Immunolocalization of β1 integrins in human gingival epithelium and cultured keratinocytes

Beta 1 integrins are cell surface receptors for extracellular matrix binding. We have recently shown that these receptors may also play a role in cell-cell binding of human epidermal keratinocytes. In this study we used immunofluorescence and confocal laser scanning microscopy to localize beta 1 integrins in frozen sections of human gingiva and cultures of human gingival keratinocytes. The results show that beta 1 integrin polypeptides, localized by monoclonal and polyclonal antibodies, were detected mainly in the basal layer of the keratinized epithelium. There was also scattered staining in connective tissue fibroblasts, nerves, and blood vessel walls. In the basal layer, the integrins were found around the entire periphery of the basal keratinocytes. Furthermore, confocal laser scanning microscopy (CLSM) revealed that most of the staining was in fact localized in dot-like structures at the lateral cell membranes of neighboring basal cells. In cultured human gingival keratinocytes maintained in low calcium (0.15 mM) conditions β1 integrins were localized in several different structures: trails which were left behind when the cells moved in culture, dots underneath the cells, around the nucleus, and in cell-cell contacts. The trails were also found to contain fibronectin and type IV collagen but not laminin. Switching the keratinocytes to high calcium (1.2 mM) conditions induced the formation of cell-cell contacts which were strongly positive for β1 integrins. No fibronectin or type IV collagen was found in cell-cell contact sites. The results indicate that β1 integrins are localized to cell-cell junctions of human gingival keratinocytes both in vivo and in vitro. Cultured cells also express these receptors in cell-matrix contacts indicating a dual role for β1 integrins in cell-matrix and cell-cell contacts of human gingival keratinocytes.     

Function of laminins and laminin-binding integrins in gingival epithelial cell adhesion

BACKGROUND: In human gingiva, epithelial cells attach to their adjacent tissues by means of specialized molecular adhesion complexes and a basement membrane. Little is known about the synthesis of adhesion proteins by gingival keratinocytes; we, therefore, studied how cultured immortalized gingival epithelial cells produce laminins and express laminin-binding integrins. We presumed that different laminins and integrins would be involved in the adhesion of gingival epithelial cells. METHODS: We cultured gingival keratinocytes and studied their production of laminins and expression of integrins using immunofluorescence microscopy, immunoprecipitation, and immunoblotting methods and by quantitative cell adhesion experiments. We also studied how gingival tissue expresses these adhesion proteins in vivo by using immunofluorescence microscopy. RESULTS: In immunofluorescence microscopy, the cells were seen to organize chains of laminin-5 (alpha3beta03gamma2) to extracellular patches, whereas the alpha5 chain of laminin-10 (alpha5betalgamma1) could only be seen intracellularly. Of the laminin-binding integrin subunits, integrin a6 subunit was organized to dotted arrays, typical of prehemidesmosomal adhesions, whereas integrin alpha3 subunit was located at cell-cell junctions, in prehemidesmosomal structures, and at some locations also in small focal-contact like patches. Integrin beta1 subunit was found at cell-cell junctions and in focal contacts. Immunoprecipitation experiments showed that the cells synthesize and secrete chains of laminin-5 and laminin-10. In quantitative cell adhesion experiments, the cells adhered efficiently to these laminins by using cooperatively integrin alpha3beta1 and alpha6beta1 integrin complexes. None of the other known laminin-binding integrin subunits appeared to be significantly involved in cell adhesion to these laminin isoforms. CONCLUSIONS: Our results provide new information on gingival epithelial cell adhesion and extracellular matrix production and may thus aid in the understanding of periodontal physiology.     

