Braz J Oral Sci. 15(1):27-34

Original Article Braz J Oral Sci.
January | March 2016 - Volume 15, Number 1

Influence of factors in the oral mucosa
maturation pattern: a cross-sectional study

applying multivariate analyses
Cristina da Silva Baumgart1, Natália Batista Daroit1, Bruna Jalfim Maraschin1, Alex Haas2,

Fernanda Visioli1, Pantelis Varvaki Rados1

1Universidade Federal do Rio Grande do Sul - UFRS, School of Dentistry, Area of Oral Pathology, Porto Alegre, RS, Brazil
2Universidade Federal do Rio Grande do Sul - UFRS, School of Dentistry, Area of Periodontology, Porto Alegre, RS, Brazil

Correspondence to:
Pantelis Varvaki Rados

Universidade Federal do Rio Grande do Sul
Faculdade de Odontologia, Area de Patologia Oral.
Rua Ramiro Barcelos, 2492/503 – CEP 90035-003

Porto Alegre, Rio Grande do Sul, Brazil
E-mail: pantelis@ufrgs.br

Abstract

Aim: To evaluate the association between oral health status, socio-demographic and behavioral
factors with the pattern of maturity of normal epithelial oral mucosa. Methods: Exfoliative cytology
specimens were collected from 117 men from the border of the tongue and floor of the mouth on
opposite sides. Cells were stained with the Papanicolaou method and classified into: anucleated,
superficial cells with nuclei, intermediate and parabasal cells. Quantification was made by selecting
the first 100 cells in each glass slide. Sociodemographic and behavioral variables were collected from
a structured questionnaire. Oral health was analyzed by clinical examination, recording decayed,
missing and filled teeth index (DMFT) and use of prostheses. Multivariable linear regression models
were applied. Results: No significant differences for all studied variables influenced the pattern
of maturation of the oral mucosa except for alcohol consumption. There was an increase of cell
surface layers of the epithelium with the chronic use of alcohol. Conclusions: It is appropriate to use
Papanicolaou cytopathological technique to analyze the maturation pattern of exposed subjects,
with a strong recommendation for those who use alcohol - a risk factor for oral cancer, in which a
change in the proportion of cell types is easily detected.

Keywords: Papanicolaou Test. Mouth Mucosa. Oral Health. Multivariate Analysis.

Introduction

Cytopathology is a diagnostic method that involves removing superficial mucosal

cells by exfoliation for subsequent microscopic analysis1. With the introduction of
quantitative techniques, oral cytology has become an important preventive exam for
monitoring the oral mucosa exposed to carcinogens2-6. From these studies, it became
apparent the individual nature of this type of evaluation, suggesting that some individuals
are more susceptible than others to oral cancer development.

The cell type quantification of exfoliated oral mucosa by Papanicoloau staining is
one of the mentioned quantitative methods associated with cytopathology; this procedure
allows the assessment of epithelial maturation process. Over time, many studies have
sought to evaluate the influence of extrinsic factors (tobacco and alcohol consumption)
and intrinsic factors such as sex and age in the process of oral epithelium maturation2,7.
However, it remains uncertain whether the consumption of alcohol and tobacco, in
addition to individual parameters modify the normal process of epithelial maturation of
the oral cavity. Other factors that may change the pattern of oral epithelial maturation
are oral hygiene status and socioeconomic factors, since they have been suggested as
risk factors for the development of oral cancer8-9.

http://dx.doi.org/10.20396/bjos.v15i1.8647094

Received for publication: May 05, 2016
Accepted: May 17, 2016

Thus, the aim of this study was to evaluate the influence of
the oral health status, sociodemographic and behavioral factors
on the maturation pattern of normal oral mucosa applying
multivariate models.

Material and methods

Study design and sample
This was a cross-sectional observational study that evaluated

male patients aged 25 years or older undergoing treatment at the
School of Dentistry of the Federal University of Rio Grande do Sul,
Brazil, who were considered eligible for the study. Visible lesions
in the oral mucosa (except for periodontal disease), previous or
current histories of malignant or benign tumors, radiotherapy and/
or chemotherapy, and use of fixed orthodontic appliances were
the exclusion criteria.

The sample size was estimated from data obtained in a
previous study2. The final sample comprised 117 men. Means and
standard deviations of the number of superficial cells with nuclei in
smokers/drinkers and non-smokers/non-drinkers were considered
for sample calculation (23.17±13.78 and 18.00±11.91,
respectively). By estimation, 98 patients should be included to
conduct the study with 80% power and alpha of 5%.

