2008 Abstract : 15

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Authors: Ketonis C, Aiyer A, Adams CS, Shapiro IM, Hickok NJ, Parvizi J

Title: Bone Graft Modification with Antibiotics Confers Protection from Bacteria

Institution: Thomas Jefferson University, Philadelphia PA

Purpose: Infection is the most devastating complication associated with use of allograft bone. We have developed a new method for protecting allografts by covalently modifying them with an antibiotic to confer a stable, bactericidal surface.

Methods: Allograft modification: Morselized human bone was washed extensively and sequentially coupled: 2X with excess Fmoc-aminoethoxyethoxyacetate (Fmoc-AEEA); deprotected with 20% piperidine in DMF; and then incubated in a molar excess solution of vancomycin (VAN) for 12-16 hours. The synthesized bone was washed extensively with DMF and was incubated in PBS for at least 1 day before use. Fluorescamine staining: Fresh morselized bone was washed with acetone and was incubated in a 1mg/ml solution of fluorescamine in acetone for 40mins in the dark and visualized by confocal laser microscopy. VAN immunofluorescence: Control or VAN-derivatized allograft (VAN-bone) was washed 5X with PBS, blocked with 10% FBS (1hr), incubated with rabbit anti-VAN IgG (4?C, 12h) followed by an AlexaFluor 488-coupled goat anti-rabbit IgG (1hr), and visualized by confocal laser microscopy. Scanning Electron Microscopy (SEM): Control and VAN-bone were thoroughly washed with dH2O and surface topography visualized using a Hitachi TM-1000. SEM Antibiotic Activity. Equal dry weights of control and VAN-bone were sterilized with 70% ethanol, rinsed with PBS, and incubated with S. aureus (Ci = 10^4 cfu) in trypticase soy broth (TSB), 37?C, for 2, 5, 8 and 12 hrs. Bacterial visualization: Non- adherent bacteria were removed by washing and adherent bacteria stained with the Live/Dead BacLight Kit (viable bacteria fluoresce green) and visualized by confocal microscopy. Bacterial counts: Non- adherent bacteria were removed by washing and adherent bacteria suspended by sonication in 0.3% Tween-80 for 10mins followed by plating on 3M Petrifilms. Cell toxicity: Control and VAN-bone were sterilized in 70%EtOH, washed 3x with DMEM/F12 and treated with UV radiation (10min). U2 osteoblastic cells were seeded (150,000 cells/ml) on the bone chips and allowed to proliferate for 5 days at which time they were stained with SybrGreen (1hr) and visualized with confocal microscopy.

Results: Surface amines: Staining of primary amines with fluorescamine revealed a bright uniform signal suggesting that there is an abundance of free amines on the bone surface. Vancomycin coverage: These primary amines were used to couple two linkers and vancomycin to the allograft. After derivatization, anti-vancomycin antibodies showed intense, diffuse staining of VAN-bone whereas no specific fluorescence was detected on controls, indicating extensive vancomycin coverage. SEM: The microtopography of both control and VAN-bone were very similar suggesting that chemical modification did not alter surface characteristics of the bone. Bactericidal activity: Using fluorescent detection of live, adherent bacteria, VAN-bone showed only background fluorescence, whereas intense fluorescence with clear colony and biofilm formation was observed on the control bone (2-12hrs). By direct bacterial counts, significantly fewer bacteria survived on VAN-bone than on controls at all time points. Biocompatibility: SybrGreen-stained U2 cells were seeded on control and VAN-bone. Both samples were extensively colonized, with no clear differences between the VAN-bone and control surfaces.

Discussion and Conclusion: Bone grafting is considered the gold standard for repair of bone defects, with worldwide usage in approximately 2.2 million orthopaedic procedures annually generating sales of more than $2.5 billion a year. Of the many complications of allograft impaction, infection remains the most devastating to the patient requiring reoperation, debridement, placement of metallic implants, and, in some cases, amputation. Current techniques to prevent infection development include antibiotic supplementation as means of locally delivering treatment. Key issues that surround the antibiotic loaded bone allografts are potential for nephrotoxicity, variable elution kinetics, and related to the latter, induction of resistance. We describe a novel surface modification to bone grafts that renders them bactericidal while appearing to maintain biocompatibility. The covalent chemical bonding confers long- term antibiotic stability, with potential coverage for multiple bacterial assaults. This proposed modification in surface design serves as a starting point for the development of a new generation of bone grafts that target biological activities to sites of physiological importance.