Australian Dental Journal

Australian Dental Journal The official journal of the Australian Dental Association Australian Dental Journal 2013; 58: 478 482 doi: 10.1111/adj.12110 Role of arginine and fluoride in the prevention of eroded enamel: an in vitro model JM Yamashita,* NM Torres,* PG Moura-Grec,* JA Marsicano,* A Sales-Peres,* SHC Sales-Peres* *Department of Pediatric Dentistry, Orthodontics and Public Health, Bauru School of Dentistry, University of S~ao Paulo, Bauru, SP, Brazil. ABSTRACT Background: The aim of this study was to investigate the effect of arginine and fluoride on the reduction of erosive wear. Methods: Bovine enamel blocks were randomly allocated into four groups (n = 20) and exposed to: ESPR group (8% arginine, 1450 ppm sodium monofluorophosphate, calcium carbonate and titanium dioxide); ESen group (1450 ppm sodium monofluorophosphate, 5% potassium citrate); positive control PC group (1500 ppm sodium monofluorophosphate) and negative control NC group (water). The samples were submitted to six alternating cycles of demineralization remineralization (cola, 10 minutes; artificial saliva, 1 hour, respectively). Before and between cyclic demineralization and remineralization, blocks were treated with slurries of the respective toothpastes or water (1 minute). Erosive tissue loss was analysed by microhardness and profilometry. Data were analysed by ANOVA and Tukey tests for individual comparisons among the groups (p < 0.05). Results: In microhardness, the ESPR (217.46 55.45) group was significantly better than the other treatment groups (PC = 302.76 96.10; ESen = 315.56 74.56; p < 0.001). The ESPR group showed a similar loss to NC group (NC = 210.8 49.98; p = 0.991). The mean erosion depth (+/ SE, lm) was detected between NC (14.37 1.72) and dentifrices tested (ESPR (4.11 1.34), ESen group (7.64 1.61) and PC (8.20 2.19) (p = 0.000). Conclusions: From the results of the present study, the effectiveness of Sensitive Pro Relief in the prevention of erosive surface loss seems to be attributed to the possible effect of the arginine associated with fluoride. Keywords: Arginine, enamel wear, toothpaste. Abbreviations and acronyms: ANOVA = analysis of variance; SD = standard deviation; SMF = sodium monofluorophosphate; SMH = surface microhardness. (Accepted for publication 22 January 2013.) INTRODUCTION Dental erosion is defined as loss of tooth substance by chemical processes not involving bacteria and caused by a variety of extrinsic and intrinsic factors. 1 Acid attack leads to the irreversible loss of dental hard tissue accompanied by a progressive softening of the surface. In advanced stages, clinically detectable enamel wear and dentine exposure associated with sensitivity may occur. 2 Several products and vehicles, such as propolis, 3 neem 3 and arginine 4 have been tested to minimize or reduce dentine sensitivity. Dentine hypersensitivity is a common clinical condition experienced by the population in general and it has been defined as a sharp, but transient pain arising from exposed dentine in response to thermal, osmotic, tactile or chemical stimuli that cannot be attributed clearly to any other type of defect. 5 Changes in dietary and oral hygiene habits, oral products, and toothpastes must be made to prevent or decrease the progression of erosion. 6 As sensitivity is a common complaint among patients, desensitizing agents are increasingly being incorporated into toothpastes, and many of these recently introduced on the market contain both fluoride and desensitizing agents, e.g. the dentifrice containing 8.0% arginine and calcium carbonate. 4,7 It has been demonstrated that the application of high concentrations of fluoride increases abrasion resistance and decreases the development of tooth enamel erosion in some cases. 8 12 However, to date, only a few studies have analysed dentifrices specifically for the prevention of erosion, and the results have been contradictory. 13,14 Thus, the aim of the present study was to evaluate the protective effect against enamel erosion, of 478 2013 Australian Dental Association

