafford JR. Unprotected protein at the dentin–adhesive interface. Quintessence Int 1999;30: 501–7. [33] Spencer P, Wang Y. Adhesive phase separation at the dentin interface under wet bonding conditions. J Biomed mater Res 2002;62:447–56.
[34] Spencer P, Wang Y, Walker MP, Wieliczka DM, Swafford JR. Interfacial chemistry of the dentin/adhesive bond. J Dent Res 2000;79:1458–63. [35] Miyasaki M, Onose H, Moore BK. Analysis of the dentin–resin interface by use of laser Raman spectroscopy. Dent Mater 2002;18:576–80. [36] Hashimoto M, Ohno H, Sano H, Kaga M, Oguchi H. In vitro degradation of resin–dentin bonds analyzed by microtensile bond test, scanning and transmission electron microscopy. Biomaterials 2003;24:3795–803. [37] Hashimoto M, Ohno H, Sano H, Kaga M, Oguchi H. Degradation patterns of different adhesives and bonding procedures. J Biomed Mater Res B: Appl Biomater 2003;66:324–30. [38] Pashley DH, Ciucchi B, Sano H, Carvalho RM, Russell CM. Bond strength versus dentine structure: a modelling approach. Arch Oral Biol 1995;40:1109–18. [39] Takahashi A, Sato Y, Uno S, Pereira PNR, Sano H. Effects of mechanical properties of adhesives on bond strength to dentin. Dent Mater 2002;18:263–8. [40] Okuda M, Pereira PNR, Najakima M, Tagami J. Relationship between nanoleakage and long-term durability of dentin bonds. Oper Dent 2001;26:482–90. [41] Tay FR, Pashley DH. Water treeing—a potential mechanism for degradation of dentin adhesives. Am J Dent 2003;16:6–12. [42] Tay FR, Frankenberger R, Krejci I, Bouillaguet S, Pashley DH, Carvalho RM, et al. Single-bottle adhesives behave as permeable membranes after polymerization. I. In vivo evidence. J Dent 2004;32:611–21. [43] Li HP, Burrow MF, Tyas MJ. The effect of long-term storage on nanoleakage. Oper Dent 2001;26:609–16. [44] Guzman-Ruiz S, Armstrong SR, Cobb DS, Vargas MA. Association between microtensile bond strength and leakage in the indirect resin composite/dentin adhesively bonded joint. J Dent 2001;29:145–53. [45] Pereira PN, Okuda M, Nakajima M, Sano H, Tagami J, Pashley DH. Relationship between bond strengths and nanoleakage: evaluation of a new assessment method. Am J Dent 2001;14:100–4. [46] Loguercio AD, Reis A, Ballester RY. Polymerization shrinkage: effects of constraint and filling technique in composite restorations. Dent Mater 2004;20:236–43. [47] Kenshima S, Reis A, Uceda-Gomez N, Tancredo LLF, Filho LE, Nogueira FN, et al. Effect of smear layer thickness and pH of self-etching adhesive systems on the bond strength and gap formation to dentin. J Adhes Dent 2005;7:117–26. [48] Cenci MS, Demarco FF, Carvalho RM. Class II composite resin restorations with two polymerization techniques: relationship between microtensile bond strength and marginal leakage. J Dent 2005;33:603–10. [49] Yiu CK, King NM, Pashley DH, Suh BI, Carvalho RM, Carrilho MR, et al. Effect of resin hydrophilicity and water storage on resin strength. Biomaterials 2004;25:5789–96.
available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/dema
A 2-year evaluation of moisture on microtensile bond strength and nanoleakage A. Reis a , R.H.M. Grande b , G.M.S. Oliveira c , G.C. Lopes c , A.D. Loguercio a,∗ a b c
School of Dentistry, University of Oeste de Santa Catarina, Joac¸aba, SC, Brazil ˜ Paulo, SP, Brazil School of Dentistry, University of Sao School of Dentistry, Federal University of Santa Catarina, SC, Brazil
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objectives. This study evaluated the effect of moisture on the resin–dentin -bond strength
Received 23 August 2005
(BS) and silver nitrate uptake (SNU) of three adhesive systems (Single Bond, One-Step and
Accepted 24 May 2006
Syntac Single Component) soon after bonding (IM) and after 2 years of water storage (2Y). Methods. Dentin surfaces were bonded on a dry (D), moist (W) or over-wet surfaces (OW). After restorations were constructed, specimens were stored in water (37 ◦ C/24 h). Resin–dentin
Keywords:
sticks were prepared (0.8 mm2 ) and they were divided for immediate (IM) and 2-year stor-
Adhesive systems
age (2Y) testing. Half of the specimens from each period of time were tested in tension at
Moisture degree
0.5 mm/min and the other half was immersed in silver nitrate and examined by SEM–EDX.
Degradation
The data was analyzed by three-way repeated measures ANOVA and Tukey’s tests (˛ = 0.05).
