Early stage fatigue damage occurs in bovine tendon fascicles in the absence of changes in mechanics at either the gross or micro-structural level

Many tendon injuries are believed to result from repetitive motion or overuse, leading to the accumulation of micro-damage over time. In vitro fatigue loading can be used to characterise damage during repeated use and investigate how this may relate to the aetiology of tendinopathy. This study considered the effect of fatigue loading on fascicles from two functionally distinct bovine tendons: the digital extensor and deep digital flexor. Micro-scale extension mechanisms were investigated in fascicles before or after a period of cyclic creep loading, comparing two different measurement techniques - the displacement of a photo-bleached grid and the use of nuclei as fiducial markers. Whilst visual damage was clearly identified after only 300 cycles of creep loading, these visual changes did not affect either gross fascicle mechanics or fascicle microstructural extension mechanisms over the 900 fatigue cycles investigated. However, significantly greater fibre sliding was measured when observing grid deformation rather than the analysis of nuclei movement. Measurement of microstructural extension with both techniques was localised and this may explain the absence of change in microstructural deformation in response to fatigue loading. Alternatively, the data may demonstrate that fascicles can withstand a degree of matrix disruption with no impact on mechanics. Whilst use of a photo-bleached grid to directly measure the collagen is the best indicator of matrix deformation, nuclei tracking may provide a better measure of the strain perceived directly by the cells

Structure-function Specialisation of the Interfascicular Matrix in the Human Achilles Tendon

Tendon consists of highly aligned collagen-rich fascicles surrounded by interfascicular matrix (IFM). Some tendons act as energy stores to improve locomotion efficiency, but such tendons commonly obtain debilitating injuries. In equine tendons, energy storing is achieved primarily through specialisation of the IFM. However, no studies have investigated IFM structure-function specialisation in human tendons. Here, we compare the human positional anterior tibial tendon and energy storing Achilles tendons, testing the hypothesis that the Achilles tendon IFM has specialised composition and mechanical properties, which are lost with ageing. Data demonstrate IFM specialisation in the energy storing Achilles, with greater elasticity and fatigue resistance than in the positional anterior tibial tendon. With ageing, alterations occur predominantly to the proteome of the Achilles IFM, which are likely responsible for the observed trends towards decreased fatigue resistance. Knowledge of these key energy storing specialisations and their changes with ageing offers crucial insight towards developing treatments for tendinopathy. STATEMENT OF SIGNIFICANCE: Developing effective therapeutics or preventative measures for tendon injury necessitates the understanding of healthy tendon function and mechanics. By establishing structure-function relationships in human tendon and determining how these are affected by ageing, potential targets for therapeutics can be identified. In this study, we have used a combination of mechanical testing, immunolabelling and proteomics analysis to study structure-function specialisations in human tendon. We demonstrate that the interfascicular matrix is specialised for energy storing in the Achilles tendon, and that its proteome is altered with ageing, which is likely responsible for the observed trends towards decreased fatigue resistance. Knowledge of these key energy storing specialisations and their changes with ageing offers crucial insight towards developing treatments and preventative approaches for tendinopathy

Fascicles and the interfascicular matrix show adaptation for fatigue resistance in energy storing tendons

Tendon is composed of rope-like fascicles, bound together by interfascicular matrix (IFM). Our previous work shows that the IFM is critical for tendon function, facilitating sliding between fascicles to allow tendons to stretch. This function is particularly important in energy storing tendons, which experience extremely high strains during exercise, and therefore require the capacity for considerable inter-fascicular sliding and recoil. This capacity is not required in positional tendons. Whilst we have previously described the quasi-static properties of the IFM, the fatigue resistance of the IFM in functionally distinct tendons remains unknown. We therefore tested the hypothesis that fascicles and IFM in the energy storing equine superficial digital flexor tendon (SDFT) are more fatigue resistant than those in the positional common digital extensor tendon (CDET). Fascicles and IFM from both tendon types were subjected to cyclic fatigue testing until failure, and mechanical properties were calculated. The results demonstrated that both fascicles and IFM from the energy storing SDFT were able to resist a greater number of cycles before failure than those from the positional CDET. Further, SDFT fascicles and IFM exhibited less hysteresis over the course of testing than their counterparts in the CDET. This is the first study to assess the fatigue resistance of the IFM, demonstrating that IFM has a functional role within tendon and contributes significantly to tendon mechanical properties. These data provide important advances into fully characterising tendon structure-function relationships

