Background: Implant-associated infections contribute to patient morbidity and health care costs. We hypothesized that

Background Bone grafts are frequently used to supplement bone stock and to establish structural stability. However, graft-associated infection represents a challenging complication leading to increased patient morbidity and healthcare costs. Questions/purposes We therefore designed this study to (1) determine if increasing initial S. aureus inoculation of bone allograft results in a proportionate increase in colonization; (2) assess if antibiotics decrease colonization and if antibiotic tethering to allograft alters its ability to prevent bacterial colonization; and (3) determine if covalent modification alters the allograft topography or its biological properties.Methods Allograft bone and vancomycin-modified bone (VAN-bone) was challenged with different doses of S. aureus for times out to 24 hours in the presence or absence of solution vancomycin. Bacterial colonization was assessed by fluorescence, scanning electron microscopy (SEM), and by direct colony counting. Cell density and distribution of osteoblast-like cells on control and modified allograft were then compared. Results Bacterial attachment was apparent within 6 hours with colonization and biofilm formation increasing with time and dose. Solution vancomycin failed to prevent bacterial attachment whereas VAN-bone successfully resisted colonization. The allograft modification did not affect the attachment and distribution of osteoblast-like cells.

Bacterial contamination of bone allograft is a significant complication of orthopaedic surgery. To address this issue, we have engineered a method for covalently modifying bone allograft tissue with the antibiotic vancomycin. The goal of this investigation was to compare the biocidal properties of this new allograft material with those of vancomycin physisorbed onto graft material. The duration of antibiotic release from the vancomycin-modified allograft matrix was determined and no elution was observed. In contrast, the adsorbed antibiotic showed a peak elution at 24 h that then decreased over several days. We next used an S. aureus disk diffusion assay to measure the activity of the eluted vancomycin. Again we found that no active antibiotic was eluted from the covalently-modified allograft. Similarly, when the vancomycin-modified allograft morsel was used in the assay, no measurable elution was observed; amounts of antibiotic released from the adsorbed samples inhibited S. aureus growth for 4-7 days. Probably the most telling property of the allograft was that after two weeks, the tethered-allograft was able to resist bacterial colonization. Unlike the elution system in which vancomycin was depleted over the course of Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Ethical Board Review statement: No animals or human subjects Conflict of Interest: CK, CSA, IMS, JP, and NJH have filed an invention disclosure on this process. RESEARCH HIGHLIGHTS •Vancomycin that is covalently tethered to allograft bone retains its anti-bacterial activity for times far longer than classical elution systems.• Allograft bearing covalently-bonded vancomycin retains active antibiotic for times approximating bone allograft replacement (3-6 months)• Osteoblastic-cell morphology and expression of maturation markers are indistinguishable between control allograft and allograft bearing covalently-bonded vancomycin.

Infection is an important medical problem associated with the use of bone allografts. To retard bacterial colonization, we have recently reported on the modification of bone allografts with the antibiotic vancomycin (VAN). In this report, we examine the ability of this antibiotic-modified allograft to resist bacterial colonization and biofilm formation. When antibiotic was coupled to the allograft, a uniform distribution of the antibiotic was apparent. Following challenges with Staphylococcus aureus for 6 h, the covalently bonded VAN decreased colonization as a function of inoculum, ranging from 0.8 to 2.0 log 10 CFU. Furthermore, the VAN-modified surface resisted biofilm formation, even in topographical niches that provide a protected environment for bacterial adhesion. Attachment of the antibiotic to the allograft surface was robust, and the bonded VAN was stable whether incubated in aqueous media or in air, maintaining levels of 75 to 100% of initial levels over 60 days. While the VAN-modified allograft inhibited the Gram-positive S. aureus colonization, in keeping with VANs spectrum of activity, the VAN-modified allograft was readily colonized by the Gram-negative Escherichia coli. Finally, initial toxicity measures indicated that the VAN-modified allograft did not influence osteoblast colonization or viability. Since the covalently tethered antibiotic is stable, is active, retains its specificity, and does not exhibit toxicity, it is concluded that this modified allograft holds great promise for decreasing bone graft-associated infections.

Infection associated with inert implants is complicated by bacterial biofilm formation that renders the infection antibiotic insensitive. The goal of this investigation was to synthesize and characterize a vancomycin (VAN)-modified bone allograft that could render the tissue inhospitable to bacterial colonization and the establishment of infection. We found that the numbers of primary amines, which could serve as anchors for chemical synthesis, increased with limited demineralization. Using these amines, we coupled two linkers and VAN to bone using Fmoc chemistry. By immunohistochemistry, VAN was abundant on the surface of the allograft; based on elution and measurement of bound antibody, this coupling yielded at least approximately 26 ng VAN/mg bone. The coupled VAN appeared to be permanently bound to the allograft, as it showed no elution in a disk diffusion assay, and, importantly, resisted colonization by Staphylococcus aureus challenges. We suggest that this chimeric construct represents a new generation of antibiotic-modified allografts that provide antibacterial properties.

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