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Effects of Dentin Characteristics on Interfacial Nanoleakage
1 Department of Cariology and Operative Dentistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan; Correspondence: * corresponding author, yuanyang.ope{at}gmail.com
Water emanating from dentinal tubules during air-drying and light-curing of adhesives leads to entrapment of droplets at the resin-dentin interface and contributes to nanoleakage. This study tested the null hypothesis that characteristics of substrate dentin and type of adhesive used for bonding would not affect the occurrence of nanoleakage. Three self-etch adhesives were used to bond to 4 types of dentin with different characteristics in 12 groups. After silver challenge, nanoleakage percentage was measured within the hybrid layer of each sample. The deep dentin cut perpendicular to tubules always showed a significantly higher nanoleakage percentage compared with that of the other 3 types of dentin. The percentages of nanoleakage within the hybrid layers were not statistically different among adhesives. However, when bonding to deep perpendicular dentin, both all-in-one adhesives revealed more distinct nanoleakage within the adhesive layer compared with that achieved with Clearfil SE Bond, a two-step self-etch adhesive. The results did not support the null hypothesis.
Key Words: dentin depth • tubule orientation • nanoleakage • hybrid layer • TEM
While a hermetic seal of enamel could be achieved with use of current bonding systems, it is still a challenge to seal the resin-dentin interface perfectly, due to the heterogeneous character of the dentin structure and surface morphology (Pashley and Carvalho, 1997). Dentinal tubules are slightly tapered, with the wider portion oriented toward the pulp. Dentin permeability increases almost logarithmically with cavity depth; such an increase is attributed to the vast differences in the sizes and numbers of dentinal tubules between superficial and deep dentin (Garberoglio and Brannström, 1976). Air-drying and light-curing of the adhesive resins induce fluid movement across dentin (Hashimoto et al., 2004a,b). Water emanating from dentinal tubules leads to entrapment of fluid droplets at the resin-dentin interface, and contributes to nanoleakage (Tay et al., 2005). Some of the latest all-in-one adhesives have a comparatively mild etching effect and pH values of around 2 or higher. Mild acid-etching may modify and partially remove the smear layer. This effect could lead to a reduction of outward fluid flow during resin application (Shimada et al., 1995). However, primed smear plugs have a porous structure and cannot totally block the outward flow of dentinal fluid under air-drying (Matthews et al., 1993). Therefore, increased density and tubule size, accompanied by reduced thickness of the remaining dentin, may result in an increased wetness of the dentin surface (Pashley and Carvalho, 1997). Excessive water at the interface may hinder infiltration and the polymerization of resin, resulting in nanoleakage (Sano et al., 1995a,b). The influence of dentin characteristics on bonding mechanisms and bond strength has been studied by several researchers (Swift et al., 1995; Yoshiyama et al., 1998). An in vitro study showed a lower bonding capacity for deep dentin compared with superficial dentin (Sattabanasuk et al., 2004). Evidence of insufficient marginal sealing in bonded restorations has been presented by various microleakage studies (Kubo et al., 2001; Jayasooriya et al., 2003). In addition, effects of tubule orientation on the variations in bond strength remain controversial (Phrukkanon et al., 1999; Sattabanasuk et al., 2004). To date, no research has been published on the effects of dentin depth and tubule orientation on the occurrence of nanoleakage. The aim of this in vitro study was to determine the ability of current all-in-one adhesives to seal dentin, in comparison with a widely accepted two-step self-etch bonding system, under different dentin characteristics. The null hypothesis to be tested was that the characteristics of substrate dentin and type of adhesive used for bonding would not affect the occurrence and extent of nanoleakage.
Tooth Preparation The procedure for specimen preparation is schematically illustrated in Fig. 1  . Eighteen extracted intact human third molars were collected after the individuals’ informed consent was obtained under a protocol approved by the Institutional Review Board of TMDU. The teeth were stored at 4°C in isotonic saline saturated with thymol until the start of the experiment. The teeth were randomly distributed among 3 groups corresponding to the 3 adhesive materials. In each group, we used 3 teeth to obtain "perpendicular dentin" and the remaining 3 to obtain "parallel dentin", with regard to the orientation of dentinal tubules. The occlusal one-third and root of each tooth were removed by means of a diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under water lubrication. For perpendicular dentin subgroups, the teeth were cut perpendicular to the longitudinal axis into 1- to 1.5-mm-thick slices. The first occlusal slice represented "superficial perpendicular dentin", and the last one (cervical) represented "deep perpendicular dentin", with a remaining dentin thickness of around 0.7 mm.
For parallel dentin subgroups, each tooth was cut parallel to the longitudinal axis into mesial and distal halves. Each half provided a flat dentin surface that lay parallel to the dentinal tubules. The occlusal portion of this surface on a random half of each tooth served as "superficial parallel dentin", while the cervical potion of the other half served as "deep parallel dentin". All designated dentin surfaces were polished with 600-grit SiC papers under running water to create a standardized smear layer.
