Periosteal Mesenchymal Progenitor Dysfunction and Extraskeletally-Derived Fibrosis Contribute to Atrophic Fracture Nonunion

Atrophic nonunion represents an extremely challenging clinical dilemma for both physicians and fracture patients alike, but its underlying mechanisms are still largely unknown. Here, we established a mouse model that recapitulates clinical atrophic nonunion through the administration of focal radiation to the long bone midshaft 2 weeks before a closed, semistabilized, transverse fracture. Strikingly, fractures in previously irradiated bone showed no bony bridging with a 100% nonunion rate. Radiation triggered distinct repair responses, separated by the fracture line: a less robust callus formation at the proximal side (close to the knee) and bony atrophy at the distal side (close to the ankle) characterized by sustained fibrotic cells and type I collagen-rich matrix. These fibrotic cells, similar to human nonunion samples, lacked osteogenic and chondrogenic differentiation and exhibited impaired blood vessel infiltration. Mechanistically, focal radiation reduced the numbers of periosteal mesenchymal progenitors and blood vessels and blunted injury-induced proliferation of mesenchymal progenitors shortly after fracture, with greater damage particularly at the distal side. In culture, radiation drastically suppressed proliferation of periosteal mesenchymal progenitors. Radiation did not affect hypoxia-induced periosteal cell chondrogenesis but greatly reduced osteogenic differentiation. Lineage tracing using multiple reporter mouse models revealed that mesenchymal progenitors within the bone marrow or along the periosteal bone surface did not contribute to nonunion fibrosis. Therefore, we conclude that atrophic nonunion fractures are caused by severe damage to the periosteal mesenchymal progenitors and are accompanied by an extraskeletal, fibro-cellular response. In addition, we present this radiation-induced periosteal damage model as a new, clinically relevant tool to study the biologic basis of therapies for atrophic nonunion.