- Email: [email protected]
Influence of natural and synthetic metalloproteinase inhibitors on bonding durability of an etch-and-rinse adhesive to dentin
International Journal of Adhesion & Adhesives 47 (2013) 83–88
Contents lists available at ScienceDirect
International Journal of Adhesion & Adhesives journal homepage: www.elsevier.com/locate/ijadhadh
Influence of natural and synthetic metalloproteinase inhibitors on bonding durability of an etch-and-rinse adhesive to dentin T.M.A. Monteiro, R.T. Basting, C.P. Turssi, F.M.G. França, F.L.B. Amaral n São Leopoldo Mandic Institute and Research Center, Campinas, São Paulo, Brazil
art ic l e i nf o
a b s t r a c t
Article history: Accepted 22 August 2013 Available online 5 October 2013
The aim of the present study was to evaluate the effect of natural (green tea (Camellia sinensis) GT) and synthetic (chlorhexidine-CLX) metalloproteinase inhibitors on the microtensile bond strength (mTBS) of an etch-and-rinse adhesive to dentin, after 24 h and 6 months of water storage (WS). Thirty human dentin specimens were conditioned with 37% phosphoric acid for 15 s, rinsed for the same amount of time and dried gently. They were then divided into 3 groups, according to the solution to be applied to the dentin surface (n ¼10): GT, 2% CLX, or NT (none, as control). CLX and GT solution (20 μl) were applied for 60 s and dried gently with absorbent paper. The adhesive system (Adper Single Bond 2, 3M ESPE) was then applied according to the manufacturer's instructions, and a 4-mm composite resin block was built. After 24 h, at 37 1C, resin–dentin blocks were sectioned into 1-mm2 sticks that were assigned into two mTBS test conditions: after being stored in water for 24 h or after 6 months. Data were submitted to repeated-measures two-way ANOVA and Tukey's test, with a 5% significance level. The failure pattern was described in percentage terms. The results showed that the mTBS values in the NT group were significantly higher compared to the GT values. The application of CLX resulted in intermediate mTBS values, which were not statistically different from NT or GT. There was no significant difference between the mTBS values in the two time points of analysis for CLX and GT groups while the NT group showed a significant decrease over time. After 6 months of WS, all groups had mTBS values statistically similar among themselves. It can be concluded that in a short-term evaluation, chlorhexidine showed no interference on bond strength to dentin, while green tea did. After a long-term evaluation, both metalloproteinase inhibitors, chlorhexidine and green tea, were capable of maintaining bond strength stability. & 2013 Elsevier Ltd. All rights reserved.
Keywords: Surface treatment Adhesion by mechanical interlocking Fracture mechanics Composites Primers and coupling agents
1. Introduction The fundamental principle of adhesion to tooth substrate is based on the formation of hybrid layer [1], which is a result of (1) removal of calcium phosphates and exposition of microporosities at enamel and dentin tooth surface and (2) diffusion and polymerization of resin into those microporosities [1]. The mechanical interlocking promoted by the hybrid layer formation is responsible for adequate bond strength in adhesive restorations. Bond durability of adhesive systems to dental substrates has been reported as limited, with a significant reduction in bond strength and structural alterations in the hybrid layer over time [2,3]. This occurrence has been observed for etch-and-rinse adhesives, which
n Correspondence to: São Leopoldo Mandic Institute and Research Center, Rua José Rocha Junqueira, 13, CEP: 13045-755, Ponte Preta, Campinas, SP, Brazil. Tel.: þ 55 19 32113600; fax: þ55 19 32113712. E-mail addresses: [email protected] (T.M.A. Monteiro), [email protected] (R.T. Basting), [email protected] (C.P. Turssi), [email protected] (F.M.G. França), fl[email protected] (F.L.B. Amaral).
0143-7496/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijadhadh.2013.09.020
requires dentin conditioning with phosphoric acid [4], and also for self-etching adhesives [5]. Clinical and in vitro studies have revealed that partial demineralization of collagen fibrils occurs after acid conditioning [6]. This procedure is essential for further filling with adhesive monomers [1]. However, a decrease in pH (that occurs with acid conditioning), followed by an increase in pH (that occurs when the primer/adhesive is applied), triggers the activation of endogenous matrix metalloproteinases (MMPs) [7] and cysteine–cathepsins [3]. MMPs are noncollagenous enzymes that are trapped within the dentin extracellular matrix [3,8]. When activated, MMPs exert proteolytic action on unprotected collagen, which results in degradation of the hybrid layer and decreased bond durability [9]. Collagenolytic/gelatinolytic activity of MMPs in dentin, such as MMP-2 (MMP-2, MMP-8 and MMP-9) [10] may be inhibited by applying MMPs inhibitors, such as chlorhexidine (synthetic MMP inhibitor) [3,10]. The application of chlorhexidine after acid conditioning has been reported as not interfering in the bonding action of adhesives [11–17], in addition to preventing dentin degradation by MMPs [12,13,18]. Accordingly, decreases
84
T.M.A. Monteiro et al. / International Journal of Adhesion & Adhesives 47 (2013) 83–88
in adhesive interface degradation have been reported, lasting from 6 months to 2 years following chlorhexidine application [18–21]. Furthermore, chlorhexidine has antibacterial properties [22–24]. Another matrix metalloproteinase inhibitor is green tea (Camellia sinensis) [25]. Recently, green tea was reported as a natural MMP inhibitor, capable of reducing erosive–abrasive lesions in dentin, due to its MMP inhibitory potential [26–28]. This potential has been mainly ascribed to the polyphenols composing green tea, including epigallocatechin gallate (EGCG) [27,29]. Despite the promising MMP-inhibitor property of green tea, its influence as a therapeutic approach for increasing bond durability by decreasing adhesive interface degradation has never been reported. Accordingly, the aim of the present study was to determine the influence of green tea (natural MMP-inhibitor) and chlorhexidine (synthetic MMP-inhibitor) on the microtensile bond strength of an etch-and-rinse adhesive to dentin. The null hypothesis tested was that neither the application of a green tea solution nor chlorhexidine would have any influence on the bond strength of an etch-and-rinse adhesive to dentin, regardless of the storage time.
2. Materials and methods 2.1. Ethical aspects The present study was approved by the Research Ethics Committee of the São Leopoldo Mandic School of Dentistry and Research Institute (Protocol #2012/0103). 2.2. Experimental design Thirty dentin specimens were divided into 3 groups according to the solution applied on the dentinal surface after dentin conditioning with phosphoric acid (n¼ 10): GT: green tea; CLX: 2% chlorhexidine; NT (no treatment – control group). After the application of an etch-and-rinse adhesive, a block of resin composite was built on the dentin. Each resin–dentin block was considered as an experimental unit. From each resin–dentin block,
sticks measuring approximately 1.0 mm2 were obtained and assigned into two tensile test conditions: after being stored in water for 24 h or after 6 months. μTBS values of sticks from the same restored teeth were averaged and considered as a single value, which was considered as the outcome variable. Another variable was failure pattern, described in percentage terms. Fig. 1 describes a schematic illustration of the experiment. 2.3. Dentin slab preparation Thirty human third molars, extracted for reasons not related to those of the present research, and stored in thymol (0.1%, pH 7.0) after extraction, were used in this experiment. Teeth were submitted to debriding with scalpel blades and periodontal curettes. Teeth were cross sectioned using a water-cooled diamond saw (15 HC series, Buehler Ltd., Lake Bluff, Illinois, EUA) in a sectioning machine (Isomet 1000 Precision Diamond Saw, Buehler Ltd., Lake Bluff, Illinois, EUA), which separated the occlusal third of the crown and obtained a large dentin surface in the middle third, perpendicular to the long axis of the tooth. A second parallel section was made 4 mm from the first, to obtain 4-mm high slabs. These slabs were sectioned in the mesiodistal and buccal–lingual directions to obtain square sections measuring 5 5 mm. Dentin slabs were flattened in a water-cooled polishing machine (Politriz Aropol 2V, Arotec, São Paulo, SP, Brazil) with decreasing granulations (400, 600 and 1200) of water abrasive paper (Imperial Wetordry, 3M, USA). 2.4. Adhesive procedures Dentin slabs were submitted to conditioning with 37% phosphoric acid, and were randomly divided into three groups, according to the solution applied to the dentin surface: GT – Green Tea – Camellia sinensis or 2% CLX, or NT (none, as control) (Table 1). The etch-and-rinse adhesive system was then applied to the dentin fragments, which were restored with a microhybrid resin composite. The final resin–dentin blocks were 4-mm high. Description of the main materials and their respective instructions for use are shown in Table 1.
Fig. 1. Schematic illustration. A. Removal of enamel to obtain 4-mm high dentin fragments. B. Fragments were sectioned in the mesiodistal and buccal–lingual directions and polished to obtain square slabs. C. Fragments were randomly divided into 3 groups, according to the treatment done on dentine surface. D. After application of etch-and-rinse adhesive, a composite resin restoration was built on dentin surface, resulting in resin–dentine blocks (E). F. Resin–dentine blocks were sectioned perpendicular to the bonding surface, into 1.0-mm thick slabs. By rotating samples 901 and sectioning them again lengthwise, multiple 1.0-mm2 beam-shaped sticks were obtained. G. After 24 h or 6 months of water storage sticks were submitted to microtensile bond strength testing (H).
