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Biomaterials & Bioengineering |
Tubular Occlusion Optimizes Bonding of Hydrophobic Resins to Dentin
F.T. Sadek1,2,
D.H. Pashley3,
M. Ferrari2 and
F.R. Tay2,3,*
1 Department of Dental Materials, School of Dentistry, University of São Paulo, Brazil;
2 Department of Restorative Dentistry and Dental Materials, School of Dentistry, University of Siena, Italy; and
3 Department of Oral Biology & Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, GA 30912-1129, USA
Correspondence: * corresponding author, franklintay{at}gmail.com
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ABSTRACT
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Although hydrophobic resins may be bonded to acid-etched dentin with an ethanol wet-bonding technique, the protocol is sensitive to moisture contamination when bonding is performed in deep dentin. This study tested the hypothesis that the use of oxalate or poly(glutamic) acid-modified, diluted ceramicrete (PADC) for dentinal tubule occlusion prevents fluid contamination and improves the bonding of an experimental hydrophobic adhesive to acid-etched, ethanol-dehydrated dentin. Mid-coronal and deep acid-etched moist dentin pre-treated with oxalate or PADC was dehydrated by ethanol wet-bonding and infiltrated with the experimental three-step etch-and-rinse hydrophobic adhesive under simulated pulpal pressure. Tensile bond strengths to deep dentin without pre-treatment were severely compromised. Conversely, oxalate and PADC pre-treatments reduced dentin permeability, prevented water contamination, and improved bond strengths. Minimal nanoleakage was identified within hybrid layers created in the oxalate- and PADC-pre-treated deep dentin. The use of tubular occluding agents optimized bonding of hydrophobic resins to dentin.
Key Words: ethanol wet-bonding • hydrophobic resin • tubule occlusion • oxalate • pulpal pressure
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INTRODUCTION
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The hydrophilic monomer 2-hydroxyethyl methacrylate (HEMA) is often used as a solvent for sparingly water-soluble resin monomers used in contemporary dentin adhesive formulations. It increases the ability of these adhesives to wet dentin and reduces their sensitivity to moisture contamination (Tay and Pashley, 2003). Although the incorporation of hydrophilic and acid resin monomers has substantially improved the initial bonding of contemporary adhesives to intrinsically wet dentin substrates (Kanca, 1992), it also resulted in increases in water sorption (Hashimoto et al., 2005; King et al., 2005) and decreases in mechanical properties of the polymerized adhesives (El Zohairy et al., 2004; Yiu et al., 2004), thereby compromising the longevity of resin-dentin bonds (De Munck et al., 2003, 2005; Koshiro et al., 2005). Moreover, it has been demonstrated that resin-dentin bonds created by contemporary adhesives are susceptible to fluid permeation, with the HEMA-based adhesives being more severely affected, which significantly reduces their bond strengths (Sauro et al., 2007).
Theoretically, the use of comparatively hydrophobic resins as dentin adhesives would avoid these problems and improve the durability of resin-dentin bonds. However, it is impossible to wet or infiltrate acid-etched dentin with hydrophobic adhesives (Brudevold et al., 1956). With the use of concepts of tissue-embedding for electron microscopy, it was recently shown that it is possible to bond hydrophobic resin blends to acid-etched dentin with an ethanol wet-bonding technique (Nishitani et al., 2006; Sadek et al., 2007), attaining tensile strengths that were comparable with contemporary hydrophilic adhesives, and with minimal nanoleakage within the hybrid and adhesive layers. In this technique, the water in the acid-etched dentin is slowly replaced by an ascending concentration of ethanol, avoiding the collapse of the interfibrillar spaces within the collagen matrix. Preservation of these spaces enables the ethanol to be replaced with increasing concentrations of hydrophobic monomers dissolved in ethanol, and finally with pure hydrophobic resin. Although promising results were obtained when dentin specimens were bonded in the absence of fluid contamination from dental pulp, the ethanol wet-bonding protocol was found to be technique-sensitive in the presence of water. Since hydrophobic monomers are immiscible with water, contamination of these resin monomers with as little as 5 vol% water on the ethanol-saturated dentin substrate resulted in a 25% reduction in tensile strength of the experimental hydrophobic adhesive to dentin (Sadek et al., 2007). This renders the ethanol wet-bonding technique impractical when it is used in vital deep dentin (Lopes et al., 2006).
