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Journal of Dentistry journal homepage: www.intl.elsevierhealth.com/journals/jden
Effect of adjunctive application of epigallocatechin-3-gallate and ethanol–wet bonding on adhesive–dentin bonds Hongye Yanga,1, Jingmei Guoa,1, Donglai Denga , Zhiyong Chenb , Cui Huanga,* a The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory for Oral Biomedical Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China b College of Stomatology, Guangxi Medical University, Nanning, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 September 2015 Received in revised form 25 November 2015 Accepted 1 December 2015 Available online xxx
Objectives: To determine the effect of the combined use of epigallocatechin-3-gallate (EGCG) and ethanol–wet bonding (EWB) on resin–dentin bonds. Methods: Sixty molars were sectioned, polished, and randomly divided into six groups (n = 10) according to the following pretreatments: group 1, water–wet bonding (WWB); group 2, WWB with 0.02% (w/v) EGCG; group 3, WWB with 0.1% EGCG; group 4, EWB; group 5, EWB with 0.02% EGCG; and group 6, EWB with 0.1% EGCG. An etch-and-rinse adhesive was then used, followed by the resin composites building. The microtensile bond strength (MTBS), failure modes and interfacial nanoleakage were separately determined after 24 h water storage or 10,000 runs of thermocycling. Results: Both pretreatment method (P < 0.05) and thermocycling (P < 0.05) significantly influenced bond strength and nanoleakage. Irrespective of thermocycling, the 0.02% EGCG/ethanol (group 5) pretreated adhesive–dentin interfaces showed higher MTBS than the control group (P < 0.05). Nanoleakage expression of all groups increased after thermocycling (P < 0.05) except group 5. Adhesive failure was the main fracture pattern in all groups. Conclusion: This study showed that pretreatment with 0.02% EGCG/ethanol solutions can effectively improve immediate bond strength and bond stability of etch-and-rinse adhesives on dentin. Clinical significance: The adjunctive application of EGCG and EWB provides a new strategy for dentists to obtain the desired bond effectiveness during adhesive restoration in clinical practice. ã 2015 Elsevier Ltd. All rights reserved.
Keywords: Dentin Adhesive Epigallocatechin-3-gallate Ethanol–wet bonding
1. Introduction As the basis of esthetic restoration, contemporary dentin adhesive system has been developed and reached the eighth generation; however, the durability and stability of adhesive– dentin bonds remain limited, particularly in clinical applications [1]. Poor bonding durability may weaken retention, produce marginal deterioration, and reduce service life of restorations [2]. Costs and resources have been consumed. Therefore, methods to improve dentin bonding durability have been extensively investigated in dentistry. A decrease in bond strength is mainly attributed to the degradation of a hybrid layer at an adhesive–dentin interface [3,4]. In general, degradation is caused by incomplete infiltration of resin monomers, hydrolysis through water sorption, and
* Corresponding author. Fax: +86 27 87873260. E-mail address: [email protected] (C. Huang). These authors contributed equally to this work.
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collagenolysis by endogenous matrix metalloproteinases (MMPs) and cysteine cathepsins [5,6]. Therefore, some effective methods, such as ethanol–wet bonding (EWB), MMP inhibitor, collagen cross-linker application, or biomimetic remineralization, have been developed to protect hybrid layer integrity [7,8]. Surface moisture is necessary to achieve effective dentin bonding [9]. Although water–wet bonding (WWB) is widely applied in dentin bonding, conventional hydrophilic adhesives likely increase water sorption and accelerate bonding interface degradation [10,11]. Therefore, the “EWB” technique, a method by which ethanol is used to replace water to support collagen fiber network of demineralized dentin, has been developed [12]. EWB can prevent collagen matrix collapse, promote infiltration of hydrophobic adhesive monomers into a collagen network, and avoid phase separation [13]. This approach has been successfully applied not only with experimental hydrophobic adhesives [14,15], but also with currently commercial adhesives, which are usually a combination of hydrophobic and hydrophilic monomers [16,17]. MMP inhibitors are used to prevent the degradation of incomplete resin-infiltrated collagen fibrils [8]. Among these
http://dx.doi.org/10.1016/j.jdent.2015.12.001 0300-5712/ ã 2015 Elsevier Ltd. All rights reserved.
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inhibitors, chemical synthetics, such as chlorhexidine (CHX), are used to modify dentin adhesives; however, concerns on drug resistance have increased [18]. Epigallocatechin-3-gallate (EGCG), a green tea polyphenol, provides several beneficial functions, such as antioxidant, antimicrobial, antidiabetic, anti-inflammatory, and cancer-preventive properties [19]. EGCG induces low toxicity and inhibits MMP-2 and MMP-9 expression and activity [20]. Various concentrations of EGCG ranging from 0.0065% to 5% have been used in dentistry [21–24]. In this range, 0.02% and 0.1% EGCG/water solution can effectively facilitate dentin bonding [21]. To make dentin bonding more stable, the combined application of several well-developed methods is gaining people’s attention. Ekambaram et al. [7] were the first to incorporate CHX to EWB, which enhanced the hydrophobic adhesive’s ability to bond esthetic restorations with teeth. However, the possibility of drug resistance by CHX [18] and the complexity of self-made hydrophobic adhesive limited its clinical potential. Consequently, the combined use of EWB and EGCG, which possesses none of CHX’s shortcomings, might provide clinicians with a better alternative. To the best of our knowledge, no report was available on this topic. Therefore, the aim of this study was to explore the interactive effect of the adjunctive application of EGCG and EWB on adhesive– dentin bonds. The null hypothesis stated that the combined use of EWB and EGCG does not affect dentin bond strength, even after thermocycling is completed. 2. Materials and methods 2.1. Specimen preparation and experimental groups Sixty intact extracted human third molars were collected after the donors' informed consents were obtained in accordance with the protocols approved by the Ethics Committee for Human Studies of the School and Hospital of Stomatology, Wuhan University. The teeth were maintained in 1% chloramine T solution at 4 C for 1 month before use. A flat dentin surface was prepared by removing the occlusal crown with a low-speed water-cooled diamond saw (Isomet; Buehler, Evanston, IL, USA). The dentin surface was ground with water-irrigated 600-grit silicon-carbide paper for 60 s to create a standardized smear layer. Afterward, each dentin surface was etched with 35% phosphoric acid for 15 s, rinsed thoroughly with deionized water, and then blot-dried. The prepared teeth were randomly divided into six groups (n = 10 each group) according to the following pretreatments: group 1 (WWB); group 2 (WWB + 0.02% EGCG); group 3 (WWB + 0.1% EGCG); group 4 (EWB); group 5 (EWB + 0.02% EGCG); and group 6 (EWB + 0.1% EGCG). Table 1 shows the composition and application methods of pretreatment solutions in each group and the dentin adhesive used in this study. Briefly, the etched dentin surfaces in groups 1 were pretreated with a microbrush covered with distilled water for
