Abstract Implant-associated infections are the bane of musculoskeletal tissue engineering. With over 1.5 million total hip and total knee replacement procedures performed each year, bone infection primarily caused by Staphylococcus aureus remains among the most severe and devastating risks associated with musculoskeletal implants. It has been understood for decades that the addition of a foreign material to a biological environment provides a haven for bacterial attachment, colonization and recalcitrant biofilm formation. Based on this dogma, the concept of the “race for the surface” has been established to explains the competition between host cells and bacteria for implant colonization. To bias this competition in favor of the host, various antimicrobial biomaterials, surface coatings, drugs and immunotherapies have been tested. While many have shown promise based on in vitro findings and preliminary results in animal models, none have proven efficacy in clinical trials. While there are several explanations for the lack of clinical translation, a broadly accepted shortcoming has been the over reliance on assays (e.g. static biofilm, colony formation units (CFU), minimum inhibitory concentration (MIC)), and cross-sectional outcomes (e.g. static radiology and microscopy), which cannot faithfully assess the in vivo infection process. Thus, the Scientific Premise of this program is that real time in vivo quantification of planktonic bacterial growth on the surface of musculoskeletal implants, and the innate host response to these bacteria, is critical for the evaluation of novel prophylactic and therapeutic interventions that significantly inhibit colonization and biofilm formation. To this end, we have pioneered the use of intravital multiphoton laser scanning microscopy (IV-MLSM) with a murine model of implant-associated osteomyelitis. Our preliminary studies quantifying the proliferation and surface coverage of red fluorescent S. aureus, versus surface coverage of green fluorescent host cells on a metal implant within the femur demonstrate that the race for the surface is very dynamic and complete within 3hrs. In Aim 1 of this program, we will confirm these findings, and formally establish the real time kinetics of the race of the surface on standard of care stainless steel implants, and the efficacy of standard of care parenteral antibiotics against methicillin sensitive and resistant strains of S. aureus. We will also assess cerulean S. aureus in Ly6GCre/ROSAtdTomato/Csf1r-EGFP mice to quantify implant surface colonization, and clearance of bacteria (blue) by neutrophils (orange) and macrophages (green) in vivo. In Aim 2, we will test the hypothesis that the efficacy of previously described antimicrobial implants (“as fired” silicon nitride (Si3N4) and 3D-printed titanium) is due to their ability to favor host cells over bacterial colonization during the race for the surface. At the completion of this high risk-high reward program, we will have new in vivo outcome metrics to elucidate the molecular and cellular mechanisms that govern the race for the surface, and empirical threshold values to assess the efficacy of antimicrobial interventions for musculoskeletal tissue engineering.

Narrative Bone infection, primarily caused by Staphylococcus aureus, remains among the most severe and devastating risks associated with musculoskeletal tissue engineering. To facilitate development of antimicrobial implants and treatments that inhibit these bone infections, we propose to establish a mouse model in which this infection process can be imaged in real time with in vivo fluorescent microscopy.