Ethical considerations
The present study was conducted in accordance to the ethical

guidelines set forth in the Declaration of Helsinki. The local Ethics
Committee approved the study protocol and all patients signed
an informed consent form prior to their inclusion in the study.

Interview and Clinical examination
Participants were interviewed using a structured questionnaire

to gather demographic data, information about tobacco and alcohol
habits, oral hygiene conditions, educational status, socio-economic
variables and history of oral and systemic diseases.

A single researcher performed the oral examination, with the
participant sitting in the proper position, artificial light, buccal mirror
and explorer. The first physical examination was performed to detect
visible oral lesions for exclusion. In their absence, the DMFT was
recorded index according to the WHO criteria. Next, the prosthetic
conditions were assessed. Dentures and removable appliances were
removed for inspection. The number or lost teeth in each individual
was derived from the DMFT index.

Sample collection and cytopathological analysis
Exfoliative cytology smears were collected from the border of

tongue and floor of the mouth on opposite sides with four uniform
brush turns by Cytobrush Plus®. Smears were applied on a glass
slide and then fixed in 99.6% alcohol. Samples were stained by the
modified Papanicolaou technique2. One blinded observer examined
the maturation pattern in all fields of each slide. One hundred well-
formed and isolated cells were counted on each slide horizontally,
from left to right, at 400x magnification. Cells were classified as
anucleated, superficial with nuclei, intermediate and parabasal
(Figure 1).

Intra-examiner reproducibility for classification of cell types was

Influence of factors in the oral mucosa maturation pattern: a cross-sectional study applying multivariate analyses

Braz J Oral Sci. 15(1):27-34

measured by the kappa statistic, considered acceptable when kappa
is above 0.7. Prior to the study, intra-examiner reproducibility was
tested for quantification of 20 slides. During the study 10% of the
sample was reassessed for reproducibility analysis.

Fig.1. Cellular types of normal oral mucosa epithelium: 1) parabasal; 2) intermediate; 3)
superficial with nuclei and 4) anucleated. (Papanicolaou method, 400x).

Statistical Methods
Independent Variables: Socio-demographic, behavioral and
oral variables were evaluated in the present study. Age was
categorized as <50, 50-59 and ≥60 years old. Skin color was
dichotomized into white and non-white individuals. Marital
status was categorized as married, single/divorced and widowed.

The total number of packs of cigarettes consumed in a lifetime
was calculated by multiplying the number of cigarettes smoked
per day by the number of years of habit, divided by 20 (1 pack).
Subjects were classified into three groups: non-smokers (0 pack-
years), level 1 exposure (≤20 pack-years) and level 2 exposure
(>20 pack-years).

Daily alcohol consumption was calculated by multiplying
the number of drinks consumed weekly by the average alcohol
content of a glass of beer, wine or cachaça (a typical Brazilian drink
distilled from sugarcane), divided by 7 days. The alcohol contents
were estimated as 8 g in a glass of beer, 9.6 g in a glass of wine,
and 8 g in a drink of cachaça. Drinkers were categorized into three
groups: level 1 (≤3 g/day), level 2 (>3 g/day) and non-drinker.

Oral health conditions of the subjects were analyzed by
DMFT index (decay, missing, filled teeth) and were classified in
three categories according to the number of lost teeth: level 1 (1-19
teeth lost), level 2 (≥20 teeth lost) and no tooth loss. For caries
experience, individuals were grouped in two categories: Level 1
(DMFT=0) and level 2 (DMFT≥1).

Socio-economic status was assessed using a standard
Brazilian economy classification (CCEB), which quantifies the
home consumer goods. The subjects were grouped as low status
or medium/high status and the cut off was 12 points. Educational
level was categorized according to the number of education years,
as follows: low level (≤4 years), intermediate level (5-10 years of
education), and high level (≥11 years of education).

Statistical analyses: Cell types were expressed in mean
percentage and standard error separately for the border of the
tongue and floor of the mouth. Comparisons between means
according to the independent variables (socio-demographic,
behavioral and oral) were performed using Wald tests, adjusting
for multiple comparisons if required.

Predictive models of multivariable linear regression were

applied by entering all independent variables of interest in the
model. Interactions and multicollinearity were evaluated and
were not found. Data analysis was conducted using Stata software
(StataCorp., version 10 for Macintosh). The individual was the
analytical unit. The level of significance was set at 5%.

Results

The characteristics of the participants of the study are
presented in Table 1. The study sample comprised mostly
Caucasian men under 50 years of age, married, from low
socioeconomic status and medium educational level. Regarding
oral status, the majority of individuals reported brushing teeth at
least once a day and did not use mouthwashes. Almost half the
patients said they had never smoked. Among smokers or former
smokers, the percentage of heavy smokers was high. Regarding
alcohol consumption, almost 40% of the patients never ingested
alcohol beverages and equal amount use more than 3 g of alcohol
per day (Table 1).

