Contractile and Tensile Measurement of Molecular Artificial Muscles for Biohybrid Robotics.

A printable artificial muscle assembled from biomolecular motors, which we have recently developed, showed great potential in overcoming the design limitations of conventional biohybrid robots as a new bio-actuator. Characterizing its contractility for extending its applicability is important. However, conventional measurement methods are composed of complex operations with poor reproducibility, flexibility, and real-time responsiveness. This study presents a new method for measuring the contractile force generated by artificial muscles. A measurement system was constructed, wherein artificial muscles were patterned by UV laser scanning in an oil-sealed microchamber, and the contractile force was measured in real time using a microforce sensor extended by a 3D-printed microcantilever. The measurement accuracy of the sensor was ensured through calibration and correction. For demonstration purposes, a series of contractile measurements were carried out using the proposed system. The relationship between contractile force and the dimensions of the activation space of the artificial muscles, as well as the tensile properties of the contracted muscle chain were evaluated. The results will help characterize the contractile properties of the artificial muscle and lay the foundations for its further application in biohybrid robotics.

Feedback control of automatic navigation for cyborg cockroach without external motion capture system.

Due to their size and locomotion ability, cockroaches are favorable as hybrid robot platforms in search and rescue (SAR) missions. However, cockroaches most likely approach the corner area and stay for an uncertain time. This natural behavior will hinder the utilization of cyborg cockroaches in SAR missions under rubble, unstructured, and unknown areas. Therefore, we proposed onboard automatic obstacle avoidance and human detection that can run on the wireless backpack stimulator without an external motion capture system. A low-power and small-size Time of Flight (ToF) sensor was selected as a distance measurement sensor, while a low-resolution thermopile array sensor was applied for human presence detection. The implemented feedback control based on IMU and ToF sensors has successfully navigated the cyborg cockroach to avoid obstacles and escape from the sharp corners in the laboratory unstructured area without stopping or being trapped. It could also recognize the human presence when the human was in front of it in real-time. Due to its performance, the random forest classifier was implemented as an embedded human detection system. It could achieve the highest accuracy at a distance of around 25 cm (92.5%) and the lowest accuracy at about 100 cm (70%).

Analysis of Rowing Force of the Water Strider Middle Leg by Direct Measurement Using a Bio-Appropriating Probe and by Indirect Measurement Using Image Analysis.

Rowing force of the middle leg of a water strider is one of the important factors affecting water repellency and applications in biomimetics, biomechanics, and biology. However, many previous studies have been based on estimated leg rowing force and lack some credibility. Therefore, we tried to measure leg rowing force directly by a force transducer. In this article, we report the rowing force of water striders obtained by direct and indirect measurements. In the direct measurement, water striders were set onto a sensor system and the rowing force of a middle leg of the set water striders was directly measured using a bio-appropriating probe (BAP), a kind of hook. In the indirect measurement, water striders were not fixed and the rowing force of locomoting water striders was evaluated by image analysis using a high-speed camera. As a result, we determined the rowing force by the direct measurement to be 955 µN, while the rowing force by the indirect measurement was 493 µN. We considered that the indirect measurement might lack some credibility because half the propellant energy was lost in the indirect force measurement due to various other factors.

Movement Optimization for a Cyborg Cockroach in a Bounded Space Incorporating Machine Learning.

Cockroaches can traverse unknown obstacle-terrain, self-right on the ground and climb above the obstacle. However, they have limited motion, such as less activity in light/bright areas and lower temperatures. Therefore, the movement of the cyborg cockroaches needs to be optimized for the utilization of the cockroach as a cyborg insect. This study aims to increase the search rate and distance traveled by cockroaches and reduce the stop time by utilizing automatic stimulation from machine learning. Multiple machine learning classifiers were applied to classify the offline binary classification of the cockroach movement based on the inertial measuring unit input signals. Ten time-domain features were chosen and applied as the classifier inputs. The highest performance of the classifiers was implemented for the online motion recognition and automatic stimulation provided to the cerci to trigger the free walking motion of the cockroach. A user interface was developed to run multiple computational processes simultaneously in real time such as computer vision, data acquisition, feature extraction, automatic stimulation, and machine learning using a multithreading algorithm. On the basis of the experiment results, we successfully demonstrated that the movement performance of cockroaches was importantly improved by applying machine learning classification and automatic stimulation. This system increased the search rate and traveled distance by 68% and 70%, respectively, while the stop time was reduced by 78%.

