酪蛋白磷酸肽-无定形磷酸钙、不同玷污层的处理和储存时间对牙本质粘接的影响

Influence of casein phosphopeptide-amorphous calcium phosphate application, smear layer removal, and storage time on resin-dentin bonding 酪蛋白磷酸肽-无定形磷酸钙、不同玷污层的处理和储存时间对牙本质粘接的影响 1 first-author 1 1 1 1 1 1 2 3 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:Christian.Mehl@HarderMehl.de †E-mail:Christian.Mehl@HarderMehl.de Christian.Mehl@HarderMehl.de Department of Stomatology, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Department of Stomatology, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Institute of Medical Informatics and Statistics, University Hospital Schleswig-Holstein, Campus Kiel, 24105 Kiel, Germany Institute of Medical Informatics and Statistics, University Hospital Schleswig-Holstein, Campus Kiel, 24105 Kiel, Germany Department of Prosthodontics, Propaedeutics and Dental Materials, Christian-Albrechts University at Kiel, Arnold-Heller-Straße 16, 24105 Kiel, Germany Department of Prosthodontics, Propaedeutics and Dental Materials, Christian-Albrechts University at Kiel, Arnold-Heller-Straße 16, 24105 Kiel, Germany The aim of this study is to evaluate the influence of Tooth Mousse (TM) application, smear layer removal, and storage time on resin-dentin microtensile bond strength (μTBS). Dentin specimens were divided into two groups: (1) smear layer covered; (2) smear layer removed using 15% EDTA for 90 s. In each group, half the specimens were treated once with TM for 60 min. After bonding procedures using a two-step self-etching adhesive (Clearfil SE Bond (CSE); Kuraray Medical, Tokyo, Japan), an all-in-one adhesive (G-Bond (GB); GC Corp, Tokyo, Japan), and a total-etch adhesive (Adper Single Bond 2 (SB); 3M ESPE, St. Paul, MN, USA), the specimens were stored for 3 d or 6 months in deionized water at 37 °C, and μTBS was tested and analyzed. With the exception of SB (no TM application) and GB, the μTBS was significantly increased for CSE and SB using EDTA pre-conditioning and 3 d of storage ( P ≤0.001). Bond strength of GB decreased significantly when using EDTA (3 d storage, P < 0.05). TM application only increased the μTBS of GB (no EDTA) and SB (with EDTA) after 3 d ( P ≤0.02). Comparing the adhesives after 3 d of storage, CSE exhibited the greatest μTBS values followed by GB and SB ( P ≤0.02). The factors of adhesive, EDTA, and TM did not show any significant impact on μTBS when specimens were stored for 6 months ( P > 0.05). The additional application of TM and EDTA for cavity preparation seems only to have a short-term effect, and no influence on μTBS of dentin bonds after a period of 6 months. 研究目的 2 评价含有酪蛋白磷酸肽-无定形磷酸钙（CPP-ACP）的再矿化剂Tooth Mousse（TM）的使用、不同玷污层的处理以及样本储存时间对牙本质粘接微拉伸性能的影响。 研究方法 2 将牙本质样本分成保留玷污层组和用15%乙二胺四乙酸（EDTA）处理90秒去除玷污层组。每组根据是否使用TM处理再分亚组。每个亚组分别用三种不同的粘结剂（两步法自酸蚀Clearfil SE Bond（CSE）、一步法自酸蚀G-Bond（GB）和全酸蚀Adper Single Bond 2（SB））与牙本质样本粘接，分别经过3天和6个月的去离子水储存。样本进行切割微拉伸测试并通过扫描电镜分析断裂界面模式。 重要结论 2 经过再矿化剂TM预处理，可以减少牙齿的敏感性，并且对于这三种粘结系统经过长时间储存后的粘结性能没有影响。EDTA的处理对于长期储存的粘结性能没有显著影响。额外的TM和EDTA对短期（3天）粘接力会有效应，但对长期（6个月）的粘接力没有影响。 Tooth Mousse Ethylenediaminetetraacetic acid (EDTA) Casein phosphopeptide-amorphous calcium phosphate Smear layer Bond strength 玷污层 再矿化剂Tooth Mousse 微拉伸性能 储存时间 粘结剂 Introduction 1 Dentin hypersensitivity has a prevalence of around 25% (Que et al., 2010 2010 ). It is mainly caused by exposed dentin as a result of enamel loss and/or gingival root surface exposure from attrition, abrasion, erosion, or wedge-shaped defects (West et al., 1998 1998 ; Vanuspong et al., 2002 2002 ; Pashley et al., 2008 2008 ). Also, iatrogenically caused dentin sensitivity, after placement of direct or indirect restorations on vital teeth, is a common clinical phenomenon (Swift, 2004 2004 ; Denner et al., 2007 2007 ; Berkowitz et al., 2009 2009 ). Dentin hypersensitivity is possibly provoked by an abnormal flow of dentin tubule fluid due to external stimuli (Pashley et al., 1996 1996 ). Agents used to close patent dentinal tubules include fluoride-containing solutions/compounds, oxalates and nitrates of potassium, potassium chloride or strontium chloride (Sulieman, 2005 2005 ), amorphous calcium phosphates (Geiger et al., 2003 2003 ), resin-based bonding agents, and the placement of restorations (Adebayo et al., 2008b 2008b ). Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), introduced in the 1990s (Reynolds, 1998 1998 ), consists of casein phosphopeptides derived from milk proteins that have been reported to bind amorphous calcium phosphate; this forms nanoclusters of CPP-ACP, thereby stabilizing calcium phosphates in solution (Reynolds, 1998 1998 ; Adebayo et al., 2008b 2008b ). CPP-ACP reduces demineralization and enhances remineralization of enamel and dentin by maintaining a high concentration gradient of calcium phosphate on the tooth surface (Reynolds, 1997 1997 ; 1998 1998 ). Various studies have successfully used CPP-ACP as a supplement in acidic drinks to reduce erosivity and dentin hypersensitivity and to protect dental hard tissues (Ramalingam et al., 2005 2005 ; Piekarz et al., 2008 2008 ; Tantbirojn et al., 2008 2008 ). Moreover, CPP-ACP also reduces aggressive abrasion and dentin hypersensitivity caused by interproximal enamel reduction (Giulio et al., 2009 2009 ; Ranjitkar et al., 2009 2009 ). Additional studies confirmed reports that CPP-ACP can reduce demineralization and enhance remineralization of bovine and human dentin (Oshiro et al., 2007 2007 ; Rahiotis and Vougiouklakis, 2007 2007 ; Yamaguchi et al., 2007 2007 ). Hypersensitive cervical dentin is mostly associated with a thinner amorphous smear layer and more open and wider tubules than non-sensitive cervical dentin (Absi et al., 1995 1995 ; Rimondini et al., 1995 1995 ). Additional loss of intertubular dentin is reported (Rimondini et al., 1995 1995 ). A uniform amorphous smear layer varying in thickness and containing crystalline-coating debris has been observed on exposed cervical dentin in non-carious cervical lesions (Rimondini et al., 1995 1995 ). The presence and quality of the smear layer affect bonding of self-etching adhesives and may reduce bond strength since the dissolved smear layer components are not rinsed away (Koibuchi et al., 2001 2001 ; Tani and Finger, 2002 2002 ; Oliveira et al., 2003 2003 ). The removal of the smear layer using cavity conditioners before CPP-ACP application is a current topic in the literature, as remaining smear layer could impede penetration of the CPP-ACP paste and subsequently the adhesive resin (Adebayo et al., 2008b 2008b ). Few studies report a negligible influence of TM pretreatment and cavity conditioning using self-etch adhesives on bond strength (Adebayo et al., 2008b 2008b ; Zorba et al., 2010 2010 ). Based on the usefulness of CPP-ACP as a desensitizer and in the absence of data on bond strength after long-term storage, the aim of this study was to evaluate the influence of CPP-ACP on microtensile bond strength (μTBS) in a two-step self-etching/priming adhesive, an all-in-one adhesive, and a total-etch adhesive to dentin in combination with or without smear layer removal. The null-hypotheses of this study were that CPP-ACP, storage time, and ethylenediaminetetraacetic acid (EDTA) pretreatment have no influence on μTBS following a two-step self-etching/priming adhesive, an all-in-one adhesive, and a total-etch adhesive to dentin. Materials and methods 1 Tooth preparation 2 Seventy-two intact non-carious human third molars were extracted and stored in 0.1% (1 g/L) thymol-saturated isotonic saline solution at 4 °C. The