Tables 2 and 3 present the means and standard errors of
exfoliated squamous cells percentage from the border of the
tongue and the floor of the mouth, respectively, according to
the independent variables. On the border of tongue, 75% of the
cells were intermediate, 20% were superficial cells with nuclei,
5% were anucleated cells. Parabasal cells were seldom observed.
A significantly higher percentage of superficial and anucleated
cells was observed in drinker subjects compared to non-drinker
individuals. For intermediate cells, the percentage was significantly
lower in drinkers compared to those who did not drink alcoholic
beverages (Table 2).

Table 3 presents the pattern of maturation of the floor of the
mouth, where approximately 82.5% of the cells were intermediate,
15% were superficial cells with nuclei, 2.5% were anucleated,
whereas parabasal cells were rarely observed. Evaluating the
studied variables, again only alcohol consumption was statistically
significant, with differences between drinkers and non-drinkers.
Similar to the tongue, intermediate cells were found in smaller
proportions and anucleated cells in larger number in individuals
who drank alcohol often.

Tables 4 and 5 show the multivariate linear regression
models for the association between the percentage of cell types on
the border of tongue and floor of mouth, respectively, as well as
the independent variables. Significant associations were observed
for consumption of alcoholic beverages, which showed increase
of anucleated cells and superficial cells with nuclei, as well as
decrease of intermediate cells with increased alcohol consumption.

Discussion

Some risk factors contributing to oral cancer, such as
smoking and alcohol, are already well established in the
literature10-11. Analysis of individuals exposed to these factors
using Papanicolaou test showed alterations in maturation pattern
of cells2,12. This study aimed to evaluate other possible variables
that could influence the pattern of maturation of oral epithelial
cells, such as oral health and sociodemographic factors. To our

Influence of factors in the oral mucosa maturation pattern: a cross-sectional study applying multivariate analyses

Braz J Oral Sci. 15(1):27-34

Table 1 - Absolute distribution and percentage of socio-
demographic and behavioral status of the participants.
Variables n %
Age
<50 years 61 52.1
50-59 years 30 25.7
≥60 years 26 22.2
Skin color
White 81 69.2
Non-white 36 30.8
Marital status
Married 67 57.3
Single/divorced 48 41.0
Widowed 2 1.7
Socio-economic status
Medium/High 53 45.3
Low 64 54.7
Educational level
High (≥11 education years) 44 37.6
Intermediate (5-10 education years) 50 42.7
Low (≤4 education years) 23 19.7
Brush teeth
< 1 time/day 3 2.6
≥1 time/day 114 97.4
Use mouthwashes
Yes 25 21.4
No 92 78.6
Visited the dentist
<1year 57 48.7
≥1 year 60 51.3
Smoke
Current smoker 25 21.4
Former smoker 30 25.6
Never smoked 62 53.0
Exposed smoke
Never smoked 62 53.0
Smoked ≤ 20 pack-years 34 29.1
Smoked > 20 pack-years 21 17.9
Alcohol consumption
Never ingested alcohol 46 39.3
Drink ≤ 3g 24 20.5
Drink > 3g 47 40.2
Total 117 100.0

knowledge, this study is the first to assess by cytopathology, the
influence of oral health, behavioral and sociodemographic factors
on maturation pattern of oral mucosa using multilevel modeling.

In order to assist the early detection of this altered tissue -
even before clinical lesion, when a biopsy contraindicated, oral
exfoliative cytopathology can be used, performing tests such as
Papanicolaou stain (PAP). Oral cytopathology is a fast, easy to
perform, non-invasive and inexpensive technique; which allows
to evaluate changes in cytological pattern maturation of normal
oral mucosa, inferring risk groups and assisting in the prevention
of these factors1.

In these studies, the only statistically significant variable
associated with alterations in maturation pattern was the use of
alcohol, which resulted in an increase of surface cell layers of the
epithelium (anucleated and superficial with nuclei) in individuals
exposed to alcohol. Our results contradict the previous literature.
One study evaluated the cell maturation pattern of individuals
exposed to tobacco and alcohol observed a smaller number of

superficial cells with nuclei compared to the non-exposed group2. We
consider that these results differ from our study for some reasons:
first, because alcohol consumption was not evaluated alone, but
synergistically with tobacco; further, in our study was performed a
multivariate analysis, thus, it can control other variables that could
be biases. Another reason is that in our study a greater number of
participants was assessed compared with the above-mentioned study.