In situ integrated microrobots driven by artificial muscles built from biomolecular motors.

Microrobots have been developed for applications in the submillimeter domain such as the manipulation of micro-objects and microsurgery. Rapid progress has been achieved in developing miniaturized components for microrobotic systems, resulting in a variety of functional microactuators and soft components for creating untethered microrobots. Nevertheless, the integration of microcomponents, especially the assembly of actuators and mechanical components, is still time-consuming and has inherent restrictions, thus limiting efficient fabrications of microrobots and their potential applications. Here, we propose a method for fabricating microrobots in situ inspired by the construction of microsystems in living organisms. In a microfluidic chip, hydrogel mechanical components and artificial muscle actuators are successively photopatterned from hydrogel prepolymer and biomolecular motors, respectively, and integrated in situ into functional microrobots. The proposed method allows the fast fabrication of microrobots through simple operations and affordable materials while providing versatile functions through the precise spatiotemporal control of in situ integration and reconfiguration of artificial muscles. To validate the method, we fabricated microrobots to elicit different motions and on-chip robots with unique characteristics for microfluidic applications. This study may establish a new paradigm for microrobot integration and lead to the production of unique biohybrid microrobots with various advantages.

A printable active network actuator built from an engineered biomolecular motor.

Leveraging the motion and force of individual molecular motors in a controlled manner to perform macroscopic tasks can provide substantial benefits to many applications, including robotics. Nonetheless, although millimetre-scale movement has been demonstrated with synthetic and biological molecular motors, their efficient integration into engineered systems that perform macroscopic tasks remains challenging. Here, we describe an active network capable of macroscopic actuation that is hierarchically assembled from an engineered kinesin, a biomolecular motor, and microtubules, resembling the contractile units in muscles. These contracting materials can be formed in desired areas using patterned ultraviolet illumination, allowing their incorporation into mechanically engineered systems, being also compatible with printing technologies. Due to the designed filamentous assembly of kinesins, the generated forces reach the micronewton range, enabling actuation of millimetre-scale mechanical components. These properties may be useful for the fabrication of soft robotic systems with advanced functionalities.

Survival Rate of Cells Sent by a Low Mechanical Load Tube Pump: The "Ring Pump".

A ring pump (RP) is a useful tool for microchannels and automated cell culturing. We have been developing RPs (a full-press ring pump, FRP; and a mid-press ring pump, MRP). However, damage to cells which were sent by the RP and the MRP was not investigated, and no other studies have compared the damage to cells between RPs and peristaltic pumps (PPs). Therefore, first, we evaluated the damage to cells that were sent by a small size FRP (s-FRP) and small size MRPs (s-MRPs; gap = 25 or 50 µm, respectively). "Small size" means that the s-FRP and the s-MRPs are suitable for microchannel-scale applications. The survival rate of cells sent by the s-MRPs was higher than those sent by the s-FRP, and less damage caused by the former. Second, we compared the survival rate of cells that were sent by a large size FRP (l-FRP), a large size MRP (l-MRP) (gap = 50 µm) and a PP. "Large size" means that the l-FRP and the l-MRP are suitable for automated cell culture system applications. We could not confirm any differences among the cell survival rates. On the other hand, when cells suspended in Dulbecco's phosphate-buffered saline solution were circulated with the l-MRP (gap = 50 µm) and the PP, we confirmed a difference in cell survival rate, and less damage caused by the former.

