UNIVERSIDADE ESTADUAL PAULISTA
“JÚLIO DE MESQUITA FILHO”
Instituto de Ciência e Tecnologia
Campus de São José dos Campos
ORIGINAL ARTICLE DOI: https://doi.org/10.4322/bds.2023.e3767
1
Braz Dent Sci 2023 Apr/Jun;26 (2): e3767
The ability of different formulations of artificial saliva to protect dentin
from erosive wear
Ação protetora de diferentes formulações de saliva artificial no desgaste erosivo
Graziela Ribeiro BATISTA
1
, Rayssa Ferreira ZANATTA
2
, Marina Gullo AUGUSTO
3
, Gabriela Souza ARANTES
4
,
Alessandra Bühler BORGES
4
, Carlos Rocha Gomes TORRES
4
1 - A.T. Still University, Missouri School of Dentistry and Oral Health. City- Kirksville, Missouri, USA.
2 - Universidade de Brasília, Faculdade de Ciências da Saúde, Departamento de Odontologia. Brasília, Distrito Federal, Brazil.
3 - Centro Universitário de Cascavel. Cascavel, Paraná, Brazil.
4 - Universidade Estadual Paulista, Instituto de Ciência e Tecnologia, Departamento de Odontologia Restauradora. São José dos Campos, Brazil.
How to cite: Batista GR, Zanatta RF, Augusto MG, Arantes GS, Borges AB, Torres CRG. The ability of different formulations of articial
saliva to protect dentin from erosive wear. Braz Dent Sci. 2023;26(2):e3767. https://doi.org/10.4322/bds.2023.e3767
ABSTRACT
Objective: Evaluate the protective effect of articial saliva formulations associated or not with mucin on dentin.
Materials and Methods: Bovine dentin specimens were randomly allocated to 10 groups (n = 20) according
to the articial saliva tested and the presence or absence of mucin: Amaechi et al. (1998); Klimek et al. (1982);
Vieira et al. (2005) and Eisenburger et al. (2001) and deionized water (control). Samples were submitted to
an erosive cycle consisting of two immersions of 120 min in the saliva, followed by 1 min in hydrochloric acid
solution, and new storage in saliva for 120 min. Surface loss (μm) was measured before and after the cycle. Data
were analyzed using 2-way ANOVA and Tukey’s test (p < 0.05). Results: A signicant difference was observed
for the saliva formulation but not for the presence of mucin. The deionized water provided the highest surface
loss and the Eisenburger’s saliva formulation the lowest. The groups testing the Amaechi, Klimek, and Vieira
saliva did not present signicant differences. Conclusion: Eisenburger’s saliva formulation provides a higher
protective effect against dentin erosion. The presence of mucin did not increase the erosion-preventive effect of
articial saliva formulations.
KEYWORDS
Articial saliva; Dental erosion; Dental Wear; Prolometry; Surface loss.
RESUMO
Objetivo: Avaliar o efeito protetor de formulações de saliva articial associadas ou não à mucina sobre a dentina
submetida a erosão. Material e Métodos: Espécimes de dentina bovina foram alocados em 10 grupos (n = 20)
de acordo com a saliva testada e a presença ou ausência de mucina: . Amaechi et al. (1998); Klimek et al. (1982);
Vieira e cols. (2005), Eisenburger et al (2001) e agua deionizada (controle). As amostras foram submetidas a um
ciclo erosivo composto por duas imersões de 120 min na saliva, seguidas de 1 min em solução de ácido clorídrico
e novo armazenamento na saliva por 120 min. A perda de superfície (μm) foi medida antes e depois do ciclo. Os
dados foram analisados usando ANOVA 2 fatores e teste de Tukey (p <0,05). Resultados: Foi observada diferença
signicativa para a formulação de saliva, mas não para a presença de mucina. A água deionizada proporcionou
a maior perda de superfície e a formulação de saliva de Eisenburger a menor. Os grupos que testaram a saliva
Amaechi, Klimek e Vieira não apresentaram diferenças signicativas entre si. Conclusão: A formulação de saliva
de Eisenburger fornece o maior efeito protetor contra a erosão dentinária e a presença de mucina não aumentou
o efeito preventivo de erosão de formulações de saliva articial.
