The anisotropic and region-dependent mechanical response of wrap-around tendons under tensile, compressive and combined multiaxial loads.

The present work reports on the multiaxial region and orientation-dependent mechanical properties of two porcine wrap-around tendons under tensile, compressive and combined loads based on an extensive study with n=175 samples. The results provide a detailed dataset of the anisotropic tensile and compressive longitudinal properties and document a pronounced tension-compression asymmetry. Motivated by the physiological loading conditions of these tendons, which include transversal compression at bony abutments in addition to longitudinal tension, we systematically investigated the change in axial tension when the tendon is compressed transversally along one or both perpendicular directions. The results reveal that the transversal compression can increase axial tension (proximal-distal direction) in both cases to orders of 30%, yet by a larger amount in the first case (transversal compression in anterior-posterior direction), which seems to be more relevant for wrap-around tendons in-vivo. These quantitative measurements are in line with earlier findings on auxetic properties of tendon tissue, but show for the first time the influence of this property on the stress response of the tendon, and may thus reveal an important functional principle within these essential elements of force transmission in the body. STATEMENT OF SIGNIFICANCE: The work reports for the first time on multiaxial region and orientation-dependent mechanical properties of wrap-around tendons under various loads. The results indicate that differences in the mechanical properties exist between zones that are predominantly in a uniaxial tensile state and those that experience complex load states. The observed counterintuitive increase of the axial tension upon lateral compression points at auxetic properties of the tendon tissue which may be pivotal for the function of the tendon as an element of the musculoskeletal system. It suggests that the tendon's performance in transmitting forces is not diminished but enhanced when the action line is deflected by a bony pulley around which the tendon wraps, representing an important functional principle of tendon tissue.

A quadriphasic mechanical model of the human dermis.

The present study investigates the multiphasic nature of the mechanical behavior of human dermis. Motivated by experimental observations and by consideration of its composition, a quadriphasic model of the dermis is proposed, distinguishing solid matrix components, interstitial fluid and charged constituents moving within the fluid, i.e., anions and cations. Compression and tensile experiments with and without change of osmolarity of the bath are performed to characterize the chemo-mechanical coupling in the dermis. Model parameters are determined through inverse analysis. The computations predict a dominant role of the permeability in the determination of the temporal evolution of the mechanical response of the tissue. In line with the previous studies on other tissues, the analysis shows that an ideal model based on Donnan's equilibrium overestimates the osmotic pressure in skin for the case of very dilute solutions. The quadriphasic model is applied to predict changes in dermal cell environment and therefore alterations in what is called the "mechanome," associated with skin stretch. The simulations indicate that skin deformation causes a variation in several local variables, including in particular the electric field associated with a deformation-induced non-homogeneous distribution of fixed charges.

Sustained Physiological Stretch Induces Abdominal Skin Growth in Pregnancy.

Supraphysiological stretches are exploited in skin expanders to induce tissue growth for autologous implants. As pregnancy is associated with large levels of sustained stretch, we investigated whether skin growth occurs in pregnancy. Therefore, we combined a mechanical model of skin and the observations from suction experiments on several body locations of five pregnant women at different gestational ages. The measurements show a continuous increase in stiffness, with the largest change observed during the last trimester. A comparison with numerical simulations indicates that the measured increase in skin stiffness is far below the level expected for the corresponding deformation of abdominal skin. A new set of simulations accounting for growth could rationalize all observations. The predicted amount of tissue growth corresponds to approximately 40% area increase before delivery. The results of the simulations also offered the opportunity to investigate the biophysical cues present in abdominal skin along gestation and to compare them with those arising in skin expanders. Alterations of the skin mechanome were quantified, including tissue stiffness, hydrostatic and osmotic pressure of the interstitial fluid, its flow velocity and electrical potential. The comparison between pregnancy and skin expansion highlights similarities as well as differences possibly influencing growth and remodeling.

Discrete network models of endothelial cells and their interactions with the substrate.