Influence of factors in the oral mucosa maturation pattern: a cross-sectional study applying multivariate analyses

Variables Anucleated p* Supnuclei** p* Intermediate p* Parabasal p*
Age
<50 years 4.97±0.33 Ref. 20.37±0.38 Ref. 74.84±0.53 Ref. 0.00±0.00 Ref.
50-59 years 5.37±0.51 0.51 20.10±0.52 0.67 74.53±0.81 0.76 0.00±0.00 -
60+ years 5.57±0.53 0.33 19.77±0.66 0.43 74.58±0.84 0.80 0.08±0.05 0.15
Skin color
White 5.35±0.31 Ref. 20.37±0.34 Ref. 74.40±0.50 Ref. 0.01±0.01 Ref.
Non-white 4.89±0.38 0.35 19.72±0.48 0.27 75.36±0.59 0.22 0.03±0.03 0.61
Socio-economic status
High 5.15±0.38 Ref. 20.34±0.40 Ref. 74.51±0.58 Ref. 0.02±0.02 Ref.
Low 5.25±0.32 0.84 20.03±0.39 0.58 74.86±0.53 0.66 0.02±0.02 0.89
Educational level
High 5.39±0.38 Ref. 20.36±0.41 Ref. 74.48±0.58 Ref. 0.00±0.00 Ref.
Intermediate 5.04±0.38 0.52 20.28±0.46 0.89 74.66±0.62 0.83 0.04±0.03 0.16
Low 5.22±0.56 0.80 19.56±0.64 0.30 75.22±0.94 0.51 0.00±0.00 -
Alcohol consumption
Never ingested alcohol 4.43±0.36 Ref. 19.35±0.45 Ref. 76.22±0.58 Ref. 0.02±0.02 Ref.
Drink ≤3g 6.04±0.56 0.02 20.42±0.68 0.19 73.96±0.85 0.03 0.00±0.00 0.32
Drink > 3g 5.53±0.38 0.04 20.85±0.39 0.01 73.60±0.60 0.01 0.02±0.02 0.99
Exposed Smoke
Never smoked 5.00±0.33 Ref. 20.24±0.40 Ref. 74.74±0.52 Ref. 0.02±0.02 Ref.
Smoked ≤20 pack-years 5.47±0.42 0.38 20.21±0.49 0.95 74.35±0.72 0.66 0.00±0.00 0.32
Smoked >20 pack-years 5.38±0.67 0.61 19.90±0.65 0.66 75.14±1.04 0.73 0.05±0.05 0.53
Smoke + Alcohol
Never smoked/never drink 4.82±0.49 Ref. 19.71±0.58 Ref. 75.43±0.74 Ref. 0.04±0.04 Ref.
Smoke or Drink intermediate 5.29±0.48 0.50 19.75±0.65 0.97 75.00±0.81 0.70 0.00±0.00 0.32
Smoke or drink hard 5.34±0.35 0.39 20.57±0.35 0.21 74.23±0.55 0.20 0.02±0.02 0.62
Use mouthwashes
No 5.17±0.27 Ref. 20.17±0.31 Ref. 74.74±0.43 Ref. 0.02±0.02 Ref.
Yes 5.32±0.56 0.82 20.16±0.67 0.98 74.56±0.94 0.86 0.00±0.00 0.16
Teeth lost
0 tooth 4.08±0.45 Ref. 20.00±0.47 Ref. 75.19±0.70 Ref. 0.00±0.00 Ref.
1-19 teeth 5.38±0.30 0.30 20.31±0.37 0.61 74.46±0.50 0.39 0.01±0.01 0.32
20+ teeth 5.21±0.87 0.68 19.86±0.88 0.89 74.86±1.25 0.81 0.07±0.07 0.32
Caries experience
DMFT≤8 4.88±0.38 Ref. 20.32±0.39 Ref. 74.98±0.64 Ref. 0.02±0.02 Ref.
DMFT≥9 5.46±0.31 0.24 20.04±0.38 0.61 74.47±0.47 0.53 0.02±0.02 0.87
Use of oral prosthesis
Not use 5.17±0.28 Ref. 20.43±0.32 Ref. 74.40±0.46 Ref. 0.01±0.01 Ref.
Partial removable prosthesis 5.47±0.56 0.63 19.63±0.63 0.26 75.89±0.79 0.59 0.00±0.00 0.31
Total prosthesis 5.07±0.86 0.92 19.36±0.95 0.29 76.21±1.38 0.22 0.07±0.07 0.42
Total 5.21±0.24 20.17±0.28 74.70±0.39 0.02±0.01

* Comparisons of the reference group (Ref.).
** superficial cells with nuclei.

