Expression pattern of adhesion molecules in junctional epithelium differs from that in other gingival epithelia

BACKGROUND AND OBJECTIVE: The gingival epithelium is the physiologically important interface between the bacterially colonized gingival sulcus and periodontal soft and mineralized connective tissues, requiring protection from exposure to bacteria and their products. However, of the three epithelia comprising the gingival epithelium, the junctional epithelium has much wider intercellular spaces than the sulcular epithelium and oral gingival epithelium. Hence, the aim of the present study was to characterize the cell adhesion structure in the junctional epithelium compared with the other two epithelia. MATERIAL AND METHODS: Gingival epithelia excised at therapeutic flap surgery from patients with periodontitis were examined for expression of adhesion molecules by immunofluorescence. RESULTS: In the oral gingival epithelium and sulcular epithelium, but not in the junctional epithelium, desmoglein 1 and 2 in cell-cell contact sites were more abundant in the upper than the suprabasal layers. E-cadherin, the main transmembranous molecule of adherens junctions, was present in spinous layers of the oral gingival epithelium and sulcular epithelium, but was scarce in the junctional epithelium. In contrast, desmoglein 3 and P-cadherin were present in all layers of the junctional epithelium as well as the oral gingival epithelium and sulcular epithelium. Connexin 43 was clearly localized to spinous layers of the oral gingival epithelium, sulcular epithelium and parts of the junctional epithelium. Claudin-1 and occludin were expressed in the cell membranes of a few superficial layers of the oral gingival epithelium. CONCLUSION: These findings indicated that the junctional epithelium contains only a few desmosomes, composed of only desmoglein 3; adherens junctions are probably absent because of defective E-cadherin. Thus, the anchoring junctions connecting junctional epithelium cells are lax, causing widened intercellular spaces. In contrast, the oral gingival epithelium, which has a few tight junctions, functions as a barrier.     

A comparative light-microscopic, electron-microscopic and chemical study of human vaginal and buccal epithelium

The scarcity of sizeable specimens of normal oral mucosa for experimental purposes has hampered research on oral epithelium. Because large specimens of viable human vaginal mucosa are readily available and because vaginal and buccal epithelia are microscopically similar, vaginal mucosa has been used successfully to establish a human cyst model in experimental animals. The ultrastructure and distribution of keratin filaments in these epithelia are also similar, as is their permeability to water and a number of chemical substances. Therefore, if vaginal mucosa could be substituted for buccal mucosa it would expedite research on the epithelium of buccal mucosa. To strengthen further the concept that vaginal epithelium could replace buccal epithelium in certain experimental studies, the thickness of these epithelia, their patterns of surface keratinization, the presence or absence of intercellular lipid lamellae and their lipid contents were now compared. Thirty-three specimens of vaginal mucosa from postmenopausal women and 36 of buccal mucosa were investigated. To compare the thickness of the epithelial layers the number of cell layers in sections of 20 vaginal and 20 buccal mucosal specimens were counted in the three thickest and three thinnest regions of each specimen. Surface keratinization was evaluated on sections stained with the Picro-Mallory method. To demonstrate lipid lamellae two vaginal and two buccal mucosa specimens were examined electron microscopically after normal fixation and postfixation in ruthenium tetroxide. Following solvent extraction of 11 vaginal and 14 buccal epithelia, quantitative lipid analyses were performed using thin-layer chromatography. No statistically significant differences were found between the maximum and minimum number of epithelial cell layers. The patterns of surface keratinization and the distribution and appearance of the lipid lamellae in the intercellular spaces were similar. The lipid composition of the two epithelia corresponded, except for the cholesterol esters and glycosylceramides, which were higher in buccal epithelium. Ceramides for vaginal epithelium and triglycerides for buccal epithelium were not determined. Based on structural similarities, a similar lipid composition and earlier findings, it is concluded that vaginal epithelium can be used as a substitute for buccal epithelium in certain in vitro, and possibly for in vivo, studies.     