Endothelial cell monolayers line the inner surfaces of blood and lymphatic vessels. They are continuously exposed to different mechanical loads, which may trigger mechanobiological signals and hence play a role in both physiological and pathological processes. Computer-based mechanical models of cells contribute to a better understanding of the relation between cell-scale loads and cues and the mechanical state of the hosting tissue. However, the confluency of the endothelial monolayer complicates these approaches since the intercellular cross-talk needs to be accounted for in addition to the cytoskeletal mechanics of the individual cells themselves. As a consequence, the computational approach must be able to efficiently model a large number of cells and their interaction. Here, we simulate cytoskeletal mechanics by means of molecular dynamics software, generally suitable to deal with large, locally interacting systems. Methods were developed to generate models of single cells and large monolayers with hundreds of cells. The single-cell model was considered for a comparison with experimental data. To this end, we simulated cell interactions with a continuous, deformable substrate, and computationally replicated multistep traction force microscopy experiments on endothelial cells. The results indicate that cell discrete network models are able to capture relevant features of the mechanical behaviour and are thus well-suited to investigate the mechanics of the large cytoskeletal network of individual cells and cell monolayers.

Multiscale mechanical analysis of the elastic modulus of skin.

The mechanical properties of the skin determine tissue function and regulate dermal cell behavior. Yet measuring these properties remains challenging, as evidenced by the large range of elastic moduli reported in the literature-from below one kPa to hundreds of MPa. Here, we reconcile these disparate results by dedicated experiments at both tissue and cellular length scales and by computational models considering the multiscale and multiphasic tissue structure. At the macroscopic tissue length scale, the collective behavior of the collagen fiber network under tension provides functional tissue stiffness, and its properties determine the corresponding elastic modulus (100-200 kPa). The compliant microscale environment (0.1-10 kPa), probed by atomic force microscopy, arises from the ground matrix without engaging the collagen fiber network. Our analysis indicates that indentation-based elasticity measurements, although probing tissue properties at the cell-relevant length scale, do not assess the deformation mechanisms activated by dermal cells when exerting traction forces on the extracellular matrix. Using dermal-equivalent collagen hydrogels, we demonstrate that indentation measurements of tissue stiffness do not correlate with the behavior of embedded dermal fibroblasts. These results provide a deeper understanding of tissue mechanics across length scales with important implications for skin mechanobiology and tissue engineering. STATEMENT OF SIGNIFICANCE: Measuring the mechanical properties of the skin is essential for understanding dermal cell mechanobiology and designing tissue-engineered skin substitutes. However, previous results reported for the elastic modulus of skin vary by six orders of magnitude. We show that two distinct deformation mechanisms, related to the tension-compression nonlinearity of the collagen fiber network, can explain the large variations in elastic moduli. Furthermore, we show that microscale indentation, which is frequently used to assess the stiffness perceived by cells, fails to engage the fiber network, and therefore cannot predict the behavior of dermal fibroblasts in stiffness-tunable fibrous hydrogels. This has important implications for how to measure and interpret the mechanical properties of soft tissues across length scales.

Control of hydrostatic pressure and osmotic stress in 3D cell culture for mechanobiological studies.

Hydrostatic pressure (HP) and osmotic stress (OS) play an important role in various biological processes, such as cell proliferation and differentiation. In contrast to canonical mechanical signals transmitted through the anchoring points of the cells with the extracellular matrix, the physical and molecular mechanisms that transduce HP and OS into cellular functions remain elusive. Three-dimensional cell cultures show great promise to replicate physiologically relevant signals in well-defined host bioreactors with the goal of shedding light on hidden aspects of the mechanobiology of HP and OS. This review starts by introducing prevalent mechanisms for the generation of HP and OS signals in biological tissues that are subject to pathophysiological mechanical loading. We then revisit various mechanisms in the mechanotransduction of HP and OS, and describe the current state of the art in bioreactors and biomaterials for the control of the corresponding physical signals.

On multiscale tension-compression asymmetry in skeletal muscle.

