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Probing nano-scale adhesion force between AFM and acid demineralized intertubular dentin: Moist versus dry dentin
journal of dentistry 37 (2009) 963–969
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Probing nano-scale adhesion force between AFM and acid demineralized intertubular dentin: Moist versus dry dentin Amr S. Fawzy a,*, Ahmed M. Farghaly b a b
Dental Biomaterials Department, Faculty of Dentistry, Ain Shams University, Cairo, Egypt Operative Dentistry Department, Faculty of Dentistry, El-Azhar University, Cairo, Egypt
article info
abstract
Article history:
Objective: The aim of this study was to investigate the effect of dentin dryness on the
Received 14 April 2009
variation in the probed nano-scale adhesion force between an AFM silicon nitride tip and
Received in revised form
demineralized intertubular dentin collagen fibrils network surface.
2 August 2009
Methods: Dentin specimens were etched with 37% phosphoric acid for 15 s and then divided
Accepted 6 August 2009
into three groups. Specimens of the first two groups were air-dried for 5 or 10 s (DH5s and DH10s), respectively, whereas specimens of the third group were left in the hydrated condition (H). For each group, Force curves were characterized by contact-mode AFM
Keywords:
and the adhesion force (Fad) was calculated. The structure of the demineralized collagen
AFM
fibrils network was characterized by tapping mode AFM. The tensile bond strength (TBS) to
Adhesion force
dentin was evaluated using one alcohol-based dentin self-priming adhesive. The dentin/
Intertubular dentin
resin interface was investigated by SEM.
Collagen fibrils
Results: Dentin specimens in the wet-hydrated condition (H) showed significantly higher adhesion force and TBS values than dry-dehydrated specimens (DH5s and DH10s). AFM images showed open collagen fibrils network structure in wet-hydrated specimens (H), while the dry-dehydrated specimens (DH5s and DH10s) showed a collapsed appearance to varying degrees. SEM images revealed minimum resin infiltration in dry-dehydrated specimens. Significance: The nano-scale adhesion force between the AFM probe and demineralized intertubular dentin surface was shown to be sensitive to surface air-drying. The decrease in the nano-scale adhesion force with the increase in the time of air-dryness is related to dehydration of the demineralized collagen fibrils network surface. # 2009 Elsevier Ltd. All rights reserved.
1.
Introduction
Recent modeling experiments on the packing density of molecules within tendon collagen fibrils1 indicate that there may be significant space between the collagen molecules for tissue fluid. This water may be lost with dehydration and subsequent shrinkage of the collagen fibrils. As stated by Nakabayashi2 and others3,4 for hybrid layer formation, intertubular dentin must be demineralized to expose the open
collagen fibrils network of the dentinal matrix to create diffusion pathways for monomer infiltration. As long as the interfibrillar spaces, which form interconnected channels having 15–20 nm dimensions, are in a hydrated state, waterfilled pathways are provided for monomers diffusion through collagen fibrils network.3,5–8 In addition, 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.9–11
* Corresponding author. Tel.: +20 02 26359765; fax: +20 02 26382906. E-mail addresses: [email protected], [email protected] (A.S. Fawzy). 0300-5712/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2009.08.005
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Therefore, the precise characterization of the degree of surface hydration of demineralized intertubular dentin collagen fibrils network is essential to evaluate its monomer permeability.12 Atomic force microscope (AFM) is considered as an essential tool in almost every research dealing with the characterization or engineering of materials, including biomaterials, surfaces and interfaces in nanometer scale.13 AFM 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 vs. distance curves. Such curves, briefly called 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 provided in more specialized references.13–15 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. The vertical force on the cantilever tip is proportional to the cantilever bending, which is measured using a position sensitive photo-detector. 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. When retracting the AFM tip from the sample surface, the tip stays in contact with the surface until the cantilever force overcomes the adhesive tip–sample interaction. The first measurements of this pull-out force or adhesion force were performed by Martin et al.17 and Erlandsson et al.18. In the most general case, this adhesion force 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. At ambient conditions, a water neck forms between AFM tip and substrate due to capillary condensation and adsorption of thin water films at surfaces leading to the formation of capillary attractive forces.14 This capillary attractive interaction depends on the relative humidity and the hydrophilicity of tip and sample surface. Depending on the chemical end-groups present on tip and substrate, chemical bonds may form during contact or other specific chemical interactions may occur. The relative contributions of capillary, van der Waals and electrostatic force under
ambient conditions for the adhesion of an AFM tip to graphite, mica and MoS2 were elucidated by Ouyang et al.19 In all cases, the capillary force was found to give the largest contribution. Capillary forces are expected to be maximal for hydrophilic surfaces and to vanish for very hydrophobic surfaces. The aim of the study was to test the null hypothesis that the state of surface hydration of acid etched intertubular dentin has no effect on the nano-scale adhesion force between an AFM probe tip and the dentinal substrate. Complementary to adhesion force measurement, the variation in the structure of the demineralized intertubular dentin collagen fibril network, tensile bond strength and dentin/resin interface with dentin dryness were also evaluated.
2.
Materials and methods
Dentin specimens used in this study for both of AFM characterization and TBS testing were prepared from randomly selected non-carious third molars. All teeth were recently extracted. All patients were of age range of 21–25 years old. All extracted teeth were stored in 0.5% chloramines T solution for 2 weeks then in distilled water at 4 8C until use.
2.1.
Dentin surface preparation for AFM study
Fifteen teeth were used for AFM characterization from which 15 dentin discs, of approximately 2 mm thickness, were prepared. For the preparation of the dentin discs, occlusal enamel was removed perpendicular to the teeth long-axis with a diamond disc mounted to a milling machine using slow-speed under water cooling until the dentin surface, 1.5 mm below the DEJ, was exposed. Then dentin discs having an approximate thickness of 2 mm were cut parallel to the prepared dentin surfaces. The smear layer on the cut dentin discs were made progressively thinner by sequentially wetgrinded with 600, 800, 1000 and 1500 grit SiC polishing papers under water cooling. All prepared discs were then ultrasonically cleaned in distilled water for 15 min. Each dentin disc was divided into two equal halves and the halves were randomly divided equally into three groups to minimize the variability between the dentin discs. Each group was formed of 10 dentin discs halves (specimens). All dentin specimens were etched for 15 s with 37% phosphoric acid gel (Ivoclar/Vivadent AG, FL-9494 Schaan/Liechtenstein). According to the subsequent dentin surface preparation steps, specimens were divided into the following groups shown in Table 1.
