Microtensile Bond Strength and Micromorphology of Bur-cut Enamel Using Five Adhesive Systems Alexandra Vinagrea / João Ramosb / Ana Messiasc / Fernando Marquesd / Francisco Carameloe / António Mataf Purpose: This study compared the microtensile bond strengths (μTBS) of two etch-and-rinse (ER) (OptiBond FL [OBFL]; Prime & Bond NT [PBNT]) and three self-etching (SE) (Clearfil SE Bond [CSEB]; Xeno III [XIII]; Xeno V+ [XV+]) adhesives systems to bur-prepared human enamel considering active (AA) and passive (PA) application of the self-etching systems. Materials and Methods: Ninety-six enamel surfaces were prepared with a medium-grit diamond bur and randomly allocated into 8 groups to receive adhesive restorations: G1: OBFL; G2: PBNT; G3: CSEB/PA; G4: CSEB/ AA; G5: XIII/PA; G6: XIII/AA; G7: XV+/PA; G8: XV+/AA. After composite buildup, samples were sectioned to obtain a total of 279 bonded sticks (1 mm2) that were submitted to microtensile testing (μTBS; 0.5 mm/min) after 24-h water storage (37ºC). Etching patterns and adhesive interfacial ultramorphology were also evaluated with confocal laser scanning (CLSM) and scanning electron microscopy (SEM). Data was analyzed with one-way ANOVA (α = 0.05). Weibull probabilistic distribution was also determined. Results: Regarding μTBS, both adhesive system and application mode yielded statistically significant differences (p < 0.05) among groups. ER adhesive systems together with CSEB/AA and XIII/PA recorded the highest and statistically similar bond strength results. XV+ presented very low bond strength values, regardless of the application mode. Among self-etching adhesives, CSEB produced significantly higher μTBS values when applied actively. Qualitative evaluation by SEM and CLSM revealed substantial differences between groups both in adhesive interfaces and enamel conditioning patterns. Conclusions: ER and SE adhesive systems presented distinctive bond strengths to bur-cut enamel. The application mode effect was adhesive dependent. Active application improved etching patterns and resin interfaces micromorphology. Keywords: adhesive systems, microtensile bond strength, application mode, enamel, morphology. J Adhes Dent 2015; 17: 107–116. doi: 10.3290/j.jad.a34060

C

urrently, etch-and-rinse and self-etching adhesive systems represent the two main strategies to promote

a

Guest Professor, Faculty of Medicine, University of Coimbra, Coimbra, Portugal. Concept, hypothesis, experimental design, performed the experiments in partial fulfillment of requirements for a degree, wrote the manuscript.

b

Auxiliary Professor, Faculty of Medicine, University of Coimbra, Coimbra, Portugal. Concept, hypothesis, experimental design, contributed to discussion, proofread the manuscript.

c

PhD Student, Faculty of Medicine, University of Coimbra, Coimbra, Portugal. Performed statistical evaluation, proofread the manuscript.

d

Lecturer, Faculty of Medicine, University of Coimbra, Coimbra, Portugal. Performed experiment, proofread the manuscript.

e

Auxiliary Professor, Faculty of Medicine, University of Coimbra, Coimbra, Portugal. Performed statistical evaluation, proofread the manuscript.

f

Full Professor, School of Dental Medicine, Lisbon University, Lisbon, Portugal. Proofread the manuscript.

Correspondence: Dr. Alexandra Vinagre, Faculty of Medicine, University of Coimbra, Av. Bissaya Barreto, Blocos de Celas, 3000-075 Coimbra, Portugal. Tel: +35-191-263-8914, Fax: +35-123-985-7777. e-mail: [email protected]

Vol 17, No 2, 2015

Submitted for publication: 05.02.15; accepted for publication: 31.03.15

adhesion of composite resins to dental substrates.26,46 Phosphoric acid continues to be the approach of choice for optimization of enamel surface conditioning, 26 whereas the application of self-etching adhesive systems in either prepared or unprepared enamel is still a controversial issue. Several studies report the unpredictability of the performance of self-etching adhesives in intact enamel, presenting evidence that previous mechanical preparation could potentiate the adhesion capacity of these systems.10,15,24,27,33 Besides, some researchers report that the type of instrumentation used to prepare dental substrates could interfere with the performance of self-etching adhesives.5,21,37,48 Diamond bur preparation of cavities produces specific effects on enamel surfaces, resulting in the formation of a thick and irregular smear layer.21 Since enamel contains a high mineral fraction and larger hydroxyapatite crystals than dentin, and because the demineralization potential of self-etching systems is more limited than that provided by phosphoric acid, it is possible that more hydrogen ions 107

