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 non-expert 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. This article is protected by copyright. All rights reserved

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

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

Mechanical loading induces primary cilia disassembly in tendon cells via TGF beta and HDAC6

D. Rowson was funded through an EPSRC PhD studentship awarded via the Institute of Bioengineering at Queen Mary University of London

Viscoelastic adaptation of tendon graft material to compression: biomechanical quantification of graft preconditioning

PURPOSE: The tensile viscoelastic behaviour of tendon tissue is of central biomechanical importance and well examined. However, the viscoelastic tendon adaptation to external compression, such as when a tendon graft is fixated with an interference screw, has not been investigated before. Here, we quantify this adaptive behaviour in order to develop a new method to mechanically precondition tendon grafts and to better understand volumetric changes of tendinous tissue. The hypothesis of this study was that under compressive loads, tendon grafts will undergo a temporary volumetric (and therefore diametric) reduction, due to the extrusion of water from the tendon. METHODS: Compressive testing was performed on a material testing machine and load applied through the use of a custom-made mould, with a semi-circular cross section to accommodate the tendon graft. The effects of different compressive forces on the length, diameter and weight of tendon grafts were measured by calipers and a weighing scale, respectively. Further, different strain rates (1 vs. 10 mm/min) (n = 6, per rate), compression method (steady compression vs. creep) (n = 15 for each method) and different compression durations (1, 5, 10 min) (n = 5 for each duration) were tested to identify the most effective combination to reduce graft size by preserving its macroscopic structure. RESULTS: The effect of compression on volume reduction (75 % of initial volume and weight) reached a plateau at 6,000 N on an 8-mm tendon bundle. Length thereby increased by approximately 10 %. Both steady compression and creeping were able to reduce dimensions of the graft; however, creeping was more effective. There was no difference in effect with different durations for compression (p > 0.05) in both methods. CONCLUSION: The viscoelastic behaviour of hamstring tendon grafts under pressure allows preconditioning of the grafts for reduction of volume and diameter and therefore to drill a smaller bone tunnel, retaining more of the original bone. At the same time, the collagen content of the transplant is preserved and a tight fit of the transplant in the bone tunnel achieved

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 (RA) and osteoarthritis (OA). 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-1β (IL-1β) 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 (HA). 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 widespread adoption to understand synovial joint disease mechanisms, permit the identification of novel therapeutic targets and support pre-clinical testing of therapies.
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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

NOVEL MODELS OF LATE ONSET, PROGRESSIVE OSTEOARTHRITIS CAUSED BY POINT MUTATIONS IN THE TWO ALPHA CHAINS OF COLLAGEN TYPE I

Genetic modification is a useful tool for identifying novel genes associated with osteoarthritis (OA) and we have combined a phenotype-driven screen with ageing to identify mutations resulting in late-onset or age-related disease. As part of the Harwell Ageing Screen mutagenised mice were aged to 18 months and phenotyped at various time points, to allow for detection of late onset disease and to track its progression. This approach has identified 2 novel models of late onset, progressive OA, caused by point mutations in the genes Col1a2 or Col1a1. Defects in the alpha subunits of Collagen I are most notably associated with osteogenesis imperfecta (OI) and Ehlers Danlos syndrome, although there has been an association found between Col1a2 and OA in a founder population (Snelgrove et al, 2005). Our current work seeks to examine the causation between Collagen I defects and OA

The mechanics of flexor tendon adhesions

The mechanics of adhesions at a local tissue level have not been extensively studied. This study compared microstrains and macrostrains in adhesions of immobilized and mobilized partially lacerated flexor digitorum profundus tendons in a New Zealand White rabbit model. At 2 weeks, 50 digits were randomized to either gross tensile testing or micromechanical assessment, in which the movement of fluorescently labelled cell nuclei, acting as dynamic markers, was visualized using real-time confocal microscopy. The structural stiffness and load at failure of immobilized adhesions were 140% and 160% of that of mobilized adhesions, respectively, and both differences were statistically significant. Micromechanically, different patterns of loading and failure were observed. Mobilized adhesions exhibited over a three-fold higher local strain, which was less uniformly distributed. Confocal microscopy provided an accurate measure of local strain. For the first time, it has been possible to visualize, define, and quantify local adhesion tissue mechanics. Mobilization appears to favour the formation of sites expressing increased local strain responses or those predisposed to heterogeneity and localized failur