Soft Collagenous Tissues Research Articles

Second harmonic generation microscopy, biaxial mechanical tests and fiber dispersion models in human skin biomechanics

Advanced numerical simulations of the mechanical behavior of human skin require thorough calibration of the material’s constitutive models based on experimental ex vivo mechanical tests along with images of tissue microstructure for a variety of biomedical applications. In this work, a total of 14 human healthy skin samples and 4 additional scarred skin samples were experimentally analyzed to gain deep insights into the biomechanics of human skin. In particular, second harmonic generation (SHG) microscopy was used to extract detailed images of the distribution of collagen fibers, which were subsequently processed using a three-dimensional Fourier transform-based method recently proposed by the authors to quantify the distribution of fiber orientations. Mechanical tests under both biaxial and uniaxial loading were performed to calibrate the relevant mechanical parameters of two widely used constitutive models of soft fiber-reinforced biological tissues that account for non-symmetrical fiber dispersion. The calibration of the models allowed us to identify correlations between the mechanical parameters of the constitutive models considered. Statement of significanceConstitutive models for soft collagenous tissues can accurately reproduce the complex nonlinear and anisotropic mechanical behavior of skin. However, a comprehensive analysis of both microstructural and mechanical parameters is still missing for human skin. In this study, these parameters are determined by combining biaxial mechanical tests and SHG stacks of collagen fibers on ex vivo healthy human skin samples. The constitutive parameters are provided for two widely used hyperelastic models and enable accurate characterization of skin mechanical behavior for advanced numerical simulations.

Cracking of soft collagenous tissues under suture retention

Soft collagenous tissues consist of collagen fibers and soft matrix, such as skin, muscle, and heart valve, etc. Suture of the tissues is ubiquitous in surgery, which may cause fracture of tissues due to inevitably introduced defects. However, the cracking mechanism of soft collagenous tissues under suture retention remains elusive. Herein, we use bovine pericardium as a model tissue to study the suture related fracture considering various fiber orientations. Fracture tests reveal distinct toughness and fracture modes of the tissue depending on the fiber orientation, such as crack propagation and crack deflection. The maximum J-integral criterion has been established to predict the direction of crack propagation in the tissue. We further conduct suture retention test to evaluate the suture resistance of tissues, and find two different failure modes: fracture and cutting. Microscopic examination of the fracture surface demonstrates that the tissue can rupture with long fiber pullout, cut through matrix failure, or cut by a combination of fiber break and pullout. The cohesive element model is used to analyze the cutting mode. The predicted failure modes and force-displacement curves match well with experiments. This paper may provide guidance to surgical operations and benefit new artificial collagenous materials.

Fatigue and fracture of soft collagenous tissues mineralized in vitro

Biomineralization occurs in natural organisms, such as bones, teeth, shells and tendons. The formation mechanism and mechanical properties have been extensively studied for mineralized hard tissues, especially for bone and teeth. However, the research on the evolution of mechanical properties for mineralized soft tissues remains challenging due to the long period of mineralization. Here we use polymer-induced liquid-precursor (PILP) method to achieve accelerated in vitro mineralization of bovine pericardium with SrCO3. The modulus, tensile strength, toughness, and suture retention force of mineralized tissues have been characterized. After 30 days of mineralization, both the tensile strength and toughness decrease by two orders of magnitude. We carry out cyclic suture retention test to characterize the fatigue properties of mineralized tissues, and find that the cycle to failure of mineralized tissues is 10-100 times lower than that of unmineralized tissues. The degradation of mechanical properties results from the microstructure change of mineralized tissues, where collagen fibers change from curled to straight configuration. After mineralization, the interfibrous slippage is limited, and the fracture mode changes from fiber pullout to fiber break. Finite element analysis has been carried out to simulate the fracture and suture retention properties, and the simulation can well reproduce experimental results. The study may provide insights for tissue replacement and repairing.

