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Variations in collagen fibrils network structure and surface dehydration of acid demineralized intertubular dentin: Effect of dentin depth and air-exposure time
d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 35–43
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Variations in collagen fibrils network structure and surface dehydration of acid demineralized intertubular dentin: Effect of dentin depth and air-exposure time Amr S. Fawzy ∗ Dental Biomaterials Department, Faculty of Dentistry, Ain Shams University, Cairo, Egypt
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objective. The aim was to characterize the variations in the structure and surface dehydration
Received 18 December 2008
of acid demineralized intertubular dentin collagen network with the variations in dentin
Received in revised form
depth and time of air-exposure (3, 6, 9 and 12 min). In addition, to study the effect of these
13 May 2009
variations on the tensile bond strength (TBS) to dentin.
Accepted 22 August 2009
Methods. Phosphoric acid demineralized superficial and deep dentin specimens were prepared. The structure of the dentin collagen network was characterized by AFM. The surface dehydration was characterized by probing the nano-scale adhesion force (Fad ) between AFM
Keywords:
tip and intertubular dentin surface as a new experimental approach. The TBS to dentin was
Intertubular dentin
evaluated using an alcohol-based dentin self-priming adhesive.
Collagen fibrils network
Results. AFM images revealed a demineralized open collagen network structure in both of
Dehydration
superficial and deep dentin at 3 and 6 min of air-exposure. However, at 9 min, superficial
AFM
dentin showed more collapsed network structure compared to deep dentin that partially
Adhesion force
preserved the open network structure. Total collapsed structure was found at 12 min for both of superficial and deep dentin. The value of the Fad is decreased with increasing the time of air-exposure and is increased with dentin depth at the same time of air-exposure. The TBS was higher for superficial dentin at 3 and 6 min, however, no difference was found at 9 and 12 min. Significance. The ability of the demineralized dentin collagen network to resist airdehydration and to preserve the integrity of open network structure with the increase in air-exposure time is increased with dentin depth. Although superficial dentin achieves higher bond strength values, the difference in the bond strength is decreased by increasing the time of air-exposure. The AFM probed Fad showed to be sensitive approach to characterize surface dehydration, however, further researches are recommended regarding the validity of such approach. © 2009 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Dentin has been characterized by Marshall et al. [1] as a biologic composite of collagen matrix filled with nanometer-
∗
sized calcium-deficient, carbonate-rich apatite crystallites dispersed between parallel micron-sized hypermineralized, collagen poor, hollow cylinders (dentinal tubules containing peritubular dentin). Superficial dentin has few dentinal
Tel.: +2 2 26359765. E-mail addresses: [email protected], [email protected]. 0109-5641/$ – see front matter © 2009 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dental.2009.08.009
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tubules and composed mainly of intertubular dentin, whereas deep dentin is composed mainly of large dentinal tubules with much less intertubular dentin matrix [2]. The dentin structure and properties are changed with depth as reported by many researchers. Kinney et al. [3] reported that the intertubular dentin becomes softer as deep dentin is tested. In addition, the water content of dentin is lowest in superficial dentin compared to deep dentin [4]. However, the average bulk mineral composition may be relatively constant, as function of dentinal depth, even though the amount of collagen rich intertubular dentin gradually decreases with depth [5]. The amount of collagen fibrils per unit volume of dentin decreases from superficial dentin to deep dentin [6]. The structural integrity and mechanical properties of the collagen fibrils of acid demineralized deep and superficial dentin play an important role in the determination of bond strength and its durability [7–9]. Atomic force microscope (AFM) was used extensively, in recent years, as a tool to study the surface morphology and roughness, collagen fibrils structure, and mechanical properties of human dentin [10–13]. Atomic force microscope is not only a tool to image and measure the topography of solid surfaces at high resolution. It can also be used to measure force-versus-distance curves. Such force curves provide valuable information on local material properties such as adhesion force, elasticity, hardness and surface charge densities [14–16]. Detailed description of the techniques, interpretation and applications of force curve measurement by AFM is clarified in more specialized references [14,15,17]. However, in brief, a force vs. distance curve is a graph of the vertical force on the cantilever tip as a function of the extension of the piezoelectric scanner tube. A force curve is generated at a single location on a specimen surface by measuring how much the cantilever bends during one or more “sweeps” (up and down movements) of the scanner. Variations in the shape of force curves taken at different locations indicate variations in the local nano-scale properties of the specimen surface. The shape of the curve is also affected by contaminants and surface lubricants, as well as the water content of the surface layer of the specimen when operating an AFM in air. Pashley et al. [18] clarified the difference in the structure of the hybrid layer between superficial and deep dentin. In the first, most of hybrid layer is composed of demineralized and resin-hybridized intertubular dentin with only an occasional resin tags penetrating from the overlying adhesive layer into, relatively narrow, funnel-shaped dentinal tubules. In deep dentin the tubules are so numerous and large that little intertubular dentinal matrix is available. Consequently, resin tags represent a major fraction of bonded surfaces near the pulp. Theoretically, resin tags could contribute to resin retention if they are firmly bonded to the walls of the tubules [19–21]. In addition to resin tags, hybrid layer formation in demineralized intertubular dentin should also contribute to resin retention in proportion to the amount of etched intertubular dentin that is available for bonding in deep dentin. Therefore, the relative contribution of resin tags and hybrid layer to overall bond strength should be varied with dentin depth because the sum of the cross-sectional area of resin tags and that of the hybrid layer is constant [5,22]. Modeling experiments on the packing density of molecules within tendon collagen fibrils [23] indicate that there may
be significant space between the collagen molecules for tissue fluid as water. As long as the interfibrillar spaces, which from interconnected channels having capillary dimensions, are in hydrated state and maintaining an open-structure, pathways are provided for monomers diffusion through collagen fibrils network [21,22,24–26]. This water may be lost with dehydration, as a result of air-exposure, leading to shrinkage and collapse of the collagen fibrils network [9]. As stated by Nakabayashi [19] and others [24,27] for hybrid layer formation, intertubular dentin must be demineralized to expose the open collagen fibrils network of the dentinal matrix to create diffusion pathway for monomer infiltration. Therefore, the presence of water is crucial to the maintenance of the structure and the strength of the demineralized dentin collagen matrix and consequently higher bond strength values are obtained with wet surfaces [28–31]. The current study investigated the hypothesis that the variations in dentin depth and air-exposure time affect the performance, collagen fibrils network structure and the bonding efficacy of acid demineralized intertubular dentin. Therefore, the first aim of this study was to determine whether the structural changes of the intertubular dentin collagen fibrils network as a result of air-exposure following phosphoric acid etching differ with dentin depth and air-exposure time. The second aim was to characterize the dehydration from the surface of acid etched intertubular dentin collagen network as a function of dentin depth and air-exposure time by probing the nano-scale adhesion force between atomic force microscope (AFM) tip and intertubular dentin surface as a new experimental approach. Finally, to study the effect of the variations in the structure of the acid demineralized collagen fibrils network and surface dehydration, with the variations in dentin depth and air-exposure time, on the bond strength to dentin using an alcohol-based dentin self-priming adhesive.
2.
Materials and methods
Dentin specimens used in this study for both of AFM surface characterization and bond strength evaluation were prepared from randomly selected non-carious and non-restored mandibular third molars. All teeth were recently extracted, less than 3 months from the time of the study. All patients were of age range of 25–30 years old. All extracted teeth were stored in 0.5% chloramines T solution for 2 weeks then in distilled water at 4 ◦ C until use [8].
2.1.
Preparation of dentin specimens for AFM study
Eight teeth were used for AFM study from which eight superficial (Sd) and eight deep (Dd) dentin disks, of approximately 2 mm thickness, were prepared. From each tooth one superficial and one deep dentin disks were prepared to decrease the variation between specimens. For the preparation of the superficial dentin disks, occlusal enamel was removed perpendicular to the teeth long-axis with a diamond disk mounted to a milling machine (Nouvage AG, Fräsgerät AF 30, Switzerland) using slow-speed under water cooling until the superficial dentin surface approximately 1 mm below the DEJ was exposed. Then the superficial dentin disks, hav-
d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 35–43
ing an approximate thickness of 2 mm, were cut parallel to the exposed superficial dentin surfaces. After cutting of the superficial dentin disks, the exposed dentin surfaces, which approximately 3 mm below the DEJ, were considered as deep dentin. Then the deep dentin disks were prepared in a similar manner as in superficial dentin disks. The dentin disks were cut into two equal halves to produce 16 superficial and 16 deep dentin specimens for the AFM study. The dentin specimens were sequentially wet-grinded with 600, 800, 1000 and 1500 grit SiC polishing papers to produce relatively smooth surfaces for AFM characterization and to creates standardized smear layer. All specimens were then ultrasonically cleaned in deionized water for 15 min and then stored in distilled water at 37 ◦ C for 24 h before AFM study.
