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

Abstract

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. 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. 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. 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.