archives of oral biology 59 (2014) 1328–1333
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/aob
Etching effect of acidic fluorides on human tooth enamel in vitro Carl Hjortsjo¨ a,*, Grazyna Jonski b, Alix Young c, Erik Saxegaard a a
Department of Prosthetic Dentistry and Oral Function, Faculty of Dentistry, University of Oslo, P.O. Box 1109, Blindern, 0317 Oslo, Norway b Oral Research Laboratory, Faculty of Dentistry, University of Oslo, P.O. Box 1109, Blindern, 0317 Oslo, Norway c Department of Cariology and Gerodontology, Faculty of Dentistry, University of Oslo, P.O. Box 1109, Blindern, 0317 Oslo, Norway
article info
abstract
Article history:
Objective: This in vitro study aimed to examine the etching effect of acidic fluoride solutions
Accepted 11 August 2014
on enamel. Materials and methods: 24 human teeth divided into 48 enamel-specimens were partly
Keywords:
isolated with impression material. Specimens were exposed for 10 min to 20 ml of the
Dental erosion
following solutions: 1.6% TiF4, 3.9% SnF2, 0.2% HF and 1.8% citric acid (CA). The isolation was
Fluorides
removed and 24 specimens analysed by profilometry (Dheight: exposed/isolated enamel
Hydrofluoric acid
surfaces, surface roughness parameters). For the remaining 24 specimens [Ca2+] in the test
Stannous fluoride
solutions was analysed by atomic absorption spectroscopy.
Titanium tetrafluoride
Results: Median Dheights (mm) after exposure were: TiF4 0.07, SnF2 0.03, HF 0.14 and CA 5.92. TiF4-exposed surfaces showed both deposits and etched areas and exhibited statistically significant different surface roughness parameters compared to the HF- and SnF2exposed surfaces. Median [Ca2+] values (ppm): TiF4 1.88, SnF2 0.11, HF 0.10 and CA 2.17. Conclusion: At the [F] tested in this study it can be concluded that SnF2- and HF solutions had negligible erosive effects on enamel. TiF4 solution resulted in an incomplete surface deposition associated with calcium dissolution suggesting that TiF4 applied as solution may not be advisable. # 2014 Elsevier Ltd. All rights reserved.
1.
Introduction
Dental erosion lesions are caused by the action of extrinsic- or intrinsic acids usually in combination with abrasion and or attrition.1,2 Citric acid is a common extrinsic acid often found in beverages and food. Treatment of enamel with acidic
fluoride solutions such as titanium tetrafluoride (TiF4), stannous fluoride (SnF2) and hydrogen fluoride (HF) has in vitro and in situ shown to provide some protection against dissolution upon exposure to such acids.3–8 It has been suggested that the erosion-inhibiting effects of TiF4 and SnF2 are related to the formation of a coating or a glaze layer that prevents further erosion or due to the formation of
* Corresponding author at: Department of Prosthetic Dentistry and Oral Function, Institute of Clinical Dentistry, University of Oslo, P.O. Box 1109, Blindern, 0317 Oslo, Norway. Tel.: +47 22852368; fax: +47 22852344. E-mail address: [email protected] (C. Hjortsjo¨ ). http://dx.doi.org/10.1016/j.archoralbio.2014.08.007 0003–9969/# 2014 Elsevier Ltd. All rights reserved.
archives of oral biology 59 (2014) 1328–1333
acid-resistant calcium fluoride precipitates on the enamel surface.3,6,9–11 Some authors have reported the coating/ glaze layer to be incomplete or have cracks,6,9 and have attributed this observed lack of a complete glaze coating to microscopy preparation. However, if the formation of an incomplete coating/glaze layer is a true effect, it may influence the protective effect against erosion seen by TiF4 and SnF2. It has also been demonstrated that in vitro solutions containing stannous ions (Sn2+) may lead to the incorporation of Sn in the outer enamel surface.12,13 The higher the amount of incorporated Sn in the enamel, the less enamel tissue was shown to be lost when the surface was exposed to citric acid.12 Acidic fluoride solutions below pH 3 contain fluoride largely in the form of undissociated HF0 molecules, in addition to smaller amounts of free fluoride anions. HF0 has the ability to diffuse into intercrystalline spaces of enamel14 and penetrate the hydroxyapatite lattice in the root-dentine surface.15 Recently it has been suggested that HF0 may result in the formation of a subsurface layer of acid-resistant calcium fluoride-like material.5 However, concerns regarding the potential of acidic fluorides to remove calcium (and phosphate ions) from the enamel surface have been raised. In a study by Skartveit and co-workers where tooth root surfaces were treated with 4% TiF4, a partly demineralised zone with a depth of 8–10 mm was demonstrated after 1 min. After 4 min this zone was 5–27 mm deep.16 Different dental hard tissues react differently to erosive demineralization as demonstrated by Ganss and co-workers.17 Although one cannot assume that the above-mentioned changes seen in root dentine can be directly extrapolated to dental enamel, the changes may serve as a predictor of possible reactions. The aim of this in vitro study was therefore to investigate possible surface changes on human enamel specimens following prolonged exposure to different acidic fluoride solutions (TiF4, SnF2 and HF) using a simplified model not including a salivary pellicle. The null hypothesis to be tested was that these acidic fluoride solutions do not result in any etching of enamel surfaces as measured by profilometric- and spectrometric analyses.
2.
Materials and methods
2.1.
