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journal of dentistry xxx (2015) xxx–xxx

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1
2
3 Anti-erosive properties of solutions containing
4 fluoride and different film-forming agents

5 Q1 Taı́s Scaramucci a, Alessandra B. Borges b, Frank Lippert c,

6 Domenick T. Zero c, Idalina V. Aoki d, Anderson T. Hara c,*
a
7 Q2 Department of Restorative Dentistry, School of Dentistry, University of São Paulo, Av. Prof. Lineu Prestes 2227, São
8 Paulo, SP 05508-000, USA
b
9 Department of Restorative Dentistry, Univ. Estadual Paulista, Av. Eng. Francisco José Longo 777, Jardim São Dimas,
10 São José dos Campos, SP 12245-000, USA
c
11 Oral Health Research Institute, Department of Preventive and Community Dentistry, Indiana University School of
12 Dentistry, Indianapolis, IN, USA
d
13 Department of Chemical Engineering, Polytechnic School, University of São Paulo, Av. Prof. Luciano Gualberto,
14 travessa 3, 380, São Paulo, SP 05508-900, Brazil

article info abstract

Article history: Objectives: To evaluate the anti-erosive potential of solutions containing sodium fluoride
Received 1 August 2014 (NaF, 225 ppm F) and different film-forming agents.
Received in revised form Methods: In Phase 1, hydroxyapatite crystals were pre-treated with solutions containing NaF
10 November 2014 (F), linear sodium polyphosphate (LPP), sodium pyrophosphate tetrabasic (PP), sodium tripo-
Accepted 20 January 2015 lyphosphate (STP), sodium caseinate (SC), bovine serum albumin (BSA), stannous chloride (Sn)
Available online xxx and some combinations thereof. Deionized water was the control (C). The pH-stat method was
used to evaluate hydroxyapatite dissolution. In Phase 2, the most effective solutions were
Keywords: tested in two independent experiments. Both consisted of an erosion–remineralization cycling
Dental erosion model using enamel and dentine specimens with three solution treatments per day. In Phase
Fluoride 2a, the challenge was performed with 0.3% citric acid (pH = 3.8). In Phase 2b, 1% citric acid
Protein (pH = 2.4) was used. Hard tissue surface loss was determined profilometrically. Data were
Stannous chloride analyzed with two-way ANOVA and Tukey tests.
Phosphate polymer Results: In Phase 1, F, LPP, Sn and some of their combinations caused the greatest reduction in
hydroxyapatite dissolution. In Phase 2a, C showed the highest enamel loss, followed by LPP.
There were no differences between all other groups. In Phase 2b: (F + LPP + Sn) < (F + LPP)
= (F + Sn) < (F) = (LPP + Sn) < (LPP) < (Sn) < C. For dentine, in both experiments, only the fluo-
ride-containing groups showed lower surface loss than C, except for LPP + Sn in 2a.
Conclusions: F, Sn, LPP reduced enamel erosion, this effect was enhanced by their combina-
tion under highly erosive conditions. For dentine, the F-containing groups showed similar
protective effect.
Clinical significance: The addition of LPP and/or Sn can improve the fluoride solution protec-
tion against erosion of enamel but not of dentine.
17 # 2015 Published by Elsevier Ltd.
15
16
18
19

Q3 * Corresponding author at: Oral Health Research Institute, Department of Preventive and Community Dentistry, Indiana University School
of Dentistry, 415 N. Lansing Street, Indianapolis, IN 46202-2876, USA. Tel.: +1 317 278 0577; fax: +1 317 274 5425.
E-mail address: ahara@iu.edu (A.T. Hara).
http://dx.doi.org/10.1016/j.jdent.2015.01.007
0300-5712/# 2015 Published by Elsevier Ltd.

Please cite this article in press as: Scaramucci T, et al. Anti-erosive properties of solutions containing fluoride and different film-forming agents.
Journal of Dentistry (2015), http://dx.doi.org/10.1016/j.jdent.2015.01.007
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2 journal of dentistry xxx (2015) xxx–xxx

