The aim of this study was to correlate the degree of conversion measured inside the hybrid layer (DC) with the microtensile resin–dentin bond strength (μTBS) and silver nitrate uptake or nanoleakage (SNU) for five simplified etch-and-rinse adhesive systems.
Fifty-five caries free extracted molars were used in this study. Thirty teeth were used for μTBS/SNU [ n = 6] and 25 teeth for DC [ n = 5]. The dentin surfaces were bonded with the following adhesives: Adper Single Bond 2 (SB), Ambar (AB), XP Bond (XP), Tetric N-Bond (TE) and Stae (ST) followed by composite resin build-ups. For μTBS and SNU test, bonded teeth were sectioned in order to obtain stick-shaped specimens (0.8 mm 2 ), which were tested under tensile stress (0.5 mm/min). Three bonded sticks, from each tooth, were not tested in tensile stress and they were immersed in 50% silver nitrate, photo-developed and analyzed by scanning electron microscopy. Longitudinal 1-mm thick sections were prepared for the teeth assigned for DC measurement and evaluated by micro-Raman spectroscopy.
ST showed lowest DC, μTBS, and higher SNU ( p < 0.05). All other adhesives showed similar DC, μTBS, and SNU ( p > 0.05), except for TE which showed an intermediate SNU level. The DC was positively correlated with μTBS and negatively correlated with SNU ( p < 0.05). SNU was also negatively correlated with μTBS ( p < 0.05).
The measurement of DC inside the hybrid layer can provide some information about bonding performance of adhesive systems since this property showed a good correlation with resin–dentin bond strength and SNU values.
As defined by Nakabayashi , the hybrid layer is the structure formed in hard dental tissues by demineralization of the surface and subsurface, and subsequent infiltration of monomers and polymerization. This structure is one of the most important factors in sucessfully bonding resin-based composite to dentin .
However, several morphological studies have revealed that nanoleakage, identified by the use of silver tracers, occurs in bonded interfaces of both etch-and-rinse and self-etching adhesives. Incomplete polymerization of adhesive monomers has been suggested as one of the reasons for the occurrence of nanoleakage in adhesive systems .
Conversion of monomer into polymer (percentage of C C incorporated into polymer chains as C C) plays an important role in successful dentin bonding . To some extent it is true that increased permeability of the adhesive layer is observed when there are unreacted monomers within the hybrid layer . This results in the formation of a porous hybridoid structure with reduced sealing ability, which is prone to degradation . An inadequate degree of conversion (DC) of adhesive monomers may also cause a reduction in material properties , which may lead to reduce durability of the adhesive restoration . A high DC is correlated with improved mechanical properties in resin materials , as well as higher biocompatibility .
Nevertheless, only recently more attention has been focused on the evaluation of DC and adhesive properties . Most of the methods available to investigate the DC of resin polymers, such as differential scanning calorimetry , microhardness and Fourier transform infrared spectroscopy , are performed in specimens polymerized under an ideal condition, i.e., not in contact with the bonding structure.
It is widely known that adhesive resin polymerization in a moist environment is much more challenging than it is under laboratory conditions and thus, the quantitative measurement of DC inside the hybrid layer can provide some information to explain current adhesive performance and recently it has been used for several research groups . High-resolution analytical techniques have made it possible to detect molecular structures directly and non-destructively, allowing measurements at a spatial resolution of approximately 1–10 μm . Micro-Raman spectroscopy has proved to be well suited to the characterization of the chemical structure and character of the adhesive resins, collagen and minerals at a resolution of up to 1 μm and is also very useful in determining the DC of dental adhesives by providing a direct measurement of the percentage of converted double bonds .
Three studies tried to correlate bond strength with DC in vitro without success . Another study indicated that the increase in nanoleakage expression was associated with the lowest DC measured inside the hybrid layer, but no correlation test was performed . Therefore the aim of this study was to test whether there is any correlation between the DC of five simplified etch-and-rinse adhesives and their respective immediate resin–dentin bond strength (μTBS) and silver nitrate uptake (SNU) values. The null hypothesis to be tested was that there is no correlation between the DC and the resin–dentin bonds and SNU.
Material and methods
Teeth selection and preparation
Fifty five extracted, caries-free human third molars were used. The teeth were collected after obtaining the patients’ informed consent under a protocol approved by the local Ethics Commitee Review Board. The teeth were disinfected in 0.5% chloramine, stored in distilled water at 4 °C and used within six months after extraction. A flat dentin surface was exposed after wet grinding the occlusal enamel on # 180 grit SiC paper. The exposed dentin surfaces were further polished on wet # 600-grit silicon–carbide paper for 60 s to standardize the smear layer.
