Archives of Oral Biology 154 (2023) 105755

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Archives of Oral Biology

journal homepage: www.elsevier.com/locate/archoralbio

Erosion-inhibiting and enamel rehardening effects of different types

of saliva
Ana Paula Boteon a, Natália Mello dos Santos a, Larissa Di Bene Kandalaf Lamana a,
Isadora Messias Batista Rosa a, Camilla Cristina Lira Di Leone a, Rafaela Aparecida Caracho a,
Thiago Saads Carvalho b, Heitor Marques Honório a, Daniela Rios a, *
a
Department of Pediatric Dentistry, Orthodontics and Public Health, Bauru School of Dentistry - University of São Paulo, Bauru, SP, Brazil
b
Department of Preventive, Restorative and Pediatric Dentistry, University of Berne, Berne, Switzerland

A R T I C L E I N F O A B S T R A C T

Keywords: Objectives: The objective of this study was to assess the effects of in situ saliva compared to in vitro human saliva,
Saliva with or without mucin, on inhibiting erosion and promoting enamel rehardening.
Mucin Design: Bovine enamel blocks were randomly distributed into groups (n = 23): Gsitu (human saliva in situ), Gvitro
Dental pellicle
(collected human saliva) and GvitroM (collected human saliva with mucin). The enamel blocks underwent a 2-
Tooth erosion
hour period for the formation of salivary pellicle, based on the assigned groups. Subsequently, they were sub­
jected to three erosive cycles, each of them consisting of an erosive challenge (immersion in 0.65 % citric acid,
pH 3.5, 1 min) and saliva exposure (immersion in situ or in vitro saliva for 2 h). Microhardness measurements
were performed at each cycle, after each experimental step (erosive challenge and exposure to saliva).
Results: After the first demineralization, in vitro saliva groups presented greater hardness loss, with no statistical
difference between GVitroM and GVitro. After the third erosive demineralization the in situ saliva resulted in less
hardness loss compared to the first demineralization. In relation to surface hardness recovery, there was no
difference among types of saliva but there was a decrease in hardness as the cycles progressed.
Conclusion: Saliva groups had different behaviors between the first and third demineralization, being similar after
the third cycle in terms of hardness loss. Regarding hardness recovery, all saliva promoted enamel gain, but there
was a gradual decrease with the progression of the cycles.

1. Introduction
Saliva is the main responsible for the homeostasis in the oral envi­
ronment (Dawes, 2008). Many post-eruptive alterations affecting dental
enamel result from the imbalance between aggressive factors and the
salivary protective effect (Dawes, 2008). One of these alterations that
demands attention due to its consequences is the erosive tooth wear
(Schlueter et al., 2020). Extrinsic and intrinsic acids from nonbacterial
origin can contact enamel causing superficial demineralization without
loss of structure called dental erosion (Schlueter et al., 2020). At this
stage, demineralization can be prevented or diminished by acid dilution,
buffering or ions supersaturation (calcium, phosphate and fluoride) by
saliva (Hara & Zero, 2014). In addition, the presence of acquired enamel
pellicle can act as a semi-permeable barrier reducing direct contact of
the acid with the tooth (Hannig & Hannig, 2014; Vukosavljevic et al.,
2014). In a next stage, prolonged acid challenge with repeated softening
events combined with oral mechanical forces can result in loss of tooth
structure, corresponding to erosive tooth wear (Schlueter et al., 2020).
Furthermore, saliva can counteract the effects of acid and mechanical
impact by supplying the essential calcium, phosphate, and fluoride ions
for mineral redeposition (Hara & Zero, 2014). Additionally, it aids in
lubrication due to the presence of the acquired enamel pellicle (Hara &
Zero, 2014). Therefore, saliva plays a direct role in influencing erosive
tooth wear and is considered the most significant natural protective
factor in its multifactorial etiology (Hara & Zero, 2014).
Nowadays the change in lifestyle and nutritional habits have led to a
high prevalence of erosive dental wear (Schlueter & Tveit, 2014). The
results of an epidemiological systematic review and meta-regression

* Correspondence to: Bauru School of Dentistry- University of São Paulo (FOB-USP), Departmentof Pediatric Dentistry, Orthodontics and Public Health, Alameda
Octávio Pinheiro Brisolla, 9-75, Vila Universitária, 17012-901 Bauru, SP, Brazil.
E-mail address: daniriosop@yahoo.com.br (D. Rios).

