Cent. Eur. J. Med.

• 7(6) • 2012 • 756-760

DOI: 10.2478/s11536-012-0050-8

Central European Journal of Medicine

Histological changes of gingival epithelium in

smokers and non-smokers

Research Article

Ana S. Pejcic*1, Vesna D. Zivkovic2, Vukadin R. Bajagic3, Dimitrije S. Mirkovic4

1 Department of Periodontology and Oral Medicine, Medical Faculty,

University of Nis,
Dr Z. Djindjica 81 Blvd, 18000 Nis, Serbia

2 Institute of Pathology, Medical Faculty, University of Nis,

Dr Z. Djindjica 81 Blvd, 18000 Nis, Serbia

3 Department of Oral Surgery, Dental Faculty, University of Podgorica,

Krusevac bb Street, 81110 Podgorica, Monte Negro

4 Dental Clinic, Medical Faculty, University of Nis,

Dr Z. Djindjica 81 Blvd, 18000 Nis, Serbia

Received 21 February 2012; Accepted 14 June 2012

Abstract: Background: Smoking patients show a reduction of inflammatory clinical signs that might be associated with local vasoconstriction
and an increased gingival epithelial thickness. The purpose of this work was to evaluate the S-thickness of the marginal gingival oral
epithelium in smokers and non-smokers. Methods: Twelve biopsies were obtained from three different groups. Group I: non-smokers
with gingivitis, group II smokers, and group III health persons without any periodontal disease. These biopsies were histologically
processed, serially sectioned at 5 µm, and underwent evaluation of the major epithelial thickness, the epithelial base thickness,
and the external and internal epithelial perimeters. Differences between the groups were analyzed using ANOVA test. The criteria
for statistical significance were at the probablity level p< 0.05. Results: A greater epithelial thickness was observed in smokers.
Conclusion: The increased epithelium thickness can contribute to the reduction of inflammatory clinical signs in the gingival tissue.
Keywords: Gingiva • Epithelium • Tobacco
© Versita Sp. z o.o

ones. Conversely, high tobacco consumption seemed

1. Introduction to reduce gingival bleeding. The gingivitis experimental
model in smoking and non-smoking patients showed that
Tobacco use has been directly associated with periodon- the plaque formation rate was similar in both groups [5].
tal disease. Smokers have a higher number of diseased There is an established biologic rationale for the neg-
sites, greater loss of alveolar bone, and increased ative effect of cigarette smoking on periodontal tissues.
tooth loss. The severity of the disease increases with First and foremost, smoking has an immunosuppresive
both the extent and duration of the smoking exposure. effect on the host, adversely affecting host-parasite inter-
Former smokers are at lower risk than current smokers actions. Peripheral blood polymorphonuclear leukocyte
[1-3]. The association between tobacco smoking and motility, chemotaxis and phagocytosis are significantly
periodontal health has been studied in several clini- impaired [6] thus, compromising this very important first
cal and epidemiological investigation [4]. Same early line of defense against subgingival bacteria.
studies indicated that smoking patients showed more The net result is that periodontal organisms in cur-
intense inflammatory gingival sings than non-smoking rent cigarette smokers escape specific and nonspecific

* E-mail: [email protected]
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A. Pejcic et al.

