The maximum velocity of intermittent oscillatory flow in a pipe with circumferential grooves is greater than sinusoidal oscillatory flow in a straight pipe. Visualization of intermittent oscillatory flow of water in a pipe with circumferential grooves of several aspect ratios has been carried out on conditions of several frequencies and tidal volume of 80[mL]. In the present paper, ensemble averages of each time from the lower dead point to the upper dead point for flow velocity distributions of intermittent oscillatory flow through pipes with circumferential grooves of different aspect ratio were measured. Maximum velocity did not necessarily increased in the pipe with circumferential grooves of aspect ratio of 1.Maximum axial velocity in a pipe with circumferential grooves of aspect ratio of 0.5 was almost similar to the other pipes, but the maximum radial velocity of aspect ratio of 0.5 was larger than the other pipes.

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We demonstrate nano-bioimaging in wet-condition using cathodoluminescence (CL) of rare-earth doped phosphors. An SEM-CL imaging system is used to observe wet-condition CL, and consists of an SEM, an ellipsoidal mirror, an optical fiber bundle and a detection part based on dichroic mirrors, which can detect 3 color signals simultaneously. Specimens were placed in a capsule having a vacuum sealing thin film to maintain atmospheric pressure inside the capsule. As CL nanoprobes, Y_2O_3:Eu and Y_2O_3:Tb rare-earth doped nanophosphors (red and green emission) were synthesized by homogeneous precipitation method. Two kinds of nanophosphors were introduced into HeLa cells via endocytotic process. After the fixation, the cellular structures were stained by osmium tetroxide for making the contrast of secondary electron. Both cellular structural image (such as lipids) and CL image were simultaneously observed. The spatial resolution of CL microscopy is similar to that of SEM. Correlative imaging of SEM and CL microscopy was succeeded in wet condition. This multimodal/correlative imaging method enables us detail understanding to reveal the cellular functions. We believe that our new nanobioimaging method using CL and rare-earth doped phosphors will be a useful correlative imaging technique.

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Amyloid β (Aβ) peptides form needle-like aggregate called amyloid fibril in vivo to cause Alzheimer's disease. The aggregation reaction is mainly divided into nucleation and fibril elongation phases. Nucleation reaction takes long term in the physiological condition. This slowness of the nucleation reaction prevents us from clarifying its aggregation mechanism. Thus, there is no effective treatment for Alzheimer's disease. Recently, formation of amyloid fibril can be drastically accelerated by ultrasonic irradiation for some peptides. The acceleration mechanism, however, remains unclear. Then, we first investigate acoustic-pressure and frequency dependences of the reaction. From our experimental results, it is revealed that the reaction rate constant for aggregation reaction increased by a factor of 〜1000 as the second-harmonics acoustic pressure increases. We also discovered the optimum frequency for aggregation acceleration near 29 kHz. For explaining these results, we propose an aggregation acceleration model based on dual effects of cavitation bubble, 'local condensation' and 'local heating'. Our theoretical model succeeded in reproducing to acoustic pressure and frequency dependences, showing its essential validity.

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We have developed a line-scanning multiplex coherent anti-Stokes Raman scattering (CARS) microscope for label free imaging. The developed microscope utilizes 10 femtosecond and 5 picosecond Ti:Sapphire lasers as light sources of a broad and a narrow spectral bandwidth. The spectrum of the 10 femtosecond laser expands from 746 to 836 ran, which corresponds from 600 to 2300 cm^<-1> in Raman shift when the 5 picosecond laser is set to 714 nm. CARS spectrum of arterial vessel wall of Watanabe heritable hyperlipidemic rabbit is demonstrated using the developed system. To improve the imaging speed, we have modified the system using line-scanning for parallel excitation and detection by inserting a Powel lens on excitation laser beams. The experimental results of line-scanning CARS imaging of polystyrene beads is also presented.

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Influenza A virus is an infection source of the epidemic. Two types of protein exist on the virus surface. One is hemagglutinin (HA) and another is neuraminidase (NA). There are 16 kinds of HA and 9 kinds of NA, which makes a combination of 144 kinds of the virus subtype. Subtype H1N1 and subtype H3N2 frequently infect human every year. Some of these subtypes can be the cause of the pandemic. For sensing the various types of the virus, a new material detecting the virus which can be easily and quickly produced, is required. In this study, we focused on the heavy chain antibodies derived from camelids (VhH) from a view point of industrial applications. The size of VhH is comparatively small (15 kDa), which makes it easy to be produced. The phage library displaying VhHs was constructed and a monoclonal VhH which bound to HA of H1N1 subtype specifically was selected by bio-panning. The characteristic of the VhH was evaluated by the Enzyme-linked immuno-sorbent assay (ELISA).

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To clarify a role of the glial cells during a change in cerebral microvasculature, morphological transformation of the glial cells under chronic hypoxia was determined using in vivo two-photon laser scanning fluorescence microscopy in the mouse brain exposed to 21 days of hypoxia (8-9% oxygen). A volume of the soma and number of processes of the glial cells were quantified with custom-written software. We observed that a volume of the soma significantly increased at days 7 and 21 after initiation of hypoxia exposure. On the other hand, a number of the major processes was observed to increase monotonically. A statistically significant difference in the number density of the glial processes was found after 14 days of hypoxia, compared to pre-hypoxia control conditions. Furthermore, this increase in the number density of the major processes was remarkable around the capillary. The findings indicate that the glial processes may interact with the vascular cells during hypoxia-induced adaptation of cerebral microvasculature.

