Original article UDC: 616.314:663.

8
doi:10.5633/amm.2018.0304

EVALUATION OF ENAMEL SURFACE ROUGHNESS AND

MORPHOLOGICAL CHANGES AFTER EXPOSURE TO COCA-COLA,
ORANGE AND ARTIFICIAL GASTRIC JUICE: AN IN VITRO STUDY

Radomir Barac1, Jovanka Gašić1,2, Jelena Popović1,2, Aleksandar Mitić1,2,

Goran Radenković3, Milena Potić-Floranović4, Marija Nikolić1,2, Nenad Stošić1

Dental erosion is a pathologic, non-bacterial hard dental tissue loss induced by extrinsic
or intrinsic acids. This in vitro study aimed to evaluate and compare the morphology and
surface roughness of dental enamel after erosive challenge in some extrinsic and intrinsic acidic
substances, Coca-Cola, orange and gastric juice.
Enamel samples (n = 48), obtained by preparation of surgical extracted human third
molars, were subjected to the erosive challenge of the artificial gastric juice and commercially-
available Coca-Cola and orange juice by immersion in 50 ml of erosive solutions for 15 min,
three times daily, for 10 days. Between immersions, the samples were kept in filtered saliva.
Twenty-four samples were prepared for the surface morphology analysis using scanning-
electron microscope, and 24 for the analysis of Ra-surface roughness parameter (using a
diamond-stylus-profilometer), including the 12 control samples (which did not undergo the
erosion procedure). Results of the surface roughness were analyzed by one-way ANOVA
Student-Newman-Keuls post hoc test.
Ultrastructural analysis of enamel surface after immersion in Coca-Cola and gastric
juice showed type 1 etching pattern with the typical honeycomb appearance. After the erosive
challenge with orange juice, a nonspecific morphological model was established. Profilometric
parameter Ra was significantly increased for samples immersed in gastric juice compared to
samples immersed in Coca-Cola and orange juice, as well as, in samples with Coca-Cola-
erosion compared with orange juice-erosion. Gastric juice had higher erosive potential in
relation to Coca-Cola and orange juice, with the most intense morphological changes and the
highest roughness on the enamel surface.
Acta Medica Medianae 2018;57(3):33-40.

Key words: Enamel erosion, soft drinks, gastric juice, SEM, surface roughness

1
University of Niš, Faculty of Medicine, Department of free tooth surfaces (1). The most important extrinsic
Restorative Dentistry and Endodontics, Serbia source of acid exposure is diet, which could include
2
Clinic of Dentristry, Niš, Serbia
3
University of Niš, Faculty of Mechanical Engineering, numerous components and products with complex
Department of Production Engineering, Serbia composition and a potential for erosive damage (car-
4
University of Niš, Faculty of Medicine, Research Center for bonated and acidic drinks, acidic food, citrus pastil-
Biomedicine, Serbia
les, various medicaments), professional exposure to
corrosive agents (acid vapors from batteries and
Contact: Radomir Barac other appliances), even exposure to chlorinated wa-
Blvd dr Zoran Djindjić 81, 18000 Niš, Serbia ter in swimming pools during water sports (2-6). In
E-mail: barac_radomir@hotmail.com
addition, behavioral factors like eating and drinking
habits (holding an acid beverage in the mouth before
swallowing, swishing around the mouth or sucking
juice through the teeth) contribute to its develop-
ment (7). Intrinsic factors are the result of endogen-
ous acid, generally gastric acids that contact teeth
especially in patients suffering from anorexia, buli-
mia, chronic vomiting during pregnancy and gastro-
intestinal disturbances (8-11).
Introduction
Many laboratory studies have found carbonat-
ed drinks, especially carbonated cola drinks, to be
Dental erosion has been defined as pathologic,
associated with erosion, most likely due to their low
non-bacterial dental hard tissue loss induced by ex-
pH (2, 4, 12, 13). Further, in vitro studies have shown
trinsic or intrinsic acids or chelators acting on plaque-
www.medfak.ni.ac.rs/amm 33
Evaluation of enamel surface roughness and morphological... Radomir Barac et al.

