Architecting well-designed interfacial structures is crucial for fabrication of better bio-related devices such as bio-sensors. A dynamic nature of the interfacial structures with appropriate mechanical properties is advantageous for interactions with bio-related substances. In this short review, a new term, mechano-nanoarchitectonics, has been proposed. This terminology represents nanoarchitectonics methodology for formation of functional structures and regulation of their properties with the aid of mechanical processes. An interfacial two-dimensional environment is an ideal medium to connect macroscopic mechanical actions and nanoscale functions. The review starts with rather traditional topics on how to architect biocomponents at interfaces for bio-reactors and bio-sensors, then covers current active research on mechanical control of bio-functions at dynamic interfaces and emerging topics of mechanical control of DNA origami array and cell differentiation control.
Cells can mainly sense mechanical cues from the extracellular matrix via integrins. Because mechanical cues can strongly influence cellular functions, understanding the roles of integrins in the sensing of mechanical cues is a key for the achievement of tissue engineering. The analyses to determine the roles of integrins in the sensing of mechanical cues have been performed by many methods based on molecular- and cell-biological techniques, atomic force microscopy, and optical tweezers. Integrin-dependent cell adhesion substrates have been also used for this purpose. Additionally, the cells can adhere on several substrates via integrin-independent mechanisms. There are two types of integrin-independent cell adhesion substrates; 1) the substrates immobilized with ligands against the receptors on cell surface and 2) the substrates suppressing protein adsorption. Cells can exhibit specific functions on these substrates. Here, the examples of integrin-independent cell adhesion substrates were reviewed, and their possible applications in mechanobiology research are discussed.
Withdrawal escape response of C. elegans to nonlocalized vibration is a useful behavioral paradigm to examine mechanisms underlying mechanosensory behavior and its memory-dependent change. However, there are very few methods for investigating the degree of vibration frequency, amplitude and duration needed to induce behavior and memory. Here, we establish a new system to quantify C. elegans mechanosensory behavior and memory using a piezoelectric sheet speaker. In the system, we can flexibly change the vibration properties at a nanoscale displacement level and quantify behavioral responses under each vibration property. This system is an economic setup and easily replicated in other laboratories. By using the system, we clearly detected withdrawal escape responses and confirmed habituation memory. This system will facilitate the understanding of physiological aspects of C. elegans mechanosensory behavior in the future.
Recently, the importance of conformational changes in actin filaments induced by mechanical stimulation of a cell has been increasingly recognized, especially in terms of mechanobiology. Despite its fundamental importance, however, long-term observation of a single actin filament by fluorescent microscopy has been difficult because of the low photostability of traditional fluorescent molecules. This paper reports a novel molecular labeling system for actin filaments using fluorescent nanodiamond (ND) particles harboring nitrogen-vacancy centers; ND has flexible chemical modifiability, extremely high photostability and biocompatibility, and provides a variety of physical information quantitatively via optically detected magnetic resonance (ODMR) measurements. We performed the chemical surface modification of an ND with the actin filament-specific binding peptide Lifeact and observed colocalization of pure Lifeact-modified ND and actin filaments by the ODMR selective imaging protocol, suggesting the capability of long-term observation and quantitative analysis of a single molecule by using an ND particle.
We designed a microfluidic system comprising microfluidic channels, pumps, and valves to enable the fabrication of cellular multilayers in order to reduce labor inputs for coating extracellular matrices onto adhesive cells (e.g., centrifugation). Our system was used to fabricate nanometer-sized, layer-by-layer films of the extracellular matrices on a monolayer of C2C12 myoblasts. The use of this microfluidic system allowed the fabrication of cellular multilayers in designed microfluidic channels and on commercial culture dishes. The thickness of the fabricated multilayer using this microfluidic system was higher than that of the multilayer that was formed by centrifugation. Because cellular multilayer fabrication is less laborious and the mechanical force to the cell is reduced, this novel system can be applied to tissue modeling for cell biology studies, pharmaceutical assays, and quantitative analyses of mechanical or chemical stimuli applied to multilayers.
