Journal of Biomechanics 48 (2015) 701–707
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Effect of partially demineralized dentin beneath the hybrid layer on dentin–adhesive interface micromechanics Rodolfo Bruniera Anchieta a,g,n, Lucas Silveira Machado b,g, Renato Herman Sundfeld b, André Figueiredo Reis c, Marcelo Giannini d, Marco Antonio Luersen e, Malvin Janal f, Eduardo Passos Rocha a, Paulo G. Coelho g a
Department of Dental Materials and Prosthodontics, Sao Paulo State University, Araçatuba Dental School, Araçatuba, SP, Brazil Department of Restorative Dentistry, Sao Paulo State University, Araçatuba Dental School, Araçatuba, SP, Brazil c Department of Operative Dentistry, School of Dentistry, University of Guarulhos, Guarulhos, SP, Brazil d Department of Restorative Dentistry, Piracicaba Dental School, State University of Campinas, Piracicaba, SP, Brazil e Universidade Tecnológica Federal do Paraná, Curitiba, Brazil f New York University, College of Dentistry, Department of Biomaterial and Biomimetics, New York, NY, USA g New York University, College of Dentistry, Epidemiology and Health Promotion, New York, NY, USA b
art ic l e i nf o
a b s t r a c t
Article history: Accepted 25 August 2014
Objective: To investigate the presence of non-infiltrated, partially demineralized dentin (PDD) beneath the hybrid layer for self-etch adhesive systems, and its effect on micromechanical behavior of dentin– adhesive interfaces (DAIs). This in-vitro laboratory and computer simulation study hypothesized that the presence of non-infiltrated PDD beneath the hybrid layer does not influence the mechanical behavior of the DAI of self-etch adhesive systems. Methods: Fifteen sound third molars were restored with composite resin using three adhesive systems: Scotchbond Multipurpose (SBMP), Clearfil SE Bond (CSEB) and Adper Promp L-Pop (APLP). The thickness and length of all DAIs were assessed using scanning electron microscopy, and used to generate threedimensional finite element models. Elastic moduli of the hybrid layer, adhesive layer, intertubular dentin, peritubular dentin and resin tags were acquired using a nano-indenter. Finite element software was used to determine the maximum principal stress. Mixed models analysis of variance was used to verify statistical differences (Po 0.05). Results: Elastic moduli and morphology were found to differ between the adhesive systems, as well as the presence and extension of PDD. Significance: Both self-etch adhesive systems (APLP and CSEB) had PDD. The DAI stress levels were higher for the one-step self-etch adhesive system (APLP) compared with the etch-and-rinse adhesive system (SBMP) and the self-etch primer system (CSEB). & 2014 Elsevier Ltd. All rights reserved.
Keywords: Dentin Adhesive systems Mechanical properties Finite element Adhesion Nano-indentation Hybrid layer
1. Introduction Alterations in the mechanical properties and morphology of dentin beneath the hybrid layer (HL) have been reported when using etch-and-rinse adhesives (Hashimoto et al., 2000a, 2000b; Katz et al., 2001; Sauro et al., 2012; Schulze et al., 2005). These alterations are mainly associated with the depth of demineralization promoted by phosphoric acid conditioning of the dentin substrate (Bouillaguet et al., 2001), collapse of the collagen fibril network (Pashley et al., n Correspondence to: Departamento de Materiais Odontológicos e Prótese, Faculdade de Odontologia de Araçatuba, 1193, Araçatuba, SP, Brazil, CEP: 16015-050. Tel.: þ 1 55 18 36363290. E-mail address: [email protected] (R. Bruniera Anchieta).
http://dx.doi.org/10.1016/j.jbiomech.2014.08.021 0021-9290/& 2014 Elsevier Ltd. All rights reserved.
1993), and discrepancy between dentin demineralization and efficiency of adhesive infiltration (Oliveira et al., 2004). However, areas with nanoleakage (Carrilho et al., 2005; Reis et al., 2007a; Tay et al., 2002) or with partially demineralized dentin (PDD) beneath the hybrid layer (Oliveira et al., 2004) have been detected for self-etch adhesive systems (Carvalho et al., 2005). These defects are likely to be a consequence of the discrepancy between self-etch adhesive demineralization and the efficiency of monomer infiltration, or due to incomplete polymerization of monomers in the deepest regions of the hybrid layer (Schulze et al., 2005; Yuan et al., 2007). Morphological alterations and mechanical properties of the supposedly altered dentin beneath the hybrid layer have only been studied for etch-and-rinse adhesives and mildly acidic, two-step
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self-etch primers (Katz et al., 2001; Oliveira et al., 2004). Little information is available regarding the mechanical properties of dentin following one-step self-etch adhesive systems, which are known to be more acidic and aggressive to the dental substrate, have greater potential for demineralization, and form more complex dentin–adhesive interfaces (DAIs) (Wang and Spencer, 2004). In addition, the influence of PDD on stress behavior and distribution in DAI structures has not been evaluated, to date, for self-etch adhesive systems. This study investigated the presence of PDD beneath the hybrid layer for self-etch adhesive systems, and its effect on the micromechanical behavior of DAIs. This in-vitro laboratory and computer simulation study hypothesized that the presence of PDD beneath the hybrid layer does not influence the mechanical behavior of the DAI of self-etch adhesive systems. 2. Methods 2.1. Sample preparation Fifteen human third molars (Protocol # 2009-02142) were used to obtain DAI morphology and mechanical properties. All teeth were stored in Hank's balanced salt solution (HBSS), and used within 3 months of extraction (Habelitz et al., 2002). After exposure of the dentin surface 3 mm from the enamel–cementum junction, (Isomet 2000; Buehler, Lake Bluff, IL, USA), three non-restored teeth were used to characterize the morphology of dentin. One slab (2 mm thick) of each tooth was obtained by cross-sectioning the tooth perpendicular to the dentin tubules (Isomet 2000). For the remaining teeth (n¼ 12), a standard smear layer was created using silicon carbide paper #600 (Buehler) (Tay and Pashley, 2001). Three adhesive systems were used in accordance with the manufacturers' recommendations, followed by composite resin placement (n¼4): ScotchBond Multipurpose (SBMP; 3M ESPE, St. Paul, MN, USA), Clearfil SE Bond (CSEB; Kuraray Noritake Dental Inc., Kurashiki, Japan) and Adper Prompt L-Pop (APLP; 3M ESPE). The restored teeth were cross-sectioned mesio-distally with a diamond disc in order to obtain at least four slabs ( 2 mm thick) containing the DAI. Two slabs of each tooth were used for scanning electron microscopy (SEM) and two slabs of each tooth were used for nano-indentation. 2.2. Scanning electron microscopy SEM was used to obtain the morphological characteristics of the DAI for each adhesive system tested, including the thickness of the hybrid layer and the adhesive layer, and the length of resin tags. The non-restored dentin specimens (n¼3) were used to measure the number and diameter of dentin tubules, and the radius of peritubular dentin. These data were used to build the finite element models. All restored and non-restored specimens were prepared for SEM in accordance with previous studies, including fixation, dehydration, drying, mounting and polishing (Kaaden et al., 2003; Perdigao et al., 1995). The restored specimens were ultra-sonicated in 100% ethanol for 5 min, dried, demineralized in 6N HCl for 30 s, and protein was denatured in 2% NaOCl for 10 min (Duarte et al., 2009). After drying, all the specimens (restored and non-restored) were coated with gold using a sputter coater (Emitech K650; Emitech Products Inc., Houston, TX, USA) at 10 mA for 4 min. The slabs were observed under an SEM (Hitachi S-3500 N; Hitachi Science System Ltd., Ibaraki, Japan) at an accelerating voltage of 5 kV and a working distance of 10 mm (2000 and 4000 ). Three images were obtained from each slab (left, center and right), and linear measurements were taken using PCI 5.5 Quartz software (Quartz Imaging Corporation, Vancouver, Canada). 2.3. Nano-indentation The two restored dentin slabs that had not been prepared for SEM were embedded in epoxy resin, ground with silicon carbide papers of decreasing abrasiveness (600–1200 grit), and polished with diamond solution (9, 6, 3, 1 and 0.5 mm) under cloth paper. The imaging and indentation processes were undertaken using a Berkovich fluid cell diamond three-sided pyramid probe in a nano-indenter (950TI; Hysitron, Minneapolis, MN, USA), and tests were performed in wet conditions (HBSS) (Dos Santos et al., 2011; Habelitz et al., 2002). A loading profile was developed with a peak load of 300 mN for dentin and 100 mN for the hybrid layer and adhesive layer, at a rate of 60 mN/s and 20 mN/s, respectively, followed by a holding time of 10 s and an unloading time of 2 s. For each indentation, the reduced elastic modulus was calculated from each loaddisplacement curve, and the elastic modulus was obtained (Anchieta et al., 2014; Oliver and Pharr, 1992).
