Implant Modification with Vancomycin and RGD to Decrease Prosthesis Failure

1Ketonis, C; 1Chen, C-P; 1Shapiro, I; 1Adams, C; 1,2 Parvizi, J; +1Hickok, N 1Thomas Jefferson University, Philadelphia, PA, 2Rothman Orthopedic Institute, Philadelphia, PA

Senior author: Noreen.Hickok@jefferson.edu INTRODUCTION: Total joint arthroplasty (TJA) is a very successful procedure with 90-98% survivorship when using revision as an endpoint. By far, the main reason of failure is aseptic loosening of the prosthesis whereas the most challenging and devastating complication is periprosthetic infection (PPI). Even though many attempts have been made to combat these problems, they still cause increased patient morbidity and large economic burdens on the healthcare system. We have developed a new surface that attempts to address these issues through increased osteointegration and resistance to bacterial colonization. METHODS: Modification of Ti alloy: 1cm2 Ti90Al6V4 (Ti alloy) foils were cleaned in 1 M NaOH, MeOH/HCl (1:1), distilled deionized water (dH2O), and dimethylformamide (DMF). Surfaces were passivated by treatment with H2O2/concentrated H2SO4 (70:30) for 4 h, 4°C. Passivated foils were reacted with 5% (v/v) aminopropyltriethoxysilane (APTS) in anhydrous toluene under argon with stirring, 100oC, 5h, and the aminopropylated foils coupled with excess Fmoc-aminoethoxyethoxyacetate (Fmoc-AEEA), deprotected with 20% piperidine in DMF, and reacted with a maleimide linker. SH-modified vancomycin (VAN) and cyclic RGD (1:1 molar ratio) were reacted with the maleimide linker for 16hrs. Samples were thoroughly washed with DMF, followed by PBS and dried under vacuum before use. The same process was used to attach the neutral peptide RGE to the foils to serve as a control for the RGD foils. VAN immunofluorescence: Control or VAN/RGD derivatized foils (VR-foil) were washed 5X with PBS, blocked with 10% FBS (1hr), incubated with rabbit anti-VAN IgG (4oC, 12h) and AlexaFluor 488-coupled goat anti-rabbit IgG (1hr), and visualized by confocal laser microscopy. Antibiotic Activity: VR or control foils were sterilized with 70% ethanol, rinsed with PBS, and incubated with S. aureus (Ci=104 cfu) in trypticase soy broth (TSB), 37oC, for 4 hrs. Non-adherent bacteria were removed by washing with PBS and the remaining adherent bacteria were stained with the Live/Dead BacLight Kit (20mins, RT) to cause viable bacteria to fluoresce green. Samples were visualized by confocal microscopy. Cell Attachment: VR or control foils were sterilized with 70% ethanol and UV radiation, and 150,000 Human Fetal Osteoblasts (hFOBs) were allowed to adhere for 15 mins or 1 hr, loose cells were detached with PBS and the DNA content of the remaining attached cells were measured using a PicoGreen assay. Biocompatibility: hFOBs were allowed to attach to sterile VR, VAN-RGE and control foils for 3 days, fixed, permeabilized with 0.1% Triton-X-100. The actin cytoskeleton was stained with rhodamine-phalloidin, counterstained with SYBR-green and visualized with confocal microscopy. RESULTS: Bactericidal activity: VR foils were incubated with S.aureus, and after removing non-adherent bacteria, little to no fluorescence was detected on the VR foils, indicating that bacteria failed to colonize the surface. In contrast, control foils exhibited abundant bacterial colonization with intense fluorescence where the individual colonies had organized into biofilms.

Fig 1: VR foils resist bacterial colonization. Foils were incubated with Ci=104 cfu S. aureus for 4 hrs and viable bacteria stained with a green fluorescent dye. VR foils (right) resist bacterial colonization as opposed to controls (left) that exhibit diffuse green fluorescence indicative of abundant bacterial colonization

Cell attachment: hFOBs were allowed to attach on VR and RGE foils for 15 mins and 1 hr. Non-adherent cells were removed by washing and the DNA content of adherent cells measured using a PicoGreen assay. At 15 min, both surfaces appeared to have equal numbers of cells as determined by equivalent fluorescent intensities. At 1 h, a higher signal was measured from the VR foils, suggesting increased hFOB adhesion over the RGE foils. This result suggested that the VR foils was facilitating cellular attachment.

Fig 2: VR-Foils facilitate cellular attachment. hFOBS were allowed to attach to VR or RGE foils and DNA content measured to determine relative adherence. No apparent differences were detected between the two surfaces at 15mins but attachment was higher on VR foils at 1 hr. Biocompatibility: Finally, we assessed if the presence of VAN combined with RGD altered the biocompatibility of the Ti surface. hFOBS were seeded on foils, fixed and stained with rhodamine-phalloidin and SYBR green. Cells readily attached to control, RR and RGE surfaces and exhibited an actin cytoskeleton that was predominately stained in the cortical region, with many cells exhibiting microspikes. Overall, Control, RGE and VR surfaces were roughly equivalent in their ability to maintain normal cytoskeletal morphology.

Fig 3: VR surfaces are biocompatible. hFOBS were seeded on Control, RGE and VR surfaces and stained with rhodamine-phalloidin to reveal cytoskeletal morphology and SYBR green to visualize nuclei. All samples exhibited a normal well-spread cell morphology. The actin cytoskeleton was predominantly cortical, with no apparent differences between the three surfaces DISCUSSION: In this study, we propose an implant modification to address two of the main problems of implant failure: loosening and periprosthetic infection. This implant covalently displays antibiotics and an osteoinductive RGD peptide. We have shown that modified Ti implants are able to resist bacterial colonization when challenged with S. aureus and that they increase the attachment of musculoskeletal cells. The modification, even though noxious to bacteria, does not affect the biocompatibility of mammalian cells with these cells exhibit normal cytoskeletal architecture and morphology. This proposed modification signifies a promising start in the efforts to decrease implant failure. ACKNOLEDGEMENTS: We thank the NIH (grants DE-13319, DE-10875, and AR-051303) and the Department of Defense (grant DAMD17-03-1-0713)

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Poster No. 2251 • 55th Annual Meeting of the Orthopaedic Research Society