Preventing infection of osseointegrated transcutaneous implants: Incorporation of silver into preconditioned fibronectin-functionalized hydroxyapatite coatings suppresses Staphylococcus aureus colonization while promoting viable fibroblast growth in vitro

The success of transcutaneous implants depends on the achievement of a soft tissue seal by enabling fibroblasts to win the race for the surface against bacteria. Fibronectin-functionalized hydroxyapatite coatings (HAFn) have been shown to improve dermal tissue ingrowth and attachment. However, during the early postoperative period before a soft tissue seal has formed, bacterial colonization may occur. This study explored the incorporation of silver, a broad spectrum antimicrobial agent, into HAFn coatings with the aim of reducing bacterial colonization. Silver is known to have dose-dependent cytotoxic effects. Therefore, the effects of silver incorporation into HAFn coatings on both in vitro human dermal fibroblast viability and Staphylococcus aureus colonization were assessed. An electrochemical deposition technique was used to codeposit hydroxyapatite and silver (HAAg) and fibronectin was adsorbed onto this to produce HAAgFn coatings. Surfaces were preconditioned with serum to mimic the in vivo environment. Nonpreconditioned HAAg and HAAgFn coatings suppressed bacterial colonization but were cytotoxic. After serum-preconditioning, more than 90% of fibroblasts that grew on all HAAg and HAAgFn coatings were viable. The highest silver content coatings tested (HAAg100 and HAAgFn100) resulted in a greater than 99% reduction in biofilm and planktonic bacterial numbers compared to HA and HAFn controls. Although HAAg100 had greater antibacterial activity than HAAgFn100, the findings of this study indicate that fibroblasts would win the race for the surface against S aureus on both HAAg100 and HAAgFn100 after serum-preconditioning

In Vitro Interactions between Bacteria, Osteoblast-Like Cells and Macrophages in the Pathogenesis of Biomaterial-Associated Infections

Biomaterial-associated infections constitute a major clinical problem that is difficult to treat and often necessitates implant replacement. Pathogens can be introduced on an implant surface during surgery and compete with host cells attempting to integrate the implant. The fate of a biomaterial implant depends on the outcome of this race for the surface. Here we studied the competition between different bacterial strains and human U2OS osteoblast-like cells (ATCC HTB-94) for a poly(methylmethacrylate) surface in the absence or presence of macrophages in vitro using a peri-operative contamination model. Bacteria were seeded on the surface at a shear rate of 11 1/s prior to adhesion of U2OS cells and macrophages. Next, bacteria, U2OS cells and macrophages were allowed to grow simultaneously under low shear conditions (0.14 1/s). The outcome of the competition between bacteria and U2OS cells for the surface critically depended on bacterial virulence. In absence of macrophages, highly virulent Staphylococcus aureus or Pseudomonas aeruginosa stimulated U2OS cell death within 18 h of simultaneous growth on a surface. Moreover, these strains also caused cell death despite phagocytosis of adhering bacteria in presence of murine macrophages. Thus U2OS cells are bound to loose the race for a biomaterial surface against S. aureus or P. aeruginosa, even in presence of macrophages. In contrast, low-virulent Staphylococcus epidermidis did not cause U2OS cell death even after 48 h, regardless of the absence or presence of macrophages. Clinically, S. aureus and P. aeruginosa are known to yield acute and severe biomaterial-associated infections in contrast to S. epidermidis, mostly known to cause more low-grade infection. Thus it can be concluded that the model described possesses features concurring with clinical observations and therewith has potential for further studies on the simultaneous competition for an implant surface between tissue cells and pathogenic bacteria in presence of immune system components

Bacterial biofilm formation versus mammalian cell growth on titanium-based mono- and bi-functional coating

Biomaterials-associated-infections (BAI) are serious complications in modern medicine. Although non-adhesive coatings, like polymer-brush coatings, have been shown to prevent bacterial adhesion, they do not support cell growth. Bi-functional coatings are supposed to prevent biofilm formation while supporting tissue integration. Here, bacterial and cellular responses to poly(ethylene glycol) (PEG) brush-coatings on titanium oxide presenting the integrin-active peptide RGD (arginine-glycine-aspartic acid) (bioactive “PEG-RGD”) were compared to mono-functional PEG brush-coatings (biopassive “PEG”) and bare titanium oxide (TiO2) surfaces under flow. Staphylococcus epidermidis ATCC 35983 was deposited on the surfaces under a shear rate of 11 s-1 for 2 h followed by seeding of U2OS osteoblasts. Subsequently, both S. epidermidis and U2OS cells were grown simultaneously on the surfaces for 48 h under low shear (0.14 s-1). After 2 h, staphylococcal adhesion was reduced to 3.6±1.8 × 103 and 6.0±3.9 × 103 cm-2 on PEG and PEG-RGD coatings respectively, compared to 1.3±0.4 × 105 cm-2 for the TiO2 surface. When allowed to grow for 48 h, biofilms formed on all surfaces. However, biofilms detached from the PEG and PEG-RGD coatings when exposed to an elevated shear (5.6 s-1) U2OS cells neither adhered nor spread on PEG brush-coatings, regardless of the presence of biofilm. In contrast, in the presence of biofilm, U2OS cells adhered and spread on PEG-RGD coatings with a significantly higher surface coverage than on bare TiO2. The detachment of biofilm and the high cell surface coverage revealed the potential significance of PEG-RGD coatings in the context of the “race for the surface” between bacteria and mammalian cells

Magnetic targeting of surface-modified superparamagnetic iron oxide nanoparticles yields antibacterial efficacy against biofilms of gentamicin-resistant staphylococci

Biofilms on biomaterial implants are hard to eradicate with antibiotics due to the protection offered by the biofilm mode of growth, especially when caused by antibiotic-resistant strains. Superparamagnetic iron oxide nanoparticles (SPIONs) are widely used in various biomedical applications, such as targeted drug delivery and magnetic resonance imaging. Here, we evaluate the hypothesis that SPIONs can be effective in the treatment of biomaterial-associated infection. SPIONs can be targeted to the infection site using an external magnetic field, causing deep penetration in a biofilm and possibly effectiveness against antibiotic-resistant strains. We report that carboxyl-grafted SPIONs, magnetically concentrated in a biofilm, cause an approximately 8-fold higher percentage of dead staphylococci than does gentamicin for a gentamicin-resistant strain in a developing biofilm. Moreover, magnetically concentrated carboxyl-grafted SPIONs cause bacterial killing in an established biofilm. Thus magnetic targeting of SPIONs constitutes a promising alternative for the treatment of costly and recalcitrant biomaterial-associated infections by antibiotic-resistant strains