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Ultrastructural Correlates of in vivo/in vitro Bond Degradation in Self-etch Adhesives
1 Faculty of Dentistry, Selçuk University, Konya, Turkey; Correspondence: * corresponding author, kfctay{at}netvigator.com
The morphologic correlates of bond degradation in self-etching primers have not been fully elucidated. We hypothesized that there is no difference between the mechanism of degradation of self-etching primers in vivo and in vitro. Class I cavities prepared in vivo in 24 caries-free human molars were bonded with Clearfil SE Bond or Clearfil Protect Bond, and restored with resin composites. Eight teeth were extracted after 24 hrs, and the rest after 1 yr. The same protocol was repeated in vitro with extracted molars. Degradation of resin-dentin bonds was assessed by microtensile bond testing and TEM of interfaces after tracer immersion. Both in vivo and in vitro bond strengths decreased with time for SE Bond but not for Protect Bond, with more pronounced water treeing observed in the former adhesive under both aging conditions. There is no difference between the mechanism of degradation of self-etch adhesives in vivo or in vitro.
Key Words: self-etch adhesives • aging • microtensile bond strength • water trees
Previous in vitro studies indicated that resin-dentin bonds degrade after long-term water storage (Kiyomura, 1987; Gwinnett and Yu, 1995). The plasticizing effects of water on resin and collagen that arise from water sorption through dentin adhesives (Burrow et al., 1999; Tanaka et al., 1999; De Munck et al., 2003; Tay et al., 2003) may result in hydrolysis of hydrophilic resin components (Burrow et al., 1996; Finer and Santerre, 2004), or breakdown of unprotected collagen fibrils (Armstrong et al., 2003; Hashimoto et al., 2003; Wang and Spencer, 2003) via the activation of host-derived matrix metalloproteinases (Tjäderhane et al., 1998; Pashley et al., 2004). 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.
In vivo Sample Preparation The in vivo part of the study was performed under a protocol approved by the ethical committee of the University of Selçuk. Volunteer patients with 1 or more unrestored, erupted, non-functional, non-carious third molars that were scheduled for extraction were selected. Informed consent was received from every patient who participated in this study. Class I cavities (3 mm deep, 3 mm [buccal-lingual] x 4 mm [mesial-distal]) were prepared in 24 molar teeth under rubber dam isolation. These teeth were randomly divided into 2 groups and restored with either Clearfil SE Bond or Clearfil Protect Bond [formerly known as ABF] (Kuraray Medical Inc., Tokyo, Japan). Both are two-step, water- and ethanol-based, filled, self-etch adhesives, with the former being fluoride-free, and the latter being an antibacterial and fluoride-releasing adhesive that contains 12-methacryloyloxydodecylpyridinium bromide (MDPB) (Imazato et al., 1997, 2003) as the antibacterial monomer, and NaF crystals as the fluoride-releasing agent (Clearfil Protect Bond, MSDS data). The cavities were restored with the 2 self-etching primer systems, a thin layer of resin composite liner (Protect Liner F, Kuraray), and 2 incremental layers of a light-cured, hybrid resin composite (Clearfil AP-X, Kuraray). 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
Statistical Analyses
Transmisson Electron Microscopy (TEM)
For Clearfil SE Bond, both aging condition and aging period were significant in their influence on bond strength results (P < 0.001), but there was no significant interaction between these 2 factors (P = 0.108). For each aging period, in vivo bond strengths were significantly lower than in vitro data. Bond strengths after 1 yr were also signicantly lower than 24-hour bond strengths, regardless of in vivo or in vitro aging conditions (Table  ).
For Clearfil Protect Bond, aging conditions exerted a significant influence on bond strengths (P < 0.001), with higher bond strengths observed in the in vitro subgroups. Unlike Clearfil SE Bond, aging periods did not influence the bond strength results of Clearfil Protect Bond (P = 0.791). Under both in vivo and in vitro aging conditions, the 24-hour and one-year bond strengths were not significantly different from one another (Table  ). 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
Both the in vitro (Fig. 2A ) 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.
Both the in vitro (Fig. 3A ) 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.
Since both in vivo and in vitro samples were prepared by the same operator using the same cavity design, the overall lower in vivo bond strength results may reflect the difficulty in working under oral conditions, in terms of elevated environmental temperature, relative humidity conditions, and saliva contamination. The lower in vivo bond strengths may also be due to fluid movement from vital dentin during bonding (Purk et al., 2004). Unlike previous primate studies (Sano et al., 1999; Takahashi et al., 2002), the effects of functional stresses (fatigue) on in vivo bond strength could not be appropriately assessed in the present human study, due to the constraints imposed by the ethical committee in selecting teeth that are non-functional (i.e., non-occluding) for in vivo research involving human subjects. To facilitate meaningful comparisons, we likewise did not subject our in vitro specimens to simulated functional loading via the use of cyclic mechanical loading (Frankenberger et al., 1999). Since both in vivo and in vitro results demonstrated similar trends—in that bonds created by the fluoride-releasing self-etch adhesive remained stable after 1 yr, while those created by the fluoride-free self-etch adhesive system fell significantly—we must accept the null hypothesis that there is no difference in the mechanism of degradation of self-etching primers under in vivo and in vitro aging conditions. 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 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.
This study was based on the work partially performed by Nazyime Donmez for fulfillment of the degree of Doctor of Philosophy, University of Selçuk, Turkey. The adhesives examined were generous gifts from Kuraray Medical Inc. The TEM part of this study was supported by grant 10204604/07840/08004/324/01 from the Faculty of Dentistry, University of Hong Kong, and by grants DE 014911 and DE 015306 from the National Institute of Dental and Craniofacial Research (P.I. David Pashley). The authors are grateful to Michelle Barnes and Zinna Pang for secretarial support. Received for publication April 8, 2004. Revision received January 5, 2005. Accepted for publication January 12, 2005.
Journal of Dental Research, Vol. 84, No. 4,
355-359 (2005) This article has been cited by other articles:
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