biosis lipoidica diabeticorum.13-17 However, histological studies16-18 of ..... Yigit S, Estrada E. Recurrent necrobiosis lipoidica dia- beticorum associated with ...
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Comparison of Keratinocyte Proliferation in Diabetic and Non-Diabetic Inflamed Gingiva Gökhan Açikgoz,* ˙Inci Devrim,* and S ¸ükrü Özdamar†
Background: Keratinocytes are chiefly cells of the epidermis but also constitute 90% of the gingival cells. The molecular mechanisms of proliferative activity in keratinization whereby diabetes alters periodontal physiology have not been elucidated. In this study, we aimed to investigate the role of gingival keratinocytes in hyperglycemic subjects by examining their mitotic activities. Methods: We tested 30 patients with periodontitis, of whom 15 were type II diabetics and the remainder systemically healthy. Biopsies were obtained from the bottom of the deepest pocket in each subject by reverse beveled incision. Formalin-fixed and paraffin-embedded specimens were then processed for periodic acid-Schiff (PAS)-diastase histochemistry and proliferating cell nuclear antigen (PCNA) (P10). Immunohistochemical studies were employed to determine the presence of PCNA and were used to detect the proliferating potential of keratinocytes needed in synthesizing DNA. The expression of PCNA was evaluated using an immunoperoxidase technique and PC10 monoclonal antibody to PCNA. Mitotic index was calculated from basal cells. Statistical analysis employed the chi-square test. Results: No significant difference between the diabetic and nondiabetic patients was found in the mitotic index of the oral-gingival epithelium. Conclusion: Although the mitotic index in patients with diabetes was slightly lower, keratinization in the gingival tissues for both groups was essentially identical. J Periodontol 2004;75:989-994. KEY WORDS Diabetes mellitus; gingiva/anatomy and physiology; keratinocytes; mitosis; periodontal diseases/metabolism. * University of Ondokuz Mayis, Faculty of Dentistry, Department of Periodontology, Samsun, Turkey. † University of Hacettepe, Faculty of Medicine, Department of Pathology, Ankara, Turkey.
G
ingival epithelium plays an important role in protection against bacterial infections.1 Keratinization is one of the most important features of renewing of gingival tissue.1,2 During keratinization, while upper layers of gingiva are undergoing apoptosis, new cells move from the basal layers towards the upper layers.3,4 The oral epithelium is stratified, squamous, and separated from the lamina propria by a basal lamina. As in skin, several cell layers can be recognized. The cells which lie on the basal lamina are low columnar or cuboidal cells. Since much of the mitotic division responsible for the more superficial cells of the epithelium takes place in this layer, it is often known as the stratum germinativum. Cells in the basal layer destined to form keratin are distinguished by the presence of keratin filaments and are known as keratinocytes. The volume of the cells increases during their upward migration until they form the stratum granulosum, but the volume of the keratinized surface cells is less although they cover a larger surface area.5 Keratin produced by keratinocytes fills intercellular spaces, and normally prevents the loss of tissue fluids into the oral cavity. Therefore, keratinocytes have a unique role in the stability of gingival sulcus.3,4 Keratinocytes are chiefly cells of the epidermis and also constitute 90% of gingival cell population.3 The extent of keratinization of the oral mucosa varies in different areas, in the following order of decreasing keratinization: palate, gingiva, tongue, and cheek.1,6,7 Keratin is considered a bulk protein with several levels of structural 989
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organization. Although keratin is enormously resistant to proteolytic enzymes, it is unusually susceptible to oxidizing and reducing agents, which do not regularly dissolve cell membrane proteins. The amino acid composition of keratins shows variations, particularly among those of different tissues. The degree of gingival keratinization has been shown to diminish with age and the onset of the menopause in females.8-12 Cutaneous complications of diabetes include poor wound healing, skin infections, and several distinct skin lesions such as necrobiosis lipoidica diabeticorum.13-17 However, histological studies16-18 of diabetic changes in mucosal tissues, especially the gingiva, have been inconclusive. Keratin is an insoluble protein which fills the interior of shrunken cells and contains a high proportion of disulfide linkages, probably derived from cystine, linking different polypeptide chains together. Furthermore, the process of keratinization commences before birth on the lingual and buccal aspects of alveolar ridges, an indication that the process is genetically predetermined. Mitosis is simply described as a cell proliferation acquiring its code from the DNA molecules. Potential mitotic activity may be explained by determining the reactional structure of active molecules causing mitosis. The concentrations of cytoplasmic organelle vary among the different epithelium layers. When mitochondrial activity increases, the production of the protein used in keratinization also increases. The difference in keratinization is related to the enzyme activation and energy levels of the mitochondrial production. This may be especially important in diabetics because the deposited glycogen and intermediate products will be used instead of aerobic glycosis. In 1977, Miller et al. first reported that in streptozotocininduced diabetic rats, DNA synthesis increases and appears to be the main explanation for stimulation of mucosal cells in the small intestine.17 In the same year, Hamilton and Blackwood reported that the reduced rate of cell proliferation associated with insulin deficiency may contribute to the slower rate of wound healing in diabetic rats.18 In 1994, Werner et al. reported a decrease in keratinocyte growth factor in diabetic rats in comparison with non-diabetics.19 In 1996, Casasco et al. first suggested that since proliferating cell nuclear antigen is an endogenous cell-cycle molecule, PCNA antibodies may represent useful tools for studying cell kinetics in human oral tissues in normal as well as pathological situations.20 In 2000, Çelenligil-Nazliel et al. reported no effect of age on proliferating cell nuclear antigen in healthy and inflamed gingiva.21 In 2001, Nurmenniemi et al. studied the mitotic activity of keratinocytes in gingival overgrowth due to nifedipine usage.22 In 2002, Jarnbring et al. studied the number of apoptotic and proliferative gingival keratinocytes in patients with gingivitis and in those with periodontitis with PCNA. They found fewer apoptic keratinocytes in both groups but the proliferative ones were found to be in the most apical part of the sul990
