d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1147–1153
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Dentin-smear remains at self-etch adhesive interface Atsushi Mine a,b , Jan De Munck a , Marcio Vivan Cardoso a , Kirsten L. Van Landuyt a , André Poitevin a , Annelies Van Ende a , Mariko Matsumoto b , Yasuhiro Yoshida c , Takuo Kuboki d , Hirofumi Yatani b , Bart Van Meerbeek a,∗ a
KU Leuven BIOMAT, Department of Oral Health Sciences, KU Leuven (University of Leuven), Leuven, Belgium Department of Fixed Prosthodontics, Osaka University Graduate School of Dentistry, Osaka, Japan c Department of Biomaterials and Bioengineering, Hokkaido University Graduate School of Dental Medicine, Sapporo, Japan d Department of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan b
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objective. The bonding potential of ‘mild’ self-etch adhesives may be compromised due to
Received 4 April 2014
smear interference, as they may not dissolve/penetrate the smear layer effectively due to
Received in revised form 6 July 2014
their relatively low acidity. We observed that the thickness of the dentin smear layer differed
Accepted 11 July 2014
depending on the surface-preparation methodology used. Methods. The interaction of an (ultra-)mild self-etch adhesive (Clearfil S3 Bond, Kuraray Noritake) with human dentin, prepared either using a medium-grit diamond bur (‘thick’,
Keywords:
clinically relevant smear layer) or 600-grit SiC-paper (‘thin’ smear layer), or just fractured
Mild self-etch
(smear-free), was evaluated using high-resolution transmission electron microscopy (TEM).
Adhesion
Non-demineralized/demineralized 30–100 nm interfacial cross-sections were prepared fol-
Resin-smear complex
lowing common TEM-specimen processing and diamond-knife ultra-microtomy.
TEM
Results. The adhesive did not dissolve the bur-cut, nor the SiC-ground smear layer, but
Smear layer
impregnated it. Within this ‘resin-smear complex’, hydroxyapatite was abundantly present.
Hybrid layer
At fractured dentin, this complex was not present, while the actual layer of interaction of the adhesive was limited to about 100 nm. Non-demineralized ‘ultra-thin’ (30–50 nm) sections confirmed the interfacial ultra-structure to differ for the three surface-preparation methods. An electron dense band was consistently disclosed at the adhesive interface, most likely representing the documented chemical interaction of the functional monomer 10-MDP with Ca. Significance. The dentin surface-preparation method significantly affects the nature of the smear layer and the interaction with the ultra-mild self-etch adhesive. © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
∗ Corresponding author at: KU Leuven BIOMAT, Department of Oral Health Sciences, KU Leuven (University of Leuven), Kapucijnenvoer 7, B-3000 Leuven, Belgium. Tel.: +32 16 33 75 87; fax: +32 16 33 27 52. E-mail addresses: [email protected], [email protected] (B. Van Meerbeek). http://dx.doi.org/10.1016/j.dental.2014.07.006 0109-5641/© 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
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1.
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1147–1153
Introduction
In many laboratory studies investigating dentin bonding effectiveness, SiC-ground surfaces are used, for reasons of standardization and ease of preparation [1–3]. Nevertheless, a smear layer created during clinical procedures with a diamond or tungsten bur is rather thick and compact, and so may compromise the bonding effectiveness to dentin, in particular when a self-etch adhesive is used [4]. A thick smear layer may also be porous and thus have weak mechanical properties [5]. In this respect, the documented compromised dentin bonding effectiveness of contemporary one-step selfetch adhesives, which combine conditioning, priming and application of adhesive resin, could to a certain extent be attributed to interference of bur debris smeared across dentin during cavity preparation. However, there is very little detailed morphological data on the effect of smear interposition at the adhesive-dentin interface produced by so-called ‘ultramild’ self-etch adhesives, which are significantly less acidic (pH ≈ 2.7). Especially completeness of resin penetration and adequate resin envelopment of potential residual smear is highly important, as insufficient resin impregnation may result in more rapid interface degradation [6]. Therefore, high-resolution ultra-morphological examination is thought to give a better insight what impact potential interfacial interference of bur smear may have on the bond durability [7]. The purpose of this study was to examine the effect of different surface preparation methods on the interfacial ultrastructure of an ultra-mild self-etch adhesive bonded to dentin. For this, high-resolution transmission electron microscopy (TEM) is the method of choice, as the combination of resolution and image detail is unsurpassed.
2.
Materials and methods
Non-carious human third molars were stored in 0.5% chloramine solution at 4 ◦ C and used within one month after extraction. The teeth were randomly divided into 3 groups: ‘BUR-CUT’, ‘SiC-GROUND’ and ‘FRACTURED’ dentin (see below). All teeth were mounted in gypsum blocks in order to ease manipulation. For ‘BUR-CUT’ dentin, the occlusal third of the crown was removed at the level of mid-coronal dentin using a slow-speed diamond saw (Isomet 1000, Buehler, Lake Bluff, IL, USA), after which a standard smear layer was produced by a medium-grit (100 m) diamond bur (842, Komet, Lemgo, Germany) using a water-cooled high-speed contraangle hand piece mounted in the MicroSpecimen Former (The University of Iowa, Iowa City, IA, USA). Half of the bur-cut specimens were further processed by wet-sanding with 600grit silicone-carbide paper (‘SiC-GROUND’). For ‘FRACTURED’ (smear-free) dentin, a shallow 1–2 mm deep groove was cut circumferentially around the tooth at the level of mid-coronal dentin, after which the coronal part was fractured using a forceps to produce a fractured dentin surface free of smear debris. All dentin surfaces were carefully verified for absence of enamel and/or pulp tissue using a stereo-microscope (Wild M5A, Wild Heerbrugg AG, Heerbrugg, Switzerland).
