After the total knee arthroplasty, it is important to evaluate between the patella component and the femoral component, because patella components affect the flexion angle. There are many types of artificial knee joint. Among them, we focused on the artificial knee joint which lateral and medial condyle of femoral component is asymmetry. The purpose of this study was to investigate the mechanisms of contact between the patellofemoral joint. Femur and tibia were created by aluminum alloy. The implanted knee model was constructed to replace the posterior-stabilized artificial knee joint which lateral and medial condyle of femoral component is asymmetry to them. Artificial ligaments were used as quadriceps model and patellar tendon model. The contact pressure were measured at seven flexion angle (90 degree, 100 degree, 110 degree, 120 degree, 130 degree and 140degree) under the applied loads of 1000N. In result, the contact pressure was considered to increase with rising flexion angle, but such a phenomenon was not showed. Lateral contact area showed higher values than the medial contact area significantly in all flexion angle. But, in the average pressure and max average pressure, there were case where the medial is higher than lateral. Understanding the advantage and disadvantage of the patella component function may help to develop sophisticated component design.

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Impingement problem of femoral component and polyethylene insert after total knee arthroplasty (TKA) has been recently brought to light by contact pressure analysis using the knee joint simulator. Therefore, adequate motion analysis is important for estimating the impingement between components and improving postoperative exercise. The purpose of this paper is to analyze the impingement of TKA components during stair claiming activity in vivo. Ten patients with posterior-stabilized (PS) TKA were examined in stair claiming activity. The 3-D position and orientation of the implant components were determined using image matching technique. The 6-DOF of each component was able to be reproduced on 3-D CAD. Kinetic relation between the femoral component and polyethylene insert was calculated, and then the impingent was analyzed. In the result, impingement of the femoral intercondylar notch and anterior aspect of the tibial post was observed in extension of stair claiming activity. This study helps the surgeon be more aware about impingement between components under some conditions and is useful to improve the design of artificial knee joint.

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Quantitative evaluation of the fixation of acetabular component(cup) in pelvis is important for the clinical fixation of total hip arthroplasty(THA). The cup stability is mainly caused by the compressive stress from the inserted acetabulum, in the case of press-fit type THA. Direct measurements of the compressive stress acting between the trial cup and bone is expected to be useful for evaluation of initial stability during operation of THA. In this study, we proposed a measurement system of contact pressure distribution between the cup and the reamed acetabulum by means of a thin and flexible contact pressure sensing system. The pressure sensor unit with 78|im thickness was sealed with a polyimide film, and attached at the outer surface of the trial cup of 50mm diameter. Three bone models were drilled by hemispherical reamers 50mm, 51mm and 52mm in diameter, respectively. The contact pressure distributions were measured when the trial cup was manually inserted into the bone model. The contact pressure was measured as larger values in the acetabulum with 50mm diameter than in the 51mm and 52mm diameter. The sensing system could obviously show the differences of fixation conditions as the differences of pressure distributions with quantitative values. The system is expected to use in the intraoperative evaluation of stability during THA operations.

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Impulsive stress wave propagating from the distal end of femur was analyzed by the dynamic finite element method. In this study, two types of three-dimensional finite element model of the femur, namely the models of the femur and that after the total hip arthroplasty were prepared. The influence of the impact angle considering walking or going up and down stairs on the stress wave propagating in the femur was discussed in the present study. The propagation of the stress wave in the femur was influenced by the insertion of the femoral stem. The stress wave with large absolute value of principal stress was not found in the proximal femur around the femoral stem, and the maximum principal stress increased on posterior side of the femoral mid-diaphysis when the angle between the bone axis and the impact direction in the sagittal plane was 60 degree. As the impact angle increased, it seems that the stress wave was affected by the impact bending.

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In total hip arthroplasty, the cementless femoral stem implants have been used. Initial stability of a cementless femoral stem depends on the mechanical interlocking of the stem by press-fitting into the femoral canal. The movement of the stem relative to the femur occurs when the implant is subjected to a load. The inducible movements at the implantbone interface is defined as micromotion. It has been suggested that the use of a short stemmed implant could conserve proximal femur and reduce the extent of proximal stress shielding. However, it has little reports on the primary stability of the short stemmed implant. The aim of this study is to examine the primary stability of the short stemmed implants using the finite element analysis. The finite element model of the short stemmed implant inserted into the femur with the same alignment as the standard stemmed implant was created to compare the primary stability between the short stemmed implant and the standard stemmed implant. The maximum load during walking was applied to the stem. The micromotion was examined at both the short stemmed implant and the standard stemmed implant.

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Osteosynthesis by the use of "Mayo Clinic Congruent Elbow Plate System" in combination with "Locking Screws (LS)" or "Non-locking Screws (NLS)" was applied to the distally fractured model humeri made of polyurethane. Quasi-static compression tests on the model humeri were then performed in order to clarify their mechanical performance with respect to initial fixation. No significant difference in stiffness was found between the model humeri with NLS and that with LS. All model humeri were able to support the compressive load of 1600N at minimum independently of the type of screws, and therefore it can be concluded that the plates and the screws tested in this study provide the excellent initial fixation for distal humerus fracture.

