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Effect of fluoride gels supplemented with sodium trimetaphosphate on enamel erosion and abrasion: In vitro study
archives of oral biology 59 (2014) 336–340
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/aob
Short communication
Effect of fluoride gels supplemented with sodium trimetaphosphate on enamel erosion and abrasion: In vitro study L.P. Pancote, M.M. Manarelli, M. Danelon, A.C.B. Delbem * Faculdade de Odontologia, UNESP – Univ Estadual Paulista, Arac¸atuba, Sa˜o Paulo, Brazil
article info
abstract
Article history:
Objective: This in vitro study aims to evaluate the effect of low fluoride (F) gel associate
Accepted 20 December 2013
sodium trimetaphosphate (TMP) on erosion with or without abrasion. Design: Enamel blocks (4 mm 4 mm) selected through surface hardness (SH) is divided
Keywords:
into five groups (n = 12): gel without F and TMP (placebo), gel containing 4500 ppm F (4500),
Fluoride gels
gel containing 4500 ppm F plus TMP5% (4500 TMP5%), gel containing 9000 ppm F (9000), and
Tooth erosion
gel containing 12,300 ppm F (acid gel). Those groups were additionally subdivided into
Tooth abrasion
conditions of erosion (Ero) and of erosion plus abrasion (Ero/Abra). The blocks have under-
Topical fluorides
gone a single application of gel on the first day of the study. The erosion challenge was
Dental enamel
produced by Sprite Zero1 for five minutes four times a day and abrasion was carried out by
Polyphosphates
machine brushing for 15 s. After the challenges, the surface hardness (%SH), wear and crosssectional hardness (DKHN) were analyzed. The data were analyzed using a 2-way ANOVA test followed by a Student-Newman–Keuls ( p < 0.05). Results: Lower values of %SH, wear and DKHN were observed for erosion challenge ( p < 0.001). The %SH was lower in groups treated with fluoride gels, differing in the placebo ( p < 0.05). With addition of TMP to the gel 4500, enamel wear was lower when compared with another groups ( p < 0.05). Conclusion: In vitro conditions, the 4500 5%TMP gel showed greatest effect against erosion and erosion/abrasion. # 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
Erosion and erosive tooth wear refers to the chemical and chemical-mechanical process that has become more prevalent in 11 to 16-year-old children.1,2 As dental caries disease, the tooth erosion is a multifactorial condition (patient-related and nutritional factors) and over time, the interaction of all these factors may lead to either
progression.3 Different methods have been indicated to prevent or slow the progression of dental erosion such as the use of topical fluorides.4 Fluoride therapy has been suggested as a preventive measure against tooth erosion, and its effects are reported to be higher when applied at high concentrations.5–10 Despite the gel be a more affordable vehicle (lower cost) with high concentrations of fluoride, there are few studies where compounds are added to improve its effect against erosion.11
* Corresponding author at: Arac¸atuba Dental School, Sa˜o Paulo State University (UNESP), Department of Pediatric Dentistry and Public Health, Rua Jose Bonifacio 1193, 16015-050 Arac¸atuba, SP, Brazil. Tel.: +55 18 3636 3314; fax: +55 015 18 3636 3332. E-mail addresses: [email protected], [email protected] (A.C.B. Delbem). 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.12.007
archives of oral biology 59 (2014) 336–340
Utilisation of sodium trimetaphosphate (TMP) and fluoride (F) association has shown decreased enamel demineralisation10,12–15 in caries and dental erosion. In addition, a low fluoride topic gel (4500 ppm F) associated to TMP presented same ability to produce enamel remineralization than the fluoride acid gel (12,300 ppm F).16 However, the erosive challenge is a process that occurs at a pH of <4.0 and the pH of the dissolution of calcium fluoride, fluorapatite or any precipitate that might be formed in the presence of fluoride is exceeded during the erosive challenge. Considering the good results against dental caries and which differs from erosion process, the present in vitro study evaluated the effect of low-F gels (4500 mg F/g) plus TMP on the erosion, associated or not with abrasion, of tooth enamel.
337
water and transferred to a volumetric flask. The volume was then adjusted to 100 mL using deionized water. For each product, 3 dilutions were made. Subsequently, 2 samples of 1 mL were buffered with total ionic strength adjustment buffer II (TISAB II).
2.3.
Preparation of enamel blocks
2.
Materials and methods
Enamel blocks (n = 120, 4 mm 4 mm) were obtained from bovine incisors and polished to remove around of 200 mm of the enamel surface.12 Surface hardness (SH) was determined by performing 5 impressions in the central region of the blocks surface (Knoop diamond, 25 g, 10 s; Buehler, Lake Bluff, USA). Blocks with mean hardness between 330.0 and 370.0 kgf/mm2 were selected. To maintain a reference surface for determining the wear of enamel by profilometry, half of the surface of each block was protected with nail varnish.
2.1.
Experimental design
2.4.
