TRPV2 expression in rat oral mucosa.

The oral mucosa is a highly specialised, stratified epithelium that confers protection from infection and physical, chemical and thermal stimuli. The non-keratinised junctional epithelium surrounds each tooth like a collar and is easily attacked by foreign substances from the oral sulcus. We found that TRPV2, a temperature-gated channel, is highly expressed in junctional epithelial cells, but not in oral sulcular epithelial cells or oral epithelial cells. Dual or triple immunolabelling with immunocompetent cell markers also revealed TRPV2 expression in Langerhans cells and in dendritic cells and macrophages. Electron microscopy disclosed TRPV2 immunoreactivity in the unmyelinated and thinly myelinated axons within the connective tissue underlying the epithelium. TRPV2 labelling was also observed in venule endothelial cells. The electron-dense immunoreaction in junctional epithelial cells, macrophages and neural axons occurred on the plasma membrane, on invaginations of the plasma membrane and in vesicular structures. Because TRPV2 has been shown to respond to temperature, hypotonicity and mechanical stimuli, gingival cells expressing TRPV2 may act as sensor cells, detecting changes in the physical and chemical environment, and may play a role in subsequent defence mechanisms.

Ultrastructural study of tissues surrounding replanted teeth and dental implants.

OBJECTIVES: The aim of this study was to describe the ultrastructure of the dentogingival border at replanted teeth and implants. MATERIAL AND METHODS: Wistar rats (8 weeks old) were divided into groups for replantation and implantation experiments. In the former, the upper right first molars were extracted and then immediately replanted. In the latter, pure titanium implants were used. All tissues were fixed, demineralized and embedded in epoxy resin for ultrastructural observations. RESULTS: One week after replantation, the junctional epithelium was lost, and the oral sulcular epithelium covered the enamel surface. The amount of the epithelium increased in 2 weeks, and resembled the junctional epithelium, and the internal basal lamina and hemidesmosomes were formed in 4 weeks. One week after implantation, peri-implant epithelium was formed, and in 2 and 4 weeks, this epithelium with aggregated connective tissue cells were observed. In 8 weeks, the peri-implant epithelium receded, and aligned special cells with surrounding elongated fibroblasts and bundles of collagen fibers appeared to seal the implant interface. CONCLUSION: In replantation of the tooth, the internal basal lamina remained at the surface of the enamel of the replanted tooth, which is likely to be related to regeneration of the junctional epithelium and the attachment apparatus at the epithelium-tooth interface. Following implantation, a layer of cells with characteristics of connective tissue cells, but no junctional epithelium and attachment apparatus, was formed to seal the site of the implant.

Surface morphology of pocket epithelium.

The pocket epithelium in periodontitis differs from the clinically healthy epithelium in its increase in sulcular depth. However, closer surface morphological distinctions have not been described. To study the surface characteristics of pocket gingiva, the authors analyzed pocket and sulcular epithelium biopsies by scanning and transmission electron microscopy using cytochemical staining for visualization of bacterial adhesion. The clinically healthy and the marginal pocket epithelium were characterized by squamous epithelial cells joined by tight junctions and an inconspicuous surface lacking a distinctive papillary formation. The large quantity of bacteria that adhered to the clinically healthy and marginal pocket epithelium did not appear to elicit any significant defense response. The deeper part of the pocket epithelium revealed a wrinkled papillary relief, increased exfoliation of epithelial cells, leukocyte transmigration, and bacterial internalization, as well as internalization-induced epithelial apoptosis. The alteration of the deep pocket epithelium surface might be either genuine or due to environmental changes of the crevice, or both. Therefore, the periodontitis recovery after removing the deep pocket epithelium might now be related to the pathological alterations of the deep pocket epithelium.

Zone between plaque and attached periodontal tissues on chronic periodontitis-affected teeth: an SEM study.

