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Effects of Resin Hydrophilicity on Dentin Bond Strength

¹ Department of Operative Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan; and
² Department of Oral Biology & Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, GA 30912-1129, USA

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

	ABSTRACT

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The purpose of this study was to determine if hydrophobic resins can be coaxed into dentin wet with ethanol instead of water. The test hypothesis was that dentin wet with ethanol would produce higher bond strengths for hydrophobic resins than would dentin wet with water. This study examined the microtensile bond strength of 5 experimental adhesives (50 wt% ethanol/50% comonomers) of various degrees of hydrophilicity to acid-etched dentin that was left moist with water, moist with ethanol, or air-dried. Following composite buildups, hourglass-shaped slabs were prepared from the bonded teeth for microtensile testing. For all 3 types of dentin surfaces, higher bond strengths were achieved with increased resin hydrophilicity. The lowest bond strengths were obtained on dried dentin, while the highest bond strengths were achieved when dentin was bonded moist with ethanol. Wet-bonding with ethanol achieved higher bond strengths with hydrophobic resins than were possible with water-saturated matrices.

KEY WORDS: dentin bonding • hydrophilic resins • wet-bonding • ethanol

	INTRODUCTION

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The current generation of dentin adhesives has been criticized as being too hydrophilic (Tay and Pashley, 2003) and absorbing too much water, which lowers their stiffness when compared with that of more hydrophobic resins (Ito et al., 2005). Hydrophilic all-in-one self-etching adhesives yield lower bond strengths than do more hydrophobic etch-and-rinse adhesives (De Munck et al., 2005). The bond strengths of self-etching hydrophilic adhesives can be significantly increased when they are covered with a solvent-free, more hydrophobic adhesive (Brackett et al., 2005; King et al., 2005). Presumably, if dentin is bonded with more hydrophobic resins, they would absorb less water, plasticize less (Ito et al., 2005), and produce more durable bonds.

The challenge is how to coax hydrophobic monomers into a hydrophilic matrix without inducing phase changes (Spencer and Wang, 2002). Most of the "hydrophilicity" of acid-etched dentin matrices is due to the presence of water. After dentin is acid-etched, water replaces the volume of dentin previously occupied by mineral (ca. 50 vol%; Kinney et al., 2003). Since this water surrounds and wets the collagen fibrils, adhesive monomers must displace water from collagen if they are to develop an intimate contact with collagen fibrils. However, because the molecular weight of dental monomers ranges from 100–580 g/mol, their molar concentrations (0.3–5 mol/L) are but a fraction of the molar concentration of water (55.6 mol/L). Thus, even hydrophilic monomers such as hydroxyethyl methacrylate (HEMA) cannot displace much water from collagen fibrils. Hydrophobic dimethacrylates that create strong polymer networks by cross-linking polymer chains have very low solubilities in water. These problems may be avoided if ethanol (17.1 mol/L) is exchanged for the water within acid-etched dentin. Most dental monomers, including dimethacrylates like BisGMA, are soluble in ethanol. With ethanol filling the interfibrillar spaces, the dental matrix becomes much more hydrophobic. As ethanol replaces water, some interpeptide hydrogen bonding (H-bonding) develops, within collagen, that stiffens the matrix enough to minimize its shrinkage (Eddleston et al., 2003; Garcia et al., 2005; Becker et al., 2006). This should lead to better infiltration of hydrophobic dimethacrylates into ethanol-saturated matrices, than into water-saturated matrices. The more resin (both mono- and dimethacrylates) that infiltrates acid-etched matrices, the higher are the resin-dentin bond strengths.

The purpose of this study was to compare resin-dentin bond strengths of hydrophobic vs. hydrophilic resins bonded to acid-etched dry dentin or dentin wet with water or ethanol. The test null-hypotheses were that resin hydrophilicity has no effect on microtensile bond strengths, and that bonds made to dentin wet with alcohol are no different from those made to dentin wet with water.

	MATERIALS & METHODS

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Sixty extracted, unerupted human third molars—obtained with informed patient consent under a protocol approved by the Human Assurance Committee of the Medical College of Georgia—were stored in 0.9% NaCl containing 0.02% sodium azide at 4°C until used. Using a diamond-impregnated copper disk, we removed the occlusal enamel and superficial dentin with water irrigation (Isomet saw, Buehler Ltd., Lake Bluff, IL, USA). The tooth surface was ground with #320-grit silicon carbide paper, creating a standardized smear layer. The study design consisted of 5 experimental resins x 3 surface conditions x 4 teeth per group.