Skeletal muscle tissue shows a clear asymmetry with regard to the passive stresses under tensile and compressive deformation, referred to as tension-compression asymmetry (TCA). The present study is the first one reporting on TCA at different length scales, associated with muscle tissue and muscle fibres, respectively. This allows for the first time the comparison of TCA between the tissue and one of its individual components, and thus to identify the length scale at which this phenomenon originates. Not only the passive stress-stretch characteristics were recorded, but also the volume changes during the axial tension and compression experiments. The study reveals clear differences in the characteristics of TCA between fibres and tissue. At tissue level TCA increases non-linearly with increasing deformation and the ratio of tensile to compressive stresses at the same magnitude of strain reaches a value of approximately 130 at 13.5% deformation. At fibre level instead it initially drops to a value of 6 and then rises again to a TCA of 14. At a deformation of 13.5%, the tensile stress is about 6 times higher. Thus, TCA is about 22 times more expressed at tissue than fibre scale. Moreover, the analysis of volume changes revealed little compressibility at tissue scale whereas at fibre level, especially under compressive stress, the volume decreases significantly. The data collected in this study suggests that the extracellular matrix has a distinct role in amplifying the TCA, and leads to more incompressible tissue behaviour. STATEMENT OF SIGNIFICANCE: This article analyses and compares for the first time the tension-compression asymmetry (TCA) displayed by skeletal muscle at tissue and fibre scale. In addition, the volume changes of tissue and fibre specimens with application of passive tensile and compressive loads are studied. The study identifies a key role of the extracellular matrix in establishing the mechanical response of skeletal muscle tissue: It contributes significantly to the passive stress, it is responsible for the major part of tissue-scale TCA and, most probably, prevents/balances the volume changes of muscle fibres during deformation. These new results thus shed light on the origin of TCA and provide new information to be used in microstructure-based approaches to model and simulate skeletal muscle tissue.

Risky interpretations across the length scales: continuum vs. discrete models for soft tissue mechanobiology.

Modelling and simulation in mechanobiology play an increasingly important role to unravel the complex mechanisms that allow resident cells to sense and respond to mechanical cues. Many of the in vivo mechanical loads occur on the tissue length scale, thus raising the essential question how the resulting macroscopic strains and stresses are transferred across the scales down to the cellular and subcellular levels. Since cells anchor to the collagen fibres within the extracellular matrix, the reliable representation of fibre deformation is a prerequisite for models that aim at linking tissue biomechanics and cell mechanobiology. In this paper, we consider the two-scale mechanical response of an affine structural model as an example of a continuum mechanical approach and compare it with the results of a discrete fibre network model. In particular, we shed light on the crucially different mechanical properties of the 'fibres' in these two approaches. While assessing the capability of the affine structural approach to capture the fibre kinematics in real tissues is beyond the scope of our study, our results clearly show that neither the macroscopic tissue response nor the microscopic fibre orientation statistics can clarify the question of affinity.

Tailoring the multiscale architecture of electrospun membranes to promote 3D cellular infiltration.

Controlling the architecture of engineered scaffolds is of outmost importance to induce a targeted cell response and ultimately achieve successful tissue regeneration upon implantation. Robust, reliable and reproducible methods to control scaffold properties at different levels are timely and highly important. However, the multiscale architectural properties of electrospun membranes are very complex, in particular the role of fiber-to-fiber interactions on mechanical properties, and their effect on cell response remain largely unexplored. The work reported here reveals that the macroscopic membrane stiffness, observed by stress-strain curves, cannot be predicted solely based on the Young's moduli of the constituting fibers but is rather influenced by interactions on the microscale, namely the number of fiber-to-fiber bonds. To specifically control the formation of these bonds, solvent systems of the electrospinning solution were fine-tuned, affecting the membrane properties at every length-scale investigated. In contrast to dichloromethane that is characterized by a high vapor pressure, the use of trifluoroacetic acid, a solvent with a lower vapor pressure, favors the generation of fiber-to-fiber bonds. This ultimately led to an overall increased Young's modulus and yield stress of the membrane despite a lower stiffness of the constituting fibers. With respect to tissue engineering applications, an experimental setup was developed to investigate the effect of architectural parameters on the ability of cells to infiltrate and migrate within the scaffold. The results reveal that differences in fiber-to-fiber bonds significantly affect the infiltration of normal human dermal fibroblasts into the membranes. Membranes of loose fibers with low numbers of fiber-to-fiber bonds, as obtained from spinning solutions using dichloromethane, promote cellular infiltration and are thus promising candidates for the formation of a 3D tissue.

Visco- and poroelastic contributions of the zona pellucida to the mechanical response of oocytes.

