Cellular Response to Non-contacting Nanoscale Sublayer: Cells Sense Several Nanometer Mechanical Property

Cell adhesion is influenced not only from the surface property of materials but also from the mechanical properties of the nanometer sublayer just below the surface. In this study, we fabricated a well-defined diblock polymer brush composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) and 2-aminoethyl methacrylate (AEMA). The underlying layer of poly­(MPC) is a highly viscous polymer, and the surface layer of poly­(AEMA) is a cell-adhesive cationic polymer. The adhesion of L929 mouse fibroblasts was examined on the diblock polymer brush to see the effect of a non-contacting underlying polymer layer on the cell-adhesion behavior. Cells could sense the viscoelasticity of the underlying layers at the nanometer level, although the various fabricated diblock polymer brushes had the same surface property and the functional group. Thus, we found a new factor which could control cell spread at the nanometer level, and this insight would be important to design nanoscale biomaterials and interfaces

Slope-Dependent Cell Motility Enhancements at the Walls of PEG-Hydrogel Microgroove Structures

In recent years, research utilizing micro- and nanoscale geometries and structures on biomaterials to manipulate cellular behaviors, such as differentiation, proliferation, survival, and motility, have gained much popularity; however, how the surface microtopography of 3D objects, such as implantable devices, can affect these various cell behaviors still remains largely unknown. In this study, we discuss how the walls of microgroove topography can influence the morphology and the motility of unrestrained cells, in a different fashion from 2D line micropatterns. Here adhesive substrates made of tetra­(polyethylene glycol) (tetra-PEG) hydrogels with microgroove structures or 2D line micropatterns were fabricated, and cell motility on these substrates was evaluated. Interestingly, despite being unconstrained, the cells exhibited drastically different migration behaviors at the edges of the 2D micropatterns and the walls of microgroove structures. In addition to acquiring a unilamellar morphology, the cells increased their motility by roughly 3-fold on the microgroove structures, compared with the 2D counterpart or the nonpatterned surface. Immunostaining revealed that this behavior was dependent on the alignment and the aggregation of the actin filaments, and by varying the slope of the microgroove walls, it was found that relatively upright walls are necessary for this cell morphology alterations. Further progress in this research will not only deepen our understanding of topography-assisted biological phenomena like cancer metastasis but also enable precise, topography-guided manipulation of cell motility for applications such as cancer diagnosis and cell sorting

Enhancement of Cell Adhesion on a Phosphorylcholine-Based Surface through the Interaction with DNA Mediated by Ca²⁺ Ions

2-Methacryloyloxyethyl phosphorylcholine (MPC) has a PC group and is one of the most well-known bioinert polymers. In this study, we evaluated the interaction between MPC and DNA, which specifically interacts with the phospholipid head group via Ca²⁺ ions. A MPC monolayer and poly­(MPC) brush were fabricated to observe the effect of the structure on the interaction between MPC and DNA via Ca²⁺ ions. The poly­(MPC) brush, which shows higher MPC unit density, more efficiently interacted with DNA via Ca²⁺ ions. Also, serum protein could interact with the poly­(MPC) brush via DNA, although the brush itself hardly interacted with serum proteins. Cell adhesion was significantly provoked on poly­(MPC)/DNA compared with poly­(MPC) because serum protein adsorption was induced on poly­(MPC)/DNA

Lectin-Tagged Fluorescent Polymeric Nanoparticles for Targeting of Sialic Acid on Living Cells

In this study, we fabricated lectin-tagged fluorescent polymeric nanoparticles approximately 35 nm in diameter using biocompatible polymers conjugated with lectins for the purpose of detecting sialic acid on a living cell surface, which is one of the most important biomarkers for cancer diagnosis. Through cellular experiments, we successfully detected sialic acid overexpression on cancerous cells with high specificity. These fluorescent polymeric nanoparticles can be useful as a potential bioimaging probe for detecting diseased cells

DataSheet1_Adhesion preference of the sticky bacterium Acinetobacter sp. Tol 5.PDF

