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Effect of cyclic loading under enzymatic activity on resin–dentin interfaces of two self-etching adhesives
d e n t a l m a t e r i a l s 2 4 ( 2 0 0 8 ) 178–184
available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/dema
Effect of cyclic loading under enzymatic activity on resin–dentin interfaces of two self-etching adhesives Tissiana Bortolotto a,∗ , Ioana Onisor a , Ivo Krejci a , Marco Ferrari b , Franklin R. Tay c , Serge Bouillaguet a a b c
Division of Cariology and Endodontology, University of Geneva, School of Dental Medicine, Geneva, Switzerland Department of Dental Materials and Restorative Dentistry, Policlinico ‘Le Scotte’, University of Siena, Siena, Italy Department of Oral Biology and Maxillofacial Pathology, Medical College of Georgia, Augusta, USA
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i n f o
a b s t r a c t
Article history:
Objectives. To evaluate microtensile bond strength and micro-morphology at the resin–dentin
Received 12 June 2006
interfaces of two self-etching adhesive systems subjected to simultaneous mechanical and
Received in revised form
enzymatic stress.
16 January 2007
Methods. Sixteen enamel/dentin discs were bonded with a two-step self-etching adhesive
Accepted 16 March 2007
(AdheSE, n = 8) and a one-step self-etching adhesive (Xeno III, n = 8) to a 2 mm thick resin composite layer. One resin–dentin bar was obtained per tooth. In half of the specimens of each group TBS and micro-morphological evaluations (TEM) was performed without
Keywords:
loading. The other half was mechanically loaded in a cholinesterase-containing solution.
Dentin
TBS as well as ultra-morphological evaluations of the directly loaded areas using TEM
Composite
were performed on the loaded specimens.
Biomechanics
Results. The TBS of the specimens (non-loaded/loaded) were of 39.6 ± 14.7/35.4 ± 22.1 and
Microtensile
of 21.8 ± 29.8/15.9 ± 25.5 for AdheSE and Xeno III, respectively. Under TEM, both materials
Self-etch
presented signs of nanoleakage. However, on loaded specimens the extent of nanoleakage
Micromorphology
was slightly reduced for AdheSE and no silver staining was observed on the adhesive inter-
Cholinesterase
face of Xeno III. TEM evaluations of the specimens’ loaded area revealed no decrease in
Enzyme
the width of the adhesive interface for AdheSE. The contrary was observed in the interface
Cyclic load
created by Xeno III.
Aging
Significance. The adhesive interfaces created by the two-step self-etching adhesive (AdheSE) could better withstand both mechanical and enzymatic stresses on the long-term than the one-step self-etching system (Xeno III) tested in the present study. © 2007 Published by Elsevier Ltd on behalf of Academy of Dental Materials. All rights reserved.
1.
Introduction
Self-etching adhesives are progressively used in clinical practice. They are user-friendly, less technique sensitive than adhesive systems that require a separate etching step and have been shown to reduce the risk of postoperative sensitiv-
ity [1]. Although their short-term bonding ability has been well documented, the long-term durability of resin–dentin bonds made with such adhesives remains controversial [2]. Armstrong et al. [3] reported that both hydrolysis of the collagen matrix and/or the degradation of the synthetic components of the hybrid layer are two important factorscontributing to
∗ ´ Corresponding author at: Division of Cariology and Endodontology, School of Dentistry, University of Geneva, 19, Rue Barthelemy-Menn, CH-1205 Geneva, Switzerland. Tel.: +41 22 379 4076; fax: +41 22 379 4102. E-mail address: [email protected] (T. Bortolotto). 0109-5641/$ – see front matter © 2007 Published by Elsevier Ltd on behalf of Academy of Dental Materials. All rights reserved. doi:10.1016/j.dental.2007.03.008
d e n t a l m a t e r i a l s 2 4 ( 2 0 0 8 ) 178–184
the degradation of resin–dentin bonds over time. Hydrolysis is a major problem affecting the bond durability of self-etching adhesives, especially their simplified versions called “all in one” adhesives. With such systems, high amounts of solvents (water, acetone or ethanol) or hydrophilic monomers like 2 hydroxyethyl methacrylate (HEMA) have been added to their formulations to facilitate the diffusion of the resins inside the dental substrates. HEMA is amphoteric, i.e. it has the capacity to react either as an acid or a base, and displaces water in dentin but is also miscible with most of the monomers of the adhesives [4]. Water is also an essential component because it provides hydrogen ions that are necessary for demineralization. The problem is that these components raise the hydrophilicity of the adhesive, which may in turn increase the risk of water uptake into the matrix and decrease the polymerisation rate of the resins [5]. One method for assessing bond durability in vitro is to challenge the adhesive interface under a simulated oral environment [6,7]. In a recent study thermal and short-term mechanical stresses (100,000 cycles) have been shown to negatively affect the bond strength of one etch and rinse and three two-step self-etching adhesive systems [8]. Twostep self-etching adhesives performed better than simplified formulations due to the presence of a more hydrophobic adhesive layer that better protects the adhesive interface against hydrolytic degradation. Enzymatic attack also has been shown to contribute to the degradation process of resin composites [9]. In a recent in vitro study, resin–dentin specimens were stored either in a cholesterol esterase solution or in a collagenase-containing medium for 12 weeks [10]. The first solution was used as an enzymatic challenge to adhesive polymers contained in the adhesive system while the second one was used as an enzymatic challenge to demineralized dentin matrix. Significantly weaker bonds were found in specimens stored in cholesterol esterase when compared to those immersed in collagenase-containing medium. The authors concluded that the enzymatic hydrolysis of methacrylate resins might be of more clinical concern than the enzymatic hydrolysis of the collagen components of the resin–dentin bond.
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Even so, there is no in vitro study that investigated the degradation of resin–dentin interfaces made with self-etching adhesive systems simultaneously subjected to mechanical loading and enzymatic stress. It was therefore the purpose of this in vitro study to evaluate the effect of long-term cyclic loading under esterase activity on the microtensile bond strength of a two-step (AdheSE) and a one-step (Xeno III) self-etching adhesive, applied to dentin. Alterations in resin–dentin interfaces were further observed with TEM in order to evaluate the micro-morphological changes that occurred after aging.
