d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1245–1251
Available online at www.sciencedirect.com
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Stability of bonds made to superficial vs. deep dentin, before and after thermocycling Ling Zhang a,1 , Dan-yang Wang a,b,1 , Jing Fan c , Fang Li a , Yu-jiang Chen a , Ji-hua Chen a,∗ a
State Key laboratory of Military Stomatology, Department of Prosthodontics, School of Stomatology, Fourth Military Medical University, Xi’an, China b Department of Stomatology, Xi’an medical university, Xi’an, China c Department of Endocrine & Vascular Surgery, Xi’jing Hospital, Fourth Military Medical University, Xi’an, China
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objectives. Bonding stability of resinous adhesives to dentin is still problematic and may
Received 2 October 2013
involve regional variations in dentin composition. This study is to evaluate the effect of
Received in revised form
dentin depth on the stability of resin-dentin bonds under thermocycling challenge.
13 July 2014
Methods. Dentin slabs with two flat surfaces parallel to the tooth axis were obtained from
Accepted 8 August 2014
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 S3 Bond), and the storage treatment (thermocycling for 5000
Keywords:
times vs. no). After the adhesive treatment and composite buildup on the dentin slabs, the
Stability
micro-shear bond strength (SBS) of each group was detected. The concentrations of cross-
Dentin depth
linked carboxyterminal telopeptide of type I collagen (ICTP) were also evaluated using an
Matrix metalloproteinases
immunoassay to detect the degree of collagen degradation in each group.
Microshear bond strength
Results. Dentin depth, adhesive treatment and storage treatment all showed significant
Cross-linked carboxyterminal
effects on both the SBSs and the ICTP values (P < 0.05). Regardless of the adhesive type,
telopeptide of type I collagen
thermocycling decreased the SBSs and increased the ICTP values (P < 0.05). The DD groups showed significantly lower SBSs 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 S3 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. © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
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 [1]. Dentin bonding is created by the formation of the socalled hybird layer [2]. Degradation of hybrid layers has been considered as the major limitation to the stability of resin-
∗ Corresponding author at: School of Stomatology, Fourth Military Medical University, 145 West Changle Road, Xi’an 710032, China. Tel.: +86 29 8477 6329; fax: +86 29 8477 6329. E-mail address: [email protected] (J.-h. Chen). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.dental.2014.08.362 0109-5641/© 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
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dentin interfaces [1]. Extensive studies have been reported on the factors that related to the degradation of hybrid layers [1,3,4], 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 [5–7]. These factors may contribute to the differences in hybrid layer that is formed within different dentin layers, and thus affect their bonding performance [8]. 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 [1,3]. Our previous study has found that the distribution of MMP-2 and MMP-9 was different within different depths of coronal dentin [9], 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. depth
Isolation of coronal dentin according to dentin
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 [9]: 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 S3 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. 1a). 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 resinbonded 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
Fig. 1 – Schematic drawing of the specimens preparation.
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Table 1 – Resinous adhesives used for test. Adhesives
Composition [lot number]
Adper Single Bond 2 (3M ESPE, St. Paul, MN, USA)
Bis-GMA; polyalkenoic acid, copolymer; dimethacrylate; CQ; HEMA; ethanol; water [N377455]
Clearfil S3 Bond (Kuraray Co. Ltd., Osaka, Japan)
10-MDP, dl-Camphorquinone, Hydrophobic dimethacrylate, HEMA, Bis-GMA, water, ethyl alcohol, silanated colloidal silica [00169A]
Application procedures 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 Apply the adhesive for 20 s, dry with high-pressure air for 5 s, light cure for 10 s
Abbreviations: Bis-GMA, bis-phenol A diglycidyl-methacrylate; HEMA, 2-hydroxylethyl methacrylate; CQ, camphoroquinone; 10-MDP, 10methacryloyloxydecyl dihydrogen phosphate.