The ultra-mild one-step self-etch adhesive Clearfil S3 Bond (Kuraray Noritake, Tokyo, Japan) was applied according to the manufacturer’s instructions, followed by a thin layer of flowable composite (Clearfil Protect Liner F, Kuraray Noritake). Light-curing was performed using an Optilux 500 (Demetron/Kerr, Danbury, CT, USA) device with a light output not less than 600 mW/cm2 . After bonding procedures, specimens were stored for 1 day in distilled water at 37 ◦ C. The specimens were processed according to the procedure described in detail by Van Meerbeek et al. [8]. Nondemineralized and laboratory-demineralized thin (60–100 nm) and non-demineralized ultra-thin (30–50 nm) sections were cut (Ultracut UCT, Leica, Vienna, Austria) and examined unstained and positively stained (3% uranyl acetate for 12 min/lead citrate for 13 min) using TEM (JEM-1200EX II, JEOL, Tokyo, Japan). In order to reveal potential porous zones at the interface, additional specimens were immersed in a 50 wt% ammoniacal silver nitrate solution according to a nanoleakage-detection protocol previously described by Tay et al. [9].
3.
Results
TEM of non-demineralized sections revealed clear differences in substrate irregularity and smear-layer thickness for the different surface-preparation methods applied. At FRACTURED dentin, a smear layer could not be observed, but distinct resin tags were clearly formed (Fig. 1a and b). At SiC-GROUND dentin, the interface appeared relatively flat and smooth with a regularly thin smear layer (up to 1 m) deposited by the shearing/pushing motion during grinding (Fig. 2a and b). Due to the smear plugs in the dentin tubules, resin tags were clearly less prominent than at fractured dentin. At BUR-CUT dentin, the surface was clearly more irregular with a much thicker (up to 5 m), but also much more in thickness varying smear layer deposited (Fig. 3a and b). The adhesive was not able to visibly dissolve the underlying dentin substrate at FRACTURED dentin (Fig. 1b), nor able to dissolve the SiCGROUND (Fig. 2b) and the BUR-CUT smear layer (Fig. 3b). In the two latter conditions, a ‘resin-smear complex’ was formed, in which hydroxyapatite (HAp), often fragmented, was abundantly present. AgNO3 -immersed specimens revealed relatively small deposits of silver within the adhesive and at the adhesive-dentin interface at FRACTURED dentin (Fig. 1c), most likely due to absence of surface smear. In contrast, a varying pattern of both spot- and cluster-like appearance of nano-leakage was revealed within the resin-smear complex and within dentin for the SiC-GROUND or BUR-CUT dentin specimens. The inorganic component of dentin, and especially peritubular dentin, was demineralized by the 36-h laboratory demineralization (formic acid, formaldehyde). Laboratory-demineralized sections more clearly revealed the resin-tag structure (Figs. 1d and 2d) and smear-layer thickness (Figs. 2d and 3d), as compared to what could be observed using non-demineralized sections. Moreover, laboratory-demineralized sections disclosed better the difference between resin-impregnated smear and acidresistant hybridized dentin. At FRACTURED dentin, dentin
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1147–1153
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Fig. 1 – TEM photomicrographs of an ‘ultra-mild’ self-etch adhesive bonded to FRACTURED dentin. (a) Non-demineralized, unstained section, disclosing a tight, void-free interface and distinct resin-tags (black arrowheads). (b) Corresponding higher magnification of (a): no morphologic features of interaction or demineralization were revealed in this non-demineralized section at this high magnification (original magnification = 100 k). Hydroxyapatite (HAp) is abundantly present (white arrowheads). (c) AgNO3 -immersed specimens for nano-leakage screening revealed relatively few deposits of silver within the adhesive (arrow #1) and at the adhesive-dentin interface (arrow #2). (d) Demineralized, unstained section, illustrating a 10-m long resin tag. (e) Demineralized, stained section: a clear electron dense hybrid layer (HL) was detected, representing an acid-resistant hybrid layer with a thickness of about the width of one collagen fibril.
hybridization was mostly limited to a depth ranging up to a few hundreds of nanometres, while in most areas the infiltration did not extend beyond the width of one collagen fibril (about 100 nm) (Fig. 1e). Clearly, a much thicker interaction layer, consisting of a true hybrid layer and a resin-smear complex on top, was observed at SiC-GROUND (Fig. 2e) and BUR-CUT dentin (Fig. 3e). Collagen fibrils were hardly observed within the resin-smear complex. A partial loss of the resin-smear complex was observed at BUR-CUT dentin (Fig. 3e), suggesting that the resin-smear complex contained areas that were not fully impregnated by resin. Ultra-thin sections clearly confirmed the difference in substrate irregularity and HAp presence, as was disclosed by the conventional thickness non-demineralized sections. Nevertheless, although these ultra-thin sections were also non-demineralized sections, they appeared somewhat similar to laboratory-demineralized sections regarding the absence of peritubular dentin and the resin-tag structure. The ultramorphological differences were, however, more pronounced in the ultra-thin sections than in case of both the conventional non-demineralized and laboratory-demineralized
sections (Fig. 4). The twofold hybrid layer/resin-smear complex and the partial loss of the resin-smear complex at BUR-CUT dentin (Fig. 4f) could be distinguished without any staining.
4.
