Mild hypercholesterolemia impacts achilles sub-tendon mechanical properties in young rats.

BACKGROUND: Hypercholesterolemia is associated with tendon pathology, but the reasons underpinning this relationship are not well understood. Cholesterol can accumulate in the tendon non-collagenous matrix which may affect both global and local tissue mechanics. Changes to the local strain environment within tendon may have significant implications for mechanosensitive tenocytes. Here, we investigated the association between elevated blood cholesterol and presence of tendon lipids in the Achilles tendon. We expected lipids to be localised in the proteoglycan-rich inter-sub-tendon matrix (ISTM), therefore we also sought to examine the impact of this on the biomechanical and viscoelastic properties of the ISTM. METHODS: The Achilles tendons of 32 young wild-type (SD) and 32 apolipoprotein E knock-out rats (ApoE-/-) were harvested at 15.6 ± 2.3 weeks of age. 32 specimens underwent histological examination to assess the distribution of lipids throughout sub-tendons and ISTM. The remaining specimens were prepared for biomechanical testing, where the ISTM between the gastrocnemius and soleus sub-tendons was subjected to shear load mechanical testing. A sub-set of tests were video recorded to enable a strain analysis. RESULTS: ApoE-/- serum cholesterol was double that of SD rats (mean 2.25 vs. 1.10 mg/ml, p < 0.001) indicating a relatively mild hypercholesterolemia phenotype. Nonetheless, we found histological evidence of esterified lipids in the ISTM and unesterified lipids in the sub-tendons, although the location or intensity of staining was not appreciably different between rat strains. Despite a lack of observable histological differences in lipid content between groups, there were significant differences in the mechanical and viscoelastic behaviour of the Achilles sub-tendon matrix. CONCLUSION: Even slightly elevated cholesterol may result in subtle changes to tendon biomechanical properties and hence injury risk. The young age of our cohort and the mild phenotype of our ApoE-/- rats are likely to have limited our findings and so we also conclude that the ApoE-/- rat model is not well suited for investigating the biomechanical impact of tendon xanthomas on Achilles sub-tendon function.

A Recruitment Model of Tendon Viscoelasticity That Incorporates Fibril Creep and Explains Strain-Dependent Relaxation.

Soft tissues exhibit complex viscoelastic behavior, including strain-rate dependence, hysteresis, and strain-dependent relaxation. In this paper, a model for soft tissue viscoelasticity is developed that captures all of these features and is based upon collagen recruitment, whereby fibrils contribute to tissue stiffness only when taut. We build upon existing recruitment models by additionally accounting for fibril creep and by explicitly modeling the contribution of the matrix to the overall tissue viscoelasticity. The fibrils and matrix are modeled as linear viscoelastic and each fibril has an associated critical strain (corresponding to its length) at which it becomes taut. The model is used to fit relaxation tests on three rat tail tendon fascicles and predict their response to cyclic loading. It is shown that all of these mechanical tests can be reproduced accurately with a single set of constitutive parameters, the only difference between each fascicle being the distribution of their fibril crimp lengths. By accounting for fibril creep, we are able to predict how the fibril length distribution of a fascicle changes over time under a given deformation. Furthermore, the phenomenon of strain-dependent relaxation is explained as arising from the competition between the fibril and matrix relaxation functions.

An in vitro investigation into the effects of 10 Hz cyclic loading on tenocyte metabolism.

Tendinopathy is a prevalent, highly debilitating condition, with poorly defined etiology. A wide range of clinical treatments has been proposed, with systematic reviews largely supporting shock wave therapy or eccentric exercise. Characterizing these treatments have demonstrated both generate perturbations within tendon at a frequency of approximately 8-12 Hz. Consequently, it is hypothesized that loading in this frequency range initiates increased anabolic tenocyte behavior, promoting tendon repair. The primary aim of this study was to investigate the effects of 10 Hz perturbations on tenocyte metabolism, comparing gene expression in response to a 10 Hz and 1 Hz loading profile. Tenocytes from healthy and tendinopathic human tendons were seeded into 3D collagen gels and subjected to 15 minutes cyclic strain at 10 Hz or 1 Hz. Tenocytes from healthy tendon showed increased expression of all analyzed genes in response to loading, with significantly increased expression of inflammatory and degradative genes with 10 Hz, relative to 1 Hz loading. By contrast, whilst the response of tenocytes from tendinopathy tendon also increased with 10 Hz loading, the overall response profile was more varied and less intense, possibly indicative of an altered healing response. Through inhibition of the pathway, IL1 was shown to be involved in the degradative and catabolic response of cells to high-frequency loading, abrogating the loading response. This study has demonstrated for the first time that loading at a frequency of 10 Hz may enhance the metabolic response of tenocytes by initiating an immediate degradatory and inflammatory cell response through the IL1 pathway, perhaps as an initial stage of tendon healing.

