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The most fundamental function of an epithelial tissue is to act as a barrier, regulating interactions between the external environment and the body. This barrier function typically requires a contiguous cell layer but since teeth penetrate the oral epithelium, a modified barrier has evolved, called the junctional epithelium (JE). In health, the JE attaches to the tooth, sealing the inside of the body against oral micro-organisms. Breakdown of the JE barrier results in periodontal ligament (PDL) disintegration, alveolar bone resorption, and ultimately tooth loss. Using lineage tracing and DNA pulse-chase analyses, we identified an anatomical location in the JE that supported both fast- and slow-cycling Wnt-responsive stem cells that contributed to self-renewal of the tissue. Stem cells produced daughter cells with an extraordinarily high rate of turnover that maintained JE integrity for 1.4 y in mice. Blocking cell proliferation via a chemotherapeutic agent 5-fluorouracil (5-Fu) eliminated fast-cycling stem cells, which caused JE degeneration, PDL destruction, and bone resorption. Upon removal of 5-Fu, slow-cycling stem cells regenerated both the structure and barrier function of the JE. Taken together, our studies identified a stem cell population in the JE and have potential clinical implications for prevention and treatment of periodontitis.

The junctional epithelium (JE) is unique with regard to its wide intercellular spaces and sparsely developed intercellular junctions. Thus, knowledge of the molecular mechanisms that regulate the formation of the intercellular junctions of the junctional epithelium may be essential to understand the pathophysiology of the JE. HOK-16B cells, a normal human gingival epithelial cell line, were used to identify the molecules involved in the regulation of the formation of intercellular E-cadherin junctions between human gingival epithelial cells. Activation of c-Jun N-terminal kinase (JNK) disrupted the intercellular junctions through the dissociation of E-cadherin. The role of JNK in the formation of these E-cadherin junctions was further confirmed by demonstrating that JNK inhibition induced the formation of intercellular E-cadherin junctions. The upstream signaling of JNK was also examined. Activation of the small GTPase RhoA disrupted the formation of E-cadherin junctions between HOK-16B cells, which was accompanied by JNK activation. Disruption of these intercellular junctions upon RhoA activation was prevented when JNK activity was inhibited. In contrast, RhoA inactivation led to HOK-16B cell aggregation and the formation of intercellular junctions, even under conditions in which the cellular junctions were naturally disrupted by growth on a strongly adhesive surface. Furthermore, the JE of mouse molars had high JNK activity associated with low E-cadherin expression, which was reversed in the other gingival epithelia, including the sulcular epithelium. Interestingly, JNK activity was increased in cells grown on a solid surface, where cells showed higher RhoA activity than those grown on soft surfaces. Together, these results indicate that the decreased formation of intercellular E-cadherin junctions within the JE may be coupled to high JNK activity, which is activated by the upregulation of RhoA on solid tooth surfaces.

Amelotin (AMTN) is a relatively recently discovered enamel protein that is predominantly expressed by ameloblasts during the maturation stage of amelogenesis and is present at lower levels in the junctional epithelium of erupted teeth. Previous studies have suggested a function of this protein in enamel mineralization and cell attachment. Genetic mouse models have been instrumental in defining the role of many enamel-related proteins, but a genetic mouse model lacking the Amtn gene has not been reported. Here, we describe the generation of amelotin-deficient mice and the analysis of their enamel phenotype in comparison with that of wild-type animals. Ablation of AMTN expression resulted in mechanically inferior enamel of mandibular incisors that showed chipping and fractures at the incisal edge. Enamel mineralization was delayed, resulting in hypomineralized inner enamel and structural defects in the outer enamel. Erupted enamel close to the gingival margin showed increased surface roughness. The expression levels of the enamel matrix proteins AMEL, AMBN, ENAM, and ODAM and the enamel proteases MMP-20 and KLK-4 were not significantly altered, although the expression of KLK-4 was delayed. The morphology of ameloblasts showing prominent Tomes’ processes during the secretory stage was not altered, and there was no indication of disruption of cell structures or activities, but a residual layer, presumably consisting of organic material, remained at the enamel surface close to the gingival margin. The integrity of the dentogingival attachment at the junctional epithelium appeared unaffected by AMTN deficiency. These observations indicate that AMTN plays a subtle yet critical role in enamel biomineralization, particularly during the establishment of the outer and surface enamel layers. This role appears to be largely independent of other enamel proteins.