60 s. In groups 2 and 3, 0.02% or 0.1% (w/v) EGCG solution was prepared in advance by dissolving 0.02 or 0.1 g EGCG (Sigma– Aldrich, St. Louis, MO, USA) in 100 ml of deionized water. The etched dentin surface was then pretreated with corresponding EGCG/water solution for 60 s. Excess liquid was removed from the specimens by gently blotting with filter papers to leave a visibly moist dentin surface. For groups 4–6, the similar operation was adopted but the solvent was replaced by 100% ethanol. The specimens in each group were bonded with Adper Single Bond 2 (3M ESPE, St. Paul, MN, USA). The procedures were performed by one dentist, and the manufacturer’s instructions were strictly followed. The adhesive was polymerized using a LED light-curing unit (Bisco Inc., Schaumburg, IL, USA) at approximately 700 mW/cm2 irradiance for 10 s. A resin composite (Charisma, Heraeus Kulzer, Hanau, Germany) was formed in four increments (thickness of 3–4 mm), and each increment was polymerized for 20 s. 2.2. Microtensile bond strength (MTBS) test After the teeth were stored in deionized water at 37 C for 24 h, the bonded teeth were longitudinally sectioned under watercooling to produce slabs with a thickness of 0.9 mm. Three slabs from each tooth were sectioned again to prepare beams with a dimension of 0.9 mm 0.9 mm. After excluding unqualified beams which situated peripherally and showed presence of enamel, ten beams were obtained from each tooth. Five of them were immediately subjected to MTBS test, while the other five were thermocycled before being subjected to MTBS test. The beams were placed in a thermal cycling machine (Temperature Cycling Chambers; HUAN-S, Wuhan, China) from 5 C to 55 C for 10,000 cycles and dwell time of 15 s. The parameters (cycle times, temperature, and dwell time) were selected based on our previous study [25]. In MTBS test, each beam (50 beams for each subgroup) was attached to a jig with cyanoacrylate glue (Zapit, Dental Ventures of America, Corona, CA); the jig was placed in a universal testing machine (Microtensile Tester; Bisco, Schaumburg, IL, USA) and loaded in a tension at a crosshead speed of 1 mm/min until failure occurred. The maximum load was recorded, the fracture area of each beam was measured using a digital caliper; final MTBS values (MPa) were then calculated. 2.3. Fracture mode analysis After the MTBS test was conducted, the dentin side of the failed beams was observed under a stereomicroscope (Stemi 2000-C; Carl Zeiss Jena, Gottingen, Germany) at 50 magnification and classified into four groups [26]: A, adhesive failure; CD, cohesive failure in dentin; CC, cohesive failure in composite; or M, mixed failure.
Table 1 Adhesive system, pretreatment solutions, compositions and application modes in this study. Materials
Main components
Application modes
Adper Single Bond 2 (3M ESPE, St. Paul, MN, USA) WWB (group 1) WWB + 0.02% EGCG (group 2) WWB + 0.1% EGCG (group 3) EWB (group 4)
Bis-GMA, HEMA, dimethacrylates, polyalkenoic acid copolymer, initiators, water, and ethanol Deionized water Dissolving 0.02 g EGCG into 100 ml deionized water Dissolving 0.1 g EGCG into 100 ml deionized water 100% ethanol
EWB + 0.02% EGCG (group 5) EWB + 0.1% EGCG (group 6)
Dissolving 0.02 g EGCG into 100 ml 100% ethanol Dissolving 0.1 g EGCG into 100 ml 100% ethanol
Apply two coats of adhesive on pretreated dentin surface, gently air thin for 5 s, light-cure for 10 s 1. Dentin surface was etched with 35% phosphoric acid 15 s, rinse and blot dry 2. The etched dentin surface was pretreated with a microbrush covering with the corresponding pretreatment solutions (groups 1–6) for 60 s, respectively 3. Excess liquid on dentin surface was gentle blotted with filter papers to leave a visibly moist dentin surface
Abbreviations: Bis-GMA, bisphenol-A-diglycidylether dimethacrylate; HEMA, 2-hydroxyethyl methacrylate; EGCG, epigallocatechin-3-gallate; WWB, water–wet bonding; EWB, ethanol–wet bonding.
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2.4. Interfacial nanoleakage evaluation Non-thermocycled and thermocycled slabs (n = 2 in each subgroup) were used to evaluate nanoleakage at the adhesive– dentin interface. For nanoleakage evaluation, specimens were prepared using previously described methods [27]. Slab surfaces were coated with two layers of nail varnish 1 mm away from the bonded interface. The specimens were then dipped in 50 wt% ammoniacal AgNO3 solution in the dark for 24 h. The specimens were thoroughly rinsed with water and immersed in a photodeveloping solution with fluorescent light irradiation for 8 h. The slabs were wet-polished with 600-, 800-, and 1200-grit SiC papers and with 0.25 mm of diamond paste by using a polishing cloth. Silver-stained specimens were examined through field emission scanning electron microscopy (FESEM; Zeiss, Sigma, Germany) in a back-scattered electron mode. Five fields-of-view along the interface of one slab (10 images for each subgroup) were randomly captured. Image J (NIH, Frederick, MD, USA) was used to compute the percentage distribution of silver deposits along bonding interfaces in each image [28]. 2.5. Statistical analysis Statistical analysis was performed using SPSS17 (SPSS Inc., Chicago, IL, USA). After the normal distribution of MTBS values and nanoleakage percentage was confirmed, two-way ANOVA was conducted to analyze the effect of two variables (pretreatment method and thermocycling) on bond strength or nanoleakage expression, respectively. Post hoc multiple comparisons were conducted by using Student–Newman–Keuls test (a = 0.05). 3. Results 3.1. Microtensile bond strength Mean MTBS values were calculated from all groups and are shown in Fig. 1. Two-way ANOVA showed that the variables pretreatment method (F = 23.68, P = 0.000) and thermocycling (F = 6.19, P = 0.014) significantly influenced bond strength. The interaction of pretreatment method thermocycling was also
Fig. 1. Means and standard deviations of microtensile bond strength for each group (groups with the same superscripts are not statistically significant (P > 0.05)).