Effects of cell adhesion motif, fiber stiffness, and cyclic strain on tenocyte gene expression in a tendon mimetic fiber composite hydrogel.

We recently developed a fiber composite consisting of tenocytes seeded onto discontinuous fibers embedded within a hydrogel, designed to mimic physiological tendon micromechanics of tension and shear. This study examined if cell adhesion peptide (DGEA or YRGDS), fiber modulus (50 or 1300 kPa) and/or cyclic strain (5% strain, 1 Hz) influenced bovine tenocyte gene expression. Ten genes were analyzed and none were sensitive to peptide or fiber modulus in the absence of cyclic tensile strain. Genes associated with tendon (SCX and TNMD), collagens (COL1A1, COL3A1, COL11A1), and matrix remodelling (MMP1, MMP2, and TIMP3) were insensitive to cyclic strain. Contrarily, cyclic strain up-regulated IL6 by 30-fold and MMP3 by 10-fold in soft YRGDS fibers. IL6 expression in soft YRGDS fibers was 5.7 and 3.3-fold greater than in soft DGEA fibers and stiff RGD fibers, respectively, under cyclic strain. Our findings suggest that changes in the surrounding matrix can influence catabolic genes in tenocytes when cultured in a complex strain environment mimicking that of tendon, while having minimal effects on tendon and homeostatic genes.

A2B-Miktoarm Glycopolymer Fibers and Their Interactions with Tenocytes.

Electrospun biodegradable membranes have attracted great attention for a range of tissue engineering applications. Among them, poly(ε-caprolactone) (PCL) is one of the most widely used polymers, owing to its well-controlled biocompatibility and biodegradability. However, PCL also has a number of limitations, such as its hydrophobic nature and the lack of functional groups on its side chain, limiting its ability to interact with cells. Herein, we have designed and prepared a series of well-defined A2B-miktoarm copolymers with PCL and glycopolymer segments to address these limitations. Moreover, copolymers were electrospun to make membranes, which were studied in vitro to investigate cell affinity, toxicity, activity, and adhesion with these materials. The results indicate that incorporating glucose moieties into miktoarm polymers has improved the biocompatibility of the PCL while increasing the cellular interaction with the membrane material.

Distribution of proteins within different compartments of tendon varies according to tendon type.

Although the predominant function of all tendons is to transfer force from muscle to bone and position the limbs, some tendons additionally function as energy stores, reducing the energetic cost of locomotion. To maximise energy storage and return, energy-storing tendons need to be more extensible and elastic than tendons with a purely positional function. These properties are conferred in part by a specialisation of a specific compartment of the tendon, the interfascicular matrix, which enables sliding and recoil between adjacent fascicles. However, the composition of the interfascicular matrix is poorly characterised and we therefore tested the hypothesis that the distribution of elastin and proteoglycans differs between energy-storing and positional tendons, and that protein distribution varies between the fascicular matrix and the interfascicular matrix, with localisation of elastin and lubricin to the interfascicular matrix. Protein distribution in the energy-storing equine superficial digital flexor tendon and positional common digital extensor tendon was assessed using histology and immunohistochemistry. The results support the hypothesis, demonstrating enrichment of lubricin in the interfascicular matrix in both tendon types, where it is likely to facilitate interfascicular sliding. Elastin was also localised to the interfascicular matrix, specifically in the energy-storing superficial digital flexor tendon, which may account for the greater elasticity of the interfascicular matrix in this tendon. A differential distribution of proteoglycans was identified between tendon types and regions, which may indicate a distinct role for each of these proteins in tendon. These data provide important advances into fully characterising structure-function relationships within tendon.

Tendon Structure and Composition.

