DOI 10.1007/s00784-013-1096-y ORIGINAL ARTICLE Efficacy of TiF 4 and NaF varnish and solution: a randomized in situ study on enamel erosive abrasive wear Flávia Mauad Levy & Daniela Rios & Marília A. R. Buzalaf & Ana Carolina Magalhães Received: 13 January 2013 /Accepted: 21 August 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract Objectives This in situ/ex vivo study analysed the antierosive/abrasive effect of TiF 4 and NaF varnish and solution on enamel wear. Materials and methods Twelve subjects took part in this study which was performed in three periods (phases) with the duration of 5 days each. Each two human enamel specimens per subject were pretreated with experimental NaF varnish or solution (phase A), experimental-tif 4 varnish or solution (phase B) and placebo varnish or untreated control (phase C). The specimens were worn in palatal appliances; one enamel specimen, from each treatment, was subjected to erosion (ERO; cola soft drink, 4 90 s/day), and the other specimen was subjected to erosion plus abrasion (ERO + ABR; tooth brushing, 2 10 s/day). The tooth wear was quantified by a contact profilometer (micrometre) and analysed using two-way repeated measures ANOVA and Bonferroni s test (n =12 subjects, p <0.05). Results All fluoride varnishes and solutions reduced the enamel wear (around 25 %) significantly compared to the control and placebo varnish. There were no significant differences among the fluoride formulations and between the conditions ERO and ERO + ABR. Conclusions Therefore, it can be concluded that TiF 4 has the same protective potential as NaF formulations to reduce human enamel wear under this experimental in situ model. F. M. Levy: M. A. R. Buzalaf : A. C. Magalhães (*) Department of Biological Sciences, University of São Paulo, Al. Dr. Octávio Pinheiro Brisolla, 9-75, Bauru, SP 17012-901, Brazil e-mail: acm@fob.usp.br D. Rios Department of Pediatric Dentistry, Orthodontics and Public Health, Bauru School of Dentistry, University of São Paulo, Al. Dr. Octávio Pinheiro Brisolla, 9-75, Bauru, SP 17012-901, Brazil Clinical significance In vitro studies have indicated a better anti-erosive/abrasive effect of TiF 4 compared to NaF varnish. The present in situ study does not support the previous findings. Therefore, any of the tested professional fluoride varnishes in principle could be able to partially reduce enamel wear. Keywords Enamel. Fluoride. Titanium fluoride. Tooth abrasion. Tooth erosion Introduction Conventional fluorides whose beneficial effect against caries is already established [1] have been tested for prevention or control of dental erosion [2, 3]. The potential of conventional fluorides, such as NaF, to prevent erosive demineralisation is mainly related to the formation of a calcium fluoride (CaF 2 )- like layer [4, 5]. This layer is assumed to behave as a physical barrier hampering the contact of the acid with the underlying enamel or to act as a mineral reservoir, which is attacked by the erosive challenge, reducing the demineralisation. Afterward, calcium and fluoride released might increase the saturation level with respect to the dental hard tissue in the liquid adjacent to the enamel surface, thus promoting mineral precipitation. As the anti-erosive effect of conventional fluorides requires a very intensive fluoridation regime [6, 7], current studies have focused on fluoride compounds that might have higher efficacy. In this context, compounds containing polyvalent metal ions such as stannous fluoride or titanium tetrafluoride (TiF 4 )weretested. The potential of TiF 4 solution to prevent tooth erosive demineralisation has been investigated since 1997 [8]. Its protective effect may be related to the increased fluoride uptake due to the low ph. Additionally, titanium may also
