1http://dx.doi.org/10.20396/bjos.v20i00.8659342 Volume 20 2021 e219342 Original Article 1 Department of Operative Dentistry, Endodontics and Dental Materials, Bauru School of Dentistry, University of São Paulo, Bauru, São Paulo, Brazil. Corresponding author: Genine Guimarães Email: genine_mg@hotmail.com Received: April 29, 2020 Accepted: December 14, 2020 Dentin bond strength evaluation between a conventional and universal adhesive using etch-and-rinse strategy Genine Moreira de Freitas Guimarães1,* , Karin Cristina da Silva Modena1 , Carolina Yoshi Campos Sugio1 , Tamires de Luccas Bueno1 , Maria Angélica Silvério Agulhari1 , Maria Teresa Atta1 Aim: The aim of this study was to compare the microtensile bond strength (μTBS) and the characteristics of the adhesive interface of Scotchbond Universal - SU – etch-and-rise mode (3M ESPE) and Adper Scotchbond Multi-Purpose - MP (3M ESPE) to dentin over time. Methods: Class I cavity preparations were performed in 60 human molars that were randomly divided according to the dentin bonding system (DBS) used (n=30): (1) Acid conditioning + SU and (2) Acid conditioning + MP. For bonding strength (BS) analysis, 30 teeth (n = 15) were sectioned into sticks and submitted to the microtensile test in a universal testing machine after 24 hours and 12 months. The adhesive interface of the others 30 teeth was analyzed in a confocal microscope after 24 hours and 12 months. The data of μTBS were analyzed by two-way repeated measures ANOVA and Tukey’s HSD (α = 0.05). Results: SU presented the lowest DBS compared to MP (p=0.000). Time did not influenced DBS for both adhesive systems (p=0.177). Confocal microscopy analysis showed no cracks between both adhesive systems tested. Conclusion: The results indicate that MP - μTBS showed a better performance compared to SU in total-etch mode. Keywords: Dentin-bonding agents. Dentin. Methacrylates. Microscopy, confocal. https://orcid.org/0000-0002-4415-850X https://orcid.org/0000-0002-0926-1253 https://orcid.org/0000-0001-8934-4472 https://orcid.org/0000-0002-4912-5846 https://orcid.org/0000-0003-3945-4205 https://orcid.org/0000-0002-2449-5728 2 Guimarães et al. Introduction Since the introduction of the adhesive systems, over 50 years ago, the union interface to dentin remains the weakest link in restorative treatment1-4. Although many studies have shown excellent short-term and immediate adhesion effectiveness5,6, the dura- bility and stability of the adhesive interface on dentin remain questionable7,8 due to its inherent characteristics. The failure of adhesion may lead marginal infiltration, which may cause discoloration, secondary caries, and subsequent loss of retention3,9,10. In order to minimize adhesive failures, universal adhesives have become a trend in den- tistry due to its effectiveness and longevity, and a simplified operative technique. Conventional three-step adhesive systems are considered the “gold standard”. How- ever, as a disadvantage, if all the collagen exposed after acid etch is not completely covered by the adhesive systems, matrix metalloproteinases (MMPs) are activated, when they have free access to water, causing restoration failures and post-operative sensitivity8,11,12. The first simplified adhesive system ‘’one bottle’’ introduced on the market was the Scotchbond Universal (SU) adhesive (3M ESPE, Saint Paul - MN, USA), which can be used as an etch-and-rinse or a self-etch mode13,14, according to the clinical conditions. The self-etch mode eliminate previous application of phosphoric acid, that is a sensi- tive part of the technique. Thus, acid monomers have the ability to demineralize and penetrate the dentin substrate simultaneously, decreasing the chances of demineral- ized zones without hybridization, as can happen in etch-and-rinse mode15. However, the different applications of SU (etch-and-rise and self-etch mode) show different behaviors and reflect on the bond strength and quality of the hybrid layer16-19. The difference from SU to the others adhesive systems is the substitution of meth- acrylate monomers (UDMA and GDMA) or phosphorylated methacrylate monomer (MHP) by 10-methacryloyloxydecyl dihydrogenpho-sphate (10-MDP)15,18,20-23. Func- tional monomers, such as 10-MDP, contain carboxylic groups and phosphates that are capable of chemically interact with the calcium of the hydroxyapatite by means of primary ionic bonds, forming stable salts of calcium-phosphate and calcium-carbox- alate