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 two-dimensional (2D) monolayer culture paradigm has limited translational potential to physiological systems; chondrocytes and tenocytes in monolayer lose expression of hallmarks of differentiated status (dedifferentiation). Qualitative assessment of three-dimensional (3D) cultures in musculoskeletal biology relative to native tissues has been limited. An understanding of prevailing gene regulatory networks is required to define whether 3D culture systems faithfully restitute the native tissue phenotype (redifferentiation). Using a systems biology approach to explore the gene networks associated with de- and re-differentiation may define targetable regulators associated with phenotypic plasticity of adult musculoskeletal cells. Global transcriptomic and proteomic profiling of matrix-depleted chondrocytes and tenocytes from the rat was performed for each of three conditions (native tissue, monolayer at passage three, or tissue-appropriate 3D cultures). Differential analysis of mRNA and protein abundance, gene ontology annotation, pathway topology impact analysis, and derivation of common mechanistic networks was undertaken to define consensus expression profiles, signalling pathways, and upstream regulators for de- and re-differentiation in each cell type.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
Tendons are critical to mobility, and are susceptible to degeneration through injury and ageing. Type I collagen is the most abundant protein in vertebrates; it is the main structural protein of the extracellular matrix in numerous musculoskeletal tissues, including tendons. Type I collagen predominantly is a heterotrimer, which consists of two alpha-1 chains and one alpha-2 chain (α1)2(α2) encoded by the COL1A1 and COL1A2 genes, respectively. However, type I collagen can form homotrimers (α1)3 which are protease-resistant, and are associated with age-related musculoskeletal diseases, fibrotic and connective tissue pathologies. Transforming growth factor beta (TGFβ) enhances collagen (I) gene expression, is involved in tendon mechanobiology and repair processes, while its effect on homotrimer formation is unknown. Our aim is to investigate the relative expressions of collagen (I) α1 and α2 polypeptide chains in tenocytes (tendon fibroblasts) stimulated with TGFβ. Included RT-qPCR to measure the relative expression of COL1A1 and COL1A2 genes. [14C]-proline metabolic labelling was used to measure the expression of the collagen (I) α1 and α2 polypeptide chains. These techniques were performed in equine superficial digital flexor tendon (SDFT) tenocytes (n=3) and murine tail tendon tenocytes (n=3) with different concentrations of TGFβ (0.01 ng/ml-100 ng/ml).Introduction
Materials and Methods
The rabbit common calcanean (Achilles) tendon is a compound apparatus frequently used in studies considering novel interventions to facilitate tendon regeneration. These studies often employ complete surgical transection of the apparatus. Due consideration of the translational relevance to human tendinopathy is often lacking and refinement of this injury model, consistent with the principles of the 3Rs, has not been forthcoming. Wild rabbit cadavers (n=10) were obtained from a licensed game dealer. For gross anatomy studies the caudal crus was dissected and transverse sections obtained every 5 mm. Ultrasongraphic examination of the entire apparatus was peformed with a 15 Hz transducer in transverse sections.Introduction
Materials and Methods
The equine SDFT tendon is a complex hierarchal structure that transmits force from muscle to bone and stores energy through its stretching and recoiling action. It is a common site of pathology in athletic horses. Our aim was to describe the ultrastructural anatomy of the SDFT as part of a larger programme to understand the structure-functional relationship of this tendon. Fifteen SDFT from different aged horses, sectioned transversely (2–3 mm thickness) and then photographed using Canon EOS 5D Mark III (100 mm focal length). Images processed through ImageJ and IMOD software for 3D reconstruction. Samples were also taken from the proximal, middle and distal part of the SDFT from a foetal, one and nine years old horse, processed for H&E staining and sectioned longitudinally in series into 20 sections (5µm), additionally the mid metacarpal region of one year old was fully sectioned into 250 sections. The entire cut surface on the slide was imaged and transformed to one collated image using Inkscape. Using IMOD collated photos transformed to mrc file (Z-stack) and in order to reconstruct 3D forms.Introduction
Materials and Methods
Tendon is prone to degeneration through ageing and injury and current therapies are largely ineffective. The recent identification of a cell population within tendon with stem cell-like characteristics holds potential for regeneration of tendon. The local stem cell environment (niche) is important for stem cell maintenance and function. This study aims to characterize extracellular matrix (ECM) components of the stem cell niche in equine tendon, which is prone to age-related degeneration and rupture. Putative tendon stem cells (TSCs) were isolated from equine superficial digital flexor tendon by low-density plating and differential adhesion to fibronectin. Cells were analysed by flow cytometry using antibodies to mesenchymal stem cell markers, as well as qRT-PCR for stem cell and tenogenic markers. The multipotency of cells was assessed using tri-lineage differentiation assays. ECM components of the tenocyte and TSC niche were analysed using radio-isotope labelling, immunohistochemistry and histology.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