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The purpose of the present study is to develop an analytical method for automatic detection of the brain cell activation, imaged with two-photon microscopy in the somatosensory cortex of awake mice. The cells expressed calcium-sensitive genetic sensor (GCaMP3) under neural promoter of CaMKII, were also labeled with sulforhodamine 101 (SR101), a marker of glial cells. The software detected the active cells which showed enhanced fluorescent signal during contralateral whisker stimulation, and measured a location of the cells from the cortical surface with a distance from the nearest blood vessels which were also labeled with SR101. In addition, the cells were further classified into neurons and glial cells based on SR101 staining. The present method will be a useful technique to determine a chronological order of functional plasticity/degeneration at the cellular level based on massive cellular imaging data for learning or a development of neurological disorders, cognitive impairment in the long term changes in neurovascular unit.

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The outer hair cells (OHCs) are the sensory cells in the mammalian cochlea of the inner ear. They exhibit elongation and contraction in response to acoustical stimulation. Due to this somatic motility, the basilar membrane is subjected to force, resulting in cochlear amplification and thus leading to the high sensitivity of mammalian hearing. This motility is thought to be driven by a motor protein embedded in the lateral wall of OHCs. In 2000, this motor protein was identified and termed prestin. Prestin consists of 744 amino acids with a molecular weight of about 81.4 kDa. In a series of attempts to clarify its membrane topology, hydrophobicity analysis in conjunction with the prediction of the conserved phosphorylation site has suggested that prestin is a 10 or 12 transmembrane protein with cytoplasmic N- and C-termini. The size of prestin was reported to be about 10 nm in diameter, presumably forming tetramer. For the further progress on prestin research at molecular level, it is indispensable to develop a method of obtaining a large amount of prestin. In this study, therefore, an attempt was made to construct an expression system for prestin. For that, a mammalian expression vector containing prestin cDNA was developed. After the amount of such vector was amplified with Escherichia coli (E. coli), its existence was confirmed by electrophoresis and sequence analysis. Results showed that the mammalian expression vector of prestin was properly developed.

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In living cells, membrane proteins play important roles in cellular activities. Since cell membranes consist of a lipid bilayer, membrane proteins can be incorporated into artificial lipid bilayer, and its membrane can be applied to the analysis of membrane proteins and/or biosensor devices using membrane proteins. Therefore, a technique to form lipid bilayer membrane on a microchannel or a microchamber has been paid attention. In this paper, we propose a new technique to form lipid bilayer membrane on a mesh filter. Filling the water solution in the micropores of the mesh filter by using a microchannel, lipid bilayer membrane will be formed on the filter with contact method. This technique will be useful since the filter can be transferable to anywhere for various experiments. Presently, we develope a MEMS device which consists microchannels and films with micropores. In order to evaluate the micropores, the device is filled with water solution to form the lipid bilayer membrane on the pores, and observed under a microscope.

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The relationship between the mechanical properties and hierarchical structure in the bone tissue is essential to understand the factors determining the bone strength during aging and/or osteoporosis. This study aimed to investigate the distribution of elastic modulus at the microstructural level, i.e., osteon scale, in the cortical bone. In the experiments, block-like specimens were taken from the ocrtex in a bovine femoral diaphysis and the transverse cross-section of the diaphysis was polished. The microindentation tests were conducted by using a spherical shape head indentor at four kinds of microstructure from the outer to the inner surface: osteon and interstitial region in the osteonal bone and lamellar and interstitial regions in the plexiform bone. To evaluate the distribution of elasticic modulus, the contact stiffness was calculated from the force-depth relationship during unloading. As a result, in the posterior specimen, the contact stiffness of plexiform bone obserbed in the outer region was larger than that of osteonal bone in the inner rregion. In the anterior specimen, the valiation of contact stiffness was larger than that of the posterior specimen and there was no significant difference among the four kinds of microstructure. A further study of the radial distribution of the microstructure in the diaphysis and the effects on the elastic modulus is needed for further elucidating the difference of elastic modulus at those microstructures.

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Flapping-wing small flying robots inspired from natural flyers have a potential for high maneuverability and high aerodynamic performance in low-Reynolds-number flow regime. To generate sufficient aerodynamic force in hovering flight, the wings should not only flap but also feather (i.e. rotate around the spanwise axis of the wing) in order to maintain appropriate angle of attack. In this study, we investigated effect of passive feathering at the wing base on aerodynamic thrust and efficiency. In particular, various pre-determined limits of passive feathering angle were tested for three different aspect ratios of the wings. The time-averaged thrust, power consumption, and efficiency were measured using a hummingbird-sized electric flapping mechanism. It was found that the low-aspect-ratio wing without the passive feathering generated the largest thrust, but the power consumption was the worst too. On the other hand, the high-aspect-ratio wing like a hummingbird wing with passive feathering achieved relatively large thrust and the smallest power consumption, that is, the best efficiency. This result suggested that passive feathering at the wing base drastically improves efficiency with the minimum loss of the thrust.

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Flying insects can achieve smart maneuverability, such as rapid acceleration, deceleration, and hovering under complex environmental disturbances. Previous studies suggest that there may exist a delay of two wingbeat cycles for insects to actively tune their wing kinematics in response of disturbances. Such delay may degrade the dynamic flight stability of flapping-wing flights. And it is considerable that insects may use some passive mechanisms in terms of wing flexibility to enhance the flight stability in disturbances. In this study, we carried out a numerical study of hawkmoth hovering flight stability with a focus on how steady and unsteady disturbances affect the body attitudes by performing a fluid-structure interaction simulation of flexible wings in flapping. Our impedimentary FSI results indicate that the bending of the flexible wing in a steady flow seems to be responsible for augmenting the generation of vertical and horizontal forces while reducing the variation of the aerodynamics torques. These results point out to the importance of wing flexibility in enhancing the flapping-wing flight stability in disturbances.

