Comparative assessment of exfoliated gingival cells in healthy individuals and chronic periodontitis patients with and without tobacco chewing habit: A cytomorphometric study.

Background: Cytomorphometry is the analysis of the morphometry of exfoliated cells. Studies on exfoliated gingival epithelial cells in chronic periodontitis were not evident on the literature search. In the backdrop of this information, we were inspired to conduct a cytomorphometric study. Aim: The aim of the study is to correlate the nuclear area (NA), cytoplasm area (CA), and the nuclear: cytoplasm ratio (N/C) in the gingival cells of healthy individuals, patients with chronic periodontitis, and chronic periodontitis patients who are tobacco chewers. Materials and
Methods: Ninety subjects, aged 35-45 years, were categorized into three equal groups of 30 each. Group I had individuals with healthy gingiva, Group II patients with chronic periodontitis without any deleterious habits, while Group III patients with chronic periodontitis and tobacco chewing habit. Exfoliated gingival cells were stained with Papanicolaou stain. Analysis of cells was done using binocular light microscope under ×40 and i-progress® image analyzer software.
Results: There was statistically significant increase in CA in all the groups, but NA and N/C ratio did not show any statistical significant difference. When Group II was compared with Group III, we found significant correlation of CA with pocket probing depth and clinical attachment loss (CAL) in Group II. Similarly, we also found a correlation of CA and N/C with pocket depth and CA with CAL in Group III.
Conclusion: The results suggest that molecular level changes occur in the cells. Cytomorphometry can be used to analyze the cellular changes in chronic periodontitis patients even before clinical signs manifest. Hence, it can be used as an investigative tool. Copyright:

INTRODUCTION

Diagnosis is identification of a disease as it presents with its signs and symptoms. Data derived from symptoms offered by the patient and from examination help an investigator in arriving at a diagnosis. Oral diagnostic tests or techniques assist an examiner to diagnose the pathology. These tests could be invasive techniques such as blood tests and biopsies as well a noninvasive techniques such as radiographs, cone beam computed tomography, and exfoliative cytology, to name a few.

Exfoliative cytology has become popular as it is noninvasive and time-effective.[1] This test can be used to diagnose various oral diseases, such as squamous cell carcinoma, potentially malignant disorders, candidiasis, and hairy leukoplakia, as well as to assist in monitoring dysplastic areas.[12] Exfoliative cytology uses the scrapings of different areas of the oral cavity such as the gingiva and buccal mucosa to detect changes of the superficial epithelial cells.[3]

Quantitative assessment of the morphology of the epithelial cells is done by cytomorphometric analysis. Cellular alterations in exfoliated cells of the buccal mucosa have been used by few investigators, to assist in the diagnosis of oral mucosal changes in diabetes mellitus.[4] Cytomorphometric analysis of exfoliated gingival epithelial cells has also shown its relevance in age and sex determination in healthy individuals. These findings were used as a tool in forensic odontology for estimation of the chronological age of a person involved in judicial or legal proceedings.[5] Studies have also been done in aggressive and chronic periodontitis subjects to analyze cytological changes in the periodontal pocket lining of patients, in different clinical stages of the disease.[6] Cytomorphometric analysis has been employed to check for the cellular changes in the exfoliated epithelial cells of the buccal mucosa of tobacco chewers.[78]

Data searched seem scanty with respect to studies done with exfoliated gingival epithelial cells in chronic periodontitis patients. The lack of published data available on cytomorphometric studies in gingival exfoliated cells in chronic periodontitis patients with and without use of tobacco prompted us to conduct this study.

Hence, we conducted a cytomorphometric study on exfoliated gingival cells in healthy individuals, in chronic periodontitis patients, with and without the tobacco chewing habit, to see if any difference could be noted.

MATERIALS AND METHODS

Three groups of 30 patients each, in the age bracket of 35–45 years, constituted the study groups. Ethical clearance for the study was obtained from the institutional ethical and review board. Written informed consent was duly signed by the patients.