Discussion
Three types of dentin surface-preparation methods were used and their effect on the interfacial interaction of a representative 10-MDP-based ‘ultra-mild’ one-step self-etch adhesive (Clearfil S3 Bond, Kuraray Noritake; pH ≈ 2.7) with dentin was ultra-morphologically characterized using TEM. Previously, a so-called ‘nano-interaction zone’ without distinct demineralization was described [10]. The thickness of such a ‘nano-interaction zone’ was measured to be below 300 nm, when an experimental 10-MDP-based precursor of Clearfil S3 Bond (SSB-200, Kuraray Noritake) was employed. In that study, solely non-demineralized unstained sections were examined. We additionally prepared and examined in this study stained laboratory-demineralized and ultra-thin non-demineralized which enabled to more clearly reveal this nano-scale
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d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1147–1153
Fig. 2 – TEM photomicrographs of an ‘ultra-mild’ self-etch adhesive bonded to SiC-GROUND dentin. (a) Non-demineralized, unstained section, disclosing a tight interface. No clear resin tag was observed, compared to what was observed at fractured dentin, most likely due to the presence of a smear plug (black arrowhead). (b) Corresponding higher magnification of (a): no clear morphologic features of interaction or demineralization were revealed (original magnification = 100 k). HAp was abundantly present (white arrowheads). (c) AgNO3 -immersed specimens revealed relatively small deposits of silver within the adhesive (arrow #1), in the smear layer (arrow #2) and inside dentin underneath (arrow #3). (d) Demineralized, unstained section: porous resin tags were observed, most likely because of demineralization of residual smear particles. (e) Demineralized, stained section: a clearly electron dense hybrid layer (HL) can be detected, with on top a resin-smear complex (RSC), consisting of residual smear enveloped by resin. The collagen fibril structure can hardly be seen in the RSC.
interaction with dentin and thus to investigate potential smear interference. As nor the SiC-GROUND, nor the BUR-CUT smear layer was dissolved, but impregnated by the adhesive, a ‘resinsmear complex’ was produced, which we have described before in our previous study regarding the interaction of the adhesive with likewise differently prepared enamel surfaces [11]. Hydroxyapatite was abundantly present within this resin-smear complex, that differed in thickness and uniformity depending on the surface-preparation methodology used (Figs. 2a,b and 3a,b). The increasing gradation of HAp crystals with depth, as Koshiro et al. reported [10], could not be observed to the same degree in our study. The underlying mechanism of bonding of mild selfetch adhesives was shown to be based upon submicron micro-mechanical interlocking [12], supplemented by primary chemical interaction of the functional monomer 10-MDP with HAp that remained around the partially exposed collagen [13,14]. According to the ‘Adhesion-Decalcification concept’ (AD-concept), 10-MDP chemically bonds to HAp and thereby keeps HAp as the natural shelter around collagen [15]. While
such chemical interaction did not raise the ‘immediate’ bond strength, studies testing the biodegradation resistance of adhesive interfaces have shown that it improved the bond stability [16,17]. Additionally, the demineralization capability of ultra-mild self-etch adhesives is limited; the adhesive showed hardly any sign of demineralization at FRACTURED dentin (Fig. 1a and b). The nano-scale interaction with dentin was clearly evidenced by the stained laboratory-demineralized sections. The densely stained layer observed at FRACTURED dentin, was limited to about 100 nm (Fig. 1e). Since this substrate lacked a smear layer, the densely stained layer must be identified as ‘true’ hybrid layer. The much thicker densely stained layer observed at SiC-GROUND (Fig. 2e) and BUR-CUT dentin (Fig. 3e) must thus be attributed to resin having infiltrated residual surface smear. The resultant ‘resin-smear complex’ is positioned on top of the true hybrid layer. Some authors have already before speculated that separation of the hybridized smear layer from the true hybrid layer may occur [18,19]. In the event that the underlying dentin is demineralized and hybridized, there is a further concern that the only material
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1147–1153
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Fig. 3 – TEM photomicrographs of an ‘ultra-mild’ self-etch adhesive bonded to BUR-CUT dentin. (a) Non-demineralized, unstained section: the surface irregularity and thickness of the smear layer (SL) differed regionally. The thickness of the smear layer varied from almost 0 to 3 m. (b1) Corresponding higher magnification of (a), disclosing a ‘shallow’ smear layer of less than 1 m. Hydroxyapatite was abundantly present (white arrowheads). (b2) Corresponding higher magnification of (a), disclosing a ‘thick’ smear layer of more than 4 m. (c) AgNO3 -immersed specimens revealed a varying pattern of both spot- and cluster-like appearance of nano-leakage within the adhesive (arrow #1), in the smear layer (arrow #2) and inside dentin underneath (arrow #3). Only a few spots of silver deposition were found in the resin-smear complex (RSC). (d) Demineralized, unstained section: the irregularity of the smear layer was more evident. (e1) Demineralized, stained section, disclosing a ‘shallow’ smear layer. A clearly electron dense hybrid layer (HL) became visible, with on top a resin-smear complex, consisting of residual smear enveloped by resin. (e2) Demineralized, stained section, disclosing a ‘thick’ smear layer. Not only the thickness of the resin-smear complex, but also that of the hybrid layer was different from that at SiC-GROUND dentin (see Fig. 2e). A partial loss within the resin-smear complex was observed (hand point).
