Following euthanasia on day 14, the explanted plate and screws are processed for CFU analysis, and the infected femur is usually harvested for mCT and histology to assess osteolysis, osteogenesis, and abscess formation

Following euthanasia on day 14, the explanted plate and screws are processed for CFU analysis, and the infected femur is usually harvested for mCT and histology to assess osteolysis, osteogenesis, and abscess formation. 1-stage revision plate is fixed with the outer screw holes, which provided adequate fixation out to day 14 post-infection in all untreated mice that we analyzed. Additionally, as untreated-infected mice display all of the salient features of chronic implant associated osteomyelitis (e.g. biofilm around the implants, biofilm in necrotic bone, Brodies abscesses and bacterial colonization of the osteocytic-canalicular network), we find this terminal time point to be ideal for assessment of intervention for reinfection following 1-stage revision surgery. NIHMS1031251-supplement-Supp_figS1.tif (1.6M) GUID:?0EEC95F4-D27D-47D1-B597-8797CAA7D8C8 Supp figS2: Supplemental Figure 2. Malpositioning of the femoral plate prospects to osteolysis at the stress riser. Post-operative X-rays obtained immediately after the primary surgery (Day 0) reveal femoral plating that was too proximal (A) or too distal (B) for the model, as evidenced by osteolysis (blue arrowheads) on Day 14 (Day 7 post-revision surgery) due to stress from the end of the plate. NIHMS1031251-supplement-Supp_figS2.tif (1.3M) GUID:?9FA4D286-875F-4B5A-9D97-003E78357ADC Supp figS3: Supplemental Physique 3. Micro-CT quantification of osteolysis and osteogenesis adjacent to the screws. Plain X-rays obtain after the main (A) and revision (B) femoral plate surgeries are shown to illustrate the numbering of the screw holes (#1, #2, Warangalone Warangalone #3, and #4) from your proximal to distal femora. At post-surgery day 14 (day 7 post revision), the plated femurs were disarticulated, all implants and soft tissue were removed cautiously, and the samples were subjected to micro-CT scanning at 10.5 micron resolution (VivaCT 40; Scanco Medical AG, Basserdorf, Switzerland). To quantify osteolysis and osteogenesis in the screw holes, the micro-CT images in DICOM format were obtained for volumetric osteolysis assay. Despite great efforts to eliminate obvious bone remodeling round the plate (reddish arrow in C), these artifacts of osteolysis and osteogenesis could not be resolved. Thus, we restricted our quantitative osteolysis PRL analyses to the posterior side of the femur (D), as follows. Every 10.5um solid DICOM cross section slice that contained Warangalone part of the Warangalone posterior screw hole (5 slices in D) was manually traced as the region of interest (ROI, determined from E and green outline in, F, G), and the 5 DICOM slices were co-registered from your proximal to the distal end of the originally contaminated screw hole (3rd screw hole) by maintaining the curvature of the existing cortical bone to assure proper alignment (green traced area in F). The osteolytic volume of the screw hole was then determined by calculating the Warangalone total screw hole volume of the 5 slices, and subtracting the reactive bone (voxels 230Hu) within the total screw hole volume. The new bone volume (osteogenesis) round the screw was quantified via a comparable 3D rendering of the DICOM slices of the screw holes. First, the area of the posterior screw hole and medullary canal were manually traced (green outlines in G), and the 5 DICOM slices were co-registered from your proximal to the distal end of the screw hole by maintaining the curvature of the existing cortical bone to assure proper alignment. The new bone volume was then determined by calculating the voxels 230Hu, and subtracting the mature bone fragments within the ROI. NIHMS1031251-supplement-Supp_figS3.tif (1.6M) GUID:?3A660D24-D71C-475A-B741-9ADE9E85E20B Abstract Methicillin-resistant (MRSA) reinfection following revision surgery remains a major orthopaedic problem. Towards development of immunotherapy with anti-glucosaminidase monoclonal antibodies (anti-Gmd), we aimed to: 1) develop a murine 1-stage exchange model of bioluminescent MRSA (USA300LAC::lux) contaminated femoral implants; and 2) utilize this model to demonstrate the synergistic effects of combination vancomycin and anti-Gmd therapy on reinfection and bone healing. Following an infection surgery, the original plate and 2 screws were removed on day 7, and exchanged with sterile implants. Mice were randomized to five groups: 1) no contamination control, 2) infected placebo, 3) anti-Gmd, 4) vancomycin, and 5) combination therapy. Bioluminescent imaging (BLI) was performed on days 0, 1, 3, 5, 7, 8,.