Group I - Periodontal healthy individuals without any deleterious habits. Assessed considering absence of gingival inflammation, probing depth ≤3 mm, and no clinical attachment loss (CAL).

Group II - Chronic periodontitis patients without any deleterious habits. Assessed considering the probing depth >3 mm, CAL ≥5 mm.

Group III - Chronic periodontitis group without any deleterious habits, except tobacco chewing habit for at least 6 months and at least 4–11 times a day. Assessed considering the probing depth >3 mm, CAL ≥5 mm, and history of tobacco chewing.

In both Groups II and III, generalized periodontitis cases were considered.

Tobacco chewers were included in Group III but were excluded from Groups I and II. Pregnant and lactating women, patients with systemic conditions, patients who received any medications 3 months before sampling, patients with habit of mouth breathing/incompetent lips, patients with any cancerous or precancerous lesions, patients with any kind of deleterious habits (smoking, alcohol, areca-nut chewing, and betel quid chewing) were excluded from all the three groups.

After rinsing the oral cavity thoroughly with water which allowed the debris to wash off, under adequate illumination, the gingiva was examined for any deformities or pathology of gingiva. Samples were collected using a medium nylon toothbrush.[9] Using moderate pressure, the brush was repeatedly moved in one direction from distal of maxillary canine to canine, across the arch, so as to procure epithelial cells of the gingiva. The collected material from the brush was spread on a clean, dried glass slide. The smear was then fixed and stained using Rapid PAP kit (Papanicolaou staining). The stained slides were analyzed under Olympus BX 41 research microscope;which has an in-built standardized image analysis software( i-progress®). The cytomorphometric analysis of the cells was done at ×40. Thirty well-preserved epithelial cells were studied in each subject by a single observer. The nuclear area (NA), cytoplasm area (CA), and nuclear: cytoplasm ratio (N/C) were analyzed from the epithelial cells. The software was calibrated to micrometers (μm) from pixels.

Statistical analysis

Statistical analysis was done using t-test for inter group comparison between two groups. Intergroup comparison (>2 groups) was done using one-way ANOVA followed by pair-wise comparison using post hoc test.

RESULTS

A total of 90 subjects who participated in the study were in the age group of 35–45 years, with an average of 39.26 years. Of these, 47.8% were female and 52.2% were male. The mean duration of tobacco chewing in Group III was calculated as 14.38 years ± 10.63.

As shown in [Table 1] the healthy group (Group I), the mean value of the NA was 0.168 μm2 ± 0.02, CA was 4.696 μm2 ± 0.94, and N/C was 0.041 μm2 ± 0.01 [Figure 1]. In the chronic periodontitis group (Group II), the mean value of the NA was 0.238 μm2 ± 0.26, CA was 6.546 μm2 ± 1.55, and N/C was 0.373 μm2 ± 0.29 [Figure 2]. In the chronic periodontitis group with tobacco chewing habit (Group III) the mean value of the NA was 0.179 μm2 ± 0.15, CA was 5.108 μm2 ± 1.46, and N/C was 0.039 μm2 ± 0.00 [Figure 3].

Table 1

The inter group comparison of nuclear area, cytoplasm area, and nuclear: cytoplasm ratio

Groups	n	Mean	SD	SE	Minimum	Maximum	F	P value of one-way ANOVA
NA
I	30	0.168667	0.0231537	0.0042273	0.1100	0.2100	1.748	0.180#
II	30	0.238333	0.2651621	0.0484117	0.1400	1.6300
III	30	0.179667	0.0370911	0.0067719	0.1100	0.2700
Total	90	0.195556	0.1564573	0.0164921	0.1100	1.6300
CA
I	30	4.696333	0.9456415	0.1726497	3.6800	6.8900	18.389	0.000**
II	30	6.546000	1.5538375	0.2836906	3.5500	9.7800
III	30	5.108667	1.1428329	0.2086518	3.1600	6.9600
Total	90	5.450333	1.4626392	0.1541757	3.1600	9.7800
N/C
I	30	0.0413	0.01408	0.00257	0.03	0.10	0.311	0.733#
II	30	0.0373	0.02947	0.00538	0.02	0.19
III	30	0.0393	0.00944	0.00172	0.02	0.06
Total	90	0.0393	0.01948	0.00205	0.02	0.19