20 or in combination with F would present improved anti- 78

1. Introduction erosive potential, when evaluated under different erosive 79
conditions. 80
21 Q4 Epidemiological studies have demonstrated that erosive tooth
22 wear (ETW) is a common dental condition worldwide.1 During 81
23 the initial stages, the signs and symptoms of ETW are seldom 2. Materials and methods
24 perceived by the patients. In advanced cases, however, pain as 82
25 result of dentine exposure, as well as loss of tooth anatomy 2.1. Experimental design
26 and vertical dimension can become serious consequences.2,3
27 Considering the irreversible nature of this condition, early This study was carried out in 2 phases. In Phase 1, 83
28 diagnosis and management by implementation of preventive hydroxyapatite crystals were exposed to solutions containing 84
29 and therapeutic measures are of utmost importance. In this F (as NaF), phosphate polymers, proteins and Sn (as SnCl2); as 85
30 regard, regular application of fluoride (F) products intended to well as some of their combinations. Deionized water was used 86
31 reduce the solubility of the tooth surfaces has been one of the as negative control (C). The crystals were then tested in 87
32 most recommended approaches.4,5 However, previous studies triplicate with the pH-stat as a screening method, using a 88
33 show a large variation in efficacy, which among other factors standard citric acid solution (0.3%, pH = 3.8). The agents with 89
34 seems to be strongly related to the type of F compound the most promising results in Phase 1 (F, LPP, Sn), some of their 90
35 tested.6–8 combinations and the control were tested in Phase 2 in two 91
36 The formation of calcium fluoride (CaF2)-like deposits on independent experiments (2a and 2b), following a factorial 92
37 the tooth surfaces is assumed to be the main mechanism of 8  2 design, with rinse solutions and tooth substrates as the 93
38 protection against dental erosion provided by conventional F two experimental factors. Both experiments consisted of an 94
39 compounds, such as sodium fluoride (NaF) and amine fluoride erosion–remineralization cycling model using enamel and 95
40 (AmF).8 These deposits can act either as a physical barrier dentine specimens (n = 10 for each substrate). In experiment 96
41 against erosive acids or as a mineral reservoir for reminer- 2a, erosion was performed with 0.3% citric acid (pH = 3.8). In 97
42 alization.9 Nevertheless, comparative studies showed that 2b, a more aggressive challenge with the use of 1% citric acid 98
43 some F compounds with polyvalent metal cations, such as (pH = 2.4) was carried out. The response variable for phase 1 99
44 stannous fluoride (SnF2) have improved efficacy.6,8,10 Besides was the volume (ml) of the titrant (0.1 N HCl), which was 100
45 the formation of CaF2, the stannous ion (Sn) can interact with converted to hydroxyapatite dissolution (in mg). For phase 2 101
46 the tooth surfaces forming a Sn-rich coating, which may also the response variable was surface loss (SL in mm) measured by 102
47 be relevant for erosion prevention.6,8,11 Previous in vitro and optical profilometry. To better understand the interaction 103
48 in situ studies on solutions containing F and Sn (F as NaF and between F, LPP and Sn with the hydroxyapatite surface, an 104
49 AmF; Sn as SnCl2 – stannous chloride) seem to suggest an additional test was performed to determine the zeta potential 105
50 additive effect between these ions.12,13 of the dispersed HA particles treated with the solutions 106
51 Organic and inorganic polymers have also been evaluated containing these agents isolated. Most particles in colloidal 107
52 for their ability to reduce dental erosion. Some polymers have dispersions have an electrical charge on its surface. The zeta 108
53 been tested as active ingredients of rinse solutions or tooth- potential is the overall charge that a particle acquires in a 109
54 pastes5,14–16 and also as additives in acidic beverages.17–21 The particular environment. There are several mechanisms for the 110
55 tested polymers have shown the potential to reduce erosion appearance of this surface charge depending on the nature of 111
56 progression due to their ability to adsorb to the dental the particle and the surrounding medium. The main mecha- 112
57 surfaces, leading to the formation of a protective film. nisms are: (1) ionization of groups on the surface, (2) a 113
58 Promising results were obtained especially for milk casein,15 differential ion loss of crystal lattice and (3) adsorption or 114
59 chitosan,5 linear sodium polyphosphate (LPP),17,18,21 and a precipitation of charged species. Thus, the determination of 115
60 combination of carboxymethylcellulose, xantham gum and the zeta potential is important for the study of surface 116
61 copovidone.14 In addition, previous studies have demonstrat- interactions.23 117
62 ed that several of these polymers can interact positively with
63 fluoride compounds, thereby enhancing its protective 2.2. Phase 1 118
64 effects.5,15,22
65 There is, however, considerably more scope for further In this phase, solutions of the following agents: sodium 119
66 research as (a) none of the tested film-forming agents or fluoride (F) (Sigma –Aldrich, St. Louis, MO, USA), sodium 120
67 combinations thereof have been shown to completely inhibit polyphosphate with an average chain length of 25 phosphate 121
68 erosion, and (b) research to determine additive or synergistic units, linear structure (LPP) (Calgon 696, Thermos Inc., 122
69 anti-erosive properties of combinations of actives has been Cheshire, UK); sodium tripolyphosphate (STP) (Sigma – 123
70 very limited. Therefore, the present study was concerned Aldrich Co.); sodium pyrophosphate tetrabasic (PP) (Sigma – 124
71 with the investigation of the anti-erosive properties of some Aldrich Co.), sodium caseinate (SC) (Spectrum Chemicals, 125
72 of the above mentioned film-forming agents, and especially New Brunswick, NJ, USA), albumin from bovine serum (BSA) 126
73 in their interaction with fluoride. The pH-stat method (Phase (Sigma –Aldrich Co.), stannous chloride (Sn) (Sigma –Aldrich 127
74 1) was used as a first screening tool. The most promising test Co.) and some of their combinations were prepared, creating 128
75 solutions were then further evaluated using two in vitro the experimental groups described in Table 1. For F, the 129
76 erosion cycling models of different aggressiveness (Phase 2a concentration that is usually found in oral rinse products 130
77 and 2b). Our test hypothesis was that the additives, alone (225 ppm or approximately 0.5 g/l) was chosen.24 For the 131

