Collagen Fibrils In Tendon Research Articles

Differential effects of live bacteria and lipopolysaccharide on amino acid and 2-deoxy-d-glucose transport in rat muscle

The eggshell membrane was pierced and the chorio-allantoic membrane was dropped by suction. Tubocurarine hydrochloride (2%, w/v) in either isotonic saline or in water was injected on to the membrane. Control eggs received saline or water. Some eggs were left untouched. Eggs were then further incubated without rocking. After 9, 13, 14, 16and 19 days incubation the embryos were removed from their shells. Flexor digitorum tendons were removed and fixed in 2.5% (w/v) aqueous glutaraldehyde. The tendons were dehydrated in graded alcohols and embedded in Taab epoxy resin. Ultrathin sections were stained with phosphotungstic acid for electron microscopy. Photographs of specimens were calibrated by reference to a standard graticule. The outcome of the experiments conducted according to several different protocols is clear. The administration of tubocurarine, and subsequent paralysis of the developing chick, has little or no effect on the increase in diameter of the collagen fibrils of the flexor digitorum tendon (Table I) . This result very strongly suggests that the major part of the continual process of fibril expansion is unaffected by tension in the tendon due to muscle activity. Further work will be required to determine whether the mechanisms were triggered by muscle action before 9 days. If they were, our results exclude that the continuing function of these mechanisms was dependent on muscle activity.

Topographies of extracytoplasmic compartments in developing chick tendon fibroblasts

The complex topography of the fibroblast leading to the formation of extracellular compartments is demonstrated in the native three-dimensional state in cryofractured chick embryo tendon at different stages of development. Tendon collagen fibrils, fibril bundles, and macroaggregates are intimately associated with these extracytoplasmic domains. The localization of fibrils within narrow channels, bundles within peripheral compartments, and the coalescence of bundles into macroaggregates within larger extracytoplasmic compartments is illustrated at different stages of development. Changing patterns of matrix organization are apparent as morphogenesis proceeds. Our interpretation is that collagen fibrils, oriented along the tendon axis, appear in extracytoplasmic channels defined by the fibroblast which are added at the periphery of the cell until a row of fibrils outlines the cellular border at Day 10. By Day 14, cytoplasmic processes have partitioned the fibrils into groups and then into round bundles. New fibrils are continually added to the fibril bundles through fusion of the extracytoplasmic channels. As the bundles grow larger, the cell boundaries between the bundles retract and bundles begin to coalesce into macroaggregates. The fibroblast becomes attenuated, bundle-forming compartments disappear as processes retract, bundles coalesce, and macroaggregates predominate by Day 17. This work is consistent with the model proposed for the compartmentalization of the extracellular space by the fibroblast, providing distinct microenvironments where collagen fibrillogenesis and matrix morphogenesis occur under close cellular regulation.

An X‐ray diffraction analysis of rat tail tendons treated with Cupromeronic Blue

Cupromeronic Blue was used to stain selectively the proteoglycans in rat tail tendons under 'critical electrolyte' conditions. Earlier electron microscopical observations indicated that at least one type of proteoglycan filament is associated with tendon collagen fibrils at the positive staining band 'd'. To ensure that this was not an artefact caused by specimen preparation or the subsequent positive staining of the collagen fibrils, we have analysed low angle meridional diffraction patterns from stained but not dehydrated, embedded or counterstained tissues. Axial electron density profiles of Cupromeronic Blue-stained compared with unstained rat tail tendons revealed the axial locations and relative amounts of dye in both mature and young wet specimens. In mature tendons, the difference electron density profile contained a broad peak centred near residue 180 along the 234-residue D-period. This corresponds to the electron-optical staining band 'd'. In young tendons a similar distribution of stain was observed although in this case there was evidence of a doublet of peaks, one centred near residue 182 (band 'd') and the other near residue 165 (midway between bands d and e1). The wet proteoglycan--Cupromeronic Blue complexes distribute over about 30 nm along the collagen fibril axis. Comparison with the images of filaments seen in the electron microscope suggests that the dye complexes collapse significantly on dehydration and embedding.