Modeling of mechanosensitive remodeling processes in collagen fibers

AbstractA large number of biochemical processes such as enzymatic collagen degradation are involved in the remodeling of biological soft tissue constituents. Experiments have found that moderate strain may stabilize fibrillar collagen to enzymatic degradation. This strain stabilization is crucial for the homeostatic balance during the tissue remodeling process in healthy soft tissue. This article studies a material model applicable to the modeling of collagenous soft tissue. The ground substance properties are taken to be time‐independent, while the mechanical behavior of the fibers is modeled in terms of the Helmholtz free energy of the underlying protofibers along with a survival kernel that accounts for how the fiber density is dependent upon the deformation of the overall material. The examples illustrate the consequences of a compressively induced mechanical disruption to the homeostatic fiber remodeling, and they show the differences in the re‐approach to homeostasis when either the stress or strain remains constant.

Tendon Repair and Regeneration Using Bioinspired Fibrillation Engineering That Mimicked the Structure and Mechanics of Natural Tissue.

Replicating the controlled nanofibrillar architecture of collagenous tissue represents a promising approach in the design of tendon replacements that have tissue-mimicking biomechanics─outstanding mechanical strength and toughness, defect tolerance, and fatigue and fracture resistance. Guided by this principle, a fibrous artificial tendon (FAT) was constructed in the present study using an engineering strategy inspired by the fibrillation of a naturally spun silk protein. This bioinspired FAT featured a highly ordered molecular and nanofibrillar architecture similar to that of soft collagenous tissue, which exhibited the mechanical and fracture characteristics of tendons. Such similarities provided the motivation to investigate FAT for applications in Achilles tendon defect repair. In vitro cellular morphology and expression of tendon-related genes in cell culture and in vivo modeling of tendon injury clearly revealed that the highly oriented nanofibrils in the FAT substantially promoted the expression of tendon-related genes combined with the Achilles tendon structure and function. These results provide confidence about the potential clinical applications of the FAT.

Univariate Gauss quadrature for structural modelling of tissues and materials with distributed fibres

This work presents a method for computing the averaged free energy and constitutive relations in hyperelastic material models with distributed fibres, as they apply to soft fibre-reinforced materials and biological tissues. While these models are currently implemented through either spherical cubature of the fibre free energy or its Taylor series, we here propose a new method based on a univariate Gauss quadrature rule with integration points and weights informed by the statistical moments of the distribution of fibre stretch. As an intrinsic property, the new approach separates the integration of the fibre constitutive law from the integration of the orientation distribution, the latter leading to structural tensors of even order. Provided the latter 2n−1 tensors are computed accurately up to tensorial order 2(2n−1), the method integrates exactly any polynomial of order 2n−1 that agrees with the fibre law at the n integration points. After formally introducing the quadrature method for generally non-affine fibre deformations and arbitrary order, we focus on the important special case of affine fibre kinematics and discuss the rules with n≤3 integration points, for which the corresponding positions and weights are determined analytically. At a computational cost comparable to the existing approaches, the new method does not require the fibre law to be analytic and can thus robustly deal with piece-wise definitions of the fibre energy, in contrast to Taylor-series approaches, and it does not induce additional anisotropy as it can occur with spherical cubature rules. The 3-point rule is further investigated and illustrated in numerical examples relating to soft collagenous tissues based on a Fortran implementation of the method suitable for use in finite element analyses.

Spatiotemporal and microstructural characterization of heterotopic ossification in healing rat Achilles tendons.

