A review: Biodegradation of resin dentin bonds

Transcription

1 Japanese Dental Science Review (2011) 47, 5 12 available at journal homepage: Review article A review: Biodegradation of resin dentin bonds Masanori Hashimoto *, Futami Nagano, Kazuhiko Endo, Hiroki Ohno Division of Biomaterials and Bioengineering, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido , Japan Received 17 August 2009; received in revised form 28 December 2009; accepted 15 February 2010 KEYWORDS Adhesion; Hybrid layer; Degradation; Electron microscopy; Polymer; Resin adhesive Summary Resin dentin bonding was first achieved through mechanical hybridization between resin and collagen fibrils using a functional monomer containing resin system. In the last decade, new adhesive resin systems were frequently released onto the market within a short-period of time. Before and after commercialization, the bond integrity has been tested by bond tests, and leakage evaluation by researchers, but it is very difficult for clinicians to obtain a comprehensive, up-to-date understanding of their nature and degradation. Although newly developed adhesive resins have attempted to improve the bond strength at least in the first 24 h after bonding, the long-term durability of the bonds has not yet been established analytically. However, numerous recent studies have shown micromorphological evidence of biodegradation of resin dentin bonds, due to hydrolysis of the resin and collagen fibrils within the bonds. This review mainly summarizes the most recent work in biodegradation of resin dentin bonds based on micromorphological analyses of data obtained by scanning and transmission electron microscopy. # 2011 Japanese Association for Dental Science. Published by Elsevier Ltd. All rights reserved. Contents 1. Introduction Bond degradation of total-etch (etch-and-rinse) adhesives Bond degradation of self-etching adhesives Bond degradation of one-bottle self-etching adhesives Summary Acknowledgements References Introduction * Corresponding author. Tel.: ; fax: addresses: masanori-h@mue.biglobe.ne.jp, has@hoku-iryo-u.ac.jp (M. Hashimoto). Many resin adhesive systems and types have been developed and marketed in dentistry over the last two decades (Fig. 1). The initial attempts at adhesion of resin concentrated on enamel, the first successful attempts to achieve a micromechanical interlocking of resin tag formation with acid /$ see front matter # 2011 Japanese Association for Dental Science. Published by Elsevier Ltd. All rights reserved. doi: /j.jdsr

2 6 M. Hashimoto et al. Figure 1 Classification of adhesive resin systems. E: etchant; P: primer; B: bonding resin (adhesive). pretreatment being reported by Buonocore [1]. However, resin bonding to dentin could not be achieved due to its complicated structure involving surface moisture and the collagen network. Nakabayashi et al. reported micromechanical bonding of a functional monomer containing resin system (4-META/MMA-TBB) to collagen fibrils in demineralized dentin in 1982 [2]. Since a smear layer created during tooth preparation has an adverse effect on dentin bonding [3,4] due to weak adhesion to underling dentin [5], its removal by an acidic agent prior to application of a bonding resin require to gain stronger bond strength. Acid-etching of etch-and-rinse systems (total-etching system) demineralizes the dentin surface to expose a few-micron-thick collagen network providing space for resin infiltration. The following application of a primer and/or adhesive penetrates into the interfibrillar spaces of the collagen web, leading to the formation of a hybrid layer composed of collagen fibrils and adhesive resin (Fig. 2). Wet bonding leaves the dentin surface visibly moist after acid etching. This leads to increased resin infiltration into the exposed collagen network as a result of increasing bond strength [6,7]. However, the degree of dentin surface wetness (i.e. moist, wet or overwet) greatly affects the bond strength in both the laboratory and clinical situations. The depth of demineralization and completeness of monomer diffusion affect the quality of the hybrid layer. When the former exceeds the latter, a region of naked collagen fibers is left exposed as a bond defect (Fig. 3). For the first system of self-etching adhesives, an acidic monomer of phenyl-p diffuses through extant smear layers to reach the underlying calcium-rich dentin as a result of formation of a hybrid layer where the smear layer remains in the lower half of the hybrid layer [8,9]. The hybridization occurs among resin, collagen fibrils, and inorganic matter of the smear layer (Fig. 2). Theoretically, there are no demineralized dentin zones in the bond face because of the lack of acid pretreatment [8 10]. This system is attractive to clinicians because it is thought the application step is simple and easy compared with total-etching systems with separate etching and water rinsing step. Newly marketed one-bottle self-etching systems, have been developed with a view to further simplifying the bonding procedure, combining etching, priming, and bonding Figure 2 Schematic illustration of bond structure of a selfetching system and etch-and-rinse system (total-etching system). The structure of the hybrid layer of total-etch adhesives consists of resin and collagen fibrils. The combination of resin, collagen fibrils and inorganic matter (the smear layer) is a typical hybrid layer of self-etching adhesives. The hybrid layer of selfetching systems is thinner than that of total-etching adhesives, due to the mild acidic effect of their acidic monomers on dentin. steps into one component (Fig. 1). These adhesives can be categorized as a self-etching system. If the hybridized layer is impermeable to water or various chemical stimuli, it could make dentin stable for long-term clinical use. However, many studies have reported the longterm water storage testing of resin dentin bonds to measure bond strength [11 23]. Most adhesives had decreased bond Figure 3 Schematic illustration of demineralized dentin zone of total-etch adhesives. Inadequate resin infiltration into collagen fibrils leaves nano- or microspaces within the adhesive interface. This zone is not common for self-etching adhesive systems. Collagen hydrolysis of the demineralized dentin zone is one of the degradation patterns for total-etch adhesives. In the absence of a demineralized dentin zone within the bonds, this collagen hydrolysis does not occur because the encapusulation of cured adhesive resins or protection of the mineral matrix of dentin prevents chemical degradation of the collagen fibrils even after long-term function.

