Scalable 4D biofabrication process for the manufacture of scaffold-free bone-forming callus implants

Critical-size long bone defects remain a major clinical challenge, with treatments such as autografts or distraction osteogenesis causing donor-site morbidity, infection, or failure to restore complex bone architecture. Tissue-engineered implants that recapitulate native fracture healing provide a promising solution. However, scalability for dense cellular constructs is lacking. To address this, we bioprinted high-cell-density implants using rheologically competent sacrificial alginate bioinks. The constructs were supported by partially crosslinked alginate during bioprinting and chondrogenic differentiation, after which selective EDTA-mediated alginate dissolution generated scaffold-free implants. Quality characterization confirmed chondro-osteogenic signatures and extracellular matrix gene upregulation. Upon in vivo implantation in immunocompromised mice, implants underwent endochondral ossification, forming cortical and trabecular bone with bone marrow compartments. Integration with a suspension bioreactor enabled production of human-sized proof-of-concept implants. This work establishes a scalable 4D biofabrication process that integrates 3D bioprinting with dissolvable sacrificial alginate bioinks and results in scaffold-free, bone-forming callus implants.