Table 1 – Dentin specimens surface preparation. Dentin surface preparation steps Etched dentin was rinsed ultrasonically in distilled water for 10 min, treated by 6.5 vol.% sodium hypochlorite (NaOClaq) for 120 s,20 thoroughly rinsed in distilled water for 5 min and finally air-dried for 10 s by oil-free compressed air. Etched dentin was prepared as previously described except that the dentin surface was air-dried for 5 s by oil-free compressed air. Etched dentin was prepared as previously described except no air-dryness was done and the excess water was removed by gentle blotting with absorbent paper leaving the dentin surface visibly moist.
Surface condition
Symbols
Dried for 10 s
DH10s
Dried for 5 s
DH5s
Wet
H
journal of dentistry 37 (2009) 963–969
2.2. AFM characterization of dentin collagen fibrils network Three specimens from each group were used for AFM characterization of dentin collagen fibrils network structure. Tapping mode characterization was performed with an AFM (Autoprobe CP-II, Veeco, CA, USA). The tapping mode operates by scanning a tip, attached to the end of a vibrating cantilever, across the sample surface, so that it is in intermittent contact with the surface. The cantilever amplitude is maintained constant by altering the vertical position of the piezoelectric scanner. For the tapping mode, a gold-coated all-silicon cantilever (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. All specimens were characterized in air at a relative humidity of 45 5% and temperature of 37 0.5 8C immediately after dentin surface preparation. The Images were recorded with scan rate of 0.5– 1.0 Hz and a resolution of 512 512 pixels per image was chosen.
2.3.
Probing nano-scale adhesion force (Fad)
For the characterization of the force vs. distance curve, a contact AFM mode was preformed to scan the etched dentin surfaces using silicon nitride tips, with nominal radii of 50 nm, mounted to cantilevers (Type D MicroleversTM, Thermo-Microscopes, CA, USA). The force constant and the resonance frequency of the used cantilevers were 2.1 N/m and 160 kHz, respectively. Dentin surface scans of 10 mm 10 mm areas were first done at a scan rate of 4 kHz and then a set of force curves were measured for 14 selected points on the intertubular dentin for each specimen. 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 sample 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 surface adhesion force (Fad) between the cantilever tip and specimen surface was calculated in nano-Newton (nN) from the mean force curve of each specimen. The adhesion force (Fad) was calculated from the difference between the snap-in 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).13,14 All dentin specimens were tested for adhesion force measurement, immediately after surface preparation in a well controlled environments in term of humidity (45 5%) and temperature (37 0.5 8C) as previously described. Seven dentin specimens from each group were tested for force curve and adhesion force measurement.
2.4.
Tensile bond strength (TBS) evaluation
For TBS evaluation, 30 dentin/resin-based composite slabs were prepared from third molar teeth (10 slabs for each group). The roots of the extracted molars were embedded in self-polymer-
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ized acrylic resin 3 mm below the cervical line. The occlusal enamel was removed using a low speed diamond disc in a straight handpiece fixed to a milling machine under copious water spray to expose flat dentin surfaces 1.5 mm below the DEJ. The smear layer was prepared and standardized as previously described for specimens used for AFM characterization. The exposed dentin surfaces of all teeth were acid etched and the etched dentin surfaces were prepared to be in the dry (DH10s or DH5s) and the wet (H) conditions as described in Table 1. Single bottle alcohol-based self-priming adhesive (Excite, Ivoclar/ Vivadent AG, FL-9494 Schaan/Liechtenstein) was used to bond composite restorative material to etched dentin surfaces for all groups. 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, MecaPhysik Dental Division, D-76437 Rastatt, Germany) with a light output of 600 mW/cm2. A direct restorative composite resin material (Tetric Ceram, Ivoclar/Vivadent AG, FL-9494 Schaan/ Liechtenstein) was applied in four increments for each tooth until composite crowns of 6 mm height were built. The thickness of each increment was about 1.5 mm and each increment was light-cured for 40 s. The restored teeth were then stored for in distilled water at 37 8C for 24 h to complete the polymerization process before sectioning. Dentin/composite slabs were prepared from the central region of each composite restored tooth. The thickness of each slab was approximately 1 mm. The slabs were prepared using low speed diamond disc mounted to the milling machine under copious water spray. The teeth were vertically sectioned in a bucco-lingual direction. A fine high speed diamond bur with air/ water spray was used for trimming the slabs into an hour glass shape with cross-sectional area of approximately 1 0.05 mm2 at the bonded interface. The slabs were checked for premature debonding which could be resulted during preparation. All slabs were stored in distilled water for 24 h before mechanical testing. Each dentin/composite slab was fixed to two disposable acrylic resin jigs using cyanoacrylate adhesive. Two rounded L-shaped metallic pins 2 mm in diameter were attached to the upper and lower jaws of a universal testing machine (Lloyd Instrument LR5 series, UK). The dentin/composite slab-jig assembly was mounted to the testing machine through the openings created at both ends of the acrylic jigs. The TBS 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.
2.5.
Evaluation of dentin/resin interface by SEM
Dentin/resin-based composite slabs were prepared as previously described for the TBS testing to evaluate the dentin/ resin interface. Each slab was polished using 600, 800 and 1000 grit silicon carbide paper discs to produce smooth polished surfaces, etched with 37% phosphoric acid gel for 15 s, then rinsed for 15 s and dried with compressed oil-free air. The slabs were immersed in 5.25% sodium hypochlorite solution for 20 min, washed thoroughly under running water for 5 min, then immersed in ascending concentrations of ethanol, 50%, 70%, 90% for 20 min each and 100% for 1 h. Then
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Fig. 1 – Tapping mode AFM images of etched intertubular dentin surface showing (A) collapsed collagen fibrils network with no interfibrillar spaces in air-dried specimens (DH10) for 10 s, (B) partially collapsed collagen fibrils network in air-dried specimens (DH5) for 5 s, and (C) open collagen fibrils network structure with numerous interconnected interfibrillar spaces in hydrated specimens (H).
the slabs were gold sputtered and were examined using SEM (JEOL JXA-840A, Electron Probe Micro-analyzer, Japan), operated at 30 kV. The dentin/resin interface of the three groups (DH10s, DH5s and H) was investigated.