contribute to the formation of an acid-resistant surface coating due to the formation of a new compound (hydrated hydrogen titanium phosphate) or organometallic complexes that might act as diffusion barrier on enamel [3, 9]. In situ studies have shown that TiF 4 solution is as effective as SnF 2 or AmF solution in the prevention of erosion or combined erosion/abrasion [10, 11], while another in situ study did not show any protective effect against enamel wear [12].TheefficacyofTiF 4 is highly dependent on the ph of the agent. TiF 4 at its native ph (ph 1.2) could significantly reduced enamel erosion but not at a ph buffered to 2.1 [13] or 3.5 [14]. However, the low ph of TiF 4 solution does not allow self-application as mouth rinsing by the patient. In respect to the limitation of the solution, TiF 4 was incorporated in an experimental varnish, indicated for the professional application. In vitro studies have shown higher antierosive and anti-abrasive effects of the varnish compared to the solution [15, 16]. Moreover, the varnish is considered safe for the patient in terms of ingestion and toxicity, as it is directly applied on the tooth surface compared to a mouthrinse solution. However, further properly designed in situ or clinical studies are recommended to better understand the relative differences in performance of the various fluoride formulations. Thus, the aim of this in situ study was to analyse the effect of a single application of TiF 4 varnish/solution, compared to NaF varnish/solution, to protect human enamel against erosion with or without abrasion. The null hypothesis tested was that there is no significant difference between NaF and TiF 4 formulations on the prevention of enamel erosive and erosive abrasive wear. Materials and methods Selection of the subjects and ethical aspects Twelve healthy adult subjects (eleven females, one male; aged 23 to 35 years) who fulfilled the inclusion criteria (physiological salivary flow rates: stimulated, >1 ml/min and resting, >0.25 ml/min; good oral health: no frank cavities or significant gingivitis/periodontitis) without violating the exclusion criteria (systemic illness, pregnancy or breastfeeding, use of fixed or removable orthodontic appliances, use of fluoride mouthrinse or professional fluoride application in the last 2 months or hyposalivation) were enrolled. Sample size calculation was based on previous in situ study [12]. A sample size of 12 subjects was calculated considering α-error level of 5 % and β-error level of 30 % and a relevant difference between TiF 4 solution and control of 13 % (www.ddsresearch.com). The research was ethically conducted in accordance with the Declaration of Helsinki. The ethical approval for the study involving human subjects was granted by the local ethics committee (no. 083/2008). The study was planned as a prospective, single-centre, double-blind and six-cell study in crossover and split-mouth design with three periods/phases (two treatments and two conditions per phase) of 5 days each, with a washout time of 5 days between the periods/phases [12, 17]. The subjects received written instructions and a schedule and were extensively trained for all procedures. Informed consent was obtained from all subjects prior to the study. Figure 1 shows the study design. The description of the study design followed the guideline of the CONSORT statement. Preparation of specimens and the distribution to the experimental groups One hundred and forty-four human enamel specimens (4 4 3 mm) were prepared from the labial/lingual surface of the crowns (two specimens/crown; total, 72 teeth). The impacted third molars were collected and then stored in 2 % buffered formaldehyde solution (ph 7.0) at 4 C for 30 days [12]. Thereafter, the specimens were cut in the middle of the surfaces using an ISOMET low-speed saw (Buehler Ltd., Lake Bluff, IL, USA) with two diamond discs (Extec Corp., Enfield, CT, USA) separated by a 4-mm-thick spacer. The specimens surfaces were ground flat using water-cooled silicon carbide discs (320-, 600- and 1200-grade papers; Buehler, Lake Bluff, IL, USA) and polished using felt paper wet with diamond spray (1 μm; Buehler). The polishing removed around 200 μm of enamel. The enamel surface was checked by using the software of the profilometer. After polishing, the specimens were cleaned in an ultrasonic device with deionized water for 2 min. Prior to the experiment, two layers of nail varnish were applied on two thirds of the enamel surface to maintain