Tenocyte contraction induces crimp formation in tendon-like tissue

Tendons are composed of longitudinally aligned collagen fibrils arranged in bundles with an undulating pattern, called crimp. The crimp structure is established during embryonic development and plays a vital role in the mechanical behaviour of tendon, acting as a shock-absorber during loading. However, the mechanism of crimp formation is unknown, partly because of the difficulties of studying tendon development in vivo. Here, we used a 3D cell culture system in which embryonic tendon fibroblasts synthesise a tendon-like construct comprised of collagen fibrils arranged in parallel bundles. Investigations using polarised light microscopy, scanning electron microscopy and fluorescence microscopy showed that tendon constructs contained a regular pattern of wavy collagen fibrils. Tensile testing indicated that this superstructure was a form of embryonic crimp producing a characteristic toe region in the stress–strain curves. Furthermore, contraction of tendon fibroblasts was the critical factor in the buckling of collagen fibrils during the formation of the crimp structure. Using these biological data, a finite element model was built that mimics the contraction of the tendon fibroblasts and monitors the response of the Extracellular matrix. The results show that the contraction of the fibroblasts is a sufficient mechanical impulse to build a planar wavy pattern. Furthermore, the value of crimp wavelength was determined by the mechanical properties of the collagen fibrils and inter-fibrillar matrix. Increasing fibril stiffness combined with constant matrix stiffness led to an increase in crimp wavelength. The data suggest a novel mechanism of crimp formation, and the finite element model indicates the minimum requirements to generate a crimp structure in embryonic tendon.     

Recruitment of tendon crimp with applied tensile strain

The tensile stress-strain behavior of ligaments and tendons begins with a toe region that is believed to result from the straightening of crimped collagen fibrils. The in situ mechanical function is mostly confined to this toe region and changes in crimp morphology are believed to be associated with pathological conditions. A relatively new imaging technique, optical coherence tomography (OCT), provides a comparatively inexpensive method for nondestructive investigation of tissue ultrastructure with resolution on the order of 15 microm and the potential for use in a clinical setting. The objectives of this work were to assess the utility of OCT for visualizing crimp period, and to use OCT to determine how crimp period changed as a function of applied tensile strain in rat tail tendon fascicles. Fascicles from rat tail tendons were subjected to 0.5 percent strain increments up to 5 percent and imaged at each increment using OCT. A comparison between OCT images and optical microscopy images taken between crossed polarizing lenses showed a visual correspondence between features indicative of crimp pattern. Crimp pattern always disappeared completely before 3 percent axial strain was reached. Average crimp period increased as strain increased, but both elongation and shortening occurred within single crimp periods during the application of increasing strain to the fascicle.     

Tendon crimps and peritendinous tissues responding to tensional forces

Tendons transmit forces generated from muscle to bone making joint movements possible. Tendon collagen has a complex supramolecular structure forming many hierarchical levels of association; its main functional unit is the collagen fibril forming fibers and fascicles. Since tendons are enclosed by loose connective sheaths in continuity with muscle sheaths, it is likely that tendon sheaths could play a role in absorbing/transmitting the forces created by muscle contraction. In this study rat Achilles tendons were passively stretched in vivo to be observed at polarized light microscope (PLM), scanning electron microscope (SEM) and transmission electron microscope (TEM). At PLM tendon collagen fibers in relaxed rat Achilles tendons ran straight and parallel, showing a periodic crimp pattern. Similarly tendon sheaths showed apparent crimps. At higher magnification SEM and TEM revealed that in each tendon crimp large and heterogeneous collagen fibrils running straight and parallel suddenly changed their direction undergoing localized and variable modifications. These fibril modifications were named fibrillar crimps. Tendon sheaths displayed small and uniform fibrils running parallel with a wavy course without any ultrastructural aspects of crimp. Since in passively stretched Achilles tendons fibrillar crimps were still observed, it is likely that during the tendon stretching, and presumably during the tendon elongation in muscle contraction, the fibrillar crimp may be the real structural component of the tendon crimp acting as shock absorber. The peritendinous sheath can be stretched as tendon, but is not actively involved in the mechanism of shock absorber as the fibrillar crimp. The different functional behaviour of tendons and sheaths may be due to the different structural and molecular arrangement of their fibrils.     