The zone between plaque and attached periodontal tissues on chronic periodontitis-affected teeth was examined by a combined macroscopic and scanning electron microscopy (SEM) approach. Examined were 27 teeth with chronic periodontitis (chronic periodontitis-affected group) and three healthy teeth with no evidence of periodontal disease (control group). Both groups were collected immediately after extraction, fixed in 2% glutaraldehyde, and post-fixed in 1% osmium tetroxide. Then, teeth were macroscopically examined to identify their stained zones. Teeth were dehydrated, critical-point dried, gold coated, and examined in an SEM. Both healthy and chronic periodontitis-affected teeth showed a very similar staining pattern on their surfaces. An unstained band with a belt-like appearance was observed around the teeth, delimited by two osmium-stained zones. Some weakly stained areas were frequently observed inside the unstained band. The SEM examination showed four different regions in both groups. These regions appeared in the following coronoapical sequence: dental plaque, plaque-free zone, junctional epithelium, and attached periodontal tissues. A dental cuticle covering the cementum surface from the plaque border to the junctional epithelium was detected on chronic periodontitis-affected teeth. Some aspects of this particular zone may be involved in the pathogenesis of inflammatory periodontal disease and therefore may have some influence on treatment for chronic periodontitis-affected teeth.

Biological characteristics of the junctional epithelium.

This review summarizes the biological properties of the junctional epithelium, focusing on its developmental aspects, wide intercellular spaces and desmosomes, dense granules, permeability barrier, phagocytotic activity, adhesive structures and nerve terminals. It also discusses the morphology and functions of long junctional epithelium and peri-implant epithelium. Junctional epithelium is derived from the reduced enamel epithelium during tooth development. Apoptosis occurs in the border between oral and reduced enamel epithelia during tooth eruption. Junctional epithelium expresses a cytokeratin-19 immunoreaction, suggesting that this protein is a consistent differentiation marker. Wide intercellular spaces, which contain neutrophils and nerve endings, are formed as there are fewer desmosomes than in the oral epithelium. Dense, membrane-bound granules in the epithelium might correspond with membrane-coating granules, as revealed by their shape, components and freeze-fracture images. Junctional epithelium with high permeability contains exogenously expressed alpha-defensins, while stratified epithelia contain endogenously expressed beta-defensins. The phagocytotic activity in this epithelium remains unclear. Integrin-alpha6beta4 and laminin-5 form a complex in the tooth surface internal basal lamina. Long junctional epithelium created experimentally attaches to the cementum surface by hemidesmosomes and basal lamina. The peri-implant epithelium differs in proliferation and in adhesive structure from the normal junctional epithelium. In conclusion, wide intercellular spaces and poorly developed desmosomes are closely correlated with a permeable nature. There is still uncertainty over the phagocytotic activity of the epithelium. Integrin-alpha6beta4 and laminin-5 form a significant complex in the internal basal lamina. Junctional epithelium receives a rich sensory nerve and has a high rate of cell turnover. Long junctional epithelium can be produced rapidly during wound healing, due to high proliferative activity. Peri-implant epithelium might be a poorly adhered and permeable epithelium.

Difference in penetration of horseradish peroxidase tracer as a foreign substance into the peri-implant or junctional epithelium of rat gingivae.

Horseradish peroxidase (HRP) tracer was applied to the gingival sulcus of implants or natural teeth at 5, 25, or 50 mg/ml to investigate the sealing capacities of the peri-implant epithelium (PIE) and junctional epithelium (JE); the extent of HRP penetration was observed under electron microscopy. A Ti-6Al-4V implant was inserted either immediately (immediate implantation) or 2 weeks (delayed implantation) after extraction of the maxillary left first molar of rats. The JE of the right molar was used as a control. Although the whole PIE of undecalcified sections appeared to be attached to the implant surface, electron microscopically, the internal basement lamina (IBL) and hemidesmosomes were deficient in the coronal-middle region of the PIE. There were extensive extracellular deposits of HRP in the intercellular spaces between PIE cells, and HRP was blocked to some extent by the lamina lucida and lamina densa of the external basal lamina and basal cell junction. HRP was detected in the connective tissue under the PIE, but was not found in the connective tissue under the JE. Intracellularly, HRP was found in the vesicles and granules of PIE cells and JE cells. These were fewer in number in PIE cells than in JE cells. There were no differences between the findings for immediate and delayed implantation. The results indicate that a deficiency in the IBL permitted penetration of HRP from the gingival sulcus into the connective tissue under the PIE, and suggest that the endocytotic capacity of PIE cells is inferior to that of JE cells.