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The swimming humanoid robot "SWUMANOID" was developed in the previous study for research of human swimming. The crawl stroke and breaststroke have been already realized by the robot. However, the butterfly stroke has not been realized to date. Therefore, the objective of this study was to realize the free swimming of the butterfly stroke with the robot. The robot were developed on a half scale, and had 24 specially waterproofed motors inside the body. The swimming motion of butterfly stroke was created and examined by means of the simulation. The swimming experiment was conducted at a swimming pool. The swimming motion was filmed by a video camera. The swimming speed was estimated from the filmed images. As a result, the constant propulsion by the robot was confirmed. The swimming speed in the experiment was found to be 0.17〜0.20 m/s for the stroke cycle of 3.5 s. It was somewhat lower than that in the simulation.

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The swimming motion of Tuna type fishes has excellent ability for its speed and efficiency. In the former studies, we made 1/1 scale model of Blue Fin Tuna body and caudal fin .The body resistance in the water flow are measured, and the lift and drag force of caudal fin are also measured. By using these data, we developed swimming mechanism and mounted on float, This paper shows a performance comparison between Tuna type caudal fin and Rectangular caudal fin.

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Snow removal is an important issue in snowy cold region. Although calcium chloride powders have usually applied in snow covered area especially on the road, many kind of artificial and natural structure are damaged by chloride. Scallop shell powders are expected to use in snow removal. However the whiteness of the shell powder reduces the ability of heat generation produced by optical absorption under sunshine. In this study, the heat treatment was applied to the scallop shell powders to change the blackness of them. An ice melting testing was introduced to evaluate the snow melting ability of the shell powders. The heat treatment improved the optical absorption characteristics of shell powders. The blackness of the powders became the best state at 680 ℃ heat treatment. The ice melting ability of the 680 ℃ heated scallop shell powders showed favorably compared with charcoal powders which were used to snow removal for farm land. The results provided the efficient use of scallop shell in snow removal technique.

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The living system is thought to be one of the benchmarks for an inspiration of the artificial things, e.g. robotics, biomimetics. If we could understand the living system, we should reconstruct it by mechanical stuff. The gait analysis of normal and pathological function have been studied for the intervention to walking disruptions following neuromuscular diseases. The purpose of this study is to develop the musculoskeletal walking simulator, which actually walks with the similar coordination pattern to the neurotypical human walking. Human walking appears coodinated and efficient, and effortless. The biped robot consisted of two-dimensional motion of hip, knee and ankle pin joint. The typical muscles attributing to normal walking were altered by simple springs because the efficient walking meant spring energy change and discharge cycles. The arrangement of the muscles was almost represented in the robot. Then, the robot regenerated the walking like motion patterns without any interventions. The selected two proximal muscles per leg were altered by spring and driving motor, whose were connected serially. Although we required several fine modulations of each spring stiffness and relating moment arms, the walking simulator could walk by an appropriate on-off stimulation pattern of the muscles, which thought to be similar to the EMG pattern in normal human walking. The coordination of the walking of the robot was evaluated by the angle detectors of each joint. The result showed a similar coordination pattern to the normal human walking.

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Water bears (tardigrades) are microscopic crawlers with body lengths of 100 μm order that walk on four pairs of stubby, tubular and clawed legs without any joints called the first, second, third and fourth legs. The gait of water bears is mechanically interesting because it is unusual that such a small microorganism walks on four pairs of legs. In this study, the gait of water bears with different walking speeds was analyzed and the features of the gait were investigated. Twenty-two water bears (Milnesium tardigradum) with body lengths between 0.204 and 0.598 mm, which were obtained from Miyashiro town (Saitama, Japan), were used as samples. The results were as follows. The water bears tended to have the tripod gait for faster walking speeds, the wave gait for slower speeds and the ripple gate for intermediate speeds. These results indicated that the gait of water bears is similar to that of insects. During the directional change, the third and fourth legs supported the body, and the first and second legs were used to change the direction of movement.

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Infertility is often cited as one of the causes of the declining birthrate, which has a social problem in recent years. Roughly 10% of couples have infertility problems, and almost 50% of all cases of infertility are associated with a lack of sperm or sperm abnormalities. Therefore, motile sperm are required to increase the probability of fertilization. We previously observed of flagellar motion of bovine sperm and clarified that ambient fluid viscosity and non-Newtonian properties affected its shape and motion. However, the detail mechanics of the effect of surrounding fluid properties on the sperm motion is still unclear. Therefore, we simulated the sperm motion in various viscosity and non-Newtonian fluid to reveal the effect of the surrounding environments on the sperm motility. The simulation results indicated the decrease in the sperm velocity when the viscosity increased, because the increase in the viscosity lead to increase in the resistance of the ambient fluid. Additionally, the increase in the viscosity brought about the increase in sperm motility. We compared the present results with previous experimental data for the sperm velocity, and estimated the sperm motility for each viscosity. Consequently, we obtained the relationship of viscosity coefficient and sperm motility. These results will provide the useful information to estimate the sperm motility for various fluid properties.

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The swimming process by which mammal spermatozoa progress towards an egg within the reproductive organs is important in achieving successful internal fertilization. The viscosity of oviductal mucus is more than two orders of magnitude greater than that of water, and oviductal mucus also has viscoelasticity. In this study, we experimentally observed sperm motion in Newtonian and non-Newtonian fluids using the semen of Japanese cattle in order to investigate the effect of viscoelasticity on bovine sperm motility. Furthermore, we visualized flow around sperm by conducting PIV analysis, and investigated shear rate surrounding sperm. We obtained the following results from the experiments. Bovine sperm swim more linearly in high viscous non-Newtonian fluid than in low viscous Newtonian fluid. Bovine sperm swim generating some vortices surrounding flagellum. Especially, the vortices in low viscosity fluid were formed more largely and strongly than one of in non-Newtonian fluid. In low viscosity fluid, high shear rate area distributed wholly around sperm head, whereas in non-Newtonian fluid, high shear rate area distributed only partly around sperm head. This deference in shear rate area imply that sperm changes the way of swinging according to the surrounding fluid. The difference in the way of swimming may lead to the change in linearity of sperm motion.

