Elongational flow studies on type IV collagen: comparison with type I.

Elongation flow techniques have been used to investigate the birefringent response of monodisperse type IV collagen in dilute solution and the results compared with type I collagen. A four-roll mill apparatus was used to characterize the solutions at low strain rates, epsilon < or = 300 s-1. The birefringence is nonlocalized and rises gradually to a plateau value, in accordance with rigid-rod behavior. The gradients of the tangent to the curves at zero strain rate are estimated for types IV and I collagen. The concentrations of the solutions used were in the dilute to semidilute regimes. Using a value of 300 nm for the length of type I collagen, values of 364-408 nm were calculated for the length of the type IV collagen molecule, depending on the concentration regime chosen, which is consistent with biochemical predictions based on a rigid molecule. The results imply that the behavior of type IV collagen molecules in solution is similar to type I collagen, despite the presence of several sequence interruptions in the type IV helix.

Molecular bending and networks in a basement membrane-like collagen: packing in dogfish egg capsule collagen.

Low-angle X-ray diffraction data have been obtained from three mutually perpendicular axes through sheets of the collagenous egg capsule of the dogfish Scyliorhinus caniculus, a collagen that resembles type IV collagen. The data are interpreted in the light of the body of knowledge of the structure derived from transmission electron microscopy by Knight and Hunt. A model to account for the X-ray data is proposed incorporating the main dimensions of the Knight and Hunt model which are confirmed by the diffraction data. Several features of the diffraction patterns are not explained by the existing model however, and a new model is proposed to account for these features. This consists of antiparallel packed pairs of two mutually parallel molecules, each kinked and rotated so as to produce a four-fold helix resembling a crankshaft. This has the advantage of conferring intermolecular linkage in three dimensions throughout the structure with tetragonal symmetry and unit dimensions a = b = 22.6 nm, c (fibre axis direction) = 39.3 nm. The result is a fairly rigid open polygonal network or sponge-like architecture that is capable of accommodating large quantities of water and other molecules.

Short and long range order in basement membrane type IV collagen revealed by enzymic and chemical extraction.

Oriented bovine lens capsules give X-ray diffraction patterns suggesting a considerable degree of order in the collagenous components, predominantly type IV collagen. Here we report the effects of preliminary treatment of lens capsules before orientation. Extraction with 4 M guanidinium hydrochloride or with heparinase/hyaluronidase reveals the same collagenous diffraction patterns previously seen after extraction with 1 M NaCl. There is a four-point pattern of d-spacing 3.9 nm, indicating liquid crystal cybotactic nematic organization, along with sharp streaked meridional reflections which index as orders of 21 nm. This suggests that the removal of basement membrane proteoglycans results in a reduction in diffuse scatter and clarification of the pattern. Extraction of the lens capsules with trypsin or dithiothreitol greatly reduces the intensity of the four-point pattern while leaving the meridional pattern unaffected. This strengthens the evidence that the 21 nm period has its origins in the collagen IV helix. Reduction in the four-point pattern could arise if disruption of non-helical NC1 domains or 7S overlap regions allows slippage of the collagen molecules on orientation, weakening the proposed 1 nm intermolecular stagger. Ultra-low angle diffraction patterns of extracted lens capsules show meridional reflections which index as a long-range axial repeat of approximately 95 nm. This is consistent with a model of microfibrils of type IV collagen in which the NC1 domains bind to the collagen helix at approximately 100 nm intervals, as has been previously suggested.

Molecular organization of type IV collagen: polymer liquid crystal-like aspects.

A new X-ray diffraction pattern from type IV collagen is described, which can be interpreted on the basis of crystalline and liquid crystalline origins of the reflections. Bovine anterior lens capsules extracted with 1 M NaCl and oriented by extension of 60% under constant load gave medium angle X-ray diffraction patterns showing many of the characteristics typical of liquid crystals. Prominent features, apart from those wide angle features attributable to the collagen triple helix, are (1) a four-point pattern of broad reflections at d-spacing 3.9 nm, and layer line spacing near 5 nm. (2) A broad intense equatorial peak centred at 1.24 nm, indicative of liquid-like lateral molecular associations. (3) A set of five sharp, streaked meridional reflections (previously obscured by the broad peak near 5 nm in unextracted capsules). (4) A further six higher angle reflections of a diffuse, arced and broad appearance on the meridian. The sharp streaked meridional reflections emanate from a long-range periodicity of units 8-9 nm in diameter. These features form a self-consistent system if interpreted on the basis of a staggered liquid crystal-like array of collagen molecules, in which case the first five meridionals and remaining broad reflections, sampled on the meridian, can all be indexed as orders of 21 nm.