Expression of fibronectin-binding integrins in gingival epithelium in drug-induced gingival overgrowth

BACKGROUND AND OBJECTIVE: Gingival overgrowth is a side-effect of nifedipine and cyclosporin medications. Integrins are transmembrane glycoproteins that mediate cell adhesion, regulate cell proliferation and participate in the regulation of tissue fibrosis. The aim of this study was to investigate whether expression of epithelial cell integrins is linked to the development of drug-induced gingival overgrowth. MATERIAL AND METHODS: Human gingival biopsies of patients taking nifedipine, cyclosporin, or a combination of both medications, were used. Expression of the alpha5beta1, alphavbeta1 and alphavbeta6 integrins, and of cellular extra domain A of fibronectin, was localized in frozen sections using immunohistochemistry. RESULTS: The activated conformation of the beta1, alpha5beta1 and alphavbeta6 integrins were more frequently expressed in distinct locations in the oral epithelium in the combined drug group. Cellular extra domain A of fibronectin, a ligand for both alpha5beta1 and alphavbeta6 integrins, was expressed within the connective tissue of all groups. It was also expressed around the basal keratinocytes of the control, nifedipine and cyclosporin-induced gingival overgrowth groups, but not in the combined medication group. No relationship between the presence of inflammation and integrin expression was found. CONCLUSION: The results indicate that expression of certain integrins is up-regulated in the epithelium of drug-induced gingival overgrowth where they could participate in controlling the formation of elongated rete ridges and tissue fibrosis.     

Expression of fibronectin and integrins in cultured periodontal ligament epithelial cells.

The process of attachment of epithelial cells obtained from the porcine periodontal ligament (cell rests of Malassez) to different extracellular matrix proteins and their expression of fibronectin and integrin receptors were studied by means of immunocytochemistry, in situ hybridization, and time-lapse cinemicrography techniques. The cell lines of periodontal ligament epithelial cells (PLE cells) attached to and spread rapidly on fibronectin, vitronectin, and type I collagen. One of the cell lines also attached to laminin, while the other cell line showed poor attachment to both laminin and Matrigel, a basement membrane material. By use of the in situ hybridization technique, some PLE cells were found to express the fibronectin gene strongly. Immunocytochemical staining localized fibronectin in extracellular fibrils and intracellular granules. Fibronectin was also found in the tracks left behind by the cells migrating on the substratum. Arg-gly-asp-ser peptide inhibited the attachment of the PLE cells to fibronectin, laminin, type I collagen, and vitronectin by 47%, 43%, 83%, and 94%, respectively, suggesting that the cell-matrix interactions were partly mediated by receptors related to the integrin family. Antibodies against the beta 1-integrin subunit stained the cell bodies and the plasma membrane projections of spreading cells. After 24 h or longer in culture, beta 1-integrins were localized to the regions of cell-cell contact. Cinemicrography of the arg-gly-asp-ser-peptide-treated cells demonstrated that the spreading and migration of isolated cells were prevented by the peptide. The peptide did not appear to dissociate the cell-cell contacts or interfere with migration of spread-cell colonies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adhesion of human fibroblasts to root-end-filling materials

This study evaluated the attachment of cultured explants of human periodontal ligament fibroblasts and gingival fibroblasts to different root-end-filling materials. Although periodontal ligament and gingival fibroblasts initially attached avidly to Geristore, these same cells displayed no significant attachment to ProRoot, Tytin amalgam, or SuperEBA. With further incubation on Geristore, the attachment of both periodontal ligament and gingival fibroblasts improved and these cells proliferated. In contrast, no improvement in attachment or proliferation was observed for cells incubated for greater times with ProRoot, Tytin amalgam, or SuperEBA. Because the attachment characteristics of these two groups of fibroblasts were identical, we examined the potential role of the extracellular matrix family of receptors (integrins) on the attachment of gingival fibroblasts. Gingival fibroblast attachment to collagen type I was determined to be dependent on alpha1beta1 and alpha2beta1 integrins, whereas their attachment to the RGD-binding sequence of fibronectin and vitronectin was partially inhibited by antibodies to the beta1 and alphaV integrin subunits. However, attachment of gingival fibroblasts to Geristore was not reduced by the addition of any of the attachment-perturbing anti-integrin antibodies examined. Thus, gingival fibroblasts attach to Geristore, but this attachment was mediated by mechanisms other than integrins.     