Prior to indenting the region of interest, the interface was scanned with an in-situ scanning probe microscope with the Berkovich indenter to verify appropriate positioning. Ten indentations were made in the DAI (hybrid layer and adhesive layer) on each slab. Indentations were made horizontally following the adhesive layer and hybrid layer. For intertubular dentin, the first 10 indentations were made below the hybrid layer, moving away from the interface in a single column. Indentations were made in a straight line, at intervals of 1 mm, from the hybrid layer to the dentin with a load of approximately 300 mN (Fig. 1). A large indent of 2000 mN in the adhesive served as a fiduciary mark. Five additional indentations were made horizontally, far from the DAI, in order to assess intact intertubular dentin (Fig. 1). 2.4. Finite element analysis A micromodel of dentin restored with composite resin (41 41 82 mm3) (Fig. 2) was built using solid modelling software (SolidWorks 2010; SolidWorks Corporation, Concord, MA, USA), in accordance with previously published data, with appropriate tensile loading (0.03 N), boundary conditions (Anchieta et al., 2007, 2011; Junior et al., 2012; Martini et al., 2009) and cross-sectional area (Fig. 2) (Lin et al., 2011; Phrukkanon et al., 1998) taken into account. The morphological characteristics and dimensions of the DAI for each adhesive system were obtained from SEM analysis, and are shown in Table 1. All materials were considered to be linear, isotropic and homogenous. Elastic modulus (E) and Poisson's ratio (υ) were used in accordance with Table 2. The elastic modulus of peritubular dentin obtained from the nano-indentation test was 30 ( 7 3.6) GPa, and the Poisson ratio was 0.3 (Le et al., 2001). Composite resin had an elastic modulus of 30 GPa and a Poisson ratio of 0.3 (Junior et al., 2012). The morphological characteristics and dimensions of the DAI for each adhesive system were used to build geometrical models, as follows:
SBMP – 4-mm-thick hybrid layer, funnel-shaped resin tags with length of 30 mm
and base thickness of 3.6 mm, 15-mm-thick adhesive layer and 5-mm-thick PDD layer; CSEB – similar to SBMP, but with 0.85-mm-thick hybrid layer, funnel-shaped resin tags with length of 16 mm and base thickness of 2.5 mm, 10-mm-thick adhesive layer and 1.5-mm-thick PDD layer; and APLP – similar to SBMP, but with 3-mm-thick hybrid layer, funnel-shaped resin tags with length of 25 mm and base thickness of 3 mm, 6-mm-thick adhesive layer and 3-mm-thick PDD layer (Table 1 and Fig. 3).
The thickness of the PDD layer was subdivided into 1-mm layers, and the elastic modulus was obtained through nano-indentation in order to reproduce a graded structure. Two other models (CSEB-0 and APLP-0) were created to verify the influence of the PDD layer. These comprised the exact same features of the CSEB and APLP models, correspondingly, but did not include a PDD layer. Quadratic tetrahedral elements were used for high-quality finite element mesh generation, which was driven by the convergence of analysis (6%). On average, the models presented 1,148,776 elements and 5,077,374 nodes. The bases of all models were fixed in the x, y, and z axes, and loading conditions were applied to each model. For numerical analysis, finite element software (ANSYS Workbench 14.0; Swanson Analysis System, Canonsburg, PA, USA) was used to acquire maximum principal stress (σmax). The loading definition details are presented in Appendix A (Fig. 2). 2.5. Data analysis Preliminary analysis evaluated distributional assumptions. The elastic moduli of the hybrid layer and adhesive layer were compared using mixed model analysis of variance (ANOVA) (P o0.05). For dentin, the mechanical behavior of each adhesive system was compared at increasing distances from the hybrid layer using a mixed model ANOVA; first, with fixed factors of adhesive system (three levels) and distance (linear trend); second, with fixed factors of adhesive system and squared distance (quadratic trend); and third, with fixed factors of adhesive system, linear trend, quadratic trend and their two-way interactions. All models included a random intercept to account for multiple observations from the same samples. The model fit was summarized using Aikaike's Information Criterion, and Po 0.05 was considered to indicate significance. Statistical Package for the Social Sciences Version 21 (IBM Corp., Armonk, NY, USA) was used for all statistical analyses. Descriptive analyses were used to describe results from SEM and finite element analysis.
