Inhibition of demineralization around the enamel-dentin/ restoration interface after dentin pretreatment with TiF 4 and self-etching adhesive systems

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1 DOI /s ORIGINAL ARTICLE Inhibition of demineralization around the enamel-dentin/ restoration interface after dentin pretreatment with TiF 4 and self-etching adhesive systems Enrico Coser Bridi 1 & Flávia Lucisano Botelho do Amaral 1 & Fabiana Mantovani Gomes França 1 & Cecilia Pedroso Turssi 1 & Roberta Tarkany Basting 1,2 Received: 26 February 2015 /Accepted: 18 August 2015 # Springer-Verlag Berlin Heidelberg 2015 Abstract Objectives The objective of this study was to evaluate the inhibition of demineralization around enamel-dentin/restoration interface after dentin pretreatment with 2.5 % titanium tetrafluoride (TiF 4 ). Materials and methods Forty dental class V cavities at the cementoenamel junction were distributed into four groups (n = 10), according to the presence or absence of TiF 4 and to the adhesive system (Clearfil SE Bond/CL and Adper EasyOne/AD), and restored with a resin composite. A dynamic ph cycling model was used to induce the development of artificial caries lesions. After sectioning the dental blocks, Knoop microhardness tests were performed at different depths (20, 40, and 60 μm from the occlusal margin of the restoration) and at different distances (100, 200, and 300 μm from * Roberta Tarkany Basting rbasting@yahoo.com Enrico Coser Bridi enricobridi@gmail.com Flávia Lucisano Botelho do Amaral flbamaral@gmail.com Fabiana Mantovani Gomes França biagomes@yahoo.com Cecilia Pedroso Turssi cecilia.turssi@gmail.com the adhesive interface). Repeated measures three-way analysis of variance (ANOVA) and Tukey s testwereused(α =0.05). Results For enamel, there were no differences in the microhardness values for CL, AD, and TiF 4 -AD at depths, regardless of the distances. Considering each depth, there were no significant differences among treatments. For dentin, ANOVA showed no significant interaction among the independent variables treatment*distance*depth (p = 0.994), no significant interaction between treatment*depth (p = 0.722), no significant interaction between treatment*distance (p = 0.265),no significant interaction between depth*distance (p = 0.365), and no significant effect on treatment (p = 0.151), depth (p = 0.067), or distance (p =0.251). Conclusions Dentin pretreatment of the cavity walls with TiF 4 before self-etching adhesive systems was not effective in inhibiting demineralization around the enamel-dentin/restoration interfaces. Clinical relevance The mechanism of incorporating fluoride in enamel and dentin of the cavity walls to inhibit demineralization around restorations seems ineffective when using TiF 4 as a dentin pretreatment. Keywords Demineralization. Microhardness. Titanium tetrafluoride. Self-etching adhesive systems Introduction 1 2 São Leopoldo Mandic School of Dentistry and Research Institute, Campinas, São Paulo, Brazil Departamento de Odontologia Restauradora Dentística, Faculdade de Odontologia e Instituto de Pesquisas São Leopoldo Mandic, Rua José Rocha Junqueira, 13. Bairro Swift, Campinas, SP CEP: , Brazil Titanium tetrafluoride (TiF 4 ) has demonstrated a cariostatic effect promoted by both fluoride- and titanium-rich acid-stable cover layer formed on top of enamel surfaces exposed to it. This enables a reduction in enamel solubility that provides mechanical protection to the surface, reducing its demineralization and also preventing the threat of erosion [1 9]. TiF 4 can

2 be used for these purposes as an aqueous solution or a varnish with concentrations ranging from 0.1 to 4 % [6, 10 12]. In addition to its effects on enamel substrate, TiF 4 may also be effective in reducing dentin hypersensitivity [13]by sealing the dentinal tubules of root canal dentin [14, 15] and reducing dentin hydraulic conductance [16, 17]. Kazemi et al. [16] were the first to suggest using the acidic solution of TiF 4 in dentine cavities as a dentin pretreatment before placing restorations, in order to modify the smeared dentine surface to produce a stable, acid-resistant state similar to a glaze-like or vitreous surface [14]. No studies had been conducted until that time to evaluate the interaction between restorative materials (mainly adhesive systems) and the glaze-like dentin surface. The effects of TiF 4 as a dentin pretreatment