RESEARCH ARTICLE
Influence of Intraoral Temperature and Relative Humidity on the Dentin Bond Strength: An in Situ Study LETÍCIA O. SARAIVA, DDS, MS*, THAIANE R. AGUIAR, DDS, MS, PhD†, LEONARDO COSTA, DDS, MS‡, ANDREA N. CAVALCANTI, DDS, MS, PhD‡, MARCELO GIANNINI, DDS, MS, PhD†, PAULA MATHIAS, DDS, MS, PhD§
ABSTRACT Statement of the Problem: The effect of the intraoral environment during adhesive restorative procedures remains a concern, especially in the absence of rubber dam isolation. Objective: To evaluate the temperature and relative humidity (RH) at anterior and posterior intraoral sites and their effects on the dentin bond strength of two-step etch-and-rinse adhesive systems. Methods: Sixty human molars were assigned to six groups according to the adhesive systems (Adper Single Bond Plus and One Step Plus) and intraoral sites (incisor and molar sites). The room condition was used as a control group. Dentin fragments were individually placed in custom-made acetate trays and direct composite restorations were performed. The intraoral temperature and RH were recorded during adhesive procedures. Then, specimens were removed from the acetate trays and sectioned to obtain multiple beams for the microtensile bond strength test. In addition, the adhesive interface morphology was evaluated through scanning electron microscopy. Intraoral conditions were statistically analyzed by paired Students’ t-tests and the bond strength data by two-way analysis of variance and Tukey test (α = 0.05). Results: The posterior intraoral site showed a significant increase in the temperature and RH when compared with the anterior site. However, both intraoral sites revealed higher temperatures and RH than the room condition. In regards to the adhesive systems, the intraoral environment did not affect the bond strength, and the One Step Plus system showed the highest bond strength means. Conclusion: Despite the fact that remarkable changes in the intraoral conditions were observed for both anterior and posterior sites, the intraoral environment was not able to compromise the immediate dentin bond strength.
CLINICAL SIGNIFICANCE Some conditions of intraoral temperature and relative humidity may not impair the dentin bond strength of two-step etch-and-rinse adhesive systems. Thus, an adequate relative isolation seems to be a good alternative under the specific clinical conditions in which rubber dam isolation is either impossible or very difficult to perform. (J Esthet Restor Dent ••:••–••, 2014)
INTRODUCTION The clinical success of adhesive restorations represents a valuable parameter to evaluate the effectiveness of
resin-based dental materials. Although in-vitro studies provide important information regarding the physical and biomechanical properties of these materials, in-vivo scenarios can affect the clinical outcomes because of
*Private practice, Salvador, BA, Brazil † Postdoctoral research associate, Department of Restorative Dentistry, University of Illinois at Chicago, IL, USA ‡ Professor, Department of Integrated Clinic Rehabilitation, School of Dentistry, Bahiana School of Medicine and Public Health (EBMSP), Salvador, BA, Brazil § Associate professor, Department of Clinical Dentistry, School of Dentistry, Federal University of Bahia, Salvador, BA, Brazil
© 2014 Wiley Periodicals, Inc.
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interoperator variations, patient behaviors, and the intraoral environment.1,2
MATERIALS AND METHODS Specimen Preparation
Intraoral sites appear to be related to changes in the mechanical load, temperature, and saliva flow.1 Additionally, studies have showed that the temperature and the relative humidity (RH) may have negative effects on the performance of adhesive restorative materials.3–8 Temperature and RH are usually simulated in experimental chamber devices; however, some differences may be expected from the oral environment. Thus, adhesive restorations performed in the oral cavity may provide a more accurate understanding of the temperature and RH effects on intraoral sites. With dental tissues, extrinsic sources of moisture, such as the intraoral humidity, may impair the dentin bonding mechanism. The moisture level of the demineralized dentin can promote the collapse or over-wet of collagen fibrils and hence affect the resin-dentin infiltration.9–11 In addition, simplified etch-and-rinse systems might form semipermeable membranes even after curing, allowing water diffusion and fluid accumulation through the adhesive interface, which may jeopardize the bonding durability.12 Some studies simulated extreme conditions of temperature and RH (35–37°C and 80–100% RH) to evaluate the adhesive properties of resin-based materials.4–7,13 However, none of these investigations evaluated the effect of temperature and RH in different intraoral sites, whereas composite restorations were performed under clinical scenarios. Only one clinical trial compared the type of field isolation (absolute or relative) on the clinical performance of restorations for noncervical carious lesions; however, no significant differences were observed after 1 year.14 Thus, the aim of this in situ study was to evaluate the temperature and RH at anterior and posterior intraoral sites (upper incisor and molar) and their effects on the dentin bond strength of two-step etch-and-rinse adhesive systems. The null hypothesis tested was that the intraoral environment (temperature and RH) would not influence the dentin bond strength for both anterior and posterior intraoral sites when compared with a control group.
