Influence of Double Application Technique on the Bonding Effectiveness of Self-Etch Adhesive Systems RAJNI NAGPAL,1 PALLAVI SHARMA,1* NAVEEN MANUJA,2 SHASHI PRABHA TYAGI,1 UDAI PRATAP SINGH,1 SHIPRA SINGH,1 AND PAYAL SINGH1 1

Department of Conservative Dentistry & Endodontics, Kothiwal Denta L College & Research Centre, Moradabad, Uttar Pradesh, India 2 Department of Pediatric Dentistry, Kothiwal Dental College & Research Centre, Moradabad, Uttar Pradesh, India


double-application; single-step self-etch adhesives; microleakage; resin-dentin interfacial micromorphology

ABSTRACT Aim: To evaluate and compare the effect of double-application of single-step self-etch adhesives using microleakage study and to analyze the dentin–adhesive interfacial micromorphology. Methods: In total, 72 extracted human premolars were divided into three groups for different self-etch adhesives (G Bond, GC [GB], Optibond, Kerr [OB], and Xeno V Plus, Dentsply [XV]). Class V cavities were prepared. Each group was further divided into two subgroups (n 5 10) according to the placement technique of the adhesive, using the singleapplication [subgroup (a)] or double-application method [subgroup (b)]. Resin composite (Z 250, 3M ESPE, St. Paul, MN) was used to restore the cavities and light cured for 40 s. Twenty samples from each group were subjected to microleakage study. Two samples from both the subgroups of the three adhesives were used for scanning electron microscopic examination of the resin–dentin interfacial ultrastructure. Dye leakage scores were subjected to statistical analysis using Kruskal–Wallis and Mann–Whitney U-tests at significance level of P < 0.05. Results: GB depicted significantly more microleakage which was significantly greater than OB and XV. The double application led to significant decrease in microleakage of GB with no significant effect on the microleakage scores of other two all-in-one adhesives, that is OB and XV. Conclusion: Double application of all-in-one self-etch adhesives improves the marginal sealing ability in dentin although it appears to be product dependent. Microsc. Res. Tech. 78:489–494, 2015. V 2015 Wiley Periodicals, Inc. C

INTRODUCTION Trend toward the simplification of bonding procedures and overcoming the problem of inconsistent adhesion to dentin have led to the introduction of selfetch adhesives. The self-etch systems have been categorized as two-step and one-step adhesives. Furthermore, these one-step self-etch adhesives have been subclassified as strong, moderate (intermediary strong), and mild, based on their pH value (Van Meerbeek et al., 2003). One-step self-etch adhesives combine all the components of an adhesive system (etchant, primer, and bonding agent) into a single solution with a single-application (SA) step (Van Landuyt et al., 2005). With this approach, the rinsing and drying phase is eliminated, which does not only reduce clinical application time, but also significantly decreases technique sensitivity (Tay and Pashley, 2001; Van Meerbeek et al., 2001). In addition, the simultaneous impregnation of the exposed collagen matrix with resin up to the same depth of demineralization eliminates the risk of incomplete resin infiltration (Van Landuyt et al., 2005). Unfortunately, it seems that this simplicity relates to the extent of efficacy (Reis et al., 2007) as controversies over the performance of these adhesives with regard to their bonding potential have also been C V


reported (Batra et al., 2014; Chersoni et al., 2004; De Munck et al., 2003; Inoue et al., 2001; Ishikawa et al., 2007; Manuja and Nagpal, 2012b; Nagpal et al., 2011). The poor bonding performance of one-step self-etch adhesives reported in dentin may be attributed to different factors (Hegde and Bhandary, 2008). The etching pattern is not well defined as that provided by phosphoric acid (Manuja et al., 2012a; Miguez et al., 2003; Moura et al., 2006; Perdigao and Geraldeli, 2003). These products create very thin coatings, leading to oxygen inhibition, resulting in a suboptimal polymerization. They are prone to phase separation as the solvent evaporates from the solution and they behave as semi-permeable membranes after polymerization (Albuquerque et al., 2008). These adhesives are extremely hydrophilic as they contain high concentrations of both ionic and hydrophilic monomers (Miyazaki et al., 2000). Such hydrophilicity renders these *Correspondence to: Pallavi Sharma, Department of Conservative Dentistry & Endodontics, Kothiwal Dental College & Research Centre, Moradabad, Uttar Pradesh, India. E-mail: [email protected] REVIEW EDITOR: Prof. Alberto Diaspro Received 2 February 2015; accepted in revised form 7 March 2015 Abbreviations: DA, double application; GB, G bond; OB, optibond; SA, single application; SEM, scanning electron microscope; XV, Xeno V Plus DOI 10.1002/jemt.22499 Published online 10 April 2015 in Wiley Online Library (wileyonlinelibrary.com).


