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.

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.

Ultrasound Measurement of Local Deformation in the Human Free Achilles Tendon Produced by Dynamic Muscle-Induced Loading: A Systematic Review.

Achilles tendinopathy is the most prevalent lower limb tendinopathy, yet it remains poorly understood, with mismatches between observed structure and reported function. Recent studies have hypothesised that Achilles tendon (AT) healthy function is associated with variable deformation across the tendon width during use, focusing on quantifying sub-tendon deformation. Here, the aim of this work was to synthesise recent advances exploring human free AT tissue-level deformation during use. Following PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines, PubMed, Embase, Scopus and Web of Science were systematically searched. Study quality and risk of bias were assessed. Thirteen articles were retained, yielding data on free AT deformation patterns. Seven were categorised as high-quality and six as medium-quality studies. Evidence consistently reports that healthy and young tendons deform non-uniformly, with the deeper layer displacing 18%-80% more than the superficial layer. Non-uniformity decreased by 12%-85% with increasing age and by 42%-91% in the presence of injury. There is limited evidence of large effect that AT deformation patterns during dynamic loading are non-uniform and may act as a biomarker of tendon health, risk of injury and rehabilitation impact. Better considered participant recruitment and improved measurement procedures would particularly improve study quality, to explore links between tendon structure, function, aging and disease in distinct populations.

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.

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.

Self-reported bio-psycho-social factors partially distinguish patellar tendinopathy from other knee problems and explain patellar tendinopathy severity in jumping athletes: A case-control study.

OBJECTIVE: To determine what combinations of self-reported factors distinguish patellar tendinopathy (PT) from other knee problems, and explain PT severity variance. DESIGN: Case-control study. SETTING: Social media, private practice and National Health Service. PARTICIPANTS: An international sample of jumping athletes diagnosed with either PT (n = 132; 30.7 ± 8.9 years; 80 males; VISA-P = 61.6 ± 16.0) or another musculoskeletal knee condition (n = 89; 31.8 ± 9.9 years; 47 males; VISA-P = 62.9 ± 21.2) by a clinician in the last 6 months. MAIN OUTCOME MEASURES: We considered clinical diagnosis (case = having PT vs control = having other knee problems) as the dependent variable. Severity and sporting impact were defined by VISA-P and availability, respectively. RESULTS: A model comprising seven factors distinguished PT from other knee problems; training duration (OR = 1.10), sport type (OR = 2.31), injured side (OR = 2.28), pain onset (OR = 1.97), morning pain (OR = 1.89), condition acceptability (OR = 0.39) and swelling (OR = 0.37). Sports-specific function (OR = 1.02) and player level (OR = 4.11) explained sporting availability. 44% of PT severity variance was explained by quality of life (ß = 0.32), sports-specific function (ß = 0.38) and age (ß = -0.17). CONCLUSION: Sports-specific, biomedical and psychological factors partially distinguish PT from other knee problems. Availability is mainly explained by sports-specific factors, while psychosocial factors impact on severity. Adding sports-specific and bio-psycho-social factors into assessments could help better identification and management of jumping athletes with PT.

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.

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.

Organ-on-a-chip: current gaps and future directions.

As an emerging hot topic of the last decade, Organ on Chip (OoC) is a new technology that is attracting interest from both basic and translational scientists. The Biochemical Society, with its mission of supporting the advancement of science, with addressing grand challenges that have societal impact, has included OoC into their agenda to review the current state of the art, bottlenecks and future directions. This conference brought together representatives of the main stakeholders in the OoC field including academics, end-users, regulators and technology developers to discuss and identify requirements for this new technology to deliver on par with the expectations and the key challenges and gaps that still need to be addressed to achieve robust human-relevant tools, able to positively impact decision making in the pharmaceutical industry and reduce overreliance on poorly predictive animal models.

Are Landing Patterns in Jumping Athletes Associated with Patellar Tendinopathy? A Systematic Review with Evidence Gap Map and Meta-analysis.

