Fibronectin accelerates the growth and differentiation of ameloblast lineage cells in vitro.

During tooth development, the growth and differentiation of ameloblast lineage (AL) cells are regulated by epithelial-mesenchymal interactions. To examine the dynamic effects of components of the basement membrane, which is the extracellular matrix (ECM) lying between the epithelium and mesenchyme, we prepared AL cells from the epithelial layer sheet of mandibular incisors of postnatal day 7 rats and cultured them on plates coated with type IV collagen, laminin-1, or fibronectin. The growth of AL cells was supported by type IV collagen and fibronectin but not by laminin-1 in comparison with that on type I collagen as a reference. Clustering and differentiation of AL cells were observed on all matrices examined. AL cells showed normal growth and differentiation at low cell density on fibronectin but not on type I collagen. Furthermore, the population of cytokeratin 14-positive cells on fibronectin was lower than that on other ECM components, suggesting that fibronectin may be a modulator to accelerate the differentiation of AL cells. After the cells had been cultured for 9 days on fibronectin, crystal-like structures were observed. These structures overlaid the cell clusters and were positive for von Kossa staining. These findings indicate that each matrix component has a regulative role in the proliferation and differentiation of AL cells and that fibronectin causes the greatest acceleration of AL cell differentiation.     

Distribution of laminin and collagens during avian neural crest development

The distribution of type I, III and IV collagens and laminin during neural crest development was studied by immunofluorescence labelling of early avian embryos. These components, except type III collagen, were present prior to both cephalic and trunk neural crest appearance. Type I collagen was widely distributed throughout the embryo in the basement membranes of epithelia as well as in the extracellular spaces associated with mesenchymes. Type IV collagen and laminin shared a common distribution primarily in the basal surfaces of epithelia and in close association with developing nerves and muscle. In striking contrast with the other collagens and laminin, type III collagen appeared secondarily during embryogenesis in a restricted pattern in connective tissues. The distribution and fate of laminin and type I and IV collagens could be correlated spatially and temporally with morphogenetic events during neural crest development. Type IV collagen and lamin disappeared from the basal surface of the neural tube at sites where neural crest cells were emerging. During the course of neural crest cell migration, type I collagen was particularly abundant along migratory pathways whereas type IV collagen and laminin were distributed in the basal surfaces of the epithelia lining these pathways but were rarely seen in large amounts among neural crest cells. In contrast, termination of neural crest cell migration and aggregation into ganglia were correlated in many cases with the loss of type I collagen and with the appearance of type IV collagen and laminin among the neural crest population. Type III collagen was not observed associated with neural crest cells during their development. These observations suggest that laminin and both type I and IV collagens may be involved with different functional specificities during neural crest ontogeny. (i) Type I collagen associated with fibronectins is a major component of the extracellular spaces of the young embryo. Together with other components, it may contribute to the three-dimensional organization and functions of the matrix during neural crest cell migration. (ii) Type III collagen is apparently not required for tissue remodelling and cell migration during early embryogenesis. (iii) Type IV collagen and laminin are important components of the basal surface of epithelia and their distribution is consistent with tissue remodelling that occurs during neural crest cell emigration and aggregation into ganglia.     

Immunohistochemical localization of type I collagen, fibronectin and tenascin C during embryonic osteogenesis in the dentary of mandibles and tibias in rats

Type I collagen, fibronectin and tenascin C play an important role in regulating early osteoblast differentiation, but the temporal and spatial relationship of their localization during embryonic osteogenesis in vivo is not known. The present study was designed to localize these three molecules in the dentary of mandibles and tibias in rat embryos using immunohistochemistry. Serial paraffin sections were cut and adjacent sections were processed for von Kossa staining or immunohistochemistry for type I collagen, fibronectin and tenascin C. In the dentary, tenascin C was localized within and around the mesenchymal cell condensation in embryos at 14 days in utero. The bone matrix at 15 days showed immunoreactivity for both type I collagen and fibronectin. The immunoreactivity of type I collagen was persistent, whereas that of fibronectin decreased with age of embryos. In tibias, tenascin C was localized in the perichondral mesenchymal tissue at 17 days. Immunoreactivity for type I collagen was persistent in the bone matrix, whereas the tibial bone showed little immunoreactivity for fibronectin at any embryonic age examined. The present study demonstrated characteristic localization of type I collagen, fibronectin and tenascin C during embryonic osteogenesis in the dentary of mandibles and tibias.     