Effect of fatigue loading on structure and functional behaviour of fascicles from energy-storing tendons

Tendons can broadly be categorized according to their function: those that act purely to position the limb and those that have an additional function as energy stores. Energy-storing tendons undergo many cycles of large deformations during locomotion, and so must be able to extend and recoil efficiently, rapidly and repeatedly. Our previous work has shown rotation in response to applied strain in fascicles from energy-storing tendons, indicating the presence of helical substructures which may provide greater elasticity and recovery. In the current study, we assessed how preconditioning and fatigue loading affect the ability of fascicles from the energy-storing equine superficial digital flexor tendon to extend and recoil. We hypothesized that preconditioned samples would exhibit changes in microstructural strain response, but would retain their ability to recover. We further hypothesized that fatigue loading would result in sample damage, causing further alterations in extension mechanisms and a significant reduction in sample recovery. The results broadly support these hypotheses: preconditioned samples showed some alterations in microstructural strain response, but were able to recover following the removal of load. However, fatigue loaded samples showed visual evidence of damage and exhibited further alterations in extension mechanisms, characterized by decreased rotation in response to applied strain. This was accompanied by increased hysteresis and decreased recovery. These results suggest that fatigue loading results in a compromised helix substructure, reducing the ability of energy-storing tendons to recoil. A decreased ability to recoil may lead to an impaired response to further loading, potentially increasing the likelihood of injury

A transverse isotropic viscoelastic constitutive model for aortic valve tissue

A new anisotropic viscoelastic model is developed for application to the aortic valve (AV). The directional dependency in the mechanical properties of the valve, arising from the predominantly circumferential alignment of collagen fibres, is accounted for in the form of transverse isotropy. The rate dependency of the valve's mechanical behaviour is considered to stem from the viscous (η) dissipative effects of the AV matrix, and is incorporated as an explicit function of the deformation rate (λ˙). Model (material) parameters were determined from uniaxial tensile deformation tests of porcine AV specimens at various deformation rates, by fitting the model to each experimental dataset. It is shown that the model provides an excellent fit to the experimental data across all different rates and satisfies the condition of strict local convexity. Based on the fitting results, a nonlinear relationship between η and λ˙ is established, highlighting a ‘shear-thinning’ behaviour for the AV with increase in the deformation rate. Using the model and these outcomes, the stress–deformation curves of the AV tissue under physiological deformation rates in both the circumferential and radial directions are predicted and presented. To verify the predictive capabilities of the model, the stress–deformation curves of AV specimens at an intermediate deformation rate were estimated and validated against the experimental data at that rate, showing an excellent agreement. While the model is primarily developed for application to the AV, it may be applied without the loss of generality to other collagenous soft tissues possessing a similar structure, with a single preferred direction of embedded collagen fibres

In vivo biological response to extracorporeal shockwave therapy in human tendinopathy:Response of tendinopathy to shockwave therapy