Table 2 – Micro-shear bond strengths to different dentin depths [means (SD), MPa]. Dentin treatment
Aging treatment
Single Bond 2
No Thermocycling
54.05 (6.06)a,A 37.06 (5.17)a,B
51.09 (8.01)a,A 30.58 (7.23)b,B
Clearfil S3 Bond
No Thermocycling
39.92 (8.72)a,B 30.89 (6.72)a,C
37.69 (4.24)a,C 18.95 (7.43)b,D
Dentin depth Superficial dentin (SD)
Deep dentin (DD)
N = 50 per group. For each horizontal row, values with same lowercase letters indicate no significant difference (P > 0.05). For each vertical column, values with same uppercase letters indicate no significant difference (P > 0.05).
were 60 s and 6 s respectively. The micro-shear bond strength (SBS) was then measured using the previously described method [10]. 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 [11]: adhesive failure, mixed failure, cohesive failure in dentin, and cohesive failure in resin.
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. 1b). 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 S3 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 [12]. 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 l of sterile artificial saliva containing 50 U ml−1 of penicillin G and 1500 g 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 l 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 g/l to 250 g/l were firstly used to construct the standard curve. Only data within the confidence interval were considered to be significant and
Table 3 – ICTP values of dentin specimens from different depths [means (SD), ng telopeptide/mg dry mineralized dentin]. Dentin treatment
Aging treatment
Dentin depth
Control
No Thermocycling
0.009 (0.005)1,a,A 0.084 (0.013)1,b,A
0.048 (0.012)2,a,A 0.242 (0.017)2,b,A
Single Bond 2
No Thermocycling
0.079 (0.008)1,a,B 1.554 (0.159)1,b,B
0.331 (0.025)2,a,B 3.192 (0.312)2,b,B
Clearfil S3 Bond
No Thermocycling
0.010 (0.004)1,a,A 0.186 (0.017)1,b,A
0.050 (0.005)2,a,A 0.372 (0.031)2,b,A
SD
DD
For each horizontal row, values with identical numbers indicate no significant difference (P > 0.05). For each vertical column, values with identical lowercase letters indicate no significant difference between aging treatments within the same dentin treatment (P > 0.05), and values with identical uppercase letters indicate no significant difference between dentin treatments within the same aging treatment (P > 0.05).
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applied to the calculation. The final ICTP value for each group were presented as ng telopeptide per mg dry mineralized dentin.
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 SBS 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).
3.
Results
3.1.
Micro-shear bond strength
The three factors of dentin depth, aging treatment, and storage treatment all showed significant effects on SBSs (P < 0.05). Interaction between dentin depth and storage treatment was also significant (P < 0.05). The SBSs to different tested groups were summarized in Table 2. For the two adhesives tested, immediate SBSs between SD and DD groups showed no significant difference (P > 0.05). After thermocycling, the SBS of DD group was significantly lower than that in SD group (P < 0.05). Within each dentin layer, thermocycling treatment significantly decreased the SBSs (P < 0.05). Chi-Square test revealed that there were significant differences in failure modes within tested groups (P < 0.05). The failure distribution in each tested group was presented in Fig. 2. No cohesive failures in dentin were observed in this study. The failure mode presented a similar distribution for the two adhesives tested. The mixed failure was the most frequent mode of failure in all SD groups and the DD groups without thermocycling treatments. After thermocycling, the percentage of adhesive failures in the DD groups increased and became the most prevalent mode of failure. The representative failure modes of all groups are presented in Fig. 3. Regardless of the adhesive type, SD groups showed a similar pattern of mixed failure both before (Fig. 3a and e) and after (Fig. 3b and f) the thermocycling treatment. Without thermocycling treatment, DD specimens also showed a mixed failure at the bottom of the hybrid layer (Fig. 3c and g). After thermocycling for 5000 cycles, the characteristic pattern of adhesive failure was presented in DD groups for both Single Bond 2 and Clearfil S3 Bond (Fig. 3d and h): a fracture at the bottom of hybrid layer, with the dentin tubules opening widely and resin tags being completely pulled out.
3.2.
ICTP values
ANOVA analysis showed that the ICTP values were significantly affected by the three tested variables (P < 0.001). The interactions between dentin depths and adhesive treatments were also significant (P < 0.05). Regardless of the effect of adhesive and storage treatments, the ICTP values of DD groups
were significantly higher than those of SD groups (P < 0.05, Table 3). Thermocycling significantly increased the ICTP values of dentin specimens (P < 0.05). Compared with the control group, the treatment with Single Bond 2 could significantly increase the ICTP values (P < 0.05). On the other hand, the treatment with Clearfil S3 Bond showed no effect on the ICTP values (P > 0.05).