Please cite this article in press as: Scaramucci T, et al. Anti-erosive properties of solutions containing fluoride and different film-forming agents.
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journal of dentistry xxx (2015) xxx–xxx 3

Table 1 – Experimental groups and their compositions, means (SD) of the solution’s pH, means of the volume of titrant
needed in the pH-stat method, and means of hydroxyapatite dissolution.
Experimental groups Compounds Compounds pH Titrant Hydroxyapatite
concentration concentration volume dissolution (mg)
(g/l) (mM) (ml)
C: deionized water None – 5.70 2.13 (0.11) 26.70 (1.44)
F: sodium fluoride 0.5 11.83 6.60 0.82 (0.01) 10.34 (0.07)
LPP: polyphosphate 2 – 6.99 1.10 (0.10) 13.82 (1.19)
PP: pyrophosphate 2 7.52 10.11 1.89 (0.03) 23.80 (0.36)
STP: tripolyphosphate 2 5.43 9.33 1.83 (0.05) 22.98 (0.60)
SC: sodium caseinate* 2 – 6.42 2.03 (0.08) 25.46 (1.05)
BSA: bovine serum 2 – 6.58 1.94 (0.08) 24.41 (1.02)
albumin
Sn 1.28 10.75 4.50 0.95 (0.02) 11.93 (0.31)
LPP 20 – 7.29 0.92 (0.02) 11.51 (0.26)
PP 20 75.21 10.31 1.84 (0.00) 23.04 (0.06)
STP 20 54.36 9.59 1.82 (0.01) 22.80 (0.12)
SC** 20 – 6.88 1.66 (0.16) 20.83 (2.03)
BSA 20 – 5.21 1.77 (0.09) 22.22 (1.19)
F + LPP 0.5 + 2 6.60 0.53 (0.07) 6.69 (0.84)
F + PP 0.5 + 2 7.06 0.77 (0.04) 9.72 (0.54)
F + STP 0.5 + 2 6.90 0.64 (0.02) 7.98 (0.30)
F + SC 0.5 + 2 6.65 0.71 (0.05) 8.98 (0.64)
F + BSA 0.5 + 2 6.55 0.82 (0.05) 10.35 (0.68)
F + Sn 0.5 + 1.28 4.50 0.33 (0.02) 4.18 (0.27)
Sn + LPP 1.28 + 2 4.50 0.47 (0.01) 5.92 (0.12)
Sn + PP 1.28 + 2 4.50 0.93 (0.02) 11.68 (0.22)
Sn + STP 1.28 + 2 4.50 1.14 (0.11) 14.31 (1.41)
Sn + SC 1.28 + 2 4.50 0.69 (0.05) 8.63 (0.60)
Sn + BSA 1.28 + 2 4.50 0.81 (0.02) 10.23 (0.21)
F + LPP + Sn 0.5 + 1.28 + 2 4.50 0.23 (0.02) 2.89 (0.21)
*
65% protein content, approximately 0.7 g/l of casein.
**
65% protein content, approximately 13 g/l of casein.

