Freeze-dryable inks combining chitosan nanofibers and hyaluronic acid for extrusion bioprinting

Freeze-dryable inks offer significant advantages for bioprinting, such as long-term biochemical and biophysical stability with minimal risks of hydrolysis, denaturation, and microbial contamination. In this study, we developed inks that can be stored in a ready-to-use, freeze-dried form by incorporating chitosan nanofibers (ChitoNFs), sodium hyaluronic acid (HA-Na), and phenolated HA (HA-Ph). HA-Na and HA-Ph effectively inhibited ChitoNF aggregation during drying. Upon rehydration, the developed inks exhibited shear-thinning behavior and underwent horseradish peroxidase-catalyzed gelation. The optimization of the ChitoNF, HA-Na, and HA-Ph concentrations enabled precise control of the viscosity, gelation time, and hydrogel stiffness after rehydration. Using 0.8 w/v% HA-Na, 0.2 w/v% HA-Ph, and 1.5 w/v% ChitoNF, we achieved high-fidelity three-dimensional printing of constructs. Furthermore, the incorporation of 0.5 w/v% phenolated gelatin supported the encapsulation of 10 T1/2 fibroblasts, resulting in more than 90 % viability and a 3.1-fold increase in mitochondrial activity over 7 d. Our findings revealed that pre-mixed, freeze-dryable ChitoNF-HA-Na/HA-Ph bioinks offer a practical and innovative solution for bioprinting. By ensuring long-term storability, quantitative retention of print fidelity and cytocompatibility after rehydration, and simplified logistics for clinical translation, they address unmet needs in current bioink technologies.