Histological and ultrastructural observations on the keratinizing epithelia of the palate of the rat

The epithelia of the hard and soft palate of the rat show two different types of keratinization and the soft palate shows a higher cell renewal rate. The two epithelia presented ultrastructural differences in all their layers. In the soft palate, the cells were larger with bulky cytoplasm and often appeared to be binucleate. They had fewer tonofilaments and the keratohyalin granules often consisted of two components. Lamellated membrane-coating granules occurred in the stratum granulosum and many similar lamellae were found intercellularly at the boundary with the stratum corneum. In the hard palate, the cells were smaller, flatter and had wide bundles of electron-dense tonofilaments. Keratohyalin granules also consisted of two components but they differed from those in the soft palate. Lamellated membrane-coating granules were numerous but intercellular lamellae were rarely seen at any level. Unlike parts of the rat tongue, the hard palate did not undergo “hard” keratinization. Intercellular lamellae have been found to be more obvious in epithelia of rapid cell turnover in other sites.     

Interepithelial cells of the oral mucosa Light and electron microscopic observations in germfree, specific pathogen-free and conventionalized mice

Abstract Interepithelial cells are found in all epithelia of the internal and external surfaces of the mammalian body. The regional differences of these Interepithelial cells and their function are not completely known so far. The quantitative and qualitative changes of the interepithelial cell population were investigated in germfree, specific pathogen-free and conventionalized mice by light and electron microscopy. Germfree and specific pathogen-free animals did not show significant differences in the number of interepithelial cells. In the epithelium of the tongue a mean of 7.4 cells per 1000 basal cells is found. After conventionalization a significant increase to 14.4 interepithelial cells per 1000 basal cells is observed. The number of cells in the buccal epithelium is constantly about 20% higher than in the epithelium of the tongue. In the oral mucosa lymphocytes, cerebriform cells and Langerhans cells are an integral component of the epithelium. In contrast to the monostratified intestinal mucosal epithelium, which is considered a secondary lymphatic tissue, the interepithelial lymphocytes of the oral mucosa are not significantly decreased in germfree animals. This could indicate that the oral mucosa functions partly as a primary lymphatic tissue. Interepithelial cerebriform cells and Langerhans cells increased after conventionalization with a maximum after 10 days in response to exogenous antigens. Both cells are immunologically important. The observations prove that the oral mucosa represents a local immunologic system in which the Langerhans cell plays an important part by formation a reticulo-epithelial tissue.     

Lectin histochemistry of oral premalignant and malignant lesions: Correlation of JFL and PNA binding pattern with tumour progression

The expression of glycoconjugates specific to Jack fruit lectin (JFL) and peanut agglutinin (PNA) in various clinicopathological stages of tumour progression in the oral mucosa were studied. These included various clinical forms of dysplastic and non-dysplastic oral leucoplakias, carcinomas, normal keratinising (gingiva) and non-keratinising (buccal mucosa) epithelia. It was seen that the binding patterns of PNA and JFL in the epithelial cells of various types of oral lesions were more or less similar. Normal non-keratinising epithelium showed mild membrane staining only in the spinal layers, while normal keratinising epithelium showed a moderate membrane staining and mild cytoplasmic staining in all layers. Moderate membrane and mild cytoplasmic staining were observed in leucoplakias, irrespective of various clinical or histological types. In carcinomas, the intensity of lectin binding was high, particularly in the membrane of differentiated cells. Correlation analysis of the binding pattern of PNA and JFL showed significant correlation in the membrane and cytoplasm of all layers with histological stages of tumour progression. The present study thus showed that PNA and JFL may be used as cytochemical probes in differentiating malignancy from benign lesions of the oral mucosa.     

Cytokeratin patterns of human oral mucosae in histiotypic culture.

In a three-dimensional culture model, oral epithelial differentiation was investigated ultrastructurally and biochemically for cytokeratin expression. Epithelia from the hard palate, gingiva and alveolar mucosa grown on freely floating collagen lattices populated with fibroblasts from homotypic origins, and fed with medium containing 10% delipidized fetal calf serum for 21 days before analysis, stratified and differentiated to basal cuboidal cells, polyhydral spinous cells and elongated superficial cells. The epithelium of palatal origin had non-nucleated superficial cells resembling orthokeratinized cells. The upper spinous cells had keratohyalin-like granules. The corresponding cells of gingival and alveolar mucosal origins retained their nuclei and had smaller numbers of keratohyalin-like granules. Basal cell keratins (CK 5 and 14) and those of hyperproliferation (CK 6 and 16) were consistently found in all epithelia. Furthermore, simple epithelial keratins (CK 18 and 19) were variably expressed by cells from different oral origins. In epithelial cells from the alveolar mucosa, CK 13 and 19 formed major bands, which correlates with their expression in vivo. In contrast, these polypeptides were either absent or formed minor bands in extracts of gingival and hard palatal cells. Although in small quantities, keratins of terminal differentiation (CK 1, 2, 10 and 11) were detected in gels prepared from palatal epithelia. This expression correlates with the higher morphological differentiation of these cells in this model. The model is of interest for studies of epithelial differentiation, as the differentiation markers of keratinized epithelia (CK 1 and 10) were expressed by cells from palatal origin, and those of non-keratinized epithelia (CK 4, 13 and 19) were prominent in cells from alveolar mucosal origin.     