132 polymers and Sn, the concentrations used were within the to the procedures described elsewhere.21 For this test, a 158
133 range suggested in previous investigations.25,26 To avoid standard 0.3% citric acid solution (pH adjusted to 3.8 with 1 M 159
134 saturation and precipitation of some solutions, when the KOH solution) was used. The test was performed in triplicate 160
135 agents were added in combination, lower polymer concen- for each solution. 161
136 trations were used. In this Phase, the solutions were tested at
137 their natural pH, except for the Sn-containing solutions, 2.4. Phase 2 162
138 which had its natural pH adjusted to 4.5 with 1 M KOH
139 solution (Fisher Scientific, Pittsburgh, PA, USA) and 2.3 g/l of For this phase, the solutions that promoted the highest 163
140 gluconic acid sodium salt (Acros Organic, Geel, Belgium) reduction in HAPc dissolution: F, Sn, LPP and combinations 164
141 added, for stability purposes. All the pH values were thereof (F + Sn, F + LPP, LPP + Sn, F + LPP + Sn) were tested in 165
142 determined using a calibrated pH electrode (Accumet 13- two experiments, both consisting of an erosion–remineraliza- 166
143 620-530; Fisher Scientific). tion cycling model. To avoid having the pH as a confounding 167
factor, the pH of all the solutions was adjusted to 4.5, with 168
144 2.3. Hydroxyapatite crystals pre-treatment either KOH or HCl solutions. In addition, LPP was tested at 2%25 169
as this concentration had shown improved protection 170
145 25 mg of HAPc were weighted and placed into a 2 ml Eppendorf compared to 0.2%, in our preliminary test. 171
146 tube. 1 ml of each experimental solution was added to each
147 tube with the aid of a pipette. The tube was gently agitated for 2.5. Sample preparation 172
148 1 min and centrifuged for 30 s at 10,000 rpm. The supernatant
149 was removed with a pipette, and 1 ml of deionized water was Enamel and root dentine specimens (4 mm  4 mm  2 mm) 173
150 added to the tube and gently agitated for 10 s. The tube was were sectioned from the crowns and the roots of the bovine 174
151 centrifuged again and the supernatant removed. This washing teeth, respectively, using a microtome. The specimens were 175
152 procedure was repeated one more time, however, at this point, embedded in acrylic resin (Varidur, Buehler, Lake Bluff, IL, USA). 176
153 only 0.9 ml of the supernatant was removed. The samples The blocks were ground flat with water-cooled abrasive discs 177
154 were then vortexed to disaggregate the HAPc, allowing them to (500-, 1200-, 2400- and 4000-grit Al2O3 papers; MD-Fuga, Struers 178
155 be used in the pH-stat analysis. Inc, Cleveland, OH, USA) and polished with polishing cloth and 179
156 The pH-stat test was performed using an automatic titrator diamond suspension (1 mm; Struers Inc.). The specimens with 180
157 (Titralab 856, Radiometer Analytical, Lyon, France), according cracks or any structural defects were discarded. Adhesive 181

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4 journal of dentistry xxx (2015) xxx–xxx

