Rat Tail Tendon Fibers Research Articles

A damage model for collagen fibres with an application to collagenous soft tissues.

We propose a mechanical model to account for progressive damage in collagen fibres within fibrous soft tissues. The model has a similar basis to the pseudoelastic model that describes the Mullins effect in rubber but it also accounts for the effect of cross-links between collagen fibres. We show that the model is able to capture experimental data obtained from rat tail tendon fibres, and the combined effect of damage and collagen cross-links is illustrated for a simple shear test. The proposed three-dimensional framework allows a straightforward implementation in finite-element codes, which are needed to analyse more complex boundary-value problems for soft tissues under supra-physiological loading or tissues weakened by disease.

Effect of Low-Intensity Ultrasound Stimulation on the Attenuation of Mechanical Properties of Fibrous Tissue

The effect of low intensity ultrasound on the mechanical properties of soft tissue such as tendon is not well understood. In this study, the rat tail tendons were used to determine the effect of ultrasound on the mechanical properties of tendon fiber. Twenty two collagen fibers were extracted from rat tail tendon fibers. The collagen fibers were separated into two groups, one with and one without ultrasound stimulations. Cyclic loading test within toe-region of collagen fibers were conducted in degassed water. The intensity and frequency of the ultrasound was 70 mW/cm2 and 1 MHz respectively. The results showed the tangent modulus of the collagen fiber in the ultrasound group was reduced from 300 MPa to 250 MPa in comparison to the control group. Further mechanical test and analysis of mathematical model should be done to understand the mechanism and reaction between ultrasound and the stiffness of the soft tissue.

Role of Preferential Ions of Ammonium Ionic Liquid in Destabilization of Collagen.

Ions play a key role in the destabilization of collagen. This study explores the effect of diethyl methyl ammonium methane sulfonate (AMS), an ionic liquid (IL), on different hierarchical orderings of collagen, namely, at the molecular and fibrillar levels. The rheological behavior and secondary structural changes reveal changes in the hydrogen-bonding environment of collagen, leading to alterations in the triple helical structure of collagen. An increase in the concentration of AMS resulted in swelling of rat-tail tendon fibers, and also, decreased thermal stability signifies that ions are obliged to destabilize collagen at the fibrillar level. Molecular modeling studies confirm that anions are judiciously held responsible for structural deformities in collagen, whereas cations have a tenuous effect. Thus, the preferential role of ions present in an ammonium IL has been elucidated in this study.

Stabilization of type I collagen using dialdehyde cellulose

Type I collagen from rat tail tendon (RTT) fibres was crosslinked with dialdehyde cellulose to bring about stabilization of the matrix. Dialdehyde cellulose (DAC) was prepared by periodate oxidation of hydrolyzed cellulose. Autoclaving of DAC resulted in hydrolysis and lower molecular weight oligomeric species. The formation of the crosslinked network between DAC and the collagen fibres has brought about significant thermal and enzymatic stability to collagen. DAC crosslinked collagen fibres exhibited an increase in hydrothermal stability by 20 °C with autoclaved DAC at pH 8. The collagen matrix resulted in an increase in denaturation peak temperature ( T D) and an increase in phase change of activation energy ( E a) and enthalpy change (Δ H) for the shinking process indicating intermolecular crosslinking arising from covalent interactions. Thermal stability and crosslinking efficiency was found to increase with pH and concentration of DAC. DAC treated collagen exhibited 93% resistance to collagenolytic hydrolysis.

