Th. Nemetschek

Stress-induced molecular rearrangement in tendon collagen

Tension-induced molecular rearrangements in wet native fibres of rat-tail tendons and human finger flexor tendons are registered with the help of time-resolved diffraction spectra using synchrotron radiation. The tension-induced increase of the 67 nm D period is combined with changes in the intensities of some orders of the meridional small angle reflection. Both effects are reversible when unloading the fibre, but are preserved when the load is held constant until the fibre tears. The increase in the D period is partly due to a sliding of the triple helices relative to each other and partly due to a stretching of the triple helices themselves. The sliding of the triple helices results in an alteration of the D stagger, leading to a change in the length of the gap and overlap regions, and to a stretching of the cross-linked telopeptides. This interpretation is supported by comparison with the relative intensities derived from a model with varying length of gap and overlap regions, as well as by comparison with model calculations that include the telopeptides.

Quantitative analysis of the molecular sliding mechanisms in native tendon collagen — time-resolved dynamic studies using synchrotron radiation

Abstract The stretching of native fibres from rat tail tendons (RTT) was monitored in time-resolved X-ray measurements using synchotron radiation, by registering one meridional small angle diffraction pattern every second. The time course of this dynamic molecular process was analyzed quantitatively with the help of model calculations based on the amino acid sequence. The results show that two mechanisms contribute to the elongation of fibrils, namely the stretching of the collagen triple helices and their sliding relative to each other (increase of the D stagger). The results further show that these two processes do not take place simultaneously. The first increase of the D period from 67.0 nm to about 67.6 nm is correlated with a stretching of the triple helices. The further increase of the D period is due to a continuous increase of the D stagger. This succession is independent of the age of the animals and also independent of the stretching velocity. The stretching process is shown to be reversible at the molecular level up to a D period of about 68.4 nm.

Schachtelhalm-structure of the octafibrils in collagen

Abstract X-ray diffraction patterns suggest that the microfibrils in the collagen of native rat tail tendon are eight-stranded ropes, quarter staggered and packed together in an orthorhombic lattice with paracrystalline (liquid-like) distortions between adjacent tropocollagen molecules. These so called octafibrils react under stress like synthetic elastomers with a 135 A periodicity of hard segments.

Structural dynamic of native tendon collagen

The dynamic behaviour of collagen fibrils is revealed by time-resolved X-ray investigations of native rat tail tendon fibres in tensile tests.

New aspects of the etiology of tendon rupture

SummaryNative collagen fibers were exposed to different dynamic loads to simulate damage to tendons and ligaments relevant clinically and for sports medicine. The results suggest that the rupture of a tendon is caused at the submicroscopic fibrillar level. Not only slow or very fast elongation, but also very fast unloading of stretched fibers seems to be responsible for disseminated damage, which reduces the stability of a fiber. This damage is induced by intrafibrillar sliding processes, which occur only a few seconds before macroscopic slippage takes place. The significance of these events for the beginning and progress of repair in vivo is discussed. The conclusions are supported by simultaneous mechanical and radiological measurements, as well as by light- and electron-microscopic results.ZusammenfassungZur Simulierung klinisch bzw. sportmedizinisch relevanter Schäden an Sehnen und Bändern wurden native Kollagenfasern unterschiedlichen dynamischen Belastungen ausgesetzt. Die erzielten Ergebnisse sprechen dafür, die Ursachen einer Sehnenruptur im submikroskopischen fibrillären Bereich zu suchen. Sowohl eine langsame oder ruckartige Dehnung als auch eine ruckartige Entlastung zugbelasteter Fasern scheinen für das Auftreten disseminierter, die Faserstabilität reduzierender Gefügestörungen verantwortlich zu sein. Diese Gefügestörungen werden durch intrafibrilläre Gleitvorgänge eingeleitet, die an den noch voll belastbaren Fasern nur wenige Sekunden vor dem Einsetzen eines makroskopischen Faserfließens auftreten. Die Bedeutung dieser Ereignisse für den Beginn und Verlauf einer in vivo stattfindenden Reparationsphase wird erörtert. Die Aussagen werden gestützt durch simultane mechanische und röntgenographische Messungen sowie durch makroskopische, licht- und elektronenmikroskopische Befunde.

