Biology subjects

Thorpe, C. T.

Publications and source records attributed to Thorpe, C. T..

8 recordsLinked to original sources

bioRxiv2026

Senescent tenocytes drive an age-associated loss of cell-extracellular matrix homeostasis, which can be partially restored using dihydroresveratrol

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 individuals 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.

cell biology↗

bioRxiv2024

Evaluation of suitable reference genes for qPCR normalisation of gene expression in a Achilles tendon injury model.

Tendons are one of the major load-bearing tissues in the body; subjected to enormous peak stresses, and thus vulnerable to injury. Cellular responses to tendon injury are complex, involving inflammatory and repair components, with the latter employing both resident and recruited exogenous cell populations. Gene expression analyses are valuable tools for investigating tendon injury, allowing assessment of repair processes and pathological responses such as fibrosis, and permitting evaluation of therapeutic pharmacological interventions. Quantitative polymerase chain reaction (qPCR) is a commonly used approach for such studies, but data obtained by this method must be normalised to reference genes: genes known to be stably expressed between the experimental conditions investigated. Establishing suitable tendon injury reference genes is thus essential. Accordingly we investigated mRNA expression stability in a rat model of tendon injury, comparing both injured and uninjured tendons, and the effects of rapamycin treatment, at 1 and 3 weeks post injury. We used 11 candidate genes (18S, ACTB, AP3D1, B2M, CSNK2A2, GAPDH, HPRT1, PAK1IP1, RPL13a, SDHA, UBC) and assessed stability via four complementary algorithms (Bestkeeper, deltaCt, geNorm, Normfinder). Our results suggests that ACTB, CSNK2A2, HPRT1 and PAK1IP1 are all stably expressed in tendon, regardless of injury or drug treatment: any three of these would serve as universally suitable reference gene panel for normalizing qPCR expression data in the rat tendon injury model. We also reveal 18S, UBC, GAPDH, and SDHA as consistently poor scoring candidates (with the latter two exhibiting rapamycin- and injury-associated changes, respectively): these genes should be avoided.

molecular biology↗

bioRxiv2023

The interfascicular matrix of energy storing tendons houses heterogenous cell populations disproportionately affected by ageing

Energy storing tendons such as the human Achilles and equine superficial digital flexor tendon (SDFT) are prone to injury, with incidence increasing with ageing. The interfascicular matrix (IFM), which binds tendon fascicles, plays a key role in energy storing tendon mechanics, and ageing 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 ageing. 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 ageing, with differential expression of genes related to senescence, dysregulated proteostasis and inflammation. This is the first study to uncover the importance of the IFM niche for a diverse range of cell populations, and to identify age-related alterations specific to IFM-localised cells.

cell biology↗

bioRxiv2021

Structure-function Specialisation of the Interfascicular Matrix in the Human Achilles Tendon

ObjectiveTendon consists of highly aligned collagen-rich fascicles surrounded by interfascicular matrix (IFM). Some tendons act as energy stores to improve locomotion efficiency; these tendons are prone to debilitating injuries, the incidence of which increases with ageing. In equine tendons, energy storage is achieved primarily through specialisation of the IFM. However, no studies have investigated IFM structure-function specialisation in human tendons. Here, we compare the positional anterior tibialis and energy storing Achilles tendons, testing the hypothesis that the Achilles IFM has specialised composition and mechanical properties, which are lost with ageing. MethodsWe used a multidisciplinary combination of mechanical testing, immunolocalisation and proteomics to investigate structure-function specialisations in functionally distinct human tendons and how these are altered with ageing. ResultsThe IFM in the energy storing Achilles tendon is more elastic and fatigue resistant than the IFM in the positional anterior tibialis tendon, with a trend towards decreased fatigue resistance with age in the Achilles IFM. With ageing, alterations occur predominantly to the proteome of the Achilles IFM. ConclusionThe Achilles tendon IFM is specialised for energy storage, and changes to its proteome with ageing are likely responsible for the observed trends towards decreased fatigue resistance. Knowledge of key energy storing specialisations and their changes with ageing offers insight towards developing effective treatments for tendinopathy. Key messagesO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIEnergy storing tendons in animals and humans are particularly prone to tendinopathy and the incidence increases with increasing age. C_LIO_LIPrevious work in some animal models has shown that the specialisation of tendon properties for energy storage is achieved primarily through adaptation of the interfascicular matrix, with specialisation lost in ageing. However, the structural specialisations that provide the human Achilles tendon with its energy storing ability, and how these are affected by ageing, remain to be established. C_LI What does this study add?O_LIWe demonstrate that the interfascicular matrix in the human Achilles tendon is specialised for energy storing, with increased elastic recoil and fatigue resistance, and that these specialisations are partially lost with ageing, likely due to alterations to the proteome of the interfascicular matrix. C_LI How might this impact on clinical practice or future developments?O_LIShort term, the specialist IFM mechanics we have demonstrated can be detected with new developments in ultrasound functional imaging, offering improved opportunities for contextual tendinopathy diagnostics. Personalised rehabilitation programmes can now be explored and designed specifically to target IFM mechanics. C_LIO_LILonger term, the knowledge of key specialisations in injury prone energy storing tendons and how they are affected by ageing, offers crucial insight towards developing cell or tissue engineering treatments targeted at restoring tendon structure and function post-injury, specifically targeted at the IFM. C_LI

bioengineering↗

bioRxiv2020

Elastase treatment of tendon specifically impacts the mechanical properties of the interfascicular matrix