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After the protocol approved by the local Ethics Committee (n.38/07, CAAE 0019.0.368.000 −07 FR 144816), 60 sound extracted human third molars were collected and pumiced. Occlusal enamel and root were removed using double-faced diamond discs (#7020, KG Sorensen, Barueri, SP, Brazil) under constant water irrigation. To standardize the smear layer, the occlusal dentin surfaces were abraded (#600-grit SiC papers) under water irrigation for 60 seconds. Then, dental fragments were sterilized in an autoclave at 121°C for 40 minutes at 1 atm pressure (Practical 12 L, Odontobrás, Ribeirão Preto, SP, Brazil).15
Experimental Groups Specimens were randomly divided into six groups according to the adhesive system and intraoral environment (upper incisor, molar sites and room condition [control group]). Two-step etch-and-rinse adhesive systems were selected based on their solvent content (Adper Single Bond Plus, ethanol-based; and One Step Plus, acetone-based). Details of adhesive systems’ composition and manufacturers are described in Table 1.
TABLE 1. The manufacturers’ composition and batch number of adhesive systems Adhesive systems
Composition (batch number)
Adper Single Bond Plus (3 M/ESPE, St. Paul, MN, USA)
bis-GMA, HEMA, UDMA, ethyl alcohol, water, glycerol 1,3 dimethacrylate, copolymer of acrylic and itaconic acids, water, silane treated silica (5FE).
One Step Plus (Bisco, Inc., Schaumburg, IL, USA)
bis-GMA, BPDM, HEMA, acetone, photo-initiator, dental glass (0700003585)
bis-GMA = bisphenol A diglycidyl methacrylate; BPDM = biphenyl dimethacrylate; HEMA = 2-hydroxyethyl methacrylate; UDMA = diurethane dimethacrylate.
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Bonding Procedures To reproduce the intraoral environment, custom-made acetate trays were prepared for the upper central incisors and the first molar region. Dentin fragments were individually fixed in the acetate tray with glue (Interneed Ind. Com. Ltd, São Paulo, SP, Brazil) and positioned in the anterior or posterior site. The restorative procedures were performed under relative isolation with cotton rolls placed in the buccal vestibule and gauze over the tongue (Figure 1). The in situ procedures were carried out on 3 successive days and only during the morning, as changes in the dental office environment may affect the relative air humidity.16 In addition, the volunteer’s diet was controlled during the experiment days to avoid possible alterations of his intraoral condition. The volunteer was instructed to avoid fluid intake for 2 hours before the restorative procedure and advised to avoid salty and spicy food to reduce fluid intake during the experimental study period. The two-step etch-and-rinse adhesive systems were used after dentin etching with 35% phosphoric acid for 15 seconds, then rinsed with water. The excess water was removed with absorbent papers, leaving the dentin visibly moist. Adhesive systems were applied according to the manufacturers’ instructions and were light-cured for 10 seconds (Optilight 600, Gnatus, Ribeirão Preto, SP, Brazil). Afterwards, a 6-mm-thick layer of composite resin was incrementally placed over the dentin surface (Filtek Z350, 3 M ESPE, Irvine, CA, USA), and each increment was light-cured for 40 seconds. Then, specimens were stored for 48 hours at 100% RH at 37°C.
To monitor the intraoral experimental conditions, the temperature and RH were recorded for incisor and molar sites during the adhesive procedures using a digital thermo-hygroscope (MTH—1361, Minipa Ind. Com. Ltd, São Paulo, SP, Brazil). The bonding procedures for the control group were similar to intraoral sites; however, these procedures were performed at ambient room conditions to simulate the external environment of a dental clinic.6 Both the temperature and the RH of the ambient room were also recorded during bonding procedures. The temperature and RH data were individually analyzed by paired Student’s t-test at a 95% confidence level.
Microtensile Bond Strength Nine of 10 specimens were used for the microtensile bond strength test. After 48 hours of restorative procedures, specimens were longitudinally sectioned in buccal-lingual and mesiodistal directions with a diamond blade (11–4244, Buehler, Lake Buff, IL, USA) under constant water cooling to obtain multiple bonded beams with a cross-sectional area of 0.8 mm2. For each tooth, six beams from the inner region of the restoration were selected and individually measured with a digital caliper (727–6/150, Starret, Itu, SP, Brazil) to calculate the bonded area. Beams were fixed to the grips of a microtensile device using cyanoacrylate glue (Super Bonder; Henckel Loctite, Itapevi, SP, Brazil) and stressed to failure under tension in a universal testing machine (4411, Instron Corp, Canton, UK) at a crosshead speed of 0.5 mm/minute until failure. Bond strength data (MPa) were calculated, and the statistical analysis was
FIGURE 1. The custom-made acetate trays used to fix the dentin fragments in anterior (A) and posterior (B) sites to reproduce the intraoral conditions during the adhesive restorative procedures.
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performed using two-way analysis of variance (ANOVA) and Tukey test, at a 95% confidence level.
TABLE 2. Mean (SD) of the temperature (°C) and relative humidity (%) Incisor site
Molar site
Room condition
Temperature
26.18 (1.77) b*
27.28 (1.35) a*
21.10
Relative humidity
84.84 (3.84) b*
90.79 (5.87) a*
50.30
Failure Mode Analysis Fractured surfaces of tested specimens were dried and mounted on aluminum stubs and sputter-coated with gold (Balzers-SCD 050 Sputter Coater, Fürstentum, Liechtenstein). Ultrastructure observations were performed in the scanning electron microscope (JSM 5900, JEOL, Peabody, MA, USA) at ×500 to ×2,000 magnifications. Failure patterns were classified into one of four categories as described as follows:17 Type 1: adhesive failure between bonding system and dentin, and partially cohesive failure within the layer of adhesive system; Type 2: cohesive failure within adhesive resin; Type 3: partially cohesive failure within dentin; Type 4: partially cohesive failure within composite resin or adhesive failure between composite resin and adhesive system.
