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Influence of the Number of Adhesive Layers on

Adhesive Interface Properties Under Cariogenic

Challenge Using Streptococcus Mutans

Nashalie Andrade de Alencara / Tatiana Kelly da Silva Fidalgob / Marlus Roberto Rodrigues Cajazeirac / Lucianne Cople Maiad

Purpose: To test the hypothesis that the number of adhesive layers influences the adhesive interface properties under cariogenic challenge conditions using a Streptococcus mutans model.

Materials and Methods: Bovine teeth (n = 90) were sectioned into blocks of 5 mm and divided into two groups for microleakage testing (n = 60) and tensile bond strength testing (n = 30). In each group, the samples were subdivided into subgroups according to the number of adhesive layers applied on the dentin: one (SB1), two (SB2), and three adhesive layers (SB3). The samples of the control groups were placed in BHI broth medium supplemented with 2% sucrose without microorganisms, and the experimental groups were submitted to Strepto-coccus mutans American Type Culture Collection (ATCC) for 5 days. For the tensile strength test, samples were sectioned into 1-mm-thick slices and submitted to a constant load of 0.5 mm/min in a universal testing ma-chine. Fractured surfaces were analyzed and characterized as adhesive, cohesive, or mixed. The microleakage test was performed with silver nitrate solution.

Results: In experimental groups, the tensile test revealed a statistically signifcant difference between the one- (18.59 ± 5.3) and three-layer (11.28 ± 5.0) groups (p < 0.001; ANOVA and Tukey’s test). The adhesive failure mode was slightly more frequent in the one- (60%) and three-layer (80%) adhesive application groups. On the other hand, the microleakage levels of all experimental groups were statistically similar (Kruskal-Wallis; p > 0.05).

Conclusion: The experimental conditions influenced tensile properties and failure modes of different adhesive interfaces; however, they did not influence microleakage.

Keywords: dentin bonding agent, tensile strength, dental leakage, Streptococcus mutans, dental caries.

J Adhes Dent 2014; 16: 339–346. Submitted for publication: 27.06.13; accepted for publication: 14.02.14 doi: 10.3290/j.jad.a32569

a Graduate Student, Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. Performed the experiments, performed statistical analysis, wrote the manuscript.

b Postdoctoral Fellow, Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. Performed the experiments, proofread the manuscript, performed statistical evaluation, contributed substantially to discussion.

c PhD Student, Associate Professor, Department of Specific Formation, School of Dentistry, Universidade Federal Fluminense – Nova Friburgo, Rio de Janeiro, Brazil. Experimental design, proofread the manuscript, performed statistical evaluation, and contributed substantially to discussion.

d Full Professor, Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. Idea, hypothesis, experimental design, proofread the manuscript, performed statistical evaluation, and contributed substantially to discussion.

Correspondence: Professor Lucianne Cople Maia, Disciplina de Odontopedia-tria da FO-UFRJ, Caixa Postal: 68066, Cidade Universitária, CCS, CEP 21941-971, Rio de Janeiro, RJ, Brazil. Tel: +55-21-2562-2101. e-mail: rorefa@terra.com.br

The hybrid layer is an important structure for the ad-hesion of composite resins to dentin.25 It is formed

by the replacement of dissolved hydroxyapatite crystals with adhesive monomers that infiltrate in dentin and copolymerize in intimate contact with the collagen fiber network.9

Typically, the etch-and-rinse technique is based on an acid conditioner, usually phosphoric acid at concentra-tions from 30% to 40%, for removal of smear layer and superficial demineralization of the underlying dentin. After rinsing off the phosphoric acid, the conditioned surface is prepared to receive the primer and adhesive, which may be in separate containers or together in the same vial.9

Some demineralized dentin remains that is not com-pletely infiltrated by the adhesive during etching, resulting in a persistent area of unprotected collagen located under the hybrid layer.17,35 The existence of this unprotected

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area is considered an important drawback of the etch-and-rinse technique, and it may present a risk to bonding longevity, since unprotected collagen fibers can be hydro-lyzed15,16 or degraded by endogenous metalloproteinases in the dentin.10,27 However, the monomer infiltration level into dentin may be increased by the technique with which the adhesive is applied. Some authors suggest increasing the application time,32 applying friction during applica-tion,8 and using a consecutive multilayer adhesive ap-proach.18,19 The multilayer application increases the bond strength with dentin and produces a less porous hybrid layer which is less susceptible to degradation.19

The long-term maintenance of adhesive interface in-tegrity is still a challenge in adhesive dentistry.9 This difficulty usually occurs in clinical practice, since failure due to marginal leakage23 and secondary caries20,23 are directly associated with the adhesive interface degrada-tion process. Simple changes to bonding technique can improve resin-dentin bond strengths and keep the restor-ation intact.11 Therefore, the effect of multilayer adhesive application on bonding and the quality of the resin/dentin interface is of crucial interest in clinical practice.