2.2.
Atomic force microscope (AFM) study
An atomic force microscope (Autoprobe CP-II, Veeco, CA, USA) was used to characterize the etched intertubular dentin collagen fibrils network structure and to probe the nano-scale adhesion force between AFM silicon nitride tip and etched intertubular dentin surface to characterize surface dehydration as a function of both of dentin depth and air-exposure time. The dentin specimens were etched for 15 s with 37% phosphoric acid gel (Ivoclar Vivadent AG, FL-9494 Schaan/Liechtenstein) and rinsed ultrasonically in distilled water for 5 min. Then the etched dentin specimens were treated by 6.5 vol.% sodium hypochlorite (NaOClaq) for 120 s [32] as a deproteinizing agent to remove non-collagenous proteins from the extracellular organic matrix and to reveal the collagen fibril network structure. Then all specimens were thoroughly rinsed in distilled water for 5 min and the excess water was removed by gentle blotting with absorbent paper leaving the dentin surface visibly moist. Immediately after surface preparation, dentin specimens were characterized by AFM.
2.2.1. Characterization of collagen fibrils network structure by tapping-mode AFM Eight specimens of each of superficial and deep dentin were used for the tapping-mode AFM study. For the tapping mode, gold-coated all-silicon cantilevers (UltraleversTM , Thermo-Microscopes, CA, USA) with integrated high aspect ratio conical tips were used. The typical radius of curvature of the scanning tip was 10 nm. The force constant and the resonance frequency of the used cantilevers were 17 N/m and 320 kHz, respectively. The Images were recorded with scan rate of 2 Hz at a scanning area of 3 m × 3 m and a resolution of 512 × 512 pixels per image. All dentin specimens were characterized in air under well-controlled laboratory environments in term of humidity (45 ± 5%) and temperature (21 ± 0.5 ◦ C). Immediately after surface preparation, each specimen was mounted to the AFM and characterized immediately at relatively hydrated state. The average time for specimen mounting and operating the AFM was approximately 3 min. Therefore, the first scan taken for each specimen was preformed approximately at 3 min (t1) of air-exposure. After the first scan at time (t1), a series of three consecutive scans were preformed at 3 min time inter-
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vals from the end of the first scan. Therefore, each specimen, prepared either from superficial or deep dentin, was characterized at 3 (t1), 6 (t2), 9 (t3), and 12 (t4) min of air-exposure from the end dentin surface preparation steps. Accordingly, the variations in the structure of the acid demineralized intertubular dentin collagen fibrils network with variations in dentin depth and air-exposure time were characterized.
2.2.2. Probing nano-scale adhesion force (Fad ) by contact-mode AFM The remaining superficial (n = 8) and deep (n = 8) dentin specimens were used to probe the nano-scale adhesion force between AFM silicon nitride tip and etched intertubular dentin surface as a function of time of air-exposure and dentin depth. Immediately after surface preparation, superficial and deep dentin specimens were characterized by contact-mode AFM at the same environmental conditions and air-exposure time intervals (t1–t4) as previously described. AFM contact-mode was operated to characterize the forceversus-distance curve (force curve) between the AFM tip and specimen surface. Silicon nitride tips, of 50 nm nominal radii, mounted to contact-mode cantilevers (Type D MicroleversTM , Thermo-Microscopes, CA, USA) were used to scan specimens surfaces. The force constant and the resonance frequency of the used cantilevers were 2.1 N/m and 160 kHz, respectively. For each specimen, surface scan of 3 m × 3 m area was done at a scan rate of 2 Hz and then a set of force curves were measured for selected 14 points at the intertubular dentin. The same process was repeated for each specimen at each previously described time intervals such that a set of force curves as a function of time of air-exposure (t1–t4) and dentin depth (Sd and Dd) were recorded. The force curves typically show the deflection of the free end of the AFM cantilever as the fixed end of the cantilever is brought vertically towards and then away from the specimen surface. The deflection of the free end of the cantilever is measured and plotted at many points as the z-axis scanner extends the cantilever towards the surface and then retract it again. Then the nano-scale surface adhesion force (Fad ) between the AFM tip and intertubular surface was calculated in nano-Newton (nN) from the mean force curve of each specimen at each air-exposure time interval. The adhesion force (Fad ) was calculated from the difference between the snapin point (the point of contact between the cantilever tip and specimen surface) and the snap-out point (the point of separation or detachment between the cantilever tip and specimen surface) [14,17].
2.3.
Tensile bond strength (TBS) testing
Dentin/resin-based restorative composite specimens were prepared from freshly extracted mandibular third molars for the tensile bond strength (TBS) testing. The roots of the extracted molars were embedded in self-polymerized resin 3 mm below the cervical line. The occlusal enamel was removed using diamond disk mounted to a milling machine as previously described in the preparation of the specimens for the AFM study under copious water spray to expose flat dentin surface. Half of the teeth were prepared to expose the superficial dentin surfaces, while in the other half the deep
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dentin surfaces were exposed. The smear layer was standardized as described previously. The exposed dentin surfaces were etched for 15 s with 37% phosphoric acid gel, thoroughly rinsed in distilled water for 5 min and the excess water was removed by gentle blotting with absorbent paper leaving the dentin surface visibly moist. A single bottle alcohol-based self-priming adhesive (Excite, Ivoclar Vivadent AG, FL-9494 Schaan/Liechtenstein) was applied to the conditioned dentin surfaces. One coat of Excite adhesive was applied over the etched dentin surface and gently agitated for 10 s; excess solvent was removed using a gentle stream of air for 3 s. The Excite was then light-cured for 20 s using a light-curing unit (Cromalux-E, Meca-Physik Dental Division, D-76437 Rastatt, Germany) with a light output of 600 mW/cm2 . According to the dentin depth and the time of application of the self-priming adhesive (airexposure time), teeth were divided into the following test groups to simulate what was done in AFM study. Superficial dentin (Sd): Sd(t1), Sd(t2), Sd(t3), and Sd(t4). Deep dentin (Dd): Dd(t1), Dd(t2), Dd(t3), and Dd(t4). After the application of the self-priming adhesive at the desired air-exposure times (t1–t4), a light cured resin-based restorative composite material (Tetric Ceram, Ivoclar Vivadent AG, FL-9494 Schaan/Liechtenstein) was applied and cured in four increments for each tooth. The restored teeth were then stored in distilled water at 37 ◦ C for 48 h to complete the curing process. After storage, each restored tooth was sectioned with diamond disk fixed to a milling machine under copious water spray to prepare dentin/composite slabs of 1 mm thickness from the central part of the tooth. A fine high-speed diamond stone with air/water spray was used to trim the slabs into an hour-glass shape with cross-sectional area of approximately 1 mm2 at the bonded interface which checked and confirmed by a digital caliper. Twelve (n = 12) hour-glass shape slabs were tested for each group. All slabs were stored in distilled water at 37 ◦ C for 24 h before mechanical testing. Each slab was fixed to two disposable acrylic resin jigs using cyanoacrylate adhesive. The acrylic jigs were mounted to a universal testing machine (Lloyd Instruments, LR5 series, UK). The test was run at a cross-head speed of 0.5 mm/min until failure. The cross-sectional area of each fractured slab was confirmed using a digital caliper and the respective load was divided over the cross-sectional area to calculate the TBS in MPa.
2.4.
Statistical analysis
All data of nano-scale adhesion force (Fad ) and tensile bond strength (TBS) were expressed as means and standard deviations (SD). Statistical analysis was carried out using SPSS program (Release 15, 2006). Two-way ANOVA was preformed to test the effect of dentin depth, air-exposure time and their interaction on either of Fad and TBS. Tukey–Kramer multiple-comparison post hoc test was used to compare each of Fad and TBS between the tested groups. The correlation between Fad and TBS through the different air-exposure times (t1–t4) was tested using Pearson’s correlation coefficient. P-values less than 0.05 were considered statistically significant.
3.