BACKGROUND: Patellar tendinopathy (PT) is common and debilitating for jumping athletes. Intriguingly, despite its high prevalence and many research studies, a causal explanation for PT presence remains elusive. OBJECTIVE: Our objective was to investigate whether landing biomechanics among jumping athletes are associated with PT and can predict onset. METHODS: We conducted a systematic review with evidence gap map and meta-analysis. We searched three databases from inception to May 2021 for observational studies or trials evaluating landing biomechanics in jumping athletes with PT (JPTs). We assessed quality with a modified Downs and Black checklist, risk of bias with the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool, and evidence levels with van Tulder's criteria and provided an evidence gap map. RESULTS: One prospective cohort (moderate quality), one cross-sectional cohort (moderate quality), and 14 case-control (four high-, seven moderate-, and three low-quality) studies, including 104 JPTs, 14 with previous PT, 45 with asymptomatic patellar tendon abnormality (PTA), and 190 controls were retained. All studies had a high risk of bias. Meta-analysis showed an association between lower ankle dorsiflexion and the presence of tendinopathy during drop and spike landings, and JPTs had reduced knee joint power and work during volleyball approach or drop landings (moderate evidence). Limited evidence suggested that JPTs had lower patellar tendon loads during drop landings. Strong or moderate evidence showed no relation between PT and sagittal plane peak knee and hip angles or range of motion; hip, knee, or ankle angles at initial contact (IC); knee angular velocities, peak trunk kinematics, or trunk angles at IC; sagittal plane hip, knee, or ankle moments; and peak vertical ground reaction force (vGRF) and vGRF impulse. Identified gaps were that no study simultaneously investigated athletes with previous PT, current PT, and PTA, and studies of joint angular velocities at IC, ankle and hip angular velocities after touchdown, leg stiffness, loading rate of forces, and muscle activation are lacking. CONCLUSION: Despite the voluminous literature, large number of participants, multitude of investigated parameters, and consistent research focus on landing biomechanics, only a few associations can be identified, such as reduced ankle dorsiflexion-plantarflexion range. Further, the quality of the existing literature is inadequate to draw strong conclusions, with only four high-quality papers being found. We were unable to determine biomechanical factors that predicted PT onset, as longitudinal/prospective studies enabling causal inference are absent. The identified gaps indicate useful areas in which to explore causal relationships to inform intervention development. Therefore, high-quality prospective studies are essential to definitively determine whether landing biomechanics play a part in the development, recurrence, or management of PT and represent a potential therapeutic or preventive target alongside non-biomechanical factors.

The effects of cholesterol accumulation on Achilles tendon biomechanics: A cross-sectional study.

Familial hypercholesterolemia, a common genetic metabolic disorder characterized by high cholesterol levels, is involved in the development of atherosclerosis and other preventable diseases. Familial hypercholesterolemia can also cause tendinous abnormalities, such as thickening and xanthoma (tendon lipid accumulation) in the Achilles, which may impede tendon biomechanics. The objective of this study was to investigate the effect of cholesterol accumulation on the biomechanical performance of Achilles tendons, in vivo. 16 participants (10 men, 6 women; 37±6 years) with familial hypercholesterolemia, diagnosed with tendon xanthoma, and 16 controls (10 men, 6 women; 36±7 years) underwent Achilles biomechanical assessment. Achilles biomechanical data was obtained during preferred pace, shod, walking by analysis of lower limb kinematics and kinetics utilizing 3D motion capture and an instrumented treadmill. Gastrocnemius medialis muscle-tendon junction displacement was imaged using ultrasonography. Achilles stiffness, hysteresis, strain and force were calculated from displacement-force data acquired during loading cycles, and tested for statistical differences using one-way ANOVA. Statistical parametric mapping was used to examine group differences in temporal data. Participants with familial hypercholesterolemia displayed lower Achilles stiffness compared to the control group (familial hypercholesterolemia group: 87±20 N/mm; controls: 111±18 N/mm; p = 0.001), which appeared to be linked to Achilles loading rate rather than an increased strain (FH: 5.27±1.2%; controls: 4.95±0.9%; p = 0.413). We found different Achilles loading patterns in the familial hypercholesterolemia group, which were traced to differences in the centre of pressure progression that affected ankle moment. This finding may indicate that individuals with familial hypercholesterolemia use different Achilles loading strategies. Participants with familial hypercholesterolemia also demonstrated significantly greater Achilles hysteresis than the control group (familial hypercholesterolemia: 57.5±7.3%; controls: 43.8±10%; p<0.001), suggesting that walking may require a greater metabolic cost. Our results indicate that cholesterol accumulation could contribute to reduced Achilles function, while potentially increasing the chance of injury.

Can Achilles tendon xanthoma be distinguished from Achilles tendinopathy using Dixon method MRI? A cross-sectional exploratory study.