Topochemistry of the binding of phosphotungstic acid to collagen.

Abstract The interaction between collagen and phosphotungstic acid can be divided into two specific reaction steps, dependent on the steric arrangement. 1. (1) A relatively quick, firm intersubfibrillar binding (within ≈ 10 min) of the polyanions PW 12 O 40 3− to easily accessible basic groups. This occurs even when the units become more compact through tension, as indicated by meridional layer-line reflections. 2. (2) A less tight, intrasubfibrillar bond to relaxed incubated fibres (≧ 20 min) with participation of relatively heavy accessible amino groups, indicated by an equatorial reflection at ~19 A. The intrasubfibrillar PW 12 O 40 3− deposition is restricted by previous aldehyde cross-linking of the fibres, whereas it continues further with non-drawn and non-crosslinked fibres. In the latter case, a contraction of the fibre and a shortening of the 670 A period are observed. The polyanion PW 11 O 39 7− present at pH 5.5 to 7 is evidently bound to relaxed fibres by the guanido group only. The scattering reflections, the contraction of phosphotungstic acid-treated fibres and the associated changes of the long period, are interpreted on the basis of an eight-stranded unit for the subfibrils. These results have been obtained with native and chemically modified collagen fibres using conventional and synchrotron X-ray radiation as well as mechanical measuring devices.

Die Viskoelastizität parallelsträngigen Bindegewebes und ihre Bedeutung für die Funktion

A limited glide of overlapping subunits of collagen fibres under stretching forces represents a characteristic feature of the system. The viscoelastic behaviour of human and animal tendon collagen is described simultaneously by mechanical, morphological and X-ray data and correlated with the structural principle of the fibres. The existence of gliding subunits is proven by retardation- and relaxation-experiments in combination with short-time-x-ray-measurements by synchrotron-radiation. This behaviour may explain the damping properties of tendons. The retractive force of deformed helical and nonhelical molecular chains is generally thought to be responsible for the elasticity of the system. Length-increase of tendons during growth may be also correlated with the gliding processes. The so called conditioning-effect is due to an improved arrangement of possible reacting groups by cyclic stretch which can be well demonstrated on low ordered samples and is therefore greater in the case of juvenile specimens than in older ones. A quick first stretch causes greater molecular deformations than further stretches of the same strength, and may lead to disordered domains in the structure. A mathematical model for the simulation of the mechanical properties is given and was used for the calculation of some examples. Eine unter Zugbelastung begrenzte Parallelverschieblichkeit sich überlappender Untereinheiten (Fibrillen und Subfibrillen) wird als ein wesentliches Merkmal des Ordnungsprinzips kollagener Fasern angegeben. Das viskoelastische Verhalten von menschlichem und tierischem Sehnenkollagen wird anhand kombinierter mechanischer, röntgenographischer und morphologischer Meßdaten beschrieben und mit dem Ordnungsprinzip der Fasern korreliert. Das Vorliegen parallel verschieblicher Untereinheiten wird durch Retardations- und Relaxationsversuche in Kombination mit Kurzzeitbeugungsmessungen unter Einsatz der Synchrotronstrahlung bewiesen und mit der dämpfenden Eigenschaft der Sehnenfasern in Zusammenhang gebracht. Für die elastische Eigenschaft der Sehnenfasern wird die Rückstellkraft deformierter helikaler und nichthelikaler Molekülketten verantwortlich gemacht. Das Längenwachstum von Sehnenfasern wird mit dem Gleitvermögen von Untereinheiten korreliert. Der auf eine Verbesserung der Zueinanderausrichtung potentieller Bindungspartner beruhende Konditionierungseffekt einer zyklischen Faserbelastung ist an weniger gut formierten Einheiten besonders auffällig und entsprechend an jugendlichen Sehnenfasern größer als an alten. Die stoßartige Belastung einer Sehnenfaser aus der Ruhephase führt zu größeren molekularen Deformationen als nachfolgende gleichhohe Belastungen und kann fibrilläre Gefügestörungen hervorrufen. Es wird eine Modellkonstruktion zur Simulation mechanischer Eigenschaften von Kollagen beschrieben und für einige Beispiele durchgerechnet.

