The Effect of Smoking on EpithelialProliferation in Healthy and PeriodontallyDiseased Marginal Gingival EpitheliumSibel Elif Gultekin,* Burcu Senguven,* and Burcu Karaduman†

Background: Smoking causes an increase in the thicknessof gingival epithelium, which is the outcome of increased ke-ratinocyte proliferation or loss. Smoking-related changes inthe proliferative activity of the gingival epithelium are largelyuncharacterized for periodontal diseases. The aim of the pres-ent study was to determine the effects of smoking on the pro-liferation of the epithelium in periodontally diseased marginalgingiva by comparing the expression patterns of two differentproliferation markers.

Methods: Gingival biopsies (N = 60) were obtained fromsmokers who had clinically healthy gingiva (n = 10), smokerswith gingivitis (n = 10), smokers with periodontitis (n = 10),non-smokers with clinically healthy gingiva (n = 10), non-smokers with gingivitis (n = 10), and non-smokers with peri-odontitis (n = 10). The quantitative measurement of maximumepithelial thickness was performed on hematoxylin and eosin–stained sections. The expression patterns for proliferating cellnuclear antigen (PCNA) and Ki67 were evaluated immunohis-tochemically.

Results: The percentage of PCNA-positive cells was higherthan the percentage of Ki67-positive cells in all groups(P <0.001). When the mean values of PCNA and Ki67 werecompared in each group, a statistically significant differencewas observed only in the healthy smoker group (P = 0.003).Significant differences in PCNA proliferation indices wereonly found between the smoker group and the non-smokerhealthy group (P = 0.015).

Conclusions: Smoking had an affect on the proliferation ofcells in the oral gingival epithelium, regardless of periodontalstatus. The increase in thickness of the epithelium was not as-sociated with smoking; periodontal status and inflammationseemed to be more important factors. Smoking induced thereplication activity of gingival epithelium and induced DNArepair. J Periodontol 2008;79:1444-1450.

KEY WORDS

Cell proliferation; epithelium; gingiva; smoking.

Periodontal disease is an infectious,chronic, inflammatorydiseasechar-acterized by the loss of connective

tissue attachment and alveolar bonein association with the formation of apocket and subsequent tooth loss.1 Cur-rent knowledge suggests that smoking isan important risk factor for the develop-ment of periodontal disease, especiallysevere periodontitis.2,3 A clear associa-tion has been demonstrated betweensmoking and periodontal disease, whichwas first studied by Pindborg in 19474 andthen by Arno et al.5 in 1958. Clinical,epidemiologic, and laboratory studies6,7

indicated an association between smok-ing and the prevalence and severity ofperiodontal disease. The results of thestudies6-9 revealed tobacco smoking tobe strongly associated with loss of attach-ment and bone. Probing depth10 andgingival recession are greater in smokersthan in non-smokers.11,12 In addition,smokers are more susceptible than non-smokers to advanced and aggressiveforms of periodontitis.8,13 All of theseclinical findings reflect the biologic effectsof smoking on the periodontal tissues. Thetobacco products affect variousaspectsofthe host response6,14 and are capable ofcausing alterations in the cells of theperiodontium.15 The alterations in thegingival epithelium and connective tissueinclude an increase in the thickness ofgingival epithelium and keratosis;16,17

induction of gingival keratinocytes toproduce increased levels of mediators;18

* Department of Oral Pathology, Faculty of Dentistry, Gazi University, Ankara, Turkey.† Department of Periodontology, Faculty of Dentistry, Gazi University.

doi: 10.1902/jop.2008.070645

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fibroblast dysfunction;16 and an increase in thenumber of inflammatory cells, blood vessels, andcollagen fibers in connective tissue.17 Although it iswell established that smoking increases the prolifera-tion of epithelial cells in healthy and dysplastic oralmucosa,16,19 the effect of smoking on the proliferativeactivity of gingival keratinocytes has not been exten-sively evaluated in periodontally diseased patients.