3 A review: Biodegradation of resin-dentin bonds 7 strength to various extents after long-term water storage such as for 6 months or 1 year. All such degradation is accelerated in the presence of water [24,25]. The hydrolytic effect of water on the adhesives itself is of great importance when considering the bond degradation. Although the extent of the bond strength reduction is similar for all types of adhesives after aging, micromorphological examination is able to demonstrate the various degradation phases for each adhesive system in in vivo and in vitro. Since the bond structure of the resin dentin bond depends on the type of adhesive, micromorphological analysis reveals various degradation patterns of bonds after aging. The objective of this article is to provide a critical review of the degradation of resin dentin bonds for all types of adhesive systems. 2. Bond degradation of total-etch (etch-and-rinse) adhesives Figure 4 Scanning electron micrographs of fractured surface of resin dentin bonded beams. A three-step total-etch adhesive were used for both specimens. Fractured surface were obtained in the dentin side of specimen after microtensile bond test. A fractured surface of control specimens (after 24 h of bonding) (a) and that had been functioning in human oral cavity for 10 months (b). The presence of lateral branches of dentinal tubules is morphological symptom of collagen depletion. Acidic solutions (i.e. 35% phosphoric acid) are used to demineralize the smear layer and the underlying intact dentin to expose the collagen network. The incomplete impregnation of the exposed collagen space by subsequent application of bonding resin is due to imperfect resin monomer infiltration (Fig. 3). The discrepancy between the depth of the collagen layer and resin infiltration creates an exposed demineralized dentin zone under the hybrid layer (Fig. 3) [26 35]. These zones correspond with the sites of different modes of silver nitrate staining within the hybrid layer. Spencer and Wang reported resin monomer distribution in the demineralized dentin zone, especially evaluating the heterogeneity of the monomer collagen interaction, using micro-raman spectroscopy [34,35]. In their publications, the differentiation of resin monomer diffusion was reveal to depend on the molecular weight of the monomer at the collagen network of the hybrid layer [34,35]. Using atomic force microscopy (AFM), Marshall et al. observed in situ collagen morphology before and after dentin surface treatment [36 38]. The zone of demineralized dentin and the degradation phase were found morphologically using AFM analysis [29]. The exposed collagen fibrils here underwent structural deterioration due to hydrolytic degradation, resulting in decreasing bond strength [12,14 16]. In vivo morphological evidence of collagen hydrolysis was first obtained using extracted human primary teeth with resin restorations [12,39]. Fig. 4 shows collagen degradation within the demineralized dentin in the fractured surface of a resin dentin bonded specimen (Scotchbond Multi-Purpose) that functioned in the human oral environment for 10 months (Fig. 4b). Although an intact hybrid layer is visible in the control specimen at 24 h after bonding (Fig. 4a), hydrolytic degradation of collagen fibrils (Fig. 4b) is clearly observed after aging. There are many lateral branches of dentinal tubules at the fractured surfaces of the dentin side of a specimen in the aged specimen (Fig. 4b), whereas there are none in the control (Fig. 4a). The peritubular matrix of the dentinal tubules is richer in inorganic compounds than the lateral branches and the lateral branches are readily widened by collagen hydrolysis [12,39]. The degradation of the collagen suggests that the resin dentin bonds made with this adhesive system remained a demineralized dentin zone due to the incomplete encapsulation of the dentin by the resin. The same degradation has also been morphologically found using SEM and TEM in in vitro testing of a total-etching system with resin dentin bonded beams after 1 year of water storage [12,14 16]. Several in vitro studies using morphological analysis have shown hydrolytic degradation of the collagen mesh in the resin dentin interface in specimens stored in water for over 1 year [29,39,40]. Transmission electron microscopic examinations have shown the deformation of less stainable collagen fibrils as an indicator of collagen chemical degradation [16,41]. This hydrolysis of collagen greatly affects the longterm bond stability of total-etch adhesive systems. One reason for this collagen hydrolysis may be the effects of saliva or oral bacteria [42,43] in the human oral environment. However, little information is available on the mechanism of collagen degradation in vitro. Recently, Pashley et al. established a different concept, degradation of naked collagen fibrils by host-derived matrix metalloproteinases (MMPs) in the dentin matrix [44 50]. Matrix metalloproteinases are a family of zinc-dependent proteolytic enzymes that are capable of degrading the organic dentin matrix after demineralization [51]. Although collagenolytic or gelatinolytic activity identified from oral