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released from the adhesive are neutralized by the enamel smear, limiting their interaction with the subsurface enamel.6,21,36 These conditions could compromise the effectiveness of adhesion to ground enamel conditioned by self-etching adhesives, particularly the least acidic ones. Active application of self-etching adhesives seems to favor the interaction with substrates by improving bond strength.2,7,8,19,22,30,39,47 This is considered a simple and rapid technique to increase mixture entropy and improve solvent evaporation before the light-curing step by increasing the kinetics of the moieties.31 Moreover, active rubbing application is thought to improve depth of demineralization, infiltration, and chemical interaction as it promotes a more effective contact of monomers at the surface.7,8,19 Thus, an active application of self-etching adhesive by rubbing it into ground enamel is expected to produce significant improvements in bonding efficacy. To date, no studies have evaluated the effect of different application modes of self-etching adhesive systems to bur-treated enamel surfaces on the bond strength and surface micromorphology. The present study aimed to evaluate the resin-enamel bond strength of two etch-and-rinse systems and three self-etching systems applied with different modes, either active or inactive, to bur-cut enamel, and to examine the enamel surface micromorphology and resin/enamel interfaces produced under the same conditions. The null hypotheses tested in this study are that (1) no differences in bur-cut enamel bond strength are found between etch-and-rinse and self-etching bonding systems; (2) surface application modes do not affect bur-cut enamel bond strength of self-etching adhesive systems.

MATERIALS AND METHODS Specimen Preparation Forty-eight noncarious human molars were collected after patients’ informed consent, as approved by the Ethics Committee of the Faculty of Medicine, University of Coimbra, Portugal (CE-001/2013). The teeth were cleaned of all surrounding soft tissues or debris and stored in a 10% buffered formalin solution (pH 7.0) at room temperature for up to six months after extraction. All molars were sectioned mesiodistally parallel to the long axis with a water-cooled diamond saw (Accutom 50, Struers; Ballerup, Denmark) in order to obtain two specimens per tooth. The buccal and lingual surfaces were carefully identified by labelling the corresponding tooth and surface. Roots were then partially removed and pulp debris detached from the remnant root canal pathway, ensuring sufficient retention for subsequent tooth embedding. Each sample was individually embedded in an acrylic resin (Vertex, Vertex-Dental; Zeist, Netherlands) for better handling, leaving the coronal portion above the acrylic resin. All buccal and lingual enamel surfaces were flattened using a medium-grit (105 to 125 μm) diamond bur (G837314-018-8-ML, Diatech, Coltène Whaledent; Altstätten, Switzerland) for 5 s with a water-cooled high-speed turbine. For each sample, the maximum surface area of flat 108

enamel was exposed. Prepared specimens were carefully observed under a stereomicroscope at 20X magnification to ensure absence of dentin, cracks, or defects. Enamel surfaces were then demarcated to outline the flattest prepared area for bonding using a flowable blue light-curing resin (LC Block-Out Resin, Ultradent; South Jordan, UT, USA), which also served to facilitate, a posteriori, the identification of useful and eligible samples for testing. An even number of buccal- and lingual-faced specimens was randomly divided into groups according to the adhesive system tested and protocol of application (AA – active application, or PA – passive application mode). Twelve halves were used for each experimental group. Two etch-and-rinse (OptiBond FL [OBFL] and Prime & Bond NT [PBNT]) and three self-etching adhesive systems (Clearfil SE Bond [CSEB], Xeno III [XIII], and Xeno V+ [XV+]) were used in this study. Their composition and application methods are described in Table 1. A single operator applied the adhesives according to the specific group protocol. An LED light-curing unit (Bluephase, Ivoclar Vivadent; Schaan, Liechtenstein) was set to the low power mode with a light intensity of 650 mW/cm2 and used throughout the adhesive application and restorative procedure. Following adhesive application, a microhybrid resin composite (Esthet·X HD, A2, Dentsply DeTrey; Konstanz, Germany) was used to create resin composite buildups in three layers of 1.5 mm each. Each layer was light cured for 20 s, followed by a final polymerization of 60 s. The specimens were then stored at 100% humidity at 37ºC for 24 h. Microtensile Bond Strength Test (μTBS) After storage, specimens were sectioned horizontally with a low-speed cutting saw (Accutom 50) perpendicular to the adhesive/tooth interface under water cooling in order to obtain three or four 1-mm parallel slices. The spaces between slices were dried and filled with a light-body polyvinylsiloxane (Aquasil Ultra XLV; Dentsply DeTrey; Konstanz, Germany) for specimen stabilization prior to the next set of cuts. Then, specimens were sectioned vertically, dividing the horizontal parallel slices into sticks with a cross-sectional area of approximately 1.00 ± 0.2 mm2. Each specimen yielded a set of 3 to 6 useful sticks. The interface perimeter of all sticks was inspected under an optical microscope (Leica CLS 150 MR; Heerbrugg, Switzerland) set at 40X magnification to exclude those with any kind of defect or failure. Additionally, each stick was measured with a digital caliper (Mitutoyo; Kawasaki, Japan) to determine the mean bonding area within each group. All sticks were constantly stored in tap water. For microtensile measurement, each stick was fixed on a testing jig with a cyanoacrylate adhesive (Permabond 735, Permabond International; Englewood, NJ, USA) and stressed at a crosshead speed of 0.5 mm/min until failure in a specific device (Od04-Plus, Odeme Prod. Med. Odont.; Luzerna, SC, Brasil) linked to a universal testing machine (Model AG-I, Shimadzu; Kyoto, Japan). The μTBS values were expressed in MPa and were calculated by dividing the imposed force (N) at the time of fracture by the bonded area (mm2). The occurrence of failure prior to The Journal of Adhesive Dentistry

