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J Dent Res 84(8):736-740, 2005
© 2005 International and American Associations for Dental Research

RESEARCH REPORT
Biomaterials & Bioengineering

EDTA Treatment Improves Resin-Dentin Bonds’ Resistance to Degradation

R. Osorio1, M.C.G. Erhardt2, L.A.F. Pimenta2, E. Osorio1, and M. Toledano1,*

1 Department of Dental Materials, School of Dentistry, University of Granada, Granada, Spain; and
2 Department of Restorative Dentistry, School of Dentistry. University of Campinas, Piracicaba, São Paulo, Brazil;

* corresponding author, Avda. de las Fuerzas Armadas n°1, 1°B, 18014 Granada, Spain, toledano{at}ugr.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The existence of unprotected collagen fibrils within the hybrid layer compromises the longevity of restorations. This phenomenon may be avoided if solutions other than strong acids are used for dentin demineralization. The hypothesis to be tested is that bond durability may be improved by EDTA demineralization. Dentin surfaces (human and bovine) were bonded: (1) after phosphoric-acid-etching, and after EDTA demineralization with (2) a total-etch adhesive and (3) a self-etching adhesive. After the teeth were sectioned into beams, half of the specimens were immersed in NaOCl, while the other half was immersed in water. Beams were tested to failure in tension. ANOVA and multiple-comparisons tests were used (P < 0.05). No differences in bond strength were found among the 3 bonding procedures, although bonds made to human molars were 43-61% higher than those to bovine incisors. After NaOCl immersion, only specimens subjected to EDTA demineralization maintained the initial bond strength. We conclude that the collagen network is better-preserved after EDTA demineralization.

KEY WORDS: resin • EDTA • adhesion • dentin • degradation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The durability of bonds between adhesive resins and dentin is crucial, and little is known regarding the stability of hybridized dentin. Dentin bond strength decreases in vivo (Sano et al., 1999) and during water storage over time (Hashimoto et al., 2000; Okuda et al., 2002; De Munck et al., 2003). This decline is the result of hydrolytic degradation of the resin and proteolysis of unprotected collagen fibrils within the decalcified dentin (Hashimoto et al., 2000; Okuda et al., 2002; De Munck et al., 2003). It is not always uniform for different adhesives (De Munck et al., 2003). The chief reason for resin-dentin bond degradation, when a total-etch bonding approach is used, is the impaired resin infiltration of the collagen web, due to incomplete expansion of collagen, after dentin etching and priming (Pashley et al., 2003), and both the loss of the adhesive resin (Sano et al., 1999; Yamauti et al., 2003) and the solubilization of unprotected collagen fibrils within the decalcified dentin, when some hydrophilic self-etching adhesives are used (Sano et al., 1999; Osorio et al., 2005). Thus, the hydrolytic degradation of the hybrid layer seems to be the weakest component of modern adhesive systems. It is thought that milder dentin demineralization may leave more residual apatite crystallites in the collagen matrix, thus improving its durability (Takarada, 1990).

The purpose of this study was to determine the effect of ethylenediaminetetraacetic acid (EDTA) demineralization of human and bovine dentin on resin-dentin bond durability, by means of a microtensile bond strength test. The results were compared with those produced by the use of a mild self-etching primer adhesive. The null hypothesis to be tested was that there was no difference in dentin bond strength stability when 3 different adhesive procedures—phosphoric-acid-etching, self-etching, and EDTA demineralization of dentin—were used.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Twenty-four caries-free extracted human third molars and 18 freshly extracted bovine incisors, stored at 4°C in 0.5% chloramine T for up to 1 mo, were used. The use of animal tissues was approved by the institutional review board (IRB), and human specimens were obtained following a protocol that was reviewed and approved by the IRB and with the informed consent of the donors. Occlusal portions of the molars and labial surfaces of the incisors were sectioned to remove enamel and ground flat (180-grit) under running water to provide uniform dentin surfaces. Table 1Go displays modes of application, components, and manufacturers of the adhesives. A total-etch self-priming adhesive system, Adper Scotchbond 1 (SB), and a self-etching adhesive, Clearfil SE Bond (SEB), were applied following the manufacturers’ instructions. In group 1, the dentin was etched with phosphoric acid and bonded with SB. In group 2, 0.1 M EDTA (pH 7.4) was applied for 60 sec and rinsed for 10 sec prior to application of the bonding resin of SB. In group 3, untreated dentin was bonded with SEB primer, followed by its adhesive. Resin build-ups, each 6 mm in height, were constructed incrementally (1.5 mm) with Tetric Ceram (Vivadent, Schaan, Liechtenstein) resin composite. Each layer of the composite was light-activated for 40 sec with a Translux EC halogen light-curing unit (Kulzer GmbH, Bereich Dental, Wehrheim, Germany). Light intensity output was monitored with a Demetron Curing Radiometer (Model 100, Demetron Research Corporation, Danbury, CT, USA) of at least 600 mW/cm2.


