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Four-year Water Degradation of Total-etch Adhesives Bonded to Dentin

¹ Leuven BIOMAT Research Cluster, Department of Conservative Dentistry, School of Dentistry, Oral Pathology and Maxillo-Facial Surgery, Catholic University of Leuven, Kapucijnenvoer 7, B-3000 Leuven, Belgium;
² Department of Biomaterials, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8525, Japan;
³ Department of Oral Health Science, Hokkaido University Graduate School of Dental Medicine, Kita 13 Nishi 7, Kita-ku, Sapporo 060-8586, Japan; and
⁴ Department of Operative Dentistry, The University of Iowa, Iowa City, USA;

*corresponding author, bart.vanmeerbeek{at}med.kuleuven.ac.be

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Resin-dentin bonds degrade over time. The objective of this study was to evaluate the influence of variables like hybridization effectiveness and diffusion/elution of interface components on degradation. Hypotheses tested were: (1) There is no difference in degradation over time between two- and three-step total-etch adhesives; and (2) a composite-enamel bond protects the adjacent composite-dentin bond against degradation. The micro-tensile bond strength (µTBS) to dentin of 2 three-step total-etch adhesives was compared with that of 2 two-step total-etch adhesives after 4 years of storage in water. Quantitative and qualitative failure analyses were conducted correlating Fe-SEM and TEM. Indirect exposure to water did not significantly reduce the µTBS of any adhesive, while direct exposure resulted in a significantly reduced µTBS of both two-step adhesives. It is concluded that resin bonded to enamel protected the resin-dentin bond against degradation, while direct exposure to water for 4 years affected bonds produced by two-step total-etch adhesives.

KEY WORDS: adhesion • dentin • total-etch • bond strength • durability

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Most current total-etch adhesives perform well in bond strength tests, at least when tested shortly after application and under controlled in vitro conditions (Inoue et al., 2001). However, the oral cavity—with temperature changes, chewing loads, and chemical attacks by acids and enzymes—forms a rather severe challenge for tooth-composite bonds to survive for a reasonably long time. Clinically, marginal deterioration of composite restorations remains problematic and forms the major reason that dramatically shortens the lifetime of adhesive restorations (Van Meerbeek et al., 1998a). A factor known to degrade tooth-composite bonds is exposure to water (Gwinnett and Yu, 1995; Sano et al., 1999; Armstrong et al., 2001b). Among different forms of marginal leakage, nano-leakage, or the ingress of oral fluids through nanometer-sized channels along collagen fibrils within the hybrid layer, is considered very detrimental to bond integrity (Sano et al., 1995; Hashimoto et al., 2000, 2002). As part of a total-etch procedure, the rather aggressive phosphoric-acid-etching nearly completely deprives collagen of hydroxyapatite (Van Meerbeek et al., 1998b). Consequently, adequate infiltration into, wetting of, and molecular interaction with hydroxyapatite-depleted collagen by resin monomers is challenging. It may result in incomplete hybridization, leaving collagen unprotected and vulnerable to hydrolytic degeneration (Hashimoto et al., 2000). Other degradation-promoting factors are, e.g., residual solvent of the adhesive or insufficiently removed surface water. Eventually, resin itself degrades over time and leaches out, causing the restoration-tooth bond to deteriorate (Santerre et al., 2001).

The objective of this laboratory study was to test the hypotheses that: (1) two-step total-etch adhesives resist water degradation as well as do three-step total-etch adhesives, and that (2) an adjacent composite-enamel bond protects the composite-dentin bond against degradation. Therefore, the micro-tensile bond strength (µTBS) to dentin of 2 three-step total-etch adhesives was compared with that of 2 two-step total-etch adhesives after 4 yrs of storage in water. Quantitative and qualitative failure analysis was conducted correlating field-emission scanning (Fe-SEM) and transmission electron microscopy (TEM).