Five simplified etch-and-rinse adhesive systems were tested and a total of eleven teeth were used for each adhesive system. Of these, six were used for μTBS/SNU evaluation while the remaining teeth were used for DC measurement. The teeth were randomly allocated according to the adhesive system to be used.
Materials and restorative procedures
The simplified etch-and-rinse adhesive systems tested were: Adper Single Bond 2 (SB – 3M ESPE, St. Paul, MN, USA), Ambar (AB, FGM, Joinville, SC, Brazil), Stae (ST, SDI Limited, Bayswater, Victoria, Australia), Tetric N-Bond (TE, Ivoclar Vivadent AG, Schaan, Liechtenstein, Germany) and XP Bond (XP, Dentsply DeTrey, Konstanz, Germany). The composition, application mode and batch number are described in Table 1 .
|Adhesive system (manufacturer)||Composition (batch number)||Application mode|
|Adper Single Bond 2 (3M ESPE) SB||• Etchant: 35% H 3 PO 4
• HEMA, Bis-GMA, DMAs, methacrylate functional copolymer of polyacrylic and polyitaconic acids, water, ethanol, nanofiller, photo-initiator. (BFBR)
|(1) Acid etching (15 s); (2) rinsing (15 s); (3) air drying leaving dentin visible moist (5 s); (4) application of 2–3 coats of adhesive (15 s) with gentle agitation; (5) gently air-thin for 10 s to evaporate solvent; (6) light curing (10 s).|
|Ambar (FGM) AB||• CondAc 37: 37% phosphoric acid
• Methacrylic monomers 10-MDP, photo-initiators, co-initiators, stabilizer, silica nano-particulates, ethanol. (04122010)
|(1) Acid etching (15 s); (2) rinsing (15 s); (3) air drying leaving dentin visible moist (5 s); (4) application of one coat of adhesive (10 s) vigorously; (5) application of other coat of the adhesive (10 s) with gentle agitation; (6) air drying (10 s) at distance; (7) light curing (10 s).|
|Stae (SDI) ST||• Etchant: 37% H 3 PO 4
• Acetone, water, proprietary hydrophilic/hydrophobic monomer, HEMA, CQ, stabilizer. (100830)
|(1) Acid etching (15 s); (2) rinsing (5 s); (3) air drying leaving dentin visible moist (5 s); (4) application of one coat of adhesive (10 s) to saturate the surface; (5) air drying (10 s) at distance; (6) Light curing (10 s).|
|Tetric N-Bond (Ivoclar Vivadent) TE||• Total Etch: 37% H 3 PO 4
• Phosphonic acid acrylate, HEMA, BisGMA, UDMA, ethanol, nanofiller, catalysts and stabilizer. (L50568)
|(1) Acid etching (15 s); (2) rinsing (5 s); (3) air drying leaving dentin visible moist (5 s); (4) application of 1 thick layer of adhesive gently for at least 10 s; (5) gently air-thin for 10 s to evaporate solvent; (6) light curing (10 s).|
|XP Bond (Dentsply) XP||• Etchant: H 3 PO 4
• PENTA, TCB, HEMA, TEGDMA, UDMA, tert-butanol, nanofiller, CQ, stabilizer. (0804002269)
|(1) Acid etching (15 s); (2) rinsing (15 s); (3) air drying leaving dentin visible moist (5 s); (4) application of 1 coat of adhesive uniformly and leave the surface undisturbed (20 s); (5) gently air-thin for 10 s to evaporate solvent; 6. Light curing (10 s)|
After acid etching with the respective etchants of each adhesive system and water rinsing, the adhesives were applied in accordance with the manufacturers’ directions ( Table 1 ). The adhesives were light polymerized for 10 s using a quartz-tungsten halogen light with an intensity of 600 mW/cm 2 (VIP, Bisco, Schaumburg, IL, USA) ( Table 1 ). Resin composite build-ups (Opallis, shade A2, FGM, Joinville, SC, Brazil) were constructed on the bonded surfaces in 3 increments of 1 mm each, which were individually light polymerized for 30 s at the same light intensity. The light intensity was checked with a curing radiometer (VIP, Bisco, Schaumburg, IL, USA) before start to light-curing the adhesive and resin composite for each five tooth. All the bonding procedures were carried out by a single operator at a room temperature of 20 °C and constant relative humidity.
