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Evaluation of the micro-mechanical strength of resin bonded–dentin interfaces submitted to short-term degradation strategies
journal of the mechanical behavior of biomedical materials 15 (2012) 112 –120
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Research Paper
Evaluation of the micro-mechanical strength of resin bonded–dentin interfaces submitted to short-term degradation strategies Victor P. Feitosaa,n, Salvatore Saurob, Timothy F. Watsonb, Ame´rico B. Correra, Raquel Osorioc, Manuel Toledanoc, Lourenc- o Correr-Sobrinhoa, Ma´rio Alexandre C. Sinhoretia a
Department of Restorative Dentistry, Dental Materials Division, Piracicaba Dental School, State University of Campinas, Limeira Avenue 901, 13414-903 Piracicaba, Brazil b Biomaterials, Biomimetics and Biophotonics, King’s College London Dental Institute, Guy’s, King’s College and St. Thomas’ Hospital, London SE1 9RT, UK c Department of Dental Materials, School of Dentistry, University of Granada, Colegio Maximo, Campus de Cartuja, Avda. de las Fuerzas Armadas 1, 1B, 18014 Granada, Spain
ar t ic l e in f o
abs tra ct
Article history:
The aim of this study was to evaluate the microtensile bond strength (mTBS) and confocal
Received 18 April 2012
micropermeability of resin bonded–dentin specimens created using two representative
Accepted 18 June 2012
two-step/self-etch adhesives submitted to short-term period degradation strategies such
Available online 28 June 2012
as simulated pulpal pressure, thermo- or mechanical-cycling challenges. Clearfil SE Bond
Keywords:
(CSE) and Silorane adhesive (SIL) were bonded to flat deep dentin from seventy extracted
Hydrostatic pulpal pressure
human molars and light-cured for 10 s. Composite build-ups were constructed using with
Dental adhesives
Filtek Z350 XT and Filtek P90 respectively. The specimens of each adhesive group were
Confocal microscopy
subjected to three different accelerated aging methods: (1) thermo-cycling challenge (5000
Thermo-cycling
cycles); (2) mechanical-cycling load (200,000 cycles); (3) experiment and (4) conventional
Mechanical loading
method for simulated pulpal pressure (20 cm H2O). Control resin-bonded specimens were stored in distilled water for 24 h. mTBS and confocal microscopy (CLSM) micropermeability evaluation were performed and the results were analyzed using Two-way ANOVA and Tukey’s tests (a¼0.05). The CLSM evaluation revealed micro-cracks within the Siloranebonded dentin subsequent to mechanical-cycling load, whereas, the simulated pulpal pressure induced evident micropermeability in both bonding agents. Mechanical loading provides discernible bonding degradation in a short-term period in resin–bonded dentin created using two-step/self-etch adhesives. However, simulated pulpal pressure may reduce the sealing ability of self-etch adhesives causing greater water uptake within the resin–dentin interface. & 2012 Elsevier Ltd. All rights reserved.
n
Corresponding author. Tel./fax: þ55 19 21065345. E-mail addresses: [email protected] (V.P. Feitosa), [email protected] (S. Sauro), [email protected] (T.F. Watson), [email protected] (A.B. Correr), [email protected] (M. Toledano), [email protected] (L. Correr-Sobrinho), [email protected] (M.A.C. Sinhoreti). 1751-6161/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jmbbm.2012.06.010
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journal of the mechanical behavior of biomedical materials 15 (2012) 112 –120
1.
Introduction
Direct and indirect adhesive restorations are commonly realized in practice using resin-based materials (Ferracane, 2011). Self-etch adhesives have become increasingly popular due to more user-friendly and less technique-sensitive characteristics (Van Meerbeek et al., 2003). They do not require a separate etching step because they contain acidic monomers that simultaneously demineralize and infiltrate the dentin to create a chemical/micro-mechanical bond; thus, no discrepancy could be expected between the demineralization depth ~ et al., 2006). Neverand the resin infiltration depth (Perdigao theless, a decrease in bond strength has also been reported for this type of dental adhesive subsequent to challenge conditions (Cadenaro et al., 2005; De Munck et al., 2003; Osorio et al., 2008). Novel dentin bonding agents (DBA) are being continuously developed and launched in the market without full knowledge of their bonding efficacy and durability (Amaral et al., 2007; Feitosa et al., 2010) due to a lack of long-term in vitro investigations and/or in vivo clinical trials. Simple water immersion over ‘‘few’’ weeks may not provide sufficient degradation to challenge the bonding performance of these bonding agents bonded to dentin and enamel. Therefore, alternative in vitro short-term period degradation strategies to reveal valuable clinical information are needed (Hashimoto et al., 2004). It has been suggested that a reliable method to challenge the bonding durability should employ accelerated degradation strategies (Amaral et al., 2007), such as chemical challenge (Sauro et al., 2009) thermo- (Bedran-de-Castro et al., 2004; Feitosa et al., 2010; Ulker et al., 2010), and mechanicalcycling (Bedran-de-Castro et al., 2004; Feitosa et al., 2010; Osorio et al., 2008). Mechanical-cycling load and thermocycling testing performed in water at temperatures between
5 and 55 1C which subject the adhesive interface to water infiltration, mechanical and expansion/contraction strain (Ferracane et al., 1998; Gale and Darvell, 1999) may be considered suitable aging methods for dental adhesive materials. A further suitable strategy to challenge the resin bonded– dentin is based on the use of a simulated pulpal pressure (20 cm H2O) in order to induce water seepage, polymer degradation and droplets formation within the resin–dentin interface jeopardizing the mTBS (Bakry et al., 2009; Hosaka et al., 2007; Sauro et al., 2007; Sauro et al., 2007). The purpose of this study was to evaluate the microtensile bond strength (mTBS) and confocal micropermeability of resin bonded–dentin created with two representative ‘‘gold-standard’’ self-etch adhesive when submitted to short-term period degradation strategies such as simulated pulpal pressure, thermo- or mechanical-cycling challenges. SEM ultra-morphological analysis of the fractured specimens subsequent to challenge strategies used in this study was also performed. The null hypotheses to be tested is the strategies used in this study to accelerate degradation in a short-term period have no effect on the micro-mechanical adhesive properties (mTBS) or on the ultra-morphology/micropermeability of the resin–dentin interfaces created using self-etch adhesives.
2.
Materials and methods
2.1.
Sample preparation
Seventy human third molars extracted under the approved protocol of the appropriate institutional Research Ethics Committee (protocol 167/2009) were used in this study. The teeth were stored in 0.5% chloramine water solution for a period not exceeding 4 months at a temperature of 4 1C. Deep dentin specimens with a remaining dentin thickness (RDT) of approximately 0.9 mm were obtained by removing
Table 1 – Materials, compositions, application procedures and batches. Materials
Composition
Application procedure
Batch no.
P90 System adhesive
Self-etch primer: Vitrebond copolymer, phosphorylated methacrylates, BisGMA, HEMA, water, ethanol, silica filler, stabilizers, photoinitator Bond: Hydrophobic methacrylates, phosphorylated methacrylates, TEGDMA, silica filler, initiators, stabilizers
Apply one coat of the self-etch primer for 15 s with gentle agitation. Gently air dry. Light curing for 10 s. Apply the Bond. Gently air thin until the Bond is spread to an even film and does not move any longer. Light curing for 10 s.
9BN
Clearfil SE Bond
-Primer: MDP, HEMA, water, photoinitator -Bond: MDP, BisGMA, HEMA, hydrophobic dimethacrylates, photoinitator
Apply primer for 20 s, gently air-dry; apply bond. Light cure for 10 s
896A 1321A
Filtek P90 Shade A3
Matrix: TEGDMA, ECHCPMS Filler: Quartz filler
Apply in 1–2 mm increments and light cure for 40 s
N110333
Filtek Z350 XT Shade A3
Matrix: BisGMA, BisEMA, TEGDMA, UDMA. Filler: Silica and zirconia nanofiller
Apply in 1–2 mm increments and light-cure for 40 s
9BK
9XN
BiSGMA: bisphenol A diglycidylmethacrylate; HEMA: hydroxyethylmethacrylate; MDP: 10-methacryloyloxi decyl phophate; TEGDMA: triethylene glycol dimethacrylate; ECHCPMS: 3,4-epoxycyclohexylcyclopolymethylsiloxane; BisEMA: ethoxylated bisphenol A diglycicylmethacrylate.
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journal of the mechanical behavior of biomedical materials 15 (2012) 112 –120
the roots 2 mm below cemento–enamel junction (CEJ) and with a parallel cut at 2 mm above CEJ using a slow-speed water-cooled diamond saw (Isomet, Buehler, Lake Bluff, IL, USA). When the RDT was more than 0.9 mm, the flat dentin surface was abraded with 600-grit silicon carbide paper until obtaining the standardized RDT. The pulpal tissue was removed from the pulp chamber with small forceps without altering or scratching the pre-dentin surface along the walls of the pulpal chamber. The dentin surface was wet-polished with 600-grit SiC papers for 1 min to create a standard smear layer prior to bonding procedures (Hamouda et al., 2011).
2.2.
