by Keyword: Patterning

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Casanellas, Ignasi, Lagunas, Anna, Vida, Yolanda, Pérez-Inestrosa, Ezequiel, Andrades, J. A., Becerra, J., Samitier, Josep, (2020). The Janus role of adhesion in chondrogenesis International Journal of Molecular Sciences 21, (15), 5269

Tackling the first stages of the chondrogenic commitment is essential to drive chondrogenic differentiation to healthy hyaline cartilage and minimize hypertrophy. During chondrogenesis, the extracellular matrix continuously evolves, adapting to the tissue adhesive requirements at each stage. Here, we take advantage of previously developed nanopatterns, in which local surface adhesiveness can be precisely tuned, to investigate its effects on prechondrogenic condensation. Fluorescence live cell imaging, immunostaining, confocal microscopy and PCR analysis are used to follow the condensation process on the nanopatterns. Cell tracking parameters, condensate morphology, cell–cell interactions, mechanotransduction and chondrogenic commitment are evaluated in response to local surface adhesiveness. Results show that only condensates on the nanopatterns of high local surface adhesiveness are stable in culture and able to enter the chondrogenic pathway, thus highlighting the importance of controlling cell–substrate adhesion in the tissue engineering strategies for cartilage repair.

Keywords: Dendrimer, Nanopatterning, RGD, Mesenchymal cell condensation, Cell–cell interactions, YAP, Chondrogenesis

Gállego, Isaac, Manning, Brendan, Prades, Joan Daniel, Mir, Mònica, Samitier, Josep, Eritja, Ramon, (2017). DNA-origami-driven lithography for patterning on gold surfaces with sub-10 nm resolution Advanced Materials 29, 1603233

Schieber, R., Lasserre, F., Hans, M., Fernández-Yagüe, M., Díaz-Ricart, M., Escolar, G., Ginebra, M. P., Mücklich, F., Pegueroles, M., (2017). Direct laser interference patterning of CoCr alloy surfaces to control endothelial cell and platelet response for cardiovascular applications Advanced Healthcare Materials 6, (19), 1700327

The main drawbacks of cardiovascular bare-metal stents (BMS) are in-stent restenosis and stent thrombosis as a result of an incomplete endothelialization after stent implantation. Nano- and microscale modification of implant surfaces is a strategy to recover the functionality of the artery by stimulating and guiding molecular and biological processes at the implant/tissue interface. In this study, cobalt-chromium (CoCr) alloy surfaces are modified via direct laser interference patterning (DLIP) in order to create linear patterning onto CoCr surfaces with different periodicities (≈3, 10, 20, and 32 μm) and depths (≈20 and 800 nm). Changes in surface topography, chemistry, and wettability are thoroughly characterized before and after modification. Human umbilical vein endothelial cells' adhesion and spreading are similar for all patterned and plain CoCr surfaces. Moreover, high-depth series induce cell elongation, alignment, and migration along the patterned lines. Platelet adhesion and aggregation decrease in all patterned surfaces compared to CoCr control, which is associated with changes in wettability and oxide layer characteristics. Cellular studies provide evidence of the potential of DLIP topographies to foster endothelialization without enhancement of platelet adhesion, which will be of high importance when designing new BMS in the future.

Keywords: CoCr, Direct laser interference patterning, Endothelial cells, Linear surface pattern, Platelets

Gállego, Isaac, Manning, Brendan, Prades, Joan Daniel, Mir, Mónica, Samitier, Josep, Eritja, Ramon, (2017). DNA-Origami-Aided Lithography for Sub-10 Nanometer Pattern Printing Proceedings Eurosensors 2017 , MDPI (Paris, France) 1, (4), 325