T.M.A. Monteiro et al. / International Journal of Adhesion & Adhesives 47 (2013) 83–88
85
Table 1 Main composition and application mode of materials used. Materials
Manufacturer and batch number
Composition
Application mode
Condac 37
FGM (Dentscare Ltda – Joinville, SC, Brazil) #020211 FGM (Dentscare Ltda – Joinville, SC, Brazil) #270911 Leão Júnior (Fazenda Rio Grande, PR, Brazil) #8325
37% Phosphoric acid
Applied to dentin surface for 15 s, rinsed for the same amount of time and dried gently with absorbent paper.
Chlorhexidine gluconate 2% solution
Twenty microliters of the solution were actively applied to dentin for 60 s. Dentin was dried gently with absorbent paper.
Chlorhexidine S 2% Green Tea
Adper™ Single Adper™ Single Bond 2 Bond 2 (3M ESPE, Minnesota, USA) #N211104BR Filtek Z250 3M ESPE, Minnesota, Shade B2 USA 1109500117
Camellia sinensis
Infusion of 2 g of herb in 180 ml of boiled water for 1 min. (1.1%). Twenty microliters of the solution were actively applied to dentin for 60 sn. Dentin was dried gently with absorbent paper. The adhesive system was applied in two consecutive layers; Bis-GMA, HEMA, copolymer of acrylic and itaconic acids, water, ethyl alcohol, glycerol 1, 3-dimethacrylate, diurethane remaining solvent was evaporated with a brief, gentle, dry air jet for 10 s and light polymerized for 20 s. dimethacrylate, silane treated silica, water Aluminum oxide, silica, zirconium oxide, Bis-GMA, Each 1-mm increment was light polymerized for 20 s. Bis-EMA, UDMA
n
According to a protocol adopted by Kato et al. [29].
at 30 magnification to assess the failure modes, which were classified as adhesive (lack of adhesion), cohesive in dentin (failure of the dental substrate), cohesive in composite resin (failure of the resin composite) or mixed (adhesive and cohesive failures).
Table 2 Mean (standard deviation) of experimental groups. Treatment
Bond strength 24 h
6 months
22.6 Aba 16.2 Ba 26.0 Aa
14.6 Aa 16.6 Aa 16.3 Ab
2.6. Statistical analysis Chlorhexidine Green tea No treatment (control)
Means followed by the same letters (capitals within each column and lower case within each row) are not significantly different (α ¼ 0.05).
Light polymerization was performed on the adhesive system (Adper™ Single Bond 2) and microhybrid composite (Filtek Z250) for the time recommended by manufacturers (Table 1), using a visible light-curing unit (Ultralux EL, Dabi Atlante, Ribeirão Preto, SP, Brazil). The output of the light-curing unit was periodically measured with a radiometer (Newdent Equipamentos Ltda., Ribeirão Preto, SP, Brazil), and was found to have a mean range of 620 mW/cm². Resin–dentin blocks were kept in relative humidity at 37 1C for 24 h, and sectioned perpendicular to the bonding surface, into 1.0mm thick slabs, using a water-cooled diamond saw. By rotating samples 901 and sectioning them again lengthwise, multiple beam-shaped sticks were obtained, each with a cross-sectional surface area of 1.0 mm2. Half of sticks from a same resin–dentin block were submitted to mTBS testing after 24 h. The other half was kept in distilled water that was changed every 2 days, and kept in a bacteriological oven for 6 months. 2.5. mTBS testing Specimens were attached to a specific testing device for mTBS testing, with a cyanoacrylate adhesive (Super Bonder Gel, Henkel Ltda., Brazil). They were subjected to tensile stress in a universal testing machine, at a crosshead speed of 0.5 mm/min and a 50 N load cell until fracture. The bond strength values were expressed in kgf/cm2, and converted to MPa after measuring the crosssectional area at the fracture site with a digital caliper (Mitutoyo, Tokyo, Japan). The comparison was made using the average value of each tooth (n ¼10). After bond strength testing, the failure pattern of each stick was analyzed under a stereomicroscope (EK3ST, CQA, São Paulo, Brazil)
Based on the normal distribution of the data, repeated-measures two-way analysis of variance (ANOVA) and Tukey's test were applied. Statistical calculations were performed with SPSS 20 (SPSS Inc., Chicago, IL, USA). Significance level was set at 5%. Failure pattern was described with descriptive statistics (percentage).
3. Results Table 2 describes the means and standard deviations of experimental groups. One-way ANOVA revealed that the P-value for the interaction between “solutions applied on dentin” and “time” was borderline for statistical significance (p ¼0.065). So, the interaction evaluation was performed. Results demonstrated that, in a 24-h evaluation, microtensile bond strength values in the NT group were significantly higher compared to GT values. The application of CLX resulted in intermediate mTBS values, which were not statistically different from NT or GT. The GT group showed lower microtensile bond strength values that were statistically different from the NT group. After 6 months of WS, all groups had mTBS values statistically similar among themselves. There was no significant difference in bond strength means after 24 h and 6 months of water storage for the CLX and GT solutions. However, for the group in which any solution was applied (NT group), bond strength values significantly decreased after 6 months of water storage. Fig. 2 illustrates the failure pattern observed after the microtensile bond strength test. There was a predominance of adhesive failures in the NT (38.9%) and the CLX (41.2%) groups. However, mixed failures were predominant (33.3%) in the GT group; moreover, a greater frequency of cohesive failures in dentin was also observed (23.8%) in this group. After 6 months of water storage, CLX group showed a predominance of mixed (33.3%) and adhesive (33.3%) failures, while in NT group, a greater frequency of adhesive (35%) and cohesive failures in resin (35%) were found. For the GT group, a predominance of failure patterns could not be observed, as the percentage was the same for the adhesive, mixed or cohesive in dentin failures.
86
T.M.A. Monteiro et al. / International Journal of Adhesion & Adhesives 47 (2013) 83–88
Fig. 2. Failure pattern percentage of each experimental group.
4. Discussion The fact that adhesive-based restorations fail and undergo degradation over time has increased the concern over the longevity of these materials. Reduction in bond strength is often accompanied by hybrid layer degradation, responsible for the reduction in restoration survival [2]. The hybrid layer degradation occurs at some stages [2,30]: the polymer absorbs water, possibly causing hydrolysis and plasticizing of the resin components, and their subsequent leaching and degradation; collagen fibrils (especially those that are demineralized and not fully impregnated by resin monomers) are degraded by host-derived enzymes exhibiting collagenolytic activity, such as metalloproteinases (MMPs) [9] cysteine cathepsins [31] and exogenous proteinases from saliva. MMPs are secreted as inactive proenzymes and must be activated to act on the degradation of collagen fibrils. It is known that MMPs are denaturized when pH decreases, as is the case of cariogenic bacteria fermenting sucrose to form lactic acid, or when dentinal tissue is conditioned with phosphoric acid. When the pH recovers its value (for example, during the adhesive system application, or neutralization of pH by the buffering system), MMPs are activated [31–33]. Recently, it was reported that both MMPs and cysteine cathepsins act synergistically, in such a way that the cysteine cathepsins participate in MMPs activation [31]. Knowledge regarding the effect of MMPs and cysteine cathepsins on resin–dentin bond degradation has led to the focusing of attention on substances capable of inhibiting these enzymes. Among these substances, 2% chlorhexidine has been used with the purpose of inhibiting MMP [11–17,34] and cysteine cathepsins [35]. In fact, its application has resulted in the enhanced longevity of adhesive-based restorations [13,15,31, 34,36]. Green tea is another substance that has been described as a natural MMPs inhibitor [8,37] particularly because it contains flavanoids, such as the catechins, specially the epigallocatechin gallate (EGCG). This property has contributed to the use of green tea in preventing erosive–abrasive lesions, with promising results [28,29,38]. Also, there is evidence that green tea may reduce dental caries through different mechanisms including antimicrobial properties against cariogenic microflora [39–41] and enzymes activity inhibition [39,41].
