Infection poses one of the greatest medical challenges, one further complicated by bacterial biofilm formation that renders the infection antibiotic insensitive. The goal of this investigation was to covalently link the antibiotic vancomycin (VAN) to a bone allograft so as to render the tissue inhospitable to bacterial colonization and the subsequent establishment of infection. We could achieve uniform tethering of the antibiotic to the allograft with minimal disruption of the underlying bone structure. The tethered VAN remained active against gram-positive organisms with no detectable S.aureus colonization. Additionally, the grafted VAN prevented biofilm formation, even in protected topographical niches. Attachment of the antibiotic to the allograft surface was robust-the stabilized VAN remained active for long time periods. Osteoblasts cultured on the VAN-allograft evidenced no changes in cellular phenotype. We opine that this new chimeric construct represents a superior transplantable substrate with a plethora of applications in medicine, dentistry and surgery.

Use of allograft bone has become standard for bridging defects unlikely to heal by simple fixation and routinely used in revision arthroplasties for implant stabilization. Unfortunately, this decellularized allograft provides an ideal surface for bacterial colonization, necessitating repeated surgeries, extensive debridement and lengthy antibiotic treatments. With up to 18% infection rate following allograft surgeries, a need for more effective means to prevent this process is evident. We describe a novel modification of native bone allografts that renders their surface bactericidal while increasing the effectiveness of systemic antibiotic treatments.

Allograft modification: Morselized human bone was washed extensively and sequentially coupled: 2X with Fmoc-aminoethoxyethoxyacetate (Fmoc-AEEA); deprotected with 20% piperidine in Dimethylformamide (DMF); and then coupled with vancomycin (VAN) for 12–16 hours. The VAN-bone was washed extensively with DMF and PBS for at least 1 day. VAN immuno-fluorescence: Control or VAN-bone was washed 5X with PBS, blocked with 10% FBS (1hr), incubated with rabbit anti-VAN IgG (4oC, 12h) followed by an Alexa-Fluor 488-coupled goat anti-rabbit IgG (1hr), and visualized by confocal laser microscopy. Antibiotic Activity. Equal dry weights of control and VANbone were sterilized with 70% ethanol, rinsed with PBS, and incubated with either Staphylococcus aureus (S. aureus) or Escherichia Coli (Ci=104 cfu) in TSB, 37oC, for 2, 5, 8 and 12 hrs. Antibiotic treatment: Clinical grade vancomycin was added to the solution with bacteria or following infection at a final concentration of 10μg/ml. 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. Bacterial visualization: Non-adherent bacteria were removed by washing extensively with PBS and adherent bacteria stained with the Live/Dead BacLight Kit (20mins, RT) to cause viable bacteria to fluoresce green. Samples were visualized by confocal microscopy.

In comparison to controls, VAN-bone consistently reduced the graft bacterial load by ~90% at all time points. After staining and visualization of adherent bacteria, biofilm formation was apparent on controls by 12 hrs and absent from VAN-bone. E.coli, a gramnegative organism that is not sensitive to VAN, readily colonized both control and VANbone, confirming retention of VAN specificity. We then evaluated VAN-bone activity in a system that modeled systemic antibiotic therapy and antibiotic prophylaxis. In the absence of solution antibiotics, VAN-bone exhibited a significant decrease in bacterial colonization as compared to controls. When 10 μg/ml VAN was added to the medium for the last 4 h (modeling systemic antibiotic therapy), colonization of control surfaces was reduced, while colonization of VAN-allograft was almost eliminated. When 10 μg/ml VAN was added concomitantly with S. aureus, VAN-bone colonization was undetectable, while colonization of control surfaces still occurred.

We have previously described an antibiotic-tethered allograft that resists bacterial colonization. In this abstract, we test this technology with an vitro model of bone implantation in the presence of solution antibiotics. In these models, solution antibiotics failed to prevent infection of control bone while completely clearing the bacteria on VAN-bone. Furthermore, VAN bone exhibited high activity against S. aureus, a gram positive organism, whereas it was ineffective against E. coli, a gram negative organism. The specificity of the tethered antibiotic supported the view that the antibacterial properties of the allograft were related to the tethered antibiotic and not to undefined aspects of the attachment chemistry. In terms of antibacterial activity, when challenged with 104 CFU S. aureus (with concentrations reaching > 107 CFU by 24 h), the antibiotic -modified allograft consistently decreased bacterial colonization by > 90%; S. aureus inocula < 102 CFU resulted in no detectable colonization of the VAN-allograft. Thus, development of these allografts may not only combat allograft colonization but increase the effectiveness of prophylactic antibiotics to ultimately result in a new therapy for allograft-associated infection.

One of the routinely used intraoperative tests for diagnosis of periprosthetic infection (PPI) is Gram stain that is reported to carry a very high specificity and a poor sensitivity. However, it is not known if the result of this test can vary according to the type of joint affected or the number of specimen samples collected. This study intended to examine the role of this diagnostic test in a large cohort of patients from single institution.

A review of our joint registry database revealed that 453 total knee arthroplasty (TKA) and 551 total hip arthroplasty (THA) of which 171 and 150 cases were respectively infected underwent revision surgery during 2000–2005 and had intraoperative cultures available for interpretation. A positive gram stain was defined as the visualisation of bacterial cells or ‘many leukocytes’ (> 5 per high power field) under the smear. The sensitivity, specificity, and predictive values of each individual diagnostic arm of Gram stain were determined. Combinations were performed in series that require both tests to be positive to confirm infection and in parallel that necessitate both tests to be negative to rule out infection. This analysis was performed for THA and TKA separately and later compared for each joint type.

The presence of organism cells and ‘many’ neutrophils on a Gram smear had high specificity (98%–100%) and positive predictive value (89%–100%) in both THA and TKA. The sensitivities (30%–50%) and negative predictive values (70%–79%) of the two tests were low as expected among both joint types. When the two tests were combined in series the specificity and positive predictive value were absolute (100%). The sensitivity (43%–64%) and the negative predictive value (82%) improved among both THA and TKA.

The presence of organisms or ‘many’ leukocytes on the Gram smear can confirm PPI in TJA. As expected, the sensitivity and negative predictive value of the two tests were low, and therefore infection could not be safely ruled out. Although the two diagnostic arms of Gram stain can be combined to achieve improved negative predictive value (82%), Gram stain continues to have poor value in ruling out PPI. With the advances in the field of molecular biology, novel diagnostic modalities need to be designed that can replace these traditional and poor tests.