Experimental design and bonding procedures
The dentin specimens were divided randomly into two principal groups (n¼ 35) based on the bonding agents used in this study: Clearfil SE Bond (CSE: Kuraray Medical Inc., Tokyo, Japan) and Silorane adhesive (SIL: 3M ESPE, St. Paul, MN, USA). The composition, application procedures and batch numbers are listed in Table 1. All the bonding and restorative procedures were performed in absence of positive pulpal pressure. Resin composite build-ups were constructed in 6 layers (1 mm thick) up to 6 mm [Filtek Z350 XT (3M ESPE)/Clearfil SE Bond; Filtek P90 (3M ESPE)/Silorane adhesive]. Photoactivation of the resin-based materials was performed using a quartz–tungsten–halogen lamp XL-2500 (3M ESPE). The output intensity was monitored with a Demetron Radiometer (Model 100, Demetron Research, Danbury, CT, USA) to maintain a minimal light output intensity of 600 mW/cm2 throughout all the experiments. All materials were used following the manufacturers’ recommendations (Table 1). Subsequent to the bonding procedures, the specimens of each principal group were divided into sub-groups (n¼ 7), based on the challenge test: (1) CTRL: water immersion 24 h; (2) TC: thermocycling; (3) MCL: mechanical-cycling; (4) CPP: conventional simulated pulpal pressure; and (5) EPP: experimental simulated pulpal pressure (Fig. 1).
2.3.
Simulated pulpal pressures challenge
The bonded-dentin specimens were submitted to a simulated hydrostatic intra-pulpal pressure by using two different techniques (Fig. 1). A conventional simulated pulpal pressure (CPP) required that the specimens were submitted to simulated pulpal pressure where the water pressure was implemented 1 h after the restorative procedure in order to simulate the vasoconstrictor effect of local anesthesia on pulpal tissue (Sauro et al., 2007). The CPP was performed according to a protocol previously described (Sauro et al., 2007). Briefly, the crown segments were fixed to plexiglass plates by using cyanoacrylate glue which was perforated by an 18-gauge stainless steel tube (Fig. 1). Distilled water was perfused into the pulpal chamber using a pressure of 20 cm H2O via a hydraulic pressure device (Fig. 1). The water pressure was maintained for one week and subsequently disconnected and sectioned for CLSM and mTBS evaluation. An experimental simulated pulpal pressure method (EPP) was also used. This method required that the resin-bonded specimens were covered with two coats of nail varnish and positioned sideways on the lid of a cylindrical receptacle using dental wax (Fig. 1). The pulp chamber of each specimen was glued on the
Fig. 1 – Schematic drawing of the four aging treatments. Conventional simulated pulpal pressure and the experimental method to simulate pulpal pressure are illustrated in the upper two boards. Mechanical- and thermo-cycling methods are illustrated in the lower two boards. Description of the new method of simulated pulpal pressure: section the teeth to obtain a flat dentin surface with a mean remaining dentin thickness of 0.9 mm, similar to the protocol used in the conventional method. Undertake bonding and restorative procedures. Apply two coats of nail varnish on enamel–composite border. Fix the samples by sideways in a container lid with wax without obstructing the pulpal chamber opening. Fill the cylindrical container with distilled water to reach 20 cm and close the container with samples attached in the lid. Turn the container upside down to submit the samples to 20 cm H2O pulpal pressure.
base of the system and filled with distilled water. The lid was subsequently sealed to a cylindrical receptacle containing 20 cm of distilled water and turned upside down. Thus, the samples had a 20 cm water column over them according to the classic method and hydrostatic pressure equation: P¼gdh, where P is the hydrostatic pressure, g is the gravity, d is the liquid density and h is the liquid height. The water pressure was maintained for one week, prepared and evaluated as previously described.
2.4.
Thermal and mechanical-cycling challenges
The resin-bonded specimens were submitted to thermalcycling challenge using a thermo-cycling machine (MSCT-3, ~ Carlos, SP, Brazil) proMarcelo Nucci ME Instrument, Sao grammed to perform 5000 thermal-cycles during one week at temperatures between 5 1C and 55 1C, with a dwell time of 30 s at each bath temperature (Fig. 1) (Ulker et al., 2010). The mechanical cycling was executed using the MSCM equipment (Marcelo Nucci ME Instrument) which has a stainless steel tip of 4 mm in diameter in contact with the central part of the
journal of the mechanical behavior of biomedical materials 15 (2012) 112 –120
restored specimens. All the resin-bonded specimens were submitted to 200,000 mechanical cycles under a load of 50 N, at a rate of 2 Hz for one week (Ulker et al., 2010).
2.5.
Microtensile bond strength testing (lTBS)
The resin-bonded specimens prepared for this group were sectioned occluso-gingivally in serial slabs (1 mm thick) using a diamond-embedded blade under continuous water irrigation (Buehler, Lake Bluff, IL, USA) and subsequently in 1 mm2 match-sticks. Five specimens were used for each group (n¼ 5). The match-sticks from the most peripheral area presenting residual enamel were excluded. The selected match-sticks were fixed to a jig with cyanoacrylate glue and tested to failure under tension in the universal testing machine EZ-test (Shimadzu Co., Kyoto, Japan) with a 500N load cell, at a crosshead speed of 1.0 mm/min. The exact cross-sectional area of each tested sticks was measured after failure with a digital caliper (Mitutoyo Co. Tokyo, Japan). Means and standard deviation were calculated and expressed in MPa. Five restored teeth (experimental unit, n¼ 5) were evaluated in each group, the bond strength of sticks from the same restored teeth was averaged and the mean bond strength was used as one statistical unit for the statistical analysis. The microtensile bond strength (mTBS) data were statistically analyzed using twoway ANOVA (DBA and aging regime) to identify differences among groups. When significant differences were found, they were compared using Tukey’s test at a¼ 0.05.
2.6. Failure mode analysis and scanning electron microscopy ultra-morphology Subsequent to the mTBS tests, the mode of failure of the fractured specimens was determined by stereomicroscopy at 60x magnification. The fractures were classified as follows: Type A: Adhesive failure at the interface among resin composite, adhesive resin and hybrid layer. Type M: Mixed failure. Both adhesive and cohesive failures observed in the same fractured stick. Type C: Cohesive failure in resin composite. Type D: Cohesive failure in dentin. Five representative samples exhibiting the most frequently observed failure pattern (i.e. mixed or cohesive) and the mTBS close to the mean, were prepared for scanning electron microscopy (SEM) ultra-morphology analysis. The parts of the fractured samples were paired and mounted on aluminum stubs, coated with gold (Balzers SCD 050 sputter coater, B.U.A. Co., Fu¨rstentum, Germany) and examined by SEM, JSM-5600LV (JEOL, Tokyo, Japan), operated at 15 kV, with 20 mm as work distance.
2.7.
Confocal micropermeability evaluation (CLSM)
Two specimens for each sub-group were prepared as previously described and prepared for confocal micropermeability evaluation (Sauro et al., 2012). Briefly, the evaluation of micropermeability of resin–dentin interfaces was performed using a
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0.1 wt% lucifer yellow water solution (LY: Sigma Chemicals, St. Louis, MO, USA). This fluorescent dye was used to trace the water-filled spaces and the sealing ability from the pulp chamber through the tubules to the resin–dentin interface for 3 h. The specimens were subsequently cut into 1 mm slabs, slightly polished with wet 1200 SiC-grit paper and ultrasonicated for 5 min. Three slabs from each specimen were selected for confocal microscopy (6 slabs/group). Three images were obtained from each slice resulting in nine images per specimen and 18 images per group. These images were intended to be representative of the most common features regarding the micropermeability observed along the interfaces. The microscopy examination was performed using a laser confocal scanning microscope (CLSM: Leica SP2 CLSM, Heidelberg, Germany) equipped with a 63x/1.4 NA oil immersion lens using 514 nm argon/helium ion laser illumination both in reflection and fluorescence mode. Reflected and fluorescence signals were detected with a photomultiplier tube to a depth of 20 mm and then converted to single-projection images for better visualization and qualitative analysis (Sauro et al., 2012).
3.
Results
3.1.
Microtensile bond strength testing (lTBS)
Mean bond strength was affected by dental adhesives (po0.001) and aging groups (p ¼0.001). Interactions were not significant between both factors (p¼ 0.173). Therefore, the comparison between the DBAs has no correlation with the accelerated degradation strategies; thus, the comparisons between the degradation strategies should be performed regardless the DBA. The differences between treatments were highly significant as well as the tested DBAs (po0.001). Microtensile bond strength results are depicted in Table 2. Clearfil SE Bond (CSE) showed higher mTBS values than Silorane adhesive (SIL) for all aging treatments (po0.001). Regarding the accelerated degradation strategies, the statistical analysis showed significant changes in mechanicalcycling load as well as, thermal-cycling and mechanicalcycling compared to the control groups (p¼ 0.002), (Table 2). Despite the absence of significant interactions in the CSE group, no difference was found between the accelerated bonding degradation strategies (p40.05). SIL showed statistical difference between control and mechanical-cycling Table 2 – Means (standard deviations) lTBS of each group in MPa. Aging regimen
Silorane adhesive
Clearfil SE Bond
Mean
Control Thermal cycling Conventional PP Experimental PP Mechanical cycling
40.8 41.1 34.2 33.8 32.5
47.1 46.9 46.5 46.8 40.7
43.97a 44.03a 40.35ab 40.30ab 36.61b
Mean
36.50B
(4.3) (4.5) (4.3) (4.6) (4.5)
(3.4) (4.4) (4.0) (3.5) (3.1)
45.60A
Different capital letters represent statistical significant difference between adhesives (po0.05). Different lowercase letters represent
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journal of the mechanical behavior of biomedical materials 15 (2012) 112 –120
strain (p¼ 0.021) and between thermo-cycling and mechanical-cycling strain (p¼ 0.016); no difference was found between the conventional and the experimental pulpal pressure as well as thermo-cycling and control.