4.
Discussion
According to the results of the present study, dentin depth, adhesive treatment and thermocycling treatment all showed significant effects on the micro-shear bond strengths and the ICTP values. Thus, the null hypothesis should be rejected. The bonding performance at different dentin depths remains controversial in literature. Some studies reported that deep dentin was difficult to bond due to the low contents of intertubular dentin and collagen fibrils, as well as the high water content [13,14]. Other studies reported that deep dentin presented higher bond strength than superficial dentin [15,16]. It was considered that dentinal tubules in deep dentin were denser and the orientations are almost radial, which may benefit the infiltration of resin monomer, resulting in higher bond strength to deep dentin than superficial dentin [17]. In the present study, there was no significant difference in the immediate SBSs between SD and DD groups, which was in accordance with the previous study [15]. The difference in type and chemical composition of the tested adhesives may be the major reason for the discrepancy within studies. It has been reported that the hydrophobicity, wetting ability, and solvent type of the adhesives may all affect their bonding performance to different dentin depths [18]. The testing methods applied should also be taken into account. Micro-shear bonding strength test was applied in the present study. Using the longitudinally sectioned dentin slab, a complete dentin surface around the pulp chamber was exposed and it was easy to define the region of dentin (Fig. 1). The bond strength to different dentin depths could therefore be simply tested using the same specimen [9]. Furthermore, the specimen preparation for the microshear test was relatively simple compared with the microtensile test [19,20], which would produce more stress or damage during the sectioning, trimming and shaping of the specimens. To our knowledge, this is the first study to evaluate the bonding stability to different dentin depths. According to the results, the SBSs in both DD and SD groups significantly decreased after thermocycling treatment, indicating the bonding degradation in different dentin depths. The SBSs in DD groups were significantly lower than that in SD groups after thermocycling, indicating more severe bonding degradation in DD groups. The special structure of deep dentin may contribute to its significant bonding degradation [5]. Hybrid layers in deep dentin are composed mainly of resin tags with little surrounding intertubular dentin to hybridize the resin tags to surrounding collagen [21]. The comparable large content of water in deep dentin may interfere with the polymerization of resin tags and make it weak under the thermocycling challenge. The results of failure modes detection confirmed the weakness of the hybrid layer in DD groups. The percentage of
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Fig. 2 – Percentage distribution of failure modes among tested groups: (A) Single Bond 2; (B) Clearfil S3 Bond.
adhesive failure in DD groups was significantly increased after thermocycling. Regardless of the adhesive type, this characteristic failure was located at the bottom of the hybrid layer: most of the dentin tubules opened widely and the resin tags were completely pulled out, leaving thin shell of intertubular dentin surrounding the dentin tubules (Fig. 3d and h). The high amount of ICTP released from deep dentin may also be an important reason for the significant bonding degradation in DD groups. Our previous study [9] has found that the distribution of dentinal MMPs decreased from deep dentin to superficial dentin, which was not in accordance with the distribution of their specific tissue inhibitors of metalloproteinases (TIMPs). We speculated that there might be a high gelatinolytic potential in deep dentin due to the distribution character of MMPs and TIMPs here. In the present study, the results of the ICTP release confirmed the speculation. ICTP is a product liberated during the degradation of telopeptide from
mature type I collagen by MMPs. Among known collagenolytic proteinases relevant to hard tissue sorption, only MMPs can generate ICTP [22]. Thus, the ICTP values of dentin specimens directly demonstrated the MMPs activity of dentin matrix and the degree of collagen degradation mediated by these MMPs [23]. For the two adhesives tested, the ICTP values in DD groups were always higher than those in SD groups, which exhibited a higher level of MMPs activity and collagen degradation originated from deep dentin during the bonding and thermocycling process than those from superficial dentin. Besides MMPs, dentinal cysteine cathepsins have also been reported as proteases that identified in human dentin and showed activities in the degradation of dentin matrix [24,25]. Further studies will be continued on the distribution of cysteine cathepsins within coronal dentin and their effects on the degradation of collagen matrix under histological or pathological conditions.