reference surfaces for determining tooth wear after the experiment, leaving 1.5 mm of central exposed area. The specimens were maintained in 100 % humidity until the in situ experiment was conducted. Each two enamel specimens per subject were randomly allocated to each treatment (total, 24 specimens/treatment): experimental NaF varnish and NaF solution (both were located in phase A, FGM-Dentscare/Brazil, 2.45 % F, ph 5.0), experimental TiF 4 varnish and TiF 4 solution (both in phase B, FGM-Dentscare/Brazil, 2.45 % F, ph 1.0) and placebo varnish and untreated control (both in phase C, FGM-Dentscare/ Brazil, ph 5.0). The composition of the varnishes was previously reported [15]. In each treatment, half of the specimens were subdivided into erosion only (ERO, n =12) and erosion plus abrasion (ERO + ABR, n =12). For each phase (A, B and C), 48 new enamel specimens were used. Enamel specimens were fixed with wax into the recesses of each individual acrylic palatal appliance. The treatment (solution or varnish) was randomly divided in rows (at least 1 cm apart from each other), and the conditions (ERO or ERO + ABR) were randomly divided in lines (at least 1 cm apart) for
Fig. 1 Flowchart of the study Selection of 12 study subjects Information about the study and written informed consent Collection of saliva (Salivary flow rate and ph) - Inclusion of subjects (n=12) Impressions and preparation of the intraoral appliances for each study phase 5 days washout period before starting the study 1 st study period: Phase A (n=4, subjects 1-4), phase B (n=4, subjects 5-8) and phase C (n=4, subjects 9-12) (5 days each phase) 5 days washout period 2 nd study period: Phase C (n=4, subjects 1-4), phase A (n=4, subjects 5-8) and phase B (n=4, subjects 9-12) (5 days each phase) 5 days washout period 3 rd study period: Phase B (n=4, subjects 1-4), phase C (n=4, subjects 5-8) and phase A (n=4, subjects 9-12) (5 days each phase) Profilometric assessment and statistical analysis (n=12) each volunteer. This split-mouth palatal appliance design has been previously used for testing the effect of TiF 4 on enamel caries lesion [17]. Treatment and in situ experiment Five days prior to and throughout the entire experiment, the subjects brushed their teeth with experimental fluoride-free toothpaste (Crest, Procter & Gamble, USA) to allow the protective effect of fluoride to be only due to the solution/ varnish treatments. The subjects wore the appliance for 2 h to allow the formation of a salivary pellicle on the enamel surface. After the 2-h lead-in period, the treatments were performed only once at the beginning of each experimental phase. The fluoride solutions were applied on two enamel specimens (one row) using a microbrush for 1 min ex vivo. The excess of the solution was gently removed with a cotton swab [15, 16, 18, 19]. The fluoride varnishes were applied on the surface of the two other enamel specimens (the other row) using a microbrush ex vivo. During 6 h, the subjects were not allowed to remove the appliance from the mouth. Thereafter,
the varnishes were removed with a scalpel and a cotton swab soaked in acetone diluted in water (1:1) ex vivo. The varnishes were removed to allow the chemical rather than mechanical effect of the tested products. The nail varnish was reapplied after this procedure to protect the control areas again [15, 16, 20]. At the next day, the erosive and abrasive regimens started. The erosive challenges were performed four times daily (morning, midday, afternoon and evening at least 3 h apart). For erosion, the subjects were instructed to immerse the appliance containing all treated enamel specimens in a cup with 150 ml of cola drink (ph 2.6, 0.32 ppm F, Coca-Cola Company) at room temperature for 90 s. Immediately after erosion, the appliances were washed in tap water and reinserted into the mouth. Two times a day, after the first and last erosive challenges, the subjects performed toothbrush abrasion on one line (one enamel specimen for each varnish and solution treatment). Each specimen was brushed ex vivo with an electrical toothbrush (Colgate Motions Multi-action) using one drop (around 35 μl) of fluoridefree dentifrice slurry (Experimental Crest, Procter & Gamble, USA; ratio dentifrice: water=1:3, ph 6.8) for 10 s (166 oscillations per second, 1.5 N) [16]. The erosive and abrasive challenges were repeated daily for 5 days. The subjects were previously trained by the researchers to perform the toothbrushing. The erosive and abrasive protocols were applied according to the Guideline for Erosion Studies to appropriately simulate the clinical condition [21]. The appliances were worn day and night (20 h/day) and were stored in humidity during the main meals and oral hygiene procedures (four times daily, 1 h each, total of 4 h). The subjects were advised not to eat or to drink while the appliances were into the mouth. A minimum of 30 min elapsed between individual oral hygiene and the experiment. Wear analysis Enamel wear (micrometer) was defined as outcome parameter. After the end of each experimental period/phase, the enamel specimens were removed from the appliances. The nail varnish on the reference surfaces was carefully removed with acetone/water-soaked cotton swab [15, 16, 20]. The specimens were stored in 100 % humidity until the analysis. All specimens were analysed at the end of the study. The wear was quantitatively determined by a contact profilometer (Hommel Tester T1000, VS, Schwenningen, Germany). The diamond stylus (2 μm in diameter; angulation, 90 ) moved 2.5 mm from the first reference across the exposed area onto the other reference area. Four profile measurements were randomly performed in the centre of each specimen. The vertical distance between the midpoints of regression lines on the references and experimental areas was defined as tooth wear using the software of the device. The values were averaged (micrometre) and submitted to statistical analysis. The detection limit for this contact profilometer is about 0.5 μm of enamel wear. The standard deviation of repeated analysis (the reproducibility of the method) of a given enamel sample from the control group, without removing it from the holder, was 0.2 μm (mean wear of 2.5 μm). Statistical analysis The assumptions of equality of variances and normal distribution of errors were checked for all the variables tested using the Bartlett and Kolmogorov Smirnov tests (GraphPad Instat for Windows version 4.0, San Diego, CA, USA), respectively. Since the assumptions were satisfied, two-way repeated measures, ANOVA and Bonferroni post hoc test, were used (GraphPad Prism 4 version 4.0 for Windows, GraphPad Software, San Diego, CA, USA). Thereby, the different treatments (fluoride) were considered as dependent and the conditions (ERO and ERO+ABR) as independent variables and vice versa. The subjects were considered as statistical unit (n = 12), and the significance level was set at 5 %. Results All participants completed the study, and all specimens could be measured profilometrically. No specimens were lost and the nail varnish was still protecting the control areas during whole in situ phase. No participants reported adverse events or side effects. Two-wayrepeatedmeasureANOVArevealednosignificant difference between the conditions ERO and ERO + ABR (p >0.05). In respect to the treatments, all fluoride varnishes and solutions significantly reduced (around 25 %) the enamel wear when compared to the untreated control and placebo varnish (p <0.001), which in turn did not significantly differ from each other. There were no significant differences among the fluoride formulations against enamel erosion and erosion plus abrasion as well (Table 1). There was no interaction between the factors (p >0.05). Discussion The null hypothesis tested was accepted. In the present study, all fluoridated varnishes and solutions were able to significantly reduce enamel erosion and erosion plus abrasion compared to placebo varnish and control. Differently from previous results obtained from in vitro studies [15, 16], TiF 4 has the same protective potential as NaF to reduce human enamel wear in situ. Considering the same fluoride salt, there was also no significant difference between varnish and solution, showing that the distinct contact times between the vehicles (solution, 1 min vs. varnish, 6 h) seem not to be an important factor in the present model. It is important to discuss that no cross-effect between solution and varnish treatments could be expected by using the present experimental design (split mouth appliance).
