Expression of basement membrane components in odontogenic cysts

OBJECTIVE: To compare the expression of basement membrane components (BMCs), including laminins 1 and 5, collagen type IV, and fibronectin in odontogenic keratocysts (OKCs) with dentigerous cysts (DCs) and radicular cysts (RCs). MATERIALS AND METHODS: Basement membrane components were analysed in 20 OKCs, 20 DCs and 20 RCs using an immunohistochemical technique. RESULTS: Odontogenic keratocysts, DCs and RCs showed positive reaction to all BMCs studied, with different distributions and intensity. OKCs showed continuous linear deposits for laminins 1 and 5 but two staining patterns (continuous and discontinuous) for collagen type IV and fibronectin. DCs exhibited continuous linear deposits for laminins 1 and 5 and collagen type IV but a discontinuous linear deposit for fibronectin. RCs displayed similar results to DCs for laminin 1, collagen type IV and fibronectin. Laminin 5 in RCs had two staining patterns. Constant results in all cysts were strong intensity for laminin 1 and moderate intensity for laminin 5. CONCLUSIONS: Substantial differences in the expression of BMCs among studied cysts were not observed, suggesting that the separation of the epithelial lining in OKCs is not associated with the existence of these proteins.     

Immunohistochemical expression of basement membrane proteins of verrucous carcinoma of the oral mucosa

Squamous cell carcinoma (SCC) of the oral cavity is an extremely invasive tumour of stratified squamous epithelium that spreads throughout degradation of the basement membrane (BM) and extra-cellular matrix. Oral verrucous carcinoma (VC) is a rare low-grade variant of oral SCC that penetrates into the subepithelial connective tissue. It also has a different clinical behaviour from classical oral SCC. We investigated the immunohistochemical expression of laminin, laminin-5, collagen IV and fibronectin in VC, severe epithelial dysplasia (SED) and SCC in order to analyse if the patter of these molecules expression contributes to the differences in the biological behaviour of these diseases. The staining pattern of laminin was less intensive in SCC compared with SED and VC, and collagen IV expression was increased in VC compared with SED. Discontinuities of laminin, collagen IV and fibronectin were more evident in SED than in VC. This study indicates that VC has a biological behaviour different from SED or SCC, observable by immunohistochemistry in the BM zone.     

Expression of type IV collagen and laminin at the interface between epithelial cells and fibroblasts from human periodontal ligament

The present study was undertaken to examine whether synthesis of type IV collagen and laminin around the epithelial rests of Malassez (ERM) requires direct contact between cells from ERM and periodontal ligament fibroblasts. Human periodontal ligament (HPDL) explants produced outgrowths containing both ERM cells and fibroblasts when cultured in a modified serum-free medium. The interface between ERM cells and fibroblasts was examined using phase-contrast microscopy (PCM) and scanning electron microscopy (SEM). Expression of type IV collagen and laminin was studied by immunohistochemistry and in situ hybridization. It was observed that ERM cells grew underneath fibroblasts or attached to them. At the interface, type IV collagen and laminin and their respective mRNAs were abundant in both ERM cells and fibroblasts, while these proteins and mRNAs showed little if any staining in cells further away from the interface. Hence, these findings indicate that synthesis of type IV collagen and laminin is induced by direct interaction between ERM cells and periodontal ligament fibroblasts.     

Kaposi's sarcoma in AIDS: basement membrane and endothelial cell markers in late-stage lesions

Early-stage lesions of Kaposi's sarcoma (KS) are composed of single-layered, highly flattened cells lining collagen bundles, whereas late-stage lesions contain densely packed, spindle-shaped cells. We examined the progression of KS lesions in oral mucosa and lymph nodes from patients with AIDS, using antibodies specific for blood vascular endothelial cells (Factor VIII-related antigen) and their basement membrane (Type IV collagen and laminin). In addition, the plant lectin Ulex europaeus, which selectively stains blood vessels, was also used. In early-stage KS lesions, fibronectin, laminin and Type IV collagen were co-distributed at the interface between KS cells and collagen bundles; Factor VIII-related antigen and Ulex europaeus lectin staining was present in vascular channels and in the KS cells. However, in late-stage lesions, few if any KS cells stained with antibody to Factor VIII-associated antigen, although endothelial cells lining blood vessels were positive. Strong staining for laminin and Type IV collagen was present in a pericellular pattern throughout the nodular late-stage lesions. Since lymphatic capillary endothelium does not produce basement-membrane-specific macromolecules, these results support the conclusion that KS cells are related to blood vascular endothelium but eventually lose certain endothelium-specific markers as the cells are transformed into the spindle-shaped cell type.     