4 8 M. Hashimoto et al. bacteria [52] may contribute to the hydrolysis of organic matter of the dentinal matrices in the caries process, recent studies have reported host-derived proteinases in the form of different types of MMPs present and released from the dentin matrix [51,53,54]. When a region of naked collagen remains in the demineralized dentin zone, the gradual and slow release of active MMPs dissolves the collagen during the long-term, even in in vitro conditions. In addition, by SEM and TEM micromorphological evidence of self-destruction of collagen has been found in the human dentin matrix in vivo and in vitro [44 50]. Elution of resin from hybrid layers due to hydrolysis of the resin is a further possible explanation for the bond degradation of total-etching adhesives [17,55 58]. This degradation phase has been found in all type of adhesive systems. 3. Bond degradation of self-etching adhesives The combination of an etchant and primer into a self-etching primer is advantageous in that it eliminates one application step. For etch-and-rinse systems, factors affecting sensitivity include the surface wetness of the acid etched dentin, acidetching time, light irradiation time, thickness of the bonding resin layer, consecutive coating methods, and the method of air blowing for the adhesive-coated dentin surface, etc. [6,7,59 61]. For self-etching adhesives, the surface wetness of acid-etched dentin can be theoretically eliminated because there is no water rinsing or dentin moisture retention. The fewer application steps of self-etching adhesives are thought to require less skill by the operator and be easier to implement. Self-etching primer adhesives differ from the etch-andrinse systems in that the self-etching adhesives partially involve original smear layers in the hybrid layer [8,9]. The acidic monomer penetrates into the smear layer, smear plug or underlying intact dentin but can be neutralized to stop the demineralized reaction due to ph change [62]. Therefore, the hybrid layer of self-etching systems is a combined structure of resin, collagen fibrils, and minerals. The strong acid of etch-and-rinse systems (i.e % phosphoric acid) completely dissolves the matrix of the dentin surface, including the smear layer, exposing the collagen network approximately 3 7 mm in depth. For self-etching adhesives, the smear layer is completely or partially enveloped into the bond structure, providing simultaneous demineralization and infiltration during the application of the acidic monomer, resulting in formation of a hybrid layer. The market-driven simplification of adhesive systems of self-etching primers that combine the conditioning and priming is thought to have overcome the shortcomings of the formation of an exposed collagen network within the bonds of the total-etching adhesives. However, incomplete infiltration was also found as nanoleakage within the hybrid layer [63,64]. Therefore, there is a route for water impregnation into bond faces of self-etching systems after bonding. One of the beneficial characteristics of a microtensile bond test is that the bond strength measurement can be done for specimens with small adhesive areas (i.e. 1mm 2 ). Using a microtensile bond test, Sano et al. measured resin dentin bond strengths in in vivo specimens after long-term function in monkeys [11]. This small adhesive area of the microtensile bond test allowed them to make specimen beams from resin dentin bonded teeth that had functioned orally. The study revealed evidence of hydrolysis of bonding resin within the hybrid layer of a self-etching adhesive after 1 year of functioning. Later, similar morphological evidence of degradation was confirmed by in vitro tests [13,14]. However, hydrolysis of collagen fibrils is not common as a degradation phase with self-etching adhesive systems. Regions of incomplete resin infiltration or incomplete resin polymerization within the hybrid layer or bonding resin termed nanoleakage have been shown by silver tracer deposition. Although nanoleakage can theoretically be eliminated by using self-etching adhesives, many studies have shown that all self-etching adhesives had a spot- or reticular-mode of nanoleakage within the hybrid layer [63 67]. Although a naked collagen zone was absent within the interface of the self-etching adhesives, it is possible that a loss of resin may initiate and promote proteolytic hydrolysis of collagen. However, this aging pattern is uncommon and which of the structures (collagen or resin) contributes to the bond degradation is unclear. According to the results of these previous studies, resin hydrolysis may be more damaging to long-term bonding effectiveness than collagen hydrolysis in the case of the hybrid layer of self-etching adhesives. 4. Bond degradation of one-bottle selfetching adhesives Recently, one-bottle self-etching adhesive systems are widely used in clinics because of their simple and easy application. Self-etching adhesive systems are currently available as two-step and single-step types. The single-step self-etching systems can be further divided into two types, the all-in-one and one-bottle types, depending on whether they require mixing or not. The recently introduced all-inone adhesives are supplied as two-bottles that are mixed together immediately before use. One-bottle self-etching adhesives that combine the etchant, primer, and bonding resin into one bottle with single-step application have been developed, allowing simultaneous etching and priming with one adhesive component. One drop of the adhesive is applied to the dentin/enamel surface with the smear layer covered, resulting in the combination of resin, collagen, and hard tissue as a bonding substrate. This system is generally thought to be less technique-sensitive and time-consuming than traditional adhesive resins (two-step self-etching and etch-and-rinse adhesives). This system is thus attractive for