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significant (F = 2.74, P = 0.003), indicating that the changes in MTBS were dependent on these two factors. Irrespective of thermocycling, the EGCG/ethanol solution pretreated adhesive–dentin showed higher MTBS than the control group (P < 0.05). The highest MTBS was observed in group 5, in which the dentin surface was pretreated with 0.02% EGCG/ethanol solution, while the lowest MTBS was detected in group 3 (WWB + 0.1% EGCG). After 10,000 cycles of thermocycling, significant reduction of MTBS was observed for the control group (P < 0.05), whilst no significant reduction was observed when EGCG was pretreated (P > 0.05). 3.2. Failure mode analysis The frequency distribution of fracture modes is shown in Fig. 2. The dominant fracture pattern in all groups was adhesive failure. After thermocycling was completed, the frequency of mixed failure increased in most groups. 3.3. Nanoleakage evaluation Statistical analysis results of nanoleakage expression are shown in Fig. 3. Nanoleakage statistical results of two-way ANOVA showed that both factors were significant: pretreatment method (F = 20.75, P = 0.000) and thermocycling (F = 146.71, P = 0.000). The interaction of pretreatment method and thermocycling was also significant (F = 1.95, P = 0.039). Irrespective of pretreatment method, the nanoleakage expression significantly increased after thermocycling (P < 0.05). The only exception was the group 5 (EWB + 0.02% EGCG), for which no significant difference in nanoleakage expression was found between immediate and thermocycled groups. Irrespective of thermocycling, the EGCG/ ethanol pretreated adhesive–dentin (groups 4–6) showed less nanoleakage expression than the control group (P < 0.05). The respective FESEM images of nanoleakage expression are shown in Fig. 4 (immediate) and Fig. 5 (after thermocycling). Most silver particles precipitated on the hybrid layer or on the whole adhesive layer. No evident silver uptake was observed in dentinal tubules. 4. Discussion This study determined the effect of the combined use of EGCG and EWB on resin–dentin bonds, especially after thermocycling. As a mimic of temperature variations under intraoral conditions, thermocycling is widely used to test the performance of adhesive restorations by exposing samples to repetitive cycles of hot and cold alterations [29,30]. The MTBS results demonstrated that the adjunctive application of EWB and EGCG, particularly at 0.02% (w/ v), could improve not only immediate adhesive–dentin bonds but also bonding stability after thermocycling was completed. This finding was also confirmed by nanoleakage expression. As such, the null hypothesis was rejected. Since the concept of “EWB” strategy was proposed, various ethanol–wet approaches have been developed to optimize the technique [12,31]. Among these approaches, the stepwise dehydration protocols (from 70%, 80%, 95% to 100%) with a self-made hydrophobic adhesive yield the most encouraging results [32,33]. However, these approaches still exhibit some limitations in clinical applications. Dehydration with a series of increasing ethanol concentrations is time consuming and impractical. Commercial available adhesives always contain hydrophilic monomers. Therefore, the possibility of simplified EWB protocol with commercial etch-and-rinse adhesives for clinical applications should be investigated. Although Sadek et al. [32,34] suggested that simplified dehydration protocol using 100% ethanol should be avoided for the EWB technique, our study demonstrated the
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Fig. 2. Distribution of failure modes after microtensile bond strength test.
feasibility of the proposed method. In group 4, when the specimen was pretreated with 100% ethanol for 60 s, the MTBS of Adper Single Bond 2 did not decrease compared with control group (group 1). This finding might be attributed to two main reasons. First, Adper Single Bond 2 is composed of hydrophobic (e.g., bisGMA, bisphenol A glycerolate dimethacrylate) and hydrophilic (e.g., HEMA, 2-hydroxyethyl methacrylate) monomers; the former is well miscible with ethanol, while the latter is more soluble in ethanol than in water [31]. Second, commercial hydrophilic adhesive (Adper Single Bond 2 in this study) may be more tolerant to residual water when used with EWB; as a result, the adhesive is less sensitive to the technique. These findings are consistent with those observed in previous studies [16,17]. In addition to the hydrolysis of adhesive resin, the enzymolysis of collagen matrix plays a critical role in the degradation of the hybrid layer [35–37]. Among collagen enzymes, endogenous enzymes, particularly MMP-2 and MMP-9, are implicated in
Fig. 3. The statistical analysis of nanoleakage expression (groups with the same superscripts are not statistically significant (P > 0.05)).