PALAVRAS-CHAVE
Erosão dental; Desgaste dental; Perda de estrutura; Perlometria; Saliva articial.
2
Braz Dent Sci 2023 Apr/Jun;26 (2): e3767
Batista GR et al.
The ability of different f ormulations of artificial saliva to pr otect dentin from er osive wear
Batista GR et al.
The ability of different formulations of artificial saliva to protect
dentin from erosive wear
INTRODUCTION
Erosive tooth wear (ETW) is a multifactorial
condition resulting from a chemical-mechanical
process involving the dissolution of enamel and
dentin by non-bacterial acids and modulated by
several behavioral and biological aspects [1].
Dental surface loss due to erosive conditions is
considered a frequent condition, mainly among
children and young adults [2-4], enhancing the
scientic community’s interest in this topic and
encouraging the development of preventive
measures and treatment options.
Saliva is considered the main biological factor
responsible for modulating the development of
ETW lesions [5]. It acts as a reservoir of ions
responsible for remineralizing the dental tissues
softened by extrinsic or intrinsic acids. It also
functions as a diluent of them and promotes their
buffering, reducing surface loss [6,7]. Most of the
evidence in dental erosion is obtained by
in vitro
studies. These set-ups allow standardization of the
study variables under controlled situations, reduce
costs compared to clinical studies, and enable
a rapid assessment of products or treatments
without considering ethical aspects [8].
Ideally,
in vitro
experiments should mimic
the clinical conditions to produce results
comparable to the clinical situations [9];
therefore, in 1982, Klimek et al. [10] described
an artificial saliva formulation to be used in
erosion experiments [10], and later, different
formulations were proposed, such as the ones from
Amaechi et al. (1998) [11], Vieira et al. (2005) [12]
and Eisenburger et al. (2001) [13]. To make the
articial saliva consistency more similar to natural
human saliva, some formulas add mucin to the
mixture, an important component of the salivary
pellicle and the main lubricant constituent of
saliva [5]. This aims to increase the representativity
of the
in vitro
experiments and facilitate the
performance of such experiments since there
would be no need to collect human saliva from
volunteers. However, there are a few suggestions
that using articial saliva formulations and human
saliva in
in vitro
experiments does not reect the
actual intraoral situation [8] and that the presence
of mucin in the articial saliva formulation may
increase its remineralizing effect [14].
Therefore, this study aimed to evaluate the
dentin erosion-preventive effect of articial saliva
formulations used in previous studies [11-13]
with or without mucin. The null hypotheses
tested were that the different articial saliva
formulation does not influence the dentin
erosive surface loss and that the presence of
mucin in the formulation does not improve its
protective effect.
MATERIAL AND METHODS
Specimen preparation and allocation to the
groups
Fresh and sound bovine incisors were
collected for this study. The crowns were separated
from the roots and stored in 0.5% thymol solution
at 4
o
C until use [15]. Two hundred cylindrical
dentin specimens (3 mm diameter) were obtained
from the roots using a diamond-coated trephine
mill. The specimens were embedded in acrylic
resin (diameter: 6 mm, height: 3 mm; JET,
Classico, Sao Paulo, Brazil). The external surfaces
of the specimens were ground at and polished
using silicon carbide paper in sequential grits of
1200, 2400, and 4000 (Extec Corp, CT, USA) in
a polishing device (DP-10, Panambra Industrial
e Técnica SA, Sao Paulo, SP, Brazil) under water
irrigation for 30, 60 and 120 s, respectively.
After each paper grit change, specimens were
kept in ultrasonic baths in distilled water for
10 min to remove debris and abrasive grains.
The prepared samples were examined under a
stereomicroscope (Discovery V20, Karl Zeiss,
Jena, Germany) to ensure the absence of cracks
or other surface defects. After preparation, the
specimens were stored at 100% relative humidity
at 4
o
C to avoid dehydration.
Articial saliva formulations were prepared
according to the descriptions in the previous
studies: Amaechi et al. (1998) [11], Klimek et al.
(1982) [10], Vieira et al. (2005) [12], and
Eisenburger et al. (2001) [13]. Table I shows the
composition of the articial saliva formulations.
Each formulation was made with and without
mucin, as shown in Figure 1.
