Emergent Synchronous Volumetric Oscillation in Hierarchically Structured Self-Oscillating Gel Clusters.

Emergent properties accompanying synchronization among oscillators are vital characteristics in biological systems. Belousov-Zhabotinsky (BZ) oscillators are an artificial model to study the emergence and synchronization in life. This research represents a self-oscillating gel system with clusterable properties to experimentally examine synchronous and emergent properties at a fundamental hierarchical level. Incorporating acrylic acid (AAc) moieties within the gel network facilitates cluster formation through hydrogen bonding in an acidic BZ substrate solution. Upon clustering, both homogeneous and heterogeneous gel assemblies─ranging from double to quadruple clusters─exhibit increased and synchronized periods and amplitudes during the BZ reaction. Notably, in heterogeneous clusters, gel units with initially short periods and small volumetric amplitudes display a significant increase, aligning with the lonfger periods and larger amplitudes of other elements within the cluster, an emergent property. This research can pave the way for a better understanding of synchronous and emergent properties in biological oscillators such as cardiomyocytes.

Temperature-Adaptative Self-Oscillating Gels: Toward Autonomous Biomimetic Soft Actuators with Broad Operating Temperature Region.

Self-oscillating gel systems exhibiting an expanded operating temperature and accompanying functional adaptability are showcased. The developed system contains nonthermoresponsive main-monomers, such as N,N-dimethylacrylamide (DMAAm) or 2-acrylamido-2-methylpropane sulfonic acid (AMPS) or acrylamide (AAm) or 3-(methacryloylamino)propyl trimethylammonium chloride (MAPTAC). The gels volumetrically self-oscillate within the range of the conventional (20.0 °C) and extended (27.0 and 36.5 °C) temperatures. Moreover, the gels successfully adapt to the environmental changes; they beat faster and smaller as the temperature increases. The period and amplitude are also controlled by tuning the amount of main-monomers and N-(3-aminopropyl) acrylamide. Furthermore, the record amplitude in the bulk gel system consisting of polymer strand and cross-linker at 36.5 °C is achieved (≈10.8%). The study shows new self-oscillation systems composed of unprecedented combinations of materials, giving the community a robust material-based insight for developing more life-like autonomous biomimetic soft robots with various operating temperatures and beyond.

Anisotropically self-oscillating gels by spatially patterned interpenetrating polymer network.

Here we introduce sub-millimeter self-oscillating gels that undergo the Belousov-Zhabotinsky (BZ) reaction and can anisotropically oscillate like cardiomyocytes. The anisotropically self-oscillating gels in this study were realized by spatially patterning an acrylic acid-based interpenetrating network (AA-IPN). We found that the patterned AA-IPN regions, locally introduced at both ends of the gels through UV photolithography, can constrain the horizontal gel shape deformation during the BZ reaction. In other words, the two AA-IPN regions could act as a physical barrier to prevent isotropic deformation. Furthermore, we controlled the anisotropic deformation behavior during the BZ reaction by varying the concentration of acrylic acid used in the patterning process of the AA-IPN. As a result, a specific directional deformation behavior (66% horizontal/vertical amplitude ratio) was fulfilled, similar to that of cardiomyocytes. Our study can provide a promising insight to fabricating robust gel systems for cardiomyocyte modeling or designing novel autonomous microscale soft actuators.

Fabrication of submillimeter-sized spherical self-oscillating gels and control of their isotropic volumetric oscillatory behaviors.

In this study, we established a fabrication method and analyzed the volumetric self-oscillatory behaviors of submillimeter-sized spherical self-oscillating gels. We validated that the manufactured submillimeter-sized spherical self-oscillating gels exhibited isotropic volumetric oscillations during the Belousov-Zhabotinsky (BZ) reaction. In addition, we experimentally elucidated that the volumetric self-oscillatory behaviors (i.e., period and amplitude) and the oscillatory profiles depended on the following parameters: (1) the molar composition of N-(3-aminopropyl)methacrylamide hydrochloride (NAPMAm) in the gels and (2) the concentration of Ru(bpy)3-NHS solution containing an active ester group on conjugation. These clarified relationships imply that controlling the amount of Ru(bpy)3 in the gel network could influence the gel volumetric oscillation during the BZ reaction. These results of submillimeter-sized and spherical self-oscillating gels bridge knowledge gaps in the current field because the gels with corresponding sizes and shapes have not been systematically explored yet. Therefore, our study could be a cornerstone for diverse applications of (self-powered) gels in various scales and shapes, including soft actuators exhibiting life-like functions.

Capsule self-oscillating gels showing cell-like nonthermal membrane/shape fluctuations.