2.
Materials and methods
The set-up used in this study is schematically represented in Fig. 1. Sixteen intact caries-free human molars were stored in 0.1% thymol solution for a maximum of 2 months until being used. The teeth were cut perpendicular to their longitudinal axis using a slow-speed diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under water cooling in order to obtain 2 mm thick dentin discs. A standard smear layer was created at the dentin surface by the use of a 40 m dia` ¨ mond bur (Coltene-Whaledent, Altstatten, Switzerland) and subsequently bonded using either AdheSE (IvoclarVivadent, Schaan, Liechtenstein) or Xeno III (DeTrey-Dentsply, Konstanz, Germany). The composition and application procedure of each material are detailed in Table 1. The adhesives were applied onto the entire dentin surface and light cured for 20 s (Optilux 501, KerrHawe, Boggio, Switzerland). A 2 mm thick layer of composite resin (Durafill, Heraeus Kulzer, Dormagen, Germany) was applied to the bonded surface and light-cured for 40 s. Each disc was then sectioned with a diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) to produce a bar specimen of 4 mm thick. Thus, each bar was made of 2 mm of composite resin bonded to 2 mm of dentin. For each material, half of the bars were subjected to microtensile testing without being loaded. The other half of the bars were challenged with mechanical and chemical stresses and then a bond strength test of the loaded area was performed. Chemical and mechanical stresses were applied to
Fig. 1 – Representation of the set-up of the study. The non-loaded groups are represented on the upper and the loaded groups on the lower part of the schema. ChE, cholinesterase; TBS, microtensile bond strength; TEM, transmission electron microscopy; mio, million; r-c bar, resin-composite bar; n, number of specimens (for microtensile evaluation) per group.
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d e n t a l m a t e r i a l s 2 4 ( 2 0 0 8 ) 178–184
Table 1 – Materials, batch numbers, composition and application procedures as used in the present investigation Material, manufacturer, batch # AdheSE; IvoclarVivadent, Schaan, Liechtenstein, F40620
Xeno III; DeTrey-Dentsply, Konstanz, Germany, 0311001490
Durafill; Heraeus Kulzer, Dormagen, Germany, 010143
Composition Primer: phosphonic acid acrylate, Bis-acrylamide, water, initiators and stabilizers Bond: Dimethacrylates, Hydroxyethyl methacrylate, highly dispersed silicon dioxide, initiators and stabilizers Liquid A: 2-hydroxyethyl methacrylate, purified water, ethanol, 2,6-Di-tertbutyl-p-hydroxy-toluene, nanofiller Liquid B: Pyro-EMA-SK (tetra-methacryl-ethyl-pyrophosphate), PEM-F (Penta-methacryl-oxy-ethyl-cyclo-phosphazenmonofluoride), UDMA, BHT (2,6-di-tert-butyl-p-cresol), camphorquinone, EPD (p-dimethyl amine ethyl benzoate) Urethanedimethacrylate, highly disperse silicon dioxide, splinter polymer
the bar specimens using a chewing machine [11,12] filled with a cholinesterase-containing solution (Cholinesterase Acetyl type VI-S, Sigma–Aldrich Chemie GmbH, Schnelldorf, Germany). The concentration of the enzyme was of 1 Unit/ml and dilution was performed in 0.02 M sodium phosphate (pH 7.0). The specimens were simultaneously submitted to a 1.2 million cyclic stress at a frequency of 1.7 Hz with a maximum loading force of 49 N. To avoid the displacement of the specimen during loading, the set-up consisted of two supports holding both ends of the specimen with a single loading pin (6 mm wide, rounded tip) located in the middle of the bar, where the force was introduced (Fig. 2). At the end of the mechanical test, the area where the load was applied was identified with an insoluble color marker. Within this marked area, six slabs were prepared by sectioning the specimen perpendicularly to the resin–dentin interface with a diamond saw. The dimension of each slab was 4.0 mm × 1.0 mm × 1.0 mm and was controlled with a digital calliper. The slabs were hand-trimmed under stereo-microscopic observation (ensuring that the narrowest area was located at the bonding interface) and the final bonded area was of about 0.8 mm2 . Four slabs were used for microtensile bond strength test and the two remaining slabs were used for TEM analysis. The same procedure was followed with the non-loaded specimens.
Fig. 2 – Schematic representation of bars’ loading.
Handling procedure Apply primer, leave for 30 s, air-dry Apply bonding, air-blow, light-cure 20 s Mix A + B for 5 s. Apply 3 layers, leave for 30 s. Dry carefully until seeing a shiny surface, then dry well to evaporate the solvent. Light-cure 20 s
Apply one layer of 2 mm thickness, light cure 40 s
Twenty-four hours after cutting the slabs for microtensile testing, they were attached to the grips of a custom-made holder with cyanoacrylate adhesive (Zapit, DVA Inc., Corona, CA, USA) and stressed to failure at a crosshead speed of 1 mm/min using a universal testing machine (Vitrodyne V1000 Universal Tester, John Chatillon and Sons, Greensboro, NC, USA). The tensile bond strength value of each slab was calculated as the force (N) at failure divided by the bonded area (mm2 ) and expressed in MPa. Slabs that failed during the sectioning process were assigned a bond strength value of 0 MPa and were included into the results. The micro-morphological evaluations were carried out on the two remaining slabs (from the six that were initially obtained) of both non-loaded and loaded specimens. The presence of silver staining and the width of adhesive interfaces created by the two adhesive systems were observed from around 24 micrographs obtained from both non-loaded and loaded specimens. The samples for TEM observation were prepared according to the protocol of Tay et al. [5]. The slabs were immersed in a tracer solution (an aqueous solution of 50 wt% ammoniacal silver nitrate) for 24 h. The silver impregnated slabs were then removed form the tracer solution and thoroughly rinsed in distilled water. They were then placed in photo developing solution for 8 h under a fluorescent light to reduce silver nitrate to metallic silver. The slabs were fixed in Karnovsky’s fixative (2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3) for a minimum of 4 h and rinsed thoroughly with 0.1 M sodium cacodylate buffer. Post-fixation was performed with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7) for 1 h at room temperature. The slabs were then rinsed three times in cacodylate buffer, dehydrated in ascending ethanol series (30–100%) and immersed in propylene oxide as a transition fluid. After resin embedding (TAAB 812 resin, TAAB Laboratories, Aldermaston, UK), 1 mm × 1 mm ultra thin sections about 90 nm thick were prepared with an ultra microtome (Reichert Ultracut S, Leica, Viena, Austria). Without any further staining the ultra thin sections were observed using a TEM (EM208S, Philips, Eindhoven, The Netherlands) operated at 80 kV.