clinicians because of its easy handling and short application time. Nanoleakage was first visualized in SEM interfacial observations in 1995 [68] and a water tree was first found as an indicator of a leakage pathway by TEM analysis in 2003 [63]. Several studies have shown unfavorable bond defects such as nanoleakage, water trees, bubbles, and phase separation in the bond faces of all-in-one and one-bottle adhesives due to their characteristically high amounts of water [69,70], which is needed for demineralization of dentinal hard tissue by the acid-effect of the monomers of self-etching systems. The hydrophilic nature of bonding resins easily induces water absorption as a result of replacement of the hydrophilic resin monomers even after curing, leading to hydrolytic degradation in the long term [71 77]. Several studies have shown an

5 A review: Biodegradation of resin-dentin bonds 9 increased amount of silver staining in the hybrid layer or bonding resin layer as a function of time using total-etch and self-etching adhesives [78,79]. Recent studies have shown water sorption of adhesive resin to be proportional to the hydrophilic characteristics of the resin [71 77]. The selfetching ability is commonly achieved by incorporation of water in resin monomers that enables ionization of acidic monomers. In addition to the water in the compounds, the ionizable moieties of acidic monomers are also hydrophilic [80]. The presence of such a hydrophilic layer may thus induce water sorption and uptake, in turn, plasticizing the polymer network [17,55 58]. Phase separation of the adhesive or micro-size bubble formation in the bonding resin layer is a typical morphology in one-bottle self-etching adhesive systems [69,70]. A recent study has shown the effect of dentinal surface wetness before bonding on the bond strengths of one-bottle adhesives [81]. In that study, the bonding strengths of resins to dentin were measured using two different surface wetnesses for the dentin substrate before bonding, wet-dentin with short air blowing and dentin dried in a desiccator for 24 h. The hydrophilic one-bottle adhesives with high contents of solvents and water exhibit high water sorption. This water sorption further contributes to a large decrease in the bond strength in wet-dentin compared to dry bonding. An interesting finding was that this result indicated the adverse effect of etch-and-rinse systems on surface wetness. Fig. 5 shows nanoleakage expression (silver staining) in a two-step self-etching specimen (Fig. 5a) and one-bottle self-etching adhesives (Fig. 5b and c) using the back-scatter electron mode of SEM. Although silver staining was observed in the bonding resin layer of onebottle self-etching adhesives, there is no silver tracer present in the bonding resin when using two-step self-etching adhesives, as shown in Fig. 5a. Based on this, it may be concluded that the nanoleakage expression in bonding resin layers is a special characteristic of one-bottle self-etching adhesives (Fig. 5b and c). Recently, a typical type of degradation of one-bottle selfetching adhesives was found at the border between adhesives and the resin composite border [82,83]. Although, there was little or no silver staining after 24 h (not shown) of bonding in one-bottle self-etching adhesive specimens, silver particles were present around filler particles of the resin composite between the bonding resin and resin composite border after more than 300 days (Fig. 6) inwater. Fractured surface observations also showed a similar degradation pattern (detached filler particles and gap formation) at the same region (adhesive/composite border) in in vitro tests using one-bottle adhesives, despite the fact that similar morphology cannot be found in other adhesive systems [82,83]. Aschematicillustrationoftheregionoftypical degradation at the adhesive/composite border is shown in Fig. 7.Oxygeninhibitsfreeradicalpolymerizationandyields a thin unpolymerized and/or hydrogel layer on cured surfaces [84 86]. Large amounts of water and/or solvent are responsible for a decrease in viscosity, and lead to oxygen transport to the top surface of the cured adhesive layer, and the depth of the uncured layer with one-bottle adhesives may be more severe than for hydrophobic adhesives. Furthermore, the remaining monomer around the filler around inadequately polymerized monomers will become a pathway for environmental water invasion and entry into Figure 5 Longitudinal cross-section of the resin dentin interface of control specimens that stained silver tracer (a: backscatter electron mode of two-step self-etching adhesive; b and c: back-scatter electron mode one-bottle self-etching systems). There is an electron lucent layer bonding resin with no silver staining in the specimens of two-step self-etching systems. Spotmode of silver impregnation (b) and balloon-shaped staining (c) are visible within the hybrid layer or adhesive layers for onebottle self-etching adhesives. White arrows in (c) indicate a balloon-shaped nanoleakage at the hybrid layer/bonding resin junction. These regions are enough large for water infiltration. The degree of silver staining of bonding resin layer of one-bottle self-etching system is greater than the other type of adhesive systems. B: bonding resin; C: composite resin; D: dentin. the bulk polymer, leading to hydrolysis of the filler-adhesive junction after aging. This poorly polymerized hydrophilic polymer domain is deteriorated rapidly by environmental water and thus is sensitive to interfacial attack by water.