Basement membrane collagen--evidence for a novel molecular packing.

Type IV collagen is the major structural protein of basement membranes but very little is known about its molecular organisation in vivo. We have used X-ray diffraction of a thick basement membrane, bovine lens capsule, to provide information. Under constant load, lens capsule gave a collagen diffraction pattern of a similar quality to unstretched rat rail tendon. In addition there were clear meridional reflections which indexed as orders of 10 nm, and equatorial reflections at 2.1 and 3.8 nm. These results suggest the ordering of type IV collagen molecules in fibrils, with a 10 nm periodicity along the length of the fibrils.

In-vitro mechanics of intrusive loading in porcine cheek teeth with intact and perforated root apices.

Cyclic intrusive loads were applied to deciduous cheek teeth and monitored by radiography in dissected mandibles in three successive states: (A) intact, (B) vertically bisected in a bucco-lingual plane, (C) with perforation of the apices by lateral drilling of the mandible subsequent to (B). Loading was in a compression cage in an Instron mechanical testing-machine utilizing cross-head speeds of 0.5 and 5 mm/min, leading to loading rates of approximately 2 and approximately 20 N/s. Peak loads were approximately 100 N, causing an intrusion of approximately 200 microns. Load/recovery curves for each tooth in the three states were recorded and the data treated assuming: (a) a simple shear on the periodontal ligament, (b) tensile loading on the fibres of the collagen fraction of the ligament alone. Treatment (b) gave the best agreement with published mechanical data on isolated mammalian periodontal ligament. The small effect of apicectomy on the mechanical behaviour indicates that a mobile fluid-like support mechanism is unlikely to operate for major loads in vitro and tensile support by the collagen fibres of the periodontal ligament is the most likely dominant mechanism.

Morphology of human palmaris longus tendon.

A systematic morphological investigation of human palmaris longus tendons by polarisation microscopy and low angle x-ray diffraction is reported. It is shown that contrary to some previously reported observations, and in common with other tension bearing soft collagenous tissues, the fibres in this tendon are crimped. A new method of preparation of the tissue enabling one to see directly the crimped organisation in scanning electron microscopy was used to reinforce the findings by other methods.

The Ehlers-Danlos syndrome: an analysis of the structure of the collagen fibres of the skin.

The structure of the collagen fibres of the skin in the Ehlers-Danlos syndrome (ED-S) was studied in eight patients with ED-S Type I, three patients with ED-S Type II and three patients with the X-linked Type V. The results show that the reducible cross-links are present and undergo the same maturation process to non-reducible cross-links as in normal skin. Transmission electron microscopy revealed a normal ultrastructure of the collagen fibrils. At a higher morphological level of organization scanning electron microscopy demonstrated progressive increase in fibre bundle disorder from the X-linked to mitis, to gravis, in which the fibres making up the large fibre bundles demonstrated a considerable inability to aggregate.

X-ray diffraction effects related to superstructure in rat tail tendon collagen.

A number of unusual X-ray diffraction effects concerning the meridional low angle reflections of collagen are presented which are due to structural inhomogeneities in the 100 micron range within the tendon such as are expected to be associated with the planar crimp structure of tendon units. These newly observed effects provide diffraction confirmation for such a crimped morphology. The effects consist of splitting of the reflections both azimuthally and radially. Azimuthal split varied with rotation of the tendon unit around its own axis and is directly related to the existence of the crimp, its angle and planarity. Radial splitting is a consequence of the limited sampling of the micro-structure by the collimator, which provides further support for the crimp morphology and conveys added information about the crimp form. It is shown that this limited sampling can provide a kind of "micro-X-ray topography" technique for the study of micro-textures on the appropriate dimensional level, while ti could be the source of misrepresentation if its origin remains unrecognized. The effect of stretching is examined and interpreted in terms of crimp straightening. Simultaneous examination of medium angle equatorial reflections, when combined with some electron micrographs point to further issues concerning the relation between the large scale and fibril substructure. It is believed that the present findings have general implications for structure research on collagen.

Low temperature electron diffraction and beam effects in tendon collagen.

In the course of experiments to obtain electron diffraction from forzen rat tail tendon (RTT) collagen, a characteristic development of perforations relating to the banding pattern was observed under conditions of moderate beam intensity. This is most likely to be due to an etching process resulting from ionization or sublimation of bound or preferentially retained water molecules in the band-interband region and could provide a means of visualization of the location of such water.