3.2. Failure mode analysis and scanning electron microscopy ultra-morphology The percentages of fracture types for each group are summarized in Fig. 2. Representative SEM micrographs are shown in Fig. 3. The type A (adhesive failure at the resin composite/ adhesive/dentin interface) failure pattern was predominantly observed in the de-bonded specimens of the CSE control, mechanical- and thermo-cycling groups. SIL showed type A failure in the de-bonded specimens submitted to both simulated pulpal pressure methods. The specimens of the SIL group submitted to thermo- and mechanical-cycling strains showed prevalently a type M failure (mixed failures, partially adhesive and cohesive) as well as those of the CSE specimens submitted to both simulated pulpal pressure. The control group in SIL resulted in 50% adhesive and 50% mixed failure. Fractured match-sticks of SIL after mechanical-cycling load showed micro-cracks within the hybrid and adhesive layers (Fig. 3B). Specimens subjected to both methods of simulated pulpal pressure showed a characteristic de-bonding at the interdiffusion layer (IDL), (Fig. 3C).
3.3.
Confocal micropermeability evaluation (CLSM)
CLSM results are presented in Figs. 4 and 5. The high dye penetration was observed within the resin–dentin interfaces of all the specimens submitted to both methods of simulated pulpal pressure (Figs. 4B and 5B). Thermo- and mechanicalcycling strain induced similar micropermeability within the
Coh Dentine
Coh Comp
Mixed
Adhesive
SIL - CPP
SIL - EPP
SIL - MC
SIL - TC
SIL - Control
CSE - CPP
CSE - EPP
CSE - MC
CSE - TC
CSE - Control
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
Fig. 2 – Graph presenting the fractures patterns (%) of debonded specimens. CSE means Clearfil SE Bond. SIL means Silorane adhesive. MC, TC, CPP and EPP mean mechanical cycling, thermo-cycling, conventional simulated pulpal pressure and experimental simulated pulpal pressure respectively.
adhesive and IDL of Clearfil SE Bond (CSE) compared to the control groups (24 h) (Fig. 5A, C and D). Similarly, Thermo- and mechanical-cycling strain induced dye penetration (micropermeability) only into dentinal tubules for CSE (Fig. 5C and D). Both methods for the simulation of the pulpal pressure promoted a slight increase in micropermeability within the IDL of CSE (Fig. 5B). The resin–dentin interface of Silorane adhesive group (SIL) reduced adhesive layer thickness subsequent to mechanical-cycling load strain (Fig. 4C). This phenomenon was not observed in the resin–dentin interface created with Clearfil SE Bond adhesive. Thermo-cycling strain slightly increased the micropermeability within the IDL of SIL (Fig. 4D). Mechanicalcycling load led to the formation of micro-cracks along IDL and the adhesive layer revealing visible micropermeability (Fig. 4C1). Both methods of simulated pulpal pressure increased the micropermeability within the IDL and adhesive layer of Silorane adhesive (Fig. 4B and B1).
4.
Discussion
The outcomes of the present study showed a statistical difference (po0.05) in mTBS results between control (24 h/H2O) and the mechanical-load (200,000 cycles) groups but no significant difference (p40.05) by the other degradation strategies. Furthermore, both methods for simulated pulpal pressure and the mechanical-cycling load strain induced important changes in the micropermeability within the resin–dentin interfaces created with the two two-step/self-etching adhesives tested in this study. Therefore, the first null hypothesis that there is no difference between the strategies used in this study to accelerate degradation in a short-term period have no effect on the micromechanical adhesive properties (mTBS) or on the ultra-morphology/micropermeability of the resin–dentin interfaces created using self-etch adhesives must be partially rejected. The most popular approach to induce bonding degradation is submerging resin–dentin match-sticks in water or phosphate buffer solutions for a minimal period of 3 months in order to contrast significant differences between groups (Van Meerbeek et al., 2010). Despite the water degradation strategy results are time demanding, a thermo-cycling approach may be a suitable method to induce bonding degradation in a short-term period (Amaral et al., 2007). This latest strategy might simulate an in vivo challenge where the restorations are submitted to thermal changes in oral environment occurring during eating and drinking (Amaral et al., 2007; Feitosa et al., 2010). Moreover, the aging effect induced by thermo-cycling relies on the ability of hot water to accelerate the hydrolysis of nonprotected collagen and leaching of poorly polymerized resin monomers (Ferracane et al., 1998). The interface undergoes the process known as plasticization, with a decrease in its mechanical properties (De Munck et al., 2005). However, Van Meerbeek et al. (2010) have recently stated that the thermo-cycling strategy to accelerate bonding degradation must be performed using a number of cycles up to 100,000 to discriminate significant differences. They also highlighted that the ISO’s recommendations of thermocycling regimens (500 cycles) are of little use, since most dental bonding agents (DBAs) would not reveal significant bonding degradation. Nikaido et al. (2002) obtained decreased
journal of the mechanical behavior of biomedical materials 15 (2012) 112 –120
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Fig. 3 – SEM micrographs showing representative fractures of debonded specimens. c—composite, hy—hybrid layer and a—adhesive. (A) Resin side of a de-bonded stick bonded with CSE after one week of conventional simulated pulpal pressure. The predominant fracture pattern in this group was mixed, and this SEM image illustrates this pattern. (B) Dentin side of a de-bonded match-stick of SIL after mechanical cycling loading. A mixed failure (predominant pattern) is shown in the micrograph. Note some microcracks along hybrid and adhesive layer (white circle) in agreement with confocal microscopy outcomes. (C) SEM image of the dentin side of a de-bonded stick of SIL after experimental simulated pulpal pressure. The adhesive failure image illustrates deterioration of hybrid layer above and around dentinal tubules (white circle) with resin remnants above intertubular dentin whereas no resin remnant could be observed above dentinal tubules and peritubular dentin. (D) Image of a mixed failure (predominant pattern) in the dentin side from a de-bonded specimen of SIL after thermo-cycling.
bond strength only after a higher number of thermo-cyclings in higher C-factor Class I cavities. The results of this study showed that a thermo-cycling challenge performed with 5000 cycles (5 1C and 55 1C) may not be an appropriate strategy to induce statistical change in mTBS in specimens bonded with gold-standard two-step selfetch bonding agents (Table 2). Moreover, the thermo-cycling regimen used in this study provoked no change in micropermeability within the resin–dentin interface created both with SIL (Fig. 4D) and CSE (Fig. 5D) compared to those of the control group (Figs. 4A and 5A). These types of adhesives require a separate application of a hydrophobic resin layer which provides a substantial improvement for bonding stability (Lodovici et al., 2009; Reis et al., 2008; Va´squez et al., 2009) and it likely contributes for bonding preservation against thermal challenge; a longer thermocycling regime is required to create discernible bonding degradation and significant differences in mTBS (De Munck et al., 2005; Van Meerbeek et al., 2010). Conversely, the short-term mechanical-cycling challenge (200,000 cycles in 7 days/2 Hz frequency) was the exclusive artificial aging regimen to promote significant decrease in bond strength (Table 2). The CLSM micropermeability results obtained within the resin–dentin interface created with both the tested DBAs submitted to mechanical-cycling challenge showed no dissimilarity compared to those obtained in the control group (24 h/H2O).
However, a thinner adhesive layer could be observed for SIL after mechanical loading (Fig. 4C), whereas no striking alteration was found for CSE. Since the DBA–Silorane requires a double photo-activation both for the primer and for the bond, we believe that remaining primer was incompletely polymerized due to its hydrophilic and water permeable nature and may have been deformed by the mechanical-cycling (Sauro et al., 2011). Indeed, the application of the Silorane bond layer after primer photo-activation may not overcome the compression stresses of mechanical loading, yielding reduction in adhesive layer thickness without altering the micropermeability (Frankenberger et al., 2002). Conversely, the primer of CSE is not separately light-cured, thus, this phenomenon did not occur (Fig. 5). This hypothesis may be confirmed by the results obtained during the confocal micropermeability evaluation which showed the presence of some micro-cracks within this thinner Silorane adhesive layer as well as the IDL (Fig. 4C1). These micro-cracks (Fig. 3B) may be also responsible for the reduced bond strength and higher mixed failures of debonded specimens obtained in the mechanical-cycling group (Fig. 2). Furthermore, despite the immediate acceptable results of Silorane adhesive, this DBA was more prone to degradation under artificial aging regimes. The SIL degradation may be explained by the presence of silane agents and siloxane groups in the adhesive composition, which are more easily hydrolytically degradable (Duarte et al., 2009). The intervening zone in the adhesive layer between the cured primer
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Fig. 4 – 3D confocal-single projections (reflection/fluorescence) of Silorane adhesive showing the representative micropermeabilities of tested groups. c—composite, a—adhesive and dt—dentin. (A) Reflection/fluorescence image of lucifer yellow uptake in control group. (A1) Higher magnification (fluorescence image) of control group. Visible micropermeability into the tubules and within the interdiffusion layer (IDL). (B) Reflection/fluorescence image observed from experimental simulated pulpal pressure group. Note the higher micropermeability under hydrostatic pressure (open arrow). (B1) Fluorescence image of new method of simulated pulpal pressure with higher magnification. Visible lucifer yellow uptake into the dentinal tubules (open arrow), IDL and reaching the adhesive layer. (C) Resin–bonded dentin interface represented by reflection/fluorescence image after the mechanical loading stress. Note the reduced thickness of adhesive layer (double arrows) due to compression induced by mechanical cyclings. (C1) Higher magnification of Silorane adhesive under loading (fluorescence image). Visible micropermeability into the tubules, IDL and into the microcracks along the adhesive/primer layer (arrows). (D) Resin–bonded dentin interface after thermocyclings (reflection/fluorescence image). Visible micropermeability in IDL. Note the similarity of this group and control group. (D1) Higher magnification of Silorane adhesive under thermocycling (fluorescence image). Visible micropermeability into the tubules, IDL and adhesive/primer layer.