Characterization of human gingival keratinocytes cultured in a serum-free medium

Primary cultures of keratinocytes were established from gingival tissue explanted on the surface of type I collagen gels and fed a serum-containing medium. Cells could be routinely subcultured for at least five passages in a basal nutrient medium (MCDB 153) containing low calcium (0.1 mM), and supplemented with ethanolamine, phosphoethanolamine, hydrocortisone, insulin, epidermal growth factor and protein of bovine pituitary extract. Cells seeded at low densities doubled exponentially in number every 24-30 h and formed a confluent monolayer within 10-14 days. Phase-contrast light and transmission electron microscopy showed that the keratinocyte cultures had features typical of epithelial cells, including desmosomes and perinuclear tonofilament bundles. Immunofluorescent microscopy showed the presence of specific keratin proteins in basal cells of proliferating cultures. Gel electrophoresis of the insoluble cytosolic proteins of gingival and skin keratinocytes showed several differences. Suspension of dividing gingival keratinocytes in 1.3% methylcellulose medium induced greater than 50% cross-linked envelopes, suggesting the existence of a terminal differentiation pathway in gingival basal cells. Clonal growth experiments showed that both insulin and epidermal growth factor were required for optimal clonal growth. The growth of subcultures was arrested and the unstratified epithelial monolayer induced to form a stratified sheet by replacing the growth medium with basal MCDB 153 medium depleted of growth factors and containing 2 mM calcium. Sheets of stratified gingival epithelium formed on and later released from the dish by enzymatic treatment may be suitable for a variety of experimental and clinical uses.     

Lutheran blood group antigen as a receptor for alpha5 laminins in gingival epithelia

BACKGROUND: Lutheran blood group glycoprotein (Lu) is a transmembrane receptor of the immunoglobulin superfamily. Lu serves as a receptor for alpha5 laminins (Lm). The Lm alpha5 chain is a constituent of Lms-511 and -521. Lm-511 is found in most human basement membranes (BMs) and also is detected in BM of gingival epithelia. Recent studies indicated that Lu mediates cell adhesion to Lms-511/521 independently or in concert with integrins. This study focused on the expression of Lu in gingival epithelia and on cultured immortalized gingival keratinocytes. The role of Lu and alpha(3) and beta(1) integrin subunits in the adhesion of oral epithelial cells to Lms-511/521 was also studied. METHODS: Immunofluorescence microscopy was used to study the expression of Lu in gingival tissues and in cultured gingival keratinocytes. Immunoprecipitation of radioactively metabolically labeled cells was used to detect Lu. Cell adhesion to Lm-511/521 preparation and to pure Lm-511 was studied in quantitative cell adhesion experiments. Morphological adhesion assays were carried out for visualization of the morphology and adhesion structure formation of the adhering cells. RESULTS: Immunofluorescence studies on gingiva showed complete coalignment of Lu on basal epithelial cells with the BM Lm alpha5 chain. A surface-confined, punctate immunoreaction for Lu was detected throughout cell surfaces on cultured gingival cells. Immunoprecipitation showed a broad polypeptide with molecular weight 85,000. In quantitative cell adhesion assays, the adhesion of cells to both Lm alpha5 preparations was diminished with monoclonal antibodies (MAbs) against integrin alpha(3) and even more effectively with MAbs against the beta(1) subunit. When the adhesion sites were blocked with soluble recombinant Lu (sol-Lu), the adhesion of gingival epithelial cells to Lms-511/521 was reduced significantly, and sol-Lu increased the inhibition with integrin alpha(3) antibody. Lm-511 did not induce lamellipodia or focal contacts in cultured gingival keratinocytes. CONCLUSIONS: Lu was in coalignment with Lm alpha5 chain in gingival epithelia. Lu also seemed to have a role in gingival epithelial cell adhesion together with integrin alpha(3)beta(1).     

Expression of integrins in human gingiva

The distribution of the alpha 1-alpha 6 subunits of beta 1 integrins was studied by using a panel of monoclonal antibodies in indirect immunofluorescence microscopy. The results showed that the beta 1 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 and, more faintly, in inflammatory cells in gingival connective tissue. The alpha 4 subunit was expressed selectively in inflammatory cells, and the alpha 5 subunit was expressed in cells throughout gingival connective tissue. An overall cell membrane immunoreactivity for the alpha 2 and alpha 3 subunits was shown in basal cells of gingival epithelium and in cells of JE, corresponding to the epithelial localization of the beta 1 subunit. The alpha 6 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 beta 4 integrin subunit was mainly expressed at the basal aspects of basal cells in gingival epithelium and JE. The results indicate that the alpha 2/beta 1, alpha 3/beta 1, alpha 6/beta 1, and alpha 6/beta 4 integrins are all expressed in human gingival epithelium. Of these, the alpha 6/beta 4 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.     

