Environmental factors play an important role in the pathogenesis of carcinomas of the head and neck, and tobacco smoking is considered as one of the major risk factors. ¹ Exposure to tobacco can be in the form of smoking or smokeless habits. ² Betel quid chewing is an ancient practice common in many countries of Asia and among migrated communities in Africa, Europe, and North America. ³ The term areca nut is used to denote the unhusked whole fruit of the areca nut tree, and term betel nut is used exclusively to refer to the inner kernel or seed which is obtained after removing husk. ⁴

Histological changes of the oral mucosa have been reported in association with exposure to tobacco, and betel nut chewers along with exfoliative cytological techniques have also been applied to examine the effects of tobacco and betel nut chewing on the oral mucosa. ² Exfoliative cytology, a simple, noninvasive diagnostic technique to microscopically examine shed or desquamated cells from the epithelial surface, usually the mucous membrane, could increase the chances of early detection of premalignant and malignant lesions. ¹ , ⁵

In the past, histopathological and cytological examinations of lesions were only used for the diagnosis of all carcinomas. However, recent advances in cancer research include the use of electron microscopy. Scanning electron microscopy (SEM) cannot contribute to routine diagnostic procedures, but it can add to the understanding of the biology of disturbances of epithelial differentiation in malignancy. Studies in the past under SEM have graded the malignant cells as abnormal on the basis of their size, shape, and general architectural and surface features. ⁶ The oral epithelium exhibits regional variations in the degree of keratinization. The surface ultrastructure has been described as specific to the type of keratinization; fully keratinized cells being described as having pitted surface appearance and nonkeratinized cells having surface micro-ridges. ⁶

The present study was undertaken to correlate epithelial surface pattern changes of oral exfoliated cells of tobacco smokers and betel nut chewers and also to compare them with oral squamous cell carcinoma (OSCC) and healthy individuals, under SEM.

Selection of subjects

This cross-sectional, analytical study was conducted in the Department of Oral Pathology and Microbiology and Birbal Sahani Institute of Paleobotany, Lucknow, India after ethical approval (Ethical No. CPGIDSH/IEC-2/0016/2013).

Inclusion criteria

Persons with habits of betel nut chewing 5-10gms per day for 5 years and above

Exclusion criteria

Persons with combination habits of tobacco smoking and betel nut chewing

Exfoliated oral epithelial cells were taken only from the buccal mucosa of all the 15 patients intobacco smokers group (Group III) ; the 15 patients in betel nut chewers group (Group IV); the ten patients with histologically confirmed OSCC in positive control group; (Group I) and the ten individuals with normal buccal mucosa in negative control group (Group II).

Smear procedure

Cells were scraped from the buccal mucosa of all the four groups individually with the help of wooden spatula and were placed on coverslips and immediately fixed with 2.5% glutaraldehyde solution at pH of 7.4 for 2 h.

Scanning electron microscopy

The specimens were mounted on aluminum stubs using double-sided adhesive tape; after drying under vacuum, they were coated with gold-palladium coating by Polaron Sputter for 2 min and then placed into the SEM (LEO 430 Oberkochen, Germany) after mounting over the metallic stub. Each specimen was scanned at an accelerating voltage of 15–30 kV at different magnifications. The areas selected for photography were those where maximum regularly shaped polygonal cells were observed with raised central contours indicating the position of cell nucleus; only the images of the representative areas were photographed. At higher magnification, the surface was seen to be composed of fine granular protrusions with a tendency to form numerous short discontinuous parallel micro-ridges along the cell surface. During visualization of specimens, the different surface morphologies of the cells of interest were marked in master chart of SEM findings of respective groups and appropriate images were stored and later photographed.

The normal samples (control group) were scanned first to form a base for the interpretation of samples for different SEM characteristics of exfoliated oral epithelial cells. Then, the samples of tobacco smokers and betel nut chewers were subjected to a preliminary scan to note the different SEM characteristics of exfoliated oral epithelial cells. Based on preliminary trial scan and review of literature, cell modifications, intercellular relationships, and surface characteristics such as micro-ridges, microvilli, and surface structures such as blebs, spherical, or cylindrical structures were looked in the cases of tobacco smokers and betel nut chewers. Comparisons were made between the study and control groups.

The statistical analysis was determined using ANOVA, Tukey honestly significant difference, Chi-square test, and Statistical Package (window version 18.0: PSAW). P < 0.05 (significant) was determined and analysis was performed on SPASS software

The present SEM study of surface exfoliated epithelial cells helped us analyze the general architectural and surface structural differences between tobacco smokers, betel nut chewers, patients with OSCC, and normal oral epithelial cells.

The cell modifications are observed in the groups and summarized in Table 1. The frequency (%) of cell modifications varied significantly (P < 0.05 or P < 0.01 or P < 0.001) among the groups, except degenerative changes. The frequency of abnormal size variation (χ ²= 10.82, P = 0.013), aberrant forms (χ ²= 11.49, P = 0.009), and surface pattern irregularity (χ ²= 19.44, P < 0.001) was found significantly higher in Groups I, III, and IV as compared to Group II.{Table 1}

The intercellular relationships between the groups are summarized in Table 2. The frequency (%) of intercellular relationships varied significantly (P < 0.05 or P < 0.01 or P < 0.001) among the groups, except dense grouping of cells and loss of close intercellular adherence. The frequency of both surface pattern irregularity (χ ²= 9.50, P = 0.023) and absence of distinct cell borders (χ ²= 13.33, P = 0.004) was found significantly higher in Groups I, III, and IV than Group II. In contrast, the frequency of apparent cell cannibalism in both Groups III and IV was found to be significantly higher as compared to both Groups I and II (χ ²= 26.73, P < 0.001).{Table 2}

The SEM surface characteristics are summarized in Table 3. The frequency (%) of most of the SEM surface characteristics were found similar (P > 0.05) among the groups, except cylindrical structures, surface evaginations, slender strands, and filopodia. The frequency of cylindrical structures was found significantly higher in Groups I, III, and IV than Group II (χ ²= 12.43, P = 0.006). In contrast, the frequency of surface evaginations was found significantly different and higher in Group I as compared to both Group III and IV (χ ²= 20.24, P < 0.001). Conversely, the frequency of both slender strands (χ ²= 17.39, P = 0.001) and filopodia (χ ²= 8.33, P = 0.040) was found significantly higher in Group I as compared to Groups II, III, and IV.{Table 3}

This study demonstrated that general architectural and surface structural differences existed between tobacco smokers, betel nut chewers, patients with OSCC, and normal oral epithelial cells and these could not be solely attributed to keratinizing changes. ⁷

When changes in a cell as whole were observed, surface pattern irregularity was a characteristic feature for both tobacco smokers and betel nut chewers where cells often showed regional variation in surface appearance, whereas normal exfoliated oral epithelial cell showed regular polygonal outline Figure 1. ⁶ , ⁷ The frequency of cell modifications varied significantly among the groups, except degenerative changes. Abnormal size variation, aberrant forms, and surface pattern irregularity were found higher in OSCC > tobacco smokers >betel nut chewers >normal mucosa. Figure 1

Scanning electron microscope of Group II showing cells with regular polygonal outline, distinct cell border, and granular surface (×1.33).

The study could be concluded as individual cell modifications, intercellular relationships and surface characteristics observed by scanning electron microscopy between OSCC, tobacco smokers, betel nut chewers compared to normal oral mucosa have been tabulated and further it could prove helpful in detecting any carcinomatous change at its incipient stage and also give an insight into the ultra-structural details of cellular differentiations in epithelial tissues.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non.financial in this article.