https://doi.org/10.1016/j.archoralbio.2023.105755
Received 11 April 2023; Received in revised form 9 June 2023; Accepted 20 June 2023
Available online 22 June 2023
0003-9969/© 2023 Published by Elsevier Ltd.
A.P. Boteon et al.
analysis estimated that the prevalence of erosive tooth wear in perma­
nent teeth of children and adolescents is around 30 % (Salas et al.,
2015). This scenario raises concern and has intensified the demand for
strategies to control and prevent erosive tooth wear.
Well-designed randomized clinical trials are considered the gold
standard for studying treatment and prevention of diseases, aiming to
address specific questions. However, conducting such trials in erosive
tooth wear is challenging due to the limited availability of validated
alternatives for quantitative evaluation of dental tissues over time
(Arends & ten Bosch, 1992; Brevik, Lussi, & Rakhmatullina, 2013).
Although profilometric measurements of casts have been used in
research, together with software to align the reading areas, to ultimately
analyze wear change, these methods still show some limitations (Alar­
audanjoki et al., 2017; Marro et al., 2018; Marro et al., 2020; O’Toole
et al., 2019). In addition, intra-oral scanners are a promising instrument
Fig. 1. Experimental Design.
2
Archives of Oral Biology 154 (2023) 105755
to be potentially used for the clinical diagnosis and monitoring of ETW
(Alaraudanjoki et al., 2017; Marro et al., 2018). However, these alter­
natives are not yet a reality for clinical use. Therefore, most of the evi­
dence on strategies for the prevention and control of dental erosion are
originated from in vitro and in situ studies (Buzalaf et al., 2014; Huys­
mans et al., 2011; Huysmans et al., 2014; West et al., 2011). However,
both of them are not able to mimic the oral environment with all of the
biological variations known to influence erosive tooth wear (West et al.,
2011).
In vitro models are suitable for the initial stages of hypothesis testing
or product development because they allow testing isolated experi­
mental factors in a relatively short experiment time, with low operating
cost and few staff, and not depending on participant compliance (Hara
et al., 2008; West et al., 2011). Their main limitation is the impossibility
to expose the specimens to the oral environment and saliva (Ionta et al.,
A.P. Boteon et al.
2014). To overcome this restriction, some studies use pooled human
saliva between erosive challenges. Nevertheless, collection and storage
may alter the salivary effect because of protein degradation (Schipper
et al., 2007). Mucins are important components of saliva and acquired
enamel pellicle (Buzalaf et al., 2014; Nieuw Amerongen et al., 1987).
They have been identified as an acid-resistant protein, since they were
still present in the acquired pellicle after exposure to citric acid (Dele­
crode et al., 2015). In addition, when submandibular/sublingual saliva
was depleted of mucins, acquired enamel pellicle protection against
demineralization by citric acid diminished 70 % (Nieuw Amerongen
et al., 1987). Therefore, mucin might be involved on the salivary pro­
tective effect. This protein has been added to artificial saliva (Ionta et al.,
2014; Jordão et al., 2017), but was not previously tested when added to
pooled human saliva. Accordingly, the present study aimed to investi­
gate the erosion-inhibiting and enamel rehardening effects of in situ
saliva compared to in vitro pooled human saliva with or without addi­
tional mucin.
2. Materials and methods
2.1. Experimental design
This study was conducted according to the Declaration of Helsinki.
The protocol was approved by the local Research Ethics Committee
(Protocol 49810015.8.0000.5417). The volunteers signed an informed
consent form before the confirmation of their eligibility for the study.
Bovine enamel blocks were selected from the initial superficial
microhardness (initial SMH) and randomly distributed into three groups
(n = 23): Gsitu (human saliva in situ, positive control), Gvitro (collected
human saliva – in vitro) and GvitroM (collected human saliva with mucin
– in vitro) (Fig. 1). A sample size of 18 blocks per group was estimated
considering a α error of 5 %, β error of 20 %, 7,35 % standard deviation
(Jordão et al., 2017) and a 10 % minimum detectable difference in the
means of enamel hardening. Five additional blocks were used in case of
eventual losses, totaling 69 enamel blocks. The blocks were subjected to
a salivary pellicle formation period of 2 h, according to the group (Gsitu,
Gvitro, GvitroM). Then they were subjected to 3 erosive cycles, each of
them composed of an erosive challenge and a saliva exposure period. For
the erosive challenges, the enamel blocks were individually immersed in
1.8 ml of 0.65 % citric acid solution (Sigma-Aldrich, Germany), pH 3.5,
for 1 min (ratio between the enamel block and the solution was 0.03),
under constant agitation, at controlled room temperature (24 ◦ C). After
that, the enamel blocks were washed with deionized water for two mi­
nutes to interrupt the demineralization. The erosive challenges were all
made in vitro. For the saliva exposure, the blocks were either placed in
contact with saliva in situ, for the Gsitu group, or in vitro, for the Gvitro
