Biology subjects

Thorpe, C. T.

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

3 recordsLinked to original sources

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↗