Results
AFM images revealed a demineralized open collagen fibrils network structure with numerous interconnected interfibrillar spaces and porosities in both of superficial (Fig. 1A and B) and deep (Fig. 1E and F) intertubular dentin at 3(t1) and 6(t2) min of air-exposure. However, at 9(t3) min of air-exposure, superficial intertubular dentin collagen fibrils network showed more collapsed structure (Fig. 1C) compared to deep dentin that partially preserved the open fibrils network structure (Fig. 1G). Total collapsed structure of the collagen fibrils network was shown at 12(t4) min of air-exposure in both of superficial and deep dentin specimens (Fig. 1D and H). Fig. 2A–C shows selected cantilever-specimen surface interactions (force curves) between AFM tip and etched deep intertubular dentin at 3(t1) min and 9(t3) min; and etched superficial intertubular dentin at 9(t3) min of air-exposure, respectively. The force is represented on the vertical axis in nN and the AFM piezoelectric scanner z-position is represented on the horizontal axis in nm. The scanner extension (approach) path is demonstrated by the solid line. While the scanner retraction path, is demonstrated by the dashed line. The point of contact between the tip and specimen surface is represented by the snap-in point. The point of separation or detachment between the cantilever tip and specimen surface is represented by the snap-out point. The nano-scale adhesion force (Fad ) is represented by the difference between the snap-in and snap-out points (black arrow). Two-way ANOVA (Tables 1 and 2) showed the significant effect (P ≤ 0.001) of either of dentin depth and air-exposure time on AFM-probed adhesion force (Fad ) and tensile bond strength (TBS). In addition, a significant interaction was found between the two independent variables (dentin depth and airexposure time) on either of Fad (P = 0.015) and TBS (P = 0.002). The variations of the mean values of Fad and TBS with air-exposure time for both of superficial and deep dentin specimens are showing Figs. 3 and 4, respectively. For each of superficial (Sd) and deep dentin (Dd), the values of both of Fad (Table 3) and TBS (Table 4) are significantly decreased with the increase in the air-exposure time from t1 to t4. In addition, Pearson’s correlation analysis revealed a significant positive correlation (P < 0.001) between Fad and TBS within each of superficial and deep dentin. No significant difference was found in Fad between superficial and deep dentin at 3(t1) and 6(t2) min of air-exposure (Table 3). However, at the same air-exposure times (t1 and t2), superficial dentin shows significantly higher TBS (Table 4). In contrast, at 9(t3) and 12(t4) min of air-exposure, deep dentin showed significant increase in Fad (Table 3), however, no significant difference in TBS was found between superficial and deep dentin (Table 4).
4.
Discussion
Atomic force microscope (AFM) was selectively used in this study to characterize the structure of acid demineralized collagen fibrils network because it has an advantage over the ultra-high vacuum measuring equipments, such as SEM, as specimens were imaged in air at humidity of 45 ± 5% and tem-
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Fig. 1 – Tapping-mode AFM images showing the structure of the acid demineralized dentin collagen fibrils network at the different air-exposure times of both of superficial (A–D) and deep (E–H) dentin specimens. Open collagen network structure with numerous interconnected interfibrillar spaces and porosities are observed at 3 and 6 min of air-exposure for both of superficial (A and B) and deep (E and F) dentin. At 9 min of air-exposure superficial dentin shows collapsed structure (C), however, deep dentin specimens partially preserved the open network structure (G). Total collapsed network structure is observed at 12 min of air-exposure for both of superficial (D) and deep (H) dentin. Arrows point out collagen fibrils and open interfibrils spaces.
Table 1 – Two-way ANOVA for the effect of dentin depth, air-exposure time and their interaction on the AFM probed nano-scale adhesion force (Fad ). Source
Type III sum of squares
Dentin-depth Air-exposure time Dentin-depth × air-exposure time
3.238 60.878 0.516
d.f.
Mean square
1 3 3
3.238 20.293 0.172
F
Sig.
56.527 354.303 3.002
0.000 0.000 0.015
R Squared = 0.972 (adjusted R Squared = 0.966).
Table 2 – Two-way ANOVA for the effect of dentin depth, air-exposure time and their interaction on the tensile bond strength (TBS). Source
Type III sum of squares
Dentin depth Air-exposure time Air-exposure time × dentin depth
19.182 1064.487 25.327
d.f.
Mean square 1 3 3
19.182 354.829 8.442
F
Sig.
14.338 265.218 6.310
0.001 0.000 0.002
R Squared = 0.963 (adjusted R Squared = 0.955).
Table 3 – Nano-scale adhesion force (Fad ) of superficial (Sd) and deep (Dd) dentin at the different air-exposure times (t1–t4). Sd Dd
t1
t2
t3
t4
4.77 (0.26)aA 5.23 (0.30)aA
3.53 (0.24)bA 3.92 (0.27)bA
2.05 (0.10)cA 2.99 (0.13)cB
1.47 (0.35)dA 2.11 (0.12)dB
Mean values with dissimilar small letters within each row and capital letters within each column are statistically significant different at P ≤ 0.05.
Table 4 – Tensile bond strength (TBS) to superficial (Sd) and deep (Dd) dentin at the different air-exposure times (t1–t4).
Sd Dd
t1
t2
t3
t4
27.40 (4.76)aA 22.34 (5.32)aB
19.88 (5.58)bA 17.43 (3.94)bB
14.68 (4.38)cA 14.12 (3.51)cA
11.02 (3.62)dA 10.15 (2.03)dA
Mean values with dissimilar small letters within each row and capital letters within each column are statistically significant different at P ≤ 0.05.
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Fig. 3 – Showing the variations in the mean values of nano-scale adhesion force (Fad ) with the time of air-exposure of both of superficial and deep dentin specimens.
Fig. 2 – Selected force curves showing the interaction between AFM tip and etched deep intertubular dentin at 3 min (A), etched deep intertubular dentin at 9 min (B), and etched superficial intertubular dentin at 9 min (C) of air-exposure. The AFM scanner approach and retraction paths are represented by the solid and the dashed lines, respectively. The nano-scale adhesion force (Fad ) in nN is represented by the black arrow between the snap-in and snap-out points.
perature of 21 ± 0.5 ◦ C. Therefore, by such way the dehydration of the dentin specimens as a result of air-exposure could be minimized and relatively standardized. It was reported by El Feninat et al. [9] that considerable water loss and observable structure change occur within few minutes to etched dentin collagen fibrils network when characterized by AFM in air. The progressive dehydration effect by increasing the time of air-exposure (from 3 to 12 min) on the structure of the intertubular dentin collagen fibrils network is demonstrated by the consecutive AFM images for
both of superficial (Fig. 1A–D) and deep (Fig. 1E–H) dentin specimens. These structural changes associated with airdehydration effect are in agreement with previous findings reported that the presence of water is essential to prevent the collapse of the demineralized dentin collagen network [33,34]. However, from the AFM images (Fig. 1), the integrity of the structure of the demineralized intertubular dentin collagen fibrils network of deep dentin seems to be less affected by the air-dehydration process. This could be attributed to the higher water content in deep dentin compared to superficial dentin which might be responsible for the higher structural resistance to collapse by increasing the time of air-dehydration [4]. The phenomena of shrinkage and collapse of the demineralized soft collagen fibrils network by air dehydration could be clarified by more than one explanation. The demineralized collagen network could be assumed to be suspended in water that occupies the spaces that were previously occupied by apatite crystals between collagen fibrils. Therefore, by water loss due to air dehydration, collagen fibrils become more close together resulting in passive collapse [5]. Alternatively, at molecular level, the intermolecular spaces between collagen molecules are preserved by water that surrounds the triple helices of collagen molecule. Consequently, hydrogen bonds are formed preventing direct inter-chain contact and
Fig. 4 – Showing the variations in the mean values of tensile bond strength (TBS) with the time of air-exposure of both of superficial and deep dentin specimens.
