Topographic cell instructive patterns to control cell adhesion,polarization and migration

Topographic patterns are known to affect cellular processes such as adhesion, migration and differentiation. However, the optimal way to deliver topographic signals to provide cells with precise instructions has not been defined yet. In this work, we hypothesize that topographic patterns may be able to control the sensing and adhesion machinery of cells when their interval features are tuned on the characteristic lengths of filopodial probing and focal adhesions (FAs). Features separated by distance beyond the length of filopodia cannot be readily perceived; therefore, the formation of new adhesions is discouraged. If, however, topographic features are separated by a distance within the reach of filopodia extension, cells can establish contact between adjacent topographic islands. In the latter case, cell adhesion and polarization rely upon the growth of FAs occurring on a specific length scale that depends on the chemical properties of the surface. Topographic patterns and chemical properties may interfere with the growth of FAs, thus making adhesions unstable. To test this hypothesis, we fabricated different micropatterned surfaces displaying feature dimensions and adhesive properties able to interfere with the filopodial sensing and the adhesion maturation, selectively. Our data demonstrate that it is possible to exert a potent control on cell adhesion, elongation and migration by tuning topographic features’ dimensions and surface chemistry.     

Analysing semiconductor manufacturing big data for root cause detection of excursion for yield enhancement

With the shrinking feature size of integrated circuits driven by continuous technology migrations for wafer fabrication, the control of tightening critical dimensions is critical for yield enhancement, while physical failure analysis is increasingly difficult. In particular, the yield ramp up stage for implementing new technology node involves new production processes, unstable machine configurations, big data with multiple co-linearity and high dimensionality that can hardly rely on previous experience for detecting root causes. This research aims to propose a novel data-driven approach for Analysing semiconductor manufacturing big data for low yield (namely, excursions) diagnosis to detect process root causes for yield enhancement. The proposed approach has shown practical viability to efficiently detect possible root causes of excursion to reduce the trouble shooting time and improve the production yield effectively.     

Role of contact electrification and electrostatic interactions in gecko adhesion

Geckos, which are capable of walking on walls and hanging from ceilings with the help of micro-/nano-scale hierarchical fibrils (setae) on their toe pads, have become the main prototype in the design and fabrication of fibrillar dry adhesives. As the unique fibrillar feature of the toe pads of geckos allows them to develop an intimate contact with the substrate the animal is walking on or clinging to, it is expected that the toe setae exchange significant numbers of electric charges with the contacted substrate via the contact electrification (CE) phenomenon. Even so, the possibility of the occurrence of CE and the contribution of the resulting electrostatic interactions to the dry adhesion of geckos have been overlooked for several decades. In this study, by measuring the magnitude of the electric charges, together with the adhesion forces, that gecko foot pads develop in contact with different materials, we have clarified for the first time that CE does contribute effectively to gecko adhesion. More importantly, we have demonstrated that it is the CE-driven electrostatic interactions which dictate the strength of gecko adhesion, and not the van der Waals or capillary forces which are conventionally considered as the main source of gecko adhesion.     

Near-infrared (NIR) controlled reversible cell adhesion on a responsive nano-biointerface

Light-activated dynamic variations have promoted the development of smart interfaces,especially nano-biointerfaces.In this article,the near-infrared (NIR)-responsive surface for controlling cell adhesion was designed by grafting a thermal responsive polymer (poly(N-isopropylacrylamide),PNIPAM) onto silicon nanowires (SiNWs) instead of the traditional photosensitive moieties.NIR induced the photothermal effect of the SiNWs,and the local heat induced thermodynamic phase transformation of PNIPAM.With the application of NIR radiation,the surface turned to a hydrophobic state,and restored to the hydrophilic state when NIR was switched off,leading to reversible cell adhesion and release.The switchable wettability of the surface and cell adhesion/release occurred efficiently even after 20 cycles.Proteins were anchored on the surface via hydrophobic interactions using NIR;further connection of a cell-capture agent helped in achieving specific cell capture.This dynamic control of cell adhesion via NIR may provide new clues for designing functional nano-biointerfaces.     