The possibility that green tea could inhibit host-derived MMPs from dentin drove the authors of the present study to evaluate the influence of a green tea solution on the bond strength of etch-andrinse adhesives to dentin, compared with a chlorhexidine solution or no treatment (control group). In this respect, the null hypothesis was rejected, since the results of this study revealed statistical differences among the experimental groups. When 2% chlorhexidine was applied after conditioning with phosphoric acid and prior to the application of an etch-and-rinse adhesive, a decrease was observed in bond strength values, but with no statistical difference when compared with the control group (in which any solution was applied). This result is in agreement with previous studies showing that the use of chlorhexidine had no effect on immediate adhesion, and did not compromise bond strength of adhesive systems [11–16,42]. Even in a 24-h evaluation, Osorio et al. [43] reported that the chlorhexidine application was able to decelerate collagen degradation, in a methodology that proposed to quantify the C-terminal telopeptide concentration (which indicates the amount of collagen degradation). After 6 months of water storage, results of the present study indicate that chlorhexidine is capable of maintaining bond strength stability since there were no differences among bond strength values after 24 h and 6 months of water storage. Other reports also found that the benefits of chlorhexidine are not limited to the short-term, since its application frequently results greater bond longevity after long-term water storage [19,21]. This behavior can also be expected in vivo [12]. Chlorhexidine has the benefit of being an antimicrobial agent that possesses substantivity and binds to mineralized dentin for at least 12 weeks [44,45]. On the other hand, the 24-h period of evaluation showed that the green tea solution used in this study had an undesirable effect on the bond strength of the tested adhesive to dentin, compared to the group to which no solution was applied. According to Mirkarimi et al. [46] the green tea solution must have allowed the formation of a surface deposit of organic materials on the dentin and the formation of newly induced collagen crosslinks. According to them, there is a substance widely present in green tea, namely proanthocyanidin, a flavanol, that might interact with the organic portion of dentin. It is unknown if these interactions may have jeopardized, in a first moment, hybrid layer formation within dentin, but further analysis must confirm such condition. Nevertheless, the green tea values were statistically similar
T.M.A. Monteiro et al. / International Journal of Adhesion & Adhesives 47 (2013) 83–88
to those of chlorhexidine after 24 h of storage. Surprisingly, after 6 months, green tea application led to the maintenance of bond strength values being those values statistically similar to those found in no treated and chlorhexidine groups. Although there are no reports that have evaluated green tea's substantivity in dentin, i.e., the time this solution would be bound to mineralized dentin, some reasons may explain the bond stability observed with the group treated with green tea. Ephasinghe et al. [47] showed that proanthocyanidin (the above mentioned green tea component) inactivated around 90% MMP-2, -8 and -9 and around 75–90% of cysteine cathepsin, which was significantly higher than chlorhexidine. In addition green tea's composition includes (besides flavonoids/catechins) proteins/enzymes, carbohydrates, lipid components, vitamins (B, C, E), xanthic bases such as caffeine and theophylline, volatile components (aldehydes and alcohols), minerals and other elements such as Ca, Mg, Cr, Mn, Fe, Cu, Zn, Mo, Se, Na, P, Co, Sr, Ni, K, F and Al [41]. It is known that Zn has an important hole against collagen degradation [48] by binding to it and leading to a new conformation that protects the cleavage sites from metalloproteinases [48]. These properties therefore could explain why green tea application led to more stable bond strength values. Also, in the present study, the green tea was applied in a concentration of approximately 1.1%. Magalhães et al. [28] tested an experimental green tea extract that was diluted to a final concentration of 0.61%. At this concentration, the green tea was also able to reduce erosive–abrasion wear. Moreover, in the present study, green tea was applied as a solution, whereas Kato et al. [49] proposed the use of green tea as a gel vehicle. Buzalaf et al. [27] revealed that green tea gel showed a more pronounced effect over erosive–abrasive lesions than green tea incorporated to dentifrices or green tea solutions. In future bond strength studies, other concentrations/ vehicles of green tea could be used to test whether better results could be achieved. According to the failure pattern, the results of the present study demonstrated that most failures in the chlorhexidine groups were adhesive, regardless of the time points of analysis. Other studies evaluating the effect of chlorhexidine on bond strength have produced similar failure patterns [14,19,42]. However, an increase in mixed failures and decrease in adhesive failures were observed in the green tea solution group. After 6 months, a predominance of failure patterns could not be observed, as the percentage was the same for the adhesive, mixed or cohesive in dentin failures. According to the results of the present study, it can be verified that chlorhexidine solution may be applied to the dentin surface after acid conditioning to boost its inhibitory potential against MMPs and take advantage of its antimicrobial properties. A further consideration is that chlorhexidine has the potential of establishing adhesion over time by decreasing bond degradation. In addition, the results indicate that the application of green tea solution has a potential to maintain bond stability, but the results and the reasons for that need to be further evaluated.
5. Conclusions It can be concluded that in a short-term evaluation, chlorhexidine showed no interference on bond strength to dentin, while green tea did. After a long-term evaluation, both metalloproteinase inhibitors, chlorhexidine and green tea, were capable of maintaining bond strength stability. References [1] Van Meerbeek B, De Munck J, Yoshida Y, Inoue S, Vargas M, Vijay P, et al. Buonocore memorial lecture. Adhesion to enamel and dentin: current status and future challenges. Operative Dentistry 2003;3:215–35.
87
[2] Amaral FL, Colucci V, Palma-Dibb RG, Corona SA. Assessment of in vitro methods used to promote adhesive interface degradation: a critical review. Journal of Esthetic and Restorative Dentistry 2007;19:340–53, http://dx.doi. org/10.1111/j.1708-8240.2007.00134.x. [3] Tjäderhane L, Nascimento FD, Breschi L, Mazzoni A, Tersariol IL, Geraldeli S, et al. Optimizing dentin bond durability: control of collagen degradation by matrix metalloproteinases and cysteine cathepsins. Dental Materials 2013;29:116–35, http://dx.doi.org/10.1016/j.dental.2012.08.004. [4] De Munck J, Mine A, Vivan Cardoso M, Van Landuyt KL, Lührs AK, Poitevin A, et al. Hydrolytic stability of three-step etch-and-rinse adhesives in occlusal class-I cavities, Clinical Oral Investigations, http://dx.doi.org/10.1007/ s00784-012-0884-0, in press. [5] Cardoso MV, de Almeida Neves A, Mine A, Coutinho E, Van Landuyt K, De Munck J, et al. Current aspects on bonding effectiveness and stability in adhesive dentistry. Australian Dental Journal 2011;56:31–44, http://dx.doi. org/10.1111/j.1834-7819.2011.01294.x. [6] Boushell LW, Swift Jr. EJ. Critical appraisal. Dentin bonding: matrix metalloproteinases and chlorhexidine. Journal of Esthetic and Restorative Dentistry 2011;23:347–52, http://dx.doi.org/10.1111/j.1708-8240.2011.00464.x. [7] Mazzoni A, Scaffa P, Carrilho M, Tjäderhane L, Di Lenarda R, Polimeni A, et al. Effects of etch-and-rinse and self-etch adhesives on dentin MMP-2 and MMP9, Journal of Dental Research. 92, 2013, 82–6, http://dx.doi.org/10.1177/ 0022034512467034. [8] Chaussain-Miller C, Fioretti F, Goldberg M, Menashi S. The role of matrix metalloproteinases (MMPs) in human caries. Journal of Dental Research 2006;85:22–32, http://dx.doi.org/10.1177/154405910608500104. [9] Pashley DH, Tay FR, Yiu C, Hashimoto M, Breschi L, Carvalho RM, et al. Collagen degradation by host-derived enzymes during aging. Journal of Dental Research 2004;83:216–21, http://dx.doi.org/10.1177/154405910408300306. [10] Moon PC, Weaver J, Brooks CN. Review of matrix metalloproteinases' effect on the hybrid dentin bond layer stability and chlorhexidine clinical use to prevent bond failure. Open Dentistry 2010;4:147–52, http://dx.doi.org/10.2174/ 1874210601004010147. [11] Erhardt MC, Osorio R, Toledano M. Dentin treatment with MMPs inhibitors does not alter bond strengths to caries-affected dentin. Journal of Dentistry 2008;36:1068–73, http://dx.doi.org/10.1016/j.jdent.2008.09.002. [12] Brackett WW, Tay FR, Brackett MG, Dib A, Sword RJ, Pashley DH. The effect of chlorhexidine on dentin hybrid layers in vivo. Operative Dentistry 2007;32:107–11, http://dx.doi.org/10.2341/06-55. [13] Carrilho MR, Carvalho RM, de Goes MF, di Hipólito V, Geraldeli S, Tay FR, et al. Chlorhexidine preserves dentin bond in vitro. Journal of Dental Research 2007;86:90–4, http://dx.doi.org/10.1177/154405910708600115. [14] Mobarak EH. Effect of chlorhexidine pretreatment on bond strength durability of caries-affected dentin over 2-year aging in artificial saliva and under simulated intrapulpal pressure. Operative Dentistry 2011;36:649–60, http: //dx.doi.org/10.2341/11-018-L. [15] Ricci HA, Sanabe ME, de Souza Costa CA, Pashley DH, Hebling J. Chlorhexidine increases the longevity of in vivo resin–dentin bonds. European Journal of Oral Sciences 2010;118:411–6, http://dx.doi.org/10.1111/j.1600-0722.2010.00754.x. [16] Soares CJ, Pereira CA, Pereira JC, Santana FR, do Prado CJ. Effect of chlorhexidine application on microtensile bond strength to dentin. Operative Dentistry 2008;33:183–8, http://dx.doi.org/10.2341/07-69. [17] Stape TH, Menezes MS, Barreto BC, Aguiar FH, Martins LR, Quagliatto PS. Influence of matrix metalloproteinase synthetic inhibitors on dentin microtensile bond strength of resin cements. Operative Dentistry 2012;37:386–96, http://dx.doi.org/10.2341/11-256-L. [18] Breschi L, Mazzoni A, Nato F, Carrilho M, Visintini E, Tjäderhane L, et al. Chlorhexidine stabilizes the adhesive interface: a 2-year in vitro study. Dental Materials 2010;26:320–5, http://dx.doi.org/10.1016/j.dental.2009.11.153. [19] Campos EA, Correr GM, Leonardi DP, Barato-Filho F, Gonzaga CC, Zielak JC. Chlorhexidine diminishes the loss of bond strength over time under simulated pulpal pressure and thermo-mechanical stressing. Journal of Dentistry 2009;37:108–14, http://dx.doi.org/10.1016/j.jdent.2008.10.003. [20] Carrilho MR, Carvalho RM, Sousa EN, Nicolau J, Breschi L, Mazzoni A, et al. Substantivity of chlorhexidine to human dentin. Dental Materials 2010;26: 7779–85, http://dx.doi.org/10.1016/j.dental.2010.04.002. [21] Stanislawczuk R, Reis A, Loguercio AD. A 2-years in vitro evaluation of a chlorhexidine-containing acid on the durability of resin–dentin interface. Journal of Dentistry 2011;39:40–7, http://dx.doi.org/10.1016/j. jdent.2010.10.001. [22] Borges FM, de Melo MA, Lima JP, Zanin IC, Rodrigues LK. Antimicrobial effect of chlorhexidine digluconate in dentin: in vitro and in situ study. Journal of Conservative Dentistry 2012;15:22–6, http://dx.doi.org/10.4103/09720707.92601. [23] Pashley DH, Tay FR, Imazato S. How to increase the durability of resin–dentin bonds. Compendium of Continuing Education in Dentistry 2011;32:60–4. [24] Thompson JM, Agee K, Sidow SJ, McNally K, Lindsey K, Borke J, et al. Inhibition of endogenous dentin matrix metalloproteinases by ethylenediaminetetraacetic acid. Journal of Endodontics 2012;38:62–5, http://dx.doi.org/10.1016/j. joen.2011.09.005. [25] Mandal M, Mandal A, Das S, Chakraborti T, Sajal C. Clinical implications of matrix metalloproteinases. Molecular and Cellular Biochemistry 2003;252: 305–29. [26] Barbosa CS, Kato MT, Buzalaf MA. Effect of supplementation of soft drinks with green tea extract on their erosive potential against dentine.
