Pathological calcification in canine tendon-derived cells is modulated by extracellular ATP.

Tendon calcification is a commonly associated with degenerative tendinopathy of the Achilles tendons in dogs. It is characterised by the formation of calcific deposits and is refractory to treatment, often re-forming after surgical removal. Little is known about its pathogenesis and therefore the aims of this study were to develop an in vitro model of canine tendon calcification and use this model to investigate mechanisms driving calcification. Cells from the canine Achilles tendon were cultured with different calcifying media to establish which conditions were best able to induce specific, cell-mediated calcification. Once optimum calcification conditions had been established, the effect of ATP treatment on calcification was assessed. Results revealed that 2 mM di-sodium phosphate combined with 2 mM calcium chloride provided the optimum calcifying conditions, increasing calcium deposition and expression of osteogenic-related genes similar to those observed in tendon calcification in vivo. ATP treatment inhibited calcification in a dose-dependent manner, reducing calcium deposition and increasing cell viability, while osteogenic-related genes were no longer upregulated. In conclusion, the in vitro model of canine tendon calcification developed in this study provides the ability to study mechanisms driving tendon calcification, demonstrating that ATP plays a role in modulating tendon calcification that should be explored further in future studies.

The Interfascicular Matrix of Energy Storing Tendons Houses Heterogenous Cell Populations Disproportionately Affected by Aging.

Energy storing tendons such as the human Achilles and equine superficial digital flexor tendon (SDFT) are prone to injury, with incidence increasing with aging, peaking in the 5th decade of life in the human Achilles tendon. The interfascicular matrix (IFM), which binds tendon fascicles, plays a key role in energy storing tendon mechanics, and aging alterations to the IFM negatively impact tendon function. While the mechanical role of the IFM in tendon function is well-established, the biological role of IFM-resident cell populations remains to be elucidated. Therefore, the aim of this study was to identify IFM-resident cell populations and establish how these populations are affected by aging. Cells from young and old SDFTs were subjected to single cell RNA-sequencing, and immunolabelling for markers of each resulting population used to localise cell clusters. Eleven cell clusters were identified, including tenocytes, endothelial cells, mural cells, and immune cells. One tenocyte cluster localised to the fascicular matrix, whereas nine clusters localised to the IFM. Interfascicular tenocytes and mural cells were preferentially affected by aging, with differential expression of genes related to senescence, dysregulated proteostasis and inflammation. This is the first study to establish heterogeneity in IFM cell populations, and to identify age-related alterations specific to IFM-localised cells.

Small RNA signatures of the anterior cruciate ligament from patients with knee joint osteoarthritis.

Introduction: The anterior cruciate ligament (ACL) is susceptible to degeneration, resulting in joint pain, reduced mobility, and osteoarthritis development. There is currently a paucity of knowledge on how anterior cruciate ligament degeneration and disease leads to osteoarthritis. Small non-coding RNAs (sncRNAs), such as microRNAs and small nucleolar RNA (snoRNA), have diverse roles, including regulation of gene expression. Methods: We profiled the sncRNAs of diseased osteoarthritic ACLs to provide novel insights into osteoarthritis development. Small RNA sequencing from the ACLs of non- or end-stage human osteoarthritic knee joints was performed. Significantly differentially expressed sncRNAs were defined, and bioinformatics analysis was undertaken. Results and Discussion: A total of 184 sncRNAs were differentially expressed: 68 small nucleolar RNAs, 26 small nuclear RNAs (snRNAs), and 90 microRNAs. We identified both novel and recognized (miR-206, -365, and -29b and -29c) osteoarthritis-related microRNAs and other sncRNAs (including SNORD72, SNORD113, and SNORD114). Significant pathway enrichment of differentially expressed miRNAs includes differentiation of the muscle, inflammation, proliferation of chondrocytes, and fibrosis. Putative mRNAs of the microRNA target genes were associated with the canonical pathways "hepatic fibrosis signaling" and "osteoarthritis." The establishing sncRNA signatures of ACL disease during osteoarthritis could serve as novel biomarkers and potential therapeutic targets in ACL degeneration and osteoarthritis development.

The tendon interfascicular basement membrane provides a vascular niche for CD146+ cell subpopulations.

Introduction: The interfascicular matrix (IFM; also known as the endotenon) is critical to the mechanical adaptations and response to load in energy-storing tendons, such as the human Achilles and equine superficial digital flexor tendon (SDFT). We hypothesized that the IFM is a tendon progenitor cell niche housing an exclusive cell subpopulation. Methods: Immunolabelling of equine superficial digital flexor tendon was used to identify the interfascicular matrix niche, localising expression patterns of CD31 (endothelial cells), Desmin (smooth muscle cells and pericytes), CD146 (interfascicular matrix cells) and LAMA4 (interfascicular matrix basement membrane marker). Magnetic-activated cell sorting was employed to isolate and compare in vitro properties of CD146+ and CD146- subpopulations. Results: Labelling for CD146 using standard histological and 3D imaging of large intact 3D segments revealed an exclusive interfascicular cell subpopulation that resides in proximity to a basal lamina which forms extensive, interconnected vascular networks. Isolated CD146+ cells exhibited limited mineralisation (osteogenesis) and lipid production (adipogenesis). Discussion: This study demonstrates that the interfascicular matrix is a unique tendon cell niche, containing a vascular-rich network of basement membrane, CD31+ endothelial cells, Desmin+ mural cells, and CD146+ cell populations that are likely essential to tendon structure and/or function. Contrary to our hypothesis, interfascicular CD146+ subpopulations did not exhibit stem cell-like phenotypes. Instead, our results indicate CD146 as a pan-vascular marker within the tendon interfascicular matrix. Together with previous work demonstrating that endogenous tendon CD146+ cells migrate to sites of injury, our data suggest that their mobilisation to promote intrinsic repair involves changes in their relationships with local interfascicular matrix vascular and basement membrane constituents.

