Hydrogel delivery of antibiotics in a mouse model of methicillin-resistant Staphylococcus aureus osteomyelitis

Author(s):
Christopher Dussik; Kristin Yu; Francis Lee; Sean Cahill; Hyuk-Kwon Kwon ; Jungho Back

Background:

Osteomyelitis is difficult and expensive to treat, with below optimal treatment rates. The leading agent of osteomyelitis is S. aureus. Standard management is limited to surgical debridement and systemic antibiotic therapy, which may increase susceptibility to opportunistic infection, organ toxicity and antibiotic resistance.

Hypothesis:

We created a contiguous infection, femoral fracture model of MRSA osteomyelitis and hypothesized that targeted application of antibiotic-laden hydrogels would reduce bacterial growth and inflammation.

Methods:

Transverse femoral osteotomies were induced in the femurs of 10-week-old male C57BL/6 mice. An incision was on the lateral right leg, a 1” 23G needle drilled through the length of the femur, and a transverse fracture was induced with a Gigli wire saw. 10⁴ CFU of USA300 was applied to the fracture site. Wounds were washed with saline 10 minutes after inoculation. Empty, vancomycin, rifampin, or vancomycin-rifampin (VR)-loaded hydrogels were applied.

Mice were sacrificed 1, 7, and 28 days post-op. Weekly CBC, cytokine, and histologic analyses were performed. Serum isolates and harvested implants were plated and analyzed for MRSA.

Results:

Tissue samples and implants demonstrated the presence of MRSA infection. Gram and H&E staining of untreated, MRSA-infected mice revealed extensive bacterial colonization of muscle surrounding the fracture site and intramedullary space, neutrophil recruitment to areas of infection, and the spread of MRSA to the hip and knee joints within three days of infection. Lower bacterial burden was seen on samples from the antibiotic-treated groups. CBC revealed neutrophil elevation in all infected groups within 24 hours of infection. Average neutrophil percentage was suppressed in the antibiotic-treated groups compared to the infection-only mice by 18% (p<0.01). Cytokine arrays within one day of infection revealed reduced levels of G-CSF and IL-1b between the infection only and antibiotic-treated groups (p<0.01), IL-1a between the MRSA and rifampin and VR groups (p<0.01), and KC between the MRSA and vancomycin and rifampin-only groups (p<0.01).

Conclusions:

Targeted injection of antibiotic-containing hydrogels reduced circulating levels of neutrophils and pro-inflammatory cytokine production. Reduction in the expression of G-CSF and KC in treated groups may contribute to lowered levels of neutrophil activation and indicate a lesser bacterial burden. The observed reductions in cytokines that promote inflammation and osteoclastogenesis and suppress osteoblast proliferation suggest that the hydrogel treatments have the potential to mitigate the osteolytic effects of MRSA.