Assessment of the quality of resin–dentin bonded interfaces: An AFM nano-indentation, μTBS and confocal ultramorphology study



The aim of this study was to assess by using confocal microscopy (CLSM), AFM nano-indentation and microtensile bond strength test (μTBS) the quality of the resin–dentin interfaces created with selected bonding parameters.


Dentin conditioned with H 3 PO 4 or EDTA was bonded in ethanol- or water-wet condition using a HEMA-free or HEMA-containing adhesive. The resin-bonded teeth were stored in distilled water (24 h) and sectioned as match-sticks (0.9 mm 2 ) for μTBS. Further resin-bonded teeth were sectioned and analyzed using CLSM, and AFM nano-indentation. The AFM imaging and nano-indentation processes were undertaken using a Berkovich diamond indenter. The modulus of elasticity (Ei) and hardness (Hi) across the interface were evaluated with the specimens in a fully hydrated status. The AFM imaging was performed both in dry and wet conditions for evaluating the shrinkage of the hybrid layer on dehydration.


The HEMA-containing adhesive applied onto H 3 PO 4 -etched ethanol or water-wet dentin created hybrid layers with the lowest biomechanical nano-properties ( p < 0.05); no significant differences in μTBS were found between the two wet-bonding techniques ( p > 0.05). However, the ethanol-wet bonding reduced the dye penetration into the adhesive layer created with the HEMA-containing adhesive. Hybrid layers with high biomechanical properties, low micropermeability and no shrinkage were only possible when using HEMA-free adhesive applied in ethanol wet-dentin. In particular, a significant increase in Ei and Hi was achieved at the hybrid layer and underneath the resin–dentin interface of ethanol-wet EDTA-treated dentin.


The use of HEMA-free adhesives applied onto ethanol-wet dentin may be considered as an alternative and suitable bonding strategy to achieve high quality resin–dentin interfaces.


The demineralization of dentin and the exposure of the collagen matrix is a crucial step in adhesive dentistry to achieve a micromechanical interlocking between resin monomers and dentin . The water-wet bonding technique is commonly used in etch-and-rinse bonding procedures to suspend the demineralized collagen fibrils and prevent shrinkage caused by the electrostatic attraction (i.e. hydrogen bonds formation) of dentinal proteoglycans and glycosaminoglycans . Unfortunately this bonding technique does not allow a complete resin infiltration of the demineralized dentin, leaving unprotected collagen fibrils below and within the hybrid layer . The imperfect resin infiltration is mainly due to the incomplete replacement of water from the demineralized collagen network, especially when vital dental pulps perfuse dentinal fluid . Unprotected collagen fibrils within the hybrid layer may be degraded by the action of endogenous matrix metalloproteinases (MMPs) derived from the demineralized dentin . Moreover, poor infiltrated hybrid layers have an evident attitude to water-sorption that contributes to the hydrolytic degradation of the resin–dentin interface . The ethanol-wet bonding technique has been shown to increase the longevity of resin-bonded H 3 PO 4 -etched dentin . In this technique, absolute ethanol, a polar solvent with less hydrogen bonding capacity than water , may be used to chemically dehydrate the demineralized collagen matrix, reduce the hydrophilicity of the collagen matrix and create wider interfibrillar spaces for a better resin infiltration .

It has been demonstrated that “poor-quality” hybrid layers are characterized by an excessive presence of water are also affected by nano/micro porosities phase separation and low monomer polymerization . The assessment of the hardness and modules of elasticity along the resin–dentin interface and the hybrid layer resistance to dry shrinkage may be suitable to achieve further knowledge on the quality of hybrid layers created using the etch-and-rinse technique. Nevertheless, there is still little information available regarding the quality of formed resin–dentin interfaces with etch-&-rinse adhesives applied with water or ethanol-wet bonding techniques and with HEMA-free or HEMA-containing resins .

The aim of this study was to evaluate the bond strength (μTBS), the ultramorphology/micropermeability and the biomechanical nano-properties of the resin–dentin interface created with two experimental etch-&-rinse adhesives applied onto 37%-H 3 PO 4 or 0.5 M-EDTA conditioned dentin when using the water-wet or ethanol-wet bonding techniques (5 min.). The null hypotheses to be tested are that the use of: (1) H 3 PO 4 vs. EDTA to condition dentin, (2) water-wet vs. ethanol-wet bonding techniques and (3) HEMA-free vs. HEMA-containing adhesives do not affect the quality of the created resin–dentin interfaces.

Materials and methods

Specimen preparation

Caries-free human molars (age: 18–40 yr) extracted for surgical reasons under an informed consent, reviewed and approved by the Institutional Ethics Committee, were stored in 0.5% chloramine-T at 4 °C for no more than 1 month. The teeth were sectioned 1 mm beneath the cemento-enamel junction using a diamond wafering blade (Isomet 11/1180, Buehler, Coventry, UK). The occlusal enamel was removed to expose the middle coronal dentin and a standard smear layer was created using 500 grit SiC paper (Struers LaboPol-4. Struers, Copenhagen, Denmark).