and GvitroM groups. For Gsitu, the volunteers used an intraoral device
for 2 h always in the morning. For the human saliva in vitro groups
(Gvitro and GvitroM), the enamel blocks were individually placed in
Eppendorfs containing 1.8 ml of saliva for 2 h, at controlled room
temperature (24 ºC). It is important to emphasize that in each remi­
neralization cycle, freshly collected saliva was used. Microhardness
measurements were performed at each cycle, after each experimental
step (erosive challenge and exposure to saliva).
2.2. Enamel blocks preparation
One hundred and ten enamel blocks (4×4×3 mm) were prepared
from the labial surfaces of bovine incisors crowns. They were cut with an
ISOMET low speed machine (ISOMET Low Speed Saw, Buehler Ltd.,
Lake Bluff, IL, USA) with two double-sided diamond discs (XL 12205,
“High concentration”, 102 × 0.3 × 12.7 mm3 Extec Corp., Enfield, CT,
USA/Ref: 12205), which were separated by a 4-mm thick spacer. Then,
the enamel blocks were ground flat with water-cooled silicon carbide
discs (320, 600, and 1200 grade papers; Buehler, Lake Bluff, IL, USA),
and polished with felt paper wet by 1-μm diamond spray (Buehler, Ltd.,
3
Archives of Oral Biology 154 (2023) 105755
Lake Bluff, IL, USA). The blocks were cleaned using an ultrasonic device
(T7 Thornton, Unique Ltda., São Paulo, SP, Brazil) with deionized water
for 2 min between each disc change and for 10 min at the end of the
polishing procedures. The enamel blocks were sterilized with ethylene
oxide.
2.3. Saliva groups
Ten healthy volunteers (aged 19–30 years) residing in the same
fluoridated area (0.70 mg F / l) participated in the study, after satisfying
the following inclusion criteria: physiological stimulated salivary flow
rate (> 1 ml / min) and physiological unstimulated salivary flow rate (>
0.25 ml / min). The volunteers did not present erosive tooth wear, un­
treated carious lesions, periodontitis, systemic diseases, pregnancy or
breastfeeding, gastro-esophageal reflux, frequent regurgitation and/or
vomiting, smoking, or high concentration fluoride topical application in
the past two months.
For the in situ human saliva group (Gsitu), an intraoral palatal device
was custom-made using acrylic resin for each volunteer. The device had
two sites (6 × 6 × 3 mm) where one enamel block could be fixed per
site. Only three volunteers had three blocks on their device. An ortho­
dontic wire was attached to the ends of the sites, positioned over the
enamel blocks without making direct contact, to prevent abrasion of the
enamel by the tongue or oral mucosa (Mendonça et al., 2017).
For the in vitro human saliva groups (Gvitro and GvitroM), the same
volunteers donated stimulated saliva. They were informed not to eat or
drink (except water) for 2 h before the saliva collection, performed in
the morning. The volunteers chewed on a paraffin wax for 10 min, and
all the stimulated saliva was collected in chilled vials. After the collec­
tion, the saliva was pooled and centrifuged for 15 min at 4 ◦ C (4000g).
The supernatant was divided in small aliquots and stored at − 80 ◦ C until
use (Brevik et al., 2013). The saliva samples were collected in cooled
flacons, and the amount of saliva from each volunteer varied according
to the number of enamel blocks present in their intraoral device (7
volunteers donated 30 ml, and 3 volunteers donated 45 ml). Immedi­
ately after collection, saliva was centrifuged at 4000 rpm / 6 ◦ C for
15 min (Brevik et al., 2013). In the human saliva with mucin group
(GvitroM), after centrifugation, 2.7 g of porcine mucin (Mucin from
Porcine Stomach – type II, Sigma-Aldrich, Saint Louis, MO, USA) (Ionta
et al., 2014; Jordão et al., 2017) per liter of solution were added to its
supernatant portion.
2.4. Percentage of surface hardness loss and recovery
Surface Knoop hardness was measured initially and after every
experimental step: (1) after exposure to saliva and (2) after erosion
(MicroMet 5114 Hardness Tester; Buehler Ltd., Lake Bluff, IL, USA). For
every measurement, five indentations were carried out (25 g for 10 s)
100 µm apart each other. The surface hardness value corresponded to
the average of these five indentations. Initial SH values averaged at
358.71 ± 30.07 KHN. Additionally, one reference indentation was made
at the start of the experiment, 200 µm away from the initial SH in­
dentations. This mark was performed using an indentation of 500 g for
10 s.
The percentage of surface hardness recovery (% HR) or surface
hardness loss (% HL) was calculated after each erosive cycle according
to the two following formulas: (1) % HR = [(hardness after redeposition
- hardness after erosion) / (Initial hardness - hardness after erosion)] X
100 and (2) % HL = [(initial hardness - hardness after erosion) / (initial
hardness)] X 100. It is important to highlight that there will be three
surface hardness values after redeposition and three hardness values
after erosion, corresponding to the hardness after rehardening and
erosion in each of the three cycles. However, the initial hardness will be
the one measured immediately before each cycle. For instance, the
initial hardness for % HL of the second erosive cycle corresponds to the
hardness value after the redeposition in cycle 1. On the other hand, the
A.P. Boteon et al. Archives of Oral Biology 154 (2023) 105755