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collapse [35–37]. Furthermore, the surface tension force acting on air-collagen fibrils interface [38], and the shortening of the interconnected collagen fibrils [39] associated with water loss are among the possible explanations. Therefore, we could assume that the dehydration from the surface of the acid demineralized intertubular collagen fibrils network as a result of air-exposure is lower in deep dentin than in superficial dentin. The results of the nano-scale adhesion force (Fad ) are in agreement with the previous assumption (Table 3). The values of the Fad was significantly higher for deep dentin specimens than for superficial dentin specimens at 9(t3) and 12(t4) min of air-exposure. In addition, the results of the two-way (Table 1) and one-way (Table 3) ANOVA and the AFM images (Fig. 1) partially prove the sensitivity of the AFM probed nano-scale adhesion force (Fad ) as an approach to characterize the dehydration from the surface of the demineralized intertubular dentin collagen fibrils network. Fig. 3 shows simultaneous decrease in the mean values of Fad , in both of superficial and deep dentin specimens, with the increase in the time of air dehydration. When retracting the AFM tip from the dentin surface, the tip stays in contact with the surface until the cantilever force overcomes the adhesion force between the tip and specimen surface. In the most general case the adhesion force (Fad ) is a combination of the electrostatic force, the van der Waals force, the meniscus or capillary force, and forces due to chemical bonds or acid–base interactions [14,40,41]. At ambient conditions, a water neck forms between AFM tip and specimen surface due to capillary condensation and adsorption of thin water films at surfaces leading to the formation of capillary attractive forces. This capillary attractive interaction depends on the relative humidity and the hydrophilicity of tip and sample surface. Therefore, capillary forces are expected to be maximal for hydrophilic surfaces and to vanish for very hydrophobic surfaces [14]. The relative contributions of capillary, van der Waals and electrostatic force under ambient conditions for the adhesion of an AFM tip to several materials surfaces were previously studied. In all cases, the capillary force was found to give the largest contribution [14,42]. Therefore, in this study, every effort was done to standardize the experimental conditions during probing the nano-scale adhesion force (Fad ) in order to dominate the effect of the capillary attractive force and to standardize the contribution of other forces in changing the values of the probed Fad . This is done through using of identical silicon nitride AFM tips without any chemical modification. In addition, AFM characterization was done at the same well-controlled experimental conditions in term of temperature, humidity and air-exposure time. Furthermore, through the using of randomly divided specimens prepared from extracted non-carious and nonrestored mandibular third molars of the same age range, extraction period and storage conditions. Consequently, we could suggest that the variations in the values of the probed Fad are mainly attributed to the dehydration from the surface layer of the demineralized intertubular dentin collagen fibrils network. The significant decrease in the TBS with increase in the time of air-exposure from 3(t1) to 12(t4) min within both of superficial and deep dentin specimens recorded in the current
41
study (Table 4) and (Fig. 4) could be explained by increasing the time of air-exposure the demineralized soft collagen fibrils network suffers from dehydration effect leading structural collapse which might interfered with the infiltration of the Excite monomers to form hybrid layer. It was found in previous studies that, for successful bonding to dentin, proper resin infiltration of the demineralized intertubular dentin collagen fibrils network to form hybrid layer is required. This is achieved by the creation of open collagen fibrils network structure with water-filled interfibril spaces [5,19,22,24]. As Excite is an alcohol-based dentin self-priming adhesive, open collagen fibrils network structure in hydrated condition should be available for proper monomers infiltration and hybrid layer formation. This is in agreement with other previous studies which recommended to not dry etched dentin surface before the application of acetone or alcohol-based bonding primers to achieve higher bond strength values [28,29,38,43,44]. This previous explanation is in consistence with and support the finding of the nano-scale adhesion force (Fad ) (Table 3) and (Fig. 3); as with increase in the time of air-exposure, within both of superficial and deep dentin specimens, there are a significant decrease in the value of the recorded Fad . Therefore, the assumed sensitivity of the nano-scale adhesion force (Fad ) to detect surface dehydration from the demineralized intertubular dentin collagen fibrils network is further supported. In addition, Pearson’s correlation confirmed the positive correlation between Fad and TBS. The significantly higher TBS (Table 4) for superficial dentin specimens over deep dentin specimens recorded at 3(t1) and 6(t2) min of air-exposure could be explained by the following. Bonding to dentin is mainly depends on the amount of intertubular dentin matrix available to be infiltrated with the resin monomers to form hybrid layer [5,18]. However, resin tags could contribute, but to less extant, in the bonding to dentin if they are firmly bonded to dentinal tubules walls [18–21]. Superficial dentin has an advantage over deep dentin as more intertubular dentin matrix is available in superficial dentin for hybrid layer formation [2]. Therefore, provided all other variables are standardized, the higher bond strength values recorded for superficial dentin specimens over deep dentin specimens are justified. However, as previously mentioned, the demineralized collagen fibrils network of the intertubular dentin should be in hydrated state to preserve the open collagen fibrils structure for the Excite monomers infiltration and hybrid layer formation. In this respect, based on AFM images (Fig. 1) and the results of the nano-scale adhesion force (Fad ) (Table 3) and (Fig. 3), deep dentin might has an advantage over superficial dentin in resisting surface dehydration and partially preserving the integrity of intertubular dentin open collagen fibrils network structure with the increase in air-exposure time. This could explain the non-significant difference in TBS at more prolonged time of air-exposure (t3 and t4) between deep and superficial dentin specimens (Table 4). It should be mentioned that adhesion force (Fad ) measurement by AFM is affected with many variables which should be taken in consideration in any future research. Capillary forces, which contribute in the overall value of the Fad between AFM and specimen surface, are important factor in force measurement taken in ambient air, but not in aqueous environment or in high vacuum [17]. Variables related to AFM tip geometry,
42
d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 35–43
cantilever force constant and the tip surface condition such as water content should also be critically considered [14]. In addition, the dentin specimen surface roughness and heterogeneity should be taken in account. Furthermore, studying the interactions between chemically modified AFM tip and specific functional group(s) in dentinal substrate is recommended in future research.
5.
Conclusions
Based on the results of this study, the ability of the demineralized intertubular dentin soft collagen fibrils network to resist air-dehydration and to preserve the integrity of open network structure with the increase in air-exposure time is increased with dentin depth. Although superficial dentin achieves higher bond strength values than deep dentin with an alcohol-based self-priming adhesive (Excite), this difference in the bond strength between superficial and deep dentin is decreased by increasing the time of air-exposure before the application of the dentin self-priming adhesive to the demineralized dentin. The AFM probed nano-scale adhesion force showed to be sensitive approach to characterize surface dehydration, resulted from air-exposure, from the demineralized intertubular dentin collagen fibrils network. The value of the nano-scale adhesion force is decreased with increasing the time of air-exposure and is increased with dentin depth at the same time of air-exposure. However, further researches regarding the validity of such approach and the quantitative correlation between the degree of water loss as result of airdehydration from the demineralized collagen fibrils network and the nano-scale adhesion force are recommended.
references
[1] Marshall GW, Marshall SJ, Kinney JH, Balooch M. The dentin substrate structure and properties related to bonding. J Dent 1997;25:441–58. [2] Garberoglio R, Brannstrom M. Scanning electron microscopic investigation of human dentinal tubules. Arch Oral Biol 1972;21:355–62. [3] Kinney JH, Balooch Marshall SJ, Marshall GW, Weihs TP. Atomic force microscope measurements of the hardness and elasticity of peritubular and intertubular human dentin. J Biomech Eng 1996;118:133–5. [4] Pashley DH. The clinical correlation of dentin structure and function. J Prosthet Dent 1991;66:777–81. [5] Nakabayashi N, Pashley DH. Hybridization of dental hard tissues. Tokyo: Quintessence Publishing Co.; 1998. pp. 16–76. [6] Levine RS. The distribution of hydroxyproline in sound human coronal dentine. Arch Oral Biol 1971;16:473–8. [7] Eliades G, Palaghias G, Vougiouklakis G. Effect of acidic conditioners on dentin morphology, molecular composition and collagen conformation in situ. Dent Mater 1997;13:24–33. [8] Yang B, Adelungb R, Ludwiga K, Bobmann K, Pashley DH, Kern M. Effect of structural change of collagen fibrils on the durability of dentin bonding. Biomaterials 2005;26:5021–31. [9] El Feninat F, Ellis T, Sacher E, Stangel I. A tapping mode AFM study of collapse and denaturation in dentinal collagen. Dent Mater 2001;17:284–8. [10] Nakabayashi N, Watanabe A, Igarashi K. AFM observation of collapse and expansion of phosphoric acid-demineralized dentin. J Biomed Mater Res A 2004;68A:558–65.