Experimental adhesives and bonding procedures

Two experimental etch-and-rinse bonding systems were prepared: (i) a HEMA-containing resin blend (HEMA-containing) was formulated using UDMA-60%/BisGMA-10%/TEGDMA-30% (Esstech Essington, PA, USA). A hydrophilic monomer (2-hydroxyethyl methacrylate, Aldrich Chemical Co, Gillingham, UK) [20–40%] and absolute ethanol (Aldrich Chemical), [70% for the primer and 10% for the bond] were subsequently added to the neat resin blend. (ii) a HEMA-free resin blend (HEMA-free) was formulated using UDMA-60%/BisGMA-10%/TEGDMA-30% (Esstech Essington, PA, USA) and dissolved in absolute ethanol (Aldrich Chemical), [70% for the primer and 10% for the bond]. The experimental resin blends were finally mixed with 0.5 wt% of camphoroquinone (Aldrich Chemical) and 1.0 wt% of ethyl 4-dimethylaminobenzoate (Aldrich Chemical). Forty-eight dentin specimens were divided into two main groups. Five-minute sonication (Model QS3, Ultrawave Ltd, Cardiff, UK) and 2-day shaking (Orbital Shakers PSU-20i, Cole Fisher Scientific Ltd, Loughborough, UK) were required to yield well-mixed resin solutions. The specimens ( n = 12) of the first group were etched using a 37% phosphoric acid gel (H 3 PO 4 ; Bisco, Itasca, IL, USA) for 15 s and the specimens of the second group ( n = 12) were conditioned for 60 s using a 0.5 M water solution of ethylenediaminetetraacetic acid (EDTA: 99.995%, Lot. 431788 -Aldrich Chemical).

The specimens of each group were copiously rinsed with water for 1 min and immediately immersed in absolute ethyl alcohol (EtOH), (Aldrich Chemical) for 5 min ( n = 6) or in deionized water (H 2 O) for 1 min ( n = 6), . The dentin surface was always covered by ethanol to avoid surface tension forces, keeping it visibly moist prior to the application of the resins. The water-wet bonding substrate was achieved by water-rinsing the dentin surfaces and gently blowing off the excess water to leave a wet reflective surface . The primer and the bond were applied within a period of 20 s and light-cured for 30 s using a halogen light-curing unit (Translux EC Kulzer GmBh, Bereich Dental, Werheim, Germany). The output intensity was monitored with a Demetron Radiometer (Model 100, Demetron Research, Danbury, CT, USA) to maintain a minimal light output intensity of 600 mW/cm 2 throughout all the experiment. A flowable resin composite (X-Flow™, Dentsply, Caulk, UK) was placed incrementally in two 1 mm layers and light-cured for 40 s (Demetron Research).

AFM imaging and nano-indentation

The resin-bonded specimens were left undisturbed in water for 3 h and then cut perpendicularly to the bonding zone using a diamond saw (Isomet 11/1180) to obtain 3 resin–dentin slabs with a thickness of 2 mm. The resin–dentin slabs were polished through SiC abrasive papers from 800 up to 4000 grit (Struers LaboPol-4) followed by final polishing steps performed using diamond pastes (Buheler-MetaDi, Buheler Ltd. Lake Bluff, IL, USA) through 1 μm down to 0.25 μm . The specimens were treated in ultrasonic bath (Model QS3, Ultrawave Ltd, Cardiff, UK) containing deionized water for 5 min at each polishing step. The specimens were finally stored in buffered deionized water [pH: 7.1] for no more than 12 h.

An atomic force microscope (AFM Nanoscope V, Digital Instruments, Veeco Metrology group, Santa Barbara, CA, USA) equipped with a Triboscope indentor system (Hysitron Inc., Minneapolis, MN, USA) was employed in this study. The imaging and indentation processes were undertaken using a water immersion Berkovich diamond cube corner indenter with a tip radius of approximately 20 nm. The AFM ultramorphology analysis was performed in dry and wet conditions in order to evaluate the shrinkage of the hybrid layer. The modulus of elasticity (Ei) and hardness (Hi) across the interface were evaluated with the specimens in a fully hydrated status . After a pre-experimental pilot study, it was observed that six indentations performed in a straight line with a load of 4000 nN and a time function of 10 s were suitable for a standardized evaluation of the biomechanical nano-properties along the resin–dentin interface. To determine the location and width of the hybrid layer the confocal laser scanning microscopy (CLSM) was also employed ( Fig. 1 A and B). The number of indentations in hybrid layers with different thicknesses (i.e. EDTA or H 3 PO 4 ) was standardized by performing for each line only one indentation in the middle of the hybrid layer (HL). Three indentation lines (two lines over the pulpal horns and one in the middle of the specimen) were undertaken for each resin–dentin interface starting from the hybrid layer down to the intertubular dentin as seen in Fig. 1 C. The distance between each indentation was kept constant by adjusting the distance intervals in 5 (±1) μm steps and the load function. Moreover, three disks (: 0.3 mm) for each adhesive and resin composite used in this study were created and analyzed to obtain reference Ei and Hi values ( Fig. 1 C and D). These values were used during the nano-indentation test to correlate and give comparative values for the results produced on testing the hybrid layer. An image was captured at the end of each indentation in order to analyze the shape and the position of the indentations. If premature interface debonding occurred during specimen preparation, a value “0” was arbitrarily assigned and included in the statistical analysis.

Nov 28, 2017 | Posted by in Dental Materials | Comments Off on Assessment of the quality of resin–dentin bonded interfaces: An AFM nano-indentation, μTBS and confocal ultramorphology study
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