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Table 1

Adhesive system: groups, composition and application mode

Group

Material (abbreviation), manufacturer, batch No.

Composition and pH

Application mode

1

Optibond FL (OBFL) Kerr; Orange, CA, USA

Etchant: 37.5% phosphoric acid (Kerr Gel Etchant) FL Primer: HEMA, GPDM, PAMA, ethanol, water, CQ, BHT (pH ca 1.9) FL Adhesive: bis-GMA, HEMA, GDMA, CQ, ODMAB, filler (fumed silica, barium aluminoborosilicate glass, disodium hexafluorosilicate), coupling factor A174

Apply etchant for 15 s; rinse for 15 s; air dry for 5 s; scrub surface for 15 s with primer; gently air blow for 5 s; apply bonding agent; gently air blow for 3 s; light cure for 20 s

Etchant: 36% phosphoric acid (Conditioner 36) Adhesive: PENTA, UDMA, resin R5-62-1, T-resin (cross-linking agent), D-resin (small hydrophilic molecule), cetylamine hydrofluoride, acetone, butylated hydroxytoluene, 4-ethyl dimethyl aminobenzoate, silica nanofiller (pH ca 2.7)

Apply etchant for 15 s; rinse for 15 s; air dry for 5 s; apply adhesive on surface and wait 20 s; gently air blow for 5 s; light cure for 10 s; apply second coat; gently air blow for 5 s; light cure for 10 s

Primer: 10- MDP, HEMA, hydrophilic aliphatic dimethacrylate, CQ, DET, water (pH ca 2.1)

Passive (CSEB/PA)

Apply primer and leave undisturbed for 20 s; gently air blow for 5 s; apply bonding agent; gently air blow for 5 s; light cure for 10 s (manufacturer’s directions)

Active (CSEB/AA)

Apply primer with a rubbing motion for 20 s; gently air blow for 5 s; apply bonding agent; gently air blow for 5 s; light cure for 10 s

Passive (XIII /PA)

Mix liquids A and B for 5 s; apply adhesive on surface and leave undisturbed for 20 s; gently air blow for 5 s; light cure for 10 s (manufacturer’s directions)

Active (XIII/AA)

Mix liquids A and B for 5 s; apply adhesive on surface and scrub it in a rubbing motion for 20 s; gently air blow for 5 s; light cure for 10 s

Passive (XV+/PA)

Apply adhesive on surface and leave undisturbed for 20 s; gently air blow for 5 s; light cure for 10 s

Active (XV+/AA)

Apply adhesive on surface and scrub it in a rubbing motion for 20 s; gently air blow for 5 s; light cure for 10 s (manufacturer’s directions)

4677483

2

Prime&Bond NT (PBNT) Dentsply DeTrey; Konstanz, Germany 1206000730

3

Clearfill SE Bond (CSEB) Kuraray Medical; Tokyo, Japan

4

041931

Bond: 10-MDP, bis-GMA, HEMA, hydrophobic aliphatic dimethacrylate, DET, silanated colloidal silica

5

Xeno III Dentsply DeTrey

Bottle A: HEMA, ethanol, purified water, BHT, stabilizers, nanofiller

1302000019

Bottle B: Pyro-EMA, PEM-F; UDMA, BHT, CQ, EPD (mixture pH < 1)

6

7

Xeno V + Dentsply DeTrey 1209000038

Bifunctional acrylic amides, acryloamido alcylsulfonic acid, “inverse” functionalized phosphoric acid esters, camphorquinone, butylated benzenediol, water, tert-butanol (pH ca 1.3)

