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.2024.e4273
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Braz Dent Sci 2024 July/Sept;27 (3): e4273
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
The effect of ozone and silver diamine fluoride combination on
enamel microhardness: an
in vitro
study
Efeito da combinação de ozônio e diamino fluoreto de prata na microdureza do esmalte: um estudo
in vitro
Asmaa Mohammed KHAMMAS1 , Shatha A ABBAS1 , Aseel H MJ AL HAIDAR1 , Mohamed Saeed M ALI2
1 - University of Baghdad, College of Dentistry, Department of Pedodontics and Preventive Dentistry. Baghdad, Iraq.
2 - University of Baghdad, College of Dentistry, Department of Periodontics. Baghdad, Iraq.
How to cite: Khammas AM, Abbas SA, Al-Haidar AHMJ, Ali MSM. The effect of ozone and silver diamine uoride combination on enamel
microhardness: an
in vitro
study. Braz Dent Sci. 2024;27(3):e4273. https://doi.org/10.4322/bds.2024.e4273
ABSTRACT
Objective: This study aims to evaluate ozone’s impact on initially formed enamel caries before applying silver
diamine uoride, utilizing a Vickers microhardness tester as the excitation source. Material and Methods: Sixty
premolars were exposed to demineralization to form articial incipient caries. Then, the teeth were arbitrarily
distributed among four investigational groups (group 1 received silver diamine uoride, group 2 received ozone,
group 3 received ozone then silver diamine uoride, while group 4 received no treatment as a control group).
Enamel microhardness was evaluated at three stages: before exposure to demineralization solution, after exposure,
and following treatment using a Vickers microhardness tester. A one-way ANOVA test was utilized to calculate
the difference in microhardness values across the four groups, followed by Tukey’s test and paired t-tests were
accomplished before and after treatment. 5% was established as the signicance level. Results: The data shows
a clear and statistically signicant difference in surface microhardness between the control and experimental
groups. Tukey’s test indicates that Group 3 and Group 1 did not have a signicant difference. However, paired
t-tests conducted before and after treatment revealed signicant differences in the experimental groups, while
non-signicant differences were found before and after in the control group. Conclusion: This investigation
disclosed that SDF is still an excellent option whereas ozone alone has no impact on remineralizing enamel
incipient caries, and there is no advancement in combining both approaches.
KEYWORDS
Dental caries; Fluorides; Hardness; Ozone; Silver diamine uoride.
RESUMO
Objetivo: Esse estudo tem como objetivo avaliar o impacto do ozônio na formação de cárie inicial em esmalte
previamente a aplicação de diamino uoreto de prata, foi utilizado microdurômetro Vickers como fonte de análise.
Material e Métodos: Sessenta pré-molares foram expostos a desmineralização para formação articial de lesões
incipientes de cárie. Então, os dentes foram arbitrariamente distribuídos entre quatro grupos de investigação
(grupo 1 – diamino uoreto de prata; grupo 2 – ozônio; grupo 3 – ozônio seguido de diamino uoreto de prata;
grupo 4 – sem tratamento, grupo controle). A microdureza do esmalte foi avaliada em três estágios: antes da
desmineralização, após a desmineralização e após a aplicação dos tratamentos, utilizando um microdurômetro
Vickers. Teste ANOVA a um fator foi utilizado para avaliar a diferença nos valores de microdureza entre os
quatro grupos, seguido do teste de Tukey e teste T pareado para contemplar antes e após o tratamento. 5% foi
estabelecido como nível de signicância. Resultados: Os dados mostram clara e signicante diferença estatística
na microdureza supercial entre os grupos controle e experimentais. Teste de Tukey indica que o grupo 3 e grupo
1 não tiveram diferença signicativa. No entanto, o teste T pareado conduzido antes e após o tratamento revelou
signicante diferenças entre os grupos experimentais, enquanto nenhuma diferença signicante foi encontrada
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Braz Dent Sci 2024 July/Sept;27 (3): e4273
Khammas AM et al.
The effect of ozone and silver diamine fluoride combination on enamel microhardness: an in vitro study
Khammas AM et al. The effect of ozone and silver diamine fluoride combination on
enamel microhardness: an
in vitro
study
INTRODUCTION
Fluoride has been the most effective
remineralization method in clinical settings
for several decades. However, in the last few
years, new remineralizing approaches have been
created that integrate phosphates and calcium
at the level of demineralized tooth surfaces [1].
