In vitro and in vivo biocompatibility and osteogenesis of graphene-reinforced nanohydroxyapatite polyamide66 ternary biocomposite as orthopedic implant material

Graphene and its derivatives have been receiving increasing attention regarding their application in bone tissue engineering because of their excellent characteristics, such as a vast specific surface area and excellent mechanical properties. In this study, graphene-reinforced nanohydroxyapatite/polyamide66 (nHA/PA66) bone screws were prepared. The results of scanning electron microscopy observation and X-ray diffraction data showed that both graphene and nHA had good dispersion in the PA66 matrix. In addition, the tensile strength and elastic modulus of the composites were significantly improved by 49.14% and 21.2%, respectively. The murine bone marrow mesenchymal stem cell line C3H10T1/2 exhibited better adhesion and proliferation in graphene reinforced nHA/PA66 composite material compared to the nHA/PA66 composites. The cells developed more pseudopods, with greater cell density and a more distinguishable cytoskeletal structure. These results were confirmed by fluorescent staining and cell viability assays. After C3H10T1/2 cells were cultured in osteogenic differentiation medium for 7 and 14 days, the bone differentiation-related gene expression, alkaline phosphatase, and osteocalcin were significantly increased in the cells cocultured with graphene reinforced nHA/PA66. This result demonstrated the bone-inducing characteristics of this composite material, a finding that was further supported by alizarin red staining results. In addition, graphene reinforced nHA/PA66 bone screws were implanted in canine femoral condyles, and postoperative histology revealed no obvious damage to the liver, spleen, kidneys, brain, or other major organs. The bone tissue around the implant grew well and was directly connected to the implant. The soft tissues showed no obvious inflammatory reaction, which demonstrated the good biocompatibility of the screws. These observations indicate that graphene-reinforced nHA/PA66 composites have great potential for application in bone tissue engineering.