|Year : 2014 | Volume
| Issue : 2 | Page : 59-65
Oral epithelium in diabetics: A cytomorphometric correlation
KP Nandita, Karen Boaz, N Srikant, Amitha J Lewis, Nidhi Manaktala
Department of Oral Pathology and Microbiology, Manipal College of Dental Sciences, Mangalore, Karnataka, India
|Date of Web Publication||2-Jun-2014|
Department of Oral Pathology and Microbiology, Manipal College of Dental Sciences, Light House Hill Road, Mangalore - 575 001, Karnataka
Source of Support: None, Conflict of Interest: None
Objectives: The study aims to establish an etiological association between diabetes and precancerous lesions of the oral cavity by cytomorphometric analysis of the oral epithelium. Study Design: Smears were obtained from three distinct oral sites - buccal mucosa, dorsum of the tongue and the floor of the mouth in ten controls and ten patients previously diagnosed with type II diabetes. The oral smears were stained with Papanicolaou SA-36 solution. An eye - piece graticule was used to obtain the cytoplasm and nuclear dimension; where larger dimension was denoted as "D" and the smaller dimension was denoted as "d". The nuclear area (NA), nuclear diameter (ND), cytoplasmic area (CA) and the cytoplasmic / nuclear ratio (C/N) were evaluated from 50 cells predominant in each oral site. Statistical Analysis: The cytomorphometric data obtained was compared between the group of diabetic patients and the control groups using the student's t- test (SPSS version 11.0). Results: Results showed that the nuclear area and the nuclear diameter of oral epithelial cells were increased in diabetic patients, as compared to non- diabetics, while the non- diabetic patients demonstrated an increase in nuclear ratio. Conclusions: The results from this study suggest that diabetes mellitus can cause alterations in oral epithelial cells that are detectable with exfoliative cytology.
Keywords: Cytomorphometry, diabetes mellitus, oral exfoliative cytology
|How to cite this article:|
Nandita K P, Boaz K, Srikant N, Lewis AJ, Manaktala N. Oral epithelium in diabetics: A cytomorphometric correlation. Dent Hypotheses 2014;5:59-65
| Introduction|| |
Diabetes mellitus (DM) represents a heterogeneous group of disorders that have hyperglycemia as a common feature. It may arise secondarily from any disease causing extensive destruction of pancreatic islets such as pancreatitis, tumors, certain drugs, iron overload, certain acquired or genetic endocrinopathies, and surgical excision of pancreas. 
DM is a chronic disorder of carbohydrate, fat, and protein metabolism characterized by a defective or deficient insulin secretory response, which translates into impaired carbohydrate (glucose) use. 
DM can be divided into two major variants; "type I DM" also called as "insulin-dependent DM" and previously referred to as juvenile onset diabetes". This variant accounts for 10-20% of all cases of primary diabetes. "Type II DM" also called as "noninsulin-dependent DM" and previously referred to as "adult onset diabetes" comprises the majority (80-90%) of all cases of DM. 
Type II DM is the fifth most common chronic condition and sixth leading cause of mortality among the elderly. The disease affects 9% of adults over the age of 65 years and its prevalence has increased 30-40% during the past 20 years. There are many associations between diabetes and oral health. 
Exfoliative cytology is a noninvasive technique and the smears obtained can be analyzed quantitatively and qualitatively. Quantitative cytomorphometric assessments of exfoliated buccal mucosal cells have shown measurable changes in the cells obtained from malignant and premalignant lesions. 
The main complications associated with DM are retinopathy, neuropathy, nephropathy, and macro/microangiopathy. Further, DM damages tissue repair processes and causes dysfunction of oral mucosa.  Several studies suggest a higher prevalence and severity of some pathologies in the oral tissues of patients with DM like gingivitis, periodontitis, dental caries, candidiasis, and other oral manifestations such as alteration of salivary flow and oral burning sensation.
Several studies have examined the deleterious effects of diabetes on the oral mucosa. It was reported by Caldeira et al., (2004) that diabetes adversely affects the histomorphology of cheek mucosa, which may compromise tissue function to favor the occurrence of oral infections and probably neoplasia.
The most accepted clinical technique for the diagnosis of lesions in the oral mucosa is incisional or excisional biopsy. 
However, in specific clinical conditions, such as DM, a great many invasive techniques are relatively contraindicated and necessitate the use of oral exfoliative cytology which may be suitable.  As exfoliative cytology is considered a moderate and noninvasive technique when compared to conventional anatomopathological examination, it is necessary to make an assessment of the application of oral exfoliative cytological methods in patients with DM. 
The study aimed to quantitatively and qualitatively assess changes in oral epithelial cells by exfoliative cytology to determine the effects of DM on oral cavity, where the smears were obtained from the buccal mucosa (BM), floor of mouth, and dorsum of tongue.
| Materials and Methods|| |
The study group consisted of 20 patients; 10 patients with known history of type II DM and 10 controls; healthy individuals without any history of DM (assessed by random blood sugar). Ethical clearance was obtained from the Institutional Ethics Committee. Subjects of both the study and control groups were informed of the procedure and a written consent was obtained.
