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ORIGINAL RESEARCH Table of Contents   
Year : 2007  |  Volume : 18  |  Issue : 2  |  Page : 63-66
Image analysis of normal exfoliated gingival cells


1 Department of Oral and Maxillofacial Pathology, St. Joseph Dental College, Eluru, India
2 Department of Oral and Maxillofacial Pathology, Meenakshi Ammal Dental College, Chennai, India

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Date of Submission07-Mar-2006
Date of Decision21-Jan-2007
Date of Acceptance23-Jan-2007
 

   Abstract 

Objective: The present study was undertaken to evaluate the nuclear diameter (ND), cell diameter (CD) and nuclear-cytoplasmic ratio (N:C) and their variation with age and sex in normal gingival smears. Study Design: Gingival smears were collected from 320 apparently healthy individuals. After fixation in 95% alcohol, the smears were stained using standard papanicolaou laboratory procedure. The cell and nuclear diameters were measured using image analysis software (KS lite 2.0). Statistical analysis of the data was done using one-way ANOVA, Students 't' test and Tukey-HSD procedure. Results: The results showed an increase in ND from the 1-20 group to 21-40 age group in males. Above 40 years, there was a decrease in ND. In females, the ND was high in 21-40 age group; and then from 41 years, ND gradually decreased but the difference was not significant. The CD in males and females was low in the 1-20 age group and then it gradually increased. However, the difference was not significant between the ages 20 and 60 years. After 60, there was a decrease in CD. Similar changes are also seen in the ratio N:C in both males and females. The ND, CD and N:C irrespective of the age were high in females. The difference in CD was insignificant, except in the 0-20 age group, where females had significantly more cell diameters. Irrespective of the gender, the ND, CD and N:C increased from 0-20 age group to 20-40 age group. After 40, there is a steady decrease in ND, CD and N:C. Conclusion: Age-related and sex-related alterations are observed in gingival smears.

Keywords: Cell diameter, normal exfoliated gingival cells, nuclear diameter, nuclear-cytoplasmic ratio

How to cite this article:
Anuradha A, Sivapathasundharam B. Image analysis of normal exfoliated gingival cells. Indian J Dent Res 2007;18:63-6

How to cite this URL:
Anuradha A, Sivapathasundharam B. Image analysis of normal exfoliated gingival cells. Indian J Dent Res [serial online] 2007 [cited 2019 Dec 6];18:63-6. Available from: http://www.ijdr.in/text.asp?2007/18/2/63/32422

   Introduction Top


Epithelial cells, as a part of normal physiologic turnover, undergo continuous renewal. They move from the basal layer to the surface and are exfoliated. Exfoliative cytology is a noninvasive technique, which allows simple and pain-free collection of intact cells from different layers within the epithelium for microscopic examination.

The use of exfoliative cytology as a diagnostic aid accentuates the need for establishing an accurate baseline. However studies on oral epithelium are largely done in pathological state. But secrets of pathology can be explored only when the fundamental observations in normal oral mucosal cells are established. Since the initial studies of normal epithelial smears by Miller and Montgomery in 1951,[1] there are only few instances where the normal buccal mucosal smears are studied. Moreover, there are virtually no studies on normal exfoliated gingival cells in this part of the country.

Donne, in 1945[2],[3] first proposed that the size of microscopic objects could be detected. From then on, measuring cells and their components has been an intellectual challenge. Image analysis technology is programmed to analyze cells, replacing the human eyes. In 1960, Prewitt and Mendelson first conceived image analysis system for studying the leukocyte; subsequently, it was extended by Weid and his co-workers to study the cells in the cervical smears.[2],[3]

The quest for diagnosis with the simplest tool and with reasonable accuracy has led to various advancements in the field of exfoliative cytology. The present study uses exfoliative cytology and image analysis for scrutinizing the gingival cells to estimate the cell and nuclear diameter and to quantify the age- and sex-related changes in the above variables.


   Materials and Methods Top


Three hundred twenty apparently healthy subjects with no known history of any systemic disease or therapeutic medication were selected for the study. All the subjects were free of any deleterious habits like smoking, alcohol consumption or tobacco use. No detailed biochemical, radiological or other laboratory investigations were carried out to establish their normal status.

