Anna Lagunas

Stem cell differentiation by functionalized micro- and nanostructured surfaces.

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 processes 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.

Cell adhesion and focal contact formation on linear RGD molecular gradients: study of non-linear concentration dependence effects

UNLABELLED 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/cm(2)), with a threshold value of 4.0 pmol/cm(2) 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. FROM THE CLINICAL EDITOR The authors observed by AFM nonlinear dependence of cell adhesion on RGD gradient surfaces with different surface densities. The nonlinear characteristics may be explained by non-homogeneous RGD surface distribution at the nanometer scale, conditioned by the stochastic nature of the hydrolysis process.

Universal Chemical Gradient Platforms Using Poly(methyl methacrylate) Based on the Biotin Streptavidin Interaction for Biological Applications

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) (PMMA), 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 (ToF-SIMS) 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 (SAV) 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.

Facile Modification of Silica Substrates Provides a Platform for Direct‐Writing Surface Click Chemistry

Please click here: a facile two-step functionalization strategy for silicon oxide-based substrates generates a stable platform for surface click chemistry via direct writing. The suitability of the obtained substrates is proven by patterning with two different direct-writing techniques and three different molecules.

Protein patterning on hydrogels by direct microcontact printing: application to cardiac differentiation

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™ 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.

Continuous bone morphogenetic protein-2 gradients for concentration effect studies on C2C12 osteogenic fate.

UNLABELLED 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/cm(2)) with a shallow slope (0.9 pmol/cm(3)). 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. FROM THE CLINICAL EDITOR The authors propose the use of surface-applied continuous chemical gradients for in-depth study of dose-dependent effects on cells. The method is demonstrated using BMP-2 proteins on C2C12 cells as a model system.

Large-scale dendrimer-based uneven nanopatterns for the study of local arginine-glycine-aspartic acid (RGD) density effects on cell adhesion

AbstractCell 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.

Surface-Bound Molecular Gradients for the High-Throughput Screening of Cell Responses

Chemical gradient surfaces are described as surfaces with a gradually varying composition along their length. Continuous chemical gradients have recently been proposed as an 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.

Mesopattern of immobilised bone morphogenetic protein-2 created by microcontact printing and dip-pen nanolithography influence C2C12 cell fate

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.

Fabrication of bioactive polypyrrole microelectrodes on insulating surfaces by surface-guided biocatalytical polymerization

Although promising, organic microelectronics lack 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 μm 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.