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A method for estimating three dimensional (3D) position of knee joint in vivo was presented. Volume data of a knee joint were generated using magnetic resonance imaging (MRI) scanning of the knee. The volume data was rendered as the mean voxel values along the ray drawn from the focal point to each pixel of the image plane. The 3D positions of the femur and tibia were recovered by matching the rendered image with calibrated bi-plane x-ray images of the knee. To validate this method, in vitro experiments were carried out using a cadaveric knee. To define the local coordinate systems, 4 polymeric markers containing contrast media were attached to the femur and the tibia, respectively. The knee underwent MRI scan to generate volume data. Then, bi-plane x-ray images of the knee were taken at 2 different flexion angles. The relative position (6 degree-of-freedom parameters) between the femur and tibia was compared with that measured using a 3D coordinate measuring machine. The maxi-mum errors of the rotation and translation parameters were about 3.2° and 10.9 mm.

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The technique to estimate three-dimensional position and posture to elucidate the mechanism of a joint in the knees is effective. As one of the technique, the Template Pattern Matching (TPM) method based on 3D bone data is known. A prepared library based on 3D bone data is necessary for the TPM method. Our final goal is to alter the library,3D bone data constructed from simple X-rays image. In this study, proposed method were adapted to tibia and fibula. Tibia and fibula bone model comprised of voxel are prepared. Tibia and fibula bone model are transformed based on X-rays image respectively. When bone models were transformed, there was often a gap between two models. The two models were connected by new voxels. As the result posture estimate precision was θx=1.50 ± 1.29 deg, θy=0.98 ± 0.95 deg. Posture estimate precision of the conventional method with CT data was θx=l.05 ± 1.05 deg, θy=0.25 ± 0.51 deg. θx was inferior than θy to conventional method. The result suggests that the sign of 0k cannot be determined from extracted outlines.

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Many of the 2D/3D registration for joint use 3D bone model obtained by MRI and CT. Making a bone model only use the X-ray image (lateral view) contribute to reducing the cost and exposure dose of X-ray. We have developed the reconstruction methods of subject's 3D bone model from X-ray lateral image and prepared 3D bone model that consists of voxels. In this study, the proposal method was adopted to the cervical spine. Using the 3D bone models made from CT and 3D bone model that was reconstructed the proposed method, template pattern matching was carried out, and the accuracy of pose estimation was compared. Estimation accuracy of the proposed method was θ_x=2.60±2.00deg, θ_y=1.68±1.43deg. Estimation accuracy of the conventional method was θ_x=0.36±0.13deg, θ_y=0.83±0.44deg. Because the present method is to modify the prepared 3D bone model based on only lateral X-ray image, bone shape, especially about the depth direction, cannot exactly reflect subject's bone shape. The method can't reproduction the bone shape with a recessed shape, such as bottom of vertebral body. These problems affect the estimation accuracy.

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The purpose of this study is to elucidate of cancer growth phenomenon and to support to the development of treatment. The paper presents the development of a simulator that can be expressed in a short period of time, the tumor growth observed over long time and difficult. In order to tumor growth, it is necessary to produce new blood vessels to supply nutrients. Tumor angiogenesis has stimulated the production of vascular endothelial growth factor by cells. In this way, there is a close relationship between tumor growth and angiogenesis. In this study, we analyze for cancer growth and angiogenesis using the particle model. Particle model is analysis method that particle is treated as cancer cells or blood vessels, phenomenon is expressed by the interaction of the particles. We study necrosis that occurs in tumor inside.

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In this study, the indentation tests of soft materials were performed to predict the internal distributions of the materials which are assumed as the human soft tissues. These results of tactile tests were applied to the finite element analysis which is combined with the genetic algorithm search technique. In the results of these trials, the predicted distributions and the size of the foreign particles by the simulations have good agreements with the specimens in especially using the hyperelastic definitions.

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In order to investigate the human body by palpation, three-dimensional evaluation is useful for precise diagnosis. Here, the result of the systems development for this palpation is reported for realization of the threedimensional evaluation. This system is developed using the 3DOF desktop robot. For palpation, a tactile force sensor was adopted as a probe of this robot. The tactile data can be measured as 3D forces by using this sensor. The measured force is analyzed using the extended Hertian contact theory, and will be applied to investigate the state of human body. The hardness distribution inside any organization can be inspected by this result.

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The morbidity rate of arteriosclerosis is high in cardiovascular disease. The diagnosis and operations method of the artery diseases generally have been done by the feeling and experience of medical doctors. The highaccurate identification of the diseased part of artery is important in the excision and substitution operation of the hardened artery. Then, the establishment of the method which can identify the diseased part of the artery is needed for the high-accurate operation. This identification method is developed by haptical technology based on the mechanics of palpation. The mechanics of the palpation is analyzed using Hertian contact theory and derives the objective data of the artery stiffness simply by pinching the artery with the probe imitating human's fingers. Here, the pinching method on the identification process is modified by the pulsatile blood pressure for the realization of in situ observation. The developed method is verified by using an artificial system of circulatory organ, which was newly developed for this research. The introduced technique was validated by the comparison among some results measured by different method, and then its usefulness was verified.

