Characterization of the extracellular matrix of the primate temporomandibular joint.

The distribution of type I and II collagens, fibronectin and the fibronectin-integrin receptor, tenascin, laminin, link protein, and cartilage-specific glycosaminoglycans was examined in the primate temporomandibular joint complex using an immunohistochemical approach. In general, type I collagen, fibronectin, and the fibronectin-integrin receptor were found to co-distribute throughout the joint complex. Immunostaining for these proteins was notably intense in the prechondroblastic and mineralization zones of the articular cartilages of the joint. Tenascin was identified in several structures of the joint, including the articular cartilages, where intense staining was observed in the prechondroblastic and cartilagenous zones. Laminin was detected only in the adventitia of blood vessels located in the attachment tissues of the disc and joint synovium. Cartilage-specific glycosaminoglycans and type II collagen were observed in the cartilagenous zones of the articular cartilages of the mandibular condyle and temporal bone. In addition, immunostaining for cartilage-specific glycosaminoglycans also was detected throughout the extracellular matrix surrounding "chondrocyte-like" cells located in the joint disc. Despite the localization of cartilage-specific glycosaminoglycans in the disc, type II collagen was not detected in this structure. It is suggested that a fibronectin-integrin receptor mechanism may be involved in the regulation of growth of the articular cartilages of the temporomandibular joint.     

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.     

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.     

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.     

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.     

Changes in condylar cartilage after anterior mandibular displacement in juvenile pigs

Adaptive remodelling of the mandibular condyle in response to mandibular advancement is the mechanism exploited by orthodontic forward displacement devices.ObjectiveThis work investigated the expression of collagens, matrix metalloproteinases and vascular endothelial growth factor during this process.DesignTwenty juvenile pigs were randomly divided into two experimental groups, where the treatment group was fitted with mandibular advancement splints, and the control group was not. Changes in the mRNA content of condylar cartilage tissue was then were measured by real-time PCR using specific primers after 4 weeks of treatment.ResultsThe temporal pattern of the expression of Col1 and MMP13 during condylar adaptation coincided with that during natural condylar growth. The amount of the expression of Col10 during condylar adaptation was significantly lower (p < 0.05), whereas the expression of Col2, MMP8 and VEGF was significantly higher compared to natural growth (p < 0.05).ConclusionsIt is suggested that condylar adaptation in growing pigs triggered by mandibular forward positioning results not only from passive adaptation of cartilage, but also involves growth affected processes. Our results showed that mechanical strain produced by mandibular advancement induced remodelling and revascularization in the posteriocranial mandibular condyle. These results are mostly consistent with former published histological and histomorphometrical analyses.     

Effects of transforming growth factor beta 1 on the plasminogen activation system,collagen and integrin synthesis,and proliferation of rabbit mandibular condylar chondrocytes

The objective of this study was to identify the mechanism by which mandibular condyle chondrocytes regulate the extracellular matrix. Primary rabbit condylar chondrocytes were isolated, cultured, and treated with transforming growth factor beta 1 (TGF-β1). Cells were then assayed for the following: urokinase-type plasminogen activator (uPA) and its inhibitor (PAI-1), collagen types I and II, β1 integrin expression, and proliferative activity. TGF-β1 induced synthesis of collagen type II, αVβ1 integrin, and PAI-1. TGF-β1 induced the growth of chondrocytes and suppressed the synthesis of uPA. Chondrocyte regulation of the extracellular matrix is mediated by TGF-β1. Synthesis of collagen type II, αVβ1 integrin, and PAI-1 is induced, while uPA is suppressed. Also, TGF-β1 induces cellular growth.     

雌激素受体与Ⅱ型胶原在人胚髁突软骨中的表达

目的探讨雌激素受体（ER）和Ⅱ型胶原在人胚髁突软骨中的表达情况。方法 采用苏木精-伊红（HE）及免疫组织化学染色法检测ERα、ERβ和Ⅱ型胶原在人胚颞下颌关节髁突软骨各层的表达。结果 Ⅱ型胶原主要表达于髁突软骨的过渡层及肥大层。ERα主要表达于髁突软骨的过渡层及肥大层,均匀分布于细胞内;ERβ集中表达于肥大层,且以细胞核内分布为主。纤维层及增殖层未见Ⅱ型胶原和ER表达,钙化软骨层有少量表达。结论 ER与Ⅱ型胶原在髁突软骨中分布一致,雌激素可选择性地结合不同亚型的ER,从而调节髁突软骨细胞分泌Ⅱ型胶原的能力。     

Adhesion of a chondrocytic cell line (USAC) to fibronectin and its regulation by proteoglycan.

