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Influence of chemical and natural cross-linkers on dentin bond strength of self-etching adhesives
International Journal of Adhesion & Adhesives 60 (2015) 117–122
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International Journal of Adhesion & Adhesives journal homepage: www.elsevier.com/locate/ijadhadh
Influence of chemical and natural cross-linkers on dentin bond strength of self-etching adhesives Cristiane Franco Pinto a, Sandrine Bittencourt Berger b, Vanessa Cavalli c, Ana Karina Bedran-Russo d, Marcelo Giannini e,n a
Department of Restorative Dentistry, São Francisco University, Avenida São Francisco de Assis, 218, 12916-350 Bragança Paulista, SP, Brazil Department of Restorative Dentistry, University of Northen Parana, Rua Marselha, 183, 86041-140 Londrina, PR, Brazil c Department of Restorative Dentistry, São Leopoldo Mandic Dental School and Research Center, Rua José Rocha Junqueira, 13, Bairro Swift, 13045-755 Campinas, SP, Brazil d Department of Restorative Dentistry, Chicago College of Dentistry, University of Illinois, 801 South Paulina Street, 60612 Chicago, IL, USA e Department of Restorative Dentistry, Piracicaba School of Dentistry, State University of Campinas, Avenida Limeira, 901, 13414-018 Piracicaba, SP, Brazil b
ar t ic l e i nf o
a b s t r a c t
Article history: Accepted 10 April 2015 Available online 23 April 2015
Objectives. The objective of this study was to evaluate the effect of chemical collagen cross-linkers on the bond strength of three self-etching adhesives to dentin. Materials and methods. Exposed dentin from the buccal surface of 45 incisors bovine was used to analyze the effect of chemical cross-linkers. The control groups were tested with self-etching adhesives (Clearfil SE Bond, Clearfil SE Protect and One-up Bond F Plus) according to the manufacturer instructions. Two cross-linkers agents were tested: 5% glutaraldehyde and 6.5% proanthocyanidin-rich grape seed extract (both for 10 min). After surface treatments with the agents, the surfaces were washed with distilled water, followed by the bonding/build-up procedures. Restored teeth were prepared for microtensile bond strength test and specimens tested in a universal testing machine (0.5 mm/min) after 24 h storage. Fracture sites of the bonded interface qualitatively evaluated. Results. According to two-way ANOVA and Tukey test (p o0.05) glutaraldehyde pretreatment did not affect the microtensile bond strength of any of two self-etching systems (p4 0.05). However, when the grape seed extract was used with Clearfil SE Bond, the dentin bond strength values increased (po 0.05), but decrease for the One-up Bond F Plus treated-group (po 0.05). For the Clearfil SE Protect there was no difference between treatments (p 40.05). Conclusions. The effect of the application of grape seed extract cross-linker was product-dependent and glutaraldehyde did not affect the bond strength to dentin. & 2015 Elsevier Ltd. All rights reserved.
Keywords: Self-etch adhesives Dentine Micro-tensile Cross-linkers
1. Introduction Reliable long-term dentin bonding remains a clinical challenge as the adhesive interface can be porous and behave as a permeable membrane over time [1]. As a consequence, water diffusion through the hybrid layer promotes elution of unreacted monomers [2], polymer swelling, water sorption, resin hydrolysis [3] and degradations of type I collagen fibrils as a result of enzymatic activity [4,5].
n Correspondence to: Department of Restorative Dentistry-Operative Dentistry Division, Piracicaba Dental School-State University of Campinas, Avenida Limeira, 901-Piracicaba, PO Box 52, 13414-903, SP, Brazil. Tel.: þ 55 19 2106 5340; fax: þ55 19 2106 5218. E-mail addresses: [email protected] (C.F. Pinto), [email protected] (S.B. Berger), [email protected] (V. Cavalli), [email protected] (A.K. Bedran-Russo), [email protected] (M. Giannini).
http://dx.doi.org/10.1016/j.ijadhadh.2015.04.008 0143-7496/& 2015 Elsevier Ltd. All rights reserved.
Hence, the two major methods under investigations to improve dentin adhesion rely on the development of new adhesive systems and improvements of the intrinsic properties of dentin through a tissue engineering approach [5,6]. One of the aspects investigated by tissue engineering is the enhancement of inter- and intramolecular collagen cross-links [7,8]. Extrinsic collagen cross-linking agents may induce additional inter- and intra-molecular crosslinks, enhancing collagen mechanical properties and its resistance to enzymatic degradation, which is an advantageous to dentin bonding [6,7,9]. Type I collagen, one of the main component of the hybrid layer, is a triple-helix molecular structure containing three polypeptide chains. These chains are intertwined to one another and folded into a ropelike right-handed structure [10]. The bonds between the side chains of amino acids of the collagen molecules constitute the cross-links and these bonds improve the tensile properties of collagen fibrils [6,11,12]. Glutaraldehyde and proanthocyanidin are two chemical crosslinking agents frequently used to test the mechanical properties of
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the hybrid layer [9,13]. The first is a synthetic cross-linker widely used as a fixative agent. It has been reported to improve mechanical properties of various collagen-based tissues [14,15]. The second is a natural cross-linker present in fruits, vegetables, nuts, seeds and flowers. The grape seed extract, for instance, is a proanthocyanidin-rich solution that has been shown to improve the mechanical properties of demineralized dentin [9,13] and dentin–resin interface [16]. Recent investigations have shown the ability of glutaraldehyde solution and grape seed extract to enhance dentin collagen stability and bond strength to dentin treated with conventional etchand-rinse adhesives both immediately [6, 17,18] and in long-term analyzes of the quality of the bonded interface [5,11,16]. Other reports have demonstrated a significant improvement of bond strengths of deep dentin treated with proanthocyanidin (6.5% grape seed extract) prior to the application of conventional etchand-rinse [18] and self-etching adhesives [19]. Although the application of cross-linkers to dentin seems valuable to bonding, it was also reported that 6.5% grape seed extract incorporated to the primer of a self-etching adhesive decreased immediate bond strength when compared to the dentin treated with other agents (0.5% chlorhexidine and 0.5% hesperidin) [20]. Although controversy exists among the studies concerning type, time of application and concentration of the cross-linker as well as the adhesive systems used, most investigations showed promising results of the application of these agents. Therefore, the objective of this study was to evaluate bond strength to dentin treated with grape seed extract and glutaraldehyde and bonded with self-etching adhesives. The null hypothesis tested was that chemical cross-linker would not increase dentin bond strength.