Periodontal-derived cells attach to cementum attachment protein via α5β1 integrin

A specific collagenous cementum attachment protein (CAP) has been identified in human cementum which promotes selective cell migration towards and attachment of various periodontal derived cell populations to root surfaces in vitro. The CAP is known to support attachment of periodontal-derived cell via an RGD motif, which suggests an integrin-mediated mode of attachment. The purpose of the present study was to ascertain which integrin(s) are involved in the attachment of periodontal-derived cells to CAP. The integrins examined comprised subunits of the major receptors for fibronectin (alpha 5) and collagen (alpha 2, alpha 3), as well as the common beta 1 subunit which is present in many extracellular matrix receptors. The wells of 48-well non-tissue culture treated plates were coated with CAP (2 micrograms/ml). For negative and positive controls the wells were coated with bovine serum albumin and fibronectin (5 micrograms/ml), respectively. Human gingival fibroblasts and periodontal ligament fibroblasts were labeled with [3H]-proline, incubated with anti-integrin antibodies and added to the precoated wells. Attachment was assessed after incubating the cells for 1 h at 37 degrees C in the presence of the antibodies. Antibodies to alpha 5 and beta 1 inhibited the attachment of both human gingival fibroblasts and human periodontal ligament fibroblasts to CAP, while anti alpha 2 and alpha 3 antibodies did not affect the attachment. The binding of the fibroblasts to fibronectin was also inhibited by anti-alpha 5 and beta 1 antibodies, both of which are components of the "classical" fibronectin receptor and remained unaffected by the addition of anti-alpha 2 and alpha 3 antibodies. Proteins migrating in SDS-polyacrylamide gels in positions similar to the alpha 5 and beta 1 integrin subunits were present in fractions bound to a column of CAP coupled to Sepharose CL-4B. These results indicate that the attachment to CAP of the periodontal-derived cells, human gingival fibroblasts and human periodontal ligament fibroblasts, is mediated primarily via the integrin alpha 5 beta 1.     

Epithelial cell-fibroblast interactions: modulation of extracellular matrix proteins in cultured oral cells

A model system involving co-cultures of human gingival or periodontal ligament fibroblasts with mouse epithelial root sheath cells or human gingival eipthelial cells was used to study epithelial cell-fibroblast interactions. Double-labeled immunofluorescence and microfluorometry were used to investigate the expression of extracellular martix molecules of collagen type I (collagen I), type III (collagen III) and fibronectin in fibroblasts. When fibroblasts from either source were cultured alone, the fluorescence for collagen I and fibronectin ranged from strongly positive to almost negative. Collagen III staining was relatively weak compared with that of collagen I. After 2-3 days of co-culture, gingival fibroblasts and ligament fibroblasts adjacent to the mouse sheath cells exhibitied enhanced intracellular fluorescence for collagen I and fibronectin. Very little change was observed for collagen III. Gingival fibroblasts cultured with gingival epithelial cells showed increased fluorescence for collagen I but decreased fluorescence for fibronectin. In contrast, the fluorescence intensity for both collagen I and fibronectin in ligament fibroblasts were reduced after 3 days of co-culture with gingival epithelial cells. Ultrastructural changes in fibroblasts co-cultured with mouse root sheath cells included increased Golgi cisternae and vesicles and an increased abundance of rough endoplasmic reticulum, polyribosomes, secretory vesicles and pinocytotic vesicles. Thus, the expression of extracellular martix proteins and the metabolic activity of fibroblasts can be modulated by oral epithelial cells.     