(control) are shown in Table 2. Because the room conditions did not change, those temperature and RH data were used as a reference value (21.10°C and 50.30% RH). Significant statistical differences were observed for the temperature and RH among the experimental conditions (p < 0.01). Significant higher temperatures and RH were found at molar site when compared with the incisor site. However, the room condition demonstrated significantly lower temperatures and RH than both the incisor and the molar intraoral sites.
Morphology of the Bonded Interface
Microtensile Bond Strength
To investigate the morphology of the dentin-resin bonded interface, one specimen from each group was sectioned in a mesiodistal direction, obtaining three 2-mm-thick slices. These slices were ground using SiC abrasive papers (#600, 1200, and 2,000-grit) under running water and polished with soft cloths and diamond pastes (6, 3, and 1 and ¼ μm). Afterwards, specimens were demineralized with 37% phosphoric acid solution for 5 seconds, washed with distilled water, and deproteinized with a 5% sodium hypochlorite solution for 5 minutes. Then, specimens were dried, gold sputter-coated, and observed in the scanning electron microscope at ×1,500 magnifications.
The statistical interaction between the main factors studied—“adhesive system” and “experimental condition”—was not significant (p = 0.63). Thus, the effects of these factors were evaluated separately (Table 3). There were no significant differences in dentin bond strengths among experimental conditions (room versus incisor versus molar sites) for either adhesive system (p = 0.67). However, dentin bond strengths were significantly higher for One Step Plus than for Adper Single Bond Plus in all experimental conditions (p = 0.001).
RESULTS Temperature and RH The temperature and RH measurements for experimental intraoral conditions and room condition
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Distinct letters indicate statistically significant differences for each row (Student’s t-test, α = 5%). Asterisks denote statistically significant differences between intraoral sites (incision and molar) when compared with reference value (room condition).
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Failure Pattern Mode The distribution of failure patterns is depicted in Figure 2. Representative photomicrographs of each failure are illustrated in Figure 3. Adhesive failure (type 1) was the most predominant failure for all groups, followed by type 4 and type 2. Interestingly, the highest percentage of cohesive failure within adhesive resin (type 2) was observed in the molar site for both
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TABLE 3. Mean (SD) of the dentin bond strength (MPa) at the different experimental conditions Adhesive Systems
Experimental conditions Incisor site
Molar site
Room condition
Adper Single Bond Plus
23.01 (6.47) Ab
22.70 (6.04) Ab
20.17 (7.55) Ab
One Step Plus
29.69 (4.90) Aa
27.67 (4.77) Aa
29.02 (5.82) Aa
Distinct upper and lower case letters indicate statistically significant differences within each row and column, respectively (two-way analysis of variance/Tukey test, α = 5%).
FIGURE 2. Distribution of failure patterns among experimental groups.
adhesive systems (Adper Single Bond Plus: 23%; and One Step Plus: 28%). A lower percentage of type 3 (partially cohesive failure within dentin) was noted in both groups restored at the incisor site (Adper Single Bond Plus: 5%; and One Step Plus: 2%).
Morphology of the Bonded Interface Representative images of the resin-dentin interface were depicted in Figure 4. Both adhesive systems showed an authentic hybrid layer with long and dense resin tags.
DISCUSSION In this investigation, the choice of an in situ analysis aimed to assess the effect of temperature and RH on the bond strength of adhesive systems to dentin in
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conditions that were close to clinical scenarios. For this purpose, restorative procedures were conducted on dentin fragments fixed in an acetate tray and placed in the upper incisor or molar region of a volunteer. Changes in the temperature and RH were observed between intraoral environments; however, the dentin bond strength was not affected by the intraoral environment. Thus, the null hypothesis was accepted. Moisture control during adhesive procedures is essential to achieve adequate bond to dental tissues. In some clinical conditions, the use of rubber dam isolation is difficult, if not impossible (i.e., technique issues, patients’ intolerance). Therefore, adequate relative isolation should be considered in these clinical situations.14,18,19 Regarding the use of rubber dam isolation, the intraoral temperature and RH were compared to the dental surgery conditions.16 In this
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FIGURE 3. A, Photomicrography representing failure type 1 (Adper Single Bond Plus [SB], intraoral condition, molar region). B, Photomicrography representing failure type 2 (One Step Plus [OSP], intraoral condition, molar region). C, Photomicrography representing failure type 3 (OSP, intraoral condition, incisor region). D, Photomicrography representing failure type 4 (SB, ambient condition). AS = adhesive system; CR = composite resin; D = dentin; HL = bottom of the hybrid layer; TAG = extensions of adhesive system.
FIGURE 4. Representative images illustrating the adhesive interface of Adper Single Bond Plus A, and One Step Plus B. Scanning electron microscopy micrograph shows the hybrid layer (asterisks) and long resin tags (arrows) for both adhesive systems.
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study, rubber dam isolation was compared with the room environment, which showed a lower temperature (21.1°C) than incisor and molar sites.
scenarios.16,20 In addition, it seems that the elevated and constant humidity level present in the chamber may impair water evaporation.