and the bonding layer also contributes to the bond instability of SIL after aging regimes, since this zone is more prone to hydrolysis and silver uptake (Duarte et al., 2009). The presence of a physiological pulpal pressure plays an important role in adhesive dentistry (Hosaka et al., 2007; Lin et al., 2010) thus its simulation for in vitro studies concerns a fundamental approach to challenge bonding of resin-based materials to dentin (Sauro et al., 2007; Van Meerbeek et al., 2010). Nevertheless, few investigations have been performed using this methodology, probably due to the laborious methodology required during the simulation of the pulpal pressure. An easier and standardized protocol to simulate pulpal pressure and test adhesive permeability is currently necessary (Van Meerbeek et al., 2010). The conventional simulated PP method requires the use of plexiglass or acrylic plate perforated by an 18-gauge stainless steel tube where the dentin specimen is attached using cyanoacrylate glue and a water column apparatus with water level 15–20 cm above the specimen (Fig. 1). However, when the water column creates the hydrostatic pressure inside the pulp the cyanoacrylate cementation might allow water seepage through the glue compromising the stability of water pressure inside the pulpal chambers.
The experimental methodology presented in this study to simulate PP requires no use of cyanoacrylate glue cementation, plexiglass plate and stainless steel tube providing an easier and effortless maintenance of hydrostatic pressure (Fig. 1, upper board). The specimens are attached to a cylindrical receptacle lid this way, and the receptacle is filled with distilled water until the water level reaches a height of 20 cm. Moreover, many specimens can be included in the same container and subjected to simulated pulpal pressure together. The mTBS and confocal micropermeability results obtained in this in vitro study ratify the resemblance between the two methods, since the microtensile bond strengths were numerically and statistically similar for both DBAs; the micropermeabilities were also comparable regardless of the DBA. The confocal micropermeability evaluation revealed discernible higher micropermeability for both the simulated pulpal pressure methods in comparison with control groups. However, although the presence of a positive pressure induced micropermeability within the interdiffusion layers (Figs. 4B and 5B) in the one week period-time, it induced no significant statistical differences in bond strength reduction for CSE and SIL. Sauro et al. (2009) showed acceptable dentin
journal of the mechanical behavior of biomedical materials 15 (2012) 112 –120
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Fig. 5 – 3D confocal-single projections (reflection/fluorescence) of Clearfil SE Bond showing the representative micropermeabilities. (A) Reflection/fluorescence image of lucifer yellow uptake in control group. Visible micropermeability into the tubules (open arrow) and within the interdiffusion layer (IDL). (B) Reflection/fluorescence image observed after one-week underconventional simulated pulpal pressure. Note the higher micropermeability in IDL under hydrostatic pressure. (C) Resin–bonded dentin interface represented by fluorescence image after the 200,000 mechanical loading regimen. Note visible micropermeability into the tubules (open arrow) and IDL. (D) Resin–-bonded dentin interface after 5000 thermo-cyclings (fluorescence image). Visible micropermeability into dentinal tubules and IDL, especially at the bottom of the IDL (open arrow). Note the similarity of this group, mechanical loading and control groups.
sealing ability of Silorane adhesive bonded to dentin while, further investigations depicted the low permeability of CSE (Kim et al., 2010; Sauro et al., 2007; Sword et al., 2011). Also in this case, the low permeability of these DBAs is likely to be provided by the hydrophobic adhesive resin that is applied as a separate step (Kim et al., 2010). This hydrophobic layer may achieve maintenance of bond strengths (Hosaka et al., 2007) and preservation of bonding integrity, even with the observed higher lucifer yellow uptake. Two DBAs were used in the present investigation to exclude potential misinterpretation of outcomes related with usage of a single adhesive. However, the micropermeability can reveal resistance against water penetration of IDL and adhesive layers and may predict the relative durability of the bonded dentin interface.
5.
Conclusion
In conclusion, mechanical-cycling strain performed under a regimen of 200,000 cycles (50 N-load/2 Hz-rate) for one week in combination with the use of simulated pulpal pressure performed with the ‘‘easy’’ method proposed in this study may be a suitable strategy for an effective short-term artificial aging to test the bonding performance of ‘‘high-performance’’ DBAs. Further investigations are ongoing to evaluate the effects of the cycling-load in combination of simulated pulpal pressure on other types of DBAs. It is also under investigation whether
a longer period under simulated pulpal pressure (20 cm H2O) may affect the mTBS of two-step/self-etching adhesives.
Acknowledgments This work was supported by the CAPES—Brazil, the Center of Excellence in Medical Engineering funded by the Wellcome trust and the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy’s & St Thomas’ NHS Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust. The authors also thank Dr. Flavia Gouvea da Costa for providing sound teeth in this investigation. The authors have no financial affiliation or involvement with any commercial organization with direct financial interest in the materials discussed in this manuscript. Any other potential conflict of interest is disclosed.
r e f e r e nc e s
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Bakry, A.S., Nakajima, M., Otsuki, M., Tagami, J., 2009. Effect of Er:YAG laser on dentin bonding durability under simulated pulpal pressure. Journal of Adhesive Dentistry 11, 361–368. Bedran-de-Castro, A.K., Pereira, P.N., Pimenta, L.A., Thompson, J.Y., 2004. Effect of thermal and mechanical load cycling on microtensile bond strength of a total-etch adhesive system. Operative Dentistry 29, 150–156. Cadenaro, M., Antoniolli, F., Sauro, S., Tay, F.R., Di Leonarda, R., Prati, C., Biasotto, M., Contardo, L., Breschi, L., 2005. Degree of conversion and permeability of dental adhesives. European Journal of Oral Sciences 113, 525–530. De Munck, J., Van Landuyt, K., Peumans, M., Poitevin, A., Lambrechts, P., Braem, M., Van Meerbeek, B., 2005. A critical review of the durability of adhesion to tooth tissue: methods and results. Journal of Dental Research 84, 118–132. De Munck, J., Van Meerbeek, B., Yoshida, Y., Inoue, S., Vargas, M., Suzuki, K., Lambrechts, P., Vanherle, G., 2003. Four-year water degradation of total-etch adhesives bonded to dentin. Journal of Dental Research 82, 136–140. Duarte Jr., S., Phark, J., Varjao, F.M., Sadan, A., 2009. Nanoleakage, ultramorphological characteristics, and microtensile bond strengths of a new low-shrinkage composite to dentin after artificial aging. Dental Materials 25, 589–600. Feitosa, V.P., Medina, A.D., Puppin-Rontani, R.M., Correr-Sobrinho, L., Sinhoreti, M.A., 2010. Effect of resin coat technique on bond strength of indirect restorations after thermal and load cycling. Bulletin of Tokyo Dental College 51, 111–118. Ferracane, J.L., Berge, H.X., Leung, B.W., 1998. In vitro aging of dental composites in water—effect of degree of conversion, filler volume, and filler/matrix coupling. Journal of Biomedical Materials Research 42, 465–472. Ferracane, J.L., 2011. Resin composite—state of the art. Dental Materials 27, 29–38. ~ J., Ambrose, W.W., Rosa, B.T., Frankenberger, R., Lopes, M., Perdigao, 2002. The use of flowable composites as filled adhesives. Dental Materials 18, 227–238. Gale, M.S., Darvell, B.W., 1999. Thermal cycling procedures for laboratory testing of dental restorations. Journal of Dentistry 27, 89–99. Hamouda, I.M., Samra, N.R., Badawi, M.F., 2011. Microtensile bond strength of etch and rinse versus self-etch adhesive systems. Journal of the Mechanical Behavior of Biomedical Materials 4, 461–466. Hashimoto, M., Ito, S., Tay, F.R., Svizero, N.R., Sano, H., Kaga, M., Pashley, D.H., 2004. Fluid movement across the resin–dentin interface during and after bonding. Journal of Dental Research 83, 843–848. Hosaka, K., Nakajima, M., Monticelli, F., Carrilho, M., Yamauti, M., Aksornmuang, J., Nishitani, Y., Tay, F.R., Pashley, D.H., Tagami, J., 2007. Influence of hydrostatic pulpal pressure on the microtensile bond strength of all-in-one self-etching adhesives. Journal of Adhesive Dentistry 9, 437–442. Kim, S.Y., Ferracane, J.L., Kim, H.Y., Lee, I.B., 2010. Real-time measurement of dentinal fluid flow during amalgam and composite restoration. Journal of Dentistry 38, 343–351. Lin, J., Mehl, C., Yang, B., Kern, M., 2010. Durability of four composite resin cements bonded to dentin under simulated pulpal pressure. Dental Materials 26, 1001–1009. Lodovici, E., Reis, A., Geraldeli, S., Ferracane, J.L., Ballester, R.Y., Rodrigues Filho, L.E., 2009. Does adhesive thickness affect
