Mechanical properties of collagen fascicles from the rabbit patellar tendon

Tensile and viscoelastic properties of collagen fascicles of approximately 300 microns in diameter, which were obtained from rabbit patellar tendons, were studied using a newly designed micro-tensile tester. Their cross-sectional areas were determined with a video dimension analyzer combined with a CCD camera and a low magnification microscope. There were no statistically significant differences in tensile properties among the fascicles obtained from six medial-to-lateral locations of the patellar tendon. Tangent modulus, tensile strength, and strain at failure of the fascicles determined at about 1.5 percent/s strain rate were 216 +/- 68 MPa, 17.2 +/- 4.1 MPa, and 10.9 +/- 1.6 percent (mean +/- S.D.), respectively. These properties were much different from those of bulk patellar tendons; for example, the tensile strength and strain at failure of these fascicles were 42 percent and 179 percent of those of bulk tendons, respectively. Tangent modulus and tensile strength of collagen fascicles determined at 1 percent/s strain rate were 35 percent larger than those at 0.01 percent/s. The strain at failure was independent of strain rate. Relaxation tests showed that the reduction of stress was approximately 25 percent at 300 seconds. These stress relaxation behavior and strain rate effects of collagen fascicles differed greatly from those of bulk tendons. The differences in tensile and viscoelastic properties between fascicles and bulk tendons may be attributable to ground substances, mechanical interaction between fascicles, and the difference of crimp structure of collagen fibrils.     

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.     

The relationship between crimp pattern and mechanical response of human patellar tendon-bone units

The objectives of this study are twofold. First, to further develop the understanding of the relationship between the observed mechanical response and changes in the crimp pattern in human patellar tendon bone units. This is accomplished through the use of a specially constructed test frame and microscope system that permits observation and measurement of the crimp patterns as a function of load. Second, the results of the experimental study are used to develop a constitutive equation that includes spatial variation in the crimp pattern. The results of both the experimental and analytical study imply that local strain in the proximity of the attachment site is significantly larger than the strain in the central region of the tendon. The experimental and histological results are for specimens taken from four human bone-patellar tendon-bone units.     

Collagen fibril diameter distribution does not reflect changes in the mechanical properties of in vitro stress-deprived tendons

The purpose of this study was to determine if an association exists between the tensile properties and the collagen fibril diameter distribution in in vitro stress-deprived rat tail tendons. Rat tail tendons were paired into two groups of 21 day stress-deprived and 0 time controls and compared using transmission electron microscopy (n = 6) to measure collagen fibril diameter distribution and density, and mechanical testing (n =6) to determine ultimate stress and tensile modulus. There was a statistically significant decrease in both ultimate tensile strength (control: 17.95+/-3.99 MPa, stress-deprived: 6.79+/-3.91 MPa) and tensile modulus (control: 312.8+/-89.5 MPa, stress-deprived: 176.0+/-52.7 MPa) in the in vitro stress-deprived tendons compared to controls. However, there was no significant difference between control and stress-deprived tendons in the number of fibrils per tendon counted, mean fibril diameter, mean fibril density, or fibril size distribution. The results of this study demonstrate that the decrease in mechanical properties observed in in vitro stress-deprived rat tail tendons is not correlated with the collagen fibril diameter distribution and, therefore, the collagen fibril diameter distribution does not, by itself, dictate the decrease in mechanical properties observed in in vitro stress-deprived rat tail tendons.     

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.     

Self-crimping, biodegradable, electrospun polymer microfibers

Semicrystalline poly(l-lactide-co-ε-caprolactone) (P(LLA-CL)) was used to produce electrospun fibers with diameters on the subcellular scale. P(LLA-CL) was chosen because it is biocompatible and its chemical and physical properties are easily tunable. The use of a rotating wire mandrel as a collection device in the electrospinning process, along with high collection speeds, was used to align electrospun fibers. Upon removal of the fibers from the mandrel, the fibers shrunk in length, producing a crimp pattern characteristic of collagen fibrils in soft connective tissues. The crimping effect was determined to be a result of the residual stresses resident in the fibers due to the fiber alignment process and the difference between the operating temperature (T(op)) and the glass-transition temperature (T(g)) of the polymer. The electrospun fibers could be induced to crimp by adjusting the operating temperature to be greater than that of the polymer glass-transition temperature. Moreover, the crimped fibers exhibited a toe region in their stress-strain profile that is characteristic of collagen present in tendons and ligaments. The crimp pattern was retained during in vitro degradation over 4 weeks. Primary bovine fibroblasts seeded onto these crimped fibers attached, proliferated, and deposited extracellular matrix (ECM) molecules on the surface of the fiber mats. These self-crimping fibers hold great promise for use in tissue engineering scaffolds for connective tissues that require fibers similar in structure to that of crimped collagen fibrils.     