Electron-microscopic study of the collagen fibrils of the rat tail tendon as revealed by freeze-fracture and freeze-etching techniques

The ultrastructure of the collagen of rat tail tendon was investigated by the freeze-fracture technique. Collagen fibers were pretreated with the digestive enzymes, alpha-amylase, elastase and collagenase to remove matrix substances. Some of the samples were etched for 20 min. Fibrils had an average diameter of 318 +/- 12 nm and a banded structure with a mean periodicity of 64.2 +/- 0.9 mm; the banding was most marked in alpha-amylase/elastase-treated specimens, although the periodicity was independent of pretreatment. Microfibrils were well-displayed following alpha-amylase/elastase and collagenase pretreatments. A difference in the diameters of microfibrils was, however, observed between etched specimens (8.3 +/- 0.3 nm) and those prepared by other experimental methods (11.4 +/- 0.5 nm). In replicas of collagenase-treated and etched specimens, the interconnecting filaments in the interfibrillar region formed a network that was continuous with the microfibrils of collagen fibrils. The diameter of the interconnecting filaments was the same as that of microfibrils. Microfibrillar bundles were observed in the interfibrillar region.

Three-dimensional organization of fibroblasts and collagen fibrils in rat tail tendon.

The orderly arrangement of fibroblasts and collagen in tendons and ligaments suggests that these cells may have precise relationships with one another and with the collagen fibrils. The spatial organization of rat tail tendon was therefore examined using scanning and transmission electron microscopy and by reconstructing a 35-microns long segment of tendon from serial transmission electron micrographs. Fibroblasts were regularly arranged in columns and showed more intimate association in the longitudinal than in the transverse plane. Thin cytoplasmic sheets extended up to 3 microns transversely, frequently forming junctional attachments with similar processes from adjacent cells or from the same cell. Longitudinal processes were longer, often extending for more than 20 microns and forming junctional attachments with other cells in the same column. Such processes often exhibited invaginations in which there were single fibrils or small groups of fibrils; this arrangement may be indicative of fibril elongation or may serve to transmit tension between the fibroblast and the collagen fibrils. This organization has interesting implications for the growth and function of other fibrous connective tissue, such as the periodontal ligament.

Adhesion of Human Platelets to Collagen: Evidence for Two States of Platelet Activation

Normal human platelets suspended in buffer were exposed to a suspension of bovine tendon collagen fibrils and the rate of adhesion observed over a period of 60 minutes. In fresh platelet preparations, approximately 15–20 0/o of the platelets adhered within one minute, and the total percent adhesion, following pseudo first order kinetics after the first minute, approached 60 % after 60 minutes. Pre-treatment with N-ethylmaleimide, dithiothreitol, and glutaraldehyde reduced adhesion at 1 minute to less than 5 %, but only N-ethylmaleimide completely blocked adhesion at 60 minutes. Thus, freshly collected, washed human platelets contain two distinct subpopulations exhibiting greatly differing rates of adhesion, reflecting different levels of reactivity toward collagen.

Arrangement of microfibrils in collagen fibrils of tendons in the rat tail. Ultrastructural and x-ray diffraction investigation.

The microfibrillar arrangement in collagen fibrils of tendons in the tail of the rat was examined by electron microscopy and X-ray diffraction. Fresh and air-dried collagen fibers were examined in unstretched and stretched conditions. The results demonstrate that the microfibrils have a course parallel to the longitudinal axis of the collagen fibrils. The influence of stretching and hydration of the samples on the orientation of fibrils and microfibrils is also assessed.

The banding pattern of rat tail tendon freeze-etched collagen fibrils.

Banding patterns of freeze-etched and replicated rat tail tendon collagen fibrils were studied. Better banding definition was obtained by freezing the samples without using a cryoprotectant and by prolonging etching. Under these conditions, the banding pattern was characterized by a sequence of elevated and depressed segments with a D-period of 65 nm, by two ridges at the margins of the elevations and by a third ridge at an intermediate point in the depressions. A comparison between replicas and isolated negatively stained collagen fibril micrographs as well as densitometric determinations have allowed interpretation of the elevations and depressions as overlap and gap zones and of the three ridges as the main bands with higher molecular density. Estimates, carried out on densitometric diagrams obtained from replicas, gave values which may represent a new parameter in the study of collagen banding and led to the calculation of a 0.55 D long gap zone and of a 0.45 D long overlap zone.

Second harmonic generation and orientational order in connective tissue: a mosaic model for fibril orientational ordering in rat-tail tendon

Orientational ordering of collagen fibrils in rat-tail tendon is studied by means of second harmonic generation (SHG). The polarization dependence of this nonlinear optical process is shown to provide information about the orientation distribution of the fibrils which is comparable to that obtainable from X-ray diffraction. It is also shown that SHG provides new information about the spatial dependence of this distribution which is not easily obtainable by other means. In rat-tail tendon the collagen fibrils are ordered in units whose dimensions are large compared to the wavelength of light, and within each such unit there exists a high degree of fibril parallelism. The major source of orientational disorder is due to the relative misalignment of these units. It is thus demonstrated for the first time that the correct description of orientational order in this widely studied model system requires the use of a mosaic model.

