journal of dentistry 42 (2014) 298–304
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Effect of smear layer deproteinizing on resin–dentine interface with self-etch adhesive Ornnicha Thanatvarakorn a,b, Masatoshi Nakajima a,*, Taweesak Prasansuttiporn c, Shizuko Ichinose d, Richard M. Foxton e, Junji Tagami a,b a Cariology and Operative Dentistry, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan b Global Center Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan c Department of Restorative Dentistry and Periodontology, Faculty of Dentistry, Chiang Mai University, T. Suthep, A. Muang, Chiang Mai 50200, Thailand d Instrumental Analysis Research Center, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan e Division of Conservative Dentistry, King’s College London Dental Institute at Guy’s, King’s and St Thomas’ Hospitals, King’s College London, Floor 25, London Bridge, London SE1-9RT, UK
article info
abstract
Article history:
Objectives: This study aimed to investigate deproteinizing effect of sodium-hypochlorite
Received 18 August 2013
(NaOCl) and mild acidic hypochlorous-acid (HOCl) pretreatment on smear layer-covered
Received in revised form
dentine and to evaluate their effects on morphological characteristics of resin–dentine
29 November 2013
interface with self-etch adhesive.
Accepted 30 November 2013
Methods: Human coronal-dentine discs with standardized smear layer were pretreated with 6% NaOCl or 50 ppm HOCl for 15 s or 30 s. Their deproteinizing effects at the treated smear layer-covered dentine surfaces were determined by the measurement of amide:phosphate
Keywords:
ratio using ATR-FTIR analysis. In addition, using TEM, micromorphological alterations of
Sodium-hypochlorite
hybridized complex and nanoleakage expression were evaluated at the interface of a self-
Hypochlorous-acid
etch adhesive (Clearfil SE Bond) to the pretreated dentine surface with or without subse-
Smear layer deproteinizing
quent application of a reducing agent ( p-Toluenesulfinic acid salt; Accel1).
Attenuated total reflection Fourier
Results: Both pretreatments of NaOCl and HOCl significantly reduced the amide:phosphate
transform infrared
ratio as compared with the no-pretreated group ( p < 0.05), coincident with the elimination of
Hybridized smear layer
the hybridized smear layer on their bonded interfaces. Nanoleakage within the hybrid layer
Nanoleakage
was found in the no-pretreated and NaOCl-pretreated groups, whereas the subsequent reducing agent application changed the reticular nanoleakage to spotted type. HOCl-pretreated groups showed less nanoleakage expression in a spotted pattern, regardless of reducing agent application. Conclusions: NaOCl and HOCl solutions could remove the organic component on the smear layer-covered dentine, which could eliminate the hybridized smear layer created by selfetch adhesive, leading to the reduction of nanoleakage expression within hybrid layer. Clinical significance: Smear layer deproteinizing could modify dentine surface, giving an appropriate substrate for bonding to self-etch adhesive system. # 2013 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +81 3 5803 5483; fax: +81 3 5803 0195. E-mail address: [email protected] (M. Nakajima). 0300-5712/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdent.2013.11.026
journal of dentistry 42 (2014) 298–304
1.
Introduction
Self-etch adhesive cannot completely remove smear layer due to its mild acidity. Accordingly, the resin-impregnated smear layer (so-called hybridized smear layer) is formed on the hybrid layer by incorporating remnant of smear layer.1 Smear layer does not have physiological and morphological continuous connection to the underlying dentine, therefore, hybridized smear layer is regarded as a weak link at the interface.2 Eliminating hybridized smear layer might be required to improve the quality of the adhesive interface of self-etch adhesive. Dentine smear layer is composed of disorganized organic debris with hydroxyapatite minerals.3,4 The apparent denatured and gel-like collagen in the outermost of smear layer is likely to act as a selective barrier for monomer infiltration.5 Some researchers have demonstrated that oxidizing/deproteinizing agent, such as sodium-hypochlorite (NaOCl) and hypochlorous-acid (HOCl) solutions, can thin the smear layer by dissolving the collagen fibrils.6,7 Additionally, pretreatment of these deproteinizing agents can improve bond strength of self-etch adhesives to caries-affected dentine with a thick organic-rich smear layer.6,7 It has been speculated that removal of the organic phase of the smear layer might promote further infiltration of resin monomer into the underlying dentine,8 which might lead to a reduction in nanoleakage. However, the residual oxidization effect within NaOCl-treated dentine reversely affected the adhesive performance by impairing resin polymerization, leading to compromised dentine bonding9,10 and an increase in nanoleakage.11 These negative effects on oxidized dentine can be reversed by the subsequent application of a reducing agent.9,11,12 Some studies have investigated the effect of collagen deproteinizing on acid-etched dentine,13,14 however, few information was available for the effect of smear layer deproteinizing on the dentine interface bonded to self-etch adhesive. Attenuated total reflection Fourier transform infrared (ATR-FTIR) microscopy is a useful method for identifying the presence of some function groups. It can be used to quantitatively investigate the change in amide:phosphate ratio on dentine surface, representing organic:inorganic components, from a certain pretreatment.15 The aim of this study was to evaluate the deproteinizing effect of NaOCl and HOCl solutions on smear layer-covered dentine by using ATR-FTIR. In addition, the micromorphological alterations in the hybridized complex and nanoleakage expression at the interface of self-etch adhesive bonded to the pretreated dentine surface with or without subsequent application of reducing agent were observed by transmission electron microscopy (TEM). The null hypothesis was that there was no difference neither in amide:phosphate ratio on the NaOCl-, HOCl-pretreated and nopretreated smear layer-covered dentine nor in morphology and nanoleakage expression at the adhesive interface of each pretreatment, and those interfaces that were further neutralized with the reducing agent, p-Toluenesulfinic acid salt.
