Modulation of gingival epithelial phenotypes by interactions with regionally defined populations of fibroblasts.

BACKGROUND AND OBJECTIVE: The unusual structure and functions of junctional epithelium, together with its pattern of migration in periodontal disease, raise interesting questions about the factors associated with the maintenance of its unique phenotype. To explore the effects of regionally differing fibroblast populations on the growth and patterns of differentiation of oral epithelia, this study used an organotypical in vitro model in an attempt to detect interactions occurring between populations of human oral fibroblasts and keratinocytes. MATERIAL AND METHODS: Keratinocytes and fibroblasts, isolated from the gingival region and periodontal ligament, were characterized by their patterns of growth and by their expression of known differentiation markers. Changes in cell behaviour and phenotypic marker expression were examined during in vitro passage as an indication of the maintenance of in vivo phenotypic traits. Using early passage cells, organotypical cultures were generated and patterns of epithelial growth and expression of phenotypic markers were examined. RESULTS: Phenotypically different populations of junctional and oral-gingival keratinocytes, and of oral-gingival and periodontal ligament fibroblasts, were successfully isolated, cultured and characterized. In the organotypic culture system, oral-gingival fibroblasts were found to have a markedly greater ability than periodontal ligament fibroblasts to support and maintain the growth of either type of epithelium. Shifts of epithelial phenotype were induced by different fibroblasts. CONCLUSION: Periodontal and gingival fibroblast subpopulations have differential effects on the growth and patterns of differentiation of oral and junctional epithelia. By modulating the epithelial phenotype, regionally differing fibroblasts can influence the stability and behaviour of the gingival attachment apparatus in health and disease.

The in vitro behaviour and patterns of colony formation of murine epithelial stem cells.

OBJECTIVE: The mechanisms of renewal of skin and mucosal epithelia in vivo are associated with hierarchies of stem and amplifying cells organized in distinct spatial patterns. Stem and amplifying characteristics persist after isolation and growth of human keratinocytes in vitro but the pattern for murine keratinocytes has been less clear. MATERIALS AND METHODS: Murine keratinocytes were grown in low calcium media and examined for their patterns of colony morphologies. RESULTS: We consistently identified three types of colonies, one of which contains concentric zones of amplifying and differentiated cells surrounding a central zone of cells that have patterns of expression and behavioural characteristic of stem cells. This zonal organization facilitated analysis of stem cell formation and loss. Cells in the central stem cell zone undergo rapid symmetric divisions but expansion of this population is partially limited by their peripheral transition into amplifying cells. A striking feature of central zone cells is their enhanced apoptotic susceptibility and stem cell expansion limited by consistently high background rates of apoptosis. This occasionally reaches catastrophic levels with elimination of the entire central zone. CONCLUSION: In vitro amplification of stem cells for the generation of engineered tissue has tended to focus on control of asymmetric division but these findings suggest that development of mechanisms protecting stem cells from apoptotic changes are also likely to be of particular value.

Expression of Ricinus communis receptors on epithelial cells in oral carcinomas and oral wounds.

The histological distribution of receptors for Ricinus communis Fraction 1 (RCA1) in oral carcinomas and in oral epithelial cells during wound healing has been studied by use of fluorescein-tagged RCA1. Biopsies from 15 human oral carcinomas and adjacent normal mucosa showed RCA1 receptors at the cell membranes in the basal and spinous layer of the normal epithelium, whereas receptors could not be demonstrated in invading islands of the tumors. In healing oral wounds from eight humans and three monkeys, RCA1 receptors were demonstrated both in normal epithelium adjacent to the wounds and in the epithelial outgrowth from the wound margin. Titrations, however, showed that the epithelial outgrowth reacted more weakly than did the normal adjacent epithelium. These results support previous in vitro studies showing changes in carbohydrate composition of moving normal cells and of malignant cells, a finding that may be of interest in relation to formation of metastases.

Retroviral transduction of murine epidermal stem cells demonstrates clonal units of epidermal structure.