that fruit juices may also be potentially erosive, due Material and methods
to their high content of titratable acid (2, 4, 12).
On the other hand, acidic stomach contents The material for this research included 12
refluxed into the oral cavity can dissolve tooth stru- human impacted mandibular third molars (from pati-
ctures and cause erosive tooth wear (14, 15) be- ents aged 18-25 years) disinfected in 1% thymol
cause contact between the hydrochloric acid from solution and kept in 1% sodium hypochlorite for 24
the stomach (with pH from 1.5 to 3,5) and the oral h. Organic debris was removed by carefully using a
cavity occurs for a few seconds, several times a day dentist’s set of instruments (17).
(16). After the removal of the roots, at least 2 mm
The aims of the present in vitro study were below cementoenamel junction, the crowns were cut
twofold: (1) to analyze the experimental models of (using a diamond saw under water irrigation) from
enamel erosion after exposure to Coca-Cola, orange the distal, mesial, buccal, and lingual side. Out of the
juice and artificial gastric juice at the ultrastructural total of 48 samples, 24 were used for SEM analysis
level, and (2) to evaluate enamel surface roughness and 24 were used for the analysis of enamel surface
after erosive challenge in the same acidic solutions. roughness (Table 1).

Table 1. Distribution of samples used in experimental protocols.

Number of samples Number of samples for

Samples Total
for SEM analysis surface roughness analysis
Control 6 6 (3 measurements) 12
Immersed in Coca-Cola 6 6 (3 measurements) 12
Immersed in orange juice 6 6 (3 measurements) 12
Immersed in artificial gastric juice 6 6 (3 measurements) 12
Total 24 24 48

Erosion solutions and human saliva whereas orange juice had pH 3.73 ± 0.03, requiring
5.70 ml of NaOH to reach pH 7.0 (4).
The erosion models caused by soft drinks The model of enamel erosion with GERD was
were obtained by immersing the samples in Coca- created using artificial gastric juice according to the
Cola (HBC - Serbia A.D. Zemun) and orange juice methodology of Stefaniak et al. (18) and it was mo-
(NECTAR’ D.O.O. Backa Palanka, Serbia). dified in accordance with the stablished goals of the
In the previous study it was established that research. Its initial pH was 2,1 (Table 2.).
Coca-Cola had pH 2.67 ± 0.06 and TA 1.87 ± 0.09,

Table 2. The contents of artificial gastric juice (primary electrolytes and ionic compounds)

Contents Concentration

Calcium chloride dihydrate

0.264 g/L
(CaCl2×2H2O)
Magnesium chloride hexahydrate
0.152 g/L
hexahydrat (MgCl2×6H2O)
Potassium chloride (KCl) 0.864 g/L

Sodium chloride (NaCl) 2.855 g/L

Hydrochloric acid (HCl) 1.426 (3.38 ml; 36.2%)

Human saliva was collected from healthy vo- before saliva collection (19). Filtrates were obtained
lunteers in the morning, 2 hours after fasting. Volun- with Whatman filter papers grade 1: 11 μm (Sigma-
teers rinsed their mouths twice with distilled water Aldrich, USA).
34
Acta Medica Medianae 2018, Vol.57(3) Evaluation of enamel surface roughness and morphological...

Erosive Challenge lometer, the statistical analysis took into account

only one, the most frequently used parameter, Ra,
This study was approved by the institution’s which is defined as the average distance from the
Ethics Committee. Tooth samples planned for analy- profile to the mean line over the length of assess-
sis using SEM, immediately after cutting, rinsing and ment. A detailed description of the measurement
drying, were distributed into one of three erosive method using a diamond stylus profilometer has al-
challenges, while the samples planned for analysis ready been published in our recent study (4). Sta-
using profilometer, before exposure to acidic solu- tistical analysis was carried out using one-way
tions, were prepared as follows: circular molds of 16 ANOVA Student-Newman-Keuls post hoc test.
mm in diameter and 3 mm deep were filled by self- On enamel surfaces not exposed to the ero-
cured resin. Each sample was embedded in resin, sive challenge by Coca-Cola, orange and gastric
with labial (oral) surfaces uppermost, and was clean- juice (control group), the typical structures of sound
ed with nonfluoridated pumice, rinsed with water and enamel (grooves and perichimata lines) were appa-
dried with oil-free compressed air. rent. Additionally, small depressions or ditches or
All of the enamel samples were exposed to grinding marks were observed and they were found
acidic solution according to the following protocol to be indicative of the cumulative mechanical effects
(10): immersion in 50 ml acidic solution (Coca- the teeth have experienced. (Figure. 1)
Cola/orange juice/gastric juice) for 15 minutes with
occasional shaking, rinsing with distilled water, and
immersion in human saliva.
The cycle was repeated three times a day for
10 days. During the night, the samples were placed
in human saliva, including the 12 control samples
(which did not undergo the demineralization proce-
dure). The experiment was conducted at room tem-
perature.