88
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
T.M.A. Monteiro et al. / International Journal of Adhesion & Adhesives 47 (2013) 83–88
Australian Dental Journal 2011;56:317–21, http://dx.doi.org/10.1111/j.18347819.2011.01338.x. Buzalaf MA, Kato MT, Hannas AR. The role of matrix metalloproteinases in dental erosion. Advances in Dental Research 2012;24:72–6, http://dx.doi.org/ 10.1177/0022034512455029. Magalhães AC, Wiegand A, Rios D, Hannas A, Attin T, Buzalaf MA. Chlorhexidine and green tea extract reduce dentin erosion and abrasion in situ. Journal of Dentistry 2009;37:994–8, http://dx.doi.org/10.1016/j.jdent.2009.08.007. Kato MT, Magalhães AC, Rios D, Hannas AR, Attin T, Buzalaf MAR. Protective effect of Green tea on dentin erosion and abrasion. Journal of Applied Oral Science 2009;17:560–4, http://dx.doi.org/10.1590/S167877572009000600004. Sanabe ME, Costa CA, Hebling J. Exposed collagen in aged resin–dentin bonds produced on sound and caries-affected dentin in the presence of chlorhexidine. Journal of Adhesive Dentistry 2011;13:117–24, http://dx.doi.org/ 10.3290/j.jad.a19239. Nascimento FD, Minciotti CL, Geraldeli S, Carrilho MR, Pashley DH, Tay FR, et al. Cysteine cathepsins in human carious dentin. Journal of Dental Research 2011;90:506–11, http://dx.doi.org/10.1177/0022034510391906. Tjäderhane L, Larjava H, Sorsa T, Uitto VJ, Larmas M, Salo T. The activation and function of host matrix metalloproteinases in dentin matrix breakdown in caries lesions. Journal of Dental Research 1998;77:1622–9, http://dx.doi.org/ 10.1177/00220345980770081001. Mazzoni A, Pashley DH, Nishitani Y, Breschi L, Mannello F, Tjäderhane L, et al. Reactivation of inactivated endogenous proteolytic activities in phosphoric acid-etched dentine by etch-and-rinse adhesives. Biomaterials 2006;27: 4470–6, http://dx.doi.org/10.1016/j.biomaterials.2006.01.040. Hebling J, Pashley DH, Tjäderhane L, Tay FR. Chlorhexidine arrests subclinical degradation of dentin hybrid layers in vivo. Journal of Dental Research 2005;84:741–6, http://dx.doi.org/10.1177/154405910508400811. Scaffa PM, Vidal CM, Barros N, Gesteira TF, Carmona AK, Breschi L, et al. Chlorhexidine inhibits the activity of dental cysteine cathepsins. Journal of Dental Research 2012;91:420–5, http://dx.doi.org/10.1177/ 0022034511435329. Komori PC, Pashley DH, Tjäderhane L, Breschi L, Mazzoni A, de Goes MF, et al. Effect of 2% chlorhexidine digluconate on the bond strength to normal versus caries-affected dentin. Operative Dentistry 2009;34:157–65, http://dx.doi.org/ 10.2341/08-55. Demeule M, Brossard M, Pagé M, Gingras D, Béliveau R. Matrix metalloproteinase inhibition by green tea catechins. Biochimica et Biophysica Acta 2000;1478:51–60, http://dx.doi.org/10.1016/S0167-4838(00)00009-1.
[38] Bassiouny MA, Kuroda S, Yang J. Topographic and radiographic profile assessment of dental erosion. Part III: effect of green and black tea on human dentition. General Dentistry 2008;56:451–61. [39] Wu CD, Wei GX. Tea as a functional food for oral health. Nutrition 2002;18: 443–4, http://dx.doi.org/10.1016/S0899-9007(02)00763-3. [40] Ferrazzano GF, Roberto L, Amato I, Cantile T, Sangianantoni G, Ingenito A. Antimicrobial properties of green tea extract against cariogenic microflora: an in vivo study. Journal of Medicinal Food 2011;14:907–11, http://dx.doi.org/ 10.1089/jmf.2010.0196. [41] Narotzki B, Reznick AZ, Aizenbud D, Levy Y. Green tea: a promising natural product in oral health. Archives of Oral Biology 2012;57:429–35, http://dx.doi. org/10.1016/j.archoralbio.2011.11.017. [42] Dalkilic EE, Aris HD, Kivanc BH, Uctasli MB, Omurlu H. Effect of different disinfectant methods on the initial microtensile bond strength of a self-etch adhesive to dentin. Lasers in Medical Science 2012;27:819–25, http://dx.doi. org/10.1007/s10103-011-0987-x. [43] Osorio R, Yamauti M, Osorio E, Ruiz-Requena ME, Pashley D, Tay F, et al. Effect of dentin etching and chlorhexidine application on metalloproteinasemediated collagen degradation. European Journal of Oral Sciences 2011;119: 79–85, http://dx.doi.org/10.1111/j.1600-0722.2010.00789.x. [44] Liu Y, Tjäderhane L, Breschi L, Mazzoni A, Li N, Mao J, et al. Limitations in bonding to dentin and experimental strategies to prevent bond degradation. Journal of Dental Research 2011;90:953–68, http://dx.doi.org/10.1177/ 0022034510391799. [45] Mohammadi Z, Abbott PV. Antimicrobial substantivity of root canal irrigants and medicaments: a review. Australian Endodontic Journal 2009 Dec;35 (3):131–9, http://dx.doi.org/10.1111/j.1747-4477.2009.00164.x. [46] Mirkarimi M, Toomarian L. Effect of green tea extract on the treatment of dentin erosion: an in vitro study. Journal of Dentistry 2012;9:224–8. [47] Epasinghe DJ, Yiu CK, Burrow MF, Hiraishi N, Tay FR. The inhibitory effect of proanthocyanidin on soluble and collagen-bound proteases, Journal of Dentistry; 2013 [in press]. 41, 2013, 832–839. http://dx.doi.org/10.1016/j.jdent. 2013.06.002. [48] Osorio R, Yamauti M, Osorio E, Román JS, Toledano M. Zinc-doped dentin adhesive for collagen protection at the hybrid layer. European Journal of Oral Sciences 2011;119(5):401–10, http://dx.doi.org/10.1111/j.16000722.2011.00853.x. [49] Kato MT, Leite AL, Hannas AR, Buzalaf MA. Gels containing MMP inhibitors prevent dental erosion in situ. Journal of Dental Research 2010;89:468–72, http://dx.doi.org/10.1177/0022034510363248.
