Longitudinal Intravital Imaging of the Bone Marrow (LIMB) for Analysis of the Race for the Surface in a Murine Osteomyelitis Model.

Critical knowledge gaps of orthopaedic infections pertain to bacterial colonization. The established dogma termed the Race for the Surface posits that contaminating bacteria compete with host cells for the implant post-op, which remains unproven without real time in vivo evidence. Thus, we modified the murine longitudinal intravital imaging of the bone marrow (LIMB) system to allow real time quantification of GFP + host cells and ECFP + or RFP + methicillin-resistant Staphylococcus aureus (MRSA) proximal to a transfemoral implant. Following inoculation with ~10 5 CFU, an L-shaped metal implant was press-fit through the lateral cortex at a 90° angle ~0.150mm below a gradient refractive index (GRIN) lens. We empirically derived a volume of interest (VOI) = 0.0161 ± 0.000675 mm 3 during each imaging session by aggregating the Z-stacks between the first (superior) and last (inferior) in-focus LIMB slice. LIMB post-implantation revealed very limited bacteria detection at 1hr, but by 3hrs 56.8% of the implant surface was covered by ECFP+ bacteria and the rest covered by GFP + host cells. 3D volumetric rendering of the GFP + and ECFP + or RFP + voxels demonstrated exponential MRSA growth between 3-6hrs in the Z plane, which was validated with cross-sectional ex vivo bacterial burden analyses demonstrating significant growth by ~2 x 10 4 CFU/hr on the implant from 2 to 12hrs post-op (p<0.05; r 2 >0.98). Collectively, these results show the competition at the surface is completed by 3hrs in this model and demonstrate the potential of LIMB to elucidate mechanisms of bacterial colonization, the host immune response, and efficacy of antimicrobials. This article is protected by copyright. All rights reserved.