Macrophage phagocytic activity toward adhering staphylococci on cationic and patterned hydrogel coatings versus common biomaterials.

Biomaterial-associated-infection causes failure of biomaterial implants. Many new biomaterials have been evaluated for their ability to inhibit bacterial colonization and stimulate tissue-cell-integration, but neglect the role of immune cells. This paper compares macrophage phagocytosis of adhering Staphylococcus aureus on cationic-coatings and patterned poly(ethylene)glycol-hydrogels versus common biomaterials and stainless steel in order to identify surface conditions that promote clearance of adhering bacteria. Staphylococci were allowed to adhere and grow on the materials in a parallel-plate-flow-chamber, after which murine macrophages were introduced. From the decrease in the number of adhering staphylococci, phagocytosis-rates were calculated, and total macrophage displacements during an experiment determined. Hydrophilic surfaces had the lowest phagocytosis-rates, while common biomaterials had intermediate phagocytosis-rates. Patterning of poly(ethylene)glycol-hydrogel coatings increased phagocytosis-rates to the level of common biomaterials, while on cationic-coatings phagocytosis-rates remained relatively low. Likely, phagocytosis-rates on cationic coatings are hampered relative to common biomaterials through strong electrostatic binding of negatively-charged macrophages and staphylococci. On polymeric biomaterials and glass, phagocytosis-rates increased with macrophage displacement, while both parameters increased with biomaterial surface hydrophobicity. Thus hydrophobicity is a necessary surface condition for effective phagocytosis. Concluding, next-generation biomaterials should account for surface effects on phagocytosis in order to enhance the ability of these materials to resist biomaterial-associated-infection.

Conditions of lateral surface confinement that promote tissue-cell integration and inhibit biofilm growth.

Surfaces with cell adhesiveness modulated at micro length scales can exploit differences between tissue/bacterial cell size, membrane/wall plasticity, and adhesion mechanisms to differentially control tissue-cell/material and bacteria/material interactions. This study explores the short-term interactions of Staphylococcus aureus and osteoblast-like cells with surfaces consisting of cell-adhesive circular patches (1-5 µm diameter) separated by non-adhesive electron-beam patterned poly(ethylene glycol) hydrogel thin films at inter-patch distances of 0.5-10 µm. Osteoblast-like U2OS cells both bind to and spread on the modulated surfaces, in some cases when the cell-adhesive area comprises only 9% of the total surface and in several cases at least as well as on the continuously adhesive control surfaces. In contrast, S. aureus adhesion rates are 7-20 times less on the modulated surfaces than on the control surfaces. Furthermore, the proliferation of those bacteria that do adhere is inhibited by the lateral confinement imposed by the non-adhesive boundaries surrounding each patch. These findings suggest a new approach to create biomaterial surfaces that may promote healing while simultaneously reducing the probability of infection.

Phagocytosis of bacteria adhering to a biomaterial surface in a surface thermodynamic perspective.

Bacterial biofilms can increase the pathogenicity of infection and constitute a major problem in modern health-care, especially on biomaterial implants and devices. Biofilms are difficult to eradicate by the host immune system, even with antibiotics, and have been the number one cause of biomaterial implant and device failure for decades. Therefore, it is important to understand how immune cells interact with adhering pathogens. This study firstly aims to develop a simple method to quantify phagocytosis of six different strains of staphylococci adhering on a surface with phase-contrast-microscopy. Phagocytosis of adhering staphylococci to a glass surface by phagocytes was quantified in a parallel plate flow chamber, and expressed as a phagocytosis rate, accounting for the number of adhering staphylococci initially present and for the duration of phagocytosis. Murine macrophages were more effective in clearing staphylococci from a surface than human phagocytes, which require differentiation from their monocyte or promyelocytic state during an experiment. Direct visualization of internalization of a GFP-modified S. aureus strain inside phagocytes confirmed the validity of the method proposed. As a second aim, the differences in phagocytosis rates observed were investigated on a surface thermodynamic basis using measured contact angles of liquids on macroscopic lawns of staphylococci and phagocytes, confirming that phagocytosis of adhering pathogens can be regarded as a surface phenomenon. In addition, surface thermodynamics revealed that phagocytosis of adhering pathogens is determined by an interplay of physical attraction between pathogens and phagocytes and the influence of chemo-attractants. For future studies, these results will help to place in vitro experiments and murine infection models in better perspective with respect to human ones.

Macrophage response to staphylococcal biofilms on crosslinked poly(ethylene) glycol polymer coatings and common biomaterials in vitro.

Biomaterial-associated-infections (BAI) are serious clinical complications that threaten the longevity of implanted devices and lead to high morbidity and mortality. Poly(ethylene)glycol (PEG) coatings have been studied as a strategy to reduce the incidence of BAI by reducing protein deposition that promotes pathogen adhesion and growth on device surfaces. Despite their effectiveness to reduce protein adsorption and a hundred-fold reduction in bacterial adhesion, PEG-based coatings still facilitate weak bacterial adhesion that can form an initial basis for biofilms. Here, we describe a methodology enabling direct, quantitative and detailed qualitative in situ observation of macrophage morphology, migration and phagocytosis of bacteria. In vitro interaction of macrophages with Staphylococcus epidermidis 3399 adhering to commercial, crosslinked PEG-based coatings (OptiChem®) was compared with fluorinated ethylene propylene, silicone rubber and glass. Adhesion, phagocytosis and migration were studied real-time in a parallel-plate-flow-chamber. Macrophages cultured on OptiChem® coatings showed enhanced migration and phagocytosis of bacteria compared to common biomaterials. Bacterial clearance per macrophage on both inert and reactive OptiChem® coatings were about three times higher than on the common biomaterials studied, corresponding with up to 70% reduction in bacterial numbers on OptiChem®, whereas on the biomaterials less than 40% bacterial reduction was obtained. These findings show that bacterial clearance from cross-linked PEG-based coatings by macrophages is more effective than from common biomaterials, possibly resulting from weak adhesion of bacteria on Optichem®. Moreover, macrophages exhibit higher mobility on Optichem® retaining an improved capability to clear bacteria from larger areas than from other common biomaterials, where they appear more immobilized.