Microtensile bond strength
Results. The overall BS (MPa) in the IM group under W condition was higher than in D and
EDX–SEM
OW groups. After 2Y, the BS in W was lower than in the IM group, however higher than in the D and OW for OS and SB. The overall silver nitrate deposition (%) in the IM group under D, W and OW were similar. In the 2Y groups, the nanoleakage was higher than IM groups, however the increase was less pronounced in the W condition. Significance. Higher BS and a significantly lower nitrate uptake were observed for IM groups, for OS and SB. Under W conditions, the BS reduction over time was less pronounced and less nitrate uptake occurred. © 2006 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
The immediate bond strength of etch&rinse adhesive systems is dependent on a proper combination of surface moisture and solvent type [1–3]. It is not a new issue that low immediate bond strengths values of etch&rinse adhesives are obtained when the demineralized dentin is subjected to overdry or overwet conditions prior to adhesive application [3,4]. In both circumstances, the permeability of demineralized dentin is reduced, restricting the diffusion of resin monomers within a shrunken overdried collagen matrix [5] or within an excessively water-filled dentin that occurs on overwet surfaces [6].
Several studies have demonstrated that resin–dentin bond strengths of adhesives decrease after water storage [7–11]. This degradation may result from water movement within the hybrid and adhesive layer. Although some authors claim that the hydrolysis of collagen fibrils, not encapsulated by resin monomers at the base of the hybrid layer is responsible for such reduction [12], there are much more evidence showing that the water movement within the adhesive–dentin interface may extract unconverted monomers from the hybrid layer, rendering the interface weak [13,14]. One could also credit this reduction to inadequate management of surface moisture, however a recent study comparing the immedi-
∗ Corresponding author at: Universidade do Oeste de Santa Catarina, Campus Joac¸aba, SC, Faculdade de Odontologia, Disciplina de Mate´ ´ riais Dentarios e Dent´ıstica, Rua Getulio Vargas, 2125, Bairro Flor da Serra, CEP 89600-000 Joac¸aba, SC, Brazil. Tel.: +55 49 5512041. E-mail address: [email protected] (A.D. Loguercio). 0109-5641/$ – see front matter © 2006 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dental.2006.05.005
863
d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 862–870
ate and 6-month bond strength values of three two-step etch&rinse adhesives under extreme and ideal moisture conditions demonstrated reductions on the bond strengths values, after 6 months only when the adhesives were applied under ideal moisture conditions [15]. One hypothesis for this surprising finding is that the presence of water, from wet bonding technique, can render the polymer weaker than the one formed in a free-water environment, due to a poorly and incomplete polymerization [16]. This sub-polymerized polymer is therefore, more prone to the plasticizing effects of water over time. However, we cannot rule out the fact that the water storage period could have been too short (6 months) to explain the effects of surface moisture on the durability of resin–dentin bonds. Besides that, the aforementioned study has not evaluated the adhesive interfaces ultra morphologically. The evaluation of silver uptake (i.e. nanoleakage evaluation) provides good spatial resolution of submicron defects in resin infiltration or inadequate polymerization [13,17–19]. So far, no study has attempted to address the effects of moisture degrees on the microstructure of resin–dentin adhesive interfaces, after 2 years of water storage. Therefore, the objective of this study was to determine the effects of different surface moisture degrees on the immediate and 2-year specimens by means of microtensile bond strength test and silver nitrate uptake of three different solvent-based adhesive systems to dentin. The null hypothesis was that there is no influence of the surface moisture degree on the bond strengths or silver nitrate uptake of the adhesives, regardless of the type of solvent in their composition and the period of evaluation.
2.
Materials and methods
2.1. Selection of teeth, adhesive systems, experimental design and teeth preparation Three solvent-based, etch&rinse adhesive systems were tested: Single Bond (SB—3M ESPE, St. Paul, MN, USA),
an ethanol/water-based, One-Step (OS—Bisco, Schaumburg, IL, USA), an acetone-based, and Syntac Single Component (SC—Vivadent, Schaan, Liechtenstein, Germany), a waterbased system. The composition, application mode and batch number are described in Table 1. Forty-five extracted, caries-free human third molars were used. The teeth were collected after obtaining the patient’s informed consent under a protocol approved by the Univer˜ Paulo Institutional Review Board. The teeth were sity of Sao disinfected in 0.5% chloramine, stored in distilled water [20] and used within 6 months after extraction. A flat dentin surface was exposed after wet grinding the occlusal enamel on a # 180 grit SiC paper. The exposed dentin surfaces were further polished on wet # 600-grit silicon-carbide paper for 60 s to standardize the smear layer. The bonding area was demarcated by placing a piece of masking tape with an 8.45 mm punched hole in diameter on the center of the surface, yielding a standard area for bonding (52 mm2 ).
2.2.
