3D printing of multicomponent polymeric inks enables hierarchical hybrid architectures for bone tissue regeneration

Bone defects arising due to trauma, disease, or surgical resections continue to constitute serious clinical challenges, demanding more advanced and customizable solutions that not only ensure structural stability, but also replicate the anisotropy and biomimetic complexity of the native bone tissue. In this context, we present a solution that is based on 3D printed scaffolds made of gellan gum (GG) and cellulose nanofibrils (CNFs), reinforced with electrospun chitosan/polyethylene oxide nanofibers (NFs). First, we optimized the base ink by selecting the proper GG/CNFs ratio as 3/1 after evaluating its rheological properties, printability, and scaffold morphology. Next, various concentrations of NFs (0.5, 1, 2, and 4 wt%) were added to the base ink, and the resulting formulations were again evaluated in terms of rheology and printability, while the subsequent printed scaffolds were investigated by scanning electron microscopy and micro-computed tomography, showing a well-organized internal architecture with interconnected pores and improved volumetric fidelity, especially for 2 % NFs addition (GCN-2.0), which recorded the highest total porosity (90.7 %). Structural characterization confirmed the successful integration of NFs within the polymeric matrix, while nanomechanical indentation revealed an increase in storage modulus (72.5 kPa for GCN-2.0) compared to the control (47.5 kPa) and also mechanical anisotropy due to filament orientation and NFs alignment within the printed structures. In vitro swelling and degradation studies showed the highest increase in water uptake capacity and long-term stability (up to 12 weeks) for GCN-2.0, while biological evaluation demonstrated good cytocompatibility with MG-63 cells for all samples and increased mineralization in the scaffolds with higher NFs content. Overall, the results indicate that 2 % NFs reinforcement constitutes the ideal balance between printability, nanomechanical properties, and biological response, indicating a great potential for this hybrid formulation to be used successfully in bone tissue engineering. • Developed gellan-gum/cellulose nanofibrils scaffolds reinforced with electrospun chitosan/polyethylene oxide nanofibers. • Nanofibers addition tailored rheology, print fidelity, and pore interconnectivity. • Achieved tunable swelling and degradation via nanofibers concentration control. • Morpho-structural characterization revealed optimal properties for scaffolds with 2 % nanofibers reinforcement. • 3D printed scaffolds enhanced cell adhesion and proliferation for bone repair.