Identity of TUNEL-positive cells in the oral buccal epithelium of normal mucosa and lichen lesions

BACKGROUND: In situ detection of DNA fragmentation by TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick end labeling (TUNEL) is a widely used technique to identify apoptotic cells in the terminal phases of cell death. Several studies have shown that there are statistically increased numbers of TUNEL(+) cells within the epithelium of oral lichen (OL). It was suggested that this indicates an increased rate of apoptosis among basal and suprabasal keratinocytes in OL epithelium. The aim of this study was to identify the TUNEL(+) cells in the epithelium of erythematous (ERY) OL and normal oral mucosa (NOM). METHODS: Sections of biopsies from NOM and ERY OL were processed for TUNEL combined with immunostaining for pan-cytokeratin or for cell markers specifically expressed by different leukocytes. RESULTS: In NOM, TUNEL(+) keratinocytes were almost exclusively seen in the outermost epithelial layers. This labeling was absent in ERY OL. In the basal and lower spinous layers, more TUNEL(+) cell nuclei were seen in ERY OL as compared with NOM, in accordance with previous studies. The present observations show, however, that only very few of these cells were keratinocytes, but rather were CD4(+) lymphocytes and CD68(+) macrophages. There was no difference between the numbers of TUNEL(+) keratinocytes in basal and lower spinous layers in ERY OL and NOM epithelium. No intraepithelial CD8(+) lymphocytes, Langerhans cells, or mast cells were found to be TUNEL(+). CONCLUSION: The findings indicate that the pathologic changes in ERY OL epithelium cannot be explained by increased prevalence of terminal keratinocyte cell death identified by TUNEL.     

Studies related to reaction of supporting soft tissue to denture wear: the histological response of vervet monkey oral epithelium to a -80 mmHg vacuum

The histological response of vervet monkey oral epithelium to a negative force similar to that experienced under dentures was investigated. Using impression trays with adherent impressions linked to a vacuum pump, the epithelia of the hard palate, attached gingiva and alveolar mucosa of 16 monkeys were subjected to a continuous vacuum of -80 mmHg, and then fixed by perfusion and immersion while in situ. After processing for light microscopy, sections were measured to obtain the rete peg length, supra-papillary width, epithelial width and basal, spinous and superficial cell density 700 microns-2. Mean values and standard deviations were calculated for each measurement and, using Student's t-test, these data were compared with results obtained for normal tissue from nine additional monkeys. The vacuum caused an increase in epithelial width in the palate and attached gingiva, and a decrease in epithelial width in the alveolar mucosa. The cell density 700 microns-2 decreased significantly in all layers of the palate, but increased in the basal layer of the attached gingiva and the basal and superficial layers of the alveolar mucosa. The alveolar mucosa within 0.5 mm of the mucogingival junction showed a variable response. This study demonstrates that a vacuum of -80 mmHg modifies the structure of the oral epithelium, and this response is directly related to the functional demands of the tissue.     

In vitro and in vivo cytokeratin patterns of expression in bioengineered human periodontal mucosa

Background and Objective:  Development of human oral mucosa substitutes by tissue engineering may provide new therapeutic tools for the management of periodontal diseases. In this study we evaluated a fibrin–agarose human oral mucosa substitute both in vitro and in vivo .
Material and Methods:  In vitro bioengineered oral mucosa substitutes were developed from irrelevant biopsy samples of human oral gingiva. In vivo evaluation of the constructed tissues was performed by implantation into athymic nude mice. The expression of several epithelial markers was assessed by microarray analysis and immunohistochemistry.
Results:  Bioengineered oral mucosa samples kept in vitro developed a multilayered epithelium that expressed several cytokeratins, including some markers of simple epithelia (cytokeratins 7, 8 and 18), along with markers of stratified epithelia (cytokeratins 5 and 13) and of cell proliferation (proliferating cell nuclear antigen). Bioengineered tissues grafted in vivo onto nude mice exhibited very good biointegration with the host, showing a cytokeratin expression pattern that was very similar to that of normal native oral mucosa controls. Histological analysis of the artificial tissues demonstrated that oral mucosa substitutes evaluated in vivo were structurally mature, showing some typical structures of human native oral mucosa such as rete ridges and chorial papillae, along with numerous blood vessels at the fibrin–agarose stromal substitute. These structures were absent in samples evaluated in vitro .
Conclusion:  The results indicate that this model of human oral mucosa, constructed using fibrin–agarose scaffolds, shows similarities to native oral mucosa controls and imply that bioengineered oral mucosa substitutes could eventually be used clinically.     

