Calcified apoptotic vesicles from PROCR+ fibroblasts initiate heterotopic ossification

Heterotopic ossification (HO) comprises the abnormal formation of ectopic bone in extraskeletal soft tissue. The factors that initiate HO remain elusive. Herein, we found that calcified apoptotic vesicles (apoVs) led to increased calcification and stiffness of tendon extracellular matrix (ECM), which initiated M2 macrophage polarization and HO progression. Specifically, single‐cell transcriptome analyses of different stages of HO revealed that calcified apoVs were primarily secreted by a PROCR+ fibroblast population. In addition, calcified apoVs enriched calcium by annexin channels, absorbed to collagen I via electrostatic interaction, and aggregated to produce calcifying nodules in the ECM, leading to tendon calcification and stiffening. More importantly, apoV‐releasing inhibition or macrophage deletion both successfully reversed HO development. Thus, we are the first to identify calcified apoVs from PROCR+ fibroblasts as the initiating factor of HO, and might serve as the therapeutic target for inhibiting pathological calcification. Calcified apoVs increased calcification and stiffness of tendon extracellular matrix, and initiated the pro‐osteogenic microenvironment during HO development. PROCR+ fibroblasts released calcified apoptotic vesicles (apoVs) in the early stage of HO and calcium was continuously enriched in calcified apoVs via Annexin channels. When the crystallites were produced within the calcified apoVs, the mechanical stress within the calcified apoVs resulted in the puncture of the membrane and the release of crystallites. The increased stiffness of ECM caused by calcium and phosphorus deposition promoted M2 macrophage polarization, which initiated the osteogenic cascade in HO.