Achilles tendon rupture is a common debilitating medical condition. The healing process is slow and can be affected by heterotopic ossification (HO), which occurs when pathologic bone-like tissue is deposited instead of the soft collagenous tendon tissue. Little is known about the temporal and spatial progression of HO during Achilles tendon healing. In this study we characterize HO deposition, microstructure, and location at different stages of healing in a rat model. We use phase contrast-enhanced synchrotron microtomography, a state-of-the-art technique that allows 3D imaging at high-resolution of soft biological tissues without invasive or time-consuming sample preparation. The results increase our understanding of HO deposition, from the early inflammatory phase of tendon healing, by showing that the deposition is initiated as early as one week after injury in the distal stump and mostly growing on preinjury HO deposits. Later, more deposits form first in the stumps and then all over the tendon callus, merging into large, calcified structures, which occupy up to 10% of the tendon volume. The HOs were characterized by a looser connective trabecular-like structure and a proteoglycan-rich matrix containing chondrocyte-like cells with lacunae. The study shows the potential of 3D imaging at high-resolution by phase-contrast tomography to better understand ossification in healing tendons.

Flaw-insensitive fatigue resistance of chemically fixed collagenous soft tissues.

Bovine pericardium (BP) has been used as leaflets of prosthetic heart valves. The leaflets are sutured on metallic stents and can survive 400 million flaps (~10-year life span), unaffected by the suture holes. This flaw-insensitive fatigue resistance is unmatched by synthetic leaflets. We show that the endurance strength of BP under cyclic stretch is insensitive to cuts as long as 1centimeter, about two orders of magnitude longer than that of a thermoplastic polyurethane (TPU). The flaw-insensitive fatigue resistance of BP results from the high strength of collagen fibers and soft matrix between them. When BP is stretched, the soft matrix enables a collagen fiber to transmit tension over a long length. The energy in the long length dissipates when the fiber breaks. We demonstrate that a BP leaflet greatly outperforms a TPU leaflet. It is hoped that these findings will aid the development of soft materials for flaw-insensitive fatigue resistance.

Fracture toughness determination of porcine muscle tissue based on AQLV model derived viscous dissipated energy

The ability of soft collagenous tissue (SCT) to withstand propagation of a defect in the presence of a macroscopic crack is termed the ’fracture toughness parameter’. In soft tissues not undergoing significant plastic deformation, it is purported that a considerable amount of additional energy is dissipated during failure processes, due to viscoelasticity. Hence the total work, measured experimentally during failure, is the sum of fracture and viscoelastic energies. Previous authors have aimed to apply constitutive modeling to describe viscoelastic hysteresis for fracture toughness determination with a tendency of models to either over or underestimate the viscous energy. In this study, the fracture toughness of porcine muscle tissue is determined using two strategies. Firstly, it was determined experimentally by calculation of the difference in dissipated energy of notched and unnotched tissue specimens undergoing cyclic ’triangular wave’ excitation with increasing strain levels in uniaxial tension. The second strategy involved the extension and use of the adaptive quasi-linear viscoelastic model (AQLV) to model cyclic loading (model parameters were obtained from a previous study) and sequentially the dissipated energy was calculated. The mean value of the dissipated energy based on the AQLV approach was then subtracted from the total dissipated energy of notched porcine muscle tissue samples to determine the fracture toughness. The mean experimental viscous dissipated energy ratio was 0.24 ± 0.04 in the experimental approach, compared to 0.28 ± 0.03 for the AQLV model. Fracture toughness determined experimentally yielded 0.84 ± 0.80 kJ/m2, and 0.71 ± 0.76 kJ/m2 for the AQLV model, without a significant difference (p = 0.87). Hence, the AQLV model enables a reasonable estimation of viscous dissipated energy in porcine muscle tissue with the advantage to perform tests only on notched specimens, instead of testing additional unnotched samples. Moreover, the AQLV model will help to better understand the constitutive viscoelastic behaviour of SCTs and might also serve as a basis for future fracture toughness determination with constitutive model simulations.

