In situ cell-only bioprinting of patterned prevascular tissue into bioprinted high-density stem cell-laden microgel bioinks for vascularized bone tissue regeneration

Recently, microgels have been widely used in three-dimensional (3D) bioprinting as both supporting baths and bioinks. As a bioink, microgels have several unique properties, such as shear-thinning and self-healing behaviors with tunable mechanics, making them useful in 3D bioprinting. While cell encapsulated microgels offer many advantages in 3D bioprinting, they also have some limitations. It is still challenging to produce large quantities of cell encapsulated microgels with consistent quality and properties due to processes that are often complex and time-consuming. In this study, stem cell encapsulated, photocrosslinkable, shear-thinning and self-healing alginate microgel (SSAM) bioinks have been successfully fabricated via simple mixing of an oxidized and methacrylated alginate solution with suspended stem cells and a supersaturated calcium sulfate slurry solution through a custom-made spiral mixing unit. The SSAM bioinks can be bioprinted into complex 3D structures with both high resolution and shape fidelity due to their shear-thinning and self-healing properties. The 3D bioprinted SSAM bioinks can then serve as a supporting bath for the creation of prevascular network patterns using an individual cell-only prevasculogenic bioink within the 3D printed constructs. The prevascular network patterned 3D bioprinted constructs can be further stabilized by secondary photocrosslinking of the SSAMs, which enables long-term culture of the printed constructs for functional vascularized osteogenic tissue formation by differentiation of the bioprinted cells. The SSAM bioinks and individual cell-only printing technique enable in situ bioprinting of prevascularized tissue constructs in a mouse calvarial bone defect, achieving mechanical stability and ensuring the in situ bioprinted constructs remain within the defect.