Bonding procedures
After acid etching with the respective etchants, the surfaces were rinsed with distilled water for 20 s and extensively airdried for 30 s with oil-free compressed air. The adhesives were applied on the surface that was either kept dried (0 l of water) or rewetted for 10 s (Fig. 1) with different amounts of distilled water (2.5 or 4.0 l) pipetted on the surface (Micropipet, Pipetman, Gilson, NY, USA). The adhesives were light cured for the respective recommended time using a VIP light unit set at 600 mW/cm2 (Bisco, Schaumburg, IL, USA). The time elapsed between rewetting and adhesive application was the time required to open the bottles, pour the adhesive on the microbrush and take it to the tooth surface. Resin composite build-ups (Z250, 3M ESPE, St. Paul, MN, USA) were constructed on the bonded surfaces in increments of 1 mm that were individually light cured for 30 s with the same light intensity. All the bonding procedures were carried out by a single operator at a room temperature of 24 ◦ C and 75% relative humidity [21]. Five teeth were used for each combination of adhesive system and surface moisture.
Table 1 – Adhesive systems: composition, application mode and batch number Adhesive systems Single Bond (3M ESPE)
One Step (Bisco)
Syntac Single Component (Vivadent)
Composition Scotchbond—37% phosphoric acid Adhesive—bis-GMA, HEMA, dimethacrylates, polyalquenoic acid copolymer, initiators, water and ethanol Uni-Etch—32% phosphoric acid Adhesive—bis-GMA, BPDM, HEMA, initiator and acetone Total Etch—37% phosphoric acid Maleic acid, HEMA, polyacrylic acid modified by methacrylates, initiators and water
Application mode
Batch number
a, b, c, da , e1, f1, g1
9CX
a, b, c, da , e1, f1, g1
CE0459
a, b, c, da , e2, f2, g2, e2, f2, g2
D56201
a—acid-etch (15 s); b—rinse (15 s); c—air-dry (30 s); d—dentin kept dry or rewetted with different amounts of water; e1—two coats of adhesive systems, brushed for 10 s each; e2—one adhesive coat kept for 20 s, brushed for 10 s; f1—air-dry for 10 s at 20 cm; f2—air-dry for 20 s at 5 cm; g1—light-cure (10 s − 600 mW/cm2 ); g2—light-cure (20 s − 600 mW/cm2 ). a
According to each experimental condition (as shown in Fig. 1).
864
d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 862–870
Fig. 1 – Experimental design.
2.3.
Specimen’s preparation and storage
After storage of the restored teeth in distilled water at 37 ◦ C for 24 h, they were longitudinally sectioned in both “x” and “y” directions across the bonded interface with a diamond saw in a Labcut 1010 machine (Extec Corp., Enfield, CT, USA) to obtain 30 bonded sticks per tooth, each with a crosssectional area of approximately 0.8 mm2 . The number of premature debonded sticks (D) per tooth during specimen preparation was recorded. Specimens originated from the areas immediately above the pulp chamber had their remaining dentin thickness (RDT) measured with a digital caliper and recorded (Absolute Digimatic, Mitutoyo, Tokyo, Japan). The cross-sectional area of each stick was measured with the digital caliper to the nearest 0.01 mm and recorded for posterior calculation of the bond strength (BS). The bonded sticks originated from the same teeth were randomly divided and assigned to be tested immediately or after 24 months of storage in distilled water containing 0.4% sodium azide [22] at 37 ◦ C. The storage solution was not changed [23] and its pH was monitored monthly. In each period of time, half of the specimens were divided to be tested by microtensile bond strength, while the other half was ultra morphologically examined by SEM. The experimental design of this study is shown in Fig. 1.
2.4.
Resin–dentin microtensile bond strength (BS)
At each storage time period, individual bonded sticks were attached to a modified device for microtensile testing with cyanoacrylate resin (Zapit, Dental Ventures of North America, Corona, CA, USA) and subjected to a tensile force in a ˜ Jose´ dos Pinhais, PR, universal testing machine (EMIC, Sao Brazil) at a crosshead speed of 0.5 mm/min. The failure modes were evaluated at 400× (HMV-2, Shimadzu, Tokyo, Japan) and classified as cohesive (failure exclusive within dentin or resin 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).
2.5.
Ultramorphological analysis by SEM/EDX
Bonded sticks were coated with two layers of nail varnish applied up to within 1 mm of the bonded interfaces. The specimens were re-hydrated in distilled water for 10 min prior to immersion in the tracer solution for 24 h. Ammoniacal silver nitrate was prepared according to the protocol previously described by Tay et al. [24]. The sticks were placed in the ammoniacal silver nitrate in darkness for 24 h, rinsed thoroughly in distilled water, and immersed in photo developing solution for 8 h under a fluorescent light to reduce silver ions
865
d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 862–870
for every testing group was expressed as the average of the five teeth used per group and expressed in MPa. The amount of the silver penetration (%) was determined for every testing group was expressed as the average of the five teeth. A three-way repeated measurement ANOVA (material versus moisture degree versus time) and Tukey’s multiple comparisons test was used to analyze the data at ˛ = 0.05 obtained in both methods. The correlation between the bond strength measurements and silver nitrate uptake of each experimental condition was analyzed by simple linear regression analysis. The strength of the association between these two variables (bond strength measurements versus silver nitrate uptake) was estimated with the Pearson product–moment correlation statistics (˛ = 0.05).