Tooth samples, specimen preparation and isolation
Twenty-four intact, surgically extracted human third molar teeth were collected from private dental practitioners in Oslo and surrounding areas, or from the Department of Oral Surgery and Oral Medicine, Institute of Clinical Dentistry, Faculty of Dentistry, University of Oslo. All tooth donors gave their consent to the use of their teeth for this research project. It was not known whether the teeth crowns (or parts of the crowns) had been exposed to the oral cavity prior to extraction. Teeth were cleaned and stored in a moist thymol environment (99.5% thymol, Sigma–Aldrich, St. Louis, MO, USA). Prior to the experiment, the teeth were prepared using a saw with water cooling (Exakt-apparatgebau, Norderstedt, Germany) and were cut vertically into two halves thus providing 48 enamel specimens. The teeth that were to be
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analysed by profilometry also had their roots removed by use of the same saw with water cooling. All specimens were then glued to a plastic object plate (Technovit 7210 VLC; Heraeus Kulzer GmbH, Wehrheim, Germany). A flat enamel surface was ground wet on each specimen with an abrasive paper to grit 2500 (P2500, silicon carbide grinding paper, Buehler, Du¨sseldorf, Germany) (Exakt-apparatebau, Norderstedt, Germany) until a surface area of approximately 9 mm2 was exposed. The size of this area was dependent on the original size, position and form of each tooth, and approximately 200 mm of the surface enamel was removed during this process. One half of the enamel specimens (n = 24) were prepared for profilometric analysis. Half of the ground enamel surface area of each specimen was isolated using a thin layer of light body polyvinyl siloxane (PVS) impression material (Express II light body, 3M-ESPE, Seefeld, Germany). The border between the isolated and exposed area of enamel was situated at the centre of the ground enamel surface. After approximately 6 min the impression material was set and the specimens were ready for exposure to one of the test solutions. The other half of the specimens (n = 24) were prepared for calcium analysis. Specimens were completely covered with a thin layer of the same light body PVS impression material. After setting a custom-made punching instrument with a circular cutting edge (diameter 2.5 mm) was used to carefully punch a hole through the impression material on each specimen, exposing a 4.9 mm2 circular area at the centre of the ground enamel surface.
2.2.
Test solutions and treatment of the specimens
The following fluoride solutions and citric acid solution were included in the study: TiF4 solution (1.6% w/v, 0.5 M F, pH 1.42) was prepared from titanium tetrafluoride (Sigma–Aldrich Chemie, Steinheim, Germany), SnF2 solution (3.9% w/v, 0.5 M F, pH 2.65) was prepared from tin II fluoride (Sigma–Aldrich Chemie, Steinheim, Germany), and HF solution (0.20% w/v, 0.1 M F, pH 3.02) was prepared from 40% hydrogen fluoride (Rectapur, Prolabo, Paris, France). Citric acid solution (1.8%, pH 2.27) was included as a positive control (Sigma–Aldrich Chemie, Steinheim, Germany). Deionised water was used in the preparation of all solutions. The choice of concentration for the fluoride solutions was based on previous studies reporting the beneficial effects of these fluoride solutions.4,5,18,19 The HF solution had a lower fluoride concentration than the TiF4 and SnF2 solutions since previous in vivo and in vitro studies have demonstrated HF toxicity at higher concentrations.20 The pH of the solutions was measured at room temperature (pH-meter Orion Star, Thermo Electron Corporation, Beverly, MA, USA). Following the isolation procedures, pellicle-free enamel specimens were exposed for 10 min to one of the test solutions (n = 6 specimens/solution) by submerging each specimen in separate plastic containers (Coulter, Kartell spa, Noviglio, Italy) each containing 20 ml of the solutions under constant, gentle agitation (100 rpm, IKA KS 260 Basic, Stuafen, Germany). The large volume of solution was used in order to avoid the possibility of re-precipitation of minerals onto the
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enamel specimens. All specimens were removed from the test solutions after 10 min, and rinsed with deionised water for approximately 5 s.
2.3.
Profilometric analysis
Following exposure to the test solutions and prior to profilometry, excess water was gently removed from all specimens using pressurised air, and the isolating PVS material was removed. The specimens were examined to confirm that the uncovered ground area of enamel had in fact been exposed to the test solution. Analysis was performed using a high power LED blue light profilometer (PLm2300 Optical Imaging Profiler, Sensofar-tech Terrassa, Spain) at 50 magnification and a wavelength of 470 nm. One measurement per sample was performed including both exposed and isolated enamel surfaces for each specimen. If the sample is etched during the F-solution exposure, the exposed surface will have lost height compared to the adjacent isolated surface (Dheight < 0). Conversely, the lack of etching will be shown either by no height difference (Dheight = 0) or possibly even by a net height gain for the F-exposed surfaces (Dheight > 0). All data was stored on the profiler computer until statistical analysis. Tooth surface areas the size of 254.64 190.90 mm2 were analysed for each sample. Depth measurements included 233–239 planes/sample, each of 0.2 mm (total a maximum of 47.6 mm). The data were analysed using Sensomap plus 4.1 software (Sensofar-tech Terrassa, Spain). Following processing, the plane of the image for each sample was first levelled using the levelling function (three points of measurement on the reference area). The step height function was then used to calculate the height differences (Dheight in mm) between the exposed enamel surfaces and isolated enamel surfaces following exposure to the solutions. The following surface parameters were also registered for the exposed and isolated areas: Sa = average roughness, Sp = maximum height (height between the mean level and the highest peak), Sv = maximum depth (depth between the mean level and the deepest valley), St = total height (height between the highest peak and the deepest valley).
2.4.
Atomic absorption spectrometric analysis
Solutions were collected following exposure of the enamel specimen and stored at 4 8C until calcium analyses were performed. An Analyst 400 Atomic Absorption Spectrometer (Perkin Elmer Analytical Instrument, Norwalk, CT, USA) was used for the analyses. Prior to analysis, 0.55 ml of 36% HCl (Rectapur, Prolabo, Paris, France) was added to each of the TiF4 and SnF2 test solution samples. In order to counteract the negative effect of phosphorus on the calcium sensitivity of the spectrometer lanthanum chloride (VWR International, Fontenay sous Bois, France) was added to all test solutions to a final concentration of 1% (10% LaCl3 in 5% HCl stock). Calcium analyses were performed directly after adding HCl and LaCl3.
2.5.
Statistical analysis
The raw data from the spectrometric and profilometric Dheight measurements failed the Shapiro–Wilk normality
test (SigmaPlot version 12.0, Systat software, San Jose´, California, USA). The Kruskal–Wallis test was therefore performed and pairwise multiple comparison procedures (Student Newman–Keuls test) were used to compare differences between groups. The profilometric surface parameters data comparing surface parameters between exposed and isolated areas passed the Shapiro–Wilk normality test, and mean differences were tested using a t-test. All differences were considered significant at the 0.05 level.
3.