collagenolytic activity [37]. EGCG, a natural plant extract, is an excellent inhibitor of MMP-2 and MMP-9 activities [20,38]. In the present study, when EGCG solution was used as a primer, such as group 2 (WWB + 0.02% EGCG) and 5 (EWB + 0.02% EGCG), a satisfactory bonding effectiveness was obtained. The results could be attributed to the following phenomena. EGCG can stabilize collagen depending on hydrogen bond and hydrophobic interactions with collagenases [39]; stabilization of collagen may be one of the mechanisms that inhibit MMPs. Although molecular mechanisms remain ambiguous, this finding may be attributed to zinc chelation [40]. EGCG exhibits high affinity to metal ions [21], which are usually combined with collagen; metal ions also participate in MMP recognition [37]. Once the combination is changed by EGCG, MMP recognition is likely prevented, and collagen degradation may be inhibited [37]. Furthermore, based on a previous research [24], we speculated that the mechanical property of dentin might be improved by the use of EGCG, resulting in higher resistant ability of adhesive–dentin interface against thermocycling. This was all great, but the real kicker was that, the adjunctive application of EWB and EGCG, particularly at 0.02% (w/v), improved not only immediate adhesive–dentin bonds but also bonding stability after thermocycling. This phenomenon could possibly occur because the adjunctive use of EWB and EGCG could display superior performances and elicit a synergistic effect on dentin bonding. Another interesting phenomenon is that, regardless of WWB or EWB, 0.02% EGCG groups (group 2 or 5) showed higher immediate and aged bonding strength compared with 0.1% EGCG groups (group 3 or 6), while the former presented less nanoleakage expression than the latter, respectively. All these data indicated that the effect of EGCG pretreatment on adhesive–dentin bonds is concentration-dependent. This results echo several recent research. Hiraishi et al. [24] evaluated the effect of various plantderived agents on the stability of dentin collagen matrix to resist collagenase degradation, and a dose-dependent effect of EGCG was revealed. Vidal et al. [23] proved that, even at the concentration as low as 0.0065%, EGCG was able to inhibit MMP-9. One of our previous studies showed that the bonding strength tended to
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Fig. 4. Representative FESEM images (1000) of interfacial nanoleakage expression from immediate groups. A, group 1 (WWB); B, group 2 (WWB + 0.02% EGCG); C, group 3 (WWB + 0.1% EGCG); D, group 4 (EWB); E, group 5 (EWB + 0.02% EGCG); and F, group 6 (EWB + 0.1% EGCG). The silver uptake in A and C was higher than B. Images D–E showed similar nanoleakage expression. c, composite resin; d, dentine; pointers, silver deposits.
increase with the concentration of EGCG. However, instead of continually increasing, the bonding strength dropped unexpectedly once the concentration of EGCG rose over a certain point, which might be a threshold [22]. Santiago et al. [21] also obtained similar result which showed the bonding strength of dentin pretreated with 0.5% EGCG/water solutions was surprisingly lower than that of another lower concentration group. Based on these research, some clues were provided to explain our results. With the pretreatment of EGCG solution on dentin surface, EGCG could be trapped within the linear chains of adhesive after later curing. However, a relatively high EGCG concentration might interfere the formation of linear polymer chains [22]. Furthermore, the freeradical scavenging effect of high EGCG concentration may disrupt adhesive polymerization [21]. The combined application of EGCG and EWB may provide several advantages. First, the proposed strategy may improve bonding durability and stability of adhesive–dentin bonds by overcoming hybrid layer hydrolysis and enzymolysis. Second,
EGCG/ethanol solution was used as a pretreatment of demineralized dentine in one step. This technique combines simplified ethanol dehydration with EGCG and prevents the use of additional steps; hence, the method can be conveniently used for clinical applications. Third, EGCG may allow improved biosafety and reduced drug resistance because this substance possesses characteristics of a natural extract compared with CHX. Fourth, EGCG is an effective antimicrobial agent against various pathogenic microorganisms [22]. The occurrence of secondary caries may be prevented by applying our approach. Despite these advantages, the limitations of this study should be considered. Additional artificial aging methods, such as long-term storage, sodium hypochlorite treatment, and pH cycling, should be applied to confirm whether the adjunctive use of EWB and EGCG is valid. Additional commercial dentin adhesives should also be used to evaluate the application range of the proposed strategy. Further in vivo experiments should be performed to confirm the efficacy and safety of the proposed method.
Fig. 5. Representative FESEM images (1000) of interfacial nanoleakage expression from thermocycled groups. A, group 1 (WWB); B, group 2 (WWB + 0.02% EGCG); C, group 3 (WWB + 0.1% EGCG); D, group 4 (EWB); E, group 5 (EWB + 0.02% EGCG); and F, group 6 (EWB + 0.1% EGCG). The silver deposits in A, C and F were extensive and high. However, slight nanoleakage was observed in E. c, composite resin; d, dentine; pointers, silver deposits.
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5. Conclusions This study showed that the combined use of EGCG and EWB can effectively improve immediate dentin bond strength and bond stability. The proposed strategy recommended the use of 0.02% EGCG/ethanol solutions as a useful primer during adhesive restoration. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgment This work was financially supported by National Natural Science Foundation of China (No. 81371191). References [1] C. Chen, L.N. Niu, H. Xie, Z.Y. Zhang, L.Q. Zhou, K. Jiao, et al., Bonding of universal adhesives to dentine—old wine in new bottles? J. Dent. 43 (2015) 525–536. [2] D.H. Pashley, F.R. Tay, C. Yiu, M. Hashimoto, L. Breschi, R.M. Carvalho, et al., Collagen degradation by host-derived enzymes during aging, J. Dent. Res. 83 (2004) 216–221. [3] M.R. Carrilho, R.M. Carvalho, M.F. de Goes, V. di Hipolito, S. Geraldeli, F.R. Tay, et al., Chlorhexidine preserves dentin bond in vitro, J. Dent. Res. 86 (2007) 90–94. [4] J. De Munck, P.E. Van den Steen, A. Mine, K.L. Van Landuyt, A. Poitevin, G. Opdenakker, et al., Inhibition of enzymatic degradation of adhesive–dentin interfaces, J. Dent. Res. 88 (2009) 1101–1106. [5] A.P. Manso, R.H. Grande, A.K. Bedran-Russo, A. Reis, A.D. Loguercio, D.H. Pashley, et al., Can 1% chlorhexidine diacetate and ethanol stabilize resin– dentin bonds? Dent. Mater. 