The stiffness of elastomeric surfaces influences the mechanical properties of endothelial cells

Optimal characterization of the mechanical properties of both cells and their surrounding is an issue of major interest. Indeed, cell function and development are strongly influenced by external stimuli. Furthermore, a change in cell mechanics might, in some cases, associate with diseases or malfunctioning. In this work, atomic force microscopy (AFM) was applied to examine the mechanical properties of the silicone elastomer polydimethylsiloxane (PDMS) a common substrate in cell culture. Force spectroscopy analysis was done over different specimens of this elastomeric material containing varying ratios of resin to cross-linker in its structure (5:1, 10:1, 20:1, 30:1 and 50:1), which impacts the final material properties (e.g., stiffness, elasticity). To quantify the mechanical properties of the PDMS, factors as the modulus of Young, the maximum adhesive forces as well as both relaxation amplitudes and times upon constant height contact of the tip (dwell time different of zero) were calculated from the different segments forming the force curves. It is demonstrated that the material stiffness is increased by prior oxygen plasma treatment of the sample, required for hydrophilic switching, contrarily to what observed for its adhesiveness. Subsequent incubation of endothelial HUVEC cells on top of these plasma treated PDMS systems yields minor variation in cell mechanics in comparison to those obtained on a glass reference, on which cells show much higher spreading tendency and, by extension, a remarkable membrane hardening. Thus, surface wettability turns a factor of higher relevance than substrate stiffness inducing variations in the cell mechanics.Comment: manuscript (12 pages, 4 figures, 2 tables), supplementary information (2 pages and 3 figures), the main results of the manuscript are based on a master thesi

Synchronized cell attachment triggered by photo-activatable adhesive ligands allows QCM-based detection of early integrin binding

The Quartz Crystal Microbalance with dissipation (QCM-D) technique was applied to monitor and quantify integrin-RGD recognition during the early stages of cell adhesion. Using QCM-D crystals modified with a photo-activatable RGD peptide, the time point of presentation of adhesive ligand at the surface of the QCM-D crystal could be accurately controlled. This allowed temporal resolution of early integrin-RGD binding and the subsequent cell spreading process, and their separate detection by QCM-D. The specificity of the integrin-RGD binding event was corroborated by performing the experiments in the presence of soluble cyclicRGD as a competitor, and cytochalasin D as inhibitor of cell spreading. Larger frequency change in the QCM-D signal was observed for cells with larger spread area, and for cells overexpressing integrin avb3 upon stable transfection. This strategy enables quantification of integrin activity which, in turn, may allow discrimination among different cell types displaying distinct integrin subtypes and expression levels thereof. On the basis of these findings, we believe the strategy can be extended to other photoactivatable ligands to characterize cell membrane receptors activity, a relevant issue for cancer diagnosis (and prognosis) as other several pathologies.Fil: Iturri, Jagoba. Max Planck Institute for Polymer Research; AlemaniaFil: García Fernández, Luis. Max Planck Institute for Polymer Research; AlemaniaFil: Reuning, Ute. Technische Universitat Munchen; AlemaniaFil: García, Andrés J.. Georgia Institute Of Techology; Estados UnidosFil: del Campo, Aránzazu. Max Planck Institute for Polymer Research; AlemaniaFil: Salierno, Marcelo Javier. Max Planck Institute for Polymer Research; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Antibacterial Strategies from the Sea: Polymer-Bound Cl-Catechols for Prevention of Biofilm Formation

Inspired by the amino acid 2-chloro-4,5-dihydroxyphenylalanine (Cl-DOPA), present in the composition of the proteinaceous glue of the sandcastle worm Phragmatopoma californica, a simple strategy is presented to confer antifouling properties to polymer surfaces using (but not releasing) a bioinspired biocide. Cl-Dopamine is used to functionalize polymer materials and hydrogel films easily, to prevent biofilm formation on themThe authors thank Uwe Rietzler from the Max-Planck-Institut für Polymerforschung for the SFM-based thickness measurements

Nucleotides-Induced Changes in the Mechanical Properties of Living Endothelial Cells and Astrocytes, Analyzed by Atomic Force Microscopy