Involucrin expression in epithelial tumors of oral and pharyngeal mucosa and skin

Involucrin has been recognized recently as a marker of terminal differentiation of squamous epithelial cells and also as a useful marker for keratinization; its expression in epithelial tumors of oral and pharyngeal mucosa and skin was examined. Involucrin in normal oral mucosa and skin was restricted to the granular and upper spinous layers and was absent in the basal layer. Hyperkeratosis was characterized by strong positive staining for involucrum in spinous and granular cell layers. A similar pattern was noted in seborrheic keratosis and verruca vulgaris. Condyloma acuminatum specimens revealed slight staining, whereas Paget cells were negative. Calcifying epitheliomas of Malherbe were usually unreactive. Papillomas exhibited the regular distribution of involucrin, as found in normal squamous epithelium. Basal cell carcinomas were generally negative, whereas squamous cell carcinomas showed an irregular distribution of involucrin. Immunohistochemical staining for involucrin may be useful for identification of keratinizing cells in epithelial tumor foci, just as is the use of monoclonal antibody to keratin KL1.     

Thin-layer chromatographic analyses of lipids in different layers of porcine epidermis and oral epithelium.

Frozen cryosections were cut parallel to the surface of porcine skin and palatal, buccal and floor-of-mouth mucosa so as to provide separate samples representing various epithelial layers. The samples were dried, extracted with chloroform:methanol, and the lipids were chromatographed on silica gel plates in various solvent systems. After charring, lipids were quantified with a scanning densitometer. Overall, greater differences in proportions and distributions of lipid components were evident between keratinized and non-keratinized epithelia than between epidermis and keratinized oral epithelium. For epidermis and palate there was an increase in neutral lipids, including ceramides, from the deeper layers to the surface; ceramides were most abundant in surface layers. In buccal epithelium there was a distinct increase in glycosylceramides toward the surface, and in both non-keratinized regions ceramides were present in only very small amounts. The results suggest that although neutral lipids may be associated with a superficial barrier layer in skin and oral mucosa, there are differences in the composition of this barrier between keratinized and non-keratinized epithelia.     

以L929细胞为饲细胞的口腔粘膜上皮细胞体外培养

目的:探索口腔粘膜上皮细胞体外培养的技术和方法,为进一步采用组织工程技术构建口腔粘膜组织奠定基础。方法:取刚离乳的新西兰幼兔口腔粘膜组织小块,酶消化成单细胞悬液,接种后以L929细胞为饲细胞培养, 定期换液、传代。以相差显微镜每天动态观察细胞形态变化及生长增殖情况,细胞进行常规HE染色、免疫组化染色及流式细胞仪检查,并以扫描电镜、透射电镜观察其超微结构。结果:整个上皮细胞生长期内无成纤维细胞混杂生长,均为单一的上皮细胞,并证实为二倍体细胞。细胞可传11~13代,成活50~60 d。结论:新西兰幼兔口腔粘膜上皮细胞可在体外进行培养,在一定时间内保持增殖能力。这不仅为组织工程化口腔粘膜构建奠定了基础,而且为口腔粘膜的体外研究提供了实验模型。     

Histopathologic and ultrastructural studies of oral mucosa with Candida infection

Eighteen oral mucosal biopsies with Candida infection were studied with light and electron microscopy. Under light microscopy, candidal infected oral mucosa was classified with epithelial hyperplasia, 15 cases and epithelial dysplasia, three cases. Four of 15 epithelial hyperplasias showed marked parakeratosis, and high grade acanthosis with many eosinophilic cells in the spinous cell layers. Epithelial dysplasia was characterized by atrophy of the spinous cell layers and increased nucleocytoplasmic ratio in the basal cell layers. Ultrastructurally, candidal infected oral mucosa showed numerous small desmosomes and the interdigitation of cytoplasmic membranes between spinous cells in both epithelial hyperplasia and epithelial dysplasia. Moreover, eosinophilic spinous cells, observed predominantly in epithelial hyperplasia showed intricate arrangement of dense tonofibrils. These ultrastructural findings seemed to give rise to mechanical strength between spinous cells in oral mucous epithelium with Candida infection. Results in this study suggest that excessive hyperplasia of candidal infected oral mucosa might be a protective reaction to the invasion of candidal pseudohyphae, but not associated with precancerous conditions.     