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  		Table 1. Mode of Application, Compositions, and Manufacturers of Tested Adhesives
 
After distilled water storage for 24 hrs at 37°C, the bonded specimens were sectioned into multiple beams, each giving a cross-sectional area of 1 mm2. Bovine specimens were sectioned into slabs, and trimmed with a fine diamond bur, giving a cross-sectional area of 0.8 mm2. Half of the beams and/or slabs in each group were immersed in 10% NaOCl solution (Panreac Química SA, Barcelona, Spain) for 5 hrs, then rinsed in water for 1 hr. All beams/slabs were attached to a modified Bencor Multi-T testing apparatus (Danville Engineering Co., Danville, CA, USA) with a cyanoacrylate adhesive (Zapit, Dental Venture of America Inc., Corona, CA, USA) and stressed to failure in tension with the use of a universal testing machine (Instron 4411, Instron Corporation, Canton, MA, USA) at a crosshead speed of 0.5 mm/min. The fractured specimens were carefully removed from the apparatus, and the cross-sectional area at the site of failure was measured to the nearest 0.01 mm with a pair of digital calipers (Sylvae Ultra-Cal, Fowler Co., Inc., Newton, MA, USA). The bond strength values were calculated in MPa and analyzed by two-way ANOVA (adhesive procedure, NaOCl challenge) and Student-Newman-Keuls multiple comparisons (P < 0.05). Data from human and bovine dentin were analyzed separately. We examined fractured specimens using a stereomicroscope (Olympus SZ-CTV, Olympus, Tokyo, Japan) at 40X magnification to determine the mode of failure. Failure modes were classified as adhesive (A) or mixed (M). Representative specimens of each group were maintained for 48 hrs in a desiccator (Sample Dry Keeper Simulate Corp., Tokyo, Japan) and then mounted on aluminum stubs with carbon cement. They were then sputter-coated with pure gold by means of a sputter-coating Unit E500 (Polaron Equipment Ltd., Watford, England), and observed with a scanning electron microscopy (SEM) (Zeiss DSM-950, Karl-Zeiss, Göttingen, Germany) at an accelerating voltage of 20 kV, so that microscopic fracture patterns and the morphology of the debonded interface could be studied.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mean bond strength values (MPa) obtained on human and bovine dentin are shown in Tables 2Go and 3Go. In both cases, mean bond strength was affected by the NaOCl challenge (bovine, F = 42.37, P < 0.001; human, F = 28.03, P < 0.001), but the dentin bonding procedure (phosphoric acid/Scotchbond 1 resin, EDTA/Scotchbond 1 resin and Clearfil SE Bond) did not affect the bond strength (bovine, F = 0.38, P > 0.05; human, F = 0.44, P > 0.05). Interactions between both variables were significant in both types of dentin (bovine, F = 4.74, P < 0.01; human, F = 4.22, P < 0.01). The power of the ANOVA was 74% for bovine dentin and 78% for human dentin.


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  		Table 2. Microtensile Bond Strength (MPa) Obtained on Bovine Dentin for Each Tested Group and Failure Modes (%)
 

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  		Table 3. Microtensile Bond Strength (MPa) Obtained on Human Dentin for Each Tested Group and Failure Modes (%)
 
After specimen storage in water for 24 hrs, the mean bond strengths in the 3 groups were not significantly different (Tables 2Go, 3Go). Dentin bonds were 43 to 61% higher in human dentin compared with bovine dentin. NaOCl challenge diminished bond strength values in both types of dentin, except when EDTA was used for dentin decalcification prior to bonding (Tables 2Go, 3Go).