Oxalate desensitizers have been shown—in both in vitro (Gillam et al., 2001; Tay et al., 2003, 2005) and in vivo studies (Muzzin and Johnson, 1989; Chang et al., 1996; Gillam et al., 2004)—to be effective in reducing the permeability of deep, acid-etched dentin. When oxalate desensitizers are applied to acid-etched dentin, the oxalate reacts with the calcium ions within the dentin tubules, to form sparingly soluble precipitates of calcium oxalate that occlude the tubules and minimize outward fluid movement to the dentin surface (Gillam et al., 2001; Tay et al., 2003). Similar to the use of oxalate desensitizers (Pashley et al., 2001), a poly(glutamic) acid-modified diluted ceramicrete (PADC) was recently found to be able to reduce dentin permeability by occluding dentinal tubules with very fine agglomerates of MgKPO4.6H2O crystallites (Tay and Pashley, unpublished results). Rinsing of the ceramicrete-treated dentin surface for bonding procedures may remove enough of these crystallites from the surface, while blocking the tubules to prevent fluid contamination during the application of hydrophobic adhesives to acid-etched dentin.
Although dentin bonding with hydrophobic resins by the ethanol wet-bonding technique shows initially favorable results (Nishitani et al., 2006; Sadek et al., 2007), the protocol must be further tested in vitro when bonding is performed under dentin perfusion, before it may be recommended for clinical testing. Thus, the objectives of this study were: (1) to compare the microtensile bond strength of an experimental hydrophobic adhesive to mid-coronal and deep-dentin, when bonding was performed under physiological pulpal pressure by the ethanol wet-bonding technique; (2) to identify the effect of subsurface tubular occlusion with oxalate and PADC desensitizer during the ethanol wet-bonding technique; and (3) to examine, with transmission electron microscopy, the ultrastructure of the bonded interfaces. The null hypothesis tested was that there is no difference in the bonding of an experimental hydrophobic adhesive to oxalate or PADC desensitizer-treated acid-etched mid-coronal and deep dentin with the use of an ethanol wet-bonding technique under simulated physiological pulpal pressure.
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MATERIALS & METHODS
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Tooth Preparation
Forty-eight human third molars were collected after the individuals’ informed consent was obtained under a protocol reviewed and approved by the Human Assurance Committee of the Medical College of Georgia. The teeth were sectioned 3 mm below the cemento-enamel junction with a slow-speed diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under water-cooling. They were initially divided into two groups according to the remaining dentin thickness: mid-coronal dentin [MD] or deep dentin [DD]. Flat dentin surfaces were then prepared perpendicular to the longitudinal axes of the teeth with the diamond saw under water cooling, leaving 2.5–3.0 mm of dentin in the MD group, and 0.5–1.0 mm in the DD group. Each surface was ground with 180-grit silicon carbide paper under running water for 20 sec to create a clinically relevant smear layer. The final thickness of the remaining dentin was precisely measured with a dial caliper gauge (0.01 mm, Renfert GmbH, Hilzingen, Germany).
Experimental Design
Two experimental tubular occlusion materials were prepared: 3% potassium tetroxalate (JT Baker, Phillipsburg, NJ, USA) and PADC (0.05 µL of 10% phosphoric acid, 0.05 µL of 16 µg/mL polyglutamic acid in de-ionized water) and 50 µg of ceramicrete. The latter is a "chemically bonded phosphate ceramic" consisting of MgO and KH2PO4 (Wagh et al., 1999). We used a comonomer resin blend comprised of 70 wt% Bis-GMA, 28.75 wt% TEGDMA, 0.25 wt% camphorquinone, and 1 wt% ethyl N,N-dimethyl-4-aminobenzoate to formulate the experimental three-step, etch-and-rinse, hydrophobic adhesive. We prepared the primer solution by diluting the neat comonomer blend with 50 wt% of absolute ethanol. The neat comonomer resin blend was used as the adhesive component.
