Exfoliative Cytology of Oral Mucosa Epithelium: Cytochemical Study and Morphologic Analysis of Patients with Type 2 Diabetes

Background: Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia. An association between diabetes mellitus and periodontal disease has been reported. However, few comprehensive reports have examined the impact of chronic hyperglycemia on oral mucosal epithelial cells. This study clarified the effect of diabetes on oral mucosal epithelium cells. Methods: Cells from 44 patients with type 2 diabetes and 10 healthy controls were collected from the buccal mucosa, gingiva, and lateral margin of the tongue by exfoliation. Samples were subjected to Papanicolaou staining; immunochemical staining using anti-AGE antibody, anti-Ki-67 antibody, and anti-p53 antibody; and lectin chemical staining. Furthermore, the nuclear area and cytoplasmic area were measured, and the nuclear/cytoplasm ratio was calculated. Results: A chromatin condensation-like substance was found in samples from some diabetic patients. In AGE staining, positive cells were found in both groups, although the rate of positive cells tended to be higher in diabetic patients. No positive findings were found for Ki-67 and p53 anti-bodies. PNA and RCA120 showed differences in staining between diabetic patients and controls. The nuclear area was significantly expanded in diabetic patients (P < 0.05). There was no significant difference between groups in the cytoplasmic area. The nuclear/cytoplasm ratio was significantly increased in diabetic patients (P < 0.05). Conclusion: These findings suggest that in patients with persistent hyperglycemia, changes in the sugar chain terminal of the cell surface may influence mucosal epithelial cell differentiation. The appearance of chromatin-like substances may be an indicator of the need for better management of type 2 diabetes.


Introduction
It is estimated that the total number of people with diabetes mellitus (DM) and incipient diabetes surpasses 20 million in Japan [1]. In addition, there are 420 million people with DM worldwide. It has been calculated that the worldwide population with DM will reach 700 million by 2025 if effective preventive measures are not taken [2]. DM is a metabolic disease characterized by chronic hyperglycemia in many patients; this hyperglycemia is associated with multiple complications [3]. Recently, several reports indicated that DM was associated with an increased risk for cancer [4] [5] [6] [7], including a 1.2 times increased risk for all cancers [8] [9], 1.85 times increased risk for pancreatic cancer [10], 2.5 times increased risk for liver cancer [11], and 1.30 times increased risk for colon cancer [12]. In terms of oral effects, it is reported that DM influences dry mouth, delay in healing, and periodontal disease [13] [14] [15] [16]. In addition, several researchers are conducting morphological searches for cells using exfoliative cytology, which is less invasive to patients, Seifi et al. [17] reported that the nuclear area and cytoplasmic area of oral mucosal epithelial cells in controls were significantly larger than in patients with DM. Jajarm et al. [18] reported that the nuclear and cytoplasmic areas, as well as the N/C ratio, were significantly larger in patients with DM than in controls. Alberti et al. [19] Shareef et al. [20], Sankhla et al. [21], Suvarna et al. [22], Sonawane et al. [23], and Sahu et al. [24] reported that the nuclear area and N/C ratio of oral mucosal epithelial cells were significantly increased compared with control in patients with DM, but no significant differences in the area of the cytoplasm was seen.
However, few comprehensive reports have examined the effects of DM on mucosal epithelial cells, including cell proliferation and malignant transformation.
In this study, we aimed to clarify the influence of type 2 DM on the oral mucosal epithelium by conducting cytochemical and morphologic research using exfoliative samples.

2) Cell Collection Method
The mouth was washed, and the absence of food residues in the oral cavity was confirmed. Cells were collected from the gingival, buccal mucosa, and lateral margin of the tongue using a cytobrush (Orcellex ® Brush; Medical & Biological Laboratories (MBL) Co., Ltd. Aichi, Japan).