on microtensile bond strength to dentin have been studied recently. Although Dündar et al. [18] found that TiF 4 application decreased bond strength values following acid etching in 4-META/MMAbased and Bis-GMA-based luting cements and following acidic monomer application for MMA cement, Devabhaktuni and Manjunath [19] showedthattif 4 before and after acid etching with a conventional three-step adhesive system did not significantly affect the bond strength of a resin composite to dentin. Another observation was that dentin pretreated with titanium tetrafluoride did not adversely affect bond strength of one- or two-step self-etching [20, 21] and also increased bond strength to dentin in a conventional two-step adhesive system [22]. Despite the highly acidic ph (about 1.2) of the TiF 4 solution, partial demineralization is limited to 1 5 μm of the dentin surface and it may provide a modified smear layer prior to application of the adhesive systems and also reduce microleakage by preventing further dissolution and disintegration of the smear layer [14]. Accordingly, dentin pretreatment with TiF 4 may be a good choice when trying to extend the longevity of adhesive systems by inhibiting hybrid layer degradation and the microleakage and/or demineralization at the tooth/restoration interface. The mechanism by which TiF 4 may inhibit enamel and dentin demineralization around composite resin restorations may be based on the following posits: (a) metal-rich surface precipitates of TiF 4 may induce the formation of a glaze-like protective layer, minimizing the demineralization of enamel around restorations; (b) the incorporation of TiF 4 in the hybrid layer formed by self-etching adhesives results in a biomodified layer that could strengthen the interface between composite resin and enamel/dentin [15]. In relation to fluoride-containing adhesive systems, the fluoride ion released from the adhesive system could inhibit the development of caries formation by inhibiting demineralization and providing remineralization of the affected dentin around the restoration [23]. Although the amount of fluoride released from the dental adhesives appears to be insufficient for preventing caries formation [24, 25], fluoride indeed plays a role in increasing the acid resistance of the cavity margins and is capable of maintaining the integrity of the cavity walls [23, 26 29]. It is believed that the application of TiF 4 as a dentin pretreatment before applying self-etching adhesive systems may inhibit demineralization at the interface of the enamel-dentin/resin composite restoration since the incorporation of TiF 4 in the hybrid layer formed by self-etching adhesives may result in a biomodified hybrid layer. Thus, the aim of the present study was to evaluate the inhibition of demineralization around the enamel-dentin/restoration interface after dentin pretreatment with TiF4 and self-etching adhesive systems. The null hypothesis to be tested was that application of TiF 4 on the dentin cavity before one- or two-step self-etching adhesive systems would not exert any effect on the inhibition of enamel and dentin demineralization around composite resin restorations. Materials and methods Cavity preparations and restorations After approval by the Research Ethics Committee (process number 2011/0105), 20 completely unerupted human third molars, stored in 0.1 % aqueous thymol solution with no coronal cracks or enamel malformations, were used in this study. The teeth were submitted to debridement with scalpel blades and periodontal curettes. The cervical portions of the buccal and palatal/lingual surfaces of the teeth were used. For this, the tooth was cut midregion to separate the buccal from the palatal/lingual surfaces. Both sides were then reduced to the size of a fragment measuring 5 mm 5 mm 4 mm thick to include the cervical region. A digital caliper (Mitutoyo Corporation, Kawasaki, Japan) was used to verify the measurements. Class V cavity preparations were made with a standardized flat cylindrical bur # 2292 (KG Sorensen Ind. e Com. Ltda, Barueri, SP, Brazil) with a stop, 2.0 mm in diameter and 2.0 mm in depth, at a high speed and under cooling with distilled water spray on both tooth faces. The occlusal region of the preparation margins was located in the enamel, and the cervical region was located in the dentin. The burs were changed after every 10 cavity preparations to maintain uniformity of the preparations. Forty dental preparations that appeared centralized on the buccal and palatal/lingual surfaces were used. The treatments were randomly applied to four groups (n =10),accordingto the presence or absence of dentin pretreatment with TiF 4 and also to the adhesive system to be used, in an experimental design of 2 2. In relation to the groups receiving dentin pretreatment, TiF 4 P.A. (pro-analysis) was dissolved in deionized distilled water to achieve a concentration of 2.5 % (wt/v; ph 1.2), as suggested by Dündar et al. [18] and used by Bridi et al. [20]. The TiF 4 solution was applied on cavity walls