Fig. 2 – Schematic drawing of a resin–dentin interface, showing the areas where the silver nitrate uptake was measured by SEM–EDX.
into metallic silver grains within voids along the bonded interface. All sticks were wet-polished with 600 SiC paper to remove the nail varnish. Then, the specimens were placed inside an acrylic ring, which was attached to a double-sided adhesive tape, and embedded in epoxy resin. After the epoxy resin had set, the thickness of the embedded specimens was reduced to approximately half by grinding with silicon-carbide papers under running water. Specimens were polished with a 1000grit SiC paper and 6, 3, 1 and 0.25 m diamond paste (Buehler Ltd., Lake Bluff, IL, USA) using a polish cloth. They were ultrasonically cleaned, air-dried, mounted on stubs, and carbon coated (MED 010, Balzers Union, Balzers, Liechtenstein). Resin–dentin interfaces were analyzed in a field-emission scanning electron microscope operated in the backscattered electron mode and using energy dispersive X-ray spectrometry (EDX) (LEO 435 VP, LEO Electron Microscopy Ltd., Cambridge, UK). The amount of silver nitrate within the adhesive layer, hybrid layer and resin tags, in each stick, was measured with EDX in three regions (5 m × 5 m) of the bonded stick (left, center and right). The total length of the hybrid layer scanned for silver nitrate uptake measurement was approximately 75 m2 . The silver nitrate uptake was expressed as a percentage of the total area evaluated (Fig. 2).
2.6.
Data treatment
The average BS of each tooth was calculated and expressed as an index that assumes the relative contribution of the different failure modes. The value attributed to specimens that failed prematurely during preparation and could not be tested was arbitrary and corresponded to approximately half of the minimum bond strength value that could be measured in this study [3]. The formula assumed the cohesive strength of the resin composite (CR ) as the average value of all specimens (from a single tooth) that failed in the resin composite. Similarly, the cohesive strength of dentin (CD ) was calculated. The BS index
3.
Results
The remaining dentin thickness (RDT) for all specimens ranged from 2.3 to 2.7 mm. Regression analysis between RDT and BS revealed no influence of RDT in bond strengths. The mean cross-sectional area ranged from 0.78 to 0.82 mm2 and no differences among the treatment groups were detected (p > 0.05).
3.1.
Resin–dentin microtensile bond strength
The overall bond strength values for the experimental groups are expressed in Table 2. No stick, from the SC adhesive, survived the 24-month water storage and this material was not included in the statistical model. The interaction adhesive × moisture degree × time was statistically significant (p < 0.05). While SB achieved higher BS at 0 and 2.5 l of water, OS bond strength was higher at 2.5 and 4.0 l of water in the immediate time (Table 2). After 24 months of water storage, significant reductions in BS were observed for SB and OS when
Table 2 – Means and standard deviations (MPa) of bond strength indexes for each moisture degree at each time period Adhesive systems/ moisture (l)
Time Immediate
24 months
SB 0 2.5 4.0
26.1 (1.1) a,b 32.6 (1.5) a 21.3 (1.2) a,b
5.3 (4.9) c 21.8 (7.2) a,b 7.5 (6.5) c
OS 0 2.5 4.0
18.0 (1.9) b 32.0 (2.1) a 31.4 (6.2) a
5.8 (3.9) c 19.8 (7.6) a,b 13.5 (5.3) b,c
SCa 0 2.5 4.0
24.0 (3.6) 24.3 (0.7) 12.5 (2.6)
0.0 0.0 0.0
Groups with the same letter are not significantly different using Tukey’s test (p > 0.05). a
The bond strength values of this material were not included in the ANOVA model.
866
d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 862–870
Table 3 – Means and standard deviations (%) of silver penetration for each adhesive system in each moisture degree at each time period
Table 4 – Means, standard deviations (%) and statistical significance of silver penetration for each adhesive system at each moisture degree
Adhesive systems/ moisture (l)
Moisture (l)
Time Immediate 5.1 (1.8) 7.8 (2.5) 3.0 (2.2)
46.9 (4.7) 35.1 (2.6) 50.9 (5.8)
OS 0 2.5 4.0
11.1 (6.7) 2.0 (3.4) 9.3 (6.4)
57.5 (10.1) 33.5 (8.9) 49.6 (6.3)
SCa 0 2.5 4.0 a
13.4 (9.6) 9.9 (6.7) 10.8 (9.0)
SB
24 months
SB 0 2.5 4.0
0.0 0.0 0.0
The silver penetration values of this material were not included in the ANOVA model.
the moisture degree was set at the extreme conditions of moisture (i.e. 0 and 4.0 l) (p < 0.05) (Table 2). No significant reduction in BS was observed when the moisture degree was set at 2.5 l, regardless of the adhesive system employed (Table 2).
Adhesive systems
0 2.5 4.0
26.0 (22.5) a,b 21.4 (15.4) a 27.0 (25.2) a,b
OS 34.3 (24.8) b,c 18.0 (20.6) a 29.5 (23.4) b,c
Groups with the same letter are not significantly different using Tukey’s test (p > 0.05).