Increased elafin expression in cystic, dysplastic and neoplastic oral tissues

Expression of human leukocyte elastase inhibitor, elafin, otherwise known as skin-derived antileukoproteinase inhibitor (SKALP). was investigated in normal and abnormal oral tissues using a specific anti-SKALP rabbit antiserum. Weak staining was observed in keratinizing epithelia of normal oral mucosa but not in non-keratinizing mucosa. Increased expression was also observed in the suprabasal layers of dysplastic oral epithelia and in well-differentiated squamous cell carcinoma, but not in basal cell carcinoma. A uniform strong expression was observed in all supra-basal layers of odontogenic keratocyst epithelia, except in regions where inflammatory infiltrate was adjacent to keratocyst epithelia. In contrast, elafin expression in a small number of dentigerous cysts and ameloblastomas was more patchy. The increased levels of elafin in keratocyst epithelia and dysplastic tissue may be a cellular homoeostatic response to generate a protective barrier preventing proteolytic degradation of underlying elastic tissue.     

细胞角蛋白19和间隙连接蛋白43在4-亚基硝氧喹啉诱发大鼠舌黏膜癌变过程中表达的研究

目的 研究4-亚基硝氧喹啉（4NQO）诱发大鼠口腔黏膜癌变过程中细胞角蛋白19（CK19）和间隙连接蛋白43（Cx43）的表达,探讨口腔黏膜癌变过程中CK19与Cx43的相关性。方法 利用4NQO诱导SD大鼠的口腔黏膜癌变,运用免疫组织化学的方法检测CK19、Cx43在口腔黏膜癌变过程中各阶段的动态变化。结果 在大鼠正常舌黏膜组织中,CK19阳性染色的细胞散在分布于黏膜基底层;随着大鼠舌黏膜上皮异常增生程度的增加,CK19表达于黏膜基底上层;在口腔鳞状细胞癌（OSCC）组织中,CK19阳性染色细胞分布在黏膜各层。CK19在正常舌黏膜、轻度上皮异常增生、中度上皮异常增生、重度上皮异常增生、OSCC组织中阳性表达率分别为30.00%、50.00%、58.33%、80.00%、91.67%,差异有统计学意义（P<0.05）。在正常舌黏膜中Cx43蛋白主要表达于大鼠舌黏膜上皮细胞的细胞膜上,上皮的基底层、棘层和颗粒层呈阳性染色。随着大鼠舌黏膜上皮异常增生程度的增加,Cx43的表达明显下降。Cx43在正常舌黏膜、轻度上皮异常增生、中度上皮异常增生、重度上皮异常增生、OSCC组织中阳性表达率分别为100.00%、85.71%、66.67%、40.00%、33.33%,差异有统计学意义（P<0.05）。结论 在大鼠舌黏膜癌变过程中,CK19蛋白表达水平随病变程度加重显著升高,提示CK19与口腔上皮细胞的癌变有关;Cx43蛋白表达水平随病变程度加重显著下降,Cx43表达下降是口腔黏膜癌变的早期事件。CK19与Cx43蛋白表达呈负相关,CK19和Cx43的联合检测对OSCC的早期诊断有重要的作用,有利于提高OSCC早期诊断的灵敏度和特异性。     

Quantitative evaluation of regional differences between epithelia in the adult mouse

The composition and proliferation of skin from the ear, tail and footpad and oral mucosa from the palate, cheek and tongue of adult mice were examined. The thickness of the nucleated cell layer of skin snowed an approximately two-fold variation; a similar range was found between that of oral mucosae but that was considerably thicker than skin. No direct correlation between epithelial thickness and the number of nucleated cells was observed. Proliferative activity, assessed following the administration of vinblastine sulphate, and turnover of the epithelium showed a broad range of activities but more rapid in the oral epithelia than in the epidermis, suggesting a relationship between functional stress and proliferative activity. The criteria used clearly distinguish between morphologically different epithelia and should prove useful in examining experimentally produced changes in epithelial histodifferentiation.     