Microscopic, immunocytochemical, and ultrastructural properties of peri-implant mucosa in humans.

BACKGROUND: Microscopic and immunocytochemical studies have demonstrated both similarities and differences between the gingival mucosa and the peri-implant mucosa restored around dental implants. METHODS: This study was performed on 10 samples of peri-implant mucosa from 10 patients who had undergone implant treatment 16 to 18 months before. Microscopic, ultrastructural, and immunocytochemical investigations were performed to characterize the epithelial and connective layers of the peri-implant mucosa. RESULTS: All specimens showed the morphologic characteristics of well restored tissues. The immunocytochemical reactions used to detect cytokeratins demonstrated that the restored peri-implant mucosa had a non-keratinized epithelium. The apical part of the inner epithelium was a few cell layers thick, like the corresponding junctional epithelium, but it stained positively with the markers for the cytokeratins expressed by the stratified epithelia. Ultrastructurally the keratinocytes adjacent to the implant displayed nuclei containing a rich cytoplasm with filaments and specialized intercellular junctions right up to the more superficial strata. Immunocytochemical reactions and ultrastructural observations demonstrated the presence of non-keratinocyte cells; i.e., Langerhans cells, melanocytes, and Merkel cells. Several cells were found to be proliferating by immunoreaction with mAb anti-PCNA, while immunoreactions with mAbs to detect von Willebrand factor, CD34, and vascular endothelial growth factor marked the well-developed networks of blood and lymphatic vessels in the connective tissue. S-100 and protein gene product 9.5 positive nerve fibers were marked. Immunocytochemical reactions with mAbs anti-vimentin, anti-laminin were also performed. CONCLUSIONS: Our results demonstrated that all the epithelial and connective components of the mucosa are involved in the substantial regrowth of the peri-implant tissue and subsequently in the success of the implant.

The dento-epithelial junction: cell adhesion by type I hemidesmosomes in the absence of a true basal lamina.

BACKGROUND: The junctional epithelium (JE) is a unique structure that makes contact with both a non-renewable hard tooth surface and with a basement membrane (BM) facing the connective tissue. Ultrastructurally, this attachment occurs through hemidesmosomes (HD) and a basal lamina-like extracellular matrix which, on the tooth side, is termed the internal basal lamina. In this study we investigated the expression of basal cell markers in the tooth-facing (TF) cells of JE. METHODS: Samples of healthy marginal gingiva were removed by careful dissection. The expression of laminin-5 was used to indicate TF cell preservation in double immunofluorescence labeling and confocal laser scanning microscopy. RESULTS: The results show that integrin alpha6beta4 and laminin-5 colocalize unequivocally in the TF cells. The results also show the specific expression of the basal cytokeratin 14 and the alpha(v) integrin subunit in the TF cells. All 3 major hemidesmosomal components BP180, BP230, and HD1 antigen are likewise present. On the other hand, type IV collagen, laminin-1/10, type VII collagen, and the BM proteoglycan perlecan are all absent from the dento-epithelial junction. CONCLUSIONS: The results indicate that the epithelium-tooth interface is a unique structure wherein epithelial cells adhere by means of bona fide hemidesmosomes to an epithelium-derived extracellular matrix lacking most of the common BM components. Moreover, TF cells differ from connective tissue facing (CTF) cells, not only by their cell surface molecules and their production of extracellular matrix, but also by their cytoskeletal architecture.

High resolution ultrastructural reevaluation of dental cuticle in monkey tooth.