[11] Silikas N, Watts DC, England KR, Jandt KD. Surface fine structure of treated dentine investigated with tapping mode atomic force microscopy (TMAFM). J Dent 1999;27:137–44. [12] Balooch G, Marshall G, Marshall S, Warren O, Asif S, Balooch M. Evaluation of a new modulus mapping technique to investigate microstructural features of human teeth. J Biomech 2004;37(8):1223–32. [13] Balooch M, Habelitz S, Kinney J, Marshall S, Marshall G. Mechanical properties of mineralized collagen fibrils as influenced by demineralization. J Struct Biol 2008;162(3):404–10. [14] Butt H, Cappella B, Kappl M. Force measurements with the atomic force microscope: technique, interpretation and applications. Surf Sci Rep 2005;59:1–152. [15] Vinckiera A, Semenza G. Measuring elasticity of biological materials by atomic force microscopy. FEBS Lett 1998;430:12–6. [16] Martin Y, Williams CC, Wickramasinghe H. Atomic force microscope-force mapping and profiling on a sub 100 Å-scale. J Appl Phys 1987;61(10):4723–9. [17] Jandt KD. Atomic force microscope of biomaterials surfaces and interfaces. Surf Sci 2001;491:303–32. [18] Pashley DH, Sano H, Yoshiyama M, Ciucchi B, Carvalho M. Dentin, a dynamic bonding substrate. The effects of dentin variables on resin adhesion. In: Shimono M, Maeda T, Suda H, Takahashi K, editors. Dentin/pulp complex. Tokyo: Quintessence; 1996. p. 11–21. [19] Nakabayashi N. Bonding of restorative materials to dentin: the present status in Japan. Int Dent J 1985;35:145–54. [20] Titly K, Chernecky R, Chan A, Smith D. The composition and ultrastructure of resin tags in etched dentin. Am J Dent 1995;8:224–30. [21] Pashley DH, Ciucchi B, Sano H, Carvalho RM, Russell CM. Bond strength versus dentin structure: a modeling approach. Arch Oral Biol 1995;40:1109–18. [22] Gwinnett AJ. Quantitative contribution of resin infiltration/hybridization to dentin bonding. Am J Dent 1993;6:7–9. [23] Hulmes DJS, Wess TJ, Prockop DJ, Fratzl P. Radial packing, order, and disorder in collagen fibrils. Biophys J 1995;68:1661–70. [24] Van Meerbeek B, Dhem A, Goret-Nicaise M, Braem M, Lambrechts P, Vanherle G. Comparative SEM and TEM examination of ultrastructure of resin–dentin interdiffusion zone. J Dent Res 1993;72:495–501. [25] Marshall GW, Wu-Magidi IC, Watanabe LG, Inai N, Balooch M, Kinney JH, et al. Effect of citric acid concentration on dentin demineralization, dehydration, and rehydration: atomic force microscopy study. J Biomed Mater Res 1998;42:500–7. [26] El Feninat F, Stangel I, Sacher E, Ellis TH. Moisture-dependent renaturation of collagen in phosphoric acid-etched dentin. J Biomed Mater Res 1998;42:549–53. [27] Watanabe I, Nakabayashi N, Pashley DH. Bonding to ground dentin using a self-etching phenyl-P primer. J Dent Res 1994;73:1212–20. [28] Kanca J. Effect of resin primer solvents and surface wetness on resin composite bond strength to dentin. Am J Dent 1992;5:213–5. [29] Kanca J. Improving bond strength through acid etching of dentin and bonding to wet dentin surfaces. JADA 1992;123:35–43. [30] Gwinnett AJ. Moist versus dry dentin: its effect on shear bond strength. Am J Dent 1992;5:127–9. [31] Swiff EJ, Triolo PT. Bond strengths of Scotchbond multi-purpose to moist dentin and enamel. Am J Dent 1992;5:318–20. [32] Marshall GW, Yucel N, Balooch M, Kinney JH, Habelitz S, Marshall SJ. Sodium hypochlorite alterations of dentin and dentin collagen. Surf Sci 2001;491:444–55.
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[33] Pashley DH, Ciucchi B, Sano H, Horner JA. Permeability of dentin to adhesive agents. Quintessence Int 1993;24:618–31. [34] Kanca J. Resin bonding to wet substrate. I. Bonding to dentin. Quintessence Int 1992;23:39–41. [35] Brodsky B, Ramshaw AM. The collagen triple-helix structure. Matrix Biol 1997;15:545–54. [36] Bella J, Eaton M, Brodsky B, Berman HM. Crystal and molecular structure of a collagen-like peptide at 1.9 Angstrom resolution. Science 1994;266(5182):75–81. [37] Bella J, Brodsky B, Berman HM. Hydration structure of collagen peptide. Structure 1995;3(9):893–906. [38] Gwinnett AJ. Dentin bond strength after air drying and rewetting. Am J Dent 1994;7:144–8. [39] Carvalho RM, Zhang Y, Shono H, Pashley DH. Shrinkage force of demineralized dentin during air-drying [abstract 1762]. J Dent Res 1996;76(Special issue):238.
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Variations in collagen fibrils network structure and surface dehydration of acid demineralized intertubular dentin: Effect of dentin depth and air-exposure time Amr S. Fawzy ∗ Dental Biomaterials Department, Faculty of Dentistry, Ain Shams University, Cairo, Egypt
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objective. The aim was to characterize the variations in the structure and surface dehydration
Received 18 December 2008
of acid demineralized intertubular dentin collagen network with the variations in dentin
Received in revised form
depth and time of air-exposure (3, 6, 9 and 12 min). In addition, to study the effect of these
13 May 2009
variations on the tensile bond strength (TBS) to dentin.
Accepted 22 August 2009
Methods. Phosphoric acid demineralized superficial and deep dentin specimens were prepared. The structure of the dentin collagen network was characterized by AFM. The surface dehydration was characterized by probing the nano-scale adhesion force (Fad ) between AFM
Keywords:
tip and intertubular dentin surface as a new experimental approach. The TBS to dentin was
Intertubular dentin
evaluated using an alcohol-based dentin self-priming adhesive.
Collagen fibrils network
Results. AFM images revealed a demineralized open collagen network structure in both of
Dehydration
superficial and deep dentin at 3 and 6 min of air-exposure. However, at 9 min, superficial
AFM
dentin showed more collapsed network structure compared to deep dentin that partially
Adhesion force
preserved the open network structure. Total collapsed structure was found at 12 min for both of superficial and deep dentin. The value of the Fad is decreased with increasing the time of air-exposure and is increased with dentin depth at the same time of air-exposure. The TBS was higher for superficial dentin at 3 and 6 min, however, no difference was found at 9 and 12 min. Significance. The ability of the demineralized dentin collagen network to resist airdehydration and to preserve the integrity of open network structure with the increase in air-exposure time is increased with dentin depth. Although superficial dentin achieves higher bond strength values, the difference in the bond strength is decreased by increasing the time of air-exposure. The AFM probed Fad showed to be sensitive approach to characterize surface dehydration, however, further researches are recommended regarding the validity of such approach. © 2009 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Dentin has been characterized by Marshall et al. [1] as a biologic composite of collagen matrix filled with nanometer-
∗
sized calcium-deficient, carbonate-rich apatite crystallites dispersed between parallel micron-sized hypermineralized, collagen poor, hollow cylinders (dentinal tubules containing peritubular dentin). Superficial dentin has few dentinal
Tel.: +2 2 26359765. E-mail addresses: [email protected], [email protected]. 0109-5641/$ – see front matter © 2009 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dental.2009.08.009
36
d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 35–43
tubules and composed mainly of intertubular dentin, whereas deep dentin is composed mainly of large dentinal tubules with much less intertubular dentin matrix [2]. The dentin structure and properties are changed with depth as reported by many researchers. Kinney et al. [3] reported that the intertubular dentin becomes softer as deep dentin is tested. In addition, the water content of dentin is lowest in superficial dentin compared to deep dentin [4]. However, the average bulk mineral composition may be relatively constant, as function of dentinal depth, even though the amount of collagen rich intertubular dentin gradually decreases with depth [5]. The amount of collagen fibrils per unit volume of dentin decreases from superficial dentin to deep dentin [6]. The structural integrity and mechanical properties of the collagen fibrils of acid demineralized deep and superficial dentin play an important role in the determination of bond strength and its durability [7–9]. Atomic force microscope (AFM) was used extensively, in recent years, as a tool to study the surface morphology and roughness, collagen fibrils structure, and mechanical properties of human dentin [10–13]. Atomic force microscope is not only a tool to image and measure the topography of solid surfaces at high resolution. It can also be used to measure force-versus-distance curves. Such force curves provide valuable information on local material properties such as adhesion force, elasticity, hardness and surface charge densities [14–16]. Detailed description of the techniques, interpretation and applications of force curve measurement by AFM is clarified in more specialized references [14,15,17]. However, in brief, a force vs. distance curve is a graph of the vertical force on the cantilever tip as a function of the extension of the piezoelectric scanner tube. A force curve is generated at a single location on a specimen surface by measuring how much the cantilever bends during one or more “sweeps” (up and down movements) of the scanner. Variations in the shape of force curves taken at different locations indicate variations in the local nano-scale properties of the specimen surface. The shape of the curve is also affected by contaminants and surface lubricants, as well as the water content of the surface layer of the specimen when operating an AFM in air. Pashley et al. [18] clarified the difference in the structure of the hybrid layer between superficial and deep dentin. In the first, most of hybrid layer is composed of demineralized and resin-hybridized intertubular dentin with only an occasional resin tags penetrating from the overlying adhesive layer into, relatively narrow, funnel-shaped dentinal tubules. In deep dentin the tubules are so numerous and large that little intertubular dentinal matrix is available. Consequently, resin tags represent a major fraction of bonded surfaces near the pulp. Theoretically, resin tags could contribute to resin retention if they are firmly bonded to the walls of the tubules [19–21]. In addition to resin tags, hybrid layer formation in demineralized intertubular dentin should also contribute to resin retention in proportion to the amount of etched intertubular dentin that is available for bonding in deep dentin. Therefore, the relative contribution of resin tags and hybrid layer to overall bond strength should be varied with dentin depth because the sum of the cross-sectional area of resin tags and that of the hybrid layer is constant [5,22]. Modeling experiments on the packing density of molecules within tendon collagen fibrils [23] indicate that there may
be significant space between the collagen molecules for tissue fluid as water. As long as the interfibrillar spaces, which from interconnected channels having capillary dimensions, are in hydrated state and maintaining an open-structure, pathways are provided for monomers diffusion through collagen fibrils network [21,22,24–26]. This water may be lost with dehydration, as a result of air-exposure, leading to shrinkage and collapse of the collagen fibrils network [9]. As stated by Nakabayashi [19] and others [24,27] for hybrid layer formation, intertubular dentin must be demineralized to expose the open collagen fibrils network of the dentinal matrix to create diffusion pathway for monomer infiltration. Therefore, the presence of water is crucial to the maintenance of the structure and the strength of the demineralized dentin collagen matrix and consequently higher bond strength values are obtained with wet surfaces [28–31]. The current study investigated the hypothesis that the variations in dentin depth and air-exposure time affect the performance, collagen fibrils network structure and the bonding efficacy of acid demineralized intertubular dentin. Therefore, the first aim of this study was to determine whether the structural changes of the intertubular dentin collagen fibrils network as a result of air-exposure following phosphoric acid etching differ with dentin depth and air-exposure time. The second aim was to characterize the dehydration from the surface of acid etched intertubular dentin collagen network as a function of dentin depth and air-exposure time by probing the nano-scale adhesion force between atomic force microscope (AFM) tip and intertubular dentin surface as a new experimental approach. Finally, to study the effect of the variations in the structure of the acid demineralized collagen fibrils network and surface dehydration, with the variations in dentin depth and air-exposure time, on the bond strength to dentin using an alcohol-based dentin self-priming adhesive.
