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Background: Three-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 utilising 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. Results: Using fluorescent immunolabelling and optical clearing, we visualised the expression of the novel crossspecies marker of tendon basement membrane, laminin-α4 in 3D throughout whole rat Achilles tendons and equine superficial digital flexor tendon 5 mm segments. This revealed a complex network of laminin-α4 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. Conclusions: Whole-mount imaging of tendon allowed us to identify the presence of an extensive network of laminin-α4 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.

The interfascicular matrix (IFM) binds tendon fascicles and contains a population of morphologically distinct cells. However, the role of IFM-localised cell populations in tendon repair remains to be determined. The basement membrane protein laminin-α4 also localises to the IFM. Laminin-α4 is a ligand for several cell surface receptors, including CD146, a marker of pericyte and progenitor cells. We used a needle injury model in the rat Achilles tendon to test the hypothesis that the IFM is a niche for CD146+ cells that are mobilised in response to tendon damage. We also aimed to establish how expression patterns of circulating non-coding RNAs alter with tendon injury and identify potential RNA-based markers of tendon disease. The results demonstrate the formation of a focal lesion at the injury site, which increased in size and cellularity for up to 21 days post injury. In healthy tendon, CD146+ cells localised to the IFM, compared with injury, where CD146+ cells migrated towards the lesion at days 4 and 7, and populated the lesion 21 days post injury. This was accompanied by increased laminin-α4, suggesting that laminin-α4 facilitates CD146+ cell recruitment at injury sites. We also identified a panel of circulating microRNAs that are dysregulated with tendon injury. We propose that the IFM cell niche mediates the intrinsic response to injury, whereby an injury stimulus induces CD146+ cell migration. Further work is required to fully characterise CD146+ subpopulations within the IFM and establish their precise roles during tendon healing.

9BACKGROUND: 3-dimensional imaging modalities for optically dense connective tissues 10 such as tendons are limited and typically have a single imaging methodological endpoint. Here, 11we have developed a bimodal procedure that utilises fluorescence-based confocal microscopy 12 and x-ray micro-computed tomography for the imaging of adult tendons to visualise and 13 analyse extracellular sub-structure and cellular composition in small and large animal species. 14 RESULTS: Using fluorescent immunolabelling and optical clearing, we visualised the 15 expression of the basement membrane protein laminin-α4 in 3D throughout whole rat Achilles 16 tendons and equine superficial digital flexor tendon 5 mm segments. This revealed a complex 17 network of laminin-α4 within the tendon core that predominantly localises to the interfascicular 18 matrix compartment. Furthermore, we implemented a chemical drying process capable of 19 creating contrast densities enabling visualisation and quantification of both fascicular and 20 interfascicular matrix volume and thickness by x-ray micro-computed tomography. We also 21 demonstrated that both modalities can be combined using reverse clarification of fluorescently 22 labelled tissues prior to chemical drying to enable bimodal imaging of a single sample. 23 CONCLUSIONS: Whole-mount imaging of tendon allowed us to identify the presence of an 24 extensive network of laminin-α4 within tendon, the complexity of which cannot be appreciated 25 using traditional 2D imaging techniques. Creating contrast for x-ray micro-computed 26 tomography imaging of tendon using chemical drying is not only simple and rapid, but also 27 markedly improves on previously published methods. Combining these methods provides the 28 ability to gain spatio-temporal information and quantify tendon substructures to elucidate the 29 relationship between morphology and function. 30

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.