Stepwise Control of Crosslinking in a One-Pot System for Bioprinting of Low-Density Bioinks

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.