Adhesive interface deterioration in vivo is a complex phenomenon, occurring as a result of a combination of physical factors, such as chewing stress26 and dimen-sional changes resulting from thermal alterations,10 chemical enzymatic action,14,27 hydrolytic effects,10 and microbiological effects.5

Knowledge about the effects of cariogenic challenge on the adhesive interface is still limited.2 Cariogenic chal-lenge consists of longer exposure to demineralizing con-ditions (acid exposure) than to remineralization (neutral exposure). To simulate cariogenic challenge, previous

laboratory studies have largely been performed using chemical models such as pH cycling.30,38 Collagen un-protected by the adhesive is susceptible to enzymatic degradation by microorganisms.21,35 However, microbio-logical methods to induce cariogenic challenge are still rarely applied.

Thus, the aim of this study was to determine whether the different experimental hybridization protocols based on the number of adhesive layers influence the adhesive interface properties under cariogenic challenge conditions using a Streptococcus mutans microbiological model. The hypothesis was that different numbers of adhesive layers alter the tensile bond strength and the microleakage after submission to cariogenic challenge.

MATERIALS AND METHODS

Sample Preparation and Groups

Ninety bovine incisors without signs of hypoplasia, cracks, or fractures were selected for this study. The central portions of the buccal surfaces of the teeth were sectioned into blocks of 25 mm2 surface area (5 mm x 5 mm) and a thickness that varied as a func-tion of enamel and dentin layer thickness, using a double-faced diamond saw under water cooling (Isomet, Buehler; Lake Bluff, IL, USA). Then the 90 enamel-den-tin blocks were divided into two main groups according to the test performed (Fig 1): the tensile bond strength test (n = 60; 10 control and 10 submitted to the car-iogenic challenge for each subgroup) and the micro-leakage test (n = 30; 5 control and 5 submitted to the cariogenic challenge for each subgroup).

Fig 1 Design of cariogenic challenge experiments.

90 bovine incisors

SB 2 20 blocks

SB 3 20 blocks

Cariogenic challenge

BHI + sucrose + S. mutans (15 blocks)

Control

BHI + sucrose (15 blocks)

Cariogenic challenge

BHI + sucrose + S. mutans (30 blocks)

Control

BHI + sucrose (30 blocks)

SB 1 10 blocks

SB 2 10 blocks

SB 3 10 blocks

SB 1 20 blocks

Tensile bond strength 30 blocks

Microleakage 60 blocks

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Cavity Preparations

Figure 2 illustrates the sample preparations and ex-perimental design. For tensile bond strength specimens, a groove 5 mm wide, 5 mm long, and approximately 0.5 mm deep into the dentin was prepared on the enamel surface of the blocks using a cylindrical bur (#4103, KG Sorensen; São Paulo, SP, Brazil) mounted in a high-speed handpiece under copious water cooling. For the microleakage test, cavities with a diameter of 10 mm and a depth of 0.5 mm in dentin were prepared with cylindrical diamond burs (#4103, KG Sorensen) mounted in a high-speed handpiece under copious water cooling (Fig 2).

Adhesive Application Techniques

For both tensile bond strength and microleakage speci-mens, the enamel margins of the prepared cavities were conditioned with a 37% phosphoric acid gel (Dentsply; Petrópolis, RJ, Brazil) for 30 s and dentin walls for 15 s. After the conditioner was rinsed off for 30 s, excess moisture was removed with a cotton pellet. The adhe-sive used was Adper Single Bond 2 (3M ESPE; St Paul, MN, USA), applied in one, two, or three layers as fol-lows: One coat, subgroup SB1: One thin layer of Adper Sin-

gle Bond 2 adhesive was passively applied to dentin using a microbrush (KG Sorensen). After thinning with a slow air stream for 5 s, the adhesive was polymer-ized for 10 s using a halogen light-curing unit set at 600 mW/cm2 (Elipar Highlight, 3M ESPE).