Morphological and immunocytochemical characterization of cultured rat incisor cervical epithelial cells.

Epithelial cells from the cervical loop of the rat incisor were isolated by co-culture of apical explants with growth-arrested 3T3 fibroblasts. The epithelial phenotype of the expanding outgrowths was confirmed 10 days after the seeding of the explants by phase-contrast microscopy and immunocytochemical identification of cytokeratins. After 3 weeks in culture, the epithelial cells covered the entire surface of the coverslips and were then passaged. Subcultures gave rise to a confluent sheet within 10-12 days. Light and electron microscopy showed that confluent cervical epithelial cells generally reconstituted a bi-layered structure similar to Hertwig's epithelial sheath. Epithelial cells from the rat palate, cultured and subcultured according to the same procedure, organized themselves in 5-6 cell layers, the upper cells having generally a squamous morphology. Synthesis of extracellular matrix molecules by rat incisor cervical epithelial cells was studied with specific antibodies. These cells failed to produce type I collagen, but synthesized all the major basement membrane components (type IV collagen, laminin, heparan sulphate proteoglycan and fibronectin). These observations suggest that the culture conditions allowed the reconstitution of a typical Hertwig's epithelial sheath by rat incisor cervical epithelial cells.     

Kaposi's sarcoma in AIDS: basement membrane and endothelial cell markers in late-stage lesions

Early-stage lesions of Kaposi's sarcoma (KS) are composed of single-layered, highly flattened cells lining collagen bundles, whereas late-stage lesions contain densely packed, spindle-shaped cells. We examined the progression of KS lesions in oral mucosa and lymph nodes from patients with AIDS, using antibodies specific for blood vascular endothelial cells (Factor VHI-related antigen) and their basement membrane (Type IV collagen and laminin). In addition, the plant lectin Ulex europaeus , which selectively stains blood vessels, was also used. In early-stage KS lesions, fibronectin, laminin and Type IV collagen were co-distributed at the interface between KS cells and collagen bundles; Factor VHI-related antigen and Ulex europaeus lectin staining was present in vascular channels and in the KS cells. However, in late-stage lesions, few if any KS cells stained with antibody to Factor VHI-associated antigen, although endothelial cells lining blood vessels were positive. Strong staining for laminin and Type IV collagen was present in a pericellular pattern throughout the nodular late-stage lesions. Since lymphatic capillary endothelium does not produce basement-membrane-specific macromolecules, these results support the conclusion that KS cells are related to blood vascular endothelium but eventually lose certain endothelium-specific markers as the cells are transformed into the spindle-shaped cell type.     

Enamel matrix proteins bind to wound matrix proteins and regulate their cell-adhesive properties

Enamel matrix proteins (EMP) induce periodontal regeneration and accelerate dermal wound healing, but the cellular mechanisms of these processes are unclear. We investigated the binding of EMP to the wound matrix proteins fibronectin, laminin-1, collagen type I, and collagen type IV and analyzed the interaction of epithelial cells and periodontal ligament fibroblasts (PDLF) with EMP and composite matrices of EMP + fibronectin or EMP + collagen. The adhesion of PDLF to EMP was concentration- and integrin-dependent and did not require de novo protein synthesis. EMP supported PDLF migration. In contrast, keratinocytes did not adhere to EMP if their protein synthesis was blocked. EMP showed concentration-dependent binding of fibronectin, peaking at 100 microg ml(-1) (before the precipitation point) of EMP. Type I collagen binding to EMP peaked at a low (1 microg ml(-1)) and narrow concentration range. Neither laminin-1 nor type IV collagen bound to EMP. Collagen and fibronectin, bound to EMP, showed significantly reduced (> 50%) binding of both epithelial cells and PDLF compared with the equivalent concentration of these proteins alone. PDLF, but not epithelial cell, adhesion was rescued by increasing the EMP concentration. These findings show that EMP binds to wound extracellular matrix proteins and regulates their adhesive properties. Such interactions may favor fibroblast adhesion over epithelial cells, potentially promoting connective tissue regeneration.     

