Heterotopic ossification in intact rat Achilles tendons is characterized by unique mineralized collagen fiber structures

Heterotopic ossification (HO) entails pathological mineral formation inside soft tissues. In human Achilles tendons, HO is often associated with tendinopathies, tendon weakness and pain. One hypothesis is that HO occurs in response to inflammation and by either intramembranous ossification, endochondral ossification, or a combination of both. However, refined details regarding HO deposition and microstructure are still unknown. In this study, we characterize HO in intact rat Achilles tendons through high-resolution phase contrast enhanced synchrotron X-ray tomographic imaging. Furthermore, we test the potential of using a procedure to induce local tissue injury by needling to study the relation between microdamage and formation of HO. The results show that HO occurs in all intact rat tendons occupying up to 1% of the total volume at 16 weeks of age. The HOs are characterized by an elongated ellipsoidal shape and by a distinctive fiber-like internal structure which suggests that some collagen fibers have become mineralized. The data indicates that the deposition along the fibers initiates in the pericellular area, and propagates into the intercellular area. The results also show that multiple HO deposits may merge into bigger structures with time by accession along unmineralized fibers. Furthermore, the presence of unmineralized regions within the deposits may indicate that HOs are not only growing, but mineral resorption can also occur. Additionally, phase contrast enhanced synchrotron X-ray tomography allowed to distinguish microdamage at the fiber level due to needling and it could in the future enable to elucidate the relation between local inflammation, microdamage, and HO deposition. ### Competing Interest Statement The authors have declared no competing interest.