BACKGROUND: Familial hypercholesterolemia is a genetic condition characterized by life-long elevations of plasma low-density lipoprotein cholesterol. In addition to life-threatening cardiovascular complications, intratendinous cholesterol deposits (xanthomas) can lead to pain and tendon thickening, particularly in the Achilles. Clinical detection of xanthomas currently relies upon visual assessment and palpation, or ultrasound-based measures of tendon thickening or echotexture. Misdiagnosis of xanthoma can delay the commencement of potentially life-saving lipid-lowering therapy. Our primary purpose was to determine whether analysis of separated fat and water magnetic resonance images may be able to differentiate between xanthomatic and nonxanthomatic Achilles tendons through quantification of intratendinous fat content. The main hypothesis was that Achilles tendon xanthomas will demonstrate greater lipid content than Achilles tendinopathy or healthy control tendons. METHODS: Bilateral MRI scans of Achilles tendons from 30 participants (n = 10 Achilles tendon xanthoma, n = 10 Achilles overuse tendinopathy, n = 10 healthy controls) were analyzed for total lipid content using the Dixon method of fat and water signal separation. Secondary outcome measures included tendon water content, as well as ultrasound characterization of tendon tissue organization and thickness. RESULTS: Fat content was greater in Achilles tendon xanthomas compared to the tendinopathy (p < 0.0001) and control groups (p < 0.0001). Water content was also greater in Achilles tendon xanthomas compared to the tendinopathy (p < 0.0001) and control groups (p = 0.0002). Ultrasound tissue characterization revealed worse tissue organization in Achilles tendon xanthoma tendons compared to Achilles tendinopathy (p < 0.05) but demonstrated largely overlapping distributions. Achilles tendon xanthoma tendons were, on average, significantly thicker than the tendons of the other two groups (p < 0.01 and p < 0.001, respectively). CONCLUSION: MRI-derived measures of Achilles tendon fat content may be able to distinguish xanthomas from control and tendinopathic tissue. Dixon method MRI warrants further evaluation in an adequately powered study to develop and test clinically relevant diagnostic thresholds.

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.

Elastase treatment of tendon specifically impacts the mechanical properties of the interfascicular matrix.

The tendon interfascicular matrix (IFM) binds tendon fascicles together. As a result of its low stiffness behaviour under small loads, it enables non-uniform loading and increased overall extensibility of tendon by facilitating fascicle sliding. This function is particularly important in energy storing tendons, with previous studies demonstrating enhanced extensibility, recovery and fatigue resistance in the IFM of energy storing compared to positional tendons. However, the compositional specialisations within the IFM that confer this behaviour remain to be elucidated. It is well established that the IFM is rich in elastin, therefore we sought to test the hypothesis that elastin depletion (following elastase treatment) will significantly impact IFM, but not fascicle, mechanical properties, reducing IFM resilience in all samples, but to a greater extent in younger tendons, which have a higher elastin content. Using a combination of quasi-static and fatigue testing, and optical imaging, we confirmed our hypothesis, demonstrating that elastin depletion resulted in significant decreases in IFM viscoelasticity, fatigue resistance and recoverability compared to untreated samples, with no significant changes to fascicle mechanics. Ageing had little effect on fascicle or IFM response to elastase treatment. This study offers a first insight into the functional importance of elastin in regional specific tendon mechanics. It highlights the important contribution of elastin to IFM mechanical properties, demonstrating that maintenance of a functional elastin network within the IFM is essential to maintain IFM and thus tendon integrity. STATEMENT OF SIGNIFICANCE: Developing effective treatments or preventative measures for musculoskeletal tissue injuries necessitates the understanding of healthy tissue function and mechanics. By establishing the contribution of specific proteins to tissue mechanical behaviour, key targets for therapeutics can be identified. Tendon injury is increasingly prevalent and chronically debilitating, with no effective treatments available. Here, we investigate how elastin modulates tendon mechanical behaviour, using enzymatic digestion combined with local mechanical characterisation, and demonstrate for the first time that removing elastin from tendon affects the mechanical properties of the interfascicular matrix specifically, resulting in decreased recoverability and fatigue resistance. These findings provide a new level of insight into tendon hierarchical mechanics, important for directing development of novel therapeutics for tendon injury.

Postnatal mechanical loading drives adaptation of tissues primarily through modulation of the non-collagenous matrix.

