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Table of Contents   
ORIGINAL RESEARCH  
Year : 2021  |  Volume : 24  |  Issue : 2  |  Page : 179-183
Hybrid layer formation and bond strength to dentin impregnated with endodontic sealer after cleaning protocols


Department of Restorative Dentistry, School of Dentistry, Araraquara, São Paulo State University – UNESP, Araraquara, São Paulo, Brazil

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Date of Submission08-Jan-2021
Date of Decision18-Feb-2021
Date of Acceptance23-May-2021
Date of Web Publication09-Oct-2021
 

   Abstract 


Aims: This study evaluated the hybrid layer formation and bond strength of two adhesive systems, Scotchbond Universal (U) or Adper Scotchbond Multi Purpose (M), after cleaning protocols using ethanol (E) or xylol (X), to dentin impregnated with an epoxy-resin based endodontic sealer.
Settings and Design: The study design was an Experimental in vitro study.
Methodology: One hundred bovine dentin specimens were randomly allocated into five groups (n = 10): Computed tomography (CT) (control): Only acid etching + M; E+U; X+U; E+M. After the specimen preparation, images were obtained using confocal laser scanning microscopy to evaluate the hybrid layer formation. For microshear bond strength test, the dentin specimens were included in polyvinyl chloride tubes and four resin composite cylinders were placed on the surface. The analysis was performed 24 h after storage.
Statistical Analysis Used: For parametric and nonparametric data, analysis of variance followed by Tukey test and Kruskal–Wallis, followed by Dunn test were, respectively, used at a significance level of 5%.
Results: Regarding hybrid layer formation, all experimental groups were similar to each other (P > 0.05). However, CT showed higher hybrid layer formation than other groups (P < 0.05), except in relation to X+M (P > 0.05). Bond strength was statistically similar among all groups (P > 0.05).
Conclusions: Hybrid layer formation in dentin impregnated with epoxy resin-based sealer and submitted to different cleaning protocols was similar to the control group only for X+M. No differences were found among the experimental groups. Regarding the bond strength, no effect was observed for any group.

Keywords: Adhesives, confocal, dentin, ethanol, solvents

How to cite this article:
Zaniboni JF, Besegato JF, Guiotti FA, Vitoria MS, Lima RO, Kuga MC. Hybrid layer formation and bond strength to dentin impregnated with endodontic sealer after cleaning protocols. J Conserv Dent 2021;24:179-83

How to cite this URL:
Zaniboni JF, Besegato JF, Guiotti FA, Vitoria MS, Lima RO, Kuga MC. Hybrid layer formation and bond strength to dentin impregnated with endodontic sealer after cleaning protocols. J Conserv Dent [serial online] 2021 [cited 2021 Nov 30];24:179-83. Available from: https://www.jcd.org.in/text.asp?2021/24/2/179/327838



   Introduction Top


Epoxy resin-based sealer (AH Plus) has been extensively used as the choice-material for root canal obturation.[1] It has adequate bond strength since it reacts with any exposed amino groups in collagen dentin matrix.[2] However, sealer residues may persist within the pulp chamber after obturation.[3],[4] These residues impregnate on the dentin surface reducing the bond strength of adhesives to dentin and can result in tooth discoloration.[5],[6],[7]

Nevertheless, the bond strength to dentin is adhesive-dependent. Studies have reported that self-etching adhesive systems exhibit lower bond strength values than etch-and-rinse strategies.[8],[9] Furthermore, universal adhesive systems combine the advantages of these two etching strategies, which simplify and accelerate the adhesive application.[10] However, the behavior of universal adhesives to dentin impregnated with endodontic sealers is still uncertain.

Dentin cleaning protocols are crucial to remove sealer residues and to provide an adequate surface for bonding. Although several cleaning substances have been proposed, sealer residues may persist over the dentin.[3],[4] For instance, chemicals agents such as 95% ethanol (E), acetone, isopropyl alcohol, and amyl acetate are unable to remove sealer residues successfully.[11],[12],[13] Xylol (X) is the most effective endodontic sealer solvent and it is recommended to clean instrumentals impregnated with epoxy resin-based materials.[12],[14] However, the dentin cleaning potential of X after obturation and its effects on the adhesive interface are still uncertain.