6 10 M. Hashimoto et al. Figure 6 The resin dentin interfacial aspects of specimens (a one-bottle self-etching adhesive) after 300 days of water storage (back-scatter electron mode of silver staining specimen). There were filler particles of resin composite stained at the border of bonding resin and composite resin. A continuous crack formation was visible within the interface due to the high vacuum chamber of electron microscope. The region of crack formation is in accordance with weakest portion of adhesive interface. White arrows indicate silver staining around filler particles of resin composite. This silver staining pattern is typical for aged specimens, but uncommon in control group or other type of adhesive systems. B: bonding resin; C: composite resin; D: dentin aspect [89 91] and morphological nature in the future. Degradation of the resin composite at the filler matrix junction (hydrolysis of the silane coupling agent) was easily observed in teeth that functioned in vivo in monkeys and in humans over 1 year [11,12], though the same morphological results are not available for in vitro aging tests such as longterm water storage or thermal cycling. Many studies have reported the enzyme-catalyzed hydrolysis of ester linkage in metharylate-based monomers of resin composite [43,92 94]. It is well known that esterases (i.e. cholesterol esterase, salivary esterases, or porcine liver esterases) induce ester hydrolysis. In contrast to resin or collage hydrolysis, careful comparison between in vivo and in vitro resin composite degradation is needed. In addition, analysis of the degradation of bonding resin by esterases in vitro will be an important research topic in the future. A variety of chemical and physiological factors affect the durability of resin dentin bonds. This review article concentrates on the morphological evidence of bond degradation. The long-term durability and degradation patterns of resin adhesives have changed with the types of adhesives during the last two decades. There are clear differences among total-etching systems (etch-and-rinse system), self-etching system, and one-bottle self-etching systems. Typical morphological evidence of degradations is provided by collagen hydrolysis of total-etch adhesive systems, resin elution from the hybrid layers of all systems, and hydrolytic degradation at the border between the adhesive/composite junction of onebottle self-etching adhesives. In addition, the results in many previous studies give promise for the translation of in vitro into in vivo results for bond testing and morphological analysis of resin and collagen hydrolysis. This biodegradation research will provide an avenue of progress for newly developed resin adhesives. Acknowledgements Figure 7 Schematic representation of typical bond degradation of one-bottle self-etching adhesive. Some filler particles of resin composite are occasionally floated in the upper part of bonding resin layer. This morphological phase suggested that the imperfect polymerization layer was presented onto the top surface of bonding resin, the filler particles can penetrate. The degradation at the adhesive/composite border is common for most one-bottle self-etching adhesives, but none or very rare for other type of adhesives. The staining pattern could not be clearly observed in control specimens such as 24 h bonding. However, in vivo test results are required in further research. 5. Summary Although clinical performance depends on the respective adhesive systems, recent long-term clinical trials of resin composites in non-carious cervical lesions demonstrated good clinical performance [87,88]. However, laboratory studies of bond degradation will open the way for the development of new adhesive resin systems that are more stable and have compatible components from the chemical polymer This work was supported, in part, by Grants-in-Aid for Scientific Research No , and for High-Performance Biomedical Materials Research from the Ministry of Education, Science, Sports and Culture, Japan. References [1] Buonocore MG. A simple method of increasing the adhesion of acrylic filling materials to enamel surfaces. J Dent Res 1955;34: [2] Nakabayashi N, Kojima K, Masuhara E. The promotion of adhesion by the infiltration of monomers into tooth substrates. J Biomed Mater Res 1982;16: [3] Prati C, Biagini G, Rizzoli C, Nucci C, Zucchini C, Montanari G. Shear bond strength and SEM evaluation of dentinal bonding systems. Am J Dent 1990;3: [4] Yu XY, Davis EL, Joynt RB, Wieczkowski G. Bond strength evaluation of a class V composite resin restoration. Quintessence Int 1991;22: [5] Pashley DH. Dentin bonding: overview of the substrate with respect to adhesive material. J Esthet Dent 1991;3: [6] Kanca 3rd J. Resin bonding to wet substrate. 1. Bonding to dentin. Quintessence Int 1992;23: [7] Swift Jr EJ, Triolo Jr PT. Bond strengths of Scotchbond Multi- Purpose to moist dentin and enamel. Am J Dent 1992;5:

