To evaluate the influence of storage time on the elastic modulus, micromorphology, nanoleakage, and micromechanical behavior of the dentin–adhesive interfaces of five adhesive systems (Scotchbond Multi-Purpose, Clearfil SE Bond, One Up Bond F, Adper Easy One, and Filtek LS Adhesive) after 24 h (T0) and 12 months (T1).
Fifty teeth were restored and distributed according to each adhesive system ( n = 10). At least four specimens were obtained from each tooth. One specimen was evaluated under SEM to obtain the micromorphology of dentin–adhesive interface (DAI). Two specimens were used to assess nanoleakage, one tested in T0 and the other in T1. The last specimen was used for nanoindentation, in T0 and T1, to obtain the initial and final mechanical properties of DAI structures. Two non-restored teeth were evaluated under SEM to obtain the dentin morphology. Laboratorial data were used to build 15 finite element models to assess the maximum principal stress in each time of analysis.
Storage resulted in hydrolysis of the dentin–adhesive interfaces for all groups. Silver impregnation increased for all groups after 1 year storage ( p < .05), except for Clearfil SE Bond. In general, a decrease in elastic modulus values was observed for all groups from T0 to T1 ( p < .05), mainly at the hybrid layer. The FEAs showed higher stress levels at T1 than T0 simulations for all adhesives.
At T1, degradation occurred at the dentin–adhesive interface formed by all adhesives, and the intensity of degradation differed depending on the type of adhesive system used. The interface formed by the self-etching primer containing the 10-MDP functional monomer showed the highest stability among the adhesive systems after 12 months of storage.
Contemporary adhesive systems have shown good mechanical properties and adequate bonding in the short term , however the stability of the dentin–adhesive interface (DAI) is still questionable in long-term studies . The long-term retention and stability of resin composite to dentin substrate is only possible with high quality micromechanical/chemical interaction between adhesive and substrate by the formation of hybrid layer (HL) , characterized as a three-dimensional collagen–resin biopolymer that provides a continuous and stable link between the adhesive and dentin substrate .
The formation of a hybrid layer may be achieved by two approaches, namely etch and rinse (ER) and self-etching (SE) bonding agents . ER adhesive strategy involves the smear layer removal and superficial demineralization by phosphoric acid etching, following rinsing and partial drying to keep the dentin moist. Afterwards, the clinician can use either a priming/hydrophobic resin adhesive application (3-step etch-and-rise adhesive) or one-bottle, 2-step etch-and-rinse adhesive . SE adhesives incorporates or partially removes the smear layer by the application of an acidic primer on dentin, that is not subsequently rinsed, and is followed by the application of bonding resin (2-step self-etch adhesive) or not (all-in-one self-etch adhesives) .
Basically, two mechanisms of bonded interface degradation have been related for “etch-and-rinse” and the self-etching adhesives: (1) Hydrolysis of exposed collagen not infiltrated by the adhesive resin; (2) hydrolysis of the resin-based polymeric matrix .
Degradation of exposed collagen fibrils not infiltrated by the adhesive resin has been most frequently associated after the use of ER adhesive systems . For this category of bonding agents, the phosphoric acid etching of dentin step is considered one of the main reasons for the DAI degradation , since the incomplete resin infiltration through the demineralized dentin leaves exposed collagen fibrils which are vulnerable to enzymatic degradation . For SE adhesive systems, degradation of collagen fibrils also occurs ; however, the DAI degradation of SE adhesives is considered lower when compared to ER systems. SE adhesives leave no or less amounts of exposed collagen fibrils below the hybrid layer, since the depth of demineralization and adhesive resin infiltration tends to occur simultaneously .
On the other hand, hydrolysis of the polymeric matrix occurs for both types of bonding protocols, mainly in the hybrid layer, and has been considered the main DAI degradation mechanism for SE systems . To simplify the clinical steps of SE, higher concentrations of hydrophilic acidic monomers and water have been added to the material formulation, resulting in a more hydrophilic and complex adhesive solution . This is supposed to be critical for the so called “all-in-one” or “one-step SE” adhesives, where all components are present in a single bottle, increasing the susceptibility to water attraction and absorption, creating a permeable layer shortly after the restoration is in place .
