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RESEARCH REPORTS |
1 Faculty of Dentistry, Selçuk University, Konya, Turkey;
2 Department of Oral Biology and Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, GA 30912-1129, USA; and
3 Pediatric Dentistry and Orthodontics, Faculty of Dentistry, The University of Hong Kong, Pokfulam, 34 Hospital Road, Hong Kong SAR, China;
* corresponding author, kfctay{at}netvigator.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: self-etch adhesives • aging • microtensile bond strength • water trees
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Although in vivo studies on the degradation of resin-dentin bonds are scanty, they generally support in vitro reports that progessive decreases in microtensile bond strengths occurred after aging. To date, there is only one in vivo human study on bond degradation in a total-etch adhesive (Hashimoto et al., 2000). With the increase in acceptance of self-etch adhesives, bond durability in adhesives that etch and prime simultaneously is a clinically significant issue that warrants confirmation in in vivo human studies. An in vivo long-term study reported an increase in interfacial porosity in monkey teeth bonded with a self-etching primer (Sano et al., 1999). Similarly, increases in porosity within hybrid layers were identified after in vitro and in vivo aging of teeth bonded by 2 self-etching primers in monkeys (Takahashi et al., 2002). The exhibition of voids provided significant clues for the leaching of resinous components from bonded interfaces. However, the morphologic correlates that precipitate this increase in porosity within self-etch adhesives were beyond the limits of the instrumentation used in the 2 in vivo primate studies, and require further elaboration at an ultrastructural level.
Fluoride-releasing self-etch adhesives were found to exhibit the potential to remineralize exposed demineralized dentin that is not completely encapsulated by adhesive resins (Itota et al., 2003). Another study that examined in vitro durability of a fluoride-free vs. a fluoride-containing, antibacterial, two-step, self-etch adhesive to human dentin reported a decrease in bond durability only in the fluoride-free, but not in the fluoride-containing, adhesive after 6 mos of water storage (Nakajima et al., 2003). It is of clinical significance to see if these favorable in vitro results reported for the fluoride-releasing self-etching primer/adhesive can be confirmed in an in vivo human study. Thus, the objectives of this study were: (1) to evaluate long-term in vivo and in vitro bond durability of 2 self-etch adhesive systems (fluoride-releasing, antibacterial monomer-containing, and fluoride-free) using microtensile bond testing, and (2) to characterize morphological changes in the resin-dentin bond structures aged in the oral environment and under laboratory conditions for 1 yr. The null hypothesis tested was that there is no difference in the mechanism of degradation of self-etching primers under in vivo and in vitro aging conditions.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Each adhesive group was further divided into 2 subgroups (N = 6) according to the aging (extraction) periods (24 hrs vs. 1 yr). After extraction at the designated periods, 4 teeth from each subgroup were used for microtensile bond testing. Each tooth was sectioned with a slow-speed saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under water cooling into multiple 0.9 x 0.9-mm beams, with the ‘non-trimming’ version of the microtensile test (Shono et al., 1999). Five beams were selected from each tooth, resulting in 20 beams for each subgroup. Bond testing was performed in tension with use of a universal testing machine (Testometric 500, Lancashire, UK) at a crosshead speed of 1 mm/min until failure. The cross-sectional area at the site of failure was measured to the nearest 0.01 mm with a digital caliper (Model CD-6BS; Mitutoyo, Tokyo, Japan), from which the microtensile bond strength was calculated and expressed in MPa. Failure modes were evaluated at 30X magnification with a stereoscopic microscope and classified as adhesive, cohesive within the resin composite or dentin, or mixed failures.
In vitro Sample Preparation
To compare bond degradation under in vivo and in vitro aging conditions, we repeated the in vivo experiment using 24 extracted caries-free third molars. The bonded teeth were stored at 37°C in artificial saliva to prevent the depletion of calcium and phosphate ions from the resin-dentin interfaces that may occur when specimens are stored in water. The artificial saliva contained (mmol/L): CaCl2.2H2O (0.7), MgCl2.6H2O (0.2), KH2PO4 (4.0), KCl (30), HEPES buffer (20), and NaN3 (3.0) as an antibacterial agent. The teeth were retrieved after 24 hrs or 1 yr. Microtensile bond testing and failure mode evaluation were repeated in the manners previously described.
Statistical Analyses
For each adhesive, the in vivo and in vitro bond strength data were analyzed by a two-way ANOVA design for evaluation of the effect of aging condition (in vivo vs. in vitro) and aging period (24 hrs vs. 1 yr), and the interaction of these 2 factors on bond strength of the 2 self-etching primers. Post hoc comparisons were performed by Tukey’s tests. Statistical significance was set in advance at 
= 0.05.
Transmisson Electron Microscopy (TEM)
Two teeth from the in vivo and in vitro subgroups of each adhesive were fixed in half-strength Karnovsky’s fixative (2% paraformaldehyde and 2.5% glutaraldehyde) with phosphate buffer (0.1 mol/L, pH 7.2) for 1 wk. A 1-mm-thick slab was prepared mesio-distally from each tooth. The slabs were immersed in a tracer solution consisting of 50 wt% ammoniacal silver nitrate for 24 hrs, according to the nanoleakage evaluation protocol described by Tay and Pashley (2003). The silver-impregnated slabs were dehydrated in an ascending ethanol series (30–100%), immersed in propylene oxide as a transition fluid, and embedded in epoxy resin (TAAB 812 resin, TAAB Laboratories, Aldermaston, UK) according to the TEM processing protocol described by Tay et al.(1999). After the slabs were embedded in epoxy resin, 2 2x1-mm blocks were obtained with an ultramicrotome (Ultracut S, Leica, Vienna, Austria) and a diamond knife (Diatome, Bienne, Switzerland). Undemineralized, 90- to 100-nm-thick sections were prepared and examined without additional staining, by a TEM (Philips EM208S, Philips, Eindhoven, The Netherlands) operating at 80 kV.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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).
