Packed Collagen Fibrils Research Articles

Flexilin: a new extracellular matrix glycoprotein localized on collagen fibrils.

We have immunopurified and characterized a new glycoprotein of the extracellular matrix, using a monoclonal antibody obtained after immunization with fibril-associated collagens extracted from bovine tendon. In polyacrylamide gels, the protein migrates at about 350 kDa molecular mass. The protein is insensitive to bacterial collagenase, and no disulfide-linked aggregates could be detected; sugars were stained with periodic acid-Schiff's reagent. Amino acid analysis and sequencing of tryptic peptides failed to detect any similarity with known proteins. By rotary shadowing experiments, the protein was observed as flexible, unbranched structures, approximately 150 nm long, with a small globule at one end. Investigation of the tissue distribution of the protein in fetal bovine tissues by immunofluorescence resulted in labeling in extracellular matrices with loosely packed collagen fibrils, such as the peritendineum, embryonic skin and kidney glomeruli; cornea, cartilage matrix and bone were not labeled. Ultrastructural immunolocalization in dermis and in mesangium of glomeruli showed that the protein always occurred in the vicinity of collagen fibrils. In view of its tissue distribution and molecular shape, we postulate that this protein is important in the properties of the extrafibrillar environment. By reference to its shape as observed by rotary shadowing, we propose the name 'flexilin' for this extracellular matrix glycoprotein.

Distribution of undulin, tenascin, and fibronectin in the human periodontal ligament and cementum: comparative immunoelectron microscopy with ultra-thin cryosections.

We studied the ultrastructural localization of three distantly related glycoproteins of the extracellular matrix, undulin, tenascin and fibronectin, in decalcified sections of human periodontal ligament (PL) and cementum. Undulin was associated with tightly packed major collagen fibrils and not with microfibrils, indicating that this protein may be involved in the supramolecular and functional organization of collagen fibrils into flexible bundles. Tenascin was found on globular masses between less densely packed collagen fibrils, thus displaying a pattern quite distinct from that of undulin. Fibronectin was noted in bulky material between the cross-striated fibrils, often surrounding individual fibrils like garlands, and in the microfibrillar meshwork extending from cross-striated fibrils. The three glycoproteins displayed a distinct and unique pattern of distribution in PL that can be correlated with their molecular structure and potential functions.

Scanning Electron Microscopic Study of the Collagen Networks of the Normal Mucosa, Hyperplastic Polyps, Tubular Adenoma, and Adenocarcinoma of the Human Large Intestine.

The three-dimensional structure of the collagen network of normal mucosa, hyperplastic polyps, tubular adenoma, villous adenoma and adenocarcinoma of the human large intestine was examined by scanning electron microscopy after cell-maceration with a low temperature NaOH solution. In the normal large intestine, the surface of the sub-basal laminar collagen sheath lining the crypts was generally smooth. In hyperplastic polyps, the crypts extended and the sub-basal collagen network protruded from the surrounding normal tissue. The sub-basal laminar collagen sheath was more densely arranged near the openings of the crypts and on the luminal surface than in deeper regions of the crypts. In tubular adenoma, the tumor glands showed meandering, bending, and divisions. The sub-basal laminar collagen sheath was composed of densely packed collagen fibrils. In villous adenoma, the sub-basal laminar collagen sheath formed foliate or linguiform villous cores projecting sharply into the lumen. In adenocarcinoma, the sub-basal laminar collagen sheath exhibited a dense arrangement of fibrils in the central region of the tumor. Toward the marginal region, the collagen sheaths surrounding tumor glands became thinner, and at the edge of the marginal region, basket-like collagen sheaths were observed around individual tumor cells infiltrating the interstitium. Such a tumor lesion was frequently surrounded by a thick collagen fiber zone.

