Monitoring Bacterial Burden, Inflammation and Bone Damage Longitudinally Using Optical and μCT Imaging in an Orthopaedic Implant Infection in Mice

Abstract

BackgroundRecent advances in non-invasive optical, radiographic and μCT imaging provide an opportunity to monitor biological processes longitudinally in an anatomical context. One particularly relevant application for combining these modalities is to study orthopaedic implant infections. These infections are characterized by the formation of persistent bacterial biofilms on the implanted materials, causing inflammation, periprosthetic osteolysis, osteomyelitis, and bone damage, resulting in implant loosening and failure.Methodology/Principal FindingsAn orthopaedic implant infection model was used in which a titanium Kirshner-wire was surgically placed in femurs of LysEGFP mice, which possess EGFP-fluorescent neutrophils, and a bioluminescent S. aureus strain (Xen29; 1×103 CFUs) was inoculated in the knee joint before closure. In vivo bioluminescent, fluorescent, X-ray and μCT imaging were performed on various postoperative days. The bacterial bioluminescent signals of the S. aureus-infected mice peaked on day 19, before decreasing to a basal level of light, which remained measurable for the entire 48 day experiment. Neutrophil EGFP-fluorescent signals of the S. aureus-infected mice were statistically greater than uninfected mice on days 2 and 5, but afterwards the signals for both groups approached background levels of detection. To visualize the three-dimensional location of the bacterial infection and neutrophil infiltration, a diffuse optical tomography reconstruction algorithm was used to co-register the bioluminescent and fluorescent signals with μCT images. To quantify the anatomical bone changes on the μCT images, the outer bone volume of the distal femurs were measured using a semi-automated contour based segmentation process. The outer bone volume increased through day 48, indicating that bone damage continued during the implant infection.Conclusions/SignificanceBioluminescent and fluorescent optical imaging was combined with X-ray and μCT imaging to provide noninvasive and longitudinal measurements of the dynamic changes in bacterial burden, neutrophil recruitment and bone damage in a mouse orthopaedic implant infection model.

d-amino Acid Inhibits Biofilm but not New Bone Formation in an Ovine Model

“…Longitudinal studies investigating bacterial burden, bone formation, and bone remodeling in contaminated defects [34] …”

Combined <em>In vivo</em> Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

“…Insert a medical-grade titanium Kirschner-wire (0.8 mm diameter) by using a press-fit technique, which entails manually pushing it using a pin holder, in a retrograde direction into the intramedullary canal. NOTE: Titanium K-wires were used as there were fewer artifacts seen on the μCT images with titanium K-wires compared with stainless steel K-wires 16 . 8.…”

“…In the present study, the protocol is described for this previously published model of an orthopaedic prosthetic joint infection in mice [14][15][16][17][18][19] , which involves the surgical placement of a titanium K-wire implant that extends from an intramedullary canal in the femur into the joint space [14][15][16][17][18][19] . S. aureus bioluminescent strain Xen29 (1 x 10 3 CFU in 2 μl PBS) was pipetted directly on top of the end titanium implant in the knee joint prior to closing the surgical site 16 .…”

“…S. aureus bioluminescent strain Xen29 (1 x 10 3 CFU in 2 μl PBS) was pipetted directly on top of the end titanium implant in the knee joint prior to closing the surgical site 16 . To visualize and quantify the bacterial burden and neutrophil influx noninvasively in anesthetized LysEGFP mice, in vivo whole animal optical imaging was performed to sequentially image the bioluminescent signals from the bacteria and the EGFP fluorescent signals from the infiltrating neutrophils using the IVIS Spectrum optical whole animal in vivo imaging system on three postoperative days (i.e., days 2, 14 and 28).…”

“…This model involved placing a titanium Kirschner-wire (K-wire) in such a manner that the cut end of the implant extended in the knee joint from the femurs of mice [14][15][16][17][18][19] . An inoculum of Staphylococcus aureus (bioluminescent strain Xen29 or Xen36) was then pipetted onto the surface of the implant in the knee joint before the surgical site was closed [14][15][16][17][18][19] . In vivo optical imaging was used to detect and quantify the bioluminescent signals, which corresponded to the number of bacteria in the infected joint and bone tissue…”