After preparing artificial saliva, the
specimens were randomly assigned to ten
groups (n = 20) according to the storage media
(Figure 1). The control group was deionized
water (negative control).
Surface loss assessment and erosive cycling
In each specimen, two parallel groves were
made to provide a reference for the surface loss
3
Braz Dent Sci 2023 Apr/Jun;26 (2): e3767
Batista GR et al.
The ability of different f ormulations of artificial saliva to pr otect dentin from er osive wear
Batista GR et al.
The ability of different formulations of artificial saliva to
protect dentin from erosive wear
Table I - Composition of tested artificial saliva formulations
Compound
Artificial saliva formulations
Klimeketal. [10] Vieiraetal. [12] Amaechietal. [11] Eisenburgeretal. [13]
C
6
H
8
O
6
2 mg/l
C
6
H
12
O
6
30 mg/l
NaCl 580 mg/l
CaCl
2
170 mg/l
NH
4
Cl 160 mg/l
KCl 1270 mg/l 11182.50 mg/l 624.73 mg/l 2236.50 mg/l
NaSCN 160 mg/l
KH
2
PO
4
330 mg/l 326.62 mg/l 544.36 mg/l
CH
4
N
2
O 200 mg/l
Na
2
HPO
4
340 mg/l
Ca(NO
3
)
2
.4H
2
O 60.12 mg/l
NaF 0.066 mg/l
NaH
2
PO
4
.2H
2
O 160.19 mg/l
C
4
H
11
NO
3
Tris Buffer 12114.00 mg/l
K
2
HPO
4
804.712 mg/l
CaCl
2.
2H
2
O 166.130 mg/l 77.690 mg/l
C
8
H
8
O
3
2000 mg/l
CMC-Na 10000 mg/l
MgCl
2
.6H
2
O 58.96 mg/l
MgCl
2
19.04 mg/l
C
8
H
18
N
2
O
4
S HEPES 4766.20 mg/l
Mucin* 2700 mg/l 2700 mg/l 2700 mg/l 2700 mg/l
Deionized water 1000 ml 1000 ml 1000 ml 1000 ml
pH 6.4 7.0 6.75 7.0
*Only in the groups containing mucin.
Figure 2 – Schematic chart of the steps performed in the study.
Figure 1 – Specimen allocation according to the storage media.
determination (prolometry), as shown in Figure 2.
The baseline proles of each specimen were obtained
using a contact prolometer (MaxSurf XT 20,
Mahr-Goettingen, Germany). The diamond stylus
moved from the rst groove in the acrylic resin
to the second one (4.2 mm long). Three prole
measurements were performed for each specimen
at intervals of 0.25 mm.
4
Braz Dent Sci 2023 Apr/Jun;26 (2): e3767
Batista GR et al.
The ability of different f ormulations of artificial saliva to pr otect dentin from er osive wear
Batista GR et al.
The ability of different formulations of artificial saliva to protect
dentin from erosive wear
After obtaining the baseline profiles,
the erosive cycling was performed under
agitation (40 rpm) and at room temperature.
The specimens of each group were stored in the
respective articial saliva for 120 min. Then,
they were immersed for 1 min in a hydrochloric
acid solution (2.5 mmol/l, pH = 2.6).
The specimens were washed with deionized
water (pH = 5.5) for 10 s. Additional storage
in the respective articial saliva (120 min) and
erosive challenge (1 min) were performed [8].
After the second erosive challenge, the nal
proles of each specimen were obtained using
the same parameters previously described.
Figure 2 shows a schematic chart illustrating
the erosive cycle.
At the end of the cycling, a second prole
reading was performed in each specimen using
the same parameters described above. The initial
and final profiles were overlapped, and the
dentin loss was calculated by the difference in
height between them using dedicated software
(MarSurf XCR 20 4.50-07 SP3, 2011).
Statistical analysis
The assumption of normal distribution
(Kolmogorov-Smirnov test) was checked for
the variable tested. Descriptive and inferential
statistical analyzes were performed using
SigmaPlot 13 (Systat Software Inc, San Jose, CA, EUA).
Two-way ANOVA was performed for prolometry
analysis, followed by Tukey’s test with 5%
signicance.
