Cryostorable Callus Mimetics Support Endochondral Bone Regeneration in a Large-Animal Maxillofacial Defect

Endochondral bone regeneration (EBR) is a developmentally inspired strategy based on a pre-formed cartilage template that remodels in vivo into vascularized bone. Although EBR has shown promise in orthopedic settings, its application to maxillofacial reconstruction remains largely unexplored in large animal models, particularly because maxillofacial bone is classically formed by intramembranous ossification. Here, we present soft callus mimetics (CMs) as bioactive, devitalized cartilage modules that preserve extracellular matrix-encoded cues capable of instructing endochondral ossification without viable donor cells. Their regenerative performance was evaluated in a split-mouth bilateral goat alveolar cleft model (n = 10), in which CMs were implanted in one cleft and autologous iliac crest bone graft in the contralateral cleft. Across microCT, histomorphometry, and fluorochrome labeling, CM-treated defects supported bone regeneration and remodeling without evidence of inferior early structural outcomes compared with autograft. In a separate rat subcutaneous implantation study, CMs retained osteoinductive capacity after 9 months of cryostorage compared with previously reported fresh CMs implanted under matched, non-concurrent conditions. Together, these findings support the biological feasibility of CM-driven endochondral bone regeneration in a large animal maxillofacial environment and the further development of cryostorable CMs for bone repair.