Bovine enamel blocks (n = 120) were selected through analysis of surface hardness (SH) and randomly divided into 5 groups, according to the following treatments: (a) gel without F and TMP (placebo), (b) gel containing 4500 mg F/g (4500), (c) gel containing 4500 mg F/g + TMP5% (4500 TMP5%), (d) gel containing 9000 mg F/g (9000), and (e) gel containing 12,300 mg F/g (Acid gel). Based on a previous study with similar methodology12 and considering an a-error of 5% and a b-error of 20%, twelve enamel blocks were determined for each experimental group. Enamel blocks were protected in their half with nail varnish (control area), so that half of their surface was exposed to the treatment with the gels and to the erosive (Ero) or to the erosive/abrasive (Ero+Abra) challenges. Ero was produced in all blocks by immersion in soda (Sprite Zero, pH 2.8, 4 times/ day, 5 min each time), while ERO+ABR was done in half of the blocks by brushing after each erosive challenge. The protocol was tested for 3 days. Enamel blocks were analyzed by profilometry and cross-sectional hardness. The factors studied were: type of gel (5 types) and type of challenges (Ero and Ero+Abra).
2.2. Gel formulation and determination of fluoride in products Experimental gel of neutral pH was prepared in a laboratory and had the following ingredients: carboxymethylcellulose, sodium saccharin, glycerol, peppermint oil, and water. The fluoride (NaF; Merck, Darmstadt, Germany) was added to the gel in a concentration of 0, 4500, or 9000 mg F/g. Subsequently, TMP (Sigma–Aldrich Co., St. Louis, MO, USA) was added at a concentration of 5% to gels with F concentration of 0 and 4500 ppm F. A commercial acidic gel was used as a positive control (12,300 mg F/g, Acid gel, pH = 4.5, DFL; Indu´stria e Come´rcio S.A, Rio de Janeiro, RJ, Brazil). The F concentration in the gels was determined using a specific electrode for the F ion (9609 BN; Orion Research Inc., Beverly, MA, USA) attached to an ion analyzer (Orion 720 Aplus; Orion Research Inc., Beverly, MA, USA) and calibrated with standards containing 0.125–2.000 mg F/g. Approximately 100 mg of each product was dissolved in deionized
Experimental protocol
A thin coat of gel was applied on the exposed area of enamel blocks using a microbrush. Each block was subjected to 1 min of treatment in 3 g of gel and removed with deionized water. Ero was performed every 2 h, by dipping the enamel blocks in Sprite Zero (Companhia de Bebidas Ipiranga, Ribeira˜o Preto, Brazil), pH 2.8, 4 times/day, during 5 min each time.17 Ero+Abra was performed on half of the blocks by using a mechanical brushing machine (250 g axial load, 5 strokes/s; Elquip Maq Escovac¸a˜o, Sa˜o Carlos, Brazil) immediately after the erosive challenges (4 times/day). Brushing was performed for 15 s each time, using a placebo dentifrice slurry (1:3, weight:weight). The other half of the blocks (Ero only) was immersed in the placebo dentifrice slurry for 15 s after the erosive challenges.
2.5.
Determination of surface wear
The nail varnish on the reference surfaces was removed carefully with acetone-soaked cotton wool. Enamel loss was determined in relation to the reference surfaces by profilometry (Surftest SJ 401 – Mitutoyo American Corporation), by scanning the surface of each block from the reference surfaces (control) across the exposed surfaces. The mean value of 5 readings was calculated for each block.
2.6.
Analysis of cross-sectional hardness
Blocks were sectioned at the center, and half of each block was included in acrylic resin and subsequently polished. Crosssectional hardness (SH) was determined (Knoop diamond, 5 g, 10 s, Buehler, Lake Bluff, USA). A sequence of eight prints at distances of 10, 15, 20, 25, 30, 40, 50 and 70 mm from the external surface of the enamel was performed in the center of blocks, for both the control and the test areas. The integrated area of hardness (KHN mm) of the demineralized and sound enamel was calculated using the trapezoidal rule (GraphPad Prism, version 3.02) and subtracted from the integrated area of the hardness of sound enamel loss resulting integrated hardness (DKHN).10
338
2.7.
archives of oral biology 59 (2014) 336–340
Statistical analysis
For the statistical analysis, GMC software, Campos, 200318 was used and the significance limit was set at 5%. Data (log transformed) passed normality (Kolmogorov– Smirnov) and homoscedasticity (Cochran) tests, and were submitted to 2-way ANOVA, considering the type of gel (5 formulations) and challenge (Ero and Ero+Abra) as fixed factors. Two-way ANOVA was used to analyze the data of hardness as a function of depth. Pearson’s correlation test was used to evaluate the relationship between DKHN and wear.
3.
Results
The mean (SD) of fluoride concentration (ppm F) in the placebo, 4500, 4500 TMP5%, 9000 and Acid gel groups was 114.7 (5.4), 4509.8 (65.2), 4583.3 (164.4), 9467.7 (76.2) and 11130.4 (239.9), respectively.
The Placebo group showed a higher softening (%SH) from the other groups ( p < 0.001) after the erosive challenges (Table 1). The erosion promoted higher enamel surface softening than Ero+Abra ( p < 0.001). Lower softening was observed in enamel treated with Acid gel ( p < 0.001). The gel with 4500 ppm F and 4500 ppm F plus TMP presented similar enamel softening ( p > 0.610). Lower values of wear were observed with the erosive than Ero+Abra challenge ( p < 0.001). With the addition of TMP to gel 4500, enamel wear was lower when compared to the other groups ( p < 0.001), both in the Ero and Ero+Abra (Table 1). The fluoride groups (4500, 9000 and Acid gel) presented similar enamel wear ( p > 0.608). Placebo group exhibited the highest mineral loss deep into enamel (DKHN) when compared with the other groups ( p < 0.001) (Table 1). The gel with TMP showed similar values of DKHN than Acid gel group ( p > 0.051), regardless of the challenge types. Fig. 1 shows the hardness profile of erosion and erosion/abrasion as a function of depth according to the groups and the others comparisons are shown.
