MICROSCOPY RESEARCH AND TECHNIQUE 77:37–43 (2014)
Long-Term Chlorhexidine Effect on Bond Strength to Er: YAG Laser Irradiated-Dentin 3 DANIEL GALAFASSI,1 CAMILA SCATENA,2 VIVIAN COLUCCI,1 ANTONIO LUIZ RODRIGUES-JUNIOR, ^ MONICA CAMPOS SERRA,1 AND SILMARA APARECIDA MILORI CORONA1* 1
Department of Restorative Dentistry, Ribeir~ ao Preto Dental School, University of S~ ao Paulo, Ribeir~ ao Preto, S~ ao Paulo, 14040-904, Brazil 2 Department of Paediatric Clinic, Ribeir~ ao Preto Dental School, University of S~ ao Paulo, Ribeir~ ao Preto, S~ ao Paulo, 14040-904, Brazil 3 Department of Social Medicine, Ribeir~ ao Preto Medicine School, University of S~ ao Paulo, Ribeir~ ao Preto, S~ ao Paulo, 14040-904, Brazil
KEY WORDS
bond strength; dentin; chlorhexidine; thermocycling; laser; hybrid layer
ABSTRACT This study evaluates the bond strength of dentin prepared with Er:YAG laser or bur, after rewetting with chlorhexidine on long-term artificial saliva storage and thermocycling. One hundred and twenty human third molars were sectioned in order to expose the dentin surface (n 5 10). The specimens were randomly divided in 12 groups according to treatment and aging: Er:YAG laser rewetting with deionized water (LW) and 24 h storage in artificial saliva (WC); LW and 6 months of artificial saliva storage 1 12.000 thermocycling (6M), LW and 12 months of artificial saliva storage 1 24.000 thermocycling (12M), Er:YAG laser rewetting with 2% chlorhexidine (LC) and WC, LC and 6M, LC and 12M, bur on high-speed turbine rewetting with deionized water (TW) and WC, TW6M, TW12M, bur on high-speed turbine 1 2% chlorhexidine (TC) and WC, TC and 6M, TC and12M. The specimens were etched with 35% phosphoric acid, washed, and dried with air. Single Bond 2 adhesive was applied and the samples were restored with a composite. Each tooth was sectioned in order to obtain 4 sticks, which were submitted to microtensile bond strength test (mTBS). The two-way ANOVA, showed no significant differences for the interaction between the factors and for the aging factor. Tukey 5% showed that the LC group had the lowest mTBS. The rewetting with chlorhexidine negatively influenced the bond strength of the preparation with the Er:YAG laser. The artificial saliva aging and thermocycling did not interfere with dentin bond strength. Microsc. Res. Tech. 77:37–43, 2014. V 2013 C
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INTRODUCTION The advances of adhesive dentistry and new dental technologies used in the preparation, promote minimally invasive procedures, beyond improved patient comfort. The Er:YAG laser has proven to be an alternative tool to conventional preparation with a bur (Hibst, 2002). The laser was shown to be effective in the ablation of hard tissue (Corona et al., 2007), once it emits a wavelength of 2.94 mm, coinciding with the peak absorption of water and OH-radicals present in dental tissue (Hibst and Keller, 1989; Ramos et al., 2002). This causes the vaporization of water and hydrated tissue components, causing a rapid warming, followed by microbursts resulting from the increased internal pressure of molecules, which, in turn, lead to ejection of the substrate in the form of microscopic fragments (Aoki et al., 1998; Hibst and Keller, 1989; Hossain et al., 1999; Matsumoto et al., 1996). The laser irradiation of dentin has been described as to favorable for the bond between the restorative material and dental tissue due to the increased surface area for adhesion with the presence of microretentions (Armengol et al., 1999; Trajtenberg et al., 2004) and the opening of dentinal tubules without smear layer formation (Trajtenberg et al., 2004). This would make C V
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adhesion more favorable (Armengol et al., 1999) giving a bond strength similar to preparation with a bur (Oliveira et al., 2005). However, obtaining a stable adhesion over time remains a challenge due to hydrolytic and enzymatic degradation (by matrix metalloproteinases (MMPs)) from the components in this bond (Carrilho et al., 2007a; Pashley et al., 2004). Endogenous enzymes known as MMPs are capable of breaking down the extracellular matrix (Carrilho et al., 2007a; Hebling et al., 2005; Pashley et al., 2004). These enzymes are involved in the process of collagen remodeling during amelogenesis and dentinogenesis and remain latent, trapped in the structures formed (Sulkala et al., 2002). The reactivation of MMPs can occur when there is a metabolic imbalance with the *Correspondence to: Silmara Aparecida Milori Corona, Department of Dentistry, Ribeir~ ao Preto School of Dentistry—USP, Restorative Av. do Cafe, S/N, Monte Alegre, Ribeir~ ao Preto, SP 14040-904, Brazil. E-mail: [email protected] Received 17 September 2013; accepted in revised form 21 October 2013 REVIEW EDITOR: Dr. Chuanbin Mao Contract grant sponsor: FAPESP; Contract grant numbers: 2008/09042-0, 2009/11225-8; Contract grant sponsor: CAPES; Contract grant number: AEX 18542/12-3.. DOI 10.1002/jemt.22310 Published online 1 November 2013 in Wiley Online Library (wileyonlinelibrary.com).
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release of acid as in the carious lesions, through etching before adhesive application, or the low pH of self-etching adhesive systems (Chaussain-Miller et al., 2006; Tay et al., 2006). During the adhesive procedure, some collagen fibrils of the hybrid layer may remain exposed due to imperfect resin impregnation into the demineralized dentin matrix (Breschi et al., 2010) and these MMPs may be affected, leading to enzymatic degradation, which can result in reduced bond strength. CHX is an inhibitor of MMPs 22, 28, and 29 (Gendron et al., 1999) and thus, can increase the bond strength of restorations (Carrilho et al., 2007a; Pashley et al., 2004), acting in its own confinement between the collagen fibres. Recently discovered in normal and carious dentin, the cysteine proteases from cathepsin family have been show to participate in various conditions, which result in the degradation of extracellular matrix components, increases the list of potential endogenous proteases in dentin matrices (Nascimento et al., 2011; Scaffa et al., 2012; Tezvergil-Mutluay et al., 2013). Chlorhexidine is an antimicrobial agent able to inhibit some MMPs and cysteine cathepsins (Gendron et al., 1999; Scaffa et al., 2012) and, according to some in vitro studies, do not adversely affect the bond strength of adhesive systems immediately (Carrilho et al., 2007a; Hebling et al., 2005; Pashley et al., 2004). Furthermore, in vivo (Carrilho et al., 2007b; Hebling et al., 2005) and in vitro studies (Castro et al. 2003; Hebling et al., 2005; Pashley et al., 2004) showed that the application of chlorhexidine in dentin rewetting can slow down or even prevent the degradation of collagen fibres exposed, resulting in a more stable resin-dentin adhesion over time. Given the inexistence of studies assessing the rewetting with chlorhexidine in the preservation of hybrid layer of Er:YAG laser irradiated dentin, the aim of this study was to evaluate the influence of chlorhexidine dentin rewetting on the bond strength of irradiated dentin subjected to aging through storage in artificial saliva and thermocycling. MATERIALS AND METHODS Experimental Design The factors studied were the treatment of dentin on four levels [Er:YAG laser 1 rewetting with deionized water, Er:YAG laser 1 rewetting with chlorhexidine, high-speed turbine 1 rewetting with water, and highspeed turbine 1 rewetting with chlorhexidine (TC)] and aging at three levels: 24 h of storage in artificial saliva/without cycle, 6 months of storage in artificial saliva/12,000 cycles, and 12 months of storage in artificial saliva/24,000 cycles. The sample of the experiment consisted of 120 teeth randomly divided into 12 groups (n 5 10). The design was made up of random blocks. The quantitative response variable was the bond strength (MPa), obtained through microtensile bond strength test (mTBS) testing. Selection of Teeth One hundred and twenty caries-free, freshly extracted human third molars were selected for this study (obtained from the bank of teeth FORP-USP). The following protocol was prescribed, revised and approved by the Committee of Ethics in Human Research (2009.1.165.58.1). The teeth were stored in 0.2% thymol solution at 4 C for 48 h.