	MATERIALS & METHODS

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Micro-tensile Bond Strength Testing
Twenty-eight non-carious human third molars (gathered following informed consent approved by the Commission for Medical Ethics of the Catholic University of Leuven) were stored in 0.5% chloramine in water at 4°C and used within 1 mo after extraction. The occlusal third of the molar crowns was removed by means of an Isomet diamond saw (Isomet 1000, Buehler, Lake Bluff, IL, USA). We produced a standard smear layer by wet-sanding (60 sec) the dentin surface with 600-grit silicon-carbide paper. All specimens were randomly divided into 4 groups of 7 teeth each, and subjected to a bonding treatment strictly according to the manufacturer’s instructions, with 2 two-step total-etch adhesives, Optibond Solo (Kerr, Orange, CA, USA) and Scotchbond 1 (Single Bond outside Europe, 3M ESPE, St. Paul, MN, USA), and their three-step precursors, respectively, Optibond Dual-Cure (Kerr) and Scotchbond Multi-Purpose (3M ESPE). The surface was built up with resin composite (Z100, 3M ESPE) in 3 or 4 layers to a height of 5 to 6 mm. From each group, 3 teeth were subjected to µTBS-testing after 24 hours’ storage in water (indirect exposure of resin-dentin interface, 24hr-IE) at 37°C. The 4 remaining teeth were stored for 4 yrs at 37°C in water that contained 0.5% chloramine to prevent bacterial growth (Burrow et al., 1996). Prior to water storage, 2 of these teeth were sectioned in half for direct exposure of the resin-dentin interface to water (4yr-DE). The remaining teeth were kept intact, with the resin-dentin interface entirely surrounded by resin bonded to the outer enamel rim, and consequently only indirectly exposed to water (4yr-IE). After storage, the teeth were sectioned perpendicular to the adhesive-tooth interface, by means of the Isomet saw, yielding rectangular sticks (2 x 2 mm wide; 8-9 mm long). Specimens were trimmed at the biomaterial-tooth surface to a cylindrical hourglass shape (diameter of about 1.2 mm), by means of the MicroSpecimen Former (De Munck et al., 2003) and fine cylindrical diamond burs (835KREF, Komet, Lemgo, Germany) under continuous air/water spray. Specimens were then fixed to Ciucchi’s jig with cyanoacrylate glue (Model Repair II Blue, Dentsply-Sankin, Ohtawara, Japan) and stressed at a crosshead speed of 1 mm/min until failure in an LRX testing device (LRX, Lloyd, Hampshire, UK). The µTBS was expressed in MPa, as we derived from dividing the imposed force (N) at the time of fracture by the bond area (mm²). The means were evaluated by a two-way ANOVA test, with the type of product and the degree of water exposure as predicting factors. An additional random factor, with the µTBS-samples from the same tooth grouped, was added to the statistical model as correction for the multiple samples gathered from one tooth. The means of all groups were compared by a Tukey-Kramer multiple-comparisons test. All statistical analyses were carried out with Statistica software (StatSoft, Tulsa, OK, USA).

Failure Analysis
All µTBS-specimens exhibiting mixed adhesive-cohesive failures were processed for Fe-SEM (Philips XL30, Eindhoven, The Netherlands) by common specimen-processing procedures, including fixation, dehydration, chemical drying, and gold-sputter-coating (Perdigão et al., 1995). The proportional prevalence of different fracture modes was determined for each µTBS-specimen by image analysis (Image Pro Plus, Media Cybernetics, Silver Spring, MD, USA) applied to digitally recorded Fe-SEM images. For statistical analysis, only the percentage of adhesive failures was taken into account. The data were re-ordered dichotomously and evaluated by logistic regression with the type of product and the degree of water exposure as predicting factors. We also used logistic regression to test for association of µTBS and failure mode.