first hardness refers to the value of the sound enamel used for Table 2
randomization. Means values and standard deviation ( ± SD) of percentage of enamel hardness
loss (kgf/mm2) after each of the three demineralization cycles.

2.5. Statistical analysis Group % HL 1 % HL 2 % HL 3

Gsitu 29.84 (±4.78)b 24.94 (±8.72)a,b 18.42 (±10.19)a

Statistical analysis was performed with SigmaPlot version 12.3 Gvitro 44.35 (±6.11)c 5.99 (±6.27)d 23.38 (±7.70)a
GvitroM 44.29 (±4.68)c -3.24 (±9.57)e 19.47 (±7.13)a
(2011 Systat Software, Germany). The analysis of hardness recovery (%
HR) and hardness loss (% HL) was conducted separately based on two *Different letters indicate statistically difference between the studied groups and
criteria: the type of saliva and the progression of erosive cycling. erosive cycling evolution (Two-way ANOVA and Tukey’s Test, p < 0.05).
Therefore, two-way ANOVA test and Tukey’s Test were adopted. The
significance level was set at 5 %.
Table 3
Means values and standard deviation (±SD of percentage of enamel hardness
3. Results
recovery (kgf/mm2) after each of the three demineralization cycles.

Table 1 shows the mean values of enamel hardness before and after Group % HR 1 % HR 2 % HR 3