A primary interest in cell membrane and shape fluctuations is establishing experimental models reflecting only nonthermal active contributions. Here we report a millimeter-scaled capsule self-oscillating gel model mirroring the active contribution effect on cell fluctuations. In the capsule self-oscillating gels, the propagating chemical signals during a Belousov-Zhabotinsky (BZ) reaction induce simultaneous local deformations in the various regions, showing cell-like shape fluctuations. The capsule self-oscillating gels do not fluctuate without the BZ reaction, implying that only the active chemical parameter induces the gel fluctuations. The period and amplitude depend on the gel layer thickness and the concentration of the chemical substrate for the BZ reaction. Our results allow for a solid experimental platform showing actively driven cell-like fluctuations, which can potentially contribute to investigating the active parameter effect on cell fluctuations.

Region-dependent volumetric oscillation of self-oscillating gels with gradient transducers.

Heartbeats with different ventricular contractions vary with heart regions, which can be described as anisotropy. Herein, we report self-oscillating gels which exhibit region-dependent anisotropic volumetric oscillation behavior similar to that of the heart. We installed a (Ru(bpy)3) gradient transducer on self-oscillating gels by employing slow and unidirectional diffusion in the gels and dipping part of the gel into a Ru(bpy)3-NHS solution. We found that the spatial distribution of Ru(bpy)3 in the gel caused region-dependent swelling/deswelling behavior depending on the redox state. We also confirmed that gel regions with smaller Ru(bpy)3 amounts exhibit lower amplitudes than those with larger amounts of Ru(bpy)3 during the Belousov-Zhabotinsky (BZ) reaction. These results are important in the design of self-oscillating soft actuators or machines, such as a biomimetic pump with desirable anisotropic oscillating behavior.

Self-oscillating chemoelectrical interface of solution-gated ion-sensitive field-effect transistor based on Belousov-Zhabotinsky reaction.

The Belousov-Zhabotinsky (BZ) self-oscillation reaction is an important chemical model to elucidate nonequilibrium chemistry in an open system. However, there are only a few studies on the electrical behavior of pH oscillation induced by the BZ reaction, although numerous studies have been carried out to investigate the mechanisms by which the BZ reaction interacts with redox reactions, which results in potential changes. Needless to say, the electrical characteristic of a self-oscillating polymer gel driven by the BZ reaction has not been clarified. On the other hand, a solution-gated ion-sensitive field-effect transistor (ISFET) has a superior ability to detect ionic charges and includes capacitive membranes on the gate electrode. In this study, we carried out the electrical monitoring of self-oscillation behaviors at the chemoelectrical interface based on the BZ reaction using ISFET sensors, focusing on the pH oscillation and the electrical dynamics of the self-oscillating polymer brush. The pH oscillation induced by the BZ reaction is not only electrically observed using the ISFET sensor, the electrical signals of which results from the interfacial potential between the solution and the gate insulator, but also visualized using a large-scale and high-density ISFET sensor. Moreover, the N-isopropylacrylamide (NIPAAm)-based self-oscillating polymer brush with Ru(bpy)3 as a catalyst clearly shows a periodic electrical response based on the swelling-deswelling behavior caused by the BZ reaction on the gate insulator of the ISFET sensor. Thus, the elucidation of the electrical self-oscillation behaviors induced by the BZ reaction using the ISFET sensor provides a solution to the problems of nonequilibrium chemistry.

Construction of a nano-phase-separated structure on a hydrogel surface.

A hydrogel surface with a nano-phase-separated structure was successfully fabricated by grafting a fluorine-containing polymer using activators regenerated by electron transfer atom transfer radical polymerisation (ARGET ATRP). The modified hydrogel surface exhibits water repellency and high elasticity with maintaining transparency.

Selective capture and non-invasive release of cells using a thermoresponsive polymer brush with affinity peptides.

Tissue engineering and cell transplantation therapy have become promising therapies for intractable diseases. These approaches require cell separation technology without cell modification. Accordingly, in this study, we developed a novel cell separation method using a thermoresponsive block copolymer brush with an affinity peptide. A block copolymer brush with bottom poly(2-hydroxyethyl methacrylate [HEMA]-co-propargyl acrylate) and top poly(N-isopropylacrylamide-co-HEMA) segments was prepared through two steps of atom transfer radical polymerization. Then, cell affinity peptides were conjugated to the bottom segment of the copolymer brush through a click reaction. Using cRGD as a cell-affinity peptide, enhancement of cell adhesion with rapid adhesion on the copolymer brush was observed at 37 °C, whereas the copolymer brush without cRGD did not exhibit cell adhesion. Temperature-modulated cell adhesion and detachment were performed with a relatively long upper segment because the affinity between peptides and cells was modulated by the swelling and shrinking of the upper thermoresponsive segment. Selective endothelial cell adhesion was performed at 37 °C using GGGREDV as an affinity peptide. Smooth muscle cells and fibroblasts did not adhere to the copolymer brush. Adhered human umbilical vein endothelial cells (HUVECs) were successfully recovered by reducing the temperature to 20 °C. Based on the properties of the copolymer brush, HUVECs could be purified using a mixture of cells simply by changing the temperature. These results demonstrated that the prepared copolymer brush with cell affinity peptides could be a useful cell separation tool because the cells could be separated with specificity and without cell modification using a simple procedure.