2.4. Grape seed extract treatment The dentin surface was treated with 6.5% w/v grape seed extract (94% proanthocyanidins, MegaNatural; Polyphenolics, Madera, CA, USA, pH ¼7.4) [9,17] for 10 min. After the treatment, the surface was washed with distilled water, followed by the bonding/build-up procedure described previously. 2.5. Microtensile bond strength test The restored teeth were stored for 24 h in distilled water at 37 °C. Afterwards, the samples were serially sectioned with diamond saw (Isomet 1000, Bueheler, Lake Bluff, IL, USA) parallel to the long axis into 1 mm-thick slabs under water-cooling. The slabs were further sectioned perpendicularly to produce 6 sticks per tooth with a cross-sectioned area of 1.0 mm2. The sticks were attached to a microtensile-testing device with cyanoacrylate adhesive (Super Bonder, Henkel/Loctite; Diadema, SP, Brazil) and tested in tension in a universal testing machine (EZ-test, Shimadzu, Kyoto, Japan) at a crosshead speed of 1 mm/min until failure. After testing, the specimens were carefully removed and the cross-sectional area at the site of fracture was measured with a digital caliper (727-6/150, Starret; Itu, SP, Brazil) to the nearest 0.01 mm. The cross-sectional area of each specimen was divided by the maximum tensile load at failure to calculate the stress at fracture (MPa). Exploratory analysis of data regarding the homoscedasticity and normality was performed and the assumptions to the parametric test were fulfilled. Therefore, data was analyzed by twoway ANOVA and Tukey test with the significance limit set at 5% by SAS software. 2.6. Fracture mode analysis and adhesive interface observation
2. Materials and methods 2.1. Specimen preparation for microtensile testing Forty-five freshly extracted bovine incisors were used as experimental units. The teeth were cleaned, and the buccal surfaces were ground flat with silicon carbide paper (#180 and #320) to remove enamel and expose mid-depth dentin. Dentin surfaces were then ground with 600-grit abrasive paper to create a standard smear layer. The samples were randomly assigned to nine groups (n ¼5), according to the factors: adhesive system (Clearfil SE Bond, Kuraray Noritake Dental, Kurashiki, Japan; Clearfil SE Protect, Kuraray Noritake Dental; One-up Bond F Plus, Tokuyama Dental, Tokyo, Japan) and dentin cross-linking treatment (5% glutaraldehyde or 6.5% grape seed extract).
The specimens were polished with a 1000-grit SiC paper and 6, 3, 1, and 0.25 μm diamond paste (Buehler Ltd, Lake Bluff, IL, USA) using a polish cloth and ultrasonically cleaned. The fractured sites of the tested specimens were dried overnight (at 37 °C) and then sputter coated with gold (MED 010, Balzers; Balzer, Liechtenstein). The fracture mode and the resin–dentin interfaces were observed using a scanning electron microscope (VP 435 Leo; Cambridge, UK) at 100–2000 magnification. The quality of adhesive interface and hybrid layer was analyzed and the failure patterns were classified as type 1, adhesive failure between adhesive system and dentin and partially cohesive in the adhesive system; type 2, cohesive failure within the adhesive layer; type 3, cohesive failure within the dentin; or type 4, mixed failure, when simultaneously exhibiting dentin, hybrid and/or adhesive layer and remnants of composite.
2.2. Control group Bonding protocol followed the manufacturers' instruction for the three self-etching systems used as a control group. Specimens were light-cured for 20 s and a composite build-up (Filtek Supreme Plus, 3M ESPE, St. Paul, MN, USA) was constructed with two 2-mm increments, each light-cured for 40 s. 2.3. Glutaraldehyde The dentin surface was treated with 5% v/v glutaraldehyde (Fisher Chemical, Pittsburg, PA, USA, pH 7.4) [9,17] for 10 min. After the treatment, the dentin surface was washed with distilled water, followed by the bonding/build-up procedure described previously.
3. Results Two-way ANOVA demonstrated a significant interaction between factors: adhesive system and crosslinking treatment (p o0.0001) (Table 1). The application of 6.5% grape seed extract before Clearfil SE Bond self-etching adhesive system increases the bond strength, when compared to its control. Dentin bond strength for One-up Bond F Plus adhesive in combination with glutaraldehyde treatment promoted similar bond strength to its control group, but it was significantly higher than that obtained by grape seed treatment. No difference in bond strength were observed for both 5% glutaraldehyde and 6.5% grape seed extract treatments compared to the control group for Clearfil SE Protect, in which cross-linker agents had no effect.
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The summary of failures modes for all groups is depicted in Fig. 1. Photomicrographs of the fracture modes of the dentin bonding with the Clearfil SE Bond adhesive without previous cross-linking treatment (control, Fig. 2A) showed mostly adhesive Table 1 Mean and standard deviations of bond strength measurements for the adhesive systems tested as a function of the chemical cross-linking agent treatments. Adhesive
Chemical cross-linking agent Control
5% Glutaraldehyde 6.5% Grape seed extract
Clearfil SE Bond 23.3 (11.8) a B 36.4 (11.2) a AB Clearfil SE Protect 32.4 (5.1) a A 35.8 (5.1) a A One-up Bond F Plus 30.0 (3.7) a AB 33.5 (9.9) a A
37.9 (4.9) a A 39.6 (5.9) a A 18.5 (0.6) b B
Groups identified by distinct letters (capital letters compare crosslinking treatments – lines and lower case letters compare adhesive systems – columns) are different according to two-way ANOVA and Tukey test (p r 0.05).
Fig. 1. Bar graphs of failure mode percentages according to the adhesive systems and cross-linkers tested (GD: 5% glutaraldehyde, GSE: 6.5% grape seed extract, OP: One-up Bond F Plus, PB: Clearfil SE Protect, SE: Clearfil SE Bond). Failures modes: (1) adhesive; (2) cohesive within the adhesive layer; (3) cohesive within the dentin and (4) mixed.
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failures (type 1 – 53%) and some mixed failures (type 4 – 40%, Fig. 2A). Glutaraldehyde and grape seed extract treatments previously to Clearfil SE Bond bonding agent promoted mostly mixed failures exhibiting dentin, adhesive layer and/or occasionally remnants of composite (type 4 – 59–61%, respectively) and few type 1 failures (36% and 33%, respectively) (Fig. 2B and C). The Clearfil SE Protect adhesive demonstrated mostly mixed failures exhibiting adhesive, dentin and sometimes remnants of the composite on the dentin surface (type 4: Clearfil SE Protect – 69.5%; with glutaraldehyde – 50% and with grape seed extract – 62.5%, Fig. 3A–C, respectively). The One-up Bond F Plus adhesive promoted mostly adhesive failures (type 1) regardless of dentin treatment (Fig. 4A–C); however, it was the only adhesive that presented cohesive failures within the adhesive layer (type 2), mostly in the control group (33%). Representative images of the dentin interface bonded with Clearfil SE Bond, Clearfil SE Protect and One-up Bond F Plus adhesives are displayed in Figs. 5–7, respectively. Clearfil SE Bond (Fig. 5A) was able to create a uniform hybrid layer, regardless cross-linking treatments (Fig. 5A–C); however, the layer was not as thick as the ones observed by Clearfil SE Protect (Fig. 6A–C). A homogeneous hybrid layer pattern can be observed in SEM images of Clearfil Protect Bond interface without cross-linking treatment (Fig. 6A) similar to 5% glutaraldehyde (Fig. 6B) and 6.5% grape seed extract treatment (Fig. 6C). The interfaces of One-Up Bond F Plus (Fig. 7) were not as uniform as the previous adhesives (Figs. 5 and 6). The untreated group (Fig. 7A) and grape seed extract treatment (Fig. 7C) presented the hybrid layer thicker than the glutaraldehyde treatment (Fig. 7B) but the thickness found was not related to the bond strengths results obtained.