3 actively with a disposable brush for 60 s followed by air drying briefly for 5 s. The application was restricted to the cavity walls and limited to the cavosurface angle; moreover, the adhesive systems those that do not release fluorides were applied according to their corresponding groups, following the manufacturers instructions (Table 1). After applying the adhesive systems, a non-releasing fluoride nanoparticle resin composite (Filtek Z350, 3 M ESPE, Saint Paul, MN, USA) was placed using the incremental insertion technique (two oblique layers) followed by polymerization for 20 s each. The light polymerization appliance used was a halogen light (Demetron Research Corporation, Danbury, CT, USA) with a medium light power of 552 mw/cm 2 (520 to 603 mw/cm 2 ), gauged by a radiometer (Newdent Equipamentos Ltda., Ribeirão Preto, SP, Brazil) at each five restorations. Polishing of the resin restorations was performed using aluminum oxide discs of medium and fine granulation (Sof-Lex, 3M, Saint Paul, MN, USA) and avoiding residues of resin composite, adhesive systems, and application of TiF 4 beyond the cavity margins. Artificial caries induction The inhibition of demineralization around the enamel-dentin/ restoration interface after dentin pretreatment with TiF 4 and self-etching adhesive systems was evaluated with a dynamic ph cycling model to induce development of artificial caries lesions [30, 31]. This was done by sealing dental blocks with three layers of enamel varnish (Colorama, Procosa Produtos de Beleza Ltda, São Paulo, SP, Brazil) leaving 1 mm unsealed around the restoration margins. An adhesive paper (Contact, Rio de Janeiro, Rio de Janeiro, Brazil) 4 mm in diameter was placed on the restorations to prevent application of the enamel varnish on dental margins where the cariogenic challenge would have to be produced. The blocks were immersed individually in 14.1 ml demineralizing solution (ph 4.3, 2 mm calcium, 2 mm phosphorous, M acetate buffering) for 6 h followed by washing in deionized and distilled water and immersion in 14.1 ml remineralizing solution (ph 7, 1.5 mm calcium, 0.9 mm phosphorous, 0.15 M potassium chlorate, 0.02 M tris buffer) for 18 h, for 14 days, at 37 C [30, 31]. Microhardness tests After artificial caries induction, the blocks were sectioned longitudinally through the middle of the restoration, using a metallographic cutter (Isomet 1000 Precision Diamond Saw, Buehler Ltd., Lake Bluff, IL, USA). The cut fragments were embedded in epoxy resin (Varidur, Düsseldorf, Germany) using acrylic rods. The fragments were then ground/polished using a grinder-polisher (250-Ecomet and Automet 250-Head, Buehler Ltd., Lake Bluff, IL, USA) to obtain smooth surfaces for the microhardness tests. Slabs were wet-flattened with aluminum oxide abrasive papers (600 and 1200 grit) and polished with 0.3-mm alumina suspension (Alpha Micropolish, k- Buehler Ltd., Lake Bluff, IL, USA). The slabs were then cleansed ultrasonically in deionized water for 10 m to remove any residues of the polishing procedure. The microhardness test was performed to evaluate the presence of demineralization around the resin restoration. Nine measurements were made in each fragment, both in enamel and in dentin (18 indentations per fragment). The indentations were made at the occlusal/gingival margin of each restoration in enamel or dentin at standardized distances for all the restorations evaluated (100-, 200-, and 300-μm distance from the occlusal/ gingival margin of the restoration in enamel/dentin and at a 20-, 40- and 60-μm depth of the cavosurface angle in the direction of the dentin-enamel junction for enamel margins or in the direction of the canal for dentin margins) [25, 29](Fig.1). Table 1 Materials used in this study, composition, and protocol for use Materials (manufacturer, city, state, country) Composition Protocol for use Titanium tetrafluoride 2.5 % (Sigma Aldrich, Saint Louis, MO, USA) Adper EasyOne (3M ESPE AG, Seefeld, Germany) Clearfil SE Bond (Kuraray Medical Inc., Okayama, Japan) Titanium tetrafluoride P.A. + distilled and deionized water/ph 1.2 HEMA, Bis-GMA, methacrylated phosphoric esters, 1,6 hexanediol dimethacrylate, methacrylate functionalized polyalkenoic acid (Vitrebond Copolymer), finely dispersed bonded silica filler with 7 nm primary particle size, ethanol, water, initiators based on camphorquinone, stabilizers/ ph 2.3 Primer: MDP, HEMA, hydrophilic dimethacrylate, photo-initiator, water Bond: MDP, Bis-GMA, HEMA, hydrophobic dimethacrylate, photoinitiators, silanized colloidal silica/ph 2.0 Apply the product actively with a microbrush for 60 s [6, 20, 21] Dry cavity, apply actively for 20 s, gently apply air, photoactivation for 10 s Dry cavity, apply primer for 20 s, gently apply air, apply bond, gently apply air, photoactivation for 10 s Bis-GMA bisphenol A-glycidyl methacrylate, HEMA 2-hydroxyethyl methacrylate, MDP 10-methacryloyloxydecyl dihydrogen phosphate

4 Fig. 1 Schematic representation of the microhardness indentations (depths and distances) in enamel or dentin The microhardness analyses obtained in kilogram-force/ square millimeter were performed with a microhardness tester and the Knoop indenter (Digital Microhardness Tester HVS 1000, PanTec, SP, Brazil) with a 10-gf static load and application duration of 5 s. Statistical analysis Before performing the data analyses, the normality of distribution and homogeneity of variance for enamel and dentin outcomes were verified with Shapiro-Wilk s and Levene s tests. The data recorded for dentin did not show normal distribution and homogeneity of variance. Four outliers identified in the box-plot were excluded, so that the presuppositions of the statistical analysis could be satisfied adequately. Repeated measures three-way analyses of variance (ANOVA), according to the general linear model, were used to assess both the enamel and the dentin data. Post hoc comparisons were performed using Tukey s test. The significance level was set at 5 %, and the statistical procedures were carried out using SPSS 20 (SPSS Inc., Chicago, IL, USA). Results The mean microhardness values and the standard deviation for the groups, according to the distance and depths for enamel and dentin, are presented in Table 2. For enamel, the repeated measures three-way ANOVA showed no significant interaction among the independent variables treatment*distance*depth (p = 0.997), no significant interaction between treatment*distance (p =0.187), and no significant interaction between depth*distance (p = 0.479). There was a significant effect on the interaction treatment*depth (p = 0.016), but no significant effect on the variable distance (p = 0.258). Tukey s test showed that there were no differences in microhardness values for Adper, Clearfil, and Ti-Adper at different depths, regardless of the distance. For Ti-Clearfil, there was a significant higher microhardness mean value at 60 μm depth than at 20 μm; microhardness values at 40 μm did not differ from those obtained at 20 μm orat60μm. Considering each depth, there were no significant differences among treatments (Table 3). Table 2 Mean microhardness values and standard deviation (in kgf/mm 2 ) for enamel and dentin according to treatment, distance, and depth Distance Depth Enamel Adper (104.2) (98.9) (93.9) 92.9 (93.1) (92.3) (106.5) (112.3) (91.7) (70.7) Clearfil 88.1 (58.3) (117.6) (150.0) (124.2) (96.7) (136.8) (129.7) (104.4) (149.0) Ti-Adper (145.0) (98.1) (135.6) (140.4) (148.9) (114.4) (101.3) (134.8) (117.8) Ti-Clearfil 71.8 (47.1) (120.1) (180.6) (124.6) (114.2) (147.0) (125.8) (134.6) (174.0) Dentin Adper 29.1 (22.3) 27.4 (10.0) 30.1 (11.5) 23.4 (8.0) 26.5 (10.3) 31.0 (11.3) 28.3 (18.1) 26.4 (9.0) 33.0 (10.1) Clearfil 27.7 (19.4) 41.2 (44.0) 33.9 (12.7) 22.9 (18.5) 25.6 (17.9) 30.9 (18.2) 26.5 (19.6) 33.3 (21.9) 31.5 (16.6) Ti-Adper 56.7 (78.3) 28.0 (12.6) 34.9 (15.0) 23.8 (9.9) 31.9 (13.6) 38.4 (20.6) 27.6 (10.6) 30.8 (14.4) 35.7 (11.0) Ti-Clearfil 25.5 (14.9) 27.0 (10.0) 32.2 (9.6) 25.4 (15.2) 30.4 (14.5) 36.7 (10.9) 28.1 (12.7) 32.5 (11.0) 46.5 (21.4)

