Ligaments and tendons are vital musculoskeletal soft tissues, which are commonly injured due to overuse and trauma. Their distinct functions are well known however their unique structure and biochemical composition and how they change with disease is poorly described. The most commonly injured ligament in the dog and man is the cranial cruciate (CCL) and anterior cruciate ligament (ACL) respectively. Therefore, the structure, function and pathophysiology of disease of this ligament has been most commonly studied in both species. Canine cranial cruciate ligament rupture (CCLR) most commonly occurs following gradual ligament degeneration or disease (CCLD) followed by a non-contact injury or a minor trauma. Several studies have described marked degenerative histological changes in ligament structure prior to and following rupture which consist of loss of the collagen fascicular structure, areas of poor collagen fibril staining, a marked increase in “chondroid” type cells and mineralisation. The ECM protein profile is also altered with increased sulphated glycosaminoglycans content, increased immature collagen cross-links as well as enzymes involved in collagen remodelling. In man, similar findings have been described in the ACL with age and in osteoarthritis (OA). Previously it had been thought that ligament degeneration occurred following OA but these more recent studies suggest that ligament degeneration can lead to joint destabilisation and OA. Being able to determine early degenerative ligament changes in spontaneous clinical cohorts and the mechanisms which cause them are ideal starting points to determine targets for future therapies in the prevention of ligament degradation and rupture. Further identification of ligament cell types in terms of degenerative, responsive and regenerative (stem) types is essential to try and alter ligament cellular and extracellular matrices harnessing their therapeutic potential.
Obesity is one of risk factors of anterior cruciate ligament tear in man or cranial cruciate ligament (CCL) tear in dog. Adipokines are biologically active mediators released from adipocytes, and correlate with changes in body mass index. In order to study the possibility that adipocytes play a role in the pathogenesis of CCL disease, we investigated alterations of the matrix degradation biomarker genes (matrix metalloproteinase-13 [MMP-13], aggrecan) in CCL cells after stimulating with adipokines. We collected CCLs from 6 dog cadavers that had been euthanased for reasons other than musculoskeletal disease. CCL cells were isolated and treated with key adipokines including of adiponectin, leptin and visfatin at different concentration (0.1 ng/mL, 1 ng/mL and 10 ng/mL), and at three different time points (1 h, 6 h and 24 h). Real-time PCR was used to determine gene expression for MMP-13 and aggrecan in CCL cells comparing with negative control. In addition, lipopolysaccharide was used as a positive control. The statistical significance of differences between groups was determined using non-parametric Friedman test, followed by the Conover post-hoc test, and data were considered statistically significant at P<0.05.Introduction
Materials and Methods
Tendons and ligaments (TLs) play key roles in the musculoskeletal system. However, they are commonly damaged due to age-related wear and tear or torn in traumatic/sport related incidents resulting in pain and immobility. TLs contain cells and extracellular matrix (ECM) comprised of collagen, elastin, glycoproteins and proteoglycans. Although TLs are composed of similar components, their precise composition and arrangement of matrix macromolecules differ to provide specific mechanical properties and functions. To date little is known about how the main ECM proteins are arranged between the two tissue types. This data will provide essential information on fundamental structure of TLs leading to increased understanding of the function relationship between these tissue types. The aim of this study was to compare tendon-ligament differences in their ECM distribution of collagens, proteoglycans and elastic fibres. Anterior cruciate ligament (ACL) and long digital extensor tendon (LDET) were harvested from disease free cadaveric canine knee joints (n=3). Distributions of the main ECM components were assessed on longitudinal sections of ACL and LDET mid-substance. Antibody staining were assessed for collagen type I, III, VI, agreccan, versican, decorin, biglycan, elastin, fibrillin 1 and fibrillin 2.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