resin–dentin bond strength after thermal/load cycling?. Operative Dentistry 34, 58–64. Nikaido, T., Kunzelmann, K.H., Chen, H., Ogata, M., Harada, N., Yamaguchi, S., Cox, C.F., Hickel, R., Tagami, J., 2002. Evaluation of thermal cycling and mechanical loading on bond strength of a self-etching primer system to dentin. Dental Materials 18, 269–275. Osorio, R., Pisani-Proenca, J., Erhardt, M.C., Osorio, E., Aguilera, F.S., Tay, F.R., Toledano, M., 2008. Resistance of ten contemporary adhesives to resin–dentin bond degradation. Journal of Dentistry 36, 163–169. ~ J., Gomes, G., Gondo, R., Fundingsland, J.W., 2006. In vitro Perdigao, bonding performance of all-in-one adhesives. Part I— microtensile bond strengths. Journal of Adhesive Dentistry 8, 367–373. Reis, A., Albuquerque, M., Pegoraro, M., Mattei, G., Bauer, J.R.O., Grande, R.H., Klein-Junior, C.A., Baumhardt-Neto, R., Loguercio, A.D., 2008. Can the durability of one-step self-etch adhesives be improved by double application or by an extra layer of hydrophobic resin?. Journal of Dentistry 36, 309–315. Sauro, S., Mannocci, F., Toledano, M., Osorio, R., Pashley, D.H., Watson, T.F., 2009. EDTA or H3PO4/NaOCl dentin treatments may increase hybrid layers’ resistance to degradation: a microtensile bond strength and confocal-micropermeability study. Journal of Dentistry 37, 279–288. Sauro, S., Watson, T.F., Thompson, I., Toledano, M., Nucci, C., Banerjee, A., 2012. Influence of air-abrasion executed with polyacrylic acid-Bioglass 45S5 on the bonding performance of a resin-modified glass ionomer cement. European Journal of Oral Sciences 120, 168–177. Sauro, S., Pashley, D.H., Montanari, M., Chersoni, S., Carvalho, R.M., Toledano, M., Osorio, R., Tay, F.R., Prati, C., 2007. Effect of simulated pulpal pressure on dentin permeability and adhesion of self-etch adhesives. Dental Materials 23, 705–713. Sauro, S., Toledano, M., Aguilera, F.S., Mannocci, F., Pashley, D.H., Tay, F.R., Watson, T.F., Osorio, R., 2011. Resin–dentin bonds to EDTA-treated vs. acid-etched dentin using ethanol wet-bonding. Part II: effects of mechanical cycling load on microtensile bond strengths. Dental Materials 27, 563–572. Sword, R.J., Sword, J.J., Brackett, W.W., Tay, F.R., Pashley, D.H., Garcı´a-Godoy, F., 2011. New method of measuring water permeability of adhesive resin films. American Journal of Dentistry 24, 20–24. Ulker, M., Ozcan, M., Sengu¨n, A., Ozer, F., Belli, S., 2010. Effect of artificial aging regimens on the performance of self-etching adhesives. Journal of Biomedical Materials Research Part B: Applied Biomaterials 93, 175–184. Van Meerbeek, B., De Munck, J., Yoshida, Y., Inoue, S., Vargas, M., Vijay, P., Van Landuyt, K., Lambrechts, P., Vanherle, G., 2003. Buonocore memorial lecture. adhesion to enamel and dentin: current status and future challenges. Operative Dentistry 28, 215–235. Van Meerbeek, B., Peumans, M., Poitevin, A., Mine, A., Van Ende, A., Neves, A., De Munck, J., 2010. Relationship between bond strength tests and clinical outcomes. Dental Materials 26, 100–121. Va´squez, V.Z., Ozcan, M., Kimpara, E.T., 2009. Evaluation of interface characterization and adhesion of glass ceramics to commercially pure titanium and gold alloy after thermal- and mechanical-loading. Dental Materials 25, 221–231.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jmbbm
Research Paper
Evaluation of the micro-mechanical strength of resin bonded–dentin interfaces submitted to short-term degradation strategies Victor P. Feitosaa,n, Salvatore Saurob, Timothy F. Watsonb, Ame´rico B. Correra, Raquel Osorioc, Manuel Toledanoc, Lourenc- o Correr-Sobrinhoa, Ma´rio Alexandre C. Sinhoretia a
Department of Restorative Dentistry, Dental Materials Division, Piracicaba Dental School, State University of Campinas, Limeira Avenue 901, 13414-903 Piracicaba, Brazil b Biomaterials, Biomimetics and Biophotonics, King’s College London Dental Institute, Guy’s, King’s College and St. Thomas’ Hospital, London SE1 9RT, UK c Department of Dental Materials, School of Dentistry, University of Granada, Colegio Maximo, Campus de Cartuja, Avda. de las Fuerzas Armadas 1, 1B, 18014 Granada, Spain
ar t ic l e in f o
abs tra ct
Article history:
The aim of this study was to evaluate the microtensile bond strength (mTBS) and confocal
Received 18 April 2012
micropermeability of resin bonded–dentin specimens created using two representative
Accepted 18 June 2012
two-step/self-etch adhesives submitted to short-term period degradation strategies such
Available online 28 June 2012
as simulated pulpal pressure, thermo- or mechanical-cycling challenges. Clearfil SE Bond
Keywords:
(CSE) and Silorane adhesive (SIL) were bonded to flat deep dentin from seventy extracted
Hydrostatic pulpal pressure
human molars and light-cured for 10 s. Composite build-ups were constructed using with
Dental adhesives
Filtek Z350 XT and Filtek P90 respectively. The specimens of each adhesive group were
Confocal microscopy
subjected to three different accelerated aging methods: (1) thermo-cycling challenge (5000
Thermo-cycling
cycles); (2) mechanical-cycling load (200,000 cycles); (3) experiment and (4) conventional
Mechanical loading
method for simulated pulpal pressure (20 cm H2O). Control resin-bonded specimens were stored in distilled water for 24 h. mTBS and confocal microscopy (CLSM) micropermeability evaluation were performed and the results were analyzed using Two-way ANOVA and Tukey’s tests (a¼0.05). The CLSM evaluation revealed micro-cracks within the Siloranebonded dentin subsequent to mechanical-cycling load, whereas, the simulated pulpal pressure induced evident micropermeability in both bonding agents. Mechanical loading provides discernible bonding degradation in a short-term period in resin–bonded dentin created using two-step/self-etch adhesives. However, simulated pulpal pressure may reduce the sealing ability of self-etch adhesives causing greater water uptake within the resin–dentin interface. & 2012 Elsevier Ltd. All rights reserved.
n
Corresponding author. Tel./fax: þ55 19 21065345. E-mail addresses: [email protected] (V.P. Feitosa), [email protected] (S. Sauro), [email protected] (T.F. Watson), [email protected] (A.B. Correr), [email protected] (M. Toledano), [email protected] (L. Correr-Sobrinho), [email protected] (M.A.C. Sinhoreti). 1751-6161/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jmbbm.2012.06.010
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journal of the mechanical behavior of biomedical materials 15 (2012) 112 –120
1.
Introduction
Direct and indirect adhesive restorations are commonly realized in practice using resin-based materials (Ferracane, 2011). Self-etch adhesives have become increasingly popular due to more user-friendly and less technique-sensitive characteristics (Van Meerbeek et al., 2003). They do not require a separate etching step because they contain acidic monomers that simultaneously demineralize and infiltrate the dentin to create a chemical/micro-mechanical bond; thus, no discrepancy could be expected between the demineralization depth ~ et al., 2006). Neverand the resin infiltration depth (Perdigao theless, a decrease in bond strength has also been reported for this type of dental adhesive subsequent to challenge conditions (Cadenaro et al., 2005; De Munck et al., 2003; Osorio et al., 2008). Novel dentin bonding agents (DBA) are being continuously developed and launched in the market without full knowledge of their bonding efficacy and durability (Amaral et al., 2007; Feitosa et al., 2010) due to a lack of long-term in vitro investigations and/or in vivo clinical trials. Simple water immersion over ‘‘few’’ weeks may not provide sufficient degradation to challenge the bonding performance of these bonding agents bonded to dentin and enamel. Therefore, alternative in vitro short-term period degradation strategies to reveal valuable clinical information are needed (Hashimoto et al., 2004). It has been suggested that a reliable method to challenge the bonding durability should employ accelerated degradation strategies (Amaral et al., 2007), such as chemical challenge (Sauro et al., 2009) thermo- (Bedran-de-Castro et al., 2004; Feitosa et al., 2010; Ulker et al., 2010), and mechanicalcycling (Bedran-de-Castro et al., 2004; Feitosa et al., 2010; Osorio et al., 2008). Mechanical-cycling load and thermocycling testing performed in water at temperatures between
5 and 55 1C which subject the adhesive interface to water infiltration, mechanical and expansion/contraction strain (Ferracane et al., 1998; Gale and Darvell, 1999) may be considered suitable aging methods for dental adhesive materials. A further suitable strategy to challenge the resin bonded– dentin is based on the use of a simulated pulpal pressure (20 cm H2O) in order to induce water seepage, polymer degradation and droplets formation within the resin–dentin interface jeopardizing the mTBS (Bakry et al., 2009; Hosaka et al., 2007; Sauro et al., 2007; Sauro et al., 2007). The purpose of this study was to evaluate the microtensile bond strength (mTBS) and confocal micropermeability of resin bonded–dentin created with two representative ‘‘gold-standard’’ self-etch adhesive when submitted to short-term period degradation strategies such as simulated pulpal pressure, thermo- or mechanical-cycling challenges. SEM ultra-morphological analysis of the fractured specimens subsequent to challenge strategies used in this study was also performed. The null hypotheses to be tested is the strategies used in this study to accelerate degradation in a short-term period have no effect on the micro-mechanical adhesive properties (mTBS) or on the ultra-morphology/micropermeability of the resin–dentin interfaces created using self-etch adhesives.
