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Covalent Antibiotic Attachment to Bone Allografts Provides Protection Against Bacterial Colonization and Biofilm
Constantinos Ketonis, MD, PhD1; Isabelle Mortalena, DDS1; Javad Parvizi, MD1; Christopher Adams, PhD2; Noreen Hickok, PhD1
1Thomas Jefferson University Hospital, Philadelphia, PA, 2College of Osteopathic Medicine, Philadelphia, PA
Introduction: Bone allograft continues to be used extensively in orthopaedics to help restore bony defects and to provide structural stability. Infection of this initially foreign implant remains the most devastating complication, with a reported incidence of up to 37% in the setting of massive bone allograft implantation following tumor resections. We propose that covalently-modifying allografts with antibiotics can effectively inhibit bacterial colonization and far outlast current technologies.
Materials & Methods: Human bone allograft was coupled with two Fmoc-AEEA linkers and doxycycline (DOX). Scanning Electron Microscopy (SEM) was used to assess topography during the stages of synthesis, and immunofluorescence was used to confirm doxycycline coverage and uniformity. Antibiotic activity was determined by Staphylococcus aureus (SA) and Escherichia coli (E. coli) challenges followed by either direct immunofluorescence and SEM visualization or sonication and plating for bacterial counts. To assess biocompatibility, an osteoblastic cell line was cultured on the antibiotic bone and cell nuclei/ actin was immunostained.
Results: Immunofluorescence demonstrates uniform and stable attachment of antibiotics on the grafts without alteration of surface topography. After elution of adsorbed antibiotic, the antibiotic constructs did not allow colonization by SA or E. coli. Few bacteria remained on the surface of these bone constructs but SEM imaging did not detect any evidence of biofilm formation. Modified constructs consistently decreased bacterial burden by >95% as compared to unmodified allografts that were heavily colonized with SA or E. coli encased in a glycocalyx matrix consistent with biofilm (Figure). Importantly, these antibacterial bone grafts remained biocompatible. Cell morphology and cytoskeletal architecture was maintained and appeared similar to cells grown on unmodified bone grafts suggesting little to no cytotoxicity.
Conclusion: We demonstrate the successful attachment of doxycycline on bone grafts with activity against polymicrobial infections often found in patients undergoing chemotherapy after resection of large bony tumors, immunocompromised individuals and other atypical osteomyelitis. Such constructs hold a promise in eliminating bacterial colonization associated with transplanted orthopaedic tissues.
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