Molecular mechanisms of osteogenesis and antibacterial activity of Cu-bearing Ti alloy in a bone defect model with infection in vivo

The antibacterial activity of copper (Cu)-alloy biomaterials has shown a great potential in clinical application. Here, we evaluated the osteogenesis and antibacterial effects of Ti6Al4V-6.5wt%Cu alloy in an in vivo model of infected bone defects and determine their responsible proteins and pathways using proteomics.After bone defects were filled with Ti6Al4V and Ti6Al4V-6.5wt%Cu implants for 6 week, the tissue and bone samples around the implants were harvested for radiographic, micro-CT, histological, and bone-related gene expression analyses. An iTRAQ-based protein identification/quantification approach was used to analyze the osteogenic and antibacterial effects of Ti6Al4V-6.5wt%Cu alloy.Imaging and histological results showed Ti6Al4V alloy induced a stronger inflammatory response than Ti6Al4V-6.5wt%Cu alloy; imaging results and osteogenic protein levels showed Ti6Al4V-6.5wt%Cu alloy exerted a stronger osteogenic effect. In vitro experiment, we found the Ti6Al4V-6.5wt%Cu had significant antibacterial effects and inhibited the activity of Staphylococcus aureus in the early stage. In addition, the bacterial biofilm formed in Ti6Al4V-6.5wt%Cu group was significantly lower than that in Ti6Al4V group. Proteomic screening of 4279 proteins resulted in 35 differentially expressed proteins for further examination which were mainly associated with the cellular process, metabolic process, stimulus response, and cellular component organization. In further exploration of the mechanism of osteogenic mineralization of Ti6Al4V-6.5wt%Cu alloy, we found out SDC4 and AGRN were the top two target proteins associated with osteogenic differentiation and bone mineralization.Ti6Al4V-6.5wt%Cu alloy shows a great potential as a bone implant material due to its positive effects against bacterial infection and on bone formation.At present, titanium alloys and other non-antibacterial metal materials are used in orthopedic internal fixation operations. Our study demonstrates that Ti6Al4V-6.5wt%Cu alloy has good antibacterial and osteogenic effects in vivo and in vitro. This means that Ti6Al4V-6.5wt%Cu alloy may become a new kind of antimicrobial metallic material as internal fixation material to continuously exert its antimicrobial effects and reduce the infection rate after clinical internal fixation.