Extracellular matrix analysis of nifedipine-induced gingival overgrowth: immunohistochemical distribution

The purpose of this study was to demonstrate the localization of collagen types I, III, IV, V, VI and VII as well as the glycoprotein fibronectin in nifedipine-induced gingival overgrowth. The slices, after the use of indirect immunofluorescence (incubation with antibodies against these extracellular matrix components), showed a diffuse distribution with the anit-types I and III in the stroma and fluorescent staining of the basement membranes of the epithelium, blood vessels and nerves with collagen type IV antibodies. The increased number of vessels was localized near the surface of the lesion. Collagen tyep V - seen as a filamentous - and collagen type VI - as microfibrillar - components were also localized in the tissue, showing completely different patterns of distribution. Collagen type V appeared "crater"-like and type VI displayed a "honeycomb"-shaped structural model. The blood vessels were not stained but the area around their walls demonstrated an intense fluorescence with these antibodies. Collagen type VII showed a characteristic linear staining near to the epithelial basement membrance. In contrast to this, fibronectin localized with a varied intensity in the different areas of the tissues and presented a "could"-like structure. This shows differences between the matrix components in nifedipine-induced hyperplasia and confirms the heterogeneity of the matrix in health and in gingival alterations.     

Qualitative study of collagenous and noncollagenous glycoproteins of the human healthy keratinized mucosa surrounding implants

The purpose of this study was to analyse the distribution of interstitial collagenous and noncollagenous glycoproteins of keratinized mucosa surrounding successful endosseous implants. Biopsies were incubated with highly purified antibodies against types I. III. IV collagen. laminin and fibronectin and routinely observed by immunofluorescence staining. Whereas no significative difference in the distribution of collagenous components was observed in comparison with healthy human gingiva, the collagen fibers of the connective tissue attachment ran parallel to the long axis of the implant. In 50% of the biopsies the gingival connective tissue underlying the junctional epithelium was rich in inflammatory cells and poor in collagenous components. However, the increased staining of type III collagen and the intense presence of fibronectin in this area reflect the very important remodeling ability of the local keratinized mucosa.     

Immunohistochemical localisation of extracellular matrix proteins in the periodontium during cementogenesis in the rat molar

OBJECTIVE: The development of the periodontium involves the coordinated expression of numerous extracellular matrix (ECM) macromolecules and their receptors (integrins). The aim of this study was to determine the expression of selected hard and soft tissue matrix molecules and the integrin alpha5beta1 in the periodontal tissues, during cementogenesis in the rat molar. METHODS: Using immunohistochemical methods, the distribution of the extracellular matrix proteins, fibronectin, tenascin, and bone sialoprotein (BSP), as well as the integrin subunits alpha5 and beta1 were studied in rats aged 3, 5 and 8 weeks. RESULTS: Fibronectin was widely distributed in the gingival epithelium, gingival connective tissue and in the periodontal ligament. Tenascin expression was less marked compared with fibronectin, but was more distinctly associated with cells and peri-cellular areas of the epithelial-connective tissue interface, the gingiva and within the periodontal ligament. The fibronectin-receptor alpha5beta1 integrins were expressed by epithelial cells, periodontal ligament cells and gingival fibroblasts. A notable finding was the increased staining intensity of fibronectin, tenascin and alpha5beta1 integrin in all 5-week old molar sections in the periodontal ligament matrix and cells, apical to the cemento-enamel junction (CEJ) along the alveolar crest (AC) ridge height. Bone sialoprotein was distinctly associated with the hard tissues of the periodontium as acellular cementum and alveolar bone matrix expressed bone sialoprotein throughout all sections, in all age groups. CONCLUSIONS: In conclusion, this study has demonstrated the selective distribution of several hard and soft tissue matrix molecules during periodontogenesis. The results highlight the complex nature of interactions of various proteins and molecules during development. The interactions between these molecules and their specific roles in development and regeneration await further investigation.     

Immunolocalization of integrin alpha 6 beta 4 in mouse junctional epithelium suggests an anchoring function to both the internal and the external basal lamina.

The localization of the integrin alpha 6 beta 4, a transmembrane adhesion molecule associated with hemidesmosomes, was studied in mouse junctional epithelium (JE) by the use of monoclonal antibodies in indirect immunofluorescence microscopy. The results showed that the integrin a6 subunit was expressed throughout the JE and was localized to the cell membranes, including the aspects facing the internal and external basal laminae. The beta 4 subunit had a more restricted distribution. It was expressed only in cells facing the internal and the external basal laminae and had a basally polarized distribution. In other parts of gingival epithelium, both integrin subunits were mainly expressed at the basal aspects of basal epithelial cells. The basement membrane components, type IV collagen and laminin, could be detected only in the external basal lamina and in other basement membranes of gingival epithelium. The results indicate that the a6 beta 4 integrin, expressed in mouse JE, has a role in mediating the attachment of the cells to the basement membranes facing the connective tissue and the tooth.     