Deformation-Dependent Enzyme Mechanokinetic Cleavage of Type I Collagen

Collagen is a key structural protein in the extracellular matrix of many tissues. It provides biological tissues with tensile mechanical strength and is enzymatically cleaved by a class of matrix metalloproteinases known as collagenases. Collagen enzymatic kinetics has been well characterized in solubilized, gel, and reconstituted forms. However, limited information exists on enzyme degradation of structurally intact collagen fibers and, more importantly, on the effect of mechanical deformation on collagen cleavage. We studied the degradation of native rat tail tendon fibers by collagenase after the fibers were mechanically elongated to strains of epsilon=1-10%. After the fibers were elongated and the stress was allowed to relax, the fiber was immersed in Clostridium histolyticum collagenase and the decrease in stress (sigma) was monitored as a means of calculating the rate of enzyme cleavage of the fiber. An enzyme mechanokinetic (EMK) relaxation function T(E)(epsilon) in s(-1) was calculated from the linear stress-time response during fiber cleavage, where T(E)(epsilon) corresponds to the zero order Michaelis-Menten enzyme-substrate kinetic response. The EMK relaxation function T(E)(epsilon) was found to decrease with applied strain at a rate of approximately 9% per percent strain, with complete inhibition of collagen cleavage predicted to occur at a strain of approximately 11%. However, comparison of the EMK response (T(E) versus epsilon) to collagen's stress-strain response (sigma versus epsilon) suggested the possibility of three different EMK responses: (1) constant T(E)(epsilon) within the toe region (epsilon<3%), (2) a rapid decrease ( approximately 50%) in the transition of the toe-to-heel region (epsilon congruent with3%) followed by (3) a constant value throughout the heel (epsilon=3-5%) and linear (epsilon=5-10%) regions. This observation suggests that the mechanism for the strain-dependent inhibition of enzyme cleavage of the collagen triple helix may be by a conformational change in the triple helix since the decrease in T(E)(epsilon) appeared concomitant with stretching of the collagen molecule.

Role of Storage on Changes in the Mechanical Properties of Tendon and Self-Assembled Collagen Fibers

Fibrous collagen networks are the major elements that provide mechanical integrity to tissues; they are composed of fiber forming collagens in combination with proteoglycans (PGs). Using uniaxial tensile tests we have studied the viscoelastic mechanical properties of rat tail tendon (RTT) fibers and self-assembled collagen fibers that were stored at 22°C and 1 atm of pressure. Our results indicate that storage of RTT and self-assembled type I collagen fibers results in increased elastic and viscous components of the stress-strain behavior consistent with the hypothesis that storage causes the introduction of crosslinks.Analysis of the elastic and viscous mechanical data suggests that the elastic constant of the collagen molecule in RTT is about 7.7 GPa. Measurement of the viscous component of the stress-strain curves for RTTs and self-assembled collagen fibers suggests that PGs may increase the viscous component and effectively increase the collagen fibril length.

Chromium(III) hydrolytic oligomers: their relevance to protein binding

The nature of chromium(III) complexes has been found to show a profound influence in its interaction with collagen. The hydrothermal stability of rat tail tendon (RTT) fibres treated with dimeric, trimeric and tetrameric species of chromium(III) has been found to be 102, 87 and 68°C, while that of native RTT is 62°C. This shows that the efficiency of crosslinking of collagen by chromium(III) species is dimeric>trimeric>tetrameric. This order of stabilisation is again confirmed by cyanogen bromide (CNBr) cleavage of RTT collagen treated with dimeric, trimeric and tetrameric chromium(III) species. CNBr has been found to cleave the collagen treated with tetrameric chromium(III) species extensively. On the other hand, dimer-treated collagen does not undergo any cleavage on CNBr treatment. The equilibrium constants for the reaction of a nucleophile like NCS − to the dimeric, trimeric and tetrameric species of chromium(III) have been found to be 15.7±0.1, 14.6±0.1 and 1.2±0.1 M −1, respectively. These equilibrium constant values reflect the relative thermodynamic stability of the chromium(III) species-nucleophile complex. The low stabilising effect of the tetrameric species can be traced to its low thermodynamic affinity for nucleophiles.

Mapping strain exerted on blood vessel walls using deuterium double-quantum-filtered MRI.

A technique is described for displaying distinct tissue layers of large blood vessel walls as well as measuring their mechanical strain. The technique is based on deuterium double-quantum-filtered (DQF) spectroscopic imaging. The effectiveness of the double-quantum filtration in suppressing the signal of bulk water is demonstrated on a phantom consisting of rat tail tendon fibers. Only intrafibrillar water is displayed, excluding all other signals of water molecules that reorient isotropically. One- and two-dimensional spectroscopic imaging of bovine aorta and coronary arteries show the characteristic DQF spectrum of each of the tissue layers. This property is used to obtain separate images of the outer layer, the tunica adventitia, or the intermediate layer, the tunica media, or both. To visualize the effect of elongation, the average residual quadrupole splitting <delta nu q> is calculated for each pixel. Two-dimensional deuterium quadrupolar splitting images are obtained for a fully relaxed and a 55% elongated sample of bovine coronary artery. These images indicate that the strong effect of strain is associated with water molecules in the tunica adventitia whereas the DQF NMR signal of water in the tunica media is apparently strain-insensitive. After appropriate calibration, these average quadrupolar splitting images can be interpreted as strain maps.