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The concentration of platelets in flowing blood is known to attain maximum near the vessel wall, and this phenomenon is referred to as "Near Wall Excess (NWE)". Although NWE is evidently important and expedient in hemostatic functions of the platelets, its mechanism has not been fully elucidated yet. In order to understand NEW from a mechanical point of view, we measured the distribution of platelet-sized latex beads in a mixed suspension with human red cells flowing through microchannels, by using a confocal laser scanning microscope. The results indicated that the distributions of beads of various diameters were nonuniform in the presence of red cells, whereas their distributions in the absence of red cells were uniform in the channel cross-section. In the mixed suspensions of beads and red cells, the concentration profiles of beads of 3μm diameter showed sharp peaks near the channel walls at hematocrits of 20% and 44%, and the extent of this NWE was larger for larger hematocrits in the range examined. On the other hand, the beads of 1μm diameter exhibited NWE only slightly at similar hematocrits, and instead, they showed a distinct tendency to concentrate near the channel centerline.

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Shock wave irradiations induce intracellular Ca^<2+> increase in vitro. Shock wave irradiations also induce fluid flow, which could itself induce intracellular Ca^<2+> increase. However, it is obscure whether fluid flow induced by shock wave irradiation involves intracellular Ca^<2+> increase induced by shock wave irradiation. In this study, fluid flow nearby cells was inhibited by PDMS chamber to understand the involvement of fluid flow on the intracellular Ca^<2+> increase.

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In the present study, a novel microfluidic device capable of selectively isolating dead cells from live cells utilizing the difference in their electro-mechanical response to AC electric field was proposed. The device uses a planar electrode and a planar micro-electrode array in a separation flow channel, where the positive dielectrophoretic (DEP) cells are attracted toward the high-voltage micro-electrodes while the negative DEP cells are concentrated in the central streamline in the flow channel. The effect of different voltages and flow velocities in the flow channel on the device performance was investigated numerically. Molecular dynamics (MD) simulations based on the Langevin equation of particle kinetics were used to compute the trajectories of individual cells under the action of DEP, dipole-dipole intercellular, viscous, and gravitational forces in suspension media. It was found that this devise was able to achieve greater than 50% removal efficiency of dead cells at an applied voltage of 10V and 0.1 mm/s flow velocity.

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Augmented dispersion in the trachea during high frequency oscillation is known to be mainly due to an interaction of axial convection and lateral mixing. Visualizing experiments of intermittent oscillatory flow and sinusoidal oscillatory flow using water dynamically similar to air through a straight pipe or a pipe with circumferential grooves have been carried out and the velocity distributions during high frequency oscillation have been obtained by means of the 2 dimensional PIV flow analyzing system. The RMS radial velocity during the stationary period tends to decrease gradually in the case of intermittent oscillatory flow in a pipe with circumferential grooves. Results show that the obtained correlation coefficient between the effective diffusivity and the mean values of the RMS radial velocity during the stationary period for same conditions are very strong.

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The purpose of this study is to compare aerodynamic characteristics of flight motion between rigid and elastic wing. Especially, in Elastic wing we investigate about wing of partially different Young ' s modulus. In order to this investigation, we used Fluid-structure interaction (FSI) analysis. In FSI anaiysis , fluid analysis employ finite difference method (FDM) and structure analysis employ finite element method (FEM). Computation of flapping wing motion are performed for various Young ' s modulus condition and Poisson's ratio.

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The present study is aimed to investigate if aerodynamic valving works in the junction between the primary bronchus and vertebral bronchi in a real avian lung during inspiration. Airways of a male quail were scanned with a micro CT, and airways, such as the primary bronchus and vertebral bronchi, were reconstructed from CT images. Flow in the airways was numerically solved under a condition of all outlets having the same pressure. Results showed flow separation at the first junction between the primary bronchus and the ventrobronchus due to the constriction located just proximal to the junction. The separated flow caused formation of vortices in the root of vertebral bronchi after the peak of an inspiration phase. They worked like a lid helping prevent air from going into the ventrobronchi and directing it to the mesobronchi. It was also shown that an increase in the acceleration of inflow induced quicker formation of vortices, and resulted in leading more air volume to the mesobronchi.