Results
The results are presented in Table 1 and Fig. 1A–D. Profilometric analysis demonstrated that exposure of the enamel samples to acidic fluoride solutions resulted in negligible net surface height changes. The citric acid had a clear etching effect as shown by the result for the positive controls. Fig. 1 shows representative profilometric images for enamel specimens exposed to the different solutions as generated by the Sensomap plus 4.1 software. The border between the exposed and isolated areas was clearly visible in all samples. The samples exposed to TiF 4 solution showed a clear surface deposition layer (Fig. 1A), in contrast to the citric acid positive control samples that showed a clearly etched surface (Fig. 1D). The SnF 2 and HF samples showed neglible height differences between exposed and isolated areas, indicating no surface changes (Fig. 1B and C). The surface parameters are presented in Table 1. Surface parameter analysis revealed statistically significant differences between the exposed and the isolated areas for both citric acid and TiF4 solutions. Following exposure to these solutions the surface differences were significant for Sa (indicating greater average surface roughness), Sp (indicating higher peaks on the surface), Sv (indicating deeper valleys) and St (indicating a greater distance between the highest peak and deepest valley). The spectrometric analysis showed that exposure of the enamel specimens to the TiF4- and citric acid solutions clearly resulted in calcium dissolution (Table 1). Exposure of the enamel specimens to the SnF2- and HF solutions resulted in lower levels of calcium release.
4.
Discussion
This in vitro study revealed obvious differences in the interaction of the different acidic fluoride solutions on the bare enamel surfaces. While negligible effects on the enamel surfaces were observed for specimens exposed to the SnF2and HF solutions, the TiF4-exposed specimens showed a combination of both a surface deposition layer and enamel dissolution. Under the present experimental conditions, the null hypothesis was therefore rejected. As expected the profilometric and spectrometric calcium analyses clearly showed that exposure to the citric acid solution resulted in an obvious etching of the exposed surfaces. However, regarding the effect of TiF4, although
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Table 1 – Median values (range). Dheight = profilometric height difference between exposed and isolated surfaces. Surface parameters of the specimens exposed to the solutions (isolated surfaces in brackets). Sa = average roughness, Sp = maximum height (height between the mean level and the highest peak), Sv = maximum depth (height between the mean level and the deepest valley), St = total height (height between the highest peak and the deepest valley). Test solution
Spectrometric analysis 2+
TiF4 SnF2 HF Citric acid
z
Profilometric analysis z
Median [Ca ] (ppm)
Median Dheight (mm)
1.88a (1.10–1.96) 0.11b (0.10–0.35) 0.10b (0.05–0.13) 2.17c (2.03–2.66)
0.07d (2.25 to 1.61) 0.03d (0.35 to 0.18) 0.14d (0.17 to 0.03) 5.92e (7.46 to 5.20)
Sa (mm) 1.20 0.57 0.67 0.84
(0.76) * (0.55) (0.65) (0.54) *
Sp (mm) 10.50 2.28 1.52 3.00
(2.37) * (2.89) (1.46) (1.47) *
Sv (mm) 4.14 2.59 1.63 4.14
(1.95) * (1.59) (1.58) (1.41) *
St (mm) 14.64 4.87 3.15 7.14
(4.32) * (4.47) (3.03) (2.87) *
*
T-test shows a significant difference between exposed and isolated areas. All differences were considered significant at the 0.05 level. Within columns: [Ca2+] and Dheight values sharing the same superscript letter were not significantly different from each other (Student Newman–Keuls test). z
Fig. 1 – Profilometric images of representative enamel specimens exposed to the different solutions. For each image, the area to the left of the ‘borderline’ is the enamel surface that was exposed to the solutions, and the area to the right is the isolated enamel surface. (A–D) are specimens immediately post exposure: (A) TiF4 specimen showing an obvious, but incomplete surface deposition on the exposed surface, (B) SnF2 specimen showing no obvious surface deposition, and some slight signs of etching along the borderline, (C) HF specimen showing a slight tendency to etching on the exposed surface, and with some deposition along the borderline, (D) citric acid specimen (CA) showing a typical enamel etching pattern on the exposed surface. All images are 50T magnification.
profilometric height analyses indicated a height difference close to zero between TiF4-exposed and non-TiF4-exposed surfaces, further analysis of the surface parameters revealed large differences between the highest ‘peaks’ and lowest
‘valleys’ on the TiF4-exposed surfaces. An interpretation of this result suggests the presence of an incomplete surface deposition layer as well as the presence of etched areas. Standard profilometric height analysis alone was therefore
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archives of oral biology 59 (2014) 1328–1333
not sufficient to detect possible important changes occurring on the enamel surface. The formation of a superficial layer following treatment of the dental enamel with fluorides has been reported previously and is often described as a ‘glaze’.6,9,21 The continuity of the glaze layer will without doubt depend very much upon the conditions under which the layer is formed. The present finding that the TiF4 solution resulted in greater dissolution of calcium than was observed for the SnF2- and HF-solutions supports the profilometry results, as well as previous findings that the TiF4-initiated glaze layer is not necessarily 100% complete.6,9 Furthermore, those parts of the enamel surface that were not covered by the glaze were susceptible to dissolution. While the erosive effect seen for the TiF4 solution may be partly explained by the low pH of the native solution (pH 1.42), the erosive effect seen by the citric acid can be explained by the combination of low pH (pH 2.27) and chelator properties. Given that pH, concentration and application method (solution, gel, toothpaste) are important factors in the reaction of fluoride solutions with the enamel, other concentrations and application methods of TiF4 may give rise to a more complete glaze than was observed in the present study. Recently it was demonstrated that 4% TiF4 varnish reduced enamel dissolution due to acidic beverages far more effectively than 4% TiF4 solutions 22,23. The authors suggested that the superior protective effect of the TiF4 varnish is due to its ability to adhere to the tooth surface and through this increase the reaction time between enamel and TiF4 varnish. Although these findings cannot be extrapolated to the in vivo situation, the results reflect different chemical compositions of