2.6.
Statistical analysis
All data of adhesion force (Fad) in nN and TBS in MPa was expressed as mean standard deviation (SD) and was analyzed by WinSTAT v. 2007.1. One-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple-comparison post hoc test was used for the comparison between the different tested groups. The correlation between Fad and TBS was tested using Pearson’s correlation coefficient. P < 0.05 was considered significant.
3.
Results
The structure of the collagen fibrils network of the etched intertubular dentin at the dry-dehydrated conditions (DH10s
and DH5s) and the wet-hydrated (H) condition are shown in Fig. 1(A–C), respectively. The etched dentin surface revealed a demineralized open collagen fibril network structure with numerous interconnected interfibrillar spaces and porosities in the wet-hydrated (H) specimens when scanned immediately by AFM after dentin surface preparation (Fig. 1C). Specimens dried with air for 10 s (DH10s) showed totally collapsed collagen fibrils network with no interfibrillar spaces (Fig. 1A). However, specimens dried with air for 5 s (DH5s) showed less collapsed appearance and more open structure of the collagen fibrils network (Fig. 1B) compared to specimens dried with air for 10 s (DH10s). Fig. 2 shows a selected cantilever–specimen interaction, force curve, of etched intertubular dentin at the wet-hydrated condition (H). The force is represented on the vertical axis in nN and the piezoelectric scanner z-position is represented on the horizontal axis in nm. The scanner extension (approach) path is demonstrated by the solid line, whiles 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 adhesive force (black arrow) is represented by the difference between the snap-in and snap-out points. Table 2 shows the results of the adhesion force (Fad) and the tensile bond strength (TBS) of the three groups used in the study. The etched dentin specimens in the wet-hydrated condition (H) showed significantly higher (P < 0.001) adhesion force and TBS values than etched air-dried dentin specimens for both of 10 and 5 s (DH10s and DH5s). In addition, the
Table 2 – Adhesion force (Fad) and tensile bond strength (TBS) at dry (DH10s and DH5s) and wet (H) conditions.
Fig. 2 – Selective force curve of etched intertubular dentin surface in the wet-hydrated condition (H). The AFM scanner approach and retraction paths are represented by the solid and the dashed lines, respectively. The adhesion force (Fad) in nN is represented by the black arrow between the snap-in and snap-out points.
Adhesion force (nN) Tensile bond strength (MPa)
DH10s
DH5s
H
1.79 0.23a
2.77 0.29b
7.98 0.74c
4.91 0.97a
5.39 0.89a
17.51 1.38b
Mean values with dissimilar letters in the same row are statistically significant different at P < 0.05.
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Fig. 3 – SEM images of dentin/resin interface showing limited resin infiltration (hybrid layer), resin tags formation and separation at the bonded interface of air-dried dentin specimens for 10 s (A) and 5 s (B) compared to specimens left in the wet-hydrated condition (C).
adhesion force was significantly higher (P < 0.01) for etched air-dried dentin specimens for 5 s (DH5s) than for etched airdried dentin specimens for 10 s (DH10s). In contrast, no significant difference in TBS was found between etched airdried specimens for both of 10 and 5 s (DH10s and DH5s). Pearson’s correlation analysis showed a significant positive correlation (P < 0.001) between Fad and TBS. SEM investigation (Fig. 3A and B) showed limited resin infiltration of the demineralized dentin (hybrid layer formation), very few small-sized resin tags formation and separation at the bonded interface of the dry-dehydrated specimens for 10 and 5 s (DH10s and DH5s) compared to wet-hydrated specimens (Fig. 3C).
4.
Discussion
Atomic force microscopy5,21–24 was used in this study to characterize the structure of acid demineralized collagen fibrils network because it has an advantage over measuring devices requiring high vacuums, as the specimens were imaged in ambient air at humidity and temperature of 45 5% and 37 0.5 8C, respectively. As previously mentioned, the value of the adhesion force between an AFM tip and specimen surface depends on the electrostatic force, the van der Waals force, the meniscus or capillary force, and the formation of chemical bonds.14 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 adhesion force, identical silicon nitride AFM tips of the same type were used in this study, randomly, without any chemical modification to characterize the force curve of the etched intertubular dentin specimens under the same well controlled environmental conditions. In addition, dentin specimens used in this study were prepared randomly from non-carious third molars of nearly the same age range, extraction period and storage conditions. El Feninat et al.21 reported that considerable water loss and observable structure change occur within few minutes to etched dentin surface when characterized by AFM in air. Therefore, in this study the etched dentin specimens was imaged immediately after specimens surface preparation by AFM to study the structure of the demineralized intertubular dentin collagen fibrils network and to characterize the nanoscale adhesion force (Fad) at the dry-dehydrated and the wethydrated conditions. However, water dehydration is expected during the time required for specimens mounting, image acquisition and force curve measurement by AFM in air which was approximately equal to 3 min for each investigated specimen which could significantly affect the structure of the collagen network and the results of force curve measurement. It should be clarified that, AFM characterization of both of collagen fibril network and nano-scale adhesion force (Fad) for wet specimens (H) were done in air not under water to dominate the effect of the capillary attractive force in determining the value of the non-scale adhesion force. It
