Highlights
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We compared the bonding stability between superficial dentin and deep dentin.
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Bonds to deep dentin are more prone to degradation than superficial dentin.
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MMPs distribution is related to degradation of dentin bonds to different depths.
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Total-etch adhesive may lead to more MMPs activities and bonding degradation.
Abstract
Objectives
Bonding stability of resinous adhesives to dentin is still problematic and may involve regional variations in dentin composition. This study is to evaluate the effect of dentin depth on the stability of resin-dentin bonds under thermocycling challenge.
Methods
Dentin slabs with two flat surfaces parallel to the tooth axis were obtained from extracted human third molars. The slabs were randomized into eight groups according to the location of dentin [deep dentin (DD) or superficial dentin (SD)], the adhesive treatment (Single Bond 2 or Clearfil S 3 Bond), and the storage treatment (thermocycling for 5000 times vs. no). After the adhesive treatment and composite buildup on the dentin slabs, the micro-shear bond strength (μSBS) of each group was detected. The concentrations of cross-linked carboxyterminal telopeptide of type I collagen (ICTP) were also evaluated using an immunoassay to detect the degree of collagen degradation in each group.
Results
Dentin depth, adhesive treatment and storage treatment all showed significant effects on both the μSBSs and the ICTP values ( P < 0.05). Regardless of the adhesive type, thermocycling decreased the μSBSs and increased the ICTP values ( P < 0.05). The DD groups showed significantly lower μSBSs and higher ICTP values than SD groups after thermocycling aging ( P < 0.05). The treatment with Single Bond 2 significantly increased the ICTP values ( P < 0.05), whereas Clearfil S 3 Bond showed no effect on the ICTP values ( P > 0.05).
Significance
Deep dentin showed significantly more bond degradation after thermocycling than did superficial dentin.
1
Introduction
Dentin adhesives have been well developed during the past two decades and result in high immediate bonding performance. However, the bonding stability of certain contemporary resinous adhesives to dentin remains problematic . Dentin bonding is created by the formation of the so-called hybird layer . Degradation of hybrid layers has been considered as the major limitation to the stability of resin-dentin interfaces . Extensive studies have been reported on the factors that related to the degradation of hybrid layers , including the percent conversion and hydrophilicity of adhesive resins, and the host-derived proteinases as well. The effect of differences in the dentin substrate itself, which may directly affect the quality of hybrid layer, should also be taken into account and studied.
Dentin different depths present differences in morphology, structure, and chemical composition . These factors may contribute to the differences in hybrid layer that is formed within different dentin layers, and thus affect their bonding performance . Different adhesive restorations (filling, inlay, veneer, or crown) may be applied at different dentin depths according to various clinical requirements. Prolonging the clinical lifetime of adhesive restorations requires improving the stability of adhesion to dentin in different depths. Therefore, it is of great interest to evaluate the effect of dentinal substrates from different depths on the bonding stability.
Host-derived matrix metalloproteinases (MMPs) have been reported to play an important role in the collagen degradation within the hybrid layer during aging . Our previous study has found that the distribution of MMP-2 and MMP-9 was different within different depths of coronal dentin , indicating their different proteolytic potentials. However, the relationship between MMPs activity and the bonding stability in different dentin depths is still unknown and needs to be studied.
The present study was designed to evaluate the effect of dentin depth on the bonding stability of two adhesive systems (a two-step total-etch adhesive and a one-step self-etch adhesive) under thermocycling challenge. The null hypothesis to be tested were that dentin depth, adhesive treatment and storage treatment would all have no effects on either bonding strength or collagen degradation within dentin.
2
Materials and methods
Extracted, intact human third molars ( N = 187) were collected after the patients’ informed consent had been obtained under a protocol approved by the Institution Review Board. The teeth were stored in 0.9% physiological saline at 4 °C and used within 1 week after extraction.
2.1
Isolation of coronal dentin according to dentin depth
Two sections along the longitudinal axis of the tooth were first made to remove the lingual and buccal tooth tissues with 2 mm thickness, including the enamel and a thin layer of superficial dentin. The remaining middle portion of each crown was then sectioned into 3 slabs (1.3–1.5 mm thick) along the same direction. One dentin surface of the slab was marked by a line to delineate two zones of equal width to represent different dentin depths as previously described : deep dentin (DD) and superficial dentin (SD) ( Fig. 1 ). A total of five hundred and eighty seven slabs were obtained.
2.2
Micro-shear bond strength test
Eighty slabs were randomly divided into eight groups according to the type of the dentin (DD or SD), the adhesive treatments [Single Bond 2 (3M ESPE, St. Paul, MN, USA), or Clearfil S 3 Bond (Kuraray Co. Ltd., Osaka, Japan)], and the storage treatment (with or without thermocycling, Table 2 ). One dentin surface was polished with 600-grit SiC paper to create a standard smear layer and then treated with the adhesive strictly according to the manufactures’ instructions ( Table 1 ). Five micro-bore tygon tubes (inside diameter of 0.8 mm, height of 0.6 mm) were placed at different positions on the treated surfaces within the defined DD or SD layer ( Fig. 1 a). After light curing of the adhesive, the composite resin (Z250, 3M ESPE, St. Paul, MN, USA) were carefully inserted into the tubes and light-irradiated. After the storage in deionized water at 37 °C for 24 h, the tubes were carefully removed leaving the resin-bonded composite cylinders. In the thermocycling group, the specimens were immersed in a sealed metal pipe filled with sterile artificial saliva (pH 7.0) and thermo-circulated between 5 °C and 55 °C for 5000 times. The dwell time and transfer time were 60 s and 6 s respectively. The micro-shear bond strength (μSBS) was then measured using the previously described method . The failure modes of debonded specimens were assessed under a field emission scanning electron microscope (FE-SEM, S-4800, Hitachi, Tokyo, Japan). The failure modes were classified as the following : adhesive failure, mixed failure, cohesive failure in dentin, and cohesive failure in resin.
