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RESEARCH REPORT |
1 Paediatric Dentistry and Orthodontics, Faculty of Dentistry, University of Hong Kong, Prince Philip Dental Hospital, 34 Hospital Road, Hong Kong SAR, China;
2 College of Dental Medicine, Nova Southeastern University, 3200 South University Drive, Ft. Lauderdale, FL, USA;
3 Department of Oral Biology and Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, GA, USA; and
4 Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan;
* corresponding author, kfctay{at}hknet.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: sodium hypochlorite • sodium ascorbate • nanoleakage • microtensile bond strength • ultrastructure
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Compromised bond strengths were observed for some single-bottle adhesives when dentin was treated with sodium hypochlorite either before or after acid-etching (Perdigão et al., 2000; Lai et al., 2001). This drop in bond strength was attributed to the oxidizing instead of the deproteinizing effect of sodium hypochlorite, since the compromised bond strength may be reversed by the application of a reducing agent such as sodium ascorbate to the oxidized dentin (Lai et al., 2001). However, a correlation between the drop in bond strength and ultrastructural changes along the oxidized resin-dentin interface has not been established. Removal of demineralized collagen layer by sodium hypochlorite eliminated nanoleakage formation (Pioch et al., 2001a). Nanoleakage that occurred along the bonded interfaces of oxidized acid-etched dentin and those that were subsequently neutralized with a reducing agent were assessed by means of a silver-staining technique and compared by transmission electron microscopy. The null hypothesis of this study was that there is no difference in the distribution of nanoleakage patterns and tensile bond strengths of single-bottle adhesives bonded to sodium-hypochlorite-treated, acid-etched dentin and those that were further neutralized with sodium ascorbate.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design
Two single-bottle adhesives—One-Step (OS, Bisco, Inc., Schaumburg, IL, USA), an acetone-based adhesive, and Gluma Comfort Bond + Desensitizer (GCB, Heraeus Kulzer, Inc., South Bend, IN, USA), an ethanol-based adhesive—were used in this study. Each adhesive consisted of 3 experimental groups with 10 teeth each. Eight restored teeth were used for bond strength evaluation by the microtensile bond test. The other 2 were prepared for nanoleakage evaluation by transmission electron microscopy (TEM). Bonding was performed on the occlusal surfaces of the deep coronal dentin. The 3 experimental groups were shown in the Appendix (www.dentalresearch.org).
The treated teeth were bonded visibly moist with either 2 coats of OS or 3 coats of GCB according to the manufacturers’ instructions. Bonded surfaces were air-dried, then light-cured for 10 sec. Composite build-ups were performed with the use of a light-cured composite (Renamel Sculpt, Cosmedent, Inc., Chicago, IL, USA) in 5 1-mm increments. The teeth were stored in distilled water at 37°C for 24 hrs.
Tensile Bond Strength Evaluation
Bonded teeth were sectioned occluso-gingivally into serial slabs, and further sectioned into 0.9 x 0.9 mm composite-dentin beams, according to the "non-trimming" technique of the micro-tensile test (Shono et al., 1999). Specimens were stressed to failure under tension in a Bencor Multi-T device (Danville Engineering, San Ramon, CA, USA) in a universal testing machine Model 4440 (Instron, Inc., Canton, MA, USA) at a crosshead speed of 1 mm per min. For each adhesive, bond strength data from the 3 experimental groups were statistically analyzed with the software package Instat V3.0 for Windows 95 (GraphPad Software, Inc., San Diego, CA, USA). Differences between adhesives were determined by one-way ANOVA and Tukey’s multiple-comparisons tests, with statistical significance set at 
= 0.05.
Nanoleakage Evaluation
A modified silver-staining technique (Tay et al., 2002) was used with basic 50 wt% ammoniacal silver nitrate (pH = 9.5) to avoid the possibility of artifactual dissolution of mineralized dentin along the bases of the hybrid layers (Pashley et al., 2002). We prepared the solution by dissolving 25 g of silver nitrate crystals (Sigma Chemical Co., St. Louis, MO, USA) in 25 mL of distilled water. Concentrated (28%) ammonium hydroxide (Sigma Chemical Co.) was used to titrate the black solution until it became clear as ammonium ions complexed the silver into diamine silver ([Ag(NH3)2]+) ions (Tay et al., 2002). This solution was diluted to 50 mL with distilled water to achieve a 50 wt% solution.
