Staff member

Anna Lagunas Targarona

Senior Researcher
+34 934 037 178
Staff member publications

Lagunas, Anna, Tsintzou, Iro, Vida, Yolanda, Collado, Daniel, Pérez-Inestrosa, Ezequiel, Pereira, Cristina Rodríguez, Magalhaes, Joana, Andrades, José A., Samitier, Josep, (2017). Tailoring RGD local surface density at the nanoscale toward adult stem cell chondrogenic commitment Nano Research 10, (6), 1959-1971

Arginine-glycine-aspartic acid (RGD) dendrimer-based nanopatterns on poly(L-lactic acid) were used as bioactive substrates to evaluate the impact of the RGD local surface density on the chondrogenic induction of adult human mesenchymal stem cells. During chondrogenic commitment, active extracellular matrix (ECM) remodeling takes place, playing an instructive role in the differentiation process. Although three-dimensional environments such as pellet or micromass cultures are commonly used for in vitro chondrogenic differentiation, these cultures are rather limited with respect to their ability to interrogate cells in cell–ECM interactions. In the present study, the nanopatterns of the tunable RGD surface density were obtained as a function of the initial dendrimer concentration. The local RGD surface density was quantified through probability contour plots for the minimum interparticle distance, constructed from the corresponding atomic force microscopy images, and correlated with the cell adhesion and differentiation response. The results revealed that the local RGD surface density at the nanoscale acts as a regulator of chondrogenic commitment, and that intermediate adhesiveness of cells to the substrates favors mesenchymal cell condensation and early chondrogenic differentiation.

Lagunas, A., Sasso, B., Tesson, N., Cantos, C., Martinez, E., Samitier, J., (2016). Synthesis of a polymethyl(methacrylate)-polystyrene-based diblock copolymer containing biotin for selective protein nanopatterning Polymer Chemistry 7, 212-218

Protein patterning is of interest in high-throughput screening. Due to an increase in demand for further miniaturization of protein assays, block copolymers (BCPs) that can undergo large-area phase separation into nanometer-size domains have attracted great attention as substrates for protein nanopatterning. Here we report the synthesis of a polymethyl(methacrylate)-polystyrene-based diblock copolymer which, once spin-coated, is capable of self-segregating into cylindrical polystyrene (PS) domains. In this copolymer, the PS block was modified to introduce biotin below 10% molar in order to achieve molecular recognition of streptavidin. The PMMA matrix used to introduce poly(ethylene glycol) enabled us to obtain an antifouling environment that prevents unspecific protein adsorption outside the domains. The use of the biotin-streptavidin pair in this BCP makes it suitable for nanopatterning of other biotinylated proteins of interest for the purposes of cell biology, biosensors, and tissue engineering.

Lagunas, A., Caballero, D., Samitier, J., (2016). Influence of controlled micro- and nanoengineered environments on stem cell Advanced Surfaces for Stem Cell Research (ed. Tiwari, A., Garipcan, B., Uzun, L.), Wiley (San Francisco, USA) , 85-140

The book outlines first the importance of Extra Cellular Matrix (ECM), which is a natural surface for most of cells. In the following chapters the influence of biological, chemical, mechanical, and physical properties of surfaces in micro and nano-scale on stem cell behavior are discussed including the mechanotransduction. Biomimetic and bioinspired approaches are highlighted for developing microenvironment of several tissues, and surface engineering applications are discussed in tissue engineering, regenerative medicine and different type of biomaterials in various chapters of the book. This book brings together innovative methodologies and strategies adopted in the research and development of Advanced Surfaces in Stem Cell Research. Well-known worldwide researchers deliberate subjects including: Extracellular matrix proteins for stem cell fate The superficial mechanical and physical properties of matrix microenvironment as stem cell fate regulator Effects of mechanotransduction on stem cell behavior Modulation of stem cells behavior through bioactive surfaces Influence of controlled micro and nanoengineered surfaces on stem cell fate Nanostructured polymeric surfaces for stem cells Laser surface modification techniques and stem cells applications Plasma polymer deposition: a versatile tool for stem cell research Application of bioreactor concept and modeling techniques in bone regeneration and augmentation treatments Substrates and surfaces for control of pluripotent stem cell fate and function

Lagunas, Anna, Martinez, Elena, Samitier, Josep, (2015). Surface-bound molecular gradients for the high throughput screening of cell responses Frontiers in Bioengineering and Biotechnology 3, Article 132

Chemical gradient surfaces are described as surfaces with a gradually varying composition along their length. Continuous chemical gradients have recently been proposed as alternative to discrete microarrays for the high throughput screening of the effects of ligand concentration in cells. Here we review some of the most recent examples in which gradients have been used to evaluate the effect of a varying ligand concentration in cell adhesion, morphology, growth and differentiation of cells, including some of our recent findings. They show the importance of the organization of ligands at the nanoscale, which is highlighted by abrupt changes in cell behavior at critical concentration thresholds.