3. Results The DAI linear measurements from SEM [mean and standard deviation (SD)] for each adhesive system are presented in Table 1, and Fig. 3 shows the features of each adhesive system interface. The mean number of dentin tubules counted inside a square of 40 mm2 was 36 (SD 1.3), and the peritubular dentin radius was
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Fig. 1. (A) In-situ scanning probe microscope (SPM) image (40 40 mm2) for Adper Promp L-Pop (APLP), showing a schematic region where the indentations were made for the adhesive layer (ADH), hybrid layer (HL) and dentin in all tested adhesives [Scotchbond Multipurpose (SBMP), Clearfil SE Bond (CSEB), APLP]. The indents in the HL were made in the middle of the layer. In dentin, the first 10 indents were made 1-mm apart in a straight line perpendicular to the interface, starting just below the HL. The other five indents were made far from the dentin–adhesive interface. (B and C) In-situ SPM images (20 20 mm2) showing the indentations made (red square) using linear regression (B) and 3D topographical (C). A large indent (2000 mN) in the adhesive served as a fiduciary mark. In the column of indentations, the values obtained from the adhesive and hybrid layers were not recorded, serving only to identify the first indentation made in the intertubular dentin (white arrow). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Table 2 Mechanical properties of the dentin–adhesive interface (DAI). Mean (standard deviation) elastic modulus (GPa) of DAI structures obtained from nano-indentation. Poisson's ratio based on Le et al. (2001).
SBMP CSEB APLP Poisson ratio
Adhesive layer
Hybrid layer
Intertubular dentin*
3.8 (0.48)A 3.9 (0.48)A 1.0 (0.37)B 0.28
2.9 (0.96)B 3.6 (0.70)A 1.5 (0.55)C 0.28
18.1 (2.3)A 18.1 (3.3)A 19.0 (3.6)A 0.3
SBMP, Scotchbond Multipurpose; CSEB, Clearfil SE Bond; APLP, Adper Promp L-Pop. *Intertubular dentin at a distance from the DAI. Different uppercase letters indicate significant differences between columns. Fig. 2. Determination of dimension and loading intensity of micromodels. (A) Hourglass-shaped macrospecimen with 1.1-mm2 cross-sectional area. (B) A microfragment of the dentin–adhesive interface was removed from inside the macrospecimen. (C) Micromodel of 1.681-mm2 cross-sectional area; 0.03 N loading was applied, proportional to force applied in the macrospecimen. Table 1 Mean (standard deviation) thickness of adhesive layer and hybrid layer, and length of resin tags for three adhesive systems. Thickness of adhesive layer (mm) SBMP 15.4 (0.6) CSEB 10.7 (0.4) APLP 5.9 (0.4)
Thickness of hybrid layer (mm)
Length of resin tags (mm)
4.0 (0.7) 0.85 (0.2) 3.2 (0.4)
31.3 (12.9) 15.6 (5.1) 27.8 (7.2)
SBMP, Scotchbond Multipurpose; CSEB, Clearfil SE Bond; APLP, Adper Promp L-Pop.
0.9 mm. The diameter of exposed dentin tubules in finite element models followed the diameter of the base thickness of resin tags, as measured through SEM. The elastic modulus of the hybrid layer was higher for CSEB compared with SBMP and APLP (P o0.05) (Table 2). The elastic modulus of the adhesive layer was also higher for CSEB compared with APLP (P o0.05), but was similar compared with SBMU (P 40.05) (Table 2). For all adhesive systems, the elastic modulus for dentin beneath the hybrid layer differed from the elastic modulus for dentin far from the DAI, suggesting a gradient due to the presence of non-infiltrated PDD (Fig. 4). Fig. 4A shows the mean (standard error) elastic modulus for each adhesive system at increasing distance from the DAI, unadjusted for repeated observations. The mean elastic modulus for each adhesive system was relatively similar when measured close to (approximately 10 GPa)
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Fig. 3. (A) Scanning electron microscope images of Scotchbond Multipurpose (SBMP), Clearfil SE Bond (CSEB) and Adper Promp L-Pop (APLP). SBMP had a 4-mm hybrid layer (HL) and longer resin tags (30 mm) for the dentin–adhesive interface. CSEB had a 1-mm HL and resin tags of 15 mm, and APLP had a 3-mm HL and resin tags of 25 mm. (B) In-situ scanning probe microscope images (3D topographical 3D) of SBMP, CSEB and APLP interfaces obtained from a nano-indenter. ADH, adhesive layer; D, dentin; rt, resin tags.
Fig. 4. (A) Mean (standard error) elastic modulus for dentin at increasing distance from the dentin–adhesive interface, unadjusted for repeated observations. (B) Values predicted from the linear model. (C) Values predicted from the linear/quadratic model. Attempts to fit models of increasing complexity showed that the linear model fit better [Aikaike's Information Criterion (AIC) ¼ 563.5] than the quadratic model (AIC¼ 583.4), but did not different from the linear/quadratic model (AIC¼ 562.4). SBMP, Scotchbond Multipurpose; CSEB, Clearfil SE Bond; APLP, Adper Promp L-Pop.
or distant from (approximately 20 GPa) the DAI, but more variation was seen at intermediate distances from the DAI. In other words, all three adhesive systems showed similar elastic moduli at the beginning and end of the transition zone, but appeared to follow different trajectories through the middle of the zone. Values predicted from the linear model are shown in Fig. 4B. The analysis indicated that distance had a major effect on elastic modulus, as the elastic modulus increased by approximately 1 GPa for every 1 μm from the DAI (P o0.001), and the elastic modulus was lower (P o0.05) for SBMP compared with APLP and CSEB, which did not differ from one another. The reference line suggests the earliest recovery of intact intertubular dentin (15 GPa) at approximately 3 mm for CSEB, at approximately 4 mm for APLP, and at approximately 6 mm for SBMP. The model provided no evidence to suggest different slopes for the three adhesive systems (P ¼0.65). This analysis suggests a similar trajectory for each adhesive system, but overall lower levels of elastic modulus in teeth treated with SBMP. As the linear model did not detect a difference in trajectories between the adhesive systems, as was suggested in the raw data,
and the linear/quadratic model fit as well (although not as parsimoniously) as the linear model, predicted values from the more complex linear/quadratic model are shown in Fig. 4C. This model shows greater fidelity to the raw data, while smoothing trends. Simply, the change with distance appears linear for APLP, smaller for CSEB and greater for SBMP. While elastic moduli at the beginning and end of the transition zone were similar for each adhesive system, behavior between approximately 3 and 6 mm was predicted to differ. For example, the horizontal line shows that the earliest recovery of intact intertubular dentin (15 GPa) occurred at approximately 3 mm for CSEB, at approximately 4 mm for APLP and at approximately 6 mm for SBMP. The analysis indicated a linear effect of distance (P ¼0.003) for SBMP unlike the other adhesive systems, and the linear/quadratic model identified a longer recovery for APLP compared with CSEB. The numerical simulation output, presented in Figs. 5 and 6, demonstrated that the highest stress levels were found in the hybrid layer for APLP. The highest stress level for PDD occurred for SBMP, followed by APLP and CSEB. CSEB showed the lowest
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stress level at the DAI. Models without a PDD layer (CSEB-0 and APLP-0) had lower stress levels compared with their corresponding PDD layer counterparts (CSEB and APLP). The bottom of the hybrid layer in contact with the PDD was the location where higher stresses were found for SBMP and APLP (Figs. 5 and 6).
