Novel in vivo mouse model of shoulder implant infection

Abstract

Background: Animal models are used to guide management of periprosthetic implant infections. No adequate model exists for periprosthetic shoulder infections, and clinicians thus have no preclinical tools to assess potential therapeutics. We hypothesize that it is possible to establish a mouse model of shoulder implant infection (SII) that allows noninvasive, longitudinal tracking of biofilm and host response through in vivo optical imaging. The model may then be employed to validate a targeting probe (1D9-680) with clinical translation potential for diagnosing infection and image-guided débridement.

Methods: A surgical implant was press-fit into the proximal humerus of c57BL/6J mice and inoculated with 2 μL of 1 × 10³ (e3), or 1 × 10⁴ (e4), colony-forming units (CFUs) of bioluminescent Staphylococcus aureus Xen-36. The control group received 2 μL sterile saline. Bacterial activity was monitored in vivo over 42 days, directly (bioluminescence) and indirectly (targeting probe). Weekly radiographs assessed implant loosening. CFU harvests, confocal microscopy, and histology were performed.

Results: Both inoculated groups established chronic infections. CFUs on postoperative day (POD) 42 were increased in the infected groups compared with the sterile group (P < .001). By POD 14, osteolysis was visualized in both infected groups. The e4 group developed catastrophic bone destruction by POD 42. The e3 group maintained a congruent shoulder joint. Targeting probes helped to visualize low-grade infections via fluorescence.

Discussion: Given bone destruction in the e4 group, a longitudinal, noninvasive mouse model of SII and chronic osteolysis was produced using e3 of S aureus Xen-36, mimicking clinical presentations of chronic SII.

Conclusion: The development of this model provides a foundation to study new therapeutics, interventions, and host modifications.

Keywords: Shoulder; arthroplasty; implant; infection; osteolysis; osteomyelitis.

Figure 1.. Surgical Procedure (Right Arm).

(A) A midline incision was made extending laterally over the deltoid. (B) The delto-pectoral groove was exposed. (C) The pectoralis muscle was dissected away from the humerus. (D) Posterior pressure was applied to an externally rotated and extended humerus to expose and anteriorly sublux the shoulder joint. (E) Using a 25g needle, the head of the humerus was reamed to make a canal for implant placement. (F) A 0.6mm wide steel implant (5mm in length) with a slight 15-degree bend at the most proximal end (1mm) was implanted into the humerus. (G) Deep sutures were placed prior to inoculation and 2ul of bioluminescent Xen-36 S. aureus was added to the exposed end of the implant. (H) Deep and superficial layers were closed with 5–0 Vicryl sutures.

Figure 2.. X-ray Confirmation of Implant Placement.

(A) AP view showing implant placement within the humerus. (B) Lateral view confirming implant placement within the shoulder joint space.

Figure 3A.. Quantifying Bacterial Burden.

(i) In Vivo Longitudinal Tracking of Xen 36: Measuring Bacterial Burden Using Photon Emission. Data from the IVIS Lumina was obtained and plotted. Averages of each group with respect to the POD (postoperative day) were used to produce the curve (photons/sec/cm3/sr). Error bars represent the standard error of the mean. (ii) Visualizations of peak photon emissions and intensities are shown for each group. Infected Group e3 was inoculated with 1,000 bacterial cells and compared to the Sterile Group which received sterile saline. Images are shown through POD 42, with peak photon emission on POD 5.

Figure 3B.. Quantifying Bacterial Burden.

(i) In Vivo Longitudinal Tracking of Xen 36: Measuring Bacterial Burden Using Photon Emission. Data from the IVIS Lumina was obtained and plotted. Averages of each group with respect to the POD (postoperative day) were used to produce the curve (photons/sec/cm3/sr). Error bars represent the standard error of the mean. (ii) Visualizations of peak photon emissions and intensities are shown for each group. Infected Group e4 was inoculated with 10,000 bacterial cells and compared to the Sterile Group which received sterile saline. Images are shown through POD 42, with peak photon emission between POD 7.

Figure 4.. POD 42 Colony Forming Unit (CFU) Harvest from Tissue and Implant.

(A) Shows Infected Groups e3, e4, and the Sterile Group’s respective average CFU data; representing the amount of Xen-36 harvested from these sites 42 days after initial operation. Error bars represent the standard error of the mean. (B) IVIS Lumina imaging to confirm growth of Xen-36 from CFU harvest.

Figure 5.. Longitudinal Monitoring of Osteolytic Changes.

X-rays taken every week (POD 0, 7, 14, 21, 28, 35, 42) from the AP view. All 3 groups are shown, with osteolytic changes noted in both infected groups on POD 14. Catastrophic osteomyelitis was noted in Infected Group e4 on POD 42.

Figure 6.. Live-Dead Imaging Using Confocal Microscopy and Fluorescent Stain.

Implants were removed in sterile fashion from Infected Group e3 and the Sterile Group, then placed in dying solution for 15 min. GREEN represents bacteria with an intact cell membrane (living), whereas RED represents bacteria with a ruptured cell membrane (dead). Orange represents an overlap. Central bright green image represents artifact from the light reflecting off the implant. The surrounding of this artifact represents biofilm formation on an infected implant (A), and lack thereof on a sterile implant (B).

Figure 7.. Histology of the Head of the Humerus After Implant Explantation.

(A) Humerus from Infected Group e3 shows formation of thicker capsule surrounding the canal left by the implant after explantation. (B) Humerus from the Sterile Group shows minimal capsule formation after pin explantation.

Figure 8.. 2D and 3D optical imaging to confirm fluorescent probe targeting of bioluminescent S. aureus implant infections.

Mice with POD 7 bioluminescent S. aureus infected or sterile shoulder implants were intravenously injected with fluorescently labelled 1D9–680 staphylococcal targeting antibody probe. 48h post-injection of the probe, animals were imaged both two- and three-dimensionally in the IVIS Spectrum-CT, showing that the probe could readily delineate between infected and sterile implants. CT co-registration showed both bioluminescent S. aureus and fluorescent probe to be associated with the implant in the shoulder.