Table 1 – Mean (SD) values of surface hardness (%SH), wear and DKHN of the enamel according to conditions of challenge (Ero and Ero+Abra) and groups. Analysis
Groups Placebo
%SH
Wear (mm)
DKHN
Ero Ero+Abra
A B
4500 A B
4.71a (0.80) 5.81a (0.47)
Ero Ero+Abra Ero Ero+Abra
91.2a (1.3) 84.8a (1.6)
83.6b (1.6) 79.0b (1.4)
A
A
B
B
A
A
B
3609.6a (139.6) 2240.1a (129.9)
B
9000 A B
4.19b (0.70) 5.15b (0.35)
2712.9b (81.0) 1749.3b (158.2)
A B
77.9c (1.6) 74.1c (2.3)
4500 5%TMP A B
3.95b (0.69) 5.22b (0.34)
83.7b (2.2) 78.6b (1.6)
Acid gel A B
A
A
A
B
B
B
2320.3c (122.9) 1466.1c (128.2)
2.95c (0.51) 3.78c (0.36)
74.0d (1.5) 68.3d (1.4)
A
A
B
2215.5d (64.5) 1161.6d (102.0)
B
4.11b (0.91) 5.33a,b (0.57)
2321.5c,d (78.5) 1253.4d (100.5)
Different letters show significant differences in each analysis between groups of gel (ANOVA 2-way, Student–Newman–Keuls’s test; p < 0.05).
Fig. 1 – Graphical representation of mean hardness as a function of depth according to the erosive challenges. Symbols indicates comparison among groups in each depth (Student–Newman–Keuls, p < 0.05). Vertical bars indicate the standard deviations of means. (*) indicates no statistical difference among groups. (a) g: indicates equality between the groups healthy, 9000, 4500 5%TMP and Acid gel ( p > 0.163). v: indicates difference among group ( p = 0.037). b: indicates equality between the groups healthy, 4500, 9000 and Acid gel ( p > 0.116), 4500 5%TMP and Acid gel ( p = 0.070). l: indicates equality between the groups healthy, 4500, 9000, 4500 5%TMP and Acid gel ( p > 0.478). &: indicates difference between Placebo and the other groups ( p < 0.001). (b) V: indicates equality between the groups healthy, 4500 5%TMP and Acid gel ( p = 0.286). C: indicates equality between the groups healthy, 4500 5%TMP and Acid gel ( p = 0.248). ¥: indicates that the healthy groups, 4500 5%TMP and Acid gel are similar statistically ( p = 0.339). §: indicates equality between the groups healthy, Placebo and 4500 ( p = 0.158), 4500 and 9000 ( p = 0.074), 9000 and Acid gel ( p = 0.189).
archives of oral biology 59 (2014) 336–340
There was no correlation between wear (Ero and Ero+Abra) and surface hardness. (Pearson’s r = 0.072; p = 0.587 and Pearson’s r = 0.067; p = 0.611). Positive correlation was noted between wear (Ero and Ero+Abra) and DKHN (Pearson’s r = 0.532; p < 0.001 and Pearson’s r = 0.703; p < 0.001).
4.
Discussion
As the erosive challenge is a process that occurs at a pH of <4.0, the pH of the dissolution of calcium fluoride that might be formed in the presence of fluoride is exceeded during the erosive challenge.19,20 It knows that the effect of professionally applied topical fluoride is based on CaF2 deposits on enamel.21 This means a limit effect from topical fluoride products and to enhance its action the addition of new anti-erosive compounds can be necessary.4 In the present study was tested a TMP supplemented gel with reduced F concentration that showed a greater capacity to promote enamel remineralization.16 However this is a chemical model and the data obtained using this model (or any other in vitro protocol) should be considered carefully due to limitations in reproducing the oral environment with all of the biological variations known to influence erosion.22 The presence of saliva and acquired pellicle can influence the adsorption of TMP on enamel and the erosive wear. However, in a recent in vitro study (unpublished data) the presence of the acquired pellicle leads to lower erosive wear but the effect of TMP and fluoride agents is maintained with or without pellicle. Nonetheless, the present study provides interesting data that may be used in more efficient product development in the future by using other compounds than fluoride. As the blocks were kept stored in artificial saliva after the last erosive/treatment process would be more correct to state that the hardness testing (superficial and in depth) analyzed the remineralizing ability of the formulations. In the present study, it was possible observed a eroded enamel less softened with rising fluoride concentrations in the topical gels (Erosion or Erosion/Abrasion). Despite surface hardness not to be related to wear the treatment with fluoride have a remineralizing action in the remaining enamel.23–25 This remineralization is related with CaF2 deposits on enamel.4,16 Thus, the greater calcium fluoride deposition observed in the study of Danelon et al.,16 explain the better results (%SH) with acidulated fluoride treatment (Acid gel). Nevertheless, the acidulated topic gel show a few effect against the enamel wear. It means the ability of calcium fluoride deposition from fluoride product is a few related to the enamel wear, however; it is related to a greater remineralization at the outer part of the enamel demineralization. The addition of TMP in the fluoride gel gave a superior results and it was not related to calcium fluoride deposition on enamel. As observed in a recent study that utilized a topical gel with 4500 ppm F, the TMP did not influence the adsorption of calcium fluoride and neither enhance it or reduce its precipitation.16,26 Based in previously studies, TMP interacts with the enamel, which may produce a protective layer on the enamel surface thereby hindering acid diffusion.14,27 TMP is a cyclic phosphate and although it is soluble in water, it seems not to be spontaneously hydrolyzed.13 The interaction of Ca2+