Participation of hepatocytes in the production of basement membrane components in human and rat liver during the perinatal period

Little is known about the role of the extracellular matrix in cellular growth, migration and differentiation in the developing liver. The distribution and origin of the main constituents of the hepatic extracellular matrix have never been studied during liver differentiation. We have investigated the extracellular and intracellular distribution of fibronectin, laminin and types I, III and IV collagen in both rat and human liver during the perinatal period by light and electron microscopy, using the indirect immunoperoxidase method. All these components were demonstrated extracellularly, located mainly in portal spaces and, to a lesser extent, surrounding central veins. In perisinusoidal spaces, variations in distribution were observed depending on the matrix protein, the age of the donor and the species. In fetal rat liver, fibronectin formed a continuous layer around hepatocyte clusters while laminin and type III procollagen were present in small amounts. Collagens and laminin were visualized more easily in newborn rat liver. Fetal and newborn human liver contained higher amounts of matrix components than their rat counterparts. Fibronectin also reacted strongly in the sinusoid, and laminin and collagens formed discontinuous deposits. The source of this extracellular matrix was demonstrated to be of mixed origin. The major finding was the presence of immunoreactive laminin in the rough endoplasmic reticulum of hepatocytes irrespective of the age or species. In addition, hepatocytes contained large amounts of fibronectin and little of type I collagen. Another basement membrane component, type IV collagen, was also found in hepatocytes from all groups except fetal rat. Perisinusoidal cells also contained various matrix components including laminin, type III procollagen and, again with the exception of fetal rat liver, type IV collagen. The greater amounts of basement membrane components in the sinusoids of developing liver than in adult tissue and the participation of immature hepatocytes in the production of laminin and to a lesser degree of type IV collagen suggest that these matrix proteins play a critical role during liver differentiation.     

Immunohistochemical studies on the tissue localization of collagen types I, III, IV, V and VI in schwannomas. Correlation with ultrastructural features of the extracellular matrix

The distinctive tissue localization of collagen types in typical schwannomas with Antoni type A and B areas was demonstrated immunohistochemically using affinity-purified antibodies against types I, III, IV, V and VI collagen and comparative ultrastructural studies were made on the extracellular matrix components. Antoni type A tissue, which was composed of tightly packed spindle cells with long cytoplasmic processes surrounded by a continuous basement membrane and a few fibrillar components of the extracellular matrix, was almost exclusively immunoreactive for type IV collagen, presumably representing the basement membrane. Verocay bodies, which are organoid structures of Antoni type A tissue, had a variety of more abundant extracellular fibrous components, such as banded collagen fibrils, fibrous long-spacing fibrils and microfibrils. These were positive for type I and III, as well as type IV collagen. In Antoni type B areas, where two types to tumor cells designated Schwann cell-like and fibroblast-like were scattered in large amounts of amorphous extracellular matrix containing microfibrils and thick banded collagen fibrils, type VI collagen as well as types I, III and IV collagen were consistently detected. Type V collagen was localized in dense fibrous tissue areas and around blood vessels. These findings indicate that the differently organized cellular patterns of schwannomas, identified as Antoni types A and B, are characterized not only by the ultrastructural features of the extracellular matrix, but also by the distinctive collagen types produced by neoplastic Schwann cells.     

Extracellular matrix (ECM) synthesis in muscle cell cultures: quantitative and qualitative studies during myogenesis

When the synthesis of extracellular matrix components was examined in G8-1 murine skeletal muscle cells as a function of differentiation, non-collagen and to an even greater extent collagen synthesis was increased. Specifically, collagen types I, III, IV, laminin and fibronectin were identified by SDS-PAGE. Immunoprecipitation, with specific antibodies revealed that both the cell layer and medium of differentiated multinucleated myotubes contained increased levels of type IV collagen and laminin, decreased levels of type III collagen and fibronectin and equivalent levels of type I collagen compared to mononuclear myoblasts.     