The overexpression of the intracellular signaling molecule of the transforming growth factor–beta family (TGF-β) Smad2 was found to induce apoptosis and inhibit the proliferation rate of oral epithelial cells. Therefore, the aim of this study was to investigate in vivo the effect of Smad2 overexpression on the proliferation rate of the junctional epithelium (JE). Smad2 overexpression was driven by the cytokeratin 14 promoter (K14-Smad2) in transgenic mice. The K14-Smad2 mice were compared with wild-type (WT) mice selected as the control group. Samples were stained with hematoxylin and eosin stains and analyzed by image analysis. Immunohistochemistry was conducted for proliferating cell nuclear antigen (PCNA) and c-Myc as markers of cell proliferation. The expression of cyclin-dependent kinase inhibitors (P15, P21, and P27) was determined by real-time polymerase chain-reaction (RT-PCR). The quantity of phosphorylated retinoblastoma (pRB) was determined with Western blots. The overexpression of Smad2 altered the area of the junctional epithelial cells in one-year-old K14-Smad2 mice. The area was 32,768 (± 3,473) μm2 for the WT and 24,937.25 (± 1,965) μm2 for the K14-Smad2 mice. There was a significant difference in the proliferation rates of the JE (PCNA-positive cells) between the WT and K14-Smad2 mice, 20.7% (± 1.1) and 2.1% (± 0.5), respectively. A significant difference in c-Myc expression occurred between experimental and control samples. The K14-Smad2 mice had a mean of 2.3% (± 0.6), and the WT mice had a mean of 20.1% (± 3.6). Smad2 overexpression up-regulated the mRNA expression of P15 by 2.3-fold and that of P27 by 5.5-fold in the K14-Smad2 mice. Finally, the pRB protein showed a 2.3 (± 0.5)-fold increase in K14-Smad2 mice when compared with WT mice. Smad2 overexpression inhibits the proliferation of JE cells by down-regulating c-Myc and up-regulating P15 and P27, which resulted in an increase in pRB, leading to cell-cycle arrest.

Adhesion of epithelium to the extracellular matrix is crucial for the maintenance of systemic and oral health. In the oral cavity, teeth or artificial dental implants penetrate the soft tissue of the gingiva. In this interface, gingival soft tissue needs to be well attached via the epithelial seal to the tooth or implant surface to maintain health. After injury or wounding, epithelial tissue rapidly migrates to form the initial epithelial cover to restore the barrier against infection. These events are crucially dependent on deposition of extracellular matrix and proper activation and function of integrin receptors in the epithelial cells. Recent experimental evidence suggests that epithelial integrins also participate in the regulation of periodontal inflammation. In this review, we will discuss the structure and function of epithelial integrins and their extracellular ligands and elaborate on their potential role in disease and repair processes in the oral cavity.

Toothbrushing exposes epithelia and other tissues of the oral cavity to mechanical stress. Here, we investigated whether brushing induces cell wounding—plasma membrane disruption—in epithelial and other cell types in the oral cavity. Brushing of the gingivae and tongues of rats resulted in a striking increase in the number of cells positive for a marker of disruption injury. These cells included those in all strata of the gingival epithelium, and in the skeletal muscle of the tongue. Additionally, we found that brushing resulted in an increase in c-fos expression by junctional epithelial and skeletal muscle cells. Epithelial barrier function, however, was not overtly affected by brushing, despite the observed individual injuries to cells. We concluded that brushing disrupts cell plasma membrane barriers in the oral cavity and activates gene expression events that may lead to local adaptive changes in tissue architecture beneficial to gingival health.

The aim of the present study was to examine differences in cellular characteristics of human peri-implantitis and periodontitis lesions. Two groups of patients were included: 40 patients with generalized severe chronic periodontitis and 40 patients presenting with severe peri-implantitis. Soft tissue biopsies were obtained from diseased sites (probing pocket depth ≥ 7 mm with bleeding on probing) and prepared for histologic and immunohistochemical analysis. In contrast to periodontitis samples, peri-implantitis lesions were more than twice as large and contained significantly larger area proportions, numbers, and densities of CD138-, CD68-, and MPO-positive cells than periodontitis lesions. Peri-implantitis lesions also extended to a position that was apical of the pocket epithelium and not surrounded by noninfiltrated connective tissue. They further presented with significantly larger densities of vascular structures in the connective tissue area lateral to the infiltrated connective tissue than within the infiltrate. This study suggests that peri-implantitis and periodontitis lesions exhibit critical histopathologic differences, which contribute to the understanding of dissimilarities in onset and progression between the 2 diseases.