In vitro effect of transforming growth factor-beta on adhesion molecule expression by human gingival fibroblasts cultured in the presence of a titanium abutment.

BACKGROUND: There is little information in the literature on the structural basis mediating gingival cell adhesion to the surface of titanium abutments. We cultured gingival fibroblasts on a titanium abutment creating as closely as possible the in vivo state. We analyzed the constitutive and transforming growth factor (TGF) beta-induced expression of the adhesion molecules CD44, CD49b, CD49c, CD51, CD54, and CD61 and extracellular matrix (ECM) components fibronectin, laminin and collagen IV. METHODS: Three totally edentulous patients underwent implant treatment to anchor the mandibular denture on 2 implants. Gingival mucosa cell specimens were collected from the mandible during the first surgical stage and the gingival fibroblast cultures were prepared. Cells were cultured for 48 hours with or without isoforms TGF-beta1, TGF-beta2, and TGF-beta3. The expression of adhesion molecules and ECM components was analyzed by immunofluorescence staining and flow cytometry. RESULTS: The addition of TGF-beta isoforms to the cell culture over the incubation period had little effect on cell growth rate, but significantly influenced cell orientation, which changed from a sun-burst pattern in control conditions to a more elongated organization and perpendicular to abutment surface. In all fibroblast preparations, a marked expression of CD44 and a moderate positivity for anti-CD49b and CD49c were found. By contrast, CD51, CD54, and CD61 expressions were negligible. When fibroblasts were cultured for 48 hours in the presence of TGF-beta, the expression of most of the receptor molecules increased. The cells expressed constitutively moderate levels of laminin and fibronectin and low amounts of collagen IV. By contrast, treatment with any one of the 3 TGF-beta isoforms greatly enhanced the expression levels of fibronectin, laminin, and, especially, collagen IV. CONCLUSIONS: TGF-beta not only seems to affect the orientation of the cultured gingival fibroblasts, but also to induce a clear-cut modification of their adhesion molecule expression.     

涎腺腺样囊性癌间质成分的电镜组织化学及免疫组织化学研究

采用Ⅳ型胶原及纤维连接蛋白的免疫组织化学技术研究２２例涎腺腺样囊性癌（筛孔型７例,管状型１５例）,同时采用电镜组织化学技术研究４例筛孔型腺样囊性癌。结果表明,腺样囊性癌筛状结构中分布有复层基板、蛋白多糖和胶原原纤维。免疫组化显示,筛孔中的间质成分富含Ⅳ型胶原和纤维连接蛋白。在管状型肿瘤中,小梁和条索周边的间质成分也富含Ⅳ型胶原及纤维连接蛋白。因此提示,Ⅳ型胶原及纤维连接蛋白是腺样囊性癌的主要间质成分,可能由肿瘤性肌上皮细胞分泌产生。     

Immunohistochemical studies on colloid bodies (Civatte bodies) in oral lesions of discoid lupus erythematosus

By electron microscopy colloid bodies have been shown to be derived from epithelial cells. It has been suggested, however, that connective tissue cells or components from the basement membrane zone contributed to the formation of colloid bodies. In order to examine these possibilities we stained oral lesions of discoid lupus erythematosus (DLE) with antibodies against intermediate filaments (keratin, vimentin), basement membrane components (laminin, collagen type IV) and fibronectin. IgM was used as a marker for colloid bodies. Colloid bodies were stained positive for keratin, whereas vimentin was never found in colloid bodies. Laminin and collagen type IV were occasionally seen in their periphery probably owing to adherence of basement membrane fragments during apoptosis. Fibronectin was frequently seen at the entire periphery of colloid bodies which may facilitate their elimination by macrophages. In conclusion, connective tissue cells or basement membrane components do not seem to contribute to the formation of colloid bodies in oral DLE.     