Cell mechanical properties that depend on cytoskeleton architecture are critical to the mechanotransduction process, and have great potential for cancer diagnosis and therapy. In this study, the morphological and mechanical properties of typical osteosarcoma microenvironment cells, including mesenchymal stem cells (MSC), normal human osteoblast cells (NHOst) and osteosarcoma cells (MG-63), were compared using atomic force microscopy (AFM). The MG-63 cells were smaller and thicker than the MSC and NHOst cells. The membrane roughness of MG-63 cells was higher than that of MSC and NHOst cells. The MG-63 cells had lower stiffness than their normal counterparts due to their reduced organization of the cytoskeleton structure. The cell stiffness influenced the mechanotransduction. The MG-63 cells had a lower percentage of nuclear YAP/TAZ compared with the MSC and NHOst cells. The F-actin assembly was disrupted by the cytochalasin D (cyto D) treatment used to investigate its influence on mechanotransduction. Disruption of the cytoskeleton leaded to a decrease of the cell stiffness, and reduced the nuclear YAP/TAZ percentage, indicating its inhibition in the cell mechanotransduction process. This study would shed light on the development of a novel cancer diagnosis strategy and would contribute to reveal the relationship between the cytoskeleton structure and the cell mechanical properties.
This paper describes a facile method for the preparation of photoactivatable substrates with tuned surface density of an extracellular matrix peptide to resolve the impacts of biochemical and mechanical cues on collective cell migration. The controllability of surface ligand density was validated by cell adhesion and migration tests, complemented with fluorescence observation of an alternative ligand. Depending on the surface ligand density, HeLa cells either kept or lost collective characteristics. The present materials will be useful to address mechanobiology of collective cell migration.
Detection of cellular forces plays an important role in investigating the mechanical basis of cells. As nanomechanical sensors can directly detect surface stress, they can be utilized to detect cellular forces. In the present study, we perform quantitative simulations of nanomechanical sensors for the detection of cellular forces using finite element analyses (FEA). We focus on two types of nanomechanical sensors: a cantilever-type sensor and a membrane-type surface stress sensor (MSS). It is found that sensing signals can be obtained when cells on the nanomechanical sensors synchronize their motions. To effectively detect cellular forces on the nanomechanical sensors, we discuss the optimization scheme for a coating layer on the surface of the sensors.
Immobilization of functional peptides on polymer material is necessary to produce cell-selective scaffolds. However, the expected effects of peptide immobilization differ considerably according to the properties of selected polymers. To understand such combinational effects of peptides and polymers, varieties of scaffolds including a combination of six types of poly(ε-caprolactone-co-D,L-lactide) and four types of cell-selective adhesion peptides were fabricated and compared. On each scaffold, the scaffold properties (i.e. mechanical) and their biological functions (i.e. fibroblast-/endothelial cell-/smooth muscle cell-selective adhesion) were measured and compared. The results showed that the cell adhesion performances of the peptides were considerably enhanced or inhibited by the combination of peptide and polymer properties. In the present study, we illustrated the combinational property effects of peptides and polymers using multi-parametric analyses. We provided an example of determining the best scaffold performance for tissue-engineered medical devices based on quantitative data-driven analyses.
We synthesized poly(N-isopropylacrylamide) gel containing cell-adhesive peptides, RGDS pendants, and crosslinked by a hydrophilic polymer. Since gelation occurs by simply mixing the polymers, cells can be simultaneously encapsulated inside the gel during gelation. This gel has roughly a 15% volume change between 25 and 37°C, and is transparent at both temperatures. Moreover, adhesion of encapsulated C2C12 cells to the gel could be observed. This thermoresponsive gel would be potentially useful as a cell culture material that can control cell fate by changing mechanical properties of the cell’s external environment.
