Start, Stop, Rewind, Repeat-Cyclic Exposure of Adipose Stromal Cells-derived Cartilage Organoids to Chondrogenic and Proliferative Cues to Achieve Scaled-up and Customizable Bone Formation by Endochondral Ossification.

Developmental tissue engineering (TE) strategies recapitulating endochondral ossification (ECO)-the major ossification pathway in bone development and repair- are a promising avenue for the treatment of critical size and congenital bone defects. In this work, we develop a customizable approach using adipose stromal cells (ASC)-derived cartilage organoids as building blocks to generate clinically relevant grafts. Our hypothesis is that progenitor cells rather than mature chondrocytes would enable robust cartilage organoids fusion allowing graft tunability and scaling up. Using a cyclic approach alternating chondrogenic and proliferative cues we produce cartilage organoids surrounded by stromal chondrogenic progenitors. When re-exposed to chondrogenic medium, this perichondrial layer forms new cartilage tissue and acts as a biological cement in between cartilage organoids. A key feature of our approach is the iterative aspect of the protocol, where scaled up cartilage tissues obtained can themselves be used as building blocks to create larger tissue. Finally, in vivo, these grafts remodel efficiently into functional and mechanically apt bone organs mirroring ECO during skeletal development over the course of 24 weeks. Collectively, our findings provide a strong proof of concept of the envisioned TE strategy paving the way for a clinical application in the near future.