Dentin Preparation The root was fixed in polyester resin, through a polyvinyl chloride (PVC) cylinder—1.7 3 1.5 mm (Tigre, Rio Claro, SP, Brazil). Then, the occlusal surface was removed with a diamond saw to exposing the coronal dentin surface (Isomet 1000, Buehler, Like Bluff, IL). The teeth were grounded in a polishing machine (Beta, Buehler, Lake Bluff, IL) with sandpaper # 600. The teeth were randomly divided according to the treatment of dentin (laser rewetting with deionized water (LW), LC, high-speed turbine rewetting with deionized water (TW), and TC). For the irradiated groups, the Er:YAG laser (Twin Light, Fotona Medical Lasers, Ljubljana, Slovenian)—260 mJ/4 Hz with energy density (47 J/cm2) and power density (0.81 W) was applied, scanning across the surface of the transversally and horizontally exposed dentin using a device that holds the laser pen in place. The laser beam was applied using a noncontact method, focused at a distance of 12 mm from the substrate (Amaral et al., 2008), with water spray of 1.5 mL/min (Amaral et al., 2008). For the nonirradiated group, the dentin preparation was performed with carbide bur (#245, Jota AG Rotatory instruments, R€ uthi, Switzerland) in a high-speed turbine (605C, Kavo do Brazil Ind. Com. LTDA., Joinville, SC, Brazil) under constant refrigeration. The same steps used in the Er:YAG laser group (transversally and horizontally) were carried out. Restorative Procedures The specimens were etched with 35% phosphoric acid (Etching gel, 3M ESPE Dental Products, St. Paul, MN) for 15 s, rinsed for 30 s, and dried for 30 s with air. After drying the dentin surface, the specimens in groups LC and TC were rewetting with 1.5 mL of 2% chlorhexidine (Clorhexidina S, FGM Prod. Odontologicos, Joinville, SC, Brazil) with the aid of a micropipette (Labmate Soft, PZ HTL S.A., Warsaw, Poland) at a time of 60 s, the excess being removed with absorbent paper and for specimens of the LW and TW groups dentin rewetting with deionized water was carried out in the same way as the previous groups. Next, the Single Bond 2 adhesive system (Single Bond 2, 3M ESPE Dental Products, St. Paul, MN) was applied, as instructed by the manufacturer, in two layers and cured with halogen light (XL-3000, 3M ESPE Dental products, Germany) for 10 s. After, a plateau of composite resin (Filtek Z350, shade A3, 3M ESPE Dental Products, St. Paul, MN) using the incremental technique in four layers each containing a 1 mm, 4 mm total, was placed over the dentin surface. Aging of the Adhesive Interface The restored specimens were divided according to the technique of storage/aging interface: 24 h of storage in artificial saliva/no cycle, 6 months of storage in artificial saliva/12,000 cycles, and 12 months of storage in artificial saliva/24,000 cycles. Aging consisted of 500 cycles of thermocycling (Etica Equip. Cient. S.A., SP, Brazil) (5 C and 55 C) per week until they completed 12,000 and 24,000 cycles. At weekly intervals, the groups remained immersed in 100 mL of artificial saliva in an oven at a temperature of 37 C (ECB 1.3, Microscopy Research and Technique
CHLORHEXIDINE EFFECT ON IRRADIATED DENTIN TABLE 1. Average resistance to mTBS and SD for the treatment factor Treatments TW TC LW LC
Average of MPa 24,3 (8,11)a 24,1 (9,49)a 20,1 (8,39)a,b 12,1 (6,56)b
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TABLE 2. Average resistance to mTBS and SD for the aging factor Aging WC 6M 12M
Average of MPa 22,32 (10,06)a 19,20 (10,32)a 19,01 (7,76)a
Same letters indicate statistical similarity.
Same letters indicate statistical similarity.
Odontobras, Ribeir~ ao Preto, SP, Brazil) which was substituted weekly. Microtensile Bond Strength Test After 24 h of storage in artificial saliva, the specimens of the no cycles/24 h of storage in artificial saliva group were fixed in a metallographic cutter and sectioned in both mesiodistal and buccal-lingual directions to obtain multiple sticks of 1.0 mm2 (60.2 mm2) for the mTBS test. From each tooth, the central sticks were collected. The sticks of similar length and dentin thickness remaining were tested (four sticks from each tooth was selected, results in a total of 40 sticks for each group). The sticks were properly measured with digital caliper, identified, and fixed with cyanoacrylate glue gel in the universal testing machine (EMIC, Equipamentos e Sistemas de Ensaios LTDA, S~ ao Jose dos Pinhais, PR, Brazil) at a cross-head speed of 0.5 mm/min. After fixation, they were stressed in tension until failure occurred. An average of values was obtained for the sticks of the same tooth and this was taken as the mean value of the specimen. The same procedure was performed for specimens from 6 to 12 months of aging. The sticks that failed before testing were counted as 0 MPa. The fractured sides of all specimens were inspected under stereo macroscopic (KL200, Leica Microsystems, Heerbrugg, Switzerland) at 103 magnification and classified into four types: adhesive, cohesive in dentin, cohesive in resin, and mixed. The frequency of each mode was calculated in percentage. SEM Evaluations For the scanning electron microscopy (SEM) evaluation of interfacial morphology, another 36 molars were used and their roots were fixed in PVC cylinder with polyester resin. The occlusal surface was removed with a diamond disk so as to expose the coronal dentin surface. The dentin was treated according to the described protocols for different groups TWWC, TW6M, TW12M, TCWC, TC6M, TC12M, LWWC, LW6M, LW12M, LCWC, LC6M, LC12M (n 5 3). All teeth were vertically sectioned into 2.0-mm-thick slabs which were hand polished with 600 and 1,200 grit SiC paper followed by diamond paste (3 mm and 1 mm). The slabs were ultrasonically cleaned during 10 min after each polishing step. After the slabs were treated with ethylenediamine tetraacetic acid (EDTA) 17% for 10 min and rinsed for 10 min. Slabs were dehydrated in ascending alcohol concentrations followed hexamethyldisilazane (HMDS), sputter-coated with gold, and examined under SEM. Representative areas were photographed at 20003. Statistical Analysis The R software was used in the analysis. The data was statistically analyzed by the two-way ANOVA and Tukey test at a significance level of 5%. Microscopy Research and Technique
RESULTS The two-way ANOVA showed significant difference for the Treatment factor (P 5 0.0000) (Table 1). For the Aging factor (P 5 0.1289) (Table 2) and the interaction of Treatment and Aging factors (P 5 0.4519), there was no significant difference. The mean and standard deviation (SD) of mTBS for dentin prepared with the Er:YAG laser or high-speed turbine (bur #245), after aging conducted by storing in deionized water (24 h, 6 and 12 months)/thermocycling (without cycles, 12,000 and 24,000 thermal cycles) are summarized in Table 3. Tukey post hoc test demonstrated that, for the Treatment factor, the LC group showed lower bond strength differing from groups TW and TC. The LW group did not differ statistically from the other groups (Table 1). Analysis of the Adhesive Interface by SEM The analysis of the adhesive interface by SEM showed an irregular hybrid layer formation, with the presence of cracks, for the irradiated groups. There was the formation of regular tags and bigger deposits of adhesive in the regions of the valleys and the peaks were covered with a thin layer of adhesive. However, we could observe the formation of uniform resin tags (Figs. 1g–1l). The groups prepared with the high-speed turbine there was a regular hybrid layer formation with the presence of tags and uniform funnel shapes (Figs. 1a– 1e). Image analysis of the high-speed turbine groups 1c and 1f showed micro cracks at the interface and no tags, which were also observed in the images 1i and 1l. Analysis of Fracture Pattern There was a predominance of a type of mixed fracture for all groups studied (Fig. 2). DISCUSSION The high-speed turbine is the main method for cavity preparation in dentistry. The Er:YAG laser has been indicated as an alternative to high-speed turbine, it offers some advantages which promote patient comfort, such as lower noise, pain, and pressure (Hibst, 2002). Considering the factor adhesion, some studies show that the laser provides similar adhesive resistance to the turbine after short periods of time (Trajtenberg et al., 2004) due to an increase in surface area for adhesion, due to the presence of micro retentions (Armengol et al., 1999). However, besides causing a scaly surface with the presence of rough areas (Ceballos et al., 2001), laser irradiation alters the levels of calcium and phosphate which would lead to the formation of components that are more stable and less soluble to acid conditioning through reducing the effectiveness of etching (De Moor and Delme, 2010). de
