Journal of Biomechanical Science and Engineering
Online ISSN : 1880-9863
ISSN-L : 1880-9863
Volume 3, Issue 2
Special Issue on Cardiovascular Flow and Cell Biomechanics
Displaying 1-21 of 21 articles from this issue
Special Issue on Cardiovascular Flow and Cell Biomechanics
Papers(Special Issue)
  • Toshiro OHASHI, Kazuhiko HANAMURA, Daisaku AZUMA, Naoya SAKAMOTO, Masa ...
    2008 Volume 3 Issue 2 Pages 63-74
    Published: 2008
    Released on J-STAGE: April 08, 2008
    JOURNAL FREE ACCESS
    Cell Nuclei play a critical role in controlling gene expression and replicating DNA, and is known to deform in association with cell shape changes in response to external forces. This study dealed with morphological analysis to quantitatively assess the effect of three different mechanical stimuli including fluid shear stress, cyclic stretching, and hydrostatic pressure on nucleus morphology. Fluorescence images showed that fluid shear stress and cyclic stretching induced cell elongation and orientation very specifically to the direction of flow and stretch, respectively. In contrast, hydrostatic pressure induced cell elongation at non-preferred orientation. The nuclei were also found to deform in the same manner as that of the cells, which was, in particular, dependent on the type of mechanical stimuli, possibly suggesting the direct mechanical linkages between cell surface receptors, cytoskeletal meshworks, and nuclei. It was also shown that cytoskeletal meshworks may contribute to pre-existing strain of the nuclei.
    Download PDF (2595K)
  • Jie JI, Shunichi KOBAYASHI, Hirohisa MORIKAWA, Dalin TANG, David N. KU
    2008 Volume 3 Issue 2 Pages 75-84
    Published: 2008
    Released on J-STAGE: April 08, 2008
    JOURNAL FREE ACCESS
    High-grade stenoses can limit blood flow and produce conditions in which an artery may collapse. The resultant compression may be important in the development of atherosclerotic plaque fracture and subsequent thrombosis or distal embolization. We have developed stenosis models made of polyvinyl alcohol hydrogel, which closely approximate an arterial disease situation, and performed pulsatile flow experiments. Valsalva's maneuver and cough cause a sharp increase in jugular venous pressure to greater than 50 mmHg. Such transient pressure increases within the carotid sheath may augment the external pressure around the carotid artery. We applied external pressure to a stenosis model in two different forms and discussed the influences of external pressure on pulsatile flow and deformation in the stenosis model.
    Download PDF (1740K)
  • Kiyoshi KUMAHATA, Masahiro WATANABE, Teruo MATSUZAWA
    2008 Volume 3 Issue 2 Pages 85-100
    Published: 2008
    Released on J-STAGE: April 08, 2008
    JOURNAL FREE ACCESS
    For the simulation of the fluid-structure interaction (FSI) between the blood flow and blood vessel walls, we have examined the voxel-based FSI method. This method uses a Cartesian grid, called voxel, made from medical images. Further, we have tested the accuracy and reliability of this simple method and have observed its features. In this document, we discuss the background, kinetic models of the blood vessel, a numerical method, and the result of an experiment conducted using an artificial identical shape and an actual realistic shape.
    Download PDF (2510K)
  • Lei LIU, Kenichi FUNAMOTO, Toshiyuki HAYASE
    2008 Volume 3 Issue 2 Pages 101-115
    Published: 2008
    Released on J-STAGE: April 08, 2008
    JOURNAL FREE ACCESS
    Many studies have been carried out on the relationship between the occurrence and progression of circulatory diseases and hemodynamics. However, it is difficult to obtain accurate and detailed information on blood flow in the living body with existing experimental and numerical methods. The authors have previously proposed the Ultrasonic-Measurement-Integrated (UMI) simulation and shown that the blood flow in an aortic aneurysm can be accurately reproduced when feedback signals derived from the difference between measured and computed Doppler velocities are fed back to the numerical simulation. In the present study, we performed a fundamental numerical experiment in which UMI simulation was applied to a developed laminar pipe flow using an axisymmetric model in order to understand the effect of the feedback law on the accuracy of UMI simulation systematically. The effect of two types of ultrasonic probes, the linear scanning type and the sector scanning type, and the effect of 70° and 110° irradiating angles of the ultrasonic beam in the linear probe were investigated. It was confirmed that the result of UMI simulation asymptotically approached the standard solution of developed laminar flow downstream of the feedback domain in all cases using the linear probe and the sector probe with axisymmetric feedback. Under the present conditions, a linear probe with a radiation angle of 70° was most effective, whereas there was not so much improvement in the accuracy in the case using the sector probe. The effect of the singularity of the axisymmetric coordinate on the pipe axis was observed in the axial velocity profile near the entrance of the feedback domain, but disappeared some distance downstream in that domain.