Tooth decay has been described to be a
multifactorial sugar-driven, biolm-mediated,
multifactorial, dynamic disease [2], posing a
signicant economic challenge to governments’
healthcare systems [3]. Management of tooth caries
concentrates on applying remineralizing agents to
the initial caries for promoting remineralization
and controlling demineralization. Remineralizing
means creating a super-saturated situation close
to the dissolved carious lesion, consequently, it
would stop mineral loss and ll up the empty
spaces with calcium and phosphate ions [4,5].
Fluoride is known to be a remineralizing
agent that forms uorapatite by incorporating
calcium and phosphate ions and reacting with
saliva on the interface between the enamel and
subsurface areas of teeth [6,7]. One of these
agents is silver diamine uoride (SDF) which is
a cariostatic agent [8]. Its use might be widely
encouraged as a preventive substitute for the
traditional invasive decay treatment approach,
particularly for children’s patients with special
care needs, those too little to receive dental
treatment or patients who have trouble accessing
and paying for traditional dental treatment [9].
Since the advancement of minimal dental
intervention in caries treatment, Ozone has
been presented as an alternate stratagem to
support the reversal of the initial caries. It is
assumed that ozone-induced oxidative damage
opens dentinal tubules, subsequently aiding
in the remineralization of decay [10]. Low
certainty of evidence and poor support from
published articles especially systematic reviews
concerning the capability of ozone in supporting
remineralization with or without the existence
of further remineralizing agents demands
further research [11,12]. Thus, the current study
aimed to weigh the effect of applying ozone on
articially formed initial enamel caries before
applying SDF in the microhardness values.
MATERIAL AND METHODS
Tooth preparation
The research ethics committee at the College
of Dentistry /University of Baghdad approved
the protocol of this study (project No.723322)
on December 28, 2022. Caries-free extracted
premolars were selected for orthodontic purposes
with informed consent obtained beforehand.
Before testing, the premolars were polished
with pumice to remove contaminants or debris
on the tooth surfaces and then kept at room
temperature in a 0.1% thymol solution. Using a
stereomicroscope at ×40 magnication, the teeth
were examined, and any tooth with white spot
lesions, stains, or cracks was excluded. Finally,
sixty premolars were coated with acid-resistant
nail paint, leaving a window of approximately
6mm in diameter in the middle of each buccal
enamel surface.
Demineralization and treatment procedure
Articial lesions were created on the teeth
by using a demineralizing solution containing
2.2 mM NaH2 PO4, 2.2 mM CaCl2, and 0.05 M
acetic acid for 96 hours [13]. During this process,
the pH of the solution was adjusted to 4.4 with 1M
KOH and refreshed daily to maintain the pH level.
After air-drying the demineralized surfaces,
the teeth were arbitrarily distributed among four
investigational groups (n=15):
antes e após o tratamento para o grupo controle. Conclusão: Essa investigação mostra que o diamino uoreto de
prata ainda é uma excelente opção enquanto o ozônio sozinho não tem impacto na remineralização do esmalte
em lesões incipiente de cárie, e não apresentou melhora nos resultados com a combinação das técnicas.
PALAVRAS-CHAVE
Cárie dentária; Fluoretos; Dureza; Ozônio; Diamino uoreto de prata.
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Braz Dent Sci 2024 July/Sept;27 (3): e4273
Khammas AM et al.
The effect of ozone and silver diamine fluoride combination on enamel microhardness: an in vitro study
Khammas AM et al. The effect of ozone and silver diamine fluoride combination on
enamel microhardness: an
in vitro
study
Group 1, the SDF group, was treated by rubbing
SDF solution on the exposed enamel surface
for two minutes using a micro brush. The
SDF solution used was 38% SDF Advantage
Arrest™ from Elevate Oral Care LLC., West
Palm Beach. After the treatment, the teeth
were ushed with distilled water for thirty
seconds [14]. A few days later after SDF
application, grayish staining was seen. The
staining was caused by SDF because of the
presence of the silver ion.