each erosion (DE) and saliva exposure (RE) steps. When considering the
percentage of hardness loss (Table 2), a significant effect was found for
the type of saliva (p < 0.001), the progression of erosive cycling and
their interaction. The first erosive demineralization resulted in greater
enamel loss for the in vitro saliva groups, with no difference between
GvitroM and Gvitro. After the second erosive demineralization, the
enamel blocks previously immersed in GvitroM saliva did not exhibit
mineral loss, unlike those immersed in Gvitro and Gsitu. The latter
groups showed the highest degree of enamel loss. The types of studied
saliva promoted similar enamel hardness losses after the third erosive
demineralization. In the first demineralization, a higher percentage of
enamel hardness loss was observed for the in vitro studied saliva when
compared to the second and third demineralization. After the third
erosive demineralization the in situ saliva resulted in less hardness loss
compared to the first demineralization.
Table 3 shows the enamel surface hardness recovery, a significant
effect was found only for the progression of erosive cycling (p < 0.001).
There was no difference among types of saliva. However, hardness re­
covery significantly decreased as the cycles progressed, meaning that
the recovery in cycle 2 was lower than in cycle 1, and in cycle 3 it was
lower than in cycles 1 and 2.
4. Discussion
Saliva is a key factor on erosion development. Therefore, in situ and
in vitro studies must simulate saliva clinical behavior as close as possible.
Previously study tested the erosion-preventive effect of different artifi­
cial saliva formulations and human saliva in vitro compared to human
saliva in situ against erosive cycles (Batista et al., 2016) and found no
statistical difference between groups. The authors, however, presented
only the absolute hardness values, not showing percentage calculations
of enamel hardness loss or recovery (Batista et al., 2016), so the actual
effect of saliva on enamel rehardening was not clearly demonstrated.
The present study is the first that assess the percentage of enamel
hardness loss and recovery after three erosive cycles by comparing
human saliva in vitro (with or without mucin) and human saliva in situ.
These calculations are important because they show the impact of saliva
in protecting the enamel from mineral loss after an acid challenge, as
well as reflecting how saliva might be capable of promoting hardness
recovery.
Regarding its impact on hardness loss, our results show that after the
first erosive challenge, the in situ human saliva presented greater
Gsitu 35.95 (±17.49)a,A 25.39 (±10.18)a,B 15.57 (±11.75)a,C
Gvitro 42.22 (±15.60)a,A 23.08 (±20.26)a,B 12.89 (±7.72)a,C
GvitroM 45.09 (±15.56)a,A 22.49 (±13.70)a,B 15.99 (±4.01)a,C
*Different capital letters indicate statistically significant differences for the
erosive cycling evolution (Two-way ANOVA and Tukey’s Test, p < 0.05).
Similar lowercase letters indicate no statistically significant differences among
types of saliva (Two-way ANOVA and Tukey’s Test, p < 0.05).
protection, whereas after second demineralization, the in vitro human
saliva with mucin also protected the enamel from demineralization.
After the third erosive cycle, no more differences were observed be­
tween the three types of saliva. Regarding the impact on hardness re­
covery, saliva generally increased enamel hardness, but the gain
gradually decreased with the progression of the erosive cycles, indi­
cating saliva’s limited capacity for mineral redeposition.
The main effect of saliva on hardness loss is associated with the
salivary pellicle. This is a protein-rich semipermeable layer made up of
mucins and peptides, and, to a lesser extent, enzymes, glycoproteins,
carbohydrates and lipids (Hannig et al., 2009; Hannig & Hannig, 2014).
The proteins in saliva can strongly adhere to the tooth surface, initially
forming a basal layer that subsequently undergoes maturation and
modulation processes, increasing its thickness (Hannig & Balz, 1999;
Hannig et al., 2009; Hannig & Hannig, 2014). So, after the 2 h exposure
to human saliva, our enamel specimens probably had protective pellicle,
that hindered the contact of the acid with the tooth, and thus hindered
demineralization. Interestingly, the pellicle formed in situ (Gsitu group)
was probably different to that formed in vitro (Gvitro or GvitroM), since
it led to better protection and less enamel hardness loss. Despite the
protection, we still observed an overall loss of more than 50 % in enamel
hardness for all groups after the third demineralization (DE3), sug­
gesting that the protective effect of the salivary pellicle is limited,
especially under prolonged acid exposure.
When considering the impact of saliva on hardness recovery, it is
believed that this phenomenon may be attributed to mineral deposition
onto the enamel. When enamel demineralizes in milder conditions, like
caries, the partially demineralized crystals can be replaced by a super­
saturated solution, such as saliva, where a complete remineralization
not only restores the mechanical strength of the mineral, but also im­
proves its resistance to subsequent acid challenges (Shellis et al., 2014).
However, when the demineralization is strong, like in erosion, the
crystals can be totally lost, and the supersaturated solution will not be
able to completely re-form the crystals (Shellis et al., 2014). In such

Table 1
Means values and standard deviation ( ± SD) of enamel hardness (kgf/mm2) before and after each demineralization (DE) and hardness recovery (RE) of the 3 erosive
cycles.
Group INITIAL DE 1 RE 1 DE 2 RE 2 DE 3 RE 3