The overall silver penetration percentages for the experimental groups are expressed in Table 3. No stick, from the SC adhesive, survived the 24-month water storage and this material was not included in the statistical model. A significant effect of moisture degree on the silver nitrate penetration was observed among the materials (p = 0.01). The lowest percentages of silver nitrate occurred in the wet condition, for SB and OS (Table 4). A significant interaction between the moisture degree and the storage periods was also detected (p = 0.0001). The silver nitrate penetration in the immediate period was similar for all moisture conditions (Figs. 3 and 4), regardless the adhesive employed. After 24 months the silver nitrate pen-
Fig. 3 – Backscattered SEM images of the interface bonded with Single Bond to dentin.
d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 862–870
867
Fig. 4 – Backscattered SEM images of the interface bonded with One Step to dentin.
Table 5 – Means, standard deviations (%) and statistical significance of silver penetration for each moisture degree at each time period Time
Moisture (l) 0
Immediate 24 months
8.1 (4.3) a 52.2 (7.4) c
2.5 4.9 (3.0) a 34.3 (5.8) b
component and the moisture degree on the dentin surface, which lead us to reject the null hypothesis of the study. Although not statistically significant in the immediate time, SB and SC showed high bond strength means when the moisture degree was set at 0 or 2.5 l of water. Other previous
4.0 6.2 (4.3) a 50.3 (6.1) c
Groups with the same letter are not significantly different using Tukey’s test (p > 0.05).
etration increased when compared to baseline measurements (immediate period) (Figs. 3 and 4), however this increase was less pronounced when the moisture degree was set at 2.5 l (Table 5). Regression analysis revealed a linear and negative relationship between the mean bond strength and the mean silver nitrate uptake (Fig. 5) (R2 = 0.87, p = 0.001).
4.
Discussion
The results of this study suggests that the bond strengths of etch&rinse adhesive systems is dependent on the solvent
Fig. 5 – Regression analysis between the mean microtensile bond strength values (MPa) and the mean silver nitrate penetration (%).
868
d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 862–870
investigations have confirmed that water or water/ethanol based systems have higher immediate bond strength when applied in a rather dried surface [3,15]. It is likely that the number of variables involved in this study and the substantial reduction on the bond strengths values after 24 months could explain the lack of statistical significance observed in this study. The water component, in water-based adhesive systems, is able to break the undesirable interpeptide bonds formed between the collagen fibrils when in an overdry condition [25]. Water has a higher solubility parameter for hydrogen bonding (37.3 (J/cm3 )1/2 ) than that formed between the collagen fibrils (19.0 (J/cm3 )1/2 ) [25]. Consequently, the shrunken and stiffened air-dried demineralized dentin [26] can be re-expanded allowing the penetration of resin monomers. Acetone and resin monomers are not capable of re-expanding air-dried demineralized dentin [25,27]. Under overdry conditions, the infiltration ratio of the bonding resin within the hybrid layer for acetone-based systems was reduced by 50% when applied to dry instead of wet dentin [28], which must explain the inferior performance of OS, an acetone-based system. On the other hand, the OS system showed high bond strengths values when the moisture degree was set 4.0 l of water, contrary to what was observed for SB and SC. Presumably the high vapor pressure of acetone (ca. 200 mmHg) in the OS system allows removal of residual water in a higher extent than the other solvents like water (ca 47.1 mmHg) [29] or mixture of water and ethanol presented in the SC and SB adhesives. The more residual water entrapped in the hybrid and adhesive layer, the lower the mechanical properties of the polymer formed reducing the bond strength values. It was already demonstrated that the conversion degree of adhesives is substantially reduced in the presence of water [16,30]. A previous study, that evaluated the management of surface moisture on the durability of resin–dentin bonds, concluded that the specimens bonded to adverse moisture conditions were not more prone to degradation over time [15]. This finding was not corroborated in the present investigation. The differences between these two studies could be due to the differences in the period of evaluation. While the cited study evaluated the durability of resin–dentin bonds after 6 months of water storage [15], the present investigation stored the specimens for 24 months. Actually the present investigation observed reductions on bond strengths for all experimental groups albeit only those bonded to adverse conditions of moisture was statistically different from the immediate values, which explains why no experimental group from the present investigation showed interfaces free of silver nitrate uptake. This situation was even worse for extreme moisture conditions. It was already demonstrated that the infiltration ratio of resin monomers into collapsed demineralized dentin [5,28] and into overwet dentin [5,6] is substantially reduced compared to ideal conditions. For instance, recent studies have shown that collagen collapse means that adhesive does not infiltrate as much as one half of the zone of demineralized dentin [31,32]. Under overwet conditions, phase separation of the adhesive occurs and leads to a very porous hybrid layer characterized by hydrophobic bis-GMA-rich particles distributed in a hydrophilic HEMA-rich matrix [33].