We report the first DNA-based origami technique that can print addressable patterns on surfaces with sub-10 nm resolution. Specifically, we have used a two-dimensional DNA origami as a template (DNA origami stamp) to transfer DNA with pre-programmed patterns (DNA ink) on gold surfaces. The DNA ink is composed of thiol-modified staple strands incorporated at specific positions of the DNA origami stamp to create patterns upon thiol-gold bond formation on the surface (DNA ink). The DNA pattern formed is composed of unique oligonucleotide sequences, each of which is individually addressable. As a proof-of-concept, we created a linear pattern of oligonucleotide-modified gold nanoparticles complementary to the DNA ink pattern. We have developed an in silico model to identify key elements in the formation of our DNA origami-driven lithography and nanoparticle patterning as well as simulate more complex nanoparticle patterns on surfaces.

Keywords: DNA nanotechnology, Lithography, Nanopatterning, Gold nanoparticles, Metasurfaces

Ladoux, B., Mège, R. M., Trepat, X., (2016). Front-rear polarization by mechanical cues: From single cells to tissues Trends in Cell Biology 26, (6), 420-433

Directed cell migration is a complex process that involves front-rear polarization, characterized by cell adhesion and cytoskeleton-based protrusion, retraction, and contraction of either a single cell or a cell collective. Single cell polarization depends on a variety of mechanochemical signals including external adhesive cues, substrate stiffness, and confinement. In cell ensembles, coordinated polarization of migrating tissues results not only from the application of traction forces on the extracellular matrix but also from the transmission of mechanical stress through intercellular junctions. We focus here on the impact of mechanical cues on the establishment and maintenance of front-rear polarization from single cell to collective cell behaviors through local or large-scale mechanisms.

Keywords: Cell forces, Cell polarity, Collective cell migration, Mechanobiology, Micropatterning, Substrate stiffness

Credi, C., De Marco, C., Molena, E., Pla Roca, M., Samitier, J., Marques, J., Fernàndez-Busquets, X., Levi, M., Turri, S., (2016). Heparin micropatterning onto fouling-release perfluoropolyether-based polymers via photobiotin activation Colloids and Surfaces B: Biointerfaces 146, 250-259

A simple method for constructing versatile ordered biotin/avidin arrays on UV-curable perfluoropolyethers (PFPEs) is presented. The goal is the realization of a versatile platform where any biotinylated biological ligands can be further linked to the underlying biotin/avidin array. To this end, microcontact arrayer and microcontact printing technologies were developed for photobiotin direct printing on PFPEs. As attested by fluorescence images, we demonstrate that this photoactive form of biotin is capable of grafting onto PFPEs surfaces during irradiation. Bioaffinity conjugation of the biotin/avidin system was subsequently exploited for further self-assembly avidin family proteins onto photobiotin arrays. The excellent fouling release PFPEs surface properties enable performing avidin assembly step simply by arrays incubation without PFPEs surface passivation or chemical modification to avoid unspecific biomolecule adsorption. Finally, as a proof of principle biotinylated heparin was successfully grafted onto photobiotin/avidin arrays.

Keywords: Antifouling, Heparin, Malaria, Microcontact arrayer, Microcontact printing, Micropatterning, Perfluoropolyether, Photobiotin, Polymers, Soft lithography

Álvarez, Z., Sena, E., Mattotti, M., Engel, E., Alcántara, S., (2014). An efficient and reproducible method to culture Bergmann and cortical radial glia using textured PMMA Journal of Neuroscience Methods , 232, 93-101