In this study the detailed ultrastructure of the dental cuticle at the dentogingival border in monkeys (macaques) was observed. The deciduous molars and permanent premolars with associated gingiva were prepared for thin section electron microscopy. The dental cuticle, seen as a dense amorphous, usually unlaminated layer, was localized between the tooth enamel and the junctional epithelium of the gingiva in close association with the internal basement membrane of the junctional epithelium. High resolution electron microscopy showed that its basic structure was a fine network of irregular anastomosing strands identified as the cord network of the basement membrane which was continuous with that of the lamina densa of the internal basement membrane of the junctional epithelium. In the cuticle. openings of most of the network were filled with a dark amorphous material. These observations suggest that an additional layer of a cord network formed from the basement membrane components, which is probably secreted by the cells of the junctional epithelium during formation of the cuticle, is added to the cord network of the lamina densa of the basement membrane. In addition, a dark amorphous material is deposited within the newly added cord network at the enamel side of the basement membrane. The origin of this dark material still remains to be clarified, but the possibility is that it originates, as has previously been suggested, from either serum protein in gingival exudate or from hemoglobin produced by the degradation of red blood cells.

Regulated expression of nullo is required for the formation of distinct apical and basal adherens junctions in the Drosophila blastoderm.

During cellularization, the Drosophila embryo undergoes a large-scale cytokinetic event that packages thousands of syncytial nuclei into individual cells, resulting in the de novo formation of an epithelial monolayer in the cortex of the embryo. The formation of adherens junctions is one of the many aspects of epithelial polarity that is established during cellularization: at the onset of cellularization, the Drosophila beta-catenin homologue Armadillo (Arm) accumulates at the leading edge of the cleavage furrow, and later to the apicolateral region where the zonula adherens precursors are formed. In this paper, we show that the basal accumulation of Arm colocalizes with DE-cadherin and Dalpha-catenin, and corresponds to a region of tight membrane association, which we refer to as the basal junction. Although the two junctions are similar in components and function, they differ in their response to the novel cellularization protein Nullo. Nullo is present in the basal junction and is required for its formation at the onset of cellularization. In contrast, Nullo is degraded before apical junction formation, and prolonged expression of Nullo blocks the apical clustering of junctional components, leading to morphological defects in the developing embryo. These observations reveal differences in the formation of the apical and basal junctions, and offer insight into the role of Nullo in basal junction formation.

Ultrastructural and immunoelectron microscopic studies of the peri-implant epithelium-implant (Ti-6Al-4V) interface of rat maxilla.

BACKGROUND: The role played by the internal basal lamina (IBL) and hemidesmosomes between an implant and the peri-implant epithelium (PIE) in the adherence of the epithelium to the implant is controversial. This study used rat maxilla implantation models to clarify the ultrastructure of the PIE-implant interface. METHODS: Ti-6Al-4V implants were inserted either immediately or 2 weeks after the extraction of the upper left first molar of 6- or 4-week-old rats, respectively. The junctional epithelium (JE) of the upper right molars in the same animals was used as a control. Four weeks after implantation, the animals were sacrificed to prepare specimens for light and immunoelectron microscopy. RESULTS: Under light microscopy, the PIE appeared to attach to the implant surface. Ultrastructurally, IBL, consisting of the lamina densa and lamina lucida, and hemidesmosomes were formed only in the lower region, and rarely in the middle region, of the PIE-implant interface. In control teeth, the IBL and hemidesmosomes formed throughout the dento-JE interface. Laminin-1 was found in the IBL and also in the vesicles and vacuoles of the PIE and JE cells. Statistical analysis showed that there was also a significant difference in the amount of IBL between the PIE-implant and dento-JE interfaces. CONCLUSIONS: PIE attached to the implant via hemidesmosomes and IBL in the lower region of the PIE-implant interface. Although PIE cells may secrete laminin-1, which contributes to epidermal cell adhesion, the PIE which attaches to implants only in the lower region of the interface is considered to be the poorly adhered epithelium.

Ultrastructural immunohistochemical study of interstitial collagenous components of the healthy human keratinized mucosa surrounding implants.