R. NAGPAL ET AL TABLE 1. Manufacturers, compositions, and application procedures of the tested self-etch adhesives Application procedure





GB (GC, Tokyo, Japan)

4-MET,UDMA, TEGDMA methacrylic acid ester, acetone, water, fumed silica fillers, photoinitiator GPDM, HEMA, mono- and difunctional methacrylate monomers, ethanol, acetone, and water as solvents, camphorquinone photoinitiators, nanofillers, and fluoride-releasing fillers Bifunctional acrylate, acidic acrylate, functionalized phosphoric acid ester, water, tertiary butanol, initiator, stabilizer

1. Application of one coat of adhesive (5–10 s) 2. Gentle air stream (5 s) 3. Light activation (10 s–600 mW/cm2) 4. Composite placement curing (40 s) 1. Application of one coat of adhesive (20 s) 2. Gentle air stream (10 s) 3. Light activation (10 s–600 mW/cm2) 4. Composite placement curing (40 s)

1. Steps 1–2 from (SA) 2. Repeat Steps 1–2 3. Step 3 4. Step 4 1. Steps 1–2 from (SA) 2. Repeat Steps 1–2 3. Step 3 4. Step 4

1. Application of one thick coat of adhesive under pressure 2. Gently agitate the adhesive for 20 s 3. Gentle air stream (5 s) 4. Light activation (10 s–600 mW/cm2) 5. Composite placement curing (40 s)

1. Steps 1–3 from (SA)

OB all-in-one (Kerr, Orange, CA)

XV (Dentsply DeTrey, Konstanz, Germany)

2. Repeat Steps 1–3 3. Step 4 4. Step 5

Abbreviations: Bis-GMA, bisphenol-A diglycidyl methacrylate; HEMA, 2-hydroxyethyl methacrylate; MDP, 10-methacryloyloxydecyl dihydrogen phosphate; TEGDMA, triethylene glycol dimethacrylate; 4-MET, 4-methacryloyloxy–ethyl trimellitic phosphate; UDMA, urethane dimethacrylate.

adhesives very permeable and denies their ability to hermetically seal dentin surfaces. This water sorption plasticizes polymers and lowers their mechanical properties (Bastioli et al., 1990). Although hydrophobic dimethacrylates are added to all-in-one adhesives to produce stronger crosslinked polymer networks, the hydrophilic monomers tend to cluster together before polymerization to create hydrophilic domains (Eliades et al., 2001; Spencer and Wang, 2002) and microscopic water-filled channels called water trees (Ferrari and Tay, 2003; Tay et al., 2002). Several authors have reported that self-etch adhesive systems did not improve bonding effectiveness to dentin in spite of their purported reduction in technique sensitivity (Manuja et al., 2012c; Tay and Pashley, 2003; Toledano et al., 2001). Infiltration of adhesives into the dentin and the thickness of the adhesive layer are directly correlated to rheological and chemical characteristics (Albuquerque et al., 2008; Moszner et al., 2005), but they could also be influenced by the mode of application (Belli et al., 2011). To offset the limitations of self-etching adhesives, altered bonding protocols that increase resin–dentin bond quality were suggested (Pashley et al., 2002; Toledano et al., 2007). Among the different clinical approaches are the use of an additional layer of hydrophobic resin agent (King et al., 2005), multiple-layer application of additional coats of adhesive (Erhardt et al., 2009; Hashimoto et al., 2004; Ito et al., 2005; Pashley et al., 2002; Wei et al., 2009), enhanced solvent evaporation (Van Landuyt et al., 2005), use of collagen crosslinkers and matrix metalloproteinase inhibitors (Nagpal et al., 2013, 2014; Sharma et al., 2015; Verma et al., 2013), and prolonged curing-time intervals (Breschi et al., 2007; Cadenaro et al., 2005; Erhardt et al., 2009). It has been reported that double application (DA) of onestep self-etch adhesives may result in a more uniform infiltration of the adhesive into smear layer-covered dentin if a one-step self-etch adhesive is applied in two layers (Pashley et al., 2002). Different studies have reported the effect of additional application of one-step self-etch adhesives to dentin. However, most of these studies have been