2.
Materials and methods
2.1.
Specimen preparation
299
Extracted human third molars were collected after ethical approval by the Ethics Committee of Tokyo Medical and Dental University under protocol number 725. For evaluation of deproteinzing effect by ATR-FTIR, twenty mid-coronal dentine discs were prepared by using a slow-speed water-cooled diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA). In order to enable a reproducible FTIR spectra measurement on the selected area of occlusal side of each dentine disc, the pulpal side was marked by a high-speed handpiece using a fine diamond bur. For TEM observation of micromorphology alterations and nanoleakage expression on resin–dentine interface, the occlusal enamel perpendicular to the long axis of the tooth was removed by using a model trimmer under water lubrication and forty-two flat dentine surfaces were prepared. The standardized smear layer was created to all specimens by polishing dentine disc or flat dentine surface with 600-grit SiC paper under running water for 30 s. The pretreatments employed in this study were the application of 6% NaOCl (Jiaen 6%, Yoshida Co., Tokyo, Japan) or 50 ppm HOCl (Comfosy1, Haccpper Advantec Co., Tokyo, Japan) solution for 15 s or 30 s on smear layer-covered dentine surface, then rinsing with water for 30 s.
2.2.
FTIR spectroscopy
This study was designed as a before-and-after study. The control spectra were previously collected from all of the dentine discs; using an ATR-FTIR microscope (FTIR-8300, Shimadzu, Tokyo, Japan) under the condition of 750–4000/ cm 1 at 4/cm 1 resolution by using 100 scans. After pretreatments as described above (n = 5), the spectra were, again, collected on the same region. The deproteinizing effect of NaOCl or HOCl solution was evaluated by using the collagen to apatite ratio (the ratio of C O stretching vibrations of amide I at 1643 cm 1 to v3 P–O stretching vibrations at 1026 cm 1),15 and statistically analysed by two-way ANOVA and post hoc Tukey HSD’s multiple comparisons at the significance level of 0.05.
2.3.
Hybridized complex observation
Fifteen flat dentine surfaces with standardized smear layer were randomly divided into 4 groups of pretreatments as described above and one group of no pretreatment which served as control (n = 3). All specimens were then bonded by applying a two-step self-etch adhesive (Clearfil SE Bond, Kuraray Noritake Dental Inc., Tokyo, Japan) according to the manufacturer’s instructions (Table 1). After that, low-viscosity composite (Protect Liner F; Kuraray Noritake Dental Inc., Tokyo, Japan) was built up and light-cured for 40 s. After 24 h of water storage, two 0.9-mm thick slabs from the centre of each tooth were obtained using a slow-speed water-cooled diamond saw cut perpendicular to the bond interface. Fixing and 70-nm thick ultrathin sectioning processes were
300
journal of dentistry 42 (2014) 298–304
Table 1 – Materials used in this study. Materials Accel
1
Clearfil SE bond (pH = 2)
Manufacturer
Batch number
Composition
Application
Sun Medical Co. Ltd., Kyoto, Japan
FF1
p-Toluenesulfinic acid sodium salt, ethanol, water
Apply on dentine (5 s); gentle air dry (10 s)
Kuraray Noritake Dental Inc., Tokyo, Japan
01042A
Primer: 10-MDP, HEMA, Hydrophilic aliphatic dimethacrylate, N,N-diethanol-p-toluidine, CQ, water Bond: Bis-GMA, HEMA, DMA, 10-MDP, toluidine, silanated silica, CQ
Apply primer (20 s); high-pressure air (5 s);
01553A
Apply adhesive; gentle air stream until it becomes a thin film; light-cure (20 s)
Abbreviations: 10-MDP: 10-methacryloyloxydecyl dihydrogen phosphate; HEMA: 2-hydroxyethyl methacrylate; CQ: camphorquinone; Bis-GMA: 2,2-bis[4-(2-hydroxy-3-methacryloyloxypropoxy)phenyl]propane; DMA: dimethacrylate.
achieved,16 and then collected on 150-mesh nickel grids. The specimens were demineralized and stained by 1% phosphotungstic acid followed by 2% uranyl acetate17 for examining the status of collagen within the resin–dentine bond interface using a transmission electron microscope (TEM; H-7100, HITACHI, Tokyo, Japan) operating at 75 kV.
2.4.
Nanoleakage evaluation
Twenty-seven flat dentine surfaces with standardized smear layer were used and allocated into 9 groups as follow (n = 3); a group of no pretreatment (control), 4 groups of pretreatments as described above, and the 4 additional groups of each pretreatment followed by the 5-s application of reducing agent ( p-Toluenesulfinic acid salt; Accel1, Sun Medical Co. Ltd., Kyoto, Japan). After bonding procedure as described above, all specimens were subjected to nanoleakage procedure, according to the protocol previously described.18 The ultrathin sections were prepared and collected on copper grids. Without demineralization or staining, the nanoleakage expression of the bonded interface was examined under TEM.
3.
Results
3.1.