Tendons are soft, fibrous tissues that connect muscle to bone. Their main function is to transfer muscle generated force to the bony skeleton, facilitating movement around a joint, and as such they are relatively passive, inelastic structures, able to resist high forces. Tendons are predominantly composed of collagen, which is arranged in a hierarchical manner parallel to the long axis of the tendon, resulting in high tensile strength. Tendon also contains a range of non-collagenous proteins, present in low amounts, which nevertheless have important functional roles. In this chapter, we describe general tendon composition and structure, and discuss how variations in composition and structure at different levels of the tendon hierarchy confer specific mechanical properties, which are related to tendon function.

Proteomic analysis reveals age-related changes in tendon matrix composition, with age- and injury-specific matrix fragmentation.

Energy storing tendons, such as the human Achilles and equine superficial digital flexor tendon (SDFT), are highly prone to injury, the incidence of which increases with aging. The cellular and molecular mechanisms that result in increased injury in aged tendons are not well established but are thought to result in altered matrix turnover. However, little attempt has been made to fully characterize the tendon proteome nor determine how the abundance of specific tendon proteins changes with aging and/or injury. The aim of this study was, therefore, to assess the protein profile of normal SDFTs from young and old horses using label-free relative quantification to identify differentially abundant proteins and peptide fragments between age groups. The protein profile of injured SDFTs from young and old horses was also assessed. The results demonstrate distinct proteomic profiles in young and old tendon, with alterations in the levels of proteins involved in matrix organization and regulation of cell tension. Furthermore, we identified several new peptide fragments (neopeptides) present in aged tendons, suggesting that there are age-specific cleavage patterns within the SDFT. Proteomic profile also differed between young and old injured tendon, with a greater number of neopeptides identified in young injured tendon. This study has increased the knowledge of molecular events associated with tendon aging and injury, suggesting that maintenance and repair of tendon tissue may be reduced in aged individuals and may help to explain why the risk of injury increases with aging.

Eccentric and concentric loading of the triceps surae: an in vivo study of dynamic muscle and tendon biomechanical parameters.

Triceps surae eccentric exercise is more effective than concentric exercise for treating Achilles tendinopathy, however the mechanisms underpinning these effects are unclear. This study compared the biomechanical characteristics of eccentric and concentric exercises to identify differences in the tendon load response. Eleven healthy volunteers performed eccentric and concentric exercises on a force plate, with ultrasonography, motion tracking, and EMG applied to measure Achilles tendon force, lower limb movement, and leg muscle activation. Tendon length was ultrasonographically tracked and quantified using a novel algorithm. The Fourier transform of the ground reaction force was also calculated to investigate for tremor, or perturbations. Tendon stiffness and extension did not vary between exercise types (P = .43). However, tendon perturbations were significantly higher during eccentric than concentric exercises (25%-40% higher, P = .02). Furthermore, perturbations during eccentric exercises were found to be negatively correlated with the tendon stiffness (R2 = .59). The particular efficacy of eccentric exercise does not appear to result from variation in tendon stiffness or extension within a given session. However, varied perturbation magnitude may have a role in mediating the observed clinical effects. This property is subject-specific, with the source and clinical time-course of such perturbations requiring further research.

The Interfascicular Matrix of Energy Storing Tendons Houses Heterogenous Cell Populations Disproportionately Affected by Aging.

Energy storing tendons such as the human Achilles and equine superficial digital flexor tendon (SDFT) are prone to injury, with incidence increasing with aging, peaking in the 5th decade of life in the human Achilles tendon. The interfascicular matrix (IFM), which binds tendon fascicles, plays a key role in energy storing tendon mechanics, and aging alterations to the IFM negatively impact tendon function. While the mechanical role of the IFM in tendon function is well-established, the biological role of IFM-resident cell populations remains to be elucidated. Therefore, the aim of this study was to identify IFM-resident cell populations and establish how these populations are affected by aging. Cells from young and old SDFTs were subjected to single cell RNA-sequencing, and immunolabelling for markers of each resulting population used to localise cell clusters. Eleven cell clusters were identified, including tenocytes, endothelial cells, mural cells, and immune cells. One tenocyte cluster localised to the fascicular matrix, whereas nine clusters localised to the IFM. Interfascicular tenocytes and mural cells were preferentially affected by aging, with differential expression of genes related to senescence, dysregulated proteostasis and inflammation. This is the first study to establish heterogeneity in IFM cell populations, and to identify age-related alterations specific to IFM-localised cells.

Fatigue loading of tendon.