Electron microscopy shows periodic structure in collagen fibril cross sections.

X-ray diffraction was used to monitor the effects of electron microscope fixation, staining, and embedding procedures on the preservation of the three-dimensional crystalline order in collagen fibrils of rat tail tendon. A procedure is described in which the characteristic 3.8-nm lateral spacing is preserved, with increased contrast, in the diffraction pattern of the embedded fiber. This spacing is correlated with the separation between the tangentially oriented equally spaced lines of density observed in electron microscope ultrathin fibril cross sections of the same material. Optical diffraction of electron micrographs gives an objective measure of the periodicity and suggests that the fibril is composed of concentrically oriented crystalline domains. These observations, when combined with a recent interpretation of the native x-ray diffraction data [Hulmes, D. J. S. & Miller, A. (1979) Nature (London) 282, 878-880] suggest a tentative model for the three-dimensional structure of collagen fibrils.

Collagen phagocytosis by stimulated mouse periotoneal macrophages in vitro

Thioglycollage‐stimulated mouse periotoneal macrophages were cultured in the presence of minced rat tail tendon collagen fibrils. Electron microscopic examination of the cultures after 24 hours revealed phagocytosis and intracellular degradation of collagen within phagocytic vacuoles. This ability of macrophages to phagocytose collagen may be an important mechanism in the destruction of collagen that occurs during chronic inflammation.

The crystalline structure of collagen fibrils in tendon

New data have been collected on the crystalline structure of collagen fibrils in tendon. The unit cell in decrimped tendon has been determined by measurements of the Bragg reflections in the X-ray diffraction pattern. The results are consistent with a triclinic cell with b = 75.5 A ̊ , β = 93 °, a = b sin β , a = 90 °, c = n × 668 A ̊ , where n is probably 4 and γ = 90 °. A selection rule observed for prominent reflections is explicable either in terms of a specific orientation of the microfibrils on the lattice, or by a helical distortion of the microfibril axis. The cell parameter β can be varied by changing the ionic envirionment.

Der Einfluß von D-Penicillamin auf den Gehalt einiger Organe an Spurenelementen sowie auf die mechanischen Eigenschaften von Kollagen / Trace Elements in Several Organs and Mechanical Properties of Collagen under the Influence of D-Penicillamin

The content of trace elements in several organs of rats under the influence of D-penicillamine (D-PA) was investigated by the neutronactivation-analysis. It could be shown an diminution of Cu, and Co under D-PA-treatment, the content of Fe, Mn, Rb and Zn was not influenced. The investigated organs didn't show any submicroscopic alterations under D-PA. On isolated collagen fibrils of tail tendon was seen a significantly diminuition of E-moduls. In accordance with Siegel the principal effect of D-PA is thought to block the synthesis of functional groups from Schiff-base crosslink precursors but not to inhibit lysyloxidase by loss of Cu-ions of connective tissue. The thermostability of D-PA influenced fibrils is changed in stretched state only and will be due to the lack of crosslink Schiff-bases; where as the shrinking point of not stretched fibrils shows only aging dependent changes.

Negative staining of rat tail tendon collagen fibrils with uranyl formate

Negative staining of rat tail tendon collagen fibrils with uranyl formate appears to reveal more detail in the axial banding pattern than any other positive or negative staining method hitherto employed. In addition, uranyl formate and other uranyl solutions appear to reveal fine, closely spaced, longitudinal filaments which may represent the individual tropocollagen molecules.

Structure and function of bone collagen fibrils

The intermolecular volume of fully hydrated collagen fibrils from a number of mineralized and non-mineralized tissues of adult rats has been determined both by an exclusion technique and by a method which involves the monitoring of specific X-ray diffraction parameters. The intermolecular volume of either bone or dentinal fibrils is approximately twice that of either tail or achilles tendon, and the most frequent intermolecular distance in bone or dentine fibrils is approximately 3 Å larger than of the tendons. A number of fibrillar structures are most compatible with the intermolecular volume of rat tail tendon. These include hexagonal molecular packing and orthogonal arrays of microfibrils comprising seven parallel molecular strands. The intermolecular volume of bone or dentinal collagen fibrils, on the other hand, appears to arise from structures having a disordered or pseudo-hexagonal molecular packing, in which the most frequent intermolecular distance is about 19 Å. The space associated with collagen fibrils in adult bone is such that 70 to 80% of the mineral is located within the intermolecular space of the fibrils—approximately equal amounts of mineral being in spaces having lateral dimensions of 25 to 75 Å and 6 to 12 Å, respectively. Particles located in the latter kind of intermolecular space probably constitute, to a large extent, the non-crystalline mineral phase of adult bone. The stereo-chemical constraints on the transport of mineral ions into and within collagen fibrils of bone and tendon support the postulate that bone collagen is an in vivo catalyst for mineral deposition and further suggests that its catalytic activity may be partially regulated through its molecular packing.