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Oliveira et al. (2007) found a reduction in irradiated dentin bond strength due to the formation of a modified layer that would be related to mineral dissolution through etching and diffusion of the adhesive system
TABLE 3. Average of the mTBS (standard deviation) of dentin prepared with the Er:YAG laser or high-speed turbine, rewetting with deionized water or chlorhexidine after aging (MPa) Treatment Aging Preparation/Rewetting TW TC LW LC
WC
6M
12M
27,89 (10,11) 29,30 (5,66) 19,67 (9,85) 12,42 (4,99)
21,16 (7,43) 21,82 (5,97) 21,38 (9,10) 12,42 (6,74)
23,85 (7,43) 21,29 (13,35) 19,32 (6,67) 11,59 (8,24)
Fig. 1. SEM images of adhesive interfaces. (a) TWWC; (b) TW6M; (c) TW12M; (d) TCWC; (e) TC6M; (f) TC12M; (g) LWWC; (h) LW6M; (i) LW12M; (j) LCWC; (k) LC6M; (l) LC12M. A 5 adhesive;
in the dentin surface area, leading to a deficiency in the hybridization of the substrate. Although studies (Aoki et al., 1998; Ceballos et al., 2001; Hibst, 2002) have evaluated the bond strength of dentin irradiated with an Er:YAG for short periods of time, the bond strength after aging was evaluated by a single study (Amaral et al., 2008), which found that the bond strength of dentin prepared with an Er:YAG after 6 months of water storage and thermocycling, showed lower values due to the degradation the adhesive interface. However, the literature does not have mTBS studies of rewetting irradiated dentin with water or chlorhexidine in the long term. The pulse duration of the Er:YAG is an important factor in the bond strength of dentin and is directly related to tissue ablation and surface morphology
D 5 dentin; H 5 hybrid layer; R 5 resin; * 5 gaps; 5 “Rings” around tag. Arrows indicate the peaks and valleys of irregular dentin pattern by Er:YAG laser preparation.
Microscopy Research and Technique
CHLORHEXIDINE EFFECT ON IRRADIATED DENTIN
Fig. 2. Graph of fracture pattern. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
(Firat et al., 2012). Although the Er:YAG short pulse is more effective in the ablation of dental tissue (Firat et al., 2012; Staninec et al., 2006), the long pulse is widely used and gives satisfactory results (Amaral et al., 2008; Corona et al., 2007; Ramos et al., 2002). In this study, dentin preparation with the Er:YAG long pulse seem not to have influenced the bond strength, considering that the adhesion values, for the preparations with the Er:YAG and rewetting with water, were similar to the other groups tested. The results of this study indicate that the use of 2% chlorhexidine for the rewetting of dentin in preparations carried out with an Er:YAG negatively affected the mTBS when compared to preparations with a highspeed turbine. A possible explanation for the lower levels of union in the LC group compared to the groups prepared with a bur may be related to the thermo mechanical ablation process (Hibst and Keller, 1989), which causes denaturation and fusion of collagen fibers, resulting in decreased interfibrillar space (Barceleiro et al., 2005; Ceballos et al., 2001), restricting the diffusion of resin into the subsurface of the intertubular dentin (Ceballos et al., 2002). As the molecular size of chlorhexidine (molecular weight 5 897.8 g21 mol) is greater than the molecule of some components of the adhesive system employed, for example, 2hydroxyethyl methacrylate (HEMA) (MW 5 130.0 g mol21) or bisphenol A-glycidyl methacrylate (BisGMA) (MW 5 512.0 g mol21) (Ricci et al., 2010), it could hinder complete infiltration of the monomer and promote the formation of gaps in the hybrid layer, decreasing the adhesive strength. In the SEM analysis of the adhesive interface, the dentin prepared with an Er:YAG showed the formation of “rings” around the tag (Figs. 1j and 1k) which occur due to the irradiated surface display “cracks,” which would be infiltrated by the adhesive system (de Oliveira et al., 2007). In Figure 1, arrows indicate an irregular hybrid layer, which occurs due to the buildup of adhesive in the region of the valleys, which correspond to the region of highest energy pulse, confirming the findings of de Oliveira et al. (2007). The peak area was filled with a thin layer of adhesive, which can lead to the formation of defects in the hybrid layer, corroborating other findings (Cardoso et al., 2008; Moretto et al., 2011). In the images 1g–1l, the presence of Microscopy Research and Technique
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cracks in the adhesive interface are observed, which could be the result of the breakdown of collagen by the laser and these findings were also reported in the literature (Aranha et al., 2007; Moretto et al., 2011). The SEM showed the formation of a more regular hybrid layer and the formation of longer tags for the preparations carried with a high-speed turbine (Figs. 1a–1f). Considering the aging factor, rewetting with chlorhexidine or deionized water in preparations carried out with a laser or turbine, resulted in similar bond strength values after 24 h storage without thermal cycling, after 6 months of storage and 12,000 thermal cycles and after 12 months storage and 24,000 thermal cycles, independent of rewetting with water or chlorhexidine. The data indicates that there was no degradation of the adhesive interface, in the aging periods tested, in both types of preparation. This result may have occurred due to the adhesive system containing ethanol as a solvent which facilitates their use in wet bonding, because the solvent facilitates the evaporation of water (Dantas et al., 2008). According Yiu et al. (2004), preparation with carbide burs produces a fine and uniform smear layer, which can be easily removed through etching. The etching which precedes some adhesive systems, as used in this study, removes the smear layer and smear plugs caused by the cut of the burs and demineralizes dentin surface (Pashley et al., 2004). The preparation with an Er:YAG laser produces a surface free of a smear layer to promote the thermal ablation of dentin tissue (Ceballos et al., 2001). The absence of a smear layer would explain the similarity between the specimens prepared with the turbine and those prepared with the laser and rewetting with water, given that the adhesive system employed is preceded by etching, which would make the dentin surface similar. The degradation mechanism of the components of the bonding interface can be defined as a complex phenomenon that involves both resin and dentin. This degradation occurs through the action of the water as much as dentin matrix enzyme (MMPs). It is known that MMPs may be activated in environments with low pH (Chaussain-Miller et al., 2006), and etching previously applied to the adhesive contributes to the activation process of MMPs during demineralization, transforming MMPs into pro-MMP-soluble actives via separation of low molecular weight peptides (Tay et al., 2006), thereby increasing the activity of MMPs and contributing to the degradation of the adhesive interface in the long term (Gendron et al., 1999; Pashley, 1992; Tay et al., 2006). In this study, the choice of the concentration of chlorhexidine was 2% based on the results reported in the literature indicating that the use of 2% chlorhexidine prevented a decrease in clamping force (Breschi et al., 2010; Carrilho et al., 2007a,b; Castro et al. 2003; De Munck et al., 2009; Hebling et al., 2005), due to inhibition of enzymatic degradation (MMPs) (Gendron et al., 1999). The samples rewetting with chlorhexidine considered the hypothesis of it to present increased bond strength in the long term when compared with the groups rewetting with water, which was not observed in our study. In addition to chlorhexidine, it is speculated that the presence of hydrophilic monomers such as HEMA, which are present in Single Bond could also