Ultrastructure of the bone-titanium interface in rabbits

The interface zone between cortical bone and threaded non-alloyed titanium implants inserted in the rabbit tibia for 12 months was examined by light and electron microscopy. The implants were removeden bloc with the perfusion-fixed surrounding bone and the undecalcified specimens were, after osmification, dehydrated and embedded in plastic resin (LR White). In ground sections (about 10 µm thick) cortical bone appeared to be in direct contact with the implant surface and the implants were thus “osseointegrated”. Sections for light microscopy (1 µm thick) and electron microscopy (40 nm to 0.5 µm) were prepared by using an electropolishing technique by which the bulk part of the metal was electrochemically removed and a fracture technique by which the implant was separated from the embedded tissue before sectioning. In the electropolished specimens an unmineralized zone, 2–10 µm wide, was observed at the interface. The interface zone contained osteoid-like tissue (densely packed collagen fibrils, osteocyte canaliculi) but in general no deposits of calcium mineral. This feature of the interface could not be observed in specimens prepared by the fracture technique, indicating that the electropolishing technique had induced serious artefacts, including decalcification of the interface bone. In sections prepared by the fracture technique, mineralized bone was present very close to the implant surface. No gradient of mineral was observed. A thin layer of amorphous material (100–200 nm wide) was present peripheral to the mineralized bone. An electron dense line about 100 nm wide was formed at the border between the mineralized bone and the amorphous layer. The dense layer had the same characteristics as the lamina limitans observed around osteocyte lacunae and canaliculi or the zone between areas of bone with different degree of mineralization.

Beta-D xyloside alters dermatan sulfate proteoglycan synthesis and the organization of the developing avian corneal stroma.

Corneal transparency is dependent upon the development of an organized extracellular matrix containing small diameter collagen fibrils with regular spacing, organized as orthogonal lamellae. Proteoglycan-collagen interactions have been implicated in the regulation of collagen fibrillogenesis and matrix assembly. To determine the role of dermatan sulfate proteoglycan in the development and organization of the secondary corneal stroma, its synthesis was disrupted using beta-D xyloside. The secondary corneal stroma contains two different proteoglycans, dermatan sulfate and keratan sulfate proteoglycan. beta-D xyloside interferes with xylose-mediated O-linked proteoglycan synthesis, and thus disrupts dermatan sulfate proteoglycan synthesis. Corneal keratan sulfate proteoglycan, a mannose-mediated N-linked proteoglycan, should not be altered. Biochemical analysis of corneas treated both in vitro and in ovo revealed a reduced synthesis of normally glycosylated dermatan sulfate proteoglycans and an increased synthesis of free xyloside-dermatan sulfate glycosaminoglycans. Keratan sulfate proteoglycan synthesis was unaltered in both cases. Corneal stromas were studied using histochemistry and electron microscopy after in ovo treatment with beta-D xyloside. The observed biochemical alterations in dermatan sulfate proteoglycans translated into disruptions in the organization of beta-D xyloside-treated stromas. There was a reduction in the histochemical staining of proteoglycans, but no alteration in collagen fibril diameter. In addition, focal alterations in collagen fibril packing, and a disruption of lamellar organization were observed in beta-D xyloside-treated corneas. These data suggest that dermatan sulfate proteoglycans are not involved in the regulation of corneal collagen fibril diameter, but are important in the fibril-fibril spacing as well as in lamellar organization, and cohesiveness.

Liquid crystallinity in condensed type I collagen solutions: A clue to the packing of collagen in extracellular matrices

We recently described a new type of assembly of collagen molecules, forming typical liquid crystalline phases in highly concentrated solutions after sonication. The present work shows that intact 300 nm long collagen molecules also form cholesteric liquid crystalline domains, but the time required is much longer, several weeks instead of several days. Differential calorimetry and X-ray diffraction show that sonication does not alter the triple-helical structure of the collagen fragments. In the viscous solutions, observed between crossed polars in optical microscopy, the textures vary as a function of the concentration. Molecules first align near the air interface at the coverslip edge, then as the concentration increases by slow evaporation of the solvent, the birefringence extends inwards and liquid crystalline domains progressively appear. For concentrations estimated to be above 100 mg/ml, typical textures and defects of cholesteric phases are obtained, at lower concentrations zig-zag extinction patterns and banded patterns are observed; all these textures are described and interpreted. The cholesteric packing of collagen fibrils in various extracellular matrices is known, and the relationship that can be made between the ordered phases obtained with collagen molecules in vitro and the related geometrical structures observed between fibrils in vivo is thoroughly discussed.