182 unplasticised polyvinyl chloride (UPVC) tapes were then placed 2.9. Additional test (determination of the zeta potential) 221
183 on their polished surface, leaving a central area of 4 mm  1 mm
184 exposed to subsequent testing. The zeta potentials of dispersed HAPc treated with the 222
solutions F, LPP and Sn were measured by a zeta potential 223
185 2.6. Experiment 2a analyzer (Zetasizer Nano ZS90, Malvern Instruments, Malvern, 224
UK), equipped with a Helium–Neon Laser of 633 nm and 225
186 Eighty enamel and 80 dentine specimens were randomly maximum of 4 mW. Electrophoretic light scattering (ELS) was 226
187 allocated to the eight experimental groups (n = 10). They were used to measure the electrophoretic mobility of dispersed 227
188 then submitted to an erosion–remineralization cycling model, HAPc in 0.040 mol/L phosphate buffer (pH = 7.0), and this was 228
189 consisting of: 5 min immersion in 0.3% citric acid, pH = 3.8 later converted to zeta potential using the Helmholtz– 229
190 (5 ml/specimen), with no agitation; and 60 min immersion in Smoluchowski formula. Ten mg of pretreated HAPc were 230
191 5 ml of artificial saliva,21 under gentle agitation. This cycle was dispersed in 5 ml of the phosphate buffer assisted by 8 min 231
192 repeated six times per day, over 5 d. After the demineralization under sonication in a common ultrasonic bath containing 232
193 and remineralization periods, the specimens were rinsed with water. Samples were pipetted and introduced in the sample 233
194 distilled water and gently dried with paper towel. Exposure to cell and examined at 20 8C. Five measurements were per- 234
195 the rinse solution was performed 30 min after the 1st, the 3rd formed for each sample (after a minimum of 10 and a 235
196 and the 6th remineralization periods, for 2 min. After the maximum of 40 runs) and the mean value recorded with its 236
197 exposure, no rinse was performed and the specimens were re- standard deviation. 237
198 immersed into artificial saliva for the completion of the
199 30 min. The specimens were stored in artificial saliva (under 2.10. Data analysis 238
200 agitation of 150 rpm) during the overnight period. Fig. 1 show
201 the daily cycling sequence. The experiment was conducted at Means (SD) of the triplicates were calculated for the pH-stat 239
202 room temperature. test. For each experiment in Phase 2, homoscedasticity and 240
normal distribution of the data were checked by the Hartley 241
203 2.7. Experiment 2b and Shapiro–Wilks tests. The data were analyzed by two-way 242
ANOVA followed by Tukey tests. The significance level was set 243
204 The same procedures performed in experiment 2a were at 5%. SigmaPlot 11 software (Systat Software Inc., Chicago, IL, 244
205 repeated in experiment 2b, however, this time, the erosive USA) was used for the calculations. Descriptive statistics were 245
206 challenge was performed with a 1% citric acid solution, natural used to present the results of the zeta potential test. 246
207 pH of approximately 2.4, with the intent of simulating more
208 aggressive erosive conditions. 247
3. Results
209 2.8. Erosion assessment 248
3.1. Phase 1
210 After cycling, the tapes were removed from the specimens and
211 the surface analyzed. An area 2 mm long (X)  1 mm wide (Y) Table 1 shows the mean values of HAPc dissolution obtained 249
212 was scanned with an optical profilometer (Proscan 2000, in the pH-stat method. The negative control (C) resulted in the 250
213 Scantron, Venture Way, Tauton, UK). The scan covered the highest HAPc dissolution. In general, the higher concentration 251
214 treated area and protected reference surfaces on both sides. of the polymers (20 g/l) presented slightly less HAPc dissolu- 252
215 The step size was set at 0.01 mm and the number of steps at tion than the lower ones (2 g/l). Pre-treatments with F, LPP (at 253
216 200 in the X-axis; and at 0.1 mm and 10, respectively, in the Y- 20 g/l) and Sn reduced HAPc dissolution approximately 60% in 254
217 axis. The depth of the treated area was calculated based on the comparison to C. SC, BSA, STP and PP demonstrated less ability 255
218 subtraction of the average height of the test area from the to reduce HA dissolution, with 22%, 17%, 15% and 14% of 256
219 average height of the two reference surfaces (Proscan reduction at the higher concentration, respectively. Fluoride in 257
220 Application software v. 2.0.17). combination with LPP, Sn, STP or SC, reduced HAPc dissolution 258
further. PP and BSA only showed small or no effect. When 259
221 stannous chloride was used in combination with LPP, SC or 260
BSA, additional reduction in HAPc dissolution was observed, 261
but no additional benefits were observed with PP and STP. The 262
combination F + Sn + LPP showed the lowest value of HAPc 263
dissolution. 264

3.2. Phase 2 265

The surface loss results of experiments 2a and 2b are shown in 266

Tables 2 and 3, respectively. In 2a, for enamel, C showed 267
significantly the highest SL, followed by LPP. The other groups 268
did not differ among each other (p > 0.05). For dentine, Sn and 269
LPP were not significantly different from C. The other groups 270
Fig. 1 – Daily cycling sequence. showed significantly lower SL: F and F + Sn offered the greatest 271

Please cite this article in press as: Scaramucci T, et al. Anti-erosive properties of solutions containing fluoride and different film-forming agents.
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journal of dentistry xxx (2015) xxx–xxx 5

Table 2 – Experiment 2a (less aggressive erosion model): means (SD) of surface loss for enamel and dentine, expressed in
mm, and percentage of surface loss reduction in comparison to the control (C).
C F F + LPP F + LPP + Sn F + Sn LPP LPP + Sn Sn
Enamel 2.35 (0.74) A 0.39 (0.14) A 0.43 (0.21) A 0.21 (0.11) A 0.08 (0.07) A 1.05 (0.47) A 0.32 (0.12) A 0.41 (0.16) A
c* a a a a b a a
– 84** 82 91 97 55 86 83

Dentine 6.68 (0.56) B 1.82 (0.65) B 3.17 (0.68) B 2.95 (0.44) B 1.99 (0.64) B 6.59 (0.66) B 6.03 (0.51) B 6.80 (0.88) B
d a b b a d c d
– 73 53 56 70 2 10 2%
*
Different lower case letters imply significant differences among groups, within substrate (p < 0.05). Different capital letters imply significant
differences between substrates within groups (p < 0.05).
**
Percentage of surface loss in comparison to the control, within each substrate.