Contents lists available at ScienceDirect
International Journal of Adhesion & Adhesives journal homepage: www.elsevier.com/locate/ijadhadh
Influence of natural and synthetic metalloproteinase inhibitors on bonding durability of an etch-and-rinse adhesive to dentin T.M.A. Monteiro, R.T. Basting, C.P. Turssi, F.M.G. França, F.L.B. Amaral n São Leopoldo Mandic Institute and Research Center, Campinas, São Paulo, Brazil
art ic l e i nf o
a b s t r a c t
Article history: Accepted 22 August 2013 Available online 5 October 2013
The aim of the present study was to evaluate the effect of natural (green tea (Camellia sinensis) GT) and synthetic (chlorhexidine-CLX) metalloproteinase inhibitors on the microtensile bond strength (mTBS) of an etch-and-rinse adhesive to dentin, after 24 h and 6 months of water storage (WS). Thirty human dentin specimens were conditioned with 37% phosphoric acid for 15 s, rinsed for the same amount of time and dried gently. They were then divided into 3 groups, according to the solution to be applied to the dentin surface (n ¼10): GT, 2% CLX, or NT (none, as control). CLX and GT solution (20 μl) were applied for 60 s and dried gently with absorbent paper. The adhesive system (Adper Single Bond 2, 3M ESPE) was then applied according to the manufacturer's instructions, and a 4-mm composite resin block was built. After 24 h, at 37 1C, resin–dentin blocks were sectioned into 1-mm2 sticks that were assigned into two mTBS test conditions: after being stored in water for 24 h or after 6 months. Data were submitted to repeated-measures two-way ANOVA and Tukey's test, with a 5% significance level. The failure pattern was described in percentage terms. The results showed that the mTBS values in the NT group were significantly higher compared to the GT values. The application of CLX resulted in intermediate mTBS values, which were not statistically different from NT or GT. There was no significant difference between the mTBS values in the two time points of analysis for CLX and GT groups while the NT group showed a significant decrease over time. After 6 months of WS, all groups had mTBS values statistically similar among themselves. It can be concluded that in a short-term evaluation, chlorhexidine showed no interference on bond strength to dentin, while green tea did. After a long-term evaluation, both metalloproteinase inhibitors, chlorhexidine and green tea, were capable of maintaining bond strength stability. & 2013 Elsevier Ltd. All rights reserved.
Keywords: Surface treatment Adhesion by mechanical interlocking Fracture mechanics Composites Primers and coupling agents
1. Introduction The fundamental principle of adhesion to tooth substrate is based on the formation of hybrid layer [1], which is a result of (1) removal of calcium phosphates and exposition of microporosities at enamel and dentin tooth surface and (2) diffusion and polymerization of resin into those microporosities [1]. The mechanical interlocking promoted by the hybrid layer formation is responsible for adequate bond strength in adhesive restorations. Bond durability of adhesive systems to dental substrates has been reported as limited, with a significant reduction in bond strength and structural alterations in the hybrid layer over time [2,3]. This occurrence has been observed for etch-and-rinse adhesives, which
n Correspondence to: São Leopoldo Mandic Institute and Research Center, Rua José Rocha Junqueira, 13, CEP: 13045-755, Ponte Preta, Campinas, SP, Brazil. Tel.: þ 55 19 32113600; fax: þ55 19 32113712. E-mail addresses: [email protected] (T.M.A. Monteiro), [email protected] (R.T. Basting), [email protected] (C.P. Turssi), [email protected] (F.M.G. França), fl[email protected] (F.L.B. Amaral).
0143-7496/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijadhadh.2013.09.020
requires dentin conditioning with phosphoric acid [4], and also for self-etching adhesives [5]. Clinical and in vitro studies have revealed that partial demineralization of collagen fibrils occurs after acid conditioning [6]. This procedure is essential for further filling with adhesive monomers [1]. However, a decrease in pH (that occurs with acid conditioning), followed by an increase in pH (that occurs when the primer/adhesive is applied), triggers the activation of endogenous matrix metalloproteinases (MMPs) [7] and cysteine–cathepsins [3]. MMPs are noncollagenous enzymes that are trapped within the dentin extracellular matrix [3,8]. When activated, MMPs exert proteolytic action on unprotected collagen, which results in degradation of the hybrid layer and decreased bond durability [9]. Collagenolytic/gelatinolytic activity of MMPs in dentin, such as MMP-2 (MMP-2, MMP-8 and MMP-9) [10] may be inhibited by applying MMPs inhibitors, such as chlorhexidine (synthetic MMP inhibitor) [3,10]. The application of chlorhexidine after acid conditioning has been reported as not interfering in the bonding action of adhesives [11–17], in addition to preventing dentin degradation by MMPs [12,13,18]. Accordingly, decreases
84
T.M.A. Monteiro et al. / International Journal of Adhesion & Adhesives 47 (2013) 83–88
in adhesive interface degradation have been reported, lasting from 6 months to 2 years following chlorhexidine application [18–21]. Furthermore, chlorhexidine has antibacterial properties [22–24]. Another matrix metalloproteinase inhibitor is green tea (Camellia sinensis) [25]. Recently, green tea was reported as a natural MMP inhibitor, capable of reducing erosive–abrasive lesions in dentin, due to its MMP inhibitory potential [26–28]. This potential has been mainly ascribed to the polyphenols composing green tea, including epigallocatechin gallate (EGCG) [27,29]. Despite the promising MMP-inhibitor property of green tea, its influence as a therapeutic approach for increasing bond durability by decreasing adhesive interface degradation has never been reported. Accordingly, the aim of the present study was to determine the influence of green tea (natural MMP-inhibitor) and chlorhexidine (synthetic MMP-inhibitor) on the microtensile bond strength of an etch-and-rinse adhesive to dentin. The null hypothesis tested was that neither the application of a green tea solution nor chlorhexidine would have any influence on the bond strength of an etch-and-rinse adhesive to dentin, regardless of the storage time.
2. Materials and methods 2.1. Ethical aspects The present study was approved by the Research Ethics Committee of the São Leopoldo Mandic School of Dentistry and Research Institute (Protocol #2012/0103). 2.2. Experimental design Thirty dentin specimens were divided into 3 groups according to the solution applied on the dentinal surface after dentin conditioning with phosphoric acid (n¼ 10): GT: green tea; CLX: 2% chlorhexidine; NT (no treatment – control group). After the application of an etch-and-rinse adhesive, a block of resin composite was built on the dentin. Each resin–dentin block was considered as an experimental unit. From each resin–dentin block,
sticks measuring approximately 1.0 mm2 were obtained and assigned into two tensile test conditions: after being stored in water for 24 h or after 6 months. μTBS values of sticks from the same restored teeth were averaged and considered as a single value, which was considered as the outcome variable. Another variable was failure pattern, described in percentage terms. Fig. 1 describes a schematic illustration of the experiment. 2.3. Dentin slab preparation Thirty human third molars, extracted for reasons not related to those of the present research, and stored in thymol (0.1%, pH 7.0) after extraction, were used in this experiment. Teeth were submitted to debriding with scalpel blades and periodontal curettes. Teeth were cross sectioned using a water-cooled diamond saw (15 HC series, Buehler Ltd., Lake Bluff, Illinois, EUA) in a sectioning machine (Isomet 1000 Precision Diamond Saw, Buehler Ltd., Lake Bluff, Illinois, EUA), which separated the occlusal third of the crown and obtained a large dentin surface in the middle third, perpendicular to the long axis of the tooth. A second parallel section was made 4 mm from the first, to obtain 4-mm high slabs. These slabs were sectioned in the mesiodistal and buccal–lingual directions to obtain square sections measuring 5 5 mm. Dentin slabs were flattened in a water-cooled polishing machine (Politriz Aropol 2V, Arotec, São Paulo, SP, Brazil) with decreasing granulations (400, 600 and 1200) of water abrasive paper (Imperial Wetordry, 3M, USA). 2.4. Adhesive procedures Dentin slabs were submitted to conditioning with 37% phosphoric acid, and were randomly divided into three groups, according to the solution applied to the dentin surface: GT – Green Tea – Camellia sinensis or 2% CLX, or NT (none, as control) (Table 1). The etch-and-rinse adhesive system was then applied to the dentin fragments, which were restored with a microhybrid resin composite. The final resin–dentin blocks were 4-mm high. Description of the main materials and their respective instructions for use are shown in Table 1.
Fig. 1. Schematic illustration. A. Removal of enamel to obtain 4-mm high dentin fragments. B. Fragments were sectioned in the mesiodistal and buccal–lingual directions and polished to obtain square slabs. C. Fragments were randomly divided into 3 groups, according to the treatment done on dentine surface. D. After application of etch-and-rinse adhesive, a composite resin restoration was built on dentin surface, resulting in resin–dentine blocks (E). F. Resin–dentine blocks were sectioned perpendicular to the bonding surface, into 1.0-mm thick slabs. By rotating samples 901 and sectioning them again lengthwise, multiple 1.0-mm2 beam-shaped sticks were obtained. G. After 24 h or 6 months of water storage sticks were submitted to microtensile bond strength testing (H).
