Tendon Fibrils Research Articles

Scanning force microscopy reveals structural alterations in diabetic rat collagen fibrils: role of protein glycation.

The main functional property of collagen is to provide a supporting framework to almost all tissues: the effects of non-enzymatic glycation on this protein are deleterious and in diabetes mellitus contribute to the mechanism of late complications. The aim of this work is to provide evidence by scanning force microscopy of modifications in collagen structure caused by high glucose concentration, in vivo and in vitro, and to correlate the data with markers of non-enzymatic glycation. Tendon fibrils were obtained from the tails of 8-month-old rats (BB/WOR/MOL¿BB) which developed diabetes spontaneously at least 12 weeks before they were killed, and from diabetes-resistant rats of the same strain (BB/WOR/MOL¿WB). A scanning force microscope (SFM; Nanoscope III) equipped with a Contact Mode Head was used for imaging. Band interval, diameter and depth of D-band gap were measured in non-diabetic and diabetic tail tendon fibrils and in fibrils incubated with glucose (0.5 M for 2 weeks). Fructosamine was determined in the tendon fibrils by a colorimetric method and pentosidine was evaluated in acid-hydrolyzed samples by coupled reverse phase-ionic exchange column HPLC. Incubated fibrils revealed modifications in radius (228+/-5 nm) and gap depth (3.65+/-0.10 nm) that closely reproduce diabetes-induced damage (236+/-3 and 3.20+/-0.04 nm respectively) and were significantly different from the pattern seen in non-diabetic fibrils (151+/-1 and 2.06+/-0.03 nm; p<0.001). Both fructosamine and pentosidine were higher in diabetic (3.82+/-1.43 nmol/mg and 2.23+/-0.24 pmol/mg collagen respectively) and in glucose-incubated fibrils (9.27+/-0.55 nmol/mg and 5.15+/-0.12 pmol/mg collagen respectively) vs non-diabetic tendons (1.29+/-0.08 nmol/mg and 0.88+/-0.11 pmol/mg collagen respectively; p<0.01); during the time course of incubation, an early increase in fructosamine was seen, whereas pentosidine increased later. The D-band parameter was similar in all three groups, indicating that axial organization is not modified by non-enzymatic glycation. This is the first description obtained with SFM of diabetes-induced ultrastructural changes in collagen fibrils. Moreover, the data presented are consistent with the concept that chronic exposure of collagen to glucose in vivo or in vitro leads to similar structural modifications in collagen fibrils, probably through crosslinks. The correlation between morphologic parameters and both markers of glycation provides strong evidence for a crucial role of this non-enzymatic modification.

Ultrasound assessment of large joint amyloidosis in haemodialysis

SUMMARY:Dialysis‐related amyloidosis (DRA), associated with beta‐2 microglobulin deposition, is a common cause of morbidity in patients with renal failure undergoing chronic dialysis. The non‐invasive diagnosis of DRA in its early stages is difficult. High resolution ultrasound (US) was used to examine the shoulder, hip and knee joints of 25 chronic haemodialysis patients, and features distinctive of DRA were determined. Distinctive US features were associated with increased total duration of dialysis (15 ± 5 vs 4 ± 2 years), carpal tunnel syndrome (9/10 cases) and shoulder joint discomfort (19 vs three joints). These features include altered rotator cuff echogenicity (hyperechoic and hypoechoic areas; 20/48 joints), hypoechoic material in the biceps sheath (16/48), erosions of bone (15/48) and reduced biceps tendon fibril echogenicity (12/48). These changes are unique in the experience of the current authors, but histological proof that they are definitely the result of DRA has not yet been possible. Tears of the rotator cuff tendons (14/50) were not consistently associated with symptoms (7/14). Tendon thickness was also increased (as has been described previously). The hip capsule thickness was increased in patients with signs of DRA in the shoulder, but no specific changes in texture were seen. Asymptomatic knee joint effusions were present in 40/50 joints (80%). These features detectable on high resolution US allow the non‐invasive early detection of DRA and are useful in the diagnosis and management of this condition.