Table 2 - Mean distribution (standard error) the percentage of the type of cells on the border of
tongue according to independent variables.

Braz J Oral Sci. 15(1):27-34

Another study evaluated the proportion of cell types of the
oral mucosa of individuals who used daily Listerine® mouthwash
with 26.9% of alcohol for 6 months compared to the group
that used the mouthwash without alcohol. Only outer cells of
the epithelium (superficial and intermediate) were found in
both groups, with no statistical differences between the pattern

31Influence of factors in the oral mucosa maturation pattern: a cross-sectional study applying multivariate analyses

* Comparisons of the reference group (Ref.).
** superficial cells with nuclei.

Variables Anucleated p* Supnuclei** p* Intermediate p* Parabasal p*
Age
<50 years 2.31±0.30 Ref. 15.46±0.53 Ref. 82.06±0.65 Ref. 0.00±0.00 Ref.
50-59 years 2.17±0.35 0.75 14.33±0.81 0.25 83.53±0.97 0.21 0.03±0.03 0.31

60+ years 2.40±0.39 0.82 15.88±0.87 0.68 81.65±0.97 0.72 0.04±0.04 0.32
Skin color
White 2.21±0.24 Ref. 15.17±0.48 Ref. 82.48±0.56 Ref. 0.03±0.02 Ref.
Non-white 2.50±0.37 0.51 15.47±0.71 0.72 82.06±0.89 0.69 0.00±0.00 0.16
Socio-economic status
High 2.06±0.25 Ref. 15.64±0.55 Ref. 82.30±0.59 Ref. 0.04±0.03 Ref.
Low 2.50±0.30 0.26 14.95±0.56 0.38 82.39±0.71 0.93 0.00±0.00 0.16
Educational level
High 2.43±0.33 Ref. 15.41±0.68 Ref. 82.16±0.73 Ref. 0.02±0.02 Ref.
Intermediate 2.34±0.33 0.84 15.14±0.55 0.76 82.34±0.74 0.86 0.02±0.02 0.93
Low 1.96±0.35 0.32 15.26±1.01 0.90 82.73±1.14 0.67 0.00±0.00 0.32
Alcohol consumption
Never ingested alcohol 1.63±0.25 Ref. 14.74±0.68 Ref. 83.61±0.75 Ref. 0.02±0.02 Ref.
Drink ≤3g 2.75±0.49 0.04 15.08±0.72 0.73 81.79±1.04 0.16 0.04±0.04 0.67
Drink > 3g 2.72±0.33 0.01 15.87±0.63 0.22 81.40±0.72 0.04 0.00±0.00 0.32
Exposed Smoke
Never smoked 2.18±0.28 Ref. 15.18±0.54 Ref. 82.65±0.65 Ref. 0.02±0.02 Ref
Smoked ≤20 pack-years 2.47±0.38 0.53 16.47±0.70 0.15 80.76±0.80 0.07 0.00±0.00 0.32
Smoked >20 pack-years 2.38±0.47 0.71 13.57±0.90 0.13 84.05±1.13 0.29 0.05±0.05 0.53
Smoke + Alcohol
Never smoked/never drink 1.82±0.35 Ref. 15.46±0.86 Ref. 82.64±0.98 Ref. 0.04±0.04 Ref.
Smoke or Drink intermediate 2.29±0.42 0.40 15.14±0.70 0.77 82.28±0.93 0.79 0.00±0.00 0.32
Smoke or drink hard 2.52±0.28 0.12 15.23±0.57 0.82 82.25±0.67 0.74 0.02±0.02 0.62
Use mouthwashes
No 2.39±0.23 Ref. 15.04±0.42 Ref. 82.43±0.53 Ref. 0.02±0.02 Ref.
Yes 1.96±0.34 0.31 16.08±1.04 0.36 82.04±1.05 0.74 0.00±0.00 0.16
Teeth lost
0 tooth 1.81±0.32 Ref. 15.45±0.65 Ref. 82.74±0.73 Ref. 0.00±0.00 Ref.
1-19 teeth 2.44±0.27 0.13 15.29±0.55 0.85 82.11±0.66 0.52 0.03±0.02 0.16
20+ teeth 2.64±0.58 0.21 14.71±1.00 0.54 82.71±1.31 0.99 0.00±0.00 -
Caries experience
DMFT≤8 2.32±0.27 Ref. 15.15±0.60 Ref. 82.51±0.68 Ref. 0±0 Ref.
DMFT≥9 2.27±0.28 0.89 15.35±0.60 0.80 82.21±0.65 0.75 -0.30±-0.21 0.15
Use of oral prosthesis
Not use 2.35±0.25 Ref. 15.51±0.48 Ref. 82.12±0.56 Ref. 0.02±0.02 Ref.
Partial removable prosthesis 1.68±0.36 0.13 15.26±1.04 0.83 82.53±1.26 0.76 0.00±0.00 0.16
Total prosthesis 2.79±0.53 0.47 13.79±0.90 0.09 83.5±1.26 0.32 0.00±0.00 0.16
Total 2.30±0.20 15.26±0.40 82.35±0.47 0.02±0.01