Fig. 2. Representative photomicrographs of the predominant failure modes of dentin bonded with the Clearfil SE Bond (SE) adhesive. (A) control group; failure mode type 1. (B) 5% glutaraldehyde treatment prior to SE bonding; failure mode type 4. (C) 6.5% grape seed extract treatment prior to SE bonding; type 4 (A – adhesive system; D – dentin; RC – resin composite).
Fig. 3. Representative photomicrographs of the predominant failure modes of the dentin bonded with the Clearfil Protect Bond (PB) adhesive. (A) control group; failure mode type 4. (B) 5% glutaraldehyde treatment prior to PB bonding; failure mode type 4. (C) 6.5% grape seed extract treatment prior to PB bonding; type 4 (A – adhesive system; D – dentin; RC – resin composite).
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Fig. 4. Representative photomicrographs of the predominant failure modes of the dentin bonding with the One-up Bond F Plus (OP) adhesive. (A) control group; failure mode type 1. (B) 5% glutaraldehyde treatment prior to OP bonding; failure mode type 1. (C) 6.5% grape seed extract treatment prior to OP bonding; type 1 (A – adhesive system; D – dentin; RC – resin composite).
Fig. 5. Representative scanning electron microscopy images of the adhesive interface. (A) Clearfil SE Bond. (B) Clearfil SE Bond after 5% glutaraldehyde; (C) Clearfil SE Bond after 6.5% grape seed extract (A – adhesive system; D – dentin; RC – resin composite).
Fig. 6. Representative scanning electron microscopy images of the adhesive interface. (A) Clearfil Protect Bond. (B) Clearfil Protect Bond after 5% glutaraldehyde; (C) Clearfil Protect Bond after 6.5% grape seed extract (A – adhesive system; D – dentin; RC – resin composite).
Fig. 7. Representative scanning electron microscopy images of the adhesive interface. (A) One-Up Bond F Plus. (B) One-Up Bond F Plus after 5% glutaraldehyde; (C) One-Up Bond F Plus after 6.5% grape seed extract (A – adhesive system; D – dentin; RC – resin composite).
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4. Discussion It has been assumed that if collagen fibrils are chemically engineered by the creation of additional internal and external cross-linking, the consequence would be a stronger and more durable hybrid layer that is less prone to degrade [5]. Grape seed extract, a proanthocyanidin (PA)-rich mixture of polyphenols, is able to increase collagen cross-linking by covalent [21], ionic [22], hydrogen bonding [23] and hydrophobic interactions [8]. A number of investigations have reported that these interactions increase the mechanical properties of resin– dentin bonded interface [5,16,24] and, as a result, increase dentin bond strengths [11,17–19]. This study demonstrated that the 6.5% grape seed extract enhanced dentin bond strengths, but this improvement was adhesive-dependent. This is in agreement with previous studies that reported that the stability of cross-linking treatment depends mostly on the source/type of PA-rich extract and the adhesive system employed [11]. The 6.5% grape seed extract cross-linker increased bond strengths of Clearfil SE Bond adhesive, but decreased One-up Bond F Plus’s bonding and did not enhance bond strengths of the Clearfil SE Protect adhesive. The reasons for this bonding behavior could be attributed to the composition of the adhesives. The three adhesives tested are self-etching systems with similar adhesion strategies. However, Clearfil SE Bond and Clearfil Protect Bond are two-bottle adhesive self-etching primers, both containing 10-MDP as functional monomers, with Clearfil Protect Bond also containing MDPB (12-methacryloyloxydodecylpyridinium bromide) as an additional anti-bacterial monomer [25,26]. 10-MDP is reported to form a strong chemical bond to the hydroxyapatite, contributing to the adhesion of the enamel and dentin [27]. As a two-bottle primer/adhesive system, the application of the solvent-free hydrophobic layer over the primed dentin surface reduces the hydrophilicity of the hybrid layer, increasing the polymerization rate of the adhesive systems as well as increasing the dentin bond strength [28]. One-Up Bond F Plus, a single-step self-etching adhesive, is a complex mixture of acidic primers and bonding resins with six hydrophobic and hydrophilic monomers. The functional monomers are MAC-10 (methacryloyloxydecamethlene malonic acid), HEMA (2-hydroxyethyl methacrylate), and methacryloyloxyalkyl acid phosphate. MAC-10 is the adhesion-promoting monomer and, like 10-MDP, is hydrolytically stable and its molecular structure makes this monomer hydrophobic. The chemical structures of monomers determine the viscosity, solubility, hydrolysis susceptibility, wetting, and penetration behavior [29]. Less promising results were obtained with the 6.5% grape seed extract/One-up Bond F Plus-adhesive group, in which 6.5% grape seed extract significantly decreased dentin bond strength. Singlestep self-etching adhesives are frequently associated with low bond strengths and more adhesive failures than the two-step selfetching primers [30]. These single-step adhesives have been correlated to water tree, water blister formation and adhesive phase separation at the interface [29,31–33]. In a recent nanoleakage study, it was demonstrated that these agents allow higher water diffusion in the polymerized adhesive compared to conventional etch-and-rinse systems. The high degree of nanoleakage in the adhesive layer could be attributed to the hydrophilic nature of the simplified self-etching adhesives, especially in HEMA-containing systems [34] and to the increased concentrations of water and solvents (acetone, ethanol) [35,36]. It has also been observed that the hydrophilic adhesive may entrap the water from dentin during and after bonding, which compromises bond strength [37]. Another possibility is that 6.5% grape seed extract may have promoted a poor interaction between the adhesive system and the dentin due to reduced dentin wettability, leading to less adhesive
