Project Summary Bone fractures and non-union defects often require surgical intervention where devices are used to correct the defect, and 1-5% of these procedures are compromised by bacterial infection with annual hospital costs exceeding $1.9B. Current treatments are limited to sustained, high systemic doses of antibiotics and surgical debridement of affected tissue. These corrective procedures significantly drive healthcare costs and have sub- optimal patient outcomes as effective antibiotic doses are difficult to attain at the infection site due to the presence of a biofilm and toxicity considerations. Furthermore, the emergence of antibiotic-resistant bacteria raises concerns regarding the effectiveness of current antibiotics. The objective of this renewal application is to engineer synthetic hydrogels co-delivering lysostaphin and antibiotics to eliminate established bacterial infections and promote bone repair in murine and ovine models. Our central hypothesis is that hydrogel-based delivery of lysostaphin and antibiotics will synergize to eliminate staphylococcal infections and result in bone healing in murine and ovine models of implant-associated bone infection. This research will establish a localized and translatable strategy to effectively eliminate established bacterial infections to support bone repair. Aim 1. Engineer antimicrobial hydrogels for the treatment of infected bone fractures in murine models of bone repair. Aim 2. Engineer hydrogels co-delivering antimicrobial agents and BMP-2 to eliminate established bacterial infection and repair non-healing segmental bone defects in mice. Aim 3. Evaluate the ability of antimicrobial hydrogels to mitigate bacterial infection and support bone repair in a plate-stabilized osteotomy infection model in sheep. The proposed research is innovative because it focuses on engineering hydrogels that locally deliver antimicrobial agents to eliminate bacterial infection, modulate inflammation, and support bone repair. This novel strategy represents a more effective and safer alternative to current systemic antibiotic regimens and antibiotic- releasing bone cements. This research is expected to yield the following significant outcomes. We will establish an injectable antimicrobial hydrogel for the repair of infected bone fractures and non-healing bone defects. We will analyze the efficacy, safety, and biological mechanisms of these biomaterials in murine and ovine models of implant-associated bone infections.