2.
Materials and methods
Dentin specimens used in this study for both of AFM surface characterization and bond strength evaluation were prepared from randomly selected non-carious and non-restored mandibular third molars. All teeth were recently extracted, less than 3 months from the time of the study. All patients were of age range of 25–30 years old. All extracted teeth were stored in 0.5% chloramines T solution for 2 weeks then in distilled water at 4 ◦ C until use [8].
2.1.
Preparation of dentin specimens for AFM study
Eight teeth were used for AFM study from which eight superficial (Sd) and eight deep (Dd) dentin disks, of approximately 2 mm thickness, were prepared. From each tooth one superficial and one deep dentin disks were prepared to decrease the variation between specimens. For the preparation of the superficial dentin disks, occlusal enamel was removed perpendicular to the teeth long-axis with a diamond disk mounted to a milling machine (Nouvage AG, Fräsgerät AF 30, Switzerland) using slow-speed under water cooling until the superficial dentin surface approximately 1 mm below the DEJ was exposed. Then the superficial dentin disks, hav-
d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 35–43
ing an approximate thickness of 2 mm, were cut parallel to the exposed superficial dentin surfaces. After cutting of the superficial dentin disks, the exposed dentin surfaces, which approximately 3 mm below the DEJ, were considered as deep dentin. Then the deep dentin disks were prepared in a similar manner as in superficial dentin disks. The dentin disks were cut into two equal halves to produce 16 superficial and 16 deep dentin specimens for the AFM study. The dentin specimens were sequentially wet-grinded with 600, 800, 1000 and 1500 grit SiC polishing papers to produce relatively smooth surfaces for AFM characterization and to creates standardized smear layer. All specimens were then ultrasonically cleaned in deionized water for 15 min and then stored in distilled water at 37 ◦ C for 24 h before AFM study.
2.2.
Atomic force microscope (AFM) study
An atomic force microscope (Autoprobe CP-II, Veeco, CA, USA) was used to characterize the etched intertubular dentin collagen fibrils network structure and to probe the nano-scale adhesion force between AFM silicon nitride tip and etched intertubular dentin surface to characterize surface dehydration as a function of both of dentin depth and air-exposure time. The dentin specimens were etched for 15 s with 37% phosphoric acid gel (Ivoclar Vivadent AG, FL-9494 Schaan/Liechtenstein) and rinsed ultrasonically in distilled water for 5 min. Then the etched dentin specimens were treated by 6.5 vol.% sodium hypochlorite (NaOClaq) for 120 s [32] as a deproteinizing agent to remove non-collagenous proteins from the extracellular organic matrix and to reveal the collagen fibril network structure. Then all specimens were thoroughly rinsed in distilled water for 5 min and the excess water was removed by gentle blotting with absorbent paper leaving the dentin surface visibly moist. Immediately after surface preparation, dentin specimens were characterized by AFM.
2.2.1. Characterization of collagen fibrils network structure by tapping-mode AFM Eight specimens of each of superficial and deep dentin were used for the tapping-mode AFM study. For the tapping mode, gold-coated all-silicon cantilevers (UltraleversTM , Thermo-Microscopes, CA, USA) with integrated high aspect ratio conical tips were used. The typical radius of curvature of the scanning tip was 10 nm. The force constant and the resonance frequency of the used cantilevers were 17 N/m and 320 kHz, respectively. The Images were recorded with scan rate of 2 Hz at a scanning area of 3 m × 3 m and a resolution of 512 × 512 pixels per image. All dentin specimens were characterized in air under well-controlled laboratory environments in term of humidity (45 ± 5%) and temperature (21 ± 0.5 ◦ C). Immediately after surface preparation, each specimen was mounted to the AFM and characterized immediately at relatively hydrated state. The average time for specimen mounting and operating the AFM was approximately 3 min. Therefore, the first scan taken for each specimen was preformed approximately at 3 min (t1) of air-exposure. After the first scan at time (t1), a series of three consecutive scans were preformed at 3 min time inter-
37
vals from the end of the first scan. Therefore, each specimen, prepared either from superficial or deep dentin, was characterized at 3 (t1), 6 (t2), 9 (t3), and 12 (t4) min of air-exposure from the end dentin surface preparation steps. Accordingly, the variations in the structure of the acid demineralized intertubular dentin collagen fibrils network with variations in dentin depth and air-exposure time were characterized.
2.2.2. Probing nano-scale adhesion force (Fad ) by contact-mode AFM The remaining superficial (n = 8) and deep (n = 8) dentin specimens were used to probe the nano-scale adhesion force between AFM silicon nitride tip and etched intertubular dentin surface as a function of time of air-exposure and dentin depth. Immediately after surface preparation, superficial and deep dentin specimens were characterized by contact-mode AFM at the same environmental conditions and air-exposure time intervals (t1–t4) as previously described. AFM contact-mode was operated to characterize the forceversus-distance curve (force curve) between the AFM tip and specimen surface. Silicon nitride tips, of 50 nm nominal radii, mounted to contact-mode cantilevers (Type D MicroleversTM , Thermo-Microscopes, CA, USA) were used to scan specimens surfaces. The force constant and the resonance frequency of the used cantilevers were 2.1 N/m and 160 kHz, respectively. For each specimen, surface scan of 3 m × 3 m area was done at a scan rate of 2 Hz and then a set of force curves were measured for selected 14 points at the intertubular dentin. The same process was repeated for each specimen at each previously described time intervals such that a set of force curves as a function of time of air-exposure (t1–t4) and dentin depth (Sd and Dd) were recorded. The force curves typically show the deflection of the free end of the AFM cantilever as the fixed end of the cantilever is brought vertically towards and then away from the specimen surface. The deflection of the free end of the cantilever is measured and plotted at many points as the z-axis scanner extends the cantilever towards the surface and then retract it again. Then the nano-scale surface adhesion force (Fad ) between the AFM tip and intertubular surface was calculated in nano-Newton (nN) from the mean force curve of each specimen at each air-exposure time interval. The adhesion force (Fad ) was calculated from the difference between the snapin point (the point of contact between the cantilever tip and specimen surface) and the snap-out point (the point of separation or detachment between the cantilever tip and specimen surface) [14,17].
2.3.