A histological and scanning electron microscopy study of the muco-gingival junction in the vervet monkey

Specimens of clinically healthy oral epithelium from the vervet monkey ( Cercopithecus aethiops ) containing gingiva, alveolar mucosa and muco-gingival junction were examined histologically and with the scanning electron microscope.
The gingiva was covered by a stratified squamous keratinized epithelium and the alveolar mucosa by a non-keratinized stratified squamous epithelium. A groove was present between the two epithelia in approximately half the specimens, the floor of which was lined by keratinized epithelium. Non-keratinized epithelial cells were seen to interdigitate with keratinized cells at the base of the alveolar mucosa side of the groove.     

Intrinsic regulation of differentiation markers in human epidermis, hard palate and buccal mucosa

Different epithelia show extensive variation in differentiation. Epidermis and epithelium from the hard palate are both typical examples of orthokeratinized epithelia whereas buccal mucosa is an example of a non-keratinized epithelium. Each of these tissues can be distinguished morphologically and also by the expression of a number of structural proteins. Tissue explants derived from epidermis, hard palate or buccal mucosa were cultured at the air-liquid interface on collagen gels containing human dermal fibroblasts. Reconstructed epithelia that retained many of the morphological and immunohistochemical characteristics of the original tissue were formed. Cultures derived from epidermis and the hard palate both had a well-defined stratum basale, stratum spinosum, stratum granulosum and stratum corneum whereas cultures derived from buccal mucosa had no stratum granulosum or corneum and the cells retained their nuclei. Significantly more living cell layers were observed in both types of epithelia obtained from the mouth than in epidermis. The specific localization of proliferation and differentiation markers (Ki67, loricrin, involucrin, SPRR2, SPRR3 and keratin 10) closely resembled that of the tissue from which the cultures were derived. As identical three-dimensional culture models were used here, it is concluded that the differences observed between these epithelia were due to intrinsic properties of the keratinocytes.     

Ultrastructure of rat oral epithelium in long-term cell culture

abstract — The purpose of the present study was to describe the ultrastructural appearance of squamous epithelium formed in vitro . The epithelium was formed by outgrowing cells from original explants of rat oral mucosa. Fifty-four explants from the lower surface of the tongues of 12 rats were cultured in a modified Eagle's basal medium, pH 7.2, at 32°C for up to 110 d. Subsequently the cells were prepared for electron microscopic examination. Epithelium from the lower surface of the tongues of eight rats served as control material. The in vitro specimens consisted of an extremely flattened stratified squamous epithelium. The basal cells varied from those of the control specimens by the lack of distinct bundles of tonofibrils. The intermediate cell layer exhibited a marked decrease in the number of organelles and an increase in the number of tonofilaments. No lamellated bodies or keratohyalin granules were found. The intercellular spaces were usually narrow with only a few, small desmosomes, which showed a simplified structure. The superficial cell layers showed, as did the corresponding cells of the control tissue, a distinct keratin pattern and an electron dense cytoplasmic zone along the cell membrane. It is concluded that rat oral epithelial cells grown in long-term cell culture are able to form a differentiating squamous epithelium.     

Localization of bromodeoxyuridine-incorporating, p63- and p75(NGFR)- expressing cells in the human gingival epithelium

The objective of this study was to find the sites with proliferative activity in the human gingival epithelium, where stem cells are likely to exist. Gingival tissues were excised from 16 adult patients and immunohistochemically examined for the presence of bromodeoxyuridine (BrdU)-incorporating, p63- and low affinity nerve growth factor receptor (p75(NGFR))-expressing cells. BrdU-incorporating cells were rarely present in the junctional epithelium. The number of BrdU-incorporating cells in the sulcular and oral gingival epithelia was significantly higher than that found in the junctional epithelium (ANOVA, P < 0.01). A considerable number of p63-positive nuclei were detected in the basal layer to lower spinous layers in the sulcular and oral gingival epithelia, but only few p63-positive cells were present in the junctional epithelium. p75(NGFR)-positive cells were exclusively located in the basal layer in the sulcular and oral gingival epithelia, and in limited basal area in the junctional epithelium neighboring the sulcular epithelium. In the oral gingival epithelium, intense immunostaining of BrdU, p63 and p75(NGFR) was correspondingly observed on the base and side of the rete ridges. These areas probably exhibit high proliferative activity owing to the presence of stem cells.