After Fe-SEM, representative µTBS samples of each adhesive were further processed for TEM. The µTBS samples were immersed for 12 hrs in epoxy resin prior to being embedded in molds (Robinson and Gray, 1996). Non-demineralized 70- to 90-nm sections through the fracture plane were cut by means of a diamond knife (Diatome, Bienne, Switzerland) in an ultramicrotome (Ultracut UCT, Leica, Vienna, Austria). For evaluation of collagen, TEM sections were positively stained with 5% uranyl acetate (UA) for 20 min and saturated lead citrate (LC) for 3 min prior to TEM examination (Philips CM10, Eindhoven, The Netherlands).

	RESULTS

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The mean µTBSs are graphically presented per experimental group in Fig. 1. After 24 hrs of indirect water exposure (24hr-IE), the µTBS of the three-step total-etch adhesive OptiBond Dual Cure to dentin (53.6 ± 8.2 MPa)(mean ± standard deviation) was significantly (p < 0.001) higher than that of the two-step successor OptiBond Solo (34.8 ± 9.7 MPa). No significant difference (p > 0.1) in 24h-µTBS was found between the two-step adhesive Scotchbond 1 (52.2 ± 9.1 MPa) and the three-step precursor Scotchbond Multi-Purpose (45.6 ± 11.1 MPa). Indirect exposure of the resin-dentin interface to water for 4 yrs (4yr-IE) did not significantly decrease the µTBS of all 4 adhesives. However, in the two-way ANOVA model, both the variables (water exposure and type of adhesive) significantly (p < 0.0001) influenced the µTBS to dentin. The p-value for the interaction term of the model was also significant (p < 0.0001), indicating that not all 4 adhesives were sensitive to the same degree of degradation. Direct exposure to water did not significantly (p = 0.62) affect the µTBS of OptiBond Dual Cure, while the decrease in µTBS of Scotchbond Multi-Purpose was nearly significant (p = 0.069): The µTBS (4yr-DE) of both two-step total-etch adhesives dropped significantly (Fig. 1).

View larger version (13K): [in this window] [in a new window]   Figure 1. Mean µTBS to dentin of the three- and two-step total-etch adhesives investigated for each level of water exposure (mean and 95% confidence interval corrected for the multiple samples gathered from the same tooth).

View larger version (68K): [in this window] [in a new window]   Figure 2. Graphed presentation of proportional prevalence of fracture modes for all experimental groups. *All failures cohesive in the bonding layer and resin composite were combined.

View larger version (144K): [in this window] [in a new window]   Figure 3. Fractographic failure analysis of Scotchbond MP and Scotchbond 1 (3M Espe). (a) Fe-SEM overview photomicrographs of the fracture surfaces (left = dentin side; right = composite counterpart) of a representative µTBS sample prepared with Scotchbond MP that was stored for 4 yrs with the resin-dentin interface directly exposed to water (4yr-DE). Scratches remaining from smear-layer preparation confirmed that the interface failed adhesively (A) at the level between dentin and the bonding layer for an area of 0.84 mm2 or 84% of the total surface area. A small area of 0.16 mm2 or 16% of the total surface area represents a cohesive (C) failure in the bonding resin. (b) Magnification of the adhesive failure area at the composite side of the same sample as in (a) shows a typical pattern of islands of hybrid layer (H) fragments still attached to the composite (Comp.) and detached from dentin. (c) TEM photomicrograph (non-demineralized, unstained section) of the adhesive failure area sectioned from the same sample as in (a). The thin black line covering the fracture plane (arrows) and underneath the embedding resin (E) represents the gold coating applied for the Fe-SEM examination conducted beforehand. The hybrid layer (H) was pulled from unaffected dentin (U) either at the base (left) or close to the top (right). (d) High-magnification Fe-SEM photomicrograph of the composite site of a fractured four-year-stored Scotchbond 1 sample with direct exposure of the interface to water (4yr-DE). The sample failed within the hybrid layer, part of which remained attached to the composite. A resin tag (T) within a dentinal tubule is surrounded by loosely organized collagen fibrils (Coll.), the typical cross-banding of which can be observed. This suggests either that this hybrid layer collagen was inadequately enveloped by resin or that resin was eluted.