The use of Hoy’s solubility parameters to define the degree of hydrophilicity or hydrophobicity of solvated comonomer blends (Miller et al., 1998; Chappelow et al., 2000; Pashley et al., 2001, 2002; Eddleston et al., 2003) provides a useful method for ranking dentin adhesive hydrophilicity. Since manufacturers refuse to reveal the exact composition of their products, the Hoy’s solubility parameters of commercially available dentin adhesives cannot be calculated. In this study, 5 comonomer blends were formulated based on known concentrations of all ingredients, including the solvents, so that their Hoy’s solubility parameters could be calculated. The Hoy’s solubility parameters of 50 wt% ethanol/resin mixtures were also calculated. Similar calculations were made for Hoy’s solubility parameters for dentin matrices saturated with water or ethanol that occupied 30% of the volume of the matrix, with collagen occupying the remaining 70%. The details of how Hoy’s solubility parameters for collagen were calculated have been previously published (Agee et al., 2006; Becker et al., 2006).

Experimental Resins
The compositions of the 5 experimental resins, together with the Hoy’s solubility parameters of these comonomers solvated in 50 wt% ethanol, are given in Table 1. We calculated the latter by summing the group molar attraction constants of their structures (Fig. 1) according to the method of Hoy, using commercially available software (Computer Chemistry Consultancy, www.compchemconsul.com). We used Hoy’s solubility parameters to rank the degree of hydrophilicity of solvent/monomer mixtures. Resins 1 and 2 represent non-solvated hydrophobic resins used in the final step of three-step, etch-and-rinse, and two-step, self-etching adhesives. Resin 3 represents the formulation of typical two-step, etch-and-rinse adhesives, while resins 4 and 5 correspond to very hydrophilic one-step, self-etching adhesives containing carboxylic- or phosphate-substituted methacrylates, respectively.

View larger version (9K): [in this window] [in a new window]   Figure 1. Chemical structures of the monomers used in the solvated comonomer blends listed in Table 1. Definition of abbreviations is in the Table 1 footnote. n = 3 for E-BisADM.

During bonding, a generous amount of each experimental adhesive was applied to the dentin, by means of a microbrush, with agitation for 15 sec. A second application of fresh adhesive was made, giving a total application time of 30 sec. Excess solvent was evaporated with a gentle air stream for 10 sec at a distance of 15 cm, and then the adhesive was light-cured for 40 sec by means of an Optilux 500 halogen light-curing unit (Demetron/Kerr, Danbury, CT, USA) with a power output of 600 mW/cm². Resin composite build-ups were made with three 1.5-mm increments of AP-X composite (Kuraray Medical Inc., Tokyo, Japan) that were individually light-cured for 40 sec. All bonded teeth were incubated in 37°C water for 24 hrs.

Tensile Testing
Using the Isomet saw, we vertically sectioned the bonded teeth into 0.7-mm-thick serial slabs. The central region of each slab, in turn, was trimmed into hourglass-shaped specimens (0.49 mm² cross-section). Only the central 4 slabs were used from each tooth. Thus, there were 4 slabs x 4 teeth = 16 specimens in each subgroup. Modes of failure were classified as adhesive (A), cohesive in resin (C), or mixed (M) failures when the failed bonds were examined at 30X by stereroscopic microscopy.

All tensile testing was performed with the use of a Bisco testing jig mounted in a Vitrodyne V-1000 universal tester at a cross-head speed of 1 mm/min. The specimens were glued to the testing jig by cyanoacrylate (Zapit, Dental Ventures of America, Corona, CA, USA).

Statistical Analyses
We used a two-way ANOVA design with the general linear model for examining the effects of dentin surface condition and resin hydrophilicity on microtensile bond strengths (µTBS). The least-squares means (LSM) analysis was used, due to significant interactions between the 2 factors. Multiple comparisons of the LSM were performed by the Holm-Sidak method. LSM are the expected value of group or subgroup means that one expects for a balanced design involving the group variable, with all covariates at the mean value. The variances in LSM values are given in standard error of the mean (SEM) instead of standard deviation (SD). With an = 0.05, the power of the performed test was 1.00 for resins, 1.00 for surface conditions, and 1.00 for their interaction. Correlations between bond strengths and solubility parameters were done by regression analysis. Statistical significance was set in advance at = 0.05.

