Immunolocalisation of collagens in the developing rat molar tooth

This study identifies different types of collagens during tooth development, maturation and ageing. Tissues from the rat first molar (from animals ranging in age from E14 to 104 wk postnatally) were immunostained using a panel of mono- and polyclonal antibodies against types I, II, III, IV, VI, IX and X collagen, fibronectin and laminin. During tooth development, types I and III collagens were expressed in the dental papilla at all stages but were also unexpectedly observed in the stellate reticulum of the enamel organ. Transient expression of type II collagen was also observed in the stellate reticulum during the late bell stage. Types IV and VI collagens, with laminin and fibronectin, were located within the basement membranes of the tooth germ. Collagen types I and III were observed within the developing follicle/periodontal ligament, type III predominating where collagen fibres were inserting into the alveolar bone and cementum. The pattern of types I and III collagen labelling within the periodontal ligament and the dental pulp did not change with age. Thus, some unusual collagen localisations were observed in the tooth germ, particularly within the stellate reticulum.     

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.     

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.     

Expression of extracellular matrix in human mandibular condyle

ObjectivesThe age-related expression of the extracellular matrices in human mandibular condyle was examined.Study designThe distribution patterns of types I to V collagens, laminin, fibronectin, fibronectin receptor, and transforming growth factor β in 34 human mandibular condyles dissected from autopsy specimens were studied by immunohistochemical procedure with special attention on the age-related changes.ResultsType I collagen was detected in the full layer of the condylar cartilage, and a stronger immunoreaction was delineated in the articular and cartilage zone. Types II and III collagen were mainly localized in the fibrocartilage zone. Type IV collagen and laminin were detected not only in the basement membrane of the blood vessels but also in the degenerated lesion where the expression of transforming growth factor β was also detected. Immunostaining of type V collagen and fibronectin was noted in the perichondrocytic area, whereas that of fibronectin receptor was seen in the chondrocytes. In materials from younger cadavers types I, II, IV, and V collagens, fibronectin, its receptor, and laminin showed stronger expression in the degenerative lesions than in the normal portions. In the sections from cadavers over the seventh decade, the immunoreaction of extracellular matrices was weak compared with the younger materials, and no increased reaction of extracellular matrices in the degenerative lesions was detected. In addition, severe osteoarthrosis was frequently seen in the older materials in macroscopic findings.ConclusionsThese results suggest that the expression of extracellular matrices thus seems to be closely related to agingand degenerative changes in the condyle.     

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.     

A study of the extracellular matrix in salivary gland tumors

BACKGROUND: Epithelial-mesenchymal interactions constitute fundamental phenomena in the development and maintenance of the characteristic branching pattern seen in salivary glands. This study was undertaken to discuss the extracellular matrix (ECM) role in morphogenesis and cellular differentiation of salivary gland tumors originating from the intercalated duct. METHODS: The ECM components, laminin (LN), type IV collagen, fibronectin (FN), and tenascin (TN) were revealed using a streptoavidin-biotin immunohistochemical technique and analyzed in 34 cases of salivary gland tumors: pleomorphic adenoma (PA); myoepithelioma; basal cell adenoma (BCA); polymorphous low grade adenocarcinoma (PLGA); and adenoid cystic carcinoma (ACC). RESULTS: LN and type IV collagen were present in all tumors, confining well-organized duct-like structures, separating them from the stroma, or surrounding cell clusters. In PA and myoepithelioma, the basement membrane (BM) fragmentation was observed through LN and type IV collagen staining around each individual spindle-shaped cell, which was strictly related to the cell modification. Interestingly, BM interruption could not be seen in the malign tumors, however, was frequently augmented in some cases. LN, type IV collagen, and FN were also found in the stroma of all tumors studied, except for the pseudocystic spaces of ACC, which were only delimited by replicated LN and type IV collagen. TN exhibited a variable expression, being more intense in solid ACC. CONCLUSIONS: LN and type IV collagen were always present around morphologically well-differentiated duct-like structures in all tumors studied. BM interruption could not be seen in the malign tumors, on the contrary BM production was evident, which is probably related to invasion. FN was present in the stroma of all tumors, but TN was mostly observed in less differentiated and higher degree of malignancy tumors, such as solid ACC.     

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.     

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.     

Electron immunolocalization of type IV collagen, laminin and fibronectin synthesized by multilayered cells cultured from human oral epithelium

Human palatal epithelial cells were grown on lethally irradiated 3T3 fibroblasts. Under these conditions, epithelial cultures become stratified. The basal cells have a cuboïdal shape and suprabasal layers are flattened. The reconstituted epithelium exhibits markers of differentiation: intracytoplasmic bundles of keratin-like filaments and abundant desmosmes present in every layer. Type IV collagen fibronectin and laminin are synthesized by these cultures. Basal cells grip the culture substratum via an extracellular matrix made up of type IV collagen, laminin and fibronectin.     

Immunolocalization of beta 1 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 beta 1 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 beta 1 integrins. No fibronectin or type IV collagen was found in cell-cell contact sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Connective tissue organization of healthy human gingiva

Healthy human gingival connective tissue appears to be arranged in two patterns of organization at the ultrastructural level: Pattern I (PI) and Pattern II (PII). PI is a dense pattern of organization mainly constituted of large, dense bundles of thick collagen fibers, whereas PII is a loose pattern of organization, mainly constituted of short, thin collagen fibers mixed with a fine reticular network, especially located under or around basement membranes. Ultrastructural immunoperoxidase labelling of types I, III, and IV collagen demonstrates that gingival connective tissue is made of an intricate pattern of type I and III collagen where type I collagen fibers are preferentially organized in large dense bundles in PI, whereas a fibrous and fibrillar type III collagen network is predominant in PII. Type IV collagen, which does not exist in fibrous or fibrillar form, appears to be the main collagenous component of the basement membranes.     