It has been suggested that the number and position of epidermal stem cells are related to the units of columnar structure in the upper epidermal strata and that the cells of each unit are derived from a single stem cell. Studies of cell lineage in developing tissues have been facilitated by the use of retroviral transduction to provide inherited expression of a histochemically demonstrable foreign gene product. To provide direct evidence about the clonal nature of epidermal units, murine epidermal keratinocytes were transduced with a replication-deficient retroviral vector carrying the beta-galactosidase gene. Subepidermal injection of virus in vivo led to infrequent transduction with only transient presence of beta-gal-staining keratinocytes within the epidermis. Transduction of keratinocytes in vitro and transplantation back to in vivo sites permitted demonstration of the transduced gene in clusters of cells within the reformed epidermis throughout a 12-wk period. The epidermis redeveloped an ordered columnar structure with restriction of transduced cells to individual columnar units. This clonal appearance is compatible with derivation of each epidermal unit from a single stem cell but is not compatible with a random pattern of cell proliferation. Transduced epidermal sheets that were recombined with oral mucosal connective tissue also redeveloped normal columnar structure with restriction of beta-gal staining to individual columnar units. These data suggest that the establishment of an epidermal stem cell pattern related to units of structure is an intrinsic property of the epithelium and is not dependent on regionally-specific connective tissue influences.

Ordered structure of the epidermis.

Recent reports on the ordered structure of the epidermis are reviewed. The regular relationship between Langerhans cells and cell columns in the mouse is not typical of the epidermis of other animals. The pattern of mitotic activity previously described in mouse epidermis has also been demonstrated in the hamster, which suggests control of keratinocyte activity rather than the exclusion of keratinocytes from the central region beneath cell columns. These findings are discussed in relation to mechanisms which may be responsible for the formation of cell columns.

Regeneration of organized epithelial structure.

The role of connective tissue in facilitating and directing the growth of epithelia during adult life is uncertain. The basic processes associated with maintenance of epithelial structure and previous work concerning the role of mesenchyme in this process in the embryo and adult are reviewed. A series of experiments examining the role of connective tissue in facilitating epithelial growth and development in vitro and after transplantation in vivo is described. These confirm the requirement for dermal elements if normal structure is to be reestablished and point to the requirement of dermal, as opposed to deep, connective tissues for facilitation of the growth of adult epithelia in vivo. The in vitro experiments suggest the presence of diffusible dermally produced factors that facilitate epithelial growth.

Identification and localization of label-retaining cells in hamster epithelia.

A subpopulation of basal epithelial cells which retains tritiated thymidine label for extended periods was previously demonstrated in skin and oral mucosae of mice. The present study examined the presence of similar cells in hamsters. Five-day-old hamsters were labeled with tritiated thymidine and the rate at which label was diluted from the basal cells observed. A small percentage of basal cells was found to retain label for up to 69 days. The location of such label-retaining cells ( LRCs ) in the palatal epithelium and in tongue papillae was examined. Thirty and 69 days after labeling, approximately 80% of LRCs in palate were located in the proximal halves of papillae and 80% of LRCs in tongue were positioned basally with approximately 30% of such LRCs occupying positions previously suggested to be stem cell locations. The finding that slowly cycling keratinocytes are related to patterns of tissue architecture is compatible with a function of these cells as epithelial stem cells.

The diurnal variation of epidermal metabolism.

In previous studies, the rates of epidermal glycolysis and amino acid incorporation were found to parallel rates of mitosis in both the hypoplasia associated with starvation and the hyperplasia produced by treatment with hexadecane. To examine whether similar changes in epidermal metabolism accompany the known diurnal variation in epidermal mitotic activity, the rates of glycolysis and incorporation of amino acids in epidermal sheets from skin samples at various times during a 24-hr period were assayed. These activities were found to parallel the diurnal variation of mitosis demonstrated by metaphase counts. Diurnal variation of metabolism in the suprabasal layers was investigated by examining the relative rates of incorporation of histidine and leucine: it was found that histidine showed a broader peak of activity than that seen for the other measured activities. When epidermal samples were prepared form tissue treated with hexadecane to induce hyperplasia, an increase in metabolic activity and decrease in the amplitude of diurnal variation was observed. The results indicate that the general metabolic activities of the epidermis follow a diurnal pattern similar to that found for mitosis.

