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Implant-associated Staphylococcus aureus (S. aureus) osteomyelitis is a severe challenge in orthopedics. While antibiotic-loaded bone cement is a standardized therapeutic approach for S. aureus osteomyelitis, it falls short in eradicating Staphylococcus abscess communities (SACs) and bacteria within osteocyte-lacuna canalicular network (OLCN) and repairing bone defects. To address limitations, we developed a borosilicate bioactive glass (BSG) combined with ferroferric oxide (Fe3O4) magnetic scaffold to enhance antibacterial efficacy and bone repair capabilities. We conducted comprehensive assessments of the osteoinductive, immunomodulatory, antibacterial properties, and thermal response of this scaffold, with or without an alternating magnetic field (AMF). Utilizing a well-established implant-related S. aureus tibial infection rabbit model, we evaluated its antibacterial performance in vivo. RNA transcriptome sequencing demonstrated that BSG + 5%Fe3O4 enhanced the immune response to bacteria and promoted osteogenic differentiation and mineralization of MSCs. Notably, BSG + 5%Fe3O4 upregulated gene expression of NOD-like receptor and TNF pathway in MSCs, alongside increased the expression of osteogenic factors (RUNX2, ALP and OCN) in vitro. Flow cytometry on macrophage exhibited a polarization effect towards M2, accompanied by upregulation of anti-inflammatory genes (TGF-β1 and IL-1Ra) and downregulation of pro-inflammatory genes (IL-6 and IL-1β) among macrophages. In vivo CT imaging revealed the absence of osteolysis and periosteal response in rabbits treated with BSG + 5%Fe3O4 + AMF at 42 days. Histological analysis indicated complete controls of SACs and bacteria within OLCN by day 42, along with new bone formation, signifying effective control of S. aureus osteomyelitis. Further investigations will focus on the in vivo biosafety and biological mechanism of this scaffold within infectious microenvironment.

Abstract Eradication of MRSA osteomyelitis requires elimination of distinct biofilms. To overcome this, we developed bisphosphonate-conjugated sitafloxacin (BCS, BV600072) and hydroxybisphosphonate-conjugate sitafloxacin (HBCS, BV63072), which achieve “target-and-release” drug delivery proximal to the bone infection and have prophylactic efficacy against MRSA static biofilm in vitro and in vivo. Here we evaluated their therapeutic efficacy in a murine 1-stage exchange femoral plate model with bioluminescent MRSA (USA300LAC::lux). Osteomyelitis was confirmed by CFU on the explants and longitudinal bioluminescent imaging (BLI) after debridement and implant exchange surgery on day 7, and mice were randomized into seven groups: 1) Baseline (harvested at day 7, no treatment); 2) HPBP (bisphosphonate control for BCS) + vancomycin; 3) HPHBP (hydroxybisphosphonate control for HBCS) + vancomycin; 4) vancomycin; 5) sitafloxacin; 6) BCS + vancomycin; and 7) HBCS + vancomycin. BLI confirmed infection persisted in all groups except for mice treated with BCS or HBCS + vancomycin. Radiology revealed catastrophic femur fractures in all groups except mice treated with BCS or HBCS + vancomycin, which also displayed decreases in peri-implant bone loss, osteoclast numbers, and biofilm. To confirm this, we assessed the efficacy of vancomycin, sitafloxacin, and HBCS monotherapy in a transtibial implant model. The results showed complete lack of vancomycin efficacy while all mice treated with HBCS had evidence of infection control, and some had evidence of osseous integrated septic implants, suggestive of biofilm eradication. Taken together these studies demonstrate that HBCS adjuvant with standard of care debridement and vancomycin therapy has the potential to eradicate MRSA osteomyelitis.

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.

S. aureus infection of bone is difficult to eradicate due to its ability to colonize the osteocyte-lacuno-canalicular network (OLCN), rendering it resistant to standard-of-care (SOC) antibiotics. To overcome this, we proposed two bone-targeted bisphosphonate-conjugated antibiotics (BCA): bisphosphonate-conjugated sitafloxacin (BCS) and hydroxybisphosphonate-conjugate sitafloxacin (HBCS). Initial studies demonstrated that the BCA kills S. aureus in vitro. Here we demonstrate the in vivo efficacy of BCS and HBCS versus bisphosphonate, sitafloxacin, and vancomycin in mice with implant-associated osteomyelitis. Longitudinal bioluminescent imaging (BLI) confirmed the hypothesized “target and release”-type kinetics of BCS and HBCS. Micro-CT of the infected tibiae demonstrated that HBCS significantly inhibited peri-implant osteolysis versus placebo and free sitafloxacin (p < 0.05), which was not seen with the corresponding non-antibiotic-conjugated bisphosphonate control. TRAP-stained histology confirmed that HBCS significantly reduced peri-implant osteoclast numbers versus placebo and free sitafloxacin controls (p < 0.05). To confirm S. aureus killing, we compared the morphology of S. aureus autolysis within in vitro biofilm and infected tibiae via transmission electron microscopy (TEM). Live bacteria in vitro and in vivo presented as dense cocci ~1 μm in diameter. In vitro evidence of autolysis presented remnant cell walls of dead bacteria or “ghosts” and degenerating (non-dense) bacteria. These features of autolyzed bacteria were also present among the colonizing S. aureus within OLCN of infected tibiae from placebo-, vancomycin-, and sitafloxacin-treated mice, similar to placebo. However, most of the bacteria within OLCN of infected tibiae from BCA-treated mice were less dense and contained small vacuoles and holes >100 nm. Histomorphometry of the bacteria within the OLCN demonstrated that BCA significantly increased their diameter versus placebo and free antibiotic controls (p < 0.05). As these abnormal features are consistent with antibiotic-induced vacuolization, bacterial swelling, and necrotic phenotype, we interpret these findings to be the initial evidence of BCA-induced killing of S. aureus within the OLCN of infected bone. Collectively, these results support the bone targeting strategy of BCA to overcome the biodistribution limits of SOC antibiotics and warrant future studies to confirm the novel TEM phenotypes of bacteria within OLCN of S. aureus-infected bone of animals treated with BCS and HBCS.