Concentration-Dependent Effects of Carboxymethyl Chitosan-rGO Scaffolds for Oral Bone Regeneration

To address alveolar bone defects and the limitations of conventional grafts, tissue-engineered scaffolds have emerged as a promising alternative. Carboxymethyl chitosan (CMC) is a biocompatible and biodegradable polysaccharide with potential for bone regeneration; however, its brittleness and poor mechanical strength restrict its application. Here, we developed CMC-reduced graphene oxide (rGO) composite scaffolds with rGO concentrations of 0%, 0.5%, 1%, and 2% to overcome these drawbacks. The scaffolds were systematically characterized for their morphological, crystallographic, spectroscopic, and biomechanical properties, as well as their in vitro cytocompatibility and in vivo osteogenic performance. The incorporation of rGO enhanced structural homogeneity, optimized pore architecture, and significantly improved mechanical strength in a concentration-dependent manner, with tensile strength increasing from 1.64 to 8.13 MPa and elastic modulus from 1.14 to 25.05 MPa. In vitro, when MC3T3-E1 cells were grown in osteogenic medium, scaffolds loaded with 0.5%-1% rGO led to better cell survival and higher ALP activity-both pointing to stronger osteogenic differentiation. The 2% rGO scaffolds, however, turned out to be toxic to cells. Structural analyses confirmed the preservation of CMC crystallinity and revealed hydrogen bonding between rGO and CMC, elucidating the reinforcement mechanism. In a rat cranial defect model, the 1% rGO scaffold group demonstrated superior new bone formation, mineralization, and trabecular maturation. These findings underscore the dual function of rGO in simultaneously improving the mechanical integrity and osteogenic capacity of CMC-based scaffolds, with 0.5%-1% rGO identified as the optimal concentration window for bone tissue engineering applications.