Fig. 3 – Representative FE-SEM micrographs of the debonded interfaces of specimens: (a–d), specimens treated with Single Bond 2; (e–h), specimens treated with Clearfil S3 Bond; SD, superficial dentin; DD, deep dentin.
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Aging methods such as long-time water storage, thermocycling, and pH cycling, have been widely used to evaluate the bonding stability of resin-dentin bonds [26]. Thermocycling for 5000 cycles showed a significant effect on both the SBSs and the ICTP values in the present study, indicating it an effective method to accelerate the degradation of hybrid layer. One important reason for using thermocycling instead of water storage is the testing time. Thermocycling for 5000 times in this study required approximately 10 days. Among the reported studies, the longest storage time for ICTP detection using tooth specimens was four weeks [25]. ICTP detection is a sensitive immunoassay. The storage time for at least 24 weeks which is usually applied for water storage aging would be too long to accurately reflect the ICTP levels of dentin specimens. According to the ICTP results of this study, thermocycling could activate the dentinal MMPs and result in the increase of ICTP levels. Therefore, a negative control was necessary to differentiate the real positive effect. Two types of resinous adhesives were applied in this study. The treatment with Single Bond 2 significantly increased the ICTP values, regardless of the thermocycling aging. Some previous studies [27,28] reported the treatment of adhesive resin to demineralized dentin could reduce collagen degradation and presented a decreased ICTP concentration. All these studies used dentin beams that demineralized by either EDTA for up to 6 days or phosphoric acid for 12 h. The demineralized dentin beams were then immersed in adhesive resin for 12 h to ensure a good infiltration of resin monomer. Therefore, all the six dentin surfaces of the beam were completely sealed by polymerized adhesive resin, which may prevent the ICTP telopeptide fragments from leaching out of the dentin. Different from these studies, only four surfaces of the dentin beams were treated by adhesive in the present study, leaving two dentin surfaces uncoated and the bonding interfaces exposed on these surfaces. These may facilitate the liberation of ICTP from the dentin beams. Furthermore, the dentin treatment in the study is the same with the clinical method. Single Bond 2 is a two-step etch-and-rinse adhesive system. The incompatibility between the strong demineralizing ability of the 37% phosphoric acid (pH = 0.4) used for acid-etching and the imperfect infiltration of the hydrophilic resin monomers may cause a defective zone at the bottom of hybrid layer, with a certain number of collagen fibrils and matrix-bound MMPs being exposed [3]. Theses MMPs may be activated by the acidic adhesive monomers [29] or the thermal changes originated from the thermocying treatment and induce the degradation of the denuded collagen. Different from Single bond 2, the treatment with self-etching Clearfil S3 Bond (pH = 2.5) showed no effect on the ICTP values, indicating less MMPs activation and less collagen degradation after the adhesive treatment. Clearfil S3 Bond is a self-etch adhesive which can simultaneously condition prime and bond dentin with the use of non-rinse acidic monomers. Due to its mild aggressiveness, limited collagen and MMPs may be exposed after bonding procedure and resulted in less collagen degradation [12]. As a HEMA-rich “all-in-one” adhesive, this hydrophilic adhesive may cause enhanced water sorption from the host dentin, leading to incomplete curing of the adhesive resin [30]. This may impair the physical and mechanical characters of the adhesive within the hybrid layer, leading to resin hydrolysis
and the decrease of bonding strength under thermocycling challenge. In conclusion, deep dentin showed more significant resin-dentin bond degradation than superficial dentin after thermocycling. This may relate to the special structure and high level of MMPs activities in deep dentin. Different from the self-etch adhesive-Clearfil S3 Bond, the phosphoric acid treatment with total-etch adhesive-Single Bond 2 may activate dentinal MMPs and contribute to the bonding degradation. Further studies are needed to evaluate the effect of MMPs inhibitor on improving the bonding stability of deep dentin and its potential clinic use.
Acknowledgements This study was supported by Program for Changjiang Scholars and Innovative Research Team in University to Chen JH (IRT13051) and the Natural Science Foundation of China: No. 51373198 to Zhang L (P.I.), No. 81130078 to Chen JH (P.I.), No. 81100772 to Li F (P.I.).
