Immunohistochemical localization of collagenous components in healthy periodontal tissues of the rat and marmoset (Callithrix jacchus). II. Distribution of collagen types IV, V and VI

The immunohistochemical distribution of collagen types IV, V and VI has been demonstrated in healthy periodontal tissues of rats and marmosets following decalcification of the maxillae and mandibulae in 0.2 N HCl. An intense fluorescence with anti-collagen type IV antibodies was demonstrated in the basement membranes of the epithelium and of the blood vessels and nerves. In the alveolar bone stroma and in the periodontal ligament (PL) collagen type IV was present only in the basal membranes of the blood vessels and nerves. In comparison, collagen type V was observed in a fibrillar pattern in the gingival connective tissue, as well as the PL. In the PL, type V collagenous fibers demonstrated a parallel distribution with stronger fluorescence near the cementum surface. Collagen type VI could be demonstrated in fine fibers present in the gingival connective tissue and the PL. Blood vessels and nerves were not stained in the marmoset, but were in the rat, where a localization of collagen type VI was demonstrated in these areas. Alveolar bone and cementum, as well as the Sharpey's fibers embedded in these tissues, were not stained with antibodies against collagen type V and type VI, but a pericellular localization of these collagenous components could be observed. Collectively, these results provide basic information on the relative distribution of different collagen types in normal tissues of rats and marmosets that will be required for future studies on the effects of pathological, reparative and regenerative processes.     

Immunohistochemical characterization of the basement membranes of the human oral mucosa

Type IV and V collagens, laminin and heparan sulphate proteoglycan were localized in vascular and subepithelial basement membranes. Fibronectin was distributed in a reticular pattern throughout the lamina propria under the oral epithelium. The uniform distribution of basement membrane components and type V collagen in different regions suggests a similar molecular composition for the basement membranes under functionally-different oral epithelia. The more intense reaction in the vascular than in the subepithelial basement membranes, with diluted antibodies to type IV collagen and laminin apparently reflects chemical differences in these basement membranes. Occasional discontinuities in the subepithelial basement membranes were seen in inflamed gingival sulci and in tonsillar crypts. The destruction responsible affected all basement membrane components, except fibronectin, which maintained a reticular distribution even in the deep tonsillar tissue. The immunohistochemical method is useful in demonstrating different degrees of destruction in basement membranes associated with inflammation.     

Distribution of collagens type V and VI in the normal human alveolar mucosa: An immunoelectronmicroscopic study using ultrathin frozen sections

The ultrastructural localization of collagens type V and VI in normal human gingival mucosa was investigated by immunoelectron microscopy. Twenty biopsies were fixed in dimethylsuberimidate and shock-frozen in slush nitrogen. Collagen type V was mainly located to meshworks of uniform nonstriated microfibrils of 12 to 20 nm width, which preferentially appeared in larger spaces between cross-striated major collagen fibrils. Occasionally single microfibrils of collagen type V fanned out from the ends of major collagen fibrils, which may indicate a role as a core fibril. Collagen type V was not found in the subepithelial basement membrane and the immediately adjacent stroma. Collagen type VI was detected in a loose reticular network of unhanded microfilaments that were morphologically distinguishable by knoblike protrusions every 100–110 nm. These microfilaments were found in the vicinity, but not as an intrinsic component, of the subepithelial basement membrane. Single filaments of collagen type VI filaments appeared to form bridges between neighboring cross-striated major collagen fibrils, suggesting an interconnecting role for this collagen type. The method presented appears to be excellently suited to study the normal and pathological supramolecular organization of the oral extracellular matrix.     

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.     

The immunohistochemical distribution of collagens type IV, V, VI and of laminin in the human oral mucosa

The distribution of collagens type V (form AB2) and VI was investigated on cryostat sections of normal human oral mucosa by indirect immunofluorescence. For comparison, antibodies to fragments of type IV collagen and laminin were also used to delineate basement membrane containing structures. All antibodies used were raised against human proteins. Type V collagen appeared as a microfibrillar structure throughout the interstitium, apparently touching but not being present within epithelial or vascular basement membranes. Microfibrils in blood vessel walls were limited to the intimal layer. Pericellular areas were not specifically stained. Type VI collagen appeared as an almost amorphous stromal structure becoming more prominent and more fibrillar in the upper connective tissue papillae. Intense staining was observed in the media of blood vessels and around smooth muscle cells. A possible role of type VI collagen in tissue stabilization may be expected from this ubiquitous and abundant distribution. The findings identify types V and VI collagen as important structures in the oral mucosa and serve as a basis for understanding morbid changes.     

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.     