Tendon injuries, often called tendinopathies, are debilitating and painful conditions, generally considered to develop as a result of tendon overuse. The aetiology of tendinopathy remains poorly understood, and whilst tendon biopsies have provided some information concerning tendon appearance in late-stage disease, there is still little information concerning the mechanical and cellular events associated with disease initiation and progression. Investigating this in situ is challenging, and numerous models have been developed to investigate how overuse may generate tendon fatigue damage and how this may relate to tendinopathy conditions. This article aims to review these models and our current understanding of tendon fatigue damage. We review the strengths and limitations of different methodologies for characterizing tendon fatigue, considering in vitro methods that adopt both viable and non-viable samples, as well as the range of different in vivo approaches. By comparing data across model systems, we review the current understanding of fatigue damage development. Additionally, we compare these findings with data from tendinopathic tissue biopsies to provide some insights into how these models may relate to the aetiology of tendinopathy. Fatigue-induced damage consistently highlights the same microstructural, biological and mechanical changes to the tendon across all model systems and also correlates well with the findings from tendinopathic biopsy tissue. The multiple testing routes support matrix damage as an important contributor to tendinopathic conditions, but cellular responses to fatigue appear complex and often contradictory.

The role of the non-collagenous matrix in tendon function.

Tendon consists of highly ordered type I collagen molecules that are grouped together to form subunits of increasing diameter. At each hierarchical level, the type I collagen is interspersed with a predominantly non-collagenous matrix (NCM) (Connect. Tissue Res., 6, 1978, 11). Whilst many studies have investigated the structure, organization and function of the collagenous matrix within tendon, relatively few have studied the non-collagenous components. However, there is a growing body of research suggesting the NCM plays an important role within tendon; adaptations to this matrix may confer the specific properties required by tendons with different functions. Furthermore, age-related alterations to non-collagenous proteins have been identified, which may affect tendon resistance to injury. This review focuses on the NCM within the tensional region of developing and mature tendon, discussing the current knowledge and identifying areas that require further study to fully understand structure-function relationships within tendon. This information will aid in the development of appropriate techniques for tendon injury prevention and treatment.

Organ-on-a-Chip and Microfluidic Platforms for Oncology in the UK.

Organ-on-chip systems are capable of replicating complex tissue structures and physiological phenomena. The fine control of biochemical and biomechanical cues within these microphysiological systems provides opportunities for cancer researchers to build complex models of the tumour microenvironment. Interest in applying organ chips to investigate mechanisms such as metastatsis and to test therapeutics has grown rapidly, and this review draws together the published research using these microfluidic platforms to study cancer. We focus on both in-house systems and commercial platforms being used in the UK for fundamental discovery science and therapeutics testing. We cover the wide variety of cancers being investigated, ranging from common carcinomas to rare sarcomas, as well as secondary cancers. We also cover the broad sweep of different matrix microenvironments, physiological mechanical stimuli and immunological effects being replicated in these models. We examine microfluidic models specifically, rather than organoids or complex tissue or cell co-cultures, which have been reviewed elsewhere. However, there is increasing interest in incorporating organoids, spheroids and other tissue cultures into microfluidic organ chips and this overlap is included. Our review includes a commentary on cancer organ-chip models being developed and used in the UK, including work conducted by members of the UK Organ-on-a-Chip Technologies Network. We conclude with a reflection on the likely future of this rapidly expanding field of oncological research.

The impact of mild hypercholesterolemia on injury repair in the rat patellar tendon.

Hypercholesterolemia is associated with tendon pathology and injury prevalence. Lipids can accumulate in the tendon's extracellular spaces, which may disrupt its hierarchical structure and the tenocytes physicochemical environment. We hypothesized that the tendon's ability to repair after injury would be attenuated with elevated cholesterol levels, leading to inferior mechanical properties. Fifty wild-type (sSD) and 50 apolipoprotein E knock-out rats (ApoE-/ - ) were given a unilateral patellar tendon (PT) injury at 12 weeks old; the uninjured limb served as a control. Animals were euthanized at 3-, 14,- or 42-days postinjury and PT healing was investigated. ApoE-/ - serum cholesterol was double that of SD rats (mean: 2.12 vs. 0.99 mg/mL, p < 0.001) and cholesterol level was related to the expression of several genes after injury; notably rats with higher cholesterol demonstrated a blunted inflammatory response. There was little physical evidence of tendon lipid content or differences in injury repair between groups, therefore we were not surprised that tendon mechanical or material properties did not differ between strains. The young age and the mild phenotype of our ApoE-/ - rats might explain these findings. Hydroxyproline content was positively related to total blood cholesterol, but this result did not translate to observable biomechanical differences, perhaps due to the narrow range of cholesterol levels observed. Tendon inflammatory and healing activity is modulated at the mRNA level even with a mild hypercholesterolemia. These important initial impacts need to be investigated as they may contribute to the known consequences of cholesterol on tendons in humans.