teeth were collected after informed consent under a protocol approved by the Faculty of Dentistry Ethics Committee of Zhejiang University, China. The teeth were equally divided into 3-d and 6-month storage groups. All teeth were used within three months of extraction. Fig. 1 1 shows a flow chart of the process from tooth to μTBS beam. The occlusal enamel was removed perpendicular to the longitudinal axis of each tooth by a slow-speed diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) with water cooling. The resulting flat dentin surface was checked with a stereomicroscope (Wild Makroskop M420, Heerbrugg, Switzerland) at 10× magnification for the absence of enamel. The bonding surfaces were further polished with a 600-grit silicon carbide paper to create a standardized smear layer in mid-coronal dentin (Yang et al., 2006 2006 ). For all specimens, the dentin was kept wet during these preparations by storage in deionized water or placement on a wet dish. In order to more closely resemble a clinical situation where dentin is normally exposed and hydrated naturally, the apex of the root was not sealed to allow intrusion of the storage media (de Munck et al., 2003 2003 ). This figure is modified from Lin et al. ( 2010 2010 ) Tooth conditioning and bonding procedures 2 After smear layer preparation, the short- and long-term storage groups were randomly divided again. In Group 1, the smear layer was left for the subsequent treatment and bonding procedures, while in Group 2 the smear layer was removed using 15% EDTA for 90 s and gently rinsing for 15 s. Prior to the bonding procedures, half of the specimens in each group were treated with CPP-ACP paste (Tooth Mousse (TM); GC Corp., Tokyo, Japan). TM was applied by an applicator brush covering the entire dentin surfaces. The TM-treated specimens were placed in a sealed pot at 37 °C and (95±5)% relative humidity for 1 h. The TM paste was then rinsed off in water for 15 s and the specimens gently dried using water and oil-free air. The resulting 24 experimental and control groups (three adhesives, EDTA, TM, storage time) were submitted to the bonding protocols using a two-step self-etching adhesive (Clearfil SE Bond (CSE); Kuraray Medical, Tokyo, Japan), an all-in-one adhesive (G-Bond (GB); GC Corp, Tokyo, Japan) and a total-etch adhesive (Adper Single Bond 2 (SB); 3M ESPE, St. Paul, MN, USA) according to the manufacturers’ instructions. Composite resin (Valux Plus, 3M ESPE) was placed directly on the bonded surfaces in 2.5 mm increments up to a height of 5 mm with each increment being light-cured for 40 s (Optilux 500 (550 mW/cm 2 ); Kerr, Danbury, CT, USA). All chemical compositions and application techniques are shown in Table 1 1 . After completing the adhesive procedures, all specimens were kept in deionized water at 37 °C for 3 d or 6 months. 10-MDP: 10-methacryloyloxydecyl dihydrogen phosphate; HEMA: 2-hydroxyethyl methacrylate; bis-GMA: bisphenol A-glycidyl methacrylate; TEGDMA: tetraethylene glycol dimethacrylate Material Composition Batch No. Application/bonding procedure 2 Clearfil SE Bond (CSE; Kuraray Medical, Japan) Primer: 10-MDP, HEMA, hydrophilic dimethacrylate, DL -camphorquinone, N , N -diethanol- p -toluidine, water; 00923A Apply primer and leave for 20 s, dry thoroughly with mild air flow; Bond: 10-MDP, bis-GMA, HEMA, hydrophilic dimethacrylate, DL -camphorquinone, N , N -diethanol- p -toluidine, silanated colloidal silica 01366A Apply bond, gently air-flow, light-cured for 10 s G-Bond (GB; GC Corp., Japan) 4-META, urethane dimethacrylate, triethylene glycol dimethacrylate, acetone, distilled water 0907271 Apply bond, leave for 10 s, strongly air-flow for 5 s, light-cured for 10 s Adper Single Bond 2 (SB; 3M