Tendon Extracellular Matrix Assembly, Maintenance and Dysregulation Throughout Life.

In his Lissner Award medal lecture in 2000, Stephen Cowin asked the question: "How is a tissue built?" It is not a new question, but it remains as relevant today as it did when it was asked 20 years ago. In fact, research on the organization and development of tissue structure has been a primary focus of tendon and ligament research for over two centuries. The tendon extracellular matrix (ECM) is critical to overall tissue function; it gives the tissue its unique mechanical properties, exhibiting complex non-linear responses, viscoelasticity and flow mechanisms, excellent energy storage and fatigue resistance. This matrix also creates a unique microenvironment for resident cells, allowing cells to maintain their phenotype and translate mechanical and chemical signals into biological responses. Importantly, this architecture is constantly remodeled by local cell populations in response to changing biochemical (systemic and local disease or injury) and mechanical (exercise, disuse, and overuse) stimuli. Here, we review the current understanding of matrix remodeling throughout life, focusing on formation and assembly during the postnatal period, maintenance and homeostasis during adulthood, and changes to homeostasis in natural aging. We also discuss advances in model systems and novel tools for studying collagen and non-collagenous matrix remodeling throughout life, and finally conclude by identifying key questions that have yet to be answered.

Structure-function specialisation of the interfascicular matrix in the human achilles tendon.

Tendon consists of highly aligned collagen-rich fascicles surrounded by interfascicular matrix (IFM). Some tendons act as energy stores to improve locomotion efficiency, but such tendons commonly obtain debilitating injuries. In equine tendons, energy storing is achieved primarily through specialisation of the IFM. However, no studies have investigated IFM structure-function specialisation in human tendons. Here, we compare the human positional anterior tibial tendon and energy storing Achilles tendons, testing the hypothesis that the Achilles tendon IFM has specialised composition and mechanical properties, which are lost with ageing. Data demonstrate IFM specialisation in the energy storing Achilles, with greater elasticity and fatigue resistance than in the positional anterior tibial tendon. With ageing, alterations occur predominantly to the proteome of the Achilles IFM, which are likely responsible for the observed trends towards decreased fatigue resistance. Knowledge of these key energy storing specialisations and their changes with ageing offers crucial insight towards developing treatments for tendinopathy. STATEMENT OF SIGNIFICANCE: Developing effective therapeutics or preventative measures for tendon injury necessitates the understanding of healthy tendon function and mechanics. By establishing structure-function relationships in human tendon and determining how these are affected by ageing, potential targets for therapeutics can be identified. In this study, we have used a combination of mechanical testing, immunolabelling and proteomics analysis to study structure-function specialisations in human tendon. We demonstrate that the interfascicular matrix is specialised for energy storing in the Achilles tendon, and that its proteome is altered with ageing, which is likely responsible for the observed trends towards decreased fatigue resistance. Knowledge of these key energy storing specialisations and their changes with ageing offers crucial insight towards developing treatments and preventative approaches for tendinopathy.

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.

Expression of purinergic P2X receptor subtypes 1, 2, 3 and 7 in equine laminitis.

Tissue sensitisation and chronic pain have been described in chronic-active laminitis in the horse, making treatment of such cases difficult. Purinergic P2X receptors are linked to chronic pain and inflammation. The aim of this study was to examine the expression of purinergic P2X receptor subtypes 1, 2, 3 and 7 in the hoof, palmar digital vessels and nerve, dorsal root ganglia and spinal cord in horses with chronic-active laminitis (n=5) compared to non-laminitic horses (n=5). Immunohistochemical analysis was performed on tissue sections using antibodies against P2X receptor subtypes 1-3 and 7. In horses with laminitis, there was a reduction in the thickness of the tunica media layer of the palmar digital vein as a proportion of the whole vessel diameter (0.48±0.05) compared to the non-laminitic group (0.57±0.04; P=0.02). P2X receptor subtype 3 was expressed in the smooth muscle layer (tunica media) of the palmar digital artery of horses with laminitis, but was absent in horses without laminitis. There was strong expression of P2X receptor subtype 7 in the proliferating, partially keratinised, epidermal cells of the secondary epidermal lamellae in the hooves of horses with laminitis, but no immunopositivity in horses without laminitis.