The instability of the DAI as a consequence of hydrolysis and enzymatic processes can be identified by evaluating the bond strength, elastic modulus throughout the DAI, as well as DAI nanoleakage. Observing the changes of morphological and elastic modulus that occur over time aids in observing the modification of stress distribution across the DAI, suggesting its modified mechanical and clinical behavior . Thus, the creation of finite element models based on real mechanical and micromorphological characteristics allows the analysis of the behavior of all components of the DAI and identifies the factors that may contribute to restoration failure over time.
The aim of this study was to evaluate the effects of storage (24 h and 12 months) on the elastic modulus, micromorphology, nanoleakage expression, and micromechanical behavior of the DAI formed by five contemporary adhesive systems. The following null hypotheses were tested: (1) There would be no reduction in elastic modulus after 12 months of storage in Hanks balanced salt solution (HBSS) for any adhesive tested, (2) the storage time does not increase the level of silver impregnation at DAI, and (3) there would be no increase of stress levels after 12 months of storage in HBSS.
Material and methods
Fifty-two intact human third molars, obtained from local clinics according to a protocol approved by the institutional review board (IRB) of the Sao Paulo State University—UNESP (protocol 2009-02142), were used. All teeth were cleaned and immediately stored in saline solution (0.09%) and 0.1% thymol solution at 37 °C for up to 3 months after extraction.
The dentin substrate was exposed (Isomet 2000–Buehler Ltd., Lake Bluff, IL, USA) 3 mm above the cement–enamel junction (CEJ). A standardized smear layer was created on all dentin surfaces using 600-grit silicon carbide (SiC) papers for 1 min .
Ten teeth were used for each adhesive system ( n = 10) ( Table 1 ). The adhesives were applied following the manufactures guidelines ( Table 1 ). Two increments (1 mm thickness) of composite resin were placed over the hybridized dentin, and each layer was light-cured for 40 s (Radii Cal Dental SDI Limited, Bayswater, VIC, Australia) at a 1200 mW/cm 2 intensity. The Filtek Z350 XT (3M ESPE, St. Paul, MN, USA) composite resin was used for all adhesives, except for the Filtek LS adhesive, in which the silorane-based resin composite was used (Filtek LS, 3 M ESPE, Seefeld, BY, Germany). Each tooth was then sectioned in a mesio-distal direction (Isomet 2000, Buehler, Lake Bluff, IL, USA) to obtain at least 4 specimens, each with a 2 mm thickness. All restored specimens were aged by storage (24 h and 12 months) in HBSS, which was changed weekly to avoid dentin demineralization .
|Group||Product||pH||Classification||Application procedure||Chemical composition||Batch number|
|SBMP||Scotchbond Multi-Purpose, 3 M ESPE, St. Paul, MN, USA||A:0.1
|ER 3 steps||Apply etching gel for 15 s; air and filtre paper to keep the moisture; apply Primer for 10 s; air-dry; apply Bond for 10 s; air dry; 10 s light-cure.||A: Etchant gel: water, phosphoric acid, synthetic amorphous silica, fumed, crystalline free, polyethilene glycol, aluminum oxide
B: Primer: water, HEMA, Copolymer of acrylic and itaconic acids
C: Bond: BisGMA, HEMA.
|CSEB||Clearfil SE Bond, Kuraray Noritake Dental Inc., Tokyo, Japan||2||SE 2 steps||Apply Primer for 20 s on dentin; air dry; apply Bond for 10 s; 10 s light-cure.||Primer: HEMA, 10-MPD, Hydrophilic aliphatic dimethacrylate, dl-Camphorquinone, Water, Accelerators, Dyes, Others.
Bond: BisGMA, HEMA, 10-MDP, Hydrophobic aliphatic dimethacrylate,9 Colloidal silica, dl -Camphorquinone, Water, Accelerators, Initiators, Others.
|OUB||One Up Bond F Tokuyama Dental, Tokyo, Japan||A: 0.7
|SE 1 step||Mix Agent A and Agent B, apply for 10 s on dentin; 10 s light-cure.||Agent A: MAC-10, bisphenol A polyethoxy dimethacrylate, dubutylhydroxy toluene, methacryloxyalkyl acid phosphate (phosphoric acid monomer)m methyl methacrylate.