The aim of this study was to investigate the ultrastructural and immunohistochemical organization of the main collagenous components of healthy human keratinized mucosa surrounding endosseous implants. Eight patients with completely edentulous mandibles were selected. Four endosseous implants were placed in the mandible of each patient, connected with a bar to support a complete overdenture, and loaded 4 months later. Two years after placement, biopsies of surrounding soft tissue, including the sulcular and junctional epithelium with the underlying and supracrestal connective tissue, were routinely prepared for standard electron microscopy and for ultrastructural immunolabeling of Types I, III, and IV collagen. The connective tissue located under the junctional epithelium comprised Types I and III collagen, whereas the supracrestal connective tissue was composed mainly of Type I collagen. Type IV collagen was located exclusively in the basement membrane of the junctional epithelium.

Cyclosporin A-induced gingival overgrowth in the rat: a histological, ultrastructural and histomorphometric evaluation.

This investigation was undertaken to further study cyclosporin A (CsA)-induced gingival overgrowth. Thirty mg/kg/d of vehicle or CsA solutions were given orally to 6-wk-old male Sprague-Dawley rats. After 4, 9, 14 and 19 wk 2 control and 2 experimental rats were anaesthetized, tissues fixed by intracardiac perfusion of fixative solution and jaws processed for Epon inclusion. Histological and ultrastructural studies conducted in a gingival portion (free gingiva) revealed the presence of hyalinization areas and of multinucleated cells (MCs) containing collagen fibrils (connective tissue), of amorphous areas and disorders of keratinization (epithelia). Histomorphometric evaluation indicated that in the CsA rats the mean cross-sectional area of the free gingiva was 2.52-fold increased compared to the controls. The connective tissue comprised 41.43% of this area (instead of 31.49% in controls). Additional histomorphometric evaluation was performed in 3 groups of free gingival portions: control (C group), CsA-non-respondent (CsA-nR) and CsA-respondent (CsA-R). The cross-sectional gingival areas studied were slightly lower than the mean area of all the control sites previously defined (groups C and CsA-nR) or showed the higher degrees of enlargement (CsA-R). In the CsA-R group the mean cross-sectioned area of the vessel profiles was increased and the number of fibroblast profiles decreased. In the CsA-nR group the number of vessel profiles and that of MCs profiles were increased. In the epithelia of the CsA-R group were increased (a) keratinized epithelia: thickness; thickness of the inner and of the outer compartments; surface area of spinous cell profiles; (b) oral gingival epithelium: number of cell layers (inner compartment); (c) oral sulcular epithelium: surface area of granular cell profiles; (d) junctional epithelium: thickness; number of cell layers. These results indicate that (a) the CsA induced modifications are not limited to enlarged gingiva (b) the overgrowth of the GCT is the result of a vasodilatation and of an increase in the volume of the extracellular matrix and (c) the increase of the epithelial thickness is mainly the result of a cell hypertrophy in the keratinized epithelia and of a cell hyperplasia in the junctional epithelium.

Evidence that laminin-5 is a component of the tooth surface internal basal lamina, supporting epithelial cell adhesion.

Laminin-5 (Ln-5) is an extracellular matrix (ECM) glycoprotein found in epithelial basal laminae. We studied its expression on the surface of rat molars, in relationship to the location of the internal basal lamina (IBL) of the junctional epithelium (JE). In order to avoid disruption of the JE-tooth interface as much as possible, the surface of molars was prepared by mechanical removal of tissue debris and detergent/osmotic lysis of epithelial cell layers, and directly stained by immunohistochemistry, without sectioning. Antibodies to Ln-5 specifically stained a narrow band in the region of the cemento-enamel junction (CEJ), consistent with the expected location of the IBL. Western blotting of ECM material detergent--solubilized from the prepared tooth surfaces confirmed the molecular nature of Ln-5 identified by immunohistochemistry. By the use of a high-definition 3-D microscope, it appeared that Ln-5 coated the most apical part of the enamel and the most coronal portion of the cementum, on either side of the CEJ. In adhesion assays performed directly on tooth surfaces, epithelial cells adhered preferentially to the Ln-5 coated area of the tooth compared to the root surface, which is coated by other ECM components. Adhesion to the Ln-5 coated surface was specifically inhibited by a function-blocking monoclonal antibody to Ln-5. These results suggest that Ln-5 is a component of the IBL, and that it may be important in promoting adhesion of JE cells onto the tooth surface.