Bulging of inflated membranes made of fiber reinforced materials with different natural configurations

In this article, we study the inflation and bulging of fiber reinforced hyperelastic membranes in which fibers are symmetrically arranged in two helically distributed families that are mechanically equivalent. The isotropic ground substance behavior is described by a neo-Hookean model and the mechanical behavior of fibers is described by the so-called standard reinforcing model. The two constituents, fiber and matrix, may possess different natural configurations to account for different characteristics. For example, in collagenous soft tissue synthesized collagen fibrils are integrated at a certain distribution of pre-stretches, or fibers may be crimped, and different natural configurations of the constituents can be used to model these mechanisms. In addition, fibers are taken to be dispersed with respect to a mean fiber direction within their respective fiber families. It is shown that the arrangement and the natural configuration of fibers have a strong effect on bulging of inflated cylindrical membranes. The modeling at hand can be used to understand the mechanical behaviour of arterial soft tissue and the formation of aneurysms.

How suture networks improve the protective function of natural structures: A multiscale investigation

Myriad natural protective structures consist of bone plates joined by convoluted unmineralized (soft) collagenous sutures. Examples of such protective structures include: shells of turtles, craniums of almost all animals (including humans), alligator armour, armadillo armour, and others. The function of sutures has been well researched. However, whether, and if so how, sutures improve protective performance during a predator attack has received limited attention. Sutures are ubiquitous in protective structures, and this motivates the question as to whether sutures optimize the protective function of the structure. Hence, in this work the behaviour of structures that contain sutures during predator attacks is investigated. We show that sutures decrease the maximum strain energy density that turtle shells experience during predator attacks by more than an order of magnitude. Hence, sutures make turtle shells far more resilient to material failure, such as, fracture, damage, and plastic deformations. Additionally, sutures increase the viscous behaviour of the shell causing increased dissipation of energy during predator attacks. Further investigations into the influence of sutures on behaviour during locomotion and breathing are also presented. The results presented in this work motivate the inclusion of sutures in biomimetically designed protective structures, such as helmets and protective clothing. Statement of significanceMyriad bony protective structures contain networks of sutures, that is con- voluted soft collagenous tissue. Their ubiquity motivates the question, whether, and if so how, sutures improve protective performance. Hence, this work inves- tigates how sutures affect protective performance using computational experi- ments. Due to the length scale of sutures being far smaller than the structures in which they reside, classical modelling approaches are prohibitively expensive. Hence, in this work, a multiscale approach is taken. To our knowledge, this is the first multiscale investigation of structures that contain sutures.Among other insights, we show that sutures decrease the maximum strain energy density in structures during predator attacks by over an order of mag- nitude. Hence, sutures make structures far more resilient to failure.

Optical Imaging of Dynamic Collagen Processes in Health and Disease

Collagen is a major structural component of nearly every tissue in the human body, whose hierarchical organization imparts specific mechanical properties and defines overall tissue function. Collagenous soft tissues are dynamic structures that are in a constant state of remodeling but are also prone to damage and pathology. Optical techniques are uniquely suited for imaging collagen in these dynamic situations as they allow for non-invasive monitoring with relatively high spatiotemporal resolution. This review presents an overview of common collagen dynamic processes associated with human health and disease and optical imaging approaches that are uniquely suited for monitoring, sensing, and diagnosing these changes. This review aims to 1) provide researchers with an understanding of the underlying optical properties of collagen that can be leveraged for extracellular matrix visualization and 2) present emerging opportunities for machine learning approaches to drive multiscale and multimodality solutions.

Experimental Investigation of the Anisotropic Mechanical Response of the Porcine Thoracic Aorta.

Knowledge of the mechanical properties of blood vessels and determining appropriate constitutive relations are essential in developing methodologies for accurate prognosis of vascular diseases. We examine the directional variation of the mechanical properties of the porcine thoracic aorta by performing uniaxial extension tests on dumbbell-shaped specimens cut at five different orientations with respect to the circumferential direction of the aorta. Specimens in all the orientations considered exhibit a nonlinear constitutive response that is typical of collagenous soft tissues. Shear strain under uniaxial extension demonstrates clearly discernible anisotropy of the mechanical response of the porcine aorta, and samples oriented at 45[Formula: see text] and 60[Formula: see text] with respect to the circumferential direction show a peculiar crescent-shaped shear strain-nominal stretch response not displayed by axial and circumferential specimens. Failure stress indicates decreasing tensile strength of the porcine aortic wall from the circumferential direction to the longitudinal direction. Furthermore, we determine the material parameters for the four-fiber-family and Gasser-Holzapfel-Ogden models from the mechanical response data of the circumferential and longitudinal specimens. It is shown how the material parameters derived from the uniaxial tests on circumferential and longitudinal specimens are insufficient to characterize the response of off-axis specimens.