performed on flat surfaces which do not take into account the influence of C-factor on bonding. Thus, the objective of this study was to evaluate the influence of DA technique on the bonding effectiveness of self-etch and to observe the morphological characteristics of the resin–dentin interface after such treatment under scanning electron microscope (SEM). The null hypothesis tested was that the DA of adhesives on the prepared dentin surface will not affect the microleakage of onestep self-etch adhesives. MATERIALS AND METHODS This study was performed on 72 intact caries-free human premolars extracted for orthodontic purpose. After debridement and disinfection in 1% of thymol solution, teeth were stored in distilled water until use. Buccal Class V cavities centered on the cementoenamel junction were prepared with 3-mm mesiodistal width, 3 mm occlusogingival dimensions, and 1.5 mm depth, using ISO 012 straight fissure diamond point (Dentsply Detrey, USA) in an air–water-cooled highspeed handpiece. The bur was changed after every five preparations. The gingival margins on dentin were maintained as butt joint. The teeth were randomly divided into six groups according to three one-step selfetch adhesives used: G Bond (GB) (GC, Tokyo, Japan), optibond (OB) (Kerr, Orange, CA, USA), and Xeno V Plus (XV) (Dentsply DeTrey, Konstanz, Germany) and two application modes (Table 1). Group 1a; GB-SA: Dentin was etched with 37% of phosphoric acid (Etchant, 3M ESPE, St. Paul, MN) for 15 s prior to SA of GB application according to the manufacturer’s directions. Group 1b; GB-DA: The GB adhesive applied as in Group 1, followed by DA of self-etch adhesive. Group 2a; OB-SA: Dentin was etched with 37% of phosphoric acid (Etchant, 3M ESPE, St. Paul, MN) for 15 s prior to SA of OB application according to the manufacturer’s directions. Group 2b; OB-DA: The OB adhesive applied as in Group 1, followed by DA of self-etch adhesive. Microscopy Research and Technique



TABLE 2. Mean score for dye penetration of all the three self-etch adhesives Adhesives and application modes Mean microleakage score GB Group 1a; GB-SA Group 1b; GB-DA OB Group 2a; OB-SA Group 2b; OB-DA XV Group 3a; XV-SA Group 3b; XV-DA

2.6 1.6 1.6 1.4 1.4 1.0

TABLE 3. Intergroup comparisons using Mann–Whitney U-test Group comparison


Groups 1a and 1b Groups 1a and 2a Groups 1a and 3a Groups 1b and 2b Groups 1b and 3b Groups 2a and 2b Groups 2a and 3a Groups 2b and 3b Groups 3a and 3b

0.023a 0.023a 0.011a 0.639 0.203 0.639 0.639 0.431 0.431


Statistically significant difference between the two groups at a significance level of P < 0.05.

Group 3a; XV-SA: Dentin was etched with 37% of phosphoric acid (Etchant, 3M ESPE, St. Paul, MN) for 15 s prior to SA of XV application according to the manufacturer’s directions. Group 3b; XV-DA: The XV adhesive applied as in Group 1, followed by DA of self-etch adhesive. The cavities were bulk filled with resin composite (Z 250, 3M ESPE, St. Paul, MN), light cured using Spectrum 800 (Dentsply, Caulk, Milford, DE) for 40 s at 600 mW/cm2 and polished. The restored teeth in each group were thermocycled for 500 cycles at 5 and 55  C with 30 s of dwell time. For microleakage test, 10 samples from each group were coated with two layers of sticky wax, leaving a 1-mm window around the cavity margins. All the samples were then immersed in freshly prepared 2% methylene blue dye for 48 h. The teeth were then rinsed with water, the sticky wax was removed, and the teeth were left to air dry at room temperature for 24 h. After drying the samples at room temperature, the teeth were sectioned longitudinally in a buccolingual direction with the help of diamond disk by a cut through the center of the restoration. Dye penetration at the tooth–restoration interface was assessed by a stereomicroscope (Olympus 2.5X) at a magnification of 103. The following ranking systems were used to score the degree of dye penetration (Nagpal et al., 2007; Silveira et al., 2006). Dye leakage score 0 1 2 3