The epithelium-tooth interface--a basal lamina rich in laminin-5 and lacking other known laminin isoforms.

The attachment of the marginal gingiva to the tooth surface is mediated by a thin nonkeratinized epithelium termed the junctional epithelium (JE). Ultrastructural studies have revealed that the attachment of the JE to the tooth surface occurs through hemidesmosomes (HD) and a basal lamina-like extracellular matrix termed the internal basal lamina (IBL). We have previously shown that neither type IV collagen nor prototypic laminin, two common components of basement membranes (BM), is present in the IBL between the epithelium and the tooth. In the present study, we show that laminin-5 is a major component of the IBL in both rodent and human tissues. By using in situ hybridization, we also show that the cells of the JE express the LAMC2 gene of laminin-5. In other parts of gingival epithelium, LAMC2 gene expression is less prominent. Our results indicate that the epithelium-tooth interface is a unique structure wherein epithelial cells are induced to secrete a basal lamina containing laminin-5 and no other presently known laminin isoform.

A fluid-phase endocytotic capacity and intracellular degradation of a foreign protein (horseradish peroxidase) by lysosomal cysteine proteinases in the rat junctional epithelium.

We investigated the co-localization of lysosomal cathepsins B, H and L, and horseradish peroxidase (HRP) in junctional epithelial (JE) cells both as a fluid-phase endocytotic marker to demonstrate the fluid-phase endocytotic capacity of JE cells, and to understand the morphological relationships of the endocytosed foreign substances to lysosomal cathepsins in these cells. The diaminobenzidine (DAB) histochemical and cytochemical methods and immunohistochemical avidin-biotin-peroxidase complex and immunocytochemical post-embedding colloidal gold methods were used. Under light microscopy, DAB reaction products based on HRP were found in JE but were rare or absent in the oral sulcular epithelium and oral epithelium. Immunolabeling for cathepsins B and H was found in the granular structures of the cells, but no cathepsin L was identified. With electron microscopy, DAB reaction products, which indicated both HRP and the azurophil granules of neutrophils, were endocytosed into JE cells. Using a post-embedding technique, gold particles indicating HRP were present on the plasma membrane of JE cells, at the periphery of electronlucent vacuoles, and in the electrondense granules. Gold particles indicating cathepsin B or H were found in the electrondense granules. With different sizes of colloidal golds, the co-localization of cathepsin B or H with HRP was indicated only in the electrondense portion of the larger vacuoles consisting of electronlucent and -dense parts. This study provided the first morphological data which indicate that JE has a fluid phase endocytotic capacity, and which suggest that the lysosomal cathepsins B and H are involved in the intracellular degradation of foreign substances invading through the gingival sulcus in JE cells.

Maintenance of new cementum formed during cyclosporin A administration after suspension of the treatment.

The aim of the present investigation was to examine if new cementum (NC) formed during cyclosporin A (CsA) administration was maintained after suspension of the treatment. Thirty mg/kg/d of CsA were given to 3 male Sprague-Dawley rats. Three control rats received oil-based vehicle solution. Nine wk later the drug and vehicle administration were stopped and the rats continued to be fed with the same standard laboratory diet and water ad libitum for 5 months. The rats were anaesthetized, the tissues fixed by intracardiac perfusion of fixative solution and the mandibles processed for Epon inclusion. Histological, histomorphometric and ultrastructural analysis revealed that (a) NC covered extensive areas of the root surfaces; its structural characteristics were identical to those observed in the rats killed during CsA administration. (b) collagen fibres of the adjacent connective tissue were functionally inserted into the NC. (c) In the presence of cervical NC spurs the extent of the apical downgrowth of the junctional epithelium, measured parallel to the cemento-dentinal junction, was decreased (up to 64%) compared to the one occurring in areas devoid of NC deposits. These results suggest that (a) NC deposition and its functional relations with the adjacent connective tissue are not reversible after cessation of CsA treatment and (b) in the presence of cervical NC spurs the amount of connective tissue attachment on the root surfaces is increased.