2.
Materials and methods
2.1.
Sample preparation
Seventy human third molars extracted under the approved protocol of the appropriate institutional Research Ethics Committee (protocol 167/2009) were used in this study. The teeth were stored in 0.5% chloramine water solution for a period not exceeding 4 months at a temperature of 4 1C. Deep dentin specimens with a remaining dentin thickness (RDT) of approximately 0.9 mm were obtained by removing
Table 1 – Materials, compositions, application procedures and batches. Materials
Composition
Application procedure
Batch no.
P90 System adhesive
Self-etch primer: Vitrebond copolymer, phosphorylated methacrylates, BisGMA, HEMA, water, ethanol, silica filler, stabilizers, photoinitator Bond: Hydrophobic methacrylates, phosphorylated methacrylates, TEGDMA, silica filler, initiators, stabilizers
Apply one coat of the self-etch primer for 15 s with gentle agitation. Gently air dry. Light curing for 10 s. Apply the Bond. Gently air thin until the Bond is spread to an even film and does not move any longer. Light curing for 10 s.
9BN
Clearfil SE Bond
-Primer: MDP, HEMA, water, photoinitator -Bond: MDP, BisGMA, HEMA, hydrophobic dimethacrylates, photoinitator
Apply primer for 20 s, gently air-dry; apply bond. Light cure for 10 s
896A 1321A
Filtek P90 Shade A3
Matrix: TEGDMA, ECHCPMS Filler: Quartz filler
Apply in 1–2 mm increments and light cure for 40 s
N110333
Filtek Z350 XT Shade A3
Matrix: BisGMA, BisEMA, TEGDMA, UDMA. Filler: Silica and zirconia nanofiller
Apply in 1–2 mm increments and light-cure for 40 s
9BK
9XN
BiSGMA: bisphenol A diglycidylmethacrylate; HEMA: hydroxyethylmethacrylate; MDP: 10-methacryloyloxi decyl phophate; TEGDMA: triethylene glycol dimethacrylate; ECHCPMS: 3,4-epoxycyclohexylcyclopolymethylsiloxane; BisEMA: ethoxylated bisphenol A diglycicylmethacrylate.
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journal of the mechanical behavior of biomedical materials 15 (2012) 112 –120
the roots 2 mm below cemento–enamel junction (CEJ) and with a parallel cut at 2 mm above CEJ using a slow-speed water-cooled diamond saw (Isomet, Buehler, Lake Bluff, IL, USA). When the RDT was more than 0.9 mm, the flat dentin surface was abraded with 600-grit silicon carbide paper until obtaining the standardized RDT. The pulpal tissue was removed from the pulp chamber with small forceps without altering or scratching the pre-dentin surface along the walls of the pulpal chamber. The dentin surface was wet-polished with 600-grit SiC papers for 1 min to create a standard smear layer prior to bonding procedures (Hamouda et al., 2011).
2.2.
Experimental design and bonding procedures
The dentin specimens were divided randomly into two principal groups (n¼ 35) based on the bonding agents used in this study: Clearfil SE Bond (CSE: Kuraray Medical Inc., Tokyo, Japan) and Silorane adhesive (SIL: 3M ESPE, St. Paul, MN, USA). The composition, application procedures and batch numbers are listed in Table 1. All the bonding and restorative procedures were performed in absence of positive pulpal pressure. Resin composite build-ups were constructed in 6 layers (1 mm thick) up to 6 mm [Filtek Z350 XT (3M ESPE)/Clearfil SE Bond; Filtek P90 (3M ESPE)/Silorane adhesive]. Photoactivation of the resin-based materials was performed using a quartz–tungsten–halogen lamp XL-2500 (3M ESPE). The output intensity was monitored with a Demetron Radiometer (Model 100, Demetron Research, Danbury, CT, USA) to maintain a minimal light output intensity of 600 mW/cm2 throughout all the experiments. All materials were used following the manufacturers’ recommendations (Table 1). Subsequent to the bonding procedures, the specimens of each principal group were divided into sub-groups (n¼ 7), based on the challenge test: (1) CTRL: water immersion 24 h; (2) TC: thermocycling; (3) MCL: mechanical-cycling; (4) CPP: conventional simulated pulpal pressure; and (5) EPP: experimental simulated pulpal pressure (Fig. 1).
2.3.
Simulated pulpal pressures challenge
The bonded-dentin specimens were submitted to a simulated hydrostatic intra-pulpal pressure by using two different techniques (Fig. 1). A conventional simulated pulpal pressure (CPP) required that the specimens were submitted to simulated pulpal pressure where the water pressure was implemented 1 h after the restorative procedure in order to simulate the vasoconstrictor effect of local anesthesia on pulpal tissue (Sauro et al., 2007). The CPP was performed according to a protocol previously described (Sauro et al., 2007). Briefly, the crown segments were fixed to plexiglass plates by using cyanoacrylate glue which was perforated by an 18-gauge stainless steel tube (Fig. 1). Distilled water was perfused into the pulpal chamber using a pressure of 20 cm H2O via a hydraulic pressure device (Fig. 1). The water pressure was maintained for one week and subsequently disconnected and sectioned for CLSM and mTBS evaluation. An experimental simulated pulpal pressure method (EPP) was also used. This method required that the resin-bonded specimens were covered with two coats of nail varnish and positioned sideways on the lid of a cylindrical receptacle using dental wax (Fig. 1). The pulp chamber of each specimen was glued on the
Fig. 1 – Schematic drawing of the four aging treatments. Conventional simulated pulpal pressure and the experimental method to simulate pulpal pressure are illustrated in the upper two boards. Mechanical- and thermo-cycling methods are illustrated in the lower two boards. Description of the new method of simulated pulpal pressure: section the teeth to obtain a flat dentin surface with a mean remaining dentin thickness of 0.9 mm, similar to the protocol used in the conventional method. Undertake bonding and restorative procedures. Apply two coats of nail varnish on enamel–composite border. Fix the samples by sideways in a container lid with wax without obstructing the pulpal chamber opening. Fill the cylindrical container with distilled water to reach 20 cm and close the container with samples attached in the lid. Turn the container upside down to submit the samples to 20 cm H2O pulpal pressure.
base of the system and filled with distilled water. The lid was subsequently sealed to a cylindrical receptacle containing 20 cm of distilled water and turned upside down. Thus, the samples had a 20 cm water column over them according to the classic method and hydrostatic pressure equation: P¼gdh, where P is the hydrostatic pressure, g is the gravity, d is the liquid density and h is the liquid height. The water pressure was maintained for one week, prepared and evaluated as previously described.
2.4.
Thermal and mechanical-cycling challenges
The resin-bonded specimens were submitted to thermalcycling challenge using a thermo-cycling machine (MSCT-3, ~ Carlos, SP, Brazil) proMarcelo Nucci ME Instrument, Sao grammed to perform 5000 thermal-cycles during one week at temperatures between 5 1C and 55 1C, with a dwell time of 30 s at each bath temperature (Fig. 1) (Ulker et al., 2010). The mechanical cycling was executed using the MSCM equipment (Marcelo Nucci ME Instrument) which has a stainless steel tip of 4 mm in diameter in contact with the central part of the
journal of the mechanical behavior of biomedical materials 15 (2012) 112 –120
restored specimens. All the resin-bonded specimens were submitted to 200,000 mechanical cycles under a load of 50 N, at a rate of 2 Hz for one week (Ulker et al., 2010).
2.5.
Microtensile bond strength testing (lTBS)
The resin-bonded specimens prepared for this group were sectioned occluso-gingivally in serial slabs (1 mm thick) using a diamond-embedded blade under continuous water irrigation (Buehler, Lake Bluff, IL, USA) and subsequently in 1 mm2 match-sticks. Five specimens were used for each group (n¼ 5). The match-sticks from the most peripheral area presenting residual enamel were excluded. The selected match-sticks were fixed to a jig with cyanoacrylate glue and tested to failure under tension in the universal testing machine EZ-test (Shimadzu Co., Kyoto, Japan) with a 500N load cell, at a crosshead speed of 1.0 mm/min. The exact cross-sectional area of each tested sticks was measured after failure with a digital caliper (Mitutoyo Co. Tokyo, Japan). Means and standard deviation were calculated and expressed in MPa. Five restored teeth (experimental unit, n¼ 5) were evaluated in each group, the bond strength of sticks from the same restored teeth was averaged and the mean bond strength was used as one statistical unit for the statistical analysis. The microtensile bond strength (mTBS) data were statistically analyzed using twoway ANOVA (DBA and aging regime) to identify differences among groups. When significant differences were found, they were compared using Tukey’s test at a¼ 0.05.
