Abstract
This study presents the development of nanocomposite hydrogels that integrate functionalized reduced graphene oxide (rGO) derivatives carboxylated (CBX) and aminated (AMN) in combination with gellan gum, gelatin, and cellulose nanofibrils (CNFs) to enhance osteogenesis for bone tissue engineering. Their synthesis involves ultrasound exfoliation, genipin crosslinking, and freeze-drying. Micro-computed tomography (µCT) reveals highly porous, interconnected architectures facilitating nutrient diffusion and cell infiltration. CBX/AMN scaffolds exhibit superior hydration capacity and a biphasic degradation profile. Spectroscopically, enhanced interfacial interactions were confirmed, contributing to the overall structural stability of the hydrogels. In vitro, all scaffolds are cytocompatible, sustain cell viability, and promote osteogenic differentiation consistent with adaptive cellmaterial interactions. Subcutaneous implantation in mice confirms scaffold biocompatibility, showing no signs of inflammation or foreign body response. Histological and immunofluorescence analyses reveal osteogenic potential, with enhanced mineralization and elevated expression of osteopontin and osteocalcin. µCT imaging of explants quantifies ectopic mineralization, spatial deposition patterns, and bone mineral density, correlating scaffold architecture and composition with osteogenic performance. These findings suggest that the synergistic integration of functionalized rGO, CNFs, and biopolymers results in scaffolds with enhanced structural and biological properties, highlighting their potential for design optimization and clinical translation in bone regenerative medicine.