Fig. 5. Maximum principal stress (MPa) in hybrid layer (HL), adhesive layer (ADH), resin tag (RT), hybrid layer, intertubular dentin (ID) and non-infiltrated partially demineralized dentin (PDD) for all models: Scotchbond Multipurpose (SBMP), Clearfil SE Bond (CSEB), CSEB without a PDD layer (CSEB-0), Adper Promp L-Pop (APLP), and Adper Promp L-Pop without a PDD layer (APLP-0).
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4. Discussion From a theoretical standpoint, self-etch adhesive systems are expected to minimize limitations of the etch-and-rinse hybridization technique, such as the presence of a PDD layer beneath the hybrid layer (Van Meerbeek et al., 2003). However, studies have demonstrated that partial or total exposure of collagen fibrils beneath the hybrid layer remains (Schulze et al., 2005), which may result in failure due to interfacial degradation (Oliveira et al., 2004; Reis et al., 2007b). While the majority of investigations concerning PDD have concentrated on its potential deleterious effect on the restoration of marginal integrity, the current study aimed to test the hypothesis that the presence of PDD in both etch-and-rinse and self-etch adhesive systems would also result in biomechanically unfavorable scenarios. Unique to the present investigation, the extension and mechanical properties of the different adhesive layers and the PDD region in the etch-andrinse and self-etch adhesive systems were acquired in situ through nanomechanical testing, with real dimension computer models used to test the hypothesis. The elastic modulus of the hybrid layer differed according to the type of adhesive used. The main reason for this can be attributed to differences in chemical composition (Van Landuyt et al., 2007) and interactions with the tooth substrate (Sauro et al., 2012). The elastic modulus of the hybrid layer was lower for APLP than SBMP and CSEB, which may be related to the complex chemical composition of all-in-one adhesives (Van Landuyt et al., 2009). It has been hypothesized that one-step adhesives may not polymerize properly due to a low degree of conversion (Nunes
Fig. 6. Cross-section images from Adper Promp L-Pop (APLP), APLP without a partially demineralized dentin (PDD) layer (APLP-0), Clearfil SE Bond (CSEB) and CSEB without a PDD layer (CSEB-0) showing the stress levels inside the models. The higher stress levels were found at the bottom of the hybrid layer in contact with the PDD for CSEB and APLP. The horizontal black lines show the dentin–adhesive interface layers (adhesive layer, hybrid layer, PDD and dentin) for better visualization.
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et al., 2006), which may lead to the formation of a polymer with lower physical–mechanical strength (Van Landuyt et al., 2009). In addition to lower physical–mechanical strength, low conversion results in more water sorption, hydrolysis and subsequent DAI degradation. Furthermore, adhesives with strong acidity have uncured residual monomers and excessive presence of residual water that may be responsible for the formation of a poor-quality hybrid layer (Sauro et al., 2012). The presence of water can result in a non-resin-infiltrated demineralized collagen layer beneath the resin–hybridized dentin, due to the continuing demineralization effect of non-polymerized acidic monomers (Nunes et al., 2006). Another factor that may explain the higher elastic modulus of the hybrid layer for CSEB and SBMP is the application of a hydrophobic resin as the last step of the bonding procedure. This has a high level of cross-links, such as Bis-GMA, that provide mechanical strength to the adhesive system by forming densely cross-linked polymers that prevent water uptake after curing (Van Landuyt et al., 2009). This is in direct agreement with the elastic moduli of the hybrid layer and the adhesive layer observed for these adhesive systems. Another factor that may explain the higher elastic modulus of the hybrid layer for CSEB is the presence of an acid–base resistance zone (ABRZ). This zone is mainly described for mild self-etch adhesives, such as Clearfil SE Bond and Clearfil Protect Bond (Inoue et al., 2006). The formation of an ABRZ is a result of hydroxyapatite crystals that are not completely demineralized, and remain within inside and around collagen fibrils within the hybrid layer than an ABRZ beneath the hybrid layer (Waidyasekera et al., 2009). Hydroxyapatite crystal remnants within the hybrid layer have been reported to form calcium salts with functional monomers in mild self-etch adhesive systems, especially MDP or MDPB (Yoshida et al., 2004, 2012), and contribute to bonding durability. ABRZs are not commonly found for etch-and-rinse adhesives or aggressive self-etch adhesives (Nurrohman et al., 2012). The nano-indentation results showed differences in the mechanical properties of dentin beneath the hybrid layer and far from the DAI interface, confirming the presence of a PDD layer for all systems tested, as reported previously (Oliveira et al., 2004; Schulze et al., 2005). Previous studies regarding the elastic modulus of sound dentin have reported values ranging from 15 to 19 GPa (Kinney et al., 2003; Ryou et al., 2011). Fitting a linear model to the data suggested differences in the average elastic modulus between the adhesive systems but a common trajectory. A more complex, linear/quadratic model suggested a similar overall elastic modulus, but different trajectories between the adhesive systems. The more complex model provided a better representation of the differing elastic moduli of dentin, given differences in bonding material and distance from the transition zone, and highlighted differences in the recovery of normal elastic modulus more clearly; this model is consistent with the expectations of variable degrees of etching. Despite this, the linear/quadratic model did not have a better fit than the simple linear model, and the authors are not confident that the solution could be replicated given the relatively small sample. Thus, both models identified the longer recovery of normal elastic modulus for SBMP compared with the other adhesive systems, but more data are needed to verify if stiffness is regained at a shorter distance from the DAI for CSEB than SBMP. From a theoretical standpoint, the mechanical competence of the DAI for the etch-and-rinse adhesive system was expected to be lower compared with the self-etch adhesive systems. However, when the real dimension and mechanical property 3D finite element model output was evaluated, stress levels were higher for the one-step self-etch adhesive system (APLP) than for the selfetch primer system (CSEB) or the etch-and-rinse adhesive system (SBMP). These differences in computer model outputs pertain to a
combination of features, including differences in the thicknesses of the adhesive and hybrid layers (Anchieta et al., 2007), differences in elastic moduli, and differences in the morphology of resin tags between systems. The PDD at the bottom of the hybrid layer is a critical part of the resin–dentin interface. This was the region of maximum stress for all systems, but this was most pronounced for APLP. It has been shown previously that high stress levels in this region jeopardize the mechanical integrity of the DAI, possibly accelerating premature failure (Garcia et al., 2009; Goracci et al., 2004; Van Landuyt et al., 2009). While the results of this study demonstrate differences in the overall biomechanical behavior of the DAI between adhesive systems, future investigations must consider the effect of degradation time on PDD behavior, as it is anticipated that degradation will occur at different rates for the different adhesive systems.
5. Conclusion Use of CSEB and APLP resulted in non-infiltrated PDD beneath the hybrid layer. The presence of a PDD layer increased the stress levels for the self-etch adhesive models (APLP and CSEB), compared with the etch-and-rinse adhesive system (SBMP), showing the detrimental effect of a PDD layer for overall endurance of the DAI.
Conflict of interest statement None declared.
Acknowledgments This study was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo – FAPESP (Grant #2008/002900-9; Scholarships 2010/12926-7 and 2012/07012-1).