Immunohistochemical studies on colloid bodies (Civatte bodies) in oral lesions of discoid lupus erythematosus

Abstract – By electron microscopy colloid bodies have been shown to be derived from epithelial cells. It has been suggested, however, that connective tissue cells or components from the basement membrane zone contributed to the formation of colloid bodies. In order to examine these possibilities we stained oral lesions of discoid lupus erythematosus (DLE) with antibodies against intermediate filaments (keratin, vimentin), basement membrane components (laminin, collagen type IV) and fibronectin. IgM was used as a marker for colloid bodies. Colloid bodies were stained positive for keratin, whereas vimentin was never found in colloid bodies. Laminin and collagen type IV were occasionally seen in their periphery probably owing to adherence of basement membrane fragments during apoptosis. Fibronectin was frequently seen at the entire periphery of colloid bodies which may facilitate their elimination by macrophages. In conclusion, connective tissue cells or basement membrane components do not seem to contribute to the formation of colloid bodies in oral DLE.     

Pseudocyst formation by adenoid cystic carcinoma cells in collagen gel culture and in SCID mice

In order to reconstruct the characteristic three-dimensional architecture of adenoid cystic carcinoma, we cultured ACC2 cells, a cell system established from a human adenoid cystic carcinoma of the palate, in collagen gel matrix and transplanted them in SCID mice. In the collagen gel culture, the cells formed spherical colonies measuring 75.6 ± 14.6 urn in diameter by 6 days after seeding. The tumor cell nests contained vacuolar structures that were immunopositive for heparan sulfate proteoglycan, type III collagen, type IV collagen, and fibronectin. The rim of the nests was argyrophilic and immunopositive for type I collagen, type IV collagen, laminin, and fibronectin. Transplants of ACC2 cells in SCID mice grew to form tumor masses in which pseudocysts were formed. The results indicate that our collagen gel culture system provides physiological conditions for ACC2 cells to secrete particular extracellular matrix molecules and form pseudocystic spaces.     

Immunohistochemical assessment of extracellular matrix components in syndrome and non-syndrome odontogenic keratocysts

OBJECTIVE: Investigate the immunohistochemical distribution of fibronectin, tenascin, laminin and collagen IV in syndrome (SOKC) and non-syndrome odontogenic keratocysts (NSOKC). MATERIALS AND METHODS: Ten cases of SOKC and five of NSOKC were selected and streptoavidin-biotin technique was applied. The specimens were analyzed taking into account the following evaluation parameters: presence, continuity and thickness in the basement membrane and intensity, distribution and association with inflammatory cells in the cyst wall. RESULTS: Differences could be detected regarding tenascin, fibronectin and collagen IV between the SOKC and NSOKC. Tenascin was present in all cases along the basement membrane in SOKC and in five cases of NSOKC predominated negative areas. Furthermore, tenascin distribution was focal in the cyst wall in SOKC whereas in NSOKC it was diffuse. Concerning fibronectin, it was detected as a discontinuous band when present in SOCK and as a continuous band in NSOKC. Collagen IV was not present in the majority of the cases in SOKC. Negative areas for laminin predominated in the basement membrane in both groups. CONCLUSIONS: These findings show differences between the immunohistochemical expression of tenascin, fibronectin and collagen IV which might indicate a more aggressive biological behavior of SOKC as compared with NSOKC.     

Chemotaxis of human gingival epithelial cells to laminin. A mechanism for epithelial cell apical migration

Laminin, a large glycoprotein (Mr = 10(6)) and a major component of basement membrane, is shown here to be a potent chemoattractant for human gingival epithelial cells. Laminin stimulated chemotaxis and chemokinesis of gingival epithelial cells in the modified Boyden chamber assay. This effect appeared to be laminin receptor mediated. Gingival epithelial cells were shown to bind laminin (Kd = 2.0 nM) with 10,000 to 30,000 binding sites per cell. Antilaminin antibody, which inhibited laminin binding, inhibited the chemotactic response of epithelial cells to laminin, while antifibronectin was without effect. Fibronectin was not as potent a chemoattractant as laminin. Other biological response modifiers were also tested; of these, Type IV collagen and epidermal growth factor were active as chemoattractants, although not as effective in inducing chemotaxis as laminin. The data indicate that laminin and other components of basement membrane may be important in regulating the migration and growth of gingival epithelial cells.     