Table 3 - Mean distribution (standard error) the percentage of the type of cells on the floor
of the mouth according to independent variables.

of epithelium maturation using a mouthwash with or without
alcohol13. However, that study13 does not detail the criteria used
for cell type analysis, nor how many cells were analyzed. Another
factor that explains the difference observed in our study was that
individuals evaluated in our study were chronic alcohol users for
years and several times a day.

Braz J Oral Sci. 15(1):27-34

32 Influence of factors in the oral mucosa maturation pattern: a cross-sectional study applying multivariate analyses

Variables Anucleated Supnuclei* Intermediate Parabasal
β±SE** p β±SE** p β±SE** p β±SE** p

Age 0.02±0.01 0.21 0.06±0.03 0.07 -0.08±0.04 0.05 0.01±0.01 0.25
Skin color
White Ref. Ref. Ref. Ref.
Non-white 0.41±0.45 0.36 0.48±0.91 0.59 -0.63±1.08 0.55 -0.01±0.02 0.53
Exposed Smoke
Never smoked Ref. Ref. Ref. Ref.
Smoked ≤20 pack-years 0.21±0.47 0.64 1.07±0.95 0.26 -1.49±1.12 0.18 -0.01±0.02 0.51
Smoked >20 pack-years 0.02±0.61 0.97 -1.68±1.23 0.17 1.77±1.45 0.22 0.03±0.03 0.35
Alcohol consumption
Never ingested alcohol Ref. Ref. Ref. Ref.
Drink ≤3g 1.10±0.54 0.04 0.26±1.10 0.80 -1.70±1.30 0.19 0.02±0.03 0.51
Drink > 3g 1.16±0.47 0.01 1.24±0.94 0.19 -2.29±1.12 0.04 -0.01±0.02 0.92
Use mouthwashes
No Ref. Ref. Ref. Ref.
Yes -0.32±0.49 0.51 0.69±0.99 0.48 -0.05±1.17 0.96 -0.02±0.03 0.47
DMFT index -0.01±0.03 0.68 -0.02±0.06 0.72 0.04±0.07 0.60 0.01±0.01 0.12
Use of oral prosthesis
Not use Ref. Ref. Ref Ref
Partial removable prosthesis -0.89±0.63 0.16 -1.06±1.28 0.41 1.12±1.51 0.42 -0.06±0.03 0.47
Total prosthesis 0.20±0.72 0.77 -1.63±1.46 0.26 1.30±1.73 0.45 -0.08±0.04 0.06
R2 0.02 0.01 0.03 0.01

Table 5 - Multivariate linear regression model of the association between the percentage of cells on the
floor of the mouth and the independent variables.

* superficial cells with nuclei.
** Beta ± Standard Error.

Table 4 - Multivariate linear regression model of the association between the percentage of cells at the
border of tongue and the independent variables.

* superficial cells with nuclei.
** Beta ± Standard Error.

Variables Anucleated Supnuclei* Intermediate Parabasal
β±SE** p β±SE** p β±SE** p β±SE** p

Age 0.01±0.02 0.85 0.01±0.02 0.67 -0.27±0.03 0.44 0.01±0.01 0.16
Skin color
White Ref. Ref. Ref. Ref.
Non-white -0.25±0.56 0.66 -0.57±0.65 0.37 0.61±0.88 0.49 0.01±0.02 0.61
Exposed Smoke
Never smoked Ref. Ref. Ref. Ref.
Smoked ≤20 pack-years 0.05±0.58 0.92 -0.33±0.68 0.62 0.27±0.92 0.76 -0.01±0.02 0.68
Smoked >20 pack-years 0.14±0.75 0.84 -0.16±0.88 0.85 0.29±1.19 0.80
Alcohol consumption
Never ingested alcohol Ref. Ref. Ref. Ref.
Drink ≤3g 1.69±0.67 0.01 1.03±0.78 0.19 -2.31±1.07 0.03 -0.01±0.03 0.66
Drink > 3g 1.40±0.58 0.01 1.37±0.67 0.04 -2.92±0.92 0.00 0.01±0.02 0.69
Use mouthwashes
No Ref. Ref. Ref. Ref.
Yes 0.24±0.61 0.69 -0.04±0.70 0.95 -0.19±0.96 0.84 -0.01±0.03 0.59
DMFT index 0.06±0.04 0.09 -0.02±0.04 0.53 -0.05±0.06 0.38 -0.01±0.01 0.84
Use of oral prosthesis
Not use Ref. Ref. Ref. Ref.
Partial removable prosthesis -0.03±0.78 0.96 -0.46±0.91 0.61 0.82±1.24 0.50 -0.03±0.03 0.33
Total prosthesis -0.33±0.89 0.71 -0.85±1.04 0.41 2.05±1.42 0.15 0.02±0.04 0.63
R2 0.02 -0.01 0.03 -0.03