immune clearance mechanisms allowing them to estab- Group I (n-2): patients with clinically healthy gingivae
lish as subgingival inhabitants. Alteration in the physical as control; group II (n-5): non–smokers with gingivitis;
subgingival environment, such as decreased oxygen group III (n-5): smokers with gingivitis. The study proto-
tension, would allow the overgrowth of anaerobic flora col was approved by the Ethical committee of Medical
[7] Cigarette smoking appears to trigger an anaerobic Faculty in Nis, Serbia (No:01-2800-5). According to rule
subgingival infection, leading to greater serverity of The Ethices committee in research, all patients gave a
periodontal disease and impaired wound healing. Those informed consent for all phases of the research.
studies suggest that by-products, originated from to- The samplies were immediately fixed in 10% phos-
bacco oxidation, modify the clinical characteristics and phate-buffered formalin, pH-7.4 and later embedded
the progression of periodontal diseases and described in paraffin and serially sectioned at 5µm. The samples
smoking habit as a risk factor for periodontal desease were cut at right angles to the oral vestibular epithelium,
[8]. Smokers displayed a less pronounced gingival resulting in a section exhibiting both sulcular and oral
inflammatory reaction as compared with non-smokers. epithelium. The slides were stained with hematoxylin
The reduction of clinical inflammatory signs is confirmed and eosin (HE) within 4 minutes and were observed in
by the decrease in gingival bleeding and suppuration on a light microscope at 10 X magnification. Hematoxylin
probing, tissue redness, edema and the amount of blood stains are commonly employed for histological studies,
vessels in the marginal gingival tissue. The reduction of often employed to color the nuclei of cells (and a few
clinical inflammatory signs in smokers can be attributed other objects, such as keratohyalin granules) blue.
to the cotinine, a nicotine metabolic by-product, which Eosin is a fluorescent red dye resulting from the ac-
has a peripheral constrictive action on gingival vessels tion of bromine on fluorescein. It can be used to stain
[9]. Although the literature indicates an increase in oral cytoplasm, collagen and muscle fibers for examination
mucosa epithelium thickness in smokers, there is no under the microscope [10].
morphometric study assessing the oral gingival epithe- Histologic assessment was carried at Institute for
lial thickness in those patients. Pathology Medical Faculty in Nis.
The objective of this work was to investigate the The external epithelial (EE), internal epithelial (IE)
relation between the thickness of the marginal gingival perimeters, the major epithelial thickness (MET–dis-
oral epithelium in smokers and non–smokers, with tance between the external epithelial surface and the
clinically healthy gingivae or with gingivitis and to better epithelial crista tip) and the epithelial base thickness
understand the role of smoking in the relationship with (EBT–distance between the external epithelial surface
periodontal disease. and basal membrane located between two cristae)
were evaluated.

2. Materials and methods

3. Statistical methods
Study population – Twelve patients (27 to 55 years old)
were selected with clinical signs of gingival health (n–2) The data showed homogeneity and the differences
or gingivitis/periodontitis (n-10) with clinical indication between the three groups were analyzed using the
for periodontal surgery, at one intraoral site per patient. ANOVA test. The difference between groups II and III
The periodontal surgeries were carried at Department was analyzed using the Student`s t-test. The criteria for
of Periodontology and Oral Medicine, Medical Faculty statistical significance was accepted at the probability
in Nis. Among the 10 patients with periodontitis, were level p < 0.005.
that had smoked an average of 15 or more cigarettes
per day for at least 10 years, were considered smok-
ers. Pregnant women, former smokers, individuals with 4. Results
systemic and immunologic abnormalitiers or those had
used any drug on the 4 weeks before the experiment Smokers with gingivitis (Group III) had consumed ap-
were excluded from the sample. proximately 15 or more cigarettes per day during about
Tissue preparation – All gingival biopsies (0.4 cm 10 years (Table 1).
to 0.2 cm) from different parts of the oral gingival tissue Of the 12 sections evaluated, 5 sections were from
were obtained during periodontal surgery as part of a current smokers with gingivitis, 5 sections were from
routine periodontal treatment independent of this study. non-smokers with gingivitis and 2 from healthy persons.
The gingival biopsies were divided into three groups, ac- There was no statistical significant difference in MET
cording to the donor`s gingival health and smoking habit. between smokers and non-smokers, regardles of the

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 Pathohistology of gingival epithelium

Table 1. Smoking habits characteristics Figure 3. Normal gingival epithelium

Smokers Non-smokers Healthy

Number of patients 5 5 2
Daily consumption 15 ± 1.1 - -
Habit duration-years 10 ± 2.1 - -

Table 2. Major epithelial thickness and epithelial base thickness,

expressed in µm

Clinical
Smokers Non-smokers Healthy p value
condition
MET* (µm) 422.1 ± 66.1 418.5 ± 33.1 430.5 ± 12 (p<0.05)
EBT** (µm) 260.1 ± 19.2 15.87 ± 16 216.9 ± 11 (p<0.05)
Footnotes: MET*–distance between the external epithelial surface and
the epithelial crista tip
EBT**–distance between the external epithelial surface and basal
membrane located between two cristae