In addition, reduced dentin bond strength under extreme temperature and RH conditions were previously reported.4,5,7,16,20 Nonetheless, the temperature and RH found did not affect the dentin bond strength. A possible explanation for such divergent results is that they are related to the use of a constant temperature/humidity chamber. During inhalation and exhalation, the oral cavity might become drier, whereas the constant temperature/humidity chamber cannot replicate the natural inhalation, down time, and exhalation cycles presented in clinical
Although this study did not attempt to record the temperature and RH after the beginning of each restorative procedure, Kameyama and colleagues21 demonstrated significant alterations in the temperature and RH during the 20 minutes recorded. Moreover, the intraoral conditions may be highly unstable and influenced by environmental weather conditions.
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Two-step etch-and-rinse adhesive systems seem to be more affected by temperature and RH conditions than self-etching adhesive systems.4,20 This susceptibility may
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be due to the hydrophilic nature of simplified systems, which increases permeability and allows for fluid movements within the interface adhesive.22,23 Although the bond strength of both two-step etch-and-rinse adhesive systems tested were not influenced by the temperature and RH, the acetone-based system showed higher bond strength than the ethanol-based system. These findings are in accordance with previous studies.24,25 It can be attributed to the greater ability of acetone to join and evaporate the water in the demineralized collagen (high vapor pressure = 200 mmHg), which leads to achieving optimal bond strength when excess water remains after the etching step. The failure pattern distribution varied according to the experimental conditions. The most common failure for restorations performed under room conditions and at the incisor site was the adhesive one (type 1). Under higher temperature and RH as observed in the molar region, type 2 and type 4 were also highly frequent. Increased RH may lead to microscopic water condensation on the dentin surface and impair the polymerization of resin-based materials.26 Therefore, we can speculate that the dentin surface was contaminated during the application of the adhesive systems, which may have increased the adhesive and cohesive failures within the adhesive. On the other hand, it seems that if contamination had occurred during the composite resin insertion, predominant cohesive failures in the resin or between the composite and the adhesive layer might have occurred. Furthermore, the use of dry-field techniques provided a reduction in the intraoral temperature and RH.21 Although the present study was performed in situ, the use of an acetate tray may have led to the partial retention of the RH from the gingival and teeth area. This could be observed by the presence of fog formation inside the tray (see Figure 1). Thus, higher RH may be found in the in vivo environment (i.e., gingival crevicular fluid, saliva, and blood contamination). In addition, in this investigation, the adhesive procedures were performed in only one patient to standardize the intraoral conditions. However, differences between patients’ oral
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environments should be considered in further studies. Although adequate relative isolation is a good alternative to adhesive aesthetic anterior restorations,27,28 the contamination of the operative field with fluids can easily occur in the absence of rubber dam isolation. This study demonstrated that the intraoral temperature and RH did not influence the immediate dentin bond strength of two-step etch-and-rinse adhesive systems. This is particularly relevant for those specific clinical conditions in which the use of rubber dam isolation is restricted; however, long-term studies should evaluate the potential impact of intraoral environments on the durability of adhesive restorations.
CONCLUSION Considering the limitations of the present study, the intraoral temperature and RH in the molar site were higher when compared with the incisor site; however, the differences in intraoral environments did not affect the immediate dentin bond strength of two-step etch-and-rinse adhesive systems.
DISCLOSURE AND ACKNOWLEDGEMENTS The authors do not have any financial interest in the companies whose materials are included in this article. This study was partially supported by CAPES, Brazil.
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Miyazaki M, Rikuta A, Tsubota K, et al. Influence of environmental conditions on dentin bond strengths of recently developed dentin bonding systems. J Oral Sci 2001;43:35–40. Besnault C, Attal JP. Influence of a simulated oral environment on dentin bond strength of two adhesive systems. Am J Dent 2001;14:367–72. Besnault C, Attal JP. Influence of a simulated oral environment on microleakage of two adhesive systems in Class II composite restorations. J Dent 2002;30:1–6. Chiba Y, Miyazaki M, Rikuta A, Moore BK. Influence of environmental conditions on dentin bond strengths of one-application adhesive systems. Oper Dent 2004;29:554–9. Jacquot B, Durand JC, Farge P, et al. Influence of temperature and relative humidity on dentin and enamel bonding: a critical review of the literature. Part 1. Laboratory studies. J Adhes Dent 2012;14:433–46. Tay FR, Gwinnett AJ, Wei SH. The overwet phenomenon: a transmission electron microscopic study of surface moisture in the acid-conditioned, resin-dentin interface. Am J Dent 1996;9:161–6. Manso AP, Marquezini L Jr, Silva SM, et al. Stability of wet versus dry bonding with different solvent-based adhesives. Dent Mater 2008;24:476–82. Grégoire G, Guignes P, Nasr K. Effects of dentine moisture on the permeability of total-etch and one-step self-etch adhesives. J Dent 2009;37:691–9. Tay FR, Frankenberger R, Krejci I, et al. Single-bottle adhesives behave as permeable membranes after polymerization. I. In vivo evidence. J Dent 2004;32:611–21. Werner JF, Tani C. Effect of relative humidity on bond strength of self-etching adhesives to dentin. J Adhes Dent 2002;4:277–82. Daudt E, Lopes GC, Vieira LC. Does operatory field isolation influence the performance of direct adhesive restorations? J Adhes Dent 2013;15:27–32. Dominici JT, Eleazer PD, Clark SJ, et al. Disinfection/sterilization of extracted teeth for dental student use. J Dent Educ 2001;65:1278–80. Plasmans PJ, Creugers NH, Hermsen RJ, Vrijhoef MM. Intraoral humidity during operative procedures. J Dent 1994;22:89–91. Figueiredo Reis A, Giannini M, Ambrosano GM, Chan DC. The effects of filling techniques and a low-viscosity
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Reprint requests: Paula Mathias, DDS, MS, PhD, Department of Clinical Dentistry, School of Dentistry, Federal University of Bahia, Salvador, BA, Brazil., Av. Araújo Pinho, 62, Canela, Salvador 40110-150, Brazil; Tel.: 55-71-3336-5776; Fax: 55-71-3336-5976; email: [email protected]
DOI 10.1111/jerd.12098
© 2014 Wiley Periodicals, Inc.