Tensile bond strength (TBS) testing
Dentin/resin-based restorative composite specimens were prepared from freshly extracted mandibular third molars for the tensile bond strength (TBS) testing. The roots of the extracted molars were embedded in self-polymerized resin 3 mm below the cervical line. The occlusal enamel was removed using diamond disk mounted to a milling machine as previously described in the preparation of the specimens for the AFM study under copious water spray to expose flat dentin surface. Half of the teeth were prepared to expose the superficial dentin surfaces, while in the other half the deep
38
d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 35–43
dentin surfaces were exposed. The smear layer was standardized as described previously. The exposed dentin surfaces were etched for 15 s with 37% phosphoric acid gel, thoroughly rinsed in distilled water for 5 min and the excess water was removed by gentle blotting with absorbent paper leaving the dentin surface visibly moist. A single bottle alcohol-based self-priming adhesive (Excite, Ivoclar Vivadent AG, FL-9494 Schaan/Liechtenstein) was applied to the conditioned dentin surfaces. One coat of Excite adhesive was applied over the etched dentin surface and gently agitated for 10 s; excess solvent was removed using a gentle stream of air for 3 s. The Excite was then light-cured for 20 s using a light-curing unit (Cromalux-E, Meca-Physik Dental Division, D-76437 Rastatt, Germany) with a light output of 600 mW/cm2 . According to the dentin depth and the time of application of the self-priming adhesive (airexposure time), teeth were divided into the following test groups to simulate what was done in AFM study. Superficial dentin (Sd): Sd(t1), Sd(t2), Sd(t3), and Sd(t4). Deep dentin (Dd): Dd(t1), Dd(t2), Dd(t3), and Dd(t4). After the application of the self-priming adhesive at the desired air-exposure times (t1–t4), a light cured resin-based restorative composite material (Tetric Ceram, Ivoclar Vivadent AG, FL-9494 Schaan/Liechtenstein) was applied and cured in four increments for each tooth. The restored teeth were then stored in distilled water at 37 ◦ C for 48 h to complete the curing process. After storage, each restored tooth was sectioned with diamond disk fixed to a milling machine under copious water spray to prepare dentin/composite slabs of 1 mm thickness from the central part of the tooth. A fine high-speed diamond stone with air/water spray was used to trim the slabs into an hour-glass shape with cross-sectional area of approximately 1 mm2 at the bonded interface which checked and confirmed by a digital caliper. Twelve (n = 12) hour-glass shape slabs were tested for each group. All slabs were stored in distilled water at 37 ◦ C for 24 h before mechanical testing. Each slab was fixed to two disposable acrylic resin jigs using cyanoacrylate adhesive. The acrylic jigs were mounted to a universal testing machine (Lloyd Instruments, LR5 series, UK). The test was run at a cross-head speed of 0.5 mm/min until failure. The cross-sectional area of each fractured slab was confirmed using a digital caliper and the respective load was divided over the cross-sectional area to calculate the TBS in MPa.
2.4.
Statistical analysis
All data of nano-scale adhesion force (Fad ) and tensile bond strength (TBS) were expressed as means and standard deviations (SD). Statistical analysis was carried out using SPSS program (Release 15, 2006). Two-way ANOVA was preformed to test the effect of dentin depth, air-exposure time and their interaction on either of Fad and TBS. Tukey–Kramer multiple-comparison post hoc test was used to compare each of Fad and TBS between the tested groups. The correlation between Fad and TBS through the different air-exposure times (t1–t4) was tested using Pearson’s correlation coefficient. P-values less than 0.05 were considered statistically significant.
3.
Results
AFM images revealed a demineralized open collagen fibrils network structure with numerous interconnected interfibrillar spaces and porosities in both of superficial (Fig. 1A and B) and deep (Fig. 1E and F) intertubular dentin at 3(t1) and 6(t2) min of air-exposure. However, at 9(t3) min of air-exposure, superficial intertubular dentin collagen fibrils network showed more collapsed structure (Fig. 1C) compared to deep dentin that partially preserved the open fibrils network structure (Fig. 1G). Total collapsed structure of the collagen fibrils network was shown at 12(t4) min of air-exposure in both of superficial and deep dentin specimens (Fig. 1D and H). Fig. 2A–C shows selected cantilever-specimen surface interactions (force curves) between AFM tip and etched deep intertubular dentin at 3(t1) min and 9(t3) min; and etched superficial intertubular dentin at 9(t3) min of air-exposure, respectively. The force is represented on the vertical axis in nN and the AFM piezoelectric scanner z-position is represented on the horizontal axis in nm. The scanner extension (approach) path is demonstrated by the solid line. While the scanner retraction path, is demonstrated by the dashed line. The point of contact between the tip and specimen surface is represented by the snap-in point. The point of separation or detachment between the cantilever tip and specimen surface is represented by the snap-out point. The nano-scale adhesion force (Fad ) is represented by the difference between the snap-in and snap-out points (black arrow). Two-way ANOVA (Tables 1 and 2) showed the significant effect (P ≤ 0.001) of either of dentin depth and air-exposure time on AFM-probed adhesion force (Fad ) and tensile bond strength (TBS). In addition, a significant interaction was found between the two independent variables (dentin depth and airexposure time) on either of Fad (P = 0.015) and TBS (P = 0.002). The variations of the mean values of Fad and TBS with air-exposure time for both of superficial and deep dentin specimens are showing Figs. 3 and 4, respectively. For each of superficial (Sd) and deep dentin (Dd), the values of both of Fad (Table 3) and TBS (Table 4) are significantly decreased with the increase in the air-exposure time from t1 to t4. In addition, Pearson’s correlation analysis revealed a significant positive correlation (P < 0.001) between Fad and TBS within each of superficial and deep dentin. No significant difference was found in Fad between superficial and deep dentin at 3(t1) and 6(t2) min of air-exposure (Table 3). However, at the same air-exposure times (t1 and t2), superficial dentin shows significantly higher TBS (Table 4). In contrast, at 9(t3) and 12(t4) min of air-exposure, deep dentin showed significant increase in Fad (Table 3), however, no significant difference in TBS was found between superficial and deep dentin (Table 4).
4.
Discussion
Atomic force microscope (AFM) was selectively used in this study to characterize the structure of acid demineralized collagen fibrils network because it has an advantage over the ultra-high vacuum measuring equipments, such as SEM, as specimens were imaged in air at humidity of 45 ± 5% and tem-
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Fig. 1 – Tapping-mode AFM images showing the structure of the acid demineralized dentin collagen fibrils network at the different air-exposure times of both of superficial (A–D) and deep (E–H) dentin specimens. Open collagen network structure with numerous interconnected interfibrillar spaces and porosities are observed at 3 and 6 min of air-exposure for both of superficial (A and B) and deep (E and F) dentin. At 9 min of air-exposure superficial dentin shows collapsed structure (C), however, deep dentin specimens partially preserved the open network structure (G). Total collapsed network structure is observed at 12 min of air-exposure for both of superficial (D) and deep (H) dentin. Arrows point out collagen fibrils and open interfibrils spaces.
Table 1 – Two-way ANOVA for the effect of dentin depth, air-exposure time and their interaction on the AFM probed nano-scale adhesion force (Fad ). Source
Type III sum of squares
Dentin-depth Air-exposure time Dentin-depth × air-exposure time
3.238 60.878 0.516
d.f.
Mean square
1 3 3
3.238 20.293 0.172
F
Sig.
56.527 354.303 3.002
0.000 0.000 0.015
R Squared = 0.972 (adjusted R Squared = 0.966).
Table 2 – Two-way ANOVA for the effect of dentin depth, air-exposure time and their interaction on the tensile bond strength (TBS). Source
Type III sum of squares
Dentin depth Air-exposure time Air-exposure time × dentin depth
19.182 1064.487 25.327
d.f.
Mean square 1 3 3
19.182 354.829 8.442
F
Sig.
14.338 265.218 6.310
0.001 0.000 0.002
R Squared = 0.963 (adjusted R Squared = 0.955).
Table 3 – Nano-scale adhesion force (Fad ) of superficial (Sd) and deep (Dd) dentin at the different air-exposure times (t1–t4). Sd Dd
t1
t2
t3
t4
4.77 (0.26)aA 5.23 (0.30)aA
3.53 (0.24)bA 3.92 (0.27)bA
2.05 (0.10)cA 2.99 (0.13)cB
1.47 (0.35)dA 2.11 (0.12)dB
Mean values with dissimilar small letters within each row and capital letters within each column are statistically significant different at P ≤ 0.05.
Table 4 – Tensile bond strength (TBS) to superficial (Sd) and deep (Dd) dentin at the different air-exposure times (t1–t4).
Sd Dd
t1
t2
t3
t4
27.40 (4.76)aA 22.34 (5.32)aB
19.88 (5.58)bA 17.43 (3.94)bB
14.68 (4.38)cA 14.12 (3.51)cA
11.02 (3.62)dA 10.15 (2.03)dA
Mean values with dissimilar small letters within each row and capital letters within each column are statistically significant different at P ≤ 0.05.
40
d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 35–43
Fig. 3 – Showing the variations in the mean values of nano-scale adhesion force (Fad ) with the time of air-exposure of both of superficial and deep dentin specimens.
