Publications

Mesenchymal condensation is a prevalent morphogenetic transition that is essential in chondrogenesis. However, the current understanding of condensation mechanisms is limited. In vivo, progenitor cells directionally migrate from the surrounding loose mesenchyme towards regions of increasing matrix adherence (the condensation centers), which is accompanied by the upregulation of fibronectin. Here, we focused on the mechanisms of cell migration during mesenchymal cell condensation and the effects of matrix adherence. Dendrimer-based nanopatterns of the cell-adhesive peptide arginine-glycine-aspartic acid (RGD), which is present in fibronectin, were used to regulate substrate adhesion. We recorded collective and single-cell migration of mesenchymal stem cells, under chondrogenic induction, using live-cell imaging. Our results show that the cell migration mode of single cells depends on substrate adhesiveness, and that cell directionality controls cell condensation and the fusion of condensates. Inhibition experiments revealed that cell-cell interactions mediated by N-cadherin (also known as CDH2) are also pivotal for directional migration of cell condensates by maintaining cell-cell cohesion, thus suggesting a fine interplay between cell-matrix and cell-cell adhesions. Our results shed light on the role of cell interactions with a fibronectin-depositing matrix during chondrogenesis in vitro, with possible applications in regenerative medicine. This article has an associated First Person interview with the first author of the paper.© 2022. Published by The Company of Biologists Ltd.

JTD Keywords: alpha-v-beta-3, arginine-glycine-aspartic acid, chondrogenesis, dynamics, expression, fibronectin, gastrulation, involvement, mechanisms, mesenchymal condensation, model, nanopatterned substrates, rgd, Arginine-glycine-aspartic acid, Cell migration, Chondrogenesis, Mesenchymal condensation, N-cadherin, Nanopatterned substrates, Rgd

The influence of the properties of different solid substrates on the tethering of two antibodies, IgG1-CR3022 and IgG1-S309, which were specifically engineered for the detection of SARS-CoV-2, has been examined at the molecular level using conventional and accelerated Molecular Dynamics (cMD and aMD, respectively). Two surfaces with very different properties and widely used in immunosensors for diagnosis, amorphous silica and the most stable facet of the face-centered cubic gold structure, have been considered. The effects of such surfaces on the structure and orientation of the immobilized antibodies have been determined by quantifying the tilt and hinge angles that describe the orientation and shape of the antibody, respectively, and the dihedrals that measure the relative position of the antibody arms with respect to the surface. Results show that the interactions with amorphous silica, which are mainly electrostatic due to the charged nature of the surface, help to preserve the orientation and structure of the antibodies, especially of the IgG1-CR3022, indicating that the primary sequence of those antibodies also plays some role. Instead, short-range van der Waals interactions with the inert gold surface cause a higher degree tilting and fraying of the antibodies with respect to amorphous silica. The interactions between the antibodies and the surface also affect the correlation among the different angles and dihedrals, which increases with their strength. Overall, results explain why amorphous silica substrates are frequently used to immobilize antibodies in immunosensors. © 2022 The Authors

JTD Keywords: amorphous silica, antibody immobilization, enzyme, gol d, gold, immobilization, immunosensor, molecu l a r dynamics, molecular dynamics, protein adsorption, sars-cov-2 immunosensor, simulations, spike protein, surface interactions, target, vaccine, Amorphous silica, Antibodies, Antibody engineering, Antibody immobilization, Antibody structure, Article, Chemical detection, Computer model, Controlled study, Dihedral angle, Gold, In-silico, Molecular dynamics, Molecular levels, Molecular-dynamics, Nonhuman, Property, Sars, Sars-cov-2 immunosensor, Severe acute respiratory syndrome coronavirus 2, Silica, Silico studies, Silicon dioxide, Solid substrates, Structure analysis, Substrate chemistry, Substrates, Van der waals forces, Virus detection

Abstract The development of nanostructured plasmonic biosensors has been widely widespread in the last years, motivated by the potential benefits they can offer in integration, miniaturization, multiplexing opportunities, and enhanced performance label-free biodetection in a wide field of applications. Between them, engineering tissues represent a novel, challenging, and prolific application field for nanostructured plasmonic biosensors considering the previously described benefits and the low levels of secreted biomarkers (?pM–nM) to detect. Here, we present an integrated plasmonic nanocrystals-based biosensor using high throughput nanostructured polycarbonate substrates. Metallic film thickness and incident angle of light for reflectance measurements were optimized to enhance the detection of antibody–antigen biorecognition events using numerical simulations. We achieved an enhancement in biodetection up to 3× as the incident angle of light decreases, which can be related to shorter evanescent decay lengths. We achieved a high reproducibility between channels with a coefficient of variation below 2% in bulk refractive index measurements, demonstrating a high potential for multiplexed sensing. Finally, biosensing potential was demonstrated by the direct and label-free detection of interleukin-6 biomarker in undiluted cell culture media supernatants from bioengineered 3D skeletal muscle tissues stimulated with different concentrations of endotoxins achieving a limit of detection (LOD) of ? 0.03 ng/mL (1.4 pM).