together with a limited effect of descaling24. The chemical adhesion promoted by 10-MDP seems to be not only effective but also more stable in water than that promoted by other functional monomers such as 4-MET and phenyl P23. Despite the favorable chemical reaction from MDP, the factors that interfere with the longevity of the bonding interface are still complex. There are several mechanisms that favor the degradation of the hybrid layer. One of the most relevant factors is related to simplified adhesive systems that have hydrophilic characteristics25-27. In addition, there is a big difference in μTBS for SU when used in the etch-and-rise or self-etch strategy9-16. Therefore, the objective of this study is to evaluate the difference between mecha- nisms of adhesion and to compare the dentin bonding system (DBS), as well as to evaluate the characteristic of the bonding interface between a universal and a conven- tional three-step adhesive system with using etch-and-rise mode. 3 Guimarães et al. The null hypothesis of this study is that both DBS evaluated did not show differences between the adhesives and through the time. Material and Methods Specimens preparation Sixty sound human molars with no fracture, cracks or caries lesions extracted due to periodontal or orthodontic reasons were used according to the protocol of Ethics and Research Committee of the Bauru School of Dentistry, University of São Paulo (CAAE nº 336.286). Teeth were cleaned removing any residue of periodontal and gingival tis- sues adhered to the dental surface with manual curettes and stored in a 0.1% thymol solution at room temperature for less than 6 months. Using a low-speed diamond saw (Isomet Low Speed Saw; Buehler Ltda., Lake Bluff/IL - EUA) under water lubrica- tion, the crowns were separated from the roots. Class I cavities28-34 were prepared 4.0 mm deep in dentin, with 3.0 mm buccal extension and 5.0 mm mesiodistal extension using carbide drills (#245, KG Sorensen). Microtensile bond strength test (μTBS) Bonding procedures The experimental unit considered was the tooth, so the sticks of each tooth (n=30) were randomly divided according to DBS (n=15): 1 - SU or 2 - MP using Excel’s “ran- domization” tool. Each Class I cavity were etch-and-rinse with 35% phosphoric acid etchant (Condac, FGM, Brazil) for 30s (enamel) and 15s (dentin). The two adhesives (SU and MP) were carefully applied according to the manufacturer’s instructions (Table 1). All teeth were restored by incremental technique with Filtek™ Z250 resin composite (3M ESPE, Saint Paul - MN, USA) and photoactivated for 40s with 1200mV/cm2 of irradiation (Radii-cal®, SDI, SP, Brazil). After the restorative procedures, the specimens were immersed in deionized water at 37 ° C for 24h or 12 months, according to the tested group. Table 1. Adhesive systems: composition and protocol. Material Composition Protocol Adper Single Bond Universal (SU) (n=15) 3M ESPE Saint Paul - MN, USA 10-MDP phosphate monomer, Vitrebond Copolymer HEMA BISGMA, dimethacrylate resins Filler, silane, initiators Ethanol, water 1. Acid etch (Scotchbond Etchant 35%- Condac, FGM, Brazil) of the enamel for 30s and dentin for 15s followed by washing with “spray” air/water for 30s. Excess water removed with absorbent paper. 2. Application of the adhesive for 20s with slight movements with the application applicator. Light dry with air for 5s to evaporate the solvent. Polymerization for 10s. Adper Scotchbond Multi-Purpose (MP) (n=15) 3M ESPE Saint Paul - MN, USA Primer: HEMA, polyalkanoic acid copolymer, water Adhesive: Bis-GMA, HEMA, Camphorquinone 1. Acid etch (Scotchbond Etchant 35%) of the enamel for 30s and dentin for 15s followed by washing with “spray” air/water for 30s and removing excess water with absorbent paper. 2. Application of a primer layer and light drying for 5s 3. Application of the adhesive and polymerization for 10s. 4 Guimarães et al. Bonding test The restored teeth were sectioned buccal-lingually into slices with a double-sided diamond disc (Extec Corp., Enfield / CT - USA), cooled with deionized water, at a 150 rpm speed in a sectioning machine (Isomet Low Speed Saw; Buehler Ltda., Lake Bluff / IL - USA). Subsequently, each slice was cutted into sticks with a cross-sec- tional area of approximately 0.64 mm2 that were separated into 2 groups according to the test period: 24h (baseline) and 12 months and stored in water at 37ºC with frequent water exchange. After the storage, each