*Statistically significant (P<0.05); **Statistically highly significant (P<0.01); #Nonsignificant (P>0.05). N – Number of samples; F – Snedecor’s F distribution; P – Level of significance; NA – Nuclear area; CA – Cytoplasm area; N/C – Nuclear cytoplasm ratio; SD – Standard deviation; SE – Standard error

Figure 1

Photomicrograph showing gingival epithelial cells in healthy subjects (Papanicolaou staining, ×40)

Figure 2

Photomicrograph showing epithelial cells in chronic periodontitis subjects (Papanicolaou staining, ×40)

Figure 3

Photomicrograph showing gingival cells in chronic periodontitis with tobacco chewing habit subjects (Papanicolaou staining, ×40)

The intergroup comparison of NA, CA, and N/C ratio between three groups was done using Tukey's post hoc test. Although the intergroup group comparison of the NA between Groups I and Group II, Group I and Group III, and Group II and Group III showed a difference, the difference was not statistically significant [Table 2].

Table 2

The intergroup comparison of nuclear area, cytoplasm area, and nuclear cytoplasm ratio between three groups using Tukey’s post hoc test

Dependent variable	Groups A	Groups B	Mean difference (A-B)	SE	P
NA	I	II	−0.069	0.040	0.197#
	I	III	−0.011	0.040	0.959#
	II	III	0.0586	0.040	0.313#
CA	I	II	−1.849	0.320	0.000**
	I	III	−0.412	0.320	0.406#
	II	III	1.437	0.320	0.000**
N/C	I	II	−0.004	0.005	0.711#
	I	III	0.002	0.005	0.918#
	II	III	−0.002	0.005	0.918#

**Statistically highly significant (P<0.01); #Nonsignificant (P>0.05). NA – Nuclear area; CA – Cytoplasm area; N/C – Nuclear cytoplasm ratio; SE – Standard Error; P – Level of significance

Intergroup comparison of the CA is depicted in Table 2. Statistically significant difference was seen between Group I and Group II (P = 0.00) and between Groups II and III (P = 0.00). There was no statistically significant difference seen in CA between Group I and Group III (P = 0.40).

N/C was also compared between the groups as depicted in [Table 2]. There was a difference seen in N/C between the groups, but the difference was not statistically significant.

Table 3 depicts the correlation between the clinical parameters such as the probing pocket depth (PD) and CAL with the cytomorphometric changes seen in the Group II (chronic periodontitis group). We observed statistically significant correlation between the probing PD and CA (r = 0.688). Table 3 shows positive correlation between CAL (CAL) and CA (r = 0.856) which was statistically significant.

Table 3

Correlations of nuclear area, cytoplasm area, and nuclear cytoplasm ratio with probing pocket depth and clinical attachment loss for Group II

	CA	NA	N/C	PD	CAL
CA
Pearson correlation coefficient (r)	1	0.359	0.182	0.688**	0.856**
Significant (two-tailed)	0.051	0.336	0.000	0.000
n	30	30	30	30	30
NA
Pearson correlation coefficient (r)	0.359	1	0.976**	−0.040	0.120
Significant (two-tailed)	0.051	0.000	0.834	0.529
n	30	30	30	30	30
N/C
Pearson correlation coefficient (r)	0.182	0.976**	1	−0.167	−0.030
Significant (two-tailed)	0.336	0.000	0.379	0.874
n	30	30	30	30	30

**Correlation is significant at the P<0.01 level (two-tailed). NA – Nuclear area; PD – Probing depth; CA – Cytoplasm area; CAL – Clinical attachment level; N/C – Nuclear cytoplasm ratio; n – Number of samples; P – Level of significance; r – Pearson correlation coefficient

Similarly, when we studied the correlation between the clinical parameters with the cytomorphometric changes seen in Group III (chronic periodontitis patients with the tobacco chewing habit), we observed a statistically significant correlation between probing PD and CA with r = 0.733. We also observed a statistically significant positive correlation between CAL and CA with r = 0.821 [Table 4].