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was reported previously, that the capillary attractive force is important factor in force–curve measurement by AFM taken in ambient air, but not in aqueous environment.13 The open collagen fibrils network structure with the numerous interfibrillar spaces of the demineralized intertubular dentin in the wet-hydrated condition (H) compared to the dry-dehydrated (DH10s and DH5s) conditions (Fig. 1A–C) support the results of the adhesion force (Fad) measurement (Table 2). The significant decrease in the value of (Fad) could be attributed to the water evaporation from the demineralized dentin collagen network surface by air-drying. This is in agreement with previous findings,25,26 that the presence of water is essential to prevent the structural collapse of the demineralized dentin collagen network. The phenomena of shrinkage and collapse of the demineralized soft collagen fibrils network by air-drying could be due to several mechanisms. The demineralized collagen network could be assumed to be suspended in water that occupies the spaces that were previously occupied by apatite crystals between the collagen fibrils. By water loss due to air-drying, collagen fibrils could move close together resulting in passive collapse of the soft collagen network.27 Alternatively the structural stability of the demineralized collagen fibrils network could be explained at molecular level such that the intermolecular spaces between collagen molecules are preserved by water that surrounds the triple helices of collagen molecule. Owing to the polar hydrogen bonding characteristics of water molecules, hydrogen bonds are formed preventing direct inter-chain contact and collapse.28–30 Furthermore, the surface tension force acting on air–collagen fibrils interface,31 and the shortening of the interconnected collagen fibrils32 associated with water loss are among the possible explanations of the shrinkage and collapse of the demineralized collagen network by air. Within the results of this study, the high values of adhesion force (Fad) measured between AFM tip and specimen surface could be related to the increase in the water content in the outer most region of the demineralized collagen fibrils network. The increase in the adhesion force may be attributed to the increase in capillary attractive force which depends, as previously mentioned, on relative surface humidity and hydrophilicity of specimen surface.14,19 This explanation could be supported by previous study suggested that dry collagen is relatively hydrophobic and that the hydrophilicity of wet collagen is largely due to bound and unbound water.33 This could explain the descending order in the values of adhesion force measured for hydrated (H), air dehydrated for 5 s (DH5s), and air dehydrated for 10 s (DH10s) dentin specimens. For successful bonding to dentin, proper resin infiltration of the demineralized intertubular collagen fibrils network to form hybrid layer is required. This is achieved by the creation of an open collagen fibrils network with water-filled interfibrillar spaces.2,3,7,27 It was recommended to not dry etched dentin surfaces before the application of acetone or alcohol-based bonding primers and consequently higher bond strength values were reported with moist bonding.9,10,31,34,35 The previous reports are in agreement with results of the TBS of the current study (Table 2). As Excite is an alcohol-based dentin selfpriming adhesive, water containing collagen fibrils network should be available for proper monomers infiltration and hybrid layer, and resin tags formation (Fig. 3A–C). Therefore, the
tapping mode AFM images (Fig. 1), SEM images (Fig. 3), and the TBS results (Table 2) are all consistent with and support the previous assumption that the probed nano-scale adhesion force (Fad) between AFM tip and demineralized intertubular dentin surface is sensitive to dentin surface dryness. Although a significant increase in the nano-scale adhesion force (Fad) between specimens dehydrated by air for 5 s (DH5s) compared to specimens dehydrated by air for 10 s (DH10s) was recorded, no difference in TBS was found (Table 2). We speculate that this may be due to the fact that 5 s air-drying of etched dentin specimens is enough to result in structural collapse of the demineralized collagen fibrils network to an extent that interfere with monomers infiltration as shown in SEM images of dentin/resin interface (Fig. 3). It should be pointed out that, although treatment of acid etched dentin surface by 6.5 vol.% sodium hypochlorite (NaOCl) before the application of the selfpriming adhesive is not a routine clinical practice; specimens used for TBS evaluation were treated by NaOCl to simulate what were done for specimens used for AFM characterization. Therefore, it could be expected that the results of the TBS recorded in this study were affected by the NaOCl treatment.36 The proper determination of the amount of the hydration/ dehydration conditions of the demineralized dentin collagen fibrils network is of clinical importance for successful bonding to dentin especially with the wet bonding technique. It was reported that the conditions of over-drying or over-wetting are undesirable.37–39 Probing the adhesion force between AFM tip, either unmodified or chemically modified, and dentin surface could be promising in determining the amount of residual water and/or other solvents in the surface layer of dentin with nanometer resolution. However, further research is needed for more confirmation and to study in more details the correlation between the nano-scale adhesion force probed by AFM tip and the quantity of water in the dentinal substrate. In addition, the effect of many variables such as AFM tip geometry, dentin depth and location, dentin surface treatment and surrounding conditions such as humidity and temperature should be considered. In addition, studying the interactions between chemically modified AFM tip and specific functional group(s) in dentinal substrate is recommended in future research.
5.
Conclusion
The probed nano-scale adhesion force between an AFM silicon nitride tip and demineralized intertubular dentin surface showed to be sensitive to surface air-dryness. The decrease in the nano-scale adhesion force with the increase in the time of air-drying is related to water dehydration from of the demineralized collagen fibrils network surface.