The dynamics of collagen uncrimping and lateral contraction in tendon and the effect of ionic concentration

Under tensile loading, tendon undergoes a number of unique structural changes that govern its mechanical response. For example, stretching a tendon is known to induce both the progressive “uncrimping” of wavy collagen fibrils and extensive lateral contraction mediated by fluid flow out of the tissue. However, it is not known whether these processes are interdependent. Moreover, the rate-dependence of collagen uncrimping and its contribution to tendon's viscoelastic mechanical properties are unknown. Therefore, the objective of this study was to (a) develop a methodology allowing for simultaneous measurement of crimp, stress, axial strain and lateral contraction in tendon under dynamic loading; (b) determine the interdependence of collagen uncrimping and lateral contraction by testing tendons in different swelling conditions; and (c) assess how the process of collagen uncrimping depends on loading rate. Murine flexor carpi ulnaris (FCU) tendons in varying ionic environments were dynamically stretched to a set strain level and imaged through a plane polariscope with the polarizer and analyzer at a fixed angle. Analysis of the resulting images allowed for direct measurement of the crimp frequency and indirect measurement of the tendon thickness. Our findings demonstrate that collagen uncrimping and lateral contraction can occur independently and interstitial fluid impacts tendon mechanics directly. Furthermore, tensile stress, transverse contraction and degree of collagen uncrimping were all rate-dependent, suggesting that collagen uncrimping plays a role in tendon's dynamic mechanical response. This study is the first to characterize the time-dependence of collagen uncrimping in tendon, and establishes structure–function relationships for healthy tendons that can be used to better understand and assess changes in tendon mechanics after disease or injury.     

Crimping in rat tail tendon collagen: morphology and transverse mechanical anisotropy

Examination of rat tail tendon units in scanning electron microscopy (SEM) after the removal of the endotendinium by the use of swelling agents and in comparison with controls confirms and extends our knowledge of a substantially planar crimping along the fibre axis. Polarizing optical microscopy of intact units subjected to lateral compression of controlled direction indicates a definite transverse mechanical anisotropy directly related to the morphological defined crimp plane, sensitive to shear disruption but capable of reconstitution on low strain cyclin.     

Monitoring micrometer-scale collagen organization in rat-tail tendon upon mechanical strain using second harmonic microscopy

We continuously monitored the microstructure of a rat-tail tendon during stretch/relaxation cycles. To that purpose, we implemented a new biomechanical device that combined SHG imaging and mechanical testing modalities. This multi-scale experimental device enabled simultaneous visualization of the collagen crimp morphology at the micrometer scale and measurement of macroscopic strain-stress response. We gradually increased the ultimate strain of the cycles and showed that preconditioning mostly occurs in the first stretching. This is accompanied by an increase of the crimp period in the SHG image. Our results indicate that preconditioning is due to a sliding of microstructures at the scale of a few fibrils and smaller, that changes the resting length of the fascicle. This sliding can reverse on long time scales. These results provide a proof of concept that continuous SHG imaging performed simultaneously with mechanical assay allows analysis of the relationship between macroscopic response and microscopic structure of tissues.     

Collagen fibril size and crimp morphology in ruptured and intact Achilles tendons.