Enthesopathy of the radial tuberosity in two thoroughbred racehorses

Two cases of enthesopathy of the radial tuberosity in Thoroughbred racehorses are described. Soft X-ray pictures revealed separated bony fragments at the anterior aspect of the radial tuberosity, resembling the lesions of Osgood-Schlatter disease in children. Osgood-Schlatter lesions result from detachment of a portion of the apophysis of the tibial tuberosity. However, in the affected horses, the detached bony fragments consisted of cortical bone tissue composed of trabeculae with osteons similar to lamellar bone, the main component of the radial tuberosity. Tendon fibrils were inserted into the anterior parts of the detached fragments, and scar tissue filled the gap between the posterior part of the fragments and the radial tuberosity. The bone fragments may have originated from an avulsion fracture at the site of attachment of the tendinous portion of the biceps brachii muscle to the bone tissue of the radial tuberosity, a site at which high-tensile force is repeatedly exerted by muscle contraction.

A Consensus Model for Molecular Packing of Type I Collagen

In this review, recent results from X-ray diffraction studies of tendon are used to develop an understanding of the molecular packing of type I collagen in tendon fibrils. These cover the definition of the unit cell as triclinic, the lateral architecture of molecular packing in a fibril and the molecular packing topology of a structure that gives good agreement with X-ray diffraction data. The proposed model is a 1D staggered left handed microfibril; the molecular orientation of the telopeptides indicates that there are interconnections between microfibrils that may explain the difficulty in isolating individual microfibrillar structures. This is the first structure that defines the absolute molecular packing of molecular segments based on X-ray diffraction data. These results are discussed in the light of direct and indirect evidence relating to molecular packing such as mineralization, natural crosslink position, and biomechanical evidence. The ability of the proposed structure to fulfill many of the structural and biochemical criteria point towards the structure providing a basis for a consensus model of collagen packing.

Association of type VI collagen with D-periodic collagen fibrils in developing tail tendons of mice.

The process of the arrangement of D-periodic collagen fibrils and their growth in maturing tail tendon of mice were studied with the association of type VI collagen, from fetal day 10 to 10 weeks after birth. In tail tendons, the amount of collagen fibers gradually increased along with the diameters of D-periodic collagen fibrils during maturation. Type VI collagens first appeared on fetal day 10, when D-periodic collagen fibrils were not recognizable. Type VI collagens were observed around the fibroblastic cells in early stages of development, but were among thick collagen fibrils in the adult tendon. While the periodic distances of type VI collagen fibrils were over 100 nm at fetal days, they were packed to 80-90 nm after birth. The periodic bands were stained well with ruthenium red in adult but not in young tendons, indicating the close association of proteoglycans or glycosaminoglycans (PGs/GAGs) with maturing type VI collagens. Since type VI collagen in native form is known to associate with D-periodic collagen fibrils via PGs/ GAGs, ruthenium red-stainability on the surface of D-periodic collagen fibrils was also examined; results showed that ruthenium red-stainable elements were D-periodically associated. When the surface morphology of D-periodic collagen fibrils in adult animals was examined by atomic force microscopy, a large depth of the groove between elevated and depressed surfaces became prominent when the fibril surface was digested with hyaluronidase. Thus, it is possible to observe topologically the association of PGs/GAGs and probably that of type VI collagens with D-periodic collagen fibrils.

Differential expression of fibromodulin mRNA associated with tendon fibril growth: isolation and characterization of a chicken fibromodulin cDNA.

A 450 bp cDNA fragment similar to that encoding bovine fibromodulin was isolated using a screening procedure to isolate genes differentially expressed between the pre- and post-growth phases of fibril growth in the developing chicken embryo metatarsal tendon. Using this fragment, a 2.4 kb cDNA clone for chicken fibromodulin was isolated from a lambda ZAP library, and the 5' rapid amplification of cDNA ends technique was employed to clone the 5'end of the fibromodulin cDNA. The full-length cDNA contained an open reading frame coding for a 380-amino-acid protein. There was approximately 80% similarity with human, rat and bovine fibromodulins, which confirmed its identity as fibromodulin. Structural features of the deduced sequence include an 18-amino-acid signal peptide, cysteine residues in conserved positions in the N- and C-terminal regions, and a central leucine-rich domain containing eleven repeats of the sequence LXXLXLXXNXL/I. Features unique to chicken fibromodulin include an additional glycosylation site as well as a decreased number of tyrosine residues that could be sulphated, and therefore potential changes in the charge of the molecule. In addition, there was little similarity among the untranslated regions. When compared with chicken decorin and lumican, fibromodulin showed greater similarity to the other keratan sulphate-containing proteoglycan, lumican. Northern blot analysis revealed a 6-8-fold increase in the fibromodulin mRNA level from day 14 to day 19 of development. In the chicken tendon, collagen fibril growth is a process characterized by a precipitous increase in length during a short developmental period. The necessary changes would require the expression of different genes regulating fibril formation and growth, and interactions between fibromodulin and collagen fibrils may participate in the regulation of collagen fibril growth and matrix assembly.