Keywords: Cell Adhesion, Cell Differentiation, Cell growth, Cell morphology, Molecular gradient

Galan, Teresa, Lagunas, Anna, Martinez, Elena, Samitier, Josep, (2015). Fabrication of bioactive polypyrrole microelectrodes on insulating surfaces by surface-guided biocatalytical polymerization RSC Advances 5, (82), 67082-67088

Although promising, organic microelectronics lacks standard fabrication methods comparable to photolithography in terms of resolution. Here we propose a novel and easily scalable on-surface biocatalytical procedure for the fabrication of polypyrrole microelectrodes on insulating surfaces. Arrays of polypyrrole microelectrodes were obtained by surface-guided biocatalytical polymerization, achieving up to 5 [small micro]m in resolution and conductivities up to 3 S cm-1. The mild reaction conditions provided by the biocatalytical approach permit the entrapment of bioactive compounds during polymer synthesis. This system is convenient for drug release purposes, as demonstrated by the controlled release of entrapped biotin through electrical stimulation. These results pave the way for the application of polypyrrole microelectrodes produced through biocatalysis in the development of implantable devices for remotely controlled tissue interactions.

Lagunas, A., Garcia, A., Artés, J. M., Vida, Y., Collado, D., Pérez-Inestrosa, E., Gorostiza, P., Claros, S., Andrades, J. A., Samitier, J., (2014). Large-scale dendrimer-based uneven nanopatterns for the study of local arginine-glycine-aspartic acid (RGD) density effects on cell adhesion Nano Research 7, (3), 399-409

Cell adhesion processes are governed by the nanoscale arrangement of the extracellular matrix (ECM), being more affected by local rather than global concentrations of cell adhesive ligands. In many cell-based studies, grafting of dendrimers on surfaces has shown the benefits of the local increase in concentration provided by the dendritic configuration, although the lack of any reported surface characterization has limited any direct correlation between dendrimer disposition and cell response. In order to establish a proper correlation, some control over dendrimer surface deposition is desirable. Here, dendrimer nanopatterning has been employed to address arginine-glycine-aspartic acid (RGD) density effects on cell adhesion. Nanopatterned surfaces were fully characterized by atomic force microscopy (AFM), scanning tunneling microscopy (STM) and X-ray photoelectron spectroscopy (XPS), showing that tunable distributions of cell adhesive ligands on the surface are obtained as a function of the initial dendrimer bulk concentration. Cell experiments showed a clear correlation with dendrimer surface layout: Substrates presenting regions of high local ligand density resulted in a higher percentage of adhered cells and a higher degree of maturation of focal adhesions (FAs). Therefore, dendrimer nanopatterning is presented as a suitable and controlled approach to address the effect of local ligand density on cell response. Moreover, due to the easy modification of dendrimer peripheral groups, dendrimer nanopatterning can be further extended to other ECM ligands having density effects on cells.

Keywords: Arginine-glycine-aspartic acid, Atomic force microscopy, Cell adhesion, Dendrimer, Focal adhesions, Scanning tunneling microscopy

Oberhansl, S., Garcia, A., Lagunas, A., Prats-Alfonso, E., Hirtz, M., Albericio, F., Fuchs, H., Samitier, J., Martinez, E., (2014). Mesopattern of immobilised bone morphogenetic protein-2 created by microcontact printing and dip-pen nanolithography influence C2C12 cell fate RSC Advances 4, (100), 56809-56815

Dip-pen nanolithography and microcontact printing were used to fabricate mesopatterned substrates for cell differentiation experiments. A biotin-thiol was patterned on gold substrates and subsequently functionalised with streptavidin and biotinylated bone morphogenetic protein-2 (BMP-2). The feasibility of mesopatterned substrates containing immobilised BMP-2 was proven by obtaining similar differentiation outcomes compared to the growth factor in solution. Therefore, these substrates might be suitable for replacing conventional experiments with BMP-2 in solution.

Keywords: Bone morphogenetic protein-2, C2C12 cells, Dip-pen nanolithography, Micro contact printing

Garcia, A., Hortigüela, V., Lagunas, A., Cortina, C., Montserrat, N., Samitier, J., Martinez, E., (2014). Protein patterning on hydrogels by direct microcontact printing: application to cardiac differentiation RSC Advances 4, (55), 29120-29123

An extended microcontact printing technique to chemically pattern hydrogels is reported. The procedure employs standard polydimethylsiloxane stamps and requires minor pre-processing of the hydrogels by freeze-drying. Micropatterned Matrigel[trade mark sign] and gelatin hydrogels induce NIH-3T3 cell alignment and elongation. Furthermore, human embryonic stem cells cultured on fibronectin-patterned hydrogels display beating foci earlier than those cultured on non-patterned substrates.

Lagunas, A., Comelles, J., Oberhansl, S., Hortigüela, V., Martínez, E., Samitier, J., (2013). Continuous bone morphogenetic protein-2 gradients for concentration effect studies on C2C12 osteogenic fate Nanomedicine: Nanotechnology, Biology, and Medicine 9, (5), 694-701

Cells can respond to small changes in a varying concentration of exogenous signaling molecules. Here we propose the use of continuous surface chemical gradients for the in-depth study of dose-dependent effects on cells. A continuous surface gradient of bone morphogenetic protein-2 (BMP-2) is presented. The gradient covers a narrow range of surface densities (from 1.4 to 2.3 pmol/cm2) with a shallow slope (0.9 pmol/cm3). These characteristics represent a quasi-homogeneous surface concentration at the cell scale, which is crucial for cell screening studies. Cell fate evaluation at early stages of osteogenesis in C2C12 cells, indicates the potential of continuous gradients for in vitro screening applications.