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Effect of partially demineralized dentin beneath the hybrid layer on dentin–adhesive interface micromechanics Rodolfo Bruniera Anchieta a,g,n, Lucas Silveira Machado b,g, Renato Herman Sundfeld b, André Figueiredo Reis c, Marcelo Giannini d, Marco Antonio Luersen e, Malvin Janal f, Eduardo Passos Rocha a, Paulo G. Coelho g a
Department of Dental Materials and Prosthodontics, Sao Paulo State University, Araçatuba Dental School, Araçatuba, SP, Brazil Department of Restorative Dentistry, Sao Paulo State University, Araçatuba Dental School, Araçatuba, SP, Brazil c Department of Operative Dentistry, School of Dentistry, University of Guarulhos, Guarulhos, SP, Brazil d Department of Restorative Dentistry, Piracicaba Dental School, State University of Campinas, Piracicaba, SP, Brazil e Universidade Tecnológica Federal do Paraná, Curitiba, Brazil f New York University, College of Dentistry, Department of Biomaterial and Biomimetics, New York, NY, USA g New York University, College of Dentistry, Epidemiology and Health Promotion, New York, NY, USA b
art ic l e i nf o
a b s t r a c t
Article history: Accepted 25 August 2014
Objective: To investigate the presence of non-infiltrated, partially demineralized dentin (PDD) beneath the hybrid layer for self-etch adhesive systems, and its effect on micromechanical behavior of dentin– adhesive interfaces (DAIs). This in-vitro laboratory and computer simulation study hypothesized that the presence of non-infiltrated PDD beneath the hybrid layer does not influence the mechanical behavior of the DAI of self-etch adhesive systems. Methods: Fifteen sound third molars were restored with composite resin using three adhesive systems: Scotchbond Multipurpose (SBMP), Clearfil SE Bond (CSEB) and Adper Promp L-Pop (APLP). The thickness and length of all DAIs were assessed using scanning electron microscopy, and used to generate threedimensional finite element models. Elastic moduli of the hybrid layer, adhesive layer, intertubular dentin, peritubular dentin and resin tags were acquired using a nano-indenter. Finite element software was used to determine the maximum principal stress. Mixed models analysis of variance was used to verify statistical differences (Po 0.05). Results: Elastic moduli and morphology were found to differ between the adhesive systems, as well as the presence and extension of PDD. Significance: Both self-etch adhesive systems (APLP and CSEB) had PDD. The DAI stress levels were higher for the one-step self-etch adhesive system (APLP) compared with the etch-and-rinse adhesive system (SBMP) and the self-etch primer system (CSEB). & 2014 Elsevier Ltd. All rights reserved.
Keywords: Dentin Adhesive systems Mechanical properties Finite element Adhesion Nano-indentation Hybrid layer
1. Introduction Alterations in the mechanical properties and morphology of dentin beneath the hybrid layer (HL) have been reported when using etch-and-rinse adhesives (Hashimoto et al., 2000a, 2000b; Katz et al., 2001; Sauro et al., 2012; Schulze et al., 2005). These alterations are mainly associated with the depth of demineralization promoted by phosphoric acid conditioning of the dentin substrate (Bouillaguet et al., 2001), collapse of the collagen fibril network (Pashley et al., n Correspondence to: Departamento de Materiais Odontológicos e Prótese, Faculdade de Odontologia de Araçatuba, 1193, Araçatuba, SP, Brazil, CEP: 16015-050. Tel.: þ 1 55 18 36363290. E-mail address: [email protected] (R. Bruniera Anchieta).
http://dx.doi.org/10.1016/j.jbiomech.2014.08.021 0021-9290/& 2014 Elsevier Ltd. All rights reserved.
1993), and discrepancy between dentin demineralization and efficiency of adhesive infiltration (Oliveira et al., 2004). However, areas with nanoleakage (Carrilho et al., 2005; Reis et al., 2007a; Tay et al., 2002) or with partially demineralized dentin (PDD) beneath the hybrid layer (Oliveira et al., 2004) have been detected for self-etch adhesive systems (Carvalho et al., 2005). These defects are likely to be a consequence of the discrepancy between self-etch adhesive demineralization and the efficiency of monomer infiltration, or due to incomplete polymerization of monomers in the deepest regions of the hybrid layer (Schulze et al., 2005; Yuan et al., 2007). Morphological alterations and mechanical properties of the supposedly altered dentin beneath the hybrid layer have only been studied for etch-and-rinse adhesives and mildly acidic, two-step
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self-etch primers (Katz et al., 2001; Oliveira et al., 2004). Little information is available regarding the mechanical properties of dentin following one-step self-etch adhesive systems, which are known to be more acidic and aggressive to the dental substrate, have greater potential for demineralization, and form more complex dentin–adhesive interfaces (DAIs) (Wang and Spencer, 2004). In addition, the influence of PDD on stress behavior and distribution in DAI structures has not been evaluated, to date, for self-etch adhesive systems. This study investigated the presence of PDD beneath the hybrid layer for self-etch adhesive systems, and its effect on the micromechanical behavior of DAIs. This in-vitro laboratory and computer simulation study hypothesized that the presence of PDD beneath the hybrid layer does not influence the mechanical behavior of the DAI of self-etch adhesive systems. 2. Methods 2.1. Sample preparation Fifteen human third molars (Protocol # 2009-02142) were used to obtain DAI morphology and mechanical properties. All teeth were stored in Hank's balanced salt solution (HBSS), and used within 3 months of extraction (Habelitz et al., 2002). After exposure of the dentin surface 3 mm from the enamel–cementum junction, (Isomet 2000; Buehler, Lake Bluff, IL, USA), three non-restored teeth were used to characterize the morphology of dentin. One slab (2 mm thick) of each tooth was obtained by cross-sectioning the tooth perpendicular to the dentin tubules (Isomet 2000). For the remaining teeth (n¼ 12), a standard smear layer was created using silicon carbide paper #600 (Buehler) (Tay and Pashley, 2001). Three adhesive systems were used in accordance with the manufacturers' recommendations, followed by composite resin placement (n¼4): ScotchBond Multipurpose (SBMP; 3M ESPE, St. Paul, MN, USA), Clearfil SE Bond (CSEB; Kuraray Noritake Dental Inc., Kurashiki, Japan) and Adper Prompt L-Pop (APLP; 3M ESPE). The restored teeth were cross-sectioned mesio-distally with a diamond disc in order to obtain at least four slabs ( 2 mm thick) containing the DAI. Two slabs of each tooth were used for scanning electron microscopy (SEM) and two slabs of each tooth were used for nano-indentation. 2.2. Scanning electron microscopy SEM was used to obtain the morphological characteristics of the DAI for each adhesive system tested, including the thickness of the hybrid layer and the adhesive layer, and the length of resin tags. The non-restored dentin specimens (n¼3) were used to measure the number and diameter of dentin tubules, and the radius of peritubular dentin. These data were used to build the finite element models. All restored and non-restored specimens were prepared for SEM in accordance with previous studies, including fixation, dehydration, drying, mounting and polishing (Kaaden et al., 2003; Perdigao et al., 1995). The restored specimens were ultra-sonicated in 100% ethanol for 5 min, dried, demineralized in 6N HCl for 30 s, and protein was denatured in 2% NaOCl for 10 min (Duarte et al., 2009). After drying, all the specimens (restored and non-restored) were coated with gold using a sputter coater (Emitech K650; Emitech Products Inc., Houston, TX, USA) at 10 mA for 4 min. The slabs were observed under an SEM (Hitachi S-3500 N; Hitachi Science System Ltd., Ibaraki, Japan) at an accelerating voltage of 5 kV and a working distance of 10 mm (2000 and 4000 ). Three images were obtained from each slab (left, center and right), and linear measurements were taken using PCI 5.5 Quartz software (Quartz Imaging Corporation, Vancouver, Canada). 2.3. Nano-indentation The two restored dentin slabs that had not been prepared for SEM were embedded in epoxy resin, ground with silicon carbide papers of decreasing abrasiveness (600–1200 grit), and polished with diamond solution (9, 6, 3, 1 and 0.5 mm) under cloth paper. The imaging and indentation processes were undertaken using a Berkovich fluid cell diamond three-sided pyramid probe in a nano-indenter (950TI; Hysitron, Minneapolis, MN, USA), and tests were performed in wet conditions (HBSS) (Dos Santos et al., 2011; Habelitz et al., 2002). A loading profile was developed with a peak load of 300 mN for dentin and 100 mN for the hybrid layer and adhesive layer, at a rate of 60 mN/s and 20 mN/s, respectively, followed by a holding time of 10 s and an unloading time of 2 s. For each indentation, the reduced elastic modulus was calculated from each loaddisplacement curve, and the elastic modulus was obtained (Anchieta et al., 2014; Oliver and Pharr, 1992).