Gsitu 358.96 (±29.10) 250.98 (±16.62) 289.89 (±24.36) 216.29 (±18.62) 253.96 (±18.29) 207.36 (±29.40) 231.40 (±21.56)
Gvitro 357.17 (±31.87) 197.79 (±19.24) 231.07 (±15.11) 216.70 (±13.45 246.74 (±36.74) 186.64 (±23.33) 206.39 (±29.72)
GvitroM 360.02 (±29.09) 199.23 (±1.01) 225.12 (±2.28) 232.29 (±19.79) 252.78 (±20.65) 202.94 (±17.04) 228.06 (±15.37)

4
A.P. Boteon et al.
cases, exposing the enamel to saliva will most likely only result in a
deposition of mineral particles on the surface and near-surface demin­
eralized areas, rather than a remineralization (Schlueter et al., 2020;
Shellis et al., 2013). Therefore, based on our results, which demonstrate
only slight hardness recovery (Δ%RE) after exposure to saliva and a
gradual decrease in the magnitude of these recoveries in subsequent
erosive cycles, as well as the easy loss of hardness recovery upon sub­
sequent acid challenges (Δ%DE), our study confirms that the potential
for remineralization of eroded tissues is minimal. Moreover, any mineral
deposition resulting solely from saliva is likely to be negligible.
Remarkably, despite the minimal effect of saliva in hardness recov­
ery (Δ%RE), our results show that saliva has a significant effect on Δ%DE.
Again, this is probably due to the protective effect of the pellicle. One of
the main protein components in saliva is mucin, making up 20–30 % of
the total protein content, and is one of the main components of the
pellicle. It is also among the acquired pellicle precursor proteins, which,
in addition to its lubricating capacity, has a high affinity with hy­
droxyapatite, forming the acquired pellicle basal layer (Jensen et al.,
1992). Mucin is also an important factor in the control of enamel erosion
by modulating the concentrations of calcium and phosphate in the oral
cavity (Delecrode et al., 2015). In fact, along with statherin and
proline-rich proteins, mucins maintain the saturation state of calcium
and phosphate in oral fluids, by inhibiting their precipitation at neutral
pH and releasing these ions after acid challenge (Vukosavljevic et al.,
2014). Because it inhibits precipitation and it forms complexes with
calcium and phosphate ions, mucin was considered responsible for
reducing the remineralizing effect of human saliva (Hara et al., 2008).
However, other studies have shown that mucin does not affect the
mineral deposition potential of artificial saliva (Ionta et al., 2014), and
that artificial saliva with mucin, as well as human saliva (also containing
mucin), promoted greater protection against erosion in comparison to
depleted-mucin artificial saliva (Jordão et al., 2017). Moreover, recent
studies further revealed the close relationship between salivary proteins
and the calcium and phosphate ions in saliva, where salivary pellicles
formed only by salivary proteins (ions-depleted saliva) provided a better
protective effect against acid challenge (Baumann et al., 2016; Mutahar
et al., 2017) than when ions and proteins were present. Therefore, it is
essential to highlight the delicate balance between proteins and mineral
ions in the oral homeostasis, and that the combination of both play an
important role in demineralization, leading to different degrees of pro­
tection against enamel erosion.
A positive aspect of this study is the comparison of in vitro human
saliva and in situ human saliva, both from the same individuals. We
observed no statistical difference in the hardness recovery, but there
were clear differences in the hardness loss. In situ human saliva pre­
sented a more homogeneous performance in relation to hardness loss
(preventive effect) when compared to the in vitro human saliva (Gvitro
and GvitroM groups). This lack of homogeneity in the in vitro saliva is
most probably related to issues inherent to the methodology. During the
collection, storage and cycling processes, the proteins in human saliva
undergo degradation, which lead to considerable changes in the salivary
composition, decreasing its protective effect against erosion (Hall et al.,
1999). To reduce the degradation and changes to our collected saliva,
the volunteers were asked to collect saliva immediately before each
erosive cycle. In addition, salivary composition varies between in­
dividuals, and it will have different protective effects. In our study, the
influence of these factors was kept to minimum, since the same volun­
teers who performed the in situ phase (Gsitu group) were responsible for
providing the saliva for the in vitro phases (Gvitro and GvitroM).
The results of the present study show that the in vitro human saliva
groups presented comparable results to the in situ group, which was also
observed in a previous study (Batista et al., 2016). This means that using
human saliva in an in vitro set up can be a good alternative to artificial
saliva, especially in pre-clinical phases, when we need the presence of
