AVS2011 Session BI-WeM: Cells at Interfaces

Biomaterials for implant purposes are increasingly applied in modern medicine e.g. to recover human body functions or for tissue substitution in general. Infections of these implants, called Biomaterial-centered infections (BCI), are among the fundamental challenges in biomaterials science. They are primarily initiated by adhesion of bacteria on the biomaterial’s surface. The subsequent formation of a bacterial biofilm requires a total implant replacement in the majority of cases.

The adhesion of bacteria is thus the first crucial step for biofilm formation that is only incompletely understood. Interactions of bacteria with the surface are controlled by surface properties such as surface energy, surface chemistry and topography.

In the present work, a model for bacterial adhesion is introduced that describes the early stages of biofilm formation as a function of the surface properties. A two-dimensional Cellular Automaton (CA) / Finite Difference (FD) adsorption model is combined with the predictions of the extended DLVO (Derjaguin, Landau, Verwey, Overbeek) theory that accounts for the interaction energies between the bacteria and the material’s surface. The model describes the mass transport of bacteria in an aqueous solution towards the material’s surface and the adsorption and desorption process, depending on the surface properties.

The adhesion process of different human pathogenic bacteria (Enterococcus faecalis, Staphylococcus aureus, Escherichia coli) on different biomaterial surfaces (titanium, stainless steel, polyethylene, polymethylmethacrylate, polytetrafluoroethylene) has been simulated. Results are the surface coverage with bacteria and, where applicable, clustering of the bacteria due to their migration on the surface.

Excellent agreement with experimental findings from the literature and own adhesion experiments concerning the kinetics of the adsorption process is found. In addition, a realistic bond strengthening mechanism of bacteria on surfaces, as described in the literature, is reproduced by the model. By using a spatial pattern analysis of our own experimental data we show that physical processes occurring during initial stages of the adhesion process are essentially correctly incorporated in the model.

Biofilm formation leads to a 1000 times increase in antibiotic tolerance compared with planktonic bacteria and is associated with 80% of hospital acquired infections, resulting in $3.0 billion in excess health-care costs each year in the U.S alone. Thus, new materials for biomedical devices that prevent biofilm formation would offer enormous benefits to the health industry and improve patient welfare. However, our current understanding of bacteria-material interactions limits scope for rational design of such materials. Polymer microarrays are emerging as a key enabling technology for the discovery of new biomaterials.¹ A method for forming polymer microarrays has been developed using contact printing to deposit nanolitre volumes of premixed acrylate monomer and initiator to defined locations on a poly(HEMA) coated glass slide with UV photo-initiation.² This platform enables a large combinatorial space to be rapidly screened by a biological assay to identify new materials that fulfil a given performance criterion.³ A library of polymer gradients that enables the systematic investigation of biology-material interactions can be created by producing polymers from monomers mixed at hundreds of different concentrations. Utilising a high throughput surface characterisation approach the surface chemical and physical properties of each material can be characterised and related to the biological performance.⁴ We have developed a high throughput bacterial attachment assay based on three pathogens (Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli) expressing green fluorescent protein, which is compatible with the polymer microarray format. This study provides novel insights into the bacteria-material interactions, highlighting chemical moieties that both support and resist bacterial attachment. Specifically, superior efficiacy to prevent bacterial attachment has been demonstrated for hydrophobic moieties on a polyacrylate backbone that contains weakly polar ester groups, which represent an amphiphillic chemical nature.

¹ Hook, A. L. et al., High throughput methods applied in biomaterial development and discovery. Biomaterials 31 (2), 187 (2010).

² Anderson, D. G., Levenberg, S., and Langer, R., Nanoliter-scale synthesis of arrayed biomaterials and application to human embryonic stem cells. Nature Biotechnology 22 (7), 863 (2004).

³ Mei, Y. et al., Combinatorial development of biomaterials for clonal growth of human pluripotent stem cells. Nature Materials 9 (9), 768 (2010).

⁴ Urquhart, A. J. et al., High throughput surface characterisation of a combinatorial material library. Advanced Materials 19 (18), 2486 (2007).

Stem cell differentiation is sensitive to a variety of global and local environmental cues that impact cell fate decisions. Pluripotent stem cells (i.e. ESCs & iPSCs) are capable of recapitulating many aspects of early development and can serve as a robust cell source for the development of cell-based diagnostics and regenerative medicine therapies. ESCs are commonly differentiated as three-dimensional multi-cellular aggregates referred to as “embryoid bodies” (EBs), because of their ability to mimic the early morphogenic transformation of pluripotent cells into derivatives of the three germ lineages (ecto-, endo-, and mesoderm). In order to better understand and ultimately control ESC morphogenesis, w e have focused on systematically engineering biochemical and biophysical parameters of the 3D EB microenvironment via the integration of different biomaterials and examining the emergent results on stem cell differentiation. Microparticles (MPs) of varying size (1-20 µm) and chemistries (i.e. PLGA, agarose, gelatin) were incorporated within 3D stem cell aggregates in a dose-dependent manner (~1 particle / 10 cells) without adversely affecting cell viability. Interestingly, the mere presence of relatively small numbers of different types of materials alone could modulate stem cell phenotype as evidenced by gene expression profiling and immunophenotype analyses. D elivery of morphogenic factors, such as retinoic acid (RA), bone morphogenic protein 4 (BMP4) or vascular endothelial growth factor (VEGF), to ESCs from incorporated MPs significantly impacted the differentiation of the cells to different lineages and was more efficient than comparable soluble treatment methods. Altogether these results suggest that engineered biomaterials can direct the differentiation of stem cells through modulation of biochemical and/or biophysical properties of the 3D microenvironment. It is expected that the development of inherently scalable techniques to direct pluripotent stem cell differentiation will benefit the biomanufacturing of stem cell derivatives for regenerative cellular therapies and in vitro cell based diagnostic technologies, as well as enable engineering of tissues directly from stem cells.

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