Influence of Intraoral Temperature and Relative Humidity on the Dentin Bond Strength: An in Situ Study LETÍCIA O. SARAIVA, DDS, MS*, THAIANE R. AGUIAR, DDS, MS, PhD†, LEONARDO COSTA, DDS, MS‡, ANDREA N. CAVALCANTI, DDS, MS, PhD‡, MARCELO GIANNINI, DDS, MS, PhD†, PAULA MATHIAS, DDS, MS, PhD§
ABSTRACT Statement of the Problem: The effect of the intraoral environment during adhesive restorative procedures remains a concern, especially in the absence of rubber dam isolation. Objective: To evaluate the temperature and relative humidity (RH) at anterior and posterior intraoral sites and their effects on the dentin bond strength of two-step etch-and-rinse adhesive systems. Methods: Sixty human molars were assigned to six groups according to the adhesive systems (Adper Single Bond Plus and One Step Plus) and intraoral sites (incisor and molar sites). The room condition was used as a control group. Dentin fragments were individually placed in custom-made acetate trays and direct composite restorations were performed. The intraoral temperature and RH were recorded during adhesive procedures. Then, specimens were removed from the acetate trays and sectioned to obtain multiple beams for the microtensile bond strength test. In addition, the adhesive interface morphology was evaluated through scanning electron microscopy. Intraoral conditions were statistically analyzed by paired Students’ t-tests and the bond strength data by two-way analysis of variance and Tukey test (α = 0.05). Results: The posterior intraoral site showed a significant increase in the temperature and RH when compared with the anterior site. However, both intraoral sites revealed higher temperatures and RH than the room condition. In regards to the adhesive systems, the intraoral environment did not affect the bond strength, and the One Step Plus system showed the highest bond strength means. Conclusion: Despite the fact that remarkable changes in the intraoral conditions were observed for both anterior and posterior sites, the intraoral environment was not able to compromise the immediate dentin bond strength.
CLINICAL SIGNIFICANCE Some conditions of intraoral temperature and relative humidity may not impair the dentin bond strength of two-step etch-and-rinse adhesive systems. Thus, an adequate relative isolation seems to be a good alternative under the specific clinical conditions in which rubber dam isolation is either impossible or very difficult to perform. (J Esthet Restor Dent ••:••–••, 2014)
INTRODUCTION The clinical success of adhesive restorations represents a valuable parameter to evaluate the effectiveness of
resin-based dental materials. Although in-vitro studies provide important information regarding the physical and biomechanical properties of these materials, in-vivo scenarios can affect the clinical outcomes because of
*Private practice, Salvador, BA, Brazil † Postdoctoral research associate, Department of Restorative Dentistry, University of Illinois at Chicago, IL, USA ‡ Professor, Department of Integrated Clinic Rehabilitation, School of Dentistry, Bahiana School of Medicine and Public Health (EBMSP), Salvador, BA, Brazil § Associate professor, Department of Clinical Dentistry, School of Dentistry, Federal University of Bahia, Salvador, BA, Brazil
© 2014 Wiley Periodicals, Inc.
DOI 10.1111/jerd.12098
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interoperator variations, patient behaviors, and the intraoral environment.1,2
MATERIALS AND METHODS Specimen Preparation
Intraoral sites appear to be related to changes in the mechanical load, temperature, and saliva flow.1 Additionally, studies have showed that the temperature and the relative humidity (RH) may have negative effects on the performance of adhesive restorative materials.3–8 Temperature and RH are usually simulated in experimental chamber devices; however, some differences may be expected from the oral environment. Thus, adhesive restorations performed in the oral cavity may provide a more accurate understanding of the temperature and RH effects on intraoral sites. With dental tissues, extrinsic sources of moisture, such as the intraoral humidity, may impair the dentin bonding mechanism. The moisture level of the demineralized dentin can promote the collapse or over-wet of collagen fibrils and hence affect the resin-dentin infiltration.9–11 In addition, simplified etch-and-rinse systems might form semipermeable membranes even after curing, allowing water diffusion and fluid accumulation through the adhesive interface, which may jeopardize the bonding durability.12 Some studies simulated extreme conditions of temperature and RH (35–37°C and 80–100% RH) to evaluate the adhesive properties of resin-based materials.4–7,13 However, none of these investigations evaluated the effect of temperature and RH in different intraoral sites, whereas composite restorations were performed under clinical scenarios. Only one clinical trial compared the type of field isolation (absolute or relative) on the clinical performance of restorations for noncervical carious lesions; however, no significant differences were observed after 1 year.14 Thus, the aim of this in situ study was to evaluate the temperature and RH at anterior and posterior intraoral sites (upper incisor and molar) and their effects on the dentin bond strength of two-step etch-and-rinse adhesive systems. The null hypothesis tested was that the intraoral environment (temperature and RH) would not influence the dentin bond strength for both anterior and posterior intraoral sites when compared with a control group.