Migrating cells in vivo monitor the physiological state of an organism by integrating the physical as well as chemical cues in the extracellular microenvironment, and alter the migration mode, in order to achieve their unique function. The clarification of the mechanism focusing on the topographical cues is important for basic biological research, and for biomedical engineering specifically to establish the design concept of tissue engineering scaffolds. The aim of this study is to understand how cells sense and respond to the complex topographical cues in vivo by exploring in vitro analyses to complex in vivo situations in order to simplify the issue. Since the intracellular mechanical events at subcellular scales and the way of the coordination of these events are supposed to change in the migrating cells, a key to success of the analysis is a mechanical point of view with a particular focus of the subcellular mechanical events. We designed an experimental platform to explore the mechanical requirements in a migrating fibroma cell responding to micro-grooves. The micro-grooved structure is a model of gap structures, typically seen in the microenvironments in vivo. In our experiment, the contributions of actomyosin force generation can be spatially divided and analyzed in the cell center and peripheral regions. The analysis specified that rapid leading edge protrusion, and the cell body translocation coordinated with the leading edge protrusion are required for the turning response at a micro-groove.
In this study, we investigated the effect of positive dielectrophoresis (DEP) on gene expression in mesenchymal stem cells. When applying an alternating current voltage, human bone marrow derived mesenchymal stem cells (UE7T-13) exhibited a positive DEP, and were compressed onto the electrode surface. The constructed device can easily control the DEP force to the cells by changing the frequency. Interestingly, gene expressions of the cell differentiation marker in UE7T-13 cells and the mechanical stimulation-susceptible one were changed by applying a positive DEP. These results suggested that the gene expression in mesenchymal stem cells can be regulated by applying mechanical stimulation derived from DEP.
There is an urgent need to develop novel in-vitro models to mimic the disease conditions in pulmonary hypertension (PH). We developed a microfluidic cell culture device for PH studies that withstood high shear stress. Techniques were also developed for cell recovery from the microchannel and mRNA isolation from the collected cells. Using this device, we found that shear stress caused a 7.5-fold increase in the transcription levels of a PH-related molecule, Cyclin D1.
A Rhodamine-based dual chemosensor L1 for simultaneously detecting Fe3+ and Cu2+ was designed and synthesized. The spectroscopic properties of L1 were analyzed, and its recognition mechanism was speculated. We found that the addition of Fe3+ induced a great fluorescence enhancement, while Cu2+ induced a strong UV-Vis absorption enhancement. The results revealed that L1 was highly selective for recognizing Fe3+ and Cu2+ in UV-Vis spectroscopy in CH3OH–H2O (1/1, v/v, pH 7.2) with the interference of other metal ions. A good linear relationship between the fluorescence intensities of L1 and the concentration of Fe3+, as well as the UV-Vis absorption intensities of L1 and the concentration of Cu2+ was observed, respectively. The detection limit was 9.2 × 10−8 M (5.5 μg/L) for Fe3+ and 3.8 × 10−8 M (2.4 μg/L) for Cu2+, respectively. The detection capacity for targeted metal ions of Fe3+ and Cu2+ were studied, which are less than 5 min. Job’s plot method for L1 with Fe3+ and ESI-MS for L1 with Cu2+ indicated a 1:1 stoichiometry in the complex. The results may provide an effective strategy for the design of new dual chemosensors for the rapid detection of targeted metal ions.
A simple, rapid, and sensitive malachite green-based spectrophotometric method for the selective trace determination of an iodate has been developed and presented for the first time. The reaction mixture was specifically involved in the liberation of iodine in the presence of an excess of iodide in an acidic condition following an instantaneous reaction between the liberated iodine and malachite green dye. The optimum condition was obtained with a buffer solution pH of 5.2 in the presence of 40 mg L−1 potassium iodide and 1.5 × 10−5 M malachite green for a 5-min incubation time. The iodate contents in some table-salt samples were in the range of 26 to 45 mg kg−1, while those of drinking water, tap water, canal water, and seawater samples were not detectable (< 96 ng mL−1 of limits of detection, LOQ) with their satisfied method of recoveries of between 93 and 108%. The results agreed with those obtained using ICP-OES for comparison.