It is not known whether epithelial differentiation patterns are reflected in the composition of gingival basement membranes (BMs). We have investigated the expression of laminin isoforms and associated BM components in the murine dento-epithelial junction by using immunofluorescence microscopy. Our results show that chains of laminins 5/6/7/10/11 are expressed in the BM of outer gingival epithelium. The external BM between junctional epithelium (JE) and connective tissue differs from gingival BM by lacking laminin-7 and -11 chains. The internal basal lamina (IBL) between JE and tooth contains only laminin-5. Collagen chains α1,2(IV) and nidogen-1 are present in other BMs except the IBL. The dento-epithelial junction thus has a unique BM composition, suggesting that epithelial cells are able to secrete two extracellular matrices in a polarized manner. The exclusive expression of the non-self-polymerizing laminin-5 indicates that the IBL is not a BM by definition, but rather a simple extracellular matrix lacking network structure.

This study examined the directly attached cells (DAT cells) of the human junctional epithelium from teeth extracted for advanced periodontal disease. The monolayer nature of the DAT cells remaining on the tooth surface after extraction offers a unique opportunity to study their morphology and activity in situ. We collected DAT cells and examined them by transmission electron microscopy and by autoradiography of cells labeled with 3H-thymidine. Our morphological results suggest that degenerative changes associated with pocket formation affect individual cells rather than regions of the DAT cell population at one time. The epithelial attachment apparatus (EAA) appeared to be the most resistant structure persisting on the tooth surface during the degeneration of the individual DAT cells. The new technique developed for two-dimensional observation of the sheet of DAT cells labeled in situ on the tooth surface in culture showed that the attached cells, even in periodontal disease, exhibit proliferative ability, suggesting a regenerative role for the DAT cells.

Analysis of three measures of periodontal health (plaque index, gingival index, and attachment loss) was carried out on 178 individuals in 75 families examined as part of a family study of periodontal health. Original participants in this study were volunteers recruited from the University of Maryland Dental Clinic, and were selected independently of any specific dental disease or condition. Relatives were invited to participate in the family study so that the extent of familial aggregation of indices of periodontal health could be assessed. By means of an analysis of variance model for persons nested within families, evidence for familial aggregation of plaque index was found both before and after adjustment for covariates such as age, gender, race, and reported oral hygiene habits. While a substantial fraction of variance in gingival index and attachment loss was also due to differences among families, neither attained statistical significance in these data. Examination of familial correlations (e.g., parent-offspring, sib-sib, spouse correlations) confirmed that plaque index showed greater familial resemblance compared with other measures of periodontal health. Both mean gingival index and mean attachment loss showed a stronger correlation between mothers and offspring compared with fathers and offspring. This suggests that further analysis of models for separating genetic and environmental effects may be appropriate for plaque index, but complete analysis of other periodontal indices will require more flexible statistical models for separation of genetic and cultural inheritance while considering gender-specific expression and transmission, as well as incorporation of information from covariates.

This experiment was aimed at studying the intact tissue/implant interface of non-submerged dental implants with a titanium surface. Epoxy-resin replicas were fabricated from 3.05 x 8 mm cylindrical titanium implants with a plasma-sprayed apical portion and a smooth coronal collar. The replicas were coated with a 90-120-nm-thick layer of pure titanium and autoclaved. The coated replicas were inserted as non-submerged endosseous implants in the edentulous premolar region of dog mandibles and allowed to heal for three months. Jaw sections containing the implants were processed for light and electron microscopic study of the intact tissue/implant interface with and without prior demineralization. Gingival connective tissue fibers were closely adapted to the titanium layer, in an orientation more or less parallel to the implant surface. There was no evidence of any fiber insertions into the surface irregularities of the smooth or rough titanium surface. Undemineralized bone was intimately adapted to the titanium surface without any intervening space. In demineralized sections, the collagen fibers of the bone matrix tended to be somewhat thinner and occasionally less densely packed in the vicinity of the implant surface. However, they extended all the way to the titanium surface, without any intervening fibril-free layer.

The sensory innervation of junctional and oral sulcular epithelium was investigated by use of anterograde transport of wheat-germ agglutinin-horseradish peroxidase from the trigeminal ganglion. By light microscopy, labeled intra-epithelial nerve fibers were observed forming a dense plexus in the apical two-thirds of the junctional epithelium, with some fibers located near the enamel space. Occasional fibers extended coronally to the sulcus bottom. By electron microscopy, labeled intra-epithelial axon terminals or varicosities were demonstrated to be in close contact with both junctional epithelial cells and neutrophils. These varicosities, which were occasionally surrounded by the cytoplasmic processes of epithelial cells or neutrophils, frequently contained large granular and small clear vesicles. In contrast to the junctional epithelium, the oral sulcular epithelium was sparsely innervated, except for the transition region between the oral sulcular epithelium and the junctional epithelium, where a dense innervation by labeled intraepithelial fibers was found. These fibers extended as far as the stratum spinosum. Electron microscopy revealed mitochondria-filled profiles of varicosities between epithelial cells. This study shows differences in the distribution and ultrastructure of sensory nerves between the junctional and oral sulcular epithelia, and further provides morphological evidence that sensory nerves in the junctional epithelium come into contact not only with epithelial cells but also with neutrophils.