RESULTS
The results of 2-way ANOVA showed a
signicant difference (p < 0.05) for the different
articial saliva formulations (p = 0.0001) and the
interaction between the two factors (p = 0.040),
but not for the presence of mucin (p = 0.360).
Tukey’s test revealed that the deionized water
without mucin (control group) provided the highest
surface loss compared with the other formulations.
The lowest surface loss was observed for the
Eisenburger’s saliva formulation, while the groups
testing the formulas from Amaechi, Klimek, and
Vieira presented similar surface loss. The mean
surface loss (μm), standard deviations, and results
of Tukey’s test are shown in Figure 3.
DISCUSSION
This study showed that the different articial
saliva formulations signicantly inuenced the
erosive surface loss but not by the presence
of mucin. Hence, the rst null hypothesis was
denied, and the second one was accepted.
Hydrochloric acid is a strong inorganic acid,
easily ionizable in an aqueous solution and resistant
to buffering by saliva, making it a potent agent
to erode dental tissues and cause surface loss.
It was used in the present study in short erosive
events (1 min) aiming to simulate patients with
gastroesophageal disorders, such as GERD or
bulimia [16]. Also, the storage in articial saliva for
120 minutes aimed to optimize the remineralization
process as proposed previously [17].
Figure 3 – The mean surface loss (µm), standard deviations, and results of Tukey’s test for the artificial saliva, with or without the presence of mucin.
Different letters mean significant difference among groups.
5
Braz Dent Sci 2023 Apr/Jun;26 (2): e3767
Batista GR et al.
The ability of different f ormulations of artificial saliva to pr otect dentin from er osive wear
Batista GR et al.
The ability of different formulations of artificial saliva to
protect dentin from erosive wear
When acid comes into contact with dentin,
it dissolves the mineral phase, but the organic
part remains as a spongy and demineralized
structure that acts as a physical barrier for all
chemical processes, including active ingredients
from preventive measures and remineralizing
agents [18]. It is suggested that in
in vivo
situations,
the exposed collagen matrix can be broken down
by collagenases and proteolytic enzymes found
in the oral uids [18,19] or through abrasion
such as toothbrushing. However, in this study,
salivary collagenases and toothbrushing were not
performed, so this collagen barrier resultant from
the erosive process might have remained intact
and helped to modulate the remineralization
by the ions from some of the artificial saliva
formulations. Still, it is suggested that the collagen
brils of organic matrix exposed during erosive or
carious events house some prenucleation mineral
crystals that aggregate into larger amorphous
calcium phosphate (ACP) nanoparticles and
induce mineral formation [20] in the presence of
remineralizing ions, such as calcium, phosphate,
or magnesium.
From the solutions tested, the articial
saliva formulation described by Eisenburger
presented the lowest values of surface loss,
which might have been due to the presence
of-HEPES (C
8
H
18
N
2
O
4
S), an organic chemical
buffer known as zwitterionic sulfonic acid and
classied as a good buffer [21], thus able to
neutralize the H
+
ions from the dissociation
of HCl and promote an anti-erosive effect.
The groups testing the formulation from
Amaechi, Klimek, and Vieira did not differ
from each other, even though the last one
also presented a buffer in its composition
(C
4
H
11
NO
3
- Tris Buffer), and the possible
explanation for that relies on the different
degrees of saturation with respect of calcium
phosphates in each solution [8].
Regarding the groups in which mucin was
included, it has been reported that they contribute to
a large extent to the protective effect of the acquired
pellicle against enamel erosion [22]. However,
this study showed that mucin did not signicantly
reduce dentin erosive surface loss. Even for the
comparison between the control group (deionized
water) with and without mucin, the absence of
signicant differences indicated that this protein
was not able to protect dentin against erosion.
This may be because the surface of eroded enamel
presents high mineral content, whereas the mesh
of collagen matrix from eroded dentin might have
interfered with the deposition of mucin, thus reducing
its protective effect against erosion. Although, a
previous study found that the anti-erosive protection
of the acquired pellicle in dentin is lower than in
enamel [23]. Moreover, it is essential to consider
that the behavior of the synthetic mucin used in
in
vitro
studies may be different from the mucin found
in human saliva. Its stability in the formulations
and the combination with the other components
may be able to cause differences from the natural
human saliva.