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22. Marshall GW, Balooch M, Tench R, Kinney JH, Marshall SJ. Atomic force microscopy of acid effects on dentin. Dental Materials 1993;9:265–8. 23. Oliveira SA, Pugach MK, Hilton. Watanabe LG, Marshall SJ, Marshall GW. The influence of dentin smear layer on adhesion: a self-etching primer vs. total-etch system. Dental Materials 2003;19:758–67. 24. Carvalho RM, Yoshiyama M, Brewer PD, Pashley DH. Dimensional changes of demineralized human dentin during preparation for scanning electron microscopy. Archives of Oral Biology 1996;41:379–86. 25. Pashley DH, Ciucchi B, Sano H, Horner JA. Permeability of dentin to adhesive agents. Quintessence International 1993;24:618–31. 26. Kanca J. Resin bonding to wet substrate. I. Bonding to dentin. Quintessence International 1992;23:39–41. 27. Nakabayashi N, Pashley DH. Hybridization of dental hard tissues. Tokyo: Quintessence Publishing Co.; 1998. p. 47–50. 28. Brodsky B, Ramshaw AM. The collagen triple-helix structure. Matrix Biology 1997;15:545–54. 29. Bella J, Eaton M, Brodsky B, Berman HM. Crystal and ˚ molecular structure of a collagen-like peptide at 1.9 A resolution. Science 1994;266:75–81. 30. Bella J, Brodsky B, Berman HM. Hydration structure of collagen peptide. Structure 1995;3:893–906. 31. Gwinnett AJ. Dentin bond strength after air drying and rewetting. American Journal of Dentistry 1994;7:144–8. 32. Pashley DH, Agee KA, Nakajima M, Tay FR, Carvalho RM, Terada R, et al. Solvent-induced dimensional changes in EDTA-demineralized dentin matrix. Journal of Biomedical Materials Research 2001;50:273–81. 33. Pashley DH, Tay FR, Carvalho RM, Rueggeberg FA, Agee KA, Carrilho M, et al. From dry bonding to water-wet bonding to ethanol-wet bonding. A review of the interactions between dentin matrix and solvated resins using a macromodel of the hybrid layer. American Journal of Dentistry 2007;20:7–21. 34. Kanca J. A method for bonding to tooth structure using phosphoric acid as a dentin enamel conditioner. Quintessence International 1991;22:285–90. 35. Kanca J. Wet bonding: effect of drying time and distance. American Journal of Dentistry 1996;9:273–6. 36. Fawzy AS, Amer MA, El-Askary FS. Sodium hypochlorite as dentin pretreatment for etch-and-rinse single-bottle and two-step self-etching adhesives: atomic force microscope and tensile bond strength evaluation. Journal of Adhesive Dentistry 2008;10:135–44. 37. Tay FR, Gwinnett AJ, Wei SHY. Micromorphological spectrum from overdrying to overwetting acid-conditioned dentin in water-free, acetone-based, single-bottle primer/ adhesives. Dental Materials 1996;12:236–44. 38. Tay FR, Gwinnett AJ, Pang KM, Wei SHY. The over wet phenomenon: an optical, Micromorphological study of surface moisture in total etched resin–dentin interface. American Journal of Dentistry 1996;9:43–8. 39. Tay FR, Gwinnett AJ, Pang KM, Wei SHY. Resin permeation into acid-conditioned, moist and dry dentin: a paradigm using water-free adhesive primers. Journal of Dental Research 1996;75:1034–44.
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Probing nano-scale adhesion force between AFM and acid demineralized intertubular dentin: Moist versus dry dentin Amr S. Fawzy a,*, Ahmed M. Farghaly b a b
Dental Biomaterials Department, Faculty of Dentistry, Ain Shams University, Cairo, Egypt Operative Dentistry Department, Faculty of Dentistry, El-Azhar University, Cairo, Egypt
article info
abstract
Article history:
Objective: The aim of this study was to investigate the effect of dentin dryness on the
Received 14 April 2009
variation in the probed nano-scale adhesion force between an AFM silicon nitride tip and
Received in revised form
demineralized intertubular dentin collagen fibrils network surface.
2 August 2009
Methods: Dentin specimens were etched with 37% phosphoric acid for 15 s and then divided
Accepted 6 August 2009
into three groups. Specimens of the first two groups were air-dried for 5 or 10 s (DH5s and DH10s), respectively, whereas specimens of the third group were left in the hydrated condition (H). For each group, Force curves were characterized by contact-mode AFM
Keywords:
and the adhesion force (Fad) was calculated. The structure of the demineralized collagen
AFM
fibrils network was characterized by tapping mode AFM. The tensile bond strength (TBS) to
Adhesion force
dentin was evaluated using one alcohol-based dentin self-priming adhesive. The dentin/
Intertubular dentin
resin interface was investigated by SEM.
Collagen fibrils
Results: Dentin specimens in the wet-hydrated condition (H) showed significantly higher adhesion force and TBS values than dry-dehydrated specimens (DH5s and DH10s). AFM images showed open collagen fibrils network structure in wet-hydrated specimens (H), while the dry-dehydrated specimens (DH5s and DH10s) showed a collapsed appearance to varying degrees. SEM images revealed minimum resin infiltration in dry-dehydrated specimens. Significance: The nano-scale adhesion force between the AFM probe and demineralized intertubular dentin surface was shown to be sensitive to surface air-drying. The decrease in the nano-scale adhesion force with the increase in the time of air-dryness is related to dehydration of the demineralized collagen fibrils network surface. # 2009 Elsevier Ltd. All rights reserved.
1.
Introduction
Recent modeling experiments on the packing density of molecules within tendon collagen fibrils1 indicate that there may be significant space between the collagen molecules for tissue fluid. This water may be lost with dehydration and subsequent shrinkage of the collagen fibrils. As stated by Nakabayashi2 and others3,4 for hybrid layer formation, intertubular dentin must be demineralized to expose the open
collagen fibrils network of the dentinal matrix to create diffusion pathways for monomer infiltration. As long as the interfibrillar spaces, which form interconnected channels having 15–20 nm dimensions, are in a hydrated state, waterfilled pathways are provided for monomers diffusion through collagen fibrils network.3,5–8 In addition, 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.9–11
* Corresponding author. Tel.: +20 02 26359765; fax: +20 02 26382906. E-mail addresses: [email protected], [email protected] (A.S. Fawzy). 0300-5712/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2009.08.005
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Therefore, the precise characterization of the degree of surface hydration of demineralized intertubular dentin collagen fibrils network is essential to evaluate its monomer permeability.12 Atomic force microscope (AFM) is considered as an essential tool in almost every research dealing with the characterization or engineering of materials, including biomaterials, surfaces and interfaces in nanometer scale.13 AFM 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 vs. distance curves. Such curves, briefly called 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 provided in more specialized references.13–15 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. The vertical force on the cantilever tip is proportional to the cantilever bending, which is measured using a position sensitive photo-detector. 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. When retracting the AFM tip from the sample surface, the tip stays in contact with the surface until the cantilever force overcomes the adhesive tip–sample interaction. The first measurements of this pull-out force or adhesion force were performed by Martin et al.17 and Erlandsson et al.18. In the most general case, this adhesion force 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. At ambient conditions, a water neck forms between AFM tip and substrate due to capillary condensation and adsorption of thin water films at surfaces leading to the formation of capillary attractive forces.14 This capillary attractive interaction depends on the relative humidity and the hydrophilicity of tip and sample surface. Depending on the chemical end-groups present on tip and substrate, chemical bonds may form during contact or other specific chemical interactions may occur. The relative contributions of capillary, van der Waals and electrostatic force under
ambient conditions for the adhesion of an AFM tip to graphite, mica and MoS2 were elucidated by Ouyang et al.19 In all cases, the capillary force was found to give the largest contribution. Capillary forces are expected to be maximal for hydrophilic surfaces and to vanish for very hydrophobic surfaces. The aim of the study was to test the null hypothesis that the state of surface hydration of acid etched intertubular dentin has no effect on the nano-scale adhesion force between an AFM probe tip and the dentinal substrate. Complementary to adhesion force measurement, the variation in the structure of the demineralized intertubular dentin collagen fibril network, tensile bond strength and dentin/resin interface with dentin dryness were also evaluated.