Effect of lactoferrin on interaction of Prevotella intermedia with plasma and subepithelial matrix proteins

A lactoferrin-binding protein with an estimated molecular mass of 57 kDa was identified in the cell envelope of Prevotella intermedia by gel electrophoresis and Western-blol analysis. Peroxidase-labeled bovine lactoferrin and human lactoferrin showed similar specific binding to this protein. Whole cells of P. intermedia were also examined for interactions with 5 ¹²⁵I-labeled plasma and subepithelial matrix proteins. A high degree of binding was found with fibronectin, collagen type I and type IV and laminin, whereas a moderate interaction was detected with fibrinogen. The ability of bovine lactoferrin to affect the interactions of the above proteins with P. intermedia was examined. In the presence of unlabeled bovine lactoferrin, a dose-dependent inhibition of binding was observed with all 5 proteins tested. Unlabeled bovine lactoferrin also dissociated the bacterial complexes with these proteins. The complexes with laminin or collagen type I were more effectively dissociated than fibronectin or fibrinogen, whereas the interaction with collagen type IV was affected to a lesser extent. A strain-dependent variation in the effect of bovine lactoferrin was observed. These data establish the presence of a specific lactoferrin-binding protein in the cell envelope of P. intermedia. The ability of lactoferrin to inhibit the binding of some plasma and subepithelial matrix proteins to P. intermedia could be a protective mechanism against the establishment of this pathogen in the periodontal pocket.     

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.     

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.     

Immunohistochemical study of the relationship between extracellular matrix and root bifurcation in the mouse molar

In order to investigate epithelial-mesenchymal interaction during root bifurcation, the distribution of type III and I collagen, fibronectin and laminin in the epithelial-mesenchymal junction between the dental epithelia (epithelial diaphragm and interradicular process) and the cells of the dental papilla (or pre-odontoblasts) was examined, using maxillary first molar tooth germs of CF1 mice from day 1-16 after birth. Three-dimensional reconstructions of the immunofluorescent patterns were made from serial sections of tooth germs from day 3-9, stained with the antibodies against the collagens. The findings were as follows. (1) Type III collagen was first seen in the epithelial-mesenchymal junction at the tip of the interradicular process, where it sprouted from the epithelial diaphragm, and spread along the interradicular process toward its base, accompanied its extension, and then disappeared on completion of root bifurcation. No staining was seen in the epithelial-mesenchymal junction at the epithelial diaphragm during and after root bifurcation. (2) Type I collagen appeared in the epithelial-mesenchymal junction at the base of the interradicular process, where it sprouted from the epithelial diaphragm and spread toward the tip of the interradicular process, following its extension, and increased on completion of the root bifurcation. No staining was seen in the epithelial-mesenchymal junction at the epithelial diaphragm during or after root bifurcation. (3) Fibronectin and laminin remained constant in the epithelial-mesenchymal junction, both at the interradicular process and the epithelial diaphragm, during and after root bifurcation. These findings suggest that type III collagen may play a significant role in the early stage of root bifurcation in the molar.     

Maxillary fibrosarcoma with extracellular immuno-characterization

Maxillary fibrosarcoma of a 31-year-old female is described with special reference to immunofluorescent characterization of the extracellular matrix. Following extractions, endodontic treatment and occlusal adjustment the tumour was operated on. The spindle-shaped tumour cells displayed a herring-bone pattern. The tumour was diagnosed as a fibrosarcoma and graded as poorly differentiated. Immunostaining for the specific type I collagen was positive throughout the tumour matrix, whereas staining for type III collagen was faint and occasional, and did not definitely codistribute with reticulin. Staining for type IV collagen and fibronectin in the tumour matrix was negative.     

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 study of types I, III and IV collagen in fibrosis of diseased gingiva during chronic periodontitis: A light and electron microscopic study

The distribution of type I, III and IV collagen and their ultrastructural organization have been studied in fibrotic gingival connective tissue of patients with longstanding cases of chronic periodontitis. The use of an immunofluorescent procedure has shown that the diseased connective tissue was made up of both type I and type III collagen. Type I collagen was strongly fluorescent and appeared to be the main gingival collagenous component, equally distributed in all layers of the tissue, whereas type III collagen was weakly fluorescent and mostly found in gingival papillae, but not around blood vessels. Standard electron microscopy and immunolabelling using the peroxidase procedure have shown that the large and dense bundles of type I collagen of P₁, which is the main pattern of organization of the gingival connective tissue, were infiltrated by small bundles of microfilaments (10–15 nm) identified as the fibrillar form of type III collagen. However, in P_II, the second pattern of organization of the gingival connective tissue, type I collagen fibers were predominant and gave a fibrous feature to this area. Type IV collagen was exclusively located in the thickened, degraded Lamina densa of basement membranes.     

Immunohistochemical localization of types I and III collagen and fibronectin in the dentine of carious human teeth

Reparative dentine formed as a response to caries was mostly type I collagen similar to that of normal dentine. The predentine related to reparative dentine reacted positively with antisera to types I and III collagen and to fibronectin. Normal odontoblasts and their processes reacted positively both with types I and III collagen antibodies. Fibronectin was related to odontoblasts and their processes pericellularly. Odontoblasts appeared not to lose totally their developmental ability to synthesize type III-like molecules after maturation. Pulp fibroblasts reacted positively both with types I and III antibodies as well as antifibronectin. The cell-free and cell-rich zones revealed a dense layer of fibres reacting positively with type III collagen and fibronectin antibodies. The width of these zones were reduced in relation to reparative dentine.