8

Bis-GMA: bisphenol A diglycidyl methacrylate; BHT: butylhydroxytoluene; CQ: camphorquinone (photo-initiator); DET: N,N-diethanol p-toluidine; EPD: pdimethylamino ethyl benzoate; GDMA: glycerol dimethacrylate; GPDM: glycerol phosphate dimethacrylate; HEMA: 2-hydroxyethyl methacrylate; MDP: 10-methacryloyloxydecyl dihydrogenphosphate; ODMAB: 2-(ethylhexyl)-4-(dimethylamino)benzoate; PAMA: phthalic acid monoethyl methacrylated; PEM-F: pentamethacryloyloxyethyl cyclohexaphosphazene monofluoride; PENTA: dipentaerythritol pentaacrylate phosphate; pyro-EMA: tetramethacryloyloxyethyl pyrophosphate; UDMA: urethane dimethacrylate.

the actual testing was included in the calculation of the mean μTBS as 0 MPa, with an explicit note of the number of such pre-testing failures (PTF) in each group. Figure 1 shows a schematic diagram of tooth preparation, restoration, specimen sectioning, and subsequent testing. After μTBS measurement, the fractured sticks were evaluated under an optical microscope (Leica CLS 150 MR) set at 40X magnification to determine the failure mode, which was classified as follows: cohesive failure in enamel (CE), cohesive failure in composite resin (CR), adhesive (A), or mixed (M), when adhesive and cohesive failures simultaneously occurred and cohesive failures occupied more than 10% of the total area. Vol 17, No 2, 2015

Surface Enamel Micromorphology Qualitative assessment of conditioning effects created on bur-cut enamel by the different adhesive systems and methodologies described for bond strength testing was conducted under scanning electron microscopy (SEM). Enamel samples were obtained from two large molars, previously bur-prepared on all their surfaces so that substrate homogeneity could be attained across groups. For purposes of comparison, one sample was always reserved with no treatment. Self-etching systems were applied to enamel surfaces but not light cured, so that resin could be eliminated. For CSEB, only the primer was applied. Surfaces were rinsed with acetone for 109

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components. The specimens were dehydrated in ascending concentrations of ethanol and then gold sputtercoated prior to SEM observation (Hitachi S-4100, Hitachi) at an acceleration voltage of 25 kV. Micrographs were taken at magnifications of 800X and 2000X. Statistical Analysis Statistical analysis was performed with IBM SPSS Statistics 20.0 (SPSS; Chicago, IL, USA). One-way ANOVA was used to compare means of microtensile bond strength data between groups. Post-hoc pairwise comparisons were performed using the Games Howell correction. The chi-square test was used to compare between failure modes of the eight groups. The significance level was set at α = 0.05. For each group, Weibull distribution parameters were also determined using the linear regression method at a 95% confidence level. This analysis was performed using MATLAB R2012 (The Mathworks; Natick, MA, USA). Fig 1 Schematic diagram of specimen preparation. From top left to bottom right: embedding 96 buccal and lingual surfaces; grinding enamel with a diamond bur; outlining the flattest surface, bonding and restorative procedures; specimen preparation; sectioning; resin-enamel bonded sticks; microtensile bond strength.

30 s, immersed in an acetone bath and sonicated continuously for 15 min, followed by immersion for 10 min in a 95% ethanol solution and then 10 min more in a 100% ethanol solution. A 12-h acetone bath completed the dehydration process. For samples etched with 37% phosphoric acid, after the 15-s conditioning period, the acid was rinsed off with distilled water for 20 s and the sample surface dehydrated similarly. Then all samples were mounted on aluminum stubs, sputter coated with gold-paladium (Polaron E-5000, Sputter-Coater, Polaron Equipment; Watford, UK), observed and photographed in a scanning electron microscope (Hitachi S-4100, Hitachi; Tokyo, Japan) at an acceleration voltage of 25 kV. In order to standardize and compare, the micrographs were taken at magnifications of 1000X and 5000X. Resin/Enamel Interfacial Micromorphology Morphological observation of the resin/enamel interfaces was performed in 4 additional samples of each of the 8 group combinations that were prepared as for the microtensile specimens, except that two to three grains of rhodamine B were added to the adhesive to provide a fluorescent label. The teeth were sectioned longitudinally through the restoration, polished with 1200-, 2500-, and 4000-grit silicone carbide paper under refrigeration and sonicated for 15 min. The fluorescent specimens were observed with a confocal laser scanning microscope (LSM 710, emission 561 nm, pass filter 570 nm; Carl Zeiss; Göttingen, Germany). Micrographs were taken at magnifications of 400X and 800X. The same specimens were then conditioned with a 37% phosphoric acid solution for 10 s to remove the inorganic component and washed with distilled water. They were then immersed in a sodium hypochlorite solution to dissolve the organic 110

