Two-step crosslinking to enhance the printability of methacrylated gellan gum biomaterial ink for extrusion-based 3D bioprinting

Photocrosslinkable bioinks have gained interest in 3D bioprinting due to their versatility and ease of use.
However, a specific functional group, such as methacrylate or photo-click chemistry, is needed in the polymer backbone to enable photocrosslinking. Methacrylated gellan gum (GGMA) precursor has been proven to possess
good rheological properties for an injectable hydrogel due to its inherent viscosity. It can also be photo-crosslinked in situ at the target site. Unfortunately, the GGMA precursors alone are unable to maintain a stable
filament shape after extrusion from the nozzle. In this study, a two-step crosslinking technique involving ionic and photocrosslinking was used to make the GGMA biomaterial ink printable. In the presence of an ionic crosslinker (Ca2+), GGMA transformed from a liquid precursor to a weak extrudable hydrogel followed by photocrosslinking turning the weak hydrogel into true hydrogel with good shape fidelity. The printability of various GGMA ink compositions was prescreened thoroughly by characterising their fibre formation and rheo-logical properties. A quantitative approach was introduced to quantify the experimental printability of different GGMA/CaCl2 ink compositions from the printed two-layered grid structures. According to the results, 2% GGMA with 90 mM calcium chloride provided a formulation with the best printability. The optimum ink formulation was then used to print 3D structures. This optimised GGMA ink was printed with consistent fibres and provided high printability during the fabrication. The 3D printed structures still lacked high resolution compared to the control structures. In conclusion, the two-step crosslinking technique provided biomaterial ink with good printability and enabled the printing of genuine 3D constructs. Hence, pre-crosslinked GGMA may be applicable for a wide range of bioprinting applications.