The effects of alpha and beta adrenergic agonists and cyclic adenosine 3':5'-monophosphate on epidermal metabolism.

Continuously regenerating stratified squamous epithelia form an interesting model for examining mechanisms controlling the balance between rates of cell formation and cell maturation and death. In vitro assays of rates of glycolysis and amino acid incorporation of epidermal sheets free from dermal contamination were used to examine rates of metabolism in both normal and hyperplastic epidermis after treatment with various adrenergic agonists and cAMP. Epinephrine and isoproterenol over the concentration range of 1 x 10(-9) to 1 x 10(-5) M depressed the rates of glycolysis and amino acid incorporation in normal epidermis. Dibutyryl cyclic AMP produced a 73 to 78% depression in metabolic activity and its action was enhanced by the addition of theophylline. The alpha adrenergic agonist norepinephrine produced similar reductions. When epidermal samples were treated with hexadecane to induce a mild hyperplasia, depressant effects of isoproterenol and epinephrine were lost, but dibutyryl cyclic AMP and norepinephrine still reduced metabolic activity. The results suggest that adrenergic agents and their putative second messenger cAMP cause reductions in epidermal metabolic activity, an effect similar to their effects on cell proliferation, and that increased rates of proliferation are associated with loss of beta adrenergic responsiveness of the epidermis.

The development of ordered structure in neonate rat epidermis.

The establishment of a columnar pattern of organization in rat backskin and earskin was examined by using frozen sections expanded in alkaline buffer and by labeling with 3H-TdR and autoradiography. An adult columnar pattern of organization was established earlier in backskin than earskin. In both tissues the appearance of cell columns was related to a decreasing rate of cell proliferation and, for ear, to a decreasing rate of lateral growth of the epidermis.

The pattern of cellular organization of human epidermis.

Cell alignment in the stratum corneum of frozen sections of specimens of human skin was examined by light microscopy following expansion of the stratum corneum in alkaline buffer. Some degree of ordered structure was found in all specimens examined but considerable variation existed in precision of cell alignment. The typical degree of cell alignment was less precise than that typically observed in experimental animals.

Concanavalin A and ricinus communis receptor sites in normal human oral mucosa.

Fluorescein conjugates of concanavalin A (Con-A) and Ricinus communis fraction 120 (RCA120) were shown to bind to the cell surfaces of basal and spinous cell layers in oral buccal mucosa. Palatal epithelium showed distinct binding to basal and spinous cells; cell membranes in the granular layer occasionally bound Con-A and always RCA120. The ultrastructural localization of Con-A binding sites on exfoliated buccal cells was detected by the Con-A peroxidase staining method. The Con-A receptors were seen on the cell surface in association with the outer leaflet of the plasma membrane. The reaction products appeared as a homogeneous, electron-dense layer containing irregularly distributed globules.

Reactivity of epidermal Langerhans cells to a histochemical method for demonstration of beta-glucuronidase.

Sheets or sections of mouse epidermis reacted by a histochemical method for the enzyme beta-glucuronidase display a subpopulation of dendritic cells which correspond in number and spacing to Langerhans cells demonstrated by reactivity for ATPase or Ia antigens. A similar staining pattern is seen in rat, rabbit, and guinea pig epidermis. In rhesus monkey and human skin, Langerhans cells appear to be reactive for beta-glucuronidase but, as keratinocytes are also reactive, Langerhans cells are not readily identifiable by this method. The thermal stability of beta-glucuronidase differs between strains of mice. Langerhans cells of Balb/C and C3H strains can thus be distinguished by appropriate pretreatment before incubation, a method of potential value for experimental investigations of the origin of Langerhans cells.