Agent B: 2-dimethylminoethyl methacrylate, 2-hydroxyethyl methacrylate, dibutyl hydroxy toluene, MMA, water.
|AEO||Adper Easy One, 3 M ESPE, St. Paul, MN, USA||2.4||SE 1 step||Apply for 20 s on dentin; air-dry for 5 s, 10 s light-cure.||BisGMA, HEMA, Ethanol, water, phosphoric acid-6-methacryloxy-hexylesters, Silane treated silica, 1,6-Hexanediol dimethacrylate, copolymer of acrylic and itaconic acid, ethyl metacrylate, camphorquinonem 2,46-trimethylbenzoyldiphenylphosphine oxide.||1030200148|
|LS||Filtek LS adhesive, 3 M ESPE, Seefeld, Germany||2.7 (primer)||SE 2 steps||Apply Primer for 15 s; air dry; 10 s light-cure; Apply Bond and light cure for 10 s.||Primer: BisGMA, HEMA, phosphoric acid-6-methacryloxy-hexylesters, Ethanol, water, silane treated silica,6-Hexanediol dimethacrylate, ethyl metacrylate, copolymer of acrylic and itaconic acid, phosphine oxide, dl -camphorquinone, Ethy 4-dimethyl Aminobenzoate, Methyl alcohol.
Bond: Substituted dimethacrylate, silane treated silica, TEGDMA, phosphoric acid methacryloxy-hextlesters, dl -camphorquinone, 1,6-Hexanediol dimethacrylate.
Two additional teeth (non-restored) were used for observation of dentin morphology. After the use of phosphoric acid etchant and the primer agent of Clearfil SE Bond, each tooth was sectioned perpendicular to dentin tubules for dentin observation.
One specimen of each tooth was evaluated at SEM to obtain the morphological characteristics of the DAI for each group tested, including measurements of hybrid layer and adhesive layer thickness, and length and thickness of resin tags. The non-restored dentin specimens ( n = 2) were used to measure the number and diameter of dentin tubules and the radius of peritubular dentin. These data were used to build individualized finite element models.
All restored and non-restored specimens were then immersed in 2.5% glutaraldehyde with 0.1 M sodium cacodylate buffer at pH 7.4 (Electron Microscopy Science, Hatfield, PA, USA) for 12 h at 4 °C. Afterwards, the disks were rinsed with a 0.2 M sodium cacodylate buffer at pH 7.4 (Electron Microscopy Science, Hatfield, PA, USA) for 1 h with three changes, followed by distilled water for 1 min. Specimens were dehydrated in ascending concentrations of ethanol (25% for 20 min, 50% for 20 min, 75% for 20 min, 95% for 30 min, and immersion in 100% for 60 min). The specimens were dried by immersion in hexamethyldisilazane (HMDS, Electron Microscopy Science, Hatfield, PA, USA) for 10 min, placed on a filter paper inside a covered glass vial, and air-dried at room temperature . These dentin disks were embedded in self-curing epoxy resin (Epofix, Struers, Ballerup, Denmark) and stored at room temperature for 24 h . After setting, the samples were polished with silicon carbide papers (Buehler, Lake Bluff, IL, USA) that decreased in abrasiveness (600-, 800-, and 1200-grit) and then polished with diamond solution to particle sizes 9, 6, 3, 1, and 0.5 μm (Metadi, Buehler, Lake Bluff, IL, USA) using a polishing cloth (Textmet 1000 and MicroCloth, Buehler, Lake Bluff, IL, USA).
The restored specimens were ultra-sonicated in 100% ethanol for 5 min, dried, demineralized in 6 N HCl for 30 s, and protein denatured in 2% NaOCl for 10 min . After drying, all specimens (restored and non-restored) were mounted on aluminum stubs (Electron Microscopy Science, Hatfield, PA, USA). The specimens were coated with gold using a sputter-coater (Emitech K650, Emitech Products Inc., Houston, TX, USA) at 10 mA for 4 min. The slabs were observed under SEM (Hitachi S-3500N, Hitachi Science System Ltd., Japan) at an accelerating voltage of 5 kV and a working distance of 10 mm (×2000 and ×4000). Three images were obtained from each specimen (left, center, and right), and then linear measurements were performed by PCI 5.5 Quartz software.