Influence of various extracellular matrix components on the behavior of cultured chick embryo dermal cells

Dermal cells isolated from the back of 7-day chick embryos were cultured on homogeneous two-dimensional substrates consisting of one or two extracellular matrix components (type I, III or IV collagen, fibronectin and several glycosaminoglycans: hyaluronate, chondroitin-4, chondroitin-6, dermatan or heparan sulfate). The effect of these substrates on cell behavior was compared with that of culture dish polystyrene. Three parameters of cell behavior were examined: cell proliferation and patterning, spreading (cell surface) and locomotion (velocity and directionality). Data were collected by sequential microphotography and analyzed by computer assisted morphometry. Types I and III collagen, hyaluronate and heparan sulfate had a slowing down effect on cell proliferation and patterning. The inhibitory effect of type I collagen was also detected in mixtures with glycosaminoglycans. The other components had no effect. While the smallest spreading was observed on fibronectin substrate, the largest was recorded on chondroitin-6 sulfate and heparan sulfate. The slowest velocity of locomotion was measured on fibronectin, types I and IV collagen and a mixture of type I collagen and chondroitin-6 sulfate. The fastest speed was recorded on chondroitin-4 sulfate. These effects are discussed in view of our knowledge of the role of the dermis in the development of skin and cutaneous appendages, and in the light of the morphogenetically related microheterogeneous distribution of collagens, fibronectin and various glycosaminoglycans in the developing skin.     

Ultrastructural study on the specific binding of genetically engineered epidermal growth factor to type I collagen fibrils

In an attempt to develop collagen-growth factor composites for use in tissue engineering, chimeric proteins consisting of epidermal growth factor and collagen binding domains derived from von Willebrand factor or fibronectin were synthesized by means of recombinant technology. These chimeric proteins were bound to type I collagen fibrils, and the ultrastructures of composites were analyzed by transmission electron microscopy combined with the gold nanoparticle labeling technique. The results of the ultrastructural study revealed that chimeric proteins were densely assembled on collagen fibrils through the specific recognition of binding sites, producing the ordered array of chimeric proteins.     

Collagen-binding proteins secreted by type II pneumocytes in culture

The alveolar epithelial basement membrane is believed to play important roles in lung development, in maintaining normal alveolar architecture, and in guiding repair following lung injury. However, little is known about the formation of this structure, or of the mechanisms which mediate interactions between the epithelium and specific matrix macromolecules. Since type IV collagen is a major structural component of basement membranes, we investigated the production of type IV collagen-binding proteins by primary cultures of rat lung type II pneumocytes. Cultures were labeled for up to 24 h with 3H-labeled amino acids or [3H]mannose. Soluble collagen-binding proteins which accumulated in the culture medium were isolated by chromatography on collagen-Sepharose and examined by SDS-polyacrylamide gel electrophoresis. The major type IV collagen-binding protein (CBP1) was identified as fibronectin. We also identified a novel disulfide-bonded collagen-binding glycoprotein (CBP2; Mr = 45,000, reduced). This protein was not recognized by polyclonal antibodies to fibronectin, and showed no detectable binding to denatured type I collagen. The protein was resolved from fibronectin and partially purified by sequential chromatography on gelatin and type IV collagen-Sepharose. We suggest that type II pneumocyte-derived collagen-binding proteins contribute to the formation of the epithelial basement membrane and/or mediate the attachment of these cells to collagenous components of the extracellular matrix.     

Metabolic changes in the extracellular matrix during differentiation of myoblasts of the L6 line and of a Myo- non-fusing mutant

In the present study we have characterized by biochemical and immunochemical methods the changes which take place in collagen, laminin and fibronectin biosynthesis during the differentiation of clonal skeletal myoblasts of the L6 line. Time-course experiments showed that the relative rate of synthesis of collagen increased significantly during the cell-cell contact step of myogenesis and decreased later on. The major collagens synthesized were types I and III, found mainly as soluble precursors in the culture medium. Types IV and V collagens were detected exclusively in the cell layer. The relative amounts of types I and III collagens remained unchanged during myogenesis, while types IV and V collagens increased as the cells of the L6 line fused. In a non-fusing alpha-amanitin-resistant mutant of the L6 line (Ama 102), the rate of collagen synthesis was largely depressed and its rate of degradation was increased as compared with the fusing wild type. The synthesis of laminin was very low in cells of the fusing wild type, but abundant and associated with the cell layer of the Myo- mutant. The appearance of a muscle-specific extracellular matrix is a complex process involving changes in the organization, the biosynthesis and remodelling of its macromolecules of the extracellular matrix.     