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A sperm cell is a flagellated cell and it can swim in a fluid by beating flagellum. Locomotion of a sperm cell is strongly affected by surrounding fluid flow, and recently rheotaxis of sperm cells was found experimentally. Fluid mechanics then becomes a subject of growing importance in sperm motility. In this study, we numerically investigate sperm cell behavior in shear flow near a plane wall to quantitatively understand how fluid mechanics affects on swimming of sperm cell. Due to the small size of sperm cells, inertia effects of fluid motion can be neglected and we assume Stokes flow around the cell. Flow field is then expressed by boundary integral equation and which is solved by a boundary element method. As a result, the sperm cell shows reorient to upstream and keeps on swimming against the fluid flow. This result suggests that fluid mechanics plays a key role in navigation of sperm cells towards the ovum, which is located upstream in the oviduct.

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Swimming behavior of microorganism near an interface is important to understand how to prevent bio-film formation, which can be a cause of medical instrument pollution. Recently, we found that a ciliate can be trapped at a water-air interface, though swims away from a water-wall interface. We conducted numerical simulation of swimming microorganism near an interface from hydrodynamic perspective, to reveal the entrapment mechanism. By mimicking microorganism shape in detail, our microorganism model reproduced entrapment phenomena for the first time in the world. We concluded that shape of a ciliate dominates the entrapment phenomena.

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In vivo, hydrostatic pressure is one of the main mechanical stimuli cartilage cells are submitted to during joint loading. Moderate pressurization is beneficial to cartilage physiology but pressures in excess of 20 MPa induce changes in chondrocytes reminiscent of osteoarthritis. However, the molecular mechanisms involved in the detrimental effects of high pressure are largely unknown. Here, using chondrocyte progenitor cells, we show that high hydrostatic pressure leads to a marked and prolonged decrease in chondrocyte marker expression after 1 hour of pressurization and a concomitant increase in the early response gene Fos. Fos up-regulation, however, is not responsible for the decrease in chondrocyte marker expression and is mediated by different signalling pathways, including the MAP kinase ERK pathway.

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Recently, extensive researches have reported that intracellular contractile forces play pivotal roles in cell functions, such as morphogenesis, differentiation, and homeostasis. Actin stress fibers are the main contractile force generator in non-muscle cells. Actin stress fibers have a less-organized sarcomeric structure constituted of actin, myosin, α-actinin, and other contraction-associated proteins. Their localization and activity of cytoskeletal molecules are highly regulated in a spatiotemporal manner. Moreover, dynamics and contractile properties of actin stress fibers are thought to be regulated by physical cues, for example, the tension externally applied to the actin stress fibers and internally generated by actomyosin contraction. To shed light on kinetics of actin stress fibers under various actomyosin activities, here we investigate the dynamics of fluorescent protein-tagged myosin molecules by fluorescence recovery after photobleaching (FRAP). We investigate the turnover rates and fractional recovery of myosin molecules associated with phosphomimic and nonphosphorylatable myosin regulatory light chain mutants (collaborative work with Dr. Masayuki Takahashi, Hokkaido Univ.). The double phosphomimic myosin regulatory light chain mutant (DD-mutant) had low fractional recovery. It indicates that myosin molecules with DD-mutant might have the longer lifetime attached to F-actin.

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Mechanical signals from external environment of a cell are transmitted to the nucleus from actin cytoskeletons through linkers of nucleoskeletons and cytoskeletons (LINC) complex and are believed to play important role in not only cellular morphology but also nuclear mechanical properties. To address the underlying mechanisms, we explored the effect of knockdown of nesprin-1, a key component of LINC complex for the transmission of mechanical signals, on nuclear mechanical properties of fibroblasts exposed to cyclic stretching. For siRNA knockdown of nesprin-1, human dermal fibroblasts were transfected with siRNA targeting human nesprin-1. After cells were exposed to cyclic stretching for 24 h, their nuclear elastic modulus were evaluated by atomic force microscopy-based indentation tests. While the nuclear elastic moduli for non-treated wild type cells were not changed by exposure to cyclic stretching, nuclear elastic moduli for siRNA-treated cells exposed to cyclic stretching were significantly lower than those cultured statically (p < 0.01). These results indicate that mechanical signals transmitted through nesprin-1 have a critical role in maintenance of mechanical properties of nuclei.

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Mitochondria are subcellular organelles that involved in ATP synthesis, ROS generation, apoptosis and aging, etc. Their morphologies influence cellular activities. When the balance of mitochondrial fusion and fission is lost under the exposure of supra-physiologic cyclic stretches, reactive oxygen species (ROS) are released and cells apoptosis is prompted. In previous study, the changes of the mitochondrial morphology were observed in the bovine aortic endothelial cells (BAEC) under supra-physiologic cyclic stretches. However, the underlying mechanisms remain obscure. In this study, we examined the relationship between the supra-physiologic cyclic stretches and the alteration of the mitochondrial morphology in human aortic endothelial cells (HAECs). To visualize the changes of mitochondrial morphology, HAECs were stained with 1.0μM Mito Tracker Orange, and then pretreated with 10μM GdCl_3 to inhibit stretch-induce intracellular Ca^<2+> increase involved in mitochondrial fission. HAECs were subjected to supra-physiologic cyclic stretches (20% at 1 Hz) by using a stretch camber with a stepper motor for 1 hour and mitochondrial morphologies were time-lapse imaged every 12 min. In HAECs under 20% cyclic stretch, the average length of mitochondria decreased in comparison with that of the control cells (0%), in both the presence and absence of the inhibitor, GdCl_3. These results indicate that the supra-physiologic cyclic stretch causes mitochondrial fragmentation involved in apoptosis via the pathway which is different from stretch-induce intracellular Ca^<2+> increase in HAECs.