Endothelial cells and astrocytes preferentially express metabotropic P2Y nucleotide receptors, which are involved in the maintenance of vascular and neural function. Among these, P2Y1 and P2Y2 receptors appear as main actors, since their stimulation induces intracellular calcium mobilization and activates signaling cascades linked to cytoskeletal reorganization. In the present work, we have analyzed, by means of atomic force microscopy (AFM) in force spectroscopy mode, the mechanical response of human umbilical vein endothelial cells (HUVEC) and astrocytes upon 2MeSADP and UTP stimulation. This approach allows for simultaneous measurement of variations in factors such as Young’s modulus, maximum adhesion force and rupture event formation, which reflect the potential changes in both the stiffness and adhesiveness of the plasma membrane. The largest effect was observed in both endothelial cells and astrocytes after P2Y2 receptor stimulation with UTP. Such exposure to UTP doubled the Young’s modulus and reduced both the adhesion force and the number of rupture events. In astrocytes, 2MeSADP stimulation also had a remarkable effect on AFM parameters. Additional studies performed with the selective P2Y1 and P2Y13 receptor antagonists revealed that the 2MeSADP-induced mechanical changes were mediated by the P2Y13 receptor, although they were negatively modulated by P2Y1 receptor stimulation. Hence, our results demonstrate that AFM can be a very useful tool to evaluate functional native nucleotide receptors in living cells

Allergy / Prevention of allergy by viruslike nanoparticles (VNP) delivering shielded versions of major allergens in a humanized murine allergy model

Background: In highrisk populations, allergenspecific prophylaxis could protect from sensitization and subsequent development of allergic disease. However, such treatment might itself induce sensitization and allergies, thus requiring hypoallergenic vaccine formulations. We here characterized the preventive potential of viruslike nanoparticles (VNP) expressing surfaceexposed or shielded allergens. Methods: Fulllength major mugwort pollen allergen Art v 1 was selectively targeted either to the surface or to the inner side of the lipid bilayer envelope of VNP. Upon biochemical and immunological analysis, their preventive potential was determined in a humanized mouse model of mugwort pollen allergy. Results: Viruslike nanoparticles expressing shielded version of Art v 1, in contrast to those expressing surfaceexposed Art v 1, were hypoallergenic as they hardly induced degranulation of rat basophil leukemia cells sensitized with Art v 1specific mouse or human IgE. Both VNP versions induced proliferation and cytokine production of allergenspecific T cells in vitro. Upon intranasal application in mice, VNP expressing surfaceexposed but not shielded allergen induced allergenspecific antibodies, including IgE. Notably, preventive treatment with VNP expressing shielded allergenprotected mice from subsequent sensitization with mugwort pollen extract. Protection was associated with a Th1/Tregdominated cytokine response, increased Foxp3+ Treg numbers in lungs, and reduced lung resistance when compared to mice treated with empty particles. Conclusion: Viruslike nanoparticles represent a novel and versatile platform for the in vivo delivery of allergens to selectively target T cells and prevent allergies without inducing allergic reactions or allergic sensitization.DKW1248SFB F4605SFB F4609(VLID)313247

Characterization of Cell Scaffolds by Atomic Force Microscopy

This review reports on the use of the atomic force microscopy (AFM) in the investigation of cell scaffolds in recent years. It is shown how the technique is able to deliver information about the scaffold surface properties (e.g., topography), as well as about its mechanical behavior (Young’s modulus, viscosity, and adhesion). In addition, this short review also points out the utilization of the atomic force microscope technique beyond its usual employment in order to investigate another type of basic questions related to materials physics, chemistry, and biology. The final section discusses in detail the novel uses that those alternative measuring modes can bring to this field in the future

Life under Continuous Streaming: Recrystallization of Low Concentrations of Bacterial SbpA in Dynamic Flow Conditions

The well-known bacterial S-layer protein SbpA from Lysinibacillus sphaericus CCM2177 induces spontaneous crystal formation via cooperative self-assembly of the protein subunits into an ordered supramolecular structure. Recrystallization occurs in the presence of divalent cations (i.e., Ca2+) and finally leads to producing smooth 2-D crystalline coatings composed of squared (p4) lattice structures. Among the factors interfering in such a process, the rate of protein supply certainly plays an important role since a limited number of accessible proteins might turn detrimental for film completion. Studies so far have mostly focused on high SbpA concentrations provided under stopped-flow or dynamic-flow conditions, thus omitting the possibility of investigating intermediate states, in which dynamic flow is applied for more critical concentrations of SbpA (i.e., 25, 10, and 5 µg/mL). In this work, we have characterized both physico-chemical and topographical aspects of the assembly and recrystallization of SbpA protein in such low concentration conditions by means of in situ Quartz Crystal Microbalance with Dissipation (QCMD) and atomic force microscopy (AFM) measurements, respectively. On the basis of these experiments, we can confirm how the application of a dynamic flow influences the formation of a closed and crystalline protein film from low protein concentrations (i.e., 10 µg/mL), which otherwise would not be formed

Electrochemical-QCMD Control over S-Layer (SbpA) Recrystallization with Fe2+ as Specific Ion for Self-Assembly Induction

The critical role of divalent ions (M²+) in the self-assembly of SbpA S-layer proteins (from Lysinibacillus sphaericus CCM 2177) into crystalline structures has been reported in several studies. Hence, ions such as magnesium, barium, nickel and, most commonly, calcium (Ca²+) have proven to trigger both protein-protein and protein-substrate interactions involved in the two-stage non-classical pathway recrystallization followed by SbpA units. As a result, two dimensional, crystalline nanometric sheets in a highly ordered tetrameric state (p4) can be formed on top of different surfaces. The use of iron in its ferrous state (Fe2+) as self-assembly inducing candidate has been omitted so far due to its instability under aerobic conditions, tending to natural oxidation to the ferric (Fe3+) state. In this work, the potentiality of assembling fully functional S-layers from iron (II) salts (FeCl2 and FeSO4) is described for the first time. A combination of chemical (oxidation retardants) and electrical (−1 V potential) factors has been applied to effectively act against such an oxidizing trend. Formation of the respective crystalline films has been followed by means of Electrochemical Quartz Crystal Microbalance with Dissipation (EQCM-D) measurements and complementary Atomic Force Microscopy (AFM) topography studies, which prove the presence of squared lattice symmetry at the end of the recrystallization process. Both techniques, together with additional electrochemical tests performed over the ion permeability of both types of S-layer coatings formed, show the influence of the counterion chosen (chloride vs. sulphate) in the final packing and performance of the S-layer. The presence of an underlying Secondary Cell Wall Polymer (SCWP) as in the natural case contributes to pair both systems, due to the high lateral motility freedom provided by this biopolymer to SbpA units in comparison to uncoated substrates

In Vitro Characterization of the Two-Stage Non-Classical Reassembly Pathway of S-Layers

The recombinant bacterial surface layer (S-layer) protein rSbpA of Lysinibacillus sphaericus CCM 2177 is an ideal model system to study non-classical nucleation and growth of protein crystals at surfaces since the recrystallization process may be separated into two distinct steps: (i) adsorption of S-layer protein monomers on silicon surfaces is completed within 5 min and the amount of bound S-layer protein sufficient for the subsequent formation of a closed crystalline monolayer; (ii) the recrystallization process is triggered—after washing away the unbound S-layer protein—by the addition of a CaCl2 containing buffer solution, and completed after approximately 2 h. The entire self-assembly process including the formation of amorphous clusters, the subsequent transformation into crystalline monomolecular arrays, and finally crystal growth into extended lattices was investigated by quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM). Moreover, contact angle measurements showed that the surface properties of S-layers change from hydrophilic to hydrophobic as the crystallization proceeds. This two-step approach is new in basic and application driven S-layer research and, most likely, will have advantages for functionalizing surfaces (e.g., by spray-coating) with tailor-made biological sensing layers

Stick–Slip Friction of PDMS Surfaces for Bioinspired Adhesives

Friction plays an important role in the adhesion of many climbing organisms, such as the gecko. During the shearing between two surfaces, periodic stick–slip behavior is often observed and may be critical to the adhesion of gecko setae and gecko-inspired adhesives. Here, we investigate the influence of short oligomers and pendent chains on the stick–slip friction of polydimethylsiloxane (PDMS), a commonly used material for bioinspired adhesives. Three different stick–slip patterns were observed on these surfaces (flat or microstructured) depending on the presence or absence of oligomers and their ability to diffuse out of the material. After washing samples to remove any untethered oligomeric chains, or after oxygen plasma treatment to convert the surface to a thin layer of silica, we decouple the contributions of stiffness, oligomers, and pendant chains to the stick–slip behavior. The stick phase is mainly controlled by the stiffness while the amount of untethered oligomers and pendant chains available at the contact interface defines the slip phase. A large amount of oligomers and pendant chains resulted in a large slip time, dominating the period of stick–slip motion