Bonding Procedures
Nanoleakage Evaluation Nanoleakage evaluation was carried out by TEM micrography. Two images were taken from each specimen at 15k magnification. In total, 72 images were evaluated, with the use of image analysis software (Image J 1.34s, Wayne Rasband, National Institutes of Health, Bethesda, MD, USA), for calculation of the percentage distributions of silver deposits within the hybrid layers. Data were statistically analyzed among the 4 subgroups within each adhesive and also between similar dentin subgroups of the 3 adhesives. The analyses were carried out by Kruskal-Wallis ANOVA on ranks and Dunn’s multiple-comparison tests, with statistical significance set at P < 0.05.
Typical nanoleakage patterns at the resin-dentin interfaces for each adhesive group are illustrated in Figs. 2 –4. Nanoleakage occurred both within the hybrid layer and through the adhesive layer of all samples. Spotted silver penetration was the main nanoleakage pattern. Regardless of the adhesive used, the resin-dentin interface of superficial perpendicular dentin (Figs. 2A, 3A, 4A) and both parallel dentin subgroups (Figs. 2C, 2D, 3C, 3D, 4C, 4D) showed very fine silver dots within the hybrid layer. However, all deep perpendicular dentin subgroups not only presented isolated silver grains with increased size and density within the hybrid layers, but also showed increased silver deposition through the adhesive layer. The latter finding was particularly distinct for all-in-one adhesives (Figs. 3B, 4B).
In addition to nanoleakage formation within the hybrid layer and through the adhesive layer, all dentin subgroups of G-Bond presented exceptional nanoleakage directly under the hybrid layer (Fig. 4  ). Two parallel dentin subgroups of G-Bond showed hardly any nanoleakage within the hybrid layer; however, they revealed distinct nanoleakage within the dentin under the hybrid layer (Figs. 4C, 4D).
Percentages of silver penetration within the hybrid layers of Clearfil SE Bond were 0.6 ± 0.5%, 4.4 ± 1.5%, 1.6 ± 0.6%, and 1.3 ± 0.6% for superficial perpendicular dentin, deep perpendicular dentin, superficial parallel dentin, and deep parallel dentin, respectively. The percentages for Clearfil S3 Bond were, respectively, 1.6 ± 1.0%, 9.1 ± 3.8%, 1.3 ± 0.5%, and 1.5 ± 0.8%. For G-Bond, they were 2.6 ± 1.8%, 9.1 ± 3.8%, 0.4 ± 0.7%, and 2.0 ± 1.4%. The deep perpendicular dentin group presented a statistically significantly higher nanoleakage score among the 4 dentin subgroups within each adhesive (P < 0.05). For similar dentin subgroups, there was no significant difference in nanoleakage percentages through the hybrid layers among the 3 adhesive groups (P > 0.05). However, based upon observation of the adhesive layers bonded to deep perpendicular dentin (Figs. 2B
The evaporative water flux transuding from dentinal tubule orifices may affect the bonding interface, even in the absence of convective water flux (Tay et al., 2005). The dentin surface cut parallel to the dentinal tubules is partly covered by peritubular dentin of the longitudinally sectioned tubule; such a surface may include no orifices opening directly to the bonding interface. While superficial perpendicular dentin has a similarly limited surface area of tubule orifices, in deep perpendicular dentin both the diameter and density of tubules increase (Cagidiaco et al., 1997). As for deep perpendicular dentin, the larger surface area of the orifices may lead to more severe contamination by interfacial water. Excessive contamination by water could lead to phase separation of the adhesive and deterioration in the quality of the bonding interface (Spencer and Wang, 2002). Moreover, water contamination would not only impair the priming capacity of the primer, by rapidly reducing the acidity (Zheng et al., 2000), but also may lead to incomplete polymerization of the resin monomer, through decrease of the conversion rate (Jacobsen and Söderholm, 1995; Tay et al., 1996). The excessive hydrophilic monomers of adhesive, remaining due to incomplete polymerization, could be stained by ammoniacal silver nitrate during nanoleakage tests (Tay et al., 2002). Meanwhile, the elution of these residual hydrophilic components from the bonding interface during water storage might lead to the exposure of denuded collagen fibrils, which may similarly be stained by silver particles (Sano et al., 1999; Tay et al., 2002). In the current study, deep perpendicular dentin subgroups exhibited statistically significantly higher nanoleakage scores compared with the other dentin subgroups for all 3 adhesives; this finding was in accord with our predictions. All three adhesives tested could be classified as mild self-etch adhesives, according to criteria described previously (Van Meerbeek et al., 2003). Mild self-etch adhesives showed less nanoleakage in comparison with "strong" self-etch adhesives tested in other nanoleakage studies (Carvalho et al., 2005; De Munck et al., 2005). From the FE-SEM/EDS observations in our previous study, we found that Clearfil SE Bond and Clearfil S3 Bond showed less silver penetration along the resin-superficial dentin interface among 5 adhesives tested (Yuan et al., 2007); TEM observations