    Download PDF (930K)
  • Noriyuki KATAOKA, Ken HASHIMOTO, Susumu KUDO, Ryuhei YAMAGUCHI, Katsuh ...
    2008 Volume 3 Issue 2 Pages 116-123
    Published: 2008
    Released on J-STAGE: April 08, 2008
    JOURNAL FREE ACCESS
    It is well known that endothelial cells (ECs) respond to the fluid imposed shear stress and change their shapes and functions. We have focused on the importance of cytoplasmic micro-mechanical strain in mechano-sensing mechanism of ECs. To this end, the cytosolic Ca2+ responses of ECs to the mechanical stimulus by laser tweezers that can apply the micro-mechanical force to nano/micro-organisms without any physical contact, were investigated. When the laser spot focused on the nucleus of EC was slightly moved, the cytosolic Ca2+ increased immediately in the same EC, whereas there was no Ca2+ increase without laser spot movement. In the absence of extracellular Ca2+ in the medium or the blockade of stretch activated ion channels, there was also an increase of Ca2+ in stimulated ECs. Therefore, the increased Ca2+ in stimulated ECs is considered to be derived from intracellular Ca2+ store. The heterogeneous Ca2+ propagation from the stimulated EC to surrounding ECs was also observed. Two types of Ca2+ wave propagation were observed, the fast one that the velocity was more than 20 μm/sec, and the slow one that the velocity was less than 1 μm/sec. The micro-stress induced by the micro-movement of the nucleus can be a trigger of the cytosolic Ca2+ increase and the cytoplasmic micro-mechanical strain may play an important role in mechano-sensing mechanism of ECs.
    Download PDF (722K)
Papers
  • Yixiang FENG, Shigeo WADA, Takuji ISHIKAWA, Ken-ichi TSUBOTA, Takami Y ...
    2008 Volume 3 Issue 2 Pages 124-137
    Published: 2008
    Released on J-STAGE: April 08, 2008
    JOURNAL FREE ACCESS
    Study of the development of aneurysm is a difficult task due to lack of experimental and clinical data. The current study takes advantage of fluid-structure interaction (FSI) to simulate the formation and growth of aneurysms by focusing on the interplay between the wall shear stress, degeneration of the vessel wall, and the wall deformation. We construct numerical aneurysm models arisen from both straight and curved arteries, under the hypothesis that high local wall shear stress larger than a certain threshold value will lead to a linear decrease in the wall mechanical properties. In the straight model, the growth of aneurysm is small and only at the distal neck region, and the aneurysm stops growing after several steps. In contrast, in the curved model, the aneurysm continues to grow in height and width. Our computer simulation study shows that even if the wall shear stress inside an aneurysm is low, aneurysm development can occur due to degeneration of the wall distal and proximal to the aneurysm. Our study demonstrates the potential utility of rule-based numerical methods in the investigation of developmental biology of cardiovascular diseases.
    Download PDF (506K)
  • Shigenao MARUYAMA, Kazuto NAKAGAWA, Hiroki TAKEDA, Setsuya AIBA, Atsuk ...
    2008 Volume 3 Issue 2 Pages 138-150
    Published: 2008
    Released on J-STAGE: April 08, 2008
    JOURNAL FREE ACCESS
    This paper describes a flexible cryoprobe utilizing the Peltier effect. The conventional cryoprobes have problems such as low flexibility and difficulty in precise temperature control. In this study, the cryoprobe composed of a flexible tube was developed to treat the inner part of the human body. The thermoelectric elements are attached on the top of the cryoprobe, so the Peltier effect makes it possible to control the heat-transfer of the cooling tip actively and to perform both cooling and heating only by reversing the electric current. The performance of the cryoprobe was evaluated through experiments of agar gel cooling and the temperature distribution was estimated by numerical simulation. In addition, an animal experiment using a guinea pig was conducted for practical application. In the experiment of an agar gel cooling, the agar gel surface was frozen within 10 seconds, which is in good agreement with numerical simulations. Repetition of cooling and heating are also performed experimentally. In the animal experiments, the temperature of the skin surface was monitored and its histological change was examined. Although the continuous cooling for 500 sec after freezing the skin surface could induce only the focal necrosis of the epidermis, the cyclic treatment of cooling and heating induced the necrosis of the whole epidermal layer and a part of eccrine glands. From experimental and numerical results, the cryoprobe could induce the necrosis of the skin.