TACAS Method Details
Since formalin is not used for fixation, cell atrophy is mild, inflammatory cells, bacteria, necrotic cells, etc. are maintained moderately, and gene preservation is also excellent.
A special slide glass is coated with a coating agent, and the surface is positively charged. Since the cells are negatively charged, they are smeared onto the glass by attracting each other. Since there is no smearing on the part without positive charge, the sample has less cell accumulation. In addition, it has excellent hemolytic ability, and even if blood is mixed, there is little influence on observation.

a) Conventional Cytology
Oral mucosal epithelial cells were smeared on a glass slide and immersed in 95% alcohol for fixation.

b) LBC Method
Oral mucosal epithelial cells were fixed for more than 30 min with a fixative and then 1.5 mL per sample was transferred to a centrifuge tube and centrifuged at 2200 rpm for 5 min. The supernatant was removed and mixed with 3 mL distilled water (DW). Samples were centrifuged at 2200 rpm for 5 min, and the supernatant was removed again and mixed with 300 μL DW. Samples (300 μL) were placed on the slide, incubated for 10 min, and rinsed with 95% ethanol.
Then they were immersed in 95% ethanol and fixed.

4) Staining Methods a) Papanicolaou Stain
Samples prepared using conventional cytology and the LBC method were rinsed with water, immersed in 0.5% hydrochloric acid alcohol 10 to 15 times, rinsed with 95% alcohol, and underwent OG-6 (Muto Pure Chemicals Co. Ltd.  As a positive control, AGEs staining was used for diabetic kidney tissue, and Ki-67 and p53 staining was used for oral squamous cell carcinoma cases.
In AGEs immunocytochemical staining, 10 fields of view were observed with a 400 × objective lens, and the ratio of positive cells to the observed cells of patients with DM and controls was calculated.

Judgment Criteria
AGEs were positive if the cell surface and cytoplasm were stained brown, and Ki-67 and p53 were positive if the cell nucleus was stained brown.

5) Cytomorphologic analysis
Ten cells per field of view were extracted with a 400 × magnification, and the cytoplasmic area and nuclear area were calculated with the Image J (NHI Maryland USA). The N/C ratio was calculated from these results.
For statistical analysis, the Mann-Whitney U test was used with Bonferroni's adjustment. All analyses were conducted with SPSS statistics, version 22 (IBM, Tokyo, Japan). A p value < 0.05 was considered significant.
The Nihon University School of Dentistry at Matsudo Ethics Committee approved this research (approval number: EC17-031).

1) Papanicolaou Stain
Of 44 Figure 1 shows the high frequency of lightgreen stained cells in all areas. The frequency of appearance by area was gingival > buccal > lateral margin of the tongue. Almost no CCCLs were found in any samples from controls (Table 1).   The percentage of positive cells at each site was as follows: gingival area, 30%

2) Immunocytochemical
for patients with DM and 17% for controls; buccal area, 32% for patients with DM and 21% for controls; lateral margin of tongue, 26% for patients with DM and 14% for controls. The proportion of positive cells tended to be higher in patients with DM than in controls.

Ki-67
No positive findings were found in the cell nuclei of patients with DM or controls.
The positive control was positive in the nuclear in cases of OSCC (oral squamous cell carcinoma).  Table 2). In terms of the nuclear area and the cytoplasmic areas, the nuclear area increased significantly in patients with DM. There were no significant differences between groups in the cytoplasmic area. The N/C ratio was significantly increased in patients with DM. There were no significant differences between groups in terms of CCLS (+) and CCLS (−) (Figures 4-6 and Table 3).