Criteria for Scoring No evidence of microleakage Dye penetration up to half the cavity depth Dye penetration of more than half the cavity depth Dye penetration along the axial wall

SEM Evaluation The restored samples were sectioned mesiodistally, and polished with wet 210 grit SiC paper. Acid–base Microscopy Research and Technique

Fig. 1. a: SEM view showing generalized gap at resin–dentin interface after bonding with GB using SA technique (Group 1a; GB-SA). b: SEM view showing improved interfacial seal after bonding with GB using DA technique (Group 1b; GB-DA).

treatment (6 N HCl for 30 s followed by 4% NaOCl for 10 min) was done, and the samples were dehydrated in ascending ethanol concentration (50, 75, and 95% for 20 min each and 100% for 1 h), and then transferred to a critical point dryer for 30 min. All 12 specimens were then gold sputter coated and the surfaces were examined under a SEM (Leo 435 VP Cambridge, United Kingdom). Statistical Analysis Dye leakage scores obtained were subjected to statistical analysis using Kruskal–Wallis and Mann– Whitney U-tests (SPSS Base 15.0 software) at a significance level of P < 0.05. RESULTS Sealing Ability Table 2 lists the mean scores of microleakage for GB, OB, and XV and Table 3 summarizes the intergroup comparisons using Mann–Whitney U-test and corresponding P-values. When used according to the manufacturer’s instructions in SA mode, GB depicted maximum microleakage which was significantly



Fig. 2. a: SEM view showing generalized gap at resin–dentin interface after bonding with OB using SA technique (Group 2a; OB-SA). b: SEM view showing perfect interfacial seal after bonding with OB using DA technique (Group 2b; OB-DA).

Fig. 3. a: SEM view showing generalized gap at resin–dentin interface after bonding with XV using SA technique (Group 3a; XV-SA). b: SEM view showing perfect interfacial seal after bonding with XV using DA technique (Group 3b; XV-DA).

greater than OB and XV. The double-layer application of adhesive systems led to significant decrease in microleakage with GB self-etch adhesive (Groups 1a and 1b; P 5 0.023). However, DA had no significant effect on the microleakage scores of both the other allin-one adhesives, that is OB and XV.

adhesive bonding (Kantona and Winkler, 1994; Kidd, 1976; Manuja et al., 2011; Yoshikawa et al., 1999). The results of the present investigation led to the rejection of the null hypotheses as the use of a DA technique significantly improved marginal sealing for GB although improved marginal adaptation after DA was observed for all the self-etch adhesive systems regardless of the acetone-based (GB), ethanol-based (XV), or ethanol- and acetone-based (OB) nature of the adhesives. This was also observed by Hashimoto et al. (2006). Several mechanisms could account for the better performance of DA. As the first layer of the adhesive begins to etch the dentin substrate, it might become rapidly buffered by the hydroxyapatite (Camps and Pashley, 2000), so that the additional layers of unpolymerized acidic monomers may improve the etching ability of these adhesives by increasing the concentration of acidic reagents. Simultaneously to this process, more impregnation of resin might occur by additional supply of adhesive resin as hypothesized by Ito et al. (2005) and the increased thickness of the adhesive layer, which is known to reduce polymerization stress and improve the marginal seal (Choi et al., 2000).

