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Integrating Oxalate Desensitizers with Total-etch Two-step Adhesive

¹ Conservative Dentistry, University of Hong Kong, Hong Kong SAR, China;
² Department of Oral Biology and Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, GA 30912-1129, USA;
³ Bauru School of Dentistry, University of São Paulo, Brazil; and
⁴ Bisco, Inc., Schaumburg, IL, USA;

^* corresponding author, dpashley{at}mail.mcg.edu

	ABSTRACT

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Compromised bonding of total-etch adhesives to dentin treated with oxalate desensitizers results from the interference of a surface layer of acid-resistant crystals of calcium oxalate. We hypothesize that effective tubular occlusion and dentin bonding may be simultaneously achieved by depleting dentin surfaces of calcium with acids before desensitizer application. Dentin specimens treated with 4 oxalate desensitizers before or after being acid-etched were bonded with a two-step adhesive. Microtensile bond strengths (µTBS) were significantly lower, compared with the control, when oxalates were used before the specimens were acid-etched; in contrast, when oxalates were used after acid-etching. µTBS were similar to nonoxalate-treated controls. Dentin surfaces and tubular orifices were covered with a surface layer of crystals when desensitizers were applied to fractured dentin and smear-layer-covered dentin before specimens were acid-etched. However, when the dentin was acid-etched prior to the application of oxalate desensitizers, the crystals were largely limited to the subsurface of dentinal tubules, where they did not interfere with subsequent resin bonding.

KEY WORDS: dentin permeability • oxalic acid • acid-etch • adhesive

	INTRODUCTION

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Despite the improved sealing performance of contemporary total-etch adhesives (Prati et al., 2001; Ferrari and García-Godoy, 2002), bonding to vital, deep, acid-etched dentin may still be hampered by the increase in wetness following smear layer removal (Pereira et al., 1999). Incomplete sealing of dentin may be caused by shrinkage of resin tags from dentin tubular walls during composite polymerization. Transudation of dentinal fluid beneath water-immiscible resins after solvent evaporation and before the resins polymerize can also result in the entrapment of water blisters over tubular orifices (Tay et al., 1996), that induce rapid fluid shift across deep dentin (Brännström and Åström, 1972; Närhi, 1985) during mastication. These factors may account for the post-operative sensitivity occasionally reported with the use of these adhesives (Opdam et al., 1998; Akpata and Sadiq, 2001; Unemori et al., 2001).

Oxalate desensitizers are available as resin-free gels or solutions that consist of low concentrations of oxalic acid (Pashley, 1986). They are effective in reducing the hydraulic conductance of dentin with exposed tubules, both in vitro (Greenhill and Pashley, 1981; Pashley and Galloway, 1985; Camps et al., 1998) and in vivo (Hirvonen et al., 1984; Muzzin and Johnson, 1989; Cuenin et al., 1991). They react with calcium ions on dentin and in dentinal fluid to form insoluble calcium oxalate crystals (Kerns et al., 1991; Gillam et al., 2001). Surface occlusion of dentinal tubules provides only temporary reduction of dentin permeability and sensitivity, since the crystals are either partially dissolved in oral fluids or lost during toothbrushing (Gillam et al., 1999; Niazy, 1999). Entrapping calcium oxalates with resins in a resin-containing oxalate desensitizer did not significantly improve the retention of these precipitates (Zhang et al., 1998).

Total-etch adhesives do not bond well to oxalate-treated dentin, since the surface layer of acid-resistant calcium oxalate crystals interferes with resin infiltration through the demineralized collagen matrices (Pashley et al., 1993). It is possible, however, to reduce outward fluid flow from acid-etched dentin during bonding by the adjunctive use of oxalate desensitizers on acid-etched dentin prior to adhesive application (Pashley et al., 2001). When calcium ions are depleted from the smear layer and underlying dentin, oxalate ions diffuse further down the dentinal tubules until calcium is available for reaction. Reduction in dentin permeability is thus achieved via subsurface tubular occlusion that does not interfere with subsequent resin infiltration. Bond strengths of total-etch adhesives were not compromised with the adjunctive use of an experimental, resin-free oxalate desensitizing gel after acid-etching. Since this technique has potential clinical uses for improving the retention of oxalates in exposed dentin, and in reducing dentinal fluid transudation during total-etch adhesive procedures, the hypothesis of combined tubular occlusion with oxalates and resins should be further tested on different commercially available oxalate desensitizers. Thus, the objective of this study was to examine the effect of subsurface tubular occlusion with oxalates during total-etch bonding, with resin-free and resin-containing oxalate desensitizers. The null hypothesis tested was that the application of resin-free oxalate desensitizers to acid-etched dentin prior to adhesive application does not compromise the bonding of a total-etch adhesive to deep dentin.

