Lauren R. Clements

Electrochemistry-enabled fabrication of orthogonal nanotopography and surface chemistry gradients for high-throughput screening.

Gradient surfaces are emerging tools for investigating mammalian cell-surface interactions in high throughput. We demonstrate the electrochemical fabrication of an orthogonal gradient platform combining a porous silicon (pSi) pore size gradient with an orthogonal gradient of peptide ligand density. pSi gradients were fabricated via the anodic etching of a silicon wafer with pore sizes ranging from hundreds to tens of nanometers. A chemical gradient of ethyl-6-bromohexanoate was generated orthogonally to the pSi gradient via electrochemical attachment. Subsequent hydrolysis and activation of the chemical gradient allowed for the generation of a cyclic RGD gradient. Whilst mesenchymal stem cells (MSC) were shown to respond to both the topographical and chemical cues arising from the orthogonal gradient, the MSCs responded more strongly to changes in RGD density than to changes in pore size during short-term culture.

Assessing embryonic stem cell response to surface chemistry using plasma polymer gradients

The control of cell-material interactions is the key to a broad range of biomedical interactions. Gradient surfaces have recently been established as tools allowing the high-throughput screening and optimization of these interactions. In this paper, we show that plasma polymer gradients can reveal the subtle influence of surface chemistry on embryonic stem cell behavior and probe the mechanisms by which this occurs. Lateral gradients of surface chemistry were generated by plasma polymerization of diethylene glycol dimethyl ether on top of a substrate coated with an acrylic acid plasma polymer using a tilted slide as a mask. Gradient surfaces were characterized by X-ray photoelectron spectroscopy, infrared microscopy mapping and profilometry. By changing the plasma polymerization time, the gradient profile could be easily manipulated. To demonstrate the utility of these surfaces for the screening of cell-material interactions, we studied the response of mouse embryonic stem (ES) cells to these gradients and compared the performance of different plasma polymerization times during gradient fabrication. We observed a strong correlation between surface chemistry and cell attachment, colony size and retention of stem cell markers. Cell adhesion and colony formation showed striking differences on gradients with different plasma polymer deposition times. Deposition time influenced the depth of the plasma film deposited and the relative position of surface functional group density on the substrate, but not the range of plasma-generated species.

Screening rat mesenchymal stem cell attachment and differentiation on surface chemistries using plasma polymer gradients

It is well known that the surface chemistry of biomaterials is important for both initial cell attachment and the downstream cell response. Surface chemistry gradients are a new format that allows the screening of the subtleties of cell-surface interactions in high throughput. In this study, two surface chemical gradients were fabricated using diffusion control during plasma polymerization via a tilted mask. Acrylic acid (AA) plasma polymer gradients were coated on a uniform 1,7-octadiene (OD) plasma polymer layer to generate OD-AA plasma polymer gradients, whilst diethylene glycol dimethyl ether (DG) plasma polymer gradients were coated on a uniform AA plasma polymer layer to generate AA-DG plasma polymer gradients. Gradient surfaces were characterized by X-ray photoelectron spectroscopy, infrared microscopy mapping, profilometry, water contact angle (WCA) goniometry and atomic force microscopy. Cell attachment density and differentiation into osteo- and adipo-lineages of rat-bone-marrow mesenchymal stem cells (rBMSCs) was studied on gradients. Cell adhesion after 24 h culture was sensitive to the chemical gradients, resulting in a cell density gradient along the substrate. The slope of the cell density gradient changed between 24 and 6 days due to cell migration and growth. Induction of rBMSCs into osteoblast- and adipocyte-like cells on the two plasma polymer gradients suggested that osteogenic differentiation was sensitive to local cell density, but adipogenic differentiation was not. Using mixed induction medium (50% osteogenic and 50% adipogenic medium), thick AA plasma polymer coating (>40 nm thickness with ∼11% COOH component and 35° WCA) robustly supported osteogenic differentiation as determined by colony formation and calcium deposition. This study establishes a simple but powerful approach to the formation of plasma polymer based gradients, and demonstrates that MSC behavior can be influenced by small changes in surface chemistry.

Gradient technology for high-throughput screening of interactions between cells and nanostructured materials

We present a novel substrate suitable for the high-throughput analysis of cell response to variations in surface chemistry and nanotopography. Electrochemical etching was used to produce silicon wafers with nanopores between 10 and 100nm in diameter. Over this substrate and flat silicon wafers, a gradient film ranging from hydrocarbon to carboxylic acid plasma polymer was deposited, with the concentration of surface carboxylic acid groups varying between 0.7 and 3% as measured by XPS. MG63 osteoblast-like cells were then cultured on these substrates and showed greatest cell spreading and adhesion onto porous silicon with a carboxylic acid group concentration between 2-3%. This method has great potential for high-throughput screening of cell-material interaction with particular relevance to tissue engineering.

Preparation of two-directional gradient surfaces for the analysis of cell-surface interactions

The ability to evaluate and control the cellular response to substrate materials is the key to a wide range of biomedical applications ranging from diagnostic tools to regenerative medicine. Gradient surfaces provide a simple and fast method for investigating optimal surface conditions for cellular responses such as attachment and growth. By using two orthogonal gradients on the same substrate, a large space of possible combinations can be screened simultaneously. Here, we have investigated the combination of a porous silicon (pSi) based topography gradient with a plasma polymer based thickness gradient. pSi was laterally anodised on a 1.5 × 2.5cm2 silicon surface using hydrofluoric acid to form a pore size gradient along a single direction. The resulting pSi was characterised by SEM and AFM and pore sizes ranging from macro to mesoporous were found along the surface. Plasma polymerisation was used to form a thickness gradient orthogonal to the porous silicon gradient. Here, allylamine was chosen as the monomer and a mask placed over the substrate was used to achieve the thickness gradient. The analysis of this chemistry based gradient was carried out using profilometry and XPS. It is expected that orthogonal gradient substrates will be used increasingly for the in vitro screening of materials used in biomedical applications.

Preparation of chemical gradients on porous silicon by a dip coating method

Gradient surfaces have become invaluable tools for the high-throughput characterisation of biomolecule- and cellmaterial surface interactions as they allow for the screening and optimisation of surface parameters such as surface chemistry, topography and ligand density in a single experiment. Here, we have generated surface chemistry gradients on oxidised porous silicon (pSi) substrates using silane functionalisation. In these studies, pSi films with a pore size of 15- 30 nm and a layer thickness of around 1.7 ìm were utilised. The manufacture of gradient surface chemistries of silanes was performed using a simple dip coating method, whereby an increasing incubation time of the substrate in a solution of the silane led to increasing surface coverage of the silane. In this work, the hydrophobic n-octadecyldimethyl chlorosilane (ODCS) and pentafluorophenyldimethyl chlorosilane (PFPS) were used since they were expected to produce significant changes in wettability upon attachment. Chemical gradients were characterised using infrared (IR) spectroscopy, X-ray photoelectron spectroscopy (XPS) and sessile drop water contact angle measurements. In addition, the surface chemistry of the gradient was mapped using synchrotron IR microscopy. The ODCS gradient displayed sessile drop water contact angles ranging from 12° to 71°, confirming the successful formation of a gradient. IR microscopy and an XPS line scan confirmed the formation of a chemical gradient on the porous substrate. Furthermore, the chemical gradients produced can be used for the high-throughput in vitro screening of protein and cell-surface interactions, leading to the definition of surface chemistry on nanostructured silicon which will afford improved control of biointerfacial interactions.

CHAPTER 10:High Throughput Techniques for the Investigation of Cell–Material Interactions

The evaluation and control of the cellular response to substrate materials is important for a wide range of regenerative medicine applications including implants, biomaterial devices and biosensors. Gradient surfaces provide a simple and fast method for determining optimal surface conditions for cellular responses such as attachment and growth. This chapter presents an overview of high-throughput techniques available for the optimisation of cell material interactions with particular emphasis on surface topographical and chemical gradients. The gradient approaches presented throughout this chapter are expected to become useful tools for researchers that are aiming to optimise advanced materials for a variety of biomedical applications.