Abstract
Abstract Extrusion‐based 3D bioprinting is hampered by the inability to print materials of low‐viscosity. In this study, a single initiating system based on ruthenium (Ru) and sodium persulfate (SPS) is utilized for a sequential dual‐step crosslinking approach: 1) primary (partial) crosslinking in absence of light to alter the bioink's rheological profile for print fidelity, and 2) subsequent secondary post‐printing crosslinking for shape maintenance. Allyl‐functionalized gelatin (Gel‐AGE) is used as a bioink, allowing thiol‐ene click reaction between allyl moieties and thiolated crosslinkers. A systematic investigation of primary crosslinking reveals that a thiol‐persulfate redox reaction facilitates thiol‐ene crosslinking, mediating an increase in bioink viscosity that is controllable by tailoring the Ru/SPS, crosslinker, and/or Gel‐AGE concentrations. Thereafter, subsequent photoinitiated secondary crosslinking then facilitates maximum conversion of thiol‐ene bonds between AGE and thiol groups. The dual‐step crosslinking method is applicable to a wide biofabrication window (3–10 wt% Gel‐AGE) and is demonstrated to allow printing of low‐density (3 wt%) Gel‐AGE, normally exhibiting low viscosity (4 mPa s), with high shape fidelity and high cell viability (>80%) over 7 days of culture. The presented approach can therefore be used as a one‐pot system for printing low‐viscous bioinks without the need for multiple initiating systems, viscosity enhancers, or complex chemical modifications.