Three‐Dimensional Characterization of the Collagen–Hydroxyapatite Interaction During Heterotopic Ossification in Healing Rat Achilles Tendons

Pathological ossification is seen affecting different organs across the body. This includes the Achilles tendon, where the healing process after rupture can be impaired by heterotopic ossifications (HO). The formation pathways for HO in tendons are still debated, and one reason for this is our limited knowledge of their structure and composition in 3D. This study utilizes a multimodal 3D characterization approach to study HO and the surrounding healing tissue at 3‐, 6‐, and 12‐week post‐transection in rat Achilles tendons. Microcomputed tomography provided 3D microstructural information. Small‐ and wide‐angle scattering tensor tomography contributed with combined collagen and hydroxyapatite (HA) 3D nanostructural information. Combined 3D X‐ray diffraction and fluorescence provided high‐resolution structural dimensions of HA crystallites, and chemical composition of both the deposits and the surrounding healing tendon tissue. Morphologically, dense mineralized tissue appeared more fibrous, with higher co‐localized quantities of Zn present internally, while porous regions of HO deposits had Zn localization primarily at the boundary. Collagen d‐spacing was higher within HO deposits than in the surrounding collagenous tendon tissue. Collagen misalignment close to the HO boundary supports the notion of tissue mineralization occurring prior to the full assembly of the healing tendon network. This methodology can be extrapolated to investigate pathological ossification in other tissues at the nanoscale while capturing the heterogeneity of entire specimens.