FTIR spectroscopy
HOCl-pretreated (Fig. 2D), and 30-s HOCl-pretreated dentine interfaces (Fig. 2E).
3.3.
Nanoleakage evaluation
Silver deposits were observed at the adhesive interfaces of all groups, but their expression varied. A reticular nanoleakage, consisting of fine discontinuous islands of silver deposits, was found predominantly in the bonded interface of no-pretreated dentine (Fig. 3A). In the NaOCl-pretreated dentine, a reticular nanoleakage was also identified increasing with an increase in
The representative spectra of NaOCl and HOCl pretreatments which were normalized to the phosphate stretching vibration at 1026/cm 1 reveal a reduction in the amide I peak at 1643/ cm 1 (Fig. 1A), compared to control. The amide:phosphate ratio significantly decreased after the NaOCl or HOCl pretreatment ( p < 0.05), regardless of the pretreatment time ( p > 0.05) (Fig. 1B).
3.2.
Hybridized complex observation
In the no-pretreated dentine, 1 mm-thick uneven hybridized complexes consisting of hybridized smear layer (Hs) and authentic hybrid layer (Ha) were observed (Fig. 2A). The Hs was identified as loosely aligned and disorganized microfibrillar strands, whereas the banded collagen fibrils could be seen in the Ha. In the 15-s NaOCl-pretreated dentine, the Hs was thinned, but slightly remained on the Ha (Fig. 2B), whereas the Hs was completely absent in the 30-s NaOCl-pretreated (Fig. 2C), 15-s
Fig. 1 – (A) Representative FTIR spectra of smear layercovered dentine in each pretreatment. The spectra were normalized to phosphate v3 (peak at 1026/cmS1). (B) Mean and standard deviation of amide:phosphate ratio of smear layer-covered dentine without or with pretreatment by deproteinizing solutions. Different lowercase letters represent significant differences ( p < 0.05).
journal of dentistry 42 (2014) 298–304
301
Fig. 2 – Demineralized TEM micrographs of the resin–dentine interfaces showing the status of the collagen fibrils. (A) Nopretreated group; the thick hybridized smear layer (Hs) with disorganizedly-dispersed microfibrillar strands was observed on the authentic hybrid layer (Ha) which was identified by intact collagen fibrils with cross banding (hand pointer). (B) 15-s NaOCl pretreatment group; the extremely-thin Hs was seen on the Ha. (C) 30-s NaOCl pretreatment group, (D) 15-s HOCl pretreatment group, (E) 30-s HOCl pretreatment group; their groups were absence of Hs. Ld: laboratory-demineralized intertubular dentine.
the pretreatment time (Fig. 3B and C), where the application of reducing agent after 15-s and 30-s NaOCl-pretreatments entirely and partially decreased the reticular nanoleakage formation, respectively (Fig. 3D and E). In the HOCl-pretreated groups, there were less silver deposits with the spotted nanoleakage, regardless of the reducing agent application (Fig. 4).
4.
Discussion
From the result of this study, the 15- or 30-s pretreatment of NaOCl or HOCl solution resulted in the removal of organic phase on the smear layer-covered dentine surface. The micromorphological findings revealed the elimination of hybridized smear layer in both pretreatment groups. The distribution of nanoleakage patterns varied in no-pretreated, NaOCl- or HOCl-pretreated dentine and that of furtherneutralized oxidized dentine. Thus, the null hypothesis can be rejected. In the no-pretreated dentine, a thick and uneven hybridized smear layer was observed concomitant with a reticular nanoleakage within the hybrid layer. This type of nanoleakage corresponds to regions of incomplete resin infiltration.19 Selfetch adhesives can dissolve only the mineral phase, but not the organic phase of the smear layer. The undissolved
remaining organic phase, being incorporated to form a hybridized smear layer,2 might in turn hamper resin monomer infiltration into the underlying dentine.20 NaOCl solution can effectively dissolve organic substrates from biological materials. Previous FTIR studies revealed that amide:phosphate ratio significantly reduced on smear layercovered dentine by using 1% NaOCl treatment for 60 s15 or using 5% NaOCl for 40 s,21 whereas the carbonate peak or carbonate:phosphate ratio was not altered. Likewise, in the present study, application with 6% NaOCl solution for 15 or 30 s significantly reduced amide:phosphate ratio on smear layer-covered dentine. These would indicate that only organic component on smear layer-covered dentine was eliminated. NaOCl dissociates into Na+ and OCl and establishes HOCl in water. HOCl is considered to be powerful oxidizer and deproteinizer, because it readily reacts with a range of biological molecules.22 However, the proportion of HOCl and the OCl in the solution depends on the pH.23–25 That is, above pH 9, almost all HOCl ionizes into OCl ,24,25 and at an approximate pH of 6, the concentration of HOCl is optimal and its dissociation is minimal.24,26 Therefore, OCl ions exist almost exclusively in NaOCl aqueous solution, due to their extremely alkalinity (pH 10.8–13.2). The 50 ppm HOCl solution used in this study was developed at a pH of 7.0 by acidifying NaOCl with HCl, so the majority of the chlorine is in the form of HOCl. A recent study demonstrated that HOCl solution with an
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journal of dentistry 42 (2014) 298–304
Fig. 3 – Undemineralized, silver-stained TEM micrographs of nanoleakage patterns in control and NaOCl-pretreated dentine. (A) No-pretreated group; the reticular nanoleakage pattern, consisting of discontinuous silver deposits (black arrows), was observed at the bottom of the hybrid layer. (B) 15-s NaOCl; the reticular nanoleakage pattern (black arrows) were entirely observed within the hybrid layer. (C) 30-s NaOCl; the size and density of silver deposits (black arrows) increased compared with (B). (D) 15-s NaOCl followed by reducing agent application; the spotted pattern, consisting of isolated spots of silver grains (hand pointers), were observed within the hybrid layer. (E) 30-s NaOCl followed by reducing agent application; amount of silver deposits decreased compared with (C), but the reticular nanoleakage pattern (black arrows) was held.