Scanning Electron Microscope SEM observations of the resin–dentin interface are shown in Figures 1–3. All the self-etch adhesives when applied according to the manufacturer’s directions showed the presence of interfacial gap at the dentinal surface with little or no resin tag formation. The maximum interfacial gap was evident with GB. After double-layer application of these adhesives, reduction in interfacial gap was observed for all the adhesives. DISCUSSION The problem encountered with adhesive restorative procedures is the impaired marginal seal and to ensure outstanding marginal adaptation of an adhesive restoration, some restorative aspects must be considered, such as preparation design, composite shrinkage, and

Microscopy Research and Technique


SA of the adhesive has the effect of a strong etchant although the infiltration of resin to demineralized dentin may not be sufficient. In the second application, the additional supply of adhesive resin may improve the infiltration of resin monomers into the intertubular demineralized dentin. Two of the one-step self-etching adhesives are 2hydroxyethyl methacrylate (HEMA)-free: GB and XV. The HEMA monomer is commonly added to the bonding agents and improves the stability of adhesive solutions that contain hydrophobic and hydrophilic components. In this study, GB presented significantly more microleakage as compared to other two adhesives. GB is a HEMA-free, acetone/water-based mild one-step self-etch adhesive containing a high percentage of acetone (40%). Acetone being more volatile than ethanol does not form an azeotrope with water, and hence it may not promote water evaporation when compared to ethanol-based adhesives, resulting in a low water/solvent evaporation ratio (Cho and Dickens, 2004). Lower polymerization efficiency owing to residual-free water and phase separation of its components (owing to a lack of HEMA) may result in marginal gap along the tooth–adhesive junction (Van Landuyt et al., 2007; Van Meerbeek et al., 2003; Zheng et al., 2001). This can partly explain the increased microleakage after the dye penetration test. Similarly, XV is also a HEMA-free water-based unfilled adhesive with t-butanol solvent and contains functionalized phosphoric acid esters. The presence of t-butanol in XV may plays the role of HEMA and in addition may help in eliminating the high water contents present by lowering the water vapor pressure by the formation of azeotrope (Yoshida et al., 2004). Both XV and OB depicted similar leakage scores significantly lesser than GB. This may be attributed to the fact that XV and OB have similar acidity levels, which should cause similar demineralization patterns. They are known as moderately strong self-etch adhesives; unlike strong self and total-etch adhesives, they create only a submicron hybrid layer (Tay et al., 1999). In addition to micromechanical interlocking through hybridization, specific functional monomers of these adhesives may interact chemically with residual hydroxyapatite crystals that remain available in the submicron hybrid layer (Inoue et al., 2001; Tay et al., 1999). Glycerol phosphate dimethacrylate (GPDM) adhesive monomer and filled adhesive technologies coupled with OB unique ternary solvent system provide excellent adhesion to all dental substrates. Although the tested adhesives have similar mechanisms of adhesion, HEMA-containing OB adhesive depicted lower microleakage scores and this justifies the presence of HEMA to prevent organic phase separations from the water-based composition. Several mechanisms may account for the superior performance of OB. The longer adhesive application time (40 s) recommended by the manufacturer may improve the etching ability of OB by increasing the exposure time of acidic reagents. Chemical composition of OB also differs from that of the other adhesives. This adhesive uses a ternary solvent system (water, acetone, and ethanol) that, according to the product’s technical bulletin, provides effective etching, enhanced material Microscopy Research and Technique


stability, and a uniform adhesive layer. Functional acidic monomers of these adhesives also differ: OB uses GPDM as the functional monomer, OB adhesive contains nanofillers; the previous reports (Deliperi et al., 2007; Fortin and Swift, 1994) found that the collagen fibril network filters out most nanofillers, holding them at the hybrid layer surface where they act as an intermediate shock absorber. Reduced microleakage scores have been reported for filled adhesives (Deliperi et al., 2003, 2007; Fortin and Swift, 1994). These differences in chemical composition may have helped create a thicker, more homogeneous resin layer above the hybrid layer using OB, which may have improved resistance to microleakage. In addition, under the conditions of this study, the DA of this adhesive over the cured OB and XV adhesives did not reduce the microleakage scores in comparison with the application according to the manufacturers’ directions. Thus, lower leakage values can be obtained with OB and XV adhesives applied according to the manufacturers’ directions; it is not necessary to consider the alternative modes of application.

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Microscopy Research and Technique

Influence of double application technique on the bonding effectiveness of self-etch adhesive systems.

To evaluate and compare the effect of double-application of single-step self-etch adhesives using microleakage study and to analyze the dentin-adhesiv...
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