Short communication

Effect of fluoride gels supplemented with sodiumtrimetaphosphate 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, Aracatuba, Sao Paulo, Brazil

3

a r c h i v e s o f o r a l b i o l o g y 5 9 ( 2 0 1 4 ) 3 3 6 – 3 4 0

a r t i c l e i n f o

Article history:

Accepted 20 December 2013

Keywords:

Fluoride gels

Tooth erosion

Tooth abrasion

Topical fluorides

Dental enamel

Polyphosphates

a b s t r a c t

Objective: This in vitro study aims to evaluate the effect of low fluoride (F) gel associate

sodium trimetaphosphate (TMP) on erosion with or without abrasion.

Design: Enamel blocks (4 mm � 4 mm) selected through surface hardness (SH) is divided

into five groups (n = 12): gel without F and TMP (placebo), gel containing 4500 ppm F (4500),

gel containing 4500 ppm F plus TMP5% (4500 TMP5%), gel containing 9000 ppm F (9000), and

gel containing 12,300 ppm F (acid gel). Those groups were additionally subdivided into

conditions of erosion (Ero) and of erosion plus abrasion (Ero/Abra). The blocks have under-

gone a single application of gel on the first day of the study. The erosion challenge was

produced by Sprite Zero1 for five minutes four times a day and abrasion was carried out by

machine brushing for 15 s. After the challenges, the surface hardness (%SH), wear and cross-

sectional 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.

Available online at www.sciencedirect.com

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1. Introduction

Erosion and erosive tooth wear refers to the chemical and

chemical-mechanical process that has become more pre-

valent 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

* Corresponding author at: Aracatuba Dental School, Sao Paulo State

Health, Rua Jose Bonifacio 1193, 16015-050 Aracatuba, SP, Brazil. Tel.:E-mail addresses: adelbem@foa.unesp.br, adelbem@pq.cnpq.br (A

0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reservehttp://dx.doi.org/10.1016/j.archoralbio.2013.12.007

progression. 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

University (UNESP), Department of Pediatric Dentistry and Public +55 18 3636 3314; fax: +55 015 18 3636 3332..C.B. Delbem).

d.

a r c h i v e s o f o r a l b i o l o g y 5 9 ( 2 0 1 4 ) 3 3 6 – 3 4 0 337

Utilisation of sodium trimetaphosphate (TMP) and fluoride

(F) association has shown decreased enamel demineralisa-

tion10,12–15 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.

2. Materials and methods

2.1. Experimental design

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 contain-

ing 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 stu-

died were: type of gel (5 types) and type of challenges (Ero and

Ero+Abra).

2.2. Gel formulation and determination of fluoride inproducts

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. Subse-

quently, 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 Comercio 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. Approxi-

mately 100 mg of each product was dissolved in deionized

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

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 determin-

ing the wear of enamel by profilometry, half of the surface of

each block was protected with nail varnish.

2.4. 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,

Ribeirao 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 Escovacao, Sao 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 profilo-

metry (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. Cross-

sectional 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

a r c h i v e s o f o r a l b i o l o g y 5 9 ( 2 0 1 4 ) 3 3 6 – 3 4 0338

2.7. 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.

Fig. 1 – Graphical representation of mean hardness as a functio

indicates comparison among groups in each depth (Student–Ne

deviations of means. (*) indicates no statistical difference amon

healthy, 9000, 4500 5%TMP and Acid gel ( p > 0.163). v: indicate

between the groups healthy, 4500, 9000 and Acid gel ( p > 0.116)

between the groups healthy, 4500, 9000, 4500 5%TMP and Acid

the other groups ( p < 0.001). (b) V: indicates equality between th

indicates equality between the groups healthy, 4500 5%TMP an

4500 5%TMP and Acid gel are similar statistically ( p = 0.339). §: i

4500 ( p = 0.158), 4500 and 9000 ( p = 0.074), 9000 and Acid gel ( p

Table 1 – Mean (SD) values of surface hardness (%SH), wear an(Ero and Ero+Abra) and groups.

Analysis

Placebo 4500

%SH Ero A�91.2a (1.3) A�83.6b (1.6)

Ero+Abra B�84.8a (1.6) B�79.0b (1.4)

Wear (mm) Ero A4.71a (0.80) A4.19b (0.70)

Ero+Abra B5.81a (0.47) B5.15b (0.35)

DKHN Ero A3609.6a (139.6) A2712.9b (81.0)

Ero+Abra B2240.1a (129.9) B1749.3b (158.2)

Different letters show significant differences in each analysis between gr

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.

n of depth according to the erosive challenges. Symbols

wman–Keuls, p < 0.05). Vertical bars indicate the standard

g groups. (a) g: indicates equality between the groups

s difference among group ( p = 0.037). b: indicates equality

, 4500 5%TMP and Acid gel ( p = 0.070). l: indicates equality

gel ( p > 0.478). &: indicates difference between Placebo and

e groups healthy, 4500 5%TMP and Acid gel ( p = 0.286). C:

d Acid gel ( p = 0.248). ¥: indicates that the healthy groups,

ndicates equality between the groups healthy, Placebo and

= 0.189).

d DKHN of the enamel according to conditions of challenge

Groups

9000 4500 5%TMP Acid gelA�77.9c (1.6) A�83.7b (2.2) A�74.0d (1.5)B�74.1c (2.3) B�78.6b (1.6) B�68.3d (1.4)

A3.95b (0.69) A2.95c (0.51) A4.11b (0.91)B5.22b (0.34) B3.78c (0.36) B5.33a,b (0.57)

A2320.3c (122.9) A2215.5d (64.5) A2321.5c,d (78.5)B1466.1c (128.2) B1161.6d (102.0) B1253.4d (100.5)

oups of gel (ANOVA 2-way, Student–Newman–Keuls’s test; p < 0.05).

a r c h i v e s o f o r a l b i o l o g y 5 9 ( 2 0 1 4 ) 3 3 6 – 3 4 0 339

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 com-

pounds 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 remineraliza-

tion.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 reminer-

alizing action in the remaining enamel.23–25 This reminer-

alization 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+

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 CaHPO4

0 and HF0.28

The diffusion of neutral ions into the enamel is thousand

times higher than charged ions enhancing the remineraliza-

tion 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.

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