in the current study support those findings. It is speculated that mild acid-etching adhesives modify and partially remove the smear layer; thus, the bonding interface would be less influenced by evaporative water flux during air-drying or light-curing. Moreover, due to partial removal of hydroxylapatite, residual mineral crystals within the hybrid layer would be able to form intense and stable calcium salts with the acidic monomers (MDP and 4-MET). Such additional chemical bonding is believed to help prevent or retard nanoleakage (Yoshida et al., 2004). All-in-one adhesives did not show significantly higher nanoleakage percentages compared with that of the two-step self-etch adhesive under any conditions. This finding might be explained by the fact that the hybrid layers formed by all-in-one adhesives were comparatively thin. In contrast, those adhesives revealed more distinct nanoleakage within the adhesive layer in the deep perpendicular dentin subgroup. It has been reported that all-in-one adhesives were more vulnerable to excessive dentin surface water, which could cause interfacial phase separation (Van Landuyt et al., 2005), and this may especially be the case for deep perpendicular dentin. It has been suggested that prolonged air-drying may contribute to the properties of water-containing adhesives (Sadr et al., 2007), and strong air-drying was particularly recommended for all-in-one adhesives, such as G-Bond, that are HEMA-free and contain acetone solvent (Van Landuyt et al., 2005). Following those suggestions, we prolonged the air-drying duration for all-in-one adhesives in the current study. However, based on the results, it may be speculated that the strong air-drying in the case of deep perpendicular dentin may also cause diffusion of more evaporative water flux from dentinal tubules toward the bonding interface, eventually leading to droplet entrapment and nanoleakage. In contrast, Clearfil SE Bond adopted a separate bonding step with a hydrophobic bonding resin. It has been suggested that the increased concentration of hydrophobic resin component within the adhesive layer of the two-step self-etch adhesives may contribute to the sealing of the bonding interface (Nikaido et al., 2002). Such an effect might prevent water treeing and promote the longevity of the bonding (Tay et al., 2002). In addition to the nanoleakage within the hybrid layer, G-Bond also occasionally showed exceptional nanoleakage under the hybrid layer in all dentin subgroups. The existence of a porous zone beneath the hybrid layer after the application of all-in-one adhesives has recently been reported. It was conjectured that this porous zone was caused by retarded infiltration of non-acidic comonomers, due to the formation of molecular sieve-like structures from dissolved calcium and phosphate ions during self-etching (Carvalho et al., 2005). Based on our current observations, we speculated that the large-molecular-weight hydrophobic components of G-Bond did not successfully infiltrate the thinner spaces created by the acidic monomers, possibly due to the lack of HEMA. HEMA supposedly prevents phase separation of dental adhesive blends (Spencer and Wang, 2002). Multiple applications of all-in-one adhesives and continuous agitation on the tooth substrate might be an effective method of reducing such nanoleakage under the hybrid layer. The quality of sealing against the nanoleakage may greatly affect the bonding durability of restorations and, subsequently, their long-term success (Okuda et al., 2002). Clinically, adhesion problems are commonly observed at the gingival wall of class II resin-based restorations. According to the present study, these problems may be explained by the fact that tubules in the gingival area tend to be cut perpendicular to their longitudinal axes, and have the highest density values among all dentin locations within a preparation (Cagidiaco et al., 1997; Purk et al., 2004). The current findings may justify the clinical significance of in vitro nanoleakage, which is hypothesized to be the pathway for degradation of the bonding interface. It is inferred that the use of Clearfil SE Bond—a two-step self-etch adhesive—for class II cavities might provide a better prognosis for the restoration. In conclusion, we had to reject the null hypothesis. Deep perpendicular dentin showed significantly higher nanoleakage scores among the 4 dentin subgroups with different characteristics, regardless of the adhesive used. Clearfil SE Bond—a two-step self-etch adhesive—might be a more suitable choice for bonding to deep dentin cut perpendicular to the direction of dentinal tubules.
This research was supported by the COE program, FRMDRTB, at the TMDU. The adhesive materials used were generously provided by GC Company and Kuraray Medical. The assistance of Mr. Masaomi Ikeda, regarding statistical analysis, and Dr.MD. Akhtar Uzzaman is greatly appreciated.
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org. Received for publication April 7, 2006. Revision received April 16, 2007. Accepted for publication May 29, 2007.
Journal of Dental Research, Vol. 86, No. 10,
1001-1006 (2007)
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