saliva to verify how some substances will behave in the oral environ­
ment. The utilization of in situ erosive models allows for the exposure of
5
Archives of Oral Biology 154 (2023) 105755
substrates to the oral environment, thereby bringing the results closer to
clinical reality. This is particularly significant since the volume of saliva
present in the oral environment, to which the specimens are exposed, is
significantly greater than the 1.0–1.8 ml of collected saliva used in in
vitro studies (Hall et al., 1999). Consequently, in situ studies facilitate a
constant renewal of saliva, which further reduces the degradation of
salivary proteins.
In the current study, all saliva samples exhibited similar behavior
during remineralization. However, due to the observed variation in
protective potential across different cycles, it would be valuable to
conduct a study involving multiple cycles with enamel loss as a response
variable to validate this pattern. Furthermore, it would be beneficial to
explore the association between brushing and saliva, as mucin alters the
rheological properties of saliva, potentially impacting enamel protection
against mechanical forces.
Funding
This work was supported by CAPES - Coordenação de Aperfeiçoa­
mento de Pessoal de Nível Superior - Brazil - (finance code 001); FAPESP
- Fundação de Amparo a Pesquisa de São Paulo (grant number 2016/
13631-7); and CNPq - Conselho Nacional de Desenvolvimento Científico
e Tecnológico (grant number 310679/2015-0).
CRediT authorship contribution statement
Ana Paula Boteon: Responsible for the acquisition and interpreta­
tion of data, drafting the article and final approval of the version to be
published, Natália Mello dos Santos: Responsible for the acquisition
and interpretation of data, revising article critically for important in­
tellectual content, and final approval of the version to be published,
Larissa Di Bene Kandalaft Lamana: Contributed with the conception
and design, interpretation of data, revising article critically for impor­
tant intellectual content and final approval of the version to be pub­
lished, Isadora Messias Batista Rosa: Contributed with the conception
and design, revising article critically for important intellectual content
and final approval of the version to be published, Camilla Cristina Lira
Di Leone: Responsible for revising article critically for important in­
tellectual content and final approval of the version to be published,
Rafaela Aparecida Caracho: Responsible for revising article critically
for important intellectual content and final approval of the version to be
published, Thiago Saads Carvalho: Responsible for revising article
critically for important intellectual content and final approval of the
version to be published, Heitor Marques Honório: Contributed with
the conception and design, with the analysis and interpretation of data,
and final approval of the version to be published, Daniela Rios:
Contributed with the conception and design, with the analysis and
interpretation of data, and final approval of the version to be published.
Declaration of Competing Interest
We wish to confirm that there are no known conflicts of interest
associated with this publication and there has been no significant
financial support for this work that could have influenced its outcome.
We confirm that the manuscript has been read and approved by all
named authors and that there are no other persons who satisfied the
criteria for authorship but are not listed. We further confirm that the
order of authors listed in the manuscript has been approved by all of us.
We confirm that we have given due consideration to the protection of
intellectual property associated with this work and that there are no
impediments to publication, including the timing of publication, with
respect to intellectual property. In so doing we confirm that we have
followed the regulations of our institutions concerning intellectual
property.
A.P. Boteon et al. Archives of Oral Biology 154 (2023) 105755

Acknowledgments Ionta, F. Q., Mendonça, F. L., de Oliveira, G. C., de Alencar, C. R., Honório, H. M.,
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who participated in this study and the funding sources: CAPES - Coor­ Jensen, J. L., Lamkin, M. S., & Oppenheim, F. G. (1992). Adsorption of human salivary
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(finance code 001); FAPESP - Fundação de Amparo a Pesquisa de São 10.1177/00220345920710090501
Paulo (grant number 2016/13631-7); and CNPq - Conselho Nacional de Jordão, M. C., Ionta, F. Q., Bergantin, B. T., Oliveira, G. C., Moretto, M. J.,
Desenvolvimento Científico e Tecnológico (grant number 310679/ Honório, H. M., Silva, T. C., & Rios, D. (2017). The effect of mucin in artificial saliva
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