Table 4

Correlations of nuclear area, cytoplasm area, and nuclear cytoplasm ratio with probing pocket depth and clinical attachment loss for group III

	CA	NA	N/C	PD	CAL
CA
Pearson correlation coefficient (r)	1	0.363*	−0.495**	0.733**	0.821**
Significant (two-tailed)	0.049	0.005	0.000	0.000
n	30	30	30	30	30
NA
Pearson correlation coefficient (r)	0.36*	1	0.5016**	0.146	0.186
Significant (two-tailed)	0.049	0.005	0.441	0.324
n	30	30	30	30	30
N/C
Pearson correlation coefficient (r)	−0.495**	0.501**	1	−0.367*	−0.445*
Significant (two-tailed)	0.005	0.005	0.046	0.014
n	30	30	30	30	30

*Correlation is significant at the 0.05 level (two-tailed); **Correlation is significant at the 0.01 level (two-tailed). NA – Nuclear area; PD – Probing depth; n – Number of samples; CA – Cytoplasm area; CAL – Clinical attachment level; N/C – Nuclear cytoplasm ratio; r – Pearson correlation coefficient

DISCUSSION

Periodontitis is a chronic multifactorial inflammatory disease associated with a plaque biofilm and characterized by progressive destruction of the tooth-supporting apparatus.[10] Periodontal disease is influenced by various factors that can modify the host response, such as pathogenic bacteria, microbial tooth deposits, psychological stress, aging, and deleterious habits such as use of tobacco.[11] Use of tobacco has been recognized to be a significant risk factor for the development and progression of periodontal disease.[12]

Tobacco is used either in a smokeless form or by smoking. Studies state that tobacco chewing has been reported to cause increased gingival recession and attachment loss, due to irritation, at sites adjacent to mucosal lesions associated with tobacco placement.[11] A major component of tobacco is nicotine, which is presumably a contributing factor for the exacerbation of periodontal diseases.[13] In tobacco chewers, normal tissue undergoes cellular changes at the molecular level before getting noticeable clinically.[14] Nuclear changes reflect the biological activities of a cell, and the cytoplasm reflects the functional activity.[14] Any molecular changes in cell will cause a difference in the nuclear size or the cytoplasm size and the N/C.[15] Keratinocytes of buccal mucosa have been studied intensively in healthy individuals as well as tobacco chewers. We could not unearth published data on the effect of tobacco chewing on gingival keratinocytes. This prompted us to take on a cytomorphometric study of exfoliated healthy gingival cells as compared to those from chronic periodontitis with and without tobacco chewing habit.

A total of 90 subjects participated in our study, in the age group of 35–45 years, with an average of 39.26 years. Various studies have considered different age groups. Anuradha and Sivapathasundharam in their study had three groups of age 0–21 years, 40–60 years, and more than 60 years. They reported an increase in NA and N/C as age increased.[16] We standardized our age group between 35 and 45 years to take care of this variance.

In our present study, we assessed the CA, NA, and N/C ratio in the healthy group (Group I). Mean values of CA was 4.69 μm2 ± 0.94, NA was 0.16 μm2 ± 0.02, and N/C ratio was 0.04 μm2 ± 0.01. Sahu et al.[17] have done a similar study where they noted that in the gingival cells of healthy individuals, CA was 3127.90 μm2 ± 425.85, NA was 93.58 μm2 ± 12.40, and cytoplasm: nuclear ratio was 36.45 μm2 ± 6.93. The software tool used in their study was Lynx Biolux (Lawrence and Mayo) image analysis software,[17] whereas our study employed i progress® image analyzer software.

