Orthogonal Morphological Feature Size and Density Gradients for Exploring Synergistic Effects in Biology.

Gradient surfaces enable rapid screening and high-throughput investigations in various fields, such as biology and tribology. A new method is described for the preparation of material-independent morphological gradients, in which the density and height of roughness features are varied along two orthogonal axes. A polystyrene-particle-density gradient was produced by a dip-coating process on titanium-oxide-coated silicon wafers. A controlled exposure to ultraviolet light enabled the generation of a particle-height gradient in the orthogonal direction. These gradients were replicated to generate material-independent morphology gradients. MC3T3 cell proliferation studies were performed on titanium-coated replicas and showed a higher cell density on the high-feature-density region of the gradient. The cell area coverage was found to increase with decreasing particle height.

Patterning gradients.

Surface-chemical and -morphological gradients can be extremely useful in cell-biological research as high-throughput screening tools-for example, exposing a given set of cells to many different surface conditions at once, under identical ambient conditions, in order to monitor cell behavior such as proliferation or specific gene expression. They can also be used to investigate the effects of gradients themselves on cell behavior, such as migration. A number of simple, reliable techniques for both chemical- and morphological-gradient fabrication have been developed in our laboratories and are described in detail in the following.

Orthogonal nanometer-micrometer roughness gradients probe morphological influences on cell behavior.

Surface gradients facilitate rapid, high-throughput, systematic investigations in cell biology, materials science, and other fields. An important surface parameter is the surface roughness on both the micrometer and nanometer scales in the lateral direction. Two approaches have been combined to create two-dimensional roughness gradients by adding a nanoparticle density gradient onto a gradient of micro-featured roughness. All fabricated gradients were extensively characterized by SEM, AFM and optical profilometry to ensure their quality and to determine the roughness parameter Ra along the gradient. Additionally, a Fourier-transform approach was applied that allows a wavelength-dependent analysis of the surface topography. Since cell-culture assays require replicate experiments, a replica technique was used to create copies of the master gradient. Creating a negative replica in an elastomeric material served as a mold for a subsequent ceramic-casting process. A positive replica was then formed from epoxy resin, which was subsequently coated with titanium and used for cell studies. Finally, these gradients were used in cell-culture assays to determine cellular response to surface roughness. The results clearly demonstrate the influence of surface roughness on the production by osteoblasts of markers for osteogenesis. It was shown that high roughness in the micrometer range, combined with an intermediate nanofeature density (30-40 features/µm2), leads to the highest degree of osteopontin production after 14 days.

Effects of surface microtopography on the assembly of the osteoclast resorption apparatus.

Bone degradation by osteoclasts depends on the formation of a sealing zone, composed of an interlinked network of podosomes, which delimits the degradation lacuna into which osteoclasts secrete acid and proteolytic enzymes. For resorption to occur, the sealing zone must be coherent and stable for extended periods of time. Using titanium roughness gradients ranging from 1 to 4.5 µm R(a) as substrates for osteoclast adhesion, we show that microtopographic obstacles of a length scale well beyond the range of the 'footprint' of an individual podosome can slow down sealing-zone expansion. A clear inverse correlation was found between ring stability, structural integrity and sealing-zone translocation rate. Direct live-cell microscopy indicated that the expansion of the sealing zone is locally arrested by steep, three-dimensional 'ridge-like barriers', running parallel to its perimeter. It was, however, also evident that the sealing zone can bypass such obstacles, if pulled by neighbouring regions, extending through flanking, obstacle-free areas. We propose that sealing-zone dynamics, while being locally regulated by surface roughness, are globally integrated via the associated actin cytoskeleton. The effect of substrate roughness on osteoclast behaviour is significant in relation to osteoclast function under physiological and pathological conditions, and may constitute an important consideration in the design of advanced bone replacements.

Surface-chemical and -morphological gradients.

Surface gradients of chemistry or morphology represent powerful tools for the high-throughput investigation of interfacial phenomena in the areas of physics, chemistry, materials science and biology. A wide variety of methods for the fabrication of such gradients has been developed in recent years, relying on principles ranging from diffusion to time-dependent irradiation in order to achieve a gradual change of a particular parameter across a surface. In this review we have endeavoured to cover the principal fabrication approaches for surface-chemical and surface-morphological gradients that have been described in the literature, and to provide examples of their applications in a variety of different fields.

Poly(L-lysine)-grafted-poly(ethylene glycol)-based surface-chemical gradients. Preparation, characterization, and first applications.

A simple dipping process has been used to prepare PEGylated surface gradients from the polycationic polymer poly(L-lysine), grafted with poly(ethylene glycol) (PLL-g-PEG), on metal oxide substrates, such as TiO(2) and Nb(2)O(5). PLL-g-PEG coverage gradients were prepared during an initial, controlled immersion and characterized with variable angle spectroscopic ellipsometry and x-ray photoelectron spectroscopy. Gradients with a linear change in thickness and coverage were generated by the use of an immersion program based on an exponential function. These single-component gradients were used to study the adsorption of proteins of different sizes and shapes, namely, albumin, immunoglobulin G, and fibrinogen. The authors have shown that the density and size of defects in the PLL-g-PEG adlayer determine the amount of protein that is adsorbed at a certain adlayer thickness. In a second step, single-component gradients of functionalized PLL-g-PEG were backfilled with nonfunctionalized PLL-g-PEG to generate two-component gradients containing functional groups, such as biotin, in a protein-resistant background. Such gradients were combined with a patterning technique to generate individually addressable spots on a gradient surface. The surfaces generated in this way show promise as a useful and versatile biochemical screening tool and could readily be incorporated into a method for studying the behavior of cells on functionalized surfaces.