A generalized strain approach to anisotropic elasticity

This work proposes a generalized Lagrangian strain function f_alpha (that depends on modified stretches) and a volumetric strain function g_alpha (that depends on the determinant of the deformation tensor) to characterize isotropic/anisotropic strain energy functions. With the aid of a spectral approach, the single-variable strain functions enable the development of strain energy functions that are consistent with their infinitesimal counterparts, including the development of a strain energy function for the general anisotropic material that contains the general 4th order classical stiffness tensor. The generality of the single-variable strain functions sets a platform for future development of adequate specific forms of the isotropic/anisotropic strain energy function; future modellers only require to construct specific forms of the functions f_alpha and g_alpha to model their strain energy functions. The spectral invariants used in the constitutive equation have a clear physical interpretation, which is attractive, in aiding experiment design and the construction of specific forms of the strain energy. Some previous strain energy functions that appeared in the literature can be considered as special cases of the proposed generalized strain energy function. The resulting constitutive equations can be easily converted, to allow the mechanical influence of compressed fibres to be excluded or partial excluded and to model fibre dispersion in collagenous soft tissues. Implementation of the constitutive equations in Finite Element software is discussed. The suggested crude specific strain function forms are able to fit the theory well with experimental data and managed to predict several sets of experimental data.

Structural Mechanisms in Soft Fibrous Tissues: A Review

Through years of evolution, biological soft fibrous tissues have developed remarkable functional properties, unique hierarchical architectures, and -most notably, an unparalleled and extremely efficient deformation ability. Whereas the structure-function relationship is well-studied in natural hard materials, soft materials are not getting similar attention, despite their high prevalence in nature. These soft materials are usually constructed as fiber-reinforced composites consisting of diverse structural motifs that result in an overall unique mechanical behavior with large deformations. Biomimetics of their mechanical behavior is currently a significant bioengineering challenge. The unique properties of soft fibrous tissues stem from their structural complexity, which, unfortunately, also hinders our ability to generate adequate synthetic analogs, such that autografts remain the “gold standard” materials for soft-tissue repair and replacement. This review seeks to understand the structural and deformation mechanisms of soft collagenous tissues, with a particular emphasis on tendon and ligaments, the annulus fibrosus (AF) in the intervertebral disc (IVD), skin, and blood vessels. We examined and compared different mechanical and structural motifs in these different tissue types, which are subjected to complex and varied mechanical loads, to isolate the mechanisms of their deformation behavior. Herein, we focused on their composite structure from a perspective of the different building blocks, architecture, crimping patterns, fiber orientation, organization and their structure-function relationship. In the second part of the review, we presented engineered soft composite applications that used these structural motifs to mimic the structural and mechanical behavior of soft fibrous tissues. Moreover, we demonstrated new methodologies and materials that use biomimetic principles as a guide. These novel architectural materials have tailor-designed J-shaped large deformations behavior. Structural motifs in soft composites hold valuable insights that could be exploited to generate the next generation of materials. They actually have a two-fold effect: 1) to get a better understanding of the complex structure-function relationship in a simple material system using reverse biomimetics and 2) to develop new and efficient materials. These materials could revolutionize the future tailor-designed soft composite materials together with various soft-tissue repair and replacement applications that will be mechanically biocompatible with the full range of native tissue behaviors.

