Cartilaginous spheroid-assembly design considerations for endochondral ossification: towards robotic-driven biomanufacturing.

Spheroids have become essential building blocks for biofabrication of functional tissues. Spheroid formats allow high cell-densities to be efficiently engineered into tissue structures closely resembling the native tissues. In this work, we explore the assembly capacity of cartilaginous spheroids (d∼ 150µm) in the context of endochondral bone formation. The fusion capacity of spheroids at various degrees of differentiation was investigated and showed decreased kinetics as well as remodeling capacity with increased spheroid maturity. Subsequently, design considerations regarding the dimensions of engineered spheroid-based cartilaginous mesotissues were explored for the corresponding time points, defining critical dimensions for these type of tissues as they progressively mature. Next, mesotissue assemblies were implanted subcutaneously in order to investigate the influence of spheroid fusion parameters on endochondral ossification. Moreover, as a step towards industrialization, we demonstrated a novel automated image-guided robotics process, based on targeting and registering single-spheroids, covering the range of spheroid and mesotissue dimensions investigated in this work. This work highlights a robust and automated high-precision biomanufacturing roadmap for producing spheroid-based implants for bone regeneration.