Braz J Oral Sci. 15(1):27-34

Some researches using cytopathology observed the influence
of the chronic use of ethanol on the cells of oral mucosa. Reis et
al.14 (2006) showed a significant increase in micronucleus, abnormal
nucleus/cytoplasm ratio, pyknosis, karyorrhexis and karyolysis in
exposed mucosa. Webber et al.6 (2016) evaluated by Feulgen staining
nuclear changes in cells from the border of the tongue and the floor of
the mouth of alcoholics. The last cited site showed a higher frequency
of karyorrhexis and this suggests higher degrees of keratinization.
This statement agrees with our results, taking into account the alcohol
variable, we found more cells of the superficial layers, which suggests
an organism reaction as protection against aggression, maybe with
increased keratinization.

In contradiction, smoking, the greatest risk factor for oral cancer
alone did not significantly affect the maturation pattern of the oral
mucosa. The results are conflicting in the literature on this subject.
Burzlaff and Gedoz2-3 found an increased number of surface nucleated
cells in patients exposed to tobacco smoke.

The sites chosen for the collection of smears were tongue and
floor of the mouth because in Brazil these locations are the most
frequently affected by oral squamous cell carcinoma15-16. In our
results it is possible to see a clear distinction of cell types (anucleated,
superficial cells with nuclei, intermediate and parabasal) at each site,
approximately with 5%, 20%, 75%, 0% on the tongue, and 2.5 %,
15%, 82.5%, 0% on the floor of the mouth, respectively (Tables 2
and 3). It is important to know the particular pattern of each site –
under normal conditions the floor of mouth is less keratinized than
the border of tongue, so it presents a lower amount of surface and
more intermediate cells6,17 - to detect possible changes in this cellular
types proportion when exposed to a carcinogenic factor.

Some studies correlated low income with higher incidence of
oral cancer18-19,for this reason sociodemographic factors were included
as independent variables. Because cytopathology has been used as a
monitoring tool of cytogenetic changes, we decided to include these
factors in our study. Madani19 reported that individuals with low
educational level have 3.3 more chances of developing the disease than
those with higher schooling. Regarding the results of our study, the
pattern of the oral epithelium maturation had no significant differences
comparing the different ranges of economic and educational level.

The association between the status of oral health and head and
neck cancer has been widely studied; these studies suggest that poor
oral hygiene can contribute to an increased risk of this pathology20-21.
In the present study there was no change in cell maturation pattern
in relation to oral health status, which disagrees with the idea that
oral microorganisms can increase inflammatory cytokines level and
change the complex metabolic pathways, triggering the process of
carcinogenesis22.

The use of Papanicolaou cytopathological method is appropriate
to analyze the proportion of exfoliated cells as the maturation pattern
of healthy male subjects. The maturation pattern of the buccal mucosa
cells was not affected by the studied variables, except by the intake
of alcoholic beverages.

References

1. Kazanowska K, Hałoń A, Radwan-Oczko M. The role and application
of exfoliative cytology in the diagnosis of oral mucosa pathology –

contemporary knowledge with review of the literature. Adv Clin Exp Med.
2014; 23: 299-305.

2. Burzlaff JB, Bohrer PL, Paiva RL, Visioli F, Sant’Ana Filho M, da Silva
VD, et al. Exposure to alcohol or tobacco affects the pattern of maturation
in oral mucosal cells: a cytohistological study. Cytopathology. 2007; 18:
367-75.

3. Gedoz L, Lauxen I, Sant’Ana Filho M, Rados PV. Proliferative activity in
clinically healthy oral mucosa exposed to tobacco smoking and alcohol: a
longitudinal study using the AgNOR staining technique. Anal Quant Cytol
Histol. 2007; 29: 231-8.