The molecular packing of collagen in mineralized and non-mineralized tissues

Summary The collagen in adult bovine dentine, adult rat bone and tail tendon, and purified, reconstituted steerskin collagen were studied by x-ray diffraction in conjunction with a technique for assaying the intermolecular volume of collagen fibrils. These four collagens all have a geometric lateral arrangement compatible with hexagonal or near-hexagonal molecular arrays. The average intermolecular gap in bone and dentine, however, is 6A whereas in rat tail tendon it is only 3A. Phosphate ions, which have a diameter of approximately 4A, can thus penetrate the collagen fibrils of bone and dentine but not those of rat tail tendon. We conclude mineral cannot accumulate within the collagen fibrils of rat tail tendon purely for steric reasons.

Effect of the proline analogue azetidine-2-carboxylic acid on collagen synthesis in vivo II. Morphological and physical properties of collagen containing the analogue

Chick embryos were treated from day 8 to day 12 with 500 μg per day of azetidine-2-carboxylic acid. Electron microscopy indicated there were essentially no cross-striated collagen fibrils in tendon or skin of the treated embryos. Amino acid analysis of the tendons suggested that the decrease in cross-striated fibrils was accounted for by the decreased collagen content of the tissue. Acid-soluble collagen isolated from the treated embryos had essentially the same amino acid composition as normal collagen except that it contained about 4 residues of azetidine-2-carboxylic acid per 1000 residues of amino acid. The only demonstrable abnormality in the physical properties of the acid-soluble collagen from treated embryos was that the T m value for the helix-coil transition was 1.8° lower and the early part of the melting curve was less sharp than the melting curve of the control. The results did not help to resolve the question of whether collagen-containing azetidine-2-carboxylic acid is extruded from cells at a decreased rate because of the presence of the analogue or because of other changes in the molecule. The observations emphasized, however, that incorporation of proline analogues into collagen produces relatively minor changes in the structure of the molecule but that these minor changes have marked effects on the overall rate at which collagen fibrils are deposited in the extracellular matrix.

Composition of Membranous Tissues obtained from Tendon and Skin

THE presence of a sheath surrounding the collagen fibrils of skin and tendon was first shown histologically by Kaye1 in 1925, and its restrictive effects on swelling and eventual rupture were demonstrated by Jordan Lloyd and Marriott2. Recently, Kwon, Mason and Rigby3 have again directed attention to the presence of this sheath in tail tendon and have pointed out its probable effect on hydrothermal shrinkage as well as on swelling.

The attachment of the monogenean Entobdella soleae to the skin of the common sole

The muscular saucer-shaped haptor of the monogenean parasite Entobdella soleae is attached to the skin of its host Solea solea by means of a suction pressure generated within the sea-water-filled cavity enclosed between the cup-shaped haptor and the skin of the fish. The suction pressure is produced by the action of a pair of extrinsic muscles which are situated in the body of the parasite. Each extrinsic muscle communicates with the haptor by means of a long tendon which passes through a fair-lead in a prop-like accessory sclerite and is inserted on the end of a girder-like anterior hamulus embedded in the roof of the concavity of the haptor. The pull exerted by the muscles lifts the girders and the roof of the suction cup in which they are embedded relative to the props, thereby producing a suction pressure.An electron microscope was used to investigate the ultrastructure of the tendon, which was found to consist largely of unbranched banded fibrils which differed from the collagen fibrils of vertebrate tendon in their diameter and periodicity.I would like to express my thanks to Dr J. Llewellyn for suggesting the problem and for much helpful discussion. I am also grateful to Dr M. P. Osborne for tuition on the cutting of sections for electron microscopy. The work was conducted during the tenure of a Fishery Research Training Grant from the Development Commission.

The nature of the end products from the enzymic treatment of tropocollagen and collagen fibrils

AbstractTreatment of a solution of rat tail tendon collagen in acetate buffer with a proteolytic enzyme (ficin) led to a monodispersed solution of rodlike particles whose dimensions were 12.6 A. in diameter and 2400 A. in length. Similar enzymic treatment of dispersions of steer tendon collagen fibrils resulted in a disaggregation of the fibrils to a solution of particles of essentially similar dimensions. The nature of the enzymic attack in relation to the structure of the substrate is discussed.