A new wet-bonding technique using DMSO is proposed.
The mechanism relies on DMSO displacing free water from dentin.
This increases bond strength of etch-and-rinse and self-etch adhesives even after aging.
Formation of hybrid layers with higher hydrolytic stability and reduced nanoleakage.
DMSO-saturated dentin benefits the degree of conversion of self-etch adhesives.
This study evaluated a new approach, named dimethyl sulfoxide (DMSO)-wet bonding, to produce more desirable long-term prospects for the ultrafine interactions between synthetic polymeric biomaterials and the inherently hydrated dentin substrate.
Sound third molars were randomly restored with/without DMSO pretreatment using a total-etch (Scocthbond Multipurpose: SBMP) and a self-etch (Clearfil SE Bond: CF) adhesive systems. Restored teeth (n = 10)/group were sectioned into sticks and submitted to different analyses: micro-Raman determined the degree of conversion inside the hybrid layer (DC); resin–dentin microtensile bond strength and fracture pattern analysis at 24 h, 1 year and 2 years of aging; and nanoleakage evaluation at 24 h and 2 years.
DMSO-wet bonding produced significantly higher 24 h bond strengths for SBMP that were sustained over the two-year period, with significantly less adhesive failures. Similarly, DMSO-treated CF samples presented significantly higher bond strength than untreated samples at two years. Both adhesives had significant less adhesive failures at 2 years with DMSO. DMSO had no effect on DC of SBMP, but significantly increased the DC of CF. DMSO-treated SBMP samples presented reduced silver uptake compared to untreated samples after aging.
Biomodification of the dentin substrate by the proposed strategy using DMSO is a suitable approach to produce more durable hybrid layers with superior ability to withstand hydrolytic degradation over time. Although the active role of DMSO on dentin bond improvement may vary according to monomer composition, its use seems to be effective on both self-etch and etch-and-rinse bonding mechanisms.
Adhesion of resin materials to tooth structure has been a challenge in the history of adhesive dentistry. Currently, the issue of bond durability has attracted significant attention regarding resin–dentin bonding . Despite improvements in dental adhesive technology and advances in bonding knowledge, resin–dentin bonding still shows limited durability for both etch-and-rinse and self-etch adhesive systems .
Resin–dentin bonding is a unique form of tissue engineering in which an ultrafine biopolymer, known as hybrid layer, links composites to the underlying mineralized dentin by two substantially different bonding mechanisms produced by the adhesive system used: etch-and-rinse or self-etch . Nevertheless, resin–dentin bonds created by infiltration of hydrophilic resin monomers into demineralized and mineralized dentin are imperfect and unstable . Inadequate polymerization reduces the quality of the hybrid layer leading to lower dentin bond strengths and increased nanoleakage . Moreover, high permeability of the bonded interface and phase separation during adhesive application contribute to hydrolytic degradation of the adhesive resin . Insufficient resin impregnation of dentin is associated with the collagenolysis of unprotected collagen fibrils by endogenous matrix metalloproteinases (MMPs) and cysteine cathepsins . Irrespective of adhesive type, hydrolytic degradation of the adhesive resin and collagen matrix degradation occur concurrently, for resin elution from hydrolytically unstable polymeric hydrogels within the hybrid layers increases the exposure of unprotected collagen matrix over time.
Several adjunctive procedures have been suggested to prevent biodegradation of hybrid layers over time . Although encouraging results have been produced, the current available techniques do not effectively address both hydrolytic degradation of the adhesive resin and collagen degradation concurrently. The possible exception is the ethanol-wet bonding aiming to remove water from the exposed dentin collagen and to replace it with more hydrophobic resin components . Excluding water with high ethanol concentrations would reduce/eliminate the hydrolytic degradation of both the collagen and resin components of the hybrid layer . Unfortunately, ethanol-wet bonding is clinically unfeasible due to technique sensitivity and increase in application steps and treatment time . Thus, current strategies are at least partially limited in their true potential to optimize the durability of resin–dentin bonding.
Dimethyl sulfoxide (DMSO; (CH 3 ) 2 SO) is a polar aprotic solvent with a highly polar S O group and two hydrophobic CH 3 groups. Its ability to penetrate biological surfaces and tissues makes it the best penetration enhancer for medical purposes . Recent studies have indicated that DMSO may improve the penetration of adhesive into the exposed collagen matrix , and improve both immediate and long-term dentin bond strength. However, the long-term efficacy has only been demonstrated with a two-step etch-and-rinse adhesive . Therefore, this in vitro study evaluated the effect of DMSO-wet bonding on dentin bond durability, monomer conversion inside the hybrid layer and the quality of aged bonded interfaces of two-step self-etch and three-step etch-and-rinse adhesives after 1 and 2 year storage. The null hypotheses to be tested were that irrespective of adhesive type, the application of 50 vol% DMSO in water on dentin: (i) would not influence monomer conversion at the hybrid layer; (ii) would not affect immediate or long-term dentin bond strength and; (iii) would not improve the adhesive interface quality regarding the formation of nanoleakage channels.