The proliferative activity of the oral mucosal andgingival epithelium has been analyzed for many yearsby monoclonal antibodies to such antigens as prolif-erating cell nuclear antigen (PCNA) and Ki67. PCNAis one of the nuclear antigens; it is a 36-kDa auxiliaryprotein to DNA polymerase delta.20,21 Expression ofPCNA is elevated in the nucleus during the late G1

phase, immediately before the onset of DNA synthe-sis; expression becomes maximal during the S phasebefore declining during the G2 and M phases. The levelof PCNA correlates directly with rates of cellular pro-liferation and DNA synthesis.22,23

Ki67 is a non-histone nuclear protein that is ex-pressed in proliferating cells during all active phasesof the cell cycle except G0;24,25 Ki67 is an excellentmarker for determining the fraction of growing cellsin a given cell population.26 Thus, PCNA and Ki67have been used extensively as indicators of cell prolif-eration.26-28

Considering all of this knowledge together, smok-ing, which is a risk factor for the development of peri-odontal diseases,2,3,6-10 increases the thickness ofgingival epithelium.17 Furthermore, it was shown thatan increase in the cell number in dysplastic and nor-mal epithelium is correlated with smoking.15-19 Theimbalance in homeostasis of gingival epithelium, par-ticularly the increased proliferation rate of keratino-

cytes, may result in the observed thickening of thegingival epithelium in smokers. Therefore, the aimof our study was to determine the effects of smokingon the proliferation of gingival epithelium in subjectswith gingivitis and those with periodontitis, using twodifferent proliferation markers.

MATERIALS AND METHODS

Study PopulationThe study included 60 subjects, with equal numbers ofsmokers and non-smokers, who were referred to theclinics of the Department of Periodontology, Facultyof Dentistry, Gazi University, between 2004 and2006. Individuals who had smoked an average ‡10cigarettes per day for ‡3 years were consideredsmokers. Additionally, no subject presented with ahistory of systemic diseases or prescription medica-tion use for ‡3 months prior to evaluation. Pregnantsubjects were not included in the study. All subjectswere selected on a voluntary basis, and all providedoral informed consent. The study population con-sisted of 40 males and 20 females. The gender andage ranges are shown in Table 1. The University ofGazi institutional review board approved the use of hu-man subjects in this study.

Subjects were placed into six groups of 10 subjectseach: smokers with clinically healthy gingiva (SH; n =10), smokers with gingivitis (SG; n = 10), smokerswith periodontitis (SP; n = 10), non-smokers with clin-ically healthy gingiva (NSH; n = 10), non-smokerswith gingivitis (NSG; n = 10), and non-smokers withperiodontitis (NSP; n = 10).

The groups SH and NSH were recruited as the con-trol with a gingival index score of 0 for the smoker andnon-smoker groups.29 The criteria for gingivitis were

Table 1.

Age, MET, and PRI (mean – SD) and Gender Distribution of Subjects

Gender

(females/males)

(N = 60)

Age (years)

(N = 60)

MET

(N = 60)

PCNA (%)

(N = 60)

Ki67 (%)

(N = 60)

SH (n = 10) 2/8 21.7 – 1.50 7.09 – 2.32 10.95 – 8.76* 0.27 – 0.41†

SG (n = 10) 4/6 33.3 – 8.65‡ 7.75 – 2.85 5.00 – 8.81 1.99 – 1.87‡

SP (n = 10) 1/9 49.9 – 11.30 8.76 – 2.64§ 3.42 – 4.83 2.31 – 2.31§

NSH (n = 10) 5/5 25.3 – 6.60 7.36 – 2.49 1.86 – 2.21 0.21 – 0.30

NSG (n = 10) 3/7 31.9 – 15.90 9.48 – 3.30 4.76 – 7.26 1.00 – 1.49

NSP (n = 10) 5/5 44 – 12.14 9.90 – 4.38 2.23 – 3.91 2.77 – 4.33

* Paired-sample t test (P = 0.015).† Paired-sample t test (P = 0.003).‡ Pearson correlation = 0.657 (P = 0.019).§ Pearson correlation = 0.568 (P = 0.043).MET = maximum epithelial thickness; PRI = proliferation index.

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no attachment loss and a gingival index of 1 or 3.29

Criteria for periodontitis were an adult having at leastfive sites with >4 mm horizontal alveolar bone loss onradiographs. The site where the biopsy was taken hadto have a probing depth ‡5 mm. All subjects selectedfor this study met the necessary criteria for the biopsyprotocol explained below.

All gingival biopsies (one per person), with a max-imum diameter of 5 mm, were collected from a buccalsite of the mandibular posterior region. Biopsy sam-ples from gingivitis and periodontitis groups weretaken from the marginal gingiva during the periodon-tal treatment (periodontal surgery and/or prepros-thetic surgery) of mandibular posterior teeth. Healthytissue samples were obtained from attached gingivaof completely erupted mandibular third molars.

