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

Title: Antibiotic Attachment to Ti Alloy Surfaces Does Not Alter Their Topography

Institution: Thomas Jefferson University, Philadelphia PA

Purpose: We have covalently attached antibiotics to smooth Ti alloy surfaces to render anti-bacterial. We now ask if these modifications can be applied to different surfaces with retention of geometry and topography.

Methods: Synthesis: Titanium alloy (Ti6Al4V) wires and smooth/beaded discs were cleaned in Alconox,1M NaOH with intermittent shaking/sonication and HCl:MeOH. Cleaned surfaces were passivated using (1) H2SO4:H2O2 (70:30) or (2) autoclaving. These surfaces were reacted with aminopropyltriethoxysilane (APTS), and coupled with two aminoethoxyethylacetic acid (AEEA) linkers and a 4X molar excess of vancomycin (VAN). Passivation assessment: A 10ìl drop of dH2O was deposited on the surface of the discs and the static contact angle was recorded photographically..Scanning Electron Microscopy (SEM): Ti-VAN and control Ti alloy discs were extensively washed with dH2O and surface topography was visualized using a Hitachi TM-1000 SEM. Immunofluorescence: VAN coupled to discs and rods was detected with mouse anti-VAN IgG (12h, 4?C) followed by AlexaFluor 488-donkey anti-mouse IgG (1hr), and visualized using confocal microscopy. Bacterial Culture: Samples were sterilized in 70% EtOH, washed with Trypticase Soy Broth (TSB) and incubated with S. aureus in TSB (4h, 37?C) under static conditions. Non-adherent bacteria were removed by 3 TSB washes, and samples reincubated for 1 hr in fresh TSB. Non-adherent bacteria were then removed with 6 PBS washes, stained with the Live/Dead BacLightTM Viability Kit (live bacteria fluoresce green), and visualized by confocal microscopy.

Results: We asked if autoclaving successfully created a Ti oxide layer (passivation) by observing the static contact angle of a 10ìl drop of water. On controls, water remained in a drop both on smooth and beaded Ti. On autoclaved Ti, water spread to a thin film indicating an oxide layer. On piranha treated smooth Ti, the water still balled up, but with a smaller contact angle than the control. On both piranha and autoclave-treated beaded Ti, the water droplet completely spread into the crevices between the beads. Overall, autoclaving appeared to produce a better surface than piranha treatment. We then examined surface topography by SEM. Upon SEM, autoclaved discs looked identical to the controls whereas the piranha treated discs showed extensive pitting. VAN coverage caused no further topographical change for any of the surface treatments. Using autoclave passivation, we then derivatized the different Ti surfaces and tested for the presence of VAN. Uniform and complete VAN ! coverage was observed on all surfaces; no staining was apparent on controls. When challenged with S. aureus, Ti-VAN surfaces successfully inhibited bacterial adhesion, whereas control surfaces were extensively colonized with bacteria.

Discussion and Conclusion: Orthopedic implants may have a heterogeneous surface depending on their function, with smoother surfaces minimizing friction and rough surfaces providing stability/osteointegration; any modification of these surfaces that aims to render them resistant to bacterial colonization should retain these surface characteristics. In the present study, we have explored alternative methods of passivation that are both feasible on a large scale and applicable to surfaces used by implant manufacturers without alteration of topography. Importantly, these surface modifications are as efficacious as previously explored methods in rendering smooth, beaded Ti6Al4V surfaces and Kirshner wires, bactericidal. Such techniques can be applicable to other topographies and metals, such as stainless steel, to meet the many demands for orthopedic implants.

Musculoskeletal Infecton Society
Musculoskeletal Infecton Society