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penetration, as observed in Fig. 7C. In the microscopy analysis, the adhesive layer is considerably thicker and the adhesive might have not penetrated dentin, as it would be expected, promoting lower bond strength values. Adhesive failure (type I) was the predominant failure of the OP adhesive regardless 5% glutaraldehyde or 6.5% grape seed extract treatments, which confirmed the compromised performance of this adhesive, due to possible water deposits within the hybrid layer. It is possible that 6.5% grape seed extract was able to stabilize the collagen structure in dentin by increasing the amount of external cross-linking interactions (which could be observed for the 6.5% grape seed extract/Clearfil SE Bond-treated dentin). However, it is also likely that the enhanced collagen cross-linking reduced the width of interfibrillar spaces that serve as diffusion channels for monomer infiltration. The MDPB monomer and fluoride ions present in the Clearfil SE Protect adhesive may interfere with viscosity and permeation of the adhesive in the collagen-treated complex. As a result, the grape seed treated-collagen layer exhibited similar bond strengths, regardless the crosslinking treatment. The analysis of the fractured bonded area exhibited mostly adhesive failures for Clearfil SE Bond-treated dentin. Interestingly, this condition was reversed with the 5% glutaraldehyde and 6.5% grape seed extract treatments, which promoted mostly mixed failures (fractures at dentin, adhesive layer with or without remnants of composite). This might indicate that cross-linkers treatments could have increased strength at the adhesive interface and for this reason, after 6.5% grape seed extract and 5% glutaraldehyde treatments, the weakest area was not the adhesive anymore. Although 5% glutaraldehyde increased noticeably bond strengths values of Clearfil SE Bond dentin bonding, it was not statistically significant from 6.5% grape seed extract. Failure modes of Clearfil SE Protect bonding were always type 1 (adhesive) and this uniformity is also markedly related to the bond strength values. Glutaraldehyde has a molecular affinity for free amino groups of amino acids and is often used as a protein fixation agent [38,39]. 5% glutaraldehyde treated acid-etching dentin has been associated with a decrease in the rate of collagen degradation [8], enhanced mechanical properties of demineralized dentin [17] and improved resin–dentin bonded interfaces [6,39]. However, unlike these reports, in the present study 5% glutaraldehyde was unable to significantly increase dentin bond strengths of the adhesives tested compared to untreated and 6.5% grape seed extract-treated dentin. These differences could be related to the adhesive system used, as some of the studies used etch-and-rinse [6,17], instead of self-etching adhesives. The bonding strategy along with the composition of the adhesives could imply better performance in etch-and-rinse adhesives before the 5% glutaraldehyde application. Besides, 5% glutaraldehyde induces cross-linking interactions differently from that of 6.5% grape seed extract, involving Lys and Hyl amino acids [15] and this could contribute to a different adhesive behavior. Likewise, it is possible that the results of the current study were not as favorable as those previously obtained, as the incubation time of 5% glutaraldehyde and 6.5% grape seed extract cross-linkers on dentin was performed for 10 min instead of 60 min [6,16,17]. It has been observed that increased exposure time of 5% glutaraldehyde and 6.5% grape seed extract enhances the elastic modulus of demineralized dentin; therefore, the effectiveness of the treatments might be time-dependent [5,13,24]. Although a ten-minute cross-linking treatment is still not suitable for clinical application, other possibilities that are under investigation should be considered, as reduced pre-treatment time with higher concentrations or even using several cross-linkers simultaneously [6]. Clearly, the adhesive system used, its components and adhesive techniques should be considered in the use of this strategy. Furthermore, the cross-linker of choice seems to be
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6.5% grape seed extract, since 5% glutaraldehyde was not able to increase bond strengths for any of the adhesives tested, and because of the cytotoxicity of this agent [40,41].
5. Conclusions Grape seed extract (6.5%) cross-linking treatment was able to increase bond strength for one of the adhesives tested (Clearfil SE Bond), but decreased bond strength for another one-step selfetching adhesive tested (One-up Bond F Plus), whereas glutaraldehyde (5%) did not change bond strength for any of the adhesives tested. Thus, the benefits of cross-linking treatments previous to bonding were adhesive system-dependent.
Acknowledgments This investigation was supported by Research Grant 06/538282 from the State of São Paulo Research Foundation, São Paulo, Brazil.
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[16] dos Santos PH, Karol S, Bedran-Russo AK. Long-term nano-mechanical properties of biomodified dentin–resin interface components. J Biomech 2011;44:1691–4. [17] Macedo GV, Yamauchi M, Bedran-Russo AK. Effects of chemical cross-linkers on caries-affected dentin bonding. J Dent Res 2009;88:1096–100. [18] Srinivasulu S, Vidhya S, Sujatha M, Mahalaxmi S. Shear bond strength of composite to deep dentin after treatment with two different collagen crosslinking agents at varying time intervals. Oper Dent 2012;37:485–9. [19] Srinivasulu S, Vidhya S, Sujatha M, Mahalaxmi S. Effect of collagen crosslinkers on the shear bond strength of a self-etch adhesive system to deep dentin. J Conserv Dent 2013;16:135–8. [20] Islam S, Hiraishi N, Nassar M, Yiu C, Otsuki M, Tagami J. Effect of natural crosslinkers incorporation in a self-etching primer on dentine bond strength. J Dent 2012;40:1052–9. [21] Pierpoint WS. o-Quinones formed in plant extracts. Their reactions with amino acids and peptides. Biochem J 1969;112:609–16. [22] Loomis WD. Overcoming problems of phenolics and quinones in the isolation of plant enzymes and organelles. Methods Enzymol. 