Two coats, subgroup SB2: The first thin layer of Adper Single Bond 2 adhesive was passively applied to den-tin using a microbrush (KG Sorensen). An air stream

was briefly applied to the first adhesive layer, and then a second layer was applied in the same manner as the first. These adhesive layers were polymerized for 10 s using the same halogen light-curing unit set at 600 mW/cm2.

Three coats, subgroup SB3: After the application of the second layer as described for subgroup SB2, a third thin adhesive layer was applied and polymerized for 10 s using the same light-curing unit set at 600 mW/cm2.

Cavities were restored with Filtek Z350 composite resin (3M ESPE) according to the incremental technique. Each increment was polymerized for 20 s using the halogen light-curing unit set at 600 mW/cm2 (Elipar Highlight). For tensile bond strength specimens, the resin-composite buildups were standardized using a caliper rule to a height of 3.5 mm. Resin composite buildups were con-structed above the top of the restored groove.

After restoration of the samples for both tensile bond strength and microleakage testing, a layer of nail varnish was applied to the tooth to prevent demineralization of other areas, leaving only the buccal surface exposed.

Microbiological Cariogenic Challenge

Each prepared sample was placed into a well and ster-ilized in ethylene-oxide gas at 25°C to preserve the organic phase. After restoration, half of the specimens of each group were immersed in the test solution to simulate cariogenic challenge, while the other half was immersed in the control solution. For both groups, the incubation period was 7 days.

For the control groups (C), each well was filled with 1.5 ml of BHI broth medium (Brain Heart Infusion, Difco;

Fig 2 Sample preparations and experimental design.

Sample preparation sequence for microitensile bond strength test

Sample preparation sequence for microleakage test

Microleakage analysis

Score 0

Score 1

Score 2

Score 3

Tensile load (0.5 mm/min)

Control solution BHI + sucrose

Test solution BHI + sucrose + S. mutans

5.0 mm

5.0  mm

5.0 mm2.0 mm

1.0 mm

1.0 mm0.5  mm

Enamel Dentin

3.5  mm

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Franklin Lakes, NJ, USA) supplemented with 2% sucrose without microorganisms.

For the experimental groups (E), inoculation was per-formed with American Type Culture Collection (ATCC) specimens of Streptococcus mutans ATCC 25175. Bac-teria were kept at -20°C in tryptic soy broth (TSB; Oxoid, Hampshire, England) with 20% glycerol and activated by transfer into BHI agar. They were incubated under micro-aerophilic conditions at 37°C for 48 h in a candle jar. Bacterial cells were suspended according to the 0.5 Mc-Farland protocol22 and the 0.5 scale in BHI broth medium supplemented with 2% sucrose. The absorbance was de-termined at 625 nm (A625) in a spectrophotometer (Bi-oespectro model SP-220; Curitiba, PR, Brazil). The A625 was set at 0.10, which is within the range (A625 = 0.08 to 0.14, corresponding to 1.5 x 108 CFU/ml) that corre-sponds to 0.5 on the McFarland scale. Then the cells were diluted to approximately 5.0 x 106/ml (1.5 ml/well). Sub-sequently, the experimental group was placed in contact with the S. mutans cells and the culture was incubated at 37°C under microaerophilic conditions for 5 days. The BHI broth medium supplemented with 2% sucrose of all samples was replaced twice throughout the experimental period.

At the end of this period, the pH of the BHI medium (with and without inocula) was analyzed (pH-Fix test strips, Macherey-Nagel; Bethlehem, PA, USA) in both control and experimental groups.

Tensile Bond Strength Test

After a storage period in control or test solution, the specimens were sectioned into 6 sticks. The outer 2 sticks were discarded to avoid overexposure to Strepto-coccus mutans. The sticks, with dimensions of 2.0 mm (wide) x 1.0 (long) x 3.5 mm (deep), were fixed in special devices with a cyanoacrylate adhesive (Super Bonder, Loctite; São Paulo, SP, Brazil) and submitted to a tensile load at a crosshead speed of 0.5 mm/min in a univer-sal testing machine (EMIC; São José dos Campos, SP, Brazil), as shown in Fig 2. The tensile bond strength values obtained were expressed in MPa (N/mm2). After bond strength measurement, the fracture modes were viewed at 20X magnification under an optical micro-

scope (Olympus SZ-ST stereomicroscope; Tokyo, Japan) by two independent examiners, and were classified as adhesive, cohesive, or mixed.