Histopathologic and ImmunohistochemicalAnalysesHistopathologic and immunohistochemical proce-dures and evaluation were performed at the Depart-ment of Oral Pathology, Faculty of Dentistry, GaziUniversity.

Tissue samples were fixed in 10% neutral-bufferedformalin and embedded in paraffin. Three sectionswith 5-mm thickness were cut at the central regionof each specimen to obtain maximum standardizationof the cutting surface. All sections were deparaffinizedby xylene and incubated in 96% ethanol. One of theslides was stained with hematoxylin and eosin to eval-uate the maximum epithelial thickness (MET). METof oral gingival epithelium was measured from thetip of the rete to the top of epithelium at ·200 magni-fication by using an ocular grid. The measurementswere taken from three different sites of the epithelium.The mean value of each section was calculated andrecorded.

The streptavidin-biotin method was used for im-munohistochemical detection of the proliferationmarkers Ki67 and PCNA. After deparaffinization byxylene and rehydration in 96% ethanol, endogenousperoxide activity was blocked with 3% hydrogen per-oxide in phosphate buffered saline (PBS; pH 7.6) for10 minutes and then rinsed with PBS. For antigen re-trieval, the sections were boiled in citrate buffer (2.64g/l sodium citrate; pH 6.0) for 15 minutes and left tocool to room temperature. After washing with PBS,sections were incubated‡ for 5 minutes to blocknon-specific background staining. Primary antibodiesto anti-PCNA§ and anti-Ki67i were added, and thesamples were incubated for 2 hours at room temper-ature. After rising thoroughly with PBS, the slides wereoverlaid with secondary antibody¶ for 10 minutes. Theimmunoperoxidase labeling was performed usingstreptavidin peroxidase# for 10 minutes. Then, ami-noethylcarbazole** was used as a chromogen for

visualization of antibody binding. Finally, the sectionswere counterstained with Mayer’s hematoxylin,cleared, and mounted. Tonsil tissue was used as pos-itive control tissue for both antibodies.

The localization of PCNA and Ki67 immunoreactiv-ity within tissues was evaluated before cell counting.Any cell with red nuclear staining was considered pos-itive (for PCNA and Ki67), regardless of the intensity.

Scoring of PCNA and Ki67 reactivity was carriedout using a standard light microscope on a ·400 field.All nucleated cells located at the epithelium werecounted for each section. The proliferation index21

(PRI) for each section was expressed as the percent-age of positively stained cells per total number of nu-cleated epithelial cells.

Statistical AnalysisStatistical analyses were performed using the paired-sample t test to compare results of groups. The Pear-son correlation test was used in analyzing data. Allcalculations were undertaken using a statistical soft-ware package.††

RESULTS

Histopathologic and ImmunohistochemicalFindingsMET. Means – SDs for MET are shown in Table 1. In allgroups (healthy, gingivitis, and periodontitis), METwas slightly higher in non-smokers’ gingiva com-pared to the smokers. Mean MET values in periodon-titis groups were higher in smokers and non-smokerscompared to gingivitis and healthy group values.However, the differences were not statistically signif-icant.

PCNA and Ki67 expression. Cells with red nucleiwere considered positively stained for PCNA and forKi67, although there were some staining pattern dif-ferences between them. PCNA-positive cells showeda uniformly dense red staining of nuclei, whereasKi67-positive cells had much more pale and granularred nuclear staining (Figs. 1 and 2).

As shown in Figure 2, PCNA-positive cells were ob-served in basal and parabasal layers of the oral gingi-val epithelium, whereas Ki67-positive cells werelocated generally in the basal layer, and the numberof positive cells was lower compared to the PCNA-expressing cells.

‡ Ultra V block, Ultravision Detection System Anti-Polyvalent, HRP/AECKit, Neomarkers, Freemont, CA.

§ PCNA mouse monoclonal antibody, Clone PC10, Ab-1, Neomarkers.i Ki67 rabbit monoclonal antibody, Clone/sp6, Zymed, South San

Francisco, CA.¶ Ultravision Detection System Anti-Polyvalent, HRP/AEC Kit,

Neomarkers.# Ultravision Detection System Anti-Polyvalent, HRP/AEC Kit,

Neomarkers.** AEC (red) Substrate Kit, Zymed.†† Statistical Package for the Social Sciences, version 10.0 for Windows,

SPSS, Chicago, IL.