Group 2, the Ozone group, was treated by applying
ozone for 80 seconds and then rinsing it off
for 15 seconds [15]. The ozonated water used
in the treatment was obtained from an Ozone
Generator called Aqua-8.
Group 3, the Ozone and SDF group, received ozone
treatment followed by SDF application.
Group 4, Control group, received no treatment
and was only ushed with deionized water.
Surface microhardness measurement
Premolars were assessed for microhardness
of the enamel surface at three different stages.
Firstly, immediately after they were removed from
the solution (distal water+2% thymol) at room
temperature (baseline). Secondly, after being
removed from the demineralization solution.
Lastly, after being treated with the remineralizing
agents. Three different indentations were made
with a Vickers microhardness tester, and the
results were averaged under a 500 g load for 30
seconds. The hardness variables were calculated
using the following formula:
– VHN VHN remineralization VHN demineralization∆=
(1)
Statistical analysis
SPSS Statistics version 26.0 software (IBM
Corporation, Armonk, NY, USA) was utilized to
examine the data. To determine if there was a
difference in ∆VHN among the four groups (SDF,
ozone, ozone and SDF, and control groups), one-
way ANOVA tests were performed, afterwards
Tukey’s test was used. Also, paired t-tests were
accomplished before and after treatment, 5% has
been selected as the signicance level.
RESULTS
A remarkable decrease in the surface
microhardness was noticed after demineralization
then it increased again after treatment. A non-
significant difference was obtained in the
microhardness unit (VHN) between groups
(p= 0.103) at the baseline. Nevertheless, after
treatment, a significant difference was found
between them (p=0.000).
Concerning ΔVHN, group 3 exhibited the
highest mean (91.00 ± 8.13) and group 1
was close to it (86.84 ± 3.038), while the
lowest mean was for group 4 (0.93 ±5.15). A
signicant difference was obtained in the surface
microhardness among the experimental and the
control groups (p=0.000). Using Tukey’s test,
signicant differences were found between all
groups except between group 1 and group 3 where
there was no signicant difference (P=0.296).
Using paired t-tests for each group before
and after treatment, signicant differences were
obtained with the groups except for the control
group, no significant difference was found
(P=0.494), as illustrated in Table I.
DISCUSSION
This study evaluated the effectiveness of SDF,
ozone, and the combined use of ozone and SDF
in treating articial enamel caries. Two methods
were used to assess enamel remineralization: the
Knoop method and Vickers’ hardness [16,17].
Among the two, the latter was deemed more
Table I - Analysis of VHN among the study groups at different stages by mean ±SD, t-test, and Tukey’s test
Group
Baseline Demineralization Remineralization T-test ΔVHN
Mean ±SD Mean ±SD Mean ±SD p value Mean ±SD
1 301.26 ± 6.71 152.49± 2.77 239.33 ± 3.66 0.000 86.84 ± 3.038*
2 300.96 ± 6.51 144.67 ± 8.16 177.30 ± 7.99 0.000 32.62 ± 7.92
3 303.51 ± 2.03 151.36 ± 7.63 242.37 ± 1.63 0.000 91.00 ± 8.13*
4 305.33 ± 5.03 140.31 ± 6.89 141.24 ± 10.97 0.494 0.93 ±5.15
*Tukey’s test revealed non-significant difference between group 1 and group 3 only (p>0.05).
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Braz Dent Sci 2024 July/Sept;27 (3): e4273
Khammas AM et al.
The effect of ozone and silver diamine fluoride combination on enamel microhardness: an in vitro study
Khammas AM et al. The effect of ozone and silver diamine fluoride combination on
enamel microhardness: an
in vitro
study
suitable because it enables easy measurement and
detection of the pyramid-shaped marks on the
enamel surface, both visually and digitally [18].
According to research by Godoi et al. [19],
the use of SDF treatment led to an increase in
mineral density for experimentally created caries,
which aligns with the findings of the current
investigation. The analysis discovered that the
group treated with SDF had a greater effect on
remineralizing enamel caries in comparison to
the ozone alone and control groups.
However, Akyildiz and Sönmez [20]
researched the remineralization capacity of SDF
and NaF on artificial caries of the third molar
enamel and concluded that while both SDF and
NaF can remineralize carious enamel lesions,
NaF was more effective. Also, a study done by
Alcorn et al. [21] found that uoride varnish might
be a better option than SDF to treat noncavitated,
developing, white-spot, carious enamel lesions.