connecting the resin-smear complex with the true hybrid layer is resin that had to diffuse around the globular particle aggregates within the actual smear layer, and then onwards into the interfibrillar spaces of the underlying intertubular dentin, as well as into the dentinal tubules, the latter forming hybridized smear plugs. However, given the abundant presence of HAp in this resin-smear complex and the effective chemical binding potential of the 10-MDP functional monomer with this HAp, it is very likely that these residual smear particles may reinforce the resin-smear complex. Moreover, for none of our TEM sections observed, the hybrid layer separated from the resin-smear complex, corroborating our hypothesis that well resin-enveloped smear globules may reinforce the adhesive interface. At FRACTURED dentin, the acid-resistant hybrid layer, as observed in the laboratory-demineralized sections, was mostly limited to a depth ranging up to a few hundreds of nanometers, while in most areas the infiltration did not
extend beyond the width of one collagen fibril (about 100 nm). The micro-fibrillar arrangement of collagen type I, which results from the pentameric staggered organization of the molecules in a higher order supra-molecular supertwisted nanostructure, has been shown to result in dentinal collagen with an intermolecular spacing of about 1.3 nm. This zone is too small to accommodate one single molecule of common dental monomers (∼2 nm) [20]. Therefore, monomers can only infiltrate more deeply in case of considerable demineralization or fragmentation as within the smear layer. There is a common consensus that the final goal of dentin bonding is complete infiltration of resin monomers into demineralized collagen fibrils exposed by separate acid-etching or by self-etch adhesives [21]. Any discrepancy between dentin demineralization and resin infiltration may result in silvernitrate- or fluorescent-dye-detectable nanoleakage within water-rich zones of the hybrid layer [22,23], and the adhesive layer [24]. A partial loss of the resin-smear complex was
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Fig. 4 – TEM photomicrographs of non-demineralized, non-stained ‘ultra-thin’ sections of the interface between an ultra-mild self-etch adhesive and dentin covered with different smear layers. (a) Overview image of the adhesive–dentin interface at FRACTURED dentin, disclosing a tight, void-free interface. A clear resin-tag was observed (black arrowhead). The hardest dentinal tissue, namely peritubular dentin, was absent in this thin section (asterisks). (b) Overview image of the adhesive–dentin interface at SiC-GROUND dentin, disclosing more porous resin tags most likely due to residual smear (black arrowheads). Peritubular dentin could not be observed in this thin section (asterisks). (c) Overview image of the adhesive–dentin interface at BUR-CUT dentin, disclosing a smear layer (SL) regionally differing in thickness. (d) Higher magnification of the interface at FRACTURED dentin, disclosing HAp crystals scattered throughout the hybrid layer and dentin (white arrowheads). A more electron dense band was consistently observed, which might represent the interaction of the functional monomer 10-MDP with Ca. (e) Higher magnification of the interface at SiC-GROUND dentin, disclosing a resin-smear complex (RSC) on top of the hybrid layer (HL). (f) Higher magnification of the interface at BUR-CUT dentin, disclosing a fragile part of the resin-smear complex with some voids (hand pointer) that may represent areas not infiltrated well by resin. The true hybrid layer is not as distinct, as compared to that observed at FRACTURED and SiC-GROUND dentin.
observed in this study at BUR-CUT dentin (Fig. 3e); areas within the resin-smear complex were identified not to have been adequately filled with resin. This is perfectly in line with our nano-leakage assessment, where the adhesive resin was able to infiltrate the SiC-GROUND dentin surface more effectively than the BUR-CUT surface. The silver-tracer method that has been widely employed as adhesive interface evaluation method for the past 15 years, actually underestimates the retention of water within apparently well-infiltrated collagen matrices in hybrid layers after bonding with etch-and-rinse and self-etch adhesives. On the other hand, using biomimetic hybrid-layer remineralization techniques a progressive reduction in silver-tracer uptake over time and, hence, in water distribution within adhesive-dentin interfaces was reported for etch-and-rinse hybrid layers [25]. Moreover, besides incomplete resin infiltration, such regions of silver deposition may also represent sites of retained water [26], especially in case of
HEMA-containing adhesives that tend to absorb water more easily [27]. Consequently, it is not yet evident whether the partial loss of the resin-smear complex (Fig. 3e) and the fragile part (Fig. 4f) at BUR-CUT dentin are of significant concern with regard to bond durability. TEM of ultra-thin sections revealed clear differences in terms of substrate irregularity, smear-layer thickness, resin tag/smear plug formation and hybrid layer/resin-smear complex ultrastructure for the three surface-preparation methods applied. The observations are complementary to those of conventional thickness non-demineralized and laboratory-demineralized TEM sections, as especially the true interaction with underlying dentin can be disclosed, which using conventional thickness sections requires laboratorydemineralization and staining. However, as these ultrathin TEM sections were not demineralized, the crystal structure at the interface can still be detected. Since these sections
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1147–1153
were not stained, we hypothesize that the electron dense part observed at the interaction layer, represents the interaction of the functional monomer 10-MDP with Ca. It is concluded that the dentin surface-preparation method significantly affects the nature of the smear layer and thus the interaction of in particular (ultra-)mild self-etch adhesives, being more uniform at SiC-GROUND dentin as compared to the more clinically relevant BUR-CUT dentin surface. In light of the increased clinical use of (ultra-)mild self-etch adhesives, the effect of different surface-preparation methods (e.g. hand excavation, bur preparation, laser ablation, air-abrasion, chemo-mechanical methods, air-polishing, ultrasonication and sono-abrasion) on the surface receptiveness of dentin for adhesion using self-etch adhesives needs to be investigated more.