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). There was also no significant interaction between aging conditions and aging periods (P = 0.878). For both adhesives, there was a tendency for more adhesive failures to occur after long-term aging (Table).
Twenty-four hour in vitro specimens of Clearfil SE Bond (Fig. 1A) and Clearfil Protect Bond (Fig. 1B) revealed 0.5-µm-thick, partially demineralized hybrid layers in which nanoleakage was sporadically present. NaF crystals were identified from the adhesive layer in Clearfil Protect Bond. The 24-hour in vivo specimens exhibited similar features, but with 25% of the sections breaking off along the hybrid-layer-adhesive interface during sectioning.
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) and in vivo (Fig. 2B) specimens in Clearfil SE Bond after 1 yr of aging exhibited nanoleakage patterns that were similar to those of the 24-hour specimens. However, additional silver-filled water channels (water trees) that were not previously observed in the 24-hour specimens could be identified throughout the adhesive layers, being associated with silver-filled void-like structures.
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) and in vivo (Fig. 3B) interfaces in Clearfil Protect Bond also revealed water trees that were closely associated with the nanoleakage along the surface of the hybrid layers. Unlike Clearfil SE Bond, these water trees did not extend into the bulk of the adhesive.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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While the in vitro results in the present study confirm previous findings that fluoride incorporation into adhesives improved the stability of resin-dentin bonds (Saito, 1996; Nakajima et al., 2003), our in vivo results further indicate that such an improvement is clinically relevant over 1 yr. The slow fluoride release from Clearfil Protect Bond may have reduced the solubility of calcium phosphates within the hybridized smear layer and hybrid layer that resulted in comparatively more stable bond strength to dentin over time (Nakajima et al., 2003).
Our TEM results do not support the claim that fluoride-releasing adhesives are capable of remineralizing non-resin-encapsulated demineralized dentin (Itota et al., 2003), since nanoleakage in the form of silver deposits was present within the interfibrillar spaces of hybrid layers after 1 yr of in vivo clinical service, as well as 1 yr of in vitro aging in artificial saliva. Interpretation of remineralization results that are based on the identification of mineral concentration alone has recently been challenged (Kinney et al., 2003), highlighting the importance of intrafibrillar mineralization as evidence of remineralization.
The relationship between nanoleakage and long-term durability of dentin bonds with self-etching primer systems has previously been examined (Okuda et al., 2002). The authors observed that nanoleakage increased over time along the resin-dentin interfaces, and suggested that water penetration through the nanoleakage channels probably resulted in lower bond strengths and increased interfacial failure as early as 9 mos. Whereas the dentin specimens in that study were directly exposed to water, a similar phenomenon was observed in the present study, even when the bonded dentin was protected by enamel from direct exposure to the oral environment or artificial saliva.
Under our experimental conditions, water trees that were not initially observed in the adhesive layers at 24 hrs were present after 1 yr of aging, confirming the hypothesis that water trees may be a potential mechanism for degradation of resin-dentin bonds (Tay and Pashley, 2003). These water trees could have been formed by slow water sorption through the adhesives that expedited the leaching of hydrolytic resinous components, resulting in the silver-filled void-like structures (Fig. 2). These void-like structures probably account for the porosity present in the adhesive and along the surface of the hybrid layer, when fractured, aged, primate specimens were examined by FE-SEM (Sano et al., 1999).
The methacrylate-derivatives used in dental adhesives are susceptible to enzyme-catalyzed hydrolysis by salivary esterases (Munksgaard and Freund, 1990; Larsen et al., 1992). A common esterase (enzyme classification EC 3.1.1.1) is inhibited by extremely low concentrations of fluoride (Marcos and Townsend, 1995). We speculate that the fluoride-containing adhesive releases sufficient fluoride to inhibit any salivary or dentin matrix-bound esterase activity that may have been responsible for the decreased bond strength seen in the fluoride-free adhesive (i.e., Clearfil SE Bond).
Although bond stability was observed in Clearfil Protect Bond for up to 1 yr, it is interesting to observe that water trees were also present in the fluoride-releasing adhesive, although they were smaller and localized along the surface of the hybrid layer, unlike those in Clearfil SE Bond, which extended all the way through the adhesive layer. The fact that these water channels were present in both adhesives suggests that a common degradation mechanism exists in these adhesives, although these morphologic correlates of degradation were not extensive enough to cause a decline in bond strength in the fluoride-releasing adhesive. Since these morphologic correlates were similarly manifested in the in vivo and in vitro specimens, we must accept the null hypothesis that that there is no difference in the mechanism of degradation of self-etching primers under in vivo and in vitro aging conditions. This enables future results from in vitro aging studies to be predictive of the clinical scenarios.
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ACKNOWLEDGMENTS |
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Received April 8, 2004; Last revision January 5, 2005; Accepted January 12, 2005
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REFERENCES |
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