Nanomechanical control of cell rolling in two dimensions through surface patterning of receptors

We envisioned that label-free control of the transport of cells in two dimensions through receptor-ligand interactions would enable simple separation systems that are easy to implement yet retain the specificity of receptor-ligand interactions. Here we demonstrate nanomechanical control of cell transport in two dimensions via transient receptor-ligand adhesive bonds by patterning of receptors that direct cell rolling through an edge effect. HL-60 cells rolling on P-selectin receptor patterns were deflected at angles of 5-10 degrees with respect to their direction of travel. Absence of this effect in the case of rigid microsphere models of cell rolling suggests that this two-dimensional motion depends on nanomechanical properties of the rolling cell. This work suggests the feasibility of simple continuous-flow microfluidic cell separation systems that minimize processing steps and yet retain the specificity of receptor-ligand interactions.     

Adhesion formation of primary human osteoblasts and the functional response of mesenchymal stem cells to 330nm deep microgrooves.

The surface microtexture of an orthopaedic device can regulate cellular adhesion, a process fundamental in the initiation of osteoinduction and osteogenesis. Advances in fabrication techniques have evolved to include the field of surface modification; in particular, nanotechnology has allowed for the development of experimental nanoscale substrates for investigation into cell nanofeature interactions. Here primary human osteoblasts (HOBs) were cultured on ordered nanoscale groove/ridge arrays fabricated by photolithography. Grooves were 330nm deep and either 10, 25 or 100mum in width. Adhesion subtypes in HOBs were quantified by immunofluorescent microscopy and cell-substrate interactions were investigated via immunocytochemistry with scanning electron microscopy. To further investigate the effects of these substrates on cellular function, 1.7K gene microarray analysis was used to establish gene regulation profiles of mesenchymal stem cells cultured on these nanotopographies. Nanotopographies significantly affected the formation of focal complexes (FXs), focal adhesions (FAs) and supermature adhesions (SMAs). Planar control substrates induced widespread adhesion formation; 100mum wide groove/ridge arrays did not significantly affect adhesion formation yet induced upregulation of genes involved in skeletal development and increased osteospecific function; 25mum wide groove/ridge arrays were associated with a reduction in SMA and an increase in FX formation; and 10mum wide groove/ridge arrays significantly reduced osteoblast adhesion and induced an interplay of up- and downregulation of gene expression. This study indicates that groove/ridge topographies are important modulators of both cellular adhesion and osteospecific function and, critically, that groove/ridge width is important in determining cellular response.     

Nano-chessboard superlattices formed by spontaneous phase separation in oxides

The use of bottom-up fabrication of nanostructures for nanotechnology inherently requires two-dimensional control of the nanostructures at a particular surface. This could in theory be achieved crystallographically with a structure whose three-dimensional unit cell has two or more--tuneable--dimensions on the nanometre scale. Here, we present what is to our knowledge the first example of a truly periodic two-dimensional nanometre-scale phase separation in any inorganic material, and demonstrate our ability to tune the unit-cell dimensions. As such, it represents great potential for the use of standard ceramic processing methods for nanotechnology. The phase separation occurs spontaneously in the homologous series of the perovskite-based Li-ion conductor, (Nd(2/3-x)Li(3x))TiO3, to give two phases whose dimensions both extend into the nanometre scale. This unique feature could lead to its application as a template for the assembly of nanostructures or molecular monolayers.     

Stable Non‐Covalent Large Area Patterning of Inert Teflon‐AF Surface: A New Approach to Multiscale Cell Guidance

Micro‐ and nano‐patterning of cell adhesion proteins is demonstrated to direct the growth of neural cells, viz. human neuroblastoma SHSY5Y, at precise positions on a strongly antifouling substrate of technolological interest. We adopt a soft‐lithographic approach with oxygen plasma modified PDMS stamps to pattern human laminin on Teflon‐AF films. These patterns are based on the interplay of capillary forces within the stamp and non‐covalent intermolecular and surface interactions. Remarkably, they remain stable for several days upon cell culture conditions. The fabrication of substrates with adjacent antifouling and adhesion‐promoting regions allows us to reach absolute spatial control in the positioning of neuroblastoma cells on the Teflon‐AF films. This patterning approach of a technologically‐relevant substrate can be of interest in tissue engineering and biosensing.     