Two specimens of each tooth were used, one for each evaluation time (24 h and 12 months) and storage in HBSS.
The specimens were covered with two layers of nail varnish, leaving 1 mm below and above the interface exposed. Teeth were rehydrated in distilled water for 10 min, and then immersed in an ammoniacal silver nitrate solution for 24 h and stored at 37 °C in a dark room . After 24 h, each specimen was washed in water and then placed in developer solution (Kodak Professional Developer D76, Eastman Kodak Company, USA) under fluorescent light for 8 h. After the specimens were washed in running water and the varnish was removed using 600-grit sandpaper. Then, they were immersed in a pH 7.4 solution of 2.5% glutaraldehyde in 0.1 M buffered sodium cacodylate, for 12 h at 4 °C. Afterwards, they were washed in buffered 0.2 M sodium cacodylate at pH 7.4 for 1 h with 3 changes, followed by washing in distilled water for 1 min.
Dehydration was done in ascending concentrations of ethanol as described above, and then embedded in self-curing epoxy resin and stored at room temperature for 24 h . After setting, the samples were polished with silicon carbide papers of decreasing abrasiveness (600-, 800-, and 1200-grit) and then polished with diamond solutions of particle sizes 9, 6, 3, 1, and 0.5 μm using a polishing cloth. After polishing, the specimens were immersed in 10% EDTA for 5 s and then rinsed in distilled water . The specimens were mounted on aluminum stubs (Electron Microscopy Science, Hatfield, PA, USA) and coated with gold using a sputter-coater (Emitech K650, Emitech Products Inc., Houston, TX, USA) at 10 mA for 1 min. Specimens were observed under SEM (Hitachi S-3500N, Hitachi Science System Ltd., Japan) at an accelerating voltage of 15 kV, backscattered mode, and a working distance of 10 mm (×2000 and ×4000). Three images were obtained from each specimen (left, center, and right). A standard DAI area was assumed for all adhesives, and the silver impregnation area was calculated using image software (ImageJ, NIH, Bethesda, MD, USA), by averaging the silver impregnation for each adhesive, in percentage.
DAI elastic modulus
One specimen of each tooth, which was not prepared for SEM, was embedded in self-curing epoxy resin and stored at room temperature for 24 h. After setting, the samples were polished with silicon carbide papers of decreasing abrasiveness (600-, 800-, and 1200-grit) and then polished with diamond solutions of particle sizes 9, 6, 3, 1, and 0.5 μm using a polishing cloth for 3 min in each diamond solution. The specimens were ultra-sonicated in distilled water for 1 min between each step, and for 5 min as the final step.
The specimens were tested after 24 h and then stored in HBSS for 12 months, and tested again . The imaging and indentation processes were performed using a Berkovich fluid cell diamond three-sided pyramid probe in a nanoindenter (Hysitron 950TI, Minneapolis, MN, USA). The tests were performed in wet condition (thrice-distilled water, neutral pH 7.1). The equipment was calibrated with a fused silica calibration sample before the start of indentations .
A trapezoidal loading profile (5–10–5 s) was developed with a peak load of 300 μN for dentin and 100 μN for the hybrid layer and adhesive layer, at a rate of 60 μN/s and 20 μN/s followed by a holding time of 10 s and an unloading time of 5 s. The extended holding period allowed relaxation of bone surrounding the indenter and a concomitantly more linear unloading response, so that no tissue creep effect occurred during the unloading portion of the profile . Therefore, from each indentation, a load–displacement curve was obtained.
From each analyzed load–displacement curve, the reduced modulus E r (in GPa) were computed from the Hysitron TriboScan software with the following formulas, respectively:
E r = π 2 A ( h c ) × S ; H = P max A ( h c ) ;
where S is the stiffness, h c is the contact depth, P max is the maximum applied force (300- or 100 μN) and A ( h c ) is the contact area computed from the Hysitron TriboScan software taking into account the area function with respect to the contact depth . By knowing the reduced modulus E r , the correspondent elastic modulus ( E ) in GPa was calculated as follows:
1 E r = 1 − v b 2 E + 1 − v i 2 E i