Tendon injury is debilitating and recalcitrant. With limited knowledge of disease aitiology we have are lacking in effective treatments for this prevalent musculoskeletal complaint. This presentation will outline our findings over the past few years in which we have demonstrated the importance of the interfascicular matrix (IFM) niche in maintaining healthy tendon function and driving disease progression1,2. It will also continue to describe our progress in developing both in vivo and in vitro models to interrogate disease progression. We have developed and validated a rat Achilles tendon overload model, in order to explore the impact of loading on IFM and fascicle structure, and the resulting cell response. Data highlights that structural disruption and inflammatory response both initiate in the IFM region, and can be seen in the absence of demonstrable changes to animal gait, indicating a sub-injury response in the tendon which we hypothesis may drive increased matrix turnover and repair3. We are now looking to interrogate the pathways driving this inflammatory behaviour in an organ-chip model, exploring the interplay between IFM cells and cells within fascicles. We have demonstrated phenotypic distinction of cells from the two niche environments, localized the progenitor phenotype to the IFM region and demonstrated significant mechanosensitivity in the IFM cell population4. We are currently building appropriate niche environments to maintain cell phenotype in our in vitro models, to explore the metabolic changes associated with disease progression.
Proteomic analysis has the ability to reveal both the different types and abundances of proteins in a sample. To date, proteomic analysis has received limited attention in the field of tendon research, with mainly Six microdialysis samples were obtained from human subjects before (controls) or after shock wave therapy on their achilles tendon. Samples were concentrated and intefering substances removed using StrataClean™ resin. Reduction, alkylation and an In-solution tryptic digestion was performed with the prior addition of 1% Rapigest SF solution. Samples were then analysed by Liquid Chromatography Mass Spectrometry/Mass Spectrometry. Data files were searched using IPI-human database using Mascot Search Engine. Relative quantification was performed between groups by ProgenesisQI.Introduction
Materials and Methods
The hierarchical structure of tendon results in a complex mechanical strain environment, with tenocytes experiencing both tension and shear during loading. The mechanotransduction mechanisms involved in sensing these environments is currently unclear. To better understand the effects of shear and tension on cell behaviour, a fibre composite system able to recapitulate the physiological shear-tension ratio found in tendons, was used. Cell attachment within the composite was achieved by using either a collagen type I mimetic peptide, DGEA, or a fibronectin associated peptide, YRGDS, and the gene expression response analysed after loading. Fibre composites with 4 different shear-tension (S-T) ratios were made using both PEG-DGEA and PEG-YRGDS fibres. 4 composites were made for each S-T ratio, of which 2 were loaded and 2 used as non-strained controls. Bovine digital extensor tendon tenocytes were seeded within composites, with 3 biological repeats from different donors. Loaded samples were exposed to 5% cyclic strain (1Hz) for 24 hours maintained in an incubator. The gene expression of 14 matrix related genes were analysed after loading via RT-qPCR.Introduction
Materials and Methods
Energy storing tendons such as the equine superficial digital flexor tendon (SDFT) stretch and recoil with each stride and therefore require a high degree of compliance compared to tendons with a purely positional function, such as the equine common digital extensor tendon (CDET). This extra extensibility is provided by a specialised interfascicular matrix (IFM), which provides greater sliding and recoil between adjacent fascicles in energy storing tendons. However, the composition of the IFM remains largely undefined. We hypothesised that the IFM in the SDFT has a distinct composition, with a greater abundance of proteoglycans and elastin which facilitate extension and recoil. Transverse and longitudinal sections were cut from the mid-metacarpal regions of SDFTs and CDETs from 5 horses aged 3–7 years. Sections were stained using Alcian blue/Periodic acid Schiff to detect proteoglycans, elastic Van Giesson's to detect elastin, and immunohistochemistry was performed using antibodies for decorin, biglycan, fibromodulin, lumican and lubricin. Resultant images were graded by blinded observers to assess staining intensity in the IFM and fascicular matrix (FM), and statistical significance determined using ANOVA.Introduction
Materials and Methods
Tendinopathies are debilitating and painful conditions. They are believed to result from repetitive overuse, which can create micro-damage that accumulates over time, and initiates a catabolic cell response. The aetiology of tendinopathy remains poorly understood, therefore the ideal treatment remains unclear. However, current data support the use of eccentric exercise as an effective treatment. In a previous study, we have shown that eccentric loading generates perturbations in the tendon at 10Hz, which is not present during other less effective loading regimes. Consequently, we hypothesis that 10Hz loading initiates an increased anabolic response in tenocytes, that can promote tendon repair. Human tenocytes from healthy hamstring tendons and tendinopathic Achilles tendons were derived by collagenase digest and outgrowth respectively. Tenocytes were seeded into 3D collagen gels. The gels were fixed in custom-made chambers and placed in an incubator for 24hrs whilst gene expression stabilised. After 24hrs, cyclic uniaxial strain at 1% ± 1% was applied to the cells, at either 1Hz (n=4) or 10Hz (n=4) using a Bose loading system. After 15 minutes of cyclic strain, the samples were maintained in chambers under 1% static strain for 24 hrs after which gene expression was characterised using RT-PCR.Introduction