Most of the bond failures were mixed in human dentin, but adhesive in bovine dentin. Different paterns of dentin demineralization could be observed (Figs. A,B,CGo). After phosphoric-acid-etching, failures were frequently found at the base of the hybrid layer, and partial cohesive fractures of demineralized dentin could sometimes be observed just below the hybrid layer (Figs. A,DGo). After EDTA treatment, failures were mostly encountered at the top of the hybrid layer (Figs. B,EGo). Images from Clearfil SE Bond specimens showed failures found either at the top, at the base of the hybrid layer, or within the hybridized smear layer, and often both within the same section (Figs. C,FGo). After NaOCl challenge, most failures were adhesive in both types of dentin (Tables 2Go, 3Go). The microstructural aspect of intertubular dentin was modified by NaOClaq, only in specimens that underwent phosphoric-acid-etching (Figs. D,E,FGo).



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  		Figure. SEM images of the fractured interface on the dentin side of debonded specimens (A) after 24 hrs of water storage and bonded with phosphoric-acid-etching and Adper Scotchbond 1. Note failure at the base of the hybrid layer, showing partial cohesive fractures (CF) of demineralized dentin just below the hybrid layer. (B) After 24 hrs of water storage and bonded with EDTA treatment and Adper Scotchbond 1. Note failure at the top of the hybrid layer. No cohesive fracture of dentin is evidenced, and a mild dentin demineralization pattern not affecting peritubular dentin is observed. (C) After 24 hrs of water storage and bonded with Clearfil SE Bond. Failure is found either at the adhesive resin layer (AR), at the base of the hybrid layer (BHL), or within the hybridized smear layer (HSL) in the same image. (D) Bonded with phosphoric-acid-etching and Adper Scotchbond 1, and debonded after NaOClaq immersion. The intertubular dentin appearance is that of etched and NaOClaq-treated dentin, showing no collagen and enlarged tubules with many interconnecting canals. (E) Bonded with EDTA treatment and Adper Scotchbond 1, and debonded after NaOClaq immersion, intertubular dentin is not affected by NaOClaq, peritubular dentin is observed, and some tubule entrances remain resin-occluded. (F) Bonded with Clearfil SE Bond, debonded after NaOClaq immersion. Main failure is visible within the hybridized smear layer (HSL), and intertubular dentin remains unaltered by the NaOClaq.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Scotchbond 1 (SB) is a total-etch adhesive based on a Bis-GMA/HEMA/ethanol/water mixture that is able to wet the etched dentin surface and maintain the collagen in an expanded condition after the evaporation of solvents, improving monomer infiltration. High bond strength values to dentin were obtained with SB in comparison with those achieved with other total-etch adhesives (Tay et al., 1996; De Munck et al., 2003). SB also gave bond strengths that were comparable with those of SEB (Toledano et al., 2003). SEB is a two-step self-etching primer containing both HEMA and the highly hydrophilic 10-MDP monomer, which is believed to improve the wetting of the tooth surface and chelate to calcium ions of dentin (Kubo et al., 2001; Yoshida Y et al., 2004). SEB causes minimal dissolution of smear plugs and facilitates penetration, impregnation, polymerization, and entanglement of monomers with the underlying dentin, to form a very thin (ca. 1 µm) hybrid layer (Inoue et al., 2000; Osorio et al., 2003; Toledano et al., 2003).

When EDTA, instead of phosphoric acid, was used to decalcify dentin, the bond strength values were similar (Tables 2Go, 3Go) to those obtained when phosphoric-acid-etching of dentin was performed (Blömlöf et al., 2001; Cederlund et al., 2002). Similar results have been reported with the bonding of SB and other resins (including those of self-etching systems) to EDTA-demineralized bovine dentin (Miyasaka and Nakabayashi, 1999; Torii et al., 2003). EDTA is a molecule containing 4 carboxylic acid groups, and it has the ability to chelate calcium. It has been widely used to dissolve the mineral phase of dentin without altering dentin proteins, avoiding major alterations of the native fibrillar structure of dentin collagen (Sano et al., 1995; Carvalho et al., 2000; Habelitz et al., 2002; Fuentes et al., 2004). These unaltered collagen fibrils are thought to contain most of their intrafibrillar mineral, and hence are less affected by dehydration, since the structural support by the mineral is not missing (Habelitz et al., 2002), and resin infiltration may be facilitated (Sano et al., 1995). When phosphoric-acid-etching of dentin is performed, both the extrafibrillar and the intrafibrillar mineral are dissolved. These demineralized fibrils are very sensitive to dehydration. Shrinking of the fibril’s long axis is compensated by a widening of its diameter. If the individual collagen fibrils touch each other, the solvated co-monomers must diffuse around the microfibrils to break the weak forces that stabilize the matrix, thereby tending to cause them to regain their original state (Marshall et al., 2001; Habelitz et al., 2002). Wider interfibrillar spaces correlate to higher bond strength if these spaces are properly infiltrated with resin (Pashley et al., 2003).