The subjects were divided into eight groups, with 40 patients per group, based on their age and sex [Table - 1].

The microscopic slides were cleaned with water, dried and numbered. The subjects were asked to rinse their mouth thoroughly with water. Using a wet wooden spatula, the attached gingiva was scraped in the upper anterior region. Scrapings were smeared on to the slide. Slides were fixed in 95% alcohol for half an hour. Papanicolaou technique was used for staining the slides.

The cells were examined under 40x compound microscope (Olympus BX 40). They were projected onto the monitor via CCD/JVC TK-C1380 color video camera. Unfolded cells with clear outline were only selected for the study. The sampling was done in a stepwise manner, moving the slide from left upper corner to right and then down in order to avoid measuring the same cells again.

The cells were captured and the cell and nuclear diameters were measured in X and Y axis using image analysis software (KS lite 2.0). The average of X and Y values was taken as the final diameter. Mean of nuclear diameters and mean of cell diameters of all eight groups were calculated separately. Statistical analysis of the data was done using one-way ANOVA to evaluate the difference in nuclear diameter (ND), cell diameter (CD) and nuclear-cytoplasmic ratio (N:C) with age in males and females, and Tukey-HSD procedure was used to identify the significance between various age groups at 5% level. Students independent 't' test was used to evaluate the difference in ND, CD and N:C in males and females in various age groups.


   Results Top


There was a significant difference in ND, CD and N:C in males of different age groups [Table - 2]. There was a significant difference in ND, CD and N:C in females in different age groups [Table - 3]. The difference in ND between males and females was significant in 1-20, 41-60 and >60 age groups [Table - 4]. The difference in ND, CD and N:C with age irrespective of gender was significant [Table - 5]. There was a significant difference between males and females with respect to ND, CD and N:C irrespective of age [Table - 6].


   Discussion Top


The results of this study showed significant difference in the ND, CD and N:C in females of various age groups [Table - 3]. This may be attributed to the hormonal imbalances occurring during the lifetime in females. Both estrogen and progesterone promote protein anabolism and growth.[4],[5] At puberty, there is a dramatic increase in the sex hormones, which is followed by cyclic increase and decrease in the levels. An increase in these hormones may be responsible for the increased CD and ND in females and a decrease in their levels at menopause may be the reason for the decreased CD and ND in >55 years age group.

During 20-40 age, females are subjected to the use of contraceptives, most commonly the hormonal ones, which may have their effect on gingiva. Moreover, there are remarkable endocrine alterations accompanying pregnancy, which usually falls in the 20-40 age bracket. Our study sample involved clinically normal individuals, with no systemic illness or deleterious habits. A detailed history of contraceptive use or pregnancy was not established. These factors may also play an important role in age-related alterations seen in females.

Estrogen influences the cytodifferentiation of stratified squamous epithelium, as well as the synthesis and maintenance of fibrous collagen.[4],[5] The hormones have a potential effect on the gingival vasculature. Studies have shown an increase in gingival crevicular fluid during various phases of reproductive cycles, caused by the increased vascular permeability, which is in turn influenced by the increase in female sex hormones. The increased vascular permeability results in edema in the connective tissue, which has a definitive impact on the overlying epithelium.[4],[5]

Hormonal influence happens to be the differentiating factor for the cytomorphometric trends of males and females [Table - 4]. Testosterone has been frequently associated with metabolism and maintenance of bone and connective tissue matrix unlike the female sex hormones, which have their effect on the epithelium. Like females, males also showed an increase in ND and CD from ages 1-21 to ages 41-60, after which there was a significant decrease [Table - 2]. The cause for this is unclear presently.

Age-related variation of ND, CD, and N:C irrespective of gender can be ascribed to cellular senescence [Table - 5]. A basal cell can only divide for a set number; then the renewal capacity of tissues declines with age, resulting in accumulation of senescent cells.[6],[7] The cells which stay for a longer duration in the oral cavity succumb to the effect of various local environmental factors.

Sexual dimorphism is observed in human as well as in animals. For example, on an average, a male skeleton is larger with a large and robust mandible. The dentition is larger in males. Apart from the skeletal differences, even the brain shows sexual dimorphism, such as the anterior commissures being larger in females and males, having greater mean cerebral asymmetry than females and much more.[8] Considering all these aspects, it is not surprising to note differences in CD and ND between males and females [Table - 6].

