In siturheological monitoring of diffusion-controlled hydrogel crosslinking for embedded 3D bioprinting

In embedded 3D bioprinting, biomaterial inks are extruded into sacrificial support baths to facilitate the fabrication of complex shapes, even from soft, liquid-like materials. Post-printing, the diffusion of small molecules into or out of the support bath can facilitate ink crosslinking to stabilize the printed structure. In these coupled reaction-diffusion systems, the rheological properties of the ink will change over time. Despite the importance of tuning the mechanical properties of these inks for biological applications, there are currently no methods to accurately predict ink stiffness over time throughout the crosslinking process. Here, we use a custom-developed magnetic stress rheometer to continuously monitor diffusion-driven crosslinkingin situ. Our approach reveals how gelation kinetics depend on the thickness of the ink layer, and enables predictive estimation of mechanical evolution in these reaction- and diffusion-driven systems. With these insights, we fabricate specimens with predetermined mechanical properties and observe changes in cell phenotype as a response. These insights help inform the design of inks and timing of bioprinting protocols to achieve prints with desirable mechanical properties and further allow the fabrication of prints with patterned mechanical properties.