Abstract
Objectives
This study examined the effect of a dimethyl sulfoxide (DMSO) wet bonding technique on the resin infiltration depths at the bonded interface and dentin bond strength of different adhesive systems.
Methods
Flat dentin surfaces of 48 human third molars were treated with 50% DMSO (experimental groups) or with distilled water (controls) before bonding using an etch-and-rinse (SBMP: Scotchbond Multi-Purpose, 3M ESPE) or a self-etch (Clearfil: Clearfil SE Bond, Kuraray) adhesive system. The restored crown segments ( n = 12/group) were stored in distilled water (24 h) and sectioned for interfacial analysis of exposed collagen using Masson’s Trichrome staining and for microtensile bond strength testing. The extent of exposed collagen was measured using light microscopy and a histometric analysis software. Failure modes were examined by SEM. Data was analyzed by two-way ANOVA followed by Tukey Test ( α = 0.05).
Results
The interaction of bonding protocol and adhesive system had significant effects on the extension of exposed collagen matrix ( p < 0.0001) and bond strength ( p = 0.0091). DMSO-wet bonding significantly reduced the extent of exposed collagen matrix for SBMP and Clearfil ( p < 0.05). Significant increase in dentin bond strength was observed on DMSO-treated specimens bonded with SBMP ( p < 0.05), while no differences were observed for Clearfil ( p > 0.05).
Significance
DMSO-wet bonding was effective to improve the quality of resin–dentin bonds of the tested etch-and-rinse adhesives by reducing the extent of exposed collagen matrix at the base of the resin–dentin biopolymer. The improved penetration of adhesive monomers is reflected as an increase in the immediate bond strength when the DMSO-wet bonding technique is used with a water-based etch-and-rinse adhesive.
1
Introduction
Modern dental restorations rely on the adhesives providing adequate bonds between the tooth and the restorative composite. Several approaches to enhance the bond strength of adhesive system to dentin have been studied in the past decade . Even though they have produced promising in vitro results, some concepts defy the principles of user friendliness and technique simplification. Clinically feasible acceptable methods to enhance dentin adhesion improving the collagen–resin biopolymer are still needed.
Current bonding systems to dentin rely on effective adhesive penetration into dentin substrate to form the hybrid layer and on chemical interactions between residual hydroxyapatite and specific functional monomers found mainly in self-etch adhesives . The resultant micro-mechanical interlocking formed after dentin hybridization is a prerequisite to achieve adequate dentin bonding . Ideally, such resin–dentin interfusion zone should form a continuous and stable tooth-restoration interconnection. However, this objective is not achieved by contemporary adhesives .
Dimethyl sulfoxide (DMSO; (CH 3 ) 2 SO) is a polar aprotic solvent that dissolves both polar and non-polar compounds. It is a polyfunctional molecule with a highly polar S O group and two hydrophobic CH 3 groups. DMSO is fully miscible in solvents and most adhesive monomers used in adhesive dentistry. Moreover, DMSO has the ability to dissociate the highly cross-linked collagen into a sparser network of apparent fibrils also in dentin matrix , most likely by the suppression of hydrogen bond-mediated attractive forces within the collagen . This allows DMSO to efficiently penetrate biological surfaces, which makes it perhaps the best currently known penetration enhancer for medical purposes .
In a recent study, a considerably low concentration of DMSO was shown to reduce dentin bond strength loss after aging . Considering DMSO properties, higher concentrations might have a positive effect on dentin bonding. Since adhesive resins cannot completely infiltrate collagen matrices in demineralized dentin , and since monomer diffusion into the dentin substrate is critical to proper dentin bonding , the aim of this study was to investigate the effect of high DMSO concentration on dentin bonding. The hypothesis set were that dentin pretreatment with DMSO would: (i) reduce the amount of exposed collagen matrix at the base of the hybrid layer and (ii) increase immediate dentin bond strength values of commercially available adhesive systems.
2
Materials and methods
2.1
Tooth preparation
Forty-eight recently extracted non-carious human third molars were obtained after patient informed consent under a protocol approved by the Ethical Committee of the Piracicaba Dental School, University of Campinas, Brazil (protocol 017/2013). Teeth were cleaned, ultrasonicated in water for 5 min for cleaning, disinfected for 1 week in 0.5% chloramine-T solution at 4 °C, and stored in distilled water at 4 °C for up to 1 month before use. A flat coronal dentin surface was obtained by sectioning off the occlusal surface (Isomet 1000 Precision Saw, Buehler, Lake Bluff, IL, USA). The surface roughness was standardized with 600-grit silicon carbide paper (BuehlerMet, Buehler) for 1 min under water cooling.