Bonding Procedures
We constructed a Plexiglass platform by inserting 18-gauge stainless steel tubing into holes created in a 2 x 2 x 0.6-cm piece of Plexiglass. After pulpal tissues were removed from crown segments, they were attached to the Plexiglass with a cyanoacrylate adhesive (Zapit, Dental Ventures of America, Anaheim Hills, CA, USA). Each Plexiglass-crown segment assembly was attached via a piece of polyethylene tubing (Fisher Scientific, Pittsburgh, PA, USA) to a syringe barrel filled with deionized water. The latter was raised to deliver 20 cm of water pressure to the pulp chamber.
The MD and DD groups were each divided into 3 subgroups (n = 8), according to the dentin tubule-occluding material used prior to adhesive application (3% potassium tetroxalate; PADC or de-ionized water as the control group). Each tooth was etched with 37% phosphoric acid gel (Etch 37, Bisco Inc., Schaumburg, IL, USA) for 15 sec, rinsed thoroughly with de-ionized water, and left moist. The tubule-occluding materials were applied by means of a microbrush with agitation for 30 sec, left undisturbed for 60 sec, and rinsed meticulously; the substrate was left moist with deionized water.
A chemical dehydration protocol previously described by Sadek et al.(2007) was used for ethanol wet-bonding. Briefly, acid-etched and occluded wet dentin surfaces were treated with a series of increasing ethanol concentrations (50%, 70%, 80%, 95%, and 100% three times for 30 sec each). We performed this procedure meticulously to ensure that the dentin surface was always immersed in a liquid phase by keeping it visibly moist prior to the application of the subsequent solution with a higher ethanol concentration. Two consecutive coats of the experimental hydrophobic primer were then applied to ethanol-saturated dentin. Excess ethanol solvent was evaporated with a gentle air stream for 10 sec. Then, a layer of the neat comonomer adhesive was applied, spread thin with moisture-free air, and light-cured for 20 sec with the use of an Optilux 500 halogen light-curing unit (Demetron/Kerr, Danbury, CT, USA) with a power output of 600 mW/cm2. Composite build-ups were constructed with a light-cured resin composite (Clearfil APX, Kuraray Co., Okayama, Japan) in five 1-mm-thick increments.
Tensile Testing
After storage in de-ionized water at 37°C for 24 hrs, each tooth was vertically sectioned into 0.9-mm-thick serial slabs by means of the Isomet saw under water cooling. The central slab of each tooth was utilized for subsequent morphologic examination. The adjacent slabs were sectioned into 0.9 x 0.9-mm beams, according to the "non-trimming" technique of the microtensile test. Each beam was stressed to failure under tension in a testing jig (Bisco Inc.) mounted in a Vitrodyne V1000 universal tester (Liveco Inc., Burlington, VT, USA) at a cross-head speed of 1 mm/min. Bond strength data from the 6 subgroups were statistically analyzed with a two-way ANOVA design (tubular occlusion vs. dentin depth). Post hoc multiple comparisons were performed with the Tukey test, with statistical significance set at  = 0.05.
Transmission Electron Microscopy (TEM)
Three central slabs from each of the 6 subgroups were used for TEM examination of nanoleakage along the dentin hybrid layers. These slabs were immersed for 24 hrs in a tracer solution containing 50 wt% ammoniacal silver nitrate, according to a method described previously (Tay et al., 2002). The silver-impregnated slabs were then rinsed thoroughly in distilled water and placed in photodeveloping solution for 8 hrs under a fluorescent light. Undemineralized, epoxy-resin-embedded, 90- to 100-nm-thick sections were prepared and examined, without further staining, by TEM (Philips CM-100, Eindhoven, The Netherlands) operated at 80 kV.
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RESULTS
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No premature failure occurred in any of the 6 subgroups. The bond strength data were normally distributed (Kolmogorow-Smirnoff test) and exhibited equal variances (Levene test). Two-way ANOVA revealed significant differences in the factors "tubular occlusion" (p < 0.001), dentin depth (p < 0.001), and their interactions (p < 0.001). Post hoc comparisons revealed significant differences between bond strengths of mid-coronal vs. deep dentin (p < 0.05), between oxalate pre-treatment and the control (p < 0.05), and between PADC pre-treatment and the control (p < 0.05) (Fig. 1 ). Under TEM examination, the resin-dentin interface in the DD control subgroup (i.e., de-ionized water, no tubular occlusion material) separated, and resin tags pulled out of the tubules. Extensive silver leakage was present (Fig. 2). Unlike DD, the MD control subgroup was not sensitive to the effect of dentin perfusion (not shown).