Table 3. External and internal epithelial parameter in smokers and

non-smokers (µm)

Clinical
Smokers Non-smokers Healthy p value
condition
EE* (µm) 618.2 ± 3.1 601.5 ± 2.7 611.4 ± 2.1 (p<0.05)
IE** (µm) 2442.3 2290.6 2372.7 (p<0.05)
Footnotes: EE*–external epithelium
IE**–internal epithelium

Figure 1. Spinous stratum of gingival epithelium

clinical gingival condition. However, EBT was larger in

smoking patients (p< 0.05) (Table 2).
The marginal gingival epithelium was classified as
keratinized stratified squamous epithelium with small
intercellular spaces. The spinous stratum occupied
about 50% of the total epithelial thickness (Figure 1).
The stratum corneum was more exuberant in smoking
patient samples (Figure 2). On the figure 3 is the epithe-
lial thickness at health (Figure 3).
There was no significant statistical difference in both
EE and IE between smoker and non-smokers, regard-
Figure 2. Stratum corneum of gingival epithelium
less of the clinical condition (Table 3)

5. Discussion
Tobacco use has been directly associated with a variety
of medical conditions including various types of cancer,
pulmonary and cardiovascular diseases, and low birth
weight [11,12]. Although gingivitis and periodontitis
are elicited by bacteria, cigarette smoking has been
strongly implicated as a risk factor for the initiation and
progression of periodontal disease [13]. Smoking has
been associated with increased calculus deposition,
deeper pockets and greater attachment loss, more

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A. Pejcic et al.

pronounced radiographic evidence of furcation involve- In ours samples, we found, too, an increase in the
ment and increased alveolar bone loss. Variable levels gingival EBT in smoking with the spinous stratum wich
of plaque and inflammation with evidence of decreased occupied about 50% of total epithelium thickness and
signs of clinical inflammation have also been noted in stratum corneum wich was more pronounced. Ours re-
smokers. It is possible that the reduced intensity of the sults were similar with the results the others studies [18].
gingival response is due to the vascular changes and Table 3. show that IE was larger in gingivitis cases smok-
the thicknes of marginal gingival epithelium externed by ers but there was no significant statistical difference. The
smoking [14]. epitheluim is a non-vascular tissue that depends on the
The inflammatory response induced by dental subjacent connective tissue. The inflammation causes
plaque accumulation can be modified by tobacco by- connective disorganization, modifying the blood avail-
products, such as cotinine, a by-product of nicotine that ability and impending the elimination of metabolites from
has a peripheral vasoconstriction action that reduces the epithelium. Epithelium projection are more frequent
gingival clinical signs of bleeding, redness and edema and protuberant during gingival inflammation [19]. The
[15]. In the samples evaluated throughout this study, smoking patients showed increased epithelial base and
the spinous stratum occupied about 50% of total epi- stratum corneum thicknes. The increased epithelium
thelium thickness and the keratinocytes were apart by S-thickness can contribute to the reduction of inflamma-
small intercellular spaces. In the smokers samples, tory clinical signs in the gingival tissue.
the stratum corneum was more pronounced. These
evants were similar to the ones already described in
literature where the increase in local temperatures and 6. Conclusion
by-product from tobacco oxidation induce an increase
in oral mucosa and in the oral gingival epithelium Our results suggest that among all the negative conse-
thickness [16]. quences of tobacco on the periodontium, the tobacco
Analysis showed an increase in the MET in influence on epithilium S-tickness and so influence on
clinically healthy gingival samples when compared to signs and symptoms of gingival inflammation induced by
inflamed samples, in both smoking and non-smoking plaque accumulation. Although the exact mechanism of
patients, but this difference did not achieve statistical its influence is still unclear, smoking must be considered
significance (p<0.05). Gingival inflammation reduces as a high risk factor for chronic periodontal disease.
the epithelial tickness and can potentially cause clinical
ulceration [17].
Acknowledgements
The authors have no any conflict of interests.

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