2.2
Dentin bonding
The teeth were randomly assigned to four groups ( n = 12) according to the adhesive/bonding technique: (i) three-step etch-and-rinse adhesive (Adper Scotchbond Multi-Purpose, 3M ESPE, St. Paul, MN, USA) (SBMP); (ii) DMSO-wet bonding with SBMP; (iii) two-step self-etch adhesive (Clearfil SE Bond, Kuraray, Osaka, Japan) (Clearfil); and (iv) DMSO-wet bonding with Clearfil. DMSO-wet bonding 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. For SBMP, DMSO was applied after dentin etching; for Clearfil, DMSO was applied onto smear layer-covered dentin. Control groups consisted of distilled water application for 60 s instead of DMSO. Table 1 displays mode of application, components and manufacturers of the adhesives. 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 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 an LED device (Bluephase 20i, Ivoclare Vivadent, Schaan, Liechtenstein). All procedures were carried out by a single operator.
| Adhesive system | Components | Mode of application (Control/DMSO wet-bonding) |
|---|---|---|
| Adper Scotchbond Multi-Purpose 3M/ESPE—Batch #N451356 | ||
| Etchant | 37% phosphoric acid, fumared silica (pH 0.6) | H 3 PO 4 conditioning for 15 s. Rinse with water spray 30 s. Blot drying leaving dentin slight moist. Active application of either distilled water for 60 s (control), or 50% DMSO for 60 s (DMSO wet-bonding). Blot drying. Active Primer application with a fully saturated brush tip 10 s. Gently blow dry 5 s. Active Bond application 10 s. Light cure for10 s. |
| Primer | HEMA, polyalkenoic acid methacrylate copolymer, water | |
| Bond | bis-GMA, HEMA, dimethacrylates photoinitiators | |
| Clearfil SE Bond Kuraray—Batch #41491 | ||
| Primer | 10-MDP; HEMA; CQ; hydrophilic dimethacrylate; water (pH 2.0) | Blot drying until no sign of excess visible moisture was observed. Active application of either distilled water for 60 s (control), or 50% DMSO for 60 s (DMSO wet-bonding). Blot drying. active Primer application with a fully saturated brush tip for 20 s . Mild air stream for 5 s. Active Bond application. Gentle air stream 5 s. Light cure for 10 s. |
| Bond | 10-MDP; N , N -diethanol- p -toludine; HEMA; bis-GMA; silanated colloidal silica; hydrophobic dimethacrylate; CQ; MDPB | |
2.3
Specimen preparation
After storage in distilled water at 37 °C for 24 h, the restored segments were sectioned (Isomet 1000 Precision Saw, Buehler) occluso-gingivally into four slabs measuring approximately 0.9 mm. One composite-dentin slab was randomly reserved for morphologic analysis of the adhesive interface with an optical microscope to evaluate the presence of demineralized but unprotected collagen matrix at the base of the hybrid layer. The remaining slabs were further sectioned into composite-dentin sticks, 0.8 mm 2 cross-sectional area, in accordance with the “non-trimming” technique for bond strength testing. A minimum of nine sticks were obtained from each tooth.
2.4
Optical microscopy
One slab from each tooth measuring 0.9 mm in thickness was selected ( n = 12/group). The slabs were fixed in a glass slide with a cyanoacrylate adhesive (Super Bonder, Loctite, São Paulo, SP, Brazil) and hand-polished with SiC papers of increasingly fine grits (600, 1000, 1200 and 4000) under water cooling until its thickness was approximately 10 μm. Specimen were treated with Masson’s trichromic acid staining technique. Briefly, the polished specimen were immersed in Bouin’s solution for 1 h at 56 °C, rinsed with distilled water, and incubated in Weigert’s hematoxylin for 10 min. They were then washed with distilled water for 10 min, incubated in acidic scarlet fuchsine for 3 min, rinsed with distilled water, immersed in phosphomolybdic acid for 10 min, and immediately stained with light green for 10 min. Finally, the specimen were rinsed with distilled water and submerged in glacial acetic acid for 3 min.
Masson dye has a high affinity for cationic elements of normally mineralized type I collagen, staining collagen green. Etching with phosphoric acid removes these elements from collagen resulting in different coloration, generally red. Specimens were examined in an optical microscope (Leica DMLP, Leica Microsystems, Heerbrugg, Switzerland) at 400× magnification. In each slab the width of a red band corresponding to demineralized dentin with exposed collagen was analyzed. Minimum of five digital images of the entire bonded interface of each slab were obtained using an open-source image software (ImageJ, National Institute of Health, Bethesda, MD, USA). From each image, six measurements of the width of the red band were performed. To evenly distribute measurements across the entire bonding interface, each image was divided into three parts and two measurements were performed in each equidistant from each other. All in all, over 1440 measurements of the width of exposed collagen were done in this study. Measurements were performed by one single-blinded examiner. The average width of the red band for all images in each slab was calculated and corresponded for the extent (μm) of exposed collagen layer at the base of the hybrid layer for the corresponding tooth.