Conflicts of interest The authors declare that they have no conflicts of interest regarding the products investigated.
references
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Dentin-smear remains at self-etch adhesive interface Atsushi Mine a,b , Jan De Munck a , Marcio Vivan Cardoso a , Kirsten L. Van Landuyt a , André Poitevin a , Annelies Van Ende a , Mariko Matsumoto b , Yasuhiro Yoshida c , Takuo Kuboki d , Hirofumi Yatani b , Bart Van Meerbeek a,∗ a
KU Leuven BIOMAT, Department of Oral Health Sciences, KU Leuven (University of Leuven), Leuven, Belgium Department of Fixed Prosthodontics, Osaka University Graduate School of Dentistry, Osaka, Japan c Department of Biomaterials and Bioengineering, Hokkaido University Graduate School of Dental Medicine, Sapporo, Japan d Department of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan b
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objective. The bonding potential of ‘mild’ self-etch adhesives may be compromised due to
Received 4 April 2014
smear interference, as they may not dissolve/penetrate the smear layer effectively due to
Received in revised form 6 July 2014
their relatively low acidity. We observed that the thickness of the dentin smear layer differed
Accepted 11 July 2014
depending on the surface-preparation methodology used. Methods. The interaction of an (ultra-)mild self-etch adhesive (Clearfil S3 Bond, Kuraray Noritake) with human dentin, prepared either using a medium-grit diamond bur (‘thick’,
Keywords:
clinically relevant smear layer) or 600-grit SiC-paper (‘thin’ smear layer), or just fractured
Mild self-etch
(smear-free), was evaluated using high-resolution transmission electron microscopy (TEM).
Adhesion
Non-demineralized/demineralized 30–100 nm interfacial cross-sections were prepared fol-
Resin-smear complex
lowing common TEM-specimen processing and diamond-knife ultra-microtomy.
TEM
Results. The adhesive did not dissolve the bur-cut, nor the SiC-ground smear layer, but
Smear layer
impregnated it. Within this ‘resin-smear complex’, hydroxyapatite was abundantly present.
Hybrid layer
At fractured dentin, this complex was not present, while the actual layer of interaction of the adhesive was limited to about 100 nm. Non-demineralized ‘ultra-thin’ (30–50 nm) sections confirmed the interfacial ultra-structure to differ for the three surface-preparation methods. An electron dense band was consistently disclosed at the adhesive interface, most likely representing the documented chemical interaction of the functional monomer 10-MDP with Ca. Significance. The dentin surface-preparation method significantly affects the nature of the smear layer and the interaction with the ultra-mild self-etch adhesive. © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
∗ Corresponding author at: KU Leuven BIOMAT, Department of Oral Health Sciences, KU Leuven (University of Leuven), Kapucijnenvoer 7, B-3000 Leuven, Belgium. Tel.: +32 16 33 75 87; fax: +32 16 33 27 52. E-mail addresses: [email protected], [email protected] (B. Van Meerbeek). http://dx.doi.org/10.1016/j.dental.2014.07.006 0109-5641/© 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
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1.
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1147–1153
Introduction
In many laboratory studies investigating dentin bonding effectiveness, SiC-ground surfaces are used, for reasons of standardization and ease of preparation [1–3]. Nevertheless, a smear layer created during clinical procedures with a diamond or tungsten bur is rather thick and compact, and so may compromise the bonding effectiveness to dentin, in particular when a self-etch adhesive is used [4]. A thick smear layer may also be porous and thus have weak mechanical properties [5]. In this respect, the documented compromised dentin bonding effectiveness of contemporary one-step selfetch adhesives, which combine conditioning, priming and application of adhesive resin, could to a certain extent be attributed to interference of bur debris smeared across dentin during cavity preparation. However, there is very little detailed morphological data on the effect of smear interposition at the adhesive-dentin interface produced by so-called ‘ultramild’ self-etch adhesives, which are significantly less acidic (pH ≈ 2.7). Especially completeness of resin penetration and adequate resin envelopment of potential residual smear is highly important, as insufficient resin impregnation may result in more rapid interface degradation [6]. Therefore, high-resolution ultra-morphological examination is thought to give a better insight what impact potential interfacial interference of bur smear may have on the bond durability [7]. The purpose of this study was to examine the effect of different surface preparation methods on the interfacial ultrastructure of an ultra-mild self-etch adhesive bonded to dentin. For this, high-resolution transmission electron microscopy (TEM) is the method of choice, as the combination of resolution and image detail is unsurpassed.
2.
Materials and methods
Non-carious human third molars were stored in 0.5% chloramine solution at 4 ◦ C and used within one month after extraction. The teeth were randomly divided into 3 groups: ‘BUR-CUT’, ‘SiC-GROUND’ and ‘FRACTURED’ dentin (see below). All teeth were mounted in gypsum blocks in order to ease manipulation. For ‘BUR-CUT’ dentin, the occlusal third of the crown was removed at the level of mid-coronal dentin using a slow-speed diamond saw (Isomet 1000, Buehler, Lake Bluff, IL, USA), after which a standard smear layer was produced by a medium-grit (100 m) diamond bur (842, Komet, Lemgo, Germany) using a water-cooled high-speed contraangle hand piece mounted in the MicroSpecimen Former (The University of Iowa, Iowa City, IA, USA). Half of the bur-cut specimens were further processed by wet-sanding with 600grit silicone-carbide paper (‘SiC-GROUND’). For ‘FRACTURED’ (smear-free) dentin, a shallow 1–2 mm deep groove was cut circumferentially around the tooth at the level of mid-coronal dentin, after which the coronal part was fractured using a forceps to produce a fractured dentin surface free of smear debris. All dentin surfaces were carefully verified for absence of enamel and/or pulp tissue using a stereo-microscope (Wild M5A, Wild Heerbrugg AG, Heerbrugg, Switzerland).