The present study examined the hypothesis that collagen fibril diameter and crimp angle in ruptured human Achilles tendons differed from that of intact ones. Tissue samples were obtained from the central core (distal core) and the posterior periphery (distal superficial) at the rupture site, and the proximally intact (proximal superficial) part of the tendon in 10 subjects (38+/-8 years) with a complete tendon rupture. For comparisons corresponding tissue samples were procured from age (38+/-7 years) and gender matched intact Achilles tendons during routine forensic autopsy. The cross-sectional area density and diameter distribution of fibrils were analyzed using stereological techniques of digitized electron microscopy biopsy cross-sections, while crimp angle was measured by the changing banding pattern of collagen fibers when rotated between crossed polars. Nine of 10 persons with tendon ruptures reported that the injury did not occur during exceedingly large forces, and none experienced any symptoms in the days or months prior to the injury. Fibril diameter distribution showed no region-specific differences in either the ruptured or intact tendons for either group. However, in the distal core there were fewer fibrils in the ruptured compared to the intact tendons in 60-150 nm range, P<0.01. Similarly, in the distal superficial portion there were fewer fibrils in the ruptured compared to the intact tendons in the 90-120 nm range, 2P<0.05, while there were no differences in the proximal superficial tendons. Crimp angle did not display any region-specific differences, or any difference between the rupture and intact tendons. In conclusion, these data suggest that although crimp morphology is unchanged there appears to be a site-specific loss of larger fibrils in the core and periphery of the Achilles tendon rupture site. Moreover, the lack of symptoms prior to the rupture suggests that clinical tendinopathy is not an etiological factor in complete tendon ruptures.     

Estimation of finger muscle tendon tensions and pulley forces during specific sport-climbing grip techniques

The present work displayed the first quantitative data of forces acting on tendons and pulleys during specific sport-climbing grip techniques. A three-dimensional static biomechanical model was used to estimate finger muscle tendon and pulley forces during the "slope" and the "crimp" grip. In the slope grip the finger joints are flexed, and in the crimp grip the distal interphalangeal (DIP) joint is hyperextended while the other joints are flexed. The tendons of the flexor digitorum profundus and superficialis (FDP and FDS), the extensor digitorum communis (EDC), the ulnar and radial interosseus (UI and RI), the lumbrical muscle (LU) and two annular pulleys (A2 and A4) were considered in the model. For the crimp grip in equilibrium conditions, a passive moment for the DIP joint was taken into account in the biomechanical model. This moment was quantified by relating the FDP intramuscular electromyogram (EMG) to the DIP joint external moment. Its intensity was estimated at a quarter of the external moment. The involvement of this parameter in the moment equilibrium equation for the DIP joint is thus essential. The FDP-to-FDS tendon-force ratio was 1.75:1 in the crimp grip and 0.88:1 in the slope grip. This result showed that the FDP was the prime finger flexor in the crimp grip, whereas the tendon tensions were equally distributed between the FDP and FDS tendons in the slope grip. The forces acting on the pulleys were 36 times lower for A2 in the slope grip than in the crimp grip, while the forces acting on A4 were 4 times lower. This current work provides both an experimental procedure and a biomechanical model that allows estimation of tendon tensions and pulley forces crucial for the knowledge about finger injuries in sport climbing.     

Biomechanical properties of the crimp grip position in rock climbers

Rock climbers are often using the unique crimp grip position to hold small ledges. Thereby the proximal interphalangeal (PIP) joints are flexed about 90 degrees and the distal interphalangeal joints are hyperextended maximally. During this position of the finger joints bowstringing of the flexor tendon is applying very high load to the flexor tendon pulleys and can cause injuries and overuse syndromes. The objective of this study was to investigate bowstringing and forces during crimp grip position. Two devices were built to measure the force and the distance of bowstringing and one device to measure forces at the fingertip. All measurements of 16 fingers of four subjects were made in vivo. The largest amount of bowstringing was caused by the flexor digitorum profundus tendon in the crimp grip position being less using slope grip position (PIP joint extended). During a warm-up, the distance of bowstringing over the distal edge of the A2 pulley increased by 0.6mm (30%) and was loaded about 3 times the force applied at the fingertip during crimp grip position. Load up to 116N was measured over the A2 pulley. Increase of force in one finger holds by the quadriga effect was shown using crimp and slope grip position.     