339
from artificial saliva and CaF+ with TMP (crosslinking) leads the reticular formation on enamel by the binding sites on the negative PO4 in the cyclic structure. At acidic pH these linkages are ruptured releasing Ca2+ and CaF+ which can react with H2PO4 leading to the formation of CaHPO40 and HF0.28 The diffusion of neutral ions into the enamel is thousand times higher than charged ions enhancing the remineralization process.28 With the reticular formation broken probably TMP retains its structure H+ reducing the acid diffusion into the enamel decreasing enamel demineralization. Thus, the major effect of TMP during the erosive challenge is to prevent acid diffusion into the enamel. The data from cross-sectional hardness analysis showed that the addition of TMP improved the remineralization at the inner part of the enamel in the same degree when compared with Acid gel. As these data are based in an in vitro protocol, additional in situ and clinical studies must be conducted to confirm these results. According to the results of this study, we can conclude that the addition of TMP to the fluoride gel showed a higher effect against erosion and erosion/abrasion of the enamel.
Conflict of interests The corresponding author has filed an application for patenting a product used in the study at the National Institute of Industrial Property (INPI/SP) on 04/29/2008 under the number 018080026091, PI0801811-1 and published on January 11, 2011. The other authors have no financial or personal conflicts of interest in relation to this study. All authors approved the publication of the manuscript.
Funding The study was supported by CNPq/PIBIC (ID: 21613), a Brazilian Agency for Research.
Ethical approval There was no need to subnet the study to the Committee of Ethics in Research. This manuscript is an in vitro study.
references
1. Shellis RP, Ganss C, Ren Y, Zero DT, Lussi A. Methodology and models in erosion research: discussion and conclusions. Caries Res 2011;45(1):69–77. 2. Huysmans MC, Chew HP, Ellwood RP. Clinical studies of dental erosion and erosive wear. Caries Res 2011;45(1): 60–8. 3. Lussi A, Jaeggi T. Erosion – diagnosis and risk factors. Clin Oral Investig 2008;12(1):5–13. 4. Magalha˜es AC, Wiegand A, Rios D, Hono´rio HM, Buzalaf MAR. Insights into preventive measures for dental erosion. J Appl Oral Sci 2009;17(2):75–86. 5. Sorvari R, Meurman JH, Alakuijala P, Frank RM. Effect of fluoride varnish and solution on enamel erosion in vitro. Caries Res 1994;28(4):227–32.
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archives of oral biology 59 (2014) 336–340
6. Ganss C, Klimek J, Brune V, Schumann A. Effects of two fluoridation measures in erosion progression on enamel and dentine in situ. Caries Res 2004;38(6):561–6. 7. Saunders JGS, McIntyre JM. The ability of 1.23% acidulated phosphate fluoride gel to inhibit simulated endogenous erosion in tooth roots. Aust Dent J 2005;50(4):263–6. 8. Lagerweij MD, Buchalla W, Kohnke S, Becker K, Lennon AM. Prevention of erosion and abrasion by a high fluoride concentration gel applied at high frequencies. Caries Res 2006;40(2):148–53. 9. Magalha˜es AC, Comar LP, Rios D, Delbem ACB, Buzalaf MAR. Effect of a 4% titanium tetrafluoride (TiF4) varnish on demineralization and remineralization of bovine enamel in vitro. J Dent 2008;36(2):158–62. 10. Manarelli MM, Moretto MJ, Sassaki KT, Martinhon CCR, Pessan JP, Delbem AC. Effect of fluoride varnish supplemented with sodium trimetaphosphate on enamel erosion and abrasion. In vitro study. Am J Dent 2013;26(6):307–12. 11. Bueno MG, Marsicano JA, Sales-Peres SHC. Preventive effect of iron gel with or without fluoride on bovine enamel erosion in vitro. Aust Dent J 2010;55(2):177–80. 12. Moretto MJ, Magalha˜es AC, Sassaki KT, Delbem AC, Martinhon CC. Effect of different fluoride concentrations of experimental dentifrices on enamel erosion and abrasion. Caries Res 2010;44(2):135–40. 13. Takeshita EM, Castro LP, Sassaki KT, Delbem ACB. In vitro evaluation of dentifrice with low fluoride content supplemented with trimetaphosphate. Caries Res 2009;43(1):50–6. 14. Takeshita EM, Exterkate RAM, Delbem AC, ten Cate JM. Evaluation of different fluoride concentrations supplemented with trimetaphosphate on enamel de- and remineralization in vitro. Caries Res 2011;45(5):494–7. 15. Manarelli MM, Vieira AEM, Matheus AA, Sassaki KT, Delbem ACB. Effect of mouth rinses with fluoride and trimetaphosphate on enamel erosion: an in vitro study. Caries Res 2011;45(6):506–9. 16. Danelon M, Takeshita EM, Sassaki KT, Delbem ACB. In situ evaluation of a low fluoride concentration gel with sodium trimetaphosphate in enamel remineralization. Am J Dent 2013;26(1):15–20.
