Calcified apoptotic bodies from PROCR+ fibroblasts initiate the tendon calcification at the early stages of heterotopic ossification

Abstract 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 bodies (CABs), which are secreted by PROCR + fibroblasts in the early stage of HO, lead to increased stiffness of the extracellular matrix. Specifically, single-cell transcriptome analyses of different stages of HO revealed a PROCR + fibroblast population that released CABs in the early stage of HO. CAB aggregation produced calcified nodules with high concentrations of calcium and phosphate, similar to those in calcified tendons. Annexin channels mediate calcium influx into CABs, which absorb to collagen I via electrostatic interaction. Functional inhibition of CABs significantly decreased the early stage microcalcification and inhibited HO of Achilles tendons. Thus, we revealed a pathological mechanism of HO initiation and identified CABs from PROCR + fibroblasts as the initiating factor of local microcalcification, creating an osteogenic microenvironment for HO.