ESPE, USA) HEMA, bis-GMA, dimethacrylates, methacrylate, functional copolymers (poly acrylic acid and poly itaconic acid), photoinitiator, ethanol, water N201833 Apply for 15 s, gently air-flow for 15 s, light-cured for 10 s Gluma Etch 35 Gel (Heraeus, China) 35% orthophosphoric acid, SiO 2 , water, pigment 562.09 Apply for 15 s, rinse for 10 s by air-water spray Valux Plus (3M ESPE, USA) Bis-GMA, TEGDMA resins, synthetic mineral of zirconia/silica, filler N168548 Apply to the dentin in three to four increments up to 4–5 mm thick and each increment light-cured for 40 s Tooth Mousse (TM; GC Corp., Tokyo, Japan) Glycerol, 5%–10% casein phosphopeptide, amorphous calcium phosphate, pure water, zinc oxide, sodium carboxyl, methyl cellulose, xylitol, D -sorbitol, silicon dioxide, phosphoric acid, titanium dioxide, guar gum, sodium saccharin, ethyl- p -hydroxybenzoate, magnesium oxide, propylene glycol, butyl- p -hydroxybenzoate, propyl- p -hydroxybenzoate 090132M Apply using a micro-brush, cover the surface of the dentin μTBS testing 2 After 3 d or 6 months of storage, the teeth were sectioned into 1.0 mm×1.0 mm×10 mm beams perpendicular to the adhesive/tooth interface using a low-speed diamond saw according to the non-trimming technique for μTBS testing (Sano et al., 1994 1994 ). Each tooth provided 10–12 beams with the peripheral beams being discarded to reduce variation. Additionally, if stereomicroscope evaluation found beams that were bonded to enamel, these were also discarded. From each of the 24 subgroups, approximately 30 beams per subgroup were used for bond strength evaluation. All beams were glued to the test apparatus with a cyanoacrylate adhesive (Zapit, Dental Ventures of America, Corona, CA, USA) and stressed in tension until failure at a crosshead speed of 1 mm/min by using a tensile testing machine (Micro Tensile Tester, Bisco Corporation, Schaumburg, IL, USA). Statistical analysis 2 The data were analyzed using a statistical software package (SPSS 18.0, Chicago, IL, USA). Since the μTBS data were not distributed normally (Shapiro-Wilks and Kolmogorov-Smirnov test), non-parametric tests were employed. A stratified analysis using Kruskal-Wallis tests followed by Dunn post-hoc tests and Mann-Whitney U -tests was utilized to analyze the data. Post-hoc testing was adjusted according to Bonferroni-Holm for multiple comparisons. All testing was performed at a confidence level of 95%. Scanning electron microscope (SEM) examination and failure mode analysis 2 After μTBS testing, the debonded dentin specimens were observed with an SEM (JSM 6400v, JEOL, Tokyo, Japan) to evaluate bond failure modes. The specimens were air-dried for 24 h, gold sputter-coated, and observed at 25 kV. Failure modes were classified into one of the following five modes (Fig. 2 2 ): A, cohesive failure located in the dentin; B, adhesive failure at the resin/dentin interface; C, mixed adhesive and cohesive failure; D, cohesive failure in the adhesive resin; and E, adhesive failure at the resin/composite interface. The portion of each failure mode on the debonded dentin surfaces was determined from the SEM micrographs with scale paper and expressed as a percentage of the total bonded surface area for each test group. This figure is modified from Lin et al. ( 2010 2010 ) Results 1 μTBS testing 2 Since the data were not distributed normally, the median, 25th and 75th percentiles, and the interquartile range (IQR) of the μTBS values are presented in Table 2 2 . Tables 3 3 and 4 4 present the statistical analysis regarding the influence of the factors of adhesive, EDTA and TM application, and storage time. With the exception of SB (no TM application) and GB, the μTBS was significantly increased for CSE and SB by using EDTA pre-conditioning and 3 d of storage ( P ≤0.001). Bond strength of GB decreased significantly when using EDTA (3 d storage, P < 0.05). N : number of beams stressed in tension until failure. Since the data were not distributed normally, the median and 25th/75th percentiles and the interquartile range (IQR) are shown. Decimals are rounded up or down to the next number 2 Adhesive 2 Storage time 2 EDTA application 2 TM application 2 N 4 μTBS (MPa) 25th percentile Median 75th percentile IQR 8 Clearfil SE Bond (CSE) 4 3 d 2 No No 25 24 31 42 18 With 28 19 34 49 30 2 With No 33 30 51 58 28 With 33 49 53 60 11 4 6 months 2 No No 9 8 14 19 11 With 10 6 19 39 33 2 With No 22 10 16 34 24 With 26 9 32 46 37 8 G-Bond (GB) 4 3 d 2 No No 34 13 17 21 8 With 38 16 24 30 14 2 With No 24 7 14 18 11 With 30 6 9 16 10 4 6 months 2 No No 19 12 24 41 29 With 19 17 24 34 17 2 With No 33 14 26 39 25 With 17 16 34 44 28 8 Adper Single Bond 2 (SB) 4 3 d 2 No No 25 15 25 35 20 With 38 16 23 32 16 2 With No 36 20 28 37 17 With 37 29 35 41 12 4 6 months 2 No No 20 8 14 51 43 With 23 9 19 35 26 2 With No 31 13 29 55 42 With 26 13 24 33 20 Statistically significant results at P < 0.05 2 Adhesive 2 Group 2 TM application 2 EDTA application 2 No EDTA vs. with EDTA 3 d 6 months 4 Clearfil SE-Bond (CSE) CSE1 No No 2 P < 0.001 * * 2 P =0.361 CSE3 No With CSE2 With No 2 P < 0.0001 * * 2 P =0.217 CSE4 With With 4 G-Bond (GB) GB1 No No 2 P =0.083 2 P =0.974 GB3 No With GB2 With No 2 P < 0.0001 * * 2 P =0.289 GB4 With With 4 Adper Single Bond 2 (SB) S1 No No 2 P =0.477 2 P =0.125 S3 No With S2 With No 2 P < 0.0001 * * 2 P =0.616 S4 With With Statistically significant results at P < 0.05 2 Adhesive 2 Group 2 EDTA application 2 TM application 2 No TM vs. with TM 2 3 d vs. 6 months 3 d 6 months 4 Clearfil SE-Bond (CSE) CSE1 No No 2 P =0.829 2 P =0.952 P =0.001 * * CSE2 No With P =0.059 CSE3 With No 2 P =0.207 2 P =0.064 P < 0.0001 * * CSE4 With With P < 0.0001 * * 4 G-Bond (GB) GB1 No No P =0.007 * * 2 P =0.670 2 P =0.025 * * GB2 No With P =0.554 GB3 With No P =0.086 2 P =0.347 2 P =0.002 * * GB4 With With P =0.001 * * 4 Adper Single Bond 2 (SB) SB1 No No P =0.762 2 P =0.947 2 P =0.458 SB2 No With P =0.453 SB3 With No P =0.014 * * 2 P =0.280 2 P =0.878 SB4 With With P =0.002 * * TM application only increased μTBS of GB (no EDTA) and SB (with EDTA) after 3 d ( P ≤0.02). Comparing the adhesives after 3 d of storage, CSE exhibited the greatest μTBS values followed by SB and GB ( P ≤0.02). The factors of adhesive, EDTA, and TM did not show any significant impact on μTBS when specimens were stored for 6 months ( P > 0.05). SEM examination 2 The percentages of the fracture modes are recorded in Table 5 5 . The failure mode definitions and examples of failures of representative beams are presented in Figs. 3 3 and 4 4 . 2 Adhesive 2 EDTA application 2 TM application 5 Failure mode of 3 d/6 months (%) A B C D E 4 Clearfil SE-Bond (CSE) No No 0/0 36/15 1/2 44/70 19/13 No With 0/0 39/50 2/2 59/9 0/39 With No 0/0 38/52 1/2 47/38 14/8 With With 0/0 35/41 2/1 44/36 19/22 4 G-Bond (GB) No No 0/0 86/64 2/1 12/15 0/20 No With 0/0 58/58 2/3 40/14 0/25 With No 0/0 78/42 1/2 21/24 0/32 With With 0/0 94/82 1/2 5/11 0/5 4 Adper Single Bond 2 (SB) No No 0/0 75/52 2/12 16/36 7/0 No With 0/0 84/43 1/10 15/47 0/0 With No 0/0 89/49 2/5 9/46 0/0 With With 0/0 81/43 1/4 11/45 7/8 (a, b) Debonded CSE specimens and a mixed adhesive (D: dentin) and cohesive (AR: adhesive resin) failure show some resin tags (RTs) remaining firmly embedded in the tubules, but also some dentin tubules (DTs) only partially closed with resin. No evidence of dentin fibers can be observed, since they remain covered by the smear layer. (c, d) Debonded GB specimens, just like CSE, show a mixed adhesive/cohesive failure and the same mix of completely