Tracking tendon fibers to their insertion - a 3D analysis of the Achilles tendon enthesis in mice.

Tendon insertions to bone are heavily loaded transitions between soft and hard tissues. The fiber courses in the tendon have profound effects on the distribution of stress along and across the insertion. We tracked fibers of the Achilles tendon in mice in micro-computed tomographies and extracted virtual transversal sections. The fiber tracks and shapes were analyzed from a position in the free tendon to the insertion. Mechanically relevant parameters were extracted. The fiber number was found to stay about constant along the tendon. But the fiber cross-sectional areas decrease towards the insertion. The fibers mainly interact due to tendon twist, while branching only creates small branching clusters with low levels of divergence along the tendon. The highest fiber curvatures were found within the unmineralized entheseal fibrocartilage. The fibers inserting at a protrusion of the insertion area form a distinct portion within the tendon. Tendon twist is expected to contribute to a homogeneous distribution of stress among the fibers. According to the low cross-sectional areas and the high fiber curvatures, tensile and compressive stress are expected to peak at the insertion. These findings raise the question whether the insertion is reinforced in terms of fiber strength or by other load-bearing components besides the fibers. STATEMENT OF SIGNIFICANCE: The presented study is the first analysis of the 3D fiber tracks in macroscopic tendon samples as determined by a combination of cell-maceration, phase-contrast µCT and template-based tracking. The structural findings change the understanding of the tendon-bone insertion and its biomechanics: (1) The insertion is not reinforced in terms of fiber numbers or sizes. Its robustness remains unexplained. (2) The orientation of fibers in the tendon center is higher than in the margins. This arrangement could inspire material development. (3) Fibers inserting at a protrusion of the insertion area stem from a distinct portion within the tendon. The results show that fibrous structure analysis can link macro- to micromechanics and that it is ready for the application to complete muscle-tendon units.

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.