d e n t a l m a t e r i a l s 2 4 ( 2 0 0 8 ) 178–184
Table 2 – Results of the number and percentage of pre-test failures as well as microtensile bond strength evaluation for the different adhesive systems Adhesive
Non-loaded specimens TBS mean in MPa (S.D.)
Loaded specimens TBS mean in MPa (S.D.)
AdheSE No. PTF (%) Xeno III No. PTF (%)
39.6 (14.7) 1 (6.3) 21.8 (29.8) 10 (63)
35.4 (22.1) 4 (25) 15.9 (25.5) 11 (69)
No significant differences were found between non-loaded and loaded specimens within both groups (Wilcoxon rank sum test, p > 0.05). TBS = microtensile bond strength, No. PTF (%) = number of pre-test failures (percentage of pre-test failures), S.D. = standard deviation.
The statistical analysis of the microtensile data was performed with SPSS 14.0 for Windows (SPSS Inc., Chicago, IL, USA). As the data was non-normally distributed (Kolmogorov–Smirnov Test), a Wilcoxon rank sum test was performed for comparisons between non-loaded and loaded specimens. The confidence level was set to 95%.
3.
Results
3.1.
Microtensile bond strength
The results of the microtensile bond strength evaluation are shown in Table 2. Bond strength of AdheSE was 39.6 ± 14.7 MPa on nonloaded specimens and 35.4 ± 22.1 MPa after aging; for Xeno III it was 21.8 ± 29.8 MPa on non-loaded specimens and 15.9 ± 25.5 MPa after aging. Bond strength of the materials tested was adversely affected by mechanical loading, as can be deduced from the mean values in each group. However, the difference between the values of non-loaded and loaded specimens was not significant (p > 0.05, Wilcoxon rank sum test). In the groups bonded with AdheSE, one pre-test failure (6.3% of the specimens tested) was counted on non-loaded specimens and four pre-test failures (25%) were observed on the loaded specimens. For Xeno III the situation was more dramatic, 10 pre-test failures (63%) were observed on non-loaded and 11 (69%) on loaded specimens.
3.2.
Transmission electron microscope analysis
Representative TEM micrographs of adhesive interfaces created by AdheSE and Xeno III are shown in Figs. 3 and 4. On both non-loaded and loaded specimens, the resin–dentin interfaces bonded with AdheSE were around 16–20 m (Fig. 3a). Some isolated silver grain deposits within both hybrid and adhesive layer were observed on non-loaded specimens (Figs. 3a and b). Although some less amount of silver grain deposits was observed on the loaded samples (Figs. 3c and d), no substantial differences could be observed in respect to the loaded specimens. TEM observations of the non-loaded adhesive interfaces made with Xeno III revealed an adhesive layer of approximately 8 m thick (Figs. 4a and b). This width was reduced to about 2 m after the specimens were loaded
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(Fig. 4d). In respect to nanoleakage patterns, non-loaded resin–dentin interfaces made of Xeno III exhibited important nanoleakage already before loading as represented by condensed amounts of silver stain within the hybrid layer (Fig. 4a–c). The amount of silver grains was reduced or even inexistent on the loaded specimens (Fig. 4d).
4.
Discussion
This study evaluated the effects of artificial loading on the mechanical behaviour of resin–dentin joints formed by two self-etching adhesive systems that differ in their composition and number of application steps. In order to simulate clinical conditions, artificial aging was performed by stressing the specimens up to 1.2 million load cycles at 49 N in cholinesterase solution. This enzyme is present in human saliva and may contribute to the degradation of polymeric materials on the long-term [13,14]. The resin–dentin bars were positioned in the chewing machine in such a way that they could bend during loading (Fig. 2). With this set up, the bottom side of the bar was in tension while the upper side was under compression. Both mechanisms are suspected to accelerate the degradation of bonded interfaces as has been previously shown for fiber-reinforced composites [15]. We expected that under the influence of both mechanical and chemical stress, a reduction in bond strength would be observed as previously shown by Frankenberger et al. [8]. Although a trend towards inferior bond strength was observable on the specimens after aging, the high number of pre-test failures precluded to demonstrate any significant difference between stressed and non-stressed specimens. However, it must be noted that in a similar study, Nikaido et al. [16] also failed to demonstrate any difference in bond strength between loaded and unloaded specimens. Although aging has not significantly influenced the mean microtensile bond strength of both adhesives, the number of pre-test failures observed with both systems was more informative. Whereas the incidence of pre-test failures was only of 6.3% for AdheSE, 63% of the Xeno III specimens failed before testing. Even the non-loaded specimens exhibited a high number of pre-test failures, indicating that hydrolysis has occurred even in the absence of any external stress. This result could be explained by the high water content of adhesive layers produced with Xeno III as previously shown by Chersoni et al. [17]. This problem was not observed with AdheSE, which contains a phosphonic acid compound. According to the information provided by the manufacturer, it is more hydrolytically stable when compared to phosphoric acid ester compounds used in Xeno III. Further, this material also includes bis-acrylamide, a more hydrolytically stable monomer than other hydrophilic monomers used in dentinal adhesive formulations [18]. In addition, as this material is a two-step self-etching adhesive, the separate bonding layer might have protected the adhesive interface against loading conditions and chemical challenge [8]. More interesting were the observations made under the TEM. All specimens of Xeno III showed a trend towards a reduction in thickness of the adhesive interface after loading. While the thickness of the adhesive layer in non-loaded spec-
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Fig. 3 – TEM micrographs of the resin–dentin interface bonded with AdheSE on non-loaded and loaded specimens. (a) Low magnification micrograph of an interface that is characteristic of a non-loaded specimen. Areas with sporadic reticular patterns of nanoleakage (arrow) can be observed within the hybrid layer. Note that the width of the adhesive interface is around 18 m. (b) Higher magnification of a non-loaded specimen with some isolated silver grains (black arrow) and silver-filled water channels (white arrow). (c) Micrograph of a loaded specimen where less silver infiltration can be observed in respect to the non-loaded specimens. The width of the adhesive interface is around 16 m. (d) A higher magnification of a loaded specimen with some isolated silver grains (black arrow) within the hybrid layer. Grey arrow: nanofiller clusters within the adhesive. C: resin composite; D: dentin; AL: adhesive layer; HL: hybrid layer.