2.6. Failure mode analysis and scanning electron microscopy ultra-morphology Subsequent to the mTBS tests, the mode of failure of the fractured specimens was determined by stereomicroscopy at 60x magnification. The fractures were classified as follows: Type A: Adhesive failure at the interface among resin composite, adhesive resin and hybrid layer. Type M: Mixed failure. Both adhesive and cohesive failures observed in the same fractured stick. Type C: Cohesive failure in resin composite. Type D: Cohesive failure in dentin. Five representative samples exhibiting the most frequently observed failure pattern (i.e. mixed or cohesive) and the mTBS close to the mean, were prepared for scanning electron microscopy (SEM) ultra-morphology analysis. The parts of the fractured samples were paired and mounted on aluminum stubs, coated with gold (Balzers SCD 050 sputter coater, B.U.A. Co., Fu¨rstentum, Germany) and examined by SEM, JSM-5600LV (JEOL, Tokyo, Japan), operated at 15 kV, with 20 mm as work distance.
2.7.
Confocal micropermeability evaluation (CLSM)
Two specimens for each sub-group were prepared as previously described and prepared for confocal micropermeability evaluation (Sauro et al., 2012). Briefly, the evaluation of micropermeability of resin–dentin interfaces was performed using a
115
0.1 wt% lucifer yellow water solution (LY: Sigma Chemicals, St. Louis, MO, USA). This fluorescent dye was used to trace the water-filled spaces and the sealing ability from the pulp chamber through the tubules to the resin–dentin interface for 3 h. The specimens were subsequently cut into 1 mm slabs, slightly polished with wet 1200 SiC-grit paper and ultrasonicated for 5 min. Three slabs from each specimen were selected for confocal microscopy (6 slabs/group). Three images were obtained from each slice resulting in nine images per specimen and 18 images per group. These images were intended to be representative of the most common features regarding the micropermeability observed along the interfaces. The microscopy examination was performed using a laser confocal scanning microscope (CLSM: Leica SP2 CLSM, Heidelberg, Germany) equipped with a 63x/1.4 NA oil immersion lens using 514 nm argon/helium ion laser illumination both in reflection and fluorescence mode. Reflected and fluorescence signals were detected with a photomultiplier tube to a depth of 20 mm and then converted to single-projection images for better visualization and qualitative analysis (Sauro et al., 2012).
3.
Results
3.1.
Microtensile bond strength testing (lTBS)
Mean bond strength was affected by dental adhesives (po0.001) and aging groups (p ¼0.001). Interactions were not significant between both factors (p¼ 0.173). Therefore, the comparison between the DBAs has no correlation with the accelerated degradation strategies; thus, the comparisons between the degradation strategies should be performed regardless the DBA. The differences between treatments were highly significant as well as the tested DBAs (po0.001). Microtensile bond strength results are depicted in Table 2. Clearfil SE Bond (CSE) showed higher mTBS values than Silorane adhesive (SIL) for all aging treatments (po0.001). Regarding the accelerated degradation strategies, the statistical analysis showed significant changes in mechanicalcycling load as well as, thermal-cycling and mechanicalcycling compared to the control groups (p¼ 0.002), (Table 2). Despite the absence of significant interactions in the CSE group, no difference was found between the accelerated bonding degradation strategies (p40.05). SIL showed statistical difference between control and mechanical-cycling Table 2 – Means (standard deviations) lTBS of each group in MPa. Aging regimen
Silorane adhesive
Clearfil SE Bond
Mean
Control Thermal cycling Conventional PP Experimental PP Mechanical cycling
40.8 41.1 34.2 33.8 32.5
47.1 46.9 46.5 46.8 40.7
43.97a 44.03a 40.35ab 40.30ab 36.61b
Mean
36.50B
(4.3) (4.5) (4.3) (4.6) (4.5)
(3.4) (4.4) (4.0) (3.5) (3.1)
45.60A
Different capital letters represent statistical significant difference between adhesives (po0.05). Different lowercase letters represent
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journal of the mechanical behavior of biomedical materials 15 (2012) 112 –120
strain (p¼ 0.021) and between thermo-cycling and mechanical-cycling strain (p¼ 0.016); no difference was found between the conventional and the experimental pulpal pressure as well as thermo-cycling and control.
3.2. Failure mode analysis and scanning electron microscopy ultra-morphology The percentages of fracture types for each group are summarized in Fig. 2. Representative SEM micrographs are shown in Fig. 3. The type A (adhesive failure at the resin composite/ adhesive/dentin interface) failure pattern was predominantly observed in the de-bonded specimens of the CSE control, mechanical- and thermo-cycling groups. SIL showed type A failure in the de-bonded specimens submitted to both simulated pulpal pressure methods. The specimens of the SIL group submitted to thermo- and mechanical-cycling strains showed prevalently a type M failure (mixed failures, partially adhesive and cohesive) as well as those of the CSE specimens submitted to both simulated pulpal pressure. The control group in SIL resulted in 50% adhesive and 50% mixed failure. Fractured match-sticks of SIL after mechanical-cycling load showed micro-cracks within the hybrid and adhesive layers (Fig. 3B). Specimens subjected to both methods of simulated pulpal pressure showed a characteristic de-bonding at the interdiffusion layer (IDL), (Fig. 3C).
3.3.
Confocal micropermeability evaluation (CLSM)
CLSM results are presented in Figs. 4 and 5. The high dye penetration was observed within the resin–dentin interfaces of all the specimens submitted to both methods of simulated pulpal pressure (Figs. 4B and 5B). Thermo- and mechanicalcycling strain induced similar micropermeability within the
Coh Dentine
Coh Comp
Mixed
Adhesive
SIL - CPP
SIL - EPP
SIL - MC
SIL - TC
SIL - Control
CSE - CPP
CSE - EPP
CSE - MC
CSE - TC
CSE - Control
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
Fig. 2 – Graph presenting the fractures patterns (%) of debonded specimens. CSE means Clearfil SE Bond. SIL means Silorane adhesive. MC, TC, CPP and EPP mean mechanical cycling, thermo-cycling, conventional simulated pulpal pressure and experimental simulated pulpal pressure respectively.
adhesive and IDL of Clearfil SE Bond (CSE) compared to the control groups (24 h) (Fig. 5A, C and D). Similarly, Thermo- and mechanical-cycling strain induced dye penetration (micropermeability) only into dentinal tubules for CSE (Fig. 5C and D). Both methods for the simulation of the pulpal pressure promoted a slight increase in micropermeability within the IDL of CSE (Fig. 5B). The resin–dentin interface of Silorane adhesive group (SIL) reduced adhesive layer thickness subsequent to mechanical-cycling load strain (Fig. 4C). This phenomenon was not observed in the resin–dentin interface created with Clearfil SE Bond adhesive. Thermo-cycling strain slightly increased the micropermeability within the IDL of SIL (Fig. 4D). Mechanicalcycling load led to the formation of micro-cracks along IDL and the adhesive layer revealing visible micropermeability (Fig. 4C1). Both methods of simulated pulpal pressure increased the micropermeability within the IDL and adhesive layer of Silorane adhesive (Fig. 4B and B1).
4.
Discussion
The outcomes of the present study showed a statistical difference (po0.05) in mTBS results between control (24 h/H2O) and the mechanical-load (200,000 cycles) groups but no significant difference (p40.05) by the other degradation strategies. Furthermore, both methods for simulated pulpal pressure and the mechanical-cycling load strain induced important changes in the micropermeability within the resin–dentin interfaces created with the two two-step/self-etching adhesives tested in this study. Therefore, the first null hypothesis that there is no difference between the strategies used in this study to accelerate degradation in a short-term period have no effect on the micromechanical adhesive properties (mTBS) or on the ultra-morphology/micropermeability of the resin–dentin interfaces created using self-etch adhesives must be partially rejected. The most popular approach to induce bonding degradation is submerging resin–dentin match-sticks in water or phosphate buffer solutions for a minimal period of 3 months in order to contrast significant differences between groups (Van Meerbeek et al., 2010). Despite the water degradation strategy results are time demanding, a thermo-cycling approach may be a suitable method to induce bonding degradation in a short-term period (Amaral et al., 2007). This latest strategy might simulate an in vivo challenge where the restorations are submitted to thermal changes in oral environment occurring during eating and drinking (Amaral et al., 2007; Feitosa et al., 2010). Moreover, the aging effect induced by thermo-cycling relies on the ability of hot water to accelerate the hydrolysis of nonprotected collagen and leaching of poorly polymerized resin monomers (Ferracane et al., 1998). The interface undergoes the process known as plasticization, with a decrease in its mechanical properties (De Munck et al., 2005). However, Van Meerbeek et al. (2010) have recently stated that the thermo-cycling strategy to accelerate bonding degradation must be performed using a number of cycles up to 100,000 to discriminate significant differences. They also highlighted that the ISO’s recommendations of thermocycling regimens (500 cycles) are of little use, since most dental bonding agents (DBAs) would not reveal significant bonding degradation. Nikaido et al. (2002) obtained decreased
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Fig. 3 – SEM micrographs showing representative fractures of debonded specimens. c—composite, hy—hybrid layer and a—adhesive. (A) Resin side of a de-bonded stick bonded with CSE after one week of conventional simulated pulpal pressure. The predominant fracture pattern in this group was mixed, and this SEM image illustrates this pattern. (B) Dentin side of a de-bonded match-stick of SIL after mechanical cycling loading. A mixed failure (predominant pattern) is shown in the micrograph. Note some microcracks along hybrid and adhesive layer (white circle) in agreement with confocal microscopy outcomes. (C) SEM image of the dentin side of a de-bonded stick of SIL after experimental simulated pulpal pressure. The adhesive failure image illustrates deterioration of hybrid layer above and around dentinal tubules (white circle) with resin remnants above intertubular dentin whereas no resin remnant could be observed above dentinal tubules and peritubular dentin. (D) Image of a mixed failure (predominant pattern) in the dentin side from a de-bonded specimen of SIL after thermo-cycling.
