In situ bioprinting of patterned prevascular tissue using cell-only bioinks and high-density stem cell-laden alginate microgel bioinks for vascularized bone tissue regeneration.

Microgels are widely used in three-dimensional (3D) bioprinting as supporting baths and bioinks due to their shear-thinning and self-healing behaviors with tunable mechanics. However, producing large quantities of cell-encapsulated microgels remains challenging due to complex, time-consuming processes. This study presents a scalable method for fabricating stem cell encapsulated, photocrosslinkable, shear-thinning, self-healing alginate microgel (SSAM) bioinks using a custom-made spiral mixing unit. Our approach enables efficient, reproducible fabrication of sterile SSAM bioinks, addressing key bioink formation challenges. To develop clinically relevant tissue constructs, establishing microvascular networks is essential. The 3D bioprinted SSAM bioinks with high-resolution and shape fidelity serve as a supporting bath for prevascular network patterning using an individual cell-only prevasculogenic bioink, enabling vascular condensation formation via secondary photocrosslinking of the SSAMs. This SSAM bioink bath facilitates vascular condensation formation while also acting as the parenchymal tissue component of the engineered construct, enhancing vascularized osteogenic tissue formation. Compared to non-patterned prevascularization, 3D printed prevascular patterning generates complex prevascular networks, potentially improving tissue anastomosis and integration. This bioprinting platform enables in situ bioprinting of mechanically stable prevascularized tissue constructs in a mouse cadaver calvarial bone defect. This work advances bioprinting and regenerative therapies by providing a scalable method for fabricating functional, vascularized tissues. STATEMENT OF SIGNIFICANCE: Although shear-thinning and self-healing microgels are attractive bioinks, scalable sterile production of cell-laden microgels with consistent quality remains challenging due to complex and time-intensive fabrication processes. Here, we present a robust and scalable one-step fabrication strategy using a custom spiral mixing unit to fabricate stem cell-laden alginate microgel bioinks. The cell-laden microgels exhibit tunable rheological properties that enable high resolution printing bioink while providing mechanical support for embedded bioprinting. This enables direct patterning of cell-only vasculogenic bioinks to spatially organize vasculogenic and osteogenic cells, enhancing prevascular network formation and osteogenic differentiation compared to non-patterned controls. Moreover, this platform supports in situ bioprinting in a mouse calvarial defect model, demonstrating its potential for clinically translatable vascularized bone tissue engineering.