3D bioprinting of thermosensitive inks based on gelatin, hyaluronic acid, and fibrinogen: Reproducibility and role of printing parameters

Thermosensitive inks are considered an attractive option for the 3D bioprinting of different tissue types, yet comprehensive information on their reliability, preparation, and properties remains lacking.This paper addresses this gap by presenting a twofold aim: firstly, characterizing the preparation, rheology, and printing aspects of two inks that have demonstrated success in skeletal muscle tissue engineering both in vitro and in vivo. The first ink is composed of fibrinogen, gelatin, hyaluronic acid, and glycerol, while the second is a sacrificial ink made of gelatin, hyaluronic acid, and glycerol. Secondly, from this analysis, we demonstrate how thermosensitive and multicomponent inks can exhibit high variability and unpredictability. Thus, we emphasize the importance of thorough ink characterization to ensure the reproducibility and reliability of scientific outcomes.We quantified the inherent variability in ink manufacturing and we proposed specific quality assessment criteria. We found storing the fibroink at 4 °C for one day did not alter fibroink properties, while significant changes were produced if the storage time was seven days. Cell viability within the fibroink was evaluated at different temperatures, identifying 9 °C as the optimal trade-off between cell viability and printability. Rheological analyses confirmed the shear-thinning behavior of both inks and identified their respective sol-gel transition temperatures. A systematic assessment of printing fidelity was performed, by varying pressure, speed, and needle offset. The methodology proposed in this study may be useful for the management of other thermosensitive bioinks, thus properly considering their inherent variability.