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Many sperms use flagella to swim, and human sperm use it as well. However, the detail mechanism of the flagella movement is not clear yet. In the previous experiment, the effect of viscosity on the motion characteristics of the bovine sperm has been considered, whereas, the viscosity using in the experiment was much lower than that in the actual oviduct. The sperm should evolve to the shape that is appropriate to the environment of the actual oviduct. In this study, therefore, we conducted the observation of the sperm motion in various viscosities. We used PVP-K90 which is the Newtonian fluid, and five types of concentrations, 0.0, 1.0, 2.5, 4.0 and 6.0 [%] were selected by measuring the viscosity. The process of the experiment is the following. First, we investigated the viscosity of the reagents using a rheometer when temperature is 38.5 [℃]. Second, we centrifuged the bovine semen, and removed the seminal plasma from the semen. Third, we mixed 0.1 [ml] sperm with a buffer solution. Fourth, we observed the mixed solution with the microscope, and recorded a movie. Finally, we conducted the PTV analysis using the image analysis software (DIPP-Motion Pro, Ditect Co.), and calculated the following parameters; the straight-line velocity, the sperm velocity, and the amplitude of the sperm head. We obtained some useful results from the experimental data. First, as the viscosity increased, the motile capability of the sperm reduced. Second, as increasing in the viscosity, the sperm with no-rotating head increased, and the no-rotating sperm had lower motile capability than the rotating one. These results suggest that the sperm swim without rotating head in the actual oviduct.

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The Plasmodium of Physarum polycephalum is a unicellular and multinuclear giant amoeba. The cell body of the Plasmodium roughly consists of two parts, namely, sheet-like locomotive front and tubular rear part. The tubular structure of the Plasmodium is a kind of transportation network, and some studies have already been done on the structure and function of the plasmodial network. However, there are some difficulties in observing the minute structure of the plasmodial network. We thus developed a new method for the observation of the plasmodium that is a macroscopic implementation of dark-field microscopy.

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The control of ice crystal growth has been an important issue in various fields. The adding of antifreeze protein (AFP) to food or organs has been focused on recently because it is appropriate for the controlled ice growth. The present authors have paid particular attention to the effects of an electric field on the freezing solution of ion antifreeze protein. Experiments were conducted using for aqueous solutions of winter flounder antifreeze protein and sodium chloride stored in a gap between two cover glasses. The dc field was given to the solution. The temperature at the ice/solution interface was measured with a fine thermocouple. The interface velocity was measured with successive images captured with a video camera. It turned out that the interface shape was changed by the electric field. The interface velocity and the supercooling degree were decrease by the electric field.

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Lignocellulosic biomass is expected to be utilized as one of the renewable energy. Lignocellulosic biomass can produce bioethanol from a series of processes; pretreatment, enzymatic hydrolysis and fermentation. However, fermentation inhibitors are generated during the process of pretreatment. Fermentation inhibitors which include Furfural, 5-Hydroxymethylfurfiiral (5-HMF), Acetic acid, Formic acid decrease the rate of growth of yeast. In this study, we analyzed the effect of these inhibitors on yeast growth. Moreover, in research on ethanol production from lignocellulosic biomass, the enzymatic Simultaneous Saccharificiaton and Fermentation (SSF) is desirable method in practical application. However, the optimum temperature of enzymatic saccharification is different from that of yeast fermentation. Therefore, we tried to analyze the effect of temperature on yeast growth. As a result, the growth rate at 40 ℃ and 45 ℃ was lower than that of the other temp. We also analyzed the effect of contact area of the air. We changed the size of flasks from 100 to 500 mL and measured of growth rate of yeast. The surface area is no effect on growth rate of yeast.

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Intracellular pH is an important measurement of cell activity. Among pH measurements, fluorescence measurement is generally used because of the advantage of non-contact, real-time and two-dimensional distribution. The other hand, this measurement itself is very delicate and tough because fluorescence is essentially the light with extremely weak intensity. In this measurement, to avoid the influence of fade on measuring accuracy, fluorescence pH ratiometry was proposed and frequently used. However, in applying this method to plant cell in accordance with the protocol of reagent company, the pH value varied widely even in same cell in the stage of calibration between pH and intensity ratio. This research was carried out to establish the practical procedure for obtaining the result with the higher reliability. Here, Allium cepa was used for plant tissue. BCECF-AM was used for pH-indicator. The experiment was made using the cryomicroscopy having cooled CCD camera. First, to decide the conditions of obtaining sufficient image to be analysed, the calibration was made many times with varying the reagent concentration, incubation-time in addition to pH concentration. Next, we examined the distributions of luminance and fluorescence intensity ratio in detail using the good images to understand the causes of scattering of value respectively. Through above discussions, the procedure to make the smallest scatter of data was found out. Further, we applied this established procedure to the plant tissue which was in cooling process to understand the difference in pH-change due to cooling rate.

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A method to enhancing supercooling is one of most promising techniques for cryopreservation. The object of this paper is screening of plant-derived compounds having supercooling-facilitating (anti-ice nucleation) activity. In this study, ice nucleation catalyzed by ice-nucleation bacteria (Xanthomonas campetris) was tested for six kinds of plant-derived compounds by using micro-droplet freezing assay. Firstly, the supercooling-facilitating activity was depicted for oxyresvertrol and taxifolin. Secondly, the activity of compounds does not depend on the number of hydroxyl site. It was found the activity increased with decreasing the molecular weight of compound. Thirdly, one-dimensional growth experiments were carried out to observe the pattern formation of ice/solution interface growing from taxifolin solution. The column structure of primary arm was observed in the leading front. The results suggests taxifolin prevent the specific surface of ice crystal.

