Optimization of gellan gum-based bioink printability for precision 3D bioprinting in tissue engineering

Three-dimensional (3D) bioprinted gellan gum is emerging as a promising material for tissue engineering applications; however, optimizing the parameters for successful 3D bioprinting remains challenging. In this study, we investigated the effects of three calcium chloride (CaCl₂) concentrations (0.5 % w/v, 1 % w/v, and 2 % w/v), dual cross-linking with 2 % v/v glutaraldehyde, bioink temperature, and cross-linking duration to optimize gellan gum bioinks. The printability of the bioprinted constructs was assessed using key metrics, including area printability, strand printability, angle accuracy, pore size consistency, and precision. The formulation containing 0.5 % w/v CaCl₂ and 2 % v/v glutaraldehyde at 25 °C demonstrated superior printability, achieving a score of 37 out of 50, and exhibited enhanced structural integrity. Fourier-transform infrared spectroscopy confirmed successful cross-linking in this optimized formulation. Contact angle measurement (75.5 ± 0.87°) and swelling capacity (853 ± 88 % after 24 h) demonstrated excellent water retention capabilities. A gel fraction of 81.20 ± 1.47 % indicated stable ionic and covalent bonding, contributing to the bioink's robust architecture. Furthermore, dual cross-linking improved bioink integrity, as shown by cross-linking degree analyses and rheological evaluations. Additionally, the bioink exhibited a favorable degradation profile (86.48 ± 3 % after 14 days) and suitable mechanical properties (Young's modulus of 19 ± 2 kPa). Cellular assays, including cell morphology observations, viability testing, and live/dead staining presented high cell viability, further confirming the bioink's biocompatibility. These findings highlight the potential of pure gellan gum 3D bioprinting using 0.5 % w/v CaCl₂ and 2 % v/v glutaraldehyde, maintained at 25 °C for tissue engineering applications.