	RESULTS

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The results of the microtensile bond tests, reported as least-squares means, are summarized in Table 2. Highly significant differences were observed among the surface treatment (p < 0.001) groups and resins (p < 0.001). There was a significant interaction (p < 0.01) between resins and surfaces. Group 1 specimens (i.e., moist water bonding) gave µTBS values of only 3.1 and 6.4 MPa (Table 2) for the hydrophobic resins 1 and 2, respectively. These values were significantly (p < 0.05) lower than those of specimens bonded with resin 3 (27.9 ± 2.0 MPa, Table 2). The bond strengths produced by resins 4 and 5 (36.7 ± 2.0 MPa and 35.5 ± 2.2, respectively, Table 2) were not significantly different from each other, but were significantly higher than the others (p < 0.05).

Resins 1 and 2 in group 3 (i.e., dry bonding) yielded bond strengths that were similar to those in group 1 (Table 2). However, resin 3 in group 3 gave significantly lower (p < 0.05) bond strengths to dry dentin, compared with dentin wet with water (Table 2). Similarly, resins 4 and 5 in group 3 gave lower bond strengths than did the same resin in group 1 (Table 2).

Failure modes were predominantly adhesive when µTBS were less than 15 MPa, mixed when they were between 15 and 30 MPa, and largely cohesive when the bond strengths exceeded 36 MPa (Table 2).

When the mean µTBS of the 5 resins in group 1 (moist water bonding) were plotted against their Hoy’s solubility parameters for polar forces (_p), a highly significant (R² = 0.85, p < 0.05) positive exponential relationship was obtained (Fig. 2A). Similar correlations with _h and _d were not significant (R² = 0.41 and 0.36, respectively). When the tensile bond strengths of resins 1–5 in group 2 (moist ethanol bonding) were plotted against _p values (Hoy’s solubility parameter for polar forces) of the polymers, a higher correlation was found (R² = 0.90, p < 0.05). Similar plots of _h or _d were not significant (R² = 0.40 and 0.36, respectively) (Fig. 2B). In group 3 (dry bonding), significant (R² = 0.86, p < 0.05) positive correlation was found between the Hoy’s solubility parameters for polar forces (_p) (Fig. 2C). Also in group 3, lower R² values were obtained with _d and _h (0.19 and 0.50, respectively).

View larger version (7K): [in this window] [in a new window]   Figure 2. Relationships between least-squares means of microtensile bond strengths and Hoy’s solubility parameters for polar forces (p) of resins 1–5 (numbers in parentheses indicate the N upon which the mean value is based). (A) Least-squares means bond strengths of 50% ethanol-solvated resins 1–5 to acid-etched dentin saturated with water. (B) Least-squares means bond strengths of 50% ethanol-solvated resins 1–5 to acid-etched dentin saturated with ethanol. (C) Least-squares means bond strengths of 50% ethanol-solvated resins 1–5 to dry acid-etched dentin.

	DISCUSSION

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Clearly, the condition of the acid-etched dentin surface exerts a strong influence on the µTBS produced by the tested experimental resins. In group 3 (i.e., dry bonding), the collagen fibrils are known to collapse and to associate so closely that there are inadequate interfibrillar spaces available for resin infiltration (Pashley et al., 1993; Tay et al., 1996, 1998; Kanca and Sandrik, 1998; Nakaoki et al., 2000). This is due to the development of interfibrillar hydrogen bonding (H-bonds) between collagen peptides that develops a calculated attractive force of 14.8 (J/cm³) (Agee et al., 2006). For solvated comonomer mixtures to break these H-bonds, the solution must have a Hoy’s solubility parameter for H-bonding forces (_h) that is close to 14.8 (J/cm³). If not, little infiltration of solvated resins can occur within the intertubular dentin (Agee et al., 2006), although they can flow down open tubules to form resin tags. Thus, the low bond strengths produced by resins 1 and 2 in group 3 are probably due only to resin tag formation. The modest bond strengths produced by resins 3–5 may have been attributed to partial infiltration of intertubular dentin, since resins 3 and 5 have respective _h values of 14.3 and 15.5 (J/cm³).

In group 1, the etched dentin was saturated with water [_h = 40.4 (J/cm³)], which breaks interpeptide H-bonds and allows for expansion of the dentin matrix (Pashley et al., 2001; Nakajima et al., 2002; Eddleston et al., 2003). This is the mechanism responsible for the success of the wet-bonding technique with hydrophilic resins (Kanca and Sandrik, 1998). However, resins 1 and 2 gave very low µTBS values. We speculate that ethoxylated BisPhenol A dimethacrylate (resin 1) and BisGMA (resin 2) underwent phase changes when applied to water-saturated dentin, thereby weakening the hybrid and adhesive layers (Spencer and Wang, 2002). Use of the same resins in a different model system produced milky phase changes in resins 1 and 2, but not in resins 3–5. These phase changes were never seen in ethanol-saturated matrices (Becker et al., 2006).