Immunolocalization of fibronectin and vitronectin receptors in human gingival fibroblasts spreading on titanium surfaces

The adhesion and spreading of human gingival fibroblasls on glass and differently processed titanium surfaces was studied by immunolocalization of vinculin and the alpha and beta subunits of the fibronectin (α₅β₁) and vitronectin (α_vβ3) receptors. Vinculin-containing focal contacts were present both at 4 and 24 h of spreading in cells grown on glass or electropolished or etched titanium surfaces but not in cells spreading on sandblasted titanium surfaces. Immunostaining for the α5 and β1 subunits of the fibronectin receptor showed only a diffuse membrane fluorescence after 4 h of cell spreading irrespective of the growth surface. The α_v and β₃ subunits of the vitronectin receptor were at this stage detected in focal contacts in cells spreading on glass or electropolished or etched titanium surfaces. In cells spreading on sandblasted titanium surfaces, however, the vitronectin receptor had only a diffuse distribution. In cells that had been allowed to spread for 24 h on glass or electropolished or etched titanium surfaces the α₅ and β₁ integrin subunits were either diffusely distributed or showed a localization typical of extracellular matrix contacts. The α_v and β₃ integrin subunits were, as earlier, localized to typical focal contacts in cells grown on glass or electropolished or etched titanium surfaces. Cells attached to sandblasted titanium surfaces still expressed all the integrin subunits only diffusely. The results show that the surface texture of the substratum can affect the expression of integrin subunits in human gingival fibroblasts. As evidenced by the recruitment of integrin subunits to focal and extracellular matrix contacts, smooth or finely grooved titanium surfaces appear to be optimal in supporting the attachment of human gingival fibroblasts.     

Attachment of Bacteroides gingivalis to collagenous substrata

The ability of Bacteroides gingivalis 381 to attach to hydroxyapatite (HA) beads, treated with either human type I or type IV collagen, or to particles of bovine bone collagen was studied. All preparations were blocked with human albumin prior to being incubated with 3H-thymidine-labeled B. gingivalis 381 cells. The presence of collagen on HA surfaces (C-HA) significantly promoted attachment of the organism. HA treated with Type IV collagen bound B. gingivalis cells more effectively than did HA treated with type I collagen. Attachment of two additional strains of B. gingivalis to HA was also promoted by collagen. Binding to type I or type IV C-HA occurred rapidly, and equilibrium was attained within 45 min. B. gingivalis 381 cells also bound to particles of bovine bone collagen, and this appeared to be biphasic. Heating the bacteria abolished their ability to bind to C-HA. Attachment of B. gingivalis 381 cells to HA treated with type I collagen was strongly inhibited by the presence of soluble type I or type IV collagen, or gelatin, but not by the presence of human albumin, salivary proline-rich protein 1, or saliva. Human serum, fibronectin, fibrinogen, certain protease inhibitors, and some peptides were also inhibitory. 3H-fibronectin bound to bovine bone collagen particles and blocked the attachment of 14C-B. gingivalis cells. Mild trypsin treatment of the fibronectin-collagen complex restored its ability to promote 14C-B. gingivalis attachment concomitant with the loss of 3H-fibronectin. We suggest that elevated levels of proteases in the gingival sulcus, such as are associated with poor oral hygiene and gingivitis, might remove fibronectin and expose collagen molecules in the basement membrane, thereby promoting the attachment of B. gingivalis cells and facilitating their invasion into gingival tissues.     