bond strength only after a higher number of thermo-cyclings in higher C-factor Class I cavities. The results of this study showed that a thermo-cycling challenge performed with 5000 cycles (5 1C and 55 1C) may not be an appropriate strategy to induce statistical change in mTBS in specimens bonded with gold-standard two-step selfetch bonding agents (Table 2). Moreover, the thermo-cycling regimen used in this study provoked no change in micropermeability within the resin–dentin interface created both with SIL (Fig. 4D) and CSE (Fig. 5D) compared to those of the control group (Figs. 4A and 5A). These types of adhesives require a separate application of a hydrophobic resin layer which provides a substantial improvement for bonding stability (Lodovici et al., 2009; Reis et al., 2008; Va´squez et al., 2009) and it likely contributes for bonding preservation against thermal challenge; a longer thermocycling regime is required to create discernible bonding degradation and significant differences in mTBS (De Munck et al., 2005; Van Meerbeek et al., 2010). Conversely, the short-term mechanical-cycling challenge (200,000 cycles in 7 days/2 Hz frequency) was the exclusive artificial aging regimen to promote significant decrease in bond strength (Table 2). The CLSM micropermeability results obtained within the resin–dentin interface created with both the tested DBAs submitted to mechanical-cycling challenge showed no dissimilarity compared to those obtained in the control group (24 h/H2O).
However, a thinner adhesive layer could be observed for SIL after mechanical loading (Fig. 4C), whereas no striking alteration was found for CSE. Since the DBA–Silorane requires a double photo-activation both for the primer and for the bond, we believe that remaining primer was incompletely polymerized due to its hydrophilic and water permeable nature and may have been deformed by the mechanical-cycling (Sauro et al., 2011). Indeed, the application of the Silorane bond layer after primer photo-activation may not overcome the compression stresses of mechanical loading, yielding reduction in adhesive layer thickness without altering the micropermeability (Frankenberger et al., 2002). Conversely, the primer of CSE is not separately light-cured, thus, this phenomenon did not occur (Fig. 5). This hypothesis may be confirmed by the results obtained during the confocal micropermeability evaluation which showed the presence of some micro-cracks within this thinner Silorane adhesive layer as well as the IDL (Fig. 4C1). These micro-cracks (Fig. 3B) may be also responsible for the reduced bond strength and higher mixed failures of debonded specimens obtained in the mechanical-cycling group (Fig. 2). Furthermore, despite the immediate acceptable results of Silorane adhesive, this DBA was more prone to degradation under artificial aging regimes. The SIL degradation may be explained by the presence of silane agents and siloxane groups in the adhesive composition, which are more easily hydrolytically degradable (Duarte et al., 2009). The intervening zone in the adhesive layer between the cured primer
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Fig. 4 – 3D confocal-single projections (reflection/fluorescence) of Silorane adhesive showing the representative micropermeabilities of tested groups. c—composite, a—adhesive and dt—dentin. (A) Reflection/fluorescence image of lucifer yellow uptake in control group. (A1) Higher magnification (fluorescence image) of control group. Visible micropermeability into the tubules and within the interdiffusion layer (IDL). (B) Reflection/fluorescence image observed from experimental simulated pulpal pressure group. Note the higher micropermeability under hydrostatic pressure (open arrow). (B1) Fluorescence image of new method of simulated pulpal pressure with higher magnification. Visible lucifer yellow uptake into the dentinal tubules (open arrow), IDL and reaching the adhesive layer. (C) Resin–bonded dentin interface represented by reflection/fluorescence image after the mechanical loading stress. Note the reduced thickness of adhesive layer (double arrows) due to compression induced by mechanical cyclings. (C1) Higher magnification of Silorane adhesive under loading (fluorescence image). Visible micropermeability into the tubules, IDL and into the microcracks along the adhesive/primer layer (arrows). (D) Resin–bonded dentin interface after thermocyclings (reflection/fluorescence image). Visible micropermeability in IDL. Note the similarity of this group and control group. (D1) Higher magnification of Silorane adhesive under thermocycling (fluorescence image). Visible micropermeability into the tubules, IDL and adhesive/primer layer.
and the bonding layer also contributes to the bond instability of SIL after aging regimes, since this zone is more prone to hydrolysis and silver uptake (Duarte et al., 2009). The presence of a physiological pulpal pressure plays an important role in adhesive dentistry (Hosaka et al., 2007; Lin et al., 2010) thus its simulation for in vitro studies concerns a fundamental approach to challenge bonding of resin-based materials to dentin (Sauro et al., 2007; Van Meerbeek et al., 2010). Nevertheless, few investigations have been performed using this methodology, probably due to the laborious methodology required during the simulation of the pulpal pressure. An easier and standardized protocol to simulate pulpal pressure and test adhesive permeability is currently necessary (Van Meerbeek et al., 2010). The conventional simulated PP method requires the use of plexiglass or acrylic plate perforated by an 18-gauge stainless steel tube where the dentin specimen is attached using cyanoacrylate glue and a water column apparatus with water level 15–20 cm above the specimen (Fig. 1). However, when the water column creates the hydrostatic pressure inside the pulp the cyanoacrylate cementation might allow water seepage through the glue compromising the stability of water pressure inside the pulpal chambers.
The experimental methodology presented in this study to simulate PP requires no use of cyanoacrylate glue cementation, plexiglass plate and stainless steel tube providing an easier and effortless maintenance of hydrostatic pressure (Fig. 1, upper board). The specimens are attached to a cylindrical receptacle lid this way, and the receptacle is filled with distilled water until the water level reaches a height of 20 cm. Moreover, many specimens can be included in the same container and subjected to simulated pulpal pressure together. The mTBS and confocal micropermeability results obtained in this in vitro study ratify the resemblance between the two methods, since the microtensile bond strengths were numerically and statistically similar for both DBAs; the micropermeabilities were also comparable regardless of the DBA. The confocal micropermeability evaluation revealed discernible higher micropermeability for both the simulated pulpal pressure methods in comparison with control groups. However, although the presence of a positive pressure induced micropermeability within the interdiffusion layers (Figs. 4B and 5B) in the one week period-time, it induced no significant statistical differences in bond strength reduction for CSE and SIL. Sauro et al. (2009) showed acceptable dentin
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Fig. 5 – 3D confocal-single projections (reflection/fluorescence) of Clearfil SE Bond showing the representative micropermeabilities. (A) Reflection/fluorescence image of lucifer yellow uptake in control group. Visible micropermeability into the tubules (open arrow) and within the interdiffusion layer (IDL). (B) Reflection/fluorescence image observed after one-week underconventional simulated pulpal pressure. Note the higher micropermeability in IDL under hydrostatic pressure. (C) Resin–bonded dentin interface represented by fluorescence image after the 200,000 mechanical loading regimen. Note visible micropermeability into the tubules (open arrow) and IDL. (D) Resin–-bonded dentin interface after 5000 thermo-cyclings (fluorescence image). Visible micropermeability into dentinal tubules and IDL, especially at the bottom of the IDL (open arrow). Note the similarity of this group, mechanical loading and control groups.
sealing ability of Silorane adhesive bonded to dentin while, further investigations depicted the low permeability of CSE (Kim et al., 2010; Sauro et al., 2007; Sword et al., 2011). Also in this case, the low permeability of these DBAs is likely to be provided by the hydrophobic adhesive resin that is applied as a separate step (Kim et al., 2010). This hydrophobic layer may achieve maintenance of bond strengths (Hosaka et al., 2007) and preservation of bonding integrity, even with the observed higher lucifer yellow uptake. Two DBAs were used in the present investigation to exclude potential misinterpretation of outcomes related with usage of a single adhesive. However, the micropermeability can reveal resistance against water penetration of IDL and adhesive layers and may predict the relative durability of the bonded dentin interface.
5.
Conclusion
In conclusion, mechanical-cycling strain performed under a regimen of 200,000 cycles (50 N-load/2 Hz-rate) for one week in combination with the use of simulated pulpal pressure performed with the ‘‘easy’’ method proposed in this study may be a suitable strategy for an effective short-term artificial aging to test the bonding performance of ‘‘high-performance’’ DBAs. Further investigations are ongoing to evaluate the effects of the cycling-load in combination of simulated pulpal pressure on other types of DBAs. It is also under investigation whether
a longer period under simulated pulpal pressure (20 cm H2O) may affect the mTBS of two-step/self-etching adhesives.
Acknowledgments This work was supported by the CAPES—Brazil, the Center of Excellence in Medical Engineering funded by the Wellcome trust and the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy’s & St Thomas’ NHS Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust. The authors also thank Dr. Flavia Gouvea da Costa for providing sound teeth in this investigation. The authors have no financial affiliation or involvement with any commercial organization with direct financial interest in the materials discussed in this manuscript. Any other potential conflict of interest is disclosed.
r e f e r e nc e s
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