In group 2, water-saturated dentin was treated with a large excess of ethanol, to create ethanol-saturated acid-etched dentin. In expanded acid-etched dentin, where collagen fibrils were never allowed to collapse and form H-bonds, µTBS correlated best with the _p values of the solvated resins. While the _p for water-saturated matrices was 15.3 (J/cm³), that of ethanol-saturated matrices was only 12.5, which is closer to the _p values of the resins. The closer the solubility parameters of monomers are to polymers, the better they are at swelling or wetting the polymers (Barton, 1991). Treatment of acid-etched dentin with 100% ethanol causes a 15% shrinkage in the matrix (Becker et al., 2006), by allowing for the formation of some matrix interpeptide H-bonds. This stiffens the matrix (Carvalho et al., 2003; Garcia et al., 2005) and prevents any further shrinkage during resin infiltration. When the experimental adhesives were applied to ethanol-saturated dentin in the current study, the bond strengths produced by resins 1 and 2 increased about six- to seven-fold, respectively, when compared with the same resins in group 1. Smaller increases were seen with resins 3 and 4, and no significant increase was seen with resin 5. Clearly, increases in the bond strengths of hydrophobic comonomer blends can be produced by the use of moist-bonding with ethanol instead of water. Saturation of the matrix with ethanol brings the _p of the matrix closer to those of the ethanol-solvated resins. We speculate that optimal wetting of collagen fibrils by these solvated resins occurs when the polar surface-free energy components are similar (Barton, 1991). Thus, the significant relationships between resin hydrophilicity and µTBS may be due to the degree of wetting and penetration of acid-etched ethanol-saturated dentin by the resins (Rosales et al., 1999; Asmussen and Peutzfeldt, 2005).

The results of this study indicate that wet-bonding with ethanol instead of water facilitates higher bond strengths with the use of relatively hydrophobic resins. This new wet-bonding concept may successfully coax hydrophobic monomers into the dentin matrix, thereby creating more hydrophobic hybrid and adhesive layers that absorb less water over time (Ito et al., 2005). More research is required to determine if this strategy will lead to more durable resin-dentin bonds.

	ACKNOWLEDGMENTS

 
We thank Bisco Inc. for supplying the experimental adhesives. This study was supported by grant R01 DE 014911 from the National Institute of Dental and Craniofacial Research (David Pashley, PI). The authors are grateful to Mrs. Michelle Barnes for secretarial support.

Received March 3, 2006; Last revision July 25, 2006; Accepted August 27, 2006

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Agee KA, Becker TD, Joyce AP, Rueggeberg FA, Borke JL, Waller JL, et al. (2006). Net expansion of dried demineralized dentin matrix produced by monomer/alcohol saturation and solvent evaporation. J Biomed Mater Res A (E-pub ahead of print).

Asmussen E, Peutzfeldt A (2005). Resin composites: strength of the bond to dentin versus surface energy parameters. Dent Mater 21:1039–1043.[ISI][Medline]

Asmussen E, Uno S (1992). Adhesion of restorative resins to dentin: chemical and physicochemical aspects. Oper Dent 17(Suppl 5):68–74.

Asmussen E, Hansen EK, Peutzfeldt A (1991). Influence of the solubility parameter of intermediary resin on the effectiveness of the Gluma bonding system. J Dent Res 70:1290–1293.[Abstract/Free Full Text]

Barton AFM (1991). Surfaces and interfaces. In: CRC handbook of solubility parameters and other cohesion parameters. Barton AFM, editor. 2nd ed. Boca Raton, FL: CRC Press, Inc., pp. 583–629.

Becker TD, Agee KA, Joyce AP, Rueggeberg FA, Borke JL, Waller JL, et al. (2006). Infiltration/evaporation-induced shrinkage of demineralized dentin by solvated model adhesives. J Biomed Mater Res B Appl Biomater (E-pub ahead of print).