From the remaining 2 teeth in each group, 0.9-mm slabs were prepared and coated with 2 layers of fast-setting nail varnish applied 1 mm from the bonded interfaces. Before these slabs could become dehydrated, they were immersed immediately in the ammoniacal silver nitrate solution for 24 hrs. The silver-stained slabs were rinsed thoroughly in distilled water and placed in photodeveloping solution for 8 hrs under a fluorescent light (Wu et al., 1983) to reduce the diamine silver ions into metallic silver grains within potential voids along the bonded interfaces. Undemineralized, epoxy-resin-embedded, 90-nm-thick ultrathin sections were prepared according to the TEM protocol of Tay et al. (1999). The unstained sections were examined by means of 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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). When sodium ascorbate, the reducing agent, was applied to the oxidized dentin, the compromised bond strength was effectively reversed to pre-treatment levels.
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). The reticular type consisted of fine, discontinuous islands of silver deposits, which were seen exclusively in the hybrid layer of OS (Fig. 1A). The spotted type, consisting of isolated silver grains, was evident throughout the hybrid and adhesive layers in OS (Fig. 1B). The adhesive layer in GCB was very thin and almost indiscernible from the hybrid layer. A denser reticular type of nanoleakage pattern was seen in the hybrid layer created by this adhesive. A sparsely distributed, spotted type of nanoleakage was evident along the base of the hybrid layer and around the dentinal tubules (Fig. 1C).
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). No hybrid layer was present. For both OS and GCB, a third vertical, shag-carpet-like, nanoleakage pattern was observed along the junction between the demineralization front and the adhesive (Fig. 2A). This pattern originated from the porosities within the underlying mineralized dentin (Fig. 2B). The vertical projections in GCB were less prominent along the dentin surface, but were clearly identifiable along the periphery of the dentinal tubules (Fig. 2C). It is speculated that phase separation of the methacrylic polycarboxylic acid component of this adhesive was observed within the adhesive layer.
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), although the spotted type of nanoleakage was still sparsely observed within the adhesive layer at high magnification (Fig. 3B). Silhouettes of rhombohedral-shaped crystals (Lai et al., 2001) that were trapped by the adhesive layer could sometimes be seen along the dentin surface and within the dentinal tubules (Fig. 3C). The spotted type of nanoleakage was occasionally observed along the junction of these crystals and the adhesive resin (not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Different types of nanoleakage patterns occurred in the resin-dentin interfaces of all experimental groups with both adhesives, depending on the treatment applied to the dentin surface. The reticular and the spotted types were identified along the resin-dentin interface in acid-etched dentin. The reticular type represents the classic nanoleakage pattern that corresponds to regions of incomplete resin infiltration within hybrid layers (Sano et al., 1995b). This type of nanoleakage was elipminated in the absence of a hybrid layer, but was replaced with the vertical shag-carpet-like pattern after sodium hypochlorite treatment. Although subsequent treatment with sodium ascorbate further eliminated the shag-carpet-like pattern, both sodium hypochlorite and sodium ascorbate treatments did not eliminate the spotted type of nanoleakage pattern. The spotted type probably represents areas of increased permeability within the resin layers (Pashley et al., 2002) that result from the interaction of the hydrophilic resin components with the basic diamine silver ions. The relationship between the hydrophilicity of resin monomers and the permeability of the adhesive layer requires further exploration.
All previous nanoleakage studies have revealed areas of incomplete resin infiltration within hybrid layers (Pioch et al., 2001b). Incomplete resin penetration of demineralized dentin may leave spaces for penetration of bacterial products and dentinal or oral fluids. This may result in the hydrolytic breakdown of either the adhesive resin or collagen fibrils within the hybrid layer, compromising the long-term durability of resin-dentin bonds (Hashimoto et al., 2000). Although incompletely infiltrated hybrid layers have been shown recently, at least for primates, to be capable of remineralization (Akimoto et al., 2001), complete removal of the demineralized collagen matrix in acid-etched dentin has been proposed as an alternative way of improving the quality of resin-dentin bonds. A recent study indicated that a one-minute application of 10% sodium hypochlorite to acid-etched dentin completely eliminated nanoleakage at the resin-dentin interface (Pioch et al., 2001a). Other studies indicated the presence of a remnant hybrid layer when a 5% sodium hypochlorite solution (Lai et al., 2001) or a 10% sodium hypochlorite gel (Perdigão et al., 2000) was used for 1 min. A combined sequential two-minute treatment of dentin with maleic acid and sodium hypochlorite was shown to remove the collagen matrix effectively and restore the etched dentin surface to its natural composition (Di Renzo et al., 2001). Pre-treatment with a 10% sodium hypochlorite solution for 2 min was shown to remove organic material from artificial dentin lesions and to increase lesion remineralization (Inaba et al., 1996).