Micro- and nano-patterned substrates to manipulate cell adhesion

Cell adhesion to material surfaces regulates host responses to implanted biomaterials and the performance of cell arrays and biotechnological cell culture supports. Therefore, the engineering of substrates that control cell adhesive interactions is critical to the development of bio-interactive interfaces and biotechnological culture supports. We describe the application of advanced fabrication techniques to engineer substrates with well defined chemistry and topography to manipulate cell adhesive interactions. Microcontact printing of self-assembled monolayers and hot embossing imprint lithography approaches were integrated to manipulate focal adhesion assembly, cell adhesion, and cellular spreading and alignment. These micro- and nanopatterned substrates provide useful tools for the analyses of structure-function relationships in adhesive interactions.     

Modern biomaterials: a review—bulk properties and implications of surface modifications

This review concerns the importance of length and time on physicochemical interactions between living tissue and biomaterials that occur on implantation. The review provides information on material host interactions, materials for medical applications and cell surface interactions, and then details the extent of knowledge concerning the role(s) that surface chemistry and topography play during the first stage of implant integration, namely protein adsorption. The key points are illustrated by data from model in vitro studies. Host implant interactions begin nanoseconds after first contact and from then on are in a state of flux due to protein adsorption, cell adhesion and physical and chemical alteration of the implanted material. The many questions concerning the conformational form and control of bound proteins and how this may impact on cell adhesion in the first instance and later on cell signalling and implant integration can be answered by systematic investigations using model materials. Only then we will be in a more informed position to design new materials for use in the body.     

Evaluation of endothelial cell adherence onto collagen and fibronectin: A comparison between jet impingement and flow chamber techniques

The study of the cell–biomaterial interface has been revolutionized by the understanding of molecular mechanisms and signaling cascades between living cells and their environment. A successful biomaterial is a product of sound knowledge of the material surface and molecular cell biology sciences, which allow us to establish accurate control of the interfacial interactions needed for biospecificity. To improve the cell–biomaterial interactions, we have functionalized polystyrene, which is a biomaterial commonly used for vascular implants, by adhesion proteins such as fibronectin and collagen. Cell adhesion remains an important goal of this study. This critical phenomenon that controls cell behavior was evaluated by two different techniques. We used a parallel plate flow chamber and jet impingement, which are effective and complementary devices for evaluating the strength of cell adhesion to the surface.     

Morphology and adhesion of mesenchymal stem cells on PLLA,apatite and apatite/collagen surfaces

Biomimetic apatite/collagen composite coating, previously reported particularly with regard to its fabrication, characterization and interaction with osteoblast-like cells, has been investigated in this study to understand the response of human mesenchymal stem cells (hMSC) to such surface. PLLA films and PLLA films with apatite coating were compared with PLLA films with apatite/collagen composite coating. The hMSC morphology in response to such conditions was first observed using fluorescence microscopy. To further understand such cell-material interactions at a molecular level, integrin expression, actin assembly and vinculin-positive focal adhesion plaques were examined. Our results demonstrated that spreading of stem cells on the apatite/collagen composite surface was determined best among the three types of surfaces, followed by the apatite surface and then the PLLA control. Integrin expression on the apatite/collagen surface was higher than those on the apatite surface and PLLA surface. Immunostaining for vinculin and actin suggested that the composite coating on PLLA enhanced the formation of focal adhesion.     