JTD Keywords: assay, crystals, drug, label-free biosensing, molecules, plasmonic nanostructures, sensors, skeletal muscle, tissue engineering, Biodetection, Biomarkers, Biosensors, Cell culture, Cells, Chemical detection, Histology, Interleukin-6, Interleukin6 (il6), Label free, Label-free biosensing, Muscle, Nano-structured, Nanocrystals, Plasmonic nanocrystals, Plasmonic nanostructures, Plasmonics, Polycarbonate substrates, Polycarbonates, Refractive index, Sensitivity, Skeletal muscle, Tissue engineering, Tissues engineerings

[Figure: see text].

JTD Keywords: activation, cell extrusion, contraction, drives, homeostasis, interface, junctions, kinase, tension, Adhesion, Article, Cell membranes, Chemical activation, Cytology, E-cadherin, Epithelial monolayers, Epithelium, Homoeostasis, Human, Mechanical instabilities, Monolayers, Morphological transformations, Morphology, Normal tissue, Oncogenics, Soft substrates, Substrates, Tissue, Tissue mechanics, Tissue morphology, Tumor development

Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon may occur. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved. Cells sense mechanical forces from their environment, but the precise mechanical variable sensed by cells is unclear. Here, the authors show that cells can sense the rate of force application, known as the loading rate, with effects on YAP nuclear localization and cytoskeletal stiffness remodelling.

JTD Keywords: Actin cytoskeleton, Actin filament, Actin-filament, Adhesion, Animal, Animals, Atomic force microscopy, Breathing, Cell, Cell adhesion, Cell culture, Cell nucleus, Cells, cultured, Cytoplasm, Extracellular-matrix, Fibroblast, Fibroblasts, Fibronectin, Frequency, Gene knockdown, Gene knockdown techniques, Genetics, Germfree animal, Integrin, Intracellular signaling peptides and proteins, Knockout mouse, Lung, Male, Mechanotransduction, Mechanotransduction, cellular, Metabolism, Mice, Mice, knockout, Microscopy, atomic force, Mouse, Optical tweezers, Paxillin, Physiology, Primary cell culture, Pxn protein, mouse, Rat, Rats, Rats, sprague-dawley, Respiration, Signal peptide, Softening, Specific pathogen-free organisms, Sprague dawley rat, Stress, Substrate, Substrate rigidity, Talin, Talin protein, mouse, Tln2 protein, mouse, Traction, Transmission, Ultrastructure, Yap1 protein, rat

Bioactive nanocomposite scaffolds with cell-adhesive surface have excellent bone regeneration capacities. Fibronectin (FN)-immobilized nanobioactive glass (nBG)/polycaprolactone (PCL) (FN-nBG/PCL) scaffolds with an open pore architecture were generated by a robotic-dispensing technique. The surface immobilization level of FN was significantly higher on the nBG/PCL scaffolds than on the PCL scaffolds, mainly due to the incorporated nBG that provided hydrophilic chemical-linking sites. FN-nBG/PCL scaffolds significantly improved cell responses, including initial anchorage and subsequent cell proliferation. Although further in-depth studies on cell differentiation and the in vivo animal responses are required, bioactive nanocomposite scaffolds with cell-favoring surface are considered to provide promising three-dimensional substrate for bone regeneration.

JTD Keywords: Bone scaffolds, Cell response, Fibronectin, Nanobioactive glass, Nanocomposites, Polycaprolactone, Bone, Cell proliferation, Cells, Cytology, Glass, Nanocomposites, Polycaprolactone, Robotics, Bone scaffolds, Bone tissue engineering, Cell response, Fibronectin, Fibronectin immobilizations, Nano bioactive glass, Nanocomposite scaffolds, Three-dimensional substrates, Scaffolds (biology)

A numerical analysis of the polarization force between a sharp conducting probe and a dielectric film of finite lateral dimensions on a metallic substrate is presented with the double objective of (i) determining the conditions under which the film can be approximated by a laterally infinite film and (ii) proposing an analytical model valid in this limit. We show that, for a given dielectric film, the critical diameter above which the film can be modeled as laterally infinite depends not only on the probe geometry, as expected, but mainly on the film thickness. In particular, for films with intermediate to large thicknesses (>100 nm), the critical diameter is nearly independent from the probe geometry and essentially depends on the film thickness and dielectric constant following a relatively simple phenomenological expression. For films that can be considered as laterally infinite, we propose a generalized analytical model valid in the thin-ultrathin limit (<20-50 nm) that reproduces the numerical calculations and the experimental data. Present results provide a general framework under which accurate quantification of electrostatic force microscopy measurements on dielectric films on metallic substrates can be achieved.