View larger version (135K): [in this window] [in a new window]   Figure 4. Fractographic failure analysis of Optibond DC and Optibond Solo (Kerr). (a). Fe-SEM overview photomicrographs of the fracture surfaces (left = dentin side, right = composite counterpart) of a representative µTBS sample prepared with Optibond Dual Cure that was stored for 4 yrs with the resin-dentin interface indirectly exposed to water (4yr-IE). A small area of 0.11 mm2 or 10% of the total surface area represents an adhesive (A, marked with black line) failure, while the major part (1.0 mm2 or 90% of the total surface area) failed cohesively (C) in resin. (b) TEM photomicrograph (non-demineralized, stained section) of a typical failure of Optibond Dual Cure after 4 yrs of water storage (4yr-DE). A thin layer of a few µm of the particle-filled adhesive resin remained attached to the hybrid layer (H), indicating that the adhesive layer failed cohesively. Although this section was stained by heavy metals (UA/LC), collagen seemed not to have picked up much of the staining solution. E = embedding resin; T = resin tag (T) packed with filler; U = unaffected dentin; arrows = gold coating. (c) TEM photomicrograph (non-demineralized, unstained section) of a sample that was prepared with Optibond solo and stored for 4 yrs with the resin-dentin interface directly exposed to water (4yr-DE). The hybrid layer (H) remained attached to unaffected dentin (U). E = embedding resin; T = resin tag (T). (d) TEM photomicrograph (non-demineralized, unstained section) of the same sample as in (c), but now at a site were the sample failed at the base of the hybrid layer. Small hybrid-layer fragments (arrows) remained attached to unaffected dentin (U). The resin tag (T) was fractured at the same level. E = embedding resin.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

Direct exposure to water resulted in a significant decrease of the µTBS of only the two- but not the three-step total-etch adhesives. Consequently, the first hypothesis was rejected, with the adhesives involving simplified application to perform significantly worse on a long-term perspective. The decrease in µTBS was in accordance with the increase in percentage of adhesive failures. While the µTBS of Optibond Dual Cure remained quite stable despite the water storage, fewer than 8% of the failure modes were recorded as adhesive. Cohesive failure of the adhesive resin above the hybrid layer typically occurred. The superior results obtained with Optibond Dual Cure were not unexpected, since this adhesive performed repeatedly favorably in several laboratory (Pilo and Ben-Amar, 1999; Armstrong et al., 2001a, b; Inoue et al., 2001; Meiers and Young, 2001; De Munck et al., 2003) as well as clinical trials (Boghosian, 1996; Van Meerbeek et al., 2001). Among other as-yet-unknown features, the three-step application procedure with a low technique-sensitive application of, successively, etchant, primer, and adhesive (Van Meerbeek et al., 2001), the apparent favorable composition with regard to hybridization efficiency (Van Meerbeek et al., 1996), the particle-filled adhesive providing elastic shock-absorbing potential (Van Meerbeek et al., 1993), the formation of a separate coupling resin layer, and the lower hydrophilicity of the cured resin as compared with the two-step version may have resulted in this low sensitivity to water degradation.

The µTBS of all other adhesives dropped when their respective interfaces with dentin were directly exposed to water during 4 yrs. The percentage of adhesive failures significantly increased accordingly. Although the basic ingredients between the three-step adhesive Optibond Dual Cure and the two-step adhesive Optibond Solo are comparable, the simplified application procedure with the less-concentrated combined primer/adhesive resin appeared to make the Optibond-Solo-produced hybrid layers more sensitive to aging. TEM disclosed adhesive failures to prevail at different depths within the hybrid layer. The results are in total agreement with those from a previous ultra-morphological study (Van Meerbeek et al., 1999) that revealed that Optibond Dual Cure more uniformly and completely infiltrated the collagen fibril network, in contrast to Optibond Solo. Such less-optimal hybridization might explain, to a large extent, why the hybrid layer produced by Optibond Solo is more prone to degradation than that produced by Optibond Dual Cure.