Probing mechanical properties of cells has been identified as a means to infer information on their current state, e.g. with respect to diseases or differentiation. Oocytes have gained particular interest, since mechanical parameters are considered potential indicators of the success of in vitro fertilisation procedures. Established tests provide the structural response of the oocyte resulting from the material properties of the cell's components and their disposition. Based on dedicated experiments and numerical simulations, we here provide novel insights on the origin of this response. In particular, polarised light microscopy is used to characterise the anisotropy of the zona pellucida, the outermost layer of the oocyte composed of glycoproteins. This information is combined with data on volumetric changes and the force measured in relaxation/cyclic, compression/indentation experiments to calibrate a multi-phasic hyper-viscoelastic model through inverse finite element analysis. These simulations capture the oocyte's overall force response, the distinct volume changes observed in the zona pellucida, and the structural alterations interpreted as a realignment of the glycoproteins with applied load. The analysis reveals the presence of two distinct timescales, roughly separated by three orders of magnitude, and associated with a rapid outflow of fluid across the external boundaries and a long-term, progressive relaxation of the glycoproteins, respectively. The new results allow breaking the overall response down into the contributions from fluid transport and the mechanical properties of the zona pellucida and ooplasm. In addition to the gain in fundamental knowledge, the outcome of this study may therefore serve an improved interpretation of the data obtained with current methods for mechanical oocyte characterisation.

Predicting muscle tissue response from calibrated component models and histology-based finite element models.

Skeletal muscle is an anisotropic soft biological tissue composed of muscle fibres embedded in a structurally complex, hierarchically organised extracellular matrix. In a recent work (Kuravi et al., 2021) we have developed 3D finite element models from series of histological sections. Moreover, based on decellularisation of fresh tissue samples, a novel set of experimental data on the direction dependent mechanical properties of collagenous ECM was established (Kohn et al., 2021). Together with existing information on the material properties of single muscle fibres, the combination of these techniques allows computing predictions of the composite tissue response. To this end, an inverse finite element procedure is proposed in the present work to calibrate a constitutive model of the extracellular matrix, and supplementary biaxial tensile tests on fresh and decellularised tissues are performed for model validation. The results of this rigorously predictive and thus unforgiving strategy suggest that the prediction of the tissue response from the individual characteristics of muscle cells and decellularised tissue is only possible within clear limits. While orders of magnitude are well matched, and the qualitative behaviour in a wide range of load cases is largely captured, the existing deviations point at potentially missing components of the model and highlight the incomplete experimental information in bottom-up multiscale approaches to model skeletal muscle tissue.

Direct measurement of the direction-dependent mechanical behaviour of skeletal muscle extracellular matrix.

This paper reports the first comprehensive data set on the anisotropic mechanical properties of isolated endo- and perimysial extracellular matrix of skeletal muscle, and presents the corresponding protocols for preparing and testing the samples. In particular, decellularisation of porcine skeletal muscle is achieved with caustic soda solution, and mechanical parameters are defined based on compressive and tensile testing in order to identify the optimal treatment time such that muscle fibres are dissolved whereas the extracellular matrix remains largely intact and mechanically functional. At around 18 h, a time window was found and confirmed by histology, in which axial tensile experiments were performed to characterise the direction-dependent mechanical response of the extracellular matrix samples, and the effect of lateral pre-compression was studied. The typical, large variability in the experimental stress response could be largely reduced by varying a single scalar factor, which was attributed to the variation of the fraction of extracellular matrix within the tissue. While experimental results on the mechanical properties of intact muscle tissue and single muscle fibres are increasingly available in literature, there is a lack of information on the properties of the collagenous components of skeletal muscle. The present work aims at closing this gap and thus contributes to an improved understanding of the mechanics of skeletal muscle tissue and provides a missing piece of information for the development of corresponding constitutive and computational models.

Predicting the macroscopic response of electrospun membranes based on microstructure and single fibre properties.

In the present paper, the three-dimensional structure and macroscopic mechanical response of electrospun poly(L-lactide) membranes is predicted based only on the geometry and elasto-plastic mechanical properties of single fibres supplemented by measurements of membrane weight and volume, and the resulting computational models are used to study the non-affine micro-kinematics of electrospun networks. To this end, statistical parameters describing the in-plane fibre morphology are extracted from scanning electron micrographs of the membranes, and computational network models are generated by matching the porosity of the real mats. The virtual networks are compared against computed tomography scans in terms of structure, and against uniaxial tension tests with respect to their macroscopic mechanical response. The obtained virtual network structure agrees well with the fibre disposition in real networks, and the rigorous prediction of the mechanical response of two membranes with mean diameters of 1.10µm and 0.70µm captures the experimental behaviour qualitatively. Favourable quantitative agreement, however, is obtained only after lowering the Young's moduli, yield stresses and hardening slopes determined in single fibre tests, and after reducing the density of inter-fibre bonds in the model of the membrane with thinner fibres. The simulations thus demonstrate the validity and merits of the approach to study the multi-scale mechanics of electrospun networks, but also point to potential discrepancies between the properties of electrospun fibres within a network and those produced for single fibre characterisation, and highlight the existing uncertainty on the density and quality of bonds between fibres in electrospun networks.