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Figure 1. Microtensile bond strength (means and standard deviations) obtained from the 6 experimental groups (N = 35). An experimental, three-step, etch-and-rinse hydrophobic adhesive was bonded to mid-coronal and deep acid-etched dentin by the ethanol wet-bonding technique under simulated pulpal pressure. The acid-etched dentin was pre-treated with de-ionized water (control), 3% potassium tetroxalate (oxalate), or poly(glutamic) acid-modified, diluted ceramicrete (PADC) prior to ethanol dehydration. Bond strengths for the 6 groups were 38.3 ± 8.2 MPa for the mid-coronal dentin control group, 19.8 ± 4.6 MPa for the deep dentin control group, 42.4 ± 8.4 MPa for the mid-coronal dentin oxalate group, 40.2 ± 9.4 MPa for the deep dentin oxalate group, 42.6 ± 7.9 MPa for the mid-coronal dentin PADC group, and 38.2 ± 7.7 MPa for the deep dentin PADC group. Groups identified with the same letter are not significantly different (p > 0.05).
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Figure 2. A representative TEM micrograph taken from an unstained, undemineralized, silver-impregnated section in the control-deep dentin subgroup. Acid-etched deep dentin was bonded with the hydrophobic adhesive by the ethanol wet-bonding technique. A, adhesive layer; C, resin composite; D, mineralized intertubular dentin; E, space created when the adhesive-resin interface separated from the dentin, showing some voids (*) indicative of incomplete polymerization of the embedding epoxy resin, due to seepage of water from the interface during polymerization. Arrow: resin tags that pulled out of the tubules. Open arrowhead: silver deposits. The demineralized collagen matrix could not be identified clearly.
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With the use of oxalate and PADC pre-treatments, only minimal nanoleakage could be identified within the hybrid layers created in deep dentin (Figs. 3, 4). For the oxalate pre-treatment subgroups, the dentin surfaces were completely devoid of calcium oxalate crystals, and the latter could be identified only within the dentinal tubules, at 4–5 µm from the tubular orifices (Fig. 3). For the PADC pre-treatment subgroups, fine electron-dense ceramicrete crystallites could be seen attached to the lamina limitans of the dentinal tubules (Fig. 4).
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Figure 3. TEM micrograph taken from an unstained, undemineralized, silver-impregnated section from the oxalate-deep dentin subgroup. A, adhesive layer; C, resin composite; D, mineralized intertubular dentin; H, hybrid layer. (A) A low-magnification view showing the presence of calcium oxalate crystals (open arrows) in the dentinal tubules, about 5–8 µm from the tubular orifices, blocking water movement during dentin perfusion. (B) At a higher magnification, the dentin surface was completely devoid of oxalate crystals. Most of the oxalate crystals inside the dentinal tubules dislodged during ultramicrotomy, leaving empty spaces. Some of them, however, remained and appeared as electron-dense aggregates (open arrowhead). Only minimal nanoleakage could be identified as isolated, round silver grains (arrows) within the hybrid layer.
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Figure 4. TEM micrograph taken from an unstained, undemineralized, silver-impregnated section of the poly(glutamic) acid-modified, diluted ceramicrete-deep dentin subgroup. A, adhesive layer; C, resin composite; D, mineralized intertubular dentin; H, hybrid layer. (A) An overall view of the resin-dentin interface, showing the generalized absence of silver nanoleakage. The dentinal tubules were coated along their peripheries with a layer of material (arrows) that had dislodged during ultramicrotomy, leaving electron-lucent spaces that were infiltrated with neither the adhesive resin nor the epoxy resin. The hybrid layer was only 2 µm thick, probably reflecting shrinkage that occurred during the stepwise chemical (ethanol) dehydration regime. (B) At a higher-magnification view, spaces occupied by the ceramicrete crystallite aggregates were seen on the dentin surface and inside the dentinal tubules (arrows). The use of poly(glutamic acid) resulted in the reduction in the sizes of these crystallites to the dimensions that were smaller than the diameters of the dentinal tubular orifices. Hence, they did not prevent resin infiltration into the tubules and demineralized collagen matrix. A fine layer of electron-dense ceramicrete crystallites could be seen within a tubule and could be readily discerned from the peritubular dentin (pointer). Only minimal nanoleakage could be identified as isolated, round silver grains (open arrowhead) within the hybrid layer. (C) At a very high magnification, electron-dense ceramicrete crystallites (open arrowheads), approaching the dimensions of dentin apatite crystals, could be seen attaching to the lamina limitans (arrows) of the dentinal tubules and trapped by resin.