30 (2014) 735–741. [6] G. Grégoire, P. Sharrock, M. Delannée, M.B. Delisle, Depletion of water molecules during ethanol wet-bonding with etch and rinse dental adhesives, Mater. Sci. Eng. C 33 (2013) 21–27. [7] M. Ekambaram, C.K. Yiu, J.P. Matinlinna, N.M. King, F.R. Tay, Adjunctive application of chlorhexidine and ethanol–wet bonding on durability of bonds to sound and caries-affected dentine, J. Dent. 42 (2014) 709–719. [8] Y. Liu, L. Tjäderhane, L. Breschi, A. Mazzoni, N. Li, J. Mao, et al., Limitations in bonding to dentin and experimental strategies to prevent bond degradation, J. Dent. Res. 90 (2011) 953–968. [9] A.P. Manso, L. Marquezini Jr., S.M. Silva, D.H. Pashley, F.R. Tay, R.M. Carvalho, Stability of wet versus dry bonding with different solvent-based adhesives, Dent. Mater. 24 (2008) 476–482. [10] J. Malacarne, R.M. Carvalho, M.F. de Goes, N. Svizero, D.H. Pashley, F.R. Tay, et al., Water sorption/solubility of dental adhesive resins, Dent. Mater. 22 (2006) 973–980. [11] M. Takahashi, M. Nakajima, K. Hosaka, M. Ikeda, R.M. Foxton, J. Tagami, Long-term evaluation of water sorption and ultimate tensile strength of HEMA-containing/-free one-step self-etch adhesives, J. Dent. 39 (2011) 506–512. [12] F.R. Tay, D.H. Pashley, R.R. Kapur, M.R. Carrilho, Y.B. Hur, L.V. Garrett, et al., Bonding BisGMA to dentin—a proof of concept for hydrophobic dentin bonding, J. Dent. Res. 86 (2007) 1034–1039. [13] T.D. Becker, K.A. Agee, A.P. Joyce, F.A. Rueggeberg, J.L. Borke, J.L. Waller, et al., Infiltration/evaporation-induced shrinkage of demineralized dentin by solvated model adhesives, J. Biomed. Mater. Res. B Appl. Biomater. 80 (2007) 156–165. [14] Y. Nishitani, M. Yoshiyama, A.M. Donnelly, K.A. Agee, J. Sword, F.R. Tay, et al., Effects of resin hydrophilicity on dentin bond strength, J. Dent. Res. 85 (2006) 1016–1021. [15] T.P. Shin, X.M. Yao, R. Huenergardt, M.P. Walker, Y. Wang, Morphological and chemical characterization of bonding hydrophobic adhesive to dentin using ethanol wet bonding technique, Dent. Mater. 25 (2009) 1050–1057. [16] R. Nagpal, N. Manuja, I.K. Pandit, Effect of ethanol wet bonding technique on the durability of resin–dentin bond with contemporary adhesive systems, J. Clin. Pediatr. Dent. 39 (2015) 133–142.
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Contents lists available at ScienceDirect
Journal of Dentistry journal homepage: www.intl.elsevierhealth.com/journals/jden
Effect of adjunctive application of epigallocatechin-3-gallate and ethanol–wet bonding on adhesive–dentin bonds Hongye Yanga,1, Jingmei Guoa,1, Donglai Denga , Zhiyong Chenb , Cui Huanga,* a The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory for Oral Biomedical Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China b College of Stomatology, Guangxi Medical University, Nanning, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 September 2015 Received in revised form 25 November 2015 Accepted 1 December 2015 Available online xxx
Objectives: To determine the effect of the combined use of epigallocatechin-3-gallate (EGCG) and ethanol–wet bonding (EWB) on resin–dentin bonds. Methods: Sixty molars were sectioned, polished, and randomly divided into six groups (n = 10) according to the following pretreatments: group 1, water–wet bonding (WWB); group 2, WWB with 0.02% (w/v) EGCG; group 3, WWB with 0.1% EGCG; group 4, EWB; group 5, EWB with 0.02% EGCG; and group 6, EWB with 0.1% EGCG. An etch-and-rinse adhesive was then used, followed by the resin composites building. The microtensile bond strength (MTBS), failure modes and interfacial nanoleakage were separately determined after 24 h water storage or 10,000 runs of thermocycling. Results: Both pretreatment method (P < 0.05) and thermocycling (P < 0.05) significantly influenced bond strength and nanoleakage. Irrespective of thermocycling, the 0.02% EGCG/ethanol (group 5) pretreated adhesive–dentin interfaces showed higher MTBS than the control group (P < 0.05). Nanoleakage expression of all groups increased after thermocycling (P < 0.05) except group 5. Adhesive failure was the main fracture pattern in all groups. Conclusion: This study showed that pretreatment with 0.02% EGCG/ethanol solutions can effectively improve immediate bond strength and bond stability of etch-and-rinse adhesives on dentin. Clinical significance: The adjunctive application of EGCG and EWB provides a new strategy for dentists to obtain the desired bond effectiveness during adhesive restoration in clinical practice. ã 2015 Elsevier Ltd. All rights reserved.
Keywords: Dentin Adhesive Epigallocatechin-3-gallate Ethanol–wet bonding
1. Introduction As the basis of esthetic restoration, contemporary dentin adhesive system has been developed and reached the eighth generation; however, the durability and stability of adhesive– dentin bonds remain limited, particularly in clinical applications [1]. Poor bonding durability may weaken retention, produce marginal deterioration, and reduce service life of restorations [2]. Costs and resources have been consumed. Therefore, methods to improve dentin bonding durability have been extensively investigated in dentistry. A decrease in bond strength is mainly attributed to the degradation of a hybrid layer at an adhesive–dentin interface [3,4]. In general, degradation is caused by incomplete infiltration of resin monomers, hydrolysis through water sorption, and
* Corresponding author. Fax: +86 27 87873260. E-mail address: [email protected] (C. Huang). These authors contributed equally to this work.
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collagenolysis by endogenous matrix metalloproteinases (MMPs) and cysteine cathepsins [5,6]. Therefore, some effective methods, such as ethanol–wet bonding (EWB), MMP inhibitor, collagen cross-linker application, or biomimetic remineralization, have been developed to protect hybrid layer integrity [7,8]. Surface moisture is necessary to achieve effective dentin bonding [9]. Although water–wet bonding (WWB) is widely applied in dentin bonding, conventional hydrophilic adhesives likely increase water sorption and accelerate bonding interface degradation [10,11]. Therefore, the “EWB” technique, a method by which ethanol is used to replace water to support collagen fiber network of demineralized dentin, has been developed [12]. EWB can prevent collagen matrix collapse, promote infiltration of hydrophobic adhesive monomers into a collagen network, and avoid phase separation [13]. This approach has been successfully applied not only with experimental hydrophobic adhesives [14,15], but also with currently commercial adhesives, which are usually a combination of hydrophobic and hydrophilic monomers [16,17]. MMP inhibitors are used to prevent the degradation of incomplete resin-infiltrated collagen fibrils [8]. Among these
http://dx.doi.org/10.1016/j.jdent.2015.12.001 0300-5712/ ã 2015 Elsevier Ltd. All rights reserved.
