Bimodal Whole-Mount Imaging of Tendon Using Confocal Microscopy and X-ray Micro-Computed Tomography

Abstract

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.

CD146 Delineates an Interfascicular Cell Sub-Population in Tendon That Is Recruited during Injury through Its Ligand Laminin-α4

“…Taken together, these results suggest that the IFM may act as a niche for several distinct progenitor cell populations. Distribution of LAMA4 was similar to CD146, supporting previous studies demonstrating localisation of LAMA4 to the IFM in both small and large animal models [ 11 ]. Anti-inflammatory macrophage marker labelling was restricted to the IFM in uninjured tendon, suggesting that IFM cell populations may have a role in the resolution of inflammation.…”

“…However, previous studies have not been able to identify an IFM in the mouse Achilles [ 27 ], likely due to its small size, and therefore this model is not suitable for studying the role of IFM cell populations in tendon healing. By contrast, our recent work has established the presence of an IFM in the rat Achilles tendon [ 11 ], indicating that the rat may be a suitable model in which to study IFM cell populations in the context of tendon healing.…”

“…Several proteins characteristic of basement membranes have been shown to localise to the IFM [ 8 , 9 , 11 ]. Basement membranes are specialised matrices that interact with resident cells via cell surface receptors and are integral to tissue maintenance and repair, housing progenitor cell niches and providing structural integrity in musculoskeletal tissues [ 12 , 13 ].…”

“…Basement membranes are specialised matrices that interact with resident cells via cell surface receptors and are integral to tissue maintenance and repair, housing progenitor cell niches and providing structural integrity in musculoskeletal tissues [ 12 , 13 ]. Specifically, our previous work has demonstrated that the basement membrane protein laminin-α4 (LAMA4) localises predominantly to the IFM both in small and large animal models [ 11 ]. LAMA4 is a component of laminin-8, laminin-9, and laminin-14, and regulates basement membrane remodelling and function in cell niches in a variety of tissues, including adipose, endothelial, hematopoietic, muscular and neuronal tissues [ 14 , 15 , 16 , 17 ].…”

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

“…Force and displacement data for all samples were continuously recorded at 100 Hz during both preconditioning and pull to failure, and where appropriate, engineering stress and strain were calculated using the CSA and effective gauge length, respectively. Whilst recent studies indicate that IFM thickness is roughly 20 μm, this is highly variable within and between tendons [26] , [27] , therefore data were maintained as force and extension to ensure consistency. Force data were smoothed, prior to any calculations, using a 9-point moving average filter, to remove noise [24] .…”