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When all subjects in the study were analyzed asa single group, the percentage of PCNA-positivecells was higher than the percentage of Ki67-positivecells in all groups, with mean values of 4.80 – 6.98and 1.45 – 2.37, respectively; the difference was stat-ically significant (P <0.001). When the mean valuesof PCNA and Ki67 were compared in each group(PCNA versus Ki67 indices in SH, SG, SP, NSH,NSG, and NSP groups), a statistically significant dif-ference was observed in the healthy smoker group(P = 0.003; Table 1).

Table 1 shows the mean values of PRI of PCNA andKi67 in smokers and non-smokers. The highest PRIvalue was observed in the SH (10.9%) PCNA group,whereas the lowest value was in the NSH (0.21%)Ki67 group.

PRI for PCNA and Ki67 was higher in smokergroups than in non-smokers. There was an increasein Ki67 PRI values according to the periodontal healthstatus, regardless of smoking habit. However, these

results were not statistically significant. When thesmoker and non-smoker groups were compared, astatistically significant difference was found only inthe PCNA PRI between the smoker and non-smokerhealthy groups (P = 0.015).

A statistical correlation was found between age andthe percentage of Ki67-labeled cells in the epitheliumof smokers with gingivitis group (P = 0.05) and be-tween MET and percentage of Ki67-labeled cells inthe smokers with periodontitis group (r = 0.568; P =0.043) (Table 1).

DISCUSSION

Thickening of marginal gingival epithelium as a resultof smoking has been demonstrated.30,31 In the pres-ent study, MET values reflected the thickness of gin-gival epithelium. MET values of periodontitis andgingivitis groups were higher in smokers and non-smokers compared to the healthy groups. However,statistically significant differences were not found be-tween smokers and non-smokers. These findings mayindicate that smoking may not have an influenceon the thickness of the gingival epithelium, whereas

Figure 1.PCNA-positive cells in the oral marginal gingival epithelium of smokers.A) Positive cells were detected in the basal and suprabasal layers.Rare stained cells were seen on the left side, which may representan area in which two phenotypically different epithelia fuse.B) Higher-magnification feature of PCNA-positive cells with strong rednuclear staining. (Immunoperoxidase; original magnification: A, ·200;B, ·400.)

Figure 2.Ki67-positive cells in the oral marginal gingival epithelium of smokers.A) Ki67-positive cells were observed mostly in the basal layer of theepithelium. B) Higher-magnification view of Ki67-positive cells withpale nuclear staining. (Immunoperoxidase; original magnification:A, ·200; B, ·400.)

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periodontal status and inflammation seemed to bemore important factors for an increase in the thick-ness of the gingival epithelium. This idea supportsthe results of other studies1,13,32 that demonstratedan increase in the thickness of epithelium in inflamedgingiva. The study by Jarnbring et al.33 suggestedthat thickening of the gingival epithelium and reteproliferation, which are two well-established charac-teristic features of periodontitis, could be due to an im-balance in homeostasis between cell division and cellloss. Furthermore, it is known that development of in-flammation in response to plaque accumulation is re-duced in smokers relative to non-smokers, both ingingivitis and periodontitis.1 Taken together, inflam-mation stimulates the thickening of the epitheliumand rete proliferation, smoking reduces developmentof inflammation, and as a result, lower gingival epithe-lial thickness with lower inflammation is expected insmokers’ gingiva. Thus, existing data also supportour finding regarding the lower MET values in smokerscompared to non-smokers.

This contrasts with the study of Villar and deLima,17 who observed a greater epithelial thicknessin smokers, independent of the gingival situation.The differences between these findings might stemfrom differences in the methodology of the histomor-phologic analysis of the studies.