1974;31:528–44. [23] Ku CS, Sathishkumar M, Mun SP. Binding affinity of proanthocyanidin from waste Pinus radiata bark onto proline-rich bovine achilles tendon collagen type I. Chemosphere 2007;67:1618–27. [24] Castellan CS, Pereira PN, Grande RH, Bedran-Russo AK. Mechanical characterization of proanthocyanidin–dentin matrix interaction. Dent Mater 2010;26:968–73. [25] Shinohara MS, De Goes MF, Schneider LF, Ferracane JL, Pereira PN, Di Hipólito V, et al. Fluoride-containing adhesive: durability on dentin bonding. Dent Mater 2009;25:1383–91. [26] Van Landuyt KL, Snauwaert J, De Munck J, Coutinho E, Poitevin A, Yoshida Y, et al. Origin of interfacial droplets with one-step adhesives. J Dent Res 2007;86:739–44. [27] Yoshida Y, Nagakane K, Fukuda R, Nakayama Y, Okazaki M, Shintani H, et al. Comparative study on adhesive performance of functional monomers. J Dent Res 2004;83:454–8. [28] De Munck J, Van Landuyt K, Peumans M, Poitevin A, Lambrechts P, Braem M, et al. A critical review of the durability of adhesion to tooth tissue: methods and results. J Dent Res 2005;84:118–32. [29] Van Landuyt KL, Snauwaert J, De Munck J, Peumans M, Yoshida Y, Poitevin A, et al. Systematic review of the chemical composition of contemporary dental adhesives. Biomaterials 2007;28:3757–85. [30] Burrow MF, Kitasako Y, Thomas CD, Tagami J. Comparison of enamel and dentin microshear bond strengths of a two-step self-etching priming system with five all-in-one systems. Oper Dent 2008;33:456–60. [31] Tay FR, Pashley DH, Suh BI, Carvalho RM, Itthagarun R. Single-step adhesives are permeable membranes. J Dent 2002;30:371–82. [32] Tay FR, Pashley DH. Have dentin adhesives become too hydrophilic? J Can Dent Assoc 2003;69:726–31. [33] Tay FR, Pashley DH, Suh BI, Hiraishi N, Yiu CK. Water treeing in simplified dentin adhesives – déjà vu? Oper Dent 2005;30:561–79. [34] Hosaka K, Nakajima M, Takahashi M, Itoh S, Ikeda M, Tagami J, et al. Relationship between mechanical properties of one-step self-etch adhesives and water sorption. Dent Mater 2010;26:360–7. [35] Tay FR, King NM, Chan KM, Pashley DH. How can nanoleakage occur in selfetching adhesive systems that demineralize and infiltrate simultaneously? J Adhes Dent 2002;4:255–69. [36] Yiu CK, King NM, Pashley DH, Suh BI, Carvalho RM, Carrilho MR, et al. Effect of resin hydrophilicity and water storage on resin strength. Biomaterials 2004;25:5789–96. [37] Hashimoto M1, Fujita S, Endo K, Ohno H. Effect of dentinal water on bonding of self-etching adhesives. Dent Mater J 2009;28:634–41. [38] Pashley DH, Agee KA, Nakajima M, Tay FR, Carvalho RM, Terada RS, et al. Solvent-induced dimensional changes in EDTA-demineralized dentin matrix. J Biomed Mater Res Part A 2001;56:273–81. [39] Cilli R, Prakki A, de Araújo PA, Pereira JC. Influence of glutaraldehyde priming on bond strength of an experimental adhesive system applied to wet and dry dentine. J Dent 2009;37:212–8. [40] Van Luyn MJA, van Wachem PB, Dijkstra PJ, olde Damink LHH, Feiijen J. Calcification of subcutaneously implanted collagen in relation to cytotoxicity, cellular interactions and crosslinking. J Mater Sci Mater Med 1995;6:288–96. [41] Sung HW, Chang Y, Chiu CT, Chen CN, Liang HC. Crosslinking characteristics and mechanical properties of a bovine pericardium fixed with a naturally occurring crosslinking agent. J Biomed Mater Res Part A 1999;47:116–26.
Contents lists available at ScienceDirect
International Journal of Adhesion & Adhesives journal homepage: www.elsevier.com/locate/ijadhadh
Influence of chemical and natural cross-linkers on dentin bond strength of self-etching adhesives Cristiane Franco Pinto a, Sandrine Bittencourt Berger b, Vanessa Cavalli c, Ana Karina Bedran-Russo d, Marcelo Giannini e,n a
Department of Restorative Dentistry, São Francisco University, Avenida São Francisco de Assis, 218, 12916-350 Bragança Paulista, SP, Brazil Department of Restorative Dentistry, University of Northen Parana, Rua Marselha, 183, 86041-140 Londrina, PR, Brazil c Department of Restorative Dentistry, São Leopoldo Mandic Dental School and Research Center, Rua José Rocha Junqueira, 13, Bairro Swift, 13045-755 Campinas, SP, Brazil d Department of Restorative Dentistry, Chicago College of Dentistry, University of Illinois, 801 South Paulina Street, 60612 Chicago, IL, USA e Department of Restorative Dentistry, Piracicaba School of Dentistry, State University of Campinas, Avenida Limeira, 901, 13414-018 Piracicaba, SP, Brazil b
ar t ic l e i nf o
a b s t r a c t
Article history: Accepted 10 April 2015 Available online 23 April 2015
Objectives. The objective of this study was to evaluate the effect of chemical collagen cross-linkers on the bond strength of three self-etching adhesives to dentin. Materials and methods. Exposed dentin from the buccal surface of 45 incisors bovine was used to analyze the effect of chemical cross-linkers. The control groups were tested with self-etching adhesives (Clearfil SE Bond, Clearfil SE Protect and One-up Bond F Plus) according to the manufacturer instructions. Two cross-linkers agents were tested: 5% glutaraldehyde and 6.5% proanthocyanidin-rich grape seed extract (both for 10 min). After surface treatments with the agents, the surfaces were washed with distilled water, followed by the bonding/build-up procedures. Restored teeth were prepared for microtensile bond strength test and specimens tested in a universal testing machine (0.5 mm/min) after 24 h storage. Fracture sites of the bonded interface qualitatively evaluated. Results. According to two-way ANOVA and Tukey test (p o0.05) glutaraldehyde pretreatment did not affect the microtensile bond strength of any of two self-etching systems (p4 0.05). However, when the grape seed extract was used with Clearfil SE Bond, the dentin bond strength values increased (po 0.05), but decrease for the One-up Bond F Plus treated-group (po 0.05). For the Clearfil SE Protect there was no difference between treatments (p 40.05). Conclusions. The effect of the application of grape seed extract cross-linker was product-dependent and glutaraldehyde did not affect the bond strength to dentin. & 2015 Elsevier Ltd. All rights reserved.
Keywords: Self-etch adhesives Dentine Micro-tensile Cross-linkers
1. Introduction Reliable long-term dentin bonding remains a clinical challenge as the adhesive interface can be porous and behave as a permeable membrane over time [1]. As a consequence, water diffusion through the hybrid layer promotes elution of unreacted monomers [2], polymer swelling, water sorption, resin hydrolysis [3] and degradations of type I collagen fibrils as a result of enzymatic activity [4,5].
n Correspondence to: Department of Restorative Dentistry-Operative Dentistry Division, Piracicaba Dental School-State University of Campinas, Avenida Limeira, 901-Piracicaba, PO Box 52, 13414-903, SP, Brazil. Tel.: þ 55 19 2106 5340; fax: þ55 19 2106 5218. E-mail addresses: [email protected] (C.F. Pinto), [email protected] (S.B. Berger), [email protected] (V. Cavalli), [email protected] (A.K. Bedran-Russo), [email protected] (M. Giannini).
http://dx.doi.org/10.1016/j.ijadhadh.2015.04.008 0143-7496/& 2015 Elsevier Ltd. All rights reserved.