On the defect tolerance of fetal membranes.

A series of mechanical experiments were performed to quantify the strength and fracture toughness of human amnion and chorion. The experiments were complemented with computational investigations using a 'hybrid' model that includes an explicit representation of the collagen fibre network of amnion. Despite its much smaller thickness, amnion is shown to be stiffer, stronger and tougher than chorion, and thus to determine the mechanical response of fetal membranes, with respect to both, deformation and fracture behaviour. Data from uniaxial tension and fracture tests were used to inform and validate the computational model, which was then applied to rationalize measurements of the tear resistance of tissue samples containing crack-like defects. Experiments and computations show that the strength of amnion is not significantly reduced by defects smaller than 1 mm, but the crack size induced by perforations for amniocentesis and fetal membrane suturing during fetal surgery might be larger than this value. In line with previous experimental observations, the computational model predicts a very narrow near field at the crack tip of amnion, due to localized fibre alignment and collagen compaction. This mechanism shields the tissue from the defect and strongly reduces the interaction of multiple adjacent cracks. These findings were confirmed through corresponding experiments, showing that no interaction is expected for multiple sutures for an inter-suture distance larger than 1 mm and 3 mm for amnion and chorion, respectively. The experimental procedures and numerical models applied in the present study might be used to optimize needle and/or staple dimensions and inter-suture distance, and thus to reduce the risk of iatrogenic preterm premature rupture of the membranes from amniocentesis, fetoscopic and open prenatal surgery.

Publisher Correction: Tear resistance of soft collagenous tissues.

The original version of this Article contained errors in the 'Computational material models' section of the Methods in which some values were displayed with incorrect units. As a result of this, a number of changes have been made to both the PDF and the HTML versions of the Article. A full description of these changes is available online and can be accessed via a link at the top of the Article.

Tear resistance of soft collagenous tissues.

Fracture toughness characterizes the ability of a material to maintain a certain level of strength despite the presence of a macroscopic crack. Understanding this tolerance for defects in soft collagenous tissues (SCT) has high relevance for assessing the risks of fracture after cutting, perforation or suturing. Here we investigate the peculiar toughening mechanisms of SCT through dedicated experiments and multi-scale simulations, showing that classical concepts of fracture mechanics are inadequate to quantify and explain the high defect tolerance of these materials. Our results demonstrate that SCT strength is only modestly reduced by defects as large as several millimeters. This defect tolerance is achieved despite a very narrow process zone at the crack tip and even for a network of brittle fibrils. The fracture mechanics concept of tearing energy fails in predicting failure at such defects, and its magnitude is shown to depend on the chemical potential of the liquid environment.

The multiscale stiffness of electrospun substrates and aspects of their mechanical biocompatibility.

In contrast to homogeneous materials, the mechanical properties of fibrous substrates depend on the probing lengthscale. This suggests that cells feel very different mechanical cues than expected from the macroscale characterisation of the substrate materials. By means of multiscale computational analyses we study here the mechanical environment of cells adhering to typical electrospun networks used in biomedical applications, with comparable macroscopic stiffness but different fibre diameters. The stiffness evaluated at the level of focal adhesions varies significantly, and the overall magnitude is strongly affected by the fibre diameter. The microscopic stiffness evaluated at cell scale depends substantially on the network topology and is about one order of magnitude lower than the macroscopic stiffness of the substrate, and two to three orders of magnitude below the fibres' elastic modulus. Moreover, the translation of stiffness over the scales is modulated by global deformations of the scaffold. In particular, uniaxial or biaxial stretching of the substrate induces nonlinear microscopic stiffening. Finally, although electrospun networks allow long-range transmission of cell-induced deformations, the comparison between the range of forces measured in cell traction force microscopy and those required to markedly deform typical electrospun networks reveals an order of magnitude difference, suggesting that these scaffolds provide a rather rigid environment for cells. All these results underline that the achievement of mechanical biocompatibility at all relevant lengthscales, and over the whole range of physiological loading states is extremely challenging. At the same time, the study shows that the diameter, length and curvature of fibre segments might be tunable towards achieving this goal. STATEMENT OF SIGNIFICANCE: Electrospun fabrics have growing use as substrates and scaffolds in tissue engineering and other biomedical applications. Based on multiscale computational analyses, this study shows that substrates of comparable macroscopic stiffness can provide tremendously different mechanical micro-environments, and that cells adhering to fibrous substrates may thus experience by orders of magnitude different mechanical cues than it would be expected from macroscale material characterisation. The simulations further reveal that the transfer of stiffness over the length scales changes with macroscopic deformation, and identify some key parameters that govern the transfer ratio. We believe that such refined understanding of the multiscale aspects of mechanical biocompatibility is key to the development of successful scaffold materials.