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Actin cytoskeleton plays critical roles in many aspects of cell functions, such as cell movement, cell division, shape maintenance, contractile force generation, and so on. Actin organization is regulated in a spatiotemporal manner by various actin-related proteins. Transgelin 1 is a 22 kDa actin binding protein of calponin family, and is known as a molecular marker for differentiated contractile smooth muscle cells. Transgelin 1 is thought to act as an actin cross-linker and stabilizer. However, the details of transgelin 1 dynamics remain unknown. In this study, we characterize transgelin 1 dynamics with fluorescence recovery after photobleaching (FRAP). First, we consider the spatial regulation of transgelin 1 dynamics. Our result reveals that transgelin 1 molecules are highly diffusive at stress fibers located in the central portion of the cell and have higher affinity to actin filaments in peripheral stress fibers than those in central stress fibers. A previous study reported that actin binding of transgelin 1 is negatively regulated by phosphorylation of serine residue Ser181. Constructing transgelin 1 phosphomimic and nonphosphorylatable mutants, we evaluate the effect of phosphorylation of Ser181 in transgelin 1. Time constants of recovery curves were not significantly different among transgelin 1 mutants. The results indicate that the phosphorylation at Ser181 does not affect transgelin 1 dynamics.

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Morphogenesis in multicellular organisms is accompanied by apoptotic cell behaviors: cell shrinkage and cell disappearance. The mechanical effects of these behaviors are spatiotemporally regulated within multicellular dynamics to achieve proper tissue sizes and shapes in three-dimensional (3D) space. To analyze 3D multicellular dynamics, 3D vertex models have been suggested, in which a reversible network reconnection (RNR) model has successfully expressed 3D cell rearrangements during large deformations. To analyze the effects of apoptotic cell behaviors on 3D multicellular morphogenesis, we modeled cell apoptosis based on the RNR model framework. Cell shrinkage was modeled by the potential energy as a function of individual cell times during the apoptotic phase. Cell disappearance was modeled by merging neighboring polyhedrons at their boundary surface according to the topological rules of the RNR model. To establish that the apoptotic cell behaviors could be expressed as modeled, we simulated morphogenesis driven by cell apoptosis in two types of tissue topology: 3D monolayer cell sheet and 3D compacted cell aggregate. In both types of tissue topology, the numerical simulations successfully illustrated that cell aggregates gradually shrank because of successive cell apoptosis. During tissue shrinkage, the number of cells in aggregates decreased while maintaining individual cell size and shape. Moreover, in case of localizing apoptotic cells within a part of the 3D monolayer cell aggregate, the cell apoptosis caused the global tissue bending by pulling on surrounding cells. In case of localizing apoptotic cells on the surface of the 3D compacted cell aggregate, the cell apoptosis caused successive, directional cell rearrangements from the inside to the surface. Thus, the proposed model successfully provided a basis for expressing apoptotic cell behaviors during 3D multicellular morphogenesis based on an RNR model framework.

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The strategies to regulate cell differentiation, growth, and proliferation are fundamental for tissue engineering in regenerative. Our aim here is to provide a basis for noninvasive and stable control of these cellular processes by clarifying the effect of extracellular microgrooved structure. We fabricated a microstructured cell culture substrate consisting of a major groove to guide a cell body penetration and the branched grooves to guide cellular protrusion into them. We seeded the cells on the substrate and evaluate the effect of the microgrooves on the cellular distribution of actin and phosphorylated myosin. About a half of the cells penetrated into the grooved structure, whereas the other cells were grown on the grooves. In both of the two groups, the cells show various morphologies. A common feature in the penetrated groups was disappearance of the stress fibers. Furthermore, these cells had a tendency to form thin actin protrusions without myosin, which is assumed to be filopodia, into the branched grooves. The result of the observation of the phosphorylated myosin together with the actin filaments indicates the downregulation of Rho GTPase in the cell body in the major groove and upregulation of cdc42 neat the base of the branched groove. Based on the findings, the microgrooved structure is expected to be used for external control of cellular distribution of actomyosin by affecting small GTPases.

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Cell traction force (CTF) is important in many aspects of cellular activities such as migration, tensional homeostasis, and shape change. Traction force microscopy (TFM) has been used widely to measure CTF. However, this method is not sutable for the cells on stiff substrates, i.e., those with physiological stiffness. Towards estimation of CTF of cells on stiff substrate, we investigated the relationship between CTF and retardance, phase shift of polarized light when it passes through birefringent material, because birefringence may change with internal stress. Vascular smooth muscle cells were cultured on a glass bottom dish and treated with calyculin A and Y-27632 to increase and decrease, respectively, CTF. Cell retardance was measured with a birefringence imaging system, pol-scope, and normalized by the retardance at 0 min. Retardance increased and decreased significantly in cells treated with calyculin A and Y-27632, respectively, compared to control cells 50 min after administration of the reagents. When measurement of cell retardance and TFM were performed simultaneously on a single cell cultured on a polyacrylamide gel substrate with elastic modulus of 15 kPa, retardance correlated significantly with CTF. These results may indicate that retardance can be used to estimate CTF of cells cultured on substrates with physiological stiffness.