even lower chlorine concentration could thin the dentine smear layer due to dissolution of the organic component of the smear layer compared with NaOCl solution.7 From FTIR analysis in this study, the deproteinizing potential of 50 ppm HOCl solution was confirmed to be as effective as 6% NaOCl solution. However, micromorphological observation showed the partially remain of hybridized smear layer after 15-s NaOCl pretreatment, whereas both the 15-s and 30-s HOCl pretreatments could completely eliminate the hybridized smear layer. These results might indicate that the HOCl solution was able to eliminate the hybridized smear layer more effectively than that of NaOCl. Removal of the organic phase in the smear layer was expected to facilitate resin monomer infiltration into the underlying dentine, leading to a reduction in nanoleakage. However, reticular nanoleakage still remained in the NaOCltreated dentine, increasing with an increase in the NaOClpretreatment time. Many researchers have reported that NaOCl pretreatment compromises the bond strength to dentine.6,9 In our previous study, bond strength significantly reduced in the 30-s NaOCl-treated dentine, but not in the 15-s treated dentine.27 This reduction was attributed to the residual oxidizing effect in NaOCl-treated dentine, causing premature chain termination and incomplete resin polymerization.28 Presumably, impaired polymerization of resin monomer would result in increased nanoleakage in NaOCl-treated
dentine. Indeed, the increased nanoleakage observed in NaOCl-treated dentine dropped back after being neutralized with the reducing agent, p-Toluenesulfinic acid salt. These results are in agreement with the previous study using an etch & rinse system to NaOCl-treated acid-etched dentine.11 Moreover, in the 15-s NaOCl-treated dentine, applying a reducing agent changed the nanoleakage pattern from the reticular pattern, as found in the no-pretreated dentine, to the spotted pattern, which represents permeable regions of the acidic/hydrophilic resin components.18 The change to a spotted nanoleakage pattern would indicate further resin monomer infiltration into the hybrid layer. A spotted nanoleakage was observed in both the 15-s and 30-s HOCl-pretreated dentine specimens, regardless of the application of a reducing agent. These results would indicate that HOCl-pretreated dentine could facilitate resin monomer infiltration into underlying dentine and possess less oxidizing environment than NaOCl-pretreated dentine. In agricultural science, it was reported that NaOCl solution left significantly higher chlorine residues on dip treated surfaces than HOCl.29 In addition, the lower concentration of HOCl solution used in this study compared to NaOCl would be another reason why the residual oxidizing effect was less on HOCl-pretreated dentine. An ability to remove the organic phase of the smear layer coupled with a less residual oxidizing effect from HOCl solution could reduce nanoleakage in the tested self-etch
journal of dentistry 42 (2014) 298–304
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Fig. 4 – Undemineralized, silver-stained TEM micrographs of nanoleakage patterns in HOCl-pretreated dentine. (A) 15-s HOCl, (B) 30-s HOCl, (C) 15-s HOCl followed by reducing agent application, (D) 30-s HOCl followed by reducing agent application. The spotted nanoleakage pattern (hand pointers) was seen throughout the hybrid layer. No differences could be observed between the different HOCl pretreatment time, and with or without reducing agent application.
adhesive compared with the no-pretreated dentine. In our previous study, bond strengths to the HOCl-pretreated dentine with both application times exhibited no significant differences from that of the no-pretreated dentine.27 Initially, there was no correlation between nanoleakage and bond strength.30 However, it was demonstrated that after long-term water storage, the strong correlation appeared to exist between them.30 The presence of nanoleakage pathways within the hybrid layer would influence the long-term stability of resin– dentine interface. NaOCl solution has a reliable profile as disinfectant and deproteinizing agent26,31 and is routinely used in dental procedure. However, NaOCl solution can irritate the mucous membrane,26 whereas HOCl has been stated to exhibit broadspectrum antimicrobial activity without irritating and sensitizing properties.31 HOCl solution seems to be a safe and effective dentine-pretreatment solution for improving the quality of the dentine interface of self-etch adhesive by elimination of the hybridized smear layer with an ability to disinfect. The inhibition of nanoleakage within the hybrid layer may lead to an improvement in the long-term stability of bonding performance. However, further studies are required
to clarify the long-term durability of resin–dentine bond with smear layer deproteinizing.
5.
Conclusions
Smear layer deproteinizing with NaOCl or HOCl solution could eliminate the hybridized smear layer of the tested self-etch adhesive. Their nanoleakage expressions varied (increased or decreased), depending upon the residual oxidizing effect in the treated dentine, however a subsequent application of a reducing agent could modify the increased nanoleakage.