The results of the present study demonstrated thatthe expression of PCNA is higher than that of Ki67 pro-tein in all subjects, regardless of their smoking status(P <0.001). This finding was described previously andmay relate to the fact that these antigens do not markthe same phases of the cell cycle.34,35 PCNA is pres-ent throughout the cell cycle during DNA synthesisand is involved in DNA repair,36 whereas Ki67 is pres-ent in all active phases of the cell cycle but not inthe resting phases of cells (G2, S, and M phases, notG0).26 Furthermore, PCNA has a longer half-lifeand, thus, is detectable in many cells that have al-ready passed through the cycle.37

Increased PRI was found in smokers for PCNA andfor Ki67 immunostainings. However, a statisticallysignificant difference was found only for PCNA inhealthy smokers compared to healthy non-smokers(P = 0.015). When the mean values for PCNA andKi67 were compared in each group (PCNA versusKi67 indices [PRI] in SH, SG, SP, NSH, NSG, andNSP groups), a statistically significant differencewas observed only in the healthy smokers group(P = 0.003). Our results revealed that smoking alonehad an effect on the proliferation of the cells in the oralgingival epithelium, regardless of the periodontal sta-tus of the subjects. This finding is in accord with thestudy carried out by da Costa Filho et al.,38 who de-tected an increase in the PCNA index in the gingivalepithelium of smokers compared to non-smokers.

This epithelial proliferation may stimulate the kerati-nocytes directly (by action of the tobacco) or indi-rectly (via growth factors produced by stimulatedstromal cells).39 Nicotine was shown to induce theproliferation of oral keratinocytes because it displacesthe local cytotransmitter acetylcholine from the nic-otinic receptors (nAChRs) expressed by oral kerati-nocytes.39 The study by Arredondo et al.40

demonstrated that chronic stimulation of oral kerati-nocytes by nicotine alters the genetically determinedprogram of the cell differentiation–dependent expres-sion of nAChR subunits. Exposure of keratinocytesto nicotine also altered the mRNA and protein levelsof cell cycle and cell differentiation markers Ki67and PCNA. Therefore, the downstream signaling fromnAChRs expressed in the oral mucosa proceeds via apathway that upregulates the expression, at transcrip-tional and translational levels, of cell cycle progres-sion regulators.39,40 Another explanation, relevantto the increasing number of proliferating epithelialcells, was reported by van Oijen et al.30 They specu-lated that the increased PRI in smokers was due to theregenerative effect that was explained by the local re-generative response to compensate for increased cellloss or damage by tobacco as well as the other pro-tracted effects of smoking.30

In the present study, when we evaluated the corre-lation of PRI with other parameters such as MET andage, a statistical correlation was only detected be-tween Ki67-labeled cells and MET in the smokers withperiodontitis group (r = 0.568; P = 0.043) and be-tween Ki67-labeled epithelial cells and age in thesmokers with gingivitis group (P = 0.05). In this con-text, we can say that Ki67 is the only proliferationmarker that showed a correlation with other parame-ters, and this positive correlation was observed in thenon-healthy smoker subjects (gingivitis and peri-odontitis groups). Celenligil-Nazliel et al.32 reporteda significant correlation between PRI in inflamedgingiva and aging in a study in which the periodontalstatus of the subjects was not mentioned. These re-searchers used PCNA as a proliferation marker in theirstudy; however, we were unable to detect a correlationwith PCNA. Instead, we observed a correlation withKi67 as a proliferation marker. As has been confirmedin various studies, PCNA has a role in DNA replication,but the protein is also involved in DNA repair. Thesemultiple roles of PCNA strongly argue against usingits expression as a reliable marker for proliferatingcells.26 These findings support our results; we de-tected a correlation of Ki67, as a proliferation marker,with other parameters, such as MET and aging. In con-trast, our study differs from the study by Celenligil-Nazliel et al.32 based on smoking and the periodontalstatus of the subjects; thus, direct comparison mayhave limited value.

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CONCLUSIONS

Smoking induced the proliferation of gingival kerati-nocytes, regardless of the periodontal status of thesubjects, because an increased PCNA index wasfound in healthy smokers. This finding may not onlyindicate the hyperproliferation of the cells, but alsohighlights the increased DNA repair in the oral gingi-val epithelium of smokers. Because the MET valueswere slightly higher in non-smokers, we can speculatethat smoking has no influence on the thickness of theoral gingival epithelium, whereas the periodontal sta-tus and inflammation may be much more importantfactors.

ACKNOWLEDGMENT

The authors report no conflicts of interest related tothis study.

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Correspondence: Dr. Sibel Elif Gultekin, Department ofOral Pathology, Faculty of Dentistry, Gazi University,Biskek cad. Emek-06510, Ankara, Turkey. Fax: 90-312-2239226; e-mail: sibelg@gazi.edu.tr.

Submitted December 12, 2007; accepted for publicationFebruary 4, 2008.

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