The result disclosed that the adjunctive
function of ozone and SDF had a comparable
remineralizing effect to SDF alone on carious
enamel lesions. These ndings were consistent
with Yu et al.’s [22] research, which demonstrated
that the supplementary use of NaF varnish and
SDF had a comparable remineralizing impact on
SDF on enamel lesions.
Conversely, Zhao et al. [23] discovered
that enamel blocks treated with a laser mixed
with SDF had signicantly higher hardness than
those receiving only SDF or laser. Furthermore,
the research of Grocholewicz et al. [24], and
Samuel et al. [25] suggested that the use of
ozone and nano-hydroxyapatite treatment has the
potential for remineralizing carious enamel, but
the combination of the two approaches delivers
the best outcome.
There was a considerable difference in
hardness before and after the application of
ozone group, which was only higher than
the control group. However, when combined
with SDF, a better effect was obtained. This is
consistent with another study that found therapy
with ozone alone was ineffective in preventing
enamel from demineralization or stimulating
remineralization unless paired with the Reductant/
Patient Kit, which had a signicant amount of
fluoride [26]. On the other hand, Atabek and
Oztas [27] confirmed that ozone, individually
and in combination with other remineralization
treatments, helped reverse incipient fissure
caries. Similarly, Floare et al. [28] observed that
ozone could enhance the enamel condition with
substantial shifts in the demineralization values
from equivalent early demineralized enamel levels
to that approximated to the values of integral
enamel, deprived of the risk of a carious lesion
procedure. Besides, Srinivasan et al. [29] found
that ozone could be regarded as an efcient agent
in the reversal of the incipient carious enamel
lesion there through preventing the dental tissue
from submitting to the recurring restorative cycle.
Nevertheless, this in vitro study had denite
restrictions, such as the challenge in replicating
the mouth and the fact that carious lesion
remineralization is different in comparison to
the complicated biological systems in the mouth
[30]. In addition, the antimicrobial effect of
the two examined agents was not considered,
although it could potentially improve the clinical
remineralization of decay in the presence of free
mineral ions and the antibacterial activeness of
the examined agent at the same time.
CONCLUSION
This investigation disclosed that SDF is still
an excellent option whereas ozone alone has no
impact on remineralizing enamel incipient caries,
and there is no advancement in combining both
approaches.
Acknowledgements
None.
Author’s Contributions
AMK: Conceptualization, Methodology,
Software, Investigation, Writing – Original
Draft Preparation, Project Administration and
Funding Acquisition. SAA: Methodology, Review
& Editing, Project Administration and Funding
Acquisition. AHMJAH: Formal Analysis, Review
& Editing, Visualization, Supervision, Project
Administration and Funding Acquisition. MSMA:
Resources, Data Curation, Writing, Project
Administration and Funding Acquisition.
Conict of Interest
The authors have no conicts of interest to
declare.
5
Braz Dent Sci 2024 July/Sept;27 (3): e4273
Khammas AM et al.
The effect of ozone and silver diamine fluoride combination on enamel microhardness: an in vitro study
Khammas AM et al. The effect of ozone and silver diamine fluoride combination on
enamel microhardness: an
in vitro
study
Funding
This research did not receive any specic
grant from funding agencies in the public,
commercial, or not-for-prot sectors.
Regulatory Statement
The research ethics committee at the College
of Dentistry /University of Baghdad approved
the protocol of this study (project No.723322)
December 28, 2022.
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Braz Dent Sci 2024 July/Sept;27 (3): e4273
The effect of ozone and silver diamine fluoride combination on
enamel microhardness: an
in vitro
study
Khammas AM et al.
The effect of ozone and silver diamine fluoride combination on enamel microhardness: an in vitro study
Khammas AM et al. The effect of ozone and silver diamine fluoride combination on
enamel microhardness: an
in vitro
study
Date submitted: 2024 Feb 19
Accept submission: 2024 Aug 06
Asmaa Mohammed Khammas
(Corresponding address)
University of Baghdad, College of Dentistry, Department of Pedodontics and
Preventive Dentistry, Baghdad, Iraq.
Email: asmaa.m @codental.uobaghdad.edu.iq
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