This means that bonding performed under extreme moisture conditions (overdry or overwet conditions) can leave more voids at the base of the hybrid layer not fully penetrated by resin that can act as pathway for extrinsic and intrinsic water penetration over time. Although nanoleakage pathway may be located within the adhesive resin, within the hybrid layer and within partially- or fully-demineralized dentin [13], it seems that porosities in the partially demineralized dentin, not infiltrated by adhesive resin, is the easiest way for penetration of water, since the energy barrier that water molecules must overcome during water uptake is much lower under this circumstance than when water needs to break intermolecular forces established among polymer chains. It is worth mentioning that even when the dentin is adequately rewetted, a full resin infiltration does not occur along the decalcified dentin for etch&rinse adhesive systems, applied according to the time recommended by the manufacturers [28,32,34,35], which explains the silver nitrate uptake under ideal moisture conditions, over a 24-month storage period. As more water is absorbed into the polymer over time, swelling of the resin by hydrogen bonding of water on hydrophilic domains of polymer chains takes place. In a second stage, residual unreacted monomers is eluted from the hybrid layer [36,37] creating new channels for water penetration, through it water diffuses even more in an auto-accelerative manner. The elution of residual and low molecular weight polymers also cause an increase in the porosity within the hybrid layer [13] and an inevitably reduction of the cohesive strength of the bonding resin in the adhesive interface. The lower the cohesive strength of adhesive, the lower the resulting bond strength values [10,38,39], which renders the resin–dentin bond weak and less stable over time [13,15,22,40]. The findings from the silver nitrate uptake from this study add further evidence on this hypothesis. By reference to Table 5, one can observe that after 24 months of water storage, the silver nitrate uptake was higher when compared to the baseline measurements, mainly for extreme conditions of moisture, where a less than optimal hybridization and polymerization could have occurred. Interestingly are the results of the groups bonded under ideal moisture conditions. Although a numerical decrease in the resin–dentin bond strength values occurred after 2 years of water storage, this reduction was not statistically different from the immediate values. From the SEM/EDX evaluation, one could observe that a significant increase in the silver nitrate uptake has occurred for ideal moisture bonded groups, which means that these groups also underwent a degradation process; however, in a slightly low rate compared to extreme moisture conditions. As the use of an air stream does not effectively remove water left in the demineralized dentin, from HEMA/water mixtures [16,29], it is fair to suppose that remaining water and solvents led to sub-polymerization [16]. The lack of specimens from the SC adhesive after 2 years can also be explained by the aforementioned hypothesis. Syntac Single Component is a water-based system and has the lowest vapour pressure among the solvents used in adhesive formulations. Consequently the evaporation of water and solvents from the adhesive after its application on demineralized dentin is rather slow, leading to their entrapment into the adhesive layer and consequent sub-optimal polymerization
d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 862–870
which caused, in turn, the premature failure of these specimens. As previously mentioned, nanoleakage pathways within resin–dentin interfaces is not only those areas of incomplete resin infiltration into demineralized dentin but also the microvoids within the sub-polymerized resin matrix. This is one of the reasons of why two-step etch&rinse adhesives function as permeable membranes [41,42] permitting the diffusion of water molecules from dentin across the adhesive layer. The retention of unbound water, both from residual water that is incompletely evaporated from the adhesive, or from the underlying dentin as a result of the high osmolarity of the hydrophilic adhesive mixture, creates water-filled channels within the adhesive that can be visualized after silver nitrate impregnation. The higher amount of silver nitrate uptake observed after storage in water for 24 months indicates that the permeability of the adhesive layer was increased due to the degradation process caused by water uptake [40,43]. Several studies have attempted to correlate the data from marginal sealing, microleakage and nanoleakage with bond strengths values [19,44–48]. However none of these studies has succeeded in obtaining a significant correlation between these two properties. In this study a negative and linear correlation was observed between these two properties. The main difference observed in this study and the aforementioned ones is that the bonded interfaces were evaluated in the short- and long-term. This allowed time for the degradation process takes place and to promote significant changes in the resin–dentin adhesive interface. One could speculate that the highly significant correlation between decreases in bond strength and increases in silver nitrate uptake reflect degradation at the resin–dentin interfaces. Okuda et al. [17] also observed an inverse relationship between bond strength and silver nitrate uptake for the Clearfil Liner Bond 2V adhesive system mainly when the 9 month’s specimens were evaluated. However, this matter still deserves further attention since another study that also evaluated the durability of bonded interfaces by means of bond strength measurements and silver nitrate uptake has not observed any correlation between these two properties [40]. Although the wet bonding has been advocated for etch&rinse adhesive systems, this technique does not avoid the degradation process that occurs to highly hydrophilic monomers included in the composition of simplified etch&rinse adhesives. New material’s formulations should focus on the use of adhesives with more hydrophobic features [49], which seems to be less prone to the damage caused by water sorption over time. Further studies should be conducted in order to evaluate the hypothesis arose in this study.
5.
Conclusions
Higher bond strength values and a significantly lower nitrate uptake were observed for the immediate specimens, for all adhesives tested. Under the ideal moisture condition, the bond strength reduction and the silver nitrate uptake was less pronounced than in the extreme moisture conditions, however it did not avoid the degradation process that adhesives interfaces are prone to.
