Impact of steam sterilization on the rheological characteristics, printability, and bioactivity of nanocellulose/alginate hydrogels for 3D bioprinting

• The impact of steam sterilization on the rheology and biological properties of nanocellulose-based hydrogels for bioinks was investigated. • Autoclaving at 121°C for 15 min was identified as the most effective sterilization method. • Increased alginate concentration (1 % to 3 %) improved printing precision and hydrogel performance. • Hydrogels exhibited high biocompatibility and low cytotoxicity, confirming their suitability for biomedical applications. Effective sterilization of bioinks is essential for the clinical translation of 3D bioprinting, though it can compromise the physical and biological properties of natural hydrogels. This study examines the impact of steam sterilization (121 °C, 15 min) on nanocellulose (CNF)/alginate hydrogels with alginate concentrations of 1 %, 1.5 %, and 3 % (w/v), focusing on rheology, printability, and biocompatibility. Hydrogels with 1.5 % and 3 % alginate retained their viscoelastic properties after sterilization, with storage modulus (G′) values above 1000 Pa. Printability analysis showed high structural fidelity, with pore factors of 0.75–0.88 and integrity factors over 0.9. In contrast, the 1 % formulation displayed poor crosslinking and structural instability. Human bone marrow-derived mesenchymal stem cells (hB MMSCs) showed over 90 % viability in all sterilized hydrogels. The 3 % alginate formulation supported uniform cell distribution and scaffold integrity over 14 days, despite slightly reduced cell proliferation. Its superior mechanical strength and dimensional stability make it the most suitable for applications requiring robust and precise scaffolds. These findings highlight the importance of optimizing alginate concentration, particularly at 3 % w/v, to ensure a balance between sterilization resilience, printability, and biocompatibility. This work provides a validated strategy for developing safe, effective, and ready-to-use bioinks for tissue engineering and regenerative medicine.