Consisting of 10 individuals aged 40 years or above with a known history of type 2 DM for a minimum period of 1 year prior to the commencement of the study. Patients were included irrespective of the mode of medical therapy for glycemic control (i.e., insulin or oral hypoglycemic therapy).
Consisted of 10 age- and sex-matched healthy individuals without any history of DM (assessed by random blood sugar) and anemia (ruled out by assessment of hemoglobin).
Patients/individuals with the following were excluded from the study:
- Habits of smoking or betel nut chewing
- Medication for systemic disease other than DM
- Known cases of anemia and malignancy
- Patients who have undergone radiation therapy and chemotherapy
- Clinical evidence of premalignancy
- Alcohol dependency
- Subjects with poor oral hygiene
- Denture wearers
- Patients aged 40 years or above with clinically healthy oral mucosa.
- Medical history of type 2 DM for a minimum period of 1 year prior to commencement of present study, irrespective of type of medication for diabetes.
- Diagnostic criteria for type 2 DM were as follows: 
- Random serum glucose concentration >200 mg/dl (11.1 mM); or
- Fasting serum glucose level 126 mg/dl (7.0 mM); or
- 2-h plasma glucose >200 mg/dl (11.1 mM)
- Control group of volunteers with following criteria:
- Clinically healthy oral mucosa
- Negative for clinical signs of systemic diseases
- Negative for presence of diabetes and anemia (proven by laboratory investigations)
Methods of collection of data
After detailed clinical examination, the subjects were requested to rinse the mouth with normal saline. Smears were obtained from the BM and the dorsum of tongue of each subject using a wooden spatula moistened in distilled water. Two smears from each site were obtained. The smears were transferred onto grease-free glass slides and fixed with 95% ethyl alcohol.
Two smears each from both the sites (BM and tongue) were stained with Papanicolaou stain to visualize under compound light microscope for cytomorphometric analysis of cells (for nuclear area (NuA), cellular area (CA), cytoplasmic area (CyA), and nucleus: Cytoplasm (N/C) ratio.
The morphometric parameters of 150 cells in each patient (50 each in the BM, floor of the mouth, and tongue) were measured using an eyepiece reticule containing 200 points of measurement in a cruciate pattern. The maximum diameter (D) and the smaller diameter (d) of the nucleus and the cell were tabulated in Microsoft Excel sheet. The CA/NuA was calculated using the formula and the perimeter was calculated [Figure 1]. The CyA was then calculated for each cell by subtracting the NuA from the CA. The mean and standard deviations of maximum nuclear diameter, minimum nuclear diameter, NuA, nuclear perimeter, CyA, and N/C ratio (of areas) of the 50 cells in each site were computed and tabulated for statistical analysis. This yields total of 12 parameters (six mean and six standard deviation parameters) for each site separately.
|Figure 1: Superimposition of the eyepiece graticule on the cytological smears direct measurements of the individual epithelial cells|
Click here to view
This was then compared with the patients with and without diabetes using independent Student's t-test via SPSS software (IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp.).
| Results|| |
The patients in the present study had type II DM for more than 1 year and at least one kind of medication was being used (oral hypoglycemic drugs or insulin). The level of plasmatic glucose was 90.6 and 137 mg/dl, respectively in the control and diabetic groups, while the glucosylated hemoglobin levels was 5.5 and 7.7%, respectively. Smears taken from the controls and diabetic group showed a wide variation in the NuA, nuclear diameter, CyA, and N/C ratio.
Multivariate analysis using conditional logistic regression analysis did not yield any single best parameter associated with diabetes. Though using independent t-test analysis showed an increase in the nuclear size and area in all the three sites. There was corresponding reduction in the NuA/CyA ratio in the diabetics group seen in the tongue and the BM specimens [Table 1].
On comparison of the results of the morphometric parameters, the mean larger diameter of the nucleus (11.14 and 2.70) and mean NuA (73.34 and 27.23) were higher in the DM group. The N/C ratio (41.15 and 20.54) was correspondingly lower in the diabetes group. All of these were significant and (P-value < 0.05).
In the tongue sample, the standard deviation CyA was much lower in the diabetic group (134.97) as compared to the control group (1,198.51).
| Discussion|| |
DM is a syndrome characterized by abnormal carbohydrate, fat, and protein metabolism that results in acute or chronic complications due to absolute or relative lack of insulin.  Type 2 DM is the fifth most common chronic condition and the sixth leading cause of mortality among the elderly worldwide. 
Proper understanding of DM enables early diagnosis that is helpful in control of blood sugar level at an early stage to prevent various complications. Several studies have examined the deleterious effects of DM on oral mucosa with reports stating its adverse effects on the morphology of oral mucosa, which in turn may compromise tissue function to favor the occurrence of oral infections and oral neoplasia. In diabetes, there is a loss of oxidation equilibrium, whereby the activities of the antioxidant scavengers and enzymes are depressed by elevated glucose concentration, excessive formation of free radicals, and protein glycation. These noxious processes can cause serious damage to the biological structures at a molecular level which can be appreciated by oral exfoliative cytology. ,,
Oral exfoliative cytology is a noninvasive diagnostic and prognostic technique that can be repeated frequently with little discomfort to the patient.