Enzyme-nanoparticle functionalization of three-dimensional protein scaffolds

Various surface modification techniques have been developed for patterning functional biomolecules in two dimensions, allowing enzymes, antibodies, and other compounds to be localized for applications in bioanalysis and bioengineering. Here, we report a strategy for extending high-resolution patterning of biomolecules to three dimensions. In this approach, three-dimensional protein scaffolds are created by a direct-write process in which multiphoton excitation promotes photochemical cross-linking of protein molecules from aqueous solution within specified volume elements. After scaffold fabrication, protein microstructures are functionalized with enzyme-gold nanoparticle conjugates via a targeting process based in part on electrostatic attraction between the low-isoelectric-point enzyme and the microstructure, fabricated from high-isoelectric-point proteins. High signal-to-background ratios (approximately 20:1) are demonstrated for fluorescent product streams created by dephosphorylation of the fluorogenic compound, fluorescein diphosphate, at microstructures decorated with alkaline phosphatase-gold nanoparticle conjugates. We also demonstrate feasibility for using such structures to quantify substrate concentrations in flowing streams with low-micromolar detection limits and to create sensor suites based on both enzyme-nanoparticle functionalization and intrinsic enzymatic activity of protein scaffolds. These topographically complex sensors and dosing sources have potential applications in microfluidics, sensor array fabrication, and real-time chemical modification of cell culture environments.     

Cell/surface interactions on laser micro-textured titanium-coated silicon surfaces

This paper examines the effects of nano-scale titanium coatings, and micro-groove/micro-grid patterns on cell/surface interactions on silicon surfaces. The nature of the cellular attachment and adhesion to the coated/uncoated micro-textured surfaces was elucidated by the visualization of the cells and relevant cytoskeletal & focal adhesion proteins through scanning electron microscopy and immunofluorescence staining. Increased cell spreading and proliferation rates are observed on surfaces with 50 nm thick Ti coatings. The micro-groove geometries have been shown to promote contact guidance, which leads to reduced scar tissue formation. In contrast, smooth surfaces result in random cell orientations and the increased possibility of scar tissue formation. Immunofluorescence cell staining experiments also reveal that the actin stress fibers are aligned along the groove dimensions, with discrete focal adhesions occurring along the ridges, within the grooves and at the ends of the cell extensions. The implications of the observed cell/surface interactions are discussed for possible applications of silicon in implantable biomedical systems.     

Multilayer micromolding of degradable polymer tissue engineering scaffolds

Precise surface geometrical morphologies have been shown to improve cellular proliferation, adhesion, and functionality. It has been found that cells respond strongly to feature dimensions a fraction of their size. In this paper, soft lithography techniques were applied to microfabricate polydimethylsiloxane molds with precisely controlled micro-scale patterns. Three-dimensional polycaprolactone (PCL) scaffolds were fabricated using a multilayer micromolding (MMM) method. Proper heating and stamping parameters were developed for micromolding PCL. This process allowed control of the size, shape, and spacing of support structures within the scaffold. The micromolding of multiple layers with independent features allowed for alignment between layers. The high porosity, abundant interconnections, and sharp features were inherent advantages of the scaffolds. Human osteosarcoma cells were seeded in the 3-D scaffolds for cell growth testing. Fluorescent microscopy and scanning electron micrographs showed that cells responded well to the 3-D scaffolds and the scaffolds regulated cell morphology and adhesion.     

Layer-by-layer assembly through weak interactions and their biomedical applications

The surface design and control of substrates with nanometer- or micrometer-sized polymer films are of considerable interest for both fundamental and applied studies in the biomedical field because of the required surface properties. The layer-by-layer (LbL) technique was discovered in 1991 by Decher and co-workers for the fabrication of polymer multilayers constructed mainly through electrostatic interaction. The scope and applicability of this LbL assembly has been extended by introducing molecularly regular conformations of polymers or proteins by employing, for the first time, weak interactions such as van der Waals interactions and biological recognition. Since these weak interactions are the sum of the attractive or repulsive forces between parts of the same molecule, they allow macromolecules to be easily arranged into the most stable conformation in a LbL film. By applying this characteristic feature, the template polymerization of stereoregular polymers, stereoregular control of surface biological properties, drastic morphological control of biodegradable nano materials, and the development of three-dimensional cellular multilayers as a tissue model were successfully achieved. It is expected that LbL assembly using weak interactions will promote further interest into fundamental and applied studies on the design of surface chemistry in the biomedical field.     

The metrology of a miniature FT spectrometer MOEMS device using white light scanning interference microscopy

Micro-optical electro-mechanical systems (MOEMS) technology, making use of existing silicon based fabrication techniques shows great potential for making complete miniaturized hybrid devices. Such technology has been used to make a Fourier transform spectrometer based on a time-scanning Michelson interferometer. An electrostatic comb drive actuator moves the scanning mirror over a distance of 40 μm. The measured resolution of the spectrometer is 6 nm at a wavelength of 633 nm. The dimensions of the device are 5×5×0.5 mm, and the depth of feature is 75 μm. During quality control of such devices it is necessary to check the dimensions of micron wide structures that are tens of microns deep, over areas of tens of square millimeters. In this work we have investigated the use of white light scanning interference (WLSI) microscopy for making rapid, non-destructive precision three-dimensional measurements. While a high axial precision can be achieved, an artifact has been observed with classical configurations that tend to extend the location of deep step discontinuities by up to 3 μm and so broaden narrow structures. With certain modifications in the optical configuration, this error can be considerably reduced. The results of this work demonstrate that WLSI shows great potential for the rapid and precise quality control of MOEMS devices.     

Pulsed plasma polymerization for controlling shrinkage and surface composition of nanopores

Solid-state nanopores have emerged as sensors for single molecules and these have been employed to examine the biophysical properties of an increasingly large variety of biomolecules. Herein we describe a novel and facile approach to precisely adjust the pore size, while simultaneously controlling the surface chemical composition of the solid-state nanopores. Specifically, nanopores fabricated using standard ion beam technology are shrunk to the requisite molecular dimensions via the deposition of highly conformal pulsed plasma generated thin polymeric films. The plasma treatment process provides accurate control of the pore size as the conformal film deposition depends linearly on the deposition time. Simultaneously, the pore and channel chemical compositions are controlled by appropriate selection of the gaseous monomer and plasma conditions employed in the deposition of the polymer films. The controlled pore shrinkage is characterized with high resolution AFM, and the film chemistry of the plasma generated polymers is analyzed with FTIR and XPS. The stability and practical utility of this new approach is demonstrated by successful single molecule sensing of double-stranded DNA. The process offers a viable new advance in the fabrication of tailored nanopores, in terms of both the pore size and surface composition, for usage in a wide range of emerging applications.     

Surface modified electrospun nanofibrous scaffolds for nerve tissue engineering

The development of biodegradable polymeric scaffolds with surface properties that dominate interactions between the material and biological environment is of great interest in biomedical applications. In this regard, poly-ε-caprolactone (PCL) nanofibrous scaffolds were fabricated by an electrospinning process and surface modified by a simple plasma treatment process for enhancing the Schwann cell adhesion, proliferation and interactions with nanofibers necessary for nerve tissue formation. The hydrophilicity of surface modified PCL nanofibrous scaffolds (p-PCL) was evaluated by contact angle and x-ray photoelectron spectroscopy studies. Naturally derived polymers such as collagen are frequently used for the fabrication of biocomposite PCL/collagen scaffolds, though the feasibility of procuring large amounts of natural materials for clinical applications remains a concern, along with their cost and mechanical stability. The proliferation of Schwann cells on p-PCL nanofibrous scaffolds showed a 17% increase in cell proliferation compared to those on PCL/collagen nanofibrous scaffolds after 8 days of cell culture. Schwann cells were found to attach and proliferate on surface modified PCL nanofibrous scaffolds expressing bipolar elongations, retaining their normal morphology. The results of our study showed that plasma treated PCL nanofibrous scaffolds are a cost-effective material compared to PCL/collagen scaffolds, and can potentially serve as an ideal tissue engineered scaffold, especially for peripheral nerve regeneration.