To overcome problems associated with surgical site infection and implant loosening, we developed a titanium (Ti)-based material employing a modified alkaline heat treatment that releases strontium (Sr) and silver (Ag) ions (CaSrAg-Ti). In this study, to determine the optimal Ag treatment concentration, we prepared four different materials—commercially pure Ti (cp-Ti) as a negative control, CaSr1mMAg-Ti, CaSr10mMAg-Ti, and CaSr50mMAg-Ti. Ion release test was performed by immersing the prepared disks in fetal bovine serum. With increased loading of Ag ions, the amount of released ions increased. Colony-forming unit count assay was performed using methicillin-susceptible Staphylococcus aureus and Escherichia coli. High antibacterial activity was observed in CaSr10mMAg-Ti and CaSr50mMAg-Ti groups. In vivo experiments were performed using the rat subcutaneous pocket infection model and evaluated by counting the attached bacteria, wound appearance, and histological evaluation. High antibacterial activity value (AAV >2) and anti-inflammatory effects were observed in the CaSr50mMAg-Ti group. However, CaSr10mMAg-Ti did not exhibit consistent antibacterial activity. For in vivo biocompatibility and bone-bonding ability evaluation, rods were implanted into the rat femur. No cytotoxicity was observed at 1 week, and good bone-bonding ability at 4 and 8 weeks was not significantly different from that of CaSr1mMAg-Ti. To evaluate in vivo bioactivity and cytotoxicity, MC3T3-E1 cells were cultured on disks. CaSr10mMAg-Ti and CaSr50mMAg-Ti significantly inhibited the proliferation and differentiation of MC3T3E1 cells, as well as the production of extracellular matrix in vivo, despite showing good biocompatibility in vivo. In conclusion, CaSr50mMAg-Ti, with increased Ag ion loading, exhibited high antibacterial activity in vivo while maintaining the bone-bonding ability and is a promising therapeutic biomaterial. Further research is needed to determine the optimal combination of therapeutic concentrations of Sr and Ag.
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