Prats-Alfonso, E., Oberhansl, S., Lagunas, A., Martínez, E., Samitier, J., Albericio, F., (2013). Effective and versatile strategy for the total solid-phase synthesis of alkanethiols for biological applications European Journal of Organic Chemistry 2013, (7), 1233-1239

Biological applications increasingly demand tailored surfaces with a range of functional groups. Herein we describe a straightforward and inexpensive method based exclusively on solid-phase synthesis for the preparation of a variety of customized alkanethiols (ATs). The technique overcomes all the difficulties encountered during the preparation of these molecules in solution. The procedure allows the use of ATs without further purification for the preparation of self-assembled monolayers on gold, typically used to achieve functional group diversity on this surface. This paper describes a straightforward and inexpensive method based exclusively on solid-phase synthesis for the preparation of a variety of customized alkanethiols (ATs). The technique allows a variety of ATs to be obtained in only three steps, overcoming the difficulties encountered during their preparation in solution.

Oberhansl, Sabine, Hirtz, Michael, Lagunas, Anna, Eritja, Ramon, Martinez, Elena, Fuchs, Harald, Samitier, Josep, (2012). Facile modification of silica substrates provides a platform for direct-writing surface click chemistry Small 8, (4), 541-545

Lagunas, Anna , Comelles, Jordi, Martínez, Elena, Prats-Alfonso, Elisabet , Acosta, Gerardo A., Albericio, Fernando , Samitier, Josep , (2012). Cell adhesion and focal contact formation on linear RGD molecular gradients: study of non-linear concentration dependence effects Nanomedicine: Nanotechnology, Biology and Medicine 8, (4), 432-439

Cell adhesion onto bioengineered surfaces is affected by a number of variables, including the former substrate derivatization process. In this investigation, we studied the correlation between cell adhesion and cell–adhesive ligand surface concentration and organization due to substrate modification. For this purpose, Arg-Gly-Asp (RGD) gradient surfaces were created on poly(methyl methacrylate) substrates by continuous hydrolysis and were then grafted with biotin-PEG-RGD molecules. Cell culture showed that adhesion behavior changes in a nonlinear way in the narrow range of RGD surface densities assayed (2.8 to 4.4 pmol/cm2), with a threshold value of 4.0 pmol/cm2 for successful cell attachment and spreading. This nonlinear dependence may be explained by nonhomogeneous RGD surface distribution at the nanometre scale, conditioned by the stochastic nature of the hydrolysis process. Atomic force microscopy analysis of the gradient surface showed an evolution of surface morphology compatible with this hypothesis.

Keywords: RGD gradient, Cell adhesion, Poly(methyl methacrylate), Hydrolysis, Biotin-streptavidin

Lagunas, A., Comelles, J., Martinez, E., Samitier, J., (2010). Universal chemical gradient platforms using poly(methyl methacrylate) based on the biotin streptavidin interaction for biological applications Langmuir 26, (17), 14154-14161

This article describes a simple method for the construction of a universal surface chemical gradient platform based on the biotin streptavidin model. In this approach, surface chemical gradients were prepared in poly(methyl methacrylate) (PM MA), a biocompatible polymer, by a controlled hydrolysis procedure. The physicochemical properties of the resulting modified surfaces were extensively characterized. Chemical analysis carried out via time-of-flight secondary ion mass spectrometry (ToRSIMS) and X-ray photoelectron spectroscopy (XPS) showed the formation of a smooth, highly controllable carboxylic acid gradient of increasing concentration along the sample surface. Atomic force microscopy (AFM) and contact angle (CA) results indicate that, in contrast with most of the chemical gradient methods published in the literature, the chemical modification of the polymer surface barely affects its physical properties. The introduction of carboxylic acid functionality along the surface was then used for biomolecule anchoring. For this purpose, the surface was activated and derivatized first with biotin and finally with streptavidin (SA V) in a directed orientation fashion. The SAV gradient was qualitatively assessed by fluorescence microscopy analysis and quantified by surface plasmon resonance (SPR) in order to establish a quantitative relationship between SAV surface densities and the surface location. The usefulness of the fabrication method described for biological applications was tested by immobilizing biotinylated bradykinin onto the SAV gradient. This proof-of-concept application shows the effectiveness of the concentration range of the gradient because the effects of bradykinin on cell morphology were observed to increase gradually with increasing drug concentrations. The intrinsic characteristics of the fabricated gradient platform (absence of physicochemical modifications other than those due to the biomolecules included) allow us to attribute cell behavior unequivocally to the biomolecule surface density changes.

Keywords: Wettability gradient, Polyethylene surface, Combinatorial, Immobilization, Biomaterials, Fabrication, Deposition, Bradykinin, Monolayers, Discharge

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

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