Microleakage Test

After the storage period, the samples were immersed in a 50% aqueous silver nitrate solution for 24 h in a light-proof container. Next, the blocks were rinsed thor-oughly in tap water and immersed in a vial containing radiographic developing solution to reveal the silver nitrate and allow the visualization of the tracer-pene-trated areas. The samples were sectioned longitudinally through the center of the restorations (Fig 2) using a diamond saw (Isomet, Buehler) under water coolant.

The sectioned blocks were viewed at 20X magnification with an optical microscope (Olympus SZ-ST stereomicro-scope) by two independent examiners who scored the extent of tracer penetration at the resin/dentin interface according to the following scoring system:33 0 = absence of dye penetration; 1 = dye penetration up to one-half of the extension of the wall; 2 = dye penetration up to one-half of the extension of the wall without reaching the axial angle; 3 = dye penetration to the whole extent of the wall.

Scanning Electronic Microscopy

Fractured specimens of dentin were dried in a vial at room temperature for 24 h. The specimens were rinsed with deionized water to remove debris, dehydrated in as-cending concentrations of ethanol (50%, 70%, and 95% for 10 min each; 100% for 30 min), and critical-point dried. The samples were positioned on a double-faced piece of adhesive tape on a sample chamber whose sequence was carefully recorded, and were then sputter-coated with gold-palladium. The samples were analyzed using an SEM (JEOL 2000 FX; Tokyo, Japan) operating at 20 kV and 2000X to 10,000X magnification (Fig 3).

Statistical Analysis

The microleakage scores, tensile bond strength val-ues, and fracture mode were entered in the the stat-istical program SPSS 16.0 (SPSS; Chicago, IL, USA). Microleakage scores were statistically analyzed using non-parametric Kruskal-Wallis and Mann-Whitney tests.

Fig 3 Illustration of fracture modes. a: Detail of adhesive fracture showing exposed collagen fibers; b: adhesive fracture; c: cohe-sive fracture.

a b c

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Tensile bond strength data were analyzed statistically by one-way ANOVA followed by Tukey’s test. All the tests were set at a significance level of 5%.

RESULTS

The proposed system was able to induce white spot le-sions in all experimental groups. The pH in the BHI me-dium of the control group was 7.0 for all groups; in the BHI medium with microorganisms, the pH was 4.0 for all experimental groups, demonstrating that 5 days in contact with S. mutans supplemented with 2% sucrose produced a cariogenic challenge.

Table 1 presents the bond strength values for the ad-hesive application protocols and cariogenic challenge. The statistical analyses revealed a significant difference among groups (p < 0.001; ANOVA). The highest mean tensile strength was obtained with single application in the control group (SB1-C). There was a reduction in the tensile strength values of the one- and two-layer experi-mental groups (p < 0.05), while in the three-layer group, this reduction was not statistically significant (p > 0.05). The three-layer group showed a tendency to have the low-est values under both conditions.

The failure analysis of debonded specimens (Table 2, Fig 3) revealed that, in the SB1-C specimens, the adhe-sive and mixed failures were distributed equally (50%), while after cariogenic challenge, SB1-E reached adhesive (60%) and cohesive (30%) failures. The remaining 10% of failures were mixed. All SB2-C specimens (100%) frac-tured adhesively, but after the cariogenic challenge, the adhesive fractures decreased (43%) and the cohesive failures increased (43%). All cohesive failures occurred in dentin, probably due to demineralization after cariogenic challenge. SB3-C specimens predominantly showed fail-ure in the adhesive layer (75%), while after the cariogenic challenge, the adhesive failures increased (80%). Thus,

after cariogenic challenge, the adhesive failure mode was slightly higher in the one- and three-layer adhesive groups.

In the microleakage test, none of the scores of any of the experimental groups differed significantly from one another (Kruskal-Wallis; p > 0.05). The results showed extensive penetration (score 3) of silver nitrate in both control and experimental groups (Table 3), indicating that the experimental conditions did not influence the micro-leakage profile.