Brackett WW, Ito S, Tay FR, Haisch LD, Pashley DH (2005). Microtensile dentin bond strength of self-etching resins: effect of a hydrophobic layer. Oper Dent 30:733–738.[ISI][Medline]

Carvalho RM, Mendonca JS, Santiago SL, Silveira RR, Garcia FC, Tay FR, et al. (2003). Effects of HEMA/solvent combinations on bond strength to dentin. J Dent Res 82:597–601.[Abstract/Free Full Text]

Chappelow CC, Power MD, Bowles CQ, Miller RC, Pinzino CS, Eick JD (2000). Novel priming and crosslinking systems for use with isocyanatomethyacrylate dental adhesives. Dent Mater 16:396–405.[ISI][Medline]

De Munck J, Van Landuyt K, Peumans M, Poitevin A, Lambrechts P, Braem M, et al. (2005). A critical review of the durability of adhesion to tooth tissue: methods and results. J Dent Res 84:118–132.[Abstract/Free Full Text]

Eddleston CL, Hindle AR, Agee KA, Carvalho RM, Tay FR, Rueggeberg FA, et al. (2003). Dimensional changes in acid-demineralized dentin matrices following the use of HEMA-water versus HEMA-alcohol primers. J Biomed Mater Res A 67:900–907.[Medline]

Finger WJ, Inoue M, Asmussen E (1994). Effect of wettability of adhesive resins on bonding to dentin. Am J Dent 7:35–38.[Medline]

Garcia FC, Otsuki M, Pashley DH, Tay FR, Carvalho RM (2005). Effects of solvents on the early stage stiffening rate of demineralized dentin matrix. J Dent 33:371–377.[ISI][Medline]

Ito S, Hashimoto M, Wadgaonkar B, Svizero N, Carvalho RM, Yiu C, et al. (2005). Effects of resin hydrophilicity on water sorption and changes in modulus of elasticity. Biomaterials 26:6449–6459.[ISI][Medline]

Kanca J 3rd, Sandrik J (1998). Bonding to dentin. Clues to mechanism of adhesion. Am J Dent 11:154–159.[ISI][Medline]

King NM, Tay FR, Pashley DH, Hashimoto M, Ito S, Brackett WW, et al. (2005). Conversion of one-step to two-step self-etch adhesives for improved efficacy and extended application. Am J Dent 18:126–134.[ISI][Medline]

Kinney JH, Marshall SJ, Marshall GW (2003). The mechanical properties of human dentin: a critical review and re-evaluation of the dental literature. Crit Rev Oral Biol Med 14:13–29.[Abstract/Free Full Text]

Miller RG, Bowles CQ, Chappelow CC, Eick JD (1998). Application of solubility parameter theory to dentin-bonding systems and adhesive strength correlations. J Biomed Mater Res 41:237–243.[ISI][Medline]

Nakajima M, Okuda M, Pereira PN, Tagami J, Pashley DH (2002). Dimensional changes and ultimate tensile strengths of wet decalcified dentin applied with one-bottle adhesives. Dent Mater 18:603–608.[ISI][Medline]

Nakaoki Y, Nikaido T, Pereira PN (2000). Dimensional changes of demineralized dentin treated with HEMA primers. Dent Mater 16:441–446.[ISI][Medline]

Pashley DH, Ciucchi B, Sano H, Horner JA (1993). Permeability of dentin to adhesive agents. Quintessence Int 24:618–631.[Medline]

Pashley DH, Agee KA, Nakajima M, Tay FR, Carvalho RM, Terada RS, et al. (2001). Solvent-induced dimensional changes in EDTA-demineralized dentin matrix. J Biomed Mater Res 56:273–281.[ISI][Medline]

Pashley DH, Carvalho RM, Tay FR, Agee KA, Lee KW (2002). Solvation of dried dentin matrix by water and other polar solvents. Am J Dent 15:97–102.[ISI][Medline]

Rosales JI, Marshall GW, Marshall SJ, Watanabe LG, Toledano M, Cabrerizo MA, et al. (1999). Acid-etching and hydration influence on dentin roughness and wettability. J Dent Res 78:1554–1559.[Abstract/Free Full Text]

Spencer P, Wang Y (2002). Adhesive phase separation at the dentin interface under wet bonding conditions. J Biomed Mater Res 62:447–456.[ISI][Medline]

Tay FR, Pashley DH (2003). Have dentin adhesives become too hydrophilic? J Can Dent Assoc 69:726–731.

Tay FR, Gwinnett AJ, Pany KM, Wei SH (1996). Resin permeation into acid-conditioned, moist, and dry dentin: a paradigm using water-free adhesive primers. J Dent Res 75:1034–1044.[Abstract/Free Full Text]

Tay FR, Gwinnett AJ, Wei SHY (1998). Micromorphological spectrum of acid-conditioned dentin following the application of a water-based adhesive. Dent Mater 14:329–338.[ISI][Medline]

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