Arginine in the prevention of dental erosion dentifrice containing arginine and to compare it with one containing potassium citrate. Further, surface microhardness testing and profilometry were the methods adopted to analyse the enamel specimens. MATERIALS AND METHODS Enamel specimen preparation Blocks of permanent tooth enamel (4 mm x 4 mm x 3 mm) were obtained from bovine incisors disinfected by storage in 0.1 thymol solution for 30 days at room temperature. Using two parallel diamond discs separated by a 4-mm spacer, 1 2 blocks were cut from the crown of each bovine incisor. The enamel surfaces of the blocks were ground flat with water-cooled abrasive discs (600-1200-1400; Extec Corp) and polished with felt papers wet with diamond spray (1 l; Buehler). For standardization purposes, blocks were preselected based on their initial microhardness. Five indentations were made in different regions of the blocks (25 g, 5 s, HMV-2000/Shimadzu Corporation, Japan) to select 80 enamel slabs considering an acceptable microhardness range of 292 397 kg/mm 2. The enamel specimens were randomized using a table of randomly generated numbers obtained from spreadsheet software (Excel 2007, Microsoft). In order to maintain reference surfaces to determine lesion depth, two layers of nail varnish (Risque, Niasi, Tabo~ao da Serra, S~ao Paulo, Brazil) were applied on half of the surface of each specimen to protect it from erosion. Experimental design Twenty specimens were allocated to each of the four groups (two experimental groups, one positive control and one negative control). After preparation, the blocks were immersed in artificial saliva for 24 hours at 37 ºC to allow superficial hydration. The artificial saliva was prepared using the following compounds: 1.5 mmol/l Ca(NO 3 ) 2.4H 2 O; 0.9 mmol/l Na 2 H- PO 4.2H 2 O; 150 mmol/l KCl; 0.1 mol/l H 2 NC (CH 2 OH) 3 (TRIS); and 0.05 lg/ml NaF, ph 7.0. Treatments Commercial toothpastes, Colgate Sensitive Pro Relief TM (8% arginine, 1450 ppm sodium monofluorophosphate, calcium carbonate and titanium dioxide CL77891 ph = 9.11); Colgate Sensitive (1450 ppm sodium monofluorophosphate, 5.04% potassium citrate, ph = 7.35); Colgate Maximum Cavity Protection (1500 ppm sodium monofluorophosphate ph = 9.35) (Colgate-Palmolive Ind. e Com. Ltd., S~ao Paulo, Brazil) (Table 1). Table 1. Commercial toothpastes, abbreviation, composition and ph Commercial tootpastes Abbreviation Composition ph Colgate Sensitive Pro Relief TM ESPR 8% arginine, 1450 ppm sodium monofluorophosphate, calcium carbonate and titanium dioxide-cl77891 Colgate Maximum PC 1500 ppm sodium Cavity Protection monofluorophosphate Colgate Sensitive ESen 1450 ppm sodium monofluorophosphate, 5.04% K citrate 9.11 9.35 7.35 The groups were: Experimental ESPR (Colgate Sensitive Pro Relief TM ); Experimental ESen (Colgate Sensitive ); positive control PC (Colgate Maximum Cavity Protection) and negative control NC (water). Treatments consisted of immersion of the blocks in a slurry of the toothpastes (3 g:10 ml of deionized water) for 1 minute, before and between cyclic demineralization and remineralization. ph cycling The blocks were submitted to a demineralization remineralization regime. They were consecutively cycled through this regimen six times. One complete cycle comprised the following steps: (1) treatment with the toothpastes as described above; (2) demineralization in 30 ml of Coca-Cola (Cia de Bebidas Spaipa, Maringa, Brazil, ph 2.7) for 10 minutes under gentle agitation; (3) treatment with the toothpaste as described above; and (4) remineralization in artificial saliva for 60 minutes at 37 C. The blocks were thoroughly washed with deionized water before the analysis. The ph cycling was done during the course of one day. Analysis of change in surface microhardness A total of 10 indentations were made on each specimen, five on the protected enamel surface (SMHC-control) and five on the experimental areas (SMHE-demineralization/remineralization cycles). The reduction in microhardness (SMH) was calculated as SMHC-SMHE. Profilometry analysis Enamel loss (lm) was quantitatively determined by contact profilometry, which presents an accuracy of around 0.5 lm. For profilometric measurement, the layer of varnish was carefully removed from the slabs. The diamond stylus was moved from the first reference to the exposed surface and then to the other 2013 Australian Dental Association 479