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Hyperthermia and diathermy in the medical application of biothermal engineering expose cells and tissues to high-temperature stress, and damage and destroy them. Response of the cells to high-temperature stress is interesting not only in the medical application, but also thermal-biologically. This study investigated the behavior of cells in response to high-temperature stress to analyze influence of the temperature on the behavior as compared with common incubation-temperature of 37℃. Morphological change and mobility of cells were investigated by time-series three-dimensional imaging using a digital holographic microscope, which gave the thickness distribution of cells based on the information of phase difference in holography. Volume, projected area, average and maximum of thickness and position for every cell were in time-series quantified and analyzed. Therefore, the characteristic changes in cell-morphology including deformation of cells, formation of blebs from the cells and collapse of cell structure with the cell-volume decreasing, and the moving velocity of cells were revealed.

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Sugars can be a significant alternative to fossil resources, not only as biofuels, but also as hydrocarbons and starting materials for the production of polymers. Agricultural residues, forestry waste, bioenergy crops and industrial waste have potential as sources of cellulosic raw materials. Cellulose is a straight chain polymer composed of glucose monomers linked by β-1,4 glycosidic bonds. In our laboratory, a magnetically separable carbon-based solid acid catalyst has been successfully synthesized, and it can convert cellulose into glucose by the hydrolysis of theβ1,4 glycosidic bonds and decomposition of hydrogen bonded β-1,4 glucan chains. However, direct conversion from cellulose into glucose with the catalyst takes too long for industrial use. Here we show that a hydrothermal preprocessing method using mixed bubbles is effective for the hydrolysis of cellulose. The new method was more effective than the conventional method at the late stage of hydrolysis reaction.

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Techniques to control motility of the targeted cells on substrates are widely useful for biomedical applications, such as biological scaffolds, co-culture systems, and on-chip cell sorting. Our previous study has suggested a possibility of label-free control of cell motility by the micro-topographical features on the substrate. Here, we prepared the micro-structured substrates consisting of microgrooves with various sizes, and did comparative analysis of the motile behaviors of different types of cells encountering a single line groove. Although groove-size-dependence of cell motile behavior was demonstrated in Swiss3T3 fibroblasts, it was ambiguous in MCF-7 cells with dispersed cell-substrate adhesions. The ambiguous response was also shown in Swiss3T3 fibroblasts whose actin cytoskeleton and focal adhesions were disrupted by myosin light chain kinase inhibitor ML-7. In the normal Swiss3T3 fibroblasts, the groove width that was able to be crossed over by the cell was closely correlated with the distance between the neighboring focal adhesions near the leading edge of the cell. The analysis demonstrated that focal adhesion distribution near the leading edge was one of the key determinants of the motility characteristics of cells on the microstructured substrate, and thus an important factor in designing the functional microstructured surfaces for cell motility manipulation.

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This study aims to make clear the effect of directional microstructure of a scaffold on the cell adhesion force. Grooved scaffold was produced by combining lithography and casting of polydimethylsiloxane (PDMS), of which pitch was 1-6 μm and height 0.4-1.1 μm. PC12 cells were cultured and fixed on the scaffold. The cultured cells were peeled-off with a cantilever fixed on a micromanipulator. The adhesion force was quantified by analyzing the deflection of cantilever based on microscopic images captured during peering-off action and the effect of the structural directionality was discussed.

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Previous studies in direct co-culture system showed that cell-cell interactions promotes ECM expression and cell alignment. However, the effect of cell-cell interaction in direct co-culture system has not been fully understood. In this study, we developed a novel direct co-culture system to study the effect of cell-cell interaction between human bone marrow mesenchymal stem cells (hBMSCc) and human articular chondrocytes (hACs). We observed that direct co-culturing produced more aggrecan expression than mono-culturing or indirect co-culturing. The experiment results showed that the expression level of aggrecan mRNA was high in the order of direct co-culturing, indirect co-culturing and mono-culturing. In addition, the mRNA expression level in hBMSCs and hACs is clearly different in the direct co-culture system. It suggested that our direct co-culture system is possible to observe the mRNA expression for each cell after co-culturing. Therefore, we concluded that the presented co-culture system provides a simple and robust model to study the cell-cell interaction of direct co-culturing.

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This work aims to describe about effects of micro-structure on cell selective adhesion. We research to explain the effect through the use of various particles. Polystyrene and silica fine particles are hexagonally packed in 20- 50 μm line-and-space micro-structures for a cell scaffold. An incubation allows most of cells to be successfully adhered on the region of micro-structured particles, while few cells are found on the rest region of glass substrate. Micro-structure composed of 1 μm diameter is effective for selective adhesion in spite of the amount of protein adsorption is equal to 2 μm diameter.

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Intercellular tension generated by actomyosin activities is transmitted between cells and induces reciprocal mechanotransduction, resulting to many biological processes such as morphogenesis, maintenance of tissue integrity and wound healing in which dynamic cell rearrangement and tissue deformation are undergone. Adherens junctions (AJs) and its component molecules are known to not only transmit the intercellular tension but also directly contribute to the intercellular mechanotransduction. According to previous researches, a-catenin would function as a mechanosensor at AJs, which mechanically response to the intercellular tension inducing vinculin, though its mechanical behaviors still remain unclear. In this study, we analyzed the mechanical properties of a-catenin by AFM-based single molecule force spectroscopy (SMFS) and revealed that a-catenin has a high mechanical responsiveness, which dynamically changes its conformation through a specific unfolding pathway under tension.

