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
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 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
