Chao Xie

The “Race for the Surface” hypothesis remains the dogma for explaining the competition between host proteins, cells, and bacteria for implant colonization immediately post‐op. Recently, we used intravital microscopy to provide direct evidence supporting this hypothesis by demonstrating that the race for the surface in a murine implant‐associated infection (IAI) model is complete within 3 h. However, this intravital imaging is impractical for early‐stage drug development research, thus warranting a cost‐effective screening platform to mechanistically assess neutrophil‐ S. aureus interaction dynamics during the early infection period. To address this, we developed an in vitro IAI model using longitudinal confocal fluorescence microscopy (LCFM) of etched titanium implants contaminated with GFP ⁺ USA300 and cultured with tdTomato ⁺ neutrophils. This model enabled reproducible quantification of neutrophil swarming, phagocytosis, and nidus formation, which were validated with scanning electron microscopy, colony‐forming unit assays, and imaging cytometry. To demonstrate the utility of these novel platforms, we evaluated an anti‐glucosaminidase (Gmd) mAb known to mediate opsonophagocytosis of aggregated S. aureus and assessed its ability to increase total neutrophil volume and S. aureus internalization by neutrophils and cytoplasts. Collectively, these results demonstrate the utility of the LCFM and imaging cytometry platforms for defining mAb functions that enhance neutrophil and cytoplast activity to prevent IAI.

Background: Acute compartment syndrome (ACS) is a critical orthopedic emergency where increased pressure in a limb compartment leads to muscle ischemia, necrosis, and lasting dysfunction. While fasciotomy is the standard treatment, it often results in complications like infection and nerve damage, underscoring the need for effective non-surgical therapies to manage the inflammation causing tissue damage. Peripheral nerve block (PNB) with ropivacaine is clinically used for analgesia, but whether it can serve as a disease-modifying adjunct intervention for ACS beyond pain control remains unexplored. Objective: This study examined the potential of PNB utilizing ropivacaine to enhance outcomes in ACS and explored the underlying mechanisms related to macrophage polarization. We aimed to distinguish two unresolved questions: (1) whether ropivacaine-based PNB confers tissue-protective effects in ACS independent of analgesia; (2) what signaling pathway mediates ropivacaine-driven macrophage phenotypic switch under ACS-relevant inflammatory conditions. Methods: Using rats, an ACS model was established, with the animals being randomly allocated to five different groups: Control, ACS, sciatic PNB, femoral PNB, and combined PNB. The study assessed functional outcomes, histopathological changes, levels of inflammatory markers (interleukin-6 (IL-6), C-reactive protein (CRP), and markers of macrophage polarization (CD86, CD206). In parallel, RAW264.7 macrophages stimulated with LPS and IFN-γ were treated with ropivacaine (1–10 μg/mL), and M1/M2 markers, pro-inflammatory cytokines (TNF-α, IL-6, IL-1β), migratory capacity, and cytotoxicity were assessed. Differentially expressed genes and enriched pathways were identified through RNA sequencing, followed by mechanistic validation of JAK-STAT signaling using Western blotting, immunofluorescence, immunohistochemistry, and rescue experiments with the JAK2 inhibitor AG490. Results: PNB significantly improved all functional and histological parameters in ACS rats, with combined blockade demonstrating the greatest efficacy. Ropivacaine at 5 μg/mL promoted M2 macrophage polarization, evidenced by upregulated CD206 and arginase-1 alongside downregulated CD86 and iNOS, while reducing pro-inflammatory cytokine production without cytotoxicity. Ropivacaine also suppressed macrophage migration. Among 83 differentially expressed genes, RNA sequencing pinpointed JAK-STAT as the pathway with the highest level of enrichment. Mechanistically, ropivacaine activated JAK2-STAT3, reflected by increased p-JAK2/JAK2 and p-STAT3/STAT3 ratios. AG490 completely reversed ropivacaine-induced JAK-STAT activation, M2 polarization, cytokine suppression, and migratory inhibition in vitro, and similarly abrogated PNB-mediated functional recovery and histological protection in vivo. Conclusions: In vitro, ropivacaine directly promoted macrophage M2 polarization via JAK-STAT activation. In vivo, ropivacaine-based PNB ameliorates ACS, which may involve both direct immunomodulatory effects of ropivacaine and indirect biological consequences of peripheral neural blockade. This study provides the first pre-clinical proof-of-concept that ropivacaine PNB can act as an adjunctive disease-modifying strategy for ACS (beyond analgesia), and identifies macrophage JAK2-STAT3 as the critical molecular cascade responsible for this immunomodulatory effect. This identifies a readily translatable adjunctive strategy for ACS and establishes JAK-STAT as a potential pharmacological target for compartment syndrome-associated inflammation.