An examination of the relationship between experimentally altered rates of epidermal proliferations and rates of epidermal metabolism assayed in vitro.

Continuously regenerating stratified squamous epithelia form an interesting model for examining mechanisms controlling the balance between rates of cell formation and cell maturation and death. Previous investigations of epidermal metabolism have been mainly based on single enzyme assays which may not form a reliable guide to changing rates of flux through metabolic pathways. Methods for in vitro assays of rates of glycolysis, protein synthesis and RNA synthesis of epidermal sheets free from dermal contamination were developed and used to examine rates of epidermal metabolism after experimental alteration of rates of epidermal proliferation. Starvation resulted in a 45-53% reduction in the in vivo epidermal labeling index and a 49-56% reduction in glycolysis and incorporation of amino acids assayed in vitro. Induction of epidermal hyperplasia with hexadecane resulted in a 4-fold increase in labeling index, a 6-fold increase in vitro glycolysis and a 3 to 4-fold increase in in vitro assays of incorporation of amino acids and uridine. Hyperplastic epidermis also showed an increased rate of incorporation of histidine (a marker for keratokyalin synthesis) relative to leucine (a marker for basal cell protein synthesis) indicating a change in maturation. The results suggest mechanisms linking rates of cell proliferation and death and indicate the possible value of such assays investigating these mechanisms.

Lipid composition of cohesive and desquamated corneocytes from mouse ear skin.

An organ culture system has been used to examine differences in the lipid compositions of materials derived from cohesive and desquamated mouse ear stratum corneum. Within this culture system, skin explants display rates of cell replication and differentiation comparable to those observed in vivo for up to 2 weeks and, during this period, loosened or dishesive material accumulates at the surface. Lipid compositions were determined for both intact and loosened stratum corneum derived from cultured skin and also for freshly prepared stratum corneum. In all 3 cases, the profiles of the nonpolar lipids and the ceramides were essentially the same; some of the nonpolar lipids appeared to be of sebaceous origin. The only changes detected upon desquamation were reductions of cholesteryl sulfate and a second unidentified lipid of similar polarity. Cholesteryl sulfate constitutes 4-5% of the polar lipid in fresh stratum corneum or stratum corneum from organ culture. This is reduced to 0.4% in the desquamated material which accumulates in the culture system. The unidentified lipid decreases from 1-2% of the polar lipid in intact fresh or cultured stratum corneum to 0.1% in the desquamated material. The possible function of cholesteryl sulfate in corneocyte cohesion is discussed.

Expression of keratinocyte growth factor in periapical lesions.

The epithelial proliferation associated with inflammatory periapical lesions and with periapical cyst formation represents an interesting but poorly understood pathological change. Keratinocyte growth factor (KGF) is a recently identified growth factor that is produced by stromal fibroblasts and acts specifically to stimulate epithelial growth and differentiation. To investigate its possible role in the activation of the normally quiescent rests of Malassez, we examined the expression of KGF by in situ hybridization of sections of normal periodontal ligament (PDL) and of 12 periapical granulomas or cysts. Normal PDL and periapical granulomas with scant inflammatory infiltration showed few cells expressing message for KGF. However, KGF-expressing cells were found in the connective tissue stroma close to dense foci of inflammatory cells and to proliferating epithelial elements and cystic epithelial linings. Examination of tissues by the reverse-transcription polymerase chain reaction (RT-PCR) showed KGF expression in 4 specimens of periapical lesions but low or undetectable levels in normal PDL. These observations suggest that the induction of KGF expression in the stromal cells of periapical lesions may play an important role in stimulating the epithelial proliferation associated with cyst formation.

Effect of corticosteroids on rabbits corneal keratocytes after photorefractive keratectomy.