    Download PDF (1681K)
  • Tsuyoshi YASUKI
    2008 Volume 3 Issue 2 Pages 151-160
    Published: 2008
    Released on J-STAGE: May 02, 2008
    JOURNAL FREE ACCESS
    This paper describes development of a lumped mass-spring model of lower leg subsystem test of the European Enhanced Vehicle-safety Committee WG17 (here after referred as to the EEVC WG17) test method in order to provide relationship among the stiffness of the vehicle under the test, knee-bending angle and upper tibia acceleration of the lower leg subsystem impactor. The lower leg subsystem test and protection criteria are intended to protect against knee joint injuries and lower leg bone fracture. The knee-bending angle of the lower leg subsystem impactor was shown as a function of the stiffness of the vehicle under the test. The lumped mass-spring model results indicated that upper bumper stiffness forms a significant component of the knee-bending angle and lower bumper stiffness forms a significant component of upper tibia acceleration similar to upper bumper stiffness.
    Download PDF (965K)
  • Lei LIU, Toshiyuki HAYASE, Makoto OHTA, Kosuke INOUE
    2008 Volume 3 Issue 2 Pages 161-175
    Published: 2008
    Released on J-STAGE: May 20, 2008
    JOURNAL FREE ACCESS
    Cardiovascular diseases are closely related to blood flow. Ultrasonic-Measurement-Integrated (UMI) simulation, in which results of ultrasonic measurement are fed back to the flow simulation, was proposed in a previous study in order to reproduce the real blood flow accurately and efficiently. The usability of the UMI simulation was confirmed by numerical experiment, but the effectiveness of this simulation was strongly affected by the accuracy of the ultrasonic measurement. In this paper, we examined the accuracy of a commercial ultrasonic measurement device by an experiment with a PVA-H straight tube phantom. By analyzing the measured color Doppler images for a developed laminar flow inside the phantom, we obtained the relationship between the color Doppler value and the Doppler velocity (C-V relationship). It was revealed that the original C-V relationship provided in the device as a color bar was not suitable for quantitative evaluation of Doppler velocity to be used in UMI simulation. Compared with the original C-V relationship, the present C-V relationship results are in far better agreement with the analytic solution. Investigation of the normalized error confirmed that the result obtained with the present C-V relationship was reliable in cases of relatively high Reynolds number in the flow domain except near the wall. The two signal conditioning factors of the device had little influence on the Doppler velocity. Finally, we investigated the effect of temporal and spatial averaging of ultrasonic measurement data to clarify the relation between the number of averagings and the level of agreement with the analytic solution.
    Download PDF (1288K)
  • Hiroko N. MATSUMOTO, Yoshihisa KOYAMA, Kazuo TAKAKUDA
    2008 Volume 3 Issue 2 Pages 176-187
    Published: 2008
    Released on J-STAGE: June 06, 2008
    JOURNAL FREE ACCESS
    Timeline factors such as the rest interval between load cycles were, as well as the many factors that characterize dynamic mechanical loading to bone, reported to have significant effects on bone formation. Noticing the possible importance of timeline-dependent phenomena in mechanotransduction, we examined the effects of both the duration of constant load and the duration of rest interval in a 3-point bending model for rat tibiae. A square-wave loading was used, in which loading waveform was composed of constant load and rest periods. Newly formed bones were labeled with fluorescent dyes, and the amounts of new bone were measured using confocal laser microscopy. Loading to the medial surface of tibiae with a waveform composed of 0.5 s of constant load and 39.5 s of rest periods induced bone formation on the medial surface, whereas that composed of 39.5 s of constant load and 0.5 sec of rest periods induced bone formation on the lateral surface. Furthermore, the length of the rest period and the number of loading cycles were found to have significant effects on the amount of bone formation. Based on these results, a hypothetical mechanism on the mechanotransduction system in bone was discussed.
    Download PDF (1129K)
  • Michihito AOKI, Yoshihiro TAKEDA, Hajime FUKUNAGA, Takuji KOIKE, Sayur ...
    2008 Volume 3 Issue 2 Pages 188-199
    Published: 2008
    Released on J-STAGE: June 13, 2008
    JOURNAL FREE ACCESS
    Mussel adhesive protein (MAP) is expected to be used as a new bioadhesive. However, the mechanical properties of MAP have not been reported, despite the potential importance of MAP in medical applications. The study aims to estimate the mechanical strength of MAP, and describe the influence of ferric ions present in MAP on the mechanical strength. Fe3+ incorporated in MAP is regarded as having an important role in the mechanical properties. Mussels were classified into two groups in order to compare the effect of Fe3+ on both the tensile and peel properties of MAP. One group was exposed to artificial seawater with a high concentration of ferric ions, and the other group is exposed to natural seawater as a control. The results revealed that the deterioration of the adhesive strength over time was different between the two groups, although significant differences in the tensile properties were not observed. While the adhesive strength in the control group decreased with elapsed time, it was increased for the iron-enriched group over the first 17 days. This suggested that ferric ions may improve the magnitude of the adhesive strength for MAP.