imens was approximately 8 m, which is in agreement with previously published results [19], the adhesive layer was only 2 m thick after loading. This result supports the idea that adhesive interfaces produced by all-in-one adhesives behave as resin-collagen “sponges” that undergo irreversible plastic deformation under load. On the contrary, this phenomenon was not observed with AdheSE specimens. The separate bonding layer together with a more hydrolytically stable chemistry protected the adhesive form water uptake; this provided more stability to the interface and could explain why almost no decrease in the width of adhesive layer was observed after loading. Additionally, less signs of nanoleakage were observed in the specimens’ loaded area, especially of the interface created by Xeno III. We speculate that this was due to compression of the collagen network which was poorly impregnated
by the resin and that contained high amounts of water. A recent study by Chandran and Barocas [20] analysed the micromechanical behaviour of collagen gels and observed a severe collapse on the compression side. This collapse was characterized by large irreversible strains and a sharp change in fibril orientation. We presume that this collapse accounts for the less silver particles uptake in loaded specimens. A recent study evaluated the molecular interactions that take place when water diffuses through epoxy networks [21]. The authors identified mobile water localized in microvoids (free water) and water molecules firmly bound to specific sites along the resin network by hydrogen-bonding interactions (bound water). They explained that water diffusion occurs by the action of two simultaneous mechanisms: a fast diffusion process that is associated with the movement of “free water” without interacting with the network
d e n t a l m a t e r i a l s 2 4 ( 2 0 0 8 ) 178–184
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Fig. 4 – TEM micrographs of the resin–dentin interface bonded with Xeno III. (a) Interface of a non-loaded specimen. Areas with condensed silver grains (arrow) can be observed within the hybrid layer. The width of the adhesive interface is around 8 m (bar = 1 m). (b) Another cut of a non-loaded specimen with signs of nanoleakage (arrow) at the hybrid layer; the width of the adhesive interface is around 8 m (bar = 2 m). (c) Non-loaded specimen, high-magnification view of the hybrid layer with areas of extense silver staining (bar = 1 m). (d) An image that is characteristic of a loaded specimen; the width of the adhesive interface is around 2 m (bar = 0.5 m) and no signs of nanoleakage can be observed. C: resin composite; D: dentin; AL: adhesive layer; HL: hybrid layer.
and a much slower diffusion process involving interactions at the hydrogen-bonding sites of the network, being the last responsible for plasticization of the system and further degradation in structural properties. We presume that repeated water sorption/desorption during load cycles (due to the high hydrophilicity in Xeno III) caused an irreversible damage to the adhesive interface, which was evidenced by a decrease in its width. The contribution of enzymatic challenge to the degradative process of both adhesive interfaces requires further investigation. In the present study an enzyme-containing solution was only used as a model of aggressive environment. Certainly, esterase activity could have played an additional role in the degradation of the resin–dentin interface, especially for the one-step adhesive Xeno III [10]. However, future studies should be performed that compare the results of the present study with non-loaded/loaded specimens stored either in enzyme-free solutions or oil as positive control storage media. It is also necessary to determine the contribution of not
only exogenous but also endogenous enzymatic challenge to the degradation of acid-exposed dentin matrix [22]. It is known that self-etching adhesives, being acidic compounds, can activate endogenous dentin matrix metalloproteinase and contribute to the degradation of incompletely infiltrated hybrid layers in aged resin–dentin interfaces [23–26]. It would be interesting to compare for example, low performing adhesives with adhesive systems that, by completely infiltrating the demineralized collagen fibrils, may limit such enzymatic activity.
5.
Conclusion
A distinct behaviour of both self-etching adhesives, AdheSE and Xeno III, was observed when confronted to simultaneous chemical and mechanical challenge. Cyclic loading under cholinesterase activity led to an irreversible deformation in the adhesive interface of the simplified adhesive Xeno III.
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Acknowledgements [13]
This study was based on a thesis submitted to the graduate faculty, University of Siena, Italy, in partial fulfilment of the requirements for the Ph.D. degree. The authors would like to express their gratitude to Mrs. Helen Andersen (Division of Periodontology, University of Geneva) for her technical assistance.