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Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.intl.elsevierhealth.com/journals/dema
Stability of bonds made to superficial vs. deep dentin, before and after thermocycling Ling Zhang a,1 , Dan-yang Wang a,b,1 , Jing Fan c , Fang Li a , Yu-jiang Chen a , Ji-hua Chen a,∗ a
State Key laboratory of Military Stomatology, Department of Prosthodontics, School of Stomatology, Fourth Military Medical University, Xi’an, China b Department of Stomatology, Xi’an medical university, Xi’an, China c Department of Endocrine & Vascular Surgery, Xi’jing Hospital, Fourth Military Medical University, Xi’an, China
a r t i c l e
i n f o
a b s t r a c t
Article history:
Objectives. Bonding stability of resinous adhesives to dentin is still problematic and may
Received 2 October 2013
involve regional variations in dentin composition. This study is to evaluate the effect of
Received in revised form
dentin depth on the stability of resin-dentin bonds under thermocycling challenge.
13 July 2014
Methods. Dentin slabs with two flat surfaces parallel to the tooth axis were obtained from
Accepted 8 August 2014
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 S3 Bond), and the storage treatment (thermocycling for 5000
Keywords:
times vs. no). After the adhesive treatment and composite buildup on the dentin slabs, the
Stability
micro-shear bond strength (SBS) of each group was detected. The concentrations of cross-
Dentin depth
linked carboxyterminal telopeptide of type I collagen (ICTP) were also evaluated using an
Matrix metalloproteinases
immunoassay to detect the degree of collagen degradation in each group.
Microshear bond strength
Results. Dentin depth, adhesive treatment and storage treatment all showed significant
Cross-linked carboxyterminal
effects on both the SBSs and the ICTP values (P < 0.05). Regardless of the adhesive type,
telopeptide of type I collagen
thermocycling decreased the SBSs and increased the ICTP values (P < 0.05). The DD groups showed significantly lower SBSs 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 S3 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. © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
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 [1]. Dentin bonding is created by the formation of the socalled hybird layer [2]. Degradation of hybrid layers has been considered as the major limitation to the stability of resin-
∗ Corresponding author at: School of Stomatology, Fourth Military Medical University, 145 West Changle Road, Xi’an 710032, China. Tel.: +86 29 8477 6329; fax: +86 29 8477 6329. E-mail address: [email protected] (J.-h. Chen). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.dental.2014.08.362 0109-5641/© 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
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dentin interfaces [1]. Extensive studies have been reported on the factors that related to the degradation of hybrid layers [1,3,4], 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 [5–7]. These factors may contribute to the differences in hybrid layer that is formed within different dentin layers, and thus affect their bonding performance [8]. 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 [1,3]. Our previous study has found that the distribution of MMP-2 and MMP-9 was different within different depths of coronal dentin [9], 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. depth
Isolation of coronal dentin according to dentin
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 [9]: 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 S3 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. 1a). 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 resinbonded 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
Fig. 1 – Schematic drawing of the specimens preparation.
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Table 1 – Resinous adhesives used for test. Adhesives
Composition [lot number]
Adper Single Bond 2 (3M ESPE, St. Paul, MN, USA)
Bis-GMA; polyalkenoic acid, copolymer; dimethacrylate; CQ; HEMA; ethanol; water [N377455]
Clearfil S3 Bond (Kuraray Co. Ltd., Osaka, Japan)
10-MDP, dl-Camphorquinone, Hydrophobic dimethacrylate, HEMA, Bis-GMA, water, ethyl alcohol, silanated colloidal silica [00169A]
Application procedures 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 Apply the adhesive for 20 s, dry with high-pressure air for 5 s, light cure for 10 s
Abbreviations: Bis-GMA, bis-phenol A diglycidyl-methacrylate; HEMA, 2-hydroxylethyl methacrylate; CQ, camphoroquinone; 10-MDP, 10methacryloyloxydecyl dihydrogen phosphate.