Radial packing, order, and disorder in collagen fibrils

Collagen fibrils resemble smectic, liquid crystals in being highly ordered axially but relatively disordered laterally. In some connective tissues, x-ray diffraction reveals three-dimensional crystallinity in the molecular packing within fibrils, although the continued presence of diffuse scatter indicates significant underlying disorder. In addition, several observations from electron microscopy suggest that the molecular packing is organized concentrically about the fibril core. In the present work, theoretical equatorial x-ray diffraction patterns for a number of models for collagen molecular packing are calculated and compared with the experimental data from tendon fibrils. None of the models suggested previously can account for both the crystalline Bragg peaks and the underlying diffuse scatter. In addition, models in which any of the nearest-neighbor, intermolecular vectors are perpendicular to the radial direction are inconsistent with the observed radial orientation of the principal approximately 4 nm Bragg spacing. Both multiple-start spiral and concentric ring models are devised in which one of the nearest-neighbor vectors is along the radial direction. These models are consistent with the radial orientation of the approximately 4 nm spacing, and energy minimization results in radially oriented crystalline domains separated by disordered grain boundaries. Theoretical x-ray diffraction patterns show a combination of sharp Bragg peaks and underlying diffuse scatter. Close agreement with the observed equatorial diffraction pattern is obtained. The concentric ring model is consistent with the observation that the diameters of collagen fibrils are restricted to discrete values.

Fibromodulin distribution and association with collagen.

Fibromodulin, an acidic 59-kDa proteoglycan, binds to collagen and inhibits collagen fibril formation in vitro. To determine whether fibromodulin is also bound to collagen in vivo, we used immunocytochemical methods to study the spatial relation of the proteoglycan to collagen fibrils in cartilage and tendon. We also studied the quantitative distribution of fibromodulin among compartments in articular cartilage at the ultrastructural level. Fibromodulin was identified with polyclonal antibodies raised in rabbits, and immunoreactivity was detected with protein-A gold. As the major proportion of fibromodulin immunoreactivity was localized along the periphery of the collagen fibrils, the relationship to the banding pattern of the collagen fibrils was mapped. The proteoglycan showed a non-random distribution, with preference to the gap region, axially within the D-period. Reactivity differed among the tissue compartments, with the lowest degree of labelling pericellularly, increasing with distance from the cell, the highest levels being observed in the interterritorial matrix. Labelling density was highest at the articular surface, gradually decreasing towards the cartilage-bone junction. The correlation between collagen fibril diameter and fibromodulin concentration also varied among compartments. Thus, the ratio of fibromodulin to collagen surface density was highest at the surface of the joint cartilage, exhibiting a gradient with higher values in the territorial matrix, decreasing towards the cell in all layers. These findings indicate that fibromodulin represents a factor used by chondrocytes to regulate assembly and function of collagen fibrils.

The post-translational chemistry and molecular packing of mineralizing tendon collagens

Using cross-linking stereochemistry as indicators, the molecular environment of two collagens in the turkey leg Achilles tendon were compared. The tendon from one year old turkeys was dissected into nonmineralized, fully mineralized and transitionally mineralized portions. Amino acid composition and cyanogen bromide peptide mapping of these portions indicated that the collagens were essentially type I throughout. The fully mineralized compartment had a lysine hydroxylation level similar to turkey or mammalian bone collagen. The non- and transitionally mineralized collagens had a significantly higher lysine hydroxylation, typical of tendon or ligament. However, unlike mammalian tendon, the collagen cross-links were essentially derived from the carboxy-terminal ends of the molecules. The predominant cross-link in this portion was pyridinoline having a high content of 0.95 +/- 0.09 res/mole of collagen. The cross-links in the fully mineralized collagen were also essentially derived from carboxy-terminal aldehyde. However, here significant amounts of the lysyl analog of pyridinoline and lysine-involved bifunctional cross-links were present. The molecular loci of pyridinoline in nonmineralized collagen and the lysyl analog of pyridinoline in mineralized collagen were found to be identical. The total trifunctional cross-link level in the mineralized collagen, 0.55 +/- 0.05 res/mole of collagen, was virtually identical to that observed in old mammalian bone and dentin, and in long term in vitro incubation studies of predentin. We have tentatively concluded that the post-translational chemistry and molecular environments are different in these two turkey tendon fibrils. However, a relative paucity of amino-terminal based cross-links is a feature they have in common. The possible involvement of the amino-terminal telopeptides in collagen mineralization is discussed.