[14]
[15]
references [16] [1] Swift Jr EJ. Dentin/enamel adhesives: review of the literature. Pediatr Dent 2002;24:456–61. [2] 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. [3] Armstrong SR, Vargas MA, Fanf Q, Laffoon JE. Microtensile bond strength of a total-etch 3-step, total-etch 2-step, self-etch 2-step, and a self-etch 1-step dentin bonding system through 15-month water storage. J Adhes Dent 2003;5:47–56. [4] Bouillaguet S. Biological risks of resin-based materials to the dentin-pulp complex. Crit Rev Oral Biol Med 2004;15:47–60. [5] Tay FR, Pashley DH, Yoshiyama M. Two modes of nanoleakage expression in single-step adhesives. J Dent Res 2002;81:472–6. ¨ [6] Krejci I. Tooth colored restorations. Munich: Hanser Verlag; 1992, ISBN 3-446-17291-2 (in german). [7] Frankenberger R, Strobel WO, Kramer N, Lohbauer U, Winterscheidt J, Winterscheidt B, et al. Evaluation of the fatigue behavior of the resin–dentin bond with the use of different methods. J Biomed Mater Res B Appl Biomater 2003;67:712–21. [8] Frankenberger R, Pashley DH, Reich SM, Lohbauer U, Petschelt A, Tay FR. Characterisation of resin–dentin interfaces by compressive cyclic loading. Biomaterials 2005;26:2043–52. [9] Jaffer F, Finer Y, Santerre JP. Interactions between resin monomers and commercial composite resins with human saliva derived esterases. Biomaterials 2002;23:1707–19. [10] Armstrong SR, Jessop JLP, Vargas MA, Zou Y, Qian F, Campbell JA, et al. Effects of exogenous collagenase and cholesterol esterase on the durability of the resin–dentin bond. J Adhes Dent 2006;8:151–60. [11] Krejci I, Reich T, Lutz F, Albertoni M. In-vitro test procedure for the evaluation of dental restorations. 1. Computer-controlled chewing simulator. Schweiz Monatsschr Zahnmed 1990;100:953–60 (in german). [12] Krejci I, Albertoni M, Lutz F. In-vitro test procedure for the evaluation of dental restorations. 2. Tooth brush/tooth-paste
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journal homepage: www.intl.elsevierhealth.com/journals/dema
Effect of cyclic loading under enzymatic activity on resin–dentin interfaces of two self-etching adhesives Tissiana Bortolotto a,∗ , Ioana Onisor a , Ivo Krejci a , Marco Ferrari b , Franklin R. Tay c , Serge Bouillaguet a a b c
Division of Cariology and Endodontology, University of Geneva, School of Dental Medicine, Geneva, Switzerland Department of Dental Materials and Restorative Dentistry, Policlinico ‘Le Scotte’, University of Siena, Siena, Italy Department of Oral Biology and Maxillofacial Pathology, Medical College of Georgia, Augusta, USA
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objectives. To evaluate microtensile bond strength and micro-morphology at the resin–dentin
Received 12 June 2006
interfaces of two self-etching adhesive systems subjected to simultaneous mechanical and
Received in revised form
enzymatic stress.
16 January 2007
Methods. Sixteen enamel/dentin discs were bonded with a two-step self-etching adhesive
Accepted 16 March 2007
(AdheSE, n = 8) and a one-step self-etching adhesive (Xeno III, n = 8) to a 2 mm thick resin composite layer. One resin–dentin bar was obtained per tooth. In half of the specimens of each group TBS and micro-morphological evaluations (TEM) was performed without
Keywords:
loading. The other half was mechanically loaded in a cholinesterase-containing solution.
Dentin
TBS as well as ultra-morphological evaluations of the directly loaded areas using TEM
Composite
were performed on the loaded specimens.
Biomechanics
Results. The TBS of the specimens (non-loaded/loaded) were of 39.6 ± 14.7/35.4 ± 22.1 and
Microtensile
of 21.8 ± 29.8/15.9 ± 25.5 for AdheSE and Xeno III, respectively. Under TEM, both materials
Self-etch
presented signs of nanoleakage. However, on loaded specimens the extent of nanoleakage
Micromorphology
was slightly reduced for AdheSE and no silver staining was observed on the adhesive inter-
Cholinesterase
face of Xeno III. TEM evaluations of the specimens’ loaded area revealed no decrease in
Enzyme
the width of the adhesive interface for AdheSE. The contrary was observed in the interface
Cyclic load
created by Xeno III.
Aging
Significance. The adhesive interfaces created by the two-step self-etching adhesive (AdheSE) could better withstand both mechanical and enzymatic stresses on the long-term than the one-step self-etching system (Xeno III) tested in the present study. © 2007 Published by Elsevier Ltd on behalf of Academy of Dental Materials. All rights reserved.
1.
Introduction
Self-etching adhesives are progressively used in clinical practice. They are user-friendly, less technique sensitive than adhesive systems that require a separate etching step and have been shown to reduce the risk of postoperative sensitiv-
ity [1]. Although their short-term bonding ability has been well documented, the long-term durability of resin–dentin bonds made with such adhesives remains controversial [2]. Armstrong et al. [3] reported that both hydrolysis of the collagen matrix and/or the degradation of the synthetic components of the hybrid layer are two important factorscontributing to
∗ ´ Corresponding author at: Division of Cariology and Endodontology, School of Dentistry, University of Geneva, 19, Rue Barthelemy-Menn, CH-1205 Geneva, Switzerland. Tel.: +41 22 379 4076; fax: +41 22 379 4102. E-mail address: [email protected] (T. Bortolotto). 0109-5641/$ – see front matter © 2007 Published by Elsevier Ltd on behalf of Academy of Dental Materials. All rights reserved. doi:10.1016/j.dental.2007.03.008
d e n t a l m a t e r i a l s 2 4 ( 2 0 0 8 ) 178–184
the degradation of resin–dentin bonds over time. Hydrolysis is a major problem affecting the bond durability of self-etching adhesives, especially their simplified versions called “all in one” adhesives. With such systems, high amounts of solvents (water, acetone or ethanol) or hydrophilic monomers like 2 hydroxyethyl methacrylate (HEMA) have been added to their formulations to facilitate the diffusion of the resins inside the dental substrates. HEMA is amphoteric, i.e. it has the capacity to react either as an acid or a base, and displaces water in dentin but is also miscible with most of the monomers of the adhesives [4]. Water is also an essential component because it provides hydrogen ions that are necessary for demineralization. The problem is that these components raise the hydrophilicity of the adhesive, which may in turn increase the risk of water uptake into the matrix and decrease the polymerisation rate of the resins [5]. One method for assessing bond durability in vitro is to challenge the adhesive interface under a simulated oral environment [6,7]. In a recent study thermal and short-term mechanical stresses (100,000 cycles) have been shown to negatively affect the bond strength of one etch and rinse and three two-step self-etching adhesive systems [8]. Twostep self-etching adhesives performed better than simplified formulations due to the presence of a more hydrophobic adhesive layer that better protects the adhesive interface against hydrolytic degradation. Enzymatic attack also has been shown to contribute to the degradation process of resin composites [9]. In a recent in vitro study, resin–dentin specimens were stored either in a cholesterol esterase solution or in a collagenase-containing medium for 12 weeks [10]. The first solution was used as an enzymatic challenge to adhesive polymers contained in the adhesive system while the second one was used as an enzymatic challenge to demineralized dentin matrix. Significantly weaker bonds were found in specimens stored in cholesterol esterase when compared to those immersed in collagenase-containing medium. The authors concluded that the enzymatic hydrolysis of methacrylate resins might be of more clinical concern than the enzymatic hydrolysis of the collagen components of the resin–dentin bond.