7 A review: Biodegradation of resin-dentin bonds 11 [8] Watanabe I, Nakabayashi N, Pashley DH. Bonding to ground dentin by a phenyl-p self-etching primer. J Dent Res 1994;73: [9] Nakabayashi N, Saimi Y. Bonding to intact dentin. J Dent Res 1996;75: [10] Hashimoto M, Ohno H, Kaga M, Endo K, Sano H, Oguchi H. Fractographial analysis of resin dentin bonds. Am J Dent 2001;14: [11] Sano H, Yoshikawa T, Pereira PN, Kanemura N, Morigami M, Tagami J, et al. Long-term durability of dentin bonds made with a self-etching primer, in vivo. J Dent Res 1999;78: [12] Hashimoto M, Ohno H, Kaga M, Endo K, Sano H, Oguchi H. In vivo degradation of resin dentin bonds in humans over 1 to 3 years. J Dent Res 2000;79: [13] Hashimoto M, Ohno H, Sano H, Tay FR, Kaga M, Kudou Y, et al. Micromorphological changes in resin dentin bonds after 1 year of water storage. J Biomed Mater Res 2002;63: [14] Hashimoto M, Ohno H, Sano H, Kaga M, Oguchi H. Degradation patterns of different adhesives and bonding procedures. J Biomed Mater Res 2003;66: [15] Hashimoto M, Ohno H, Sano H, Kaga M, Oguchi H. In vitro degradation of resin dentin bonds analyzed by microtensile bond test, scanning and transmission electron microscopy. Biomaterials 2003;24: [16] De Munck J, Van Meerbeek B, Yoshida Y, Inoue S, Vargas M, Suzuki K, et al. Four-year water degradation of total-etch adhesives bonded to dentin. J Dent Res 2003;82: [17] Armstrong SR, Vargas MA, Chung I, Pashley DH, Campbell JA, Laffoon JE, et al. Resin dentin interfacial ultrastructure and microtensile dentin bond strength after five-year water storage. Oper Dent 2004;29: [18] Frankenberger R, Strobel WO, Lohbauer U, Krämer N, Petschelt A. The effect of six years of water storage on composite bonding to human dentin. J Biomed Mater Res 2004;69: [19] Burrow MF, Harada N, Kitasako Y, Nikaido T, Tagami J. Sevenyear dentin bond strengths of a total- and self-etch system. Eur J Oral Sci 2005;113: [20] Koshiro K, Inoue S, Sano H, De Munck J, Van Meerbeek B. In vivo degradation of resin dentin bonds produced by a self-etch and an etch-and-rinse adhesive. Eur J Oral Sci 2005;113: [21] Donmez N, Belli S, Pashley DH, Tay FR. Ultrastructural correlates of in vivo/in vitro bond degradation in self-etch adhesives. J Dent Res 2005;84: [22] García-Godoy F, Tay FR, Pashley DH, Feilzer A, Tjäderhane L, Pashley EL. Degradation of resin-bonded human dentin after 3 years of storage. Am J Dent 2007;20: [23] Reis A, Albuquerque M, Pegoraro M, Mattei G, Bauer JR, Grande RH, et al. Can the durability of one-step self-etch adhesives be improved by double application or by an extra layer of hydrophobic resin? J Dent 2008;36: [24] Carrilho MR, Carvalho RM, Tay FR, Yiu C, Pashley DH. Durability of resin dentin bonds related to water and oil storage. Am J Dent 2005;18: [25] Toledano M, Osorio R, Osorio E, Aguilera FS, Yamauti M, Pashley DH, et al. Durability of resin dentin bonds: effects of direct/ indirect exposure and storage media. Dent Mater 2007;23: [26] Nakabayashi N, Watanabe A, Arao T. A tensile test to facilitate identification of defects in dentine bonded specimens. J Dent 1998;26: [27] Kato G, Nakabayashi N. The durability of adhesion to phosphoric acid etched, wet dentin substrates. Dent Mater 1998;14: [28] Spencer P, Swafford JR. Unprotected protein at the dentin adhesive interface. Quintessence Int 1999;30: [29] Yoshida Y, Van Meerbeek B, Snauwaert J, Hellemans L, Lambrechts P, Vanherle G, et al. A novel approach to AFM characterization of adhesive tooth biomaterial interfaces. J Biomed Mater Res 1999;47: [30] Spencer P, Wang Y, Walker MP, Wieliczka DM, Swafford JR. Interfacial chemistry of the dentin/adhesive bond. J Dent Res 2000;79: [31] Pioch T, Staehle HJ, Duschner H, García-Godoy F. Nanoleakage at the composite dentin interface: a review. Am J Dent 2001;14: [32] Pioch T, Kobaslija S, Huseinbegović A, Müller K, Dörfer CE. The effect of NaOCl dentin treatment on nanoleakage formation. J Biomed Mater Res 2001;56: [33] Pioch T, Staehle HJ, Wurst M, Duschner H, Dörfer C. The nanoleakage phenomenon: influence of moist vs dry bonding. J Adhes Dent 2002;4: [34] Wang Y, Spencer P. Quantifying adhesive penetration in adhesive/dentin interface using confocal Raman microspectroscopy. J Biomed Mater Res 2002;59: [35] Wang Y, Spencer P. Hybridization efficiency of the adhesive/ dentin interface with wet bonding. J Dent Res 2003;82: [36] Marshall Jr GW. Dentin: microstructure and characterization. Quintessence Int 1993;24: [37] Marshall