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We studied the molecular transfer to the nucleus in an on-chip cell electroporation using focused electric field. The microdevice consisted of a cell culturing well and eight microchannels that had micro-orifices for electric field focusing and cell trapping. The micro-orifice was integrated in a vertical wall of the microchannel to visualize the process of molecular transfer with the confocal fluorescence microscopy. HeLa cells suspended in a phosphate buffered saline including ethidium homodimer and Qdot 525 was introduced into the cell culturing well and cells were trapped at the micro-orifices. Then, pulsed voltage in the range of 1-10 V was applied for 500 ms between Ag/AgCl electrodes that were inserted in the cell culturing well and the outlet of a microchannel. We visualized pore formation and nucleus membrane using the red fluorescence from ethidium homodimer. We observed the increase in the fraction of Qdot 525 translocated into the nucleus as the distance between the orifice and the nucleus membrane decreased. This finding indicated that the molecular permeability of nucleus membrane was increased by the focused electric field in the vicinity of the micro-orifice.

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Applying kinesin-microtubule (MT) system in vitro as gliding assay provides great possibility of manipulating materials at nanometer scales. Gliding directions of MTs is controlled by electrical field and charges of cargoes. Integrating these techniques, autonomous molecular separation system corresponding to the difference between charges of cargoes is expected to be designed. In order to evaluate the influence of charges of cargoes on gliding trajectories of MTs quantitatively, therefore, we measured electrophoretic mobility of cargo-attached MTs. From this measurement, we estimated gliding trajectories of MTs and evaluated these by calculating curvatures.

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Motility of bacteria is often recognized by the visual inspection through optical microscopy. It is known as something different from Brownian motion induced by thermal fluctuation. Diffusion coefficient is a typical measure for Brownian motion, but it tells only the overall diffusive speed. Therefore, another measure is necessary to detect the nature of random yet self-propulsive motion, especially when comparing the motions having the same diffusion coefficient. We propose a novel approach to detect and characterize motility, based on the large deviation principle in mathematics. Our proposed method does not require the chemical stimuli to induce chemo-taxis, i.e., the motility does not have to be in the specific prescribed direction. Furthermore, the method does not need the information of orientation, which is advantageous for the application to amoeboid cells as well. Thus, the method is applicable to various kinds of self-propulsive motion including unknown samples from environment.

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To examine the effects of intra- and extranuclear mechanical environment on cell differentiation, we investigated the changes in the morphology and mechanical properties of the nucleus, and dynamics of intranuclear DNA in Saos-2 osteoblastic cells during their calcification process. In uncalcified control cells, their actin filaments distributed entirely in the cell body. In contrast, they rearranged their location and distributed at cell periphery in the calcified cells. The nuclear area, height, and volume were significantly decreased during the calcification. In the control cells, release of the intracellular force by the depolymerization of cytoskeletons induced an increase in the nuclear height, and it induced a decrease in the nuclear area in the calcified cells. An indentation test for nuclear surface using atomic force microscope demonstrated that the nuclei did not change their average elastic moduli in the calcification process, and they tended to be uniform in their mechanical properties. Fluorescence recovery after photobleaching of GFP-labeled histon 2B revealed that the intranuclear DNA was more stable in the calcified cells than in the control. These results may indicate that the changes in compressive force applied to the nucleus arising from the rearrangement of cytoskeletons affect the mechanical environment of the nucleus, and its change may affect gene expression and DNA replication in calcification process of Saos-2 cells.

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We fabricated quartz microchambers for performing protein synthesis using a reconstituted in vitro transcription-translation system. By using glass microchambers, the yield of the green fluorescent protein (GFP) synthesis was significantly improved compared to that obtained in the widely-used poly(dimethylsiloxane) (PDMS)microchambers. Using this device, we demonstrated that GFP can be synthesized from a single copy of DNA. Quantized and distinctive signals from proteins synthesized from 0, 1, or 2 copies of genes were obtained. The microchamber presented here can be used for not only studying the effects of compartment volume on protein synthesis but also the comprehensive analysis of complex biochemical reactions in cell-mimetic environments.

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We have developed a resonant thermal sensor system for biological samples. The measurement principle relies on the resonant frequency shift of the resonant thermal sensor due to the temperature change. Brown fat cells, which are frequently used for metabolism researches, attract our interests to solve obesity, metabolic syndrome, and so on. To measure its thermal characteristics in single cell level, a heat loss to surrounding environments and a vibration damping should be solved when resonant thermal sensors are immersed in water. To solve these problems and realize highly sensitive thermal measurements, a resonant thermal sensor was partly placed in vacuum, and another end was in microchannel as a sample stage in a microfluidic chip. They were connected physically and thermally by a heat guide. The heat from the cell is conducted to the sensor via the heat guide, and measured. A fabricated device was evaluated by using laser Doppler vibrometer. The thermal resolution of the device was 5.2 pJ. The single brown fat cell was set on the sample stage, and generated heats were measured with and without stimulation. With the stimulation, pulsed heats were observed, which have not observed in bulk measurements. With the stimulation, gradual and long heat generations were caused similarly with bulk measurements. We have succeeded in detecting the heat from single brown fat cell by using the fabricated device.