JM Yamashita et al. reference surface (LC = 2.5 mm length). Five profile measurements were randomly performed in the centre of each slab and the average wear depth was calculated. Statistical analysis Mean and standard deviation (SD) for enamel loss and surface microhardness change in each group were calculated and statistically analysed by analysis of variance (ANOVA) and Tukey tests for individual comparisons among the groups. Pearson s correlation was used for data analysis to verify the correlation of enamel loss (lm) and surface microhardness (SMH) with ph. Data analysis was performed using STATIS- TICA version 7.0 (Sat-Soft-USA). The level of significance was set at 5%. RESULTS Table 2 shows the mean erosion depths of the blocks after treatment with commercial toothpastes. The best protective effect against erosive wear was observed for Colgate Sensitive Pro Relief, when compared with the other treatment groups. Significant difference was detected between negative control and all dentifrices tested (p = 0.000). When microhardness was evaluated, the ESPR group showed similar values to those for the NC group (p = 0.991) and lower values than those of PC and ESen groups (p < 0.001). The mean ph of toothpastes was: 9.11 for ESPR group; 9.21 for PC group; 7.35 for ESen group and 7.7 for water. There is no correlation between SMH and ph (r = 0.07; p = 0.547), and between enamel wear and ph (r = 0.41; p = 0.000). DISCUSSION Numerous studies have used bovine enamel as a substitute for human enamel for erosion and erosion/ abrasion tests because it is easier to obtain a sufficient number of sound bovine teeth. 15,16 In the present study, bovine enamel was used for the experiments. Table 2. Mean erosion of bovine enamel blocks treated with different toothpastes before and between cyclic demineralization and remineralization Groups SMH (Kg/mm²) Enamel wear (lm) ESPR 217.46 55.45 a 4.11 1.34 A PC 302.76 96.10 b 8.20 5.63 B ESen 315.56 74.56 b 7.64 1.61 B Water 210.8 49.98 a 14.37 1.72 C Means SE (n = 20). Different superscript letters indicate significant differences between the groups (Tukey tests). In the present study, surface wear of enamel was measured by means of microhardness testing and surface profilometry. This method has been used in numerous other studies on dental hard tissue wear due to erosion. 17,18 The in vitro model used has been successfully applied to evaluate strategies for preventing dental erosion, 14 as it allows discrimination between different treatments. According to the present design, slurries of the products were applied on the enamel surfaces for 1 minute, a condition that more closely resembles the clinical situation. Thus, it is very important to carefully analyse the experimental design and the response variables used in different studies before making any inferences regarding their potential clinical effects. 14 The group in which Colgate Sensitive Pro Relief was used showed lower loss of hardness and enamel wear values, and this can be attributed to some of its components, such as arginine, calcium carbonate, sodium monofluorophosphate and titanium dioxide. This is a pioneer study involving the use of arginine, calcium carbonate and fluoride to inhibit the erosive effect on enamel. The effect of Colgate Sensitive Pro Relief on the enamel surface could reduce the extent of erosion. Further research is required to better explain this effect. Colgate Sensitive contains sodium monofluorophosphate (SMF) and potassium citrate in its composition, and was able to significantly reduce erosion wear. This finding was similar to the results observed for the positive control (Colgate Maximum Cavity Protection) when compared with the negative control, which had the same fluoride concentration (SMF). These results can be attributed to the low measurable levels of free fluoride ions in the supernatant, through adsorption of the toothpaste excipients of the SMF anion to the enamel surface. 19 Kato et al. 14 suggested that the beneficial effect found for Colgate Sensitive might have been due to the action of K citrate and not to the presence of fluoride. When ph is low or acid, there is a higher level of erosion. 20 However, ESen and the water groups had similar ph values (around 7) and showed different enamel loss, which was lower in ESen. This occurrence showed the protective effect of fluoride associated with the citrate potassium in the toothpaste (ESen). The ph of ESPR and PC groups showed similar values, around 9. However, the enamel loss was lower in the ESPR group, which in turn demonstrates that its components were effective in protecting the enamel. One of the components in Colgate Maximum Cavity Protection toothpaste is sodium monofluorophosphate, which is presumed to be hydrolysed to free fluoride ions and phosphate in saliva and plaque. Afterwards, these are incorporated into the tooth enamel, showing that 480 2013 Australian Dental Association