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inhibitors, chemical synthetics, such as chlorhexidine (CHX), are used to modify dentin adhesives; however, concerns on drug resistance have increased [18]. Epigallocatechin-3-gallate (EGCG), a green tea polyphenol, provides several beneficial functions, such as antioxidant, antimicrobial, antidiabetic, anti-inflammatory, and cancer-preventive properties [19]. EGCG induces low toxicity and inhibits MMP-2 and MMP-9 expression and activity [20]. Various concentrations of EGCG ranging from 0.0065% to 5% have been used in dentistry [21–24]. In this range, 0.02% and 0.1% EGCG/water solution can effectively facilitate dentin bonding [21]. To make dentin bonding more stable, the combined application of several well-developed methods is gaining people’s attention. Ekambaram et al. [7] were the first to incorporate CHX to EWB, which enhanced the hydrophobic adhesive’s ability to bond esthetic restorations with teeth. However, the possibility of drug resistance by CHX [18] and the complexity of self-made hydrophobic adhesive limited its clinical potential. Consequently, the combined use of EWB and EGCG, which possesses none of CHX’s shortcomings, might provide clinicians with a better alternative. To the best of our knowledge, no report was available on this topic. Therefore, the aim of this study was to explore the interactive effect of the adjunctive application of EGCG and EWB on adhesive– dentin bonds. The null hypothesis stated that the combined use of EWB and EGCG does not affect dentin bond strength, even after thermocycling is completed. 2. Materials and methods 2.1. Specimen preparation and experimental groups Sixty intact extracted human third molars were collected after the donors' informed consents were obtained in accordance with the protocols approved by the Ethics Committee for Human Studies of the School and Hospital of Stomatology, Wuhan University. The teeth were maintained in 1% chloramine T solution at 4 C for 1 month before use. A flat dentin surface was prepared by removing the occlusal crown with a low-speed water-cooled diamond saw (Isomet; Buehler, Evanston, IL, USA). The dentin surface was ground with water-irrigated 600-grit silicon-carbide paper for 60 s to create a standardized smear layer. Afterward, each dentin surface was etched with 35% phosphoric acid for 15 s, rinsed thoroughly with deionized water, and then blot-dried. The prepared teeth were randomly divided into six groups (n = 10 each group) according to the following pretreatments: group 1 (WWB); group 2 (WWB + 0.02% EGCG); group 3 (WWB + 0.1% EGCG); group 4 (EWB); group 5 (EWB + 0.02% EGCG); and group 6 (EWB + 0.1% EGCG). Table 1 shows the composition and application methods of pretreatment solutions in each group and the dentin adhesive used in this study. Briefly, the etched dentin surfaces in groups 1 were pretreated with a microbrush covered with distilled water for
60 s. In groups 2 and 3, 0.02% or 0.1% (w/v) EGCG solution was prepared in advance by dissolving 0.02 or 0.1 g EGCG (Sigma– Aldrich, St. Louis, MO, USA) in 100 ml of deionized water. The etched dentin surface was then pretreated with corresponding EGCG/water solution for 60 s. Excess liquid was removed from the specimens by gently blotting with filter papers to leave a visibly moist dentin surface. For groups 4–6, the similar operation was adopted but the solvent was replaced by 100% ethanol. The specimens in each group were bonded with Adper Single Bond 2 (3M ESPE, St. Paul, MN, USA). The procedures were performed by one dentist, and the manufacturer’s instructions were strictly followed. The adhesive was polymerized using a LED light-curing unit (Bisco Inc., Schaumburg, IL, USA) at approximately 700 mW/cm2 irradiance for 10 s. A resin composite (Charisma, Heraeus Kulzer, Hanau, Germany) was formed in four increments (thickness of 3–4 mm), and each increment was polymerized for 20 s. 2.2. Microtensile bond strength (MTBS) test After the teeth were stored in deionized water at 37 C for 24 h, the bonded teeth were longitudinally sectioned under watercooling to produce slabs with a thickness of 0.9 mm. Three slabs from each tooth were sectioned again to prepare beams with a dimension of 0.9 mm 0.9 mm. After excluding unqualified beams which situated peripherally and showed presence of enamel, ten beams were obtained from each tooth. Five of them were immediately subjected to MTBS test, while the other five were thermocycled before being subjected to MTBS test. The beams were placed in a thermal cycling machine (Temperature Cycling Chambers; HUAN-S, Wuhan, China) from 5 C to 55 C for 10,000 cycles and dwell time of 15 s. The parameters (cycle times, temperature, and dwell time) were selected based on our previous study [25]. In MTBS test, each beam (50 beams for each subgroup) was attached to a jig with cyanoacrylate glue (Zapit, Dental Ventures of America, Corona, CA); the jig was placed in a universal testing machine (Microtensile Tester; Bisco, Schaumburg, IL, USA) and loaded in a tension at a crosshead speed of 1 mm/min until failure occurred. The maximum load was recorded, the fracture area of each beam was measured using a digital caliper; final MTBS values (MPa) were then calculated. 2.3. Fracture mode analysis After the MTBS test was conducted, the dentin side of the failed beams was observed under a stereomicroscope (Stemi 2000-C; Carl Zeiss Jena, Gottingen, Germany) at 50 magnification and classified into four groups [26]: A, adhesive failure; CD, cohesive failure in dentin; CC, cohesive failure in composite; or M, mixed failure.
Table 1 Adhesive system, pretreatment solutions, compositions and application modes in this study. Materials
Main components
Application modes
Adper Single Bond 2 (3M ESPE, St. Paul, MN, USA) WWB (group 1) WWB + 0.02% EGCG (group 2) WWB + 0.1% EGCG (group 3) EWB (group 4)
Bis-GMA, HEMA, dimethacrylates, polyalkenoic acid copolymer, initiators, water, and ethanol Deionized water Dissolving 0.02 g EGCG into 100 ml deionized water Dissolving 0.1 g EGCG into 100 ml deionized water 100% ethanol
EWB + 0.02% EGCG (group 5) EWB + 0.1% EGCG (group 6)
Dissolving 0.02 g EGCG into 100 ml 100% ethanol Dissolving 0.1 g EGCG into 100 ml 100% ethanol
Apply two coats of adhesive on pretreated dentin surface, gently air thin for 5 s, light-cure for 10 s 1. Dentin surface was etched with 35% phosphoric acid 15 s, rinse and blot dry 2. The etched dentin surface was pretreated with a microbrush covering with the corresponding pretreatment solutions (groups 1–6) for 60 s, respectively 3. Excess liquid on dentin surface was gentle blotted with filter papers to leave a visibly moist dentin surface
Abbreviations: Bis-GMA, bisphenol-A-diglycidylether dimethacrylate; HEMA, 2-hydroxyethyl methacrylate; EGCG, epigallocatechin-3-gallate; WWB, water–wet bonding; EWB, ethanol–wet bonding.