Hence, the two major methods under investigations to improve dentin adhesion rely on the development of new adhesive systems and improvements of the intrinsic properties of dentin through a tissue engineering approach [5,6]. One of the aspects investigated by tissue engineering is the enhancement of inter- and intramolecular collagen cross-links [7,8]. Extrinsic collagen cross-linking agents may induce additional inter- and intra-molecular crosslinks, enhancing collagen mechanical properties and its resistance to enzymatic degradation, which is an advantageous to dentin bonding [6,7,9]. Type I collagen, one of the main component of the hybrid layer, is a triple-helix molecular structure containing three polypeptide chains. These chains are intertwined to one another and folded into a ropelike right-handed structure [10]. The bonds between the side chains of amino acids of the collagen molecules constitute the cross-links and these bonds improve the tensile properties of collagen fibrils [6,11,12]. Glutaraldehyde and proanthocyanidin are two chemical crosslinking agents frequently used to test the mechanical properties of
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the hybrid layer [9,13]. The first is a synthetic cross-linker widely used as a fixative agent. It has been reported to improve mechanical properties of various collagen-based tissues [14,15]. The second is a natural cross-linker present in fruits, vegetables, nuts, seeds and flowers. The grape seed extract, for instance, is a proanthocyanidin-rich solution that has been shown to improve the mechanical properties of demineralized dentin [9,13] and dentin–resin interface [16]. Recent investigations have shown the ability of glutaraldehyde solution and grape seed extract to enhance dentin collagen stability and bond strength to dentin treated with conventional etchand-rinse adhesives both immediately [6, 17,18] and in long-term analyzes of the quality of the bonded interface [5,11,16]. Other reports have demonstrated a significant improvement of bond strengths of deep dentin treated with proanthocyanidin (6.5% grape seed extract) prior to the application of conventional etchand-rinse [18] and self-etching adhesives [19]. Although the application of cross-linkers to dentin seems valuable to bonding, it was also reported that 6.5% grape seed extract incorporated to the primer of a self-etching adhesive decreased immediate bond strength when compared to the dentin treated with other agents (0.5% chlorhexidine and 0.5% hesperidin) [20]. Although controversy exists among the studies concerning type, time of application and concentration of the cross-linker as well as the adhesive systems used, most investigations showed promising results of the application of these agents. Therefore, the objective of this study was to evaluate bond strength to dentin treated with grape seed extract and glutaraldehyde and bonded with self-etching adhesives. The null hypothesis tested was that chemical cross-linker would not increase dentin bond strength.
2.4. Grape seed extract treatment The dentin surface was treated with 6.5% w/v grape seed extract (94% proanthocyanidins, MegaNatural; Polyphenolics, Madera, CA, USA, pH ¼7.4) [9,17] for 10 min. After the treatment, the surface was washed with distilled water, followed by the bonding/build-up procedure described previously. 2.5. Microtensile bond strength test The restored teeth were stored for 24 h in distilled water at 37 °C. Afterwards, the samples were serially sectioned with diamond saw (Isomet 1000, Bueheler, Lake Bluff, IL, USA) parallel to the long axis into 1 mm-thick slabs under water-cooling. The slabs were further sectioned perpendicularly to produce 6 sticks per tooth with a cross-sectioned area of 1.0 mm2. The sticks were attached to a microtensile-testing device with cyanoacrylate adhesive (Super Bonder, Henkel/Loctite; Diadema, SP, Brazil) and tested in tension in a universal testing machine (EZ-test, Shimadzu, Kyoto, Japan) at a crosshead speed of 1 mm/min until failure. After testing, the specimens were carefully removed and the cross-sectional area at the site of fracture was measured with a digital caliper (727-6/150, Starret; Itu, SP, Brazil) to the nearest 0.01 mm. The cross-sectional area of each specimen was divided by the maximum tensile load at failure to calculate the stress at fracture (MPa). Exploratory analysis of data regarding the homoscedasticity and normality was performed and the assumptions to the parametric test were fulfilled. Therefore, data was analyzed by twoway ANOVA and Tukey test with the significance limit set at 5% by SAS software. 2.6. Fracture mode analysis and adhesive interface observation
2. Materials and methods 2.1. Specimen preparation for microtensile testing Forty-five freshly extracted bovine incisors were used as experimental units. The teeth were cleaned, and the buccal surfaces were ground flat with silicon carbide paper (#180 and #320) to remove enamel and expose mid-depth dentin. Dentin surfaces were then ground with 600-grit abrasive paper to create a standard smear layer. The samples were randomly assigned to nine groups (n ¼5), according to the factors: adhesive system (Clearfil SE Bond, Kuraray Noritake Dental, Kurashiki, Japan; Clearfil SE Protect, Kuraray Noritake Dental; One-up Bond F Plus, Tokuyama Dental, Tokyo, Japan) and dentin cross-linking treatment (5% glutaraldehyde or 6.5% grape seed extract).
The specimens were polished with a 1000-grit SiC paper and 6, 3, 1, and 0.25 μm diamond paste (Buehler Ltd, Lake Bluff, IL, USA) using a polish cloth and ultrasonically cleaned. The fractured sites of the tested specimens were dried overnight (at 37 °C) and then sputter coated with gold (MED 010, Balzers; Balzer, Liechtenstein). The fracture mode and the resin–dentin interfaces were observed using a scanning electron microscope (VP 435 Leo; Cambridge, UK) at 100–2000 magnification. The quality of adhesive interface and hybrid layer was analyzed and the failure patterns were classified as type 1, adhesive failure between adhesive system and dentin and partially cohesive in the adhesive system; type 2, cohesive failure within the adhesive layer; type 3, cohesive failure within the dentin; or type 4, mixed failure, when simultaneously exhibiting dentin, hybrid and/or adhesive layer and remnants of composite.
2.2. Control group Bonding protocol followed the manufacturers' instruction for the three self-etching systems used as a control group. Specimens were light-cured for 20 s and a composite build-up (Filtek Supreme Plus, 3M ESPE, St. Paul, MN, USA) was constructed with two 2-mm increments, each light-cured for 40 s. 2.3. Glutaraldehyde The dentin surface was treated with 5% v/v glutaraldehyde (Fisher Chemical, Pittsburg, PA, USA, pH 7.4) [9,17] for 10 min. After the treatment, the dentin surface was washed with distilled water, followed by the bonding/build-up procedure described previously.
3. Results Two-way ANOVA demonstrated a significant interaction between factors: adhesive system and crosslinking treatment (p o0.0001) (Table 1). The application of 6.5% grape seed extract before Clearfil SE Bond self-etching adhesive system increases the bond strength, when compared to its control. Dentin bond strength for One-up Bond F Plus adhesive in combination with glutaraldehyde treatment promoted similar bond strength to its control group, but it was significantly higher than that obtained by grape seed treatment. No difference in bond strength were observed for both 5% glutaraldehyde and 6.5% grape seed extract treatments compared to the control group for Clearfil SE Protect, in which cross-linker agents had no effect.