17. Attin T, Knofel S, Buchalla W, Tutuncu R. In situ evaluation of different remineralization periods to decrease brushing abrasion of demineralized enamel. Caries Res 2001;35(3):216– 22. 18. Campos GM: GMC 2003 (computer program). Ribeirao Preto: School of Dentistry; 2003http://www.forp.usp.br/ restauradora/gmc/gmc.htm#gmc. 19. Fushida CE, Cury JA. In situ evaluation of enamel-dentin erosion by beverage and recovery by saliva. Rev Odontol Univ Sa˜o Paulo 1999;12(2):127–34. 20. Barbour ME, Lussi A, Shellis RP. Screening and prediction of erosive potential. Caries Res 2011;45(1):24–32. 21. Delbem AC, Danelon M, Sassaki KT, Vieira AE, Takeshita EM, Brighenti FL, et al. Effect of rinsing with water immediately after neutral gel and foam fluoride topical application on enamel remineralization: an in situ study. Arch Oral Biol 2010;55(11):913–8. 22. West NX, Davies M, Amaechi BT. In vitro and in situ erosion models for evaluating tooth substance loss. Caries Res 2011;45(1):43–52. 23. Wiegand A, Attin T. Design of erosion/abrasion studies – insights and rational concepts. Caries Res 2011;45(1): 53–9. 24. Magalha˜es AC, Rios D, Delbem ACB, Buzalaf MAR, Machado MAAM. Influence of fluoride dentifrice on brushing abrasion of eroded human enamel: an in situ/ex vivo study. Caries Res 2007;41(1):77–9. 25. 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. 26. Souza JAS, Amaral JG, Moraes JCS, Sassaki KT, Delbem ACB. In vitro effect of sodium trimetaphosphate on hydroxyapatite solubility. Braz Dent J 2013;2:4. http:// dx.doi.org/10.1590/0103-6440201302000. 27. Van Dijk JW, Borggreven JM, Driessens FC. The effect of some phosphates and a phosphonate on the electrochemical properties of bovine enamel. Arch Oral Biol 1980;25(8–9):591–5. 28. Cochrane HJ, Saranathan S, Cai F, Cross KJ, Reynolds EC. Enamel subsurface lesion remineralisation with casein phosphopeptide stabilised solutions of calcium, phosphate and fluoride. Caries Res 2008;42(2):88–97.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/aob
Short communication
Effect of fluoride gels supplemented with sodium trimetaphosphate on enamel erosion and abrasion: In vitro study L.P. Pancote, M.M. Manarelli, M. Danelon, A.C.B. Delbem * Faculdade de Odontologia, UNESP – Univ Estadual Paulista, Arac¸atuba, Sa˜o Paulo, Brazil
article info
abstract
Article history:
Objective: This in vitro study aims to evaluate the effect of low fluoride (F) gel associate
Accepted 20 December 2013
sodium trimetaphosphate (TMP) on erosion with or without abrasion. Design: Enamel blocks (4 mm 4 mm) selected through surface hardness (SH) is divided
Keywords:
into five groups (n = 12): gel without F and TMP (placebo), gel containing 4500 ppm F (4500),
Fluoride gels
gel containing 4500 ppm F plus TMP5% (4500 TMP5%), gel containing 9000 ppm F (9000), and
Tooth erosion
gel containing 12,300 ppm F (acid gel). Those groups were additionally subdivided into
Tooth abrasion
conditions of erosion (Ero) and of erosion plus abrasion (Ero/Abra). The blocks have under-
Topical fluorides
gone a single application of gel on the first day of the study. The erosion challenge was
Dental enamel
produced by Sprite Zero1 for five minutes four times a day and abrasion was carried out by
Polyphosphates
machine brushing for 15 s. After the challenges, the surface hardness (%SH), wear and crosssectional hardness (DKHN) were analyzed. The data were analyzed using a 2-way ANOVA test followed by a Student-Newman–Keuls ( p < 0.05). Results: Lower values of %SH, wear and DKHN were observed for erosion challenge ( p < 0.001). The %SH was lower in groups treated with fluoride gels, differing in the placebo ( p < 0.05). With addition of TMP to the gel 4500, enamel wear was lower when compared with another groups ( p < 0.05). Conclusion: In vitro conditions, the 4500 5%TMP gel showed greatest effect against erosion and erosion/abrasion. # 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
Erosion and erosive tooth wear refers to the chemical and chemical-mechanical process that has become more prevalent in 11 to 16-year-old children.1,2 As dental caries disease, the tooth erosion is a multifactorial condition (patient-related and nutritional factors) and over time, the interaction of all these factors may lead to either
progression.3 Different methods have been indicated to prevent or slow the progression of dental erosion such as the use of topical fluorides.4 Fluoride therapy has been suggested as a preventive measure against tooth erosion, and its effects are reported to be higher when applied at high concentrations.5–10 Despite the gel be a more affordable vehicle (lower cost) with high concentrations of fluoride, there are few studies where compounds are added to improve its effect against erosion.11