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2.4. Interfacial nanoleakage evaluation Non-thermocycled and thermocycled slabs (n = 2 in each subgroup) were used to evaluate nanoleakage at the adhesive– dentin interface. For nanoleakage evaluation, specimens were prepared using previously described methods [27]. Slab surfaces were coated with two layers of nail varnish 1 mm away from the bonded interface. The specimens were then dipped in 50 wt% ammoniacal AgNO3 solution in the dark for 24 h. The specimens were thoroughly rinsed with water and immersed in a photodeveloping solution with fluorescent light irradiation for 8 h. The slabs were wet-polished with 600-, 800-, and 1200-grit SiC papers and with 0.25 mm of diamond paste by using a polishing cloth. Silver-stained specimens were examined through field emission scanning electron microscopy (FESEM; Zeiss, Sigma, Germany) in a back-scattered electron mode. Five fields-of-view along the interface of one slab (10 images for each subgroup) were randomly captured. Image J (NIH, Frederick, MD, USA) was used to compute the percentage distribution of silver deposits along bonding interfaces in each image [28]. 2.5. Statistical analysis Statistical analysis was performed using SPSS17 (SPSS Inc., Chicago, IL, USA). After the normal distribution of MTBS values and nanoleakage percentage was confirmed, two-way ANOVA was conducted to analyze the effect of two variables (pretreatment method and thermocycling) on bond strength or nanoleakage expression, respectively. Post hoc multiple comparisons were conducted by using Student–Newman–Keuls test (a = 0.05). 3. Results 3.1. Microtensile bond strength Mean MTBS values were calculated from all groups and are shown in Fig. 1. Two-way ANOVA showed that the variables pretreatment method (F = 23.68, P = 0.000) and thermocycling (F = 6.19, P = 0.014) significantly influenced bond strength. The interaction of pretreatment method thermocycling was also
Fig. 1. Means and standard deviations of microtensile bond strength for each group (groups with the same superscripts are not statistically significant (P > 0.05)).
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significant (F = 2.74, P = 0.003), indicating that the changes in MTBS were dependent on these two factors. Irrespective of thermocycling, the EGCG/ethanol solution pretreated adhesive–dentin showed higher MTBS than the control group (P < 0.05). The highest MTBS was observed in group 5, in which the dentin surface was pretreated with 0.02% EGCG/ethanol solution, while the lowest MTBS was detected in group 3 (WWB + 0.1% EGCG). After 10,000 cycles of thermocycling, significant reduction of MTBS was observed for the control group (P < 0.05), whilst no significant reduction was observed when EGCG was pretreated (P > 0.05). 3.2. Failure mode analysis The frequency distribution of fracture modes is shown in Fig. 2. The dominant fracture pattern in all groups was adhesive failure. After thermocycling was completed, the frequency of mixed failure increased in most groups. 3.3. Nanoleakage evaluation Statistical analysis results of nanoleakage expression are shown in Fig. 3. Nanoleakage statistical results of two-way ANOVA showed that both factors were significant: pretreatment method (F = 20.75, P = 0.000) and thermocycling (F = 146.71, P = 0.000). The interaction of pretreatment method and thermocycling was also significant (F = 1.95, P = 0.039). Irrespective of pretreatment method, the nanoleakage expression significantly increased after thermocycling (P < 0.05). The only exception was the group 5 (EWB + 0.02% EGCG), for which no significant difference in nanoleakage expression was found between immediate and thermocycled groups. Irrespective of thermocycling, the EGCG/ ethanol pretreated adhesive–dentin (groups 4–6) showed less nanoleakage expression than the control group (P < 0.05). The respective FESEM images of nanoleakage expression are shown in Fig. 4 (immediate) and Fig. 5 (after thermocycling). Most silver particles precipitated on the hybrid layer or on the whole adhesive layer. No evident silver uptake was observed in dentinal tubules. 4. Discussion This study determined the effect of the combined use of EGCG and EWB on resin–dentin bonds, especially after thermocycling. As a mimic of temperature variations under intraoral conditions, thermocycling is widely used to test the performance of adhesive restorations by exposing samples to repetitive cycles of hot and cold alterations [29,30]. The MTBS results demonstrated that the adjunctive application of EWB and EGCG, particularly at 0.02% (w/ v), could improve not only immediate adhesive–dentin bonds but also bonding stability after thermocycling was completed. This finding was also confirmed by nanoleakage expression. As such, the null hypothesis was rejected. Since the concept of “EWB” strategy was proposed, various ethanol–wet approaches have been developed to optimize the technique [12,31]. Among these approaches, the stepwise dehydration protocols (from 70%, 80%, 95% to 100%) with a self-made hydrophobic adhesive yield the most encouraging results [32,33]. However, these approaches still exhibit some limitations in clinical applications. Dehydration with a series of increasing ethanol concentrations is time consuming and impractical. Commercial available adhesives always contain hydrophilic monomers. Therefore, the possibility of simplified EWB protocol with commercial etch-and-rinse adhesives for clinical applications should be investigated. Although Sadek et al. [32,34] suggested that simplified dehydration protocol using 100% ethanol should be avoided for the EWB technique, our study demonstrated the
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Fig. 2. Distribution of failure modes after microtensile bond strength test.
