Fig. 1: Digital assembly of spherical particles (DASP) made of modular double-network bio-inks.

a DASP uses spherical bio-ink droplets, not conventional one-dimensional (1D) bio-ink filaments, as the building blocks to create three-dimensional (3D) objects. In DASP printing, (i) droplets are deposited at precisely controlled locations and with prescribed volume in a sacrificial supporting matrix made of an aqueous yield-stress fluid. (ii) Under controlled swelling, the deposited droplets partially coalesce with their neighbors. (iii) Following a complete crosslinking of droplets and the removal of the sacrificial matrix, the printed particles form a free-standing 3D construct made of interconnected yet distinguishable hydrogel particles. b DASP 2.0 enables printing modular double-network (DN) bio-inks. The ink consists of alginate and poly(acrylamide) (PAM), which is functionalized with either norbornene (NB) or tetrazine (TZ) groups. (i) In an aqueous solution, the NB-PAM (blue) and TZ-PAM (red) form a network via click reaction between NB and TZ at RT without any external trigger, while alginate forms a network via ionic crosslinking. (ii) A custom-designed multi-channel print nozzle is developed to enable printing DN bio-inks. The nozzle mixes a pair of bio-inks, TZ-PAM (red arrow)/alginate (green arrow) + NB-PAM (blue arrow)/alginate (green arrow), homogeneously without damaging cells. (iii) In a DN hydrogel, the ionically crosslinked alginate network interpenetrates with the PAM network.