Dentin Phosphophoryn Binding to Collagen Fibrils

The interaction of rat incisor phosphophoryn with native turkey tendon collagen fibers has been examined by electron microscopy. The binding of phosphophoryn to the tendon fibril surfaces is quite selective. The phosphophoryn is seen as positively or negatively stained globular particles predominantly at the “e” band in the collagen gap region in transmission electron micrographs of the phosphophoryn-reacted fibrils. The selectivity of binding to the fibrils was obtained in the presence of calcium ions, which bind avidly to phosphophoryn. The specific association of phosphophoryn at the “e” band suggests a possible regulation of mineral deposition within the gap regions of the collagen fibrils.

Cross-linkage sites in type I collagen fibrils studied by neutron diffraction

Cross-links in tendon collagen are essential for the biomechanical strength of healthy tissue. The nature and position of these cross-links has long been a subject for conjecture. We have approached this problem in a non-destructive manner, by studying neutron diffraction from collagen fibrils that have been specifically deuterated by reduction at keto-amine and Schiff base groups with sodium borodeuteride (NaB2H4). The intensities of the first 23 meridional reflections were recorded for both native and reduced tendons. These data were used to calculate the neutron-scattering density profile of the 67 nm (D) repeat of type I collagen fibrils in rat tail tendon. This approach not only succeeds in determining the location of the cross-linkage sites with respect to the fibril structure, as projected onto the fibre axis, but also presents a novel form of the isomorphous derivative solution to the phase problem.

Time-resolved studies of the dynamic behavior of the tendon fibers (chordae tendineae) of the human mitral valve.

The dynamic behavior of the tendinous chords of the mitral valve of the heart was analyzed using fast x-ray diffraction combined with mechanical measurements. The tendinous chords, which have a very fine crimp, are built up of "pseudo-interwoven" fibrils [1] consisting of 80 07o type I and 20 % type III collagen. These fibrils differ f rom tendon fibrils by their twisted molecular structure [2], which is the reason for their relatively small diameter of about 45 nm. When a tendinous chord is loaded beyond the toe region of the stress/ strain curve, then the collagen fibrils also are stretched, as it is the case in tendons [3]. The stretching of the fibrils is registered via small-angle meridional x-ray diffraction spectra, whose peak intervals change with the increasing axial periodicity (D period) of the collagen fibrils (Fig. la). The tendinous chord was loaded from curves 1 to 20 and unloaded from curves 21 to 32 (Fig. 1 b). The D period increases f rom

Crystal size and organization in bone.

Size measurements of dispersed rat bone crystals show that with age a greater proportion exceeds 400 A in length. The surface fractures of more heavily mineralized bones tend to be smooth and stepped, whereas those of less mineralized bones are fibrous. These observations combined with information reported elsewhere on the crystal-collagen relations in adjacent fibrils in turkey tendon, suggest that some crystals grow out of the confines of the collagen gap regions to form extended aggregates of large flat crystals with well developed fracture planes.

Type III collagen can be present on banded collagen fibrils regardless of fibril diameter.

Monoclonal antibodies that recognize an epitope within the triple helix of type III collagen have been used to examine the distribution of that collagen type in human skin, cornea, amnion, aorta, and tendon. Ultrastructural examination of those tissues indicates antibody binding to collagen fibrils in skin, amnion, aorta, and tendon regardless of the diameter of the fibril. The antibody distribution is unchanged with donor age, site of biopsy, or region of tissue examined. In contrast, antibody applied to adult human cornea localizes to isolated fibrils, which appear randomly throughout the matrix. These studies indicate that type III collagen remains associated with collagen fibrils after removal of the amino and carboxyl propeptides, and suggests that fibrils of skin, tendon, and amnion (and presumably many other tissues that contain both types I and III collagens) are copolymers of at least types I and III collagens.