Zur Ätiologie der Kontraktur beim Morbus Dupuytren

Regarding experimental data on the multi-factorial reduction of the thermostability of collagen, the following sequence of the mechanism of Dupuytrens contracture is discussed: 1. Hereditary or acquired weakness of fibres of the palmar fascia 2. Further disturbance of the physical property of collagen by the cumulative effects of mechanical, i.e. traumatic, influence. 3. By this means an induced stepwise shrinkage of released fascial fibres in body temperature. 4. Stabilisation of the shortening (contracture) by development of collagen. This hypothesis is supported by histological, polarizing-microscopic, electron-microscopic and chemical results.Regarding experimental data on the multi-factorial reduction of the thermostability of collagen, the following sequence of the mechanism of Dupuytrens contracture is discussed: 1. Hereditary or acquired weakness of fibres of the palmar fascia 2. Further disturbance of the physical property of collagen by the cumulative effects of mechanical, i.e. traumatic, influence. 3. By this means an induced stepwise shrinkage of released fascial fibres in body temperature. 4. Stabilisation of the shortening (contracture) by development of collagen. Hereditary or acquired weakness of fibres of the palmar fascia Further disturbance of the physical property of collagen by the cumulative effects of mechanical, i.e. traumatic, influence. By this means an induced stepwise shrinkage of released fascial fibres in body temperature. Stabilisation of the shortening (contracture) by development of collagen. This hypothesis is supported by histological, polarizing-microscopic, electron-microscopic and chemical results. Anhand experimenteller Daten über eine multifaktorielle Erniedrigung der Thermostabilität des Kollagens wird für die Dupuytrensche Kontraktur der folgende Pathomechanismus zur Diskussion gestellt: 1. durch Vererbung oder Erkrankung eingeleitete Schwächung der Fasern der Palmaraponeurose; 2. durch Summation mechanischer, d.h. traumatischer Einflüsse zusätzliche Beeinträchtigung physikalischer Kollageneigenschaften: 3. hierdurch ausgelöste etappenweise Verkürzung entlasteter Fascienfasern bei Körpertemperatur; 4. Stabilisierung der eingetretenen Verkürzung (Kontraktur) durch Kollagenneubildung. durch Vererbung oder Erkrankung eingeleitete Schwächung der Fasern der Palmaraponeurose; durch Summation mechanischer, d.h. traumatischer Einflüsse zusätzliche Beeinträchtigung physikalischer Kollageneigenschaften: hierdurch ausgelöste etappenweise Verkürzung entlasteter Fascienfasern bei Körpertemperatur; Stabilisierung der eingetretenen Verkürzung (Kontraktur) durch Kollagenneubildung. Dieser Deutungsversuch wird durch licht- und polarisationsoptische, elektronenmikroskopische, röntgenographische sowie chemische Untersuchungsergebnisse gestützt.

Transformation of the structure of collagen: A time-resolved analysis of mechanochemical processes using synchrotron radiation

Mechanochemically induced molecular transformations of collagen fibres were analysed using time-resolved small-angle diffraction spectra and histomechanical measurements. In particular, the influence of aqueous and methanolic perchlorate solutions was examined. According to a transformation continuing from the periphery towards the centre, the macroscopic contraction that is completed less than five minutes after incubation with perchlorate is caused by peripherally transformed fibrils only, whereas the centrally situated fibrils first undergo an accordion-like folding, but after more than 20 minutes are transformed similarly. The triple-helical transformation is preceded by a structure-breaking effect on structural water that can be monitored in time-resolved diffraction spectra. The combined loss of meridional low-angle reflections and cross-striated fibrils in micrographs is irreversible. By dialysis of colloidally dissolved collagen against a solution of ATP, however, segment-long spacing aggregates are obtained. Under isometric conditions, an instantaneous transformation of intermittent regions leads to an increase in the long period of adjacent, still structured regions of the same fibril that is correlated with a delayed increase in tension in the fibre. Increase of tension under isometric conditions as well as the flow-properties of a fibre relaxed in perchlorate are interpreted in terms of the parallel sliding of subunits of varying lengths, which has been demonstrated by diffraction analysis.

Glutaraldehyde-induced states of stress of the collagen triple helix

Abstract Demonstration of a decrease in pitch of some turns of the collagen triple helix caused by glutaraldehyde as shown in the stress and low-angle X-ray diagram by use of synchrotron radiation.