869
Acknowledgments This study was supported, in part, by School of Dentistry, University of Oeste de Santa Catarina and by CNPq (302552/030, 551043/02-2, 474226/03-4 and 300481/95-0) and FAPESP (01/06140-1 and 02/06682-1).
references
¨ [1] Jacobsen T, Soderholm KJ. Effects of primer solvent, primer agitation, and dentin dryness on shear bond strength to dentin. Am J Dent 1998;11:225–8. [2] Van Meerbeek B, Yoshida Y, Lambrechts P, Vanherle G, Duke ES, Eick JD, et al. A TEM study of two water-based adhesive systems bonded to dry and wet dentin. J Dent Res 1998;77:50–9. [3] Reis A, Loguercio AD, Azevedo CLN, Carvalho RM, Singer JM, Grande RHM. Moisture spectrum of demineralized dentin for different solvent-based adhesive system. J Adhes Dent 2003;5:183–92. [4] Nakajima M, Kanemura N, Pereira PNR, Tagami J, Pashley DH. Comparative microtensile bond strength and SEM analysis of bonding to wet and dry dentin. Am J Dent 2000;13: 324–8. [5] Tay FR, Gwinnett JA, Wei SH. Relation between water content in acetone/alcohol-based primer and interfacial ultrastructure. J Dent 1998;26:147–56. [6] Tay FR, Gwinnett JA, Wei SH. The overwet phenomenon: a transmission electron microscopic study of surface moisture in the acid-conditioned, resin–dentin interface. Am J Dent 1996;9:161–6. [7] DeMunck J, Van Meerbeek B, Yoshida Y, Inoue S, Vargas M, Suzuki K, et al. Four-year water degradation of total-etch adhesives bonded to dentin. J Dent Res 2003;82:136–40. [8] Koshiro K, Inoue S, Tanaka T, Koase K, Fujita M, Hashimoto M, et al. In vivo degradation of resin–dentin bonds produced by a self-etch vs. a total-etch adhesive system. Eur J Oral Sci 2004;112:368–75. ¨ [9] Frankenberger R, Strobel W, Lohbauer U, Kramer N, Petschelt A. The effect of six years of water storage on resin composite bonding to human dentin. J Biomed Mater Res Part B: Appl Biomater 2004;69B:25–32. [10] Reis A, Grandi V, Carlotto L, Bortoli G, Patzlaff R, Accorinte MLR, et al. Effect of smear layer thickness and acidity of self-etching solutions on early and long-term bond strength to dentin. J Dent 2005;33:549–59. [11] Loguercio AD, Uceda-Gomez N, de Oliveira Carrilho MR, Reis A. Influence of specimen size and regional variation on long-term resin–dentin bond strength. Dent Mater 2005;21:224–31. [12] Hashimoto M, Ohno H, Kaga M, Sano H, Endo K, Oguchi H. In vivo degradation of resin–dentin bonds in humans over 1 to 3 years. J Dent Res 2000;79:1385–91. [13] Sano H, Yoshikawa T, Pereira PNR, Kanemura N, Morigami M, Tagami J, et al. Long-term durability of dentin bonds made with a self-etching primer, in vivo. J Dent Res 1999;78:906–11. [14] Carrilho MROMR, Tay FR, Pashley DH, Tjaderhane L, Carvalho RM. Mechanical stability of resin–dentin bond components. Dent Mater 2005;21:232–41. [15] Reis A, Loguercio AD, Carvalho RM, Grande RHM. Durability of resin dentin interfaces: effects of surface moisture and adhesive solvent component. Dent Mater 2004;20:669–76. [16] Paul SJ, Leach M, Rueggeberg FA, Pashley DH. Effect of water content on the physical properties of model dentine primer and bonding resins. J Dent 1999;27:209–14.
