Enhancing Cellular Interactions Through Bioactivation and Local Nanomechanical Reinforcement in Nanodiamond-Loaded 3D-Printed Gellan Gum Scaffolds

The integration of nanomaterials within hydrogel scaffolds offers significant promise in bone tissue engineering by improving mechanical performance and modulating cellular responses through mechanotransductive and biochemical signaling. Previous studies have demonstrated that nanodiamonds (NDs) incorporated in electrospun microfibrillar meshes enhance cellular adhesion, spreading, and cytoskeletal organization through localized mechanical reinforcement. However, the effects of ND loading into soft, bioinert three-dimensional hydrogel matrices remain underexplored. Here, we developed nanostructured 3D printing inks composed of gellan gum (GG) supplemented with a low content of ND nanoadditive (0-3% <i>w</i>/<i>v</i>). ND integration improved the shear-thinning properties of the formulation, enabling consistent filament formation and reliable extrusion-based 3D printing. Structural and mechanical assessments confirmed enhanced scaffold morphology, reduced deformation, and improved morphostructural integrity under compression and increased local stiffness at 2% ND loading (GG_ND2%). Biological assessments revealed that increasing ND content enhanced murine preosteoblast viability, proliferation, and attachment, particularly in GG_ND2%. Furthermore, bioactivation of the GG_ND2% formulation with icariin (ICA), a bioflavonoid known for its osteogenic and angiogenic activity, amplified the beneficial cellular responses of MG-63 cells to ND loading, promoting enhanced surface mineralization and improved cell-matrix interactions. Collectively, these findings highlight the potential of ND-reinforced GG scaffolds bioactivated with ICA, integrating structural reinforcement and biological functionalities that may support osteogenic responses.