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inhibit the degradation of collagen through the adsorption of MMPs, and facilitate the prolonged inhibitory effect of the chlorhexidine in the MMPs in the demineralized matrix (Osorio et al., 2011). The bond strength in the long term has been analyzed and provides important information about the durability of the adhesive interface. The technique of in vitro aging most commonly used is water storage (Shono et al., 1999) or artificial saliva (Kitasako et al., 2000), which in the long term, decreases the effectiveness of the adhesion through the degradation of the interface components (resin and collagen) by hydrolysis (De Munck et al., 2005). Water can also penetrate and reduce the mechanical properties of the polymer matrix by swelling and reduction of strength between the polymer chains (Ferracane et al., 1998). In addition, some interface components such as unpolymerized monomers can be broken and reduce the union (Hashimoto et al., 2002). The degradation of the adhesive interface has been observed over short periods of time (Shono et al., 1999), in small areas, where diffusion is easier. In our study, we tried to simulate the clinical situation, opting for storage in artificial saliva that has ions, which is more similar to the oral environment, as well as the aging of the restoration, for long periods of time, 6 and 12 months in total, which were the sticks obtained later. In addition to aging through storage in artificial saliva (Kitasako et al., 2000), another method has been reported in the literature, aging by thermocycling (Gale and Darvell, 1999; Miyazaki et al., 1998), which can occur in two ways: (1) hot water accelerates the hydrolysis of the interface components, and subsequently absorbs water and extracts the products of degradation or oligomers of poorly polymerized resin (Miyazaki et al., 1998) and (2) the different temperatures cause expansion and contraction of the restorative material, which is different from tooth structure, creating stress in the adhesive interface (De Munck et al., 2005). In our study, combine both means of aging the adhesive interface as proposed by Amaral et al. (2008). These methods of aging can lead to fracture propagating along the adhesive interface (Gale and Darvell, 1999), as shown in the SEM of specimens prepared with a high-speed turbine and rewetting with water or chlorhexidine, analyzed after 12 months of storage and 24,000 thermal cycles. Based on the current findings, it may be concluded that, chlorhexidine rewetting negatively influence the bond strength of dentin preparation with an Er:YAG. The aging after 6 months saliva storage/12.000 thermal cycles and 12 months saliva storage/24.000 thermal cycles did not interfere the bond strength regardless of dental method preparation. ACKNOWLEDGMENTS The authors acknowledge Patrıcia Marchi for her technical assistance. REFERENCES Amaral FLB, Colucci V, Souza-Gabriel AE, Chinelatti MA, PalmaDibb RG, Corona SAM. 2008. Adhesion to Er:YAG laser-prepared dentin after long-term water storage and thermocycling. Oper Dent 33:51–58. Aoki A, Ishikawa I, Yamada T, Otsuki M, Watanabe H, Tagami J, Ando Y, Yamamoto H. 1998. Comparison between Er:YAG laser
and conventional technique for root caries in vitro. J Dent Res 77: 1404–1414. Aranha AC, De Paula Eduardo C, Gutknecht N, Marques MM, Ramalho KM, Apel C. 2007. Analysis of the interfacial micromorphology of adhesive systems in cavities prepared with Er,Cr:YSGG, Er:YAG laser and bur. Microsc Res Tech 70:745–751. Armengol V, Jean A, Rohanizadeh R, Hamel H. 1999. Scanning electron microscopic analysis of diseased and healthy dental hard tissues after Er:YAG laser irradiation: In vitro study. J Endod 25: 543–546. Barceleiro Mde O, de Mello JB, de Mello GS, Dias KR, de Miranda MS, Sampaio Filho HR. 2005. Hybrid layer thickness and morphology: The influence of cavity preparation with Er:YAG laser. Oper Dent 30:304–310. Breschi L, Mazzoni A, Nato F, Carrilho M, Visintini E, Tj€ aderhane L, Ruggeri A, Jr, Tay FR, Dorigo ED, Pashley DH. 2010. Chlorhexidine stabilizes the adhesive interface: A 2-year in vitro study. Dent Mater 26:320–325. Cardoso MV, Coutinho E, Ermis RB, Poitevin A, Van Landuyt K, De Munck J, Carvalho RC, Lambrechts P, Van Meerbeek B. 2008. Influence of Er,Cr:YSGG laser treatment on the microtensile bond strength of adhesives to dentin. J Adhes Dent 10:25–33. Carrilho MRO, Carvalho RM, de Goes MF, Di Hip olito V, Geraldeli S, Tay FR, Pashley DH, Tj€ aderhane L. 2007a. Chlorhexidine preserves dentin Bond in vitro. J Dent Res 86:91–94. Carrilho MRO, Geraldeli S, Tay FR, de Goes MF, Carvalho RM, Tj€ aderhane L, Reis AF, Hebling J, Mazzoni A, Breschi L, Pashley DH. 2007b. In vivo preservation of the hybrid layer by chlorhexidine. J Dent Res 86:529–533. Ceballos L, Osorio R, Toledano M, Marshall GW. 2001. Microleakage of composite restorations after acid or Er:YAG laser cavity treatments. Dent Mater 17:340–346. Ceballos L, Osorio R, Toledano M, Tay F, Marshall GW. 2002. Bonding to Er:YAG laser treated dentin. J Dent Res 81:119–122. Chaussain-Miller C, Fioretti F, Goldberg M, Menashi S. 2006. The role of matrix metalloproteinases (MMPs) in human caries. J Dent Res 85:22–32. Corona SAM, Souza AE, Chinelatti MA, P ecora JD, Borsatto MC, Palma-Dibb RG. 2007. Effect of energy and pulse repetition rate of Er:YAG laser on dentin ablation ability and morphological analysis of the laser-irradiated substrate. Photomed Laser Surg 25:26–33. Dantas DC, Ribeiro AI, Lima LH, de Lima MG, Gu^ enes GM, Braz AK, Braz R. 2008. Influence of water storage time on the bond strength of etch-and-rinse and self-etching adhesive systems. Braz Dent J 19:219–223. de Castro FL, de Andrade MF, Duarte J unior SL, Vaz LG, Ahid FJ. 2003. Effect of 2% chlorhexidine on microtensile bond strength of composite to dentin. J Adhes Dent 5:129–138. De Moor RJ, Delm e KL. 2010. Laser-assisted cavity preparation and adhesion to erbium-lased tooth structure: Part 2. Present-day adhesion to erbium-lased tooth structure in permanent teeth. J Adhes Dent 12:91–102. 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. J Dent Res 84: 118–132. De Munck J, Van den Steen PE, Mine A, Van Landuyt KL, Poitevin A, Opdenakker G, Van Meerbeek B. 2009. Inhibition of enzymatic degradation of adhesive-dentin interfaces. J Dent Res 88:1101– 1106. de Oliveira MT, de Freitas PM, de Paula Eduardo C, Ambrosano GM, Giannini M. 2007. Influence of diamond sono-abrasion, air-abrasion and Er:YAG Laser irradiation on bonding of different adhesive systems to dentin. Eur J Dent 1:158–166. Ferracane JL, Berge HX, Condon JR. 1998. In vitro aging of dental composites in water-effect of degree of conversion, filler volume, and filler-matrix coupling. J Biomed Mater Res 42:465–472. Firat E, Gurgan S, Gutknecht N. 2012. Microtensile bond strength of an etch-and-rinse adhesive to enamel and dentin after Er:YAG laser pretreatment with different pulse durations. Laser Med Sci 27:15–21. Gale MS, Darvell BW. 1999. Thermal cycling procedures for laboratory testing of dental restorations. J Dent 27:89–99. Gendron R, Grenier D, Sorsa T, Mayrand D. 1999. Inhibition of the activities of matrix metalloproteinases 2, 8, and 9 by chlorhexidine. Clin Diagn Lab Immunol 6:437–439. Hashimoto M, Ohno H, Sano H, Tay FR, Kaga M, Kudou Y, Oguchi H, Araki Y, Kubota M. 2002. Micromorphological changes in resin–dentin bonds after 1 year of water storage. J Biomed Mater Res 63:306–311. Hebling J, Pashley DH, Tj€ aderhane L, Tay FR. 2005. Chlorhexidine arrests subclinical degradation of dentin hybrid layers in vivo. J Dent Res 84:741–746.