Table 2 – Micro-shear bond strengths to different dentin depths [means (SD), MPa]. Dentin treatment
Aging treatment
Single Bond 2
No Thermocycling
54.05 (6.06)a,A 37.06 (5.17)a,B
51.09 (8.01)a,A 30.58 (7.23)b,B
Clearfil S3 Bond
No Thermocycling
39.92 (8.72)a,B 30.89 (6.72)a,C
37.69 (4.24)a,C 18.95 (7.43)b,D
Dentin depth Superficial dentin (SD)
Deep dentin (DD)
N = 50 per group. For each horizontal row, values with same lowercase letters indicate no significant difference (P > 0.05). For each vertical column, values with same uppercase letters indicate no significant difference (P > 0.05).
were 60 s and 6 s respectively. The micro-shear bond strength (SBS) was then measured using the previously described method [10]. 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 [11]: adhesive failure, mixed failure, cohesive failure in dentin, and cohesive failure in resin.
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. 1b). 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 S3 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 [12]. 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 l of sterile artificial saliva containing 50 U ml−1 of penicillin G and 1500 g 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 l 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 g/l to 250 g/l were firstly used to construct the standard curve. Only data within the confidence interval were considered to be significant and
Table 3 – ICTP values of dentin specimens from different depths [means (SD), ng telopeptide/mg dry mineralized dentin]. Dentin treatment
Aging treatment
Dentin depth
Control
No Thermocycling
0.009 (0.005)1,a,A 0.084 (0.013)1,b,A
0.048 (0.012)2,a,A 0.242 (0.017)2,b,A
Single Bond 2
No Thermocycling
0.079 (0.008)1,a,B 1.554 (0.159)1,b,B
0.331 (0.025)2,a,B 3.192 (0.312)2,b,B
Clearfil S3 Bond
No Thermocycling
0.010 (0.004)1,a,A 0.186 (0.017)1,b,A
0.050 (0.005)2,a,A 0.372 (0.031)2,b,A
SD
DD
For each horizontal row, values with identical numbers indicate no significant difference (P > 0.05). For each vertical column, values with identical lowercase letters indicate no significant difference between aging treatments within the same dentin treatment (P > 0.05), and values with identical uppercase letters indicate no significant difference between dentin treatments within the same aging treatment (P > 0.05).
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applied to the calculation. The final ICTP value for each group were presented as ng telopeptide per mg dry mineralized dentin.
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 SBS 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).
3.
Results
3.1.
Micro-shear bond strength
The three factors of dentin depth, aging treatment, and storage treatment all showed significant effects on SBSs (P < 0.05). Interaction between dentin depth and storage treatment was also significant (P < 0.05). The SBSs to different tested groups were summarized in Table 2. For the two adhesives tested, immediate SBSs between SD and DD groups showed no significant difference (P > 0.05). After thermocycling, the SBS of DD group was significantly lower than that in SD group (P < 0.05). Within each dentin layer, thermocycling treatment significantly decreased the SBSs (P < 0.05). Chi-Square test revealed that there were significant differences in failure modes within tested groups (P < 0.05). The failure distribution in each tested group was presented in Fig. 2. No cohesive failures in dentin were observed in this study. The failure mode presented a similar distribution for the two adhesives tested. The mixed failure was the most frequent mode of failure in all SD groups and the DD groups without thermocycling treatments. After thermocycling, the percentage of adhesive failures in the DD groups increased and became the most prevalent mode of failure. The representative failure modes of all groups are presented in Fig. 3. Regardless of the adhesive type, SD groups showed a similar pattern of mixed failure both before (Fig. 3a and e) and after (Fig. 3b and f) the thermocycling treatment. Without thermocycling treatment, DD specimens also showed a mixed failure at the bottom of the hybrid layer (Fig. 3c and g). After thermocycling for 5000 cycles, the characteristic pattern of adhesive failure was presented in DD groups for both Single Bond 2 and Clearfil S3 Bond (Fig. 3d and h): a fracture at the bottom of hybrid layer, with the dentin tubules opening widely and resin tags being completely pulled out.
3.2.
ICTP values
ANOVA analysis showed that the ICTP values were significantly affected by the three tested variables (P < 0.001). The interactions between dentin depths and adhesive treatments were also significant (P < 0.05). Regardless of the effect of adhesive and storage treatments, the ICTP values of DD groups
were significantly higher than those of SD groups (P < 0.05, Table 3). Thermocycling significantly increased the ICTP values of dentin specimens (P < 0.05). Compared with the control group, the treatment with Single Bond 2 could significantly increase the ICTP values (P < 0.05). On the other hand, the treatment with Clearfil S3 Bond showed no effect on the ICTP values (P > 0.05).