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We developed circular cell-culture membranes which are made of SiN (silicon nitride) films of 1 μm in thickness and have multiple square or circular holes of several micrometers in size. The membranes were fabricated using SiN film formation with PECVD (plasma-enhanced chemical vapor deposition) and anisotropic wet etching of silicon with a surfactant-added TMAH (tetramethylammonium hydroxide) solution. Mechanical properties of the membranes were evaluated using a bulge test, and it was found that a circular membrane with circular holes has much larger fracture strength than a square membrane with square holes. Also, we demonstrated that optical transmittance of the membranes decreases with decreasing size of microholes and increasing porosity of the membranes, which gives useful design rules for their practical applications to cell culturing.

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A patterning technique of single cells is required for studying cell communication between multiple cells. The objective of this research is to develop a versatile cell manipulation system capable of patterning of single cells in high precision and super parallel format. Here, we investigate cell manipulation by electroosmotic (EO) flow with a micropipette and microprobe-array. Application of DC voltage (5 V) generated an EO flow through the hollow channels of the pipette and probe-array. Cells stained by fluorescent dye, Calcein-AM were captured at the tip of the pipette and probe-array. Cell damage was characterized through the observation of the cells・ In case of micropipette manipulation, a cell swelled and the fluorescence lost 180 s after the cell capture fi°om optical microscope observation. Cells moved toward some of the microprobe-array by EO flow. The cells were tracked and analyzed. Fluorescence of all the cells disappeared 28 s after the voltage application.

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The introduction of biological macromolecules such as DNA, RNA, and proteins into living cells plays a crucial role in the fundamental analysis of cellular functions and mechanisms in living systems. Therefore, we have been developing an effective platform for the in vitro manipulation and analysis of biological cells at the single-cell level based on scanning ion conductance microscopy (SICM) for non-damaging imaging of cells. In this paper, we successfully demonstrated a novel intracellular delivery method of DNA into living HeLa cells via a glass micropipette based on electrokinetically driven flow. We also proposed a vibration-assisted insertion method for penetrating a cell membrane to reduce cell damage. Preliminary insertion tests on a homemade SICM system revealed that the application of the mechanical oscillation can reduce the deformation of cells probably due to an increase in their viscous resistance. Moreover, we also found that a change in the ion current during the insertion process allows us to detect the instant when the micropipette tip penetrates the cell membrane.

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Cell migration plays an important role in many physiological and pathological processes such as morphogenesis, wound healing, and tumor metastasis. Although the majority of such events occur with cells moving as a group, called collective cell migration, the mechanism of collective cell migrations has not been well understood. This study performs traction force microscopy on migrating NIH 3T3 fibroblasts by using a microfabricated device to clarify the mechanism of collective cell migration. The device consists of a reservoir for establishing a confluent cell monolayer and 24 microchannels with arrays of micropillars that are used for traction force microscopy. Here the effect of anisotropic properties in stiffness of substrate on cell migration is tested with ellipsoidal cross section of micropillars. Cells produced higher traction forces in the direction of major axis of micropillars. Moreover, cells migrated faster when the major axis of micropillars is parallel to the direction of microchannels. These results suggest that cell can sense the stiffness of substrate and produce traction forces depending on the stiffness, leading to modulation of cell migration rate.

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Microorganisms play a vital role in many biological, medical and engineering phenomena. Some recent research efforts have demonstrated the importance of biomechanics in understanding certain aspects of microorganism behaviors such as locomotion and collective motions of cells. Previous studies had problems of high computational load in simulating many-body interaction of swimmers. In this study, we propose a boundary element method, based on the double-layer representation, for calculating interactions of many-body swimmers in Stokes flow regime. We use Graphic Processing Unit (GPU) to speed up the computation. We then analyze interactions of 100 swimmers.

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To elucidate the reason why bacteria exhibit collective motion, we first performed microscopic observation of a colony of Bacillus subtilis cells. Bacteria were inoculated on a semi-solid media, and 200 minutes after they began swarming. They swim collectively when the average distance between cells is less than 5 μm. PIV measurement instead of measuring indivisual cells revealed the flow field, from which the velocity correlation was deduced. As the colony becomes crowded with cells, the velocity correlation becomes stronger within the distance of 10 pm Furthermore, we limited a region of the correlation analysis to the tangential direction and the normal direction of the reference vector. Different characteristic lengths are found between two directions; The width and length of the swarm turned to be 13 times Bacillus subtilis cell body's average width and 10 times cell body's length, respectively. In addition, as for the maximum velocity of swarming, number density showed a peak 25 pm/s in about 1.0 X 10^6 - 1.0 X10^7 cells/mm^2.