DISCUSSION

The quality of the hybrid layer is crucial to the longevity of composite restorations.9 However, the literature has shown that hybrid layers produced from the etch-and-rinse technique are not uniform. Experimental studies have demonstrated the existence of a zone located below the hybrid layer where the adhesive monomers do

Table 3 Percentage (%) of microleakage distribution

Microleakage SB1-C SB2-C SB3-C SB1-E SB2-E SB3-E

Score 0 (%) - 10 20 30 - 10

Score 1 (%) 10 20 - 10 - 10

Score 2 (%) 20 10 10 - - 20

Score 3 (%) 70 60 70 60 100 60

Table 2 Percentage (%) of failure

Failure SB1-C SB2-C SB3-C SB1-E SB2-E SB3-E

Adhesive (%) 50 100 75 60 43 80

Cohesive (%) - - - 30 43 -

Mixed (%) 50 - 25 10 14 20

Table 1 Resin-dentin bond strength means and stan-

dard deviations (MPa) of control and experimental

groups by number of layers

LayerCa -rio genic challenge

SB1 SB2 SB3

Control (C) 18.59 ± 5.3aC 12.55 ± 4.2abE 11.28 ± 5.0bG

Experimental (E) 7.28 ± 3.0aD 6.86 ± 2.8abF 5.99 ± 3.9bG

Superscript small letters indicate same statistical group (p > 0.05) by number of layers (SB1, SB2, and SB3) and superscript capital letters indicate same statistical group (p > 0.05) by cariogenic challenge (C and E).

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not infiltrate the collagen.35,34 The presence of this area is a potential risk for failure of the restoration, as the unprotected collagen fibrils are more susceptible to hy-drolysis and the combined action of metalloproteinases, such as collagenase, that are inactive in dentin but can be activated by acid etching.10,15,18 In this study, the initial hypothesis was accepted for tensile bond strength, since different numbers of adhesive layers influenced the tensile bond strength, and rejected for microleakage, since microleakage was not influenced by different protocols.

The current protocol using multiple consecutive layers of adhesive has been previously assessed, showing a progressive increase of adhesive resistance values and reduced nanoleakage in hybrid layers proportional to the increase in the number of adhesive layers applied.18,19 However, contrary to the previous findings, the present study found bond strength values to decrease proportion-ally to the number of adhesive layers applied. Considering the high proportion of adhesive failures in both control and experimental conditions observed in groups SB1 and SB2, the decrease of the bond strength may have been related to the thickness of the adhesive layer located above the hybrid layer. Similar results were found in other studies.7,40 The formation of thicker adhesive layers due to successive application of adhesive can hinder the evaporation of sol-vents present in the adhesive composition, consequently decreasing monomer conversion and providing undesirable physical properties.40 This was probably the cause of re-duced bond strength values. Thus, more studies evaluat-ing other solvent-drying protocols in relation to monomer conversion rates should be performed.

Regarding the effects of cariogenic challenge, all groups exhibited reduced bond strengths, especially SB1E. This may have been related to the lower infiltration through the conditioned dentin when only one adhesive layer was ap-plied.18,19 The existence of an unprotected collagen zone has been associated with poly-HEMA hydrogel formation inside the collagen fibrils exposed by etching, resulting from a mixture of HEMA monomers and water molecules present in the interfibrillar spaces.36 The presence of the poly-HEMA hydrogel can allow the penetration of acids into the hybrid layer and promote its degradation.38

The reduction in bond strength may be related to the decreased cohesive strength of dentin that occurs as a result of the demineralization caused by cariogenic chal-lenge.30 This could explain, for example, the fact that the highest percentages of failures were cohesive in groups SB1-E and SB2-E.

However, in contrast to the findings of Peris et al,30

the present study found that the groups submitted to the cariogenic challenge did not demonstrate an increase in the occurrence of cohesive or mixed failures. This discrep-ancy may be related to the different method used in the two studies. Peris et al30 used pH cycling, whereas the present study employed a microbiological model, which maintained the pH below the critical value throughout the experimental period. The greatest advantage of the mi-crobiological model is that it creates conditions similar to those that occur in the oral environment.37 The formation

of white spot lesions around all restorations submitted to microbiological cariogenic challenge demonstrated the effectiveness of this model, corroborating results by other authors.11