Characterizing local collagen fiber re-alignment and crimp behavior throughout mechanical testing in a mature mouse supraspinatus tendon model

BackgroundCollagen fiber re-alignment and uncrimping are two postulated mechanisms of tendon structural response to load. Recent studies have examined structural changes in response to mechanical testing in a postnatal development mouse supraspinatus tendon model (SST), however, those changes in the mature mouse have not been characterized. The objective of this study was to characterize collagen fiber re-alignment and crimp behavior throughout mechanical testing in a mature mouse SST.Method of approachA tensile mechanical testing set-up integrated with a polarized light system was utilized for alignment and mechanical analysis. Local collagen fiber crimp frequency was quantified immediately following the designated loading protocol using a traditional tensile set up and a flash-freezing method. The effect of number of preconditioning cycles on collagen fiber re-alignment, crimp frequency and mechanical properties in midsubstance and insertion site locations were examined.ResultsDecreases in collagen fiber crimp frequency were identified at the toe-region of the mechanical test at both locations. The insertion site re-aligned throughout the entire test, while the midsubstance re-aligned during preconditioning and the test's linear-region. The insertion site demonstrated a more disorganized collagen fiber distribution, lower mechanical properties and a higher cross-sectional area compared to the midsubstance location.ConclusionsLocal collagen fiber re-alignment, crimp behavior and mechanical properties were characterized in a mature mouse SST model. The insertion site and midsubstance respond differently to mechanical load and have different mechanisms of structural response. Additionally, results support that collagen fiber crimp is a physiologic phenomenon that may explain the mechanical test toe-region.     

A model for the creep behaviour of tendon

A theoretical model is proposed to explain the viscoelastic behaviour of tendon. The model is based on the hypothesis that the mechanism of tendon deformation is one of shear of the mucopolysaccharide gel between the collagen ribbons in the ‘toe’ region¹ of the stress strain curve followed by fibrillar extension in the ‘linear’ region^2.3. Conventional linear viscoelastic parameters of the constituents of the tendon were used to describe the behaviour of the composite. Certain structural constants of the tendon and an appropriate form for the retardation spectrum of the composite also appear in the model. It was found that the following parameters play an important part in the viscoelastic deformation process; the mean value of the crimp angle of the strained fibres, 0_O; the broadness of the distribution of crimp angles $\text{α}$; the magnitude of the compliance difference for the gel, ΔJ_G and for the fibres, ΔD_F. Excellent agreement between experiment and theory has been observed for a variety of experimental circumstances. Values of 0_O, $\text{α}$ ΔJ_G and ΔD_F were determined for a number of specimens by fitting the model equations to the experimental data. The theory illustrates the expected influence on the viscoelastic properties of tendon which would result from changes in these parameters, which may arise from disease or ageing, for instance. The model also provides a challenge for future experimental work in that an independent determination of the parameters, 0_O, $\text{α}$ ΔJ_G and ΔD_F would confirm or refute the quantitative predictions of the theory presented here.     

A finite element model predicts the mechanotransduction response of tendon cells to cyclic tensile loading

The importance of fluid-flow-induced shear stress and matrix-induced cell deformation in transmitting the global tendon load into a cellular mechanotransduction response is yet to be determined. A multiscale computational tendon model composed of both matrix and fluid phases was created to examine how global tendon loading may affect fluid-flow-induced shear stresses and membrane strains at the cellular level. The model was then used to develop a quantitative experiment to help understand the roles of membrane strains and fluid-induced shear stresses on the biological response of individual cells. The model was able to predict the global response of tendon to applied strain (stress, fluid exudation), as well as the associated cellular response of increased fluid-flow-induced shear stress with strain rate and matrix-induced cell deformation with strain amplitude. The model analysis, combined with the experimental results, demonstrated that both strain rate and strain amplitude are able to independently alter rat interstitial collagenase gene expression through increases in fluid-flow-induced shear stress and matrix-induced cell deformation, respectively.     