Histomorphological and biochemical differentiation capacity in organotypic co-cultures of primary gingival cells.

To establish a three-dimensional in vitro test system mimicking the physiological situation of the oral cavity, organotypic co-cultures consisting of primary gingival cells on a collagen matrix with fibroblasts were generated. The histomorphological development after 7 and 14 d revealed close similarity with the non-keratinized gingiva epithelium. Furthermore, as epithelial specific markers synthesis and localization of keratins as well as the deposition of basement membrane components were assessed on frozen sections by immunofluorescence and keratin expression by in situ hybridization. Primary keratinocytes in conventional culture strained positive for keratin K14 and the mucosal differentiation-specific keratins K4 and K13, while primary fibroblasts, isolated from the same tissue source, and also some keratinocytes, were positive for vimentin. In organotypic co-cultures the keratinocytes formed a multilayered epithelium within 14 d containing basal cells and flattened cells in the uppermost layers. Comparable to native non-keratinized gingiva keratin 14 gene expression was clearly detectable in the basal cell compartment but showed extending immunolocalization. In addition, particularly at the early stage (7 d), basally located keratinocytes were also vimentin positive. According to morphological differentiation K4 and K13 were detectable in suprabasal position a the RNA and protein level. The major basement membrane constituents collagen type IV and laminin increased with time revealing first an interrupted and later a fully extended staining underneath the basal cells. Maintenance of basal cell function was further demonstrated by cell proliferation (BrdU incorporation) which was initially high (7 d) but declined towards the later stages (14-21 d). The results demonstrate i) that this co-culture system leads to a stratified surface epithelium with morphological and biochemical characteristics of the non-keratinized gingiva epithelium and ii) that a state of physiological tissue balance was reached, thus rendering a suitable model for tissue compatibility studies.

Culture and characterization of human junctional epithelial cells.

This study was undertaken to establish a culture of junctional epithelial cells derived from gingival tissue attached to the tooth surface and to characterize these cells immunocytochemically and ultrastructurally. Primary cultures of cells were obtained from the junctional tissue explanted on type I collagen-coated dishes and immersed in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS). Cells were subcultured with conditioned serum-free keratinocyte medium (keratinocyte-SFM + 5% FBS) on dishes coated with solubilized extract of the basement membrane. After 24 hours, the medium was changed to keratinocyte-SFM (0.09 mM Ca2+). The cell-doubling time was 40.5 hours. As a control, cells from gingival tissue were cultured by the same method. Cells from junctional tissue and gingival tissue were compared immunocytochemically using monoclonal antibodies to keratin, vimentin, and desmoplakins I and II and using Dolichos biflorus agglutinin (DBA). The keratin AE1 and AE3 was expressed by all of culture cells. The vimentin (specific for the intermediate filament of mesenchymal cells) was also expressed by all cells. The expression pattern of keratin 19 was observed not only by cells from junctional tissue but also by cells from gingival tissue. All keratin peptides were expressed in both cells. However, DBA reacted only with cells from the junctional tissue. Anti-desmoplakin I and II reacted with both cells, however, the staining patterns differed. DBA-positive cultured epithelial cells from the junctional tissue showed poor tonofilament bundles and were rich in cytoplasmic organelles. These findings suggest that junctional epithelial cells can be isolated from junctional tissue and cultured under improved conditions.

[Prognostic value of initial ultrastructural changes in squamous cell metaplasia of junction cell papilloma of the bladder]. / Prognosticheskoe znachenie rannikh ul'trastrukturnykh priznakov ploskokletochnoi metaplazii perekhodnokletochnoi opukholi mochevogo puzyria.

In 48 patients with a junction cell papilloma, in 65--with a junction cell and in 20--with nondifferentiated bladder cancer the electron-microscopic studying of the squamous cell metaplasia signs was conducted. Great prognostic importance of an early and overt signs of squamous cell metaplasia was established.