BACKGROUND: To determine the corticosteroid effect on the activity and repopulation of keratocytes after photorefractive keratectomy (PRK). METHODS: A 193-nm excimer laser (VISX Twenty/Twenty) created a central ablation depth of 22 microns (diameter:5 nm) on 22 corneas of 16 albino rabbits. Two ablated eyes were examined 6 hours following PRK. Twelve eyes received no postoperative corticosteroids and eight were treated with topical fluoromethalone for 3 months. Corneas were examined 1, 3, 6, and 12 months after PRK by immunofluorescence and transmission electron microscopy. RESULTS: Corticosteroids reduced haze (p=0.02), but all corneas (treated or untreated) cleared 6 months after PRK. Keratocytes were absent from the anterior 100 microns of the stroma 6 hours after PRK. However, the number and activity of keratocytes were significantly greater in this area in untreated corneas at 1 month and then gradually decreased. By 6 and 12 months, the number of keratocytes approached controls. Treated corneas had fewer keratocytes than either controls or untreated eyes (p<0.01) and by 3 months, a subepithelial acellular zone of 30 to 50 microns thickness appeared and persisted until at last 12 months after PRK. CONCLUSIONS: Corticosteroids have a transient effect in reducing haze and seem to inhibit keratocyte movement, leading to an acellular subepithelial region beneath the ablated area.

Immunohistochemical studies of basal cell carcinomas transplanted into nude mice.

Xenografting into nude mice forms a system for analysis of human tissues under experimental conditions. In this study, normal skin samples and basal cell carcinomas were investigated, prior to and after transplantation, using immunofluorescence methods with antibodies against keratins, laminin, and collagen type IV. Three groups of transplants were studied: intact tissue samples, human epithelium (either normal or neoplastic) recombined with normal human dermis and, human epithelium recombined with normal mouse dermis. Transplants recovered after 3 weeks showed the following characteristics. The xenograft system was satisfactory in terms of host survival and rate of successful tissue recovery except for recombinants between human epithelium and mouse dermis. Intact and recombined samples of normal skin retained their preexisting patterns of architecture, cytodifferentiation, and basement membrane staining. Solid nonfibrosing basal cell carcinomas showed altered architecture and differentiation of both the epithelium and the basement membrane zone after transplantation: the solid tumor pattern changed towards spreading of tumor cells, a more squamous differentiation pattern was apparent and was confirmed by reactivity with antibodies against large keratins. Discontinuities of the basement membrane zone were detected with antibodies against laminin and collagen type IV. These changes were seen in both intact and recombined tumor transplants.

Formation of normal gingival epithelial phenotypes around osseo-integrated oral implants in humans.

The oral, oral sulcular, and junctional epithelia of the natural gingiva each possess distinct patterns of differentiation that are demonstrable both ultrastructurally and by their individual patterns of macromolecular synthesis. The supracrestal tissues reformed around oral implants structurally resemble those of natural gingiva but little is known about phenotype changes occurring in the epithelia. To investigate whether peri-implant epithelia acquire similar patterns of differentiation to those of natural gingiva, biopsies from the supracrestal regions of five oral implants were examined by immunofluorescent methods using a panel of monoclonal antibodies with specificities for individual cytokeratins and ICAM-1, macromolecules which act as markers of the three gingival epithelial phenotypes. The observed staining patterns indicated the formation of oral, oral sulcular, and junctional epithelia which were phenotypically indistinguishable from those of natural gingival epithelia. This degree of reprogramming of epithelial gene expression is a surprising observation and the potential mechanisms leading to the development of those new epithelial phenotypes are discussed in the context of what is known about the development of natural gingiva, in terms of the possible effects of inflammation, and in relation to the known connective tissue influences on epithelial differentiation.

Examination of topographical gingival anatomy by a filter imprint technique.

The Millipore filter imprint technique was applied to oral cytology using modification of the Papanicolaou and Shorr stains. This technique has the major advantage of demonstrating on each imprint the topographic relationship between cellular zones and also allows for repeated sampling of superficial changes in the same area without tissue. This method provides a wide variety of cytological information about gingival anatomy and demonstrates a number of regions of differing cellular composition.