Preparation of samples for SEM analysis

At the end of the experimental period the

samples were dried, fixed to aluminum stubs with a
fixing agent (Dotite paint xc 12 Carbon JEOL, Tokyo,
Japan), sputter-coated with old/palladium (in the
unit JFC 1100E Ion Sputter JEOL), and examined by
scanning electron microscopy (SEM) (JEOL-JSM- Figure 1. Control samples: the surface of untreated
5300). enamel with perikymata.

Preparation of samples for surface roughness

analysis
Results
Surface roughness of the enamel samples was
measured using a profilometer (Mitutoyo Surftest SEM results are shown in micrographs 1 to 4
SJ-301) (20). and the measurements of enamel surface roughness
Although four parameters of roughness are are shown in Table 3.
registered with the stylus of the Mitutoyo type profi-

Table 3. The values of the enamel surface roughness parameter (Ra) in relation to the tested acid solutions

Roughness Exposure I II III

Control
parameter (min) Artificial gastric juice Coca-Cola Orange juice

Ra 15 0.67 ± 0.02 1.63 ± 0.25a 1.49 ± 0.08a,b 1.27 ± 0.01a,b,c

a
p < 0.05 vs control; bp < 0.05 vs. artificial gastric juice; cp < 0.05 vs. Coca-Cola;

35
Evaluation of enamel surface roughness and morphological... Radomir Barac et al.

The enamel surface of teeth exposed to the

acidic solutions clearly demonstrated deep changes
in enamel structure: scanning micrographs of ena-
mel samples eroded by Coca-Cola and gastric juice
exhibited a distinct pattern, showing hollowing of
prism centers with relatively intact peripheral re-
gions, reflecting honeycomb appearance (Figuries 2,
4).

Figure 3. Erosive changes in enamel surface caused by

orange juice with irregular erosion type.

There was a statistically significant difference

among all of the tested roughness parameter va-
lues.

Discussion

Figure 2. Erosive changes on enamel caused by Literature data point to an increase in dental
Coca Cola: type 1 erosion, central parts of erosions in the modern society and they represent a
the prisms are affected and the peripheral challenge for the researchers. Considering that there
parts are relatively preserved. is a decreasing tendency in caries instances, erosive
tooth wear is becoming a more significant element
in planning a long-term model of dental health (2).
An increase in the prevalence of various gastro-
intestinal diseases and eating disorders leads to
more frequent contacts between the teeth and
gastric acid. Together with increasing use of acidic
beverages, these conditions are considered signifi-
cant risk factors for teeth demineralization.
The goal of our research was to create the
ultrastructural experimental models of enamel ero-
sion caused by Coca-Cola, orange juice and artificial
gastric acid and to determine differences and pos-
sible similarities between the erosions caused by
external and internal factors on experimental model.
To some extent, the findings could point to the sig-
nificance of prevention of both internal and external
causes of erosive tooth wear.
In order to simulate clinical conditions, the
Figure 4. Erosive changes in enamel surface caused by present research used gastric juice formula which,
gastric juice with significant type 1 erosion, honeycomb apart from HCl with 2,1 pH, contained only the
appearance. primary electrolytes and ionic compounds, without
organic and amino acids, carbohydrates and pepsin.
Calcium, magnesium and sodium salts should act as
buffer components which could probably control the
In contrast, samples immerged in orange erosive potential of gastric juice, similar to in vivo
juice showed atypical etching: without prisms, with conditions. On the other hand, the majority of the
pitted enamel surfaces, as well as with structures results from laboratory studies regarding the enamel
which look like unfinished puzzles, maps, networks. surface changes in reflux disease were obtained
(Figure 3). based on the use of pure HCl (16, 21, 22, 23, 24).
The highest value of Ra roughness parameter Nevertheless, in studies by Barlet at al. and Braga at
was observed in the samples immersed in gastric al. (9, 10), gastric juice which was aspirated from
juice, followed by Coca-Cola and, finally, orange patients undergoing endoscopy for symptoms of ref-
juice. lux disease was used.
In the present study, the immersion cycles (3
times for 15 minutes) could imitate GERD symptoms
36
Acta Medica Medianae 2018, Vol.57(3) Evaluation of enamel surface roughness and morphological...