Background: Radial glia cells comprise the principal population of neural stem cells (NSC) during development. Attempts to develop reproducible radial glia and NSC culture methods have met with variable results, yielding non-adherent cultures or requiring the addition of growth factors. Recent studies demonstrated that a 2-μm patterned poly-methyl methacrylate (ln2 PMMA) grooved scaffold, by mimicking the biophysical and microtopographic properties of the embryonic NSC niche, induces the de-differentiation of glial cells into functional radial glia cells. New method: Here we describe a method for obtaining cultures of adherent Bergmann radial glia (BRG) and cortical radial glia (CRG). The growth substrate is ln2 PMMA and the addition of growth factors is not required. Results: Postnatal glia obtained from mouse cerebellum or cerebral cortex and grown on ln2 PMMA adopted a BRG/CRG phenotype characterized by a bipolar shape, the up-regulation of progenitor markers such as nestin and Sox2, and the down-regulation of vimentin and GFAP. Neurons cultured over the BRG/CRG aligned their processes with those of the glial shafts, thus mimicking the behavior of migrating neuronal cells. Comparison with existing methods: The ln2 PMMA culture method offers an ideal system for analyzing both the biochemical factors controlling the neurogenic potential of BRG/CRG and neuronal migration. Conclusions: The ln2 PMMA method is a reproducible system to obtain immature BRG/CRG preparations in vitro. It can be used to study the properties of CNS progenitor cells as well as the interactions between radial glia and neurons, and supports cultured progenitors for use in different applications. © 2014 Elsevier B.V.

Keywords: Astrocytes, Bergmann glia, Micro-patterning, Poly-methyl methacrylate (PMMA), Progenitors, Radial glia, Surface topography

Mattotti, Marta, Alvarez, Zaida, Ortega, Juan A., Planell, Josep A., Engel, Elisabeth, Alcántara, Soledad, (2012). Inducing functional radial glia-like progenitors from cortical astrocyte cultures using micropatterned PMMA Biomaterials 33, (6), 1759-1770

Radial glia cells (RGC) are multipotent progenitors that generate neurons and glia during CNS development, and which also served as substrate for neuronal migration. After a lesion, reactive glia are the main contributor to CNS regenerative blockage, although some reactive astrocytes are also able to de-differentiate in situ into radial glia-like cells (RGLC), providing beneficial effects in terms of CNS recovery. Thus, the identification of substrate properties that potentiate the ability of astrocytes to transform into RGLC in response to a lesion might help in the development of implantable devices that improve endogenous CNS regeneration. Here we demonstrate that functional RGLC can be induced from in vitro matured astrocytes by using a precisely-sized micropatterned PMMA grooved scaffold, without added soluble or substrate adsorbed biochemical factors. RGLC were extremely organized and aligned on 2 μm line patterned PMMA and, like their embryonic counterparts, express nestin, the neuron-glial progenitor marker Pax6, and also proliferate, generate different intermediate progenitors and support and direct axonal growth and neuronal migration. Our results suggest that the introduction of line patterns in the size range of the RGC processes in implantable scaffolds might mimic the topography of the embryonic neural stem cell niche, driving endogenous astrocytes into an RGLC phenotype, and thus favoring the regenerative response in situ.

Keywords: Polymethylmethacrylate, Micropatterning, Surface topography, Astrocyte, Nerve guide, Co-culture

Diez-Ahedo, Ruth , Normanno, Davide , Esteban, Olga,, Bakker, Gert-Jan, Figdor, Carl, Cambi, Alessandra , Garcia-Parajo, M. F., (2009). Dynamic re-organization of individual adhesion nanoclusters in living cells by ligand-patterned surfaces Small 5, (11), 1258-1263

Ligand-patterned surfaces alter the spatio-temporal organization of specific receptors on the cell membrane. Chemically confined surfaces are fabricated using microcontact patterning. The dynamic re-organization of the integrin LFA-1 in living cells is monitored at the single-molecule level using total internal reflection fluorescence. The image on the left shows individual LFA-1 nanoclusters on a single cell being recruited to ligand-rich areas of the pattern.