Relationship between upper limb motor function and activities of daily living after removing the influence of lower limb motor function in subacute patients with stroke: A cross-sectional study.

BACKGROUND: Previous studies have reported a relationship between upper limb motor function and activities of daily living. However, their relationship after removing the influence of lower limb motor function has not been clarified. OBJECTIVE: This study aimed to investigate the relationship between Fugl-Meyer assessment upper limb and total Functional Independence Measure motor score and between Fugl-Meyer assessment upper limb and each item contained in Functional Independence Measure motor score after eliminating the influence of the motor function of the affected lower limb. METHODS: This retrospective cross-sectional study included 58 subacute stroke patients. To investigate the relationship between the Fugl-Meyer assessment upper limb and total Functional Independence Measure motor score before and after removing the influence of Fugl-Meyer assessment lower limb, Spearman's rank correlation coefficient and partial correlation analysis were used. Additionally, the relationship between Fugl-Meyer assessment upper limb and each item of Functional Independence Measure motor score after removing the influence was assessed. RESULTS: Before removing the influence of Fugl-Meyer assessment lower limb, Fugl-Meyer assessment upper limb was strongly correlated with total Functional Independence Measure motor score (r = 0.74, p < 0.001). However, it became weak after removing the influence (r = 0.27, p = 0.04). Regarding each item of Functional Independence Measure motor score, Fugl-Meyer assessment upper limb was correlated with grooming (r = 0.27, p = 0.04), bathing (r = 0.28, p = 0.03), dressing upper body (r = 0.33, p = 0.01), dressing lower body (r = 0.31, p = 0.02), and stair-climbing (r = 0.31, p = 0.02) after removing the influence. CONCLUSION: These findings suggest that the relationship between the upper limb motor function and activities of daily living is strongly influenced by lower limb motor function.

Micro Vacuum Chuck and Tensile Test System for Bio-Mechanical Evaluation of 3D Tissue Constructed of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPS-CM).

In this report, we propose a micro vacuum chuck (MVC) which can connect three-dimensional (3D) tissues to a tensile test system by vacuum pressure. Because the MVC fixes the 3D tissue by vacuum pressure generated on multiple vacuum holes, it is expected that the MVC can fix 3D tissue to the system easily and mitigate the damage which can happen by handling during fixing. In order to decide optimum conditions for the size of the vacuum holes and the vacuum pressure, various sized vacuum holes and vacuum pressures were applied to a normal human cardiac fibroblast 3D tissue. From the results, we confirmed that a square shape with 100 µm sides was better for fixing the 3D tissue. Then we mounted our developed MVCs on a specially developed tensile test system and measured the bio-mechanical property (beating force) of cardiac 3D tissue which was constructed of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM); the 3D tissue had been assembled by the layer-by-layer (LbL) method. We measured the beating force of the cardiac 3D tissue and confirmed the measured force followed the Frank-Starling relationship. This indicates that the beating property of cardiac 3D tissue obtained by the LbL method was close to that of native cardiac tissue.

Analytical Model and Experimental Evaluation of the Micro-Scale Thermal Property Sensor for Single-Sided Measurement.

We report a new analytical model of the MEMS-based thermal property sensor for samples which are difficult to handle and susceptible to damage by thermal stimulus, such as living cells. Many sensor designs had been reported for thermal property measurements, but only a few of them have considered the analytical model of the single-sided measurement in which a measurement sample is placed on the sensor substrate. Even in the few designs that have considered the analytical model, their applicable limits are restricted to more than 1 mm length in practical situations. Our new model considers both the sample and the sensor substrate thermal properties and is applicable to a sensor length less than 1 µm. In order to minimize the influence of the heat stimulus to the sample, the model formulates the required heat dissipating time for different sensor geometries. We propose fast and precise detection circuit architecture to realize our model, and we discuss the sensor performance for a number of different designs.