JTD Keywords: Dielectric constant, Dielectric films, Electrostatic force microscopy, Quantification, Analytical models, Electric force microscopy, Electrostatic force, Film thickness, Permittivity, Probes, Substrates, Ultrathin films, Accurate quantifications, Electrostatic force microscopy, Finite size effect, Lateral dimension, Metallic substrate, Numerical calculation, Polarization forces, Quantification, Dielectric films

Abstract Coordinated cell movements in epithelial layers are essential for proper tissue morphogenesis and homeostasis. Microfabrication techniques have proven to be very useful for studies of collective cell migration in vitro. In this chapter, we briefly review the use of microfabricated substrates in providing new insights into collective cell behaviors. We first describe the development of micropatterned substrates to study the influence of geometrical constraints on cell migration and coordinated movements. Then, we present an alternative method based on microfabricated pillar substrates to create well-defined gaps within cell sheets and study gap closure. We also provide a discussion that presents possible pitfalls and sheds light onto the important parameters that allow the study of long-term cell culture on substrates of well-defined geometries.

JTD Keywords: Microfabricated substrates, Microcontact printing, Collective cell behavior, Geometrical constraints, Epithelial gap closure

We show that optical visualization of ultrathin mica flakes on metallic substrates is viable using semitransparent gold as substrates. This enables to easily localize mica flakes and rapidly estimate their thickness directly on gold substrates by conventional optical reflection microscopy. We experimentally demonstrate it by comparing optical images with atomic force microscopy images of mica flakes on semitransparent gold. Present results open the possibility for simple and rapid characterization of thin mica flakes as well as other thin sheets directly on metallic substrates.

JTD Keywords: Atomic force, Conductive AFM, Gold substrates, Metallic substrate, Optical image, Optical reflection, Optical visualization, Ultrathin layers, Atomic force microscopy, Geometrical optics, Gold, Mica, Optical microscopy, Substrates

Biosensors' research filed has clearly been changing towards the production of multifunctional and innovative design concepts to address the needs related with sensitivity and selectivity of the devices. More recently, waveguide biosensors, that do not require any label procedure to detect biomolecules adsorbed on its surface, have been pointed out as one of the most promising technologies for the production of biosensing devices with enhanced performance. Moreover the combination of optical and electrochemical measurements through the integration of transparent and conducting oxides in the multilayer structures can greatly enhance the biosensors' sensitivity. Furthermore, the integration of polymeric substrates may bring powerful advantages in comparison with silicon based ones. The biosensors will have a lower production costs being possible to disposable them after use ("one use sensor chip"). This research work represents a preliminary study about the influence of substrate temperature on the overall properties of ITO thin films deposited by DC magnetron sputtering onto 0,5 mm thick PMMA sheets.

JTD Keywords: ITO thin films, PMMA sheets, Waveguide biosensing devices, Biosensing devices, Conducting oxides, Dc magnetron sputtering, Electrochemical measurements, Enhanced performance, Innovative design, ITO thin films, Multilayer structures, Overall properties, PMMA sheets, Polymeric substrate, Production cost, Sensor chips, Silicon-based, Substrate temperature, Biosensors, Deposition, Design, Film preparation, Optical multilayers, Thin films, Vapor deposition, Waveguides, Substrates

Cellular microarray developments and its applications are the next step after DNA and protein microarrays. The choice of the surface chemistry of the substrates used for the implementation of this technique, that must favor proper protein immobilization while avoiding cell adhesion on the nonspotted areas, presents a complex challenge. This is a key issue since usually the best nonfouling surfaces are also the ones that retain immobilized the smallest amounts of printed protein. To quantitatively assess the amount of protein immobilization, in this study several combinations of fluorescently labeled fibronectin (Fn*) and streptavidin (SA*) were microspotted, with and without glycerol addition in the printing buffer, on several substrates suitable for cellular microarrays. The substrates assayed included chemically activated surfaces as well as Poly ethylene oxide (PEO) films that are nonfouling in solution but accept adhesion of proteins in dry conditions. The results showed that the spotted Fn* was retained by all the surfaces, although the PEO surface did show smaller amounts of immobilization. The SA*, on the other hand, was only retained by the chemically activated surfaces. The inclusion of glycerol in the printing buffer significantly reduced the immobilization of both proteins. The results presented in this article provide quantitative evidence of the convenience of using a chemically activated surface to immobilize proteins relevant for cellular microarray applications, particularly when ECM proteins are cospotted with smaller factors which are more difficult to be retained by the surfaces.

JTD Keywords: Protein immobilization, Quantification, Microarray, Substrate, Surface chemistry