Table 1 Mean enamel wear (micrometre) ± SD (95 % confidence limit) for the different treatments and enamel conditions NaF varnish a NaF solution a TiF 4 varnish a TiF 4 solution a Placebo Varnish b Control b ERO A 1.1±0.5 1.3±0.4 1.2±0.5 1.2±0.7 1.8±0.8 1.8±0.8 (0.74 1.42) (1.03 1.57) (0.87 1.51) (0.83 1.63) (1.31 2.30) (1.33 2.30) ERO + ABR A 1.5±0.6 1.6±0.6 1.2±0.5 1.5±0.7 2.1±0.6 2.2±0.8 (1.09 1.82) (1.19 1.95) (0.91 1.58) (1.02 1.88) (1.70 2.48) (1.72 2.70) Similar upper case letters indicate no difference between the enamel conditions. Distinct lower case letters indicate significant differences among the fluoride treatments and the control Attin et al. [22] have shown that fluoride acquisition could not be detected in specimens located at a distance of 1 cm from the specimens treated with fluoride varnish. Topical fluoridation induces the formation of a protective layer on dental hard tissue, which is composed of CaF 2 -like layer (in case of conventional fluorides as NaF) or of metalrich surface precipitates (in case of TiF 4 ). From the results of the present study, it can be speculated that the CaF 2 -like layer produced by NaF was as effective as the metal-rich surface precipitates created by the application of TiF 4 formulations in situ. CaF 2 -globules behave as a physical barrier inhibiting the contact of the acid with enamel and/or acting as a fluoride reservoir. On the other hand, TiF 4 -treated enamel samples presented a visible coating on the surface rich in fluoride and titanium as confirmed, using SEM and microprobe analysis, in a previous study from our group [15]. The effect of TiF 4 is related to both fluoride (as it happens for NaF) and titanium [23]. It is hypothesized that titanium might complex with phosphate groups (e.g. hydrated hydrogen titanium phosphate) producing an acid-resistant surface coating [8, 9]. Some factors have shown to influence the efficacy of fluoride products against demineralisation such as fluoride concentration, ph, and frequency of application [4]. In our study, the formulations presented the same fluoride concentration; however, NaF presented a higher ph (5.0) compared to TiF 4 (1.0). From this view, we would expect a better effect of TiF 4 compared to NaF not only due to the titanium but also to the low ph that could increase the enamel fluoride uptake. Contrasting the expectations, there were no differences between the fluoride salts and the vehicles. The present results areindisagreementwithaninsitustudy,inwhich4%tif 4 solution was unable to reduce human permanent and deciduous enamel erosive wear [12], and from some in vitro studies in that 4 % TiF 4 solution could not reduce bovine enamel erosive wear as well [15, 20]. However, in the cited works, the specimens were not covered by salivary pellicle before the treatments. We speculate that this factor could explain the differences among the studies. We allowed 2 h of pellicle formation according to previous studies about the formation and composition of the salivary pellicle [24, 25]. The presence of human saliva in situ allows the formation of salivary pellicle, which in turn might have a significant impact on the reaction between fluoride and tooth surface. There are few studies dealing with the effect of fluoride on pellicle-covered tooth. Wiegand et al. [26] showed that the efficacy of the tetrafluoride solutions was positively influenced by the presence of the salivary pellicle in vitro. On the other hand, two studies performed by Hove et al. [27, 28] pointed out that TiF 4 solution provides protection against acid attack, regardless of the presence of the pellicle. Therefore, there is still some uncertainty if salivary pellicle modulates the anti-erosive effect of fluoride, especially in case of TiF 4,whichmaybealso influenced by the time of application, fluoride concentration as