The tendon interfascicular matrix (IFM) binds tendon fascicles together. As a result of its low stiffness behaviour under small loads, it enables non-uniform loading and increased overall extensibility of tendon by facilitating fascicle sliding. This function is particularly important in energy storing tendons, with previous studies demonstrating enhanced extensibility, recovery and fatigue resistance in the IFM of energy storing compared to positional tendons. However, the compositional specialisations within the IFM that confer this behaviour remain to be elucidated. It is well established that the IFM is rich in elastin, therefore we sought to test the hypothesis that elastin depletion (following elastase treatment) will significantly impact IFM, but not fascicle, mechanical properties, reducing IFM resilience in all samples, but to a greater extent in younger tendons, which have a higher elastin content. Using a combination of quasi-static and fatigue testing, and optical imaging, we confirmed our hypothesis, demonstrating that elastin depletion resulted in significant decreases in IFM viscoelasticity, fatigue resistance and recoverability compared to untreated samples, with no significant changes to fascicle mechanics. Ageing had little effect on fascicle or IFM response to elastase treatment. This study offers a first insight into the functional importance of elastin in regional specific tendon mechanics. It highlights the important contribution of elastin to IFM mechanical properties, demonstrating that maintenance of a functional elastin network within the IFM is essential to maintain IFM and thus tendon integrity.

bioengineering↗

bioRxiv2020

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 (fibre phase) and surrounding predominantly non-collagenous matrix (matrix phase) 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 matrix phase. 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 phase of tissue.

cell biology↗

bioRxiv2020

Bimodal Whole-Mount Imaging Of Tendon Using Confocal Microscopy And X-Ray Micro-Computed Tomography

BACKGROUND3-dimensional imaging modalities for optically dense connective tissues such as tendons are limited and typically have a single imaging methodological endpoint. Here, we have developed a bimodal procedure that utilises fluorescence-based confocal microscopy and x-ray micro-computed tomography for the imaging of adult tendons to visualise and analyse extracellular sub-structure and cellular composition in small and large animal species. RESULTSUsing fluorescent immunolabelling and optical clearing, we visualised the expression of the basement membrane protein laminin-4 in 3D throughout whole rat Achilles tendons and equine superficial digital flexor tendon 5 mm segments. This revealed a complex network of laminin-4 within the tendon core that predominantly localises to the interfascicular matrix compartment. Furthermore, we implemented a chemical drying process capable of creating contrast densities enabling visualisation and quantification of both fascicular and interfascicular matrix volume and thickness by x-ray micro-computed tomography. We also demonstrated that both modalities can be combined using reverse clarification of fluorescently labelled tissues prior to chemical drying to enable bimodal imaging of a single sample. CONCLUSIONSWhole-mount imaging of tendon allowed us to identify the presence of an extensive network of laminin-4 within tendon, the complexity of which cannot be appreciated using traditional 2D imaging techniques. Creating contrast for x-ray micro-computed tomography imaging of tendon using chemical drying is not only simple and rapid, but also markedly improves on previously published methods. Combining these methods provides the ability to gain spatio-temporal information and quantify tendon substructures to elucidate the relationship between morphology and function.

cell biology↗

bioRxiv2020

Heterogeneity of proteome dynamics between connective tissue phases of adult tendon

Maintenance of connective tissue integrity is fundamental to sustain function, requiring protein turnover to repair damaged tissue. However, connective tissue proteome dynamics remain largely undefined, as do differences in turnover rates of individual proteins in the collagen and glycoprotein phases of connective tissue extracellular matrix (ECM). Here, we investigate proteome dynamics in the collagen and glycoprotein phases of connective tissues by exploiting the spatially distinct fascicular (collagen-rich) and interfascicular (glycoprotein-rich) ECM phases of tendon. Using isotope labelling, mass spectrometry and bioinformatics, we calculate turnover rates of individual proteins within rat Achilles tendon and its ECM phases. Our results demonstrate complex proteome dynamics in tendon, with ~1000-fold differences in protein turnover rates, and overall faster protein turnover within the glycoprotein-rich interfascicular matrix compared to the collagen-rich fascicular matrix. These data provide insights into the complexity of proteome dynamics in tendon, likely required to maintain tissue homeostasis.

biochemistry↗