Regional variations in discrete collagen fibre mechanics within intact intervertebral disc resolved using synchrotron computed tomography and digital volume correlation

Many soft tissues, such as the intervertebral disc (IVD), have a hierarchical fibrous composite structure which suffers from regional damage. We hypothesise that these tissue regions have distinct, inherent fibre structure and structural response upon loading. Here we used synchrotron computed tomography (sCT) to resolve collagen fibre bundles (∼5μm width) in 3D throughout an intact native rat lumbar IVD under increasing compressive load. Using intact samples meant that tissue boundaries (such as endplate-disc or nucleus-annulus) and residual strain were preserved; this is vital for characterising both the inherent structure and structural changes upon loading in tissue regions functioning in a near-native environment. Nano-scale displacement measurements along >10,000 individual fibres were tracked, and fibre orientation, curvature and strain changes were compared between the posterior-lateral region and the anterior region. These methods can be widely applied to other soft tissues, to identify fibre structures which cause tissue regions to be more susceptible to injury and degeneration. Our results demonstrate for the first time that highly-localised changes in fibre orientation, curvature and strain indicate differences in regional strain transfer and mechanical function (e.g. tissue compliance). This included decreased fibre reorientation at higher loads, specific tissue morphology which reduced capacity for flexibility and high strain at the disc-endplate boundary. Statement of significanceThe analyses presented here are applicable to many collagenous soft tissues which suffer from regional damage. We aimed to investigate regional intervertebral disc (IVD) structural and functional differences by characterising collagen fibre architecture and linking specific fibre- and tissue-level deformation behaviours. Synchrotron CT provided the first demonstration of tracking discrete fibres in 3D within an intact IVD. Detailed analysis of regions was performed using over 200k points, spaced every 8 μm along 10k individual fibres. Such comprehensive structural characterisation is significant in informing future computational models. Morphological indicators of tissue compliance (change in fibre curvature and orientation) and fibre strain measurements revealed localised and regional differences in tissue behaviour.

Biaxial mechanics of thermally denaturing skin - Part 1: Experiments

The mechanics of collagenous soft tissues, such as skin, are sensitive to heat. Thus, quantifying and modeling thermo-mechanical coupling of skin is critical to our understanding of skin’s physiology, pathophysiology, and its treatment. However, key gaps persist in our knowledge about skin’s coupled thermo-mechanics. Among them, we haven’t quantified the role of skin’s microstructural organization in its response to superphysiological loading. To fill this gap, we conducted a comprehensive set of experiments in which we combined biaxial mechanical testing with histology and two-photon imaging under liquid heat treatment at temperatures ranging from 37∘C to 95∘C lasting between 2 seconds and 5 minutes. Among other observations, we found that unconstrained skin, when exposed to high temperatures, shrinks anisotropically with the principal direction of shrinkage being aligned with collagen’s principal orientation. Additionally, we found that when skin is isometrically constrained, it produces significant forces during denaturation that are also anisotropic. Finally, we found that denaturation significantly alters the mechanical behavior of skin. For short exposure times, this alteration is reflected in a reduction of stiffness at high strains. At long exposure times, the tissue softened to a point where it became untestable. We supplemented our findings with confirmation of collagen denaturation in skin via loss of birefringence and second harmonic generation. Finally, we captured all time-, temperature-, and direction-dependent experimental findings in a hypothetical model. Thus, this work fills a fundamental gap in our current understanding of skin thermo-mechanics and will support future developments in thermal injury prevention, thermal injury management, and thermal therapeutics of skin. Statement of significanceOur work experimentally explores how skin reacts to being heated. That is, it measures how much skin shrinks, what forces it produces, and how its mechanical properties change; all as a function of temperature, but also of direction and time. Additionally, our work connects these measurements to changes in skin’s microscopic make-up. This knowledge is important to our understanding of skin’s function and dysfunction, especially during burn injuries or heat-dependent treatments.