T.M.A. Monteiro et al. / International Journal of Adhesion & Adhesives 47 (2013) 83–88
85
Table 1 Main composition and application mode of materials used. Materials
Manufacturer and batch number
Composition
Application mode
Condac 37
FGM (Dentscare Ltda – Joinville, SC, Brazil) #020211 FGM (Dentscare Ltda – Joinville, SC, Brazil) #270911 Leão Júnior (Fazenda Rio Grande, PR, Brazil) #8325
37% Phosphoric acid
Applied to dentin surface for 15 s, rinsed for the same amount of time and dried gently with absorbent paper.
Chlorhexidine gluconate 2% solution
Twenty microliters of the solution were actively applied to dentin for 60 s. Dentin was dried gently with absorbent paper.
Chlorhexidine S 2% Green Tea
Adper™ Single Adper™ Single Bond 2 Bond 2 (3M ESPE, Minnesota, USA) #N211104BR Filtek Z250 3M ESPE, Minnesota, Shade B2 USA 1109500117
Camellia sinensis
Infusion of 2 g of herb in 180 ml of boiled water for 1 min. (1.1%). Twenty microliters of the solution were actively applied to dentin for 60 sn. Dentin was dried gently with absorbent paper. The adhesive system was applied in two consecutive layers; Bis-GMA, HEMA, copolymer of acrylic and itaconic acids, water, ethyl alcohol, glycerol 1, 3-dimethacrylate, diurethane remaining solvent was evaporated with a brief, gentle, dry air jet for 10 s and light polymerized for 20 s. dimethacrylate, silane treated silica, water Aluminum oxide, silica, zirconium oxide, Bis-GMA, Each 1-mm increment was light polymerized for 20 s. Bis-EMA, UDMA
n
According to a protocol adopted by Kato et al. [29].
at 30 magnification to assess the failure modes, which were classified as adhesive (lack of adhesion), cohesive in dentin (failure of the dental substrate), cohesive in composite resin (failure of the resin composite) or mixed (adhesive and cohesive failures).
Table 2 Mean (standard deviation) of experimental groups. Treatment
Bond strength 24 h
6 months
22.6 Aba 16.2 Ba 26.0 Aa
14.6 Aa 16.6 Aa 16.3 Ab
2.6. Statistical analysis Chlorhexidine Green tea No treatment (control)
Means followed by the same letters (capitals within each column and lower case within each row) are not significantly different (α ¼ 0.05).
Light polymerization was performed on the adhesive system (Adper™ Single Bond 2) and microhybrid composite (Filtek Z250) for the time recommended by manufacturers (Table 1), using a visible light-curing unit (Ultralux EL, Dabi Atlante, Ribeirão Preto, SP, Brazil). The output of the light-curing unit was periodically measured with a radiometer (Newdent Equipamentos Ltda., Ribeirão Preto, SP, Brazil), and was found to have a mean range of 620 mW/cm². Resin–dentin blocks were kept in relative humidity at 37 1C for 24 h, and sectioned perpendicular to the bonding surface, into 1.0mm thick slabs, using a water-cooled diamond saw. By rotating samples 901 and sectioning them again lengthwise, multiple beam-shaped sticks were obtained, each with a cross-sectional surface area of 1.0 mm2. Half of sticks from a same resin–dentin block were submitted to mTBS testing after 24 h. The other half was kept in distilled water that was changed every 2 days, and kept in a bacteriological oven for 6 months. 2.5. mTBS testing Specimens were attached to a specific testing device for mTBS testing, with a cyanoacrylate adhesive (Super Bonder Gel, Henkel Ltda., Brazil). They were subjected to tensile stress in a universal testing machine, at a crosshead speed of 0.5 mm/min and a 50 N load cell until fracture. The bond strength values were expressed in kgf/cm2, and converted to MPa after measuring the crosssectional area at the fracture site with a digital caliper (Mitutoyo, Tokyo, Japan). The comparison was made using the average value of each tooth (n ¼10). After bond strength testing, the failure pattern of each stick was analyzed under a stereomicroscope (EK3ST, CQA, São Paulo, Brazil)
Based on the normal distribution of the data, repeated-measures two-way analysis of variance (ANOVA) and Tukey's test were applied. Statistical calculations were performed with SPSS 20 (SPSS Inc., Chicago, IL, USA). Significance level was set at 5%. Failure pattern was described with descriptive statistics (percentage).
3. Results Table 2 describes the means and standard deviations of experimental groups. One-way ANOVA revealed that the P-value for the interaction between “solutions applied on dentin” and “time” was borderline for statistical significance (p ¼0.065). So, the interaction evaluation was performed. Results demonstrated that, in a 24-h evaluation, microtensile bond strength values in the NT group were significantly higher compared to GT values. The application of CLX resulted in intermediate mTBS values, which were not statistically different from NT or GT. The GT group showed lower microtensile bond strength values that were statistically different from the NT group. After 6 months of WS, all groups had mTBS values statistically similar among themselves. There was no significant difference in bond strength means after 24 h and 6 months of water storage for the CLX and GT solutions. However, for the group in which any solution was applied (NT group), bond strength values significantly decreased after 6 months of water storage. Fig. 2 illustrates the failure pattern observed after the microtensile bond strength test. There was a predominance of adhesive failures in the NT (38.9%) and the CLX (41.2%) groups. However, mixed failures were predominant (33.3%) in the GT group; moreover, a greater frequency of cohesive failures in dentin was also observed (23.8%) in this group. After 6 months of water storage, CLX group showed a predominance of mixed (33.3%) and adhesive (33.3%) failures, while in NT group, a greater frequency of adhesive (35%) and cohesive failures in resin (35%) were found. For the GT group, a predominance of failure patterns could not be observed, as the percentage was the same for the adhesive, mixed or cohesive in dentin failures.
86
T.M.A. Monteiro et al. / International Journal of Adhesion & Adhesives 47 (2013) 83–88
Fig. 2. Failure pattern percentage of each experimental group.
4. Discussion The fact that adhesive-based restorations fail and undergo degradation over time has increased the concern over the longevity of these materials. Reduction in bond strength is often accompanied by hybrid layer degradation, responsible for the reduction in restoration survival [2]. The hybrid layer degradation occurs at some stages [2,30]: the polymer absorbs water, possibly causing hydrolysis and plasticizing of the resin components, and their subsequent leaching and degradation; collagen fibrils (especially those that are demineralized and not fully impregnated by resin monomers) are degraded by host-derived enzymes exhibiting collagenolytic activity, such as metalloproteinases (MMPs) [9] cysteine cathepsins [31] and exogenous proteinases from saliva. MMPs are secreted as inactive proenzymes and must be activated to act on the degradation of collagen fibrils. It is known that MMPs are denaturized when pH decreases, as is the case of cariogenic bacteria fermenting sucrose to form lactic acid, or when dentinal tissue is conditioned with phosphoric acid. When the pH recovers its value (for example, during the adhesive system application, or neutralization of pH by the buffering system), MMPs are activated [31–33]. Recently, it was reported that both MMPs and cysteine cathepsins act synergistically, in such a way that the cysteine cathepsins participate in MMPs activation [31]. Knowledge regarding the effect of MMPs and cysteine cathepsins on resin–dentin bond degradation has led to the focusing of attention on substances capable of inhibiting these enzymes. Among these substances, 2% chlorhexidine has been used with the purpose of inhibiting MMP [11–17,34] and cysteine cathepsins [35]. In fact, its application has resulted in the enhanced longevity of adhesive-based restorations [13,15,31, 34,36]. Green tea is another substance that has been described as a natural MMPs inhibitor [8,37] particularly because it contains flavanoids, such as the catechins, specially the epigallocatechin gallate (EGCG). This property has contributed to the use of green tea in preventing erosive–abrasive lesions, with promising results [28,29,38]. Also, there is evidence that green tea may reduce dental caries through different mechanisms including antimicrobial properties against cariogenic microflora [39–41] and enzymes activity inhibition [39,41].