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The summary of failures modes for all groups is depicted in Fig. 1. Photomicrographs of the fracture modes of the dentin bonding with the Clearfil SE Bond adhesive without previous cross-linking treatment (control, Fig. 2A) showed mostly adhesive Table 1 Mean and standard deviations of bond strength measurements for the adhesive systems tested as a function of the chemical cross-linking agent treatments. Adhesive
Chemical cross-linking agent Control
5% Glutaraldehyde 6.5% Grape seed extract
Clearfil SE Bond 23.3 (11.8) a B 36.4 (11.2) a AB Clearfil SE Protect 32.4 (5.1) a A 35.8 (5.1) a A One-up Bond F Plus 30.0 (3.7) a AB 33.5 (9.9) a A
37.9 (4.9) a A 39.6 (5.9) a A 18.5 (0.6) b B
Groups identified by distinct letters (capital letters compare crosslinking treatments – lines and lower case letters compare adhesive systems – columns) are different according to two-way ANOVA and Tukey test (p r 0.05).
Fig. 1. Bar graphs of failure mode percentages according to the adhesive systems and cross-linkers tested (GD: 5% glutaraldehyde, GSE: 6.5% grape seed extract, OP: One-up Bond F Plus, PB: Clearfil SE Protect, SE: Clearfil SE Bond). Failures modes: (1) adhesive; (2) cohesive within the adhesive layer; (3) cohesive within the dentin and (4) mixed.
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failures (type 1 – 53%) and some mixed failures (type 4 – 40%, Fig. 2A). Glutaraldehyde and grape seed extract treatments previously to Clearfil SE Bond bonding agent promoted mostly mixed failures exhibiting dentin, adhesive layer and/or occasionally remnants of composite (type 4 – 59–61%, respectively) and few type 1 failures (36% and 33%, respectively) (Fig. 2B and C). The Clearfil SE Protect adhesive demonstrated mostly mixed failures exhibiting adhesive, dentin and sometimes remnants of the composite on the dentin surface (type 4: Clearfil SE Protect – 69.5%; with glutaraldehyde – 50% and with grape seed extract – 62.5%, Fig. 3A–C, respectively). The One-up Bond F Plus adhesive promoted mostly adhesive failures (type 1) regardless of dentin treatment (Fig. 4A–C); however, it was the only adhesive that presented cohesive failures within the adhesive layer (type 2), mostly in the control group (33%). Representative images of the dentin interface bonded with Clearfil SE Bond, Clearfil SE Protect and One-up Bond F Plus adhesives are displayed in Figs. 5–7, respectively. Clearfil SE Bond (Fig. 5A) was able to create a uniform hybrid layer, regardless cross-linking treatments (Fig. 5A–C); however, the layer was not as thick as the ones observed by Clearfil SE Protect (Fig. 6A–C). A homogeneous hybrid layer pattern can be observed in SEM images of Clearfil Protect Bond interface without cross-linking treatment (Fig. 6A) similar to 5% glutaraldehyde (Fig. 6B) and 6.5% grape seed extract treatment (Fig. 6C). The interfaces of One-Up Bond F Plus (Fig. 7) were not as uniform as the previous adhesives (Figs. 5 and 6). The untreated group (Fig. 7A) and grape seed extract treatment (Fig. 7C) presented the hybrid layer thicker than the glutaraldehyde treatment (Fig. 7B) but the thickness found was not related to the bond strengths results obtained.
Fig. 2. Representative photomicrographs of the predominant failure modes of dentin bonded with the Clearfil SE Bond (SE) adhesive. (A) control group; failure mode type 1. (B) 5% glutaraldehyde treatment prior to SE bonding; failure mode type 4. (C) 6.5% grape seed extract treatment prior to SE bonding; type 4 (A – adhesive system; D – dentin; RC – resin composite).
Fig. 3. Representative photomicrographs of the predominant failure modes of the dentin bonded with the Clearfil Protect Bond (PB) adhesive. (A) control group; failure mode type 4. (B) 5% glutaraldehyde treatment prior to PB bonding; failure mode type 4. (C) 6.5% grape seed extract treatment prior to PB bonding; type 4 (A – adhesive system; D – dentin; RC – resin composite).
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Fig. 4. Representative photomicrographs of the predominant failure modes of the dentin bonding with the One-up Bond F Plus (OP) adhesive. (A) control group; failure mode type 1. (B) 5% glutaraldehyde treatment prior to OP bonding; failure mode type 1. (C) 6.5% grape seed extract treatment prior to OP bonding; type 1 (A – adhesive system; D – dentin; RC – resin composite).
Fig. 5. Representative scanning electron microscopy images of the adhesive interface. (A) Clearfil SE Bond. (B) Clearfil SE Bond after 5% glutaraldehyde; (C) Clearfil SE Bond after 6.5% grape seed extract (A – adhesive system; D – dentin; RC – resin composite).
Fig. 6. Representative scanning electron microscopy images of the adhesive interface. (A) Clearfil Protect Bond. (B) Clearfil Protect Bond after 5% glutaraldehyde; (C) Clearfil Protect Bond after 6.5% grape seed extract (A – adhesive system; D – dentin; RC – resin composite).
Fig. 7. Representative scanning electron microscopy images of the adhesive interface. (A) One-Up Bond F Plus. (B) One-Up Bond F Plus after 5% glutaraldehyde; (C) One-Up Bond F Plus after 6.5% grape seed extract (A – adhesive system; D – dentin; RC – resin composite).