A microstructurally motivated constitutive description of collagenous soft biological tissue towards the description of their non-linear and time-dependent properties

A versatile constitutive model for load-carrying soft biological tissue should incorporate salient microstructural deformation mechanisms and be able to reliably predict complex non-linear viscoelastic behavior. The advancement of treatment and rehabilitation strategies for soft tissue injuries is inextricably linked to our understanding of the underlying tissue microstructure and how this defines its macroscopic material properties. Towards this long-term objective, we present a generalized multiscale constitutive framework based on a novel description of collagen, the most mechanically significant extracellular matrix protein. The description accounts for the gradual recruitment of undulated collagen fibrils and introduces proteoglycan mediated time-dependent fibrillar sliding. Crucially, the proteoglycan deformation allows for the reduction of overstressed fibrils towards a preferential homeostatic stress. An implicit Finite Element implementation of the model uses an interpolation strategy towards collagen fiber stress determination and results in a memory-efficient representation of the model. A number of test cases, including patient-specific geometries, establish the efficiency of the description and demonstrate its ability to explain qualitative properties reported from macroscopic experimental studies of tendon and vascular tissue.

The effects of a simple optical clearing protocol on the mechanics of collagenous soft tissue

Optical clearing of biological tissues improves imaging depth for light transmission imaging modalities such as two-photon microscopy. In studies that investigate the interplay between microstructure and tissue-level mechanics, mechanical testing of cleared tissue may be useful. However, the effects of optical clearing on soft tissue mechanics have not been investigated. Thus, we set out to quantify the effects of a simple and effective optical clearing protocol on the mechanics of soft collagenous tissues using ovine mitral valve anterior leaflets as a model system. First, we demonstrate the effectiveness of an isotonic glycerol-DMSO optical clearing protocol in two-photon microscopy. Second, we evaluate the mechanical effects of optical clearing on leaflets under equibiaxial tension in a dependent study design. Lastly, we quantify the shrinkage strain while traction-free and the contractile forces while constrained during clearing. We found the optical clearing protocol to improve two-photon imaging depth from ~100 μm to ~500–800 μm, enabling full-thickness visualization of second-harmonic generation, autofluorescent, and fluorophore-tagged structures. Under equibiaxial tension, cleared tissues exhibited reduced circumferential (p < 0.001) and radial (p = 0.009) transition stretches (i.e. stretch where collagen is recruited), and reduced radial stiffness (p = 0.031). Finally, during clearing we observed ~10–15% circumferential and radial compressive strains, and when constrained, ~2mN of circumferential and radial traction forces. In summary, we suggest the use of this optical clearing agent with mechanical testing be done with care, as it appears to alter the tissue’s stress-free configuration and stiffness, likely due to tissue dehydration.

Collagen denaturation is initiated upon tissue yield in both positional and energy-storing tendons

Tendons are collagenous soft tissues that transmit loads between muscles and bones. Depending on their anatomical function, tendons are classified as positional or energy-storing with differing biomechanical and biochemical properties. We recently demonstrated that during monotonic stretch of positional tendons, permanent denatured collagen begins accumulating upon departing the linear region of the stress-strain curve. However, it is unknown if this observation is true during mechanical overload of other types of tendons. Therefore, the purpose of this study was to investigate the onset of collagen denaturation relative to applied strain, and whether it differs between the two tendon types. Rat tail tendon (RTT) fascicles and rat flexor digitorum longus (FDL) tendons represented positional and energy-storing tendons, respectively. The samples were stretched to incremental levels of strain, then stained with fluorescently labeled collagen hybridizing peptides (CHPs); the CHP fluorescence was measured to quantify denatured collagen. Denatured collagen in both positional and energy-storing tendons began to increase at the yield strain, upon leaving the linear region of the stress-strain curve as the sample started to permanently deform. Despite significant differences between the two tendon types, it appears that collagen denaturation is initiated at tissue yield during monotonic stretch, and the fundamental mechanism of failure is the same for the two types of tendons. At tissue failure, positional tendons had double the percentage of denatured collagen compared to energy-storing tendons, with no difference between 0% control groups. These results help to elucidate the etiology of subfailure injury and rupture in functionally distinct tendons.