Elsevier

Biomaterials

Volume 34, Issue 24, August 2013, Pages 5969-5977
Biomaterials

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

https://doi.org/10.1016/j.biomaterials.2013.04.036Get rights and content

Abstract

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.

Introduction

Titanium and titanium alloys are frequently used in orthopaedic implants because of their good biocompatibility and reliable mechanical properties [1]. However, the formation of a bacterial surface biofilm and compromised immunity at the implant/tissue interface may lead to persistent infections on and around titanium implants. Pathogens such as Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa can be acquired shortly after the surgical installation of implants or at a later stage (e.g. via a haematogenous route) [2]. The resulting infection is usually difficult to treat and in most cases, replacement of a prosthesis is the only remedy [3]. Moreover, the emergence of multi-drug resistant bacterium like methicillin-resistant S. aureus (MRSA) has critically challenged the use of conventional antibiotics [4]. Systemic administration of antimicrobial agents have several drawbacks such as the relatively low drug concentration at the target site and potential toxicity [5]. Hence, localized delivery of antimicrobial agents with time-effective handling of infection, while potentially eliminating problems associated with systemic administration, is highly desirable [6], [7].

The inhibition of organisms in a complex biofilm requires up to 1000-times the antibiotic dose necessary to combat bacteria in suspension [8]. An ideal local antibiotic release profiles should exhibit a high initial release rate within 6 h post implantation while the immune system is weakened/compromised leaving the implant susceptible to surface bacterial colonization, followed by a continuous ‘prophylactic’ slow release [8], [9]. Conventional antibiotics like vancomycin, tobramycin, and gentamicin have been incorporated in controlled release devices [9]. A serious concern regarding the use of these antibiotics is that the release at levels below the minimal inhibitory concentration (MIC) is likely to evoke bacterial resistance [10]. High doses of antibiotics often generate cell toxicity and may impair osteogenic activity [11]. A promising alternative to conventional antibiotics is the short cationic antimicrobial peptides (AMPs) [12]. AMPs have broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria, and are also known to be antifungal and antiviral [13]. Due to the complex mechanisms of AMPs bacteria are killed more rapidly than with conventional antibiotics and it is extremely difficult for bacteria to develop resistance [14], [15].

Calcium phosphate coatings and vertically aligned titania nanotubes (NT) are two platforms used for delivering drugs from orthopaedic implants [16], [17]. In our previous work, we successfully examined, in vitro and in vivo, the feasibility of using micro-porous CaP [18], [19], and self-organized and vertically oriented TiO2 nanotubes coatings [20] on Ti surfaces as carriers to deliver peptide HHC-36, one of the most potent broad-spectrum AMPs. Both strategies led to an initial burst release of HHC-36. And HHC-36 onto loaded CaP coated Ti showed no bone growth inhibition. However, the release rate in both systems was too fast, limiting the antimicrobial effects to early stage peri-implant infection [18], [19], [20]. The main objective of this study was to develop a layer-by-layer assembly of multi-layer thin films in order to encourage prolonged AMP release on Ti implants. To create a coating that had dual beneficial effects, i.e. antimicrobial and osteoconductive, thin layers of titania NT and CaP coatings were impregnated with AMPs. These films were topped with a thin phospholipid (POPC, palmitoyl-oleoyl phosphatidyl-choline) film to control the release of AMP based on a bio-inspired cell membrane [21], [22]. POPC is found naturally in eukaryotic cell membranes and offers the least support for bacteria growth (81% reduction), and the most suitable platform for bone cell attachment [23]. POPC has also been shown to exhibit clinically acceptable osteointegration [24].

Testing of the biocompatibility of coatings has generally been performed through in vitro assessment of the interaction of the coatings with recognized cell culture lines. However, this does not adequately address the acceptability of these materials in the blood interfacing environment, which consists of a fibrin film containing platelets and red blood cells and plays a significant role in osteogenesis [25], [26]. In this regard, platelet adhesion and activation on an implant surface and the surrounding fluid are critical steps in initiating osteoconduction [27], [28]. Therefore, it was also the purpose of this study to address the hemocompatibility of the multi-layer coating systems, using platelet adhesion, activation, and haemolysis studies.

Section snippets

Fabrication of TiO2 nanotubes on titanium surface

The commercially pure Ti foils (0.1 mm, 99.6% purity, Goodfellow) were consecutively sonicated in acetone, ethanol, and distilled water and then air dried. Titania nanotubes were prepared using anodization technique, in which Ti was used as the working electrode (anode), and platinum as the cathode. The TiO2 nanotubes were prepared in 75% glycerol (C3H8O3, Fisher Scientific, Canada) solution containing 0.27 m ammonium fluoride (NH4F, Fisher Scientific, Canada) at 30 V (DC power supply,

Characteristics of TiO2 nanotubes

The morphology of titania nanotubes after annealing is shown in Fig. 1a. The FE-SEM images show that titania nanotubes were about 2 μm long with a pore size of approximately 120 nm in diameter with pores oriented vertically to the sample surface. The nanotube array was uniformly distributed over the substrate. There were ripples observed on the side wall of the nanotubes due to thickness fluctuations along the nanotubes. This phenomenon is related to periodic oscillations of the current during

Discussion

The layer-by-layer technique for flat surfaces is a simple but promising method for coating biological and non-biological substrates impregnated with drugs and other biological substances to enable controlled release [41]. The ideal design of multilayered drug delivery systems as coatings on orthopaedic implants enabling the release of antimicrobal agents in a physiological environment, should meet certain requirements: (1) the selected antimicrobial agents should not promote the development of

Conclusions

Thin hydrophilic films impregnated with antimicrobial peptide were constructed in layer-by-layer films on titanium implants by creating titania nanotubes, coated with calcium phosphate crystals, and topped with a thin layer of phospholipid. Antimicrobial peptide was loaded into each layer. Utilizing a phopholipid layer as a barrier film enabled more controlled and sustained release of AMP with a first-order model providing the closest fit for the release kinetics. This means that the system was

Acknowledgements

This work was supported by the funding from Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR). The infrastructure facility at the Centre for Blood Research is supported by the Canada Foundation for Innovation, BCKDF. R.W. is incumbent of the Canada Research Chair in Biomaterials while REWH has a Canada Research Chair in Health and Genomics. JNK is a recipient of a Michael Smith Foundation for Health Research Career Scholar

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