the surface layers formed. As no significant changes were recorded in surface parameters for the specimens exposed to the SnF2 and HF solutions, one can speculate that the changes in the enamel specimens that occur after treatment with these solutions are different from those following exposure to TiF4 solution. Previous reports have shown that pretreatment of enamel with HF reduced calcium dissolution after citric acid exposure.4,5,24,25 It has been speculated that undissociated HF-molecules are able to cause deposition of acid resistant calcium fluoride-like minerals when exposed to enamel at pH 3. The HF-molecules probably dissociate under such conditions causing the released H ions to dissolve the enamel by reduction of phosphate molecules to soluble di-hydrogen phosphates (H2PO4). The di-hydrogen phosphates are subsequently lost from the enamel surface, and released F ions react with newly available calcium ions to form calcium fluoride-like material. This CaF-like material is thought to be more acid resistant than the CaF-material formed under neutral conditions, since it is formed under acidic conditions.25 At the borderline between the areas of isolated and exposed enamel a slight surface deposition was also observed for the HF-treated area (Fig. 1C). This borderline area including some form of deposits was excluded in the profilometric analysis and therefore did not affect the height recordings. In contrast, enamel samples treated with citric acid exhibited increased etching in the borderline area (Fig. 1D). A possible explanation for this phenomenon is that the level of exposure of the enamel to the acidic solutions was greater in this area due to pooling of the solutions in contact with the isolating PVS material. These findings are supported
by similar previous experiments on enamel samples using white light interferometry, which showed that the surfaces within 200 mm of an amalgam reference marker were excessively etched by both TiF4- and SnF2 solutions.6 In the present study different specimen isolation procedures were used for the profilometric- and spectrometric analyses. For the profilometric analysis it was desirable to have approximately equal-sized surface areas of exposed and isolated enamel. This was achieved for each specimen by isolating half the enamel surface area along a straight line using PVS impression material. For the spectrometric analysis it was necessary to have well-defined and equally-sized enamel surface areas on the specimens in order to permit a comparison between [Ca] of the different fluoride solutions and the control. This was achieved by first covering the entire enamel surface with the PVS impression material and then punching out a circular window through the impression material to expose a standardised test surface area. In both procedures approximately the same area of enamel was exposed for the profilometric analysis as for the AAS analysis (profilometry: approx. 4.5 mm2 = half the area of 9 mm2 and AAS: 4.9 mm2). This allowed the results from the two different methodological procedures to be compared. The choice of concentration for the fluoride solutions was based on previous studies reporting their respective beneficial effects.4,5,18,19,25 In order to avoid participation of other ions during spectrometric analyses, HCl was added to the TiF4- and SnF2 solutions prior to the addition of lanthanum chloride and before calcium analysis. Using two slightly different sample preparation techniques for the spectrometry analysis may not be optimal, however adding HCl does not affect the amount of calcium in the solution thus allowing comparison of the results from the different test solutions. Salivary pre-conditioning of the enamel surfaces in order to form a pellicle was not included in the protocol and the exposure time of the enamel specimens to the acidic fluoride solutions was relatively long. These study conditions therefore presented a ‘worst case scenario’. Further studies using other concentrations and exposure times on specimens that are preconditioned with saliva are needed to determine whether these differences are also likely to be seen in a clinical setting. In conclusion, this in vitro study demonstrated clear differences in how the acidic fluoride solutions reacted with pellicle-free enamel. Even under the extreme conditions of this study, relatively long exposure of enamel specimens to the SnF2- and HF solutions resulted in only minimal erosive effects. Exposure to the TiF4 solution resulted in a surface deposition on the enamel specimens that could protect the enamel. However, the ‘glaze’ was incomplete and was associated with a significantly greater loss of calcium. TiF4 in solution may therefore not be the best choice of acidic fluoride if this effect also occurs in vivo where a range of different factors will influence the nature of fluoride interaction with enamel surfaces.
Funding University grants.
archives of oral biology 59 (2014) 1328–1333
Competing interests None declared.
Ethical approval Not required.
references
1. Scheutzel P. Etiology of dental erosion – intrinsic factors. Eur J Oral Sci 1996;104(2):178–90. 2. Zero DT. Etiology of dental erosion – extrinsic factors. Eur J Oral Sci 1996;104(2):162–77. 3. Ganss C, Schlueter N, Hardt M, Schattenberg P, Klimek J. Effect of fluoride compounds on enamel erosion in vitro: a comparison of amine, sodium and stannous fluoride. Caries Res 2008;42(1):2–7. 4. Hjortsjo¨ C, Jonski G, Thrane PS, Saxegaard E, Young A. Effect of stannous fluoride and dilute hydrofluoric acid on early enamel erosion over time in vivo. Caries Res 2009;43(6): 449–54. 5. Hjortsjo¨ C, Jonski G, Thrane PS, Saxegaard E, Young A. The effects of acidic fluoride solutions on early enamel erosion in vivo. Caries Res 2009;43(2):126–31. 6. Hove LH, Holme B, Øgaard B, Willumsen T, Tveit AB. The protective effect of TiF4, SnF2 and NaF on erosion of enamel by hydrochloric acid in vitro measured by white light interferometry. Caries Res 2006;40(5):440–3. 7. Hove LH, Young A, Tveit AB. An in vitro study on the effect of TiF(4) treatment against erosion by hydrochloric acid on pellicle-covered enamel. Caries Res 2007;41(1):80–4. 8. Schlueter N, Ganss C, Mueller U, Klimek J. Effect of titanium tetrafluoride and sodium fluoride on erosion progression in enamel and dentine in vitro. Caries Res 2007;41(2):141–5. 9. Bu¨yu¨kyilmaz T, Øgaard B, Rølla G. The resistance of titanium tetrafluoride-treated human enamel to strong hydrochloric acid. Eur J Oral Sci 1997;105(5):473–7. 10. Mundorff SA, Little MF, Bibby BG. Enamel dissolution. II. Action of titanium tetrafluoride. J Dent Res 1972;51(6): 1567–71. 11. Wei SH, Soboroff BM, Wefel JS. Effects of titanium tetrafluoride on human enamel. J Dent Res 1976;55(3):426–31.