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We have developed a novel mesh culture method for fabricating monolayer cell sheets by culturing cells on microstructured mesh sheets suspended in a culture medium as substrates for cell attachment and growth. In this study, we evaluated cell response to mesh shape and cyclic stretching using UV-PDMS meshes microfabricated by photolithography. Using a mesh sheet with a diamond lattice, we successfully generated cell sheets of mouse C2C12 myoblasts with cells oriented primarily in the direction of the longest axis of the diamond shape in such a manner as to maximize tension in the actin stress fibers that determine cell shape. Application of unidirectional cyclic stretching (10% strain, 0.5 Hz) induced reorientation of on-mesh cell masses in the direction orthogonal to strain direction and also elicited repeated calcium responses. This suggests that although cell-substrate interaction is limited in the case of mesh culture, cells are still capable of responding to substrate stimuli and propagating these to elicit tissue-level response. Thus, this study highlights both the geometrical sensing capability of cells and mechanosensing under limited adhesion condition. Future work will consider the mechanism of function realization by in vitro fabricated naive tissues as a result of mechanical stimulation.

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We produced a cell culture platform with magnetically deformable structures that can apply mechanical stimuli to cells, and we confirmed that we can culture the cells on our platform. We used a shape magnetic anisotropic torque which causes a thin soft magnetic material (ex. permalloy) to stand on a permanent magnet. The magnetically deformable structures consist of a permalloy thin plate covered with polydimethylsiloxane (PDMS), and a double-stick tape was used for adhesion between PDMS and the permalloy. Our platform enables various stimuli such as bending, stretching and torsion. Since magnetic actuation is a non-contact actuation, a pneumatic tube or electric wiring is not required in a petri dish. We used a frog (Xenopus laevis) kidney-derived A6 cell line for cell culture on our platform. After the cell culture of A6, we performed LIVE/DEAD assay to check viability of the cells on our platform. We confirmed that 97% of cells were alive after the culture for 24 hours. This result shows that it is possible to culture cells on our platform which was made of PDMS sheet, permalloy sheet and double-stick tape.

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As regarding cellular differentiation, it has been found that elasticity of extracellular matrix determines differentiation lineage of mesenchymal stem cells (MSCs). The direct quantitative measurement of the mechanical interaction between the MSC and the matrix for differentiation, however, has not been performed. In the present work, the displacement field of the cell-adhesive matrix was observed quantitatively using digital volume correlation (DVC) method. In practice, the maximum displacement and cellular traction stress were analyzed when the MSC differentiated into neuron or osteoblast on the soft or hard elastic matrix, respectively. Then, function of non-muscle myosin II (NMM II), which plays an important role in intracellular cytoskeletal dynamics, was investigated in cellular differentiation. As a result, the mechanical interaction (maximum displacement, subjected area of the matrix, and traction stress) between the cell and the matrix was dependent upon the elasticity of the matrix. Additionally, it has been shown that mechanical interaction between intracellular cytoskeleton and cell-adhesion matrix is indispensable for cellular differentiation.

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Focal adhesions (FAs) are mechano-sensitive elements that mediate cellular response to applied forces and scaffold material properties. The microstructure of FAs has been investigated with several approaches that include super-resolution fluorescence microscopy, cryo-electron tomography, and atomic force microscopy (AFM), but the contribution of α-actinin to forming the architecture remains unclear. Here we combine AFM with fluorescence microscopy to investigate the 3D geometry of FAs together with observation of fluorescent α-actinin expressed within cells. A7r5 cells, an embryonic aortic smooth muscle cell line, are cultured on a glass-bottom culture dish and de-roofed with a hypotonic treatment to allow their FAs to be accessed by an AFM probe. Our observations show that α-actinin-1, one of the isoforms expressed at FAs within the cells, contributes to layering of actin filaments toward the direction of the height rather than the cross-linking of individual actin filaments within planes.

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Focal adhesions are force-sensitive, multiprotein complexes that anchor cells to the extracellular substrate. Previous studies have revealed the composition, ultrastructure, and dynamics of focal adhesions, and how they change upon intracellular and/or extracellular forces. However, little is known regarding how focal adhesions sense the geometry of the extracellular substrate. Here we develop a novel traction microscopy that allows for simultaneous evaluation of the cellular forces and the effect of local substrate curvature, i.e. how the substrate is curved out of the plane to have a 2.5D surface. While it is well known that force-induced elongation of focal adhesions occurs on a flat surface, here we interestingly found that the dynamics of individual focal adhesions is abrogated by a certain level of local substrate curvature, or 2.5D substrates. Thus, further development of focal adhesions is allowed only when they retain a straight shape on a planar surface.

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During brain development, neural stem cells define specific niches within different cortical layers to control their cellular environment. Generally, communication to other cells or surrounding matrix by biochemical signaling pathways such as protein-protein interactions or soluble factors have been well studied. Much less studied, however, is how physical properties of the niche can influence behavior, growth, and differentiation of cells. My previous work revealing the role of a physical crowding effect on interkinetic nuclear migration, an oscillation of nuclear positions of neural progenitors associated with the cell-cycle, led us to investigate the intimate physical interactions between cells and the surrounding tissue. Among physical properties, elasticity of the matrix has been shown to have direct effects on fate determination in certain cell types in vitro. While some studies indicate that elasticity may influence the fate of cultured neural stem cells by unknown mechanisms, no evidence exists on whether this principle holds true during the physiological development of the mammalian brain. Using the developing brain as a model system, we have started clarifying roles of tissue elasticity for determining cell fates of neural cells via mechanosignaling pathways. The research aims to establish a novel concept for mammalian neurogenesis and brain development, and the outcomes will likely influence the fields of stem cell and developmental biology by clarifying unknown mechanosignaling mechanisms of somatic stem cells.

