Nodoka Iwasaki

Ageing is a fundamental biological process marked by declining physiological function and the accumulation of stress-induced damage. It contributes to chronic disease and drives widespread alterations across organ systems, including the vasculature. Ageing of the macro- and microvasculature impairs nutrient delivery, promotes tissue degeneration, and reduces repair capacity in many tissues, including those of the musculoskeletal system. Tendons and ligaments become increasingly injury-prone with age, yet the relationship between vascular ageing and tendon and ligament in health and disease remains insufficiently understood. This review critically examines the effects of vascular ageing on tendons and ligaments, including their interfaces with muscle and bone. Emerging evidence indicates that age-related vascular decline contributes to structural and functional impairments in these tissues, accelerating musculoskeletal deterioration. We also discuss therapeutic approaches targeting vascular dysfunction, including growth factor modulation through use of platelet-rich plasma, gene-based strategies, and stem cell therapies, which show potential for mitigating age-related deficits. By synthesizing existing knowledge, this review emphasizes the need for further research into vascular ageing and its implications for tendon and ligament health. A deeper understanding of these processes could inform the development of innovative therapies aimed at preserving vascular function and improving quality of life in the ageing population.

Tendons are commonly injured, not only in athletes, but also during normal everyday activities, particularly in older age. Tendons are rich in extracellular matrix proteins, many of which are turned over extremely slowly during an individual's lifetime. As a result, tight regulation of the extracellular matrix is essential for tendons to remain resilient to the mechanical load placed on them. However, tendons are prone to age-associated functional decline, characterised by chronic inflammation, accumulation of damaged collagen, and matrix remodelling. These degenerative changes often precede injury, wherein additional inflammation and fibrotic tissue deposition make treatment difficult and reinjury highly likely. Understanding and preventing the causes of tendon functional decline is therefore vital to improving quality of life in the ageing population. Using an equine model, here we show that tendon fibroblasts, or tenocytes, isolated from aged tendons exhibit markers of senescence when cultured in vitro. Further, we describe how senescence in tenocytes drives inflammation, hypercontractility, and dysregulation of tendon matrix components. By replicatively senescing tenocytes isolated from young tendons, we observed that senescent tenocytes instigated proinflammatory signalling, had impaired capacity in wound healing assays, increased contractility, and secreted factors that induced senescence in healthy tenocytes. Further, tenocyte senescence dysregulated matrix turnover both at the gene and protein level. Finally, we demonstrate how senotherapeutic treatment of senescent tenocytes can reduce expression of senescence markers and restore proliferative capacity. Our results uncover systemic links between tendon ageing and cellular senescence, and identify mechanisms and therapeutic strategies for age-associated tendon degeneration.

The muscle-tendon junction (MTJ) is a specialised interface between muscle and tendon and transmits muscle-generated force to the tendon. The MTJ is particularly vulnerable to injuries compared to muscle and tendon and becomes more injury prone with age. Despite its clinical importance, the mechanisms driving MTJ ageing and age-related functional deterioration remain poorly understood. In this study, young (3-month-old) and old (23-month-old) male mice were used to provide the first comprehensive three-dimensional characterisation of age-related structural and cellular changes at the mouse Achilles MTJ. This was achieved using the high-resolution imaging techniques, micro-computed tomography (µCT) and confocal microscopy. µCT analysis revealed a 27% reduction in muscle fibre diameter with age, accompanied by a trend toward increased MTJ surface area and a 19% reduction in pennation angle, which may indicate diminished force generation capacity. Confocal imaging showed a 49% reduction in endothelial cell volume (VWF-labelled) in the old mouse muscle-tendon unit, suggesting a loss of vascularity. In situ hybridisation demonstrated increased expression of senescence markers p16 and p21 in endothelial and MTJ-specific cells, with MTJ-specific cells showing the greatest accumulation of p16 and p21 (270% and 310% increases, respectively) with age, and immunofluorescence also showed increased expression of p21. These findings suggest that vascular and MTJ-specific cells are particularly susceptible to ageing and may collectively contribute to the age-related functional decline of the MTJ. Understanding these mechanisms may help to develop targeted therapeutic strategies to preserve or restore MTJ integrity and function in ageing populations.