We have compared healthy group (Group I) with the chronic periodontitis group (Group II) in this study. There was increase in CA (6.54 μm2 ± 1.55) and NA (0.23 μm2 ± 0.26) in the chronic periodontitis Group II, whereas an insignificant decrease in N/C ratio (0.037 μm2 ± 0.029) was seen when compared to the healthy group. Cecilia et al.[6] in their cytological study have examined the lining of periodontal pockets. Their results indicated that surface cells showed more cell volume, vague edges, and blurry nucleus in periodontitis patients when compared to the healthy group, which was in accordance with our present study.[6]

A cytogram study was conducted by Hasiuk[18] to study cytological features of epithelial differentiation in gums in patients with generalized periodontitis. The results indicated increase in thickening of cytoplasm. Bacterial infection leads to production of hydrolytic enzymes that break intramolecular connection. These features of the intermediate stage of cellular metabolism disorders cause’ cell cytotransformation into balloons filled with fluid. Although the results obtained from Hasiuk's study indicate an increased CA, we cannot directly correlate the results with those derived from ours because there was no mention of the area of the cell in their study.[18]

In the present study, comparison of results of the healthy group (Group I) with that of chronic periodontitis with tobacco chewing habit (Group III) was also done. There was an increase seen in NA (0.179 ± 0.03) and CA (5.108 ± 1.14) in Group III when compared to healthy group. The N/C (0.039) of Group III was found insignificantly less than that seen in the healthy group. Ramaesh et al.[19] studied the nuclear diameter and cytoplasm diameter in the buccal mucosal epithelium in tobacco chewers. Increase in nuclear diameter and decrease in cytoplasm diameter were observed. They attributed these cytomorphometric changes to the effect of tobacco on the epithelial cells. Similarly, we also observed cytomorphometric changes in the NA and CA, which could be attributed to the effect of tobacco.[19]

A cytomorphometric study was conducted by Sharma et al.[20] on the epithelial cells of the buccal mucosa in health, tobacco chewers, smokers, and smokers with tobacco chewing habit. The results showed increase in NA in tobacco chewers, when compared with the healthy group. In our study, the gingival epithelial cells also showed an increase in NA in tobacco chewing group. Both the results of our study and the study by Sharma et al.[20] could be indicative of cytomorphological changes caused due to the duration, frequency, and form of tobacco products. The mean duration of tobacco use by patients seen in our study was 14.38 years, whereas in the study by Sharma et al., they included individuals who used tobacco for 10 or more years.

Clinical parameters such as probing PD and CAL were correlated to NA, CA, and N/C between Group II and Group III in our study. We found a significant cellular-level difference between Group II and Group III. In Group II, we saw a positive correlation between CA and CAL, whereas moderately positive correlation between CA and PD. A positive correlation of CA with PD and CA with CAL was seen in Group III. A negative correlation in the N/C with PD was also observed in Group III. This suggests that changes are evident at cellular level probably even before they present at clinical level. Cytomorphometric change suggests that even though, in tobacco chewers, disease severity was slightly less than that of without habit, we saw changes at the cellular level.

CONCLUSION

Oral exfoliative cytology is a simple, noninvasive, and painless procedure that consists of microscopic analysis of the cells collected from the surface of the oral mucosa. Quantitative cytological assessment has evolved as a noninvasive diagnostic tool. Cytomorphometric analysis is one of its kind, where exfoliated cells help us in detecting the changes at cellular level, hence aiding in early detection of diseases.

The results of our present study show a significant variation in the NA, CA, and N/C between all the three groups. Our study findings suggest that changes are occurring at cellular level, even before they manifest clinically, in patients with chronic periodontitis having tobacco chewing habit. With the above-reported findings, we suggest that cytomorphometric study of exfoliated gingival cells can be used as a predictive diagnostic tool in clinical practice. Few variables that could indirectly contribute to the results could be taken care of in our further research on this subject.

This discovery is very exciting and worthy of further cellular research in various grades of periodontitis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.