Micromechanical models of helical superstructures in ligament and tendon fibers predict large Poisson's ratios

Experimental measurements of the Poisson's ratio in tendon and ligament tissue greatly exceed the isotropic limit of 0.5. This is indicative of volume loss during tensile loading. The microstructural origin of the large Poisson's ratios is unknown. It was hypothesized that a helical organization of fibrils within a fiber would result in a large Poisson's ratio in ligaments and tendons, and that this helical organization would be compatible with the crimped nature of these tissues, thus modeling their classic nonlinear stress–strain behavior. Micromechanical finite element models were constructed to represent crimped fibers with a super-helical organization, composed of fibrils embedded within a matrix material. A homogenization procedure was performed to determine both the effective Poisson's ratio and the Poisson function. The results showed that helical fibril organization within a crimped fiber was capable of simultaneously predicting large Poisson's ratios and the nonlinear stress–strain behavior seen experimentally. Parametric studies revealed that the predicted Poisson's ratio was strongly dependent on the helical pitch, crimp angle and the material coefficients. The results indicated that, for physiologically relevant parameters, the models were capable of predicting the large Poisson's ratios seen experimentally. It was concluded that helical organization within a crimped fiber can produce both the characteristic nonlinear stress–strain behavior and large Poisson's ratios, while fiber crimp alone could only account for the nonlinear stress–strain behavior.     

Influence of sensor size on the accuracy of in-vivo ligament and tendon force measurements.

In-vivo tendon forces are commonly measured using transducers, which detect tension in the tendon fibers. A poorly understood source of measurement errors is the difference in stress distribution within the tendon between experimental and transducer calibration conditions. The objective of this study was to investigate this source of error, and to determine whether these errors could be minimized by proper selection of transducer size. The study was conducted using the infrapatellar ligament (patellar tendon) of New Zealand White rabbits. Tendon force was measured with two different size implantable force transducers (IFTs), one Wide and one Narrow, and by a strain gaged load cell in series with the tendon. Tests were conducted at five different loading conditions selected to produce five different stress distributions within the tendon. One loading condition corresponded to a typical post-experiment calibration, and the data from that condition were used to develop a calibration equation for the transducer. The errors that resulted from using this calibration were determined by comparing the tendon force measured by the in-series load cell with the force predicted from the IFT output using the calibration equation. Changes in stress distribution produced measurement errors up to 64 N with the Narrow IFT but only 24 N with the Wide IFT. We found the measurement error was dependent on sensor width. Our results support the hypothesis that measurement errors can be caused by differences in tendon stress distribution between calibration and experimental conditions. We further showed that these errors can be minimized by using an IFT, which samples the tension in a large percentage of the tendon fibers. Information from this study can be used for selection of an appropriately sized implantable force transducer for measuring tendon and ligament force.     

Biaxial tensile testing and constitutive modeling of human supraspinatus tendon

The heterogeneous composition and mechanical properties of the supraspinatus tendon offer an opportunity for studying the structure-function relationships of fibrous musculoskeletal connective tissues. Previous uniaxial testing has demonstrated a correlation between the collagen fiber angle distribution and tendon mechanics in response to tensile loading both parallel and transverse to the tendon longitudinal axis. However, the planar mechanics of the supraspinatus tendon may be more appropriately characterized through biaxial tensile testing, which avoids the limitation of nonphysiologic traction-free boundary conditions present during uniaxial testing. Combined with a structural constitutive model, biaxial testing can help identify the specific structural mechanisms underlying the tendon's two-dimensional mechanical behavior. Therefore, the objective of this study was to evaluate the contribution of collagen fiber organization to the planar tensile mechanics of the human supraspinatus tendon by fitting biaxial tensile data with a structural constitutive model that incorporates a sample-specific angular distribution of nonlinear fibers. Regional samples were tested under several biaxial boundary conditions while simultaneously measuring the collagen fiber orientations via polarized light imaging. The histograms of fiber angles were fit with a von Mises probability distribution and input into a hyperelastic constitutive model incorporating the contributions of the uncrimped fibers. Samples with a wide fiber angle distribution produced greater transverse stresses than more highly aligned samples. The structural model fit the longitudinal stresses well (median R(2) ≥ 0.96) and was validated by successfully predicting the stress response to a mechanical protocol not used for parameter estimation. The transverse stresses were fit less well with greater errors observed for less aligned samples. Sensitivity analyses and relatively affine fiber kinematics suggest that these errors are not due to inaccuracies in measuring the collagen fiber organization. More likely, additional strain energy terms representing fiber-fiber interactions are necessary to provide a closer approximation of the transverse stresses. Nevertheless, this approach demonstrated that the longitudinal tensile mechanics of the supraspinatus tendon are primarily dependent on the moduli, crimp, and angular distribution of its collagen fibers. These results add to the existing knowledge of structure-function relationships in fibrous musculoskeletal tissue, which is valuable for understanding the etiology of degenerative disease, developing effective tissue engineering design strategies, and predicting outcomes of tissue repair.     