Acknowledgements The authors thank to Dr. Takahiro Wada and Dr. Masahiro Takahashi for FTIR analysis. This work was supported by a grant from the Japanese Ministry of Education, Global Center of Excellence (GCOE) Program, ‘‘International Research Center for Molecular Science in Tooth and Bone Diseases’’
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ScienceDirect journal homepage: www.intl.elsevierhealth.com/journals/jden
Effect of smear layer deproteinizing on resin–dentine interface with self-etch adhesive Ornnicha Thanatvarakorn a,b, Masatoshi Nakajima a,*, Taweesak Prasansuttiporn c, Shizuko Ichinose d, Richard M. Foxton e, Junji Tagami a,b a Cariology and Operative Dentistry, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan b Global Center Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan c Department of Restorative Dentistry and Periodontology, Faculty of Dentistry, Chiang Mai University, T. Suthep, A. Muang, Chiang Mai 50200, Thailand d Instrumental Analysis Research Center, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan e Division of Conservative Dentistry, King’s College London Dental Institute at Guy’s, King’s and St Thomas’ Hospitals, King’s College London, Floor 25, London Bridge, London SE1-9RT, UK
article info
abstract
Article history:
Objectives: This study aimed to investigate deproteinizing effect of sodium-hypochlorite
Received 18 August 2013
(NaOCl) and mild acidic hypochlorous-acid (HOCl) pretreatment on smear layer-covered
Received in revised form
dentine and to evaluate their effects on morphological characteristics of resin–dentine
29 November 2013
interface with self-etch adhesive.
Accepted 30 November 2013
Methods: Human coronal-dentine discs with standardized smear layer were pretreated with 6% NaOCl or 50 ppm HOCl for 15 s or 30 s. Their deproteinizing effects at the treated smear layer-covered dentine surfaces were determined by the measurement of amide:phosphate
Keywords:
ratio using ATR-FTIR analysis. In addition, using TEM, micromorphological alterations of
Sodium-hypochlorite
hybridized complex and nanoleakage expression were evaluated at the interface of a self-
Hypochlorous-acid
etch adhesive (Clearfil SE Bond) to the pretreated dentine surface with or without subse-
Smear layer deproteinizing
quent application of a reducing agent ( p-Toluenesulfinic acid salt; Accel1).
Attenuated total reflection Fourier
Results: Both pretreatments of NaOCl and HOCl significantly reduced the amide:phosphate
transform infrared
ratio as compared with the no-pretreated group ( p < 0.05), coincident with the elimination of
Hybridized smear layer
the hybridized smear layer on their bonded interfaces. Nanoleakage within the hybrid layer
Nanoleakage
was found in the no-pretreated and NaOCl-pretreated groups, whereas the subsequent reducing agent application changed the reticular nanoleakage to spotted type. HOCl-pretreated groups showed less nanoleakage expression in a spotted pattern, regardless of reducing agent application. Conclusions: NaOCl and HOCl solutions could remove the organic component on the smear layer-covered dentine, which could eliminate the hybridized smear layer created by selfetch adhesive, leading to the reduction of nanoleakage expression within hybrid layer. Clinical significance: Smear layer deproteinizing could modify dentine surface, giving an appropriate substrate for bonding to self-etch adhesive system. # 2013 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +81 3 5803 5483; fax: +81 3 5803 0195. E-mail address: [email protected] (M. Nakajima). 0300-5712/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdent.2013.11.026
journal of dentistry 42 (2014) 298–304
1.
Introduction
Self-etch adhesive cannot completely remove smear layer due to its mild acidity. Accordingly, the resin-impregnated smear layer (so-called hybridized smear layer) is formed on the hybrid layer by incorporating remnant of smear layer.1 Smear layer does not have physiological and morphological continuous connection to the underlying dentine, therefore, hybridized smear layer is regarded as a weak link at the interface.2 Eliminating hybridized smear layer might be required to improve the quality of the adhesive interface of self-etch adhesive. Dentine smear layer is composed of disorganized organic debris with hydroxyapatite minerals.3,4 The apparent denatured and gel-like collagen in the outermost of smear layer is likely to act as a selective barrier for monomer infiltration.5 Some researchers have demonstrated that oxidizing/deproteinizing agent, such as sodium-hypochlorite (NaOCl) and hypochlorous-acid (HOCl) solutions, can thin the smear layer by dissolving the collagen fibrils.6,7 Additionally, pretreatment of these deproteinizing agents can improve bond strength of self-etch adhesives to caries-affected dentine with a thick organic-rich smear layer.6,7 It has been speculated that removal of the organic phase of the smear layer might promote further infiltration of resin monomer into the underlying dentine,8 which might lead to a reduction in nanoleakage. However, the residual oxidization effect within NaOCl-treated dentine reversely affected the adhesive performance by impairing resin polymerization, leading to compromised dentine bonding9,10 and an increase in nanoleakage.11 These negative effects on oxidized dentine can be reversed by the subsequent application of a reducing agent.9,11,12 Some studies have investigated the effect of collagen deproteinizing on acid-etched dentine,13,14 however, few information was available for the effect of smear layer deproteinizing on the dentine interface bonded to self-etch adhesive. Attenuated total reflection Fourier transform infrared (ATR-FTIR) microscopy is a useful method for identifying the presence of some function groups. It can be used to quantitatively investigate the change in amide:phosphate ratio on dentine surface, representing organic:inorganic components, from a certain pretreatment.15 The aim of this study was to evaluate the deproteinizing effect of NaOCl and HOCl solutions on smear layer-covered dentine by using ATR-FTIR. In addition, the micromorphological alterations in the hybridized complex and nanoleakage expression at the interface of self-etch adhesive bonded to the pretreated dentine surface with or without subsequent application of reducing agent were observed by transmission electron microscopy (TEM). The null hypothesis was that there was no difference neither in amide:phosphate ratio on the NaOCl-, HOCl-pretreated and nopretreated smear layer-covered dentine nor in morphology and nanoleakage expression at the adhesive interface of each pretreatment, and those interfaces that were further neutralized with the reducing agent, p-Toluenesulfinic acid salt.
