Surface-Decorated Graphene Oxide Sheets with Copper Nanoderivatives for Bone Regeneration: An In Vitro and In Vivo Study Regarding Molecular Mechanisms, Osteogenesis, and Anti-infection Potential

It has been previously reported that graphene oxide/copper nanoderivative (GO/Cu)-incorporated chitosan/hyaluronic acid scaffolds might be promising wound dressings for the management of infected wound healing. The aim of the present research is to deeply explore the potential antimicrobial mechanisms and synergistic osteogenic activity, as well as the in vivo anti-infective behavior of GO/Cu nanocomposites, making them possible candidates for establishing implantable biomaterials for the repair of infected bone defects. The antibacterial mechanisms of the nanocomposites were explored through the examination of membrane integrity, oxidative stress, and metabolic enzyme activities. Then, the cytocompatibility with bone mesenchymal stem cells (rBMSCs) and osteogenic potential were confirmed, and a subcutaneous bacterial infection model in rats was also established to verify the in vivo anti-infective property and biosafety of the nanocomposites. It was found that leakage of adenosine triphosphate, proteins, and reducing sugars from the bacterial cells, indicative of damaged permeability of bacterial membranes, and promoted production of reactive oxygen species and disordered metabolic enzyme activities in response to oxidative stress were possible molecular mechanisms responsible for the synergistic antibacterial effects of the GO/Cu nanocomposites. Additionally, good cytocompatibility with rBMSCs and promoted osteogenic differentiation were found in GO/Cu nanocomposites (mass ratio = 2:1), which also demonstrated satisfactory in vivo anti-infective performance, reduced inflammation, and acceptable biosafety. Based on our results, damaged bacterial membranes, increased ROS production, and disorders of crucial enzyme metabolism were the main antibacterial mechanisms involved in the bacterium-killing events caused by the GO/Cu nanocomposites, which also showed enhanced osteogenic activity, in vivo anti-infective capability, and acceptable cytocompatibility and biosafety. Therefore, GO/Cu (2:1) nanocomposites are a potential strategy for improving the biological performance of current bone substitutes used for combating bacterial-contaminated bone defects.