for a shorter period of time (10 days). Similarly, the parameter showed that, after exposure to gastric
time of immersion of samples into Coca-Cola and juice, the enamel surface had prominent uneven-
orange juice can imitate the frequency of consum- ness of the surface which was statistically significant
ption of soft drinks. On the samples immersed in the compared to the surface texture of samples exposed
Coca-Cola and gastric juice, the following was ob- to Coca-Cola and orange juice.
served: diffuse demineralization involved the rod According to information from the manufactu-
core, with decomposition of morphology of prisms: rers, Coca-Cola contains phosphoric acid, compared
they were severely affected, and a greater prism- to citric acid,phosphoric acid is stronger (33). The
core dissolution compared with that in the interpris- effect of phosphoric acid results in a superficial etch-
matic areas gave the enamel a “honeycomb pattern” ed zone which might be permanently lost from the
of etching, similar to the results published by Colo- tooth surface (26). On the other hand, citric acid
mbo et al. (25) and Braga at al. (10). Also, Arnold et may act as a chelator capable of binding the calcium
al. (22) showed that exposure to pure HCl results in from enamel or dentine, thus increasing the degree
four different enamel etching patterns, and the of undersaturation and favoring demineralization
depth of the surface layer was dependent upon the (35, 36).
etching time. Our research showed that the degree In our previous study the erosive potential of
of destruction of central prism parts varied depend- various soft drinks was examined by measuring ini-
ing on location, whereby the most prominent chan- tial pH and titratable acidity and enamel surface
ges were observed in the vicinity of cement-enamel roughness using different exposure times. It was
junctions. In the present study, samples immerged found that Coca-Cola had the highest erosive poten-
in orange juice showed atypical etching, which is tial in the shortest time interval exposure (15 min),
referred to as type 4 in the literature. (26). although it had the lowest titratable acidity (4).
In the current study, surface roughness was These results are in accordance with literature data
measured using a stylus profilometer that overhangs which show that cola-based drinks have a higher ero-
across the surface of the object, registering all of the sive potential than orange juices immediately after
unevenness at a certain measuring length (17, 27, exposure (12). Profilometric parameters have demo-
28, 29, 30). According to some literature data stylus nstrated that pure orange juice causes greater ena-
profilometry shows some disadvantages (the risk of mel erosion during longer exposures. A statistically
the diamond tip causing damage to the specimens, significant lower degree of roughness compared to
inability to detect valleys which are narrower than Coca-Cola in shorter exposure can be explained by
the stylus tip) but nevertheless this technique has a higher initial pH in orange juice compared to Coca-
high degree of precision (31). Moreover, the current Cola (3.73 vs. 2.67) (4).
national standards on measuring surface texture are In the current study, gastric juice was signifi-
defined using stylus profilometry (11, 31, 32). cantly more erosive to enamel than Coca-Cola and
In a number of studies (28, 29), a difference orange juice, and Coca-Cola is more erosive than
in the surface roughness of the samples examined orange juice. Other studies attest that gastric juice
on various erosive challenges was determined only (aspirated from patients undergoing endoscopy) has
on the basis of the Ra parameter, where valid con- a greater potential for erosion than orange juice (10)
clusions were drawn. The present study showed that and carbonated drinks (Bartlett and Coward, 2001).
this parameter was statistically significantly different Results by Bartlett and Coward reflect the lower pH
among all the tested samples. Likewise, all samples and titratable acidity of gastric juice compared with
of the experimental groups were also different from the carbonated drink. If this result is extrapolated to
the control samples according to the SEM analysis the clinical situation, it confirms the suspicion that
and after analysis of the Ra parameter. gastric juice has the potential to produce the severe
According to the latest literature data, the Ra pattern of erosion found in patients with eating dis-
parameter provides no information on the characte- orders and reflux disease (9).
ristics of surface irregularities, whereby both maxi-
mal and minimal irregularities may show the same Conclusion
Ra values (31, 34). Therefore, this research also in-
cluded an ultramicroscopic analysis in order to ob- Despite the limitations characteristic of in vi-
tain more precise results. tro studies, it can be concluded that experimental
Braga et al. (10) showed that the enamel sur- erosion model of enamel surface exposed to Coca-
face after orange juice had a generalized surface Cola and artificial gastric juice shows type 1 acidic
roughening with no apparent evidence of a prism erosion (honeycomb appearance) by SEM analysis.
pattern, and the surface was not completely etched. Degree of destruction of central prism parts varied
The same authors used atomic emission and FT depending on location, whereby the most prominent
Raman spectroscopy to analyze the mineral content changes were observed in the vicinity of cement-
of enamel after exposure to gastric and orange enamel junctions. Ultrastructural experimental mo-
juice, and they determined that gastric juice has a del of enamel surface erosion after exposure to
higher erosive potential than orange juice. Our re- orange juice demonstrates atypical etching with no
search analyzed surface roughness of enamel using apparent evidence of a prism pattern. Profilometric
stylus profilometry following the exposure to the parameter Ra was significantly increased for sam-
same acidic solutions, as well as to Coca-Cola. It has ples immersed in gastric juice compared to samples
been determined that gastric juice has a higher ero-
immersed in Coca-Cola and orange juice, as well as
sive effect on the enamel, which is in accordance to
in samples with Coca-Cola-erosion compared with
the results of the mentioned authors. Roughness
37
Evaluation of enamel surface roughness and morphological... Radomir Barac et al.