Thus, the result from the present study
suggested that the protection of dentin submitted
to erosive challenges is affected by the formulation
of the articial saliva tested, and future studies,
including the comparison with human saliva in
in
situ
protocols, are encouraged, aiming to nd the
best suitable formula for in vitro analysis. It can
be concluded that the protective effect of different
articial saliva against erosion must be considered
to avoid incorrect inferences in
in vitro
studies.
The presence of mucin did not provide a signicant
reduction in the erosive surface loss of dentin.
Author’s Contributions
GRB: Conceptualization. GRB: Methodology.
RFZ: Formal Analysis. GRB, MGA, GSA:
Investigation. GRB, MGA, GSA: Data Curation.
GRB, RFZ: Writing – Original Draft Preparation.
ABB, CRGT: Writing – Review & Editing. ABB,
CRGT: Visualization. ABB, CRGT: Supervision.
CRGT: Project Administration. CRGT: Funding
Acquisition.
Conict of Interest
The author’s disclosure any conflict of
interest.
Funding
This research was supported by National
Council for Scientific and Technological
Development (CNPq).
Regulatory Statement
The authors have no proprietary, nancial,
or other personal interest of any nature or kind
in any product, service, and/or company that is
presented in this article.
6
Braz Dent Sci 2023 Apr/Jun;26 (2): e3767
Batista GR et al.
The ability of different f ormulations of artificial saliva to pr otect dentin from er osive wear
Batista GR et al.
The ability of different formulations of artificial saliva to protect
dentin from erosive wear
REFERENCES
1. Carvalho TS, Colon P, Ganss C, Huysmans MC, Lussi A,
Schlueter N,etal. Consensus report of the European Federation
of Conservative Dentistry: erosive tooth wear—diagnosis
and management. Clin Oral Investig. 2015;19(7):1557-61.
http://dx.doi.org/10.1007/s00784-015-1511-7. PMid:26121968.
2. Martignon S, Bartlett D, Manton DJ, Martinez-Mier EA,
Splieth C, Avila V. Epidemiology of erosive tooth wear,
dental fluorosis and molar incisor hypomineralization in the
American continent. Caries Res. 2021;55(1):1-11. http://dx.doi.
org/10.1159/000512483. PMid:33440378.
3. Salas MMS, Nascimento GG, Huysmans MC, Demarco FF.
Estimated prevalence of erosive tooth wear in permanent teeth
of children and adolescents: an epidemiological systematic
review and meta-regression analysis. J Dent. 2015;43(1):42-50.
http://dx.doi.org/10.1016/j.jdent.2014.10.012. PMid:25446243.
4. Salas MMS, Nascimento GG, Vargas-Ferreira F, Tarquinio
SBC, Huysmans MCDNJM, Demarco FF. Diet influenced tooth
erosion prevalence in children and adolescents: results of a
meta-analysis and meta-regression. J Dent. 2015;43(8):865-75.
http://dx.doi.org/10.1016/j.jdent.2015.05.012. PMid:26057086.
5. Buzalaf MAR, Hannas AR, Kato MT. Saliva and dental erosion. J
Appl Oral Sci. 2012;20(5):493-502. http://dx.doi.org/10.1590/
S1678-77572012000500001. PMid:23138733.
6. 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.
http://dx.doi.org/10.1159/000047459. PMid:11385203.
7. Hara AT, Zero DT. The potential of saliva in protecting against
dental erosion. Monogr Oral Sci. 2014;25:197-205. http://dx.doi.
org/10.1159/000360372. PMid:24993267.
8. Batista GR, Torres CRG, Sener B, Attin T, Wiegand A. Artificial
saliva formulations versus human saliva pretreatment in dental
erosion experiments. Caries Res. 2016;50(1):78-86. http://dx.doi.
org/10.1159/000443188. PMid:26870948.
9. Attin T, Wegehaupt FJ. Methods for assessment of dental
erosion. Monogr Oral Sci. 2014;25:123-42. http://dx.doi.
org/10.1159/000360355. PMid:24993262.
10. Klimek J, Hellwig E, Ahrens G. Fluoride taken up by plaque, by
the underlying enamel and by clean enamel from three fluoride
compounds in vitro. Caries Res. 1982;16(2):156-61. http://dx.doi.
org/10.1159/000260592. PMid:6951638.
