Date of Award

January 2022

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Francis Y. Lee

Abstract

ENHANCEMENT OF MRSA-INFECTED FRACTURE HEALING BY COMBINATORIAL ANTIBIOTICS AND MODULATION OF SUSTAINED INFLAMMATIONKristin E. Yu, Hyuk-Kwon Kwon, Christopher M. Dussik, Jungho Back, and Francis Y. Lee. Department of Orthopaedics and Rehabilitation, Yale University, School of Medicine, New Haven, CT

Fracture healing is impaired under periods of protracted inflammation, as is incited by musculoskeletal infection. Infection with methicillin-resistant S. aureus (MRSA) is particularly challenging to treat and often recurs from intracellular niduses of infection within bone. This thesis investigates the translational utility of an adjunctively administered, combinatorial antibiotic and anti-inflammatory therapy following the insufficient resolution of infection and inflammation at sites of infected fracture to prevent the development of osteomyelitis and facilitate fracture healing. This dual method of treatment represents a novel paradigm in the care of musculoskeletal infection. The primary objective of this dissertation was to evaluate whether hydrogel-mediated delivery of antibiotics with concurrent modulation of infection-induced inflammatory responses would reduce bacterial growth, improve inflammation, and facilitate healing in a murine femoral fracture model of MRSA osteomyelitis.

Unbiased transcriptome screening and proteomics analysis were performed on MRSA-infected primary murine osteoblasts. Sterile and MRSA-contaminated transverse femoral osteotomies were induced in 10-week-old male C57BL/6 mice and fixed via intramedullary nailing (n=328). In the initial therapeutic cohort, empty, vancomycin- (V), rifampin- (R), vancomycin-rifampin- VR, or vancomycin-rifampin-Trametinib (VRT) loaded hydrogels were applied to the fracture site intraoperatively. Two additional cohorts were created mimicking perioperative and postoperative antibiotic administration. Systemic vancomycin or VR was administered for two weeks, followed by two weeks of VRT hydrogel or oral Trametinib therapy alone. Hematologic, histological, and cytokine analyses were performed using serum and tissue isolates obtained at distinct postoperative intervals. Radiography and micro-computed tomography (μCT) were employed to assess fracture healing.

RNA sequencing and proteomics analysis revealed upregulation of pro-inflammatory pathway extracellular signal-related kinase (ERK) and ERK-related protein expression with MRSA infection. Antibiotic treatment reduced bacterial burden. Pro-inflammatory cytokine levels remained persistently elevated within the MRSA-infected mice with antibiotic treatment alone, but normalized with oral or local Trametinib therapy. Safranin O staining showed callus formation in the uninfected and VRT groups within one week. Rigid fixation, callus, and eventually union were achieved in the control. Impaired callus formation and malunion were observed in the MRSA-infected groups and was partially salvaged with systemic antibiotic treatment. Mice that received VR alongside adjuvant ERK inhibition displayed the greatest restoration of normal bony architecture. Using a small animal model of a fixed, MRSA-infected, transverse diaphyseal femoral fracture, the combined approach involving systemic antibiotic administration and ERK inhibition reduced bacterial bioburden, diminished pro-inflammatory cytokine production, and promoted callus formation in the setting of infection. Additional work is required to further clarify the processes that underlie inflammatory bone loss and impaired fracture healing under conditions of sustained inflammation. Regardless, the concepts described in this dissertation offer therapeutic potential toward the restoration of fracture healing under infectious conditions and the improved treatment of osteomyelitis.

Comments

This thesis is restricted to Yale network users only. This thesis is permanently embargoed from public release.

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