| Adhesives | Composition [lot number] | Application procedures |
|---|---|---|
| Adper Single Bond 2 (3M ESPE, St. Paul, MN, USA) | Bis-GMA; polyalkenoic acid, copolymer; dimethacrylate; CQ; HEMA; ethanol; water [N377455] | Etch with 37% phosphoric acid for 15 s, rinse with water and keep the dentin surface moist, apply the adhesive for 15 s, gently air blow, light-cure for 10 s |
| Clearfil S 3 Bond (Kuraray Co. Ltd., Osaka, Japan) | 10-MDP, dl-Camphorquinone, Hydrophobic dimethacrylate, HEMA, Bis-GMA, water, ethyl alcohol, silanated colloidal silica [00169A] | Apply the adhesive for 20 s, dry with high-pressure air for 5 s, light cure for 10 s |
| Dentin treatment | Aging treatment | Dentin depth | |
|---|---|---|---|
| Superficial dentin (SD) | Deep dentin (DD) | ||
| Single Bond 2 | No | 54.05 (6.06) a,A | 51.09 (8.01) a,A |
| Thermocycling | 37.06 (5.17) a,B | 30.58 (7.23) b,B | |
| Clearfil S 3 Bond | No | 39.92 (8.72) a,B | 37.69 (4.24) a,C |
| Thermocycling | 30.89 (6.72) a,C | 18.95 (7.43) b,D | |
2.3
MMP-mediated collagen degradation detection
The concentration of cross-linked carboxyterminal telopeptide of type I collagen (ICTP) was detected to evaluate the degree of MMPs-mediated collagen degradation. A beam (0.75 mm × 0.75 mm × 5.0 mm) was obtained from the DD layer or SD layer of the slab ( Fig. 1 b). Nine hundred sixty beams were obtained from 480 slabs. All beams were randomly divided into twelve groups according to the region of the beam, the adhesive treatments (no treatment as control, Single Bond 2, and Clearfil S 3 Bond), and thermocycling treatment ( Table 3 ). The beams in each group were dried and divided into four samples, each sample containing approximately 20 beams with the total weight of 100 mg . In the adhesive treated groups, the adhesive was applied to the four surfaces of the acid etched beam in the Single Bond 2 group or to similar layer-covered dentin in the S3 group. Then the adhesive was light-cured. The specimens of each group were immersed in 400 μl of sterile artificial saliva containing 50 U ml −1 of penicillin G and 1500 μg ml −1 of streptomycin (pH 7.0) (Sigma–Aldrich, St. Louis, MO, USA). After agitating the incubated beams at 37 °C for the specified time-points, 200 μl of supernatants from each group were collected and diluted four-fold. Then the supernatants were immediately used to determine the concentration of the ICTP with an enzyme immunoassay kit (ICTP-EIA, Cat. No. 05892, Orion Diagnostica, Espoo, Finland). Each test was conducted in triplicate. Standards with known concentrations within range from 0.01 μg/l to 250 μg/l were firstly used to construct the standard curve. Only data within the confidence interval were considered to be significant and applied to the calculation. The final ICTP value for each group were presented as ng telopeptide per mg dry mineralized dentin.
| Dentin treatment | Aging treatment | Dentin depth | |
|---|---|---|---|
| SD | DD | ||
| Control | No | 0.009 (0.005) 1,a,A | 0.048 (0.012) 2,a,A |
| Thermocycling | 0.084 (0.013) 1,b,A | 0.242 (0.017) 2,b,A | |
| Single Bond 2 | No | 0.079 (0.008) 1,a,B | 0.331 (0.025) 2,a,B |
| Thermocycling | 1.554 (0.159) 1,b,B | 3.192 (0.312) 2,b,B | |
| Clearfil S 3 Bond | No | 0.010 (0.004) 1,a,A | 0.050 (0.005) 2,a,A |
| Thermocycling | 0.186 (0.017) 1,b,A | 0.372 (0.031) 2,b,A | |
2.4
Statistical analysis
Kolmogorov-Smirnov test and Levene’s test were first performed to confirm the normality and equal variance assumptions of the data. The results of μSBS and ICTP values were analyzed with three-way ANOVA (dentin depth, adhesive treatment, and storage treatment as tested variables). Post hoc multiple comparisons were performed using Tukey’s test. The results of failure modes were evaluated using the Chi-Square test. Statistical significances in all tests were preset at P < 0.05, using the SPSS 14.0 software package (SPSS, Chicago, IL, USA).
2
Materials and methods
Extracted, intact human third molars ( N = 187) were collected after the patients’ informed consent had been obtained under a protocol approved by the Institution Review Board. The teeth were stored in 0.9% physiological saline at 4 °C and used within 1 week after extraction.
2.1
Isolation of coronal dentin according to dentin depth
Two sections along the longitudinal axis of the tooth were first made to remove the lingual and buccal tooth tissues with 2 mm thickness, including the enamel and a thin layer of superficial dentin. The remaining middle portion of each crown was then sectioned into 3 slabs (1.3–1.5 mm thick) along the same direction. One dentin surface of the slab was marked by a line to delineate two zones of equal width to represent different dentin depths as previously described : deep dentin (DD) and superficial dentin (SD) ( Fig. 1 ). A total of five hundred and eighty seven slabs were obtained.
