Three-dimensional imaging of the fibrous microstructure of Achilles tendon entheses in Mus musculus

The whole-organ, three-dimensional microstructure of murine Achilles tendon entheses was visualized with micro-computed tomography (microCT). Contrast-enhancement was achieved either by staining with phosphotungstic acid (PTA) or by a combination of cell-maceration, demineralization and critical-point drying with low tube voltages and propagation-based phase-contrast (fibrous structure scan). By PTA-staining, X-ray absorption of the enthesial soft tissues became sufficiently high to segment the tendon and measure cross-sectional areas along its course. With the fibrous structure scans, three-dimensional visualizations of the collagen fiber networks of complete entheses were obtained. The characteristic tissues of entheses were identified in the volume data. The tendon proper was marked as a segment manually. The fibers within the tendon were marked by thresholding. Tendon and fiber cross-sectional areas were measured. The measurements were compared between individuals and protocols for contrast-enhancement, using a spatial reference system within the three-dimensional enthesis. The usefulness of the method for investigations of the fibrous structure of collagenous tissues is demonstrated.