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cus, particularly in the periodontal patients.10 As previous studies showed, the structure of DNA can be used to observe and evaluate the mitotic activity in the proliferating cell population.23-26 Monitoring the expression of PCNA, which is a polymerase-delta helper protein in DNA, provides valuable information about the reparative and regenerative potential of the cell.27-29 In diabetics, keratinocyte proliferation is probably related to the inflammatory response of the tissue and wound healing related to glycogen content. For this reason, the main aim of this study is to evaluate the keratinocyte proliferation in Type 2 diabetics. This study was planned to observe the effect of diabetes on proliferative activity in gingival epithelium in human basal cells by an immunoperoxidase technique and using a PC10 monoclonal antibody. MATERIALS AND METHODS Study Population Thirty periodontal patients (18 females, 12 males) were included in this study. Fifteen (average age 43.5 years) were type II diabetics who had been diagnosed at the University of Hacettepe Medical School at least 3 years ago and had HbA1c values of at least 6.23%, which is accepted as a baseline score for diabetes. Non-diabetic patients (average age 35.9 years) were otherwise systemically healthy. Inclusion criteria included at least 10 teeth with bleeding on probing scores ≥30% and ≥20 sites with probing depths of ≥4 mm. Exclusion criteria included history of antibiotic therapy within the previous 6 months; non-steroidal anti-inflammatory drugs within the previous 3 months; systemic conditions known to accelerate periodontal disease, such as pregnancy; certain hematological disorders; and mouth breathers. Individuals with a healthy periodontium were not included because of ethical considerations. Full-mouth clinical assessment was made, except for third molars. Dental plaque, probing depth (PD), bleeding on probing (BOP), gingival recession, and mobility scores were recorded by an electronic probe. Recordings were carried out at four sides of each tooth after scaling. Associations between diabetes and BOP and >4 mm probing depth scores were evaluated with chi-square tests. Permission for biopsies was obtained from the ethics committee. Excisional gingival biopsy specimens were taken using local anesthetics (lidocaine HCL 2%, epinephrine 1:100,000). Gingivectomy incisions were started from the top of gingival papilla with open curettage using a V-reverse beveled incision. Samples were obtained from the base of gingival papilla in the tooth with the deepest pocket.30 The dimensions of the specimens were approximately 2 × 2 mm. Formalin-fixed and paraffin-embedded specimens from all 30 subjects were processed for PAS-diastase histochemistry and PCNA (P10) immunohistochemistry.‡ Immunohistochemical ‡ Streptavidin-biotin peroxidase; chromogen: aminoethyl carbasole, DAKO, Carpinteria, CA.
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studies were done to evaluate the presence of proliferating cell nuclear antigen, which is used to assess the proliferating potential of keratinocytes and needed to synthesize DNA.18,28,29,31 PCNA immune reaction was surveyed with a light microscope at 400× magnification on 1,000 cells. The PCNA labeling index was defined as the percentage of stained cells per 1,000 keratinocytes.28,29 RESULTS Bleeding on probing, periodontal probing depth >4 mm, and plaque levels are shown in Table 1. The PCNA index scores of diabetic (0.36 ± 0.05) and non-diabetic (0.47 ± 0.12) groups showed no statistically significant difference between groups (P >0.05), although slightly higher values of PCNA were seen in the non-diabetic group (Fig. 1). PCNA+ cells were observed especially in the basal cells of the crevicular epithelium (Fig. 2). Inflamed gingiva was more frequent in the diabetic group (Figs. 3 and 4). These data are listed in Table 2. Furthermore, minimal hyalinization was found in five diabetic subjects. The mean fasting blood glucose and HbA1c values are shown in Table 3. In both values, statistically significant differences were found between the groups. DISCUSSION Variations in the levels of keratinization with respect to mitotic activity of the epithelium in diabetic patients have not been reported previously. The molecular mechanisms of proliferative activity in keratinization whereby diabetes alters periodontal physiology have not been elucidated. In this study, we aimed to investigate the role of gingival keratinocytes in hyperglycemic subjects by examining their mitotic activities. Several methods may be used to study the profile of keratinocytic proliferation.28,29,32-34 In recent years the proliferating cell nuclear antigen (PCNA) and its related protein expression have been used in gingival tissue studies. In 1992, Tsuji et al. used PCNA to study oral cancers.35 In 1996, Casasco et al. observed intense PCNA immune reactivity in the basal layer of the gingival epithelium and stated that the occurrence of positive cells in gingival connective tissue might
Figure 1. PCNA labeling of diabetic patients. PCNA reactive nuclei of basal and suprabasal cells in gingival biopsy (streptavidin-biotin peroxidase, aminoethyl carbasole [AEC]; original magnification: ×460).
Table 1.
Patient Clinical Parameters Diabetic Patients
Non-Diabetic Patients
P
BOP (%)
43.27 ± 6.83
35.93 ± 4.66
>0.05
>4 mm PD
12.60 ± 4.15
7.73 ± 2.30
>0.05
Plaque (%)
55.40 ± 6.95
51.27 ± 5.59
>0.05
Figure 2. PCNA labeling of non-diabetic periodontitis patients. Positive cells were observed in the basal and suprabasal layers (streptavidin-biotin peroxidase, AEC; original magnification ×630, immersion oil). 991
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Table 2.
Inflamed Gingiva Scores Inflammation Level Slight Moderate or severe X2
N Diabetic Patients
N Non-Diabetic Patients
5
14
10
1
= 11.63 (P
