Initial safe(r)-by-design assessment of printable konjac-agar-pectin hydrogels for future extrusion-based bioprinting applications.

INTRODUCTION: Natural polysaccharide-based hydrogels are promising materials for extrusion-based 3D bioprinting, although achieving an adequate balance between cellular response, extrudability and shape fidelity remains challenging. METHODS: In this study, hydrogel formulations based on konjac glucomannan, agar and citrus pectin were developed and evaluated under an initial safe(r)-by-design approach for future extrusion-based bioprinting applications. The materials were combined at different concentrations to assess their rheological behaviour, direct-contact cellular response and printability-related performance. RESULTS: Konjac glucomannan contributed to water retention and viscosity modulation, agar provided thermal gelation and structural reinforcement, and citrus pectin modified flow behaviour and polymer-network interactions. Rheological analysis showed shear-thinning flow for all formulations, while the four selected formulations showed G' values higher than G″ throughout the 0.1-20 Hz frequency range and re-established G' > G″ after large-amplitude deformation. Increasing agar concentration generally increased stiffness and extrusion resistance, whereas pectin reduced resistance to flow and was associated with lower mean relative pore-area values in the analysed grids. The direct-contact MTS assay was interpreted as a measure of apparent metabolic activity, rather than as an absolute quantification of viable cell number, due to the possible influence of hydrogel-assay interactions. Printability-related assays showed that the pectin-free formulations, particularly 2K0.1A and 2K0.2A, exhibited higher mean relative pore-area values in the analysed grids, whereas the pectin-containing formulations showed lower extrusion resistance and improved flowability but lower mean relative pore-area values. DISCUSSION: Overall, 2K0.1A showed the most balanced behaviour among the evaluated formulations when considering extrusion resistance, collapse performance, relative self-supporting index and the descriptive pore-area results. These results support the potential of konjac-agar-pectin hydrogels as printable natural-material platforms, while highlighting the need for further optimisation and validation under cell-laden and tissue-specific conditions.