3D bioprinting of self-strengthening living materials using cellulose nanofiber-producing bacteria in sodium alginate hydrogel

Three-dimensional (3D) bioprinting has emerged as a powerful tool for fabricating engineered living materials (ELMs). Despite recent advances in controlling the spatial distribution of bacteria in hydrogel, printing bacteria-laden hydrogels into bulk 3D structures remains a significant challenge. This study presents a partial crosslinking bioprinting strategy for fabricating bacterial cellulose (BC)-based living scaffolds using sodium alginate (SA) hydrogels embedded with Komagataeibacter xylinus. Pre-crosslinked SA was first printed to define the scaffold outline, followed by infilling with uncrosslinked, bacteria-laden SA bioink to enable in situ BC nanofiber production. As BC nanofibers formed within the hydrogel, the scaffolds exhibited self-strengthening and self-hardening property. The effects of SA concentration and culture duration on cellulose yield, rheological properties, printability, and mechanical performance were systematically evaluated. Based on the quantitative relationship between hydrogel formulation, bacterial activity, and scaffold functionality, we optimized the bioinks to enable both high-resolution printing and efficient cellulose formation. This microbial bioprinting technique provides a robust platform for constructing functional BC-based ELMs with potential applications in biomedicine and tissue engineering.