Thermoresponsive Cationic Block Copolymer Brushes for Temperature-Modulated Stem Cell Separation.

Recently, cell separation methods have become important for preparing cells for transplantation therapy. In this study, a thermoresponsive cationic block copolymer brush is developed as an effective cell separation tool. This brush is prepared on glass surfaces using two steps of activator regenerated by electron transfer-atom transfer radical polymerization (ARGET-ATRP). The cationic segment is prepared in the first step of the ARGET-ATRP of N,N-dimethylaminopropylacrylamide (DMAPAAm). In the second step, the thermoresponsive segment is prepared, attached to the bottom cationic segment, through ARGET-ATRP with N-isopropylacrylamide (NIPAAm). The cell adhesion behavior of the prepared thermoresponsive cationic copolymer, PDMAPAAm-b-PNIPAAm, brush is observed using umbilical cord-derived mesenchymal stem cells (UCMSC), fibroblasts, and macrophages. At 37 °C, all three types of cells adhere to the thermoresponsive cationic copolymer brush. Then, by reducing the temperature to 20 °C, the adhered UCMSC are detached from the copolymer brush, whereas the fibroblasts and macrophages remain adhered to the copolymer brush. Using this copolymer brush, UCMSC can be purified from the cell mixture simply by changing the temperature. Therefore, the prepared thermoresponsive cationic copolymer brush is useful as a cell separation tool for the purification of mesenchymal stem cells.

Design of two complementary copolymers that work as a glue for cell-laden collagen gels.

We designed two types of copolymers that play a role of "polymeric glue". They introduced surface adhesive functions to cell-laden collagen gels. The present study realised surface functionalisation of naturally derived hydrogels and suggested a novel method for manipulating living cells. This method is potentially useful for in vitro reconstruction of 3D living tissue since it enabled versatile and cytocompatible hydrogel surface modification by a simple procedure without any special equipment.

Antibody drug separation using thermoresponsive anionic polymer brush modified beads with optimised electrostatic and hydrophobic interactions.

Antibody drugs play an important role in biopharmaceuticals, because of the specificity for target biomolecules and reduction of side effects. Thus, separation and analysis techniques for these antibody drugs have increased in importance. In the present study, we develop functional chromatography matrices for antibody drug separation and analysis. Three types of polymers, poly(N-isopropylacrylamide (NIPAAm)-co-2-acrylamido-2-methylpropanesulfonic acid (AMPS)-co-N-phenyl acrylamide (PhAAm)), P(NIPAAm-co-AMPS-co-n-butyl methacrylate (BMA)), and P(NIPAAm-co-AMPS-co-tert-butylacrylamide (tBAAm)), were modified on silica beads through atom transfer radical polymerisation. Rituximab elution profiles were observed using the prepared beads-packed column. Rituximab adsorption at high temperature and elution at low temperature from the column were observed, as a result of the temperature-modulated electrostatic and hydrophobic interactions. Using the column, rituximab purification from contaminants was performed simply by changing the temperature. Additionally, three types of antibody drugs were separated using the column through temperature-modulated hydrophobic and electrostatic interactions. These results demonstrate that the temperature-responsive column can be applied for the separation and analysis of biopharmaceuticals through a simple control of the column temperature.

Fabrication of Self-Oscillating Micelles with a Built-In Oxidizing Agent.

Various biological behaviors are fueled by "respiration", which is an example of catabolism. So far, we have reported various self-oscillating soft materials exhibiting bioinspired dynamic movements. These autonomous polymer systems are driven by the Belousov-Zhabotinsky (BZ) reaction, which is analogous to the tricarboxylic acid (TCA) cycle that is an integral part of respiration. However, in the BZ reaction, the external addition of an oxidizing agent is necessary to initiate the oxidation process, which is realized by intracellular moieties such as ubiquinone in living systems. Herein, we realized self-oscillating micelles that are driven without the external addition of an oxidizing agent. This was achieved by embedding the oxidizing source into the structure of the self-oscillating AB diblock copolymers. This strategy introduces a new function equivalent to intracellular oxidizing moieties, and is useful for the design of completely autonomous bioinspired materials.

