Statistical analysis

Descriptive analyses were performed for checking the distribution of each variable. These
distributions were tested using Anderson-Darling statistics in Minitab. All of the other analyses
were performed using SAS/STAT software Version 9.3 considering an alpha of 0.05. Since the
study is observational, distribution of age and sex among each group was evaluated using Chi-
square and ANOVA, respectively. Also, exploratory analysis of age for both males and females
was tested using t test, 95% confidence limits were calculated whenever necessary. Genotoxic
abnormalities followed an unknown distribution and therefore descriptive analysis of these
variables included median and 95% confidence limits for free distribution. Comparisons among
groups were performed using Kruskal-Wallis test, followed by Dunn’s post-test. The presence
of certain genotoxic abnormalities (binucleated and trinucleated broken eggs) was analyzed
with Fisher’s exact test for contingency tables. Comparisons of genotoxic abnormalities
between males and females were performed using the Mann-Whitney test.

Results
Age and sex distributions for each group are presented in Table 1. Thirty-four males and 61
females participated in the study for a total of 95 individuals. Heterogeneity in the distribution
of age (p < 0.01) and sex (p = 0.0127) among the groups was observed. On average, the time
of treatment in months for G1 to G4 was, respectively, 8.1 (SD, standard deviation = 2.4), 20.3
(SD = 3.7), 38.3 (SD = 7.2), and 71.2 (SD = 13.7).
Table 1 Age and sex distribution among individuals with and without orthodontic appliances
(n = 95)
Control n = Time of orthodontic appliance use in months
21
G1 (1–12) n G2 (13–24) n G3 (25–48) n G4 (> 48) n =
= 21 = 21 = 23 9

Age (years) 24.5 (22.3– 16.4 (14.2– 16.7 (14.5– 16.9 (14.9– 18.2 (14.9–
(CL 95%) 26.7) 18.6) 18.8) 19.0) 21.5)
Sex (%)

Male (n = 34) 2 (9.5) 8 (38.1) 7 (33.3) 14 (60.9) 3 (33.3)

Female (n = 19 (90.5) 13 (61.9) 14 (66.7) 9 (39.1) 6 (66.7)
61)
95% IC 95% confidence limits
Groups: different periods of treatment (months): G1–1 and 12 months; G2–13 and 24; G3–25
and 48; G4-over 48

Table 2 Genotoxic abnormality distribution among patients without (control) and with and
orthodontic appliance
Control (n G1 (n = G2 (n = G3 (n = G4 (n = 9) p*
= 21) 21) 21) 23) Md (95%
Md (95% Md (95% Md (95% Md (95% CLfd*)
CLfd*) CLfd*) CLfd*) CLfd*)
 1 MN 2 (0–2.5) 1.5 (0.5– 1.5 (1.0– 0.5 (0.5– 1.0 (0–2.0) 0.9016
2.0) 2.0) 2.5)
2 MN 0 (0–0.5) 0 (0–0) 0 (0–0) 0 (0–0) 0 (0–0) 0.0723
Binucleated 15 (12–21) 9 (8–16) 14 (9–21) 11 (9–16) 9 (7–15) 0.4257
Pyknosis 1 (0–7) 1 (0–4) 0 (0–1) 2 (0–3) 3 (0–4) 0.3467
Karyolysis 5 (2–10) 4 (0–9) 2 (0–4) 2 (0–5) 0 (0–4) 0.0166**
Nuclear 1 (0–1) 0 (0–1) 0 (0–1) 0 (0–0) 0 (0–3) 0.6480
Buds
Total 28 (18.5– 19 (11.5– 19.5 18 (15.5– 17.5 (7.0– 0.1470
36.5) 36.5) (14.0– 24.0) 21.0)
30.0)
*p = value Kruskal-Wallis test
Md median, 95% CLfd 95% confidence limits for free distribution
MN micronuclei
**Statistical significance difference
Groups: different periods of treatment (months): G1–1 and 12 months; G2–13 and 24; G3–25
and 48; G4-over 48