Endothelial Matrix Assembly during Capillary Morphogenesis: Insights from Chimeric TagRFP-Fibronectin Matrix

Biologically relevant, three-dimensional extracellular matrix is an essential component of in vitro vasculogenesis models. WI-38 fibroblasts assemble a 3D matrix that induces endothelial tubulogenesis, but this model is challenged by fibroblast senescence and the inability to distinguish endothelial cell-derived matrix from matrix made by WI-38 fibroblasts. Matrices produced by hTERT-immortalized WI-38 recapitulated those produced by wild type fibroblasts. ECM fibrils were heavily populated by tenascin-C, fibronectin, and type VI collagen. Nearly half of the total type I collagen, but only a small fraction of the type IV collagen, were incorporated into ECM. Stable hTERT-WI-38 transfectants expressing TagRFP-fibronectin incorporated TagRFP into ~90% of the fibronectin in 3D matrices. TagRFP-fibronectin colocalized with tenascin-C and with type I collagen in a pattern that was similar to that seen in matrices from wild type WI-38. Human Umbilical Vein Endothelial Cells (HUVEC) formed 3D adhesions and tubes on WI38-hTERT-TagRFP-FN-derived matrices, and the TagRFP-fibronectin component of this new 3D human fibroblast matrix model facilitated the demonstration of concentrated membrane type 1 metalloprotease and new HUVEC FN and collagen type IV fibrils during EC tubulogenesis. These findings indicate that WI-38-hTERT- and WI-38-hTERT-TagRFP-FN-derived matrices provide platforms for the definition of new matrix assembly and remodeling events during vasculogenesis.     

Immunohistochemistry of the intercellular matrix components and the epithelio-mesenchymal junction of the human tooth germ

Summary The immunohistochemical localization of heparan sulphate, collagen type I, III and IV, laminin, tenascin, plasma- and cellular fibronectin was studied in tooth germs from human fetuses. The lamina basalis ameloblastica or membrana preformativa, which separates the pre-ameloblasts from the pre-dentin and dentin, contained heparan sulphate, collagen type IV, laminin and fibronectin. Enamel reacted with antifibronectin, but the reaction varied depending on the type of fibronectin and the source of antibody. In early pre-dentin, collagen type I, laminin, tenascin and fibronectin were present. In late pre-dentin and dentin collagen type I was found in intertubular dentin and in the zone between enamel and dentin. The close relationship between collagen type I in dentin and fibronectin in immature enamel is interesting, as it may contribute to the stabilization of the amelodentinal interface. In dental pulp, collagen type IV and laminin were found in the endothelial basement membranes. Collagen type I and III, tenascin and fibronectin were localized to the mesenchymal intercellular matrix.The results of this study have supported the assumption that the lamina basalis ameloblastica is a basement membrane, and have lead to the suggestion that ameloblasts are producers of fibronectin or a fibronectin-like substance.     

Effect of hydrocortisone on skin development in the chick embryo: ultrastructural, immunohistological, and biochemical analysis

The effect of hydrocortisone on the development of dorsal skin was analyzed in the chick embryo by (1) transmission electron microscopy, (2) indirect immunofluorescence histology of extracellular matrix components (collagen types I, III, and IV; fibronectin; and laminin), and (3) quantitative determination of collagen content and proline incorporation, between administration of the drug at 6 or 6.5 days and final retrieval of skin pieces at 11 days of incubation. Treatment caused the formation of featherless skin areas which exhibited an early maturation of the epidermis, a uniform distribution of interstitial collagen and rarefaction of fibronectin in the dermal extracellular matrix, and a significant increase of collagen content and proline incorporation in collagen noncollagen proteins, characterized by an increased hydroxyproline-to-proline ratio. The distribution of type IV collagen and of laminin was unchanged. The absence of feather formation in hydrocortisone-induced apteria is interpreted as resulting primarily from an early extinction of epidermal morphogenetic competence, and secondarily from modifications in the amount and distribution of extracellular matrix components in the dermis.     

Development of osteogenic tissue in diffusion chambers from early precursor cells in bone marrow of adult rats

Diffusion chambers containing bone marrow cells from adult rats were implanted intraperitoneally into rat hosts and cultured in vivo for up to 64 days. Biochemical and histological analyses of the contents of the chambers demonstrate that a connective tissue consisting of bone, cartilage and fibrous tissues is formed by precursor cells present in marrow stroma. The amounts of osteogenic tissue and DNA are directly correlated with time of implantation and with number of cells inoculated. In the chambers there is initial formation of fibrous tissue which is strongly reactive to collagen type III, laminin and fibronectin. In areas of osteogenesis which appear later within this fibrous anlage, expression of collagen type III, laminin and fibronectin decrease and collagen types I and II increase in association with bone and cartilage respectively. Where osteogenesis does not develop, fibrous tissue continues to express collagen type III. The sequential expression of the different extracellular matrix components is similar to that previously observed during osteogenic differentiation in embryonic and adult developmental systems. It is concluded that the formation of fibrous and osteogenic tissues in diffusion chambers by precursor cells present in adult marrow, resembles the normal developmental process.     

Differences in lectin binding patterns of normal human endometrium between proliferative and secretory phases

Summary The biological fate of a bovine collagen implant (Zyderm Collagen Implant ZCI), injected subcutaneously into rats, was studied by the immunoperoxidase technique using specific antibodies against the bovine implant and against types I, III, IV, V collagens, fibronectin and elastin. The implant remained in the animals until the end of the experiment (90 days), with no visible modification, as demonstrated by immunoperoxidase labelling and scanning electron microscopy. A slight inflammatory reaction was visible around the implant 24 h after injection and within the implant 3 days after injection. Fibroblast invasion began 7 days after injection. The chronology of the deposition in the implant of the host (rat) extracellular matrix components was as follows: by 24 h after injection, fibronectin was observed throughout the implant; types I and V collagens appeared on the 7th day, and, in contrast to surrounding connective tissue, type V collage labelling was obtained without acid pretreatment of the section. Types III and IV collagens were detected inside the implant only 30 days after injection. At the end of the experiment (90 days), there was abundant types I and V collagens after fibroblast migration, and abundant type IV collagen demonstrating an important vascularization. No elastic fibres could be detected inside the implant but they appeared as a dense network around the implant in host connective tissue.     

Changes in the distribution of type IV collagen,laminin, proteoglycan,and fibronectin during mouse tooth development

The distribution of certain basement membrane (BM) components including type IV collagen, laminin, BM proteoglycan, and fibronectin was studied in developing mouse molar teeth, using antibodies or antisera specific for these substances in indirect immunofluorescence. At the onset of cuspal morphogenesis, type IV collagen, laminin, and BM proteoglycan were found to be present throughout the basement membranes of the tooth. Fibronectin was abundant under the inner enamel epithelium at the region of differentiating odontoblasts and also in the mesenchymal tissues. After the first layer of predentin had been secreted by the odontoblasts at the epithelial-mesenchymal interface, laminin remained in close association with the epithelial cells whereas type IV collagen, BM proteoglycan, and fibronectin were distributed uniformly throughout this area. Later when dentin had been produced and the epithelial cells had differentiated into ameloblasts, basement membrane components disappeared from the cuspal area. These matrix components were not detected in dentin while BM proteoglycan and fibronectin were present in predentin. The observed changes in the collagenous and noncollagenous glycoproteins and the proteoglycan appear to be closely associated with cell differentiation and matrix secretion in the developing tooth.     

A Collagen-Binding S-Layer Protein in Lactobacillus crispatus

Two S-layer-expressing strains, Lactobacillus crispatus JCM 5810 and Lactobacillus acidophilus JCM 1132, were assessed for adherence to proteins of the mammalian extracellular matrix. L. crispatus JCM 5810 adhered efficiently to immobilized type IV and I collagens, laminin, and, with a lower affinity, to type V collagen and fibronectin. Strain JCM 1132 did not exhibit detectable adhesiveness. Within the fibronectin molecule, JCM 5810 recognized the 120-kDa cell-binding fragment of the protein, while no bacterial adhesion to the amino-terminal 30-kDa or the gelatin-binding 40-kDa fragment was detected. JCM 5810 but not JCM 1132 also bound (sup125)I-labelled soluble type IV collagen, and this binding was efficiently inhibited by unlabelled type IV and I collagens and less efficiently by type V collagen, but not by laminin or fibronectin. L. crispatus JCM 5810 but not L. acidophilus JCM 1132 also adhered to Matrigel, a reconstituted basement membrane preparation from mouse sarcoma cells, as well as to the extracellular matrix prepared from human Intestine 407 cells. S-layers from both strains were extracted with 2 M guanidine hydrochloride, separated by electrophoresis, and transferred to nitrocellulose sheets. The S-layer protein from JCM 5810 bound (sup125)I-labelled type IV collagen, whereas no binding was seen with the S-layer protein from JCM 1132. Binding of (sup125)I-collagen IV to the JCM 5810 S-layer protein was effectively inhibited by unlabelled type I and IV collagens but not by type V collagen, laminin, or fibronectin. It was concluded that L. crispatus JCM 5810 has the capacity to adhere to human subintestinal extracellular matrix via a collagen-binding S-layer.