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We previously demonstrated monitoring and imaging of intracellular temperature based on a fluorescent polymeric thermometer and quantitative fluorescence imaging techniques, showing non-homogeneous temperature variation in steady-state and unsteady state living cells. Here, we have investigated intracellular thermogenesis to understand the physiological significance of temperature change on cell functions. By heating cells with an external infra-red laser, we were able to provoke quantitative heating of localized area (〜1 μm^2) of cells. This method of manipulating intracellular temperature allowed the observation of heat diffusion in living cells and cell response to heat shock. In particular, we have investigated intracellular temperature change in stressed cells and revealed that local thermogenesis in cells serves to initiate stress granule (SG) formation, where mRNAs are accumulated and translation is repressed. Our results of imaging and manipulating intracellular temperature unveiled a novel principle of cell biology that intracellular local temperature change drives cell functions.

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Angiogenesis plays an important role in maintenance of homeostasis, such as tissue growth, or tissue regeneration. Endothelial cells (ECs), which have a key role in angiogenesis, are exposed to the increased pressure under several health-maintenance conditions. However, the relationship between angiogenesis and the pressured condition is still unclear. Here, we report a phenomenon in which hydrostatic pressure promotes angiogenesis. We show that pressure exposure induces transient phosphorylation of extracellular signal-regulated kinase (ERK) in ECs. Phosphorylated ERK translocates from cytoplasm to nucleus, and leads to EC tube formation. Inhibiting the phosphorylation of ERK, we confirm pressure-induced transient ERK phosphorylation is essential to endothelial tube formation.

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The heart is capable of remodeling in response to hemodynamic stress. However, molecular details of mechanical responses in cardiomyocytes remain unclear. Transient receptor potential vanilloid family type 2 (TRPV2) is stretch-activated Ca^<2+> channel and localized to intercalated discs in mammalian cardiomyocytes. In this study, we show that mechanical signaling via TRPV2 in cardiomyocytes is pivotal for the maintenance of cardiac function in adult mice. Stretch-induced insulin-like growth factor (IGF-1) secretion was significantly reduced in TRPV2-deficient cardiomyocytes isolated from neonatal mice. In addition, the IGF-1 receptor/PI3K/Akt signaling pathway was significantly down-regulated in TRPV2-deficient adult hearts. Furthermore, administration of IGF-1 to TRPV2-decieinet adult hearts partially prevented impairment in cardiac pump function. These results suggest that TRPV2 regulates IGF-1/PI3K/Akt signaling pathway, which is required to maintain cardiac function in physiological hearts.

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We estimated stress distribution in fresh Xenopus laevis embryos at tailbud stage by measuring surface topography and stiffness distribution on a section of embryonic tissues. When an elastic body having residual stress is cut, pops and dents appear on the section according to the direction and magnitude of the residual stress. Stress distribution necessary to restore the section to flat plane is considered to be the residual stress distribution. In reality, however, fresh embryo is very soft and sticky, and its contents flow out easily after being cut. To overcome these problems, we constructed a special setup in which tailbud embryos were cut perpendicular to the median line in Steinberg's solution with fine platinum wire electrified with high-frequency current and topography of the section was measured within 30 s with a laser-scanning microscope. We found that three sites of the section, i.e., neural tube, notochord and ventral tissue, protruded by 40-80 μm from surrounding tissues at stages 33-34. We then developed an indentation device to measure mechanical properties on the section under a microscope, and found that Young's modulus was 〜4 kPa for neural tube and 〜0.5 kPa for ventral tissue. Our findings suggest that neural tube, notochord and ventral tissue produce the force to elongate the tailbud embryo. Multiple elongation sites may prevent the embryos from bending during body elongation process that might occur when the body elongation was driven by a single site.

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Mechanical vibration at low amplitude and high frequency is a promising method to prevent osteoporotic bone loss. However, optimal conditions of the vibration have not been fully elucidated. Therefore, the aim of this study was to clarify the optimal amplitude and frequency that enhances bone metabolism. Osteocytic cell line MLO-Y4 cells were subjected to vertical vibration at magnitude of 20-100 μm and frequency of 10-100 Hz. Secretion of nitric oxide (NO) from the cells was evaluated using fluorescent indicator, DAF-2DA. Quantitative comparison of fluorescent intensity showed that NO secretion was significantly increased when the cells were exposed to the vibration at the magnitude of 40-100 μm and the frequency of 20-100 Hz. The most effective conditions of mechanical vibration were 60 μm in amplitude and 20 Hz in frequency.

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Vascular smooth muscle cells (SMCs) are suggested to make functional changes under shear stress (SS) stimulation through intercellular communication with endothelial cells (ECs). In the present study, we constructed an EC-SMC co-culture model with phenotype-controlled SMCs, which is similar to normal healthy arterial walls, and then explored relationship between EC gene (TGF-β1) expression and SMC phenotype change under SS conditions. The result shows that the expression of contractile proteins in SMCs were at same level under 0.2, 2,6 or 10 Pa when TGF-β1 expression from EC was knocked down by SiRNA. In our previous experiment, we have reported that a low SS (0.2 Pa) or a high SS (10 Pa) could suppress the expression of contractile proteins in SMCs in a co-cultured model, therefore the present result suggests that TGF-β1 expression from EC could influence SMC response to SS.