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Pashley DH, Tay FR, Yiu C, Hashimoto M, Breschi L, Carvalho RM, Ito S. 2004. Collagen degradation by host-derived enzymes during aging. J Dent Res 83:216–221. Ramos RP, Palma Dibb RG, Chimello DT, Chinelatti MA, P ecora JD. 2002. Effect of Er:YAG laser on bond strength to dentin of a selfetching primer and two single-bottle adhesive systems. Laser Surg Med 31:164–170. Ricci HA, Sanabe ME, Costa CA, Hebling J. 2010. Effect of chlorhexidine on bond strength of two-step etch-and-rinse adhesive systems to dentin of primary and permanent teeth. Am J Dent 23:128–132. Scaffa PM, Vidal CM, Barros N, Gesteira TF, Carmona AK, Breschi L, Pashley DH, Tj€ aderhane L, Tersariol IL, Nascimento FD, Carrilho MR. 2012. Chlorhexidine inhibits the activity of dental cysteine cathepsins. J Dent Res 91:420–425. Shono Y, Ogawa T, Terashita M, Carvalho RM, Pashley EL, Pashley DH. 1999. Regional measurement of resin–dentin bonding as an array. J Dent Res 78:699–705. Staninec M, Gardner AK, Le CQ, Sarma AV, Fried D. 2006. Adhesion of composite to enamel and dentin surfaces irradiated by IR laser pulses of 0.5–35 micros duration. J Biomed Mater Res Part B Appl Biomater 79:193–201. Sulkala M, Larmas M, Sorsa T, Salo T, Tj€ aderhane L. 2002. The localization of matrix metalloproteinase-20 (MMP-20, enamelysin) in mature human teeth. J Dent Res 81:603–607. Tay FR, Pashley DH, Loushine RJ, Weller RN, Monticelli F, Osorio R. 2006. Self-etching adhesives increase collagenolytic activity in radicular dentin. J Endod 32:862–868. Tezvergil-Mutluay A, Mutluay M, Seseogullari-Dirihan R, Agee KA, Key WO, Scheffel DLS, Breschi L, Mazzoni A, Tj€ aderhane L, Nishitani Y, Tay FR, Pashley DH. 2013. Effect of phosphoric acid on the degradation of human dentin matrix. J Dent Res 92:87–91. Trajtenberg CP, Pereira PN, Powers JM. 2004. Resin bond strength and micromorphology of human teeth prepared with an Erbium: YAG laser. Am J Dent 17:331–336. Yiu CK, King NM, Pashley DH, Suh BI, Carvalho RM, Carrilho MR. 2004. Effect of resin hydrophilicity and water storage on resin strength. Biomaterials 25:5789–5796.
Long-Term Chlorhexidine Effect on Bond Strength to Er: YAG Laser Irradiated-Dentin 3 DANIEL GALAFASSI,1 CAMILA SCATENA,2 VIVIAN COLUCCI,1 ANTONIO LUIZ RODRIGUES-JUNIOR, ^ MONICA CAMPOS SERRA,1 AND SILMARA APARECIDA MILORI CORONA1* 1
Department of Restorative Dentistry, Ribeir~ ao Preto Dental School, University of S~ ao Paulo, Ribeir~ ao Preto, S~ ao Paulo, 14040-904, Brazil 2 Department of Paediatric Clinic, Ribeir~ ao Preto Dental School, University of S~ ao Paulo, Ribeir~ ao Preto, S~ ao Paulo, 14040-904, Brazil 3 Department of Social Medicine, Ribeir~ ao Preto Medicine School, University of S~ ao Paulo, Ribeir~ ao Preto, S~ ao Paulo, 14040-904, Brazil
KEY WORDS
bond strength; dentin; chlorhexidine; thermocycling; laser; hybrid layer
ABSTRACT This study evaluates the bond strength of dentin prepared with Er:YAG laser or bur, after rewetting with chlorhexidine on long-term artificial saliva storage and thermocycling. One hundred and twenty human third molars were sectioned in order to expose the dentin surface (n 5 10). The specimens were randomly divided in 12 groups according to treatment and aging: Er:YAG laser rewetting with deionized water (LW) and 24 h storage in artificial saliva (WC); LW and 6 months of artificial saliva storage 1 12.000 thermocycling (6M), LW and 12 months of artificial saliva storage 1 24.000 thermocycling (12M), Er:YAG laser rewetting with 2% chlorhexidine (LC) and WC, LC and 6M, LC and 12M, bur on high-speed turbine rewetting with deionized water (TW) and WC, TW6M, TW12M, bur on high-speed turbine 1 2% chlorhexidine (TC) and WC, TC and 6M, TC and12M. The specimens were etched with 35% phosphoric acid, washed, and dried with air. Single Bond 2 adhesive was applied and the samples were restored with a composite. Each tooth was sectioned in order to obtain 4 sticks, which were submitted to microtensile bond strength test (mTBS). The two-way ANOVA, showed no significant differences for the interaction between the factors and for the aging factor. Tukey 5% showed that the LC group had the lowest mTBS. The rewetting with chlorhexidine negatively influenced the bond strength of the preparation with the Er:YAG laser. The artificial saliva aging and thermocycling did not interfere with dentin bond strength. Microsc. Res. Tech. 77:37–43, 2014. V 2013 C
Wiley Periodicals, Inc.