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12. Schlueter N, Hardt M, Lussi A, Engelmann F, Klimek J, Ganss C. Tin-containing fluoride solutions as anti-erosive agents in enamel: an in vitro tin-uptake, tissue-loss, and scanning electron micrograph study. Eur J Oral Sci 2009;117(4):427–34. 13. Yu H, Attin T, Wiegand A, Buchalla W. Effects of various fluoride solutions on enamel erosion in vitro. Caries Res 2010;44(4):390–401. 14. Brudevold F, Savory A, Gardner DE, Spinelli M, Speirs R. A study of acidulated fluoride solutions. I. In vitro effects on enamel. Arch Oral Biol 1963;8(3):167–77. 15. Tveit AB, Hals E, Isrenn R, Totdal B. Highly acid SnF2 and TiF4 solutions. Effect on and chemical reaction with root dentin in vitro. Caries Res 1983;17(5):412–8. 16. Skartveit L, Selvig KA, Tveit AB. Root surface reactions to TiF4 and SnF2 solutions in vitro. An ultrastructural study. Acta Odontol Scand 1991;49(3):183–90. 17. Ganss C, Klimek J, Schwarz N. A comparative profilometric in vitro study of the susceptibility of polished and natural human enamel and dentine surfaces to erosive demineralization. Arch Oral Biol 2000;45(10):897–902. 18. Hove LH, Holme B, Young A, Tveit AB. The erosioninhibiting effect of TiF4, SnF2, and NaF solutions on pellicle-covered enamel in vitro. Acta Odontol Scand 2007;65(5):259–64. 19. Hove LH, Holme B, Young A, Tveit AB. The protective effect of TiF4, SnF2 and NaF against erosion-like lesions in situ. Caries Res 2008;42(1):68–72. 20. Hjortsjo¨ C, Saxegaard E, Young A, Dahl JE. In vivo and in vitro irritation testing of low concentrations of hydrofluoric acid. Acta Odontol Scand 2009;67(6):360–5. 21. Wiegand A, Waldheim E, Sener B, Magalha˜es AC, Attin T. Comparison of the effects of TiF4 and NaF solutions at pH 1.2 and 3.5 on enamel erosion in vitro. Caries Res 2009;43(4):269–77. 22. Magalha˜es AC, Romanelli AC, Rios D, Comar LP, Navarro RS, Grizzo LT, et al. Effect of a single application of TiF4 and NaF varnishes and solutions combined with Nd:YAG laser irradiation on enamel erosion in vitro. Photomed Laser Surg 2011;29(8):537–44. 23. Magalha˜es AC, Levy FM, Rizzante FA, Rios D, Buzalaf MA. Effect of NaF and TiF4 varnish and solution on bovine dentin erosion plus abrasion in vitro. Acta Odontol Scand 2012;70(2):160–4. 24. Hjortsjo¨ C, Jonski G, Young A, Saxegaard E. Effect of acidic fluoride treatments on early enamel erosion lesions – a comparison of calcium and profilometric analyses. Arch Oral Biol 2010;55(3):229–34. 25. Rølla G, Jonski G, Saxegaard E. On inhibition of dental erosion. Acta Odontol Scand 2013;71(6):1508–12.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/aob
Etching effect of acidic fluorides on human tooth enamel in vitro Carl Hjortsjo¨ a,*, Grazyna Jonski b, Alix Young c, Erik Saxegaard a a
Department of Prosthetic Dentistry and Oral Function, Faculty of Dentistry, University of Oslo, P.O. Box 1109, Blindern, 0317 Oslo, Norway b Oral Research Laboratory, Faculty of Dentistry, University of Oslo, P.O. Box 1109, Blindern, 0317 Oslo, Norway c Department of Cariology and Gerodontology, Faculty of Dentistry, University of Oslo, P.O. Box 1109, Blindern, 0317 Oslo, Norway
article info
abstract
Article history:
Objective: This in vitro study aimed to examine the etching effect of acidic fluoride solutions
Accepted 11 August 2014
on enamel. Materials and methods: 24 human teeth divided into 48 enamel-specimens were partly
Keywords:
isolated with impression material. Specimens were exposed for 10 min to 20 ml of the
Dental erosion
following solutions: 1.6% TiF4, 3.9% SnF2, 0.2% HF and 1.8% citric acid (CA). The isolation was
Fluorides
removed and 24 specimens analysed by profilometry (Dheight: exposed/isolated enamel
Hydrofluoric acid
surfaces, surface roughness parameters). For the remaining 24 specimens [Ca2+] in the test
Stannous fluoride
solutions was analysed by atomic absorption spectroscopy.
Titanium tetrafluoride
Results: Median Dheights (mm) after exposure were: TiF4 0.07, SnF2 0.03, HF 0.14 and CA 5.92. TiF4-exposed surfaces showed both deposits and etched areas and exhibited statistically significant different surface roughness parameters compared to the HF- and SnF2exposed surfaces. Median [Ca2+] values (ppm): TiF4 1.88, SnF2 0.11, HF 0.10 and CA 2.17. Conclusion: At the [F] tested in this study it can be concluded that SnF2- and HF solutions had negligible erosive effects on enamel. TiF4 solution resulted in an incomplete surface deposition associated with calcium dissolution suggesting that TiF4 applied as solution may not be advisable. # 2014 Elsevier Ltd. All rights reserved.
1.
Introduction
Dental erosion lesions are caused by the action of extrinsic- or intrinsic acids usually in combination with abrasion and or attrition.1,2 Citric acid is a common extrinsic acid often found in beverages and food. Treatment of enamel with acidic
fluoride solutions such as titanium tetrafluoride (TiF4), stannous fluoride (SnF2) and hydrogen fluoride (HF) has in vitro and in situ shown to provide some protection against dissolution upon exposure to such acids.3–8 It has been suggested that the erosion-inhibiting effects of TiF4 and SnF2 are related to the formation of a coating or a glaze layer that prevents further erosion or due to the formation of
* Corresponding author at: Department of Prosthetic Dentistry and Oral Function, Institute of Clinical Dentistry, University of Oslo, P.O. Box 1109, Blindern, 0317 Oslo, Norway. Tel.: +47 22852368; fax: +47 22852344. E-mail address: [email protected] (C. Hjortsjo¨ ). http://dx.doi.org/10.1016/j.archoralbio.2014.08.007 0003–9969/# 2014 Elsevier Ltd. All rights reserved.
