4D Biofabrication of Magnetically Augmented Callus Assembloid Implants Enables Rapid Endochondral Ossification via Activation of Mechanosensitive Pathways.

The use of magnetic-driven strategies for non-contact manipulation of engineered living modules opens up new possibilities for tissue engineering. The integration of magnetic nanoparticles (MNPs) with cartilaginous microtissues enables model-driven 4D bottom-up biofabrication of remotely actuated assembloids, providing unique properties to mechanoresponsive tissues, particularly skeletal constructs. However, for clinical use, the long-term effects of magnetic stimulation on phenotype and in vivo functionality need further exploration. Magnetic-driven biofabrication includes both rapid processes, such as guided microtissue assembly, and slower biological processes, like extracellular matrix secretion. This work explores the interplay between magnetic fields and MNP-loaded cartilaginous microtissues through mathematical modeling and experimental approaches, investigating long-term stimulation effects on ECM maturation and chondrogenic hypertrophy. Transcriptomic analysis reveal that magnetic stimulation activated mechanosensitive pathways and catabolic processes, driving accelerated cartilage-to-bone transitions via endochondral ossification, outcomes not observed in non-stimulated controls. This study paves the way for pre-programmed, remotely actuated skeletal assembloids with superior bone-forming capacity for regenerating challenging bone fractures.