Implant-associated osteomyelitis is extremely difficult to treat due to bacterial colonization of the osteocyte-lacuno canalicular network, where bacteria are inaccessible from immune clearance and antibiotic treatment. The pharmacokinetic limitations of drug delivery to this compartment of bone render this infection resistant to standard-of-care antibiotics. Bone-targeted bisphosphonate-conjugated antibiotics have been developed to overcome this issue, and hydroxybisphosphonate-conjugated sitafloxacin (HBCS) has emerged as a leading therapeutic candidate based on safety and efficacy results in mice and sheep. However, potential concerns about its adverse effects on skeletal growth and bone healing remain. To address this, we assessed HBCS effects vs placebo, zoledronic acid (ZA) positive control known to inhibit osteoclasts and increase fracture callus and growth plate bone volume, and hydroxy-phenyl-ethane-hydroxy-bisphosphonate (HPHBP), the bisphosphonate component of HBCS, using a murine closed femur fracture model, and on intramembranous and endochondral ossification in the proximal tibia of 12-wk-old mice. Longitudinal X-rays and micro-CT confirmed that no treatments inhibited fracture healing, although ZA and HBCS increased callus bone volume fraction at 3 wk. Histology results suggested that this was likely due to effects on osteoclasts at 2 and 3 wk but confirmed that all soft callus was remodeled by 3 wk. HBCS did not show the increases in polar moment of inertia or maximum torque observed in the ZA group at 4 wk. Studies on unfractured fluorochrome labeled tibias demonstrated that no treatments affected mineral apposition and bone formation rates. However, HPHBP and HBCS increased growth plate bone volume over total volume (BV/TV) concomitant with a decrease in osteoclasts, while ZA further increased total bone volume and BV/TV with a dramatic increase in osteoclasts. Collectively, these data demonstrate that while HBCS decreases osteoclast numbers in vivo, it does not inhibit fracture healing or normal bone growth in mice.

Background/Objectives: Hydroxybisphosphonate-conjugated sitafloxacin (HBCS) was developed to achieve higher antibiotic concentrations within infected bone. Small animal studies supported further development, but the feasibility of HBCS treatment in a more clinically relevant and larger animal model is unknown. Methods: In this study, we present case reports on four sheep, each receiving four MRSA-contaminated tibial screws treated with different regimens of intravenous antibiotics. The first two sheep received two screws contaminated with 10³ CFU and two screws contaminated with 10⁵ CFU. Sheep 1 only received vancomycin, starting on day two. Sheep 2 received vancomycin, starting on day 2, but also received 7 doses of HBCS (2 mg/kg/48 h). The protocol for the final two sheep was revised, and both received four screws contaminated with 10³ CFU, and vancomycin was started preoperatively. Sheep 3 and 4 received 7 doses (starting on day 6) and 9 doses (starting on day 2) of HBCS (4 mg/kg/48 h), respectively. Bacteriology was performed on three screws per animal. Longitudinal radiography and histology (n = 1 screw) were assessed for signs of osteolysis and reactive bone formation. Electron microscopy (EM) was performed in the first two sheep to evaluate antibiotic-induced bacterial damage. Results: All sheep tolerated HBCS infusion without clinical signs of discomfort. In addition to a high bacterial load (~10⁴ CFU on all screws), Sheep 1 displayed extensive radiographic and histologic evidence of peri-implant osteolysis and reactive bone formation. Despite having a high bacterial load (~10⁴ CFU on all screws), Sheep 2 displayed only mild radiographic and histologic evidence of peri-implant osteolysis and periosteal reactive bone formation. Bacteriology in Sheep 3 and 4 demonstrated near MRSA eradication (3 CFU per screw, the initiation of antibiotic treatment commencing at the time of surgery, and the usability of antibiotic-killed bacteria within altered glycocalyx observed by TEM as a potential biomarker for HBCS efficacy.

Implant-associated osteomyelitis remains a major orthopaedic problem. As neutrophil swarming to the surgical site is a critical host response to prevent infection, visualization and quantification of this dynamic behavior at the native microenvironment of infection will elucidate previously unrecognized mechanisms central to understanding the host response. We recently developed longitudinal intravital imaging of the bone marrow (LIMB) to visualize host cells and fluorescent S. aureus on a contaminated transfemoral implant in live mice, which allows for direct visualization of bacteria colonization of the implant and host cellular responses using two-photon laser scanning microscopy. To the end of rigorous and reproducible quantitative outcomes of neutrophil swarming kinetics in this model, we developed a protocol for robust segmentation, tracking, and quantifications of neutrophil dynamics adapted from Trainable Weka Segmentation and TrackMate, two readily available Fiji/ImageJ plugins. In this work, Catchup mice with tdTomato expressing neutrophils received a transfemoral pin with or without ECFP/EGFP-expressing USA300 methicillin-resistant Staphylococcus aureus (MRSA) to obtain 30-minute LIMB videos at 2-, 4-, and 6-hours post-implantation. The developed semi-automated neutrophil tracking protocol was executed independently by two users to quantify the distance, displacement, speed, velocity, and directionality of the target cells. The results revealed high inter-user reliability for all outcomes (ICC > 0.96; p > 0.05). Consistent with the established paradigm on increased neutrophil swarming during active infection, the results also demonstrated increased neutrophil speed and velocity at all measured time points, and increased displacement at later time points (6 hours) in infected versus uninfected mice (p < 0.05). Neutrophils and bacteria also exhibit directionality during migration in the infected mice. The semi-automated cell tracking protocol provides a streamlined approach to robustly identify and track individual cells across diverse experimental settings and eliminates inter-observer variability.