* Corresponding author at: Arac¸atuba Dental School, Sa˜o Paulo State University (UNESP), Department of Pediatric Dentistry and Public Health, Rua Jose Bonifacio 1193, 16015-050 Arac¸atuba, SP, Brazil. Tel.: +55 18 3636 3314; fax: +55 015 18 3636 3332. E-mail addresses: [email protected], [email protected] (A.C.B. Delbem). 0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.archoralbio.2013.12.007
archives of oral biology 59 (2014) 336–340
Utilisation of sodium trimetaphosphate (TMP) and fluoride (F) association has shown decreased enamel demineralisation10,12–15 in caries and dental erosion. In addition, a low fluoride topic gel (4500 ppm F) associated to TMP presented same ability to produce enamel remineralization than the fluoride acid gel (12,300 ppm F).16 However, the erosive challenge is a process that occurs at a pH of <4.0 and the pH of the dissolution of calcium fluoride, fluorapatite or any precipitate that might be formed in the presence of fluoride is exceeded during the erosive challenge. Considering the good results against dental caries and which differs from erosion process, the present in vitro study evaluated the effect of low-F gels (4500 mg F/g) plus TMP on the erosion, associated or not with abrasion, of tooth enamel.
337
water and transferred to a volumetric flask. The volume was then adjusted to 100 mL using deionized water. For each product, 3 dilutions were made. Subsequently, 2 samples of 1 mL were buffered with total ionic strength adjustment buffer II (TISAB II).
2.3.
Preparation of enamel blocks
2.
Materials and methods
Enamel blocks (n = 120, 4 mm 4 mm) were obtained from bovine incisors and polished to remove around of 200 mm of the enamel surface.12 Surface hardness (SH) was determined by performing 5 impressions in the central region of the blocks surface (Knoop diamond, 25 g, 10 s; Buehler, Lake Bluff, USA). Blocks with mean hardness between 330.0 and 370.0 kgf/mm2 were selected. To maintain a reference surface for determining the wear of enamel by profilometry, half of the surface of each block was protected with nail varnish.
2.1.
Experimental design
2.4.
Bovine enamel blocks (n = 120) were selected through analysis of surface hardness (SH) and randomly divided into 5 groups, according to the following treatments: (a) gel without F and TMP (placebo), (b) gel containing 4500 mg F/g (4500), (c) gel containing 4500 mg F/g + TMP5% (4500 TMP5%), (d) gel containing 9000 mg F/g (9000), and (e) gel containing 12,300 mg F/g (Acid gel). Based on a previous study with similar methodology12 and considering an a-error of 5% and a b-error of 20%, twelve enamel blocks were determined for each experimental group. Enamel blocks were protected in their half with nail varnish (control area), so that half of their surface was exposed to the treatment with the gels and to the erosive (Ero) or to the erosive/abrasive (Ero+Abra) challenges. Ero was produced in all blocks by immersion in soda (Sprite Zero, pH 2.8, 4 times/ day, 5 min each time), while ERO+ABR was done in half of the blocks by brushing after each erosive challenge. The protocol was tested for 3 days. Enamel blocks were analyzed by profilometry and cross-sectional hardness. The factors studied were: type of gel (5 types) and type of challenges (Ero and Ero+Abra).
2.2. Gel formulation and determination of fluoride in products Experimental gel of neutral pH was prepared in a laboratory and had the following ingredients: carboxymethylcellulose, sodium saccharin, glycerol, peppermint oil, and water. The fluoride (NaF; Merck, Darmstadt, Germany) was added to the gel in a concentration of 0, 4500, or 9000 mg F/g. Subsequently, TMP (Sigma–Aldrich Co., St. Louis, MO, USA) was added at a concentration of 5% to gels with F concentration of 0 and 4500 ppm F. A commercial acidic gel was used as a positive control (12,300 mg F/g, Acid gel, pH = 4.5, DFL; Indu´stria e Come´rcio S.A, Rio de Janeiro, RJ, Brazil). The F concentration in the gels was determined using a specific electrode for the F ion (9609 BN; Orion Research Inc., Beverly, MA, USA) attached to an ion analyzer (Orion 720 Aplus; Orion Research Inc., Beverly, MA, USA) and calibrated with standards containing 0.125–2.000 mg F/g. Approximately 100 mg of each product was dissolved in deionized
Experimental protocol
A thin coat of gel was applied on the exposed area of enamel blocks using a microbrush. Each block was subjected to 1 min of treatment in 3 g of gel and removed with deionized water. Ero was performed every 2 h, by dipping the enamel blocks in Sprite Zero (Companhia de Bebidas Ipiranga, Ribeira˜o Preto, Brazil), pH 2.8, 4 times/day, during 5 min each time.17 Ero+Abra was performed on half of the blocks by using a mechanical brushing machine (250 g axial load, 5 strokes/s; Elquip Maq Escovac¸a˜o, Sa˜o Carlos, Brazil) immediately after the erosive challenges (4 times/day). Brushing was performed for 15 s each time, using a placebo dentifrice slurry (1:3, weight:weight). The other half of the blocks (Ero only) was immersed in the placebo dentifrice slurry for 15 s after the erosive challenges.