2.
Materials and methods
2.1.
Specimen preparation
299
Extracted human third molars were collected after ethical approval by the Ethics Committee of Tokyo Medical and Dental University under protocol number 725. For evaluation of deproteinzing effect by ATR-FTIR, twenty mid-coronal dentine discs were prepared by using a slow-speed water-cooled diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA). In order to enable a reproducible FTIR spectra measurement on the selected area of occlusal side of each dentine disc, the pulpal side was marked by a high-speed handpiece using a fine diamond bur. For TEM observation of micromorphology alterations and nanoleakage expression on resin–dentine interface, the occlusal enamel perpendicular to the long axis of the tooth was removed by using a model trimmer under water lubrication and forty-two flat dentine surfaces were prepared. The standardized smear layer was created to all specimens by polishing dentine disc or flat dentine surface with 600-grit SiC paper under running water for 30 s. The pretreatments employed in this study were the application of 6% NaOCl (Jiaen 6%, Yoshida Co., Tokyo, Japan) or 50 ppm HOCl (Comfosy1, Haccpper Advantec Co., Tokyo, Japan) solution for 15 s or 30 s on smear layer-covered dentine surface, then rinsing with water for 30 s.
2.2.
FTIR spectroscopy
This study was designed as a before-and-after study. The control spectra were previously collected from all of the dentine discs; using an ATR-FTIR microscope (FTIR-8300, Shimadzu, Tokyo, Japan) under the condition of 750–4000/ cm 1 at 4/cm 1 resolution by using 100 scans. After pretreatments as described above (n = 5), the spectra were, again, collected on the same region. The deproteinizing effect of NaOCl or HOCl solution was evaluated by using the collagen to apatite ratio (the ratio of C O stretching vibrations of amide I at 1643 cm 1 to v3 P–O stretching vibrations at 1026 cm 1),15 and statistically analysed by two-way ANOVA and post hoc Tukey HSD’s multiple comparisons at the significance level of 0.05.
2.3.
Hybridized complex observation
Fifteen flat dentine surfaces with standardized smear layer were randomly divided into 4 groups of pretreatments as described above and one group of no pretreatment which served as control (n = 3). All specimens were then bonded by applying a two-step self-etch adhesive (Clearfil SE Bond, Kuraray Noritake Dental Inc., Tokyo, Japan) according to the manufacturer’s instructions (Table 1). After that, low-viscosity composite (Protect Liner F; Kuraray Noritake Dental Inc., Tokyo, Japan) was built up and light-cured for 40 s. After 24 h of water storage, two 0.9-mm thick slabs from the centre of each tooth were obtained using a slow-speed water-cooled diamond saw cut perpendicular to the bond interface. Fixing and 70-nm thick ultrathin sectioning processes were
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Table 1 – Materials used in this study. Materials Accel
1
Clearfil SE bond (pH = 2)
Manufacturer
Batch number
Composition
Application
Sun Medical Co. Ltd., Kyoto, Japan
FF1
p-Toluenesulfinic acid sodium salt, ethanol, water
Apply on dentine (5 s); gentle air dry (10 s)
Kuraray Noritake Dental Inc., Tokyo, Japan
01042A
Primer: 10-MDP, HEMA, Hydrophilic aliphatic dimethacrylate, N,N-diethanol-p-toluidine, CQ, water Bond: Bis-GMA, HEMA, DMA, 10-MDP, toluidine, silanated silica, CQ
Apply primer (20 s); high-pressure air (5 s);
01553A
Apply adhesive; gentle air stream until it becomes a thin film; light-cure (20 s)
Abbreviations: 10-MDP: 10-methacryloyloxydecyl dihydrogen phosphate; HEMA: 2-hydroxyethyl methacrylate; CQ: camphorquinone; Bis-GMA: 2,2-bis[4-(2-hydroxy-3-methacryloyloxypropoxy)phenyl]propane; DMA: dimethacrylate.
achieved,16 and then collected on 150-mesh nickel grids. The specimens were demineralized and stained by 1% phosphotungstic acid followed by 2% uranyl acetate17 for examining the status of collagen within the resin–dentine bond interface using a transmission electron microscope (TEM; H-7100, HITACHI, Tokyo, Japan) operating at 75 kV.
2.4.
Nanoleakage evaluation
Twenty-seven flat dentine surfaces with standardized smear layer were used and allocated into 9 groups as follow (n = 3); a group of no pretreatment (control), 4 groups of pretreatments as described above, and the 4 additional groups of each pretreatment followed by the 5-s application of reducing agent ( p-Toluenesulfinic acid salt; Accel1, Sun Medical Co. Ltd., Kyoto, Japan). After bonding procedure as described above, all specimens were subjected to nanoleakage procedure, according to the protocol previously described.18 The ultrathin sections were prepared and collected on copper grids. Without demineralization or staining, the nanoleakage expression of the bonded interface was examined under TEM.
3.