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After the protocol approved by the local Ethics Committee (n.38/07, CAAE 0019.0.368.000 −07 FR 144816), 60 sound extracted human third molars were collected and pumiced. Occlusal enamel and root were removed using double-faced diamond discs (#7020, KG Sorensen, Barueri, SP, Brazil) under constant water irrigation. To standardize the smear layer, the occlusal dentin surfaces were abraded (#600-grit SiC papers) under water irrigation for 60 seconds. Then, dental fragments were sterilized in an autoclave at 121°C for 40 minutes at 1 atm pressure (Practical 12 L, Odontobrás, Ribeirão Preto, SP, Brazil).15
Experimental Groups Specimens were randomly divided into six groups according to the adhesive system and intraoral environment (upper incisor, molar sites and room condition [control group]). Two-step etch-and-rinse adhesive systems were selected based on their solvent content (Adper Single Bond Plus, ethanol-based; and One Step Plus, acetone-based). Details of adhesive systems’ composition and manufacturers are described in Table 1.
TABLE 1. The manufacturers’ composition and batch number of adhesive systems Adhesive systems
Composition (batch number)
Adper Single Bond Plus (3 M/ESPE, St. Paul, MN, USA)
bis-GMA, HEMA, UDMA, ethyl alcohol, water, glycerol 1,3 dimethacrylate, copolymer of acrylic and itaconic acids, water, silane treated silica (5FE).
One Step Plus (Bisco, Inc., Schaumburg, IL, USA)
bis-GMA, BPDM, HEMA, acetone, photo-initiator, dental glass (0700003585)
bis-GMA = bisphenol A diglycidyl methacrylate; BPDM = biphenyl dimethacrylate; HEMA = 2-hydroxyethyl methacrylate; UDMA = diurethane dimethacrylate.
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INTRAORAL EFFECTS ON THE DENTIN BOND STRENGTH Saraiva et al.
Bonding Procedures To reproduce the intraoral environment, custom-made acetate trays were prepared for the upper central incisors and the first molar region. Dentin fragments were individually fixed in the acetate tray with glue (Interneed Ind. Com. Ltd, São Paulo, SP, Brazil) and positioned in the anterior or posterior site. The restorative procedures were performed under relative isolation with cotton rolls placed in the buccal vestibule and gauze over the tongue (Figure 1). The in situ procedures were carried out on 3 successive days and only during the morning, as changes in the dental office environment may affect the relative air humidity.16 In addition, the volunteer’s diet was controlled during the experiment days to avoid possible alterations of his intraoral condition. The volunteer was instructed to avoid fluid intake for 2 hours before the restorative procedure and advised to avoid salty and spicy food to reduce fluid intake during the experimental study period. The two-step etch-and-rinse adhesive systems were used after dentin etching with 35% phosphoric acid for 15 seconds, then rinsed with water. The excess water was removed with absorbent papers, leaving the dentin visibly moist. Adhesive systems were applied according to the manufacturers’ instructions and were light-cured for 10 seconds (Optilight 600, Gnatus, Ribeirão Preto, SP, Brazil). Afterwards, a 6-mm-thick layer of composite resin was incrementally placed over the dentin surface (Filtek Z350, 3 M ESPE, Irvine, CA, USA), and each increment was light-cured for 40 seconds. Then, specimens were stored for 48 hours at 100% RH at 37°C.
To monitor the intraoral experimental conditions, the temperature and RH were recorded for incisor and molar sites during the adhesive procedures using a digital thermo-hygroscope (MTH—1361, Minipa Ind. Com. Ltd, São Paulo, SP, Brazil). The bonding procedures for the control group were similar to intraoral sites; however, these procedures were performed at ambient room conditions to simulate the external environment of a dental clinic.6 Both the temperature and the RH of the ambient room were also recorded during bonding procedures. The temperature and RH data were individually analyzed by paired Student’s t-test at a 95% confidence level.
Microtensile Bond Strength Nine of 10 specimens were used for the microtensile bond strength test. After 48 hours of restorative procedures, specimens were longitudinally sectioned in buccal-lingual and mesiodistal directions with a diamond blade (11–4244, Buehler, Lake Buff, IL, USA) under constant water cooling to obtain multiple bonded beams with a cross-sectional area of 0.8 mm2. For each tooth, six beams from the inner region of the restoration were selected and individually measured with a digital caliper (727–6/150, Starret, Itu, SP, Brazil) to calculate the bonded area. Beams were fixed to the grips of a microtensile device using cyanoacrylate glue (Super Bonder; Henckel Loctite, Itapevi, SP, Brazil) and stressed to failure under tension in a universal testing machine (4411, Instron Corp, Canton, UK) at a crosshead speed of 0.5 mm/minute until failure. Bond strength data (MPa) were calculated, and the statistical analysis was
FIGURE 1. The custom-made acetate trays used to fix the dentin fragments in anterior (A) and posterior (B) sites to reproduce the intraoral conditions during the adhesive restorative procedures.
