Bioprinting of Complex Skeletal Implants

Spheroid-based regenerative constructs offer promising solutions for restoring tissue function in compromised biological conditions, such as complex bone defects. Innovative strategies and methodologies employing bioprinting have been explored to develop complex skeletal implants that mimic the cascade of events in bone fracture healing. By leveraging progenitor cells as the driving force for tissue formation and utilizing biocompatible biomaterials to provide essential 3D cues, we aim to create spatially organized tissue constructs that facilitate bone regeneration. Our research focused on the development of in vitro engineered fracture calluses using bioprinting techniques. These constructs were based on 3D primed human periosteum-derived cells (hPDCs) and biocompatible thiol-ene alginate hydrogels, which mimic the cells and extracellular matrix present in different zones of the callus. In vitro studies confirm cell viability and maintained osteochondrogenic differentiation upon bioprinting, while in vivo assessments demonstrate that the biomaterials provide essential 3D cues guiding the cells in their tissue-forming processes without additional stimulatory molecules. Furthermore, the potential of spheroids or microtissues as building blocks for bioprinted skeletal implants has been explored. Our investigations reveal significant morphological changes, high viability, and osteogenic differentiation of spheroids formed from hPDCs and bone marrow-derived mesenchymal stromal cells (hBMSCs) in hyaluronic acid methacrylate, gelatin methacrylate and combination of these biomaterials. Notably, hPDC spheroids exhibit higher mineralization capabilities and superior hydrogel colonization compared to hBMSC spheroids, underscoring their potential for producing mineralized bone grafts. Additionally, the incorporation of pre-vascularized spheroids represents a compelling approach for enhancing the osteogenic and angiogenic potential of bioprinted constructs. Our studies on co-cultures of stem cells and human umbilical vein endothelial cells demonstrate stable spheroid formation, with endothelial cells arranged in capillary-like monolayers surrounding the spheroids. In conclusion, insights into the optimization of bioprinting parameters, co-culture ratios, and media compositions to enhance the stability, viability, and bone formation potential of bioprinted skeletal implants will be provided. The findings emphasize the importance of biomimetic approaches and advanced bioprinting methodologies in fabricating living regenerative implants, offering promising solutions for patients with critical bone defects and other orthopedic conditions.