archives of oral biology 59 (2014) 1328–1333
acid-resistant calcium fluoride precipitates on the enamel surface.3,6,9–11 Some authors have reported the coating/ glaze layer to be incomplete or have cracks,6,9 and have attributed this observed lack of a complete glaze coating to microscopy preparation. However, if the formation of an incomplete coating/glaze layer is a true effect, it may influence the protective effect against erosion seen by TiF4 and SnF2. It has also been demonstrated that in vitro solutions containing stannous ions (Sn2+) may lead to the incorporation of Sn in the outer enamel surface.12,13 The higher the amount of incorporated Sn in the enamel, the less enamel tissue was shown to be lost when the surface was exposed to citric acid.12 Acidic fluoride solutions below pH 3 contain fluoride largely in the form of undissociated HF0 molecules, in addition to smaller amounts of free fluoride anions. HF0 has the ability to diffuse into intercrystalline spaces of enamel14 and penetrate the hydroxyapatite lattice in the root-dentine surface.15 Recently it has been suggested that HF0 may result in the formation of a subsurface layer of acid-resistant calcium fluoride-like material.5 However, concerns regarding the potential of acidic fluorides to remove calcium (and phosphate ions) from the enamel surface have been raised. In a study by Skartveit and co-workers where tooth root surfaces were treated with 4% TiF4, a partly demineralised zone with a depth of 8–10 mm was demonstrated after 1 min. After 4 min this zone was 5–27 mm deep.16 Different dental hard tissues react differently to erosive demineralization as demonstrated by Ganss and co-workers.17 Although one cannot assume that the above-mentioned changes seen in root dentine can be directly extrapolated to dental enamel, the changes may serve as a predictor of possible reactions. The aim of this in vitro study was therefore to investigate possible surface changes on human enamel specimens following prolonged exposure to different acidic fluoride solutions (TiF4, SnF2 and HF) using a simplified model not including a salivary pellicle. The null hypothesis to be tested was that these acidic fluoride solutions do not result in any etching of enamel surfaces as measured by profilometric- and spectrometric analyses.
2.
Materials and methods
2.1.
Tooth samples, specimen preparation and isolation
Twenty-four intact, surgically extracted human third molar teeth were collected from private dental practitioners in Oslo and surrounding areas, or from the Department of Oral Surgery and Oral Medicine, Institute of Clinical Dentistry, Faculty of Dentistry, University of Oslo. All tooth donors gave their consent to the use of their teeth for this research project. It was not known whether the teeth crowns (or parts of the crowns) had been exposed to the oral cavity prior to extraction. Teeth were cleaned and stored in a moist thymol environment (99.5% thymol, Sigma–Aldrich, St. Louis, MO, USA). Prior to the experiment, the teeth were prepared using a saw with water cooling (Exakt-apparatgebau, Norderstedt, Germany) and were cut vertically into two halves thus providing 48 enamel specimens. The teeth that were to be
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analysed by profilometry also had their roots removed by use of the same saw with water cooling. All specimens were then glued to a plastic object plate (Technovit 7210 VLC; Heraeus Kulzer GmbH, Wehrheim, Germany). A flat enamel surface was ground wet on each specimen with an abrasive paper to grit 2500 (P2500, silicon carbide grinding paper, Buehler, Du¨sseldorf, Germany) (Exakt-apparatebau, Norderstedt, Germany) until a surface area of approximately 9 mm2 was exposed. The size of this area was dependent on the original size, position and form of each tooth, and approximately 200 mm of the surface enamel was removed during this process. One half of the enamel specimens (n = 24) were prepared for profilometric analysis. Half of the ground enamel surface area of each specimen was isolated using a thin layer of light body polyvinyl siloxane (PVS) impression material (Express II light body, 3M-ESPE, Seefeld, Germany). The border between the isolated and exposed area of enamel was situated at the centre of the ground enamel surface. After approximately 6 min the impression material was set and the specimens were ready for exposure to one of the test solutions. The other half of the specimens (n = 24) were prepared for calcium analysis. Specimens were completely covered with a thin layer of the same light body PVS impression material. After setting a custom-made punching instrument with a circular cutting edge (diameter 2.5 mm) was used to carefully punch a hole through the impression material on each specimen, exposing a 4.9 mm2 circular area at the centre of the ground enamel surface.
2.2.
Test solutions and treatment of the specimens
The following fluoride solutions and citric acid solution were included in the study: TiF4 solution (1.6% w/v, 0.5 M F, pH 1.42) was prepared from titanium tetrafluoride (Sigma–Aldrich Chemie, Steinheim, Germany), SnF2 solution (3.9% w/v, 0.5 M F, pH 2.65) was prepared from tin II fluoride (Sigma–Aldrich Chemie, Steinheim, Germany), and HF solution (0.20% w/v, 0.1 M F, pH 3.02) was prepared from 40% hydrogen fluoride (Rectapur, Prolabo, Paris, France). Citric acid solution (1.8%, pH 2.27) was included as a positive control (Sigma–Aldrich Chemie, Steinheim, Germany). Deionised water was used in the preparation of all solutions. The choice of concentration for the fluoride solutions was based on previous studies reporting the beneficial effects of these fluoride solutions.4,5,18,19 The HF solution had a lower fluoride concentration than the TiF4 and SnF2 solutions since previous in vivo and in vitro studies have demonstrated HF toxicity at higher concentrations.20 The pH of the solutions was measured at room temperature (pH-meter Orion Star, Thermo Electron Corporation, Beverly, MA, USA). Following the isolation procedures, pellicle-free enamel specimens were exposed for 10 min to one of the test solutions (n = 6 specimens/solution) by submerging each specimen in separate plastic containers (Coulter, Kartell spa, Noviglio, Italy) each containing 20 ml of the solutions under constant, gentle agitation (100 rpm, IKA KS 260 Basic, Stuafen, Germany). The large volume of solution was used in order to avoid the possibility of re-precipitation of minerals onto the
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enamel specimens. All specimens were removed from the test solutions after 10 min, and rinsed with deionised water for approximately 5 s.
2.3.