The present study evaluated the morphometric and cytologic changes in the exfoliated cells of apparently normal buccal and tongue mucosa in type 2 diabetic patients. There was a significant increase in NuA obtained from both the sites, that is, BM and tongue in the study group. This finding concurs with the studies done by Alberti et al., (2003);  Jajarm et al., (2008);  Shareef et al., (2008);  Tozoglu and Bilge (2010);  Prasad H et al., where the mean NuA was significantly higher among the diabetic group [Table 2].
The increase in NuA among the study group could be explained by delay in keratinization of oral epithelium, effects of ageing, dehydration/atrophy, and inflammatory process. ,
Delay in the keratinization is attributed to glycation changes. Sustained hyperglycemia causes greater accumulation of advanced glycation end products by abnormal glycation of proteins, lipids, and nucleic acids in the walls of large blood vessels as well as in the basement membrane of the microvasculature. The progressive narrowing of the vessel lumen leads to decreased perfusion of the affected tissue and consequently decreases cell turnover, thereby explaining the delay in the keratinization process of the epithelium. This delay in the process of epithelial differentiation leads to an increase in the number of mature cells, which show a large nucleus as a primary characteristic. ,
Nutritional deficiencies have been also associated with changes in oral mucosa, similar to those noted in type 2 diabetic patients. Deficiencies of vitamin B12 and folic acid retard the synthesis of DNA, which is the core substance of cell nuclei, and hence may alter the size of nucleus and cytoplasm. 
Type 2 DM is a disease of the elderly and generally occurs in patients over 40 years of age. Cellular ageing produces various morphologic alterations and genotoxic damages in cells in the form of pleomorphism, bilobed nuclei, micronucleus, nuclear budding, karyorrhexis, [Figure 2] which were also observed in the smears from diabetic patients in the present study. 
Diabetic patients also suffer from dehydration due to the decreased salivary flow rates that may lead to mucosal atrophy. So, when smears from atrophic oral mucosae are made, the larger basal/spinous cells are inadvertently included in the sample. Thus, the primary pattern encompasses nonkeratinized cells of parabasal layers which are smaller in cell size, but have relatively larger nuclei; thus giving an impression of nuclear enlargement, with or without pleomorphism. ,
Inflammation has been attributed to nuclear changes observed in various lesions. Reactive nuclear changes due to inflammation may erroneously mimic pleomorphism/atypia or may even suggest malignancy.  Inflammation clinically manifests as superficial erosions or ulcerations of the oral mucosa seen as diffuse stomatitis and gingivitis. Cytomorphometric changes in the buccal mucosal cells of cigarette smokers similar to those noted in diabetics have been reported by Ogden et al., (1990). 
In the present study, there was a significant increase in CA of epithelial cells obtained from both the sites, that is, BM and tongue among the study group. CyA from tongue also showed an increase in area which was statistically significant. This is similar to the findings of Prasad et al., (2010)  who found an increase in cellular diameter (CD) among poorly-controlled diabetics (PCD) and a similar study by Jajarm et al., (2008)  who also found a significant increase in CyA in the diabetic group. However, Shareef et al., (2008)  in a similar study found a statistically significant decrease in CyA, which could be due to the cell shrinkage caused by dehydration [Table 2]. This theory is supported by the findings of Ogden et al.,(1999)  who reported a similar decrease in CyA in patients with alcoholism.
This increase in CA and CyA could be because of the altered cell membrane integrity leading to excessive accumulation of lipid droplets in the cytoplasm, thus, creating an increased intercellular space as reported by Caldeira et al., (2004)  in an experimental animal study where they examined the alterations in the histology and ultrastructure of oral epithelium of diabetic mice. In another experimental study in mice with diabetic retinopathy, Pannicke et al., (2006)  indicated that altered properties of K + channels, arachidonic acid metabolites, and acute oxidative stress could be the cause for the development of cell swelling (cytotoxic edema) in the glial cells of retina under ischemic and inflammatory conditions. A similar pathogenesis could be projected for increase in CA and CyA of oral exfoliated mucosal cells observed in our study.
In the present study, an increase in N/C ratio was evident in patients with type 2 DM, a finding similar to Alberti et al., (2003);  Jajarm et al., (2008);  Shareef et al., (2008);  and Prasad et al., (2010);  though it was not statistically significant [Table 2]. The relatively greater increase in nuclear size compared to cytoplasmic volume may explain this phenomenon.
Thus, cytomorphometry may be an efficient tool to understand the extent of cellular changes that occur in oral epithelial cells in diabetics that are secondary to microscopic changes like vascular occlusion and molecular changes such as oxidative stress. Therefore, cytomorphometric analysis of oral mucosal cells could be implicated as a noninvasive technique for screening and monitoring of the disease status in diabetic patients.
| Conclusion|| |
Knowledge of both quantitative and qualitative alterations in oral epithelial cells of diabetic patients is important as these alterations are similar to that seen in precancerous and radiation-induced changes. The present study suggests a novel hypothesis for an etiological association between diabetes and oral premalignant lesions.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]