The possibility that green tea could inhibit host-derived MMPs from dentin drove the authors of the present study to evaluate the influence of a green tea solution on the bond strength of etch-andrinse adhesives to dentin, compared with a chlorhexidine solution or no treatment (control group). In this respect, the null hypothesis was rejected, since the results of this study revealed statistical differences among the experimental groups. When 2% chlorhexidine was applied after conditioning with phosphoric acid and prior to the application of an etch-and-rinse adhesive, a decrease was observed in bond strength values, but with no statistical difference when compared with the control group (in which any solution was applied). This result is in agreement with previous studies showing that the use of chlorhexidine had no effect on immediate adhesion, and did not compromise bond strength of adhesive systems [11–16,42]. Even in a 24-h evaluation, Osorio et al. [43] reported that the chlorhexidine application was able to decelerate collagen degradation, in a methodology that proposed to quantify the C-terminal telopeptide concentration (which indicates the amount of collagen degradation). After 6 months of water storage, results of the present study indicate that chlorhexidine is capable of maintaining bond strength stability since there were no differences among bond strength values after 24 h and 6 months of water storage. Other reports also found that the benefits of chlorhexidine are not limited to the short-term, since its application frequently results greater bond longevity after long-term water storage [19,21]. This behavior can also be expected in vivo [12]. Chlorhexidine has the benefit of being an antimicrobial agent that possesses substantivity and binds to mineralized dentin for at least 12 weeks [44,45]. On the other hand, the 24-h period of evaluation showed that the green tea solution used in this study had an undesirable effect on the bond strength of the tested adhesive to dentin, compared to the group to which no solution was applied. According to Mirkarimi et al. [46] the green tea solution must have allowed the formation of a surface deposit of organic materials on the dentin and the formation of newly induced collagen crosslinks. According to them, there is a substance widely present in green tea, namely proanthocyanidin, a flavanol, that might interact with the organic portion of dentin. It is unknown if these interactions may have jeopardized, in a first moment, hybrid layer formation within dentin, but further analysis must confirm such condition. Nevertheless, the green tea values were statistically similar
T.M.A. Monteiro et al. / International Journal of Adhesion & Adhesives 47 (2013) 83–88
to those of chlorhexidine after 24 h of storage. Surprisingly, after 6 months, green tea application led to the maintenance of bond strength values being those values statistically similar to those found in no treated and chlorhexidine groups. Although there are no reports that have evaluated green tea's substantivity in dentin, i.e., the time this solution would be bound to mineralized dentin, some reasons may explain the bond stability observed with the group treated with green tea. Ephasinghe et al. [47] showed that proanthocyanidin (the above mentioned green tea component) inactivated around 90% MMP-2, -8 and -9 and around 75–90% of cysteine cathepsin, which was significantly higher than chlorhexidine. In addition green tea's composition includes (besides flavonoids/catechins) proteins/enzymes, carbohydrates, lipid components, vitamins (B, C, E), xanthic bases such as caffeine and theophylline, volatile components (aldehydes and alcohols), minerals and other elements such as Ca, Mg, Cr, Mn, Fe, Cu, Zn, Mo, Se, Na, P, Co, Sr, Ni, K, F and Al [41]. It is known that Zn has an important hole against collagen degradation [48] by binding to it and leading to a new conformation that protects the cleavage sites from metalloproteinases [48]. These properties therefore could explain why green tea application led to more stable bond strength values. Also, in the present study, the green tea was applied in a concentration of approximately 1.1%. Magalhães et al. [28] tested an experimental green tea extract that was diluted to a final concentration of 0.61%. At this concentration, the green tea was also able to reduce erosive–abrasion wear. Moreover, in the present study, green tea was applied as a solution, whereas Kato et al. [49] proposed the use of green tea as a gel vehicle. Buzalaf et al. [27] revealed that green tea gel showed a more pronounced effect over erosive–abrasive lesions than green tea incorporated to dentifrices or green tea solutions. In future bond strength studies, other concentrations/ vehicles of green tea could be used to test whether better results could be achieved. According to the failure pattern, the results of the present study demonstrated that most failures in the chlorhexidine groups were adhesive, regardless of the time points of analysis. Other studies evaluating the effect of chlorhexidine on bond strength have produced similar failure patterns [14,19,42]. However, an increase in mixed failures and decrease in adhesive failures were observed in the green tea solution group. After 6 months, a predominance of failure patterns could not be observed, as the percentage was the same for the adhesive, mixed or cohesive in dentin failures. According to the results of the present study, it can be verified that chlorhexidine solution may be applied to the dentin surface after acid conditioning to boost its inhibitory potential against MMPs and take advantage of its antimicrobial properties. A further consideration is that chlorhexidine has the potential of establishing adhesion over time by decreasing bond degradation. In addition, the results indicate that the application of green tea solution has a potential to maintain bond stability, but the results and the reasons for that need to be further evaluated.
5. Conclusions It can be concluded that in a short-term evaluation, chlorhexidine showed no interference on bond strength to dentin, while green tea did. After a long-term evaluation, both metalloproteinase inhibitors, chlorhexidine and green tea, were capable of maintaining bond strength stability. References [1] Van Meerbeek B, De Munck J, Yoshida Y, Inoue S, Vargas M, Vijay P, et al. Buonocore memorial lecture. Adhesion to enamel and dentin: current status and future challenges. Operative Dentistry 2003;3:215–35.
87
[2] Amaral FL, Colucci V, Palma-Dibb RG, Corona SA. Assessment of in vitro methods used to promote adhesive interface degradation: a critical review. Journal of Esthetic and Restorative Dentistry 2007;19:340–53, http://dx.doi. org/10.1111/j.1708-8240.2007.00134.x. [3] Tjäderhane L, Nascimento FD, Breschi L, Mazzoni A, Tersariol IL, Geraldeli S, et al. Optimizing dentin bond durability: control of collagen degradation by matrix metalloproteinases and cysteine cathepsins. Dental Materials 2013;29:116–35, http://dx.doi.org/10.1016/j.dental.2012.08.004. [4] De Munck J, Mine A, Vivan Cardoso M, Van Landuyt KL, Lührs AK, Poitevin A, et al. Hydrolytic stability of three-step etch-and-rinse adhesives in occlusal class-I cavities, Clinical Oral Investigations, http://dx.doi.org/10.1007/ s00784-012-0884-0, in press. [5] Cardoso MV, de Almeida Neves A, Mine A, Coutinho E, Van Landuyt K, De Munck J, et al. Current aspects on bonding effectiveness and stability in adhesive dentistry. Australian Dental Journal 2011;56:31–44, http://dx.doi. org/10.1111/j.1834-7819.2011.01294.x. [6] Boushell LW, Swift Jr. EJ. Critical appraisal. Dentin bonding: matrix metalloproteinases and chlorhexidine. Journal of Esthetic and Restorative Dentistry 2011;23:347–52, http://dx.doi.org/10.1111/j.1708-8240.2011.00464.x. [7] Mazzoni A, Scaffa P, Carrilho M, Tjäderhane L, Di Lenarda R, Polimeni A, et al. Effects of etch-and-rinse and self-etch adhesives on dentin MMP-2 and MMP9, Journal of Dental Research. 92, 2013, 82–6, http://dx.doi.org/10.1177/ 0022034512467034. [8] Chaussain-Miller C, Fioretti F, Goldberg M, Menashi S. The role of matrix metalloproteinases (MMPs) in human caries. Journal of Dental Research 2006;85:22–32, http://dx.doi.org/10.1177/154405910608500104. [9] Pashley DH, Tay FR, Yiu C, Hashimoto M, Breschi L, Carvalho RM, et al. Collagen degradation by host-derived enzymes during aging. Journal of Dental Research 2004;83:216–21, http://dx.doi.org/10.1177/154405910408300306. [10] Moon PC, Weaver J, Brooks CN. Review of matrix metalloproteinases' effect on the hybrid dentin bond layer stability and chlorhexidine clinical use to prevent bond failure. Open Dentistry 2010;4:147–52, http://dx.doi.org/10.2174/ 1874210601004010147. [11] Erhardt MC, Osorio R, Toledano M. Dentin treatment with MMPs inhibitors does not alter bond strengths to caries-affected dentin. Journal of Dentistry 2008;36:1068–73, http://dx.doi.org/10.1016/j.jdent.2008.09.002. [12] Brackett WW, Tay FR, Brackett MG, Dib A, Sword RJ, Pashley DH. The effect of chlorhexidine on dentin hybrid layers in vivo. Operative Dentistry 2007;32:107–11, http://dx.doi.org/10.2341/06-55. [13] Carrilho MR, Carvalho RM, de Goes MF, di Hipólito V, Geraldeli S, Tay FR, et al. Chlorhexidine preserves dentin bond in vitro. Journal of Dental Research 2007;86:90–4, http://dx.doi.org/10.1177/154405910708600115. [14] Mobarak EH. Effect of chlorhexidine pretreatment on bond strength durability of caries-affected dentin over 2-year aging in artificial saliva and under simulated intrapulpal pressure. Operative Dentistry 2011;36:649–60, http: //dx.doi.org/10.2341/11-018-L. [15] Ricci HA, Sanabe ME, de Souza Costa CA, Pashley DH, Hebling J. Chlorhexidine increases the longevity of in vivo resin–dentin bonds. European Journal of Oral Sciences 2010;118:411–6, http://dx.doi.org/10.1111/j.1600-0722.2010.00754.x. [16] Soares CJ, Pereira CA, Pereira JC, Santana FR, do Prado CJ. Effect of chlorhexidine application on microtensile bond strength to dentin. Operative Dentistry 2008;33:183–8, http://dx.doi.org/10.2341/07-69. [17] Stape TH, Menezes MS, Barreto BC, Aguiar FH, Martins LR, Quagliatto PS. Influence of matrix metalloproteinase synthetic inhibitors on dentin microtensile bond strength of resin cements. Operative Dentistry 2012;37:386–96, http://dx.doi.org/10.2341/11-256-L. [18] Breschi L, Mazzoni A, Nato F, Carrilho M, Visintini E, Tjäderhane L, et al. Chlorhexidine stabilizes the adhesive interface: a 2-year in vitro study. Dental Materials 2010;26:320–5, http://dx.doi.org/10.1016/j.dental.2009.11.153. [19] Campos EA, Correr GM, Leonardi DP, Barato-Filho F, Gonzaga CC, Zielak JC. Chlorhexidine diminishes the loss of bond strength over time under simulated pulpal pressure and thermo-mechanical stressing. Journal of Dentistry 2009;37:108–14, http://dx.doi.org/10.1016/j.jdent.2008.10.003. [20] Carrilho MR, Carvalho RM, Sousa EN, Nicolau J, Breschi L, Mazzoni A, et al. Substantivity of chlorhexidine to human dentin. Dental Materials 2010;26: 7779–85, http://dx.doi.org/10.1016/j.dental.2010.04.002. [21] Stanislawczuk R, Reis A, Loguercio AD. A 2-years in vitro evaluation of a chlorhexidine-containing acid on the durability of resin–dentin interface. Journal of Dentistry 2011;39:40–7, http://dx.doi.org/10.1016/j. jdent.2010.10.001. [22] Borges FM, de Melo MA, Lima JP, Zanin IC, Rodrigues LK. Antimicrobial effect of chlorhexidine digluconate in dentin: in vitro and in situ study. Journal of Conservative Dentistry 2012;15:22–6, http://dx.doi.org/10.4103/09720707.92601. [23] Pashley DH, Tay FR, Imazato S. How to increase the durability of resin–dentin bonds. Compendium of Continuing Education in Dentistry 2011;32:60–4. [24] Thompson JM, Agee K, Sidow SJ, McNally K, Lindsey K, Borke J, et al. Inhibition of endogenous dentin matrix metalloproteinases by ethylenediaminetetraacetic acid. Journal of Endodontics 2012;38:62–5, http://dx.doi.org/10.1016/j. joen.2011.09.005. [25] Mandal M, Mandal A, Das S, Chakraborti T, Sajal C. Clinical implications of matrix metalloproteinases. Molecular and Cellular Biochemistry 2003;252: 305–29. [26] Barbosa CS, Kato MT, Buzalaf MA. Effect of supplementation of soft drinks with green tea extract on their erosive potential against dentine.