2.5.
Determination of surface wear
The nail varnish on the reference surfaces was removed carefully with acetone-soaked cotton wool. Enamel loss was determined in relation to the reference surfaces by profilometry (Surftest SJ 401 – Mitutoyo American Corporation), by scanning the surface of each block from the reference surfaces (control) across the exposed surfaces. The mean value of 5 readings was calculated for each block.
2.6.
Analysis of cross-sectional hardness
Blocks were sectioned at the center, and half of each block was included in acrylic resin and subsequently polished. Crosssectional hardness (SH) was determined (Knoop diamond, 5 g, 10 s, Buehler, Lake Bluff, USA). A sequence of eight prints at distances of 10, 15, 20, 25, 30, 40, 50 and 70 mm from the external surface of the enamel was performed in the center of blocks, for both the control and the test areas. The integrated area of hardness (KHN mm) of the demineralized and sound enamel was calculated using the trapezoidal rule (GraphPad Prism, version 3.02) and subtracted from the integrated area of the hardness of sound enamel loss resulting integrated hardness (DKHN).10
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2.7.
archives of oral biology 59 (2014) 336–340
Statistical analysis
For the statistical analysis, GMC software, Campos, 200318 was used and the significance limit was set at 5%. Data (log transformed) passed normality (Kolmogorov– Smirnov) and homoscedasticity (Cochran) tests, and were submitted to 2-way ANOVA, considering the type of gel (5 formulations) and challenge (Ero and Ero+Abra) as fixed factors. Two-way ANOVA was used to analyze the data of hardness as a function of depth. Pearson’s correlation test was used to evaluate the relationship between DKHN and wear.
3.
Results
The mean (SD) of fluoride concentration (ppm F) in the placebo, 4500, 4500 TMP5%, 9000 and Acid gel groups was 114.7 (5.4), 4509.8 (65.2), 4583.3 (164.4), 9467.7 (76.2) and 11130.4 (239.9), respectively.
The Placebo group showed a higher softening (%SH) from the other groups ( p < 0.001) after the erosive challenges (Table 1). The erosion promoted higher enamel surface softening than Ero+Abra ( p < 0.001). Lower softening was observed in enamel treated with Acid gel ( p < 0.001). The gel with 4500 ppm F and 4500 ppm F plus TMP presented similar enamel softening ( p > 0.610). Lower values of wear were observed with the erosive than Ero+Abra challenge ( p < 0.001). With the addition of TMP to gel 4500, enamel wear was lower when compared to the other groups ( p < 0.001), both in the Ero and Ero+Abra (Table 1). The fluoride groups (4500, 9000 and Acid gel) presented similar enamel wear ( p > 0.608). Placebo group exhibited the highest mineral loss deep into enamel (DKHN) when compared with the other groups ( p < 0.001) (Table 1). The gel with TMP showed similar values of DKHN than Acid gel group ( p > 0.051), regardless of the challenge types. Fig. 1 shows the hardness profile of erosion and erosion/abrasion as a function of depth according to the groups and the others comparisons are shown.
Table 1 – Mean (SD) values of surface hardness (%SH), wear and DKHN of the enamel according to conditions of challenge (Ero and Ero+Abra) and groups. Analysis
Groups Placebo
%SH
Wear (mm)
DKHN
Ero Ero+Abra
A B
4500 A B
4.71a (0.80) 5.81a (0.47)
Ero Ero+Abra Ero Ero+Abra
91.2a (1.3) 84.8a (1.6)
83.6b (1.6) 79.0b (1.4)
A
A
B
B
A
A
B
3609.6a (139.6) 2240.1a (129.9)
B
9000 A B
4.19b (0.70) 5.15b (0.35)
2712.9b (81.0) 1749.3b (158.2)
A B
77.9c (1.6) 74.1c (2.3)
4500 5%TMP A B
3.95b (0.69) 5.22b (0.34)
83.7b (2.2) 78.6b (1.6)
Acid gel A B
A
A
A
B
B
B
2320.3c (122.9) 1466.1c (128.2)
2.95c (0.51) 3.78c (0.36)
74.0d (1.5) 68.3d (1.4)
A
A
B
2215.5d (64.5) 1161.6d (102.0)
B
4.11b (0.91) 5.33a,b (0.57)
2321.5c,d (78.5) 1253.4d (100.5)
Different letters show significant differences in each analysis between groups of gel (ANOVA 2-way, Student–Newman–Keuls’s test; p < 0.05).