Mature connective tissues demonstrate highly specialised properties, remarkably adapted to meet their functional requirements. Tissue adaptation to environmental cues can occur throughout life and poor adaptation commonly results in injury. However, the temporal nature and drivers of functional adaptation remain undefined. Here, we explore functional adaptation and specialisation of mechanically loaded tissues using tendon; a simple aligned biological composite, in which the collagen (fascicle) and surrounding predominantly non-collagenous matrix (interfascicular matrix) can be interrogated independently. Using an equine model of late development, we report the first phase-specific analysis of biomechanical, structural, and compositional changes seen in functional adaptation, demonstrating adaptation occurs postnatally, following mechanical loading, and is almost exclusively localised to the non-collagenous interfascicular matrix. These novel data redefine adaptation in connective tissue, highlighting the fundamental importance of non-collagenous matrix and suggesting that regenerative medicine strategies should change focus from the fibrous to the non-collagenous matrix of tissue.

Force Transmission Between the Gastrocnemius and Soleus Sub-Tendons of the Achilles Tendon in Rat.

The Achilles tendon (AT) is comprised of three distinct sub-tendons bound together by the inter-subtendon matrix (ISTM). The interactions between sub-tendons will have important implications for AT function. The aim of this study was to investigate the extent to which the ISTM facilitates relative sliding between sub-tendons, and serves as a pathway for force transmission between the gastrocnemius (GAS) and soleus (SOL) sub-tendons of the rat AT. In this study, ATs were harvested from Wistar rats, and the mechanical behavior and composition of the ISTM were explored. To determine force transmission between sub-tendons, the proximal and distal ends of the GAS and SOL sub-tendons were secured, and the forces at each of these locations were measured during proximal loading of the GAS. To determine the ISTM mechanical behavior, only the proximal GAS and distal SOL were secured, and the ISTM was loaded in shear. Finally, for compositional analysis, histological examination assessed the distribution of matrix proteins throughout sub-tendons and the ISTM. The results revealed distinct differences between the forces at the proximal and distal ends of both sub-tendons when proximal loading was applied to the GAS, indicating force transmission between GAS and SOL sub-tendons. Inter-subtendon matrix tests demonstrated an extended initial low stiffness toe region to enable some sub-tendon sliding, coupled with high stiffness linear region such that force transmission between sub-tendons is ensured. Histological data demonstrate an enrichment of collagen III, elastin, lubricin and hyaluronic acid in the ISTM. We conclude that ISTM composition and mechanical behavior are specialized to allow some independent sub-tendon movement, whilst still ensuring capacity for force transmission between the sub-tendons of the AT.

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.

Insights into the micromechanics of stress-relaxation and creep behaviours in the aortic valve.

Viscoelastic attributes of the aortic valve (AV) tissue are, in part, reflected in stress-relaxation and creep behaviours observed in vitro. While the extent of AV time-dependent behaviour under physiological conditions is not yet fully understood, in vitro the tissue exhibits clear stress-relaxation but minimal creep under equi-biaxial loading, in contrast to uniaxial loading where creep is evidently exhibited. Tissue-level stress-relaxation behaviour follows the form of (single and double) Maxwell-type exponential decay relaxation modes, and creep occurs in the form of exponential primary followed by linear secondary creep modes. This paper aims to provide an explanation for these behaviours based on the AV microstructural (i.e. fibre-level) mechanics. The kinematics of AV microstructural reorganisation is investigated experimentally using confocal microscopy to track the interstitial cell nuclei as markers of AV microstructural reorganisation under uniaxial loading. A theoretical framework is then applied to describe the experimentally observed kinematics in mathematical terms. Using this framework it is shown that at the microstructural level, AV stress-relaxation and creep behaviours both stem from the same dissipative kinematics of fibre-fibre and fibre-matrix interactions, that occur as a consequence of microstructural reorganisation due to the applied tissue-level loads. It is additionally shown that the proposed dissipative kinematics correctly predict the nature of relaxation and creep behaviours, i.e. the type and the number of modes involved. Further analysis is presented to demonstrate that the origin of the minimal creep behaviour under equi-biaxial loading can be explained to stem from tissue-level loading boundary conditions. These key findings help to better understand the underlying causes of AV stress-relaxation and creep behaviours in vivo, and why these may differ from the behaviours observed under non-physiological in vitro loading.