A stable and uniform hybrid layer formation is crucial to obtain a suitable adhesion.[15] However, the hybrid layer may degrade over time leading to failure of the adhesive interface.[16] When it happens, microgaps, readily penetration of pathogens and deterioration of the adhesive interface can be observed.[17] Particularly, in endodontic procedures, the persistence of sealer residues over the dentin can negatively affect the hybrid layer formation or its extent into the substrate,[13] which may result in a weak adhesion. Thus, evaluate the hybrid layer formation is crucial to predict the behavior and longevity of the bonding procedure.

This study evaluated the hybrid layer formation and bond strength of two adhesive systems, using the three-step (Adper™ Scotchbond™ Multi-Purpose; 3M ESPE, St Paul, MN, USA) and the two-step (Scotchbond™ Universal; 3M ESPE, St Paul, MN, USA) etch-and-rinse strategies immediately applied after the cleaning protocol (E or X) on dentin impregnated with endodontic sealer. The null hypotheses tested were: (i) the cleaning protocols and adhesive systems do not affect hybrid layer formation; (ii) the cleaning protocols and adhesive systems do not affect bond strength to dentin.


   Methodology Top


One hundred bovine incisors without any enamel defects or cracks visible on a stereomicroscope (×10 magnification) were used. The enamel middle third was flattened at 30° with the long axis of the crown to expose dentin from the buccal surface. After that, dentin specimens (10 mm length ×5 mm width) were obtained using a water-cooled precision cutter machine (IsoMet 1000, Buehler, Lake Bluff, IL, USA) and then stored in 0.1% thymol solution at 4°C ±1°C.

The specimens were individually immersed in 10 mL of 2.5% sodium hypochlorite (Asfer, São Caetano do Sul, SP, Brazil) for 15 min. Then, they were immediately immersed in 10 ml of 17% ethylenediaminetetraacetic acid (Biodinâmica, Ibiporã, PR, Brazil) for 3 min, irrigated using 5 ml of 2.5% sodium hypochlorite and dried using absorbent papers. After, the dentin was impregnated with epoxy resin-based sealer (AH Plus; Dentsply De Trey, Konstanz, Germany). The sealer was handled according to the manufacturer's instructions and placed over the dentin surface using a microbrush (KG Sorensen, São Paulo, SP, Brazil). The sealer remained in contact with the dentin for 15 min without any changes.

Cleaning protocols

The specimens were randomly allocated to five groups according to the cleaning protocol and the type of adhesive system used, as described in [Table 1].
Table 1: Cleaning protocols and adhesive systems used

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Hybrid layer formation

The dentin specimens (n = 50) were polished using 600-grid and 1200-grid silicon carbide sandpapers (Norton, Lorena, SP, Brazil) coupled in a circular water-cooled polisher (Arotec, Cotia, SP, Brazil). Then, the specimens were washed with distilled water and polished with felt disk and aluminum oxide (Arotec, São Paulo, SP, Brazil), at 30 μm granulation at circular polishing. Then the specimens were immersed in distilled water and submitted to ultrasonic bath (Cristófoli, Campo Mourão, PR, Brazil) for 10 min.

Absorbent papers were used to dry the specimens and phosphoric acid etching for 1 min was then performed. The conditioned surfaces were rinsed using 50 ml of distilled water, dehydrated using air-spray, and fixed on glass slide at horizontal position.

The specimens were analyzed using a confocal laser scanning microscope (LEXT OLS4100; Olympus, Shinjuku-ku, Tokyo, Japan) at ×1024 of magnification. The images were obtained using the Olympus Stream (Olympus, Shinjuku-ku, Tokyo, Japan) software and posteriorly saved as TIFF format. The extension of the hybrid layer formed was measured using the Image J program, which was calibrated in micrometer scale. The intradentin extension from the hybrid layer formation was measured in 100 μm, from the buccal surface of the middle third of the dental crown. The measurement was performed every 10 μm and a total of 10 analyzes were obtained for each specimen. Hybrid layer formation was defined as the arithmetic average from these measurements.