The mesial gingiva of the maxillary first molar of SAM-R/1/Iw (senescence-accelerated mouse, resistant/1/Iwate) and the P/2/Iw (prone/2/Iwate) were studied morphologically by quantitative and qualitative methods as a function of age (one-, two-, six-, 12-, and 16-month-old). In this region, neither plaque nor periodontitis has been found (Sashima et al., 1990a). Statistical analyses of the gingival tissues in this region in both strains showed that, with age, the length of the junctional epithelium (JED) increased, the depth of the gingival sulcus (GSD) did not change, and the height of the gingival crest (GCD) decreased. Therefore, long-junctional epithelium developed, and gingival recession occurred, since a periodontal pocket did not develop with age in either strain. In particular, there was a strong correlation between the JED and age in both strains (r = 0.84, p < 0.05 for the R/l/Iw, and r = 0.92, p < 0.05 for the P/2/ Iw). The JED, GSD, and GCD of the P/2/Iw were higher than each of those in the R/l/Iw after six months of age; however, there were no significant differences between the R/l/Iw and the P/2/Iw for any age group (one-, two-, six-, 12-, and 16-month-old).

The distribution of the α 1-α6 subunits of β1 integrins was studied by using a panel of monoclonal antibodies in indirect immunofluorescence microscopy. The results showed that the β1 subunit was expressed at the cell membrane of basal cells of gingival epithelium, throughout the cells of junctional epithelium (JE), and in cells of connective tissue, including endothelial cells. The α1 subunit was expressed in vascular endothelial cells and, more faintly, in inflammatory cells in gingival connective tissue. The α4 subunit was expressed selectively in inflammatory cells, and the α5 subunit was expressed in cells throughout gingival connective tissue. An overall cell membrane immunoreactivity for the α2 and α3 subunits was shown in basal cells of gingival epithelium and in cells of JE, corresponding to the epithelial localization of the β1 subunit. The α6 subunit was polarized to the basal aspects of basal epithelial cells, but was also present in an overall cell surface distribution in basal cells and in cells of JE. The β4 integrin subunit was mainly expressed at the basal aspects of basal cells in gingival epithelium and JE. The results indicate that the α2/β1, α3/β 1, α6/β1, and α6/β 4 integrins are all expressed in human gingival epithelium. Of these, the α6/β4 integrin complex is the major candidate for mediation of the attachment of epithelial cells to the basement membrane facing the connective tissue and probably also the tooth.

Over 500 residents of Tecumseh, Michigan, were dentally examined in 1959 as part of a community-wide health study. In 1987, the dental examinations were repeated, with use of the same criteria as in 1959, for 167 dentate persons from the original group. Another 28 reported by telephone that they had become edentulous since 1959. This report uses a historical cohort analysis for exploration of the risk factors for tooth loss, both total and partial, over the 28-year period. Over that time, the edentulous lost an average of 18.0 teeth (95% confidence interval 15.5, 20. 7), whereas the age-matched 90 dentate persons lost only 3.2 (2.2, 4.2) teeth each. Descriptive data showed the edentulous to have higher baseline scores for plaque, calculus, and gingivitis, and a higher proportion of them smoked, though only loss of periodontal attachment (LPA) of 4 mm or more, early loss of first molars, and educational attainment were significant risk factors in regression analysis. Odds ratios for these three variables were 4.0 (1.2, 12.8), 2.0 (1.3, 3.1), and 0.6 (0.4, 0.9), respectively. The strongest risk factors for partial tooth loss among 116 dentate persons were baseline gingivitis (which was correlated with LPA of 4 mm or more) and the baseline number of teeth present, with odds ratios of 2.4 (1.2, 5.2) and 0. 8 (0. 7, 1.0), respectively. While the analysis had to be carried out without caries data, it was concluded that total tooth loss is a social-behavioral issue as much as it is disease-related. Social-behavioral factors were less clearly related to partial tooth loss in dentate persons; oral disease characteristics were the most prominent risk factors for partial tooth loss.