RESULTS A total of 276 specimens were available for microtensile testing. Descriptive statistics, the number of tested specimens and pre-test failures (PTF) are depicted in Table 2. One-way ANOVA revealed statistically significant differences among groups (F[7, 105.213] = 333.636, p < 0.01). Pairwise comparisons between groups indicated no significant differences among the mean μTBS values of OBFL, PBNT, CSEB/AA, and XIII/PA, which recorded the highest bond strength values. The interaction between self-etching adhesive system and application mode was only statistically significant for CSEB (CSEB/ AA vs CSEB/PA, p < 0.01). For this adhesive, the active application mode demonstrated an increase in the mean of μTBS of 9.51 MPa (4.79-14.22, 95% CI). Regarding XIII, no significant differences in bond strength were observed between application modes, although a higher result variability was recorded for XIII/PA. CSEB/ PA showed significantly lower bond strength (p < 0.01) than OBFL, PBNT, CSEB/AA, XIII/PA, and XIII/AA, and significantly higher bond strength (p < 0.01) than XV+. XV+ also revealed significant differences from all other groups (p < 0.01), regardless of the application mode. In fact, this adhesive system generated almost null bond strength values and numerous pre-test failures (PTF). As statistically significant differences were detected for both μTBS data between groups and application modes of selfetching systems, both null hypotheses were rejected. The results of the Weibull survival analysis are presented in Fig 2 and Table 3. Weibull analysis indicated the highest characteristic strength and Weibull modulus for OBFL, PBNT, and CSEB/AA among all other groups. The failure pattern frequency and distribution can be analysed in Table 4. Etch-and-rinse adhesives OBFL and PBNT followed a similar trend, in which mixed, adhesive, and enamel cohesive failure modes were more often detected. For the self-etching adhesives XIII and CSEB in either application mode, failures were predominantly adheThe Journal of Adhesive Dentistry

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Table 2

Descriptive statistics of bur-cut enamel μTBS as mean ± standard deviation for all groups

Group 1: OBFL

n

Mean ± SD (MPa)

33

Min (MPa)

Max (MPa)

PTF (n)

26.86 ±

7.71a

12.02

39.84

0

7.17a

14.74

41.19

0

2: PBNT

31

27.10 ±

3: CSEB/PA

40

15.63 ± 6.51b

5.60

29.97

0

4: CSEB/AA

41

25.14 ± 7.11a,c

11.35

41.14

0

5: XIII/PA

35

22.65 ± 8.58a,c

6: XIII/AA

32

9.03

43.62

0

4.28c

14.27

29.71

0

3.03d

0.00

12.26

14

0.00

2.37

22

21.56 ±

7: XV+/PA

36

2.38 ±

8: XV+/AA

28

0.24 ± 0.62d

Means with different superscript letters indicate significant difference (p < 0.05). n: number of specimens; Min: lowest bond strength value; Max: highest bond strength value; PTF: pretest failures. PA: passive application mode; AA: active application mode. OptiBond FL: OBFL; Prime & Bond NT: PBNT; Clearfil SE Bond: CSEB; Xeno III: XIII; Xeno V+: XV+.

1 0.9

Probability of failure

0.8 0.7 0.6 0.5

OBFL PBNT XV+/PS XV+/AT XIII/PS XIII/AT CSEB/PA CSEB/AA

0.4 0.3 0.2 0.1 0 0

5

10

15

20

Fig 2 Weibull distribution: probability of failure vs applied stress (MPa).

25

30

35

40

45

50

Applied stress

Table 3 Weibull modulus (m) and 95% confidence interval (CI) of m. characteristic strength (σ0) and 95% confidence interval (CI) of σ0, and Weibull coefficient of correlation (r) of experimental groups Group

m

m 95% CI

σ0

σ0 95% CI

r

1: OBFL

3.75

3.11; 4.88

29.60

26.17; 32.58

0.97

2: PBNT

3.14

2.83; 3.63

30.98

26.58; 36.24

0.52

3: CS:EB/PA

2.23

2.02; 2.55

18.50

15.14; 22.32

0.57

4: CSEB/AA

3.40

2.95; 4.08

28.20

24.93; 31.57

0.76

5: XIII/PA

2.59

2.29; 3.09

25.96

21.76; 30.59

0.69

6: XIII/AA

4.99

4.37; 6.03

23.62

21.39; 25.68

0.72

7: XV+/PA

1.18

0.95; 1.62

4.20

2.57; 6.08

0.94

8: XV+/AA

0.89

0.67; 1.43

1.30

0.38; 2.71

0.96

For group abbreviations, see Table 2.