Immunohistochemical distribution of collagens type I, III, IV and VI, of undulin and of tenascin in oral fibrous hyperplasia

The distribution of collagens type I, IV and VI, of procollagen type III, of undulin and of tenascin was studied in 10 lesions which were clinically and histologically diagnosed as localized oral fibrous hyperplasias. The immunohistochemical distribution of these proteins was similar to that observed for normal oral mucosa. Undulin showed a pattern of parallel fibers throughout. Collagen type VI was pronounced in the subepithelial connective tissue, whereas the collagen fiber bundles were equally reactive for collagens type I and III. Tenascin was observed close to the subepithelial basement membrane and in proximity to collagen fiber bundles in the upper connective tissue. The present findings indicate that oral fibrous hyperplasias that are probably caused by inflammation or chronic irritation show the differentiated and ordered pattern of extracellular matrix proteins characteristic of normal oral mucosa.     

Localization of fibronectin in gingival connective tissue of the beagle dog. Immunofluorescent light microscopic findings

Affinity purified antibodies to plasma fibronectin were used to localize fibronectin in the connective tissues of inflamed and noninflamed beagle gingiva. In noninfiltrated gingival connective tissue, fibronectin was demonstrated in the basement membrane beneath gingival epithelium and around blood vessels as a uniform and intensely stained band about 3 to 10 micron thick. Fibronectin was also distributed throughout the connective tissue in association with collagen fibrils as a more diffuse, less intensely stained pattern. The inflamed gingiva included in this study was characterized by proliferation of epithelial pegs, heavy infiltration of plasma cells and loss of collagen within the subepithelial connective tissue. In these sites, fibronectin was present as an intensely stained band around blood vessels and at the crest of connective tissue papillae nearest the sulcular space. The fibronectin in the basement membrane beneath the epithelium appeared diminished and less uniformly distributed. A delicate network of fibronectin was present around plasma cells and the remaining collagen fibers.     

Odontogenic myxoma—Characterisation of the extracellular matrix (ECM) of the tumour stroma

In order to elucidate the origin of the odontogenic myxoma, the composition and the structural organisation of the extracellular matrix (ECM) of this tumour were characterised. Collagen type I, VI, procollagen type III, undulin, tenascin and fibronectin were demonstrated in biopsy material of 4 cases by polyclonal antibodies. The tumour stroma showed a pronounced reaction for collagen type I. Fibroblasts displayed an intense intracytoplasmatic reaction for procollagen type III, and collagen type I was not found in the fibroblasts of the adjacent normal oral mucosa. In contrast to the surrounding connective tissue, label for collagen type VI was weak, as was the reaction for fibronectin and tenascin. Undulin was almost undetectable. The immunohistochemical results suggest that the odontogenic myxoma is characterised by an as yet unobserved structural organisation of ECM proteins, a secretion defect of fibrillar collagens type I and III with no resemblance to physiological tooth development.     

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.     

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)     

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.     

Biochemical analysis of total collagen content and collagen types I,III, IV,V and VI in gingiva of various periodontitis categories

Collagen is one of the chief components of the extracellular matrix of gingival connective tissue, where five different types have been identified to date. The molecular mechanism of collagen loss in periodontitis still needs to be explored. In the present study total collagen content was investigated in gingival connective tissue of adult periodontitis (AP) as well as early onset periodontitis patients (EOP) and clinically healthy subjects. Furthermore, collagen type I, III, IV, V andVI content was evaluated in gingival biopsies obtained from periodontitis patients. There was a statistically significant difference between AP (25.1 +/- 8.1 microg/mg) and EOP (15.6 +/- 4.0microg/mg) groups with regard to the total collagen content (P < 0.05). In the clinically healthy control group the total collagen content was 20.7 +/- 4.6microg/mg. Moreover, the distribution of collagen types exhibited variations in pooled homogenates of each periodontitis group. The total collagen loss seemed to be greater in the EOP patients than in the AP patients. When the ratio of fibril forming collagens to nonfibrillar collagens was evaluated, it seems to be decreased in AP patients in comparison to EOP patients. The findings of the present study suggest that different collagen types present in various periodontitis categories may be related with diverse pathogenic mechanisms acting in these diseases.     

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.     

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.     

Immunohistochemical localization of type IV collagen and laminin in the gingival capillary basement membrane of the diabetic rat.

Distribution of type IV collagen and laminin in the gingival capillary basement membrane from streptozotocin-induced diabetic rats was investigated using immunoelectron microscopy. Both type IV collagen and laminin were found throughout the basement membrane. Quantitative analysis revealed that the immunoreactive area for laminin did not change with age, and the width of laminin deposition remained constant, even when diabetes was induced in the animals. However, the immunoreactive area for type IV collagen thickened with age. Further, the width of type IV collagen in the basement membrane increased markedly 36 weeks after diabetes was induced. It was concluded that the thickening of the gingival capillary basement membrane in experimentally induced diabetic rats was due an increase of type IV collagen deposition.     

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.     

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.     

Extracellular matrix in gingival fibroblasts]

Primary cultures of human gingival fibroblasts from patients of different age have been established. Histotype characterization has been confirmed by ultrastructural morphology and by the positivity of intermediate filament vimentin. Extracellular matrix expression has been analyzed by immunocitochemistry. Our data demonstrate that the extracellular matrix of human gingival fibroblasts is composed of type IV collagen, other than fibronectin and type I-III collagens.