Bacterial Cellulose-Containing Alginate Inks: A Proof-of-Concept Study on Acellular 3D Printing Feasibility and Cytocompatibility

Abstract

The development of hydrogel bioinks that combine structural stability with biological compatibility remains a major challenge in extrusion-based 3D printing for tissue engineering. In this proof-of-concept study, bacterial cellulose (BC) obtained from kombucha fermentation was explored as a sustainable nanofibrillar component for alginate/chondroitin sulfate (CS)/silicon-substituted hydroxyapatite (Si-HA) composite inks. Following alkaline purification, mechanical processing, and freeze-drying, BC was characterized by scanning electron microscopy (SEM), ATR-FTIR spectroscopy, and X-ray diffraction (XRD), revealing a highly entangled nanofibrillar architecture with high crystallinity (85.4%) and strong hydrogen-bonding potential. Four hydrogel formulations were developed as a comparative 2 × 2 matrix, contrasting BC-containing systems with methylcellulose (MC)-containing reference systems at two Si-HA loadings. Reduced-viscosity measurements of the uncrosslinked precursor formulations showed higher values at the lower Si-HA loading in both formulation series. All formulations could be extruded as acellular inks into grid-like constructs and retained identifiable macroporous architectures after ionic crosslinking. Swelling increased between 24 and 48 h, while mass loss remained limited after the initial 24 h incubation period. In direct-contact testing with L929 fibroblasts, cell viability remained above 84% after 48 h, meeting the ISO 10993-5 non-cytotoxicity criterion. These findings support the feasibility of incorporating physically processed kombucha-derived BC into alginate-based composite inks.