The ultra-mild one-step self-etch adhesive Clearfil S3 Bond (Kuraray Noritake, Tokyo, Japan) was applied according to the manufacturer’s instructions, followed by a thin layer of flowable composite (Clearfil Protect Liner F, Kuraray Noritake). Light-curing was performed using an Optilux 500 (Demetron/Kerr, Danbury, CT, USA) device with a light output not less than 600 mW/cm2 . After bonding procedures, specimens were stored for 1 day in distilled water at 37 ◦ C. The specimens were processed according to the procedure described in detail by Van Meerbeek et al. [8]. Nondemineralized and laboratory-demineralized thin (60–100 nm) and non-demineralized ultra-thin (30–50 nm) sections were cut (Ultracut UCT, Leica, Vienna, Austria) and examined unstained and positively stained (3% uranyl acetate for 12 min/lead citrate for 13 min) using TEM (JEM-1200EX II, JEOL, Tokyo, Japan). In order to reveal potential porous zones at the interface, additional specimens were immersed in a 50 wt% ammoniacal silver nitrate solution according to a nanoleakage-detection protocol previously described by Tay et al. [9].
3.
Results
TEM of non-demineralized sections revealed clear differences in substrate irregularity and smear-layer thickness for the different surface-preparation methods applied. At FRACTURED dentin, a smear layer could not be observed, but distinct resin tags were clearly formed (Fig. 1a and b). At SiC-GROUND dentin, the interface appeared relatively flat and smooth with a regularly thin smear layer (up to 1 m) deposited by the shearing/pushing motion during grinding (Fig. 2a and b). Due to the smear plugs in the dentin tubules, resin tags were clearly less prominent than at fractured dentin. At BUR-CUT dentin, the surface was clearly more irregular with a much thicker (up to 5 m), but also much more in thickness varying smear layer deposited (Fig. 3a and b). The adhesive was not able to visibly dissolve the underlying dentin substrate at FRACTURED dentin (Fig. 1b), nor able to dissolve the SiCGROUND (Fig. 2b) and the BUR-CUT smear layer (Fig. 3b). In the two latter conditions, a ‘resin-smear complex’ was formed, in which hydroxyapatite (HAp), often fragmented, was abundantly present. AgNO3 -immersed specimens revealed relatively small deposits of silver within the adhesive and at the adhesive-dentin interface at FRACTURED dentin (Fig. 1c), most likely due to absence of surface smear. In contrast, a varying pattern of both spot- and cluster-like appearance of nano-leakage was revealed within the resin-smear complex and within dentin for the SiC-GROUND or BUR-CUT dentin specimens. The inorganic component of dentin, and especially peritubular dentin, was demineralized by the 36-h laboratory demineralization (formic acid, formaldehyde). Laboratory-demineralized sections more clearly revealed the resin-tag structure (Figs. 1d and 2d) and smear-layer thickness (Figs. 2d and 3d), as compared to what could be observed using non-demineralized sections. Moreover, laboratory-demineralized sections disclosed better the difference between resin-impregnated smear and acidresistant hybridized dentin. At FRACTURED dentin, dentin
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1147–1153
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Fig. 1 – TEM photomicrographs of an ‘ultra-mild’ self-etch adhesive bonded to FRACTURED dentin. (a) Non-demineralized, unstained section, disclosing a tight, void-free interface and distinct resin-tags (black arrowheads). (b) Corresponding higher magnification of (a): no morphologic features of interaction or demineralization were revealed in this non-demineralized section at this high magnification (original magnification = 100 k). Hydroxyapatite (HAp) is abundantly present (white arrowheads). (c) AgNO3 -immersed specimens for nano-leakage screening revealed relatively few deposits of silver within the adhesive (arrow #1) and at the adhesive-dentin interface (arrow #2). (d) Demineralized, unstained section, illustrating a 10-m long resin tag. (e) Demineralized, stained section: a clear electron dense hybrid layer (HL) was detected, representing an acid-resistant hybrid layer with a thickness of about the width of one collagen fibril.
hybridization was mostly limited to a depth ranging up to a few hundreds of nanometres, while in most areas the infiltration did not extend beyond the width of one collagen fibril (about 100 nm) (Fig. 1e). Clearly, a much thicker interaction layer, consisting of a true hybrid layer and a resin-smear complex on top, was observed at SiC-GROUND (Fig. 2e) and BUR-CUT dentin (Fig. 3e). Collagen fibrils were hardly observed within the resin-smear complex. A partial loss of the resin-smear complex was observed at BUR-CUT dentin (Fig. 3e), suggesting that the resin-smear complex contained areas that were not fully impregnated by resin. Ultra-thin sections clearly confirmed the difference in substrate irregularity and HAp presence, as was disclosed by the conventional thickness non-demineralized sections. Nevertheless, although these ultra-thin sections were also non-demineralized sections, they appeared somewhat similar to laboratory-demineralized sections regarding the absence of peritubular dentin and the resin-tag structure. The ultramorphological differences were, however, more pronounced in the ultra-thin sections than in case of both the conventional non-demineralized and laboratory-demineralized
sections (Fig. 4). The twofold hybrid layer/resin-smear complex and the partial loss of the resin-smear complex at BUR-CUT dentin (Fig. 4f) could be distinguished without any staining.
