Kahp-Yang Suh

Mechanosensitivity of fibroblast cell shape and movement to anisotropic substratum topography gradients

In this report, we describe using ultraviolet (UV)-assisted capillary force lithography (CFL) to create a model substratum of anisotropic micro- and nanotopographic pattern arrays with variable local density for the analysis of cell-substratum interactions. A single cell adhesion substratum with the constant ridge width (1 microm), and depth (400 nm) and variable groove widths (1-9.1 microm) allowed us to characterize the dependence of cellular responses, including cell shape, orientation, and migration, on the anisotropy and local density of the variable micro- and nanotopographic pattern. We found that fibroblasts adhering to the denser pattern areas aligned and elongated more strongly along the direction of ridges, vs. those on the sparser areas, exhibiting a biphasic dependence of the migration speed on the pattern density. In addition, cells responded to local variations in topography by altering morphology and migrating along the direction of grooves biased by the direction of pattern orientation (short term) and pattern density (long term), suggesting that single cells can sense the topography gradient. Molecular dynamic live cell imaging and immunocytochemical analysis of focal adhesions and actin cytoskeleton suggest that variable substratum topography can result in distinct types of cytoskeleton reorganization. We also demonstrate that fibroblasts cultured as monolayers on the same substratum retain most of the properties displayed by single cells. This result, in addition to demonstrating a more sophisticated method to study aspects of wound healing processes, strongly suggests that even in the presence of adhesive cell-cell interactions, the cues provided by the underlying substratum topography continue to exercise substantial influence on cell behavior. The described experimental platform might not only further our understanding of biomechanical regulation of cell-matrix interactions, but also contribute to bioengineering of devices with the optimally structured design of cell-material interface.

Capillary force lithography: Large-area patterning, self-organization, and anisotropic dewetting

This article gives an overview on a new lithographic technique called capillary force lithography for large-area patterning. The technique simply involves placing a polydimethylsiloxane mold on a polymer film, which is then heated above the glass-transition temperature of the polymer. Various useful microstructures can be obtained by sequential applications of the technique through self-organization. Dewetting, which can be observed in capillary force lithography for relatively thin films, is also described as a new pathway for realizing anisotropic dewetting.

Fabrication of nanostructures of polyethylene glycol for applications to protein adsorption and cell adhesion

A simple method was developed to fabricate polyethylene glycol (PEG) nanostructures using capillary lithography mediated by ultraviolet (UV) exposure. Acrylate-containing PEG monomers, such as PEG dimethacrylate (PEG-DMA, MW = 330), were photo-cross-linked under UV exposure to generate patterned structures. In comparison to unpatterned PEG films, hydrophobicity of PEG nanostructure modified surfaces was significantly enhanced. This could be attributed to trapped air in the nanostructures as supported by water contact angle measurements. Proteins (fibronectin, immunoglobulin, and albumin) and cells (fibroblasts and P19 EC cells) were examined on the modified surfaces to test for the level of protein adsorption and cell adhesion. It was found that proteins and cells preferred to adhere on nanostructured PEG surfaces in comparison to unpatterned PEG films; however, this level of adhesion was significantly lower than that of glass controls. These results suggest that capillary lithography can be used to fabricate PEG nanostructures capable of modifying protein and cell adhesive properties of surfaces.

Wrinkled, Dual-Scale Structures of Diamond-Like Carbon (DLC) for Superhydrophobicity

We present a simple two-step method to fabricate dual-scale superhydrophobic surfaces by using replica molding of poly(dimethylsiloxane) (PDMS) micropillars, followed by deposition of a thin, hard coating layer of a SiO(x)-incorporated diamond-like carbon (DLC). The resulting surface consists of microscale PDMS pillars covered by nanoscale wrinkles that are induced by residual compressive stress of the DLC coating and a difference in elastic moduli between DLC and PDMS without any external stretching or thermal contraction on the PDMS substrate. We show that the surface exhibits superhydrophobic properties with a static contact angle over 160 degrees for micropillar spacing ratios (interpillar gap divided by diameter) less than 4. A transition of the wetting angle to approximately 130 degrees occurs for larger spacing ratios, changing the wetting from a Cassie-Cassie state (C(m)-C(n)) to a Wenzel-Cassie state (W(m)-C(n)), where m and n denote micro- and nanoscale roughness, respectively. The robust superhydrophobicity of the Cassie-Cassie state is attributed to stability of the Cassie state on the nanoscale wrinkle structures of the hydrophobic DLC coating, which is further explained by a simple mathematical theory on wetting states with decoupling of nano- and microscale roughness in dual scale structures.

Synergistically enhanced osteogenic differentiation of human mesenchymal stem cells by culture on nanostructured surfaces with induction media.

We have examined the effects of surface nanotopography on in vitro osteogenesis of human mesenchymal stem cells (hMSCs). UV-assisted capillary force lithography was employed to fabricate a scalable (4x5 cm), well-defined nanostructured substrate of a UV curable polyurethane polymer with dots (150, 400, 600 nm diameter) and lines (150, 400, 600 nm width). The influence of osteogenic differentiation of hMSCs was characterized at day 8 by alkaline phosphatase (ALP) assay, RT-PCR, and real-time PCR analysis. We found that hMSCs cultured on the nanostructured surfaces in osteogenic induction media showed significantly higher ALP activity compared to unpatterned PUA surface (control group). In particular, the hMSCs on the 400 nm dot pattern showed the highest level of ALP activity. Further investigation with real-time quantitative RT-PCR analysis demonstrated significantly higher expression of core binding factor 1 (Cbfa1), osteopontin (OP), and osteocalcin (OC) levels in hMSCs cultured on the 400 nm dot pattern in osteogenic induction media. These findings suggest that surface nanotopography can enhance osteogenic differentiation synergistically with biochemical induction substance.

Patterning Methods for Polymers in Cell and Tissue Engineering

Polymers provide a versatile platform for mimicking various aspects of physiological extracellular matrix properties such as chemical composition, rigidity, and topography for use in cell and tissue engineering applications. In this review, we provide a brief overview of patterning methods of various polymers with a particular focus on biocompatibility and processability. The materials highlighted here are widely used polymers including thermally curable polydimethyl siloxane, ultraviolet-curable polyurethane acrylate and polyethylene glycol, thermo-sensitive poly(N-isopropylacrylamide) and thermoplastic and conductive polymers. We also discuss how micro- and nanofabricated polymeric substrates of tunable elastic modulus can be used to engineer cell and tissue structure and function. Such synergistic effect of topography and rigidity of polymers may be able to contribute to constructing more physiologically relevant microenvironment.

25th Anniversary Article: Scalable Multiscale Patterned Structures Inspired by Nature: the Role of Hierarchy

Multiscale, hierarchically patterned surfaces, such as lotus leaves, butterfly wings, adhesion pads of gecko lizards are abundantly found in nature, where microstructures are usually used to strengthen the mechanical stability while nanostructures offer the main functionality, i.e., wettability, structural color, or dry adhesion. To emulate such hierarchical structures in nature, multiscale, multilevel patterning has been extensively utilized for the last few decades towards various applications ranging from wetting control, structural colors, to tissue scaffolds. In this review, we highlight recent advances in scalable multiscale patterning to bring about improved functions that can even surpass those found in nature, with particular focus on the analogy between natural and synthetic architectures in terms of the role of different length scales. This review is organized into four sections. First, the role and importance of multiscale, hierarchical structures is described with four representative examples. Second, recent achievements in multiscale patterning are introduced with their strengths and weaknesses. Third, four application areas of wetting control, dry adhesives, selectively filtrating membranes, and multiscale tissue scaffolds are overviewed by stressing out how and why multiscale structures need to be incorporated to carry out their performances. Finally, we present future directions and challenges for scalable, multiscale patterned surfaces.

Effect of orientation and density of nanotopography in dermal wound healing

We report on the effect of synthetic extracellular matrix (ECM) scaffold in the form of uniformly-spaced nanogrooved surfaces in dermal wound healing. The rate of wound coverage was measured on various nanotopographical densities with vertical or parallel orientation using nanogrooves of 550 nm width with three different gaps of 550, 1100, and 2750 nm (spacing ratio: 1:1, 1:2 and 1:5). Guided by the nanotopographical cues in the absence of growth factors in wound healing process, the cultured NIH-3T3 cells demonstrated distinctly different migration speed, cell division, and ECM production as dictated by the topographical density and orientation, whereas the proliferation rate turned out to be nearly the same. Based on our experimental results, the nanopattern of 1:2 spacing ratio yielded the best wound healing performance in terms of migration speed, which seems similar to the natural organization of collagen fibers.

Two distinct filopodia populations at the growth cone allow to sense nanotopographical extracellular matrix cues to guide neurite outgrowth.

Background The process of neurite outgrowth is the initial step in producing the neuronal processes that wire the brain. Current models about neurite outgrowth have been derived from classic two-dimensional (2D) cell culture systems, which do not recapitulate the topographical cues that are present in the extracellular matrix (ECM) in vivo. Here, we explore how ECM nanotopography influences neurite outgrowth. Methodology/Principal Findings We show that, when the ECM protein laminin is presented on a line pattern with nanometric size features, it leads to orientation of neurite outgrowth along the line pattern. This is also coupled with a robust increase in neurite length. The sensing mechanism that allows neurite orientation occurs through a highly stereotypical growth cone behavior involving two filopodia populations. Non-aligned filopodia on the distal part of the growth cone scan the pattern in a lateral back and forth motion and are highly unstable. Filopodia at the growth cone tip align with the line substrate, are stabilized by an F-actin rich cytoskeleton and enable steady neurite extension. This stabilization event most likely occurs by integration of signals emanating from non-aligned and aligned filopodia which sense different extent of adhesion surface on the line pattern. In contrast, on the 2D substrate only unstable filopodia are observed at the growth cone, leading to frequent neurite collapse events and less efficient outgrowth. Conclusions/Significance We propose that a constant crosstalk between both filopodia populations allows stochastic sensing of nanotopographical ECM cues, leading to oriented and steady neurite outgrowth. Our work provides insight in how neuronal growth cones can sense geometric ECM cues. This has not been accessible previously using routine 2D culture systems.

Modulus- and surface energy-tunable ultraviolet-curable polyurethane acrylate: properties and applications

Since the introduction of ultraviolet (UV)-curable polyurethane acrylate (PUA) materials in 2004, our group has extensively used the material in various applications ranging from nanopatterning, biomimetics, microfluidics, to tissue engineering. In this highlight, we aim to provide a brief overview of the properties and applications of PUA with particular focus on the ability of tuning modulus (20–320 MPa) and surface energy (20–60 mJ m−2). These unique features enable us to use the material in various patterning applications as a mold and a patterned film, respectively. In addition, one can benefit from several other features of PUA such as self-replication, partial curing, flexibility, biocompatibility, and transparency. These issues are also briefly described along with their applications.