© 2014 Wiley Periodicals, Inc.
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INTRAORAL EFFECTS ON THE DENTIN BOND STRENGTH Saraiva et al.
performed using two-way analysis of variance (ANOVA) and Tukey test, at a 95% confidence level.
TABLE 2. Mean (SD) of the temperature (°C) and relative humidity (%) Incisor site
Molar site
Room condition
Temperature
26.18 (1.77) b*
27.28 (1.35) a*
21.10
Relative humidity
84.84 (3.84) b*
90.79 (5.87) a*
50.30
Failure Mode Analysis Fractured surfaces of tested specimens were dried and mounted on aluminum stubs and sputter-coated with gold (Balzers-SCD 050 Sputter Coater, Fürstentum, Liechtenstein). Ultrastructure observations were performed in the scanning electron microscope (JSM 5900, JEOL, Peabody, MA, USA) at ×500 to ×2,000 magnifications. Failure patterns were classified into one of four categories as described as follows:17 Type 1: adhesive failure between bonding system and dentin, and partially cohesive failure within the layer of adhesive system; Type 2: cohesive failure within adhesive resin; Type 3: partially cohesive failure within dentin; Type 4: partially cohesive failure within composite resin or adhesive failure between composite resin and adhesive system.
(control) are shown in Table 2. Because the room conditions did not change, those temperature and RH data were used as a reference value (21.10°C and 50.30% RH). Significant statistical differences were observed for the temperature and RH among the experimental conditions (p < 0.01). Significant higher temperatures and RH were found at molar site when compared with the incisor site. However, the room condition demonstrated significantly lower temperatures and RH than both the incisor and the molar intraoral sites.
Morphology of the Bonded Interface
Microtensile Bond Strength
To investigate the morphology of the dentin-resin bonded interface, one specimen from each group was sectioned in a mesiodistal direction, obtaining three 2-mm-thick slices. These slices were ground using SiC abrasive papers (#600, 1200, and 2,000-grit) under running water and polished with soft cloths and diamond pastes (6, 3, and 1 and ¼ μm). Afterwards, specimens were demineralized with 37% phosphoric acid solution for 5 seconds, washed with distilled water, and deproteinized with a 5% sodium hypochlorite solution for 5 minutes. Then, specimens were dried, gold sputter-coated, and observed in the scanning electron microscope at ×1,500 magnifications.
The statistical interaction between the main factors studied—“adhesive system” and “experimental condition”—was not significant (p = 0.63). Thus, the effects of these factors were evaluated separately (Table 3). There were no significant differences in dentin bond strengths among experimental conditions (room versus incisor versus molar sites) for either adhesive system (p = 0.67). However, dentin bond strengths were significantly higher for One Step Plus than for Adper Single Bond Plus in all experimental conditions (p = 0.001).
RESULTS Temperature and RH The temperature and RH measurements for experimental intraoral conditions and room condition
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Distinct letters indicate statistically significant differences for each row (Student’s t-test, α = 5%). Asterisks denote statistically significant differences between intraoral sites (incision and molar) when compared with reference value (room condition).
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Failure Pattern Mode The distribution of failure patterns is depicted in Figure 2. Representative photomicrographs of each failure are illustrated in Figure 3. Adhesive failure (type 1) was the most predominant failure for all groups, followed by type 4 and type 2. Interestingly, the highest percentage of cohesive failure within adhesive resin (type 2) was observed in the molar site for both
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TABLE 3. Mean (SD) of the dentin bond strength (MPa) at the different experimental conditions Adhesive Systems
Experimental conditions Incisor site
Molar site
Room condition
Adper Single Bond Plus
23.01 (6.47) Ab
22.70 (6.04) Ab
20.17 (7.55) Ab
One Step Plus
29.69 (4.90) Aa
27.67 (4.77) Aa
29.02 (5.82) Aa
Distinct upper and lower case letters indicate statistically significant differences within each row and column, respectively (two-way analysis of variance/Tukey test, α = 5%).
FIGURE 2. Distribution of failure patterns among experimental groups.
adhesive systems (Adper Single Bond Plus: 23%; and One Step Plus: 28%). A lower percentage of type 3 (partially cohesive failure within dentin) was noted in both groups restored at the incisor site (Adper Single Bond Plus: 5%; and One Step Plus: 2%).
Morphology of the Bonded Interface Representative images of the resin-dentin interface were depicted in Figure 4. Both adhesive systems showed an authentic hybrid layer with long and dense resin tags.