2.
Materials and methods
Dentin specimens used in this study for both of AFM characterization and TBS testing were prepared from randomly selected non-carious third molars. All teeth were recently extracted. All patients were of age range of 21–25 years old. All extracted teeth were stored in 0.5% chloramines T solution for 2 weeks then in distilled water at 4 8C until use.
2.1.
Dentin surface preparation for AFM study
Fifteen teeth were used for AFM characterization from which 15 dentin discs, of approximately 2 mm thickness, were prepared. For the preparation of the dentin discs, occlusal enamel was removed perpendicular to the teeth long-axis with a diamond disc mounted to a milling machine using slow-speed under water cooling until the dentin surface, 1.5 mm below the DEJ, was exposed. Then dentin discs having an approximate thickness of 2 mm were cut parallel to the prepared dentin surfaces. The smear layer on the cut dentin discs were made progressively thinner by sequentially wetgrinded with 600, 800, 1000 and 1500 grit SiC polishing papers under water cooling. All prepared discs were then ultrasonically cleaned in distilled water for 15 min. Each dentin disc was divided into two equal halves and the halves were randomly divided equally into three groups to minimize the variability between the dentin discs. Each group was formed of 10 dentin discs halves (specimens). All dentin specimens were etched for 15 s with 37% phosphoric acid gel (Ivoclar/Vivadent AG, FL-9494 Schaan/Liechtenstein). According to the subsequent dentin surface preparation steps, specimens were divided into the following groups shown in Table 1.
Table 1 – Dentin specimens surface preparation. Dentin surface preparation steps Etched dentin was rinsed ultrasonically in distilled water for 10 min, treated by 6.5 vol.% sodium hypochlorite (NaOClaq) for 120 s,20 thoroughly rinsed in distilled water for 5 min and finally air-dried for 10 s by oil-free compressed air. Etched dentin was prepared as previously described except that the dentin surface was air-dried for 5 s by oil-free compressed air. Etched dentin was prepared as previously described except no air-dryness was done and the excess water was removed by gentle blotting with absorbent paper leaving the dentin surface visibly moist.
Surface condition
Symbols
Dried for 10 s
DH10s
Dried for 5 s
DH5s
Wet
H
journal of dentistry 37 (2009) 963–969
2.2. AFM characterization of dentin collagen fibrils network Three specimens from each group were used for AFM characterization of dentin collagen fibrils network structure. Tapping mode characterization was performed with an AFM (Autoprobe CP-II, Veeco, CA, USA). The tapping mode operates by scanning a tip, attached to the end of a vibrating cantilever, across the sample surface, so that it is in intermittent contact with the surface. The cantilever amplitude is maintained constant by altering the vertical position of the piezoelectric scanner. For the tapping mode, a gold-coated all-silicon cantilever (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. All specimens were characterized in air at a relative humidity of 45 5% and temperature of 37 0.5 8C immediately after dentin surface preparation. The Images were recorded with scan rate of 0.5– 1.0 Hz and a resolution of 512 512 pixels per image was chosen.
2.3.
Probing nano-scale adhesion force (Fad)
For the characterization of the force vs. distance curve, a contact AFM mode was preformed to scan the etched dentin surfaces using silicon nitride tips, with nominal radii of 50 nm, mounted to cantilevers (Type D MicroleversTM, Thermo-Microscopes, CA, USA). The force constant and the resonance frequency of the used cantilevers were 2.1 N/m and 160 kHz, respectively. Dentin surface scans of 10 mm 10 mm areas were first done at a scan rate of 4 kHz and then a set of force curves were measured for 14 selected points on the intertubular dentin for each specimen. 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 sample 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 surface adhesion force (Fad) between the cantilever tip and specimen surface was calculated in nano-Newton (nN) from the mean force curve of each specimen. The adhesion force (Fad) was calculated from the difference between the snap-in 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).13,14 All dentin specimens were tested for adhesion force measurement, immediately after surface preparation in a well controlled environments in term of humidity (45 5%) and temperature (37 0.5 8C) as previously described. Seven dentin specimens from each group were tested for force curve and adhesion force measurement.
2.4.
Tensile bond strength (TBS) evaluation
For TBS evaluation, 30 dentin/resin-based composite slabs were prepared from third molar teeth (10 slabs for each group). The roots of the extracted molars were embedded in self-polymer-
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ized acrylic resin 3 mm below the cervical line. The occlusal enamel was removed using a low speed diamond disc in a straight handpiece fixed to a milling machine under copious water spray to expose flat dentin surfaces 1.5 mm below the DEJ. The smear layer was prepared and standardized as previously described for specimens used for AFM characterization. The exposed dentin surfaces of all teeth were acid etched and the etched dentin surfaces were prepared to be in the dry (DH10s or DH5s) and the wet (H) conditions as described in Table 1. Single bottle alcohol-based self-priming adhesive (Excite, Ivoclar/ Vivadent AG, FL-9494 Schaan/Liechtenstein) was used to bond composite restorative material to etched dentin surfaces for all groups. 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, MecaPhysik Dental Division, D-76437 Rastatt, Germany) with a light output of 600 mW/cm2. A direct restorative composite resin material (Tetric Ceram, Ivoclar/Vivadent AG, FL-9494 Schaan/ Liechtenstein) was applied in four increments for each tooth until composite crowns of 6 mm height were built. The thickness of each increment was about 1.5 mm and each increment was light-cured for 40 s. The restored teeth were then stored for in distilled water at 37 8C for 24 h to complete the polymerization process before sectioning. Dentin/composite slabs were prepared from the central region of each composite restored tooth. The thickness of each slab was approximately 1 mm. The slabs were prepared using low speed diamond disc mounted to the milling machine under copious water spray. The teeth were vertically sectioned in a bucco-lingual direction. A fine high speed diamond bur with air/ water spray was used for trimming the slabs into an hour glass shape with cross-sectional area of approximately 1 0.05 mm2 at the bonded interface. The slabs were checked for premature debonding which could be resulted during preparation. All slabs were stored in distilled water for 24 h before mechanical testing. Each dentin/composite slab was fixed to two disposable acrylic resin jigs using cyanoacrylate adhesive. Two rounded L-shaped metallic pins 2 mm in diameter were attached to the upper and lower jaws of a universal testing machine (Lloyd Instrument LR5 series, UK). The dentin/composite slab-jig assembly was mounted to the testing machine through the openings created at both ends of the acrylic jigs. The TBS 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.