Fig. 1 – Graphical representation of mean hardness as a function of depth according to the erosive challenges. Symbols indicates comparison among groups in each depth (Student–Newman–Keuls, p < 0.05). Vertical bars indicate the standard deviations of means. (*) indicates no statistical difference among groups. (a) g: indicates equality between the groups healthy, 9000, 4500 5%TMP and Acid gel ( p > 0.163). v: indicates difference among group ( p = 0.037). b: indicates equality between the groups healthy, 4500, 9000 and Acid gel ( p > 0.116), 4500 5%TMP and Acid gel ( p = 0.070). l: indicates equality between the groups healthy, 4500, 9000, 4500 5%TMP and Acid gel ( p > 0.478). &: indicates difference between Placebo and the other groups ( p < 0.001). (b) V: indicates equality between the groups healthy, 4500 5%TMP and Acid gel ( p = 0.286). C: indicates equality between the groups healthy, 4500 5%TMP and Acid gel ( p = 0.248). ¥: indicates that the healthy groups, 4500 5%TMP and Acid gel are similar statistically ( p = 0.339). §: indicates equality between the groups healthy, Placebo and 4500 ( p = 0.158), 4500 and 9000 ( p = 0.074), 9000 and Acid gel ( p = 0.189).
archives of oral biology 59 (2014) 336–340
There was no correlation between wear (Ero and Ero+Abra) and surface hardness. (Pearson’s r = 0.072; p = 0.587 and Pearson’s r = 0.067; p = 0.611). Positive correlation was noted between wear (Ero and Ero+Abra) and DKHN (Pearson’s r = 0.532; p < 0.001 and Pearson’s r = 0.703; p < 0.001).
4.
Discussion
As the erosive challenge is a process that occurs at a pH of <4.0, the pH of the dissolution of calcium fluoride that might be formed in the presence of fluoride is exceeded during the erosive challenge.19,20 It knows that the effect of professionally applied topical fluoride is based on CaF2 deposits on enamel.21 This means a limit effect from topical fluoride products and to enhance its action the addition of new anti-erosive compounds can be necessary.4 In the present study was tested a TMP supplemented gel with reduced F concentration that showed a greater capacity to promote enamel remineralization.16 However this is a chemical model and the data obtained using this model (or any other in vitro protocol) should be considered carefully due to limitations in reproducing the oral environment with all of the biological variations known to influence erosion.22 The presence of saliva and acquired pellicle can influence the adsorption of TMP on enamel and the erosive wear. However, in a recent in vitro study (unpublished data) the presence of the acquired pellicle leads to lower erosive wear but the effect of TMP and fluoride agents is maintained with or without pellicle. Nonetheless, the present study provides interesting data that may be used in more efficient product development in the future by using other compounds than fluoride. As the blocks were kept stored in artificial saliva after the last erosive/treatment process would be more correct to state that the hardness testing (superficial and in depth) analyzed the remineralizing ability of the formulations. In the present study, it was possible observed a eroded enamel less softened with rising fluoride concentrations in the topical gels (Erosion or Erosion/Abrasion). Despite surface hardness not to be related to wear the treatment with fluoride have a remineralizing action in the remaining enamel.23–25 This remineralization is related with CaF2 deposits on enamel.4,16 Thus, the greater calcium fluoride deposition observed in the study of Danelon et al.,16 explain the better results (%SH) with acidulated fluoride treatment (Acid gel). Nevertheless, the acidulated topic gel show a few effect against the enamel wear. It means the ability of calcium fluoride deposition from fluoride product is a few related to the enamel wear, however; it is related to a greater remineralization at the outer part of the enamel demineralization. The addition of TMP in the fluoride gel gave a superior results and it was not related to calcium fluoride deposition on enamel. As observed in a recent study that utilized a topical gel with 4500 ppm F, the TMP did not influence the adsorption of calcium fluoride and neither enhance it or reduce its precipitation.16,26 Based in previously studies, TMP interacts with the enamel, which may produce a protective layer on the enamel surface thereby hindering acid diffusion.14,27 TMP is a cyclic phosphate and although it is soluble in water, it seems not to be spontaneously hydrolyzed.13 The interaction of Ca2+
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from artificial saliva and CaF+ with TMP (crosslinking) leads the reticular formation on enamel by the binding sites on the negative PO4 in the cyclic structure. At acidic pH these linkages are ruptured releasing Ca2+ and CaF+ which can react with H2PO4 leading to the formation of CaHPO40 and HF0.28 The diffusion of neutral ions into the enamel is thousand times higher than charged ions enhancing the remineralization process.28 With the reticular formation broken probably TMP retains its structure H+ reducing the acid diffusion into the enamel decreasing enamel demineralization. Thus, the major effect of TMP during the erosive challenge is to prevent acid diffusion into the enamel. The data from cross-sectional hardness analysis showed that the addition of TMP improved the remineralization at the inner part of the enamel in the same degree when compared with Acid gel. As these data are based in an in vitro protocol, additional in situ and clinical studies must be conducted to confirm these results. According to the results of this study, we can conclude that the addition of TMP to the fluoride gel showed a higher effect against erosion and erosion/abrasion of the enamel.
Conflict of interests The corresponding author has filed an application for patenting a product used in the study at the National Institute of Industrial Property (INPI/SP) on 04/29/2008 under the number 018080026091, PI0801811-1 and published on January 11, 2011. The other authors have no financial or personal conflicts of interest in relation to this study. All authors approved the publication of the manuscript.
Funding The study was supported by CNPq/PIBIC (ID: 21613), a Brazilian Agency for Research.
Ethical approval There was no need to subnet the study to the Committee of Ethics in Research. This manuscript is an in vitro study.
references
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