4.
Discussion
Three types of dentin surface-preparation methods were used and their effect on the interfacial interaction of a representative 10-MDP-based ‘ultra-mild’ one-step self-etch adhesive (Clearfil S3 Bond, Kuraray Noritake; pH ≈ 2.7) with dentin was ultra-morphologically characterized using TEM. Previously, a so-called ‘nano-interaction zone’ without distinct demineralization was described [10]. The thickness of such a ‘nano-interaction zone’ was measured to be below 300 nm, when an experimental 10-MDP-based precursor of Clearfil S3 Bond (SSB-200, Kuraray Noritake) was employed. In that study, solely non-demineralized unstained sections were examined. We additionally prepared and examined in this study stained laboratory-demineralized and ultra-thin non-demineralized which enabled to more clearly reveal this nano-scale
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Fig. 2 – TEM photomicrographs of an ‘ultra-mild’ self-etch adhesive bonded to SiC-GROUND dentin. (a) Non-demineralized, unstained section, disclosing a tight interface. No clear resin tag was observed, compared to what was observed at fractured dentin, most likely due to the presence of a smear plug (black arrowhead). (b) Corresponding higher magnification of (a): no clear morphologic features of interaction or demineralization were revealed (original magnification = 100 k). HAp was abundantly present (white arrowheads). (c) AgNO3 -immersed specimens revealed relatively small deposits of silver within the adhesive (arrow #1), in the smear layer (arrow #2) and inside dentin underneath (arrow #3). (d) Demineralized, unstained section: porous resin tags were observed, most likely because of demineralization of residual smear particles. (e) Demineralized, stained section: a clearly electron dense hybrid layer (HL) can be detected, with on top a resin-smear complex (RSC), consisting of residual smear enveloped by resin. The collagen fibril structure can hardly be seen in the RSC.
interaction with dentin and thus to investigate potential smear interference. As nor the SiC-GROUND, nor the BUR-CUT smear layer was dissolved, but impregnated by the adhesive, a ‘resinsmear complex’ was produced, which we have described before in our previous study regarding the interaction of the adhesive with likewise differently prepared enamel surfaces [11]. Hydroxyapatite was abundantly present within this resin-smear complex, that differed in thickness and uniformity depending on the surface-preparation methodology used (Figs. 2a,b and 3a,b). The increasing gradation of HAp crystals with depth, as Koshiro et al. reported [10], could not be observed to the same degree in our study. The underlying mechanism of bonding of mild selfetch adhesives was shown to be based upon submicron micro-mechanical interlocking [12], supplemented by primary chemical interaction of the functional monomer 10-MDP with HAp that remained around the partially exposed collagen [13,14]. According to the ‘Adhesion-Decalcification concept’ (AD-concept), 10-MDP chemically bonds to HAp and thereby keeps HAp as the natural shelter around collagen [15]. While
such chemical interaction did not raise the ‘immediate’ bond strength, studies testing the biodegradation resistance of adhesive interfaces have shown that it improved the bond stability [16,17]. Additionally, the demineralization capability of ultra-mild self-etch adhesives is limited; the adhesive showed hardly any sign of demineralization at FRACTURED dentin (Fig. 1a and b). The nano-scale interaction with dentin was clearly evidenced by the stained laboratory-demineralized sections. The densely stained layer observed at FRACTURED dentin, was limited to about 100 nm (Fig. 1e). Since this substrate lacked a smear layer, the densely stained layer must be identified as ‘true’ hybrid layer. The much thicker densely stained layer observed at SiC-GROUND (Fig. 2e) and BUR-CUT dentin (Fig. 3e) must thus be attributed to resin having infiltrated residual surface smear. The resultant ‘resin-smear complex’ is positioned on top of the true hybrid layer. Some authors have already before speculated that separation of the hybridized smear layer from the true hybrid layer may occur [18,19]. In the event that the underlying dentin is demineralized and hybridized, there is a further concern that the only material
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1147–1153
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Fig. 3 – TEM photomicrographs of an ‘ultra-mild’ self-etch adhesive bonded to BUR-CUT dentin. (a) Non-demineralized, unstained section: the surface irregularity and thickness of the smear layer (SL) differed regionally. The thickness of the smear layer varied from almost 0 to 3 m. (b1) Corresponding higher magnification of (a), disclosing a ‘shallow’ smear layer of less than 1 m. Hydroxyapatite was abundantly present (white arrowheads). (b2) Corresponding higher magnification of (a), disclosing a ‘thick’ smear layer of more than 4 m. (c) AgNO3 -immersed specimens revealed a varying pattern of both spot- and cluster-like appearance of nano-leakage within the adhesive (arrow #1), in the smear layer (arrow #2) and inside dentin underneath (arrow #3). Only a few spots of silver deposition were found in the resin-smear complex (RSC). (d) Demineralized, unstained section: the irregularity of the smear layer was more evident. (e1) Demineralized, stained section, disclosing a ‘shallow’ smear layer. A clearly electron dense hybrid layer (HL) became visible, with on top a resin-smear complex, consisting of residual smear enveloped by resin. (e2) Demineralized, stained section, disclosing a ‘thick’ smear layer. Not only the thickness of the resin-smear complex, but also that of the hybrid layer was different from that at SiC-GROUND dentin (see Fig. 2e). A partial loss within the resin-smear complex was observed (hand point).