	MATERIALS & METHODS

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Fifty extracted human third molars were collected after the patients’ informed consent had been obtained under a protocol approved by the Medical College of Georgia Institutional Review Board. After extraction, 8 teeth were fractured with a mallet and a chisel, creating exposed dentin with patent dentinal tubules that were devoid of smear layers. We used these to examine the interaction of oxalate desensitizers as they are normally used on exposed mineralized dentin that is free of smear layers. Flat surfaces were prepared on deep coronal dentin of 42 teeth by means of a slow-speed diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under water-cooling, and abraded with wet 180-grit silicon carbide papers to create smear-layer-covered dentin for bonding with a total-etch adhesive (One-Step, Bisco Inc., Schaumburg, IL, USA).

Experimental Design
Three resin-free (Protect Drops, John O. Butler Co., Chicago, IL, USA; Oxagel, Art-Dent, São Paulo, Brazil; Super Seal, Phoenix Dental Inc., Fenton, MI, USA) and 1 resin-containing oxalate desensitizer (MS Coat, Sun Medical Co. Ltd., Shiga, Japan) were used in this study (Table).

After storage in distilled water at 37°C for 24 hrs, each tooth was sectioned occluso-gingivally into 0.9-mm-thick serial slabs by means of the Isomet saw under water-cooling. The slabs were further sectioned into 0.9 x 0.9 mm composite-dentin beams, according to the technique for the "non-trimming" version of the microtensile test reported by Pashley et al. (1999). After examination under a stereomicroscope at 20X, beams containing peripheral enamel or imperfections were discarded, yielding 37–46 beams per group. Each beam was stressed to failure under tension in a universal testing machine (Model 4440, Instron Inc., Canton, MA, USA) at a crosshead speed of 1 mm/min. Bond strength data from the 8 experimental groups were statistically analyzed with SigmaStat Version 2.03 (SPSS, Chicago, IL, USA), with a two-way ANOVA on ranks (surface condition vs. material). Post hoc multiple comparisons of the experimental groups were performed with the Dunn’s test, with the highest bond strength achieved in the acid-etched groups as a control, with statistical significance set at a = 0.05.

Transmission Electron Microscopy (TEM)
Ultrastructural features of oxalate-treated dentin and adhesive-bonded dentin were first determined. Each of the 4 desensitizers was applied to 2 fractured dentin specimens without the use of the adhesive. The adhesive was applied to 2 abraded acid-etched dentin specimens without the use of oxalates. Then, the 4 desensitizers were applied before or after dentin was acid-etched, followed by adhesive application, in the manner previously described. To avoid damage to the diamond knife, we replaced the hybrid composite with a light-cured, microfilled composite with pre-polymerized fillers (Metafil CX, Sun Medical Co. Ltd.). The teeth were sectioned into 0.9-mm serial slabs. The widest slab from each tooth was prepared for TEM examination, according to the protocol reported by Tay et al. (1999). Undemineralized, epoxy-resin-embedded, 90- to 120-nm-thick sections were examined unstained under a TEM (CM100, Philips, Eindhoven, The Netherlands) operated at 80 kV.

For further comparison of the extent of adhesive infiltration in hybrid layers with oxalate desensitizers applied to specimens before or after being acid-etched, additional slabs were coated with nail varnish applied 1 mm away from the bonded interfaces. They were immersed in a 50 wt% ammoniacal silver nitrate solution for 24 hrs, according to the diamine silver impregnation protocol reported by Tay et al. (2002a). After reduction of the diamine silver (I) ions ([Ag(NH₃)₂]⁺), the silver-impregnated slabs were processed for TEM examination without further demineralization and examined unstained.

	RESULTS

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Analysis of the bond strength results (Table) by a two-way ANOVA on ranks revealed significant (p < 0.001) differences within each factor. When the oxalate-containing desensitizing agents were applied to unetched dentin, there were significant (p < 0.001) differences among the experimental groups’ bond strengths (Table). Similarly, when the oxalate-containing desensitizing agents were applied to acid-etched dentin, there were also significant (p < 0.001) differences in bond strength among the groups (Table). There was also a significant (p < 0.001) interaction between the 2 factors, dentin surface condition and materials. That is, not all 4 materials showed the same sensitivity to the pre-bonding dentin surface condition. Multiple comparisons of the means of the various groups revealed that treatment of dentin with the resin-containing desensitizing agent significantly reduced bond strengths (p < 0.05) compared with those of the other oxalate-containing products, regardless of whether they were applied to unetched or etched dentin. Although both Oxagel and the resin-containing agent lowered (p < 0.05) bond strengths more than did the other agents when applied to unetched dentin, only the resin-containing desensitizing agent significantly lowered bond strengths when applied to acid-etched dentin. All oxalate desensitizing agents lowered resin-dentin bond strengths (p < 0.05) when applied to unetched dentin, compared with acid-etched dentin (Table).