Profilometric analysis
Following exposure to the test solutions and prior to profilometry, excess water was gently removed from all specimens using pressurised air, and the isolating PVS material was removed. The specimens were examined to confirm that the uncovered ground area of enamel had in fact been exposed to the test solution. Analysis was performed using a high power LED blue light profilometer (PLm2300 Optical Imaging Profiler, Sensofar-tech Terrassa, Spain) at 50 magnification and a wavelength of 470 nm. One measurement per sample was performed including both exposed and isolated enamel surfaces for each specimen. If the sample is etched during the F-solution exposure, the exposed surface will have lost height compared to the adjacent isolated surface (Dheight < 0). Conversely, the lack of etching will be shown either by no height difference (Dheight = 0) or possibly even by a net height gain for the F-exposed surfaces (Dheight > 0). All data was stored on the profiler computer until statistical analysis. Tooth surface areas the size of 254.64 190.90 mm2 were analysed for each sample. Depth measurements included 233–239 planes/sample, each of 0.2 mm (total a maximum of 47.6 mm). The data were analysed using Sensomap plus 4.1 software (Sensofar-tech Terrassa, Spain). Following processing, the plane of the image for each sample was first levelled using the levelling function (three points of measurement on the reference area). The step height function was then used to calculate the height differences (Dheight in mm) between the exposed enamel surfaces and isolated enamel surfaces following exposure to the solutions. The following surface parameters were also registered for the exposed and isolated areas: Sa = average roughness, Sp = maximum height (height between the mean level and the highest peak), Sv = maximum depth (depth between the mean level and the deepest valley), St = total height (height between the highest peak and the deepest valley).
2.4.
Atomic absorption spectrometric analysis
Solutions were collected following exposure of the enamel specimen and stored at 4 8C until calcium analyses were performed. An Analyst 400 Atomic Absorption Spectrometer (Perkin Elmer Analytical Instrument, Norwalk, CT, USA) was used for the analyses. Prior to analysis, 0.55 ml of 36% HCl (Rectapur, Prolabo, Paris, France) was added to each of the TiF4 and SnF2 test solution samples. In order to counteract the negative effect of phosphorus on the calcium sensitivity of the spectrometer lanthanum chloride (VWR International, Fontenay sous Bois, France) was added to all test solutions to a final concentration of 1% (10% LaCl3 in 5% HCl stock). Calcium analyses were performed directly after adding HCl and LaCl3.
2.5.
Statistical analysis
The raw data from the spectrometric and profilometric Dheight measurements failed the Shapiro–Wilk normality
test (SigmaPlot version 12.0, Systat software, San Jose´, California, USA). The Kruskal–Wallis test was therefore performed and pairwise multiple comparison procedures (Student Newman–Keuls test) were used to compare differences between groups. The profilometric surface parameters data comparing surface parameters between exposed and isolated areas passed the Shapiro–Wilk normality test, and mean differences were tested using a t-test. All differences were considered significant at the 0.05 level.
3.
Results
The results are presented in Table 1 and Fig. 1A–D. Profilometric analysis demonstrated that exposure of the enamel samples to acidic fluoride solutions resulted in negligible net surface height changes. The citric acid had a clear etching effect as shown by the result for the positive controls. Fig. 1 shows representative profilometric images for enamel specimens exposed to the different solutions as generated by the Sensomap plus 4.1 software. The border between the exposed and isolated areas was clearly visible in all samples. The samples exposed to TiF 4 solution showed a clear surface deposition layer (Fig. 1A), in contrast to the citric acid positive control samples that showed a clearly etched surface (Fig. 1D). The SnF 2 and HF samples showed neglible height differences between exposed and isolated areas, indicating no surface changes (Fig. 1B and C). The surface parameters are presented in Table 1. Surface parameter analysis revealed statistically significant differences between the exposed and the isolated areas for both citric acid and TiF4 solutions. Following exposure to these solutions the surface differences were significant for Sa (indicating greater average surface roughness), Sp (indicating higher peaks on the surface), Sv (indicating deeper valleys) and St (indicating a greater distance between the highest peak and deepest valley). The spectrometric analysis showed that exposure of the enamel specimens to the TiF4- and citric acid solutions clearly resulted in calcium dissolution (Table 1). Exposure of the enamel specimens to the SnF2- and HF solutions resulted in lower levels of calcium release.
4.
Discussion
This in vitro study revealed obvious differences in the interaction of the different acidic fluoride solutions on the bare enamel surfaces. While negligible effects on the enamel surfaces were observed for specimens exposed to the SnF2and HF solutions, the TiF4-exposed specimens showed a combination of both a surface deposition layer and enamel dissolution. Under the present experimental conditions, the null hypothesis was therefore rejected. As expected the profilometric and spectrometric calcium analyses clearly showed that exposure to the citric acid solution resulted in an obvious etching of the exposed surfaces. However, regarding the effect of TiF4, although
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Table 1 – Median values (range). Dheight = profilometric height difference between exposed and isolated surfaces. Surface parameters of the specimens exposed to the solutions (isolated surfaces in brackets). Sa = average roughness, Sp = maximum height (height between the mean level and the highest peak), Sv = maximum depth (height between the mean level and the deepest valley), St = total height (height between the highest peak and the deepest valley). Test solution
Spectrometric analysis 2+
TiF4 SnF2 HF Citric acid
z
Profilometric analysis z
Median [Ca ] (ppm)
Median Dheight (mm)
1.88a (1.10–1.96) 0.11b (0.10–0.35) 0.10b (0.05–0.13) 2.17c (2.03–2.66)
0.07d (2.25 to 1.61) 0.03d (0.35 to 0.18) 0.14d (0.17 to 0.03) 5.92e (7.46 to 5.20)
Sa (mm) 1.20 0.57 0.67 0.84
(0.76) * (0.55) (0.65) (0.54) *
Sp (mm) 10.50 2.28 1.52 3.00
(2.37) * (2.89) (1.46) (1.47) *
Sv (mm) 4.14 2.59 1.63 4.14
(1.95) * (1.59) (1.58) (1.41) *
St (mm) 14.64 4.87 3.15 7.14
(4.32) * (4.47) (3.03) (2.87) *
*
T-test shows a significant difference between exposed and isolated areas. All differences were considered significant at the 0.05 level. Within columns: [Ca2+] and Dheight values sharing the same superscript letter were not significantly different from each other (Student Newman–Keuls test). z
Fig. 1 – Profilometric images of representative enamel specimens exposed to the different solutions. For each image, the area to the left of the ‘borderline’ is the enamel surface that was exposed to the solutions, and the area to the right is the isolated enamel surface. (A–D) are specimens immediately post exposure: (A) TiF4 specimen showing an obvious, but incomplete surface deposition on the exposed surface, (B) SnF2 specimen showing no obvious surface deposition, and some slight signs of etching along the borderline, (C) HF specimen showing a slight tendency to etching on the exposed surface, and with some deposition along the borderline, (D) citric acid specimen (CA) showing a typical enamel etching pattern on the exposed surface. All images are 50T magnification.