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Even so, there is no in vitro study that investigated the degradation of resin–dentin interfaces made with self-etching adhesive systems simultaneously subjected to mechanical loading and enzymatic stress. It was therefore the purpose of this in vitro study to evaluate the effect of long-term cyclic loading under esterase activity on the microtensile bond strength of a two-step (AdheSE) and a one-step (Xeno III) self-etching adhesive, applied to dentin. Alterations in resin–dentin interfaces were further observed with TEM in order to evaluate the micro-morphological changes that occurred after aging.
2.
Materials and methods
The set-up used in this study is schematically represented in Fig. 1. Sixteen intact caries-free human molars were stored in 0.1% thymol solution for a maximum of 2 months until being used. The teeth were cut perpendicular to their longitudinal axis using a slow-speed diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under water cooling in order to obtain 2 mm thick dentin discs. A standard smear layer was created at the dentin surface by the use of a 40 m dia` ¨ mond bur (Coltene-Whaledent, Altstatten, Switzerland) and subsequently bonded using either AdheSE (IvoclarVivadent, Schaan, Liechtenstein) or Xeno III (DeTrey-Dentsply, Konstanz, Germany). The composition and application procedure of each material are detailed in Table 1. The adhesives were applied onto the entire dentin surface and light cured for 20 s (Optilux 501, KerrHawe, Boggio, Switzerland). A 2 mm thick layer of composite resin (Durafill, Heraeus Kulzer, Dormagen, Germany) was applied to the bonded surface and light-cured for 40 s. Each disc was then sectioned with a diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) to produce a bar specimen of 4 mm thick. Thus, each bar was made of 2 mm of composite resin bonded to 2 mm of dentin. For each material, half of the bars were subjected to microtensile testing without being loaded. The other half of the bars were challenged with mechanical and chemical stresses and then a bond strength test of the loaded area was performed. Chemical and mechanical stresses were applied to
Fig. 1 – Representation of the set-up of the study. The non-loaded groups are represented on the upper and the loaded groups on the lower part of the schema. ChE, cholinesterase; TBS, microtensile bond strength; TEM, transmission electron microscopy; mio, million; r-c bar, resin-composite bar; n, number of specimens (for microtensile evaluation) per group.
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d e n t a l m a t e r i a l s 2 4 ( 2 0 0 8 ) 178–184
Table 1 – Materials, batch numbers, composition and application procedures as used in the present investigation Material, manufacturer, batch # AdheSE; IvoclarVivadent, Schaan, Liechtenstein, F40620
Xeno III; DeTrey-Dentsply, Konstanz, Germany, 0311001490
Durafill; Heraeus Kulzer, Dormagen, Germany, 010143
Composition Primer: phosphonic acid acrylate, Bis-acrylamide, water, initiators and stabilizers Bond: Dimethacrylates, Hydroxyethyl methacrylate, highly dispersed silicon dioxide, initiators and stabilizers Liquid A: 2-hydroxyethyl methacrylate, purified water, ethanol, 2,6-Di-tertbutyl-p-hydroxy-toluene, nanofiller Liquid B: Pyro-EMA-SK (tetra-methacryl-ethyl-pyrophosphate), PEM-F (Penta-methacryl-oxy-ethyl-cyclo-phosphazenmonofluoride), UDMA, BHT (2,6-di-tert-butyl-p-cresol), camphorquinone, EPD (p-dimethyl amine ethyl benzoate) Urethanedimethacrylate, highly disperse silicon dioxide, splinter polymer
the bar specimens using a chewing machine [11,12] filled with a cholinesterase-containing solution (Cholinesterase Acetyl type VI-S, Sigma–Aldrich Chemie GmbH, Schnelldorf, Germany). The concentration of the enzyme was of 1 Unit/ml and dilution was performed in 0.02 M sodium phosphate (pH 7.0). The specimens were simultaneously submitted to a 1.2 million cyclic stress at a frequency of 1.7 Hz with a maximum loading force of 49 N. To avoid the displacement of the specimen during loading, the set-up consisted of two supports holding both ends of the specimen with a single loading pin (6 mm wide, rounded tip) located in the middle of the bar, where the force was introduced (Fig. 2). At the end of the mechanical test, the area where the load was applied was identified with an insoluble color marker. Within this marked area, six slabs were prepared by sectioning the specimen perpendicularly to the resin–dentin interface with a diamond saw. The dimension of each slab was 4.0 mm × 1.0 mm × 1.0 mm and was controlled with a digital calliper. The slabs were hand-trimmed under stereo-microscopic observation (ensuring that the narrowest area was located at the bonding interface) and the final bonded area was of about 0.8 mm2 . Four slabs were used for microtensile bond strength test and the two remaining slabs were used for TEM analysis. The same procedure was followed with the non-loaded specimens.
Fig. 2 – Schematic representation of bars’ loading.