DISCUSSION In this investigation, the choice of an in situ analysis aimed to assess the effect of temperature and RH on the bond strength of adhesive systems to dentin in
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conditions that were close to clinical scenarios. For this purpose, restorative procedures were conducted on dentin fragments fixed in an acetate tray and placed in the upper incisor or molar region of a volunteer. Changes in the temperature and RH were observed between intraoral environments; however, the dentin bond strength was not affected by the intraoral environment. Thus, the null hypothesis was accepted. Moisture control during adhesive procedures is essential to achieve adequate bond to dental tissues. In some clinical conditions, the use of rubber dam isolation is difficult, if not impossible (i.e., technique issues, patients’ intolerance). Therefore, adequate relative isolation should be considered in these clinical situations.14,18,19 Regarding the use of rubber dam isolation, the intraoral temperature and RH were compared to the dental surgery conditions.16 In this
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FIGURE 3. A, Photomicrography representing failure type 1 (Adper Single Bond Plus [SB], intraoral condition, molar region). B, Photomicrography representing failure type 2 (One Step Plus [OSP], intraoral condition, molar region). C, Photomicrography representing failure type 3 (OSP, intraoral condition, incisor region). D, Photomicrography representing failure type 4 (SB, ambient condition). AS = adhesive system; CR = composite resin; D = dentin; HL = bottom of the hybrid layer; TAG = extensions of adhesive system.
FIGURE 4. Representative images illustrating the adhesive interface of Adper Single Bond Plus A, and One Step Plus B. Scanning electron microscopy micrograph shows the hybrid layer (asterisks) and long resin tags (arrows) for both adhesive systems.
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study, rubber dam isolation was compared with the room environment, which showed a lower temperature (21.1°C) than incisor and molar sites.
scenarios.16,20 In addition, it seems that the elevated and constant humidity level present in the chamber may impair water evaporation.
In addition, reduced dentin bond strength under extreme temperature and RH conditions were previously reported.4,5,7,16,20 Nonetheless, the temperature and RH found did not affect the dentin bond strength. A possible explanation for such divergent results is that they are related to the use of a constant temperature/humidity chamber. During inhalation and exhalation, the oral cavity might become drier, whereas the constant temperature/humidity chamber cannot replicate the natural inhalation, down time, and exhalation cycles presented in clinical
Although this study did not attempt to record the temperature and RH after the beginning of each restorative procedure, Kameyama and colleagues21 demonstrated significant alterations in the temperature and RH during the 20 minutes recorded. Moreover, the intraoral conditions may be highly unstable and influenced by environmental weather conditions.
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Two-step etch-and-rinse adhesive systems seem to be more affected by temperature and RH conditions than self-etching adhesive systems.4,20 This susceptibility may
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be due to the hydrophilic nature of simplified systems, which increases permeability and allows for fluid movements within the interface adhesive.22,23 Although the bond strength of both two-step etch-and-rinse adhesive systems tested were not influenced by the temperature and RH, the acetone-based system showed higher bond strength than the ethanol-based system. These findings are in accordance with previous studies.24,25 It can be attributed to the greater ability of acetone to join and evaporate the water in the demineralized collagen (high vapor pressure = 200 mmHg), which leads to achieving optimal bond strength when excess water remains after the etching step. The failure pattern distribution varied according to the experimental conditions. The most common failure for restorations performed under room conditions and at the incisor site was the adhesive one (type 1). Under higher temperature and RH as observed in the molar region, type 2 and type 4 were also highly frequent. Increased RH may lead to microscopic water condensation on the dentin surface and impair the polymerization of resin-based materials.26 Therefore, we can speculate that the dentin surface was contaminated during the application of the adhesive systems, which may have increased the adhesive and cohesive failures within the adhesive. On the other hand, it seems that if contamination had occurred during the composite resin insertion, predominant cohesive failures in the resin or between the composite and the adhesive layer might have occurred. Furthermore, the use of dry-field techniques provided a reduction in the intraoral temperature and RH.21 Although the present study was performed in situ, the use of an acetate tray may have led to the partial retention of the RH from the gingival and teeth area. This could be observed by the presence of fog formation inside the tray (see Figure 1). Thus, higher RH may be found in the in vivo environment (i.e., gingival crevicular fluid, saliva, and blood contamination). In addition, in this investigation, the adhesive procedures were performed in only one patient to standardize the intraoral conditions. However, differences between patients’ oral
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environments should be considered in further studies. Although adequate relative isolation is a good alternative to adhesive aesthetic anterior restorations,27,28 the contamination of the operative field with fluids can easily occur in the absence of rubber dam isolation. This study demonstrated that the intraoral temperature and RH did not influence the immediate dentin bond strength of two-step etch-and-rinse adhesive systems. This is particularly relevant for those specific clinical conditions in which the use of rubber dam isolation is restricted; however, long-term studies should evaluate the potential impact of intraoral environments on the durability of adhesive restorations.
CONCLUSION Considering the limitations of the present study, the intraoral temperature and RH in the molar site were higher when compared with the incisor site; however, the differences in intraoral environments did not affect the immediate dentin bond strength of two-step etch-and-rinse adhesive systems.
DISCLOSURE AND ACKNOWLEDGEMENTS The authors do not have any financial interest in the companies whose materials are included in this article. This study was partially supported by CAPES, Brazil.
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Reprint requests: Paula Mathias, DDS, MS, PhD, Department of Clinical Dentistry, School of Dentistry, Federal University of Bahia, Salvador, BA, Brazil., Av. Araújo Pinho, 62, Canela, Salvador 40110-150, Brazil; Tel.: 55-71-3336-5776; Fax: 55-71-3336-5976; email: [email protected]
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