5 Table 3 Mean microhardness values and standard deviation (in kgf/ mm2) for enamel according to treatment and depth, regardless of the distance In relation to the dentin, the repeated measures three-way ANOVA showed no significant interaction among the independent variables treatment*distance*depth (p = 0.994),no significant interaction between treatment*depth (p = 0.722), no significant interaction between treatment*distance (p = 0.265), no significant interaction between depth*distance (p = 0.365), and no significant effect on the treatment (p = 0.151), depth (p = 0.067), or distance (p =0.251). Discussion Depth Treatment Adper (100.1) Aa (91.2) Aa (88.5) Aa Clearfil (105.9) Aa (104.1) Aa (140.5) Aa Ti-Adper (127.2) Aa (127.2) Aa (120.3) Aa Ti-Clearfil (107.5) Aa (120.2) Aab (162.8) Ab Means of microhardness values (standard deviation) in kilogram-force/ square millimeter followed by distinct letters (uppercase for vertical and lowercase for horizontal) differ among one another according to Tukey s test (p < 0.05) A secondary caries is defined as a caries lesion that develops adjacent to a dental restoration. These caries may develop as an outer lesion on the tooth surface, next to the restoration margins, or as a wall lesion, at the tooth/restoration interface. Whereas outer lesions develop similarly to primary caries on the tooth surface, wall lesions occur when there are interfacial gaps [32, 33]. Therefore, cavity walls with a more acidresistant surface could inhibit or prevent not only the demineralization of these walls but also primary lesions caries. Insofar as fluoride has been shown to have a significant effect in inhibiting enamel and dentin caries, adhesive systems that release fluoride have been developed with the purpose of aiding in the prevention of secondary caries development [23 29]. Although the hybrid layer is acid resistant, it is not capable of increasing the resistance of cavity dentin and inhibiting caries formation along the cavity walls [28, 34]. Therefore, a desirable attribute of fluoride-containing dental adhesives is that they release fluoride in the direction of the restoration margin, thus providing a beneficial effect on demineralized enamel and dentin [23]. In the present study, the adhesive systems evaluated had no fluoride in their composition. However, a modified hybrid layer was formed after the application of an aqueous solution of TiF 4 followed by application of the self-etching adhesive systems [15]. TiF 4 may modify the micromorphology of the enamel and dentin surface [20 22] and produce an erosive, resistant surface attributed to the formation of an acid-stable glass-like surface layer on the enamel [2, 4] and a precipitate surface layer on intertubular and intratubular dentin [14, 15]. Although bond strength may increase as a consequence of filler addition to adhesive systems [35], a more acid-resistant surface can be achieved after applying the adhesive, thus resulting in less demineralization around the composite resin restorations. However, the null hypothesis tested in this study was accepted, since the inhibition of enamel and dentin demineralization around the cavity margins was not observed. Although the microhardness mean values for Ti-Clearfil were significantly higher at 60 μm versus20μm depth for enamel, no beneficial results were found for inhibiting demineralization around restorations for dentin margins when applying TiF 4 as a dentin pretreatment before applying self-etching adhesive systems. The methodology used for developing artificial caries, based on the use of ph cycles, has been previously employed in other studies [25, 28, 36]. The advantages of in vitro caries models are the facility in obtaining results versus in situ or in vivo models that need volunteers to perform the study, although the latter models are encouraged to be performed in later studies. In addition, better control can be exerted over the conditions for chemical remineralization within a given exposure period, as shown by the in vitro model used in the present study [37]. The ph cycling model simulates artificial enamel decay, but does not produce an undersaturated solution, leading to problems such as erosion [38]. This model presents a fluoride dose-response effect and can be used to evaluate the anticaries potential of various fluoride products used to inhibit enamel/dentin demineralization or to enhance remineralization [39]. Skartveit et al. [40] showed that aqueous solutions of TiF 4 in dentin cause rapid uptake and long-lasting retention and release (over 28 days) of fluoride applied topically as compared with stannous fluoride (SnF 2 ) and sodium fluoride (NaF). TiF 4 may enhance the depth of penetration of fluoride ions because of its low ph [41]; as such, it promotes a structural mass and modified smear layer when applied on smearlayer-covered dentin [15, 41]. It can be assumed that TiF 4 treatment promoted a deeper penetration of fluoride, compared with NaF, which may promote a superficial fluoride bond in the outermost dentin surface [40, 41]. In relation to the enamel, titanium ions may play an important role in enhancing the protective capability of TiF 4,insofar as titanium can bind to enamel surfaces and penetrate into sound or demineralized enamel [10, 42]. The enamel surface coating after TiF 4 application is composed of organometallic complexes, leading to higher acid resistance [1, 2, 4, 5]. However, when comparing the titanium concentration to the fluoride concentration on the dentin surface, after application of the TiF 4, we observed that the latter was only slightly higher

6 than the non-treated surface, whereas the former was significantly higher [15, 43]. Magalhães et al. [8] also showed low fluoride concentrations on enamel surface after 4 % TiF 4 application, thus making it evident that TiF 4 provides mechanical protection only superficially and cannot prevent the formation of subsurface demineralization below the glaze-like surface layer completely [43]. In addition, the layer over the enamel surfaces treated with TiF 4 is not homogeneous, and has some microcracks that allow acid to penetrate into the subsurface enamel layer, leading to subsurface demineralization [1, 3, 43]. The fluoride in this glaze-like surface seems to be structurally bound to the outermost enamel rather than being contained in CaF 2 precipitates [43]. Therefore, the mechanism of how firmly bound fluoride incorporated in enamel and dentin acts in inhibiting demineralization around restorations seems ineffective when using TiF 4 as a dentin pretreatment prior to adhesive systems. Ten Cate [37] explains that higher concentrations of fluorides in solution are needed in ph cycling studies of dentine rather than in enamel in order to maintain the mineral balance or to induce remineralization. Fluoride slow-release devices, in the form of fluoride-releasing restorative materials, may serve to increase the fluoride levels in saliva and plaque to levels at which dental caries can be prevented [37], unlike fluoride incorporated directly into the enamel and dentin substrate, as observed in this investigation. Dentin pretreatment with TiF 4 has shown positive results in not influencing bond strength values of self-etching adhesive systems to dentin [20, 21], in favorably biomodifying the smear layered dentin [15], and also in promoting an antimicrobial effect against residual Streptococcus mutans and Lactobacillus casei in dentin, as showed in our previous studies [44]. However, solutions of TiF 4 alone are not stable enough to be used in treatments, since TiF 4 forms deposits at the bottom of test tubes and does not have a stable ph. In addition, no definite protocol has yet been established to regulate the use of TiF 4 [21]. Nevertheless, stability issues can be minimized by using suitable drug carriers or nanocarriers, such as β-cyclodextrins, to modify the chemical stability and other properties of TiF 4, such as solubility, dissolution rate, and bioavailability [45]. Additional studies must be performed to determine the optimal conditions for complex formation, to develop application protocols, and to evaluate the influence of TiF 4 in the longevity of the hybrid layer. Acknowledgments The authors would like to thank FAPESP foundation for providing grants (process number 2011/ ). Conflict of interest interests. The authors declare that they have no competing Disclosure statement The authors have no financial, economic, commercial, and/or professional interests related to topics presented in the manuscript. References 1. Wei SH, Soboroff DM, Wefel JS (1976) Effects of titanium tetrafluoride on human enamel. J Dent Res 55: Tveit AB, Hals E, Isrenn R, Tøtdal B (1983) Highly acid SnF2 and TiF4 solutions. Effect on and chemical reaction with root dentin in vitro. Caries Res 17: Buyukyilmaz T, Ogaard B, Rolla G (1997) The resistance of titanium tetrafluoride-treated human enamel to strong hydrochloric acid. 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