Handling procedure Apply primer, leave for 30 s, air-dry Apply bonding, air-blow, light-cure 20 s Mix A + B for 5 s. Apply 3 layers, leave for 30 s. Dry carefully until seeing a shiny surface, then dry well to evaporate the solvent. Light-cure 20 s
Apply one layer of 2 mm thickness, light cure 40 s
Twenty-four hours after cutting the slabs for microtensile testing, they were attached to the grips of a custom-made holder with cyanoacrylate adhesive (Zapit, DVA Inc., Corona, CA, USA) and stressed to failure at a crosshead speed of 1 mm/min using a universal testing machine (Vitrodyne V1000 Universal Tester, John Chatillon and Sons, Greensboro, NC, USA). The tensile bond strength value of each slab was calculated as the force (N) at failure divided by the bonded area (mm2 ) and expressed in MPa. Slabs that failed during the sectioning process were assigned a bond strength value of 0 MPa and were included into the results. The micro-morphological evaluations were carried out on the two remaining slabs (from the six that were initially obtained) of both non-loaded and loaded specimens. The presence of silver staining and the width of adhesive interfaces created by the two adhesive systems were observed from around 24 micrographs obtained from both non-loaded and loaded specimens. The samples for TEM observation were prepared according to the protocol of Tay et al. [5]. The slabs were immersed in a tracer solution (an aqueous solution of 50 wt% ammoniacal silver nitrate) for 24 h. The silver impregnated slabs were then removed form the tracer solution and thoroughly rinsed in distilled water. They were then placed in photo developing solution for 8 h under a fluorescent light to reduce silver nitrate to metallic silver. The slabs were fixed in Karnovsky’s fixative (2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3) for a minimum of 4 h and rinsed thoroughly with 0.1 M sodium cacodylate buffer. Post-fixation was performed with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7) for 1 h at room temperature. The slabs were then rinsed three times in cacodylate buffer, dehydrated in ascending ethanol series (30–100%) and immersed in propylene oxide as a transition fluid. After resin embedding (TAAB 812 resin, TAAB Laboratories, Aldermaston, UK), 1 mm × 1 mm ultra thin sections about 90 nm thick were prepared with an ultra microtome (Reichert Ultracut S, Leica, Viena, Austria). Without any further staining the ultra thin sections were observed using a TEM (EM208S, Philips, Eindhoven, The Netherlands) operated at 80 kV.
d e n t a l m a t e r i a l s 2 4 ( 2 0 0 8 ) 178–184
Table 2 – Results of the number and percentage of pre-test failures as well as microtensile bond strength evaluation for the different adhesive systems Adhesive
Non-loaded specimens TBS mean in MPa (S.D.)
Loaded specimens TBS mean in MPa (S.D.)
AdheSE No. PTF (%) Xeno III No. PTF (%)
39.6 (14.7) 1 (6.3) 21.8 (29.8) 10 (63)
35.4 (22.1) 4 (25) 15.9 (25.5) 11 (69)
No significant differences were found between non-loaded and loaded specimens within both groups (Wilcoxon rank sum test, p > 0.05). TBS = microtensile bond strength, No. PTF (%) = number of pre-test failures (percentage of pre-test failures), S.D. = standard deviation.
The statistical analysis of the microtensile data was performed with SPSS 14.0 for Windows (SPSS Inc., Chicago, IL, USA). As the data was non-normally distributed (Kolmogorov–Smirnov Test), a Wilcoxon rank sum test was performed for comparisons between non-loaded and loaded specimens. The confidence level was set to 95%.
3.
Results
3.1.
Microtensile bond strength
The results of the microtensile bond strength evaluation are shown in Table 2. Bond strength of AdheSE was 39.6 ± 14.7 MPa on nonloaded specimens and 35.4 ± 22.1 MPa after aging; for Xeno III it was 21.8 ± 29.8 MPa on non-loaded specimens and 15.9 ± 25.5 MPa after aging. Bond strength of the materials tested was adversely affected by mechanical loading, as can be deduced from the mean values in each group. However, the difference between the values of non-loaded and loaded specimens was not significant (p > 0.05, Wilcoxon rank sum test). In the groups bonded with AdheSE, one pre-test failure (6.3% of the specimens tested) was counted on non-loaded specimens and four pre-test failures (25%) were observed on the loaded specimens. For Xeno III the situation was more dramatic, 10 pre-test failures (63%) were observed on non-loaded and 11 (69%) on loaded specimens.
3.2.
Transmission electron microscope analysis
Representative TEM micrographs of adhesive interfaces created by AdheSE and Xeno III are shown in Figs. 3 and 4. On both non-loaded and loaded specimens, the resin–dentin interfaces bonded with AdheSE were around 16–20 m (Fig. 3a). Some isolated silver grain deposits within both hybrid and adhesive layer were observed on non-loaded specimens (Figs. 3a and b). Although some less amount of silver grain deposits was observed on the loaded samples (Figs. 3c and d), no substantial differences could be observed in respect to the loaded specimens. TEM observations of the non-loaded adhesive interfaces made with Xeno III revealed an adhesive layer of approximately 8 m thick (Figs. 4a and b). This width was reduced to about 2 m after the specimens were loaded
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(Fig. 4d). In respect to nanoleakage patterns, non-loaded resin–dentin interfaces made of Xeno III exhibited important nanoleakage already before loading as represented by condensed amounts of silver stain within the hybrid layer (Fig. 4a–c). The amount of silver grains was reduced or even inexistent on the loaded specimens (Fig. 4d).
4.