Table 3 – Experiment 2b (more aggressive erosion model): means (SD) of surface loss for enamel and dentine, expressed in
mm, and percentage of surface loss reduction in comparison to the control (C).
C F F + LPP F + LPP + Sn F + Sn LPP LPP + Sn Sn
Enamel 39.44 (2.99) B 22.50 (2.74) B 16.00 (2.12) A 12.72 (2.37) A 16.97 (3.41) A 27.32 (3.12) B 24.17 (2.80) A 33.01 (4.56) B
f* c b a b d c e
– 43%** 60% 68% 57% 31% 39% 16%

Dentine 24.08 (3.85) A 18.21 (1.80) A 19.84 (0.83) B 17.87 (1.04) B 19.27 (1.49) A 24.93 (3.14) A 23.17 (2.02) A 28.16 (2.99) A
b a a a a b b c
– 24% 18% 26% 20% -4% 4% -15%
*
Different lower case letters imply significant differences among groups, within substrate (p < 0.05). Different capital letters imply significant
differences between substrates within groups (p < 0.05).
**
Percentage of surface loss in comparison to the control, within each substrate.

272 protection, followed by F + LPP + Sn and F + LPP, which were with all the test solutions resulted in more negative zeta 287
273 similar, and then LPP + Sn. potentials. Sn treated HAPc had surface potential of 32.1, 288
274 In 2b, all groups presented lower enamel loss than C: which was more negative than LPP ( 29.1) and F ( 25.7). 289
275 F + LPP + Sn had the lowest values (p < 0.05), followed by
276 F + LPP and F + Sn, which were similar. F and LPP + Sn did not 290
277 differ between each other and presented significantly lower SL 4. Discussion
278 than LPP and Sn. Sn had higher SL than LPP (p < 0.05). For 291
279 dentine, Sn showed significantly the highest SL, followed by C, 4.1. Phase 1
280 LPP and LPP + Sn, with no difference among them. F, F + LPP,
281 F + LPP + Sn, F + Sn presented the lowest SL (p < 0.05). In the screening phase, F, LPP (20 g/l) and Sn provided the 292
highest reduction of HAPc dissolution, being therefore select- 293
282 3.3. Additional test ed for Phase 2. SC, BSA, PP and STP had no more than 22% of 294
reduction. The magnitude of HAPc dissolution observed for SC 295
283 The means and standard-deviations of the five measurements at the higher concentration was similar to that reported in a 296
284 of electrophoretic mobility and zeta potential determinations previous study, where a solution containing 5 g/l of whole 297
285 are shown in Table 4. The HAPc were found to have strong casein was able to reduce enamel loss by about 20%.15 298
286 negative surface potential of 24.3 mV. Pretreatment of HAPc However, in that study, a prolonged exposure time to the 299
solution was performed (10 min vs. 1 min and 30 s presently), 300
which may have positively interfered with casein’s adsorption 301
onto the surface, thereby compensating for the lower 302
Table 4 – Means of electrophoretic mobility, zeta poten- concentration used. Two processes occur during protein 303
tial and sample conductivity of HAPc (dispersed in adsorption – the number of protein molecules adsorbed onto 304
phosphate buffer pH = 7.0) previously treated with deio-
the surface increases and, in parallel and as a consequence of 305
nized water (C), F, LPP* and Sn.
higher protein density at the surface, there is also an increased 306
HAPc Zeta Electrophoretic Sample interaction between molecules.27 Thus, we can assume that 307
treatment Potential mobility conductivity
the adsorbed layer in these cases would be more stable and 308
(mV) (mm/s for V/cm) (mS/cm)
acid resistant. Nevertheless, we decided to use a shorter 309
C (control) 24.3  1.1 1.72  0.08 8.55  0.15
exposure time in an attempt to simulate the clinical situation 310
F 25.7  0.7 1.82  0.05 8.71  0.14
where the exposure time to oral rinse products is considerably 311
LPP* 29.1  1.6 2.06  0.11 8.20  0.12
Sn 32.1  1.2 2.28  0.08 8.66  0.16 shorter than 10 min. 312
* Although the higher concentration of casein offered more 313
LPP was used at the concentration of 2%.
protection than the lower, it should be mentioned that a 314