BACKGROUND: Chondrocytes produce various extracellular matrices during chondrogenesis. Fibronectin and proteoglycan are major extracellular matrix proteins in cartilage tissue, but the interactions between them are not clear. METHODS: Recently, we succeeded in establishing a cell line (USAC) with phenotypes of chondrocytes from a human osteogenic sarcoma of the mandible. Using this cell line, cell adhesion to fibronectin, the effect of proteoglycan on the cell adhesion and expression of integrin alpha5beta1 were investigated. RESULTS: Cells immediately adhered to fibronectin and then spread. Proteoglycan inhibited cell adhesion to fibronectin dose-dependently, whereas collagen did not. The expression of both mRNAs of alpha5 and beta1 subunits was detected 12 h after treatment with proteoglycan, but the expression of beta1 subunit mRNA had diminished by 24 h after treatment. CONCLUSIONS: These findings suggest that proteoglycan might modulate signal transduction from fibronectin by decreasing the expression of alpha5beta1 integrin.     

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.     

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.     

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.     

Immunocytochemical and biochemical characterization of the connective tissue component of fibrous papular lesions of oral mucosa

In an attempt to define the fibroblastic components of fibrous papular lesions of oral mucosa, 21 cases representing these lesions were selected for study. Paraffin or frozen sections of the lesions were stained, using the immunoperoxidase technique, with antibodies to: alpha-l-antitrypsin, alpha-l-antichymotrypsin, lysozyme, fibronectin, actin, myosin, and for Clq binding. Three of these cases were biochemically analyzed for collagen Types I, III, and V. This study demonstrated the presence of cytochemical markers in fibrous papules that were similar to those observed for the compartment of circumvascular fibroblasts in control normal mucosa. Analysis of the collagens present indicated that in addition to histologic similarities, the gene products of these extracellular matrices were similar to those reported for angiofibromas and Shagreen patches in tuberous sclerosis. The cells in these lesions appear to be distinct from myofibroblasts and the high affinity complement binding fibroblasts characterized by Bordin et al.(1).     

The expression of osteopontin with condylar remodeling in growing rats.

It is suggested that osteopontin may promote osteoclast binding to resorptive sites by interacting with the alphavbeta3 receptor on osteoclasts. However, the role of osteopontin in functional remodeling of bony structures remains unclear. The present study was conducted to examine the distribution of osteopontin on the condyle and explore the role in condylar remodeling in growing rats using an immunohistochemical method. Twenty Wistar strain male rats aged 7, 14, 28 and 56 days were used. In 7- and 14-day-old rats, no immunoreaction to osteopontin was detected in the cartilage cells. In 28-day-old rats initiating mastication, the thickness of condylar cartilage was decreased abruptly as compared to the younger rats. High immunoreaction to osteopontin was found in the cytoplasm of hypertrophic chondrocytes and on the trabecular bone surfaces of primary spongiosa adjacent to the osteoclasts or chondroclasts. The immunoreactions to osteopontin in the cytoplasm of hypertrophic chondrocytes were less in 56-day-old rats than in 28-day-old rats. It is shown that the alteration in mechanical loading on the mandibular condyle due to functional changes from weaning to mastication correlates with the localization of osteopontin in growing rats. Furthermore, it is suggested that osteopontin may stimulate osteoclastic resorption of calcified matrix by mediating the attachment of osteoclasts and/or chondroclasts during growth-related functional remodeling of the condyle.     

Integrin receptors and their relationship to cellular proliferation and differentiation of oral squamous cell carcinoma. A quantitative immunohistochemical study

The expression of extracellular matrix (ECM) proteins (fibronectin, laminin, collagen IV) and ECM receptors of integrin type (α₂β₁, collagen receptor; α₆ chain of the fibronectin receptor; α₆ chain of the laminin receptor) were examined in normal oral squamous epithelium and in invasive areas of oral squamous cell carcinomas with various differentiation and proliferation activities (Ki-67 antigen labelling), evaluating the presence, quantity (using an image analysis system) and distribution of the integrin subunits. In the mucosa, there was uniform immunostaining for α₂β₁ and α₆ concentrated at the cell membrane in the basal/supra basal cell zone, whereas, α₅ showed a discontinuous staining of the basal cell-basement membrane interface. α₂ and α₆ could be visualized in all carcinomas α₅ showed low expression preferentally in less differentiated carcinomas. In contrast to normal mucosa, there was an increase in α₆ staining in well-differentiated carcinomas. Dedifferentiation of oral carcinomas was accompanied by an increase in cellular proliferation and with a decrease in α₂β₁ and α₆ staining. This reduction of α₆ staining was shown to be statistically significant, suggesting that this integrin may be a valuable grading parameter for oral squamous cell carcinoma.     