stick was individually fixed with cyanoacrylate-based adhesive (Loctite Super Bonder Flex Gel, Henkel Ltda., São Paulo/SP - Brazil) in a Bencor Multi-T device (Danville Engeneering, Danville/CA - USA) and submitted to the microtensile test in a universal testing machine (Instron Model 3342, InstronCorp., Canton, MA) at a constant speed of 0.5 mm/min, with maximum load of 500N. The Mann Whitney test was realized and the results were analyzed by two-way repeated measures ANOVA and Tukey’s HSD (α = 0.05). The average µTBS value for each tooth and time based on all the sticks was calculated and the premature failures were considered as zero for calculating the mean values. Analysis of the adhesive interface - confocal laser scanning microscopy The remained 30 teeth were prepared as describe above, but the adhesive sys- tems were labeled with rhodamine B (0.02 μg/mL for SU and 0.1 μg/mL for MP)35, in order to allow the analysis of the micromorphology of the adhesive interface with greater accuracy. A confocal laser scanning microscopy (Leica TCS SPE, Leica Microsystems CMS, Mannheim, Germany) at 40X magnification microscopy software (Leica Applica- tion Suite Advanced Fluorescence, Leica Microsystems CMS) (1.0 mm, 1024 pix- els and 0.976μm in resolution) was used to evaluate the quality of the hybrid layer, through the analysis of the presence or absence of cracks after storage in water at 37ºC for 24h and 12 months. As it is a qualitative evaluation, no statistical analysis was performed. Results Microtensile bond strength (μTBS) test The means and respective standard deviations of bond strength (MPa) in the peri- ods of 24h (baseline) and 12 months are shown in Table 2. Only DBS was a statis- tically significant factor (p= 0.000). Non-significant differences were detected in the microtensile bond strength among the adhesives tested and the periods evaluated (p = 0.1772) as well as the interaction DBS/time (p=0.570). MP DBS presented the highest values of BS. Both adhesives were able to maintain DBS after 12 months. Fracture analysis revealed that the most predominant failure pattern was ‘adhesive’. The fracture pattern of each specimen (stick) was evaluated and the results obtained are listed in Table 3. 5 Guimarães et al. Adhesive interface analysis (Hybrid Layer Quality - Confocal Microscopy) The results showed that at 24h and 12 months, no difference was observed between the two adhesive systems SU and MP. No gaps were observed at the bonding inter- face (Figure 1). Table 2. Means and standard deviations of tested groups (MPa). Adhesives Baseline (24h) 12 months SU 24.09±8.46Ab (n=15) 22.58±7.35Ab (n=13) MP 29.96±9.76Aa (n=15) 28.09±11.54Aa (n=15) Different uppercase letters indicate differences between time (columns) (p≤0.05). Different lowercase letters indicate differences between μTBS (rows) (p≤0.05). Table 3. Type of fracture in each group. Type of fracture Baseline (24h) 12 months n (%) N (%) Adhesive 33 44.59 27 51.92 Mixed 28 37.83 19 36.53 Cohesive in resin 11 14.86 4 7.69 Cohesive in dentin 2 2.70 2 3.84 Total 74 100 52 100 There was no statistical significant difference between (significance level of 5%). Figure 1. Confocal Microscopy images show the hybrid layer with no gaps at all periods observed. a) 24h-SU; b) 24h-MP; c) 12 months-SU; d) 12 months-MP. a c b d 6 Guimarães et al. Discussion The microtensile bond strength test is frequently performed to evaluate in vitro adhesive systems. This study compared the μTBS of dentin, using a universal (SU) and a conven- tional adhesive (MP) in the etch-and-rinse mode. This study also evaluated the quality of the bonding interface of both adhesive systems in a confocal microscopy. The proposal was to observe the behavior of SU and MP adhesives under similar conditions. The specimens were obtained from Class I cavities, representing high C-factor which influence the values of μTBS29,31-33,36. Specimens obtained from cavities showed a sta- tistically significant reduction in bond strength values due to the high C-factor32. So, the objective was to evaluate the behavior of those adhesives in an extremely situa- tion28-34. Also was performed Class I cavities because, unlike posterior restorations, non-carious cervical lesions usually have sclerotic dentin and could reflect different results37. Studies show that cavities with low C-factor, as in non-carious cervical lesions, have underestimated values compared to values presented clinically, as