Keywords: Cell adhesion, Microcontact printing, Patterning, Single molecule studies

Ruiz, A., Mills, C. A., Valsesia, A., Martinez, E., Ceccone, G., Samitier, J., Colpo, P., Rossi, F., (2009). Large-area, nanoimprint-assisted microcontact stripping for the fabrication of microarrays of fouling/nonfouling nanostructures Small 5, (10), 1133-1137

Methods for the accurate positioning of nanometric beads on a substrate have been developed over a number of years, and range from serial atomic force microscopy (AFM)techniques for single-bead positioning to parallel techniques for the positioning of large populations of beads in monolayer or multilayer architectures, typically from a liquid suspension. For example, topographic cues have been used for bead-based protein array production, although in this case, there is a random distribution of beads within the topography. Bead patterning has also been achieved in capillaries using a micromolding in capillaries (MIMIC) technique. Line patterns with micrometer widths are possible with this technique, achieving good multilayer organization. For monolayer bead patterning at micrometer dimensions, electrostatic forces and similar electrostatic assemblies using nanoxerography, as well as patterning by selective chemical functionalization, by transfer of particles from a liquid–liquid interface, and by subtracting top–down processes, are possible.

Keywords: Microcontact stripping, Nanostructures, Poly(acrylic acid), Polystyrene, Surface patterning

Martinez, E., Lagunas, A., Mills, C. A., Rodriguez-Segui, S., Estevez, M., Oberhansl, S., Comelles, J., Samitier, J., (2009). Stem cell differentiation by functionalized micro- and nanostructured surfaces Nanomedicine 4, (1), 65-82

New fabrication technologies and, in particular, new nanotechnologies have provided biomaterial and biomedical scientists with enormous possibilities when designing customized supports and scaffolds with controlled nanoscale topography and chemistry. The main issue now is how to effectively design these components and choose the appropriate combination of structure and chemistry to tailor towards applications as challenging and complex as stem cell differentiation. Occasionally, an incomplete knowledge of the fundamentals of biological differentiation process has hampered this issue. However, the recent technological advances in creating controlled cellular microenvironments can be seen as a powerful tool for furthering fundamental biology studies. This article reviews the main strategies followed to achieve solutions to this challenge, particularly emphasizing the working hypothesis followed by the authors to elucidate the mechanisms behind the observed effects of structured surfaces on cell behavior.

Keywords: Cell pattering, Differentiation, Microcontact printing, Micropatterning, Microstructure, Nanoimprinting, Nanostructure, Stem cells

Caballero, D., Samitier, J., Errachid, A., (2009). Submerged nanocontact printing (SnCP) of thiols Journal of Nanoscience and Nanotechnology , 9, (11), 6478-6482

Biological patterned surfaces having sub-micron scale resolution are of great importance in many fields of life science and biomedicine. Different techniques have been proposed for surface patterning at the nanoscale. However, most of them present some limitations regarding the patterned area size or are time-consuming. Micro/nanocontact printing is the most representative soft lithography-based technique for surface patterning at the nanoscale. Unfortunately, conventional micro/nanocontact printing also suffers from problems such as diffusion and stamp collapsing that limit pattern resolution. To overcome these problems, a simple way of patterning thiols under liquid media using submerged nanocontact printing (SnCP) over large areas (similar to cm(2)) achieving nanosize resolution is presented. The technique is also low cost and any special equipment neither laboratory conditions are required. Nanostructured poly(dimethyl siloxane) stamps are replicated from commercially available digital video disks. SnCP is used to stamp patterns of 200 nm 1-octadecanethiol lines in liquid media, avoiding ink diffusion and stamp collapsing, over large areas on gold substrates compared with conventional procedures. Atomic force microscopy measurements reveal that the patterns have been successfully transferred with high fidelity. This is an easy, direct, effective and low cost methodology for molecule patterning immobilization which is of interest in those areas that require nanoscale structures over large areas, such as tissue engineering or biosensor applications.

Keywords: Submerged Nanocontact Printing, Replica Molding, Nanopatterning, Large Area, Dip-pen nanolithography, High-aspect-ratio, Soft lithography, Submicronscale, Nanoimprint lithography, Thin-film, Surfaces, Fabrication, Proteins, Nanofabrication