Discussion
This study evaluated the effects of artificial loading on the mechanical behaviour of resin–dentin joints formed by two self-etching adhesive systems that differ in their composition and number of application steps. In order to simulate clinical conditions, artificial aging was performed by stressing the specimens up to 1.2 million load cycles at 49 N in cholinesterase solution. This enzyme is present in human saliva and may contribute to the degradation of polymeric materials on the long-term [13,14]. The resin–dentin bars were positioned in the chewing machine in such a way that they could bend during loading (Fig. 2). With this set up, the bottom side of the bar was in tension while the upper side was under compression. Both mechanisms are suspected to accelerate the degradation of bonded interfaces as has been previously shown for fiber-reinforced composites [15]. We expected that under the influence of both mechanical and chemical stress, a reduction in bond strength would be observed as previously shown by Frankenberger et al. [8]. Although a trend towards inferior bond strength was observable on the specimens after aging, the high number of pre-test failures precluded to demonstrate any significant difference between stressed and non-stressed specimens. However, it must be noted that in a similar study, Nikaido et al. [16] also failed to demonstrate any difference in bond strength between loaded and unloaded specimens. Although aging has not significantly influenced the mean microtensile bond strength of both adhesives, the number of pre-test failures observed with both systems was more informative. Whereas the incidence of pre-test failures was only of 6.3% for AdheSE, 63% of the Xeno III specimens failed before testing. Even the non-loaded specimens exhibited a high number of pre-test failures, indicating that hydrolysis has occurred even in the absence of any external stress. This result could be explained by the high water content of adhesive layers produced with Xeno III as previously shown by Chersoni et al. [17]. This problem was not observed with AdheSE, which contains a phosphonic acid compound. According to the information provided by the manufacturer, it is more hydrolytically stable when compared to phosphoric acid ester compounds used in Xeno III. Further, this material also includes bis-acrylamide, a more hydrolytically stable monomer than other hydrophilic monomers used in dentinal adhesive formulations [18]. In addition, as this material is a two-step self-etching adhesive, the separate bonding layer might have protected the adhesive interface against loading conditions and chemical challenge [8]. More interesting were the observations made under the TEM. All specimens of Xeno III showed a trend towards a reduction in thickness of the adhesive interface after loading. While the thickness of the adhesive layer in non-loaded spec-
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Fig. 3 – TEM micrographs of the resin–dentin interface bonded with AdheSE on non-loaded and loaded specimens. (a) Low magnification micrograph of an interface that is characteristic of a non-loaded specimen. Areas with sporadic reticular patterns of nanoleakage (arrow) can be observed within the hybrid layer. Note that the width of the adhesive interface is around 18 m. (b) Higher magnification of a non-loaded specimen with some isolated silver grains (black arrow) and silver-filled water channels (white arrow). (c) Micrograph of a loaded specimen where less silver infiltration can be observed in respect to the non-loaded specimens. The width of the adhesive interface is around 16 m. (d) A higher magnification of a loaded specimen with some isolated silver grains (black arrow) within the hybrid layer. Grey arrow: nanofiller clusters within the adhesive. C: resin composite; D: dentin; AL: adhesive layer; HL: hybrid layer.
imens was approximately 8 m, which is in agreement with previously published results [19], the adhesive layer was only 2 m thick after loading. This result supports the idea that adhesive interfaces produced by all-in-one adhesives behave as resin-collagen “sponges” that undergo irreversible plastic deformation under load. On the contrary, this phenomenon was not observed with AdheSE specimens. The separate bonding layer together with a more hydrolytically stable chemistry protected the adhesive form water uptake; this provided more stability to the interface and could explain why almost no decrease in the width of adhesive layer was observed after loading. Additionally, less signs of nanoleakage were observed in the specimens’ loaded area, especially of the interface created by Xeno III. We speculate that this was due to compression of the collagen network which was poorly impregnated
by the resin and that contained high amounts of water. A recent study by Chandran and Barocas [20] analysed the micromechanical behaviour of collagen gels and observed a severe collapse on the compression side. This collapse was characterized by large irreversible strains and a sharp change in fibril orientation. We presume that this collapse accounts for the less silver particles uptake in loaded specimens. A recent study evaluated the molecular interactions that take place when water diffuses through epoxy networks [21]. The authors identified mobile water localized in microvoids (free water) and water molecules firmly bound to specific sites along the resin network by hydrogen-bonding interactions (bound water). They explained that water diffusion occurs by the action of two simultaneous mechanisms: a fast diffusion process that is associated with the movement of “free water” without interacting with the network
d e n t a l m a t e r i a l s 2 4 ( 2 0 0 8 ) 178–184
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Fig. 4 – TEM micrographs of the resin–dentin interface bonded with Xeno III. (a) Interface of a non-loaded specimen. Areas with condensed silver grains (arrow) can be observed within the hybrid layer. The width of the adhesive interface is around 8 m (bar = 1 m). (b) Another cut of a non-loaded specimen with signs of nanoleakage (arrow) at the hybrid layer; the width of the adhesive interface is around 8 m (bar = 2 m). (c) Non-loaded specimen, high-magnification view of the hybrid layer with areas of extense silver staining (bar = 1 m). (d) An image that is characteristic of a loaded specimen; the width of the adhesive interface is around 2 m (bar = 0.5 m) and no signs of nanoleakage can be observed. C: resin composite; D: dentin; AL: adhesive layer; HL: hybrid layer.
and a much slower diffusion process involving interactions at the hydrogen-bonding sites of the network, being the last responsible for plasticization of the system and further degradation in structural properties. We presume that repeated water sorption/desorption during load cycles (due to the high hydrophilicity in Xeno III) caused an irreversible damage to the adhesive interface, which was evidenced by a decrease in its width. The contribution of enzymatic challenge to the degradative process of both adhesive interfaces requires further investigation. In the present study an enzyme-containing solution was only used as a model of aggressive environment. Certainly, esterase activity could have played an additional role in the degradation of the resin–dentin interface, especially for the one-step adhesive Xeno III [10]. However, future studies should be performed that compare the results of the present study with non-loaded/loaded specimens stored either in enzyme-free solutions or oil as positive control storage media. It is also necessary to determine the contribution of not
only exogenous but also endogenous enzymatic challenge to the degradation of acid-exposed dentin matrix [22]. It is known that self-etching adhesives, being acidic compounds, can activate endogenous dentin matrix metalloproteinase and contribute to the degradation of incompletely infiltrated hybrid layers in aged resin–dentin interfaces [23–26]. It would be interesting to compare for example, low performing adhesives with adhesive systems that, by completely infiltrating the demineralized collagen fibrils, may limit such enzymatic activity.
5.
Conclusion
A distinct behaviour of both self-etching adhesives, AdheSE and Xeno III, was observed when confronted to simultaneous chemical and mechanical challenge. Cyclic loading under cholinesterase activity led to an irreversible deformation in the adhesive interface of the simplified adhesive Xeno III.
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Acknowledgements [13]
This study was based on a thesis submitted to the graduate faculty, University of Siena, Italy, in partial fulfilment of the requirements for the Ph.D. degree. The authors would like to express their gratitude to Mrs. Helen Andersen (Division of Periodontology, University of Geneva) for her technical assistance.
[14]
[15]
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