Fig. 2 – Selected force curves showing the interaction between AFM tip and etched deep intertubular dentin at 3 min (A), etched deep intertubular dentin at 9 min (B), and etched superficial intertubular dentin at 9 min (C) of air-exposure. The AFM scanner approach and retraction paths are represented by the solid and the dashed lines, respectively. The nano-scale adhesion force (Fad ) in nN is represented by the black arrow between the snap-in and snap-out points.
perature of 21 ± 0.5 ◦ C. Therefore, by such way the dehydration of the dentin specimens as a result of air-exposure could be minimized and relatively standardized. It was reported by El Feninat et al. [9] that considerable water loss and observable structure change occur within few minutes to etched dentin collagen fibrils network when characterized by AFM in air. The progressive dehydration effect by increasing the time of air-exposure (from 3 to 12 min) on the structure of the intertubular dentin collagen fibrils network is demonstrated by the consecutive AFM images for
both of superficial (Fig. 1A–D) and deep (Fig. 1E–H) dentin specimens. These structural changes associated with airdehydration effect are in agreement with previous findings reported that the presence of water is essential to prevent the collapse of the demineralized dentin collagen network [33,34]. However, from the AFM images (Fig. 1), the integrity of the structure of the demineralized intertubular dentin collagen fibrils network of deep dentin seems to be less affected by the air-dehydration process. This could be attributed to the higher water content in deep dentin compared to superficial dentin which might be responsible for the higher structural resistance to collapse by increasing the time of air-dehydration [4]. The phenomena of shrinkage and collapse of the demineralized soft collagen fibrils network by air dehydration could be clarified by more than one explanation. The demineralized collagen network could be assumed to be suspended in water that occupies the spaces that were previously occupied by apatite crystals between collagen fibrils. Therefore, by water loss due to air dehydration, collagen fibrils become more close together resulting in passive collapse [5]. Alternatively, at molecular level, the intermolecular spaces between collagen molecules are preserved by water that surrounds the triple helices of collagen molecule. Consequently, hydrogen bonds are formed preventing direct inter-chain contact and
Fig. 4 – Showing the variations in the mean values of tensile bond strength (TBS) with the time of air-exposure of both of superficial and deep dentin specimens.
d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 35–43
collapse [35–37]. Furthermore, the surface tension force acting on air-collagen fibrils interface [38], and the shortening of the interconnected collagen fibrils [39] associated with water loss are among the possible explanations. Therefore, we could assume that the dehydration from the surface of the acid demineralized intertubular collagen fibrils network as a result of air-exposure is lower in deep dentin than in superficial dentin. The results of the nano-scale adhesion force (Fad ) are in agreement with the previous assumption (Table 3). The values of the Fad was significantly higher for deep dentin specimens than for superficial dentin specimens at 9(t3) and 12(t4) min of air-exposure. In addition, the results of the two-way (Table 1) and one-way (Table 3) ANOVA and the AFM images (Fig. 1) partially prove the sensitivity of the AFM probed nano-scale adhesion force (Fad ) as an approach to characterize the dehydration from the surface of the demineralized intertubular dentin collagen fibrils network. Fig. 3 shows simultaneous decrease in the mean values of Fad , in both of superficial and deep dentin specimens, with the increase in the time of air dehydration. When retracting the AFM tip from the dentin surface, the tip stays in contact with the surface until the cantilever force overcomes the adhesion force between the tip and specimen surface. In the most general case the adhesion force (Fad ) is a combination of the electrostatic force, the van der Waals force, the meniscus or capillary force, and forces due to chemical bonds or acid–base interactions [14,40,41]. At ambient conditions, a water neck forms between AFM tip and specimen surface due to capillary condensation and adsorption of thin water films at surfaces leading to the formation of capillary attractive forces. This capillary attractive interaction depends on the relative humidity and the hydrophilicity of tip and sample surface. Therefore, capillary forces are expected to be maximal for hydrophilic surfaces and to vanish for very hydrophobic surfaces [14]. The relative contributions of capillary, van der Waals and electrostatic force under ambient conditions for the adhesion of an AFM tip to several materials surfaces were previously studied. In all cases, the capillary force was found to give the largest contribution [14,42]. Therefore, in this study, every effort was done to standardize the experimental conditions during probing the nano-scale adhesion force (Fad ) in order to dominate the effect of the capillary attractive force and to standardize the contribution of other forces in changing the values of the probed Fad . This is done through using of identical silicon nitride AFM tips without any chemical modification. In addition, AFM characterization was done at the same well-controlled experimental conditions in term of temperature, humidity and air-exposure time. Furthermore, through the using of randomly divided specimens prepared from extracted non-carious and nonrestored mandibular third molars of the same age range, extraction period and storage conditions. Consequently, we could suggest that the variations in the values of the probed Fad are mainly attributed to the dehydration from the surface layer of the demineralized intertubular dentin collagen fibrils network. The significant decrease in the TBS with increase in the time of air-exposure from 3(t1) to 12(t4) min within both of superficial and deep dentin specimens recorded in the current
41
study (Table 4) and (Fig. 4) could be explained by increasing the time of air-exposure the demineralized soft collagen fibrils network suffers from dehydration effect leading structural collapse which might interfered with the infiltration of the Excite monomers to form hybrid layer. It was found in previous studies that, for successful bonding to dentin, proper resin infiltration of the demineralized intertubular dentin collagen fibrils network to form hybrid layer is required. This is achieved by the creation of open collagen fibrils network structure with water-filled interfibril spaces [5,19,22,24]. As Excite is an alcohol-based dentin self-priming adhesive, open collagen fibrils network structure in hydrated condition should be available for proper monomers infiltration and hybrid layer formation. This is in agreement with other previous studies which recommended to not dry etched dentin surface before the application of acetone or alcohol-based bonding primers to achieve higher bond strength values [28,29,38,43,44]. This previous explanation is in consistence with and support the finding of the nano-scale adhesion force (Fad ) (Table 3) and (Fig. 3); as with increase in the time of air-exposure, within both of superficial and deep dentin specimens, there are a significant decrease in the value of the recorded Fad . Therefore, the assumed sensitivity of the nano-scale adhesion force (Fad ) to detect surface dehydration from the demineralized intertubular dentin collagen fibrils network is further supported. In addition, Pearson’s correlation confirmed the positive correlation between Fad and TBS. The significantly higher TBS (Table 4) for superficial dentin specimens over deep dentin specimens recorded at 3(t1) and 6(t2) min of air-exposure could be explained by the following. Bonding to dentin is mainly depends on the amount of intertubular dentin matrix available to be infiltrated with the resin monomers to form hybrid layer [5,18]. However, resin tags could contribute, but to less extant, in the bonding to dentin if they are firmly bonded to dentinal tubules walls [18–21]. Superficial dentin has an advantage over deep dentin as more intertubular dentin matrix is available in superficial dentin for hybrid layer formation [2]. Therefore, provided all other variables are standardized, the higher bond strength values recorded for superficial dentin specimens over deep dentin specimens are justified. However, as previously mentioned, the demineralized collagen fibrils network of the intertubular dentin should be in hydrated state to preserve the open collagen fibrils structure for the Excite monomers infiltration and hybrid layer formation. In this respect, based on AFM images (Fig. 1) and the results of the nano-scale adhesion force (Fad ) (Table 3) and (Fig. 3), deep dentin might has an advantage over superficial dentin in resisting surface dehydration and partially preserving the integrity of intertubular dentin open collagen fibrils network structure with the increase in air-exposure time. This could explain the non-significant difference in TBS at more prolonged time of air-exposure (t3 and t4) between deep and superficial dentin specimens (Table 4). It should be mentioned that adhesion force (Fad ) measurement by AFM is affected with many variables which should be taken in consideration in any future research. Capillary forces, which contribute in the overall value of the Fad between AFM and specimen surface, are important factor in force measurement taken in ambient air, but not in aqueous environment or in high vacuum [17]. Variables related to AFM tip geometry,
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d e n t a l m a t e r i a l s 2 6 ( 2 0 1 0 ) 35–43
cantilever force constant and the tip surface condition such as water content should also be critically considered [14]. In addition, the dentin specimen surface roughness and heterogeneity should be taken in account. Furthermore, studying the interactions between chemically modified AFM tip and specific functional group(s) in dentinal substrate is recommended in future research.
5.
Conclusions
Based on the results of this study, the ability of the demineralized intertubular dentin soft collagen fibrils network to resist air-dehydration and to preserve the integrity of open network structure with the increase in air-exposure time is increased with dentin depth. Although superficial dentin achieves higher bond strength values than deep dentin with an alcohol-based self-priming adhesive (Excite), this difference in the bond strength between superficial and deep dentin is decreased by increasing the time of air-exposure before the application of the dentin self-priming adhesive to the demineralized dentin. The AFM probed nano-scale adhesion force showed to be sensitive approach to characterize surface dehydration, resulted from air-exposure, from the demineralized intertubular dentin collagen fibrils network. The value of the nano-scale adhesion force is decreased with increasing the time of air-exposure and is increased with dentin depth at the same time of air-exposure. However, further researches regarding the validity of such approach and the quantitative correlation between the degree of water loss as result of airdehydration from the demineralized collagen fibrils network and the nano-scale adhesion force are recommended.
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