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)     

Structural features of incremental line-like striations in mandibular condylar cartilage of c-src-deficient mice

Mandibular condylar cartilage is sensitive to masticatory force, while mice lacking the c-src gene (c-src-deficient mice) have osteopetrosis and tooth eruption failure. The purpose of this study was to investigate the morphology of the mandibular condyle in these mice, which were maintained with a soft-food diet for 240 days after birth. The condylar head in the c-src-deficient mice showed slight deformity in shape before weaning, but showed remarkable undergrowth after weaning. No significant morphological or histological differences were detected between the mandibular condyle in wild-type mice fed soft food and those fed hard food, indicating that osteopetrosis, as well as abnormal masticatory force, influences the morphology of the mouse mandibular condyle, and that malocclusion rather than dietary consistency may have greater influence. After 70 days, incremental line-like striations consisting of cartilaginous and non-cartilaginous layers were detected in the mandibular condyle of the c-src-deficient mice, but not in the tibial growth plate. Immunostaining of aggrecan, collagen types II and X, and osteopontin was detected in the cartilaginous layers, but not in the non-cartilaginous layers showing collagen type I immunostaining. Chondrocyte lacunae were not eroded in the cartilaginous layers, and complete circumferential mineralisation around the lacunae and impaired osteoclast (chondroclast) function can account for this phenomenon. However, repeated cessation of chondrocyte differentiation may be required to completely explain the formation of the striations. These results indicate that the mandibular condyle in the c-src-deficient mice has unique structural features, adding to its deformity.     

The expression of osteopontin with condylar remodeling in growing rats

It is suggested that osteopontin may promote osteoclast binding to resorptive sites by interacting with the α_vβ₃ receptor on osteoclasts. However, the role of osteopontin in functional remodeling of bony structures remains unclear. The present study was conducted to examine the distribution of osteopontin on the condyle and explore the role in condylar remodeling in growing rats using an immunohistochemical method. Twenty Wistar strain male rats aged 7, 14, 28 and 56 days were used. In 7‐ and 14‐day‐old rats, no immunoreaction to osteopontin was detected in the cartilage cells. In 28‐day‐old rats initiating mastication, the thickness of condylar cartilage was decreased abruptly as compared to the younger rats. High immunoreaction to osteopontin was found in the cytoplasm of hypertrophic chondrocytes and on the trabecular bone surfaces of primary spongiosa adjacent to the osteoclasts or chondroclasts. The immunoreactions to osteopontin in the cytoplasm of hypertrophic chondrocytes were less in 56‐day‐old rats than in 28‐day‐old rats. It is shown that the alteration in mechanical loading on the mandibular condyle due to functional changes from weaning to mastication correlates with the localization of osteopontin in growing rats. Furthermore, it is suggested that osteopontin may stimulate osteoclastic resorption of calcified matrix by mediating the attachment of osteoclasts and/or chondroclasts during growth‐related functional remodeling of the condyle.     

Immunohistological localization of cell adhesion proteins and integrins in the periodontium.

The distribution of the cell adhesion proteins vitronectin, fibronectin, tenascin, and laminin as well as several integrin subunits, alpha 2, alpha 5, and alpha v, was studied in primate periodontal tissues. Full baboon mandibular sections were analyzed by immunohistochemical methods in order to localize the molecules studied in both soft and hard tissues. Vitronectin was associated with the connective tissue of the marginal gingiva, the periodontal ligament, as well as the endosteum and periosteum. A notable finding was the particularly high staining intensity of vitronectin in the periodontal ligament. Fibronectin was widely distributed in the periodontal connective tissue and was also localized to the pericellular matrix of osteocytes and blood vascular elements. Epithelial basement membranes stained positively for both fibronectin and tenascin. These proteins were also expressed in the periosteal and endosteal connective tissues and the periodontal ligament. The staining intensity for tenascin was higher in zones along the cementum and bone surfaces. Laminin was, characteristically, limited to basement membranes of epithelium and endothelium. The distribution of fibronectin, tenascin, and laminin is related to previous findings in other species. The localization of the several integrin alpha-subunits is also described in full baboon mandibular sections. The vitronectin receptor (alpha v) had a uniquely strong expression in osteoclasts of the alveolar bone and was found, at lesser intensity, on periodontal ligament fibroblasts. The fibronectin receptor alpha subunit, alpha 5, was also observed on osteoclasts, and, in addition, was widely distributed on fibroblasts, cementoblasts, and osteoblasts.(ABSTRACT TRUNCATED AT 250 WORDS)