Inverse poroelasticity as a fundamental mechanism in biomechanics and mechanobiology.

Understanding the mechanisms of deformation of biological materials is important for improved diagnosis and therapy, fundamental investigations in mechanobiology, and applications in tissue engineering. Here we demonstrate the essential role of interstitial fluid mobility in determining the mechanical properties of soft tissues. Opposite to the behavior expected for a poroelastic material, the tissue volume of different collagenous membranes is observed to strongly decrease with tensile loading. Inverse poroelasticity governs monotonic and cyclic responses of soft biomembranes, and induces chemo-mechanical coupling, such that tensile forces are modulated by the chemical potential of the interstitial fluid. Correspondingly, the osmotic pressure varies with mechanical loads, thus providing an effective mechanism for mechanotransduction. Water mobility determines the tissue's ability to adapt to deformation through compaction and dilation of the collagen fiber network. In the near field of defects this mechanism activates the reversible formation of reinforcing collagen structures which effectively avoid propagation of cracks.How soft tissues respond to mechanical load is essential to their biological function. Here, the authors discover that - contrary to predictions of poroelasticity - fluid mobility in collagenous tissues induces drastic volume decrease with tensile loading and pronounced chemo-mechanical coupling.

A 2.5D approach to the mechanics of electrospun fibre mats.

In this paper, a discrete random network modelling approach specific to electrospun networks is presented. Owing to the manufacturing process, fibres in these materials systems have an enormous length, as compared to their diameters, and form sparse networks since fibre contact over thickness is limited to a narrow range. Representative volume elements are generated, in which fibres span the entire domain, and a technique is developed to apply computationally favourable periodic boundary conditions despite the initial non-periodicity of the networks. To capture sparsity, a physically motivated method is proposed to distinguish true fibre cross-links, in which mechanical interaction takes place, from mere fibre intersections. The model is exclusively informed by experimentally accessible parameters, demonstrates excellent agreement with the mechanical response of electrospun fibre mats, captures typical microscopic deformation patterns, and provides information on the kinematics of fibres and pores. This ability to address relevant mechanisms of deformation at both micro- and macroscopic length scales, together with the moderate computational cost, render the proposed modelling approach a highly qualified tool for the computer-based design and optimization of electrospun networks.

Microstructure based prediction of the deformation behavior of soft collagenous membranes.

The response of human amnion (HA) and bovine Glisson's capsule (GC) to uniaxial and biaxial tensile loading is analyzed on tissue (∼mm) and collagen fiber (∼µm) length scales. The mechanical behavior of the membranes is rationalized based on a discrete fiber network model that relates model parameters with microstructural features of the tissues. Parameters were first determined for GC based on the quantity and organization of collagen fibers in the tissue. Next, parameters for HA were defined by comparing the microstructures of the two membranes, which differ in fiber organization in that collagen forms µm-thick fiber bundles in GC while 50 nm-thin fibrils constitute the network in HA. The flexural behavior of these structures is phenomenologically represented in the model, indicating that shear forces are transmitted through fibrils within GC bundles, but to a much lesser extent than in a corresponding solid cross section. The model provides excellent predictions of the uniaxial and biaxial mechanical response, as well as of the progressive reorientation of fibers associated with uniaxial loading. The results are particularly relevant since model parameters were not obtained through a fitting procedure of the tissue's tension-stretch curve. Furthermore, simulations of representative in vivo deformation states indicated that a large part of the fibers are expected to be un-crimped under physiological loading conditions. Thus, the crimped shape of collagen fibers in the initial test configuration, and typically observed in histological analyses, might be a consequence of the contraction occurring when membranes are extracted from their environment in the body.