The muscle tendon junction (MTJ) is a specialised interface between muscle and tendon and transmits muscle-generated force to the tendon. The MTJ is particularly vulnerable to injuries compared to muscle and tendon and becomes more injury prone with age. Further, current treatments for MTJ injuries are insufficient as indicated by scar tissue formation and a high re-injury rate. Despite its clinical importance, the mechanisms driving MTJ ageing and age-related functional deterioration remain poorly understood. In this study, the first comprehensive three-dimensional characterisation of age-related structural and cellular changes at the mouse MTJ was performed using the high-resolution imaging techniques, micro-computed tomography (µCT) and confocal microscopy. µCT analysis revealed a 27% reduction in muscle fibre diameter with age, accompanied by a trend toward increased MTJ surface area and a 19% reduction in pennation angle, indicating diminished force generation capacity. Confocal imaging showed a 49% reduction in endothelial cell volume (VWF-labelled) in the old mouse muscle-tendon unit, suggesting a loss of vascularity. In situ hybridisation and immunofluorescence demonstrated increased expression of senescence markers p16 and p21 in endothelial and MTJ-specific cells, with MTJ-specific cells showing the greatest accumulation of p16 and p21 (270% and 310% increases, respectively) with age. These findings suggest that vascular and MTJ-specific cells are particularly susceptible to ageing and may collectively contribute to the age-related functional decline of the MTJ. Understanding these mechanisms may help to develop targeted therapeutic strategies to preserve or restore MTJ integrity and function in ageing populations.

Tendon degeneration is common, and its risk increases with age both in humans and horses. Tendon regeneration and healing is limited due to inherent low cell density and vascularisation, and current treatments are insufficient as indicated by scar tissue formation and a high re‐injury rate. The tendon vasculature plays a crucial role in tendon homeostasis, regeneration and healing, making it a potential therapeutic target. However, the effect of ageing on the tendon microvasculature is poorly understood. Here, we provide the first comprehensive characterisation of the tendon microvasculature. We employed high‐resolution 3D imaging techniques, using micro‐computed tomography (μCT) and confocal microscopy, to investigate age‐related alterations in the vasculature within the equine superficial digital flexor tendon (SDFT), a functional equivalent of the human Achilles tendon. μCT analysis revealed a well‐developed vascular network within the interfascicular matrix (IFM) and demonstrated significant age‐associated reductions in vascular volume (70%), vessel diameter (30%) and density (74%). 3D immunolabelling showed significant reductions in MYH11‐ (96%) and desmin‐positive (78%) volumes; however, there was a pronounced age‐associated increase in von Willebrand factor (VWF)‐positive volume (220%), which was accompanied by a significantly higher (249%) pericyte density. Taken together, these results indicate a loss of larger blood vessels in the IFM but an increase in small vessel formation, suggesting that neo‐angiogenesis is induced in aged tendon alongside a loss of vascular homeostasis. These insights enhance our understanding of tendon ageing and may contribute to developing new therapeutic approaches for improving tendon health and repair in older individuals.

We report, for the first time, the full-field 3D strain distribution of the muscle-tendon junction (MTJ). Understanding the strain distribution at the junction is crucial for the treatment of injuries and to predict tear formation at this location. Three-dimensional full-field strain distribution of mouse MTJ was measured using X-ray computer tomography (XCT) combined with digital volume correlation (DVC) with the aim of understanding the mechanical behavior of the junction under tensile loading. The interface between the Achilles tendon and the gastrocnemius muscle was harvested from adult mice and stained using 1% phosphotungstic acid in 70% ethanol. In situ XCT combined with DVC was used to image and compute strain distribution at the MTJ under a tensile load (2.4 N). High strain measuring 120,000 µε, 160,000 µε, and 120,000 µε for the first principal stain (ε_p1), shear strain (γ), and von Mises strain (ε_VM), respectively, was measured at the MTJ and these values reduced into the body of the muscle or into the tendon. Strain is concentrated at the MTJ, which is at risk of being damaged in activities associated with excessive physical activity.

The muscle tendon junction (MTJ) transmits the force generated by the muscle to the tendon and ultimately to the bone. Tears and strains commonly occur at the MTJ where regeneration is limited due poor vascularisation and the complexity of the tissue. Currently treatments for a complete MTJ tear are often unsuccessful. The creation of a tissue engineered MTJ would therefore be beneficial in the development of a novel treatment. In this study, aligned electrospun polycaprolactone fibres were fabricated and human myoblasts and tenocytes were cultured on the scaffold. The effect of 10 % cyclic strain and co-culture of myoblasts and tenocytes on the MTJ formation was investigated. The application of strain significantly increased cell elongation, and MTJ marker gene expression. Co-culture of myoblasts and tenocytes with strain induced higher MTJ marker gene expression compared with myoblasts and tenocytes cultured separately. Paxillin and collagen 22, naturally found in the MTJ, were also produced when cells were combined and grown in a 10 % strain environment. For the first time these results showed that the combination of the strain and co-culture of myoblasts and tenocytes promotes gene expression and production of proteins that are found in the MTJ.