Development and characterization of an in vitro gingival epithelial model

A 3-dimensional gingival epithelial model has been developed and characterized. Oral epithelial cells and connective tissue fibroblasts were isolated from human gingival tissue and used to create an in vitro oral mucosa co-culture model. Fibroblasts were seeded on a scaffold of nylon mesh, allowed to proliferate and secrete collagen and extracellular matrix proteins to form a stroma capable of supporting the growth of epithelial cells. Epithelial cells were seeded on top of a confluent stromal layer, proliferated and differentiated to form a stratified squamous epithelium. Resident epithelial cells were stimulated, by manipulation of growth medium and culture conditions, to form a multi-layered oral mucosa-like tissue. Histologic analyses revealed cellular architecture exhibiting stromal-epithelial interaction which supports the growth and differentiation of an epithelial layer. lmmunohistochemical analyses confirmed production of types I and III collagen. Immunofluorescence of the stromal layer identified type IV collagen and fibronectin. Fibronectin was also detected on surface epithelium. Differentiation of basal, spinous and granular cells was observed, and the presence of differentiation markers, acidic (K10, 14–16, 19) and basic (Kl–8) cytokeratins were confirmed using broad spectrum cytokeratin antibodies, AE1 and AE3. Development of a discontinuous basal lamina zone, with hemidesmosomes, was observed by electron microscopy. The co-culture was metabolically active, as measured by the thiazoyl blue (MTT) assay for mitchondrial function and [³H] thymidine incorporation into DNA. The human gingival epithelial co-culture model was viable up to 35 days post-epithelial seed. This model may offer opportunities for limited study of periodontal tissue responsiveness.     

Molecules Contributing to the Maintenance of Periodontal Tissues

This review is aimed at providing a depiction of molecules and their topography which characterize native gingiva and PDL fibroblasts, to describe their function in tissue maintenance, and to discuss their possible modulation due to orthodontic tooth movement.Maintenance of the human periodontium requires the balance of proliferation and differentiation in the respective tissues' cells. Moreover, the cells must synthesize the extracellular matrix molecules and receptors that facilitate adhesion. To describe the molecules that contribute to periodontal tissue maintenance, we illustrate the localization of their expression and topography on frozen sections from native gingival tissue and primary cell cultures derived from periodontal ligament. In native gingival epithelium, proliferation is confined to basal and parabasal cells. Keratin K14, when used as structural marker, is visible in the entire epithelium, while K13, an indicator of early differentiation, is restricted to the suprabasal cell compartment. Vimentin indicates mesenchymal cells in the subgingival connective tissue. Concerning the matrix molecules, collagen type-IV is abundant at the epithelium-lamina propria interface, and fibronectin is apparent throughout the mesenchyme. The matrix receptor integrin beta1 reveals a pericellular localization in basal and parabasal cells, while focal adhesion kinase p125FAK is seen pericellularly in all epithelial layers. Cultures of primary periodontal ligament (PDL) fibroblasts (PDL-F) reveal expression of vimentin, strong proliferation, synthesis and extracellular deposition of collagen type-I and fibronectin. The integrin subunits beta1 and p125(FAK) are largely detectable at the cell periphery.     

Localization of fibronectin in gingival connective tissue of the beagle dog. Ultrastructural detection with ferritin and peroxidase-conjugated antibodies

Ferritin and peroxidase-conjugated antibodies were used in an indirect antibody method to localize fibronectin in gingival connective tissues. Fibronectin was found in the basal lamina beneath the epithelium and endothelium. Collagen fibrils associated with the basement membranes were also heavily coated by fibronectin. Amorphous patches of fibronectin were found adjacent to the plasma membrane of epithelial cells as well as free in the interepithelial spaces. Fibronectin was present throughout the connective tissue in close association with individual collagen fibrils, apparently serving as an interfibrillar cementing substance. Patches of fibronectin were located at the cell surface of fibroblasts, plasma cells, lymphocytes, endothelial cells, smooth muscle cells, and neutrophils. These amorphous patches were observed to connect adjacent cells across narrow spaces and to connect cells to collagen fibrils. The heavy labeling for fibronectin visualized by fluorescent microscopy around gingival blood vessels (Cho et al., 1985) can be accounted for by a heavy coating of fibronectin on the collagen fibrils and basal laminas associated with endothelial cells, as well as by the presence of abundant deposits of fibronectin along the cell membranes of endothelial cells and in the intercellular spaces of the vessel wall.