Prior to indenting the region of interest, the interface was scanned with an in-situ scanning probe microscope with the Berkovich indenter to verify appropriate positioning. Ten indentations were made in the DAI (hybrid layer and adhesive layer) on each slab. Indentations were made horizontally following the adhesive layer and hybrid layer. For intertubular dentin, the first 10 indentations were made below the hybrid layer, moving away from the interface in a single column. Indentations were made in a straight line, at intervals of 1 mm, from the hybrid layer to the dentin with a load of approximately 300 mN (Fig. 1). A large indent of 2000 mN in the adhesive served as a fiduciary mark. Five additional indentations were made horizontally, far from the DAI, in order to assess intact intertubular dentin (Fig. 1). 2.4. Finite element analysis A micromodel of dentin restored with composite resin (41 41 82 mm3) (Fig. 2) was built using solid modelling software (SolidWorks 2010; SolidWorks Corporation, Concord, MA, USA), in accordance with previously published data, with appropriate tensile loading (0.03 N), boundary conditions (Anchieta et al., 2007, 2011; Junior et al., 2012; Martini et al., 2009) and cross-sectional area (Fig. 2) (Lin et al., 2011; Phrukkanon et al., 1998) taken into account. The morphological characteristics and dimensions of the DAI for each adhesive system were obtained from SEM analysis, and are shown in Table 1. All materials were considered to be linear, isotropic and homogenous. Elastic modulus (E) and Poisson's ratio (υ) were used in accordance with Table 2. The elastic modulus of peritubular dentin obtained from the nano-indentation test was 30 ( 7 3.6) GPa, and the Poisson ratio was 0.3 (Le et al., 2001). Composite resin had an elastic modulus of 30 GPa and a Poisson ratio of 0.3 (Junior et al., 2012). The morphological characteristics and dimensions of the DAI for each adhesive system were used to build geometrical models, as follows:
SBMP – 4-mm-thick hybrid layer, funnel-shaped resin tags with length of 30 mm
and base thickness of 3.6 mm, 15-mm-thick adhesive layer and 5-mm-thick PDD layer; CSEB – similar to SBMP, but with 0.85-mm-thick hybrid layer, funnel-shaped resin tags with length of 16 mm and base thickness of 2.5 mm, 10-mm-thick adhesive layer and 1.5-mm-thick PDD layer; and APLP – similar to SBMP, but with 3-mm-thick hybrid layer, funnel-shaped resin tags with length of 25 mm and base thickness of 3 mm, 6-mm-thick adhesive layer and 3-mm-thick PDD layer (Table 1 and Fig. 3).
The thickness of the PDD layer was subdivided into 1-mm layers, and the elastic modulus was obtained through nano-indentation in order to reproduce a graded structure. Two other models (CSEB-0 and APLP-0) were created to verify the influence of the PDD layer. These comprised the exact same features of the CSEB and APLP models, correspondingly, but did not include a PDD layer. Quadratic tetrahedral elements were used for high-quality finite element mesh generation, which was driven by the convergence of analysis (6%). On average, the models presented 1,148,776 elements and 5,077,374 nodes. The bases of all models were fixed in the x, y, and z axes, and loading conditions were applied to each model. For numerical analysis, finite element software (ANSYS Workbench 14.0; Swanson Analysis System, Canonsburg, PA, USA) was used to acquire maximum principal stress (σmax). The loading definition details are presented in Appendix A (Fig. 2). 2.5. Data analysis Preliminary analysis evaluated distributional assumptions. The elastic moduli of the hybrid layer and adhesive layer were compared using mixed model analysis of variance (ANOVA) (P o0.05). For dentin, the mechanical behavior of each adhesive system was compared at increasing distances from the hybrid layer using a mixed model ANOVA; first, with fixed factors of adhesive system (three levels) and distance (linear trend); second, with fixed factors of adhesive system and squared distance (quadratic trend); and third, with fixed factors of adhesive system, linear trend, quadratic trend and their two-way interactions. All models included a random intercept to account for multiple observations from the same samples. The model fit was summarized using Aikaike's Information Criterion, and Po 0.05 was considered to indicate significance. Statistical Package for the Social Sciences Version 21 (IBM Corp., Armonk, NY, USA) was used for all statistical analyses. Descriptive analyses were used to describe results from SEM and finite element analysis.
3. Results The DAI linear measurements from SEM [mean and standard deviation (SD)] for each adhesive system are presented in Table 1, and Fig. 3 shows the features of each adhesive system interface. The mean number of dentin tubules counted inside a square of 40 mm2 was 36 (SD 1.3), and the peritubular dentin radius was
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Fig. 1. (A) In-situ scanning probe microscope (SPM) image (40 40 mm2) for Adper Promp L-Pop (APLP), showing a schematic region where the indentations were made for the adhesive layer (ADH), hybrid layer (HL) and dentin in all tested adhesives [Scotchbond Multipurpose (SBMP), Clearfil SE Bond (CSEB), APLP]. The indents in the HL were made in the middle of the layer. In dentin, the first 10 indents were made 1-mm apart in a straight line perpendicular to the interface, starting just below the HL. The other five indents were made far from the dentin–adhesive interface. (B and C) In-situ SPM images (20 20 mm2) showing the indentations made (red square) using linear regression (B) and 3D topographical (C). A large indent (2000 mN) in the adhesive served as a fiduciary mark. In the column of indentations, the values obtained from the adhesive and hybrid layers were not recorded, serving only to identify the first indentation made in the intertubular dentin (white arrow). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Table 2 Mechanical properties of the dentin–adhesive interface (DAI). Mean (standard deviation) elastic modulus (GPa) of DAI structures obtained from nano-indentation. Poisson's ratio based on Le et al. (2001).