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DISCUSSION
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The present work confirmed the results of our previous studies (Nishitani et al., 2006; Sadek et al., 2007), that it is possible to bond to mid-coronal dentin and achieve high tensile strengths when experimental hydrophobic adhesives are used with the ethanol wet-bonding technique. However, tensile bond strength was severely compromised when the same technique was applied under dentin perfusion to deep dentin. Thus, we have to reject our null hypothesis. Since the numbers and the diameters of dentinal tubules in deep dentin were much greater than those of mid-coronal dentin (Garberoglio and Brännström, 1976), more outward fluid movement may be expected during ethanol wet-bonding procedures. Water derived from the dental pulp probably contaminated the chemically dehydrated dentin surfaces and resulted in their poor wetting by hydrophobic adhesives.
The rationale of water contamination during the bonding of hydrophobic resins to chemically dehydrated acid-etched dentin may be explained in terms of the solubility parameter concept. Replacement of the water in a demineralized dentin matrix with ethanol results in lowering of the Hoy’s solubility parameter for hydrogen bonding forces ( h) of the matrix from 22.5 to 18.1 MPa (Becker et al., 2006). The h value of ethanol-saturated dentin matrix is close to that of the hydrophobic primer used in this study (h = 13.3). If water, with a h = 40.4 MPa, is permitted to seep into the ethanol-saturated matrix, it will increase its h value to near 22 MPA, rendering the matrix less compatible with the primer. Thus, it is critical to avoid water contamination in the ethanol wet-bonding technique after the demineralized collagen matrix is suspended in absolute ethanol, to optimize the infiltration of hydrophobic resins.
The use of tubular occlusion materials represents a possible solution to the problem of water re-contamination. The bond strengths in MD were not compromised by the adjunctive use of these products prior to ethanol dehydration. Conversely, higher bond strengths were observed in deep acid-etched dentin when these products were used. The potassium tetroxalate used is acidic enough (pH 2.5) to etch subsurface dentin, reacting with calcium further down in the dentinal tubules and forming calcium oxalate dihydrate crystals that block water movement. Its potential effectiveness in occluding dentinal tubules and reducing dentinal permeability has previously been reported (Gillam et al., 2001; Pashley et al., 2001; Pereira et al., 2005) and is illustrated in Fig. 3 . Ceramicrete is classified as a ceramic material in which the ceramic phase is derived not from powder sintering, but from acid-base reaction. The one utilized in this study is based on the reaction of magnesium oxide with dibasic potassium phosphate (Wagh et al., 2003). The ceramic phase in ceramicrete usually contains large crystals and, when they are present on the acid-etched dentin surface, would prevent infiltration of adhesive resins into the partially demineralized dentin matrix. If poly(glutamic) acid was used to control crystal nucleation and growth (Hunter and Goldberg, 1994) in diluted ceramicrete formulations, very fine MgKPO4.6H2O crystallites may be produced for dentin desensitization purposes (Tay and Pashley, unpublished results).
Within the limitations of this study, it may be concluded that the technique sensitivity of the ethanol wet-bonding technique in deep dentin may be resolved with the adjunctive use of tubular occlusion materials. This renders the bonding of hydrophobic monomers to dentin an achievable goal for shallow, mid-coronal, and deep dentin substrates. Further studies should be performed to evaluate the longevity of hydrophobic resin-dentin bonds created by the ethanol wet-bonding technique.
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ACKNOWLEDGMENTS
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This study was supported by R01 grant DE 014911 from the NIDCR, USA (PI David Pashley). The authors graciously acknowledge the technical support of Penny Roon and the secretarial support provided by Michelle Barnes.
Received for publication September 21, 2006.
Revision received January 4, 2007.
Accepted for publication January 16, 2007.
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Journal of Dental Research, Vol. 86, No. 6,
524-528 (2007)
DOI: 10.1177/154405910708600607
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ABSTRACT
INTRODUCTION