Jr GW, Balooch M, Tench RJ, Kinney JH, Marshall SJ. Atomic force microscopy of acid effects on dentin. Dent Mater 1993;9: [38] Marshall Jr GW, Balooch M, Kinney JH, Marshall SJ. Atomic force microscopy of conditioning agents on dentin. J Biomed Mater Res 1995;29: [39] Hashimoto M, Ohno H, Kaga M, Endo K, Sano H, Oguchi H. Resin tooth adhesive interfaces after long-term function. Am J Dent 2001;14: [40] Hashimoto M, Tay FR, Ohno H, Sano H, Kaga M, Yiu C, et al. SEM and TEM analysis of water degradation of human dentinal collagen. J Biomed Mater Res 2003;66: [41] Yoshida E, Hashimoto M, Hori M, Kaga M, Sano H, Oguchi H. Deproteinizing effects on resin tooth bond structures. J Biomed Mater Res 2004;68: [42] Bean TA, Zhuang WC, Tong PY, Eick JD, Yourtee DM. Effect of esterase on methacrylates and methacrylate polymers in an enzyme simulator for biodurability and biocompatibility testing. J Biomed Mater Res 1994;28: [43] Kostoryz EL, Dharmala K, Ye Q, Wang Y, Huber J, Park JG, et al. Enzymatic biodegradation of HEMA/bisGMA adhesives formulated with different water content. J Biomed Mater Res 2009;88: [44] Pashley DH, Tay FR, Yiu C, Hashimoto M, Breschi L, Carvalho RM, et al. Collagen degradation by host-derived enzymes during aging. J Dent Res 2004;83: [45] Hebling J, Pashley DH, Tjäderhane L, Tay FR. Chlorhexidine arrests subclinical degradation of dentin hybrid layers in vivo. J Dent Res 2005;84: [46] Nishitani Y, Yoshiyama M, Wadgaonkar B, Breschi L, Mannello F, Mazzoni A, et al. Activation of gelatinolytic/collagenolytic activity in dentin by self-etching adhesives. Eur J Oral Sci 2006;114: [47] Carrilho MR, Geraldeli S, Tay F, de Goes MF, Carvalho RM, Tjäderhane L, et al. In vivo preservation of the hybrid layer by chlorhexidine. J Dent Res 2007;86: [48] Mazzoni A, Mannello F, Tay FR, Tonti GA, Papa S, Mazzotti G, et al. Zymographic analysis and characterization of MMP-2 and -9 forms in human sound dentin. J Dent Res 2007;86: [49] Carrilho MR, Carvalho RM, de Goes MF, di Hipólito V, Geraldeli S, Tay FR, et al. Chlorhexidine preserves dentin bond in vitro. J Dent Res 2007;86:90 4. [50] Mazzoni A, Pashley DH, Tay FR, Gobbi P, Orsini G, Ruggeri Jr A, et al. Immunohistochemical identification of MMP-2 and MMP-9 in human dentin: correlative FEI-SEM/TEM analysis. J Biomed Mater Res 2009;88: [51] Tjäderhane L, Larjava H, Sorsa T, Uitto VJ, Larmas M, Salo T. The activation and function of host matrix metalloproteinases in

8 12 M. Hashimoto et al. dentin matrix breakdown in caries lesions. J Dent Res 1998;77: [52] Jackson RJ, Lim DV, Dao ML. Identification and analysis of a collagenolytic activity in Streptococcus mutants. Curr Microbiol 1997;34: [53] Dung S-Z, Gregory RL, Li Y, Stookey GE. Effect of lactic acid and proteolytic enzymes on the release of organic matrix components from human root dentin. Caries Res 1995;29: [54] van Strijp AJ, Jansen DC, DeGroot J, Ten Cate JM, Everts V. Hostderived proteinases and degradation of dentine collagen in situ. Caries Res 2003;37: [55] Shono Y, Terashita M, Shimada J, Kozono Y, Carvalho RM, Russell CM, et al. Durability of resin dentin bonds. J Adhes Dent 1999;1: [56] Tanaka J, Ishikawa K, Yatani H, Yamashita A, Suzuki K. Correlation of dentin bond durability with water absorption of bonding layer. Dent Mater J 1999;18:11 8. [57] Carrilho MR, Carvalho RM, Tay FR, Pashley DH. Effects of storage media on mechanical properties of adhesive systems. Am J Dent 2004;17: [58] Abdalla AI, Feilzer AJ. Four-year water degradation of a totaletch and two self-etching adhesives bonded to dentin. J Dent 2008;36: [59] Zheng L, Pereira PN, Nakajima M, Sano H, Tagami J. Relationship between adhesive thickness and microtensile bond strength. Oper Dent 2001;26: [60] Wang Y, Spencer P. Effect of acid etching time and technique on interfacial characteristics of the adhesive dentin bond using differential staining. Eur J Oral Sci 2004;112: [61] Hashimoto M, Sano H, Yoshida E, Hori M, Kaga M, Oguchi H, et al. Effects of multiple adhesive coatings on dentin bonding. Oper Dent 2004;29: [62] Maeda T, Yamaguchi K, Takamizawa T, Rikuta A, Tsubota K, Ando S, et al. ph changes of self-etching primers mixed with powdered dentine. Dent Mater 2008;36: [63] Tay FR, King NM, Chan KM, Pashley DH. How can nanoleakage occur in self-etching adhesive systems that demineralize and infiltrate simultaneously? J Adhes Dent 2002;4: [64] Tay FR, Pashley DH. Water treeing a potential mechanism for degradation of dentin adhesives. Am J Dent 2003;16:6 12. [65] Tay FR, Pashley DH, Suh BI, Carvalho RM, Itthagarun A. Single-step adhesives are permeable membranes. J Dent 2002;30: [66] Agee KL, Pashley EL, Itthagarun A, Sano H, Tay FR, Pashley DH. Submicron hiati