2.5.
Evaluation of dentin/resin interface by SEM
Dentin/resin-based composite slabs were prepared as previously described for the TBS testing to evaluate the dentin/ resin interface. Each slab was polished using 600, 800 and 1000 grit silicon carbide paper discs to produce smooth polished surfaces, etched with 37% phosphoric acid gel for 15 s, then rinsed for 15 s and dried with compressed oil-free air. The slabs were immersed in 5.25% sodium hypochlorite solution for 20 min, washed thoroughly under running water for 5 min, then immersed in ascending concentrations of ethanol, 50%, 70%, 90% for 20 min each and 100% for 1 h. Then
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Fig. 1 – Tapping mode AFM images of etched intertubular dentin surface showing (A) collapsed collagen fibrils network with no interfibrillar spaces in air-dried specimens (DH10) for 10 s, (B) partially collapsed collagen fibrils network in air-dried specimens (DH5) for 5 s, and (C) open collagen fibrils network structure with numerous interconnected interfibrillar spaces in hydrated specimens (H).
the slabs were gold sputtered and were examined using SEM (JEOL JXA-840A, Electron Probe Micro-analyzer, Japan), operated at 30 kV. The dentin/resin interface of the three groups (DH10s, DH5s and H) was investigated.
2.6.
Statistical analysis
All data of adhesion force (Fad) in nN and TBS in MPa was expressed as mean standard deviation (SD) and was analyzed by WinSTAT v. 2007.1. One-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple-comparison post hoc test was used for the comparison between the different tested groups. The correlation between Fad and TBS was tested using Pearson’s correlation coefficient. P < 0.05 was considered significant.
3.
Results
The structure of the collagen fibrils network of the etched intertubular dentin at the dry-dehydrated conditions (DH10s
and DH5s) and the wet-hydrated (H) condition are shown in Fig. 1(A–C), respectively. The etched dentin surface revealed a demineralized open collagen fibril network structure with numerous interconnected interfibrillar spaces and porosities in the wet-hydrated (H) specimens when scanned immediately by AFM after dentin surface preparation (Fig. 1C). Specimens dried with air for 10 s (DH10s) showed totally collapsed collagen fibrils network with no interfibrillar spaces (Fig. 1A). However, specimens dried with air for 5 s (DH5s) showed less collapsed appearance and more open structure of the collagen fibrils network (Fig. 1B) compared to specimens dried with air for 10 s (DH10s). Fig. 2 shows a selected cantilever–specimen interaction, force curve, of etched intertubular dentin at the wet-hydrated condition (H). The force is represented on the vertical axis in nN and the piezoelectric scanner z-position is represented on the horizontal axis in nm. The scanner extension (approach) path is demonstrated by the solid line, whiles 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 adhesive force (black arrow) is represented by the difference between the snap-in and snap-out points. Table 2 shows the results of the adhesion force (Fad) and the tensile bond strength (TBS) of the three groups used in the study. The etched dentin specimens in the wet-hydrated condition (H) showed significantly higher (P < 0.001) adhesion force and TBS values than etched air-dried dentin specimens for both of 10 and 5 s (DH10s and DH5s). In addition, the
Table 2 – Adhesion force (Fad) and tensile bond strength (TBS) at dry (DH10s and DH5s) and wet (H) conditions.
Fig. 2 – Selective force curve of etched intertubular dentin surface in the wet-hydrated condition (H). The AFM scanner approach and retraction paths are represented by the solid and the dashed lines, respectively. The adhesion force (Fad) in nN is represented by the black arrow between the snap-in and snap-out points.
Adhesion force (nN) Tensile bond strength (MPa)
DH10s
DH5s
H
1.79 0.23a
2.77 0.29b
7.98 0.74c
4.91 0.97a
5.39 0.89a
17.51 1.38b
Mean values with dissimilar letters in the same row are statistically significant different at P < 0.05.
journal of dentistry 37 (2009) 963–969
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Fig. 3 – SEM images of dentin/resin interface showing limited resin infiltration (hybrid layer), resin tags formation and separation at the bonded interface of air-dried dentin specimens for 10 s (A) and 5 s (B) compared to specimens left in the wet-hydrated condition (C).
adhesion force was significantly higher (P < 0.01) for etched air-dried dentin specimens for 5 s (DH5s) than for etched airdried dentin specimens for 10 s (DH10s). In contrast, no significant difference in TBS was found between etched airdried specimens for both of 10 and 5 s (DH10s and DH5s). Pearson’s correlation analysis showed a significant positive correlation (P < 0.001) between Fad and TBS. SEM investigation (Fig. 3A and B) showed limited resin infiltration of the demineralized dentin (hybrid layer formation), very few small-sized resin tags formation and separation at the bonded interface of the dry-dehydrated specimens for 10 and 5 s (DH10s and DH5s) compared to wet-hydrated specimens (Fig. 3C).
4.