The purpose of this epidemiological study was to estimate the degree of change in periodontal attachment level in a sample of adults examined in 1959 and 1987 in Tecumseh, Michigan. Out of 526 individuals between the ages of five and 60 years in 1959, a sample of 325 resided within an 80-km-radius area in 1987. Of those, 167 were re-examined. Loss of periodontal attachment (LPA) was determined with a Michigan #0 probe on four tooth sites (disto-buccal, mid-buccal, mesio-buccal, mid-lingual) for all teeth present. Of the individuals contacted, 28 had lost all their teeth during the 28 years. Of the 167 adults examined, two refused periodontal probing. Out of the 165 adults with LPA measurements in 1987, only 22 (13.3%) had an average increased loss of 2 mm or more per person between 1959 and 1987; five adults (3.0%) had an average LPA increase of 3 mm or more, and only two adults (1.2%) had an average LPA increase of 4 mm or more. The attachment level in 59.3% of all the tooth sites examined in 1959 in the 165 individuals either did not change or changed within ± 1.0 mm. On the basis of bivariate analyses, the individuals with high LPA increase (≥2 mm) had the following characteristics significantly different from those with low LPA increase: They were older, smoked, had tooth mobility at baseline, higher gingivitis, plaque, calculus, and tooth mobility scores at follow-up, lower education level, and irregular dental attendance. However, in logistic regression analyses, only the following risk markers remained significantly associated with high LPA increase: age, smoking, and presence of tooth mobility.

A technique utilizing sodium deoxycholate to remove junctional epithelial cells from the epithelial attachment zone is described. Light and electron micragrayhs show a simalarity in size and appearance between the extracetlutar attachment substance in controls and the extracellular substance which remains on cementurn previously in contact with junctional epithelium.

When repetitive mechanical injury was produced in combination with marginal periodontitis a significant loss of connective tissue attachment did not occur as compared with specimens in which periodontitis alone was produced. It seems unlikely, therefore, that there is a "co-destructive" factor effect on the loss of connective tissue attachment. The amount of alveolar bone lost as a result of marginal periodontitis was increased by the addition of repeated mesiodistal jiggling of the teeth. This could represent an irreversible "co-destructive" effect or could merely be a functional adaptation of the periodontium.

An external covering over the completed enamel surface is described in the literature as a cuticular formation (1), an amorphous pellicle (2), a hornlike substance (3), or a combination of an organic membrane which may be calcified and keratinous cuticle (4). This covering is presented as consisting of a single layer (3), and as consisting of two layers (1, 5, 6). Its origin is attributed to the ameloblasts (3), the entire enamel organ (1, 5), and a combination of ameloblasts and epithelial attachment (6). It has been termed "Nasmyth's membrane", "enamel cuticle" (3), or "primary" and "secondary enamel cuticle". An additional cuticle which is frequently found covering the cementum has been described as a continuation of the secondary enamel cuticle and a derivation of the epithelial attachment. It has been termed the "dental cuticle" (6). A study of approximately 1400 decalcified sections of 64 erupted teeth in situ in human adult jaws offered an opportunity to establish more definitely whether or not cuticular structures actually exist in relation to the teeth and periodontal tissues. Three unerupted teeth completely embedded in the maxilla were included in the series studied. In general, the histological technique used in the preparation of the material was that presented in a previous publication (7). The term cuticle when used here refers to a thin, strandlike, non-cellular structure with a clear eosin-staining matrix of varying widths enclosed within smooth linear margins.

The extent and duration of experimental vascular occlusion in the periodontium of squirrel monkeys was determined by India ink perfusion. Three, hours after the induction of carbonized microspheres, only vessels of the periodontal ligament contained perfusion material. The palatal gingiva was vascularized before the maxillary buccal gingiva, Gingival vessels adjacent to the epithelial attachment and crevicular epithelium vascularized first.

The gross and microscopic anatomy of the molars was studied in 2 groups of rats:A. Completely hypophysectomized. 22 animals. Age at operation: 36 to 64 days. Post-operative life: 63 to 459 days. B. Litter mate controls. Some were unoperated but most were unsuccessfully subjected to operation. Normal oestrous cycles and normal rate of growth. 15 animals.Significant alterations were observed only in group A. The gross findings in these animals were (a) retarded eruption; (b) shorter length of roots. The histologic findings are summarized below.1. The epithelial attachment is absent in a number of cases for varying lengths near the cemento-enamel junction so that the enamel is bounded directly by connective tissue or by a cementum spur of varying length and width. In 7 animals the enamel shows here some areas of resorption.2. The enamel is normal in structure except for areas of resorption near which the organic matrix is sometimes seen in decalcified sections.3. The dentin appears, as a whole, normal in str...