profilometric height analyses indicated a height difference close to zero between TiF4-exposed and non-TiF4-exposed surfaces, further analysis of the surface parameters revealed large differences between the highest ‘peaks’ and lowest
‘valleys’ on the TiF4-exposed surfaces. An interpretation of this result suggests the presence of an incomplete surface deposition layer as well as the presence of etched areas. Standard profilometric height analysis alone was therefore
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not sufficient to detect possible important changes occurring on the enamel surface. The formation of a superficial layer following treatment of the dental enamel with fluorides has been reported previously and is often described as a ‘glaze’.6,9,21 The continuity of the glaze layer will without doubt depend very much upon the conditions under which the layer is formed. The present finding that the TiF4 solution resulted in greater dissolution of calcium than was observed for the SnF2- and HF-solutions supports the profilometry results, as well as previous findings that the TiF4-initiated glaze layer is not necessarily 100% complete.6,9 Furthermore, those parts of the enamel surface that were not covered by the glaze were susceptible to dissolution. While the erosive effect seen for the TiF4 solution may be partly explained by the low pH of the native solution (pH 1.42), the erosive effect seen by the citric acid can be explained by the combination of low pH (pH 2.27) and chelator properties. Given that pH, concentration and application method (solution, gel, toothpaste) are important factors in the reaction of fluoride solutions with the enamel, other concentrations and application methods of TiF4 may give rise to a more complete glaze than was observed in the present study. Recently it was demonstrated that 4% TiF4 varnish reduced enamel dissolution due to acidic beverages far more effectively than 4% TiF4 solutions 22,23. The authors suggested that the superior protective effect of the TiF4 varnish is due to its ability to adhere to the tooth surface and through this increase the reaction time between enamel and TiF4 varnish. Although these findings cannot be extrapolated to the in vivo situation, the results reflect different chemical compositions of the surface layers formed. As no significant changes were recorded in surface parameters for the specimens exposed to the SnF2 and HF solutions, one can speculate that the changes in the enamel specimens that occur after treatment with these solutions are different from those following exposure to TiF4 solution. Previous reports have shown that pretreatment of enamel with HF reduced calcium dissolution after citric acid exposure.4,5,24,25 It has been speculated that undissociated HF-molecules are able to cause deposition of acid resistant calcium fluoride-like minerals when exposed to enamel at pH 3. The HF-molecules probably dissociate under such conditions causing the released H ions to dissolve the enamel by reduction of phosphate molecules to soluble di-hydrogen phosphates (H2PO4). The di-hydrogen phosphates are subsequently lost from the enamel surface, and released F ions react with newly available calcium ions to form calcium fluoride-like material. This CaF-like material is thought to be more acid resistant than the CaF-material formed under neutral conditions, since it is formed under acidic conditions.25 At the borderline between the areas of isolated and exposed enamel a slight surface deposition was also observed for the HF-treated area (Fig. 1C). This borderline area including some form of deposits was excluded in the profilometric analysis and therefore did not affect the height recordings. In contrast, enamel samples treated with citric acid exhibited increased etching in the borderline area (Fig. 1D). A possible explanation for this phenomenon is that the level of exposure of the enamel to the acidic solutions was greater in this area due to pooling of the solutions in contact with the isolating PVS material. These findings are supported
by similar previous experiments on enamel samples using white light interferometry, which showed that the surfaces within 200 mm of an amalgam reference marker were excessively etched by both TiF4- and SnF2 solutions.6 In the present study different specimen isolation procedures were used for the profilometric- and spectrometric analyses. For the profilometric analysis it was desirable to have approximately equal-sized surface areas of exposed and isolated enamel. This was achieved for each specimen by isolating half the enamel surface area along a straight line using PVS impression material. For the spectrometric analysis it was necessary to have well-defined and equally-sized enamel surface areas on the specimens in order to permit a comparison between [Ca] of the different fluoride solutions and the control. This was achieved by first covering the entire enamel surface with the PVS impression material and then punching out a circular window through the impression material to expose a standardised test surface area. In both procedures approximately the same area of enamel was exposed for the profilometric analysis as for the AAS analysis (profilometry: approx. 4.5 mm2 = half the area of 9 mm2 and AAS: 4.9 mm2). This allowed the results from the two different methodological procedures to be compared. The choice of concentration for the fluoride solutions was based on previous studies reporting their respective beneficial effects.4,5,18,19,25 In order to avoid participation of other ions during spectrometric analyses, HCl was added to the TiF4- and SnF2 solutions prior to the addition of lanthanum chloride and before calcium analysis. Using two slightly different sample preparation techniques for the spectrometry analysis may not be optimal, however adding HCl does not affect the amount of calcium in the solution thus allowing comparison of the results from the different test solutions. Salivary pre-conditioning of the enamel surfaces in order to form a pellicle was not included in the protocol and the exposure time of the enamel specimens to the acidic fluoride solutions was relatively long. These study conditions therefore presented a ‘worst case scenario’. Further studies using other concentrations and exposure times on specimens that are preconditioned with saliva are needed to determine whether these differences are also likely to be seen in a clinical setting. In conclusion, this in vitro study demonstrated clear differences in how the acidic fluoride solutions reacted with pellicle-free enamel. Even under the extreme conditions of this study, relatively long exposure of enamel specimens to the SnF2- and HF solutions resulted in only minimal erosive effects. Exposure to the TiF4 solution resulted in a surface deposition on the enamel specimens that could protect the enamel. However, the ‘glaze’ was incomplete and was associated with a significantly greater loss of calcium. TiF4 in solution may therefore not be the best choice of acidic fluoride if this effect also occurs in vivo where a range of different factors will influence the nature of fluoride interaction with enamel surfaces.
Funding University grants.
archives of oral biology 59 (2014) 1328–1333
Competing interests None declared.
Ethical approval Not required.
references
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