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Traditional therapy of staphylococcal osteomyelitis is ineffective in producing complete sterilization of infected bones due to the formation of the Staphylococcus aureus biofilms. The aim of this study was to develop a new drug-delivery system of antibiotics for treatment of chronic experimental osteomyelitis.In the current work, cationic liposomal gentamicin was prepared and impregnated in calcium sulfate (CS), and tested for anti-biofilm activities in vitro and in vivo.The combination of liposomal gentamicin and CS showed initial burst-release of active liposomal gentamicin and had continuous-release (12 days). Liposomal gentamicin released from CS had the same anti-biofilm activity with the liposomal gentamicin prepared freshly. Meanwhile, both agents were more effective relative to free gentamicin at low drug concentration. Therapeutic trials with antibiotics given intravenously revealed that free gentamicin for 14 days was ineffective in sterilizing bone. Treatment with liposomal gentamicin for 14 days resulted in recovery of 33.3% of treated animals, which was the lower slightly than the result treated with implantation of gentamicin-impregnated CS (66.7%). Complete sterilization of bone tissues on cultures (100% cure) was obtained only in the group of liposomal gentamicin-impregnated CS treated for 14 days. The new drug-delivery system was effective in preventing biofilm infection in a contaminated defect, and it could also be used clinically for bacterial infections in the conditions like plaque formation or in arresting biofilm formation in the implanted devices or dead bone of osteomyelitis.