Organisation of collagen fibrils in tendon: changes induced by an anabolic steroid. II. A morphometric and stereologic analysis.

Morphometric and stereologic analysis of the organisation of collagen fibrils in tendon tissue after a treatment with an anabolic steroid hormone allowed the following observations: In a short-term study stereological data revealed a potent accumulation of collagen fibrils in the extra-cellular matrix after the administration of an anabolic steroid. Compared with controls, the anabolic steroid significantly increased the number of dysplastic collagen fibrils dependent on duration of treatment. Inter- and intrafibrillary dysplastic collagen fibrils possess characteristic diameter distributions which differ considerably from those of normal collagen fibrils. The functional significance of the changes in mean diameter, diameter distribution, numerical density and volume fraction of collagen fibrils in tendons following hormone treatment may be relevant to the use of these drugs in clinical practice and in competitive sports.

Organisation of collagen fibrils in tendon: changes induced by an anabolic steroid. I. Functional and ultrastructural studies.

The organisation of tendon collagen fibrils has been studied by electron microscopy after treatment with an anabolic steroid hormone in a short-term and a long-term study. The effect of anabolic steroids is of interest because of their use in competitive sports and in clinical therapy. In a functional experimental model the unexpected occurrence of dysplastic as well as ruptured and dissociated collagen fibrils was revealed. The striking time-dependent collagen dysplasia and other pathological findings strongly suggest that in mice anabolic steroid treatment predisposes to tendon rupture especially when the animals are exercised. The possible underlying mode of action of anabolic steroid hormone on connective tissue is discussed.

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.

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.

Formula omitted] high power double magnetic resonance investigation of collagen backbone motion in fibrils and in solution

13C 1H high power double magnetic resonance spectroscopy was used to investigate the mobility of the collagen peptide backbone. [1- 13C]- and [2- 13C]-glycine-labeled collagen samples (with >50% enrichment in 13C) were prepared via chick calvaria culture. 13C n.m.r. † † Abbreviations used: n.m.r., nuclear magnetic resonance; p.p.m., parts per million; NOE, nuclear Overhauser enhancement. spectra of labeled reconstituted collagen fibrils, of labeled helical collagen in solution, and of unlabeled bovine Achilles tendon collagen were obtained with scalar decoupling and with dipolar decoupling of protons. Proton-enhanced spectra were also obtained using cross-polarization techniques. n.m.r. parameters (linewidths, lineshapes, T 1 values, nuclear Overhauser enhancements, and cross polarization enhancements) were measured for the labeled samples and for collagen in natural abundance. Comparison of 13C n.m.r. parameters for bovine Achilles tendon fibrils and for reconstituted chick calvaria collagen fibrils established that chick calvaria collagen is a good model for the molecular dynamics of collagen in vivo. Spin-lattice relaxation times and nuclear Overhauser enhancements for [1- 13C]- and [2- 13C]glycine-labeled collagen indicated that R 1 ~2 × 10 7 s −1 in solution, where R 1 is the diffusion constant for reorientation about the long axis of the molecule. A substantially smaller value for R 1 (2.6 × 10 6 s −1) was calculated for an axially symmetric ellipsoid of revolution having dimensions appropriate to the collagen helix. The discrepancy between the rigid ellipsoid and n.m.r. values of R 1 suggests that the collagen molecule undergoes torsional reorientation, as well as rod-like reorientation, about its long axis. The T 1 and NOE values measured in the glycine-labeled fibrils show that rapid axial motion ( R 1 ~ 10 7 s −1) persists in the fibrillar state. In the collagen fibril the full width of the glycyl carbonyl powder pattern is 103 p.p.m. This value is substantially smaller than the rigid lattice value, 144 p.p.m., which provides further evidence for motion in the fibril. The observed powder pattern is axially asymmetric, which shows that certain azimuthal orientations are energetically preferred in the fibril. Taken together, the n.m.r. data provide strong evidence that rapid reorientation of the helix backbone occurs in the fibrils. This result shows that formation of a fibrillar structure does not require the existence of a unique set of intermolecular interactions at the helical surfaces.

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.