A Closed System for Pico-Liter Order Substance Transport from a Giant Liposome to a Cell.

In single cell analysis, transport of foreign substances into a cell is an important technique. In particular, for accurate analysis, a method to transport a small amount (pico-liter order) of substance into the cell without leakage while retaining the cell shape is essential. Because the fusion of the cell and the giant liposome is a closed system to the outside, it may be possible to transport a precise, small amount of substances into the cell. Additionally, there is no possibility that a leaked substance would affect other systems. To develop the liposome-cell transportation system, knowledge about the behavior of substances in the liposome and the cell is important. However, only a few studies have observed the substance transport between a liposome and a cell. Here, we report observation of small amount of substance transport into a single C2C12 cell by using a giant liposome. Substance transport occurred by electrofusion between the cell and the giant liposome containing the substance, which is a closed system. First, to observe the electrofusion and substance transport from the moment of voltage application, we fabricated a microfluidic device equipped with electrodes. We introduced suspensions of cells and liposomes into the microfluidic device and applied alternating current (AC) and direct current (DC) voltages for electrofusion. We observed a small amount (22.4 ± 0.1%, 10.3 ± 0.4% and 9.1 ± 0.1%) of fluorescent substance (Calcein) contained in the liposomes was transported into the cell without leakage outside the cell, and we obtained the diffusion coefficient of Calcein in the cell as 137 ± 18 µm²/s. We anticipate that this system and the knowledge acquired will contribute to future realization of more accurate single cell analysis in a wide range of fields.

A Method of Three-Dimensional Micro-Rotational Flow Generation for Biological Applications.

We report a convenient method to create a three-dimensional micro-rotational fluidic platform for biological applications in the direction of a vertical plane (out-of-plane) without contact in an open space. Unlike our previous complex fluidic manipulation system, this method uses a micro-rotational flow generated near a single orifice when the solution is pushed from the orifice by using a single pump. The three-dimensional fluidic platform shows good potential for fluidic biological applications such as culturing, stimulating, sorting, and manipulating cells. The pattern and velocity of the micro-rotational flow can be controlled by tuning the parameters such as the flow rate and the liquid-air interface height. We found that bio-objects captured by the micro-rotational flow showed self-rotational motion and orbital motion. Furthermore, the path length and position, velocity, and pattern of the orbital motion of the bio-object could be controlled. To demonstrate our method, we used embryoid body cells. As a result, the orbital motion had a maximum length of 2.4 mm, a maximum acceleration of 0.63 m/s², a frequency of approximately 0.45 Hz, a maximum velocity of 15.4 mm/s, and a maximum rotation speed of 600 rpm. The capability to have bio-objects rotate or move orbitally in three dimensions without contact opens up new research opportunities in three-dimensional microfluidic technology.

Biofuel cell backpacked insect and its application to wireless sensing.

This study investigated an enzymatic biofuel cell (BFC) which can be backpacked by cockroaches. The BFC generates electric power from trehalose in insect hemolymph by the trehalase and glucose dehydrogenase (GDH) reaction systems which dehydrogenate ß-glucose obtained by hydrolyzing trehalose. First, an insect-mountable BFC (imBFC) was designed and fabricated with a 3D printer. The electrochemical reaction of anode-modified poly-L-lysine, vitamin K3, diaphorase, nicotinamide adenine dinucleotide, GDH and poly(sodium 4-styrenesulfonate) in the imBFC was evaluated and an oxidation current of 1.18 mAcm(-2) (at +0.6 V vs. Ag|AgCl) was observed. Then, the performance of the imBFC was evaluated and a maximum power output of 333 µW (285 µW cm(-)(2)) (at 0.5 V) was obtained. Furthermore, driving of both an LED device and a wireless temperature and humidity sensor device were powered by the imBFC. These results indicate that the imBFC has sufficient potential as a battery for novel ubiquitous robots such as insect cyborgs.