Solitary sclerotic fibroma

A 63-year-old Chinese man with a solitary sclerotic fibroma on the abdomen is described. Skin biopsy showed a well-demarcated dermal nodule composed of hypocellular, eosinophilic collagen bundles separated by prominent clefts. Retrospective electron microscopic study revealed wide collagen bundles containing tightly packed collagen fibrils, only 50 nm in diameter. There were no signs of Cowden's disease. To our knowledge, this is the first report of electron microscopic study of sclerotic fibroma. A brief review of the literature is included.

Tissue reaction to subcutaneous implantation of a collagen sponge. A histological, ultrastructural, and immunological study

The biocompatibility of a subcutaneously implanted bovine collagen sponge (Haemostagen) was studied in rats by analyzing tissue reactions up to 3 months by histological and ultrastructural methods; in addition, the level of serum antibodies to bovine type I collagen (the major implant collagen) was measured by solid-phase radioimmunoassay. By 8 h after implantation, the implant was completely filled with polymorphonuclear cells (PMNs). After 8 days, fibroblasts had developed a granulation tissue within the sponge and the PMNs had almost disappeared. The small residue that remained after 1 month consisted of some densely packed collagen fibrils containing giant cells, which had disappeared by 3 months. No antibodies to bovine type I collagen were found in the sera of implanted rats.

Biomechanical and histomorphological changes in expanded rabbit skin

In 12 rabbits, biomechanical and histomorphological properties were determined in expanded, sham-operated and non-operated control skin. Results were evaluated in paired fashion. Both expanded and sham-operated skin showed a significant loss of stiffness of 60% and 35% respectively (ϱ < 0·001 and ϱ < 0.05). In addition, maximum stiffness in expanded skin was reached at a shorter extensibility than in sham-operated or non-operated control skin. Histomorphological examination revealed thickened dermal collagen bundles with loosened packing of collagen fibrils in both expanded and sham-operated skin. It is concluded that the surgical procedure of balloon implantation is responsible for a significant part of the reported biomechanical and histomorphological findings. Wound healing, in addition to the delay phenomenon, may therefore be a mechanism involved in skin expansion.

In-vitro formation of a new fibrous attachment to human dental roots in the presence of autologous serum.

Recent studies have accomplished the establishment of a collagenous fiber-fringe matrix upon dental root surfaces in vitro. The present study was undertaken to follow the development of such a matrix in vitro and to test the possible effects of root surface treatments upon this matrix. Periodontal ligament cells, 0.1 to 0.2-mm thick dental root discs, and alveolar bone cells were derived after extraction from four partially erupted third molars and the accompanying interradicular bony septa of 1 male patient. Autologous serum was obtained by venipuncture. Cultures were initiated by delivering a 1-ml suspension of 50,000 tritiated thymidine-labeled periodontal ligament cells and 50,000 alveolar bone cells onto each of 42 culture sets. The following day, demineralized or non-demineralized root discs treated with autologous serum, fibronectin or complete medium were placed in pairs, separated by a 0.1-1.0 mm gap, upon the initial cell layer. Representative cultures were terminated after 2, 3, 4, 5 and 6 weeks, and processed for light- and electron microscopy, morphometric analysis and autoradiography. An outstanding feature of the early cultures (2, 3 and 4 weeks) was a patchwise, random distribution of matrix making a precise developmental study impossible, although collagen fibrils were produced within the first 2 weeks. Some 3-week cultures already demonstrated a mature fiber-fringe characterized electron-microscopically as oriented, densely packed collagen fibrils closely abutting the cementum-lined root discs. The treatments (including autologous serum) used in this study had no appreciable morphologic or morphometric effect upon the fiber-fringe formed. Because none of the cultures in the present or past studies have demonstrated a true cementoid matrix, this model may not be suitable for the in-vitro study of cementum formation.