Thermoresponsive anionic copolymer brush-grafted surfaces for cell separation.

Cell separation methods that do not require surface modification of cells are needed in tissue engineering and regenerative medicine. We developed thermoresponsive anionic polymer brushes for cell separation without modification of the cell surfaces. Copolymer brush poly(N-isopropylacrylamide-co-N-tert-butylacrylamide-co-tert-butyl acrylate, P(NIPAAm-co-tBAAm-co-tBA), was prepared on a cover glass plate through activator regenerated by electron transfer atom transfer radical polymerization (ARGET-ATRP). The tert-butyl group of the copolymer brush was then deprotected and a P(NIPAAm-co-tBAAm-co-acrylic acid (AAc)) brush-modified glass surface was obtained. ARGET-ATRP synthesis achieved polymers with low polydispersity. The negative surface charge of the polymer brush-modified substrates was evaluated using zeta potential measurements and the phase transition temperature of the polymer was modulated between 37-20 °C to perform cell adhesion and detachment, respectively. The adhesion and detachment behavior of cells used in cardiovascular tissue engineering on the thermoresponsive anionic polymer brushes was investigated. Normal human umbilical vein endothelial cells (HUVEC) exhibited prompt detachment from the thermoresponsive anionic polymer brush surfaces. In addition, normal human aortic smooth muscle cells (SMC) exhibited relatively high adhesion on thermoresponsive anionic polymer brush-modified surfaces compared with those modified with thermoresponsive polymer brushes without anionic groups. By utilizing the difference in the cell adhesion and detachment properties of the cell types, a mixture of HUVEC and SMC was separated simply by altering the applied temperature. This result indicated that the prepared thermoresponsive anionic polymer brush-modified glass surface could be used as a tool for the separation of cells in cardiovascular tissue engineering.

Mixed polymer brush as a functional ligand of silica beads for temperature-modulated hydrophobic and electrostatic interactions.

We developed a mixed polymer brush, which consists of temperature-responsive polymer and cationic polymer modified beads as functional chromatographic matrices, for temperature-modulated multiple hydrophobic and electrostatic interactions. The mixed polymer brush was modified on silica beads through the combination of reversible addition-fragmentation chain transfer (RAFT) polymerization of N,N-dimethylaminopropyl acrylamide (DMAPAAm) and surface initiated atom transfer radical polymerization (ATRP) of N-isopropylacrylamide (NIPAAm). Zeta potential measurement of the mixed polymer brush revealed that the zeta potential increased with increasing temperature, which was attributed to the exposed PDMAPAAm on the beads arising from the shrinking of PNIPAAm upon increasing the temperature. The prepared beads were used as the packing material of high performance liquid chromatography (HPLC) columns, and the elution behavior of steroids, adenosine nucleotides, and proteins through the column was observed. The retention time of steroids increased with increasing the column temperature because of the dehydration of PNIPAAm in the mixed polymer brush. Adenosine nucleotides were also retained by the columns, which was attributed to the electrostatic interaction with PDMAPAAm in the mixed polymer brush. Several proteins were adsorbed on the column at elevated temperatures because of the enhanced electrostatic interaction of exposed PDMAPAAm and the enhanced hydrophobic interaction resulting from the dehydration of PNIPAAm. By exploiting this unique property, mixtures of proteins could be separated by simply changing the column temperature. These results indicate that the developed mixed polymer brush modified beads would be useful as functional chromatographic packing matrices for thermally-modulated multiple hydrophobic and electrostatic interactions.

Stable and Prolonged Autonomous Oscillation in a Self-Oscillating Polymer Brush Prepared on a Porous Glass Substrate.

We developed an autonomous functional surface, named a "self-oscillating polymer brush surface", which exhibits swelling-deswelling of the modified polymer chains synchronized with the Belousov-Zhabotinsky (BZ) reaction. The grafted polymer chain is a random copolymer composed of thermoresponsive N-isopropylacrylamide, N-(3-aminopropyl)methacrylamide, and ruthenium tris(2,2'-bipyridine) [Ru(bpy)3]. To provide stable oscillations over a long period of time, suppression of the dilution of the BZ reactants inside the polymer surface and the increase in the amount of immobilized Ru(bpy)3 are important. Here, we modified the self-oscillating polymer brush on a porous glass substrate and characterized its dynamic behavior. The increased surface area of the porous glass allowed for an efficient introduction of the metal catalyst, which resulted in a stable BZ reaction observable by optical microscopy. Compared with an aqueous BZ solution and the self-oscillating polymer modified on a glass coverslip, the wave velocity and diffusion coefficient were significantly lower for the porous glass system, which suggested that the reaction-diffusion of the reactants was markedly different than those of the other two systems. Moreover, the wave velocity was unchanged on the porous glass system for 1 h, whereas that of the solution dropped by 30 µm s-1. Waveform analyses based on the Field-Körös-Noyes mechanism revealed that densely packed Ru(bpy)3 in the porous glass system affects the duration of the key processes in the BZ reaction. These findings can help with understanding the dynamic behavior of the self-oscillating polymer brush on a porous glass substrate. Stable self-oscillations on the polymer brush-grafted porous glass substrate will aid future applications such as mass transport systems.