Materials and methods
Teeth selection and preparation
Forty intact human third molars with complete root formation were extracted for surgical reasons with patients’ (age 18–25 years) informed consent and approval by the local Ethical Committee under protocol number 110/2014. Teeth were cleaned, disinfected for one week in 0.5% chloramine-T solution at 4 °C, and stored in distilled water at 4 °C for up to one month before use. A flat coronal dentin surface was obtained by sectioning off the occlusal one-third of the crown (Isomet 1000 Precision Saw, Buehler, Lake Bluff, IL, USA). The surface roughness was standardized with 600-grit silicon carbide paper (CarbiMet, Buehler Ltd., Lake Bluff, IL, USA) for 60 s under water cooling and the specimens were randomly assigned to four groups (n = 10) according to the bonding protocols.
Dentin bonding protocol
Two commercially available unaltered adhesive systems were used ( Table 1 ): a three-step etch-and-rinse adhesive system (Adper Scotchbond Multi-Purpose, 3M ESPE, St. Paul, MN, USA) (SBMP) and a two-step self-etch adhesive (Clearfil SE Bond, Kuraray, Osaka, Japan) (CF). Table 1 lists the mode of application, components and manufacturers of the adhesive systems.
|Adhesive system||Components||Application mode (control/DMSO wet-bonding)|
|Adper Scotchbond Multi-Purpose 3M/ESPE||(1) H 3 PO 4 conditioning for 15 s; (2) rinse with water 30 s; (3) blot drying leaving dentin slight moist; (4) active application of 50% DMSO for 60 s (DMSO wet-bonding), or no dentin treatment (control); (5) blot drying; (6) active Primer application with a fully saturated brush tip 10 s; (7) gently blow dry 5 s; (8) active Adhesive application 10s; and (9) Light cure for 10 s.|
|Etchant||35% phosphoric acid, fumed silica (pH 0.6)|
|Primer||HEMA, polyalkenoic acid methacrylate copolymer, water|
|Adhesive||Bis-GMA, HEMA, dimethacrylates, photoinitiators|
|Clearfil SE bond Kuraray||(1) Blot drying until no sign of excess visible moisture was observed; (2) active application of 50% DMSO for 60 s (DMSO wet-bonding), or no dentin treatment (control); (3) blot drying until no visible moisture was observed; (4) active Primer application with a fully saturated brush tip for 20 s; (5) mild air stream for 5 s; (6) active Adhesive application; (7) gentle air stream 5 s; and (8) light cure for 10 s.|
|Primer||10-MDP; HEMA; CQ; hydrophilic dimethacrylate; water (pH 2.0)|
|Adhesive||10-MDP; N,N-diethanol-p-toludine; HEMA; Bis-GMA; silanated colloidal silica; hydrophobic dimethacrylate; CQ|
Dentin bonding in control groups was performed according to Table 1 . In experimental groups, the DMSO-wet bonding technique was employed, which consisted of light-pressure circular scrubbing movements of a 50 μL of water-based 50% (v/v) DMSO (dimethyl sulfoxide, Sigma–Aldrich, St. Louis, MO, USA) (pH 8.2) for 60 s, using a disposable cavity brush. In SBMP groups, DMSO was applied after dentin etching and water rinsing. In CF groups, DMSO was applied onto smear layer-covered dentin before the primer application. In all groups, SBMP primer was applied onto partially wet dentin, while CF primer was applied on DMSO wetted dentin. Both adhesive systems were applied actively. Adhesive procedures were carried out in a controlled environment with a temperature of 24 °C and a relative humidity of 55–60%. Resin composite build-ups (Z250, shade A2, 3M ESPE) were built on top of the bonded dentin surfaces in four 1-mm increments that were individually light-cured for 20 s. Light curing of all resin materials was performed using a LED device (Bluephase 20i, Ivoclare Vivadent, Schaan, Liechtenstein) in high power mode (1200 mW/cm 2 ). All bonding procedures were carried out by a single operator.
The restored crown segments were stored in distilled water at 37 °C for 24 h, to allow water sorption and postoperative polymerization of the adhesive and resin composite to take place, and sectioned (Isomet 1000 Precision Saw, Buehler) occluso-gingivally across the bonded interface into slabs measuring approximately 0.8 mm. The slabs were further sectioned into composite-dentin sticks, pursuing a final cross sectional area of approximately 0.7 mm 2 in accordance with the “non-trimming” technique for bond strength testing. A minimum of 24 sticks were obtained from each tooth.
Sticks were stored for up to two years at 37 °C in artificial solution (pH 7.1) containing (mmol/L): CaCl 2 (0.7), MgCl 2 ·6H 2 O (0.2), KH 2 PO 4 (4.0), KCl (30), NaN 3 (0.3), and HEPES buffer (20) . The storage solution was prepared and changed weekly in accordance with a protocol previously described by Pashley et al. .
Degree of conversion (DC) inside the hybrid layer measurements
Two sticks from each tooth (n = 10) were randomly evaluated at 24 h. Sticks were wet-polished with 600; 1000 and 2000-grit SiC paper (Buehler Ltd., Lake Bluff, IL, USA), ultrasonically cleaned for 2 min between polishing steps and 20 min after the last step. Raman spectra were collected using a micro-Raman spectrometer (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 approximately 3 μm, spectral resolution approximately 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. One site was examined in each stick. 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 hybrid layer was calculated according to the following formula: