A comparison of the immunofluorescent localization of collagen types I,III, and V with the distribution of reticular fibers on the same liver sections of the snow monkey (Macaca fuscata)

Summary Localizations of collagen types I, III, and V in monkey liver, as determined by the indirect immunofluorescence method, were photographically superimposed on the fibers revealed by silver-staining in the same tissue sections. Immunofluorescence for type I collagen was found to correspond with the brown collagen fibers and with some of the coarse reticular fibers, while that for type III collagen was found to correspond with most, but not all, reticular fibers of the liver as well as with the brown collagen fibers. The distribution of type V collagen coincides not only with the collagen fibers in the stroma of portal triads and around the central veins, but also with the coarse and fine reticular fibers in the liver lobules. By immuno-electron microscopy, reaction products with anti-type III and V collagens antibodies were demonstrated on cross-striated collagen fibrils, about 45 nm in diameter, in the space of Disse. From these observations, it is concluded that: (1) the fine reticular fibers are mainly composed of type III and type V collagens, and (2) the collagen fibers and coarse reticular fibers in the periphery of liver lobules are composed of type I, type III and type V collagens.     

Ultrastructural visualization of carbohydrates in oxytalan fibers in monkey periodontal ligaments

Fullmer's oxytalan fibers appear to be special connective tissue fibers belonging to elastic system fibers. We have ultrastructurally examined carbohydrates in oxytalan fibers in monkey periodontal ligaments after glutaraldehyde fixation and ethylenediaminetetraacetic acid (EDTA) decalcification using: Thiéry's periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for thin-section staining of vicinal glycol-containing complex carbohydrates, and the concanavalin A-ferritin (Con A-ferritin) and Con A-horseradish peroxidase (Con-A-HRP) en bloc staining methods specific for alpha-D-mannosyl and alpha-D-glucosyl groups. PA-TCH-SP stained collagen fibrils weakly to moderately and stained oxytalan fibers moderately. Con A-ferritin and Con A-HRP stained collagen fibrils weakly or moderately and stained oxytalan fibers intensely within the superficial region of specimen blocks. The penetration of staining reagents was improved by prior saponin treatment and/or chondroitinase ABC digestion. Thus, these studies demonstrate that PA-TCH-SP and Con A staining of carbohydrates is very useful in identifying oxytalan fibers at the ultrastructural level and that more carbohydrate components are present in oxytalan fibers than in collagen fibrils.     

Ultrastructural organization of the basic substance of human dermis]

The data on ultrastructural organization of the ground substance in the human dermis obtained electron histochemically are represented. Five types of ruthenium positive structures of polysaccharide origin are detected: retinal structure (I), amorfous substance (II), membranes of collagen fibrils (III) and elastic fibres (V), fine ruthenium positive streakness of collagen fibrils (IV). These structures, except fine streakness, form a united polysaccharide system of the dermis participating in maintenance of structural-functional integrity of the connective tissue (collagen-elastic) carcass of the dermis. Two mechanisms, interconnected and oppositely directed, perform this function: the buffer mechanism preventing the connective tissue fibers and collagen fibrils to approach each other, and the binding mechanism preventing the fibrils and fibers to dissociate. The reticular structure performs mainly this function at the level of fibers, and the amorphous substance does it at the level of fibrils.     

Tip-mediated fusion involving unipolar collagen fibrils accounts for rapid fibril elongation, the occurrence of fibrillar branched networks in skin and the paucity of collagen fibril ends in vertebrates.

Collagen fibrils are the principal source of mechanical strength of connective tissues such as tendon, skin, cornea, cartilage and bone. The ability of these tissues to withstand tensile forces is directly attributable to the length and diameter of the fibrils, and to interactions between individual fibrils. Although electron microscopy studies have provided information on fibril diameters, little is known about the length of fibrils in tissue and how fibrils interact with each other. The question of fibril length has been difficult to address because fibril ends are rarely observed in cross-sections of tissue. The paucity of fibril ends, or tips, has led to controversy about how long individual fibrils might be and how the fibrils grow in length and diameter. This review describes recent discoveries that are relevant to these questions. We now know that vertebrate collagen fibrils are synthesised as short (1-3 microm) early fibrils that fuse end-to-end in young tissues to generate very long fibrils. The diameter of the final fibril is determined by the diameter of the collagen early fibrils. During a late stage of tissue assembly fibril tips fuse to fibril shafts to generate branched networks. Of direct relevance to fibril fusion is the fact that collagen fibrils can be unipolar or bipolar, depending on the orientation of collagen molecules in the fibril. Fusion relies on: (1) specific molecular interactions at the carboxyl terminal ends of unipolar collagen fibrils; and (2) the insulator function of small proteoglycans to shield the surfaces of fibrils from inappropriate fusion reactions. The fusion of tips to shafts to produce branched networks of collagen fibrils is an elegant mechanism to increase the mechanical strength of tissues and provides an explanation for the paucity of fibril tips in older tissue.     

Type V collagen in splenic reticular fibers of the macaque monkey

The distribution of type I, III and V collagens in the monkey spleen was examined by indirect immunofluorescent microscopy and immunoelectron microscopy, and compared with that of reticular fibers revealed by a silver impregnation method. Type I collagen was localized on reticular fibers in the white pulps and on coarse reticular fibers in the splenic cords. Type III collagen was localized on the reticular fibers in the white pulps, and on the coarse reticular fibers and a limited number of fine reticular fibers, in the splenic cords. The anti-type V collagen antibody reacted with annular reticular fibers around the splenic sinuses, as well as with the reticular fibers in the white pulps and with the coarse and fine reticular fibers in the splenic cords. Thus, the distribution pattern of fibers that reacted with the anti-type V collagen antibody was very similar to that of the reticular fibers revealed by the silver impregnation method. Electron-microscopically, the fine reticular fibers in the splenic cords were composed of collagen fibrils, 30-50 nm in diameter, and amorphous substances. They were covered by reticular cell processes. By immunoperoxidase labeling with the anti-type V collagen antibody, electron-dense reaction products were found over the collagen fibrils with a banding pattern. These results indicate that type V collagen is an indispensable component of the reticular fibers.     

Fine structure of the fertilization membranes of sea urchin embryos

The fine structure of the fertilization membranes from S. purpuratus embryos has been studied with the electron microscope. Isolated membranes before and after their full development and membranes formed under the influence of 10⁻³% cystine have been observed. The membrane structure was found to be trilamella: a middle layer about 200 Å thick, which originally was the vitelline membrane, and about 175 Å thick peripheral layers organized by the “crystalline material” from the cortical granules. These surface layers were again found to be trilaminated structure composed of a monolayer of parallel, closely packed flat fibrils, about 160 Å wide and 75 Å thick, adhering on both sides to parallel, 40–50 Å thick filaments separated from each other by about 100 Å and intersecting with the fibrils by an angle of about 75 °.     

Amianthoid (asbestoid) transformation: electron microscopical studies on aging human costal cartilage

The present study reports on the fine structure of human costal cartilage at different ages in order to obtain information on the morphogenesis of amianthoid fibers. Our results reveal an overall increase of collagen fibril diameter with increasing age, even in areas with no signs of amianthoid transformation. Ultrastructural evidence is presented that this increase in diameter is due to a gathering of the preexisting collagen fibrils. The age-related change in collagen fibril diameter is paralleled by changes in the composition and ultrastructural appearance of cartilage proteoglycans (as revealed by acridine orange staining). Acridine-orange-positive filaments indicative for proteoglycans are markedly reduced in size with advancing age in centrally located regions of costal cartilage. Treatment with testicular hyaluronidase previous to acridine-orange staining leaves these small proteoglycan filaments unaffected. By contrast, the filaments visible after acridine-orange staining in the extracellular matrix near to the perichondrium are susceptible to hyaluronidase treatment. Infrequently, a sharp increase in collagen fibril diameter can be observed in territorial matrix areas of degenerating chondrocytes. This observation is conspicuous at ages of 10 and 20 years. Amianthoid transformation is characterized by the appearance of collagen fibrils strictly arranged in parallel. These amianthoid fibers are embedded in a matrix rich in small acridine-orange-positive filaments similar to the proteoglycan filaments observed in centrally located matrix regions. It can be concluded that extensive remodelling not only of the collagen fibrils but also of the cartilage proteoglycans is involved in the development of amianthoid transformation.     

Collagen fibrils and elastic fibers in rat-tail tendon: an electron microscopic investigation

Earlier studies by the authors showed that the collagen fibrils in rat-tail tendon have a bi-modal distribution of fibril diameters from a time shortly after birth through to the onset of maturity at about 3–4 months. Present work has extended those observations for rats up to the age of 2 years. Histograms of the fibril diameter distributions for mature tail tendon and direct electron microscope observations show that the fibrils break down as the tendon ages. Further work on the constant diameter subfibrils of diameter 140 Å described previously, has confirmed that these are part of the elastic fibers present in tendon at all ages. It has been shown that there is relatively little variation in the collagen fibril diameter distribution as a function of the position of the specimen in the tail, and as the measured percentage of the area taken by the collagen fibrils present at any particular point. Estimation of the fibrillar collagen content of rat-tail tendon as a function of age indicates that it increases steadily from birth and reaches a maximum at the onset of maturity, beyond which the fibrillar collagen content appears to remain constant.     

Quantitative electron microscope observations of the collagen fibrils in rat-tail tendon

An electron microscope study of collagen fibrils from fixed tail tendons of rats has revealed that from some time shortly after birth until maturity, the fibril diameters have a bimodal distribution. The “two” types of fibril are indistinguishable in both transverse and longitudinal section. Unfixed specimens of eight-week-old-tail tendon showed a similar bimodal distribution of diameters though the positions of the peak values compared to fixed specimens of an eight-week-old-tail tendon were shifted upwards by about 30%. It has also been shown quantitatively that the polar collagen fibrils are directed randomly “up” and “down” with respect to their neighbors. Whilst it has been suggested by others that anastomosis is a feature of collagen structure, the results presented here do not support this hypothesis. Fibrillar units ～ 140 Å in diameter have been observed and the possibilities that these are elastic fibers or the breakdown products of collagen fibrils have been considered.     

Ultrastructural features of adult human tendon

Summary A variety of human tendons have been studied at the electron microscope level. The fibers of these tendons are composed of collagen fibrils that average 1,750 Å and 600 Å in diameter. A third population that measures 100 Å in diameter may represent immature collagen or filaments that are incorporated into tendon elastic fibers. The larger collagen fibrils vary in ratio with respect to one another, and are connected by interfibrillar bridges which in some cases appear to extend through the substance of the fibril. The collagen fibrils of the paratenon are less-well organized than those of the tendon proper and average 600 Å in diameter. Tendons that exhibit the property of lateral stretch (plantaris and palmaris) were compared at the ultrastructural level with tendons that do not have this property. No differences between the two tendon types could be determined in normal or spread preparations, indicating that the differences in physical characteristics are a result of fiber rather than fibril organization.Supported by Edward G. Schlieder Foundation GrantThe authors wish to thank Mrs. Janell Buck and Mrs. Eunice Schwartz for their excellent technical and secretarial assistance, and Mr. Garbis Kerimian for his excellent photographic work     

THE FINE STRUCTURE OF ELASTIC FIBERS

The fine structure of developing elastic fibers in bovine ligamentum nuchae and rat flexor digital tendon was examined. Elastic fibers were found to contain two distinct morphologic components in sections stained with uranyl acetate and lead. These components are 100 A fibrils and a central, almost amorphous nonstaining area. During development, the first identifiable elastic fibers are composed of aggregates of fine fibrils approximately 100 A in diameter. With advancing age, somewhat amorphous regions appear surrounded by these fibrils. These regions increase in prominence until in mature elastic fibers they are the predominant structure surrounded by a mantle of 100 A fibrils. Specific staining characteristics for each of the two components of the elastic fiber as well as for the collagen fibrils in these tissues can be demonstrated after staining with lead, uranyl acetate, or phosphotungstic acid. The 100 A fibrils stain with both uranyl acetate and lead, whereas the central regions of the elastic fibers stain only with phosphotungstic acid. Collagen fibrils stain with uranyl acetate or phosphotungstic acid, but not with lead. These staining reactions imply either a chemical or an organizational difference in these structures. The significance and possible nature of the two morphologic components of the elastic fiber remain to be elucidated.     

Crystalline fibril structure of type II collagen in lamprey notochord sheath

We report here the existence of a crystalline molecular packing of type II collagen in the fibrils of the lamprey notochord sheath. This is the first finding of a crystalline structure in any collagen other than type I.The lamprey notochord sheath has a composition similar to that of cartilage, with type II collagen, a minor collagen component with 1α, 2α and 3α chains, and cartilage-like proteoglycan. The high degree of orientation of fibrils in the notochord makes it possible to use X-ray diffraction to determine collagen fibril organization in this type II-containing tissue. The low angle equatorial scattering shows the fibrils are all about 17 nm in diameter and have an average center-to-center separation of 31 nm. These results are supported by electron microscope observations. A set of broad equatorial diffraction maxima at higher angles represents the sampling of the collagen molecular transform by a limited crystalline lattice, extending over a lateral dimension close to the diameter of one fibril. This indicates that each 17 nm fibril contains a crystalline array of molecules and, although a unit cell is difficult to determine because of the broad overlapping reflections, it is clear that the quasi-hexagonal triclinic unit cell of type I collagen in rat tail tendon is not consistent with the data. The meridional diffraction pattern showed 26 orders with the characteristic 67 nm periodicity found for tendon. However, the intensities of these reflections differ markedly from those found for tendon and cannot be explained by an unmodified gap/ overlap model within each 67 nm period. Both X-ray diffraction and electron microscope data indicate a low degree of contrast along the fibril axis and are consistent with a periodic binding of a non-collagenous component in such a way as to obscure the gap region.     

Fine structure of syzygial articulations before and after arm autotomy in florometra serratissima (Echinodermata: Crinoidea)

Summary About 1 s after appropriate stimulation, arms of Florometra serratissima break at articulations called syzygies that are specialized for autotomy. The fine structure of unreacted and of newly broken syzygies is described. The unreacted syzygy includes (1) ligament fibers consisting of collagen fibrils interconnected by interfibrillar strands and (2) axons filled with presumed neurosecretory granules. The newly broken syzygy includes (1) ruptured ligament fibers consisting of swollen collagen fibrils associated with interfibrillar globules and (2) axons containing few presumed neurosecretory granules, some of which are fixed in the act of exocytosis; moreover, the calcareous skeleton adjacent to the broken syzygy is partly eroded. The observations before and after breaking suggest that the autotomy mechanism may comprise the following sequence of events: rapid neural transmission from stimulation site to syzygy triggers a massive exocytosis of granules from presumed neurosecretory axons; the released neurosecretions (which could include chelating agents, strong acids, proteolytic enzymes or enzyme activators) etch the skeleton and lower the tensile strength of the ligament fibers by weakening the collagen fibrils and/or the interfibrillar material; breakage of the ligament fibers, the major connective tissue of the articulation, is quickly followed by rupture of all the other tissues at the syzygy.     

A re-evaluation of the cytology of cat Pacinian corpuscles

Summary The ultrastructure of cat mesenteric Pacinian corpuscles in cross and longitudinal sections has been examined. The terminal ends of lamellar cells of the inner core have been identified in longitudinal sections through the proximal portion of the inner core. These terminal bulbous expansions contain characteristic concentric membranes of rough endoplasmic reticulum and in some cases masses of oval membranous inclusions. The central axon as seen in cross section is oval in profile, having X-(short) and Y-(long) axes, and each axonal face is characterized by specializations of the axolemma. At the X-axis, the inner lamellae of the inner core tightly abut a smooth axolemma, with no intervening connective tissue matrix, in a manner reminiscent of a neuroepithelium. The axolemma of the Y-axis has numerous axonal spines (microspikes) that project into the cleft in the inner core. The extent of the axolemma having axonal spines can only be appreciated in longitudinal sections. The clefts contain a specialized connective tissue with elastic and collagen fibrils. The connective tissue compartment of fibers and matrix separating individual inner core lamellae is unique, in that it contains extremely thin collagen fibrils measuring approximately 15 nm in diameter. The diameter of collagen fibrils increases as the cleft is approached. Here the fibrils resemble typical endoneural collagen.     

Three-dimensional organization of the collagen fibrils in the rat sciatic nerve as revealed by transmission- and scanning electron microscopy

Summary The organization of collagen fibrils in the rat sciatic nerve was studied by scanning electron microscopy after digestion of cellular elements by sodium hydroxide treatment, and by conventional transmission electron microscopy. The epineurium consisted mainly of thick bundles of collagen fibrils measuring about 10–20 m in width; they were wavy and ran slightly obliquely to the nerve axis. Between these collagen bundles, a very coarse meshwork of randomly oriented collagen fibrils was present. In the perineurium, collagen fibrils occupied the interspaces between the concentrically arranged perineurial cells; in each interspace, they formed a sheet of characteristic lacework elaborately interwoven by thin (about 3 m or less in width) bundles of collagen fibrils. In the subperineurial region, there was a distinct sheet of densely woven collagen fibrils between the perineurium and underlying endoneurial fibroblasts. In the endoneurium, collagen fibrils surrounded individual nerve fibers in two layers as scaffolds: the inner layer was made up of a delicate meshwork of very fine collagen fibrils, and the outer one consisted of longitudinally oriented bundles of about 1–3 m in width. The collagen fibril arrangement described above may protect the nerve fibers against external forces.     

A role for glycosaminoglycans in the development of collagen fibrils

Extensive data on the glycosaminoglycan (GAG) composition and the collagen fibril diameter distribution have been collected for a diverse range of connective tissues. It is shown that tissues with the smallest diameter collagen fibrils (mass-average diameter less than 60 nm) have high concentrations of hyaluronic acid and that tissues with the largest diameter collagen fibrils (mass-average diameter approximately 200 nm) have high concentrations of dermatan sulphate. It is suggested that the lateral growth of fibrils beyond a diameter of about 60 nm is inhibited by the presence of an excess of hyaluronic acid but that this inhibitory effect may be removed by an increasing concentration of chondroitin sulphate and/or dermatan sulphate. It is also postulated that high concentrations of chondroitin sulphate will inhibit fibril growth beyond a mass-average diameter of approximately 150 nm. Such an inhibition may in turn be removed by an increasing concentration of dermatan sulphate such that it becomes the dominant GAG present in the tissue.     

Ultrastructure of fibroin in the silk gland of larval Bombyx mori

The fibroin molecules stored in Golgi vacuoles in the posterior silk gland cells of 72-h-old, fifth instar larvae of Bombyx mori L. were observed electron-microscopically. The fibers which float in the Golgi vacuoles often have their ends attached to the limiting membrane. The fibers are helical bundles about 130 Å in diameter composed of 5–7 threads, each 20–30 Å thick.     

Elastic deformation of mineralized collagen fibrils: an equivalent inclusion based composite model

Mineralized collagen fibrils are the basic building blocks of bone tissue at the supramolecular level. Several disease states, manipulation of the expression of specific proteins involved in biomineralization, and treatment with different agents alter the extent of mineralization as well as the morphology of mineral crystals which in turn affect the mechanical function of bone tissue. An experimental assessment of mineralized fibers' mechanical properties is challenged by their small size, leaving analytical and computational models as a viable alternative for investigation of the fibril-level mechanical properties. In the current study the variation of the elastic stiffness tensor of mineralized collagen fibrils with changing mineral volume fraction and mineral aspect ratios was predicted via a micromechanical model. The partitioning of applied stresses between mineral and collagen phases is also predicted for normal and shear loading of fibrils. Model predictions resulted in transversely isotropic collagen fibrils in which the modulus along the longer axis of the fibril was the greatest. All the elastic moduli increased with increasing mineral volume fraction whereas Poisson's ratios decreased with the exception of v12 (=v21). The partitioning of applied stresses were such that the stresses acting on mineral crystals were about 1.5, 15, and 3 times greater than collagen stresses when fibrils were loaded transversely, longitudinally, and in shear, respectively. In the overall the predictions were such that: (a) greatest modulus along longer axis; (b) the greatest mineral/collagen stress ratio along the longer axis of collagen fibers (i.e., greatest relief of stresses acting on collagen); and (c) minimal lateral contraction when fibers are loaded along the longer axis. Overall, the pattern of mineralization as put forth in this model predicts a superior mechanical function along the longer axis of collagen fibers, the direction which is more likely to experience greater stresses.     

A comparative microscopic and ultrastructural study of perichondrial tissue in cartilage of Octopus vulgaris (Cephalopoda, Mollusca)

The microscopic and submicroscopic structures of perichondrial tissues in the head cartilages of Octopus vulgaris were studied by polarized light and transmission electron microscopy. The orbital cartilages possess a birefringent layer parallel to the surface of the cartilage; ultrastructurally, this layer, which may be considered perichondrial tissue, has the typical organisation of connective tissue but does not possess the stratification of collagen laminae found in vertebrate perichondria. Perichondrial extracellular matrix is clearly distinct from that of cartilage because its collagen fibrils are of a larger diameter than collagen fibrils from cartilage. In addition, perichondrial fibroblasts are characteristically located at the center of collagen fibers. In the cerebral cartilage, the perichondrium is absent or discontinuous in relation to complex interconnections between cartilage and connective fibres, muscle fibres, blood vessels and nerve. Distinctive cartilage-lining cells, rich in electron dense cytoplasmatic granules, are stratified either along the cartilage surface or along vessels and muscle fibres that penetrate within the cartilage. The perichondrium of cephalopod cartilage, whose structure varies according to the location and function of its skeletal segments, mimics that of vertebrate perichondrium, exemplifying the high level of tissue differentiation attained by cephalopods.     

Effect of calcitonin on fibroblasts and collagen formation in rabbits: an ultrastructural and scanning electron microscopic study

Transmission and scanning electron microscopic studies demonstrate the stimulatory effect of synthetic salmon calcitonin on the fine structure of fibroblasts and on collagen formation in cutaneous wounds experimentally induced in rabbits. Long-term administration of calcitonin enhances fibroblast growth and collagen synthesis. The fibroblasts hypertrophy and exhibit a highly developed rough endoplasmic reticulum (RER), several polyribosomes, large nuclei, hypertrophic Golgi complex, and many dense granules and lysosomes. Mitochondria are elongate and ramify; intracellular as well as extracellular synthesis of collagen increases. Fibrils appear tightly packed, in large heaps or spicula, with a characteristic periodicity and striation. Scanning electron micrographs of topography and relationships with collagen fibers and fibrils and cells surface changes demonstrate an extensive network of fine fibrils between collagen fibers, marked ruffling of cell membranes as well as numerous blebs on the cell surface. The latter are significant in collagen formation and egestion.