TEM of the control group bonded with only the adhesive revealed a 5-µm-thick hybrid layer that did not contain calcium oxalate crystals (image not shown). When oxalate desensitizers were applied to fractured dentin, the silhouette of a precipitate layer could be seen over the dentin surface and around patent tubular orifices. The actual crystals were lost during creation of ultrathin sections for TEM.

When oxalate desensitizers were used before resin was acid-etched, silhouettes of fine calcium oxalate crystals were mostly confined to the dentin surface (Fig. 1A). Very few oxalate crystals remained within tubular orifices rendered patent by the phosphoric acid etchant, which was eventually rinsed off (Fig. 1B). In Fig. 1A, the dentinal tubules were oriented parallel to the treated surface, while in Fig. 1B, they were oriented perpendicular to the oxalate-treated surface. It was the tubule orientation, not the products, that was responsible for the differences in the images. When oxalate desensitizers were used after dentin was acid-etched, silhouettes of subsurface crystals (Fig. 2A) were found inside the dentinal tubules. These crystals were located 3–7 microns beneath the dentin surface and were absent from either the dentin surface or tubular orifices (Fig. 2B). No major TEM differences could be discerned between the resin-containing and resin-free desensitizers.

View larger version (292K): [in this window] [in a new window]   Figure 1. Undemineralized, unstained TEM micrographs showing (A) the application of the acetone-based adhesive to dentin that was first treated with Protect Drops, a resin-free oxalate desensitizer, before being acid-etched. The silhouette of a layer of acid-resistant precipitate (between arrows) remained on the dentin surface. Few calcium oxalate crystals were seen within the resin-infiltrated dentinal tubules (T) within the hybrid layer (H). A, adhesive; H, hybrid layer; D, intertubular dentin. (B) The application of the acetone-based adhesive to dentin that was first treated with Super Seal, another resin-free oxalate desensitizer, before being acid-etched. The silhouette of a similar layer of acid-resistant precipitate could be seen along the dentin surface (pointer). Some calcium oxalate crystals could be found within the dentinal tubules (T). A, adhesive; H, hybrid layer; D, intertubular dentin.

View larger version (314K): [in this window] [in a new window]   Figure 2. Undemineralized, unstained TEM micrographs showing the application of (A) MS Coat and (B) Oxagel on acid-etched dentin, followed by the application of the acetone-based adhesive. No calcium oxalate precipitate was observed at the tubular orifices. A thin lucent layer was observed at the demineralization front. This layer is thought to consist of small crystals of calcium oxalate. As the calcium from the acid-etched dentin was depleted, the oxalate desensitizers diffused into the dentinal tubules until calcium was available for the formation of calcium oxalate crystals (pointer). Since these crystals were formed beneath the dentin surface, they may reduce outward fluid movement during the subsequent bonding procedure. Furthermore, they did not interfere with adhesive infiltration of the demineralized collagen matrix, providing tubular occlusion in conjunction with the resin tags that were formed inside the dentinal tubules (T). A, adhesive; H, hybrid layer; D, intertubular dentin.

View larger version (652K): [in this window] [in a new window]   Figure 3. Undemineralized, unstained TEM micrographs showing silver penetration in resin-dentin interfaces with the use of Super Seal on dentin followed by adhesive application. (A,B) Desensitizer applied before dentin was acid-etched. (C,D) Desensitizer applied after dentin was acid-etched. C, composite; A, adhesive layer; H, hybrid layer; T, dentinal tubule; D, intertubular dentin. (A) When the desensitizer was used before dentin was acid-etched, extensive silver deposits could be seen within the adhesive layer and the surface zone of calcium oxalate deposits (between open arrows). (B) A higher-magnification view showing the presence of water trees (pointer) that extended from the superficial oxalate layer into the adhesive. Silver grains were also deposited around the silhouettes of the calcium oxalate crystals (arrow). Additional silver deposits could be observed as nanoleakage within the hybrid layer. (C) When the desensitizer was used after dentin was acid-etched, much less nanoleakage was seen in the form of isolated islands of silver deposits (arrows) that were identified only within the hybrid layer (between open arrows), and were absent from the adhesive layer. The dentinal tubules contained silhouettes of calcium oxalate crystals (pointers) that were located beneath the hybrid layer. (D) A higher-magnification view of the hybrid layer, showing the islands of silver deposits (open arrow), additional fine isolated silver grains (open arrowhead), and the silver deposits (solid arrow) that were formed around the silhouettes of calcium oxalate crystals (pointers).