2.5
Microtensile bond strength test (μTBS)
Sticks were individually attached to a Geraldeli Device (Odeme, Santa Catarina, Brazil) with a cyanoacrylate adhesive (Zapit, Dental Ventures of America, Corona, CA, USA) and submitted to the μTBS test (DL2000, EMIC, São José dos Pinhais, Brazil) at crosshead speed of 0.5 mm/min until failure. The cross-sectional area of each stick was measured with a digital caliper (Absolute Digimatic, Mitutoyo, Tokyo, Japan) to the nearest 0.01 mm to calculate the actual bond strength (MPa). Bond strengths for each tooth were determined by the μTBS average values of a minimum of nine sticks. Sticks with premature failures were recorded as 0 MPa for the statistical analysis.
2.6
Failure mode analysis
Both sides of fractured sticks were chemically dehydrated in ascending ethanol concentrations (50–90%) for 1 h in each and for 2 h in 100% ethanol, and finally by immersion in hexamethyldisilazane for 10 min on filter paper inside a covered glass vial, and air dried at room temperature. The specimens were mounted on aluminum stubs, gold-sputtered and analyzed under magnification of 100–6000× using a scanning electron microscope (LEO 435 VP; LEO Electron Microscopy Ltd, Cambridge, UK) operating on secondary electron mode at 15 kV. Failure modes were classified as adhesive (A), cohesive failure in dentin (CD), cohesive failure in resin composite (CC), and mixed failure (M) .
2.7
Statistical analyses
Data from the μTBS tests and the exposed collagen zone analysis were normally distributed (Kolmogorov-Smirnov Test) and homoscedastic (Levene Test). Data were analyzed separately with a two-way ANOVA design (adhesive system and bonding protocol). Post hoc multiple comparisons were performed with Tukey Studentized Range (HSD) Test, with statistical significance set at α = 0.05 using SAS statistical software (SAS 9.4 Software, SAS Institute, NC, USA). Tooth was considered the statistical unit.
2
Materials and methods
2.1
Tooth preparation
Forty-eight recently extracted non-carious human third molars were obtained after patient informed consent under a protocol approved by the Ethical Committee of the Piracicaba Dental School, University of Campinas, Brazil (protocol 017/2013). Teeth were cleaned, ultrasonicated in water for 5 min for cleaning, disinfected for 1 week in 0.5% chloramine-T solution at 4 °C, and stored in distilled water at 4 °C for up to 1 month before use. A flat coronal dentin surface was obtained by sectioning off the occlusal surface (Isomet 1000 Precision Saw, Buehler, Lake Bluff, IL, USA). The surface roughness was standardized with 600-grit silicon carbide paper (BuehlerMet, Buehler) for 1 min under water cooling.
2.2
Dentin bonding
The teeth were randomly assigned to four groups ( n = 12) according to the adhesive/bonding technique: (i) three-step etch-and-rinse adhesive (Adper Scotchbond Multi-Purpose, 3M ESPE, St. Paul, MN, USA) (SBMP); (ii) DMSO-wet bonding with SBMP; (iii) two-step self-etch adhesive (Clearfil SE Bond, Kuraray, Osaka, Japan) (Clearfil); and (iv) DMSO-wet bonding with Clearfil. DMSO-wet bonding 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. For SBMP, DMSO was applied after dentin etching; for Clearfil, DMSO was applied onto smear layer-covered dentin. Control groups consisted of distilled water application for 60 s instead of DMSO. Table 1 displays mode of application, components and manufacturers of the adhesives. 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 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 an LED device (Bluephase 20i, Ivoclare Vivadent, Schaan, Liechtenstein). All procedures were carried out by a single operator.
| Adhesive system | Components | Mode of application (Control/DMSO wet-bonding) |
|---|---|---|
| Adper Scotchbond Multi-Purpose 3M/ESPE—Batch #N451356 | ||
| Etchant | 37% phosphoric acid, fumared silica (pH 0.6) | H 3 PO 4 conditioning for 15 s. Rinse with water spray 30 s. Blot drying leaving dentin slight moist. Active application of either distilled water for 60 s (control), or 50% DMSO for 60 s (DMSO wet-bonding). Blot drying. Active Primer application with a fully saturated brush tip 10 s. Gently blow dry 5 s. Active Bond application 10 s. Light cure for10 s. |
| Primer | HEMA, polyalkenoic acid methacrylate copolymer, water | |
| Bond | bis-GMA, HEMA, dimethacrylates photoinitiators | |
| Clearfil SE Bond Kuraray—Batch #41491 | ||
| Primer | 10-MDP; HEMA; CQ; hydrophilic dimethacrylate; water (pH 2.0) | Blot drying until no sign of excess visible moisture was observed. Active application of either distilled water for 60 s (control), or 50% DMSO for 60 s (DMSO wet-bonding). Blot drying. active Primer application with a fully saturated brush tip for 20 s . Mild air stream for 5 s. Active Bond application. Gentle air stream 5 s. Light cure for 10 s. |
| Bond | 10-MDP; N , N -diethanol- p -toludine; HEMA; bis-GMA; silanated colloidal silica; hydrophobic dimethacrylate; CQ; MDPB | |
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