INTRODUCTION The advances of adhesive dentistry and new dental technologies used in the preparation, promote minimally invasive procedures, beyond improved patient comfort. The Er:YAG laser has proven to be an alternative tool to conventional preparation with a bur (Hibst, 2002). The laser was shown to be effective in the ablation of hard tissue (Corona et al., 2007), once it emits a wavelength of 2.94 mm, coinciding with the peak absorption of water and OH-radicals present in dental tissue (Hibst and Keller, 1989; Ramos et al., 2002). This causes the vaporization of water and hydrated tissue components, causing a rapid warming, followed by microbursts resulting from the increased internal pressure of molecules, which, in turn, lead to ejection of the substrate in the form of microscopic fragments (Aoki et al., 1998; Hibst and Keller, 1989; Hossain et al., 1999; Matsumoto et al., 1996). The laser irradiation of dentin has been described as to favorable for the bond between the restorative material and dental tissue due to the increased surface area for adhesion with the presence of microretentions (Armengol et al., 1999; Trajtenberg et al., 2004) and the opening of dentinal tubules without smear layer formation (Trajtenberg et al., 2004). This would make C V
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adhesion more favorable (Armengol et al., 1999) giving a bond strength similar to preparation with a bur (Oliveira et al., 2005). However, obtaining a stable adhesion over time remains a challenge due to hydrolytic and enzymatic degradation (by matrix metalloproteinases (MMPs)) from the components in this bond (Carrilho et al., 2007a; Pashley et al., 2004). Endogenous enzymes known as MMPs are capable of breaking down the extracellular matrix (Carrilho et al., 2007a; Hebling et al., 2005; Pashley et al., 2004). These enzymes are involved in the process of collagen remodeling during amelogenesis and dentinogenesis and remain latent, trapped in the structures formed (Sulkala et al., 2002). The reactivation of MMPs can occur when there is a metabolic imbalance with the *Correspondence to: Silmara Aparecida Milori Corona, Department of Dentistry, Ribeir~ ao Preto School of Dentistry—USP, Restorative Av. do Cafe, S/N, Monte Alegre, Ribeir~ ao Preto, SP 14040-904, Brazil. E-mail: [email protected] Received 17 September 2013; accepted in revised form 21 October 2013 REVIEW EDITOR: Dr. Chuanbin Mao Contract grant sponsor: FAPESP; Contract grant numbers: 2008/09042-0, 2009/11225-8; Contract grant sponsor: CAPES; Contract grant number: AEX 18542/12-3.. DOI 10.1002/jemt.22310 Published online 1 November 2013 in Wiley Online Library (wileyonlinelibrary.com).
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release of acid as in the carious lesions, through etching before adhesive application, or the low pH of self-etching adhesive systems (Chaussain-Miller et al., 2006; Tay et al., 2006). During the adhesive procedure, some collagen fibrils of the hybrid layer may remain exposed due to imperfect resin impregnation into the demineralized dentin matrix (Breschi et al., 2010) and these MMPs may be affected, leading to enzymatic degradation, which can result in reduced bond strength. CHX is an inhibitor of MMPs 22, 28, and 29 (Gendron et al., 1999) and thus, can increase the bond strength of restorations (Carrilho et al., 2007a; Pashley et al., 2004), acting in its own confinement between the collagen fibres. Recently discovered in normal and carious dentin, the cysteine proteases from cathepsin family have been show to participate in various conditions, which result in the degradation of extracellular matrix components, increases the list of potential endogenous proteases in dentin matrices (Nascimento et al., 2011; Scaffa et al., 2012; Tezvergil-Mutluay et al., 2013). Chlorhexidine is an antimicrobial agent able to inhibit some MMPs and cysteine cathepsins (Gendron et al., 1999; Scaffa et al., 2012) and, according to some in vitro studies, do not adversely affect the bond strength of adhesive systems immediately (Carrilho et al., 2007a; Hebling et al., 2005; Pashley et al., 2004). Furthermore, in vivo (Carrilho et al., 2007b; Hebling et al., 2005) and in vitro studies (Castro et al. 2003; Hebling et al., 2005; Pashley et al., 2004) showed that the application of chlorhexidine in dentin rewetting can slow down or even prevent the degradation of collagen fibres exposed, resulting in a more stable resin-dentin adhesion over time. Given the inexistence of studies assessing the rewetting with chlorhexidine in the preservation of hybrid layer of Er:YAG laser irradiated dentin, the aim of this study was to evaluate the influence of chlorhexidine dentin rewetting on the bond strength of irradiated dentin subjected to aging through storage in artificial saliva and thermocycling. MATERIALS AND METHODS Experimental Design The factors studied were the treatment of dentin on four levels [Er:YAG laser 1 rewetting with deionized water, Er:YAG laser 1 rewetting with chlorhexidine, high-speed turbine 1 rewetting with water, and highspeed turbine 1 rewetting with chlorhexidine (TC)] and aging at three levels: 24 h of storage in artificial saliva/without cycle, 6 months of storage in artificial saliva/12,000 cycles, and 12 months of storage in artificial saliva/24,000 cycles. The sample of the experiment consisted of 120 teeth randomly divided into 12 groups (n 5 10). The design was made up of random blocks. The quantitative response variable was the bond strength (MPa), obtained through microtensile bond strength test (mTBS) testing. Selection of Teeth One hundred and twenty caries-free, freshly extracted human third molars were selected for this study (obtained from the bank of teeth FORP-USP). The following protocol was prescribed, revised and approved by the Committee of Ethics in Human Research (2009.1.165.58.1). The teeth were stored in 0.2% thymol solution at 4 C for 48 h.
Dentin Preparation The root was fixed in polyester resin, through a polyvinyl chloride (PVC) cylinder—1.7 3 1.5 mm (Tigre, Rio Claro, SP, Brazil). Then, the occlusal surface was removed with a diamond saw to exposing the coronal dentin surface (Isomet 1000, Buehler, Like Bluff, IL). The teeth were grounded in a polishing machine (Beta, Buehler, Lake Bluff, IL) with sandpaper # 600. The teeth were randomly divided according to the treatment of dentin (laser rewetting with deionized water (LW), LC, high-speed turbine rewetting with deionized water (TW), and TC). For the irradiated groups, the Er:YAG laser (Twin Light, Fotona Medical Lasers, Ljubljana, Slovenian)—260 mJ/4 Hz with energy density (47 J/cm2) and power density (0.81 W) was applied, scanning across the surface of the transversally and horizontally exposed dentin using a device that holds the laser pen in place. The laser beam was applied using a noncontact method, focused at a distance of 12 mm from the substrate (Amaral et al., 2008), with water spray of 1.5 mL/min (Amaral et al., 2008). For the nonirradiated group, the dentin preparation was performed with carbide bur (#245, Jota AG Rotatory instruments, R€ uthi, Switzerland) in a high-speed turbine (605C, Kavo do Brazil Ind. Com. LTDA., Joinville, SC, Brazil) under constant refrigeration. The same steps used in the Er:YAG laser group (transversally and horizontally) were carried out. Restorative Procedures The specimens were etched with 35% phosphoric acid (Etching gel, 3M ESPE Dental Products, St. Paul, MN) for 15 s, rinsed for 30 s, and dried for 30 s with air. After drying the dentin surface, the specimens in groups LC and TC were rewetting with 1.5 mL of 2% chlorhexidine (Clorhexidina S, FGM Prod. Odontologicos, Joinville, SC, Brazil) with the aid of a micropipette (Labmate Soft, PZ HTL S.A., Warsaw, Poland) at a time of 60 s, the excess being removed with absorbent paper and for specimens of the LW and TW groups dentin rewetting with deionized water was carried out in the same way as the previous groups. Next, the Single Bond 2 adhesive system (Single Bond 2, 3M ESPE Dental Products, St. Paul, MN) was applied, as instructed by the manufacturer, in two layers and cured with halogen light (XL-3000, 3M ESPE Dental products, Germany) for 10 s. After, a plateau of composite resin (Filtek Z350, shade A3, 3M ESPE Dental Products, St. Paul, MN) using the incremental technique in four layers each containing a 1 mm, 4 mm total, was placed over the dentin surface. Aging of the Adhesive Interface The restored specimens were divided according to the technique of storage/aging interface: 24 h of storage in artificial saliva/no cycle, 6 months of storage in artificial saliva/12,000 cycles, and 12 months of storage in artificial saliva/24,000 cycles. Aging consisted of 500 cycles of thermocycling (Etica Equip. Cient. S.A., SP, Brazil) (5 C and 55 C) per week until they completed 12,000 and 24,000 cycles. At weekly intervals, the groups remained immersed in 100 mL of artificial saliva in an oven at a temperature of 37 C (ECB 1.3, Microscopy Research and Technique
CHLORHEXIDINE EFFECT ON IRRADIATED DENTIN TABLE 1. Average resistance to mTBS and SD for the treatment factor Treatments TW TC LW LC
Average of MPa 24,3 (8,11)a 24,1 (9,49)a 20,1 (8,39)a,b 12,1 (6,56)b
39
TABLE 2. Average resistance to mTBS and SD for the aging factor Aging WC 6M 12M
Average of MPa 22,32 (10,06)a 19,20 (10,32)a 19,01 (7,76)a
Same letters indicate statistical similarity.