A strategy was presented for quantitative analysis of trace elements in a glass sample by laser ablation inductively coupled plasma tandem quadrupole mass spectrometry (LA-ICP-QMS/QMS) with an octapole reaction cell (ORC). Silicon in the glass was used as the internal standard as well as the matrix for making the calibrating solutions. Sample aerosols generated by the laser ablation system were introduced into the dual pass spray chamber through the make-up gas port. Calibrating solutions were nebulized into the spray chamber using a microflow nebulizer. The silicon matrix-matched calibrating solutions produced a calibration curve capable for the quantitative analysis of trace elements in a silicate glass sample, NIST SRM 612. The analytical results agreed with the certified values, taking into consideration their expanded uncertainties. The detection limits for Cr, Mn, Fe, Ni, Cu, As, Sr, Ag, Cd, Sb, Ba, and Pb, were respectively 0.3, 0.08, 0.5, 0.4, 0.19, 1.1, 0.1, 0.02, 0.03, 0.025, 0.09, and 0.07 μg g−1.
Endonuclease V (EndoV) plays the important role of nucleotide excision repair (NER) in the maintenance of genomic stability. Highly sensitive detection of EndoV was achieved through an oligonucleotides sensitizing Tb3+ luminescent technique. We found that although both guanine-rich (G-rich) single-stranded DNA and dGMP could enhance the time-resolved luminescence of Tb3+, their efficiencies of enhancement were considerably different. Employing such interesting phenomenon, a label-free and time-resolved luminescent strategy for the sensitive detection of EndoV activity was developed based on DNA-enhanced time-resolved luminescence (TRL) of Tb3+. The EndoV was used to cut off the deoxyinosine site (dI) and convert the 3′-protruding termini to a recessed end, and Exonuclease III (Exo III) was used to enhance the signal contrast via digestion of G-rich DNA to dNTP. Combining with the natural advantages of the TRL, the proposed method exhibited a good linear response to EndoV ranging from 0.005 to 0.4 U/mL, with a low limit of detection of 0.005 U/mL.
The effects of adding desipramine-containing fluorescence polymer (poly(10,11-dihydro-5-[3-(N-methylamino)propyl]dibenz[b,f]azepine-2,8-diyl-alt-9,9-didodecylfluorene-2,7-diyl, PAzep-Fl) to resins were investigated. When composites were prepared by a reactive blend of poly(lactic acid) (PLA), PAzep-Fl, and diisocyanate, the molecular weight of the obtained composite was larger than that of the composite, which was blended without PAzep-Fl; this suggested that chemical bonding occured between PLA and PAzep-Flvia diisocyanate. The effects of adding PAzep-Fl in two kinds of resins/lysine triisocyanate (LTI) blend were also investigated. The addition of LTI to resins/PAzep-Fl blend composites afforded a shorter wavelength shift of the fluorescence λmax due to the release of the aggregation of PAzep-Fl by LTI. This suggested that the compatibility of two kinds of resins could be estimated by this simple method. The addition of PAzep-Fl to PLA/poly(ε-caprolactone) (PCL)/LTI blends improved the impact strength because of chemical bond formation between PAzep-Fl and the resins (PLA and PCL) via LTI.
Quantitative analysis of nitrilotriacetate (NTA) in detergents by titration with Cu2+ solution using a copper ion selective electrode was achieved. This method tolerates a wide range of pH and ingredients in detergents. In addition to NTA, other chelating agents, having relatively lower stability constants toward Cu2+, were also qualified with sufficient accuracy by this analytical method for model detergent formulations. The titration process was automated by automatic titrating systems available commercially.
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