in acid-etched dentin are artifacts of desiccation. Dent Mater 2003;19:60 8. [67] Carvalho RM, Chersoni S, Frankenberger R, Pashley DH, Prati C, Tay FR. A challenge to the conventional wisdom that simultaneous etching and resin infiltration always occurs in self-etch adhesives. Biomater 2005;26: [68] Sano H, Takatsu T, Ciucchi B, Horner JA, Matthews WG, Pashley DH. Nanoleakage: leakage within the hybrid layer. Oper Dent 1995;20: [69] van Landuyt KL, Snauwaert J, Peumans M, de Munck J, Lambrechts P, van Meerbeek B. The role of HEMA in one-step selfetch adhesives. Dent Mater 2008;24: [70] Sauro S, Mannocci F, Toledano M, Osorio R, Thompson I, Watson TF. Influence of the hydrostatic pulpal pressure on droplets formation in current etch-and-rinse and self-etch adhesives: a video rate/tsm microscopy and fluid filtration study. Dent Mater 2009;25: [71] Malacarne J, Carvalho RM, de Goes MF, Svizero N, Pashley DH, Tay FR, et al. Water sorption/solubility of dental adhesive resins. Dent Mater 2006;22: [72] Chersoni S, Suppa P, Grandini S, Goracci C, Monticelli F, Yiu C, et al. In vivo and in vitro permeability of one-step self-etch adhesives. J Dent Res 2004;83: [73] Yiu CK, King NM, Pashley DH, Suh BI, Carvalho RM, Carrilho MR, et al. Effect of resin hydrophilicity and water storage on resin strength. Biomaterials 2004;25: [74] Tay FR, Lai CN, Chersoni S, Pashley DH, Mak YF, Suppa P, et al. Osmotic blistering in enamel bonded with one-step self-etch adhesives. J Dent Res 2004;83: [75] Ito S, Hashimoto M, Wadgaonkar B, Svizero N, Carvalho RM, Yiu C, et al. Effects of resin hydrophilicity on water sorption and changes in modulus of elasticity. Biomaterials 2005;26: [76] Hashimoto M, Tay FR, Ito S, Sano H, Kaga M, Pashley DH. Permeability of adhesive resin films. J Biomed Mater Res 2005;74: [77] Yiu CK,KingNM, Carrilho MR,Sauro S, Rueggeberg FA, Prati C,et al. Effect of resin hydrophilicity and temperature on water sorption of dental adhesive resins. Biomaterials 2006;27: [78] Reis AF, Bedran-Russo AK, Giannini M, Pereira PN. Interfacial ultramorphology of single-step adhesives: nanoleakage as a function of time. J Oral Rehabil 2007;34: [79] Okuda M, Pereira PN, Nakajima M, Tagami J, Pashley DH. Longterm durability of resin dentin interface: nanoleakage vs. microtensile bond strength. Oper Dent 2002;27: [80] Hiraishi N, Nishiyama N, Ikemura K, Yau JYY, King NM, Tagami J, et al. Water concentration in self-etching primers affects their aggressiveness and bonding efficacy to dentin. J Dent Res 2005;84: [81] Hashimoto M, Fujita S, Kaga M, Yawaka Y. Effect of dentinal water on bonding of one-bottle self-etching adhesives. Dent Mater J 2008;27: [82] Hashimoto M, Fujita S, Kaga M, Yawaka Y. In vitro durability of one-bottle resin adhesives bonded to dentin. Dent Mater J 2007;26: [83] Hashimoto M, Fujita S, Endo K, Ohno H. In vitro degradation of resin dentin bonds with one-bottle self-etching adhesives. Eur J Oral Sci 2009;117: [84] Rueggeberg FA, Margeson DH. The effect of oxygen inhibition on an unfilled/filled composite system. J Dent Res 1990;69: [85] Gauthier MA, Stangel I, Ellis TH, Zhu XX. Oxygen inhibition in dental resins. J Dent Res 2005;84: [86] Van Landuyt KL, Snauwaert J, de Munck J, Coutinho E, Poitevin A, Yoshida Y, et al. Origin of interfacial droplets with one-step adhesives. J Dent Res 2007;86: [87] Peumans M, De Munck J, Van Landuyt K, Lambrechts P, Van Meerbeek B. Five-year clinical effectiveness of a two-step selfetching adhesive. J Adhes Dent 2007;9:7 10. [88] Van Landuyt K, Peumans M, Fieuws S, De Munck J, Cardoso MV, Ermis RB, et al. A randomized controlled clinical trial of a HEMAfree all-in-one adhesive in non-carious cervical lesions at 1 year. J Dent 2008;36: [89] Nishiyama N, Suzuki K, Yoshida H, Teshima H, Nemoto K. Hydrolytic stability of methacrylamide in acidic aqueous solution. Biomaterials 2004;25: [90] Salz U, Zimmermann J, Zeuner F, Moszner N. Hydrolytic stability of self-etching adhesive systems. J Adhes Dent 2005;7: [91] Moszner N, Salz U, Zimmermann J. Chemical aspects of selfetching enamel dentin adhesives: a systematic review. Dent Mater 2005;21: [92] Bean TA, Zhuang WC, Tong PY, Eick JD, Yourtee DM. Effect of esterase on methacrylate and methacrylate polymers in an enzyme simulator for biodurability and biocompatibility testing. J Biomed Mater Res 1994;28: [93] Armstrong SR, Jessop JL, Vargas MA, Zou Y, Qian F, Campbell JA, et al. Effects of exogenous collagenase and cholesterol esterase on the durability of the resin dentin bond. J Adhes Dent 2006;8: [94] Toledano M, Osorio R, Osorio E, Aguilera FS, Yamauti M, Pashley DH, et al. Effect of bacterial collagenase on resin dentin bonds degradation. J Mater Sci Mater Med 2007;18: