Authors: McKinley C. Lawson*, Kristi S. Anseth, Christopher N. Bowman
Title: A Novel Antibacterial Polymer: Surface Modification of Ti-6Al-4V Orthopaedic Alloy
Addresses: University of Colorado Department of Chemical and Biological Engineering, University of Colorado Medical Scientist Training Program, Campus Box 424, University of Colorado, Boulder, CO 80309
Purpose: The purpose of this report is to describe a series of experiments in which a new antibacterial monomer was synthesized, chemically and biologically characterized, and photochemically polymerized to Ti-6Al-4V alloy to produce a bactericidal coating.
Methods: Vancomycin was chemically modified by the addition of monoacrylated poly (ethylene glycol). The newly synthesized monomer (termed VPA for vancomycin-PEG-acrylate) was purified by gel filtration chromatography, and its chemical structure was characterized by 1H NMR spectroscopy. The MIC and MBC of the monomer were determined by the broth dilution method with S. epidermidis ATCC 12228.
Ti-6Al-4V alloy was oxidized by treatment with a solution of concentrated sulfuric acid and hydrogen peroxide. It was then reacted with methacryloxypropyltrimethoxysilane to form a monolayer having exposed photo-reactive groups (methacrylates). Surfaces were characterized using x-ray photoelectron spectroscopy (XPS) and scanning electron microscopy.
VPA monomer was mixed with PEG (375) monoacrylate and a suitable photoinitiator to form a grafting solution for photochemical polymerization. Aliquots of the grafting solution were applied to silanized or unsilanized Ti-6Al-4V discs and polymerized by UV-irradiation. PEG (375) monoacrylate coatings were used as negative controls for biological assays.
The ability of the silane to prevent polymer delamination was assessed by extended wash in either methanol or distilled water. Extended washes were additionally employed to elute unreacted monomer and loosely held polymer chains. A variation of the Kirby-Bauer disc diffusion assay was implemented to temporally define this elution. Polymer-coated discs having undergone differing wash cycles were inverted on bacterial lawns, and zones of inhibition were measured.
Once elution had measurably stopped, subsequent determination of surface-contact-mediated antibacterial action was made. S. epidermidis was suspended in media, and 1 x 10^5 colony forming units (cfu) were allowed to settle onto the polymer-coated surfaces. Following incubation, the suspensions were sampled, diluted, and plated for cfu counts.
Results: VPA was purified by gel filtration chromatography and found to be of ca. 5,000 molecular weight based on column elution time (single adduct product). The product demonstrated structural features of vancomycin and PEG-acrylate by 1H NMR. VPA maintained activity against S. epidermidis in terms of MIC and MBC.
XPS showed that the Ti-6Al-4V oxidation protocol thickened the oxide layer, thus increasing the sites available for silane reaction. Silanization was successful as XPS showed silicon and an attenuated Ti signal. VPA coated surfaces were found to be resistant to delamination when the alloy was silanized, but oxide-only surfaces resulted in rapid delamination.
VPA and loosely-held polymer chains were found to elute from coatings for ca. two days--no zone of inhibition was observed by the modified disc diffusion assay for discs having undergone a three day wash. Wash solutions demonstrated no antibacterial action.
The polymer-coated surfaces possessed surface-contact-mediated bacterial killing after antibacterial elution stopped. A time-dependent bactericidal action was observed. The bacterial load was reduced by ca. 99.5% with respect to the initial inoculum after 15 hours.
Discussion: Though modern surgical practice has reduced the incidence of infection following total hip arthroplasty, for example, to approximately 1-2%, those patients with periprosthetic joint infection must undergo lengthy antibiotic therapy and often surgical revision. The physiologic location of these infections makes them inherently difficult to treat due to poor antibiotic penetration into bone and joint spaces and the formation of bacterial biofilms on implant surfaces. Less than ideal treatment options present definite challenges to both surgeon and patient.
Here we describe a novel means of photochemically polymer-coating orthopaedic Ti-6Al-4V alloy with the new material vancomycin-PEG-acrylate. The covalently attached coating has bactericidal properties and does not require release of an active agent. The material offers a large increase in available drug when compared to monolayer coatings and provides multiple layers of antibacterial activity should the outermost layers be removed. The polymerization technique described can be well-controlled to yield complex, biologically active co-polymer constructs. It is anticipated that this approach will be useful in the treatment of periprosthetic infections and osteomyelitis.