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315 change in the solution’s organoleptic properties, specially the was slightly more effective, causing a reduction of approxi- 375
316 odour, was observed over time, indicating low stability. mately 89%. 376
317 Another interesting finding was the interactive effect between
318 SC and Sn and between SC and F. A previous investigation 4.2. Phase 2 377
319 reported that treating HAP and enamel surfaces with NaF and
320 SnF2 caused an increase in its negative surface charge, as well In Phase 2, F, LPP, Sn and their combinations showed different 378
321 as in its hydrophobic characteristics.28 As a result, the surface anti-erosive potentials; however, this effect was dependent 379
322 would have more affinity to the adsorption of positively on the type of substrate and aggressiveness of the erosive 380
323 charged and more hydrophobic molecules, such as casein, challenge. 381
324 which has a relatively hydrophobic nature and can bind to For enamel, in less erosive conditions (experiment 2a), all 382
325 both, positively and negatively charged surfaces.28–30 solutions presented similar protective effects, reducing the 383
326 BSA at the higher concentration was slightly less effective amount of erosion by more than 80% when compared to 384
327 than SC. Serum albumin is a protein present in all body the control. LPP was less effective, despite being able to reduce 385
328 secretions, including human whole saliva.31 Previous litera- the amount of enamel loss to less than a half. Considering 386
329 ture reports showed that albumin can influence the deminer- the more erosive condition (experiment 2b), LPP exhibited 387
330 alization rate depending on its concentration.31–33 Evidence superior protection compared to Sn (31% vs. 16% of enamel 388
331 suggests that albumin may also inhibit remineralization,34 loss reduction), which was the least effective experimental 389
332 although this effect was not evaluated in this study. At the solution. The reaction of Sn with hydroxyapatite can form 390
333 concentrations tested in this study, we could observe a crystalline precipitates, such as Sn2OHPO4,11 which may be 391
334 reduction in HAPc dissolution of 9 and 17%, for the lower responsible for protecting the enamel surface in different 392
335 and the higher concentration, respectively. In accordance to degrees, depending on the Sn concentration used.39 The zeta 393
336 the observations of a recent investigation, BSA interacted potential analysis indicates the formation of such precipitates; 394
337 positively with Sn, enhancing its protective effects.26 Although once the Sn-treated HAPc presented the most negative zeta 395
338 the mechanism involved in this interaction still needs to be potential of all, which results from preferentially released 396
339 elucidated, the authors hypothesized that Sn could have acted Sn2+, thereby increasing the overall negative surface charge of 397
340 as a linking agent for BSA, rendering a more resistant surface HAPc. Nevertheless, we may assume that the precipitates 398
341 protective layer. Nevertheless, in the present study, this effect formed with the Sn solution were less resistant to stronger 399
342 was only small. This reduced interaction could be related to erosive challenges, hence the lower protective effect observed 400
343 the shorter period of exposure to the protein, as in the previous in experiment 2b when compared to 2a (16% of enamel loss 401
344 study the proteins were added to the artificial saliva used for reduction vs. 83%, respectively). 402
345 remineralization. Thus, similar to SC, it may be suggested that The combination of both agents (Sn and LPP) showed 403
346 a higher degree of protection could be achieved by increasing improved efficacy, matching the protection of F alone. This 404
347 the exposure time to the solution. No additional benefit was fact corroborates our assumption that the availability of 405
348 found by combining BSA with F. these agents was not reduced when they were added to same 406
349 Among the phosphate polymers, LPP caused the greatest solution. Although the mechanism of protection of Sn + LPP is 407
350 reduction in HAPc dissolution (57%), which is consistent with not clear, we hypothesize that the combination of these 408
351 previous investigations and can be explained by its longer substances was able to form a more resistant protective film 409
352 chain length.17,21 LPP is a linear phosphate polymer with P–O–P on the enamel surface. Sn and LPP are likely to react with 410
353 chains of the formula Nan+2PnO3n+1.35 It was postulated that different sites of enamel, Sn with the negative charged 411
354 the phosphate groups of this polymer have the ability to molecules (phosphates); and LPP with the positive charged 412
355 adsorb to the HAP surface reducing the detachment of ions, as molecules (calcium). Thus, it is suggested that their individual 413
356 well as the area for dissolution.17 The results of the zeta action in these different sites were complementary, resulting 414
357 potential support this hypothesis, as without any treatment, in improved surface resistance. 415
358 the HAPc sustained a negative zeta potential, which was due Fluoride (F) presented lower ability to reduce enamel loss in 416
359 to the prevalence of negatively charged species close to its the more aggressive model (43% vs. 84% of reduction); 417
360 surface (most likely the phosphate groups) and a preferential however, its protection was significantly improved when it 418
361 adsorption of potential determining ions from the solu- was combined with LPP or Sn (60% and 57%, respectively). The 419
362 tion.23,36 However, when HAPc was pretreated with the LPP lower protection of F in experiment 2b may be related either to 420
363 solution, the zeta potential became more negative, indicating the dissolution of fluoride deposits under more aggressive 421
364 the presence of more phosphate ions on the surface.23 When conditions12,40 or to the more noticeable remineralization 422
365 LPP was combined with F or Sn, further reduction in HAPc enhancement effect of fluoride under the less aggressive 423
366 dissolution was observed (75% and 78%, respectively). This challenge. For F + LPP, we suspect that both agents also had a 424
367 effect implies that gluconic acid added to the LPP + Sn and complementary effect, F by the formation of CaF2-like 425
368 F + Sn solutions prevented a reaction between Sn ions and precipitates and LPP by adsorbing on the remaining available 426
369 the LPP phosphate or fluoride groups37 by complexation positive sites, establishing a more resistant barrier against 427
370 with Sn ions. This complex is believed to be stable enough dissolution. The ability of the NaF solution to form CaF2-like 428
371 to protect the stannous activity in solution and thus allowing precipitates on the surface of enamel is suggested by the zeta 429
372 these compounds (Sn, F and LPP) to be available to interact potential analysis, where an increase in the HAPc negative 430
373 with the hydroxyapatite surface.38 F + Sn reduced HA dissolu- charge may be the result of the release of Ca2+ ions from these 431
374 tion by approximately 84% and the combination of F + LPP + Sn precipitates. In addition, Camara et al.41 hypothesized that in 432