Microshear bond strength test

The specimens were polished using 320-grid and 600-grid silicon carbide sandpapers for 20s and included inside polystyrene matrix molds (16.5 mm width × 25. 0 mm length) using acrylic resin (Classic Jet, São Paulo, SP, Brazil).

After the acrylic resin polymerization, the specimens were randomly allocated in 5 groups (n = 10). After all the treatment protocols, four resin composite cylinders (two at mesial and two at distal) were prepared and light-cured for 40 s on the buccal surface. Tygon tube matrix (Tygon tube, R-3603, Saint-Gobain Performance Plastics, Miami Lakes, FL, USA) with 0.7 mm internal diameter and 1.0 mm height was used for resin composite filling (Opallis; FGM, Joinville, SC, Brazil). Afterward, the specimens were stored in a 99% relative humid environment at 37°C. The microshear bond test was performed after 24 h.

All specimens were fixed inside a metal matrix to maintain the composite cylinders of the specimens perpendicularly to the load cell of 500N. An orthodontic wire (0.2 mm diameter) was placed at the base of the resin composite cylinder and all specimens were subjected to compressive loading with a crosshead speed of 0.5 mm/min until the displacement of the cylinder. Electromechanical testing machine (EMIC DL2000, São José dos Pinhais, PR, Brazil) was used to all the analyzes. The bond strength was calculated dividing the maximum force (N) by the adhesion area (mm2) and expressed in MPa. The arithmetic average was calculated for the four resin composite cylinders, obtaining the mean for each specimen.

Statistical analysis

Normality and homoscedasticity of the data were verified by Shapiro–Wilk test. For hybrid layer formation data, analysis of variance followed by Tukey post hoc test was used. For bond strength test, Kruskal–Wallis and Dunn post hoc test was used. The analyzes were carried out using the software SPSS version 22 (IBM SPSS, New York, NY, USA) at a significance level of 5%.


   Results Top


Hybrid layer formation

Cleaning protocols and the adhesive systems negatively affected the hybrid layer formation (P < 0.05), except when the three-step strategy was used after X (P > 0.05). No differences were found among the cleaning protocols and the adhesive systems (P > 0.05). [Table 2] shows the mean and standard deviation of the hybrid layer formation according to the cleaning protocol and the type of adhesive system. [Figure 1] and [Figure 2] show representative images of the hybrid layer formation according to the groups.
Figure 1: Two dimensional and three dimensional representative images of hybrid layer formation. (a and b) Computed tomography group (control); (c and d) X+M groups (xylol, Adper™ Scotchbond™ Multi-Purpose. Arrows indicate the hybrid layer. Scale of 40 μm

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Figure 2: Two dimensional and three dimensional representative images of hybrid layer formation. (a and b) E+U group (ethanol, Scotchbond™ Universal); (c and d) X+U group (xylol, Scotchbond™ Universal); (e and f) E+M group (ethanol, Adper™ Scotchbond™ Multi-Purpose). Arrows indicate the hybrid layer. Scale of 40 μm

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Table 2: Mean and standard deviation of the of hybrid layer formation in dentin, after the cleaning protocols and of adhesive systems use (micrometers)

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Microshear bond strength

No statistical differences were found among all groups in comparison to the control group (P > 0.05). [Table 3] shows the median, minimum and maximum values, first and third quartile of the bond strength of different adhesive systems to dentin according to the cleaning protocols.
Table 3: Median, maximum and minimum values, the first and third quartile of the bond strength of the adhesive systems to dentin according to the dentin cleaning protocol (MPa)

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   Discussion Top


Herein, we evaluated different cleaning protocols and bonding strategies on the hybrid layer formation and microshear bond strength in dentin impregnated with an endodontic sealer. Epoxy resin of the endodontic sealer is chemically different from methacrylate-containing resinous monomers of the adhesive systems.[18] Due to the chemical incompatibility, both materials do not copolymerize and the contact surface cleaning must be efficient.[6],[19] Despite Kuga et al.[4],[11] have observed the persistence of sealer residues over the dentin regardless of the cleaning protocol used, our results showed that X exhibited an enhanced cleanse of the dentin surface and the three-step adhesive system (Adper™ Scotchbond™ Multi-Purpose) protocol did not negatively affect the hybrid layer formation.