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.     

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.     

Tendon strain measurements with dynamic ultrasound images: evaluation of digital image correlation

Strain is an essential metric in tissue mechanics. Strains and strain distributions during functional loads can help identify damaged and pathologic regions as well as quantify functional compromise. Noninvasive strain measurement in vivo is difficult to perform. The goal of this in vitro study is to determine the efficacy of digital image correlation (DIC) methods to measure strain in B-mode ultrasound images. The Achilles tendons of eight male Wistar rats were removed and mechanically cycled between 0 and 1% strain. Three cine video images were captured for each specimen: (1) optical video for manual tracking of optical markers; (2) optical video for DIC tracking of optical surface markers; and (3) ultrasound video for DIC tracking of image texture within the tissue. All three imaging modalities were similarly able to measure tendon strain during cyclic testing. Manual/ImageJ-based strain values linearly correlated with DIC (optical marker)-based strain values for all eight tendons with a slope of 0.970. DIC (optical marker)-based strain values linearly correlated with DIC (ultrasound texture)-based strain values for all eight tendons with a slope of 1.003. Strain measurement using DIC was as accurate as manual image tracking methods, and DIC tracking was equally accurate when tracking ultrasound texture as when tracking optical markers. This study supports the use of DIC to calculate strains directly from the texture present in standard B-mode ultrasound images and supports the use of DIC for in vivo strain measurement using ultrasound images without additional markers, either artificially placed (for optical tracking) or anatomically in view (i.e., bony landmarks and/or muscle-tendon junctions).     

Mechanical and structural changes in rat tail tendon induced by alloxan diabetes and aging

The structure and mechanical properties of tail tendons from rats made diabetic by alloxan monohydrate injection were studied. Several differences are revealed upon comparison with normal tendons from control rats of identical age. In diabetic tendon an increase in the apparent failure region moduli, decreased stress relaxation rate, alterations of the crimp structure and a tendency toward larger collagen fibril diameters are observed. Some of these changes resemble aging acceleration while others must be ascribed to other hypothetical causes.     

Mechanical strain affects dura mater biological processes: implications for immature calvarial healing

The human brain grows rapidly during the first 2 years of life. This growth generates tensile strain in the overlying dura mater and neurocranium. Interestingly, it is largely during this 2-year growth period that infants are able to reossify calvarial defects. This clinical observation is important because it suggests that calvarial healing is most robust during the period of active intracranial volume expansion. With a rat model, it was previously demonstrated that immature dura mater proliferates more rapidly and produces more osteogenic cytokines and markers of osteoblast differentiation than does mature dura mater. It was therefore hypothesized that mechanical strain generated by the growing brain induces immature dura mater proliferation and increases osteogenic cytokine expression necessary for growth and healing of the overlying calvaria. Human and rat (n = 40) intracranial volume expansion was calculated as a function of age. These calculations demonstrated that 83 percent of human intracranial volume expansion is complete by 2 years of age and 90 percent of Sprague-Dawley rat intracranial volume expansion is achieved by 2 months of age. Next, the maximal daily circumferential tensile strains that could be generated in immature rat dura mater were calculated, and the corresponding daily biaxial tensile strains in the dura mater during this 2-month period were determined. With the use of a three-parameter monomolecular growth curve, it was calculated that rat dura mater experiences daily equibiaxial strains of at most 9.7 percent and 0.1 percent at birth (day 0) and 60 days of age, respectively. Because it was noted that immature dural cells may experience tensile strains as high as approximately 10 percent, neonatal rat dural cells were subjected to 10 percent equibiaxial strain in vitro, and dural cell proliferation and gene expression profiles were analyzed. When exposed to mechanical strain, immature dural cells rapidly proliferated (5.8-fold increase in proliferating cell nuclear antigen expression at 24 hours). Moreover, mechanical strain induced marked up-regulation of dural cell osteogenic cytokine production; transforming growth factor-beta1 messenger RNA levels increased 3.4-fold at 3 hours and fibroblast growth factor-2 protein levels increased 4.5-fold at 24 hours and 5.6-fold at 48 hours. Finally, mechanical strain increased dural cell expression of markers of osteoblast differentiation (2.8-fold increase in osteopontin levels at 3 hours). These findings suggest that mechanical strain can induce changes in dura mater biological processes and gene expression that may play important roles in coordinating the growth and healing of the neonatal calvaria.     