4. Ahmed HG, Omer AS, Abd Algaffar SA. Cytological study of exfoliative
buccal mucosal cells of Qat chewers in Yemen. Diagn Cytopathol. 2011;
39: 796-800.

5. Oliveira LU, Lima CF, Salgado MA, Balducci I, Almeida JD. Comparative
study of oral mucosa micronuclei in smokers and alcoholic smokers. Anal
Quant Cytol Histol. 2012; 34: 9-14.

6. Webber LP, Pellicioli A, Magnusson AS, Danilevicz CK, Bueno CC,
Sant’Ana Filho M, et al. Nuclear changes in oral mucosa of alcoholics
and crack cocaine users. Hum Exp Toxicol. 2016; 35: 184-93.

7. Cowpe JG, Longmore RB, Green MW. Quantitative exfoliative cytology of
normal oral squames: an age, site and sex-related survey. J R Soc Med.
1985; 78: 995-1004.

8. Cowpe JG, Longmore RB, Green MW. Quantitative exfoliative cytology of
normal oral squames: an age, site and sex-related survey. J R Soc Med.
1985; 78: 995-1004.

9. Ahrens W, Pohlabeln H, Foraita R, Nelis M, Lagiou P, Lagiou A, et al. Oral
health, dental care and mouthwash associated with upper aerodigestive
tract cancer risk in Europe: the ARCAGE study. Oral Oncol. 2014; 50:
616-25.

10. Andrade JO, Santos CA, Oliveira MC. Associated factors with oral cancer:
a study of case control in a population of the Brazil’s Northeast. Rev Bras
Epidemiol. 2015; 18: 894-905.

11. Sanner T, Grimsrud TK. Nicotine: Carcinogenicity and Effects on Response
to Cancer Treatment - A Review. Front Oncol. 2015; 5: 196.

12. Abdelaziz MS, Osman TE. Detection of Cytomorphological Changes in
Oral Mucosa among Alcoholics and Cigarette Smokers. Oman Med J.
2011; 26: 349-52.

13. Bagan JV, Vera-Sempere F, Marzal C, Carcelén AP, Martí-Bonmati E,
Bagan L. Cytological changes in the oral mucosa after use of a mouth
rinse with alcohol: A prospective double blind control study. Med Oral
Patol Oral Cir Bucal. 2012; 17: e956-e961.

14. Reis SR, do Espírito Santo AR, Andrade MG, Sadigursky M. Cytologic
alterations in the oral mucosa after chronic exposure to ethanol. Braz Oral
Res. 2006; 20: 97-102.

15. Gervásio OL, Dutra RA, Tartaglia SM, Vasconcellos WA, Barbosa AA,
Aguiar MC. Oral squamous cell carcinoma: A retrospective study of 740
cases in a Brazilian Population. Braz Dent J. 2001; 12: 57-61.

16. Durazzo MD, de Araujo CE, Brandão Neto JS, Potenza AS, Costa P,
Takeda F, et al. Clinical and epidemiological features of oral cancer in a
medical school teaching hospital from 1994 to 2002: increasing incidence
in women, predominance of advanced local disease, and low incidence
of neck metastases. Clinics. 2005; 60: 293-8.

17. Braga FL, Meneguzzi RD, Paiva RL, Rados PV. [Cytopathology evaluation
of the oral mucosa of smokers and nonsmokers]. Odonto Cien. 2004; 19:
157-63. [Portuguese].

18. Boing AF, Antunes JL. [Socioeconomic conditions and head and neck
cancer: a systematic literature review]. Cien Saude Colet. 2011; 16:

33Influence of factors in the oral mucosa maturation pattern: a cross-sectional study applying multivariate analyses

Braz J Oral Sci. 15(1):27-34

615-22. [Portuguese].
19. Madani AH, Dikshit M, Bhaduri D, Jahromi AS, Aghamolaei T. Relationship

between selected socio-demographic factors and cancer of oral cavity - a
case control study. Cancer Inform. 2010; 9: 163-8.

20. Meyer MS, Joshipura K, Giovannucci E, Michaud DS. A review of the

relationship between tooth loss, periodontal disease, and cancer. Cancer
Causes Control. 2008; 19: 895-907.

21. Coussens LM, Werb Z. Inflammation and cancer. Nature 2002; 420: 860-7.
22. Rajeev R, Choudhary K, Panda S, Gandhi N. Role of bacteria in oral

carcinogenesis. South Asian J Cancer. 2012; 1: 78-83.

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Influence of factors in the oral mucosa maturation pattern: a cross-sectional study applying multivariate analyses