Arginine in the prevention of dental erosion free fluoride ions are very important in inhibiting demineralization and enhancing remineralization. 21 The presence of fluoride and other components in the dentifrice groups may explain their statistically significant differences from water with regard to enamel wear. Colgate Sensitive Pro Relief was developed through the association of arginine, an amino acid which is positively charged at physiological ph, ph 6.5 7.5 and titanium dioxide. The titanium ions can bind to enamel surfaces and penetrate into sound or demineralized enamel. 22 The titanium seemed to interact with the enamel surface, thus leading to an increased fluoride uptake by enamel. 21 Alternatively, it is speculated that titanium phosphate compounds are formed 23 or that titanium can substitute calcium in the apatite lattice, 24 leading to higher acid resistance. In vitro studies have shown that an application of TiF4 is effective to induce the formation of an acidstable glaze-like layer, 25 which is related to erosioninhibiting properties. This mechanism could have had an influence on our results, considering that titanium dioxide was included among the components of Sensitive Pro Relief. Further studies are needed to analyse whether the presence of TiF4 might allow for penetration of the solution into the subsurface zone, thus decreasing further dissolution. Moreover, the abrasion resistance of the surface layer has to be evaluated, as dental hard tissues are not only exposed to erosive, but also to abrasive influences, such as toothbrushing, under clinical conditions. The effects of toothpaste slurries are still unclear. An in vitro study showed that the eroded enamel may be influenced by the abrasivity of the slurry and increased along with relative enamel abrasion. 26 With respect to the effect of toothpaste slurries on enamel erosion, it can only be assumed that their mode of action is surface precipitation. More appropriate and more complete approaches are needed for further research. The calcium and phosphate present in the slurries could also contribute to establishing surface precipitates in the presence of very low amounts of F, and it can be assumed that protective effects depend on the amount, type and acid solubility of such deposits. 27 This hypothesis could partially explain the difference between microhardness and enamel wear for ESPR. Further research is necessary to elucidate the mode of action of toothpastes in enamel erosion. The toothpaste containing arginine was shown to provide more effective protection because the results obtained showed significant difference in the microhardness and profilometry tests. This fact may be attributed to the association of all components which are present in its formulation. Further research is needed to investigate the performance of the argininecontaining toothpastes in longer in situ studies with regard to erosive wear in enamel. Thus, it is important to emphasize that the results of this study provide indications of what actually happens in the enamel surface. However, additional in situ and clinical studies with appropriate designs should be conducted to confirm these in vitro results. Sensitive Pro Relief prevents erosive surface loss due to the possible effects of synergistic action between arginine and fluoride. We believe this dentifrice is a good clinical alternative to reduce dental erosion and sensitivity. REFERENCES 1. Mahoney E, Kilpatrick N. Dental erosion: Part 1. Aetiology and prevalence of dental erosion. N Z Dent J 2003;99:33 41. 2. Lussi A. Dental erosion clinical diagnosis and case history taking. Eur J Oral Sci 1996;104:191 198. 3. Sales-Peres S, Reinato J, Sales-Peres AC, Marsicano J. Effect of iron gel on dentin permeability. Braz Dent J 2011;22:198 202. 4. Docimo R, Montesani L, Maturo P, et al. Comparing the efficacy in reducing dentin hypersensitivity of a new toothpaste containing 8.0% arginine, calcium carbonate, and 1450 ppm fluoride to a benchmark commercial desensitizing toothpaste containing 2% potassium ion: an eight-week clinical study in Rome, Italy. J Clin Dent 2009;20:137 143. 