well as to the following erosive challenges. The human saliva might also allow better stability to the CaF 2 or metal-rich layer (in case of TiF 4 solution applied only for 1 min) on enamel compared to F-treated enamel samples exposed only to the artificial saliva in vitro [15, 20]. It is already known that the stability of the CaF 2 -like layer on tooth in vitro is substantially reduced compared to in situ condition. Human salivary proteins and phosphate may stabilize the fluoride precipitation on enamel [5]. Another important point that might explain the different results is the type of substrate (bovine vs. human enamel). According to Hove et al. [28], TiF 4 solution (0.5 M F) might better react with bovine compared to human enamel. To our knowledge, there are no other studies comparing the antierosive effect of fluoride on bovine and human enamel directly. Our studies do not allow such comparison, but from the previous in vitro studies using bovine enamel, at least in the case of 4 % TiF 4 solution, it is hypothesized that this formulation might react better with human than bovine enamel, refuting previous findings [28]. However, this hypothesis should be better addressed in further studies. In the present study, the ERO + ABR condition showed similar enamel wear mean values compared to the ERO only. The presence of human saliva might also have reduced the progression of tooth wear when erosion was associated to abrasion. Additionally, this result may be a consequence of the experimental protocol, in which the abrasion procedure (2 10 s/day) was less aggressive than the erosive challenges (4 90 s/day) [29]. Using this protocol, the toothbrushing abrasion could not have adversely affected the progression of tooth erosive wear differently from the results found by
Wiegand et al. [11] in that abrasion was performed 2 30 s/ day. The erosive and abrasive protocols applied in the present study follows the guideline for erosion research published in a special issue of Caries Research [21, 29]. The cola drink was chosen as one of the most consumed soft drink by the population and has been shown to be highly erosive [12, 20]. Under the conditions of the present study, it can be concluded that TiF 4 varnish has similar protective potential as the solution and NaF formulations to reduce human enamel erosion with or without abrasion in situ. Future in situ studies must test the durability of the effect of fluoride varnishes on tooth wear using longer experimental periods [3], which in turn shall allow a satisfactory subjects compliance. Other important point that should be further addressed is the clinical relevance of the results (25 % reduction by the application of professional fluoride products) compared to other preventive measures, as daily application of fluoride mouthrinse. It would be interesting to test the independent effect of professional and home-care fluoride applications as well as the combination of both methods. Acknowledgments This study was supported by The São Paulo Research Foundation - FAPESP (Proc. 2008/07105-4 for the last author, 2008/03727-0 for the first author). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Conflict of interest interest. References The authors declare that they have no conflict of 1. ten Cate JM (1997) Review on fluoride, with special emphasis on calcium fluoride mechanisms in caries prevention. Eur J Oral Sci 105:461 465 2. Wiegand A, Attin T (2003) Influence of fluoride on the prevention of erosive lesions a review. Oral Health Prev Dent 1:245 253 3. Magalhães AC, Wiegand A, Rios D, Buzalaf MA, Lussi A (2011) Fluoride in dental erosion. Monogr Oral Sci 22:158 170 4. Saxegaard E, Rolla G (1988) Fluoride acquisition on and in human enamel during topical application in vitro. Scand J Dent Res 96:523 535 5. Ganss C, Schlueter N, Klimek J (2007) Retention of KOH-soluble fluoride on enamel and dentine under erosive conditions. A comparison of in vitro and in situ results. Arch Oral Biol 52:9 14 6. Ganss C, Klimek J, Schaffer U, Spall T (2001) Effectiveness of two fluoridation measures on erosion progression in human enamel and dentine in vitro. Caries