Divide-and-conquer strategy with engineered ossification center organoids for rapid bone healing through developmental cell recruitment

Current approaches for bone repair predominantly target localized delivery of growth factors that are aimed at the coupling of angiogenesis and osteogenesis. However, delayed revascularization and regeneration of critical-sized bone defects are still challenging. In this study, we engineer an ossification center-like organoid (OCO) that consist of an inner-core bone morphogenetic and neurotrophic spheroid generated via MSCs-loaded 3D printing, alongside the interstitially distributed outer-shell proangiogenic neurotrophic phase. Our results demonstrate that collective implantation of OCOs achieves rapid bone bridging with successive OC-like bone ossicles formation across the bone defect in a "divide-and-conquer" way. Single-cell RNA sequencing analysis unveils a developmentally mimicking stem cell community that dominated with Krt8+ skeletal stem cells (SSCs) is uniquely recruited by the pro-regenerative in-situ organoid fusion and maturation. Particularly noteworthy is the specific expansion of Krt8+ SSCs concomitant with the simultaneous reduction of Has1+ migratory fibroblasts (MFs) post-OCO implantation. Furthermore, cross-species comparisons employing machine learning reveal high resemblance of the relative Krt8+ SSCs/Has1+ MFs composition in bone regeneration with that in public data from developmental bone tissues. Our findings advocate an approach akin to "divide-and-conquer" utilizing engineered OC-like organoids for prompt regeneration of large-sized bone defects.