it appears in Class I cavities32, and the reliability of dentin adhesives is dependent upon the quality of the dentin36. Another factor to consider is the presence of enamel on the cavity margins, which theoretically provides a good seal against the ingress of bacteria and oral fluids and thus protects the most vulnerable adhesive bonding of the underlying dentin16,38. With- out enamel protection on the periphery of the restoration, water promotes adhesive interface degradation resulting in decreased bond strength over time16,25,39. This was considered mainly because of the 12 months specimens. With the limitations of this study, the μTBS results showed that the MP values was sig- nificantly higher compared to SU, with no significance between the two times tested. Besides the Universal Single Bond promotes chemical bonding to the hydroxyapa- tite crystals present on the enamel and dentine6,40,41, some authors claim that there are no differences in the performance of adhesive systems containing 10-MDP13. One hypothesis suggests that prior acid etch may remove hydroxyapatite and hinder chemical bonding, which is the main benefit of MDP. In contrast, Hidari et al.42 (2020) show that the presence of the functional monomer MDP, even with previous phos- phoric acid conditioning, produces greater bond strength results in dentin than the absence of this functional monomer19,42,43. However, it is concluded that although the functional monomer MDP has an important role in the quality of the bonding interface, the removal of the smear layer and hydroxyapatite through prior acid conditioning can be disadvantageous related to the long-term bond strength durability. A systematic review with meta-analysis concluded that universal adhesives with etch- and-rinse strategy is more effective and produces higher values of μTBS in enamel44, and, on dentin, self-etch mode can produce better values18,45,46. However, in this study, the SU was used only with the etch-and-rinse strategy and can explain the results of the present study which showed lower statistical values (22.58±7.35) compared to MP (28.09±11.54). The quality of the hybrid layer is necessary to prevent microleakage and gap forma- tion47. Therefore, the confocal interface analysis supported the μTBS data. Regarding the interface durability of both adhesives tested, there were no statistical differences 7 Guimarães et al. on μTBS in the two periods tested (24h and 12 months). This shows that both adhe- sives were able to maintain the hybrid layer quality with no cracks (Figure 1). Adhesive systems without the application of hydrophobic compound as last step tends to present higher hydrolytic degradation and bond instability because they are semipermeable membranes48. Although the SU acquires hydrophobic characteristics due to the presence of MDP after its polymerization, the adhesive still absorbs more water compared to the two-step self-adhesives (separate bottles) because they have better hydrophobic characteristics in contrast to the one-step adhesives44. In order to assess the state of deterioration or to predict the longevity of dental adhe- sives, clinical studies are clearly the best methods25,49. However, due to the difficulty of standardizing clinical studies, in-vitro tests are performed to simulate the clinical con- ditions. Therefore, by observing several different methods and comparing the results, it may be useful to understand the degradation process that occurs in intraoral condi- tions. Thus, methods such as water storage17,50 and thermal cycle42,51,52, are the most used forms of artificial aging. Hidari et al.42 (2020) compared water storage and ther- mal cycle methods and assessed statistical differences between the adhesives. The water storage has an accelerated aging potential due to the hydrolysis capacity of hydrophilic components of the adhesive and the host-derived proteases with collag- enolytic activity53,54. Therefore, water storage for 12 months may show results that reflect what happens clinically. The null hypothesis of this study that there was no difference on DBS between the adhesives and through the time was partially rejected and these results are related to the variables adopted. Therefore, it is necessary to carry out further tests and evaluate different adhesives and their different application steps clinically. The need for long- term evaluations is also needed. 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