11. Amaechi BT, Higham SM, Edgar WM. Use of transverse
microradiography to quantify mineral loss by erosion in
bovine enamel. Caries Res. 1998;32(5):351-6. http://dx.doi.
org/10.1159/000016471. PMid:9701660.
12. Vieira AEM, Delbem ACB, Sassaki KT, Rodrigues E, Cury JA,
Cunha RF. Fluoride dose response in pH-cycling models using
bovine enamel. Caries Res. 2005;39(6):514-20. http://dx.doi.
org/10.1159/000088189. PMid:16251798.
13. Eisenburger M, Addy M, Hughes JA, Shellis RP. Effect of time
on the remineralisation of enamel by synthetic saliva after
citric acid erosion. Caries Res. 2001;35(3):211-5. http://dx.doi.
org/10.1159/000047458. PMid:11385202.
14. Ionta FQ, Mendonça FL, Oliveira GC, Alencar CRB, Honório
HM, Magalhães AC,etal. In vitro assessment of artificial saliva
formulations on initial enamel erosion remineralization. J Dent.
2014;42(2):175-9. http://dx.doi.org/10.1016/j.jdent.2013.11.009.
PMid:24269764.
15. Attin T, Meyer K, Hellwig E, Buchalla W, Lennon A. Effect of
mineral supplements to citric acid on enamel erosion. Arch
Oral Biol. 2003;48(11):753-9. http://dx.doi.org/10.1016/S0003-
9969(03)00156-0. PMid:14550377.
16. Wiegand A, Bliggenstorfer S, Magalhães AC, Sener B,
Attin T. Impact of the
in situ
formed salivary pellicle on
enamel and dentine erosion induced by different acids.
Acta Odontol Scand. 2008;66(4):225-30. http://dx.doi.
org/10.1080/00016350802183401. PMid:18607835.
17. Alencar CRB, Mendonça FL, Guerrini LB, Jordão MC, Oliveira
GC, Honório HM, et al. Effect of different salivary exposure
times on the rehardening of acid-softened enamel. Braz
Oral Res. 2016;30(1):e104. http://dx.doi.org/10.1590/1807-
3107BOR-2016.vol30.0104. PMid:27737358.
18. Ganss C, Lussi A, Schlueter N. The histological features and
physical properties of eroded dental hard tissues. Monogr Oral
Sci. 2014;25:99-107. http://dx.doi.org/10.1159/000359939.
PMid:24993260.
19. Schlueter N, Hardt M, Klimek J, Ganss C. Influence of the
digestive enzymes trypsin and pepsin in vitro on the progression
of erosion in dentine. Arch Oral Biol. 2010;55(4):294-9.
http://dx.doi.org/10.1016/j.archoralbio.2010.02.003.
PMid:20197186.
20. He L, Hao Y, Zhen L, Liu H, Shao M, Xu X,etal. Biomineralization
of dentin. J Struct Biol. 2019;207(2):115-22. http://dx.doi.
org/10.1016/j.jsb.2019.05.010. PMid:31153927.
21. Good NE, Winget GD, Winter W, Connolly TN, Izawa S, Singh
RMM. Hydrogen ion buffers for biological research. Biochemistry.
1966;5(2):467-77. http://dx.doi.org/10.1021/bi00866a011.
PMid:5942950.
22. Amerongen AVN, Oderkerk CH, Driessen AA. Role of mucins
from human whole saliva in the protection of tooth enamel
against demineralization in vitro. Caries Res. 1987;21(4):297-309.
http://dx.doi.org/10.1159/000261033. PMid:3475175.
23. Hall AF, Buchanan CA, Millett DT, Creanor SL, Strang R, Foye
RH. The effect of saliva on enamel and dentine erosion.
J Dent. 1999;27(5):333-9. http://dx.doi.org/10.1016/S0300-
5712(98)00067-0. PMid:10377607.
Graziela Ribeiro Batista
(Corresponding address)
A.T. Still University, Missouri School of Dentistry and Oral Health,
Kirksville, MO, USA.
Email: grazielabatista@atsu.edu
Date submitted: 2023 Jan 24
Accept submission: 2023 Apr 04