and only partially closed DTs. Additionally, smear layer and peritubular dentin without collagen fibers are evident. (e, f) Consequences of a different concept regarding the smear layer can be observed. Since phosphoric acid is used for the Adper SB system, the smear layer is dissolved completely, resulting in visible collagen fibers and enabling the AR to enter the DTs and form RTs (a, b) Debonded CSE specimens show similar effects to the 3-d storage photographs (Fig. 3b 3b ). No evidence of dentin (D) fibers can be observed, since they remain covered by the smear layer. (c, d) In comparison, the 3-d storage debonded GB specimens show a mixed adhesive failure, but here intertubular collagen fibers are evident. (e, f) The Adper SB system exhibits the same working mechanism as already described in Fig. 3 3 with evident collagen fibers and completely closed tubule orifices forming resin tags (RTs). R: resin; AR: adhesive resin; DT: dentin tubule For specimens bonded with CSE, a nearly even mix between adhesive failure at the adhesive resin/dentin interface and cohesive failure in the adhesive resin could be observed. A smaller proportion failed adhesively at the adhesive resin/composite interface. For GB specimens, the main failure occurred at the adhesive resin/dentin interface. After 6 months, a shift from cohesive failure in the adhesive resin to adhesive failure at the adhesive resin/composite interface was observed. The main failure mode for SB specimens occurred at the adhesive resin/dentin interface. None of the samples failed cohesively in the dentin. Discussion 1 In earlier research, TM was shown to reduce demineralization and enhance remineralization of dentin, thereby suggesting its use as a desensitizer (Oshiro et al., 2007 2007 ; Rahiotis and Vougiouklakis, 2007 2007 ). Only a few studies have examined the influence of TM on bond strength using different testing set-ups, and samples were mostly stored in deionized water for 24 h or less (Adebayo et al., 2007 2007 ; Giulio et al., 2009 2009 ; Silva-Júnior et al., 2011 2011 ; Borges et al., 2013 2013 ). One of the aims of the current study was to evaluate the long-term effects of TM and EDTA pretreatment on μTBS. The null-hypotheses of this study had to be rejected, since the choice of adhesive and EDTA and TM pretreatment influenced the μTBS. Since the data were not distributed normally, interactions of the factors were not tested. Several studies suggest that sensitive dentin mostly occurs on non-carious cervical lesions on buccal and lingual surfaces of anterior teeth (Tyas, 1995 1995 ; Osborne-Smith et al., 1999 1999 ; Adebayo et al., 2008b 2008b ). Premolar and molar involvement during grinding can lead to hypersensitive non-carious cervical lesions in that area (Ommerborn et al., 2007 2007 ; Tokiwa et al., 2008 2008 ). As it is difficult to obtain non-carious teeth with cervical lesions, the current study used extracted molars with the anticipation that the degree of opening of the dentinal tubules and the smear layer density obtained would be little different from the morphology of hypersensitive dentin of naturally occurring cervical lesions (Adebayo et al., 2008b 2008b ). Bonding to dentin was carried out with and without prior smear layer removal before treatment with CPP-ACP paste in an attempt to simulate the clinical situation obtained on exposed cervical dentin lesions (Adebayo et al., 2008b 2008b ). In this situation, a thin smear layer may cover the dentin surface as in natural lesions (Rimondini et al., 1995 1995 ; Koibuchi et al., 2001 2001 ; Adebayo et al., 2008b 2008b ). Self-etching adhesives simplify the bonding process by reducing the number of application steps and thus reduce treatment time (Tay and Pashley, 2002 2002 ). All-in-one adhesives further simplify the bonding process. However, optimizing speed and efficiency should be accomplished without major trade-offs in the quality or durability of resin bonds (Tay and Pashley, 2002 2002 ), but recent results suggest that time-saving systems may be disadvantageous with regard to bond strength (Oliveira et al., 2003 2003 ; Perdigao et al., 2006 2006 ; Lin et al., 2013 2013 ). As the results of this study revealed non-parametric data and a wide IQR, operator experience could have had an impact on results (Simonetti et al., 2006 2006 ; Giachetti et al., 2011 2011 ). While self-etching adhesive systems were refractory to operator experience (Simonetti et al., 2006 2006 ; Giachetti et al., 2011 2011 ), bond strength of a total-etching adhesive system correlated to user experience (Adebayo et al., 2008a 2008a ; Giachetti et al., 2011 2011 ). Hence, in the current study, the laboratory workers were briefed and trained. However, the learning curve of the researchers can only partially explain the variations in the performance of the adhesive resins. Three major factors influence bond strength: first, the infiltration depth of adhesive resin in the dentin (Marshall et al., 1997 1997 ; Adebayo et al., 2010 2010 ); second, the degree of polymerization of the adhesive resin (Kanehira et al., 2006 2006 ); and third, the chemical bonding of the functional monomer (e.g., 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP), 4-methacryloyloxyethyl trimellitic anhydride (META)) to hydroxyapatite (Yoshida et al., 2004 2004 ). Comparing the three adhesives in the current study, CSE seemed to infiltrate the residual smear layer best without destroying the collagen network, while GB exhibited only partial infiltration of the tubule network (Figs. 3 3 and 4 4 ) (Adebayo et al., 2010 2010 ). SB specimens showed the same amount of tubule network infiltration as CSE, but a large percentage (between 80% and 90%) failed at the adhesive resin/dentin interface. There are several possible explanations, including over-drying, over-etching, over-wetting of the collagen network, or water-induced hydrolysis of the hybrid layer. In this study, it appeared that the collagen network in contact with the adhesive was mechanically or chemically compromised. With regard to the degree of polymerization of the adhesive resins, it seems that simplified adhesives exhibited a lower extent of polymerization and showed more incomplete polymerization, even after prolonged light curing (Cadenaro et al., 2005 2005 ). Although no pulpal pressure was applied (Lin et al., 2010 2010 ), permeability, and thus water entrapment within the hybrid layer, related inversely to the degree of polymerization (Tay et al., 2002 2002 ; Cadenaro et al., 2005 2005 ). Both permeability of the hybrid layer and lesser polymerization could have led to a mechanical weakening of the bond between SB/GB and dentin. The third point, the chemical bonding of the functional monomers (e.g., 10-MDP, 4-META, 2-hydroxyethyl methacrylate (HEMA)) to hydroxyapatite could additionally be of importance to explain the results in the current study (Yoshida et al., 2004 2004 ). Recently, HEMA-free one-step adhesives have been developed, since HEMA is notorious for its high allergenic potential (allergic reaction type IV) (Katsuno et al., 1996 1996 ; Paranjpe et al., 2005 2005 ; van Landuyt et al., 2008 2008 ). These HEMA-free adhesives generally contain a dentin adhesion-promoting monomer, such as 4-META or 10-MDP. It now seems to be established that one of the most efficient functional groups is 10-MDP, followed by 4-META and then HEMA (CSE=10-MDP; GB=4-META; SB=HEMA) (Tsuchimoto et al., 2006 2006 ; Iwai et al., 2013 2013 ). CSE contains the functional monomer 