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433 the presence of fluoride, sodium polyphosphates may also mind the limitations of the present in vitro investigation, we 493
434 retain charged ions of CaF+ and Ca2+ by replacement of Na+ in can conclude that sodium polyphosphate and stannous 494
435 its structure, and these species can be released into the chloride have the potential to increase sodium fluoride 495
436 medium under acidic conditions. Thus, we may also presume protection against enamel erosion. For dentine, no additional 496
437 that this increase in saturation of Ca2+ and F ions in the benefit was observed by combining the surface agents with 497
438 enamel surroundings during the acid challenge could fluoride. These findings could add new insights for the 498
439 have reduced the driving force of dissolution. formulation of oral care products for erosion prevention. 499
440 The synergistic effect of F + Sn is consistent with other 500
441 studies12,13 and it is thought to be either related to the deposition
442 of these ions on the dental surfaces, forming an acid resistant Acknowledgment 501
443 film of insoluble compounds, such as SnF3PO4, or its incorpo-
444 ration into the tooth structure, increasing its resistance.11,42 The authors wish to thank Adam Kelly, research technician at 502
445 In experiment 2b, the solution that reduced enamel loss the OHRI-Indiana University School of Dentistry, for the 503
446 the most (68%) was F + LPP + Sn and we may attribute this technical support. 504
447 finding to an additive protective action of the three agents,
448 which formed an even more resistant surface film; whereas
449 in experiment 2a, all the experimental groups, except LPP, Appendix A. Supplementary data 505
450 showed equivalent erosion protection. The more aggressive
451 model used in experiment 2b allowed for better discernment Supplementary data associated with this article can be 506
452 of the treatment effects when compared to experiment 2a. found, in the online version, at http://dx.doi.org/10.1016/j. 507
453 Regarding dentine, all F-containing groups reduced surface jdent.2015.01.007. 508
509
454 loss when compared to the negative control in both experi-
455 ments, but no additional benefits were observed when F was
456 combined with Sn and/or LPP. This lack of improvement in F references 510
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Journal of Dentistry (2015), http://dx.doi.org/10.1016/j.jdent.2015.01.007