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We investigated the effects of nuclear deformation on the physiological function of vascular smooth muscle cells (SMCs) using polydimethylsiloxane (PDMS)-based microfabricated substrates with an array of micropillars. SMCs spread normally in the space between micropillars and completely invaded the extracellular microstructures, including parts of their cytoplasm and their nuclei. We found that not only the proliferation and migration of SMCs but also their contractile protein expression was dramatically inhibited by cultivation on the micropillar substrates. A detailed image analysis with confocal microscopy revealed that expression of lamin A/C was significantly decreased in the region deforming along the pillar surfaces, and underlying DNA distribution became more heterogeneous. These results may indicate that lamin A/C has a role of mechanosensor to detect an excessive deformation of nucleus, and they switch the cell state from an "active phase" to a "resting phase".

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We numerically investigate the velocity of an adherent cell in various sizes of microchannels. The velocity drastically decreases as decreasing the size of microchannels. When the channel size becomes smaller, the motion of the adherent cell changes from a "rolling motion" to a "bullet motion", where the cell rotates on side wall in rolling motion, while the cell adhere its circular arc to the wall in bullet motion. Larger adhesion force is generated in rear parts of rolling cell, and existed bonds experience rupturing with higher probability because of slip bond. Because cell cannot move forward unless the ligand-receptor bonds in rear parts rupture, frequent rupturing allows the rolling cell to move faster than the cell exhibiting bullet motion. As getting smaller in channel diameter, the surface area attached to the wall is larger and then the number of ligand-receptor bonds is larger for smaller microchannels, resulting in a lower velocity. Our numerical model allows us to investigate the effect of various parameters on adherent cell velocity.

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Relative decrease in the blood donor population caused by a rapidly declining birthrate and aging society has brought on a chronic shortage of preserved blood. Taking countermeasures against these problems, various types of hemoglobin based oxygen carriers (HBOCs) are under investigation. Among them, the development of the hemoglobin vesicles (HbV) is being vigorously promoted in Japan and has been recognized as a useful alternative for red blood cell (RBC). HbV encapsulate concentrated Hb solution in phospholipid vesicles, and measures approximately 200-250 nm in diameter, which is roughly 1/30 size of a human RBC. Therefore, HbV will easily transport oxygen through a narrowed microvessel whose diameter is smaller than RBC. In addition, flexible adjustment of HbV performance parameter permit effective transport of oxygen to ischemic region. However, it is difficult to determine experimentally the effective performance parameter according to the various circulatory disease. Based on these backgrounds, in this study, we constructed numerical model to investigate the oxygen delivery process of HbV in microvessel including RBC. The final aim of this study is to design numerically effective HbV for various circulatory disease.

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Thrombus often results in severe disease in heart or brain In order to develop effective prevention or treatment for thrombus, it is necessary to reproduce thrombus formation numerically and clarify the detailed mechanism of platelet activation. In this paper, therefore, numerical modeling for platelet activation process is proposed and the coupled analyses with advection-diffusion of ligands are demonstrated. The model is roughly divided into two parts, advection-diffusion of ligands in blood flow and platelet activation. The platelet activation process is simplified and consists of ligand-receptor bonding, increase of concentration of calcium ion in a platelet, secretion of ligands, GPIIb/IIIa activation, and chain-reactions of these processes. Numerical results show time course of advection-diffusion of ADP in blood flow, ADP-P2Y1 and ADP-P2Y12 bonding, successive secretion of dense granule from activated platelets, and the promotion of the activation of surrounded paltelets by secreted ligands.

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A backward facing step flow was numerically simulated in two dimensions using a differential model of the LSMPS method which is a higher-order version of the MPS method, aiming at a highly accurate fluid analysis in a particle method simulation of thrombus formation. The differential model was applied to the viscous term of Navier-Stokes equation. Reynolds number was set in the range of 50 to 400. A resolution was 26 particles in a width direction of an inlet. As a result of simulation, numerical accuracy with respect to both velocity distribution of plane Poiseuille flow and reattachment length was improved from that calculated by an original MPS method. This was mainly due to an improvement of accuracy about viscous term calculation nearby channel walls.

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We performed a computer simulation for effects of an anticoagulant agent on thrombus formation under the influence of the blood flow, assuming a rat arteriovenous shunt in which a nylon filament was inserted. A blood model consisted of a normal blood and a thrombus, and they were expressed by an assembly of particles. A normal blood particle close to the nylon filament was changed to a thrombus particle when the shear rate was lower than a threshold value. An anticoagulant effect depending on a drug concentration inhibited changes from normal blood particles to thrombus ones. As a result of computer simulation, thrombus was formed with a thickness from 0.1 mm to 0.4 mm around the nylon filament. Thrombus weight decreased with an increasing dose, which was qualitatively consistent with an experimental result, while, thrombus weight in the simulation was approximately 15-20% of that in the experiment. It is necessary to improve the simulation model toward quantitative identification of an experimentally observed anticoagulant effect.

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We developed a numerical method for mass transfer across membranes with permeability. In the proposed method, the non-conforming mesh for concentration fields with respect to the membrane shape is used to handle the multiple membranes and large deformations of the membranes easily. The proposed method includes two characteristics. One is finite element discretization including the jump values at the membrane which is capable of capturing the discontinuity sharply in one element. The other is an implicit treatment of the concentration jump which is capable of treating both the permeability and non-permeability of the membrane. The method is applied to some problems of the mass transfer by a membrane, and validity and applicability of the proposed method are demonstrated.

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This study shows a novel numerical approach for estimation of the cell traction force on a soft substrate, appeared in the cell traction force microscopy. A depth expansion technique using the Legendre polynomial is applied for expression of a displacement distribution and deformation in a depth direction and coupled with the finite element formulation applied for an in-plane deformation. Numerical results converge to a reference solution as an increase of the expansion number of the Legendre polynomial and are compatible to the reference even with using a less series expansion number, e.g., 2. This fact indicates a computational cost in an inverse estimation of the cell traction force is more improved by using the present model even in a 3D analysis.

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