Development and application of a novel in vivo overload model of the Achilles tendon in rat.

Repetitive overload is a primary factor in tendon injury, causing progressive accumulation of matrix damage concurrent with a cellular response. However, it remains unclear how these events occur at the initial stages of the disease, making it difficult to identify appropriate treatment approaches. Here, we describe the development of a new model to cyclically load the Achilles tendon (AT) of rats in vivo and investigate the initial structural and cellular responses. The model utilizes controlled dorsiflexion of the ankle joint applied near maximal dorsiflexion, for 10,000 cycles at 3 Hz. Animals were subjected to a single bout of in vivo loading under anaesthesia, and either culled immediately (without recovery from anaesthesia), or 48 h or 4-weeks post-loading. Macro strains were assessed in cadavers, whilst tendon specific microdamage was assessed through collagen-hybridizing peptide (CHP) immunohistochemistry which highlighted a significant rise in CHP staining in loaded ATs compared to contralateral controls, indicating an accumulation of overload-induced damage. Staining for pro-inflammatory mediators (IL-6 and COX-2) and matrix degradation markers (MMP-3 and -13) also suggests an initial cellular response to overload. Model validation confirmed our approach was able to explore early overload-induced damage within the AT, with microdamage present and no evidence of broader musculoskeletal damage. The new model may be implemented to map the progression of tendinopathy in the AT, and thus study potential therapeutic interventions.

Human vascularised synovium-on-a-chip: a mechanically stimulated, microfluidic model to investigate synovial inflammation and monocyte recruitment.

Healthy synovium is critical for joint homeostasis. Synovial inflammation (synovitis) is implicated in the onset, progression and symptomatic presentation of arthritic joint diseases such as rheumatoid arthritis and osteoarthritis. Thus, the synovium is a promising target for the development of novel, disease-modifying therapeutics. However, target exploration is hampered by a lack of good pre-clinical models that accurately replicate human physiology and that are developed in a way that allows for widespread uptake. The current study presents a multi-channel, microfluidic, organ-on-a-chip (OOAC) model, comprising a 3D configuration of the human synovium and its associated vasculature, with biomechanical and inflammatory stimulation, built upon a commercially available OOAC platform. Healthy human fibroblast-like synoviocytes (hFLS) were co-cultured with human umbilical vein endothelial cells (HUVECs) with appropriate matrix proteins, separated by a flexible, porous membrane. The model was developed within the Emulate organ-chip platform enabling the application of physiological biomechanical stimulation in the form of fluid shear and cyclic tensile strain. The hFLS exhibited characteristic morphology, cytoskeletal architecture and matrix protein deposition. Synovial inflammation was initiated through the addition of interleukin-1ß(IL-1ß) into the synovium channel resulting in the increased secretion of inflammatory and catabolic mediators, interleukin-6 (IL-6), prostaglandin E2 (PGE2), matrix metalloproteinase 1 (MMP-1), as well as the synovial fluid constituent protein, hyaluronan. Enhanced expression of the inflammatory marker, intercellular adhesion molecule-1 (ICAM-1), was observed in HUVECs in the vascular channel, accompanied by increased attachment of circulating monocytes. This vascularised human synovium-on-a-chip model recapitulates a number of the functional characteristics of both healthy and inflamed human synovium. Thus, this model offers the first human synovium organ-chip suitable for widespread adoption to understand synovial joint disease mechanisms, permit the identification of novel therapeutic targets and support pre-clinical testing of therapies.

Guidelines for ex vivo mechanical testing of tendon.