Distribution of tenascin-C,fibronectin and collagen types III and IV during regeneration of rat submandibular gland

The aim of this study was to determine the localization of tenascin-C, fibronectin and collagen types III and IV during regeneration of the rat submandibular gland. After 7 days’ obstruction, the regenerating glands were collected at days 0, 1, 3, 5, 7, 11 and 14 after duct release to study regeneration. Immunohistochemical staining revealed that tenascin-C was strongly expressed in the epithelial cells of duct-acinar structures at days 0–3, and down-regulated in its expression from day 5 to 11, though weak expression was detected in the intercalated duct and acinar cells of the normal gland. Strong labeling of fibronectin was detected around duct-acinar structures during days 0–3 of regeneration. Type IV collagen was expressed strongly in the thickened basement membrane of acinar cells and duct-acinar structures during days 0–3, but weakly around large ducts, though type III collagen was expressed at consistent levels. These findings suggest that tenascin-C and fibronectin affect only the duct-acinar structures, and type IV but not type III collagen is involved in the regeneration of acinar cells.     

Immunoexpression of extracellular matrix proteins in human salivary gland development

Immunoexpression of the extracellular matrix (ECM) proteins laminin, fibronectin, tenascin and types I, III and IV collagen was analyzed in the major and minor salivary glands of seven human fetuses at different gestational ages. The results showed the presence and localization of laminin, collagen IV and fibronectin around glandular structures at all stages of development. Tenascin was only detectable around excretory ducts. In the earliest stages of development, type I and type III collagen were presented as fine fibers delineating the glandular structures and delimiting the extension of the future lobule. As glandular development proceeded, the lobule was gradually filled with collagens and glandular tissue.     

Distribution of basal lamina type IV collagen and laminin in normal rat tongue mucosa and experimental oral carcinoma: Ultrastructural immunolocalisation and immunogold quantitation

The relationship of basal lamina, a form of specialised extracellular matrix which separates epithelial cells and other cell types from adjacent stroma, to the behaviour of malignant neoplasms of epithelial origin is not well understood. However, it is widely acknowledged that the properties of local invasion and metastasis of carcinomas are linked to extracellular matrix (including basal lamina) changes. In the present study, the distribution of the major basal lamina components, type IV collagen and laminin, in normal rat tongue mucosa and experimentally induced oral carcinomas was investigated using post-embedding immunogold techniques and electron microscopy. The expression of these components was also quantitatively analysed using morphometry and immunocytochemistry. Results indicated that type IV collagen and laminin were confined to the lamina densa of normal oral epithelial basal lamina, and that both components were also detected in the lamina densa of basal lamina associated with carcinomas, and in the extracellular matrix of tumours. Furthermore, laminin was detected within stromal fibroblasts in normal tissues and experimental carcinomas. Quantitative analysis indicated that expression of laminin was significantly increased in carcinomas. In contrast, type IV collagen expression was significantly decreased. The quantitative changes observed in the two basal lamina constituents may be related to the process of tumour invasion, reflecting altered metabolic activities of tumour and stromal cells. These observations may be of use in understanding the architectural characteristics of oral mucosa basal lamina and in assessing the malignant potential of epithelial dysplasias or “premalignant” lesions.     

Immunolocalization of basement membrane molecules in the stroma of salivary gland pleomorphic adenoma

In order to determine the participation of basement membrane molecules in formation of its characteristic stroma, 30 cases of salivary gland pleomorphic adenoma were examined by immunohistochemical staining for type IV collagen, laminin, heparan sulfate proteoglycan, and entactin. The stroma was histopathologically classified into four types: hyaline, fibrous, myxoid, and chondroid. Immunohistochemically, type IV collagen and laminin were more intensively localized in hyaline, fibrous and chondroid types of stroma, whereas heparan sulfate proteoglycan was more prominent in myxoid areas. The results suggest that the stroma contains these basement membrane components, which are possibly biosynthesized by epithelial tumor cells, and that histological variety of the stroma depends on proportion of local contents of each basement membrane molecule.     

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)