Inkjet Bioprinting of 3D Silk Fibroin Cellular Constructs Using Sacrificial Alginate

Silk fibroin is a natural protein which has shown great promise for tissue engineering but is not printable due to slow gelation or harsh gelation conditions which are not cell-friendly. In this study, a two-step gelation process is proposed for the printing of silk fibroin, which utilizes alginate as a sacrificial hydrogel during an inkjetting-based process. A cell-laden blend of alginate with silk fibroin is utilized to achieve rapid gelation by calcium alginate formation during printing; it is followed by horseradish peroxidase (HRP) catalyzed covalent cross-linking of the fibroin protein at tyrosine residues after printing. This two-step gelation process successfully enables 3D bioprinting of well-defined cell-laden silk fibroin constructs suitable for long-term culture. The constructs remain intact after calcium chelation to liquefy the alginate component, demonstrating the formation of silk fibroin hydrogel. NIH 3T3 fibroblasts proliferate and spread through the hydrogel after printing. Increasing metabolic activity is observed for 5 weeks after printing, and histology shows dense cell populations in cultured constructs. The proposed two-step gelation technique is expected to enable 3D silk fibroin printing for various applications.