10-MDP, which contributes to dentin bonding by forming ionic chemical bonds with surface calcium ions in dentin crystallites (Dabsie et al., 2012 2012 ). In a comparative study, the interfacial ultrastructure remained unchanged for 10-MDP after 100 000 thermal cycles, while that of 4-META contained voids and less-defined collagen and thus seemed to have a less stable chemical bonding potential (Inoue et al., 2005 2005 ). The main components of the smear layer are hydroxyapatite and denatured collagen which cover the dentin surface and reduce up to 80% of the dentin permeability (Pashley et al., 1978 1978 ). EDTA is a mild acid that forms strong complexes with Ca 2+ . It retains the mineral phase of dentin, but removes the smear layer, thus enhancing the permeation and the chemical reaction of adhesive and dentin (Monticelli et al., 2008 2008 ). In general, on account of the use of EDTA, the depth of demineralization decreased, the hybrid layers were thinner, the permeation of resin monomers was better, and the μTBS increased (Sauro et al., 2009 2009 ; 2010 2010 ). However, conventional theories can hardly explain why 15% EDTA pretreatment could reduce the bond strength of GB. With regard to TM application, the general tendency seems to be that TM could be slightly beneficial for μTBS when using self-etching adhesives (Adebayo et al., 2008b 2008b ; Sattabanasuk et al., 2009 2009 ; Silva-Júnior et al., 2011 2011 ), but should not be applied when using etch-and-rinse adhesives (Sattabanasuk et al., 2009 2009 ). It is assumed that the chemical reaction between Ca 2+ in TM and the functional monomer of adhesive resin increases bond strength to dentin to some extent (Sattabanasuk et al., 2009 2009 ). In the current study, only two out of six experimental groups showed a significant increase in μTBS when using TM pretreatment and storage for 3 d. After storage of 6 months no significant influence could be observed, indicating that the long-term effect of TM application seems to be negligible. However, regardless of all differences in working mechanisms and chemical composition, after 6-month storage in water, no significant differences could be found between the adhesive resins. In contrast to three-step etch-and-rinse adhesives, the bond strength to dentin of both one-step and two-step self-etch adhesives decreased after 6 months of water storage in a comparative study (van Landuyt et al., 2010 2010 ). The overall significant reduction in bond strength after 6-month storage in the current study could be due to factors such as insufficient polymerization, water-uptake, and subsequent plasticization, water- and enzyme-induced nanoleakage, and/or the presence of voids due to phase-separation or osmosis; these demonstrate currently unsolved issues of bonding resin to dentin (Abdalla and Feilzer, 2008 2008 ; van Landuyt et al., 2010 2010 ). Conclusions 1 Initially, the tested adhesive resins showed significantly different μTBS, but after 6-month storage no significant differences could be found. TM treatment for hypersensitive teeth can be used clinically without compromising the bond strength of a self-etching two-step adhesive resin, an all-in-one adhesive resin, and a total-etch one-step adhesive in the long term. EDTA preconditioning had no significant long-term impact on bond strength. References AI Abdalla , AJ Feilzer . 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1300216 Project supported by the National Natural Science Foundation of China (Nos. 81271955 and 30973350) and the Zhejiang Provincial Natural Science Foundation of China (No. Y2080338) 2013-08-14 2014-06-20 2014-02-12 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2014 2014 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 07 15