Simultaneous Delivery of Multiple Antibacterial Agents from Additively Manufactured Porous Biomaterials to Fully Eradicate Planktonic and Adherent Staphylococcus aureus

Abstract

Implant-associated infections are notoriously difficult to treat and may even result in amputation and death. The first few days after surgery are the most critical time to prevent those infections, preferably through full eradication of the micro-organisms entering the body perioperatively. That is particularly important for patients with a compromised immune system such as orthopedic oncology patients, as they are at higher risk for infection and complications. Full eradication of bacteria is, especially in a biofilm, extremely challenging due to the toxicity barrier that prevents delivery of high doses of antibacterial agents. This study aimed to use the potential synergistic effects of multiple antibacterial agents to prevent the use of toxic levels of these agents and achieve full eradication of planktonic and adherent bacteria. Silver ions and vancomycin were therefore simultaneously delivered from additively manufactured highly porous titanium implants with an extremely high surface area incorporating a bactericidal coating made from chitosan and gelatin applied by electrophoretic deposition (EPD). The presence of the chitosan/gelatin (Ch+Gel) coating, Ag, and vancomycin (Vanco) was confirmed by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). The release of vancomycin and silver ions continued for at least 21 days as measured by inductively coupled plasma (ICP) and UV-spectroscopy. Antibacterial behavior against Staphylococcus aureus, both planktonic and in biofilm, was evaluated for up to 21 days. The Ch+Gel coating showed some bactericidal behavior on its own, while the loaded hydrogels (Ch+Gel+Ag and Ch+Gel+Vanco) achieved full eradication of both planktonic and adherent bacteria without causing significant levels of toxicity. Combining silver and vancomycin improved the release profiles of both agents and revealed a synergistic behavior that further increased the bactericidal effects.

Keywords: additive manufacturing; antibacterial surfaces/coatings; electrophoretic deposition; hydrogels; multifunctional biomaterials; porous implants.

Figure 1

(a) Macrographs of porous titanium specimens (scale bar, 1 mm).

Figure 2

Optical microscopy image of the coated porous titanium: (a) 5×, (b) 100×. The cross section of a coated specimen. EPD enables application of hydrogels as a filler as well as a coating: (c) 5×, (d) 100× (scale bar, 1 mm).

Figure 3

SEM pictures of the coated specimens show successful deposition of the polymer: (a) 120×, (b) 1000×. A selected spot at which the coated and noncoated parts of the specimen could be concurrently seen in order to estimate the coating thickness: (c) fresh coating (1000×); (d) after 28 days (1500×).

Figure 4

FTIR spectra of chitosan and gelatin polymeric matrix.

Figure 5

XPS spectra of the specimens from (a) AsM, (b) Ch+Gel, (c) Ch+Gel+Ag, (d) Ch+Gel+Vanco, (e) Ch+Gel+Ag+Vanco groups, and (f) high resolution spectra of silver.

Figure 6

Release profile of vancomycin (a) and silver ions (b) from the different groups and comparison with the MIC line.

Figure 7

Alamar blue assay results for the specimens from different groups for up to 7 days.

Figure 8

Live/dead images on MG63 osteoblast cells for all the experimental groups after 2 days (scale bar 200 μm): (a) AsM, (b) Ch+Gel, (c) Ch+Gel+Ag, (d) Ch+Gel+Vanco, (e) Ch+Gel+Ag+Vanco.

Figure 9

Short-term (a) and long-term (b) killing effect against planktonic bacteria as well as the short-term killing effect against adherent bacteria (c).