Results
3.1.
FTIR spectroscopy
HOCl-pretreated (Fig. 2D), and 30-s HOCl-pretreated dentine interfaces (Fig. 2E).
3.3.
Nanoleakage evaluation
Silver deposits were observed at the adhesive interfaces of all groups, but their expression varied. A reticular nanoleakage, consisting of fine discontinuous islands of silver deposits, was found predominantly in the bonded interface of no-pretreated dentine (Fig. 3A). In the NaOCl-pretreated dentine, a reticular nanoleakage was also identified increasing with an increase in
The representative spectra of NaOCl and HOCl pretreatments which were normalized to the phosphate stretching vibration at 1026/cm 1 reveal a reduction in the amide I peak at 1643/ cm 1 (Fig. 1A), compared to control. The amide:phosphate ratio significantly decreased after the NaOCl or HOCl pretreatment ( p < 0.05), regardless of the pretreatment time ( p > 0.05) (Fig. 1B).
3.2.
Hybridized complex observation
In the no-pretreated dentine, 1 mm-thick uneven hybridized complexes consisting of hybridized smear layer (Hs) and authentic hybrid layer (Ha) were observed (Fig. 2A). The Hs was identified as loosely aligned and disorganized microfibrillar strands, whereas the banded collagen fibrils could be seen in the Ha. In the 15-s NaOCl-pretreated dentine, the Hs was thinned, but slightly remained on the Ha (Fig. 2B), whereas the Hs was completely absent in the 30-s NaOCl-pretreated (Fig. 2C), 15-s
Fig. 1 – (A) Representative FTIR spectra of smear layercovered dentine in each pretreatment. The spectra were normalized to phosphate v3 (peak at 1026/cmS1). (B) Mean and standard deviation of amide:phosphate ratio of smear layer-covered dentine without or with pretreatment by deproteinizing solutions. Different lowercase letters represent significant differences ( p < 0.05).
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301
Fig. 2 – Demineralized TEM micrographs of the resin–dentine interfaces showing the status of the collagen fibrils. (A) Nopretreated group; the thick hybridized smear layer (Hs) with disorganizedly-dispersed microfibrillar strands was observed on the authentic hybrid layer (Ha) which was identified by intact collagen fibrils with cross banding (hand pointer). (B) 15-s NaOCl pretreatment group; the extremely-thin Hs was seen on the Ha. (C) 30-s NaOCl pretreatment group, (D) 15-s HOCl pretreatment group, (E) 30-s HOCl pretreatment group; their groups were absence of Hs. Ld: laboratory-demineralized intertubular dentine.
the pretreatment time (Fig. 3B and C), where the application of reducing agent after 15-s and 30-s NaOCl-pretreatments entirely and partially decreased the reticular nanoleakage formation, respectively (Fig. 3D and E). In the HOCl-pretreated groups, there were less silver deposits with the spotted nanoleakage, regardless of the reducing agent application (Fig. 4).
4.
Discussion
From the result of this study, the 15- or 30-s pretreatment of NaOCl or HOCl solution resulted in the removal of organic phase on the smear layer-covered dentine surface. The micromorphological findings revealed the elimination of hybridized smear layer in both pretreatment groups. The distribution of nanoleakage patterns varied in no-pretreated, NaOCl- or HOCl-pretreated dentine and that of furtherneutralized oxidized dentine. Thus, the null hypothesis can be rejected. In the no-pretreated dentine, a thick and uneven hybridized smear layer was observed concomitant with a reticular nanoleakage within the hybrid layer. This type of nanoleakage corresponds to regions of incomplete resin infiltration.19 Selfetch adhesives can dissolve only the mineral phase, but not the organic phase of the smear layer. The undissolved
remaining organic phase, being incorporated to form a hybridized smear layer,2 might in turn hamper resin monomer infiltration into the underlying dentine.20 NaOCl solution can effectively dissolve organic substrates from biological materials. Previous FTIR studies revealed that amide:phosphate ratio significantly reduced on smear layercovered dentine by using 1% NaOCl treatment for 60 s15 or using 5% NaOCl for 40 s,21 whereas the carbonate peak or carbonate:phosphate ratio was not altered. Likewise, in the present study, application with 6% NaOCl solution for 15 or 30 s significantly reduced amide:phosphate ratio on smear layer-covered dentine. These would indicate that only organic component on smear layer-covered dentine was eliminated. NaOCl dissociates into Na+ and OCl and establishes HOCl in water. HOCl is considered to be powerful oxidizer and deproteinizer, because it readily reacts with a range of biological molecules.22 However, the proportion of HOCl and the OCl in the solution depends on the pH.23–25 That is, above pH 9, almost all HOCl ionizes into OCl ,24,25 and at an approximate pH of 6, the concentration of HOCl is optimal and its dissociation is minimal.24,26 Therefore, OCl ions exist almost exclusively in NaOCl aqueous solution, due to their extremely alkalinity (pH 10.8–13.2). The 50 ppm HOCl solution used in this study was developed at a pH of 7.0 by acidifying NaOCl with HCl, so the majority of the chlorine is in the form of HOCl. A recent study demonstrated that HOCl solution with an
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Fig. 3 – Undemineralized, silver-stained TEM micrographs of nanoleakage patterns in control and NaOCl-pretreated dentine. (A) No-pretreated group; the reticular nanoleakage pattern, consisting of discontinuous silver deposits (black arrows), was observed at the bottom of the hybrid layer. (B) 15-s NaOCl; the reticular nanoleakage pattern (black arrows) were entirely observed within the hybrid layer. (C) 30-s NaOCl; the size and density of silver deposits (black arrows) increased compared with (B). (D) 15-s NaOCl followed by reducing agent application; the spotted pattern, consisting of isolated spots of silver grains (hand pointers), were observed within the hybrid layer. (E) 30-s NaOCl followed by reducing agent application; amount of silver deposits decreased compared with (C), but the reticular nanoleakage pattern (black arrows) was held.