4.
Discussion
According to the results of the present study, dentin depth, adhesive treatment and thermocycling treatment all showed significant effects on the micro-shear bond strengths and the ICTP values. Thus, the null hypothesis should be rejected. The bonding performance at different dentin depths remains controversial in literature. Some studies reported that deep dentin was difficult to bond due to the low contents of intertubular dentin and collagen fibrils, as well as the high water content [13,14]. Other studies reported that deep dentin presented higher bond strength than superficial dentin [15,16]. It was considered that dentinal tubules in deep dentin were denser and the orientations are almost radial, which may benefit the infiltration of resin monomer, resulting in higher bond strength to deep dentin than superficial dentin [17]. In the present study, there was no significant difference in the immediate SBSs between SD and DD groups, which was in accordance with the previous study [15]. The difference in type and chemical composition of the tested adhesives may be the major reason for the discrepancy within studies. It has been reported that the hydrophobicity, wetting ability, and solvent type of the adhesives may all affect their bonding performance to different dentin depths [18]. The testing methods applied should also be taken into account. Micro-shear bonding strength test was applied in the present study. Using the longitudinally sectioned dentin slab, a complete dentin surface around the pulp chamber was exposed and it was easy to define the region of dentin (Fig. 1). The bond strength to different dentin depths could therefore be simply tested using the same specimen [9]. Furthermore, the specimen preparation for the microshear test was relatively simple compared with the microtensile test [19,20], which would produce more stress or damage during the sectioning, trimming and shaping of the specimens. To our knowledge, this is the first study to evaluate the bonding stability to different dentin depths. According to the results, the SBSs in both DD and SD groups significantly decreased after thermocycling treatment, indicating the bonding degradation in different dentin depths. The SBSs in DD groups were significantly lower than that in SD groups after thermocycling, indicating more severe bonding degradation in DD groups. The special structure of deep dentin may contribute to its significant bonding degradation [5]. Hybrid layers in deep dentin are composed mainly of resin tags with little surrounding intertubular dentin to hybridize the resin tags to surrounding collagen [21]. The comparable large content of water in deep dentin may interfere with the polymerization of resin tags and make it weak under the thermocycling challenge. The results of failure modes detection confirmed the weakness of the hybrid layer in DD groups. The percentage of
d e n t a l m a t e r i a l s 3 0 ( 2 0 1 4 ) 1245–1251
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Fig. 2 – Percentage distribution of failure modes among tested groups: (A) Single Bond 2; (B) Clearfil S3 Bond.
adhesive failure in DD groups was significantly increased after thermocycling. Regardless of the adhesive type, this characteristic failure was located at the bottom of the hybrid layer: most of the dentin tubules opened widely and the resin tags were completely pulled out, leaving thin shell of intertubular dentin surrounding the dentin tubules (Fig. 3d and h). The high amount of ICTP released from deep dentin may also be an important reason for the significant bonding degradation in DD groups. Our previous study [9] has found that the distribution of dentinal MMPs decreased from deep dentin to superficial dentin, which was not in accordance with the distribution of their specific tissue inhibitors of metalloproteinases (TIMPs). We speculated that there might be a high gelatinolytic potential in deep dentin due to the distribution character of MMPs and TIMPs here. In the present study, the results of the ICTP release confirmed the speculation. ICTP is a product liberated during the degradation of telopeptide from
mature type I collagen by MMPs. Among known collagenolytic proteinases relevant to hard tissue sorption, only MMPs can generate ICTP [22]. Thus, the ICTP values of dentin specimens directly demonstrated the MMPs activity of dentin matrix and the degree of collagen degradation mediated by these MMPs [23]. For the two adhesives tested, the ICTP values in DD groups were always higher than those in SD groups, which exhibited a higher level of MMPs activity and collagen degradation originated from deep dentin during the bonding and thermocycling process than those from superficial dentin. Besides MMPs, dentinal cysteine cathepsins have also been reported as proteases that identified in human dentin and showed activities in the degradation of dentin matrix [24,25]. Further studies will be continued on the distribution of cysteine cathepsins within coronal dentin and their effects on the degradation of collagen matrix under histological or pathological conditions.