Tendons are critical for the biomechanical function of joints. Tendons connect muscles to bones and allow for the transmission of muscle forces to facilitate joint motion. Therefore, characterizing the tensile mechanical properties of tendons is important for the assessment of functional tendon health and efficacy of treatments for acute and chronic injuries. In this guidelines paper, we review methodological considerations, testing protocols, and key outcome measures for mechanical testing of tendons. The goal of the paper is to present a simple set of guidelines to the nonexpert seeking to perform tendon mechanical tests. The suggested approaches provide rigorous and consistent methodologies for standardized biomechanical characterization of tendon and reporting requirements across laboratories.

Increased expression of IL-6 family members in tendon pathology.

OBJECTIVES: Histological examination of pathological tendon generally does not reveal signs of inflammation. However, the inflammatory cytokine IL-6 has been shown to be expressed in ruptured rotator cuff tendon. The aim of this study was to investigate the expression of IL-6 family members in painful posterior tibialis tendon (PTT) and in painful and ruptured Achilles tendon (AT) compared with normal tendon. METHODS: AT samples were obtained from cadavers (normal) or from patients undergoing surgical procedures to treat chronic painful tendinopathy or ruptured tendon. PTT samples were obtained from patients undergoing surgery for other reasons (normal) and from patients with PTT dysfunction (painful). Total RNA was extracted and mRNA expression was analysed by quantitative real-time PCR. RESULTS: Collagen type I α-chain I (COL1A1) expression was increased in both painful PTT and AT compared with normal. Ciliary neurotrophic factor levels were increased in painful PTT only. In the painful AT, cyclooxygenase-2 (COX2) and IL-6 expression increased compared with normal. In the ruptured AT, levels of VEGF A, COX2, oncostatin-M, leukaemia inhibitory factor and IL-6 expression were higher compared with both normal and painful AT. IL-6R expression decreased in both painful and ruptured AT compared with normal. CONCLUSION: Painful AT and PTT show different expression patterns, indicating a substantial difference between those two tendinopathies. Inflammatory markers are up-regulated in painful and particularly in ruptured AT, pointing towards a role of inflammation not only in rupture healing, but also in Achilles tendinopathy.

Strain transfer through the aortic valve.

The complex structural organization of the aortic valve (AV) extracellular matrix (ECM) enables large and highly nonlinear tissue level deformations. The collagen and elastin (elastic) fibers within the ECM form an interconnected fibrous network (FN) and are known to be the main load-bearing elements of the AV matrix. The role of the FN in enabling deformation has been investigated and documented. However, there is little data on the correlation between tissue level and FN-level strains. Investigating this correlation will help establish the mode of strain transfer (affine or nonaffine) through the AV tissue as a key feature in microstructural modeling and will also help characterize the local FN deformation across the AV sample in response to applied tissue level strains. In this study, the correlation between applied strains at tissue level, macrostrains across the tissue surface, and local FN strains were investigated. Results showed that the FN strain distribution across AV samples was inhomogeneous and nonuniform, as well as anisotropic. There was no direct transfer of the deformation applied at tissue level to the fibrous network. Loading modes induced in the FN are different than those applied at the tissue as a result of different local strains in the valve layers. This nonuniformity of local strains induced internal shearing within the FN of the AV, possibly exposing the aortic valve interstitial cells (AVICs) to shear strains and stresses.

A combined experimental and modelling approach to aortic valve viscoelasticity in tensile deformation.

The quasi-static mechanical behaviour of the aortic valve (AV) is highly non-linear and anisotropic in nature and reflects the complex collagen fibre kinematics in response to applied loading. However, little is known about the viscoelastic behaviour of the AV. The aim of this study was to investigate porcine AV tissue under uniaxial tensile deformation, in order to establish the directional dependence of its viscoelastic behaviour. Rate dependency associated with different mechanical properties was investigated, and a new viscoelastic model incorporating rate effects developed, based on the Kelvin-Voigt model. Even at low applied loads, experimental results showed rate dependency in the stress-strain response, and also hysteresis and dissipation effects. Furthermore, corresponding values of each parameter depended on the loading direction. The model successfully predicted the experimental data and indicated a 'shear-thinning' behaviour. By extrapolating the experimental data to that at physiological strain rates, the model predicts viscous damping coefficients of 8.3 MPa s and 3.9 MPa s, in circumferential and radial directions, respectively. This implies that the native AV offers minimal resistance to internal shear forces induced by blood flow, a potentially critical design feature for substitute implants. These data suggest that the mechanical behaviour of the AV cannot be thoroughly characterised by elastic deformation and fibre recruitment assumptions alone.