SBMP CSEB APLP Poisson ratio
Adhesive layer
Hybrid layer
Intertubular dentin*
3.8 (0.48)A 3.9 (0.48)A 1.0 (0.37)B 0.28
2.9 (0.96)B 3.6 (0.70)A 1.5 (0.55)C 0.28
18.1 (2.3)A 18.1 (3.3)A 19.0 (3.6)A 0.3
SBMP, Scotchbond Multipurpose; CSEB, Clearfil SE Bond; APLP, Adper Promp L-Pop. *Intertubular dentin at a distance from the DAI. Different uppercase letters indicate significant differences between columns. Fig. 2. Determination of dimension and loading intensity of micromodels. (A) Hourglass-shaped macrospecimen with 1.1-mm2 cross-sectional area. (B) A microfragment of the dentin–adhesive interface was removed from inside the macrospecimen. (C) Micromodel of 1.681-mm2 cross-sectional area; 0.03 N loading was applied, proportional to force applied in the macrospecimen. Table 1 Mean (standard deviation) thickness of adhesive layer and hybrid layer, and length of resin tags for three adhesive systems. Thickness of adhesive layer (mm) SBMP 15.4 (0.6) CSEB 10.7 (0.4) APLP 5.9 (0.4)
Thickness of hybrid layer (mm)
Length of resin tags (mm)
4.0 (0.7) 0.85 (0.2) 3.2 (0.4)
31.3 (12.9) 15.6 (5.1) 27.8 (7.2)
SBMP, Scotchbond Multipurpose; CSEB, Clearfil SE Bond; APLP, Adper Promp L-Pop.
0.9 mm. The diameter of exposed dentin tubules in finite element models followed the diameter of the base thickness of resin tags, as measured through SEM. The elastic modulus of the hybrid layer was higher for CSEB compared with SBMP and APLP (P o0.05) (Table 2). The elastic modulus of the adhesive layer was also higher for CSEB compared with APLP (P o0.05), but was similar compared with SBMU (P 40.05) (Table 2). For all adhesive systems, the elastic modulus for dentin beneath the hybrid layer differed from the elastic modulus for dentin far from the DAI, suggesting a gradient due to the presence of non-infiltrated PDD (Fig. 4). Fig. 4A shows the mean (standard error) elastic modulus for each adhesive system at increasing distance from the DAI, unadjusted for repeated observations. The mean elastic modulus for each adhesive system was relatively similar when measured close to (approximately 10 GPa)
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Fig. 3. (A) Scanning electron microscope images of Scotchbond Multipurpose (SBMP), Clearfil SE Bond (CSEB) and Adper Promp L-Pop (APLP). SBMP had a 4-mm hybrid layer (HL) and longer resin tags (30 mm) for the dentin–adhesive interface. CSEB had a 1-mm HL and resin tags of 15 mm, and APLP had a 3-mm HL and resin tags of 25 mm. (B) In-situ scanning probe microscope images (3D topographical 3D) of SBMP, CSEB and APLP interfaces obtained from a nano-indenter. ADH, adhesive layer; D, dentin; rt, resin tags.
Fig. 4. (A) Mean (standard error) elastic modulus for dentin at increasing distance from the dentin–adhesive interface, unadjusted for repeated observations. (B) Values predicted from the linear model. (C) Values predicted from the linear/quadratic model. Attempts to fit models of increasing complexity showed that the linear model fit better [Aikaike's Information Criterion (AIC) ¼ 563.5] than the quadratic model (AIC¼ 583.4), but did not different from the linear/quadratic model (AIC¼ 562.4). SBMP, Scotchbond Multipurpose; CSEB, Clearfil SE Bond; APLP, Adper Promp L-Pop.
or distant from (approximately 20 GPa) the DAI, but more variation was seen at intermediate distances from the DAI. In other words, all three adhesive systems showed similar elastic moduli at the beginning and end of the transition zone, but appeared to follow different trajectories through the middle of the zone. Values predicted from the linear model are shown in Fig. 4B. The analysis indicated that distance had a major effect on elastic modulus, as the elastic modulus increased by approximately 1 GPa for every 1 μm from the DAI (P o0.001), and the elastic modulus was lower (P o0.05) for SBMP compared with APLP and CSEB, which did not differ from one another. The reference line suggests the earliest recovery of intact intertubular dentin (15 GPa) at approximately 3 mm for CSEB, at approximately 4 mm for APLP, and at approximately 6 mm for SBMP. The model provided no evidence to suggest different slopes for the three adhesive systems (P ¼0.65). This analysis suggests a similar trajectory for each adhesive system, but overall lower levels of elastic modulus in teeth treated with SBMP. As the linear model did not detect a difference in trajectories between the adhesive systems, as was suggested in the raw data,
and the linear/quadratic model fit as well (although not as parsimoniously) as the linear model, predicted values from the more complex linear/quadratic model are shown in Fig. 4C. This model shows greater fidelity to the raw data, while smoothing trends. Simply, the change with distance appears linear for APLP, smaller for CSEB and greater for SBMP. While elastic moduli at the beginning and end of the transition zone were similar for each adhesive system, behavior between approximately 3 and 6 mm was predicted to differ. For example, the horizontal line shows that the earliest recovery of intact intertubular dentin (15 GPa) occurred at approximately 3 mm for CSEB, at approximately 4 mm for APLP and at approximately 6 mm for SBMP. The analysis indicated a linear effect of distance (P ¼0.003) for SBMP unlike the other adhesive systems, and the linear/quadratic model identified a longer recovery for APLP compared with CSEB. The numerical simulation output, presented in Figs. 5 and 6, demonstrated that the highest stress levels were found in the hybrid layer for APLP. The highest stress level for PDD occurred for SBMP, followed by APLP and CSEB. CSEB showed the lowest
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stress level at the DAI. Models without a PDD layer (CSEB-0 and APLP-0) had lower stress levels compared with their corresponding PDD layer counterparts (CSEB and APLP). The bottom of the hybrid layer in contact with the PDD was the location where higher stresses were found for SBMP and APLP (Figs. 5 and 6).