Table 2 demonstrates that there was no significant statistical difference for any genotoxic
abnormalities, except for karyolysis (p = 0.0166), with increased levels in the control group in
comparison to G4 (p < 0.05), which underwent the longest fixed treatment time. Binucleated
cells were present in greater quantities in all evaluated groups in comparison with other cellular
abnormalities. However, statistically significant differences were not observed (p > 0.05). In
the comparison of trinucleated cells with the other abnormal cells, the Fisher’s exact test for
contingency tables was used. There were no statistically significant differences between groups,
although in G3, there was an apparent increase in these anomalies. Broken eggs only showed
values of 0.5 and 1, with eight individuals presenting values of 0.5 and two individuals with
the value of 1. Therefore, the values were classified as present or absent, so the Fisher’s exact
test for contingency tables resulted in a p value of 0.6526. This showed that there were no
statistically significant differences between groups. The binucleated cell count was also
considered to be present or absent (Table 3). Note that in Table 2 the analysis was performed
considering the data as continuous, and the p value is similar. Since there was an imbalance of
males and females among the groups, further stratified exploratory analysis was required to
elucidate possible effect of this potential confounder in the study. Tables 3, 4, and 5 reveal that
no statistical association could be observed between males and females.
Table 3 Percentage of individuals with genotoxic abnormal cells among those with and without
orthodontic appliance
Control (n G1 (n = G2 (n = G3 (n = G4 (n = 9) p*
= 21) 21) 21) 23) n (%)
n (%) n (%) n (%) n (%)
Tetra 2 (9.5) 2 (9.5) 1 (4.8) 4 (17.3) 1 (11.1) 0.7671
nucleated
Broken 2 (9.5) 4 (19.1) 1 (4.4) 1 (4.4) 1 (11.1) 0.6526
eggs
Binucleated 7 (33.3) 2 (9.5) 1(4.8) 1 (8.7) 1 (11.1) 0.0957
*p value for Fisher’s exact test. Groups: different periods of treatment (months): G1–1 and
12months; G2–13 and
24; G3–25 and 48; G4-over 48

Table 4 Distribution of cells with genotoxic abnormalities according to the sex

Male (n = 34) Female (n = 61) p*
Md (95% CLfd*) Md (95% CLfd*)
1 MN 1 (0.5–2) 1.5 (1–2) 0.2399
2 MN 0 (0–0) 0 (0–0) 0.3199
Binucleated 13.5 (10–15) 12 (10–16) 0.8800
Pyknosis 2 (0–4) 1 (0–4) 0.4328
Karyolysis 4 (0–6) 2 (0–5) 0.4564
Nuclear Buds 0 (0–1) 0 (0–1) 0.5660
Total 19.7 (18 - 25.5) 19.5 (16–26) 0.9690
*p value for Wilcoxon rank test
Md median, 95% CLfd 95% confidence limits for free distribution MN micronuclei
Groups: different periods of treatment (months): G1–1 and 12 months; G2–13 and 24; G3–25
and 48; G4-over 48

Table 5 Occurrence of genotoxic abnormal cells among males (n = 34) and females (n = 61)
Male (n = 34) Females (n = 61) p*
n (%) n (%)
Tetra nucleated 5 (14.7) 5 (8.2) 0.4868
Broken eggs 5 (14.7) 5 (14.7) 0.4868
Binucleated 3 (8.8) 10 (16.4) 0.3666
*p value for Fisher’s exact tes

Fig. 1 Images showing differentiated cells with different types of abnormalities found in MNT.
The alterations are identified by circles and arrows: a differentiated cell; b binucleated cell
(circle); c cell with one micronuclei (arrow); d cell with two micronucleus (circle); e nuclear
buds (circle); f pyknosis; g broken eggs (arrow); h karyolitic