870
d e n t a l m a t e r i a l s 2 3 ( 2 0 0 7 ) 862–870
[17] Okuda M, Pereira PN, Nakajima M, Tagami J, Pashley DH. Long-term durability of resin dentin interface: nanoleakage vs. microtensile bond strength. Oper Dent 2002;27:289–96. [18] Li H, Burrow MF, Tyas MJ. The effect of load cycling on the nanoleakage of dentin bonding systems. Dent Mater 2002;18:111–9. [19] Hashimoto M, De Munck J, Ito S, Sano H, Kaga M, Oguchi H, et al. In vitro effect of nanoleakage expression on resin–dentin bond strengths analyzed by microtensile bond test, SEM/EDX and TEM. Biomaterials 2004;25:5565–74. [20] De Wald JP. The use of extracted teeth for in vitro bonding studies: a review of infection control considerations. Dent Mater 1997;13:74–81. [21] Asmussen E, Peutzfeldt A. The influence of relative humidity on the effect of dentin bonding systems. J Adhes Dent 2001;3:123–7. [22] Burrow MF, Sato HM, Tagami J. Dentin bonding durability after three years using a dentin bonding agent with and without priming. Dent Mater 1996;12:302–7. [23] Kitasako Y, Burrow MF, Nikaido T, Tagami J. The influence of storage on dentin bond durability of resin cement. Dent Mater 2000;16:1–16. [24] Tay FR, Pashley DH, Yoshiyama M. Two modes of nanoleakage expression in single-step adhesives. J Dent Res 2002;81:472–6. [25] Pashley DH, Agee KA, Nakajima M, Tay FR, Carvalho RM, Terada RS, et al. Solvent-induced dimensional changes in EDTA-demineralized dentin matrix. J Biomed Mater Res 2001;56:273–81. [26] Maciel KT, Carvalho RM, Ringle RD, Preston CD, Russel CM, Pashley DH. The effects of acetone, ethanol, HEMA, and air on the stiffness of human decalcified dentin matrix. J Dent Res 1996;11:1851–8. [27] Pashey DH, Carvalho RM, Tay FR, Agee KA, Lee KW. Solvation of dried dentin matrix by water and other polar solvents. Am J Dent 2002;15:97–102. [28] Hashimoto M, Ohno H, Kaga M, Sano H, Endo K, Oguchi H. Extent to which resin can infiltrate dentin by acetone-based adhesives. J Dent Res 2002;81:74–8. [29] Pashley EL, Zhang Y, Lockwood PE, Rueggeberg FA, Pashley DH. Effects of HEMA on water evaporation from water–HEMA mixtures. Dent Mater 1998;14:6–10. ¨ [30] Jacobsen T, Soderholm KJ. Some effects of water on dentin bonding. Dent Mater 1995;11:132–6. [31] Wieliczka DM, Kruger MB, Spencer P. Raman imaging of dental adhesive diffusion. Appl Spectrosc 1997;51: 1593–6. [32] Spencer P, Swafford JR. Unprotected protein at the dentin–adhesive interface. Quintessence Int 1999;30: 501–7. [33] Spencer P, Wang Y. Adhesive phase separation at the dentin interface under wet bonding conditions. J Biomed mater Res 2002;62:447–56.
[34] Spencer P, Wang Y, Walker MP, Wieliczka DM, Swafford JR. Interfacial chemistry of the dentin/adhesive bond. J Dent Res 2000;79:1458–63. [35] Miyasaki M, Onose H, Moore BK. Analysis of the dentin–resin interface by use of laser Raman spectroscopy. Dent Mater 2002;18:576–80. [36] Hashimoto M, Ohno H, Sano H, Kaga M, Oguchi H. In vitro degradation of resin–dentin bonds analyzed by microtensile bond test, scanning and transmission electron microscopy. Biomaterials 2003;24:3795–803. [37] Hashimoto M, Ohno H, Sano H, Kaga M, Oguchi H. Degradation patterns of different adhesives and bonding procedures. J Biomed Mater Res B: Appl Biomater 2003;66:324–30. [38] Pashley DH, Ciucchi B, Sano H, Carvalho RM, Russell CM. Bond strength versus dentine structure: a modelling approach. Arch Oral Biol 1995;40:1109–18. [39] Takahashi A, Sato Y, Uno S, Pereira PNR, Sano H. Effects of mechanical properties of adhesives on bond strength to dentin. Dent Mater 2002;18:263–8. [40] Okuda M, Pereira PNR, Najakima M, Tagami J. Relationship between nanoleakage and long-term durability of dentin bonds. Oper Dent 2001;26:482–90. [41] Tay FR, Pashley DH. Water treeing—a potential mechanism for degradation of dentin adhesives. Am J Dent 2003;16:6–12. [42] Tay FR, Frankenberger R, Krejci I, Bouillaguet S, Pashley DH, Carvalho RM, et al. Single-bottle adhesives behave as permeable membranes after polymerization. I. In vivo evidence. J Dent 2004;32:611–21. [43] Li HP, Burrow MF, Tyas MJ. The effect of long-term storage on nanoleakage. Oper Dent 2001;26:609–16. [44] Guzman-Ruiz S, Armstrong SR, Cobb DS, Vargas MA. Association between microtensile bond strength and leakage in the indirect resin composite/dentin adhesively bonded joint. J Dent 2001;29:145–53. [45] Pereira PN, Okuda M, Nakajima M, Sano H, Tagami J, Pashley DH. Relationship between bond strengths and nanoleakage: evaluation of a new assessment method. Am J Dent 2001;14:100–4. [46] Loguercio AD, Reis A, Ballester RY. Polymerization shrinkage: effects of constraint and filling technique in composite restorations. Dent Mater 2004;20:236–43. [47] Kenshima S, Reis A, Uceda-Gomez N, Tancredo LLF, Filho LE, Nogueira FN, et al. Effect of smear layer thickness and pH of self-etching adhesive systems on the bond strength and gap formation to dentin. J Adhes Dent 2005;7:117–26. [48] Cenci MS, Demarco FF, Carvalho RM. Class II composite resin restorations with two polymerization techniques: relationship between microtensile bond strength and marginal leakage. J Dent 2005;33:603–10. [49] Yiu CK, King NM, Pashley DH, Suh BI, Carvalho RM, Carrilho MR, et al. Effect of resin hydrophilicity and water storage on resin strength. Biomaterials 2004;25:5789–96.