Same letters indicate statistical similarity.
Odontobras, Ribeir~ ao Preto, SP, Brazil) which was substituted weekly. Microtensile Bond Strength Test After 24 h of storage in artificial saliva, the specimens of the no cycles/24 h of storage in artificial saliva group were fixed in a metallographic cutter and sectioned in both mesiodistal and buccal-lingual directions to obtain multiple sticks of 1.0 mm2 (60.2 mm2) for the mTBS test. From each tooth, the central sticks were collected. The sticks of similar length and dentin thickness remaining were tested (four sticks from each tooth was selected, results in a total of 40 sticks for each group). The sticks were properly measured with digital caliper, identified, and fixed with cyanoacrylate glue gel in the universal testing machine (EMIC, Equipamentos e Sistemas de Ensaios LTDA, S~ ao Jose dos Pinhais, PR, Brazil) at a cross-head speed of 0.5 mm/min. After fixation, they were stressed in tension until failure occurred. An average of values was obtained for the sticks of the same tooth and this was taken as the mean value of the specimen. The same procedure was performed for specimens from 6 to 12 months of aging. The sticks that failed before testing were counted as 0 MPa. The fractured sides of all specimens were inspected under stereo macroscopic (KL200, Leica Microsystems, Heerbrugg, Switzerland) at 103 magnification and classified into four types: adhesive, cohesive in dentin, cohesive in resin, and mixed. The frequency of each mode was calculated in percentage. SEM Evaluations For the scanning electron microscopy (SEM) evaluation of interfacial morphology, another 36 molars were used and their roots were fixed in PVC cylinder with polyester resin. The occlusal surface was removed with a diamond disk so as to expose the coronal dentin surface. The dentin was treated according to the described protocols for different groups TWWC, TW6M, TW12M, TCWC, TC6M, TC12M, LWWC, LW6M, LW12M, LCWC, LC6M, LC12M (n 5 3). All teeth were vertically sectioned into 2.0-mm-thick slabs which were hand polished with 600 and 1,200 grit SiC paper followed by diamond paste (3 mm and 1 mm). The slabs were ultrasonically cleaned during 10 min after each polishing step. After the slabs were treated with ethylenediamine tetraacetic acid (EDTA) 17% for 10 min and rinsed for 10 min. Slabs were dehydrated in ascending alcohol concentrations followed hexamethyldisilazane (HMDS), sputter-coated with gold, and examined under SEM. Representative areas were photographed at 20003. Statistical Analysis The R software was used in the analysis. The data was statistically analyzed by the two-way ANOVA and Tukey test at a significance level of 5%. Microscopy Research and Technique
RESULTS The two-way ANOVA showed significant difference for the Treatment factor (P 5 0.0000) (Table 1). For the Aging factor (P 5 0.1289) (Table 2) and the interaction of Treatment and Aging factors (P 5 0.4519), there was no significant difference. The mean and standard deviation (SD) of mTBS for dentin prepared with the Er:YAG laser or high-speed turbine (bur #245), after aging conducted by storing in deionized water (24 h, 6 and 12 months)/thermocycling (without cycles, 12,000 and 24,000 thermal cycles) are summarized in Table 3. Tukey post hoc test demonstrated that, for the Treatment factor, the LC group showed lower bond strength differing from groups TW and TC. The LW group did not differ statistically from the other groups (Table 1). Analysis of the Adhesive Interface by SEM The analysis of the adhesive interface by SEM showed an irregular hybrid layer formation, with the presence of cracks, for the irradiated groups. There was the formation of regular tags and bigger deposits of adhesive in the regions of the valleys and the peaks were covered with a thin layer of adhesive. However, we could observe the formation of uniform resin tags (Figs. 1g–1l). The groups prepared with the high-speed turbine there was a regular hybrid layer formation with the presence of tags and uniform funnel shapes (Figs. 1a– 1e). Image analysis of the high-speed turbine groups 1c and 1f showed micro cracks at the interface and no tags, which were also observed in the images 1i and 1l. Analysis of Fracture Pattern There was a predominance of a type of mixed fracture for all groups studied (Fig. 2). DISCUSSION The high-speed turbine is the main method for cavity preparation in dentistry. The Er:YAG laser has been indicated as an alternative to high-speed turbine, it offers some advantages which promote patient comfort, such as lower noise, pain, and pressure (Hibst, 2002). Considering the factor adhesion, some studies show that the laser provides similar adhesive resistance to the turbine after short periods of time (Trajtenberg et al., 2004) due to an increase in surface area for adhesion, due to the presence of micro retentions (Armengol et al., 1999). However, besides causing a scaly surface with the presence of rough areas (Ceballos et al., 2001), laser irradiation alters the levels of calcium and phosphate which would lead to the formation of components that are more stable and less soluble to acid conditioning through reducing the effectiveness of etching (De Moor and Delme, 2010). de
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D. GALAFASSI ET AL.
Oliveira et al. (2007) found a reduction in irradiated dentin bond strength due to the formation of a modified layer that would be related to mineral dissolution through etching and diffusion of the adhesive system
TABLE 3. Average of the mTBS (standard deviation) of dentin prepared with the Er:YAG laser or high-speed turbine, rewetting with deionized water or chlorhexidine after aging (MPa) Treatment Aging Preparation/Rewetting TW TC LW LC
WC
6M
12M
27,89 (10,11) 29,30 (5,66) 19,67 (9,85) 12,42 (4,99)
21,16 (7,43) 21,82 (5,97) 21,38 (9,10) 12,42 (6,74)
23,85 (7,43) 21,29 (13,35) 19,32 (6,67) 11,59 (8,24)
Fig. 1. SEM images of adhesive interfaces. (a) TWWC; (b) TW6M; (c) TW12M; (d) TCWC; (e) TC6M; (f) TC12M; (g) LWWC; (h) LW6M; (i) LW12M; (j) LCWC; (k) LC6M; (l) LC12M. A 5 adhesive;
in the dentin surface area, leading to a deficiency in the hybridization of the substrate. Although studies (Aoki et al., 1998; Ceballos et al., 2001; Hibst, 2002) have evaluated the bond strength of dentin irradiated with an Er:YAG for short periods of time, the bond strength after aging was evaluated by a single study (Amaral et al., 2008), which found that the bond strength of dentin prepared with an Er:YAG after 6 months of water storage and thermocycling, showed lower values due to the degradation the adhesive interface. However, the literature does not have mTBS studies of rewetting irradiated dentin with water or chlorhexidine in the long term. The pulse duration of the Er:YAG is an important factor in the bond strength of dentin and is directly related to tissue ablation and surface morphology
D 5 dentin; H 5 hybrid layer; R 5 resin; * 5 gaps; 5 “Rings” around tag. Arrows indicate the peaks and valleys of irregular dentin pattern by Er:YAG laser preparation.