Gram-negative bacterium Acinetobacter sp. Tol 5 exhibits high adhesiveness to various surfaces of general materials, from hydrophobic plastics to hydrophilic glass and metals, via AtaA, an Acinetobacter trimeric autotransporter adhesin Although the adhesion of Tol 5 is nonspecific, Tol 5 cells may have prefer materials for adhesion. Here, we examined the adhesion of Tol 5 and other bacteria expressing different TAAs to various materials, including antiadhesive surfaces. The results highlighted the stickiness of Tol 5 through the action of AtaA, which enabled Tol 5 cells to adhere even to antiadhesive materials, including polytetrafluoroethylene with a low surface free energy, a hydrophilic polymer brush with steric hindrance, and mica with an ultrasmooth surface. Single-cell force spectroscopy as an atomic force microscopy technique revealed the strong cell adhesion force of Tol 5 to these antiadhesive materials. Nevertheless, Tol 5 cells showed a weak adhesion force toward a zwitterionic 2-methacryloyloxyethyl-phosphorylcholine (MPC) polymer-coated surface. Dynamic flow chamber experiments revealed that Tol 5 cells, once attached to the MPC polymer-coated surface, were exfoliated by weak shear stress. The underlying adhesive mechanism was presumed to involve exchangeable, weakly bound water molecules. Our results will contribute to the understanding and control of cell adhesion of Tol 5 for immobilized bioprocess applications and other TAA-expressing pathogenic bacteria of medical importance.</p

Video1_Adhesion preference of the sticky bacterium Acinetobacter sp. Tol 5.MP4

Gram-negative bacterium Acinetobacter sp. Tol 5 exhibits high adhesiveness to various surfaces of general materials, from hydrophobic plastics to hydrophilic glass and metals, via AtaA, an Acinetobacter trimeric autotransporter adhesin Although the adhesion of Tol 5 is nonspecific, Tol 5 cells may have prefer materials for adhesion. Here, we examined the adhesion of Tol 5 and other bacteria expressing different TAAs to various materials, including antiadhesive surfaces. The results highlighted the stickiness of Tol 5 through the action of AtaA, which enabled Tol 5 cells to adhere even to antiadhesive materials, including polytetrafluoroethylene with a low surface free energy, a hydrophilic polymer brush with steric hindrance, and mica with an ultrasmooth surface. Single-cell force spectroscopy as an atomic force microscopy technique revealed the strong cell adhesion force of Tol 5 to these antiadhesive materials. Nevertheless, Tol 5 cells showed a weak adhesion force toward a zwitterionic 2-methacryloyloxyethyl-phosphorylcholine (MPC) polymer-coated surface. Dynamic flow chamber experiments revealed that Tol 5 cells, once attached to the MPC polymer-coated surface, were exfoliated by weak shear stress. The underlying adhesive mechanism was presumed to involve exchangeable, weakly bound water molecules. Our results will contribute to the understanding and control of cell adhesion of Tol 5 for immobilized bioprocess applications and other TAA-expressing pathogenic bacteria of medical importance.</p

Significant Heterogeneity and Slow Dynamics of the Unfolded Ubiquitin Detected by the Line Confocal Method of Single-Molecule Fluorescence Spectroscopy

The conformation and dynamics of the unfolded state of ubiquitin doubly labeled regiospecifically with Alexa488 and Alexa647 were investigated using single-molecule fluorescence spectroscopy. The line confocal fluorescence detection system combined with the rapid sample flow enabled the characterization of unfolded proteins at the improved structural and temporal resolutions compared to the conventional single-molecule methods. In the initial stage of the current investigation, however, the single-molecule Förster resonance energy transfer (sm-FRET) data of the labeled ubiquitin were flawed by artifacts caused by the adsorption of samples to the surfaces of the fused-silica flow chip and the sample delivery system. The covalent coating of 2-methacryloyloxyethyl phosphorylcholine polymer to the flow chip surface was found to suppress the artifacts. The sm-FRET measurements based on the coated flow chip demonstrated that the histogram of the sm-FRET efficiencies of ubiquitin at the native condition were narrowly distributed, which is comparable to the probability density function (PDF) expected from the shot noise, demonstrating the structural homogeneity of the native state. In contrast, the histogram of the sm-FRET efficiencies of the unfolded ubiquitin obtained at a time resolution of 100 μs was distributed significantly more broadly than the PDF expected from the shot noise, demonstrating the heterogeneity of the unfolded state conformation. The variety of the sm-FRET efficiencies of the unfolded state remained even after evaluating the moving average of traces with a window size of 1 ms, suggesting that conformational averaging of the heterogeneous conformations mostly occurs in the time domain slower than 1 ms. Local structural heterogeneity around the labeled fluorophores was inferred as the cause of the structural heterogeneity. The heterogeneity and slow dynamics revealed by the line confocal tracking of sm-FRET might be common properties of the unfolded proteins