Decreased Staphylococcus epidermis adhesion and increased osteoblast functionality on antibiotic-loaded titania nanotubes

Abstract

Bacterial infection is one of the most common problems after orthopedic implant surgery. If not prevented, bacterial infection can result in serious and life threatening conditions such as osteomyelitis. Thus, in order to reduce chances of such serious complication, patients are often subjected to antibiotic drug therapy for 6–8 weeks after initial surgery. The antibiotics are systemically delivered either intravenously, intramuscularly or topically. Systemic antibiotic delivery entails certain drawbacks such as systemic toxicity and limited bioavailability. Further, in order for the drug to be effective at the site of implantation, high doses are required, which can result in undesired side effects in patients. Thus, local antibiotic therapy is the preferred way of administering drugs. To that end, we have developed titania nanotubular arrays for local delivery of antibiotics off-implant at the site of implantation. These nanotubes were fabricated on bulk titanium using anodization techniques. The fabrication strategies allow us to precisely control the nanotube length and diameter, thus enabling us to load different amounts of drugs and control the release rates. In this work we have fabricated titania nanotubes with 80 nm diameter and 400 nm length. We have loaded these tubes with 200, 400 and 600 μg of gentamicin. The gentamicin release kinetics from these nanotubes and its effect on Staphylococcus epidermis adhesion were investigated. Further, a preosteoblastic cell line called MC3T3-E1 was cultured on gentamicin-loaded nanotubes to evaluate the effect of nanoarchitecture on cell functionality. Our results indicate that we can effectively fill the nanotubes with the drug and the drug eluting nanotubes significantly reduce bacterial adhesion on the surface. Also, there is enhanced osteoblast differentiation on nanotubes filled with gentamicin.

Introduction

The number of patients in need of an internal fixation device or artificial joint in the United States has grown rapidly in last decade. It is expected that by 2010 more than 4.4 million people will have at least one internal fixation device and more than 1.3 million people will have an artificial joint [1]. The success of these implants depends not only on the bone–implant integration, but also on the presence of a sterile environment around the implant, which will prevent bacterial infection. Infection is one of the most serious complications that may arise after orthopedic implant surgery. Acute infection or chronic osteomyelitis develops in as many as 5–33% of implant surgeries despite the use of strict antiseptic operative procedures employed [2], [3], [4], [5]. When a bone becomes infected, the soft, inner part (bone marrow) often swells. As the swollen tissue presses against the rigid outer wall of the bone, the blood vessels in the bone marrow may become compressed, which reduces the blood supply to the bone. Without an adequate blood supply, parts of the bone may die. The infection can also spread outward from the bone to form collections of pus (abscesses) in adjacent soft tissues, such as the muscle. This phenomenon drastically reduces the patient's recovery process after implant surgery [6]. Although infection is not a common reason for implant failure, antibiotic treatment is usually prescribed to patients to prevent any complications that may arise after implant surgery.

Gentamicin is a commonly used antibiotic to prevent bacterial infection around the implant. It is an aminoglycoside antibiotic, and can treat many types of bacterial infections, particularly Gram-negative infection. It works by binding the 30S subunit of the bacterial ribosome, interrupting protein synthesis. It is also one of the few heat-stable antibiotics that remain active even after autoclaving, which makes it particularly useful in the preparation of certain microbiological growth media. Like all aminoglycosides, when gentamicin is given orally, it is not effective. This is because it is absorbed from the small intestine, and then travels through the portal vein to the liver, where it is inactivated. Therefore, it can only be given intravenously, intramuscularly or topically. Delivery with these routes is often not effective because the drug cannot readily reach the infection site in bone tissue, particularly in necrotic or avascular tissue left after surgery. This limitation cannot be overcome with increased systemic doses because of organ toxicity associated with antibiotics at higher concentrations. Thus local antibiotic therapy has become an accepted and common adjunct to systemic antibiotic to prevent infection. This not only offers the advantages of a high local antibiotic concentration without any systemic toxicity but also an effective way of delivering antibiotics right at the site of implantation. Several techniques have been proposed in literature to prevent infection around the implant. For example, traditional bone cements can be loaded with antibiotics [7], [8], [9]. However, the current trend in orthopedic implants is using cementless implants. Alternatively, there has been investigation into the covalent attachment of polycationic groups [10], [11], implantation of Ca⁺, N⁺, F⁺ ions [12], [13] and coating of implant surfaces with polymers loaded with drugs [14], [15], [16]. However, there are several shortcomings of these proposed techniques including limited chemical stability, local inflammatory reactions due to material composition, and lack of controlled release kinetics from coatings.

In this work, titania nanotubular interfaces have been grown on bulk titanium using a simple anodization process. Titania (native oxide TiO₂ on the surface of Ti) has been used in prosthetic devices since the 1970s. As a biocompatible material, titanium and its alloys, particularly Ti–6Al–4V is extensively used in orthopedic and dental implants. The biocompatibility of metal-oxides has already been proven as the materials have current clinical applications in orthopedic prostheses and dental implants [17]. There have been several studies reported on using nanotubes for bone tissue engineering applications [18], [19], [20]. We have also previously demonstrated that nanotubular surfaces enhance matrix production from osteoblasts [21]. In this paper we investigate the ability to control antibiotic release from the nanotubes to prevent bacterial adhesion while maintaining the osseointegrative properties of the nanostructured surface.