Self-assembly of collagen fibers. Influence of fibrillar alignment and decorin on mechanical properties

Collagen is the primary structural element in extracellular matrices. In the form of fibers it acts to transmit forces, dissipate energy, and prevent premature mechanical failure in normal tissues. Deformation of collagen fibers involves molecular stretching and slippage, fibrillar slippage, and, ultimately, defibrillation. Our laboratory has developed a process for self-assembly of macroscopic collagen fibers that have structures and mechanical properties similar to rat tail tendon fibers. The purpose of this study is to determine the effects of subfibrillar orientation and decorin incorporation on the mechanical properties of collagen fibers. Self-assembled collagen fibers were stretched 0-50% before cross-linking and then characterized by microscopy and mechanical testing. Results of these studies indicate that fibrillar orientation, packing, and ultimate tensile strength can be increased by stretching. In addition, it is shown that decorin incorporation increases ultimate tensile strength of uncross-linked fibers. Based on the observed results it is hypothesized that decorin facilitates fibrillar slippage during deformation and thereby improves the tensile properties of collagen fibers.

Structure of rat tail tendon collagen examined by atomic force microscope.

The Atomic Force Microscope (AFM) was used to inspect collagen fibrils deposited on mica sheets at different fibrillogenesis times. Collagen was obtained from rat tail tendon fibers. Various fibril forms were observed, together with the characteristic periodic intra-fibril structure (D-bands). The fibril thickness, width, D-band periodicity and depth were measured and the statistical distribution of these parameters at 1, 2, 5, 10 and 15 days of in vitro fibril formation time was calculated. The fibrils showed an increasing size with time, but the band interval measure remained stable. The band depth, after an initial increase, exhibited a relative steadiness. The results indicate that AFM offers, at low resolution, images qualitatively similar to those obtained with electron microscopy, but with less manipulation of the sample. A quantitative evaluation of collagen structural features in the nanometer scale is made possible by AFM.

Alterations of biochemical and biomechanical properties of rat tail tendons caused by non-enzymatic glycation and their inhibition by dibasic amino acids arginine and lysine

The influence of dibasic amino acids arginine and lysine on non-enzymatic glycation of tail tendon fibers from old (900-day-old) and young (61-day-old) rats was investigated in vitro. The biomechanical changes in tendon fibers of young rats after an incubation interval of 7 or 14 days in a glucose solution were abolished by the addition of arginine or lysine (molar ratio amino acid:glucose 1:10). Glucose incorporation into rat tail tendon fibers as well as Amadori product formation was decreased significantly in the presence of the amino acids. The inhibitory effect of arginine was further confirmed by measurement of the amount of ketoamine formed during the glycation reaction using soluble albumin as a protein target. The effective inhibition of non-enzymatic glycation by arginine or lysine suggests their potential use in vivo as a means of controlling protein over-glycation.

Mechanical properties of collagen fibres: a comparison of reconstituted and rat tail tendon fibres

This study involves comparison of the mechanical properties of reconstituted collagen fibres with those of collagen fibres obtained from rat tail tendons. Reconstituted collagen fibres were cross-linked in the presence of glutaraldehyde vapour for 2 and 4 d or using a combination of severe dehydration and carbodiimide treatment. Ultimate tensile strengths for reconstituted fibres cross-linked with glutaraldehyde ranged from 50 to 66 M Pa while those cross-linked by severe dehydration and carbodiimide treatment had ultimate tensile strengths between 24 and 31 MPa. Rat tail tendon fibres had tensile strengths that ranged from 33 to 39 MPa. These results indicate that high-strength collagen fibres can be reconstituted in vitro and that these fibres may be useful in repair of dermal, dental, cardiovascular and orthopaedic defects.

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.

Water content in type I collagen tissues calculated from the generalized packing model

The generalized packing model, previously suggested to account for observed variations in the equatorial diffraction spacing of wet soft type I collagen tissues, was used to calculate the water content within the collagen fibrils of rat bone matrix, rat tail tendon fibres, and fully mineralized cow tibia. These tissues were selected because the variation of equatorial diffraction spacing with water content is known for each. A certain value of the water content, defined as the characteristic value, can be identified from the plot of the variables and is taken to be the water contained only in the collagen fibrils for that tissue. The experimentally determined characteristic value and the calculated water content based on the equatorial spacing were found to be practically identical for each of the three tissues, with a discrepancy no greater than 0.02 g/g. On the other hand, the more widely accepted hexagonal packing model gives a discrepancy of 0.34 g/g for bone matrix and 0.16 g/g for bone. Further, for the generalized packing model the closest intermolecular spacing in the dried state is about one molecular diameter, but much larger for the hexagonal packing. It is concluded that the equatorial diffraction spacing for all dried type I collagen tissues is essentially the same because in all instances the molecules are in contact. Finally, it is suggested that a single critical crosslink can account for all the observed data. The composition and configuration of the postulated critical crosslink can determine the wet tissue spacing, whether the tissue will mineralize, the density of the mineralized tissue and the spacing as the water content of the tissue varies. Such a critical crosslink makes it possible for the appropriate cells to control the tissue characteristics remotely in the excellular space.

