Multilayered coating on titanium for controlled release of antimicrobial peptides for the prevention of implant-associated infections.

Prevention of bacterial colonization and formation of a bacterial biofilm on implant surfaces has been a challenge in orthopaedic surgery. The treatment of implant-associated infections with conventional antibiotics has become more complicated by the emergence of multi-drug resistant bacteria. Antimicrobial eluting coatings on implants is one of the most promising strategies that have been attempted. This study reports a controlled release of an antimicrobial peptide (AMP) from titanium surface through a non-cytotoxic multilayered coating. Three layers of vertically oriented TiO2 nanotubes, a thin layer of calcium phosphate coating and a phospholipid (POPC) film were impregnated with a potent broad-spectrum AMP (HHC-36). The coating with controlled and sustained release of AMP was highly effective against both Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria. No cytotoxicity to osteoblast-like cells (MG-63) was observed. Moderate platelet activation and adhesion on the implant surface with no observable activation in solution, and very low red blood cell lysis was observed on the implant. This multi-layer assembly can be a potential approach to locally deliver AMPs to prevent peri-implant infection in orthopaedics without being toxic to host cells.

Local delivery of antimicrobial peptides using self-organized TiO2 nanotube arrays for peri-implant infections.

Peri-implant infections have been reported as one of the major complications that lead to the failure of orthopedic implants. An ideal solution to the peri-implant infection is to locally deliver antimicrobial agents through the implant surface. The rising problem of infections caused by multiple antibiotic-resistant bacteria makes traditional antibiotics less desirable for the prevention of peri-implant infections. One of the promising alternatives is the family of antimicrobial peptides (AMPs). In this study, we report the local delivery of AMPs through the nanotubular structure processed on titanium surface. Self-organized and vertically oriented TiO2 nanotubes, about 80 nm in diameter and 7 µm thick, were prepared by the anodization technique. HHC-36 (KRWWKWWRR), one of the most potent broad-spectrum AMPs, was loaded onto the TiO2 nanotubes via a simple vacuum-assisted physical adsorption method. Antimicrobial activity testing against Gram-positive bacterium, Staphylococcus aureus, demonstrated that this AMP-loaded nanotubular surface could effectively kill the bacteria (≈ 99.9% killing) and reduce the total bacterial number adhered to the surface after 4 h of culture. In vitro AMP elution from the nanotubes was investigated using liquid chromatography-mass spectrometry (LC-MS). The release profiles strongly depended on the crystallinity of the TiO2 nanotubes. Anatase TiO2 nanotubes released significantly higher amounts of AMP than amorphous nanotubes during the initial burst release stage. Both followed almost the same slow release profile from 4 h up to 7 days. Despite the differences in release kinetics, no significant difference was observed between these two groups in bactericidal efficiency.

Antimicrobial peptides on calcium phosphate-coated titanium for the prevention of implant-associated infections.

Prevention of implant-associated infections has been one of the main challenges in orthopaedic surgery. This challenge is further complicated by the concern over the development of antibiotic resistance as a result of using traditional antibiotics for infection prophylaxis. The objective of this study was to develop a technique that enables the loading and local delivery of a unique group of cationic antimicrobial peptides (AMP) through implant surfaces. A thin layer of micro-porous calcium phosphate (CaP) coating was processed by electrolytic deposition onto the surface of titanium as the drug carrier. The broad spectrum AMP Tet213 (KRWWKWWRRC) was selected and loaded onto the CaP coating. SEM, XRD and FTIR analyses confirmed the CaP coating to be micro-porous octacalcium phosphate. By using a luminescence spectrometer technique, it was demonstrated that a 7 µm thick porous CaP coating could load up to 9 µg of AMP/cm² using a simple soaking technique. The drug-loaded CaP coating (CaP-Tet213) was not cytotoxic for MG-63 osteoblast-like cells. The CaP-Tet213 implants had antimicrobial activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria with 106-fold reductions of both bacterial strains within 30 min as assessed by measuring colony-forming units (CFU). Repeated CFU assays on the same CaP-Tet213 specimen demonstrated retention of antimicrobial activity by the CaP-Tet213 surfaces through four test cycles. The susceptibility of bacteria to the CaP-Tet213 surfaces was also evaluated by assessing the inhibition of luminescence of P. aeruginosa containing a luxCDABE cassette at 4 h and 24 h with ∼92% and ∼77% inhibition of luminescence, respectively. It was demonstrated that CaP-Tet213 was a more efficient antimicrobial coating than CaP-MX226, CaP-hLF1-11 or CaP-tobramycin following incubation of CaP implants with equimolar concentrations of Tet213, the commercially developed antimicrobial peptide MX-226, hLF1-11 or tobramycin. A device coated with CaP-Tet213 could be a potential solution for the prevention of the peri-implant infection in orthopaedics.