Fig. 3 – Representative FE-SEM micrographs of the debonded interfaces of specimens: (a–d), specimens treated with Single Bond 2; (e–h), specimens treated with Clearfil S3 Bond; SD, superficial dentin; DD, deep dentin.
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Aging methods such as long-time water storage, thermocycling, and pH cycling, have been widely used to evaluate the bonding stability of resin-dentin bonds [26]. Thermocycling for 5000 cycles showed a significant effect on both the SBSs and the ICTP values in the present study, indicating it an effective method to accelerate the degradation of hybrid layer. One important reason for using thermocycling instead of water storage is the testing time. Thermocycling for 5000 times in this study required approximately 10 days. Among the reported studies, the longest storage time for ICTP detection using tooth specimens was four weeks [25]. ICTP detection is a sensitive immunoassay. The storage time for at least 24 weeks which is usually applied for water storage aging would be too long to accurately reflect the ICTP levels of dentin specimens. According to the ICTP results of this study, thermocycling could activate the dentinal MMPs and result in the increase of ICTP levels. Therefore, a negative control was necessary to differentiate the real positive effect. Two types of resinous adhesives were applied in this study. The treatment with Single Bond 2 significantly increased the ICTP values, regardless of the thermocycling aging. Some previous studies [27,28] reported the treatment of adhesive resin to demineralized dentin could reduce collagen degradation and presented a decreased ICTP concentration. All these studies used dentin beams that demineralized by either EDTA for up to 6 days or phosphoric acid for 12 h. The demineralized dentin beams were then immersed in adhesive resin for 12 h to ensure a good infiltration of resin monomer. Therefore, all the six dentin surfaces of the beam were completely sealed by polymerized adhesive resin, which may prevent the ICTP telopeptide fragments from leaching out of the dentin. Different from these studies, only four surfaces of the dentin beams were treated by adhesive in the present study, leaving two dentin surfaces uncoated and the bonding interfaces exposed on these surfaces. These may facilitate the liberation of ICTP from the dentin beams. Furthermore, the dentin treatment in the study is the same with the clinical method. Single Bond 2 is a two-step etch-and-rinse adhesive system. The incompatibility between the strong demineralizing ability of the 37% phosphoric acid (pH = 0.4) used for acid-etching and the imperfect infiltration of the hydrophilic resin monomers may cause a defective zone at the bottom of hybrid layer, with a certain number of collagen fibrils and matrix-bound MMPs being exposed [3]. Theses MMPs may be activated by the acidic adhesive monomers [29] or the thermal changes originated from the thermocying treatment and induce the degradation of the denuded collagen. Different from Single bond 2, the treatment with self-etching Clearfil S3 Bond (pH = 2.5) showed no effect on the ICTP values, indicating less MMPs activation and less collagen degradation after the adhesive treatment. Clearfil S3 Bond is a self-etch adhesive which can simultaneously condition prime and bond dentin with the use of non-rinse acidic monomers. Due to its mild aggressiveness, limited collagen and MMPs may be exposed after bonding procedure and resulted in less collagen degradation [12]. As a HEMA-rich “all-in-one” adhesive, this hydrophilic adhesive may cause enhanced water sorption from the host dentin, leading to incomplete curing of the adhesive resin [30]. This may impair the physical and mechanical characters of the adhesive within the hybrid layer, leading to resin hydrolysis
and the decrease of bonding strength under thermocycling challenge. In conclusion, deep dentin showed more significant resin-dentin bond degradation than superficial dentin after thermocycling. This may relate to the special structure and high level of MMPs activities in deep dentin. Different from the self-etch adhesive-Clearfil S3 Bond, the phosphoric acid treatment with total-etch adhesive-Single Bond 2 may activate dentinal MMPs and contribute to the bonding degradation. Further studies are needed to evaluate the effect of MMPs inhibitor on improving the bonding stability of deep dentin and its potential clinic use.
Acknowledgements This study was supported by Program for Changjiang Scholars and Innovative Research Team in University to Chen JH (IRT13051) and the Natural Science Foundation of China: No. 51373198 to Zhang L (P.I.), No. 81130078 to Chen JH (P.I.), No. 81100772 to Li F (P.I.).
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