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Table 4 Distribution of the failure patterns of the experimental groups in absolute number of specimens (percentage) Group Adhesive Mixed Cohesive in resin Cohesive in enamel PTF

1 OBFL

2 PBNT

3 CSEB/PA

4 CSEB/AA

5 XIII/PA

6 XIII/AA

7 XV+/PA

8 XV+/AA

9 (27.3)

7 (22.6)

18 (45)

20 (48.8)

14 (40)

15 (46.9)

22 (61.1)

6 (21.4)

13 (39.4)

9 (29)

16 (40)

17 (41.5)

11 (31.4)

13 (40.6)

0 (0)

0 (0)

3 (9.1)

1 (3.2)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

8 (24.2)

14 (45.2)

6 (15)

4 (9.8)

10 (28.6)

4 (12.5)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

14 (38.9)

22 (78.6)

For group abbreviations, see Table 2.

sive or mixed. PTF were recorded only for XV+, all of which were adhesive, which indicated a significant association between groups and failure pattern (χ2[28] = 223.76; p < 0.01). The surface micromorphology of enamel rendered by bur and conditioning with different adhesives is shown in Fig 3. The SEM micrograph of enamel prepared with a 125-μm diamond bur shows an irregular, rough surface covered with a dense smear layer that prohibits identification of exposed enamel prisms (Fig 3a). The etching pattern obtained with phosphoric acid induced a clearer dissolution of enamel prisms vs that found with self-etching systems. The enamel surface appeared very porous, and numerous enamel crystallites could be observed (Fig 3b). Morphological changes in enamel surfaces were more distinct when self-etching adhesives were applied actively than passively. Nevertheless, demineralization depth seems to be adhesive related. In specimens etched with passively applied CSEB or XIII, smear layer debris and bur scratches can still be identified on enamel surfaces (Figs 3c and 3e). A few areas revealed a discrete degree of interprismatic demineralization, which can be better recognized at higher magnifications. No distinct morphological prismatic features were observed on the enamel surface where XV+ was applied passively (Fig 3 g). The enamel surface etched with actively applied CSEB shows a mild interprismatic dissolution of surface crystallites that create microporosities within enamel prisms (Fig 3d). For actively applied XV+, a less distinct pattern was observed (Fig 3 h). In contrast, actively applied XIII seemed to show a greater demineralization depth, mainly at interprismatic areas (Fig 3f). The resin/enamel interfaces obtained with SEM and fluorescence microscopy are shown in Fig 4. For both of the etch-and-rinse adhesives OBFL and PBNT a higher level of inter- and intraprismatic resin penetration into demineralized enamel was observed, compared to the self-etching adhesives, as confirmed by fluorescence images (Figs 4 and 5). CSEB/AA, CSEB/PA, or XIII/PA resin/enamel interfaces showed a very thin hybridized layer with poorly defined resin tags (Figs 6 to 8). Nevertheless, actively applied self-etching systems seemed to enhance monomer penetration (Figs 7 and 9). For XV+ in both application modes, interfacial gaps were noticeable, probably related to cohesive failures in the hybrid layer, as some enamel interprismatic penetration was evident (Figs 10 and 11). 112

DISCUSSION Although clinical trials remain the gold standard in evaluating the performance of dental materials, useful clinical data for each new individual product is difficult to obtain. Therefore, laboratory tests are still beneficial tools to evaluate and explore methodologies concerning the use of dental adhesives. Within the bond testing literature, every research group produces individualized datasets, because of the many variables associated with this field; thus, caution is advisable when interpreting bonding data across separate studies.37,38 Because of the brittleness of the tissue, enamel microtensile specimens are intrinsically more prone to failure.34 Nevertheless, this methodology is frequently used, even for testing adhesion to enamel. Van Meerbeek et al45 proposed the use of alginate or gypsum to fill up the space between the slabs during microspecimen processing to better support the slabs during the second, 90-degree-turned cut. Due to better handling, extra-low viscosity polyvinylsiloxane was used for space filling in the present study. Considering that premature debonding only occurred in the group of the self-etching adhesive XV+ at such a high frequency, it is probably due to an intrinsic problem of adhesive. For self-etching adhesives, some original protocol variations have been proposed to improve their performance in both enamel and dentin in terms of conditioning time,28,47 previous phosphoric acid etching,11,41 passive vs active application mode,2,7,8,19,22,30,39,47 number of adhesive layers applied, or placement of a final, separate hydrophobic coating.1,32,42 In the present study, as expected, both etch-and-rinse systems yielded the highest bond strengths to bur-cut enamel. Nevertheless, they did not differ significantly from those obtained for XIII regardless of the application mode, or from CSEB when actively applied. In fact, in the present study, the active application mode only increased enamel bond strength for the mild self-etching adhesive CSEB. In contrast, noticeable changes in enamel surface micromorphology could be perceived when the active application mode was employed regardless of the selfetching system studied. Generally, greater dissolution was achieved, although to different degrees depending on the adhesive system, confirming that active application allows fresh acidic monomers to progressively reach the bottom of the smear layer and interact with subsurface enamel, The Journal of Adhesive Dentistry