Glycosaminoglycans facilitate the movement of fibroblasts through three-dimensional collagen matrices

The effect of glycosaminoglycans on the invasion of choroid fibroblasts into type I collagen gels was studied. Both hyaluronate and chondroitin sulphate, when incorporated into the gel, facilitated invasion of the collagen matrix, although hyaluronate was considerably more effective. Hyaluronate-induced fibroblast invasion was markedly concentration-dependent, being reduced at both high and low concentrations. Increased cell invasion appeared to correlate with denser packing of collagen fibrils within the gel, since the same effect could be achieved by increasing the collagen concentration of native, i.e. glycosaminoglycan-free gels. Scanning electron microscopy of the interior of the collagen gels suggested that changes in packing arrangement of fibrils in gels that had polymerized in the presence of glycosaminoglycans might account in part for different rates of cell invasion.

Ultrastructural data on the scales of the dipnoan Protoptems annectens (Sarcopterygii, Osteichthyes)

The structure and the mineralization of the scales of the living dipnoan (lungfish) Protoptems annectens have been investigated by transmission electron microscopy (TEM). The thin and imbricated scales are composed of two layers: the squamulae and the basal plate. At the outer surface, the squamulae form isolated plates superficially ornamented with spines and concretions and made up of acellular bone. After demineralization, the squamulae show a heterogeneous organic matrix composed of thin randomly oriented collagen fibrils forming a loose network within which the concretions appear as electronlucent circular areas. Abundant and aggregated concretions are located within the spines. The crystallites are oriented by the collagen fibrils except in the concretions. Anchoring bundles composed of parallel collagen fibrils arise from the squamulae and connect the scales to the overlying dermis.The basal plate, the most developed part of the scale, is made up of isopedine. Its main component consists of thick, closely packed collagen fibrils organized in a ‘double twisted plywood‐like structure’. Fibroblasts are present in the basal plate. Mineralization occurs only in few plies located beneath the squamulae. Mandl's corpuscles are found in front of the mineralization front. The mineral deposit is oriented by the collagen fibrils.The scales of Protoptems annectens differ from the typical elasmoid scales of the teleosts by the peculiar structure of the squamulae, nevertheless they show enough structural characteristics to support the hypothesis that they can be considered as scales of the elasmoid grade, which have retained some plesiomorphic characteristics.

Transmission electron microscopic study of giant tubules in bovine dentin.

Giant tubules in the axiomesiodistally extended incisal dentin of unerupted permanent bovine incisors from 1/2-1 1/2-yr-old calves were studied by TEM. The pulpal portion of each giant tubule contained two capillaries forming a loop incisally. The capillaries consisted of a delicate, continuous, or fenestrated endothelium and often had a pericyte coating. Cells with a rich RER, mitochondria, and Golgi apparatus dominated along and just incisal to the vessels. Extravasated erythrocytes and occasional macrophages were also seen in the latter location. It was suggested that the pulpal retraction of the giant tubule vessels during primary dentinogenesis might be the result of an intermittent sequestration of the most incisal portion of the loop, concomitant with endothelial proliferation in the pulpal portion of the capillaries. The major portion of the giant tubules, incisal to the vessels, contained a dense matrix of more or less axially oriented unmineralized 0.07-0.1-micron-thick collagen fibrils and single cells showing gradually increasing degenerative and necrotic changes in an incisal direction. Longitudinal sections of the mineralized tubular walls revealed transversely cut collagen fibrils. The incisally located giant tubule origins were circumvented by dentinal tubules and contained 0.10-0.22-micron-thick, densely packed collagen fibrils with an electron dense perifibrillar zone.

Thin collagenous septa in cardiac muscle.

Light and electron microscopy were used to study the structure and distribution of thin collagenous septa (sheets) in dog and rabbit cardiac muscle to determine whether they, like thick collagenous septa, could affect electrical impulse propagation. Generally, thin septa (0.2-0.5 micron) ensheathed myocytes or groups of myocytes for short distances and thicker septa partially or completely ensheathed groups of myocytes for long distances (up to several mm); together, thin, and thick septa divided the myocardial mass into myocyte cords (funicles) of 10-30 micron diameter. Septal architecture varied not only between regions and within regions at different ages but also within single bundles, precluding the assumption that the architecture found in one bundle can be applied to another. Electron microscopy demonstrated that thick septa consisted of many tightly packed collagen fibrils, often with distinct layers running at different angles; thin septa consisting largely of circumferential collagen fibrils. Thin septa in dog ventricular papillary muscle generally contained few and widely spaced collagen fibrils, whereas thin septa in dog atrial Bachmann's bundle contained tightly packed collagen fibrils. In either site, thin septa were rarely breached by nexuses and thus marked sites where lateral intercellular electrical coupling was unlikely. Serial 7 micron cross sections of dog Bachmann's bundle stained by a modification of the picrosirius red technique showed that thin septa sometimes persisted uninterrupted over several myocyte lengths. The results provide evidence that thin septa comprised of tightly packed collagen fibrils may significantly modify impulse propagation transverse to the longitudinal axis of the myocytes.

Proteoglycan-collagen interactions in intervertebral disc. A chondroitin sulphate proteoglycan associates with collagen fibrils in rabbit annulus fibrosus at the d-e bands.

Rabbit annulus fibrosus and nucleus pulposus were analysed for hydroxyproline, chondroitin sulphate, keratan sulphate and dermatan sulphate. Tissue proteoglycans were stained for electron microscopy with Cupromeronic blue, used in the critical electrolyte concentration mode, with and without prior digestion by chondroitinase AC or ABC, hyaluronidase or keratanase. Collagen bands, a-e were demonstrated with UO2++. A chondroitin sulphate proteoglycan was found orthogonally associated with loosely packed collagen fibrils in annulus fibrosus at the d and e bands. The close metabolic and structural analogies with the dermatan sulphate proteoglycans previously shown to be located at collagen d-e bands in tendon, skin, etc. (Scott and Haigh (1985) Biosci. Rep. 5:71-81), are discussed. Tightly packed annulus collagen fibrils were surrounded by axially oriented proteoglycan filaments, mostly without specific locations.

The structure of the osteoderms in the Gekko: Tarentola mauritanica.

Histological and cytological analysis reveals that the osteoderms of Tarentola mauritanica are composed of an outer part superimposed on a basal region. The structure of both parts can be related to that of the surrounding dermis. The basal part of the osteoderms, inserted in the dense dermis, is made up of abundant closely packed collagen fibrils that orient the mineral deposit. The outer part, located in the superficial loose dermis, is crossed by few bundles of mineralized collagen fibrils arising from the basal part. These bundles connect the osteoderm to the overlying loose dermis. The outer superficial part is characterized by the presence of mineralized globules surrounding the mineralized collagen bundles. In these globules, the crystals are deposited on a microfibrillar matrix rich in acidic mucosubstances and composed of radially oriented, tangled microfilaments that lie among the collagen bundles. The two different mineralizing systems in the osteoderms of Tarentola mauritanica may reflect two different organic matrices. The mineral is deposited in a preexisting dermal tissue, as a "metaplastic ossification," and is another expression of the potential retained by the reptilian dermis to form mineralized structures.

Ultrastructure of durapatite-periodontal tissue interface in human intrabony defects.

The ultrastructure of the interface between Durapatite (hydroxylapatite) and human periodontal tissues was examined. Durapatite was implanted into the intrabony periodontal defects during periodontal surgery. Reentry procedures were performed after 1 year and the tissues in the defects were biopsied and processed for transmission electron microscopy. Nineteen tissue blocks from four patients were examined, 17 contained Durapatite particles embedded in fibrous tissue and two contained particles encased in bone, all without inflammation. The fibrous connective tissue consisted of densely packed collagen fibrils surrounding the implant particles. The bone surrounding the Durapatite consisted in one case of relatively mature bone, and in the other of osteoid tissue. A granular, amorphous, collagen-free, electron-dense layer was routinely observed between implant and tissue. This layer was thicker in the bone-encased samples than in those surrounded by fibrous connective tissue. Except for the particle surrounded by mature bone, this layer was continuous with an organic meshwork located on the periphery of the implant spaces. The ultrastructural features of the interface are consistent with the existence of a mucopolysaccharide "bonding zone" described by other investigators. The organic meshwork appears to outline areas similar in size and shape to the individual crystallites of hydroxylapatite. This may indicate that the reactive surface of hydroxylapatite is much larger than merely the exterior surface of the implant, a finding which may explain the apparently good tissue adhesion to the implant.

New data on the structure and the growth of the osteoderms in the reptile Anguis fragilis L. (Anguidae, Squamata).

Light and electron microscopy shows the osteoderms of Anguis fragilis to be small, flat disks located in the dermis along the adult trunk: microradiography established the extent of the mineralization. Each osteoderm coincides exactly with an epidermal fold forming the keratinized scales characteristic of the skin of reptiles. Sections perpendicular to the surface show two mineralized layers differing in histological and histochemical characteristics and in fine structure, although both contain collagen fibrils. The structure of each layer can be related to that of the surrounding dermis. The outer superficial layer located in the loose dermis contains few collagen bundles that form a discontinuous sheet at the upper surface of the osteoderms. This superficial layer appears to be constituted of units separated by furrows and is composed of woven fibered bone. The basal plate comprises stratified lamellae formed of parallel-oriented collagen fibrils; the fibrils of successive lamellae lie at right angles. The densely packed collagen fibrils of the basal plate are distributed similarly to those of the dense dermis within which it lies. This layer exhibits structural and histochemical characteristics of a lamellar bone. The presence of two different layers in the osteoderms of Anguis fragilis may reflect their mode of formation, which consists of the deposit of mineral crystals in the preexisting dermal tissue. This mineralization process, considered as a "metaplastic ossification," may reflect the potentiality retained by the dermis of reptiles to form mineralized structures.

Electron-microscopic studies on the physiological cell loss in the gastric mucosa of the golden hamster.

Fine-structural aspects of physiological cell loss in the gastric mucosa of the golden hamster were observed. As the surface mucous cell ascends along the gastric pit, the cell becomes taller and funnel-like in shape. The interfoveolar cell located at the superficial portion of the gastric pit has many lysosomes and a few lipid droplets in the cytoplasm. The nucleus moves toward the upper region of the cytoplasm, while the Golgi apparatus moves downward toward the infranuclear region. After the rupture of the apical plasma membrane takes place, the lateral and basal plasma membranes of this cell reamin in spite of loss of the cell contents. Between the basal plasma membrane of the interfoveolar cell and the capillary endothelium is a thick connective tissue layer characterized by densely packed collagen fibrils. The remaining basal and lateral plasma membranes of the ruptured cell and the thick underlying collagenous layer might play a role in protecting the tissue from potential damage induced by the physiological cell loss.

The structure of rat tail tendon fascicles.

Evidence is presented to support the view that the surface of a fascicle reflects the wave pattern of its constituent collagen fibrils. The wave pattern shows considerable variation within and between fascicles. Smooth continuous phase shifts in crimp angles and crimp lengths across the width and along the length of a fascicle are seen as explaining many of the varied crimp patterns seen. It is proposed that collagen fibril packing is a simple side-by-side aggregation with a degree of freedom in any direction in the register of the wave patterns.