Extracorporeal shock wave therapy (ESWT) is a non-invasive treatment for chronic tendinopathies, however little is known about the in-vivo biological mechanisms of ESWT. Using microdialysis, we examined the real-time biological response of healthy and pathological tendons to ESWT. A single session of ESWT was administered to the mid-portion of the Achilles tendon in thirteen healthy individuals (aged 25.7±7.0 years) and patellar or Achilles tendon of six patients with tendinopathies (aged 39.0±14.9 years). Dialysate samples from the surrounding peri-tendon were collected before and immediately after ESWT. Interleukins (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17A, vascular endothelial growth factor (VEGF) and interferon (IFN)-γ were quantified using a cytometric bead array while gelatinase activity (MMP-2 and -9) was examined using zymography. There were no statistical differences between the biological tissue response to ESWT in healthy and pathological tendons. IL-1β, IL-2, IL-6 and IL-8 were the cytokines predominantly detected in the tendon dialysate. IL-1β and IL-2 did not change significantly with ESWT. IL-6 and IL-8 concentrations were elevated immediately after ESWT and remained significantly elevated for four hours post-ESWT (p<0.001). Pro forms of MMP-2 and -9 activity also increased after ESWT (p<0.003), whereas there were no significant changes in active MMP forms. In addition, the biological response to ESWT treatment could be differentiated between possible responders and non-responders based on a minimum 5-fold increase in any inflammatory marker or MMP from pre- to post-ESWT. Our findings provide novel evidence of the biological mechanisms underpinning ESWT in humans in vivo. They suggest that the mechanical stimulus provided by ESWT might aid tendon remodelling in tendinopathy by promoting the inflammatory and catabolic processes that are associated with removing damaged matrix constituents. The non-response of some individuals may help to explain why ESWT does not improve symptoms in all patients and provides a potential focus for future research

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

Tendinopathy is a prevalent, highly debilitating condition, with poorly defined aetiology. A wide range of clinical treatments have been proposed, with systematic reviews largely supporting shock wave therapy or eccentric exercise. Characterising these treatments has demonstrated both generate perturbations within tendon at a frequency of approximately 812Hz. Consequently, it is hypothesised that loading in this frequency range initiates increased anabolic tenocyte behaviour, promoting tendon repair. The primary aim of this study is to investigate the effects of 10Hz perturbations on tenocyte metabolism, comparing gene expression in response to a 10Hz and 1Hz loading profile. Tenocytes from healthy and tendinopathic human tendons were seeded into 3D collagen gels and subjected to 15 mins cyclic strain at 10Hz or 1Hz. Tenocytes from healthy tendon showed increased expression of all analysed genes in response to loading, with significantly increased expression of inflammatory and degradative genes with 10Hz, relative to 1Hz loading. By contrast, whilst the response of tenocytes from tendinopathy tendon also increased with 10Hz 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 10Hz 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

The interfascicular matrix enables fascicle sliding and recovery in tendon, and behaves more elastically in energy storing tendons

While 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 cost of locomotion. Energy storing tendons experience extremely high strains and need to be able to recoil efficiently for maximum energy storage and return. In the equine forelimb, the energy storing superficial digital flexor tendon (SDFT) has much higher failure strains than the positional common digital extensor tendon (CDET). However, we have previously shown that this is not due to differences in the properties of the SDFT and CDET fascicles (the largest tendon subunits). Instead, there is a greater capacity for interfascicular sliding in the SDFT which facilitates the greater extensions in this particular tendon (Thorpe et al., 2012). In the current study, we exposed fascicles and interfascicular matrix (IFM) from the SDFT and CDET to cyclic loading followed by a test to failure. The results show that IFM mechanical behaviour is not a result of irreversible deformation, but the IFM is able to withstand cyclic loading, and is more elastic in the SDFT than in the CDET. We also assessed the effect of ageing on IFM properties, demonstrating that the IFM is less able to resist repetitive loading as it ages, becoming stiffer with increasing age in the SDFT. These results provide further indications that the IFM is important for efficient function in energy storing tendons, and age-related alterations to the IFM may compromise function and predispose older tendons to injury

The relative compliance of energy-storing tendons may be due to the helical fibril arrangement of their fascicles

T.S. acknowledges funding for this work from the EPSRC through grant no. EP/L017997/1

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