The biomechanical effects of limb lengthening and botulinum toxin type A on rabbit tendon

Numerous studies have examined the effects of distraction osteogenesis (DO) on bone, but relatively fewer have explored muscle adaptation, and even less have addressed the concomitant alterations that occur in the tendon. The purpose herein was to characterize the biomechanical properties of normal and elongated rabbit (N=20) tendons with and without prophylactic botulinum toxin type A (BTX-A) treatment. Elastic and viscoelastic properties of Achilles and Tibialis anterior (TA) tendons were evaluated through pull to failure and stress relaxation tests.All TA tendons displayed nonlinear viscoelastic responses that were strain dependent. A power law formulation was used to model tendon viscoelastic responses and tendon elastic responses were fit with a microstructural model. Distraction-elongated tendons displayed increases in compliance and stress relaxation rates over undistracted tendons; BTX-A administration offset this result. The elastic moduli of distraction-lengthened TA tendons were diminished (p=0.010) when distraction was combined with gastrocnemius (GA) BTX-A administration, elastic moduli were further decreased (p=0.004) and distraction following TA BTX-A administration resulted in TA tendons with moduli not different from contralateral control (p>0.05). Compared to contralateral control, distraction and GA BTX-A administration displayed shortened toe regions, (p=0.031 and 0.038, respectively), while tendons receiving BTX-A in the TA had no differences in the toe region (p>0.05). Ultimate tensile stress was unaltered by DO, but stress at the transition from the toe to the linear region of the stress–stretch curve was diminished in all distraction-elongated TA tendons (p<0.05). The data suggest that prophylactic BTX-A treatment to the TA protects some tendon biomechanical properties.     

Biomechanics of tendon healing

The biomechanics of tendon healing was investigated with unsutured rat achilles tendons. After two, three, and four weeks of healing tensile parameters were assayed with a bone-muscle-tendon-bone preparation elongated to failure at a controlled physiological strain rate.

In the third week of healing, stiffness, strength, and energy absorbing capacity all increased approximately 50%. These changes correlated with early fibroplasia.

In the fourth week of healing, strength, energy absorbing capacity and elongation to failure all increased relatively more than stiffness. Histologically, larger fibers with better longitudinal alignment developed during this period.

At the end of four weeks the tendon's strength was approximately 25% of normal.

To summarize, the return of stiffness in a healing tendon preparation correlated with the presence of fibroplasia and the return of other tensile parameters was a function of the amount and organization of the fibroplasia.     

Mechanical properties of human patellar tendon at the hierarchical levels of tendon and fibril

Tendons are strong hierarchical structures, but how tensile forces are transmitted between different levels remains incompletely understood. Collagen fibrils are thought to be primary determinants of whole tendon properties, and therefore we hypothesized that the whole human patellar tendon and its distinct collagen fibrils would display similar mechanical properties. Human patellar tendons (n = 5) were mechanically tested in vivo by ultrasonography. Biopsies were obtained from each tendon, and individual collagen fibrils were dissected and tested mechanically by atomic force microscopy. The Young's modulus was 2.0 ± 0.5 GPa, and the toe region reached 3.3 ± 1.9% strain in whole patellar tendons. Based on dry cross-sectional area, the Young's modulus of isolated collagen fibrils was 2.8 ± 0.3 GPa, and the toe region reached 0.86 ± 0.08% strain. The measured fibril modulus was insufficient to account for the modulus of the tendon in vivo when fibril content in the tendon was accounted for. Thus, our original hypothesis was not supported, although the in vitro fibril modulus corresponded well with reported in vitro tendon values. This correspondence together with the fibril modulus not being greater than that of tendon supports that fibrillar rather than interfibrillar properties govern the subfailure tendon response, making the fibrillar level a meaningful target of intervention. The lower modulus found in vitro suggests a possible adverse effect of removing the tissue from its natural environment. In addition to the primary work comparing the two hierarchical levels, we also verified the existence of viscoelastic behavior in isolated human collagen fibrils.     

Molecular dynamics study of deformation mechanisms of poly(vinyl alcohol) hydrogel

Molecular dynamics (MD) simulations have been performed on the physically crosslinking poly(vinyl alcohol) (PVA) hydrogel to study the deformation mechanisms under uniaxial tensile conditions. The distributions of hydroxyl oxygens and dihedral angle and the number of hydrogen bonds have been analysed to study the structure of the hydrogel. The water content and temperature dependency of mechanical properties have been investigated. The energy contributions from the partially united atom potential have been calculated as a function of strain. It is found that the stress–strain curve comprises toe region, linear region and yield and failure region which is close to most biomaterials. In the toe and yield region, all the contributions to the internal energy change a little. However, in the linear region, the bond stretching and angle bending energy increase rapidly and mainly dominate the region, and the energy increases more rapidly with the increasing water content but the decreasing temperature. The degree of crosslinking decreases with the increasing deformation. The polymer chains occur significant torsional activity in the toe region. Hydrogen bonds are stable in the toe and yield region, but the hydrogen bonds between hydroxyl groups and waters decrease in the linear region.     