Res 35:325 330 7. Ganss C, Klimek J, Brune V, Schürmann A (2004) Effects of two fluoridation measures on erosion progression in human enamel and dentine in situ. Caries Res 38:561 566 8. Büyükyilmaz T, Øgaard B, Rølla G (1997) The resistance of titanium tetrafluoride-treated human enamel to strong hydrochloric acid. Eur J Oral Sci 105:473 477 9. Ribeiro CC, Gibson I, Barbosa MA (2006) The uptake of titanium ions by hydroxiapatite particles structural changes and possible mechanisms. Biomaterials 27:1749 1761 10. Hove LH, Holme B, Young A, Tveit AB (2008) The protective effect of TiF 4,SnF 2 and NaF against erosion-like lesions in situ. Caries Res 42:68 72 11. Wiegand A, Hiestand B, Sener B, Magalhães AC, Roos M, Attin T (2010) Effect of TiF(4), ZrF(4), HfF(4) and AmF on erosion and erosion/abrasion of enamel and dentin in situ. Arch Oral Biol 55:223 228 12. Magalhães AC, Rios D, Honório HM, Jorge AM Jr, Delbem AC, Buzalaf MA (2008) Effect of 4 % titanium tetrafluoride solution on dental erosion by a soft drink: an in situ/ex vivo study. Arch Oral Biol 53:399 404 13. Hove LH, Holme B, Stenhagen KR, Tveit AB (2011) Protective effect of TiF(4) solutions with different concentrations and ph on development of erosion-like lesions. Caries Res 45:64 68 14. Wiegand A, Waldheim E, Sener B, Magalhães AC, Attin T (2009) Comparison of the effects of TiF 4 and NaF solutions at ph 1.2 and 3.5 on enamel erosion in vitro. Caries Res 43:269 277 15. Magalhães AC, Kato MT, Rios D, Wiegand A, Attin T, Buzalaf MAR (2008) The effect of an experimental 4 % TiF 4 varnish compared to NaF varnishes and 4 % TiF 4 solution on dental erosion in vitro. Caries Res 42:269 274 16. Levy FM, Magalhães AC, Gomes MF, Comar LP, Rios D, Buzalaf MAR (2012) The erosion and abrasion-inhibiting effect of TiF 4 and NaF varnishes and solutions on enamel in vitro. Int J Paediatr Dent 22:11 16 17. Comar LP, Wiegand A, Moron BM, Rios D, Buzalaf MA, Buchalla W, Magalhães AC (2012) In situ effect of sodium fluoride or titanium tetrafluoride varnish and solution on carious demineralization of enamel. Eur J Oral Sci 120:342 348 18. Tezel H, Ergucu Z, Onal B (2002) Effects of topical fluoride agents on artificial enamel lesion formation in vitro. Quintessence Int 33: 347 352 19. van Rijkom H, Ruben J, Vieira A, Huysmans MC, Truin GJ, Mulder J (2003) Erosion-inhibiting effect of sodium fluoride and titanium tetrafluoride treatment in vitro. Eur J Oral Sci 111:253 257 20. Magalhães AC, Stancari FH, Rios D, Buzalaf MAR (2007) Effect of an experimental 4 % titanium tetrafluoride varnish on dental erosion by a soft drink. J Dent 35:858 861 21. Shellis RP, Ganss C, Ren Y, Zero DT, Lussi A (2011) Methodology and models in erosion research: discussion and conclusions. Caries Res 45:69 77 22. Attin T, Lennon AM, Yakin M, Becker K, Buchalla W, Attin R, Wiegand A (2007) Deposition of fluoride on enamel surfaces released from varnishes is limited to vicinity of fluoridation site. Clin Oral Investig 11:83 88 23. Wei SH, Soboroff DM, Wefel JS (1976) Effects of titanium tetrafluoride on human enamel. J Dent Res 155:426 431 24. Siqueira WL, Zhang W, Helmerhorst EJ, Gygi SP, Oppenheim FG (2007) Identification of protein components in in vivo human acquired enamel pellicle using LC-ESI-MS/MS. J Proteome Res 6: 2152 2160 25. Siqueira WL, Oppenheim FG (2009) Small molecular weight proteins/peptides present in the in vivo formed human acquired enamel pellicle. Arch Oral Biol 54:437 444 26. Wiegand A, Meier W, Sutter E, Magalhães AC, Becker K, Roos M, Attin T (2008) Protective effect of different tetrafluorides on erosion of pellicle-free and pellicle-covered enamel and dentine. Caries Res 42:247 254 27. Hove LH, Holme B, Young A, Tveit AB (2007) The erosioninhibiting effect of TiF 4,SnF 2, and NaF solutions on pelliclecovered enamel in vitro. Acta Odontol Scand 65:259 264 28. Hove LH, Young A, Tveit AB (2007) An in vitro study on the effect of TiF(4) treatment against erosion by hydrochloric acid on pelliclecovered enamel. Caries Res 41:80 84 29. Wiegand A, Attin T (2011) Design of erosion/abrasion studiesinsights and rational concepts. Caries Res 45:53 59