Discussion
Atomic force microscopy5,21–24 was used in this study to characterize the structure of acid demineralized collagen fibrils network because it has an advantage over measuring devices requiring high vacuums, as the specimens were imaged in ambient air at humidity and temperature of 45 5% and 37 0.5 8C, respectively. As previously mentioned, the value of the adhesion force between an AFM tip and specimen surface depends on the electrostatic force, the van der Waals force, the meniscus or capillary force, and the formation of chemical bonds.14 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 adhesion force, identical silicon nitride AFM tips of the same type were used in this study, randomly, without any chemical modification to characterize the force curve of the etched intertubular dentin specimens under the same well controlled environmental conditions. In addition, dentin specimens used in this study were prepared randomly from non-carious third molars of nearly the same age range, extraction period and storage conditions. El Feninat et al.21 reported that considerable water loss and observable structure change occur within few minutes to etched dentin surface when characterized by AFM in air. Therefore, in this study the etched dentin specimens was imaged immediately after specimens surface preparation by AFM to study the structure of the demineralized intertubular dentin collagen fibrils network and to characterize the nanoscale adhesion force (Fad) at the dry-dehydrated and the wethydrated conditions. However, water dehydration is expected during the time required for specimens mounting, image acquisition and force curve measurement by AFM in air which was approximately equal to 3 min for each investigated specimen which could significantly affect the structure of the collagen network and the results of force curve measurement. It should be clarified that, AFM characterization of both of collagen fibril network and nano-scale adhesion force (Fad) for wet specimens (H) were done in air not under water to dominate the effect of the capillary attractive force in determining the value of the non-scale adhesion force. It
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journal of dentistry 37 (2009) 963–969
was reported previously, that the capillary attractive force is important factor in force–curve measurement by AFM taken in ambient air, but not in aqueous environment.13 The open collagen fibrils network structure with the numerous interfibrillar spaces of the demineralized intertubular dentin in the wet-hydrated condition (H) compared to the dry-dehydrated (DH10s and DH5s) conditions (Fig. 1A–C) support the results of the adhesion force (Fad) measurement (Table 2). The significant decrease in the value of (Fad) could be attributed to the water evaporation from the demineralized dentin collagen network surface by air-drying. This is in agreement with previous findings,25,26 that the presence of water is essential to prevent the structural collapse of the demineralized dentin collagen network. The phenomena of shrinkage and collapse of the demineralized soft collagen fibrils network by air-drying could be due to several mechanisms. The demineralized collagen network could be assumed to be suspended in water that occupies the spaces that were previously occupied by apatite crystals between the collagen fibrils. By water loss due to air-drying, collagen fibrils could move close together resulting in passive collapse of the soft collagen network.27 Alternatively the structural stability of the demineralized collagen fibrils network could be explained at molecular level such that the intermolecular spaces between collagen molecules are preserved by water that surrounds the triple helices of collagen molecule. Owing to the polar hydrogen bonding characteristics of water molecules, hydrogen bonds are formed preventing direct inter-chain contact and collapse.28–30 Furthermore, the surface tension force acting on air–collagen fibrils interface,31 and the shortening of the interconnected collagen fibrils32 associated with water loss are among the possible explanations of the shrinkage and collapse of the demineralized collagen network by air. Within the results of this study, the high values of adhesion force (Fad) measured between AFM tip and specimen surface could be related to the increase in the water content in the outer most region of the demineralized collagen fibrils network. The increase in the adhesion force may be attributed to the increase in capillary attractive force which depends, as previously mentioned, on relative surface humidity and hydrophilicity of specimen surface.14,19 This explanation could be supported by previous study suggested that dry collagen is relatively hydrophobic and that the hydrophilicity of wet collagen is largely due to bound and unbound water.33 This could explain the descending order in the values of adhesion force measured for hydrated (H), air dehydrated for 5 s (DH5s), and air dehydrated for 10 s (DH10s) dentin specimens. For successful bonding to dentin, proper resin infiltration of the demineralized intertubular collagen fibrils network to form hybrid layer is required. This is achieved by the creation of an open collagen fibrils network with water-filled interfibrillar spaces.2,3,7,27 It was recommended to not dry etched dentin surfaces before the application of acetone or alcohol-based bonding primers and consequently higher bond strength values were reported with moist bonding.9,10,31,34,35 The previous reports are in agreement with results of the TBS of the current study (Table 2). As Excite is an alcohol-based dentin selfpriming adhesive, water containing collagen fibrils network should be available for proper monomers infiltration and hybrid layer, and resin tags formation (Fig. 3A–C). Therefore, the
tapping mode AFM images (Fig. 1), SEM images (Fig. 3), and the TBS results (Table 2) are all consistent with and support the previous assumption that the probed nano-scale adhesion force (Fad) between AFM tip and demineralized intertubular dentin surface is sensitive to dentin surface dryness. Although a significant increase in the nano-scale adhesion force (Fad) between specimens dehydrated by air for 5 s (DH5s) compared to specimens dehydrated by air for 10 s (DH10s) was recorded, no difference in TBS was found (Table 2). We speculate that this may be due to the fact that 5 s air-drying of etched dentin specimens is enough to result in structural collapse of the demineralized collagen fibrils network to an extent that interfere with monomers infiltration as shown in SEM images of dentin/resin interface (Fig. 3). It should be pointed out that, although treatment of acid etched dentin surface by 6.5 vol.% sodium hypochlorite (NaOCl) before the application of the selfpriming adhesive is not a routine clinical practice; specimens used for TBS evaluation were treated by NaOCl to simulate what were done for specimens used for AFM characterization. Therefore, it could be expected that the results of the TBS recorded in this study were affected by the NaOCl treatment.36 The proper determination of the amount of the hydration/ dehydration conditions of the demineralized dentin collagen fibrils network is of clinical importance for successful bonding to dentin especially with the wet bonding technique. It was reported that the conditions of over-drying or over-wetting are undesirable.37–39 Probing the adhesion force between AFM tip, either unmodified or chemically modified, and dentin surface could be promising in determining the amount of residual water and/or other solvents in the surface layer of dentin with nanometer resolution. However, further research is needed for more confirmation and to study in more details the correlation between the nano-scale adhesion force probed by AFM tip and the quantity of water in the dentinal substrate. In addition, the effect of many variables such as AFM tip geometry, dentin depth and location, dentin surface treatment and surrounding conditions such as humidity and temperature should be considered. In addition, studying the interactions between chemically modified AFM tip and specific functional group(s) in dentinal substrate is recommended in future research.
5.
Conclusion
The probed nano-scale adhesion force between an AFM silicon nitride tip and demineralized intertubular dentin surface showed to be sensitive to surface air-dryness. The decrease in the nano-scale adhesion force with the increase in the time of air-drying is related to water dehydration from of the demineralized collagen fibrils network surface.
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