even lower chlorine concentration could thin the dentine smear layer due to dissolution of the organic component of the smear layer compared with NaOCl solution.7 From FTIR analysis in this study, the deproteinizing potential of 50 ppm HOCl solution was confirmed to be as effective as 6% NaOCl solution. However, micromorphological observation showed the partially remain of hybridized smear layer after 15-s NaOCl pretreatment, whereas both the 15-s and 30-s HOCl pretreatments could completely eliminate the hybridized smear layer. These results might indicate that the HOCl solution was able to eliminate the hybridized smear layer more effectively than that of NaOCl. Removal of the organic phase in the smear layer was expected to facilitate resin monomer infiltration into the underlying dentine, leading to a reduction in nanoleakage. However, reticular nanoleakage still remained in the NaOCltreated dentine, increasing with an increase in the NaOClpretreatment time. Many researchers have reported that NaOCl pretreatment compromises the bond strength to dentine.6,9 In our previous study, bond strength significantly reduced in the 30-s NaOCl-treated dentine, but not in the 15-s treated dentine.27 This reduction was attributed to the residual oxidizing effect in NaOCl-treated dentine, causing premature chain termination and incomplete resin polymerization.28 Presumably, impaired polymerization of resin monomer would result in increased nanoleakage in NaOCl-treated
dentine. Indeed, the increased nanoleakage observed in NaOCl-treated dentine dropped back after being neutralized with the reducing agent, p-Toluenesulfinic acid salt. These results are in agreement with the previous study using an etch & rinse system to NaOCl-treated acid-etched dentine.11 Moreover, in the 15-s NaOCl-treated dentine, applying a reducing agent changed the nanoleakage pattern from the reticular pattern, as found in the no-pretreated dentine, to the spotted pattern, which represents permeable regions of the acidic/hydrophilic resin components.18 The change to a spotted nanoleakage pattern would indicate further resin monomer infiltration into the hybrid layer. A spotted nanoleakage was observed in both the 15-s and 30-s HOCl-pretreated dentine specimens, regardless of the application of a reducing agent. These results would indicate that HOCl-pretreated dentine could facilitate resin monomer infiltration into underlying dentine and possess less oxidizing environment than NaOCl-pretreated dentine. In agricultural science, it was reported that NaOCl solution left significantly higher chlorine residues on dip treated surfaces than HOCl.29 In addition, the lower concentration of HOCl solution used in this study compared to NaOCl would be another reason why the residual oxidizing effect was less on HOCl-pretreated dentine. An ability to remove the organic phase of the smear layer coupled with a less residual oxidizing effect from HOCl solution could reduce nanoleakage in the tested self-etch
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Fig. 4 – Undemineralized, silver-stained TEM micrographs of nanoleakage patterns in HOCl-pretreated dentine. (A) 15-s HOCl, (B) 30-s HOCl, (C) 15-s HOCl followed by reducing agent application, (D) 30-s HOCl followed by reducing agent application. The spotted nanoleakage pattern (hand pointers) was seen throughout the hybrid layer. No differences could be observed between the different HOCl pretreatment time, and with or without reducing agent application.
adhesive compared with the no-pretreated dentine. In our previous study, bond strengths to the HOCl-pretreated dentine with both application times exhibited no significant differences from that of the no-pretreated dentine.27 Initially, there was no correlation between nanoleakage and bond strength.30 However, it was demonstrated that after long-term water storage, the strong correlation appeared to exist between them.30 The presence of nanoleakage pathways within the hybrid layer would influence the long-term stability of resin– dentine interface. NaOCl solution has a reliable profile as disinfectant and deproteinizing agent26,31 and is routinely used in dental procedure. However, NaOCl solution can irritate the mucous membrane,26 whereas HOCl has been stated to exhibit broadspectrum antimicrobial activity without irritating and sensitizing properties.31 HOCl solution seems to be a safe and effective dentine-pretreatment solution for improving the quality of the dentine interface of self-etch adhesive by elimination of the hybridized smear layer with an ability to disinfect. The inhibition of nanoleakage within the hybrid layer may lead to an improvement in the long-term stability of bonding performance. However, further studies are required
to clarify the long-term durability of resin–dentine bond with smear layer deproteinizing.
5.
Conclusions
Smear layer deproteinizing with NaOCl or HOCl solution could eliminate the hybridized smear layer of the tested self-etch adhesive. Their nanoleakage expressions varied (increased or decreased), depending upon the residual oxidizing effect in the treated dentine, however a subsequent application of a reducing agent could modify the increased nanoleakage.
Acknowledgements The authors thank to Dr. Takahiro Wada and Dr. Masahiro Takahashi for FTIR analysis. This work was supported by a grant from the Japanese Ministry of Education, Global Center of Excellence (GCOE) Program, ‘‘International Research Center for Molecular Science in Tooth and Bone Diseases’’
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