Discussion
Different steps in DNA repair are affected by the diverse metals; however, in our study,
we were not able to observe genotoxic effect differences between the four evaluated
experimental groups in patients submitted to corrective orthodontic treatment. It is possible
that the release and exposure of the metal ions from the orthodontic fixed appliance are not
clinically significant enough to cause a damage to the patient. In fact, our results are in
agreement with Westephalen et al. (2008) [27] which found no significant alterations in
cytogenetic damage. On the other hand, Natarajan et al. (2011) [29] observed higher
cytogenetic damage during installation of orthodontic appliances when compared to the control
group, but these effects decreased after 30 days of treatment. It is worth mentioning that unlike
other studies, this work evaluated patients in long-term treatments. However, in our results, the
orthodontic appliances were not associated with genotoxic effects in patients regardless of the
treatment duration, either due to DNA repair capacity or adaptability of the patient.
Tintenko-Holland et al. (1994) [33] found an MN frequency of 0.14% in healthy
patients. Hevari et al. (2013) [30] found frequency of micronuclei before the device installation
(1.06%) and after 9 months of treatment (0.92%) with no statistically significant results. In our
study, no statistically significant differences between the groups were observed.
Toy et al. (2014) [6] found that the number of binucleated cells significantly increased
in all groups of patients undergoing orthodontic treatment and cells with karyolysis increased
during the 6 months of treatment. However, genotoxic effects were absent because of the ability
of epithelial cells to renew and regenerate in the short time span of 7–14 days. In our study, the
number of binucleated cells in the experimental groups was lower than in the control group;
however, the difference was not statistically significant. Regarding the karyolysis count,
similar results were obtained in this study with higher frequency of karyolysis cells in the
control group and the reduction of these cells in the group with long-term orthodontic treatment.
According to Ramirez and Saldanha (2002) [34], increments of karyolysis cells occur in the
keratinization process, representing the adaptive response of injuries that occurred in the oral
epithelium, and also related to necrotic cells and cytotoxic effects. This anomaly is normal in
squamous epithelia, especially for the effects caused by chewing. However, the constant action
of mutagenic agents such as alcohol, tobacco, radiotherapy in cancer patients, and orthodontic
appliances could increase the rate of cell death as indicated by a significant increase of this
anomaly. This adaptive response was not observed in our study.
Binucleated cells are associated with neurodegenerative diseases and cancer cells,
suggesting that high proportions of binucleated cells may be associated with these diseases [33,
35]. This study showed high values of binucleated cells when compared to other evaluated
nuclear alterations, but with normal distribution between the groups and no statistically
significant values, demonstrating that corrective orthodontic treatment and the time of the
treatment did not increase the number of these cells.
The literature reports that there is a tendency for females to have higher frequencies of
MN and other cell abnormalities [13, 22–25]. However, our results did not show this tendency.
Bloching et al. (2008) [22] found a higher number of MN in older patients presenting oral
problems, such as missing teeth and periodontal disease. Hevari et al. (2013) [30] concluded
that advanced age, systemic diseases, and risk factors such as tobacco and alcohol consumption
may aggravate the harmful effects of corrective orthodontic appliances. In our study, smokers,
alcoholics, and individuals with systemic or oral diseases were not included. There were no
statistically significant differences in any of the genotoxic abnormalities when comparing the
nuclear alterations with the age of the patients in all groups.
Our results should be interpreted with caution because DNA damage caused by the use
of orthodontic appliances can be repaired, but any decrease in repair capacity or changes in the
immune system may allow the DNA damage to cause future mutations in the genome [30].
Our study had some limitations. The power of the study can be seen as a limitation, in
spite of estimation that 21 individuals per group would be enough to reach a power greater than
0.80. However, in spite of having only nine individuals in G4, the actual power of the study,
assuming a standard deviation of about 1.1 and only nine individuals in each group resulted in
a relatively reasonable power of 0.714. When we considered the actual number for the other
groups (n = 21) with the exception of the last one, the power was greater than 0.80. However,
we cannot rule out the possibility of false negative results by chance. The evaluation
mechanism for the slides that was conducted through optical light microscope is also a concern.
This limited the ability to capture the complete image gallery for future reanalysis and
quality control. In addition, it increases the risk of obtaining heterogeneous values, regardless
of the evaluator’s level of training [36]. Orthodontic treatments did not cause significant
cytogenetic alterations, even in patients undergoing long-term treatments. Further studies
should be performed to evaluate the genotoxic effects of orthodontic materials as self-ligating
brackets, mainly aligners, that are made of composite or plastics.

Conclusion
The null hypothesis was accepted. Corrective orthodontic treatment did not cause genotoxic
effects in patients, regardless of the time of treatment.
Authors’ contributions
María Gabriela Flores Bracho conducted a review of literature, wrote the manuscript, collected
the data, and did the experimental procedures of the project. Catarina Satie Takahashi
supervised all the experimental procedures of the project. Willian Orlando Castillo supervised
all the experimental procedures of the project. Maria Conceição Pereira Saraiva designed the
study and performed the statistical analyses. Erika Calvano Küchler organized the data and
wrote the manuscript. Mírian Aiko Nakane Matsumoto collaborated in the selection of
experimental subjects. José Tarcísio Lima Ferreira collaborated in the selection of experimental
subjects. Fabio Lourenço Romano and Paulo Nelson-Filho conceived the study and supervised
all aspects of its implementation. All authors contributed to the interpretation of results and
manuscript review.
Funding
The work was supported by CAPES-Coordination for the Improvement of Higher Education
Personnel-Brazilian federal government.

Compliance with ethical standards

The Research Ethics Committee (CAAE 35905914.1.0000.5419) approved this project. The
patients agreed to participate in the study and they, or their guardians, signed an informed
consent.
Conflict of interest The authors declare that they have no conflict of interest.
Ethical approval The Research Ethics Committee (CAAE 35905914.1.0000.5419) approved
this project. All procedures performed in studies involving human participants were in
accordance with the ethical standards of the institutional and/or national research committee
and with the 1964 Helsinki declaration and its later amendments or comparable ethical
standards.
Informed consent Informed consent was obtained from all individual participants that were
included in the study.
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