The vertical shag-carpet-like nanoleakage pattern along the demineralization front following application of sodium hypochlorite was completely eliminated after treatment with sodium ascorbate, a reducing agent. Thus, this nanoleakage pattern may be attributed to the elution of either oxygen or residual sodium hypochlorite from the subsurface porous, oxidized mineralized dentin during bonding. A recent micro-Raman spectroscopic study demonstrated spectral shifts in human dentin that was treated with sodium hypochlorite (Tsuda et al., 1996). A previous study (Lai et al., 2001) failed to demonstrate a cause-and-effect relationship between the reduction in bond strength in sodium-hypochlorite-treated dentin and the ultrastructure of resin-dentin bonds. Since the demineralized collagen network had been completely removed from the dentin surface following application of sodium hypochlorite, denatured, remnant collagen matrix could not be responsible for the compromised bonding to sodium-hypochlorite-treated dentin. Influence of organic solvents in the adhesives was not detectable. The use of a nanoleakage approach in the present study thus provides a new perspective on distinguishing among the resin-dentin interfaces in acid-etched dentin, sodium-hypochlorite-treated acid-etched dentin, and acid-etched dentin which was sequentially treated by the oxidizing and then the reducing agent. Since mineralized dentin is porous (Uchtmann and Wilkie, 1997), it is also possible that some of the sodium hypochlorite was retained within the subsurface mineralized tissues even after immersion in water for 10 min before bonding. Elution of the residual sodium hypochlorite could result in incomplete polymerization of resin monomers at the junction between the adhesive and the demineralization front that contributed to the drop in bond strength and the vertical shag-carpet-like nanoleakage pattern at the dentin-adhesive interface. This residual sodium hypochlorite could have been neutralized by the sodium ascorbate, resulting in a return of the tensile bond strengths to those that were observed in acid-etched dentin.
Both decreases (Nikaido et al., 1999; Frankenberger et al., 2000; Perdigão et al., 2000) and increases (Wakabayashi et al., 1994; Phrukkanon et al., 2000) in bond strengths were reported following application of sodium hypochlorite to acid–etched dentin. Others showed no improvement in bond strength following pre-treatment with NaOCl (Uno and Finger, 1995; Armstrong et al., 1998). This may be due to the specificity of different adhesive systems to the oxidizing effect of sodium hypochlorite. Reduction in bond strength may also be related to changes in the physical and chemical properties of dentin after application of sodium hypochlorite. Reductions in the elastic modulus and flexural strength of dentin were reported after irrigation of the root canals with 5% sodium hypochlorite (Grigoratos et al., 2001; Sim et al., 2001). Similar reductions in the microhardness of root canal dentin were reported when 5% sodium hypochlorite was used for irrigation during endodontic therapy (Saleh and Ettman, 1999). Results from these studies showed that alterations in dentin structure and properties may affect the bonding of adhesives to the treated dentin. It would be interesting to see if physical properties that are reduced after sodium hypochlorite treatment can also be reversed with the use of sodium ascorbate.
In conclusion, the demonstration of different nanoleakage patterns provides an insight into the reduction in bond strength after sodium hypochlorite treatment and its reversal with sodium ascorbate. Although no correlation appears to exist between nanoleakage and bond strength (Okuda et al., 2001; Pereira et al., 2001), nanoleakage may have significant consequences for the long-term stability of adhesive bonds between dentin and restorative materials (Pioch et al., 2001b). Further investigations are required to clarify the relationship between the different types of nanoleakage and the long-term durability of resin-dentin bonds.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received November 20, 2001; Last revision May 28, 2002; Accepted July 3, 2002
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REFERENCES |
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