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Bioconvection patterns are often observed in microalgae suspensions of randomly, but on average upwardly, swimming microalgae which are little denser than water. When we culture microalgae in the laboratory, we usually add aeration for mixing the culture fluid and supplying oxygen and carbon dioxide. However, the effect of aeration on the bioconvection pattern and the transport phenomena in the suspension is unclear. In this study, we investigated bioconvection pattern in a suspension of Chlamydomonas reinhardtii under the condition with aeration. Bioconvection pattern in a cylindrical container was visualized by red illumination, and white light for phototaxis could be added from above. Small bubbles were injected by a needle connected to a syringe. Observation of bioconvection patterns of microalgae suspension with aeration showed three kinds of flow patterns depending on the bubble flow rate. When there was no bubble flow, conventional bioconvection was dominant. When flow rate was low, observed pattern in the cylindrical container was split into two flow regions, where the bioconvection and the bubble flow were dominant respectively. When flow rate was high, bubble flow became dominant and no clear bioconvection was observed. Such changes in the bioconvection pattern have not been reported before. These findings are important to understand the transport phenomena in algae suspensions in engineering settings.

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Little is known about the mass transport in microbial suspension in previous studies. The aim of this study is to analyze the effect of the swimming motion on the nutrient uptake. We modeled a microorganism as a squirmer and calculated its locomotion by boundary element method . Moreover we simulated the nutrient uptake by tracking nutrient particles. The results show that the nutrient uptake is strongly affected by the swimming mode of a microorganism.

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The simulation model for the swimming movement of octopus was developed. This model was developed by extending SWUM (swimming human simulation model). In SWUM, a human body is represented by 21 body segments. However, it was not enough to realize the smooth arm motion of octopus. Therefore, in the octopus model, the number of body segments was increased to 90 (the body and 8 arms consisted of 10 body segments for each). Each joint had 3 degrees of freedom to realize various motions. This study aimed to clarify the motion and shape for fast swimming to develop an octopus-inspired robot for underwater rescue and exploration by using this octopus model. Three arm motions (sculling, undulation, and their composition) were analyzed. As a result, the average velocity in the last cycle of each motions were 0.08 lm/s in the sculling motion, 0.023m/s in the undulation motion and 0.106m/s in the composition motion. In future, we will investigate the fluid force coefficients to improve the precision of the model and investigate the arm motion suitable for the octopus-inspired robot.

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Since early study of power estimation of swimming dolphins by Gray in 1936, well known as "Gray's paradox," many studies have been conducted on the high swimming capability of the dolphins. Little literature, however, investigated the detailed time variation of the velocity and acceleration of the dolphins. In this study, we focused on jumping motion of the dolphins to measure the maximum acceleration capability. The whole process of the jumping motion of a pacific white-sided dolphin, Lagenorhynchus obliquidens, from initial descending in the water to final leaping, were recorded with two high-speed video cameras for 3-D motion analysis. We also created a simple body model of the dolphin to compute its drag with fluid analysis software. By combining the measured 3-D motions of the body and computed drag, we calculated the time variation of the thrust and power in relation to the fluke oscillation. As the result, the dolphin rapidly accelerated from static state to more than 9.4 m/s in about 2 seconds. It was found that the dolphin generated thrust mainly during downstroke. On the contrary, negative thrust was observed during upstroke. Calculated power mass ratio of the dolphin was 15 times larger than that of human.

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Recently, the use of a bio-inspired oscillating elastic fin has been proposed as an alternative propulsion mechanism in water. It is thought that the new mechanism will improve efficiency and safety, relative to the screw propeller. The optimum elasticity of the fin is not constant and changes based on swimming speed and task. However, it is very difficult to exchange fins of different stiffness while moving. We have developed a propulsion mechanism using a variable stiffness fin with torsional rectangular elastic plates. In this study, we aimed to develop a boat with the variable stiffness fin and evaluated the effectiveness of variable stiffness to provide basic data for the development of boats and autonomous underwater vehicles. As a first step of this study, we designed and built a model boat, and drove it on water pool. However, drag and rolling of the boat were greater. We improved the model boat and discussed its propulsion characteristics.

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The Leaf has a complex network as leaf vein. The leaf vein supply water efficiently to the whole of own. If there is some worm hole on its leaf by bug biting, the water in leaf vein flows continuously. This network system might be applied bio-mimically for an electrical and water infrastructure in near future. We observed the flow patterns of water in the Nandina domestica's Leaf vein with three patterns of normal leaf, damaged leaf and only leaf vein. The flow in leaf vein ware visualized by fluorescent solution by own water suction of leaf. As a result, the most of sucked water was flowed in the main leaf vein in each leaves, then the flows were spread into the lateral veins gradually. In the case of damaged leaf, the flow in the main leaf vein stopped at the worm hole, the water was bypassing the hole through the other fine veins. We found that leaf veins supply the water to whole of leaf even if leaf has damage in leaf vein according as a capillary phenomenon.

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