Adsorption-Desorption Control of Fibronectin in Real Time at the Liquid/Polymer Interface on a Quartz Crystal Microbalance by Thermoresponsivity.

The cell manipulation technique using thermoresponsive polymers is currently attracting much attention for applications in the medical field. To achieve arbitrary and accurate cell control, it is necessary to intensely research fibronectin behavior. A smart surface, which has thermoresponsive wettability and which can adsorb or desorb fibronectin repeatedly without the presence of cells, was fabricated by an electrospinning method. The fabricated coating changed its structure as the temperature was changed, and this transformation could substitute for the pulling force generated by the cytoskeletal contraction of cells. Moreover, a coated quartz crystal microbalance was able to detect the fibronectin behavior as frequency shifts, which could be used in the estimation of the mass shift with the aid of suitable equations. This coating and measurement system can contribute greatly not only to the development in the medical field centered on biomaterial manipulation technologies, but also to the improvement of medical instruments.

Mesenchylmal Stem Cell Culture on Poly(N-isopropylacrylamide) Hydrogel with Repeated Thermo-Stimulation.

We prepared thermoresponsive hydrogels by mixing poly(N-isopropylacrylamide) (PNIPAAm) derivatives as the main chain components, octa-arm polyethylene glycol (PEG) as a crosslinker, and the Arg-Gly-Asp-Ser (RGDS) peptides as cell adhesion units. Human bone marrow-derived mesenchymal stem cells (hbmMSCs) were cultured on the hydrogels. The PNIPAAm gel prepared by the post-crosslinking gelation method was revealed to be cytocompatible and showed temperature-dependent changes in mechanical properties. Repeated changes in the swelling ratio of the PNIPAAm gel affected the shape of the hbmMSCs. With respect to both cytocompatibility and reversibility of changes in mechanical properties, the PNIPAAm gel system could be potentially useful for the analysis of cell mechanobiology.

Macroscopic Adhesion of Thermoreversible ABC Triblock Copolymer-Based Hydrogels Via Boronic Acid-Sugar Complexation.

Two complementary thermoreversible ABC triblock copolymers containing either phenylboronic acids with low pKa values or galactosyl groups in the hydrophilic B blocks are synthesized by sequential reversible addition-fragmentation chain transfer polymerization and subsequent modification of the functional groups. Both ABC triblock copolymers undergo reversible sol-to-gel transitions upon temperature change and form physically cross-linked hydrogels under physiological conditions. Furthermore, the spontaneous adhesion of these thermoreversible hydrogels via the formation of boronic esters between the phenylboronic acid and galactosyl groups under physiological conditions is realized for the first time.

Aspects of the Belousov-Zhabotinsky Reaction inside a Self-Oscillating Polymer Brush.

We have developed a novel polymer brush surface exhibiting autonomous swelling-deswelling changes driven by the Belousov-Zhabotinsky (BZ) reaction, that is, the self-oscillating polymer brush. In this system, the ruthenium tris(2,2'-bipyridine) [Ru(bpy)3] catalyst-conjugated polymer chains are densely packed on the solid substrate. It is expected that the BZ reaction in the polymer brush would be influenced by the immobilization effect of the catalyst. To clarify the effect of the immobilization of the catalyst on the self-oscillating polymer brush, the self-oscillating behavior of the polymer brush was investigated by comparing it with that of other self-oscillating polymer materials, the free polymer, and the gel particle under various initial substrate concentrations. The initial substrate dependency of the oscillating period for the polymer brush was found to be different from those for the free polymer and the gel particle. Furthermore, the oscillatory waveform was analyzed on the basis of the Field-Körös-Noyes model. These investigations revealed that the dense immobilization of the self-oscillating polymer on the surface restricted accessibility for the Ru(bpy)3 moiety. These findings would be helpful in understanding the reaction-diffusion mechanism in the polymer brush, which is a novel reaction medium for the BZ reaction.