Resin–dentin microtensile testing
After storage in distilled water at 37 °C for 24 h, thirty specimens were longitudinally sectioned in both the mesio-distal and buccal-lingual directions across the bonded interface, using a diamond saw in a Labcut 1010 machine (Extec Corp., Enfield, CT, USA) to obtain bonded sticks with a cross-sectional area of approximately 0.8 mm 2 . The number of premature failures per tooth during specimen preparation was recorded. The cross-sectional area of each stick was measured with a digital caliper (Absolute Digimatic, Mitutoyo, Tokyo, Japan) to the nearest 0.01 mm in order to calculate the actual μTBS.
Each bonded stick was attached to a Geraldeli’s device for μTBS testing (ODEME Biotechnology, Luzerna, SC, Brazil) with cyanoacrylate resin (Super Bonder, Loctite, SP, Brazil) and tested in tensile forces in a universal testing machine (Kratos, São Paulo, SP, Brazil) at a crosshead speed of 0.5 mm/min until failure. The failure modes were evaluated under a stereomicroscope at 40× magnification and classified as cohesive (failure exclusive within dentin or resin composite, C), adhesive (failure at resin/dentin interface, A), or adhesive/mixed (failure at resin/dentin interface that included cohesive failure of the neighboring substrates, A/M).
Silver nitrate uptake evaluation
Three resin-bonded sticks, randomly selected from each tooth, were not tested under tensile forces. These bonded sticks were coated with two layers of nail varnish applied up to within 1 mm of the bonded interfaces for nanoleakage evaluation. During this procedure, specimens got dehydrated and therefore they were re-hydrated in distilled water for 10 min prior to immersion in the tracer solution. Ammoniacal silver nitrate was prepared according to the protocol previously described by Tay et al. . The bonded sticks were placed in the ammoniacal silver nitrate in darkness for 24 h, rinsed thoroughly in distilled water, and immersed in photo-developing solution for 8 h under a fluorescent light to reduce silver ions to metallic silver grains within voids along the bonded interface.
All bonded sticks were wet-polished with 600-grit SiC paper to remove the nail varnish. Specimens were polished with a 600-, 1000-, and 2000-grit SiC paper and 6, 3, 1 and 0.25 μm diamond paste (Buehler Ltd., Lake Bluff, IL, USA) using a polishing cloth. They were ultrasonically cleaned, air dried, mounted on stubs, and sputtercoated with gold (MED 010, Balzers Union, Balzers, Liechtenstein). Resin–dentin interfaces were analyzed by scanning electron microscopy (SEM) operated in the backscattered mode and using energy dispersive X-ray spectrometry (EDX) (SSX-550, Shimadzu, Tokyo, Japan). The amount of SNU within the adhesive layer, hybrid layer and resin tags of each stick, was measured with EDX in three regions (5 μm × 5 μm) of the bonded stick . After acquisition of the first image in the center of the stick, two new images were taken 0.3 mm to the right and 0.3 mm to the left, by a technician who was blind to the experimental conditions under evaluation. The intensity of backscattered electrons generated by electron bombardment by X-rays can be correlated to the atomic number of the element within the sampling volume. The percentage of SNU was calculated based on the amount of Ag presented in the pre-selected areas.
Degree of conversion inside the hybrid layer (DC)
The remaining twenty five specimens ( n = 5 for each adhesive) were longitudinally sectioned in order to obtain two 1-mm thick resin–dentin slices. These slices were wet polished with 1500; 2000 and 2500-grit SiC paper (Buehler Ltd, Lake Bluff, IL, USA). Specimens were ultrasonically cleaned for 20 min and then stored in water for 24 h at 37 °C before taking the DC readings.
The micro-Raman spectroscopy analysis was performed using Senterra (BrukerOptik GmbH, Ettlingen, Baden-Württemberg, Germany) to investigate the DC inside the hybrid layer of the adhesive interfaces. The micro-Raman spectrometer was first calibrated for zero and then for the coefficient values using a silicon sample. Samples were analyzed using the following micro-Raman parameters: 20 mW Neon laser with 532 nm wavelength, spatial resolution of ≈3 μm, spectral resolution ≈5 cm −1 , accumulation time of 30 s with 6 co-additions, and 100× magnification (Olympus UK, London, UK) to a ≈1 μm beam diameter. The spectra were taken in the middle of the hybrid layer, in an arbitrary area of the intertubular dentin. Care was taken to select an area between two dentin tubules. A digital image of the site was recorded before each acquisition ( Fig. 1 ). One site was examined in each slice.
Spectra of uncured adhesives were taken as reference. Post-processing of spectra was performed using the dedicated Opus Spectroscopy Software version 6.5 (BrukerOptik GmbH, Ettlingen, Baden-Württemberg, Germany). The ratio of double-bond content of monomer to polymer in the adhesive was calculated according to the following formula:
DC ( % ) = 1 − R ( cured ) R ( uncured ) × 100