Our first null hypothesis was rejected. Both adhesive systems (M and U) presented similar results since no differences in hybrid layer formation were found among the cleaning protocols (P > 0.05). However, X + M exhibited hybrid layer thickness similar to the computed tomography (CT) group (CT: 13.39 ± 2.21 μm; X + M: 10.54 ± 2.51 μm; P > 0.05), which was not observed in the other cleaning protocols (E + U, X + U and E + M). Thus, the hybrid layer formation was not affected by the cleaning protocols or adhesive systems used.

Etch-and-rinse adhesive systems have promoted more uniform hybrid layer formation than self-etching adhesive systems.[20],[21] In this way, we evaluated adhesive systems (M and U) that allow the use of etch-and-rinse strategies (2 or 3-step). Universal adhesive systems were introduced into the market due to their versatility and ease of application. However, our results have shown that universal adhesive (U) did not promote an enhanced hybrid layer formation, which suggests that etch-and-rinse adhesive (M) is more prone to be used over the dentin after endodontic obturation and cleaning protocols employed in this study.

E contains water in its chemical composition while the sealer presents hydrophobic resin (epoxy resin) and low solubility in polar solvents. This relative incompatibility can lead to the persistence of residues,[11] which may negatively affect the hybrid layer formation, although the thickness mean values were statistically similar to the groups using X.[6],[22]

X is a nonpolar solvent and has been routinely recommended as obturation materials solvent during the root canals retreatment. However, X is unable to completely remove the residues of epoxy resin-based sealer in the dentin.[11],[23] Furthermore, there is a need to evaluate whether the acid etching could facilitate the removal of sealer residues.[12] Our study partially fulfilled this lack of information, since we have shown that X associated with acid etching promoted hybrid layer formation similar to the control group [Figure 1], which was not observed for the other groups.

Our second null hypothesis was accepted. The bond strength of adhesive systems to dentin was not affected by any cleaning protocols and/or adhesive system. Based on our results, we may infer that the cleaning substances and the dentin acid etching possibly contributed to removing the epoxy-resin based residues, which support similar bond strength to the control group. In addition, acid etching was reported as a promising strategy to remove the smear layer of root dentin,[24] since it provides open dentinal tubules and consequently better adhesion between the adhesive system and the dentin.[25]

Universal adhesive systems can be used either by self-etching or etch-and-rinse strategy (total-etching or enamel selective etching),[21] without prejudice to its properties. Three-step adhesives present better laboratory performance than two-step or simplified adhesives systems.[20] However, in this study, the bonding strategy and the adhesive systems did not affect the hybrid layer formation and the bond strength regardless of the cleaning protocol used.

This in vitro study has some limitations, and their results must be carefully interpreted. Both analyzes (hybrid layer formation and microshear bond strength) were performed immediately after the treatments. However, long-term analyses are crucial to predict the effect of the cleaning protocols, adhesive systems, and bond strategies, since possible degradation of the hybrid layer, and consequently lower bond strength, may occur over time.


   Conclusions Top


X associated with Adper™ Scotchbond™ Multi-Purpose exhibited hybrid layer formation similar to the control group. However, no differences in bond strength to dentin impregnated with epoxy-resin based sealer residues were found, independently of the cleaning substances or adhesive systems.

Acknowledgment

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Finance code 001.

Financial support and sponsorship

This study was financially supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior– Finance code 001.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Dr. Joao Felipe Besegato
Araraquara School of Dentistry, 1680 Humaitá Street, 3rd Floor, Araraquara 14801-903, São Paulo
Brazil
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcd.jcd_14_21

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