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4. Discussion It has been assumed that if collagen fibrils are chemically engineered by the creation of additional internal and external cross-linking, the consequence would be a stronger and more durable hybrid layer that is less prone to degrade [5]. Grape seed extract, a proanthocyanidin (PA)-rich mixture of polyphenols, is able to increase collagen cross-linking by covalent [21], ionic [22], hydrogen bonding [23] and hydrophobic interactions [8]. A number of investigations have reported that these interactions increase the mechanical properties of resin– dentin bonded interface [5,16,24] and, as a result, increase dentin bond strengths [11,17–19]. This study demonstrated that the 6.5% grape seed extract enhanced dentin bond strengths, but this improvement was adhesive-dependent. This is in agreement with previous studies that reported that the stability of cross-linking treatment depends mostly on the source/type of PA-rich extract and the adhesive system employed [11]. The 6.5% grape seed extract cross-linker increased bond strengths of Clearfil SE Bond adhesive, but decreased One-up Bond F Plus’s bonding and did not enhance bond strengths of the Clearfil SE Protect adhesive. The reasons for this bonding behavior could be attributed to the composition of the adhesives. The three adhesives tested are self-etching systems with similar adhesion strategies. However, Clearfil SE Bond and Clearfil Protect Bond are two-bottle adhesive self-etching primers, both containing 10-MDP as functional monomers, with Clearfil Protect Bond also containing MDPB (12-methacryloyloxydodecylpyridinium bromide) as an additional anti-bacterial monomer [25,26]. 10-MDP is reported to form a strong chemical bond to the hydroxyapatite, contributing to the adhesion of the enamel and dentin [27]. As a two-bottle primer/adhesive system, the application of the solvent-free hydrophobic layer over the primed dentin surface reduces the hydrophilicity of the hybrid layer, increasing the polymerization rate of the adhesive systems as well as increasing the dentin bond strength [28]. One-Up Bond F Plus, a single-step self-etching adhesive, is a complex mixture of acidic primers and bonding resins with six hydrophobic and hydrophilic monomers. The functional monomers are MAC-10 (methacryloyloxydecamethlene malonic acid), HEMA (2-hydroxyethyl methacrylate), and methacryloyloxyalkyl acid phosphate. MAC-10 is the adhesion-promoting monomer and, like 10-MDP, is hydrolytically stable and its molecular structure makes this monomer hydrophobic. The chemical structures of monomers determine the viscosity, solubility, hydrolysis susceptibility, wetting, and penetration behavior [29]. Less promising results were obtained with the 6.5% grape seed extract/One-up Bond F Plus-adhesive group, in which 6.5% grape seed extract significantly decreased dentin bond strength. Singlestep self-etching adhesives are frequently associated with low bond strengths and more adhesive failures than the two-step selfetching primers [30]. These single-step adhesives have been correlated to water tree, water blister formation and adhesive phase separation at the interface [29,31–33]. In a recent nanoleakage study, it was demonstrated that these agents allow higher water diffusion in the polymerized adhesive compared to conventional etch-and-rinse systems. The high degree of nanoleakage in the adhesive layer could be attributed to the hydrophilic nature of the simplified self-etching adhesives, especially in HEMA-containing systems [34] and to the increased concentrations of water and solvents (acetone, ethanol) [35,36]. It has also been observed that the hydrophilic adhesive may entrap the water from dentin during and after bonding, which compromises bond strength [37]. Another possibility is that 6.5% grape seed extract may have promoted a poor interaction between the adhesive system and the dentin due to reduced dentin wettability, leading to less adhesive
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penetration, as observed in Fig. 7C. In the microscopy analysis, the adhesive layer is considerably thicker and the adhesive might have not penetrated dentin, as it would be expected, promoting lower bond strength values. Adhesive failure (type I) was the predominant failure of the OP adhesive regardless 5% glutaraldehyde or 6.5% grape seed extract treatments, which confirmed the compromised performance of this adhesive, due to possible water deposits within the hybrid layer. It is possible that 6.5% grape seed extract was able to stabilize the collagen structure in dentin by increasing the amount of external cross-linking interactions (which could be observed for the 6.5% grape seed extract/Clearfil SE Bond-treated dentin). However, it is also likely that the enhanced collagen cross-linking reduced the width of interfibrillar spaces that serve as diffusion channels for monomer infiltration. The MDPB monomer and fluoride ions present in the Clearfil SE Protect adhesive may interfere with viscosity and permeation of the adhesive in the collagen-treated complex. As a result, the grape seed treated-collagen layer exhibited similar bond strengths, regardless the crosslinking treatment. The analysis of the fractured bonded area exhibited mostly adhesive failures for Clearfil SE Bond-treated dentin. Interestingly, this condition was reversed with the 5% glutaraldehyde and 6.5% grape seed extract treatments, which promoted mostly mixed failures (fractures at dentin, adhesive layer with or without remnants of composite). This might indicate that cross-linkers treatments could have increased strength at the adhesive interface and for this reason, after 6.5% grape seed extract and 5% glutaraldehyde treatments, the weakest area was not the adhesive anymore. Although 5% glutaraldehyde increased noticeably bond strengths values of Clearfil SE Bond dentin bonding, it was not statistically significant from 6.5% grape seed extract. Failure modes of Clearfil SE Protect bonding were always type 1 (adhesive) and this uniformity is also markedly related to the bond strength values. Glutaraldehyde has a molecular affinity for free amino groups of amino acids and is often used as a protein fixation agent [38,39]. 5% glutaraldehyde treated acid-etching dentin has been associated with a decrease in the rate of collagen degradation [8], enhanced mechanical properties of demineralized dentin [17] and improved resin–dentin bonded interfaces [6,39]. However, unlike these reports, in the present study 5% glutaraldehyde was unable to significantly increase dentin bond strengths of the adhesives tested compared to untreated and 6.5% grape seed extract-treated dentin. These differences could be related to the adhesive system used, as some of the studies used etch-and-rinse [6,17], instead of self-etching adhesives. The bonding strategy along with the composition of the adhesives could imply better performance in etch-and-rinse adhesives before the 5% glutaraldehyde application. Besides, 5% glutaraldehyde induces cross-linking interactions differently from that of 6.5% grape seed extract, involving Lys and Hyl amino acids [15] and this could contribute to a different adhesive behavior. Likewise, it is possible that the results of the current study were not as favorable as those previously obtained, as the incubation time of 5% glutaraldehyde and 6.5% grape seed extract cross-linkers on dentin was performed for 10 min instead of 60 min [6,16,17]. It has been observed that increased exposure time of 5% glutaraldehyde and 6.5% grape seed extract enhances the elastic modulus of demineralized dentin; therefore, the effectiveness of the treatments might be time-dependent [5,13,24]. Although a ten-minute cross-linking treatment is still not suitable for clinical application, other possibilities that are under investigation should be considered, as reduced pre-treatment time with higher concentrations or even using several cross-linkers simultaneously [6]. Clearly, the adhesive system used, its components and adhesive techniques should be considered in the use of this strategy. Furthermore, the cross-linker of choice seems to be
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6.5% grape seed extract, since 5% glutaraldehyde was not able to increase bond strengths for any of the adhesives tested, and because of the cytotoxicity of this agent [40,41].
5. Conclusions Grape seed extract (6.5%) cross-linking treatment was able to increase bond strength for one of the adhesives tested (Clearfil SE Bond), but decreased bond strength for another one-step selfetching adhesive tested (One-up Bond F Plus), whereas glutaraldehyde (5%) did not change bond strength for any of the adhesives tested. Thus, the benefits of cross-linking treatments previous to bonding were adhesive system-dependent.
Acknowledgments This investigation was supported by Research Grant 06/538282 from the State of São Paulo Research Foundation, São Paulo, Brazil.
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