88
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
T.M.A. Monteiro et al. / International Journal of Adhesion & Adhesives 47 (2013) 83–88
Australian Dental Journal 2011;56:317–21, http://dx.doi.org/10.1111/j.18347819.2011.01338.x. Buzalaf MA, Kato MT, Hannas AR. The role of matrix metalloproteinases in dental erosion. Advances in Dental Research 2012;24:72–6, http://dx.doi.org/ 10.1177/0022034512455029. Magalhães AC, Wiegand A, Rios D, Hannas A, Attin T, Buzalaf MA. Chlorhexidine and green tea extract reduce dentin erosion and abrasion in situ. Journal of Dentistry 2009;37:994–8, http://dx.doi.org/10.1016/j.jdent.2009.08.007. Kato MT, Magalhães AC, Rios D, Hannas AR, Attin T, Buzalaf MAR. Protective effect of Green tea on dentin erosion and abrasion. Journal of Applied Oral Science 2009;17:560–4, http://dx.doi.org/10.1590/S167877572009000600004. Sanabe ME, Costa CA, Hebling J. Exposed collagen in aged resin–dentin bonds produced on sound and caries-affected dentin in the presence of chlorhexidine. Journal of Adhesive Dentistry 2011;13:117–24, http://dx.doi.org/ 10.3290/j.jad.a19239. Nascimento FD, Minciotti CL, Geraldeli S, Carrilho MR, Pashley DH, Tay FR, et al. Cysteine cathepsins in human carious dentin. Journal of Dental Research 2011;90:506–11, http://dx.doi.org/10.1177/0022034510391906. Tjäderhane L, Larjava H, Sorsa T, Uitto VJ, Larmas M, Salo T. The activation and function of host matrix metalloproteinases in dentin matrix breakdown in caries lesions. Journal of Dental Research 1998;77:1622–9, http://dx.doi.org/ 10.1177/00220345980770081001. Mazzoni A, Pashley DH, Nishitani Y, Breschi L, Mannello F, Tjäderhane L, et al. Reactivation of inactivated endogenous proteolytic activities in phosphoric acid-etched dentine by etch-and-rinse adhesives. Biomaterials 2006;27: 4470–6, http://dx.doi.org/10.1016/j.biomaterials.2006.01.040. Hebling J, Pashley DH, Tjäderhane L, Tay FR. Chlorhexidine arrests subclinical degradation of dentin hybrid layers in vivo. Journal of Dental Research 2005;84:741–6, http://dx.doi.org/10.1177/154405910508400811. Scaffa PM, Vidal CM, Barros N, Gesteira TF, Carmona AK, Breschi L, et al. Chlorhexidine inhibits the activity of dental cysteine cathepsins. Journal of Dental Research 2012;91:420–5, http://dx.doi.org/10.1177/ 0022034511435329. Komori PC, Pashley DH, Tjäderhane L, Breschi L, Mazzoni A, de Goes MF, et al. Effect of 2% chlorhexidine digluconate on the bond strength to normal versus caries-affected dentin. Operative Dentistry 2009;34:157–65, http://dx.doi.org/ 10.2341/08-55. Demeule M, Brossard M, Pagé M, Gingras D, Béliveau R. Matrix metalloproteinase inhibition by green tea catechins. Biochimica et Biophysica Acta 2000;1478:51–60, http://dx.doi.org/10.1016/S0167-4838(00)00009-1.
[38] Bassiouny MA, Kuroda S, Yang J. Topographic and radiographic profile assessment of dental erosion. Part III: effect of green and black tea on human dentition. General Dentistry 2008;56:451–61. [39] Wu CD, Wei GX. Tea as a functional food for oral health. Nutrition 2002;18: 443–4, http://dx.doi.org/10.1016/S0899-9007(02)00763-3. [40] Ferrazzano GF, Roberto L, Amato I, Cantile T, Sangianantoni G, Ingenito A. Antimicrobial properties of green tea extract against cariogenic microflora: an in vivo study. Journal of Medicinal Food 2011;14:907–11, http://dx.doi.org/ 10.1089/jmf.2010.0196. [41] Narotzki B, Reznick AZ, Aizenbud D, Levy Y. Green tea: a promising natural product in oral health. Archives of Oral Biology 2012;57:429–35, http://dx.doi. org/10.1016/j.archoralbio.2011.11.017. [42] Dalkilic EE, Aris HD, Kivanc BH, Uctasli MB, Omurlu H. Effect of different disinfectant methods on the initial microtensile bond strength of a self-etch adhesive to dentin. Lasers in Medical Science 2012;27:819–25, http://dx.doi. org/10.1007/s10103-011-0987-x. [43] Osorio R, Yamauti M, Osorio E, Ruiz-Requena ME, Pashley D, Tay F, et al. Effect of dentin etching and chlorhexidine application on metalloproteinasemediated collagen degradation. European Journal of Oral Sciences 2011;119: 79–85, http://dx.doi.org/10.1111/j.1600-0722.2010.00789.x. [44] Liu Y, Tjäderhane L, Breschi L, Mazzoni A, Li N, Mao J, et al. Limitations in bonding to dentin and experimental strategies to prevent bond degradation. Journal of Dental Research 2011;90:953–68, http://dx.doi.org/10.1177/ 0022034510391799. [45] Mohammadi Z, Abbott PV. Antimicrobial substantivity of root canal irrigants and medicaments: a review. Australian Endodontic Journal 2009 Dec;35 (3):131–9, http://dx.doi.org/10.1111/j.1747-4477.2009.00164.x. [46] Mirkarimi M, Toomarian L. Effect of green tea extract on the treatment of dentin erosion: an in vitro study. Journal of Dentistry 2012;9:224–8. [47] Epasinghe DJ, Yiu CK, Burrow MF, Hiraishi N, Tay FR. The inhibitory effect of proanthocyanidin on soluble and collagen-bound proteases, Journal of Dentistry; 2013 [in press]. 41, 2013, 832–839. http://dx.doi.org/10.1016/j.jdent. 2013.06.002. [48] Osorio R, Yamauti M, Osorio E, Román JS, Toledano M. Zinc-doped dentin adhesive for collagen protection at the hybrid layer. European Journal of Oral Sciences 2011;119(5):401–10, http://dx.doi.org/10.1111/j.16000722.2011.00853.x. [49] Kato MT, Leite AL, Hannas AR, Buzalaf MA. Gels containing MMP inhibitors prevent dental erosion in situ. Journal of Dental Research 2010;89:468–72, http://dx.doi.org/10.1177/0022034510363248.