Biomaterial implants are a critical component to successful treatment of many types of orthopaedic injuries. In 2004, over 2.6 million orthopaedic devices were placed with 4% developing infections, resulting in an economic burden of nearly $2 billion. In the United States, nearly 112,000 orthopaedic device infections occur annually with Staphylococcus aureus being the most common pathogen accounting for roughly 33% of all infections. Bacterial infection of orthopaedic implants frequently results in complete removal of the implant despite aggressive antibiotic therapy. Lysostaphin is an antimicrobial enzyme specific to staphylococcal species. It is composed of a cell wall targeting domain, responsible for its specificity, and a lytic domain that catalytically degrades the peptidoglycan cell wall of bacteria, causing cell lysis. We have previously engineered a poly (ethylene glycol) (PEG)-based hydrogel system for controlled delivery of therapeutic proteins. We have shown that BMP-2 delivery facilitates bone repair in a murine radial segmental defect model. Contamination of these BMP-2 loaded hydrogels with bacteria leads to complete inhibition of bone healing, persistence of bacteria, and bone resorption. The objective of this project was to engineer PEG-based hydrogels that prevent and treat orthopaedic infections while simultaneously repairing orthopaedic injuries. The central hypothesis was that delivery of lysostaphin using a PEG-hydrogel will reduce infection and promote bone repair using mouse models of orthopaedic device infections.

We engineered injectable hydrogels for the delivery of lysostaphin to infected mouse femoral fractures. Encapsulation of lysostaphin within a PEG hydrogel carrier provided an in situ polymerizable delivery platform that maintained enzyme activity and stability while conforming and adhering to the injured tissue. Lysostaphin-delivering hydrogels were effective at eliminating infection, and provided enhanced anti-biofilm activity compared to soluble lysostaphin in vitro. Lysostaphin-delivering hydrogels eradicated mouse femoral fracture infections and out-performed soluble lysostaphin delivery as well as prophylactic antibiotic administration. Infected fractures treated with lysostaphin-delivering hydrogels healed equally well as uninfected fractures, as measured by µCT analysis and mechanical testing. The local cytokine milieu of infections treated with lysostaphin-delivering hydrogels was no different than uninfected fractures, demonstrating restoration of a normal healing microenvironment. Additionally, lysostaphin-delivering hydrogels were effective at eliminating methicillin resistant S. aureus (MRSA) infections. Taken together these results show that lysostaphin delivery via a hydrogel carrier is effective at treating biomaterial-stabilized femoral fracture infections.

Segmental bone defect injuries have infection rates reported as high as 30% and successful treatment often requires bone grafting, multiple surgeries, and unacceptably high therapeutic failure rates. To address this unmet clinical need, we extended our lysostaphin-delivering hydrogel to co-deliver both lysostaphin and BMP-2 in order to investigate simultaneous elimination of infection, and induction of growth factor-mediated bone repair of non-healing segmental defects. Lysostaphin and BMP-2 co-delivery effectively eliminated bacterial infection and promoted bone regeneration in our mouse radial segmental defect infection model. We characterized the local inflammatory response to infections treated with lysostaphin-delivering hydrogels and showed that the local cytokine profile is no different than uninfected defects at both 1 and 4 weeks postoperation. In addition, no differences between the inflammatory cell profiles were observed for infections treated with lysostaphin-delivering hydrogels one week after implantation. These results demonstrate that lysostaphin and BMP-2 co-delivery can eliminate infection and facilitate segmental bone defect regeneration.

We also investigated the activity of lysostaphin-delivering hydrogels to reduce established S. aureus infections in vivo. We developed a novel model of established S. aureus infection by modification to the mouse radial segmental bone defect model. Infections were first initiated by placing infected hydrogel implants and bacteria were allowed to colonize the injury for one week. Once the infection was established, the infected implants were removed, the wound was debrided and washed, and a lysostaphin-delivering hydrogel was injected at the injury site. Our results indicated that lysostaphin delivery alone reduced biofilm infection but significant bacteria numbers remained. However, a combined effect was seen when both lysostaphin hydrogels and systemic antibiotic therapy were delivered together. These results show that lysostaphin-delivering hydrogels effectively reduce established S. aureus orthopaedic infections.

This research is innovative because it develops bi-functional materials that prevent and treat S. aureus infections while simultaneously repairing orthopaedic injuries. Additionally, lysostaphin provides specifically targeted bacteriolytic activity, acting as an alternative to traditional antibiotic therapy, which may help to reduce the spread of antibiotic-resistant bacteria. As outcomes of this research, we have engineered a lysostaphin delivery vehicle to eradicate long bone fracture infections, prevent infection of non-healing segmental defect injuries while simultaneously regenerating bone, and reduced bacteria using a segmental defect model of established biofilm infection. This research has established a strategy for preventing and treating orthopaedic biomaterials implants that could be extended to other biomedical device infection scenarios.