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Fig 3 Representative SEM photomicrographs (magnification 5000X) of (a) a bur-prepared enamel surface where surface appears irregular and rough with a thick smear layer that inhibits identification of exposed enamel rods; (b) bur-prepared enamel surface conditioned with 37% phosphoric acid showing a geometrical, uniform and regular etching pattern with a widespread removal of apatite crystals, generating a classical honeycomb configuration. A multitude of deep intercrystallite pits could be observed; (c) bur-prepared enamel surface conditioned with CSEB/PA where globular debris on the surface resembling discrete signs of smear layer are present. Cutting scratches are mostly seen and an unclear and inhomogeneous etching effect can be observed; (d) bur-prepared enamel surface conditioned with CSEB/ AA exhibiting a superficial but regular etching effect in which selective demineralization of the interprismatic enamel was preferential. Cutting scratches could be identified; (e) bur-prepared enamel surface conditioned with XIII/PA showing a shallow, non-defined etching effect, where cutting scratches could be easily identified; (f) bur-prepared enamel surface conditioned with XIII/AA with a welldefined etching pattern, where substrate was etched to a certain depth, resulting in a “keyhole” configuration, in which the etching of prism peripheries was more clearly observed; (g) bur-prepared enamel surface conditioned with XV+/ PA showing clear signs of smear layer and cutting scratches. An indistinguishable etching effect could be observed; (h) bur-prepared enamel surface conditioned with XV+/AA revealing a slight, poorly defined etching effect, in which occasional prism periphery demineralization could be observed.

a

b

c

d

e

f

g

h

leading to more aggressive demineralization. Thus, in the present investigation, a positive correlation could be found between the active mode of application and etching capacity of the self-etching systems, but this was not always accompanied by improved enamel bond strengths. Similar results were reported by other authors.2,22,39 However, there was a great qualitative difference in the morphology of enamel surfaces conditioned by phosphoric acid and self-etching adhesive systems, as shown in the SEM images. Phosphoric acid created the most regular and deepest etching pattern involving both inter- and intraprismatic enamel, as previously reported.12,29 Vol 17, No 2, 2015

The CSEB adhesive system is a two-step self-etching system that has become one of the most promising materials regarding adhesion to dentin.9,33,44 In line with threestep etch-and-rinse systems such as the OBFL system, this system is considered a benchmark in the adhesion field.45 However, the use of CSEB according to the manufacturer’s recommendation (passive mode) on bur-cut enamel led to low μTBS values in this study. The surface preparation method can significantly affect the nature of the enamel smear layer; using a bur in clinical situations, a thicker, rougher surface is produced than that obtained in the laboratory using SiC papers.21 It is hypothesized 113

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Fig 4 SEM (left) and Confocal Laser Scanning Microscope (CLSM) fluorescent (right) photographs of the enamel/resin interfaces in a cross-sectioned specimen treated with OBFL where both inter- and intraprismatic adhesive penetration in demineralized enamel is evident.

Fig 5 SEM (left) and CLSM fluorescent (right) photographs of the enamel/resin interfaces in a cross-sectioned specimen treated with PBNT where both inter- and intraprismatic adhesive penetration in demineralized enamel is evident.

Fig 6 SEM (left) and CLSM fluorescent (right) photographs of the enamel/resin interfaces in a cross-sectioned specimen treated with CSEB/PA showing a very shallow and superficial adhesive penetration in demineralized enamel.

Fig 7 SEM (left) and CLSM fluorescent (right) photographs of the enamel/resin interfaces in a cross-sectioned specimen treated with CSEB/AA showing a shallow adhesive intaprismatic penetration in demineralized enamel.

Fig 8 SEM (left) and CLSM fluorescent (right) photographs of the enamel/resin interfaces in a cross-sectioned specimen treated with XIII/PA showing a shallow and inhomogeneous adhesive intra- and interprismatic penetration in demineralized enamel.

Fig 9 SEM (left) and CLSM fluorescent (right) photographs of the enamel/resin interfaces in a cross-sectioned specimen treated with XIII/AA showing a shallow and consistent adhesive intra- and interprismatic penetration in demineralized enamel.

Fig 10 SEM (left) and CLSM fluorescent (right) photographs of the enamel/resin interfaces in a cross-sectioned specimen treated with XV+/PA showing an inconsistent adhesive penetration with an evident interfacial gap.

Fig 11 SEM (left) and CLSM fluorescent (right) photographs of the enamel/resin interfaces in a cross-sectioned specimen treated with XV+/AA showing intra- and interprismatic adhesive penetration in demineralized enamel along with interfacial gap formation indicating debonding beyond the adhesive layer.