	DISCUSSION

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Since the resin-free oxalate desensitizers compromised adhesive bond strength only before but not after dentin was acid-etched, we have to accept the null hypothesis tested in this study. The resin-containing desensitizer followed the same trend as the other resin-free desensitizers, in that the mean tensile bond strength obtained after dentin was acid-etched was higher than that achieved before dentin was acid-etched. However, it was significantly lower than that of dentin bonded with the adhesive alone. We speculate that the copolymer cannot diffuse through the demineralized collagen matrix, due to its high molecular weight. These polymers may accumulate on the acid-etched dentin surface and weaken the overall strength of the bonded interface.

Because the variability between and among individual teeth was not evaluated, the possibility that correlated data may have contributed to the observed differences among groups cannot be excluded. However, the fact that the standard deviations (Table) associated with the means were very small, even though multiple teeth were used in each group, suggests that the variability between teeth was no greater than the variability within individual teeth.

We confirmed previous scanning electron microscopy findings that smear layers on abraded dentin treated with acidic oxalate desensitizers were completely substituted by an acid-resistant layer of crystals that interfered with resin bonding to acid-etched dentin (Pashley et al., 1993, 2001). Since nanoleakage of silver was present within these hybrid layers, the possibility of the surface deposits interfering with adhesive infiltration cannot be overruled. The extensive accumulation of silver deposits within the adhesive, and the existence of "water trees" (Tay et al., 2002a) that extended from the surface calcium oxalate layer into the adhesive in specimens that were treated with oxalates prior to being resin-bonded, suggests the existence of an osmotic gradient that permitted water to diffuse from the underlying tubules into the adhesive (Pomersheim and Nguyen, 1998; Tay et al., 2002b). Water movement through the adhesive during polymerization may have generated the "water trees" that are absent from oxalate-free resin-dentin interfaces created with this acetone-based adhesive alone (Tay and Pashley, 2002). This water movement through adhesives may occur via an osmotic gradient (Tay and Pashley, 2002) and warrants further investigation.

As oxalic acid diffused through the demineralized dentin, it finally encountered mineralized intertubular and peritubular dentin matrices. Here it solubilized sufficient calcium to form insoluble subsurface crystals of calcium oxalate in the tubules (Fig. 2B). These crystals were similar in appearance and location to those that were found to reduce the hydraulic conductance of dentin (80% reduction; Pashley et al., 2001). We speculate that such reductions in hydraulic conductance would limit osmotically induced water movement during bonding procedures. Thus, the adjunctive use of oxalate desensitizers following acid-etching of dentin is a viable means of reducing dentinal fluid transudation in total-etch bonding. Subsurface tubular occlusion with calcium oxalates may also protect the dental pulp from the potential cytotoxic properties of resin monomers. This would be particularly useful when the total-etch technique is used for restoring deep, vital dentin, especially cervical lesions with no enamel margins. When oxalates are used on acid-etched cavities that contain enamel margins, the enamel surfaces become covered by calcium oxalate crystals that can interfere with resin-enamel adhesion. These surface crystals can be removed by a second, brief (ca. 10–15 sec) etch with phosphoric acid (unpublished observations) that dissolves the apatite crystals of enamel beneath the calcium oxalate crystals that then fall off, leaving the etched enamel receptive for resin infiltration. This second acid etch has no effect on the calcium oxalate crystals below the dentin surface, because they are acid-resistant. Self-etching primer adhesives are not as effective as phosphoric acid at etching enamel, so they may not remove all calcium oxalate crystals from enamel surfaces. Further studies should be done to determine the effectiveness of various self-etching adhesives with different pHs.

	ACKNOWLEDGMENTS

 
The oxalate desensitizers used in this study were donated by Art-Dent, John O. Butler Co., Phoenix Dental Inc., and Sun Medical Co. Ltd. This study was supported by grants DE 014911-01 from the National Institute of Dental and Craniofacial Research, USA, and 300481/95-0 from CNPq, Brazil. The authors are grateful to Michelle Barnes for secretarial support and Amy Wong for technical support. Dr. Pashley receives royalty payments from the sale of Protect Drops. Drs. Pashley and Carvalho are co-authors of several patents that cover the commercial use of this technique (US Patents 6,406,529, 6,506,055 EO 1,071,393). Mr. B.I. Suh is president of Bisco, Inc., the manufacturer of the two-step adhesive used in this study. None of the other authors has any financial arrangements with any commercial organizations that could benefit from this study.

Received September 13, 2002; Last revision March 3, 2003; Accepted June 26, 2003

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This Article Abstract Figures Only Full Text (PDF) Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (13) Citing Articles via Google Scholar Google Scholar Articles by Tay, F.R. Articles by Suh, B.I. Search for Related Content PubMed PubMed Citation Articles by Tay, F.R. Articles by Suh, B.I.