orange juice-erosion. The results of this study point intense morphological changes and the highest
to a higher erosive potential of gastric juice, com- roughness on the enamel surface.
pared to Coca-Cola and orange juice, with the most

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Evaluation of enamel surface roughness and morphological... Radomir Barac et al.

Originalni rad UDC: 616.314:663.8

doi:10.5633/amm.2018.0304

EVALUACIJA HRAPAVOSTI I MORFOLOŠKIH PROMENA NA GLEĐNOJ

POVRŠINI POSLE IZLOŽENOSTI COCA-COLI, SOKU OD NARANDŽE I
VEŠTAČKOM ŽELUDAČNOM SOKU: IN VITRO STUDIJA

Radomir Barac1, Jovanka Gašić1,2, Jelena Popović1,2, Aleksandar Mitić1,2,

Goran Radenković3, Milena Potić-Floranović4, Marija Nikolić1,2, Nenad Stošić1

1
Univerzitet u Nišu, Medicinski fakultet, Departman za bolesti zuba i endodonciju, Niš, Srbija
2
Stomatološka klinika, Niš, Srbija
3
Univerzitet u Nišu, Mašinski fakultet, Katedra za proizvodno-informacione tehnologije, Niš, Srbija
4
Univerzitet u Nišu, Medicinski fakultet, Naučnoistraživački centar za biomedicinu, Niš, Srbija

Kontakt: Radomir Barac

Bulevar dr Zorana Đinđića 52, 18000 Niš, Srbija
E-mail:barac_radomir@hotmail.com

Erozija zuba je patološki gubitak tvrdih zubnih struktura izazvan spoljašnjim i unutra-
šnjim kiselinama, bez učešća bakterija. Ova in vitro studija imala je za cilj da proceni i uporedi
morfologiju i površinsku hrapavost gleđi nakon erozivnog izazova nekih eksternih i internih
kiselih supstanci kao što su Coca-Cola, sok od narandže i veštački želudačni sok.
Uzorci gleđi (n = 48), dobijeni ekstrakcijom humanih trećih molara, podvrgnuti su ero-
zivnom izazovu veštačkog želudačnog soka i komercijalno dostupnih bezalkoholnih pića
(Coca-Cola i sok od narandže) uranjanjem u 50 ml kiselog rastvora u trajanju od 15 min, tri
puta dnevno tokom 10 dana. Između potapanja, uzorci su držani u filtriranoj pljuvački.
Primljena su dvadeset i četiri uzorka za analizu površinske morfologije korišćenjem skening-
elektronskog mikroskopa i 24 za analizu Ra-parametra hrapavosti (korišćenjem stylus profi-
lometra sa dijamantskom iglom), uključujući i 12 kontrolnih uzoraka (koji nisu podvrgnuti
proceduri erozije). Rezultati površinske hrapavosti analizirani su pomoću one-way ANOVA
Student-Newman-Keuls post hoc testa.
Ultrastrukturna analiza površine gleđi posle potapanja u Coca-Colu i želudačni sok
pokazala je tip 1 model nagrizanja sa tipičnom honeycomb strukturom. Nespecifičan morfo-
loški model ustanovljen je nakon erozivnog izazova sokom od narandže. Profilometrijski
parametar Ra je značajno povećan kod uzoraka potopljenih u želudačni sok u poređenju sa
uzorcima izloženim Coca-Coli i soku od narandže, kao i u uzorcima sa Coca-Cola-erozijom u
poređenju sa erozijom izazvanom sokom od narandže. Želudačni sok je pokazao veći erozivni
potencijal u odnosu na Coca-Colu i sok od narandže, sa najintenzivnijim morfološkim pro-
menama i najvećom hrapavošću na površini gleđi.

Acta Medica Medianae 2018;57(3):33-40.

Ključne reči: gleđna erozija, bezalkoholna pića, želudačni sok, SEM, površinska
hrapavost

This work is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) Licence

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