The effect of a lathyritic diet on the sensitivity of tendon to strain rate

While the tensile failure properties of rat-tail tendon depend on strain rate, the sensitivity to strain rate decreases with age, especially during sexual maturation. The object of this study was to determine the effect of an experimental model of chronic lathyrism on age-dependent changes in the sensitivity of developing tendon strength to strain rate. Tensile failure experiments were conducted at high and low strain rate on tendons excised from test and control animals aged 1 to 6 mo. The tensile "yield" response of tendon was significantly affected by the diet resulting in a reduced tensile strength and failure strain. While the sensitivity of tendon failure to strain rate was slightly elevated by the experimental diet, age-dependent changes compared with controls. Since the diet supplement is thought to inhibit covalent crosslinking of collagen in the developing tendon, other factors are likely responsible for decrease in the sensitivity of tendon strength to strain rate during maturation.     

Nonlinear viscoelastic behaviour of the flexor tendon of the human hand

The mechanical behaviour of the flexor tendon of the human hand is here investigated from the point of view of its nonlinear viscoelasticity. The samples are subjected to several single and multiple step loading histories. A quasilinear viscoelastic constitutive relationship between strain and stress history is assumed. Its characteristic material functions are determined with the aid of simple creep results, and model predictions are compared with the experimental results of complex loading histories. The validity of the quasilinear approach to tendon behaviour is discussed in connection with the deformation mechanism suggested by it.     

X-ray diffraction study of bovine lens capsule collagen.

The wide angle X-ray diffraction pattern of air-dried lens capsule collagen under tension is the same as the tendon collagen diffraction pattern with regard to the main reflections, and indicates that lens capsule collagen has the characteristic three-stranded helical structure with an axial repeat of 0.29 nm as tendon collagen. The low angle X-ray diffraction pattern shows several weak diffraction maxima corresponding to the meridional reflections of capsule collagen which show orders of 63.0 nm periodicity. This is an evidence of quarter staggered molecular assembly typical of tendon collagen even if less ordered. The results are consistent with the existence in lens capsule collagen of clearly defined molecular units, which can be oriented by stress and are packed in a poor-ordered fibrillar assembly.     

The influence of specimen length on the tensile failure properties of tendon collagen

Connective tissues such as ligament, tendon and skin are composites of strength-bearing collagen fibers embedded in a hydrated matrix. The tensile response and failure properties of rat-tail tendon are thought to represent those of the collagen fiber itself. In this study, the tensile failure properties of rat-tail tendon (tendon collagen) were determined for specimens of various test length. The experimental results indicated that failure strain, based on the test grip-to-grip dimension, and failure strain energy density decreased as specimen length increased. The failure stress, on the other hand, did not change appreciably with specimen length. Thus, tensile failure data cannot simply be normalized by the grip-to-grip length of the test specimen. Experimental data from various laboratories must clearly document the length of the test specimen.