5. Addy M. Dentin hypersensitivity: definition, prevalence, distribution aetiology. In: Addy M, ed. Tooth wear and sensitivity clinical advances in restorative dentistry. London: Martin Dunitz, 2000:239 248. 6. Magalhaes A, Wiegand A, Rios D, Honorio H, Buzalaf M. Insights into preventive measures for dental erosion. J Appl Oral Sci 2009;17:75 86. 7. Ayad F, Ayad N, Zhang Y, DeVizio W, Cummins D, Mateo L. Comparing the efficacy in reducing dentin hypersensitivity of a new toothpaste containing 8.0% arginine, calcium carbonate, and 1450 ppm fluoride to a commercial sensitive toothpaste containing 2% potassium ion: an eight-week clinical study on Canadian adults. J Clin Dent 2009;20:10 16. 8. Attin T, Siegel S, Buchalla W, Lennon A, Hannig C, Becker K. Brushing abrasion of softened and remineralised dentin: an in situ study. Caries Res 2004;38:62 66. 9. Ganss C, Klimek J, Brune V, Schurmann A. Effects of two fluoridation measures on erosion progression in human enamel and dentine in situ. Caries Res 2004;38:561 566. 10. Lussi A, Jaeggi T, Schaffner M. Prevention and minimally invasive treatment of erosions. Oral Health Prev Dent 2004;2(Suppl 1):321 325. 11. Tezel H, Ergucu Z, Onal B. Effects of topical fluoride agents on artificial enamel lesion formation in vitro. Quintessence Int 2002;33:347 352. 12. Wiegand A, Attin T. Design of erosion/abrasion studies insights and rational concepts. Caries Res 2011;45(Suppl 1):53 59. 13. Lussi A, Megert B, Eggenberger D, Jaeggi T. Impact of different toothpastes on the prevention of erosion. Caries Res 2008;42:62 67. 14. Kato M, Lancia M, Sales-Peres S, Buzalaf M. Preventive effect of commercial desensitizing toothpastes on bovine enamel erosion in vitro. Caries Res 2010;44:85 89. 15. Vieira A, Overweg E, Ruben J, Huysmans M. Toothbrush abrasion, simulated tongue friction and attrition of eroded bovine enamel in vitro. J Dent 2006;34:336 342. 16. Oesterle L, Shellhart W, Belanger G. The use of bovine enamel in bonding studies. Am J Orthod Dentofacial Orthop 1998; 114:514 519. 2013 Australian Dental Association 481

JM Yamashita et al. 17. West N, Hughes J, Addy M. Erosion of dentine and enamel in vitro by dietary acids: the effect of temperature, acid character, concentration and exposure time. J Oral Rehabil 2000;27:875 880. 18. Sales-Peres S, Pessan J, Buzalaf MA. Effect of an iron mouthrinse on enamel and dentine erosion subjected or not to abrasion: an in situ/ex vivo study. Arch Oral Biol 2007;52:128 132. 19. Fowler C, Willson R, Rees G. In vitro microhardness studies on a new anti-erosion desensitizing toothpaste. J Clin Dent 2006;17:100 105. 20. Hughes JA, West NX, Parker DM, van den Braak MH, Addy M. Effects of ph and concentration of citric, malic and lactic acids on enamel, in vitro. J Dent 2000;28:147 152. 21. Toda S, Featherstone J. Effects of fluoride dentifrices on enamel lesion formation. J Dent Res 2008;87:224 227. 22. Chevitarese A, Chevitarese O, Chevitarese L, Dutra P. Titanium penetration in human enamel after TiF4 application. J Clin Pediatr Dent 2004;28:253 256. 23. Ribeiro C, Gibson I, Barbosa M. The uptake of titanium ions by hydroxyapatite particles-structural changes and possible mechanisms. Biomaterials 2006;27:1749 1761. 24. Leadley S, Davies M, Ribeiro C, Barbosa M, Paul A, Watts J. Investigation of the dissolution of the bioceramic hydroxyapatite in the presence of titanium ions using ToF-SIMS and XPS. Biomaterials 1997;18:311 316. 25. Wiegand A, Waldheim E, Sener B, Magalhaes A, 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; 43:269 277. 26. Wiegand A, Schwerzmann M, Sener B, et al. Impact of toothpaste slurry abrasivity and toothbrush filament stiffness on abrasion of eroded enamel an in vitro study. Acta Odontol Scand 2008;66:231 235. 27. Ganss C, Lussi A, Grunau O, Klimek J, Schlueter N. Conventional and anti-erosion fluoride toothpastes: effect on enamel erosion and erosion-abrasion. Caries Res 2011;45:581 589. Address for correspondence: Silvia H C Sales-Peres Department of Pediatric Dentistry, Orthodontics and Public Health Bauru School of Dentistry University of S~ao Paulo Al. Octavio Pinheiro Brisolla, 9-75 Bauru-SP 17012-901 Brazil Email: shcperes@usp.br 482 2013 Australian Dental Association