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.