IL-12 involvement in myogenic differentiation of C2C12 in vitro.

Recently, the extracellular microenvironment has been shown to be critical for the correct differentiation of stem cells to specific tissues. Many factors, including physical (e.g. biomaterial stiffness and topography) and biological (as growth factors, cytokines and chemokines) components, cooperate to create an ideal microenvironment for muscle stem cells, with many of these factors having been widely investigated. We previously demonstrated that the use of non-proliferating muscle-specific and unrelated cells as feeder layers for skeletal muscle progenitor cell differentiation resulted in significant differences in the ability to form myotubes, suggesting the importance of biological factors in myogenic differentiation. In this study, we investigated the biological factors involved in this process, analyzing the expression profile of 84 genes coding for cytokines and chemokines. We successfully identified a novel role for the cytokine IL-12 in the myogenic differentiation of C2C12 mouse skeletal muscle cells. Experiments involving the overexpression or silencing of the IL-12 gene in C2C12 showed that IL-12 enhanced the myogenic differentiation process. Moreover, when IL-12 was overexpressed in non-biologically related feeder cells, the new co-culture system was able to improve myogenic differentiation of C2C12 seeded on top. Although IL-12 is known to be a cytokine involved in inflammatory responses, it also appears to be involved in the myogenic differentiation process, acting as a positive regulator of this mechanism. This fact is expected to prove to be important for the development of functional biomaterials.

Optogenetic induction of contractile ability in immature C2C12 myotubes.

Myoblasts can be differentiated into multinucleated myotubes, which provide a well-established and reproducible muscle cell model for skeletal myogenesis in vitro. However, under conventional differentiation conditions, each myotube rarely exhibits robust contraction as well as sarcomere arrangement. Here, we applied trains of optical stimulation (OS) to C2C12 myotubes, which were genetically engineered to express a channelrhodopsin variant, channelrhodopsin-green receiver (ChRGR), to investigate whether membrane depolarization facilitates the maturation of myotubes. We found that light pulses induced membrane depolarization and evoked action potentials in ChRGR-expressing myotubes. Regular alignments of sarcomeric proteins were patterned periodically after OS training. In contrast, untrained control myotubes rarely exhibited the striated patterns. OS-trained and untrained myotubes also differed in terms of their resting potential. OS training significantly increased the number of contractile myotubes. Treatment with nifedipine during OS training significantly decreased the fraction of contractile myotubes, whereas tetrodotoxin was less effective. These results suggest that oscillations of membrane potential and intracellular Ca(2+) accompanied by OS promoted sarcomere assembly and the development of contractility during the myogenic process. These results also suggest that optogenetic techniques could be used to manipulate the activity-dependent process during myogenic development.

Generative force of self-oscillating gel.

We succeeded in measuring the generative force of a self-oscillating polymer gel in an aqueous solution comprising the three substrates of the Belousov-Zhabotinsky (BZ) reaction (malonic acid, sodium bromate, and nitric acid) under constant temperature. In this study, we developed an apparatus with a microforce sensor for measuring the generative force of small-sized gels (1 mm(3)). The self-oscillating polymer gel directly converts the chemical energy of the BZ reaction into mechanical work. It was determined that the generative force of the self-oscillating gel was 972 Pa, and the period of self-oscillation was 480 s at 18 °C. We demonstrated that the generative force of the gel was about a hundredth the generative force of a muscle in the body. We analyzed the time dependence of the color change in the self-oscillating polymer gel. The color of the gel changed periodically owing to the cyclic change in the redox state of the Ru moiety, induced by the BZ reaction. The peaks of the waveforms of the generative force and color change were almost identical. This result showed that the generative force was synchronized with the periodical change in the oxidation number of the Ru catalytic moiety in the gel. To understand a theoretical basis for the generative force of a self-oscillating gel, we considered a general theory that is based on the volume phase transition of gel and the two-parameter Oregonator model of the BZ reaction.