Interferometric evaluation of collagen concentration in tendon fibers.

The dry mass concentration of collagen in native rat tail tendon fibers was studied by interferometric techniques. It could be shown that the collagen concentration of rat tail tendon fibers is not constant nor does it vary uniformly with the fiber diameter. Evidence is presented that the collagen concentration shows significant oscillations as a function of diameter. In order to explain the experimental findings, a layered model is proposed for the fiber structure; age dependent changes were also studied. It seems that the fiber structure is unaltered by ageing at the supramolecular level but its functional capacities are affected and swelling is inhibited.

Axial Phase Velocity in Rat Tail Tendon Fibers at 100 MHz by Ultrasonic Microscopy

Axial Phase Velocity in Rat Tail Tendon Fibers at 100 MHz by Ultrasonic Microscopy

Density of a sample bovine cortical bone matrix and its solid constituent in various media.

The density of a bovine cortical bone matrix sample was found in water, several ethanol-water solutions, and in dried state. Previously the density of the same mineralized bone was found fresh and when desiccated. The volume in each state was estimated from the dimensional changes axially, tangentially, and radially. Confirmation was found by determining the density of dried specimens upon immersion in xylene. The amount of imbibed xylene provided an estimate of the free pore volume in the dried matrix. The volume fraction of the solid constituent, S, in the wet matrix was found to be 0.57, from which the density of S in various solutions was calculated. Density of wet matrix in 0.15 M saline: 1.180 g/cc; for dried matrix, 1.246 g/cc. Density of wet S in saline: 1.33 g/cc; for dried S, 1.42 g/cc, which matches published values for collagen molecules. Dimensional changes between wet and dried state of matrix match published values for artificially cross-linked rat tail tendon fibers. Axially: 1.04, by area: 2.27; by volume: 2.62. Estimate of intrafibrillar volume, assuming 80% of mineral is within fibrils: 0.73 cc/g dry collagen.

The tensile properties and mode of fracture of elastoidin

The tensile properties and mode of fracture of elastoidin, a collagenous protein fibre from the fins of sharks, were compared with those of rat tail tendon fibres, considered to be a pure form of collagen. Elastoidin fibres were stronger than tendon in the dry state whereas the opposite was observed for fibres tested in the wet state. However, elastoidin was stiffer than tendon whether dry or wet. Scanning electron micrographs of the crosssections and fractured surfaces revealed that elastoidin fibres consisted of fibrils of varying diameter arranged in a lamellar fashion. From the nature of the fractured surfaces, it could be deduced that the primary failure mechanism for elastoidin was probably through a fissuring of the structure.

Low angle X-ray diffraction studies on stained rat tail tendons

Low angle X-ray diffraction patterns of rat tail tendons with heavy metal stains added were examined to help clarify the effects of fixation and staining on collagen fibrils. Fixing and staining of rat tail tendon fibers gives an X-ray pattern with an intensified 3.8 nm row line and the preservation of most equatorial features found in the native pattern. The presence of the native pattern features suggests the value of fixation in preserving native structure before staining. Staining of rat tail tendon fibers without prior fixation led to the disappearance of the native equatorial features and the appearance of a new broad row line corresponding to a spacing of around 10.0–17.5 nm. This observation suggests that some alteration has taken place in the native structure and may be related to electron microscopic observations of units of 10.0–20.0 nm in collagen fibrils under some disruptive or developmental conditions.

Age factor in the maturation of collagen. Cross-links in heat-denatured collagen in tail tendon and skin of rat

The statistical stress-strain theory of elasticity was applied to heated rat tail tendon fibres and skin strips to estimate the amount of cross-links in collagen. In the tail tendon collagen the average molecular weight between the junction points ( M c ) did not change from about 50,000, which was observed at 3 months of age. In skin strips the M c decreased with advancing age from about 210,000 in rats of 3 months to 40,000 in rats of 24 months.