No-Observed-Effect Level of Silver Phosphate in Carbonate Apatite Artificial Bone on Initial Bone Regeneration

Fracture-related infections require both treatments for bacteria removal and bone reconstruction. The use of combined broad-spectrum antibacterial silver compounds and artificial bone with high osteogenic activity is considered to be an effective strategy for achieving these treatments in one surgery. However, silver compounds are toxic for living tissues even at low concentrations. Herein, we investigated the no-observed-effect level (NOEL) of silver phosphate (Ag₃PO₄) in a bone substitute composed of carbonate apatite (CO₃Ap), a bone mineral, using in vitro and in vivo experiments. In vitro experiments demonstrated that the CO₃Ap artificial bone containing ≥0.1 wt % Ag₃PO₄ exerted antibacterial effects against Staphylococcus epidermidis, while those containing ≤0.3 wt % Ag₃PO₄ did not affect cellular adhesion, proliferation, differentiation, and calcification of osteoblast-like MC3T3-E1 cells. In vivo experiments demonstrated that the CO₃Ap artificial bone containing ≤0.3 wt % Ag₃PO₄ replaced a new bone to the same levels as those without Ag₃PO₄ 4 weeks after implantation into the bone defect of the rabbit femur condyle. However, the CO₃Ap artificial bone containing 0.3 wt % Ag₃PO₄ caused an inflammatory reaction, whereas those containing ≤0.1 wt % Ag₃PO₄ did not. Thus, both bone regeneration and infection control without any adverse effects were achieved using the CO₃Ap artificial bone containing 0.1 wt % Ag₃PO₄, indicating that the NOEL of Ag₃PO₄ was 0.1 wt %. Our results provide an effective strategy for the treatments of fracture-related infections.