Fig. 5. Maximum principal stress (MPa) in hybrid layer (HL), adhesive layer (ADH), resin tag (RT), hybrid layer, intertubular dentin (ID) and non-infiltrated partially demineralized dentin (PDD) for all models: Scotchbond Multipurpose (SBMP), Clearfil SE Bond (CSEB), CSEB without a PDD layer (CSEB-0), Adper Promp L-Pop (APLP), and Adper Promp L-Pop without a PDD layer (APLP-0).
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4. Discussion From a theoretical standpoint, self-etch adhesive systems are expected to minimize limitations of the etch-and-rinse hybridization technique, such as the presence of a PDD layer beneath the hybrid layer (Van Meerbeek et al., 2003). However, studies have demonstrated that partial or total exposure of collagen fibrils beneath the hybrid layer remains (Schulze et al., 2005), which may result in failure due to interfacial degradation (Oliveira et al., 2004; Reis et al., 2007b). While the majority of investigations concerning PDD have concentrated on its potential deleterious effect on the restoration of marginal integrity, the current study aimed to test the hypothesis that the presence of PDD in both etch-and-rinse and self-etch adhesive systems would also result in biomechanically unfavorable scenarios. Unique to the present investigation, the extension and mechanical properties of the different adhesive layers and the PDD region in the etch-andrinse and self-etch adhesive systems were acquired in situ through nanomechanical testing, with real dimension computer models used to test the hypothesis. The elastic modulus of the hybrid layer differed according to the type of adhesive used. The main reason for this can be attributed to differences in chemical composition (Van Landuyt et al., 2007) and interactions with the tooth substrate (Sauro et al., 2012). The elastic modulus of the hybrid layer was lower for APLP than SBMP and CSEB, which may be related to the complex chemical composition of all-in-one adhesives (Van Landuyt et al., 2009). It has been hypothesized that one-step adhesives may not polymerize properly due to a low degree of conversion (Nunes
Fig. 6. Cross-section images from Adper Promp L-Pop (APLP), APLP without a partially demineralized dentin (PDD) layer (APLP-0), Clearfil SE Bond (CSEB) and CSEB without a PDD layer (CSEB-0) showing the stress levels inside the models. The higher stress levels were found at the bottom of the hybrid layer in contact with the PDD for CSEB and APLP. The horizontal black lines show the dentin–adhesive interface layers (adhesive layer, hybrid layer, PDD and dentin) for better visualization.
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et al., 2006), which may lead to the formation of a polymer with lower physical–mechanical strength (Van Landuyt et al., 2009). In addition to lower physical–mechanical strength, low conversion results in more water sorption, hydrolysis and subsequent DAI degradation. Furthermore, adhesives with strong acidity have uncured residual monomers and excessive presence of residual water that may be responsible for the formation of a poor-quality hybrid layer (Sauro et al., 2012). The presence of water can result in a non-resin-infiltrated demineralized collagen layer beneath the resin–hybridized dentin, due to the continuing demineralization effect of non-polymerized acidic monomers (Nunes et al., 2006). Another factor that may explain the higher elastic modulus of the hybrid layer for CSEB and SBMP is the application of a hydrophobic resin as the last step of the bonding procedure. This has a high level of cross-links, such as Bis-GMA, that provide mechanical strength to the adhesive system by forming densely cross-linked polymers that prevent water uptake after curing (Van Landuyt et al., 2009). This is in direct agreement with the elastic moduli of the hybrid layer and the adhesive layer observed for these adhesive systems. Another factor that may explain the higher elastic modulus of the hybrid layer for CSEB is the presence of an acid–base resistance zone (ABRZ). This zone is mainly described for mild self-etch adhesives, such as Clearfil SE Bond and Clearfil Protect Bond (Inoue et al., 2006). The formation of an ABRZ is a result of hydroxyapatite crystals that are not completely demineralized, and remain within inside and around collagen fibrils within the hybrid layer than an ABRZ beneath the hybrid layer (Waidyasekera et al., 2009). Hydroxyapatite crystal remnants within the hybrid layer have been reported to form calcium salts with functional monomers in mild self-etch adhesive systems, especially MDP or MDPB (Yoshida et al., 2004, 2012), and contribute to bonding durability. ABRZs are not commonly found for etch-and-rinse adhesives or aggressive self-etch adhesives (Nurrohman et al., 2012). The nano-indentation results showed differences in the mechanical properties of dentin beneath the hybrid layer and far from the DAI interface, confirming the presence of a PDD layer for all systems tested, as reported previously (Oliveira et al., 2004; Schulze et al., 2005). Previous studies regarding the elastic modulus of sound dentin have reported values ranging from 15 to 19 GPa (Kinney et al., 2003; Ryou et al., 2011). Fitting a linear model to the data suggested differences in the average elastic modulus between the adhesive systems but a common trajectory. A more complex, linear/quadratic model suggested a similar overall elastic modulus, but different trajectories between the adhesive systems. The more complex model provided a better representation of the differing elastic moduli of dentin, given differences in bonding material and distance from the transition zone, and highlighted differences in the recovery of normal elastic modulus more clearly; this model is consistent with the expectations of variable degrees of etching. Despite this, the linear/quadratic model did not have a better fit than the simple linear model, and the authors are not confident that the solution could be replicated given the relatively small sample. Thus, both models identified the longer recovery of normal elastic modulus for SBMP compared with the other adhesive systems, but more data are needed to verify if stiffness is regained at a shorter distance from the DAI for CSEB than SBMP. From a theoretical standpoint, the mechanical competence of the DAI for the etch-and-rinse adhesive system was expected to be lower compared with the self-etch adhesive systems. However, when the real dimension and mechanical property 3D finite element model output was evaluated, stress levels were higher for the one-step self-etch adhesive system (APLP) than for the selfetch primer system (CSEB) or the etch-and-rinse adhesive system (SBMP). These differences in computer model outputs pertain to a
combination of features, including differences in the thicknesses of the adhesive and hybrid layers (Anchieta et al., 2007), differences in elastic moduli, and differences in the morphology of resin tags between systems. The PDD at the bottom of the hybrid layer is a critical part of the resin–dentin interface. This was the region of maximum stress for all systems, but this was most pronounced for APLP. It has been shown previously that high stress levels in this region jeopardize the mechanical integrity of the DAI, possibly accelerating premature failure (Garcia et al., 2009; Goracci et al., 2004; Van Landuyt et al., 2009). While the results of this study demonstrate differences in the overall biomechanical behavior of the DAI between adhesive systems, future investigations must consider the effect of degradation time on PDD behavior, as it is anticipated that degradation will occur at different rates for the different adhesive systems.
5. Conclusion Use of CSEB and APLP resulted in non-infiltrated PDD beneath the hybrid layer. The presence of a PDD layer increased the stress levels for the self-etch adhesive models (APLP and CSEB), compared with the etch-and-rinse adhesive system (SBMP), showing the detrimental effect of a PDD layer for overall endurance of the DAI.
Conflict of interest statement None declared.
Acknowledgments This study was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo – FAPESP (Grant #2008/002900-9; Scholarships 2010/12926-7 and 2012/07012-1).
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