connecting the resin-smear complex with the true hybrid layer is resin that had to diffuse around the globular particle aggregates within the actual smear layer, and then onwards into the interfibrillar spaces of the underlying intertubular dentin, as well as into the dentinal tubules, the latter forming hybridized smear plugs. However, given the abundant presence of HAp in this resin-smear complex and the effective chemical binding potential of the 10-MDP functional monomer with this HAp, it is very likely that these residual smear particles may reinforce the resin-smear complex. Moreover, for none of our TEM sections observed, the hybrid layer separated from the resin-smear complex, corroborating our hypothesis that well resin-enveloped smear globules may reinforce the adhesive interface. At FRACTURED dentin, the acid-resistant hybrid layer, as observed in the laboratory-demineralized sections, was mostly limited to a depth ranging up to a few hundreds of nanometers, while in most areas the infiltration did not
extend beyond the width of one collagen fibril (about 100 nm). The micro-fibrillar arrangement of collagen type I, which results from the pentameric staggered organization of the molecules in a higher order supra-molecular supertwisted nanostructure, has been shown to result in dentinal collagen with an intermolecular spacing of about 1.3 nm. This zone is too small to accommodate one single molecule of common dental monomers (∼2 nm) [20]. Therefore, monomers can only infiltrate more deeply in case of considerable demineralization or fragmentation as within the smear layer. There is a common consensus that the final goal of dentin bonding is complete infiltration of resin monomers into demineralized collagen fibrils exposed by separate acid-etching or by self-etch adhesives [21]. Any discrepancy between dentin demineralization and resin infiltration may result in silvernitrate- or fluorescent-dye-detectable nanoleakage within water-rich zones of the hybrid layer [22,23], and the adhesive layer [24]. A partial loss of the resin-smear complex was
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Fig. 4 – TEM photomicrographs of non-demineralized, non-stained ‘ultra-thin’ sections of the interface between an ultra-mild self-etch adhesive and dentin covered with different smear layers. (a) Overview image of the adhesive–dentin interface at FRACTURED dentin, disclosing a tight, void-free interface. A clear resin-tag was observed (black arrowhead). The hardest dentinal tissue, namely peritubular dentin, was absent in this thin section (asterisks). (b) Overview image of the adhesive–dentin interface at SiC-GROUND dentin, disclosing more porous resin tags most likely due to residual smear (black arrowheads). Peritubular dentin could not be observed in this thin section (asterisks). (c) Overview image of the adhesive–dentin interface at BUR-CUT dentin, disclosing a smear layer (SL) regionally differing in thickness. (d) Higher magnification of the interface at FRACTURED dentin, disclosing HAp crystals scattered throughout the hybrid layer and dentin (white arrowheads). A more electron dense band was consistently observed, which might represent the interaction of the functional monomer 10-MDP with Ca. (e) Higher magnification of the interface at SiC-GROUND dentin, disclosing a resin-smear complex (RSC) on top of the hybrid layer (HL). (f) Higher magnification of the interface at BUR-CUT dentin, disclosing a fragile part of the resin-smear complex with some voids (hand pointer) that may represent areas not infiltrated well by resin. The true hybrid layer is not as distinct, as compared to that observed at FRACTURED and SiC-GROUND dentin.
observed in this study at BUR-CUT dentin (Fig. 3e); areas within the resin-smear complex were identified not to have been adequately filled with resin. This is perfectly in line with our nano-leakage assessment, where the adhesive resin was able to infiltrate the SiC-GROUND dentin surface more effectively than the BUR-CUT surface. The silver-tracer method that has been widely employed as adhesive interface evaluation method for the past 15 years, actually underestimates the retention of water within apparently well-infiltrated collagen matrices in hybrid layers after bonding with etch-and-rinse and self-etch adhesives. On the other hand, using biomimetic hybrid-layer remineralization techniques a progressive reduction in silver-tracer uptake over time and, hence, in water distribution within adhesive-dentin interfaces was reported for etch-and-rinse hybrid layers [25]. Moreover, besides incomplete resin infiltration, such regions of silver deposition may also represent sites of retained water [26], especially in case of
HEMA-containing adhesives that tend to absorb water more easily [27]. Consequently, it is not yet evident whether the partial loss of the resin-smear complex (Fig. 3e) and the fragile part (Fig. 4f) at BUR-CUT dentin are of significant concern with regard to bond durability. TEM of ultra-thin sections revealed clear differences in terms of substrate irregularity, smear-layer thickness, resin tag/smear plug formation and hybrid layer/resin-smear complex ultrastructure for the three surface-preparation methods applied. The observations are complementary to those of conventional thickness non-demineralized and laboratory-demineralized TEM sections, as especially the true interaction with underlying dentin can be disclosed, which using conventional thickness sections requires laboratorydemineralization and staining. However, as these ultrathin TEM sections were not demineralized, the crystal structure at the interface can still be detected. Since these sections
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1147–1153
were not stained, we hypothesize that the electron dense part observed at the interaction layer, represents the interaction of the functional monomer 10-MDP with Ca. It is concluded that the dentin surface-preparation method significantly affects the nature of the smear layer and thus the interaction of in particular (ultra-)mild self-etch adhesives, being more uniform at SiC-GROUND dentin as compared to the more clinically relevant BUR-CUT dentin surface. In light of the increased clinical use of (ultra-)mild self-etch adhesives, the effect of different surface-preparation methods (e.g. hand excavation, bur preparation, laser ablation, air-abrasion, chemo-mechanical methods, air-polishing, ultrasonication and sono-abrasion) on the surface receptiveness of dentin for adhesion using self-etch adhesives needs to be investigated more.
Conflicts of interest The authors declare that they have no conflicts of interest regarding the products investigated.
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