Functional, mechanical, and biochemical assessment of ultrasound therapy on tendon healing in the chicken toe

We studied the therapeutic effect of ultrasound treatment on the functional recovery of surgically repaired profundus tendon in the right third toe of mature White Leghorn hens. Ultrasound treatment was given daily for 5 minutes for a total of 20 treatment days, starting immediately after 4 weeks immobilization. Ultrasound (frequency 3.0 mHz; intensity 0.75 W/cm2) was delivered to the right leg by a 5-cm2 probe through distilled water in a bath kept at 35 degrees C. The flexion of the toe was measured preoperatively for 4 consecutive days and daily for 6 weeks after the start of ultrasound treatment for the calculation of percent of functional recovery of the flexor tendon. The non-ultrasound-treated group of birds went through the same manipulations except that no ultrasound was given when the right leg was immersed in the water bath. Ultrasound treatment significantly (p less than 0.01) improved the functional recovery of the repaired tendons (less than 5-mm gap) starting on the third week of treatment (94 +/- 2 percent) compared with the non-ultrasound-treated group (79 +/- 4 percent). Ultrasound treatment had no effect on gap formation or breaking (tensile) strength of the repaired tendons. It is concluded that ultrasound enhanced functional return of repaired flexor tendons in the hen, and the clinical implication is discussed.     

The molecular and fibrillar structure of collagen and its relationship to the mechanical properties of connective tissue

The conformation of type I collagen molecules has been refined using a linked-atom least-squares procedure in conjunction with high-quality X-ray diffraction data. In many tendons these molecules pack in crystalline arrays and a careful measurement of the positions of the Bragg reflections allows the unit cell to be determined with high precision. From a further analysis of the X-ray data it can be shown that the highly ordered overlap region of the collagen fibrils consists of a crystalline array of molecular segments inclined by a small angle with respect to the fibril axis. In contrast, the gap region is less well ordered and contains molecular segments that are likely to be inclined by a similar angle but in a different vertical plane to that found in the overlap region. The collagen molecule thus has a D-periodic crimp in addition to the macroscopic crimp observed visually in the collagen fibres of many connective tissues. The growth and development of collagen fibrils have been studied by electron microscopy for a diverse range of connective tissues and the general pattern of fibril growth has been established as a function of age. In particular, relationships between fibril size distribution, the content and composition of the glycosaminoglycans in the matrix and the mechanical role played by the fibrils in the tissue have been formulated and these now seem capable of explaining many new facets of connective tissue structure and function.     

Effects of stress shielding on the transverse mechanical properties of rabbit patellar tendons

With the aim of studying mechanisms of the remodeling of tendons and ligaments, the effects of stress shielding on the rabbit patellar tendon were studied by performing tensile and stress relaxation tests in the transverse direction. The tangent modulus, tensile strength, and strain at failure of non-treated, control patellar tendons in the transverse direction were 1272 kPa, 370 kPa, and 40.5 percent, respectively, whereas those of the tendons stress-shielded for 1 week were 299 kPa, 108 kPa, and 40.4 percent, respectively. Stress shielding markedly decreased tangent modulus and tensile strength in the transverse direction, and the decreases were larger than those in the longitudinal direction, which were determined in our previous study. For example, tensile strength in the transverse and longitudinal direction decreased to 29 and 50 percent of each control value, respectively, after 1 week stress shielding. In addition, the stress relaxation in the transverse direction of stress-shielded patellar tendons was much larger than that of nontreated, control ones. In contrast to longitudinal tensile tests for the behavior of collagen, transverse tests reflect the contributions of ground substances such as proteoglycans and mechanical interactions between collagen fibers. Ground substances provide lubrication and spacing between fibers, and also confer viscoelastic properties. Therefore, the results obtained from the present study suggest that ground substance matrix, and interfiber and fiber-matrix interactions have important roles in the remodeling response of tendons to stress.     

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.     

Method for Quantitative Study of Airway Functional Microanatomy Using Micro-Optical Coherence Tomography

We demonstrate the use of a high resolution form of optical coherence tomography, termed micro-OCT (μOCT), for investigating the functional microanatomy of airway epithelia. μOCT captures several key parameters governing the function of the airway surface (airway surface liquid depth, periciliary liquid depth, ciliary function including beat frequency, and mucociliary transport rate) from the same series of images and without exogenous particles or labels, enabling non-invasive study of dynamic phenomena. Additionally, the high resolution of μOCT reveals distinguishable phases of the ciliary stroke pattern and glandular extrusion. Images and functional measurements from primary human bronchial epithelial cell cultures and excised tissue are presented and compared with measurements using existing gold standard methods. Active secretion from mucus glands in tissue, a key parameter of epithelial function, was also observed and quantified.