Atmospheric-operable bioactuator powered by insect muscle packaged with medium.

Despite attempts in a number of studies to utilize muscle tissue and cells as microactuators, all of the biohybrid microdevices have been operable only in the culture medium and none have worked in air due to the dry environment. This paper demonstrates an atmospheric-operable bioactuator (AOB) fabricated by packaging an insect dorsal vessel (DV) tissue with a small amount of culture medium inside a capsule. The AOB, consisting of microtweezers and the capsule, was designed based on a structural simulation that took into account the capillary effect. The base part of the microtweezers was deformed by spontaneous contractions of the DV tissue in the medium inside the capsule, by which the front edges of the microtweezer arms projecting above the medium surface were also deformed. First, we confirmed in the medium that the DV tissue was able to reduce the gap between the arm tips of the microtweezers. After taking the AOB out of the medium, as we expected, the AOB continued to work in air at room temperature. The gap reduction in air became larger than the one in the medium due to a surface tension effect, which was consistent with the simulation findings on the surface tension by the phase-field method. Second, we demonstrated that the AOB deformed a thin-wall ring placed between its tips in air. Third, we measured the lifetime of the AOB. The AOB kept working for around 40 minutes in air, but eventually stopped due to medium evaporation. As the evaporation progressed, the microtweezers were pressed onto the capsule wall by the surface tension and opening and closing stopped. Finally, we attempted to prevent the medium from evaporating by pouring liquid paraffin (l-paraffin) over the medium after lipophilic coating of the capsule. As a result, we succeeded in prolonging the AOB lifetime to more than five days. In this study, we demonstrated the significant potential of insect muscle tissue and cells as a bioactuator in air and at room temperature. By integrating insect tissue and cells not only into a microspace but also onto a substrate, we expect to realize a biohybrid MEMS device with various functions in the near future.

Voluntary movement controlled by the surface EMG signal for tissue-engineered skeletal muscle on a gripping tool.

We have developed a living prosthesis consisting of a living muscle-powered device, which is controlled by neuronal signals to recover some of the functions of a lost extremity. A tissue-engineered skeletal muscle was fabricated with two anchorage points from a primary rat myoblast cultured in a collagen Matrigel mixed gel. Differentiation to the skeletal muscle was confirmed in the tissue-engineered skeletal muscle, and the contraction force increased with increasing frequency of electric stimulation. Then, the tissue-engineered skeletal muscle was assembled into a gripper-type microhand. The tissue-engineered skeletal muscle of the microhand was stimulated electrically, which was then followed by the voluntary movement of the subject's hand. The signal of the surface electromyogram from a subject was processed to mimic the firing spikes of a neuromuscular junction to control the contraction of the tissue-engineered skeletal muscle. The tele-operation of the microhand was demonstrated by optical microscope observations.

Cell patterning through inkjet printing of one cell per droplet.

The inkjet ejection technology used in printers has been adopted and research has been conducted on manufacturing artificial tissue by patterning cells through micronozzle ejection of small droplets containing multiple cells. However, stable injection of cells has proven difficult, owing to the frequent occurrence of nozzle clogging. In this paper, a piezoelectric inkjet head constructed with a glass capillary that enabled viewing of the nozzle section was developed, the movement of cells ejected from the nozzle tip was analyzed, and a method for stably ejecting cells was verified. A pull-push ejection method was compared with a push-pull ejection method regarding the voltage waveform applied to the piezoelectric element of the head. The push-pull method was found to be more suitable for stable ejection. Further, ejection of one cell per droplet was realized by detecting the position of the cell in the nozzle section and utilizing these position data. Thus, a method for more precise patterning of viable cells at desired position and number was established. This method is very useful and promising not only for biofabrication, 3D tissue construction, cell printing, but also for a number of biomedical application, such as bioMEMS, lab on a chip research field.