Microscopy Research and Technique
CHLORHEXIDINE EFFECT ON IRRADIATED DENTIN
Fig. 2. Graph of fracture pattern. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
(Firat et al., 2012). Although the Er:YAG short pulse is more effective in the ablation of dental tissue (Firat et al., 2012; Staninec et al., 2006), the long pulse is widely used and gives satisfactory results (Amaral et al., 2008; Corona et al., 2007; Ramos et al., 2002). In this study, dentin preparation with the Er:YAG long pulse seem not to have influenced the bond strength, considering that the adhesion values, for the preparations with the Er:YAG and rewetting with water, were similar to the other groups tested. The results of this study indicate that the use of 2% chlorhexidine for the rewetting of dentin in preparations carried out with an Er:YAG negatively affected the mTBS when compared to preparations with a highspeed turbine. A possible explanation for the lower levels of union in the LC group compared to the groups prepared with a bur may be related to the thermo mechanical ablation process (Hibst and Keller, 1989), which causes denaturation and fusion of collagen fibers, resulting in decreased interfibrillar space (Barceleiro et al., 2005; Ceballos et al., 2001), restricting the diffusion of resin into the subsurface of the intertubular dentin (Ceballos et al., 2002). As the molecular size of chlorhexidine (molecular weight 5 897.8 g21 mol) is greater than the molecule of some components of the adhesive system employed, for example, 2hydroxyethyl methacrylate (HEMA) (MW 5 130.0 g mol21) or bisphenol A-glycidyl methacrylate (BisGMA) (MW 5 512.0 g mol21) (Ricci et al., 2010), it could hinder complete infiltration of the monomer and promote the formation of gaps in the hybrid layer, decreasing the adhesive strength. In the SEM analysis of the adhesive interface, the dentin prepared with an Er:YAG showed the formation of “rings” around the tag (Figs. 1j and 1k) which occur due to the irradiated surface display “cracks,” which would be infiltrated by the adhesive system (de Oliveira et al., 2007). In Figure 1, arrows indicate an irregular hybrid layer, which occurs due to the buildup of adhesive in the region of the valleys, which correspond to the region of highest energy pulse, confirming the findings of de Oliveira et al. (2007). The peak area was filled with a thin layer of adhesive, which can lead to the formation of defects in the hybrid layer, corroborating other findings (Cardoso et al., 2008; Moretto et al., 2011). In the images 1g–1l, the presence of Microscopy Research and Technique
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cracks in the adhesive interface are observed, which could be the result of the breakdown of collagen by the laser and these findings were also reported in the literature (Aranha et al., 2007; Moretto et al., 2011). The SEM showed the formation of a more regular hybrid layer and the formation of longer tags for the preparations carried with a high-speed turbine (Figs. 1a–1f). Considering the aging factor, rewetting with chlorhexidine or deionized water in preparations carried out with a laser or turbine, resulted in similar bond strength values after 24 h storage without thermal cycling, after 6 months of storage and 12,000 thermal cycles and after 12 months storage and 24,000 thermal cycles, independent of rewetting with water or chlorhexidine. The data indicates that there was no degradation of the adhesive interface, in the aging periods tested, in both types of preparation. This result may have occurred due to the adhesive system containing ethanol as a solvent which facilitates their use in wet bonding, because the solvent facilitates the evaporation of water (Dantas et al., 2008). According Yiu et al. (2004), preparation with carbide burs produces a fine and uniform smear layer, which can be easily removed through etching. The etching which precedes some adhesive systems, as used in this study, removes the smear layer and smear plugs caused by the cut of the burs and demineralizes dentin surface (Pashley et al., 2004). The preparation with an Er:YAG laser produces a surface free of a smear layer to promote the thermal ablation of dentin tissue (Ceballos et al., 2001). The absence of a smear layer would explain the similarity between the specimens prepared with the turbine and those prepared with the laser and rewetting with water, given that the adhesive system employed is preceded by etching, which would make the dentin surface similar. The degradation mechanism of the components of the bonding interface can be defined as a complex phenomenon that involves both resin and dentin. This degradation occurs through the action of the water as much as dentin matrix enzyme (MMPs). It is known that MMPs may be activated in environments with low pH (Chaussain-Miller et al., 2006), and etching previously applied to the adhesive contributes to the activation process of MMPs during demineralization, transforming MMPs into pro-MMP-soluble actives via separation of low molecular weight peptides (Tay et al., 2006), thereby increasing the activity of MMPs and contributing to the degradation of the adhesive interface in the long term (Gendron et al., 1999; Pashley, 1992; Tay et al., 2006). In this study, the choice of the concentration of chlorhexidine was 2% based on the results reported in the literature indicating that the use of 2% chlorhexidine prevented a decrease in clamping force (Breschi et al., 2010; Carrilho et al., 2007a,b; Castro et al. 2003; De Munck et al., 2009; Hebling et al., 2005), due to inhibition of enzymatic degradation (MMPs) (Gendron et al., 1999). The samples rewetting with chlorhexidine considered the hypothesis of it to present increased bond strength in the long term when compared with the groups rewetting with water, which was not observed in our study. In addition to chlorhexidine, it is speculated that the presence of hydrophilic monomers such as HEMA, which are present in Single Bond could also
42
D. GALAFASSI ET AL.
inhibit the degradation of collagen through the adsorption of MMPs, and facilitate the prolonged inhibitory effect of the chlorhexidine in the MMPs in the demineralized matrix (Osorio et al., 2011). The bond strength in the long term has been analyzed and provides important information about the durability of the adhesive interface. The technique of in vitro aging most commonly used is water storage (Shono et al., 1999) or artificial saliva (Kitasako et al., 2000), which in the long term, decreases the effectiveness of the adhesion through the degradation of the interface components (resin and collagen) by hydrolysis (De Munck et al., 2005). Water can also penetrate and reduce the mechanical properties of the polymer matrix by swelling and reduction of strength between the polymer chains (Ferracane et al., 1998). In addition, some interface components such as unpolymerized monomers can be broken and reduce the union (Hashimoto et al., 2002). The degradation of the adhesive interface has been observed over short periods of time (Shono et al., 1999), in small areas, where diffusion is easier. In our study, we tried to simulate the clinical situation, opting for storage in artificial saliva that has ions, which is more similar to the oral environment, as well as the aging of the restoration, for long periods of time, 6 and 12 months in total, which were the sticks obtained later. In addition to aging through storage in artificial saliva (Kitasako et al., 2000), another method has been reported in the literature, aging by thermocycling (Gale and Darvell, 1999; Miyazaki et al., 1998), which can occur in two ways: (1) hot water accelerates the hydrolysis of the interface components, and subsequently absorbs water and extracts the products of degradation or oligomers of poorly polymerized resin (Miyazaki et al., 1998) and (2) the different temperatures cause expansion and contraction of the restorative material, which is different from tooth structure, creating stress in the adhesive interface (De Munck et al., 2005). In our study, combine both means of aging the adhesive interface as proposed by Amaral et al. (2008). These methods of aging can lead to fracture propagating along the adhesive interface (Gale and Darvell, 1999), as shown in the SEM of specimens prepared with a high-speed turbine and rewetting with water or chlorhexidine, analyzed after 12 months of storage and 24,000 thermal cycles. Based on the current findings, it may be concluded that, chlorhexidine rewetting negatively influence the bond strength of dentin preparation with an Er:YAG. The aging after 6 months saliva storage/12.000 thermal cycles and 12 months saliva storage/24.000 thermal cycles did not interfere the bond strength regardless of dental method preparation. ACKNOWLEDGMENTS The authors acknowledge Patrıcia Marchi for her technical assistance. REFERENCES Amaral FLB, Colucci V, Souza-Gabriel AE, Chinelatti MA, PalmaDibb RG, Corona SAM. 2008. Adhesion to Er:YAG laser-prepared dentin after long-term water storage and thermocycling. Oper Dent 33:51–58. Aoki A, Ishikawa I, Yamada T, Otsuki M, Watanabe H, Tagami J, Ando Y, Yamamoto H. 1998. Comparison between Er:YAG laser
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