Quantitative mouse model of implant-associated osteomyelitis and the kinetics of microbial growth, osteolysis, and humoral immunity

Although osteomyelitis (OM) remains a serious problem in orthopedics, progress has been limited by the absence of an in vivo model that can quantify the bacterial load, metabolic activity of the bacteria over time, immunity, and osteolysis. To overcome these obstacles, we developed a murine model of implant-associated OM in which a stainless steel pin is coated with Staphylococcus aureus and implanted transcortically through the tibial metaphysis. X-ray and micro-CT demonstrated concomitant osteolysis and reactive bone formation, which was evident by day 7. Histology confirmed all the hallmarks of implant-associated OM, namely: osteolysis, sequestrum formation, and involucrum of Gram-positive bacteria inside a biofilm within necrotic bone. Serology revealed that mice mount a protective humoral response that commences with an IgM response after 1 week, and converts to a specific IgG2b response against specific S. aureus proteins by day 11 postinfection. Real-time quantitative PCR (RTQ-PCR) for the S. aureus specific nuc gene determined that the peak bacterial load occurs 11 days postinfection. This coincidence of decreasing bacterial load with the generation of specific antibodies is suggestive of protective humoral immunity. Longitudinal in vivo bioluminescent imaging (BLI) of luxA-E transformed S. aureus (Xen29) combined with nuc RTQ-PCR demonstrated the exponential growth phase of the bacteria immediately following infection that peaks on day 4, and is followed by the biofilm growth phase at a significantly lower metabolic rate (p <0.05). Collectively, these studies demonstrate the first quantitative model of implant-associated OM that defines the kinetics of microbial growth, osteolysis, and humoral immunity following infection.