Materials and Methods
Primary cilia are organelles found singularly on almost every cell in the body, including tenocytes. Tendon is a hierarchical, composite structure, and previous work from our group has suggested that the cell populations in the inter-fascicular matrix (IFM) may be different from those within the fascicle matrix (FM). This study investigated how stress deprivation influenced the primary cilia of both cell types, and the mechanics of the IFM and the FM. Rat tail tendons were dissected and then either tested immediately (fresh), or maintained in media for 1 week, either stress deprived or at 4% static strain. Fascicles and IFM were then either, fixed and imaged to determine cilia length (n = 80–160 cilia per group from across 3 rats), or mechanically tested to determine the static and viscoelastic properties of both the fascicles and the IFM (n = 6–8 per group).Introduction
Materials and Methods
Whilst all tendons connect muscle to bone, energy storing (ES) tendons, such as the equine superficial digital flexor tendon (SDFT) play an additional role, storing energy to improve locomotion efficiency. ES tendons experience significantly higher strains during locomotion than other positional tendons, such as the common digital extensor tendon (CDET). Our previous work has demonstrated that the interfascicular matrix (IFM) is more extensible in ES tendons, allowing ES tendons to stretch further during use. However, ES tendons must also recoil efficiently to perform their energy storing function. It has not been yet established if the IFM is able to recoil and recover after loading. Thus, this project aimed to determine the recoil capacity of the IFM in both the ES and positional tendons from young and old horses. Five young (3–7 years) and five old (17–20 years) SDFTs and CDETs were dissected from the forelimbs of 10 euthanized horses. Groups of 2 intact fascicles (bounded by IFM) were dissected from each tendon. Using a custom-made dissection rig and a polarised light microscope, samples were dissected, and the opposing end of each fascicle was cut transversely, leaving a 10 mm length of IFM. IFM samples were tested in shear, by preconditioning with 10 loading cycles then pulling to failure. The hysteresis and stress relaxation that occurred during preconditioning were calculated.Introduction
Materials and Methods
Fatigue loading has an age-specific effect on tendon fascicle micro-mechanics, with greater fibre sliding in aged samples indicating a decreased mechanical integrity, and a reduced ability to withstand cyclic loading, which may partially explain the age-related risk of tendon injury. The human Achilles and equine superficial digital flexor (SDFT) tendons function as energy stores, experiencing large, repetitive stresses and strains1 and are therefore highly susceptible to injury, particularly in aged individuals. We have previously observed rotation within SDFT fascicles in response to applied strain, which indicates the presence of helical sub-structures within this tendon. Further, we have shown that this rotation decreases with ageing, suggesting alterations to the helix sub-structure and a difference in the extension mechanisms in aged tendons. We therefore hypothesise that cyclic fatigue loading (FL) will result in alterations in fascicle extension mechanisms which are age specific.Summary Statement
Introduction
Tendon micromechanics were investigated using 2 methods. When collagen deformation was measured directly, higher levels of inter-fibre sliding were observed than when tenocyte nuclei were tracked. This suggests that under high strain tenocytes become unattached from the collagen fibres. Fibre extension and inter-fibre sliding have both been reported during tendon extension, but fibre sliding is believed to be the predominant mechanism in normal healthy tendon function. Fatigue damage is known to result in structural changes and reduced mechanical properties, but its influence on micromechanics is unknown. This work aimed: To investigate the effect of fatigue loading on bovine digital extensor fascicle micromechanics, comparing fibre extension and fibre sliding, hypothesising that the relative importance of these may change due to fatigue damage. To compare two techniques for characterising micromechanics: bleaching of a grid to directly measure collagen deformation, and using the cells as fiducial markers of fibre movement.Summary Statement
Introduction
Most cases of tendinopathy are believed to be overuse injuries rather than the result of a chronic event. The investigation of the fatigue properties of tendon is therefore of critical importance. This work considered the cyclic stress-relaxation and creep behaviour of two contrasting bovine tendon types – the largely postional digital extensor and the more energy storing deep digital flexor tendon. Fascicles were cyclically loaded (1Hz), to 1800 cycles of stress relaxation or to failure in creep, stopping some tests at 300, 900 or 1200 cycles to perform quasi-static failure tests or confocal imaging using a highly concentrated Acridine Orange solution. Creep tests were cycled to 60% of the ultimate tensile strength (UTS), while for stress relaxation, cyclic deformation to the strain associated with 60% UTS was used. Flexor tendon fascicles were found to exhibit reduced stress relaxation at all time points compared to the extensor fascicles and also showed an increase in the mean cycles to failure during creep testing. Evidence of fatigue damage was clear in the confocal images with breakdown of the collagen fibre alignment evident from 300 cycles; however it appears that some damage could occur without effect on the UTS of the fascicle. Despite what appears to be superior fatigue resistance in the flexor tendon fascicles, the matrix damage, certainly at early time points, appeared visually to be as severe as that observed with the extensor tendon fascicles.