Discussion
Papanicolaou staining showed that when cells of patients with DM were compared with cells of control subjects, CCLS was observed in the cell nuclei of patients with DM. It is well known that chromatin condensation is associated with cell proliferation [30]. Cell division can be observed only in the basal cell layer in the oral mucosal epithelium. Cells in the vicinity of basal cells cannot be collected by exfoliative cytology. In this study, CCLS were found in epithelial cells in the surface layer system. Based on this finding, it was suspected that these cells might be in the active proliferating stage.
Recently, the association of cancer risk has been reported by diabetes [8] [9] [10] [11] [12]. Therefore, it was necessary to examine the cell proliferating ability and malignant transformation ability of CCLS. Immunochemical studies were conducted using Ki-67, which is an indicator of cell proliferation ability, and p53, a tumor suppressor gene whose expression is observed during malignant transformation. Both Ki-67 and p53 studies showed negative findings. Based on these findings, the observed cells did not show an increase in cell proliferation ability or any malignant transformation. In addition, when cell division occurs, Open Journal of Stomatology not only chromatin condensation, but also nuclear membrane loss occurs. The correlation with the cell cycle is considered to be low, considering the clearness of the nuclear membrane of cells with CCLS found in this study.
When the oral mucous membrane epithelial cells of patients with DM and controls were compared morphologically, the nuclear area and the N/C ratio was significantly larger in patients with DM compared to controls. The results of this study were similar to those of Alberti et al. [19], Shareef et al. [20], Sankhla et al. [21], Suvarna et al. [22], Sonawane et al. [23], and Sa-hu et al. [24]. Only nucleus increases, referred to nuclear swelling, is found in cells affected by the proliferation phase, cells affected by inflammation, and tumor cells. As a result of immunocytochemical staining of cells in this study, there was no possibility of enhancement of cell proliferation ability or malignant transformation. Therefore, it is considered that the nuclear swelling observed in the epithelial cells of the oral mucosa in patients with DM was not related to cell proliferation or malignant transformation.
In addition, because there were no clinical signs of inflammation in the oral cavity of the subjects, and the appearance of inflammatory cells was rarely observed in Papanicolaou-stained specimens of exfoliative cytology samples, it is assumed that this change was not attributable inflammation.
The appearance of CCLS in the nucleus was mainly observed in the oral mucosal epithelial cells of patients with DM with a high HbA1c (NGSP) level (≥7.0%). It was assumed that the appearance of CCLS may be affected by sustained hyperglycemia. Therefore, we performed anti-AGE antibody in for samples of DM patients.
AGEs are produced when sugars in the blood, such as glucose, and proteins undergo a glycation reaction (Maillard reaction). In DM, AGEs accumulate in the Renal tube, retinas capillary, blood vessels wall, and the like. These AGEs cause an inflammatory response by inducing oxidative stress and increasing the expression of inflammatory cytokines.
It was reported that patients with DM had higher rates of AGEs-positive cells in the gingival epithelium compared to controls [31]. In this study, the incidence of AGEs-positive cells tended to be higher, although differences did not reach statistical significance in patients with DM in all parts of the gums, the buccal mucosa, and the tongue. These results were the same as the report by Zizzi et al. [32]. However, it cannot be denied that CCLS seen in this study may be due to increased oxidative stress caused by the accumulation of AGEs.
To investigate the influence of hyperglycemia, we further examined the binding of the sugar chain end using lectin cell chemistry. Lectins are proteins that bind to specific carbohydrate structures. Many kinds of lectins are isolated from plants, animals, and microorganisms. It is well known that changes in lectin binding patterns are related to cell differentiation and malignant transformation [33] [34]. In this study, 7 types of lectins, ConA, SBA, WGA, DBA, UEA-I, RCA120, and PNA, were used. A clear difference in staining was observed be- This finding suggests that there was some change in Gal-β (13)-3GalNAc and D-Gal on the oral mucosa epithelial cells of patients with DM. As discussed above, sustained hyperglycemia is the most probable cause of this finding.
In Papanicolaou staining, CCLS appeared frequently in light-green adherent cells in all sites. From this finding, it is considered that cells with weak keratinization tend to be susceptible to hyperglycemic effects. In addition, it is assumed that the cause of the abnormality in the nucleus was also continuous exposure to the hyperglycemic state in many patients, which promotes glycation and causes changes in the sugar chain end of the oral mucosal epithelial cell surface. These results have some influence on cell differentiation.
The results observed in this study were suggested to be useful as a supplementary tool for determining the pathology of DM.