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that the compromised enamel bonding could to a certain extent be attributed to interference of bur debris smeared across enamel during cavity preparation, particularly with mild self-etching adhesives, such as CSEB, as their demineralization capacity is limited.12,20,36 This was corroborated in this study, since its application in the passive mode showed an enamel surface with persistent signs of smear layer, bur-cutting scratches, and an unclear and inhomogeneous etching effect. On the other hand, 24-h enamel bond strength of actively applied CSEB was found to be significantly higher than that obtained with the same adhesive system applied passively; the increase of bond strength was about 10 MPa. In addition, a more regular etching effect and more pronounced intraprismatic resin penetration into demineralized enamel was observed. These improved results support the active application mode on bur-cut enamel for this particular adhesive system, as it can probably help increase the quantity of hydrogen ions available at the surface, enhancing the demineralization process and the penetration of the primer into subsurface demineralized enamel.22 Further, this procedure may potentiate chemical interaction with underlying enamel by forming calcium phosphate salts as a result of the chemical bond of hydroxyapatite calcium with the functional monomer 10-MDP.18,33 Active application of XIII did not reveal an increase in enamel bond strengths values, as also reported by other authors.47 Also, when applied passively according to the manufacturer’s instructions and compared with CSEB, no significant differences in ground enamel bond strengths were reported between those two systems, despite conflicting reports of the superiority of XIII17,43 vs reported higher bond strengths for CSEB.1,3 XIII is a strong two-component, one-step self-etching adhesive system, having a pH of the mixture < 1 and the ability to moderately demineralize enamel.12,43 Although agitation of XIII produced a more defined etching pattern, the inherent demineralization effect of this system must be sufficient to promote adequate micromechanical retention of monomers. Nevertheless, less variability of μTBS values, a high Weibull modulus, and more defined interfacial morphological features were reported for XIII in the active application mode, which can indicate a more reliable and consistent adhesion ability. Moreover, when applied to dentin, significantly better immediate and long-term bond strengths were reported when this system was applied actively.7,19,30 The self-etching system XV+ yielded results that may be cause for concern. Objectively, almost no enamel μTBS was measurable, regardless of application mode, since adhesive mechanically-induced or pre-testing failures readily occurred. XV+ is a HEMA-free, water-based one-bottle self-etching adhesive that incorporates tert-butanol as a cosolvent and is considered an intermediately strong system with a pH around 1.3.4 Although limited research has been conducted with this specific adhesive system, it is considered an optimized version of its predecessor Xeno V, where the main differences lie in the absence of acrylic acid and a wettability agent, but better curing efficiency. Nevertheless, both formulations contain the most important, similar components with potential innovative functions, namely, Vol 17, No 2, 2015

“inverse” functionalized phosphoric acid esters, bifunctional acrylic amides, and tertiary butanol. These patented monomers are claimed to be more stable in aqueous acidic solution than are acrylic ester functions of methacrylates. In addition, the methyl groups surrounding the alcohol group of tert-butanol would prevent an addition chemical reaction with the polymerizable acrylic groups, keeping this function intact.23,35 Despite these potential advantages, several factors can be pointed out for the failure observed in this study for the self-etching system XV+. The first is related to the composition that, if equivalent to the predecessor Xeno V, includes a high solvent (73  wt%) and low hydrophobic (3 wt%) proportion, accounting for the highly hydrophilic behavior.13 The second is related to the absence of HEMA, which can predispose to phase separation, requiring strong air drying of the adhesive before curing.40 Nonetheless, this procedure can lead to a critical reduction of adhesive layer thickness which, combined with the low hydrophobic fraction and the presence of oxygen, reduces polymerization efficiency and, consequently, bonding effectiveness. More studies should be performed to better understand the bonding mechanism of this specific system and others like it, especially because simplified adhesives are being increasingly used in patients without any proof of clinical efficacy. Previous studies have shown that ultramorphological features on enamel depend mainly on the pH of the solution.12,14,25 Instead, the composition and mechanical properties of the adhesive layer achieved after curing may contribute in a major way to the bond strength accomplished for each specific material.16,43

CONCLUSIONS The results of the present study lead to the rejection of the null hypotheses. Both the adhesive system and the application method had a significant effect on the bond strengths to bur-prepared enamel. The etch-and-rinse adhesive systems OBFL and PBNT together with CSEB used actively and XIII used passively exhibited the highest and statistically similar bond strengths. XV+ showed significantly lower bond strengths compared to all other materials, regardless of the application mode. In respect to application mode, active application demonstrated a significant and positive influence on CSEB bond strength.

ACKNOWLEDGMENTS The authors would like to express their gratitude to Kerr, Kuraray Medical Inc, and Dentsply DeTrey for providing the materials used in this study.

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Clinical relevance: The trend in bonding has been towards simplification; nevertheless this may compromise the bonding performance of some self-etching systems to bur-prepared enamel. Active application on the enamel surface can be a valuable tool to improve the performance of some self-etching adhesive systems.

The Journal of Adhesive Dentistry