Accelerated aging tests for resin-dentin bonds in vitro have been performed by reducing the specimens into small sticks or trimmed slabs (Okuda et al., 2002; Hashimoto et al., 2003) and by immersing the sticks in 10% NaOCl solution for a short experimental time period, to determine the ability of resin monomers to protect the collagen matrix of dentin from proteolytic activity (Hashimoto et al., 2003; Yamauti et al., 2003; Yoshida E et al., 2004; Osorio et al., 2005). NaOCl is a non-specific deproteinizing agent that, in aqueous solution, forms superoxide radicals, O2–, and induces oxidation that fragments long peptide chains of proteins (Habelitz et al., 2002). NaOCl also causes chlorination of amino terminal groups and hypochlorous acid formation (Weiss et al., 1982). Some of these amino-acid-derived chloramines have also been shown to increase the proteolytic susceptibility of this modified collagen (Olszowski et al., 2003). In the present study, a significant decrease in bond strength was observed for both the phosphoric-acid-etched specimens and those bonded with the self-etching adhesive after NaOCl immersion, presumably due to a hydrolytic process on collagen that was unprotected by resin, and/or to the hydrolytic degradation of the resin, in the case of the self-etching adhesive (De Munck et al., 2003; Hashimoto et al., 2003; Yamauti et al., 2003; Osorio et al., 2005).

However, specimens that were pre-treated with EDTA instead of phosphoric acid showed no significant decrease in bond strength after immersion in NaOCl. This may be due to an improved resin infiltration into the EDTA-demineralized collagen matrix, due to residual mineral in the collagen fibrils that increases the stability of the organic matrix (Carvalho et al., 2000; Marshall et al., 2001; Oyarzun et al., 2002).

Similar qualitative results were found for both human and bovine dentin. Even though the use of bovine dentin as a substitute for human dentin is widely accepted (Reis et al., 2004), the lower bond strengths of bovine compared with human dentin are disturbing. Bovine dentin is easier to demineralize than human dentin (Puppin-Rontani and Caldo-Teixeira, 2003), and may etch more deeply by either phosphoric acid or EDTA. Deeper levels of demineralization may increase the difficulty of resin infiltration to the depth of demineralization. The uninfiltrated collagen fibrils may fail at lower tensile stress than fully infiltrated dentin.

The null hypothesis must be rejected, since resin-dentin bonds produced by both the total-etch adhesive and the self-etching adhesive were susceptible to NaOCl challenge, but bonds made to EDTA-demineralized dentin were shown to be stable in response to NaOCl challenge. Further work is required to determine if the stability of EDTA-treated dentin facing NaOCl challenge also confers long-term durability on resin-dentin bonds. Further research should be conducted, because it is possible that the observed effect of EDTA on dentin is adhesive-system-specific, being dependent upon the composition of the applied adhesive resin. Although previous studies, with other EDTA solutions, reported similar results when a different adhesive resin was used (Miyasaka and Nakabayashi, 1999; Torii et al., 2003), they did not include degradation of the bonded interfaces.

The factors involved in bond degradation in vivo are numerous and not completely known. Thus, clinical studies, even though they are time-consuming, expensive, and lack control over important variables, are necessary.


   ACKNOWLEDGMENTS
 
This investigation was supported by Grants #MAT 2004-06872-C03-02, RED CYTED VIII.J, and CAPES #BEX-0086/04-5.

Received November 18, 2004; Last revision May 10, 2005; Accepted May 11, 2005


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 ABSTRACT
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 MATERIALS & METHODS
 RESULTS
 DISCUSSION
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