In this study, microleakage was not affected by the number of adhesive layers, emphasizing the poor sealing ability of restorations made by combining a nanocompos-ite (Filtek Z350) and a one-bottle adhesive (Adper Single Bond 2), as reflected in the relatively high proportion of score 3. The bond between composite resin and tooth sub-strate was not strong enough to withstand the stresses generated by polymerization shrinkage, considered the main disadvantage associated with the composite resins based on bis-GMA. In addition, the possibility should also be considered that these effects are exacerbated in class V cavities, which present a high C-factor.6 The C-factor is a coefficient related to contraction stress and is obtained from the ratio of the number of restored to free surfaces. The C-factor and the polymerization shrinkage of the com-posite are directly related.12 Thus, the contraction that the composite underwent may have been high enough to cause disruptions in the adhesive interface and, conse-quently, the formation of gaps in the restoration margins, allowing the massive penetration of silver nitrate into the adhesive interface, where the bond strength to enamel is higher than to dentin for etch-and-rinse systems.39

Comparing the levels of microleakage between groups submitted or not to cariogenic challenge, only for the two consecutive adhesive application groups (SB2C and SB2E) was a significant increase found in the level of infil-tration in the cariogenic challenge condition. This was ex-pected, since the degradation of the restoration margins should increase the penetration of silver nitrate.

To the best of our knowledge, this is the first study that has assessed the adhesive interface properties formed by the application of one, two, and three adhesive lay-ers after being submitted to cariogenic conditions with Streptococcus mutans cells. To increase acid production and potentiate cariogenic challenge, the sucrose supply was kept continuous and at a high concentration. Contrary to many studies that are conducted without microorgan-isms,13,28,29,31 the present study used Streptococcus mutans as a cariogenic challenge. The proteinases of this bacterial species cause secondary destruction of the tooth protein, contributing to a faster expansion of white spots,24 as observed with a stereomicroscope.

This study demonstrates that the number of adhesive layers and cariogenic challenge do not influence the level of microleakage. This could be explained by the similar polymerization shrinkage of the composite in all groups. Al-though the cariogenic challenge model induced white spot lesions at restoration margins, it did not interfere with the marginal sealing ability of the different adhesive layers.

In the tensile bond strength tests, the SB1-C and SB2-C groups presented higher bond strength in comparison to SB1-E and SB2-E, which were submitted to cariogenic challenge. This indicated that the cariogenic processes influenced the results. However, no significant difference between the control (SB3-C) and experimental conditions (SB3-E) was found. The thicker adhesive layer probably

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produced a better marginal seal, preventing the penetra-tion of acid from Streptoccus mutans colonization. How-ever, the thicker adhesive layer induced a reduction in tensile values, probably because of its poor adhesion to the dentin substrate. These results are in accordance with the fractographic analysis of the sticks. A larger number of cohesive failures in dentin were found in the SB1-E and SB2-E groups than in the SB1-C and SB2-C groups. We propose that the penetration of acid along the margins of the restoration demineralized the dentin adjacent to the hybrid layer, reducing its cohesive resistance.

In addition, multiple adhesive applications may remove more water from the demineralized dentin after acid at-tack, permitting increased resin penetration into the col-lagen fibril network.1,4,19 The use of multiple applications of adhesives also allows more time for removal of water by inward diffusion of adhesive monomers and subsequent solvent evaporation from the interfibrillar spaces.18,19 In the current study, irrespective of the number of adhesive layers in the control groups, the highest bond strengths were observed when fewer layers of adhesive were ap-plied. Hashimoto et al18,19 found that increasing the number of adhesive applications resulted in the desired tensile bond strength. In contrast, D’Arcangelo et al1 demonstrated that multiple adhesive coats significantly negatively affected the bond strength to dentin; an excess of adhesive layer thickness reduced the strength and the quality of adhesion, corroborating the present findings. Multiple adhesive layers probably promote excessive re-tention of water and organic solvents, resulting in inad-equately converted polymers and unsatisfactory tensile bond strengths.

Furthermore, it is important to emphasize that most studies have assessed mechanical properties under non-physiological conditions.3,7,18,19 The present study was conducted to simulate a biological system. It seems that the cariogenic challenge using a Streptococcus mutans model affected the adhesive quality of the single and double layers, but the triple application was not influenced by the cariogenic challenge.

CONCLUSION

The different experimental hybridization protocols based on the number of adhesive layers influenced the tensile bond strength and failure mode of adhesive interfaces submitted to cariogenic challenge. However, the number of adhesive layers did not influence the microleakage.

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Clinical relevance: This study shows the efficacy of different hybridization protocols based on the number of adhesive layers, with one layer providing better ten-sile bond strength than three.

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