feasibility of the proposed method. In group 4, when the specimen was pretreated with 100% ethanol for 60 s, the MTBS of Adper Single Bond 2 did not decrease compared with control group (group 1). This finding might be attributed to two main reasons. First, Adper Single Bond 2 is composed of hydrophobic (e.g., bisGMA, bisphenol A glycerolate dimethacrylate) and hydrophilic (e.g., HEMA, 2-hydroxyethyl methacrylate) monomers; the former is well miscible with ethanol, while the latter is more soluble in ethanol than in water [31]. Second, commercial hydrophilic adhesive (Adper Single Bond 2 in this study) may be more tolerant to residual water when used with EWB; as a result, the adhesive is less sensitive to the technique. These findings are consistent with those observed in previous studies [16,17]. In addition to the hydrolysis of adhesive resin, the enzymolysis of collagen matrix plays a critical role in the degradation of the hybrid layer [35–37]. Among collagen enzymes, endogenous enzymes, particularly MMP-2 and MMP-9, are implicated in
Fig. 3. The statistical analysis of nanoleakage expression (groups with the same superscripts are not statistically significant (P > 0.05)).
collagenolytic activity [37]. EGCG, a natural plant extract, is an excellent inhibitor of MMP-2 and MMP-9 activities [20,38]. In the present study, when EGCG solution was used as a primer, such as group 2 (WWB + 0.02% EGCG) and 5 (EWB + 0.02% EGCG), a satisfactory bonding effectiveness was obtained. The results could be attributed to the following phenomena. EGCG can stabilize collagen depending on hydrogen bond and hydrophobic interactions with collagenases [39]; stabilization of collagen may be one of the mechanisms that inhibit MMPs. Although molecular mechanisms remain ambiguous, this finding may be attributed to zinc chelation [40]. EGCG exhibits high affinity to metal ions [21], which are usually combined with collagen; metal ions also participate in MMP recognition [37]. Once the combination is changed by EGCG, MMP recognition is likely prevented, and collagen degradation may be inhibited [37]. Furthermore, based on a previous research [24], we speculated that the mechanical property of dentin might be improved by the use of EGCG, resulting in higher resistant ability of adhesive–dentin interface against thermocycling. This was all great, but the real kicker was that, the adjunctive application of EWB and EGCG, particularly at 0.02% (w/v), improved not only immediate adhesive–dentin bonds but also bonding stability after thermocycling. This phenomenon could possibly occur because the adjunctive use of EWB and EGCG could display superior performances and elicit a synergistic effect on dentin bonding. Another interesting phenomenon is that, regardless of WWB or EWB, 0.02% EGCG groups (group 2 or 5) showed higher immediate and aged bonding strength compared with 0.1% EGCG groups (group 3 or 6), while the former presented less nanoleakage expression than the latter, respectively. All these data indicated that the effect of EGCG pretreatment on adhesive–dentin bonds is concentration-dependent. This results echo several recent research. Hiraishi et al. [24] evaluated the effect of various plantderived agents on the stability of dentin collagen matrix to resist collagenase degradation, and a dose-dependent effect of EGCG was revealed. Vidal et al. [23] proved that, even at the concentration as low as 0.0065%, EGCG was able to inhibit MMP-9. One of our previous studies showed that the bonding strength tended to
Please cite this article in press as: H. Yang, et al., Effect of adjunctive application of epigallocatechin-3-gallate and ethanol–wet bonding on adhesive–dentin bonds, Journal of Dentistry (2015), http://dx.doi.org/10.1016/j.jdent.2015.12.001
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Fig. 4. Representative FESEM images (1000) of interfacial nanoleakage expression from immediate groups. A, group 1 (WWB); B, group 2 (WWB + 0.02% EGCG); C, group 3 (WWB + 0.1% EGCG); D, group 4 (EWB); E, group 5 (EWB + 0.02% EGCG); and F, group 6 (EWB + 0.1% EGCG). The silver uptake in A and C was higher than B. Images D–E showed similar nanoleakage expression. c, composite resin; d, dentine; pointers, silver deposits.
increase with the concentration of EGCG. However, instead of continually increasing, the bonding strength dropped unexpectedly once the concentration of EGCG rose over a certain point, which might be a threshold [22]. Santiago et al. [21] also obtained similar result which showed the bonding strength of dentin pretreated with 0.5% EGCG/water solutions was surprisingly lower than that of another lower concentration group. Based on these research, some clues were provided to explain our results. With the pretreatment of EGCG solution on dentin surface, EGCG could be trapped within the linear chains of adhesive after later curing. However, a relatively high EGCG concentration might interfere the formation of linear polymer chains [22]. Furthermore, the freeradical scavenging effect of high EGCG concentration may disrupt adhesive polymerization [21]. The combined application of EGCG and EWB may provide several advantages. First, the proposed strategy may improve bonding durability and stability of adhesive–dentin bonds by overcoming hybrid layer hydrolysis and enzymolysis. Second,
EGCG/ethanol solution was used as a pretreatment of demineralized dentine in one step. This technique combines simplified ethanol dehydration with EGCG and prevents the use of additional steps; hence, the method can be conveniently used for clinical applications. Third, EGCG may allow improved biosafety and reduced drug resistance because this substance possesses characteristics of a natural extract compared with CHX. Fourth, EGCG is an effective antimicrobial agent against various pathogenic microorganisms [22]. The occurrence of secondary caries may be prevented by applying our approach. Despite these advantages, the limitations of this study should be considered. Additional artificial aging methods, such as long-term storage, sodium hypochlorite treatment, and pH cycling, should be applied to confirm whether the adjunctive use of EWB and EGCG is valid. Additional commercial dentin adhesives should also be used to evaluate the application range of the proposed strategy. Further in vivo experiments should be performed to confirm the efficacy and safety of the proposed method.
Fig. 5. Representative FESEM images (1000) of interfacial nanoleakage expression from thermocycled groups. A, group 1 (WWB); B, group 2 (WWB + 0.02% EGCG); C, group 3 (WWB + 0.1% EGCG); D, group 4 (EWB); E, group 5 (EWB + 0.02% EGCG); and F, group 6 (EWB + 0.1% EGCG). The silver deposits in A, C and F were extensive and high. However, slight nanoleakage was observed in E. c, composite resin; d, dentine; pointers, silver deposits.
Please cite this article in press as: H. Yang, et al., Effect of adjunctive application of epigallocatechin-3-gallate and ethanol–wet bonding on adhesive–dentin bonds, Journal of Dentistry (2015), http://dx.doi.org/10.1016/j.jdent.2015.12.001
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