In vitro and in vivo studies are critical for the preclinical efficacy assessment of novel therapies targeting musculoskeletal infections (MSKI). Many preclinical models have been developed and applied as a prelude to evaluating safety and efficacy in human clinical trials. In performing these studies, there is both a requirement for a robust assessment of efficacy, as well as a parallel responsibility to consider the burden on experimental animals used in such studies. Since MSKI is a broad term encompassing infections varying in pathogen, anatomical location, and implants used, there are also a wide range of animal models described modeling these disparate infections. Although some of these variations are required to adequately evaluate specific interventions, there would be enormous value in creating a unified and standardized criteria to animal testing in the treatment of MSKI. The Treatment Workgroup of the 2023 International Consensus Meeting on Musculoskeletal Infection was responsible for questions related to preclinical models for treatment of MSKI. The main objective was to review the literature related to priority questions and estimate consensus opinion after voting. This document presents that process and results for preclinical models related to (1) animal model considerations, (2) outcome measurements, and (3) imaging.

Musculoskeletal infections (MSKI), which are a major problem in orthopedics, occur when the pathogen eludes or overwhelms the host immune system. While effective vaccines and immunotherapies to prevent and treat MSKI should be possible, fundamental knowledge gaps in our understanding of protective, nonprotective, and pathogenic host immunity are prohibitive. We also lack critical knowledge of how host immunity is affected by the microbiome, implants, prior infection, nutrition, antibiotics, and concomitant therapies, autoimmunity, and other comorbidities. To define our current knowledge of these critical topics, a Host Immunity Section of the 2023 Orthopaedic Research Society MSKI International Consensus Meeting (ICM) proposed 78 questions. Systematic reviews were performed on 15 of these questions, upon which recommendations with level of evidence were voted on by the 72 ICM delegates, and another 12 questions were voted on with a recommendation of “Unknown” without systematic reviews. Two questions were transferred to another ICM Section, and the other 45 were tabled for future consideration due to limitations of available human resources. Here we report the results of the voting with internet access to the questions, recommendations, and rationale from the systematic reviews. Eighteen questions received a consensus vote of ≥90%, while nine recommendations failed to achieve this threshold. Commentary on why consensus was not achieved on these questions and potential ways forward are provided to stimulate specific funding mechanisms and research on these critical MSKI host defense questions.

Implant-associated osteomyelitis remains a major orthopaedic problem. As neutrophil swarming to the surgical site is a critical host response to prevent infection, visualization and quantification of this dynamic behavior at the native microenvironment of infection will elucidate previously unrecognized mechanisms central to understanding the host response. We recently developed longitudinal intravital imaging of the bone marrow (LIMB) to visualize fluorescent S. aureus on a contaminated transfemoral implant and host cells in live mice, which allows for direct visualization of bacteria colonization of the implant and host cellular responses using two-photon laser scanning microscopy. To the end of rigorous and reproducible quantitative outcomes of neutrophil swarming kinetics in this model, we developed a protocol for robust segmentation, tracking, and quantifications of neutrophil dynamics adapted from Trainable Weka Segmentation and TrackMate, two readily available Fiji/ImageJ plugins. In this work, Catchup mice with tdTomato expressing neutrophils received a transfemoral pin with or without ECFP-expressing USA300 methicillin-resistant Staphylococcus aureus (MRSA) to obtain 30-minute LIMB videos at 2-, 4-, and 6-hours post-implantation. The developed semi-automated neutrophil tracking protocol was executed independently by two users to quantify the distance, displacement, speed, velocity, and directionality of the target cells. The results revealed high inter-reader reliability for all outcomes (ICC > 0.98; p > 0.05). Consistent with the established paradigm on increased neutrophil swarming during active infection, the results also demonstrated increased neutrophil speed and velocity at all measured time points, and increased displacement at later time points (6 hours) in infected versus uninfected mice (p < 0.05). Neutrophils and bacteria also exhibit directionality during migration in the infected mice. The semi-automated cell tracking protocol provides a streamlined approach to robustly identify and track individual cells across diverse experimental settings and eliminates inter-observer variability.

LONGITUDINAL INTRAVITAL IMAGING TO QUANTIFY THE “RACE FOR THE SURFACE” BETWEEN HOST IMMUNE CELL AND BACTERIA FOR ORTHOPAEDIC IMPLANTS WITH S. AUREUS COLONIZATION IN A MURINE MODEL