The µTBS of the two-step total-etch adhesive Scotchbond 1 decreased significantly more than that of its three-step precursor in cases of direct exposure to water. However, in contrast to Optibond Dual Cure, the µTBS of the Scotchbond Multi-purpose to dentin was also reduced, thereby approaching a statistically significant difference. For both adhesives, this effect should be partly attributed to the incorporation of a high-molecular-weight (MW) polyalkenoic acid copolymer. Previously, phase separation was shown to occur with the copolymer being filtered out by the collagen network and deposited as a distinct gel on the exposed collagen network (Van Meerbeek et al., 1996; Eliades et al., 2001). In the extreme case, the gel hinders adequate resin-interdiffusion, by which the hybrid layer would be constituted of collagen infiltrated by the low-MW 2-hydroxyethylmethacrylate (HEMA) that was polymerized to linear poly-HEMA chains, and any residual water (solvent) that was insufficiently removed. Indeed, analysis of the failure planes showed abundant, unprotected collagen fibrils.

Worth mentioning is also the reduced stainability of TEM sections. In positively stained sections, the heavy metal stain (UA/LC) binds to regions along the collagen fibril that are rich in polar amino acids. The staining pattern reflects the summation of charged residues along the fibril (Weiss, 1988). The reduced stainability of collagen in the hybrid layer may then reflect a decreased quantity of polar groups caused by degeneration of collagen during the four-year water storage. Accordingly, a further in-depth study on degradation of collagen as well as degradation/leaching of resin from the interface is required.

The second hypothesis could not be rejected. Direct exposure to water significantly affected bond integrity (at least for the two-step adhesives), while the effect of indirect exposure was negligible for all 4 adhesives tested. This must be attributed to the retarding role of the longer diffusion path in the indirect-exposure groups, and/or to the protective role of the surrounding resin-enamel bond against degradation. The sealing effect at enamel must have been most determining based on the following: First, no significant difference in bond strength (p < 0.05) was found for the 4yr-IE samples at areas closer to the enamel rim (outer sample area closest to the water source) and at the central area (most remote from the water source) for all adhesives tested. This means that the length of diffusion must have been less important than the protection gathered from bonding to surrounding enamel. One could argue that regional differences in bond strength (peripheral vs. mid-coronal dentin) must have been involved as well; however, these are considered negligible (Tay et al., 2000). Second, four years is long enough to expect diffusion to have occurred throughout the entire sample. Third, for the 4yr-DE samples, a tendency existed to higher bond strengths at areas more remote from the exposure plane (no statistical analysis was done due to small sample size). This suggests that, in the absence of enamel bonding, diffusion may play a more significant role. Last, even in cases where diffusion is involved, the difference in long-term bonding performance between the three- and the two-step adhesives remains.

This means that, in the clinical situation, one can rely on durable dentin bonding using three- or two-step total-etch adhesives if all cavity margins are located in enamel. For cavities with margins ending in dentin, three-step total-etch adhesives are preferred.

In conclusion: (1) The resin-dentin bond formed by total-etch adhesives is prone to water degradation; (2) two-step total-etch adhesives are more susceptible to water degradation than three-step total-etch adhesives; and (3) a surrounding resin-enamel bond protects the resin-dentin interface against water degradation.

	ACKNOWLEDGMENTS

 
This study was supported in part by a Research Grant from the Fund for Scientific Research-Flanders (F.W.O.-grant "Krediet aan Navorsers" 1.5.054.99) and by a fund of the Toshio Nakao Chair for Adhesive Dentistry inaugurated at the Catholic University of Leuven, with G. Vanherle awarded as Chairholder. We thank Kerr and 3M Espe for the generous donation of materials. A preliminary report was presented at the IADR General Session in San Diego, CA, March, 2002.

Received May 1, 2002; Last revision August 29, 2002; Accepted October 24, 2002

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