Zitwack et al.[9] treated animals with estrogen and noticed larger and more active cells in the vagina. Similar changes were also seen in the buccal mucosa and gingiva but to a lesser degree. Our results are in conformity with their study. In the study by Nayar and Sivapathasundharam,[10] the ND was reported to increase with age and CD to decrease with age. The cell diameter in the age group 40-59 particularly was less in females than males.

Pappelis et al.[11] and Lee et al.[12] reported no significant variations in ND and CD with age. Scoot et al.[11] showed a reduction in N:C ratio with advancing age at a similar rate in both males and females. Cowpe et al.[11] showed a significant increase in ND with age but there was no variation in CD. The ND and CD varied significantly in various regions in the oral cavity.

Most of the above studies were carried out in the buccal mucosa, palate and tongue, which differ considerably from gingiva both morphologically as well as functionally. Gingiva is keratinized and is subjected to greater masticatory forces than other parts next to periodontal ligament. It is a region constantly subjected to the deleterious effects of plaque and calculus. The differences seen in gingiva when compared to other regions of oral mucosa may be the result of a cumulative effect of all these factors.

Each study differs in the number of cells being counted per slide, type of fixatives used, time lapse of staining following smear preparation, time lapse of collection of specimen and cytomorphometric analysis. Absence of established guidelines for cytological studies might have contributed to the discrepancies in these reports.

Finally, the study group selected, method of cytomorphometric analysis and the region under consideration also play an important role. Since there are no exfoliative cytology studies on gingiva in human in the literature, comparison of our study with others is not possible at present.


   Conclusion Top


The present study shows age- and sex-related variations in ND, CD and N:C in normal exfoliated gingival cells.

 
   References Top

1.Montogomery PW. A study of exfoliative cytology in normal human oral mucosa. J Dent Res 1951;30:12-8.  Back to cited text no. 1    
2.Bibbo M. Comprehensive cytopathology, 2nd ed. W B Saunders and Company: Philadelphia.  Back to cited text no. 2    
3.Koss LG. The future of cytology. Acta Cytologica 1990;34:1-9.  Back to cited text no. 3  [PUBMED]  
4.Mealey BL, Moritz AJ. Hormonal influences: Effects of diabetes mellitus and endogenous female sex steriod hormones on the periodontium. Periodontology 2000;32:59-81.  Back to cited text no. 4    
5.Mascarenhas P, Gapski R, Al-Shammari K, Wang HL. Influence of sex hormones on the perodontium. J Clin Periodontol 2003;30:671-81.  Back to cited text no. 5  [PUBMED]  [FULLTEXT]
6.Wynford-Thomas D. Cellular senescence and cancer. J Pathol 1999;187:100-11.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]
7.Coates PJ. Markers of senescence? J Pathol 2002;196:371-3.  Back to cited text no. 7  [PUBMED]  [FULLTEXT]
8.Hoffman GE. Man and women sexual behaviour and the brain. Atlas of Human Anatomy 1994.  Back to cited text no. 8    
9.Neil Jenkins G. The physiology and biochemistry of the mouth, 4th ed. Blackwell Scientific Publications: Australia.  Back to cited text no. 9    
10.Nayar AK, Sivapathasundharam B. Cytomorphometric analysis of exfoliated normal buccal mucosa cells. Indian J Dent Res 2003;14:2.  Back to cited text no. 10    
11.Cowpe JG, Longmore RB, Green MW. Quantitative exfoliative cytology of normal oral squames: An age, site and sex-related survey. J Royal Soc Med 1985;78:995-1004.  Back to cited text no. 11    
12.Lee LH, Pappelis AJ, Pappelis GA, Kaplan HM. Cellular and nuclear dryness and area changes during human oral mucosal cell development. Acta Cytologica 1973;17:214-9.  Back to cited text no. 12  [PUBMED]  

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Correspondence Address:
A Anuradha
Department of Oral and Maxillofacial Pathology, St. Joseph Dental College, Eluru
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.32422

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    Tables

  [Table - 1], [Table - 2], [Table - 3], [Table - 4], [Table - 5], [Table - 6]

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