    Download PDF (560K)
  • Vladimir JURAS, Pavol SZOMOLANYI, Zuzana MAJDISOVA, Siegfried TRATTNIG
    2008 Volume 3 Issue 2 Pages 200-208
    Published: 2008
    Released on J-STAGE: July 01, 2008
    JOURNAL FREE ACCESS
    Objective: Purpose of the paper is to present and validate a device for cartilage compression for assessment of MR parameters (T1, T2, ADC) in cartilage explants before, during and after compression operating with novel features.
    Design: This device fits into a BGA-12 micro-imaging gradient system capable of delivering 200mT/m. A 35 mm inner diameter resonator was used. The reproducibility and accuracy of cartilage compression possible with the device were evaluated. Sixteen human cartilage explants from knee joints were examined by delayed Gadolinium enhancement MRI of cartilage (dGEMRIC) for T1 mapping, T2 mapping and ADC measurements.
    Results: Cartilage compression studies demonstrated both low inter-observer (CV 4.7%) and intra-observer (CV 11.9%) variation. No undesired movements were observed. The compressive piston could be moved with high accuracy (error∼1.07%). The waterproof chamber of the compression device allowed contrast enhanced T1 mapping without repositioning the cartilage samples. Preliminary results of MR parameters depending on compression are presented.
    Conclusions: In vitro MR cartilage compression studies are feasible with the custom-build device with high reproducibility and accuracy. Valuable information about biomechanical cartilage properties can be recorded using this device.
    Download PDF (1286K)
  • Natsuko HOSODA, Nobuo SAKAI, Yoshinori SAWAE, Teruo MURAKAMI
    2008 Volume 3 Issue 2 Pages 209-220
    Published: 2008
    Released on J-STAGE: July 09, 2008
    JOURNAL FREE ACCESS
    Articular cartilage has high water content from 70 to 80% and biphasic property. The structures of the tissue are inhomogeneous and anisotropy. Consequently, cartilage tissue shows complicated viscoelastic behavior to mechanical stimuli because of depth-dependent and time-dependent interstitial fluid flux and stress-strain behaviors. Therefore it is necessary to consider not only the average tissue property but also the local one to explain mechanical and functional behaviors. The aim of this study is to consider the effect of elastic modulus distribution on the mechanical behavior of cartilage tissue using experiments with visualization and two-dimensional finite element method (FEM).
    In this study, we performed the compression test of the articular cartilage under the unconfined condition. First, on the basis of the distribution of Young's modulus in depth direction calculated from local strain at equilibrium in experiment, the stress and strain behaviors in articular cartilage were analyzed by biphasic theory.
    Immediately after loading, the FEM analysis for deformed profiles of the model with depth-dependent Young's modulus corresponded to actual profiles, while the model with average value for Young's modulus showed inadequate deformed profiles. However, the total load-carrying capacity estimated in FEM for the former model is about one order lower than the experimental one measured by a load cell. Therefore, we provided time-dependence to elastic property to understand the complicated viscoelastic behavior during stress relaxation. Thus, the deformed shape profiles of the model immediately after loading and the total load-carrying capacity could satisfactorily correspond to measured data by considering depth-dependent and time-dependent variation of elastic property.
    Download PDF (836K)
  • Koji IIDA, Michio MURAKOSHI, Shun KUMANO, Kouhei TSUMOTO, Katsuhisa IK ...
    2008 Volume 3 Issue 2 Pages 221-234
    Published: 2008
    Released on J-STAGE: August 06, 2008
    JOURNAL FREE ACCESS
    Prestin is regarded as the motor protein of cochlear outer hair cells (OHCs). Due to the conformational change of prestin, OHCs are believed to contract and elongate, this OHC motility realizing the high sensitivity, wide dynamic range and sharp tuning of the auditory system of mammals. Since its identification in 2000, prestin has been intensively investigated. As a result, knowledge about the structure and function of prestin has been gradually accumulated by studies using prestin-expressing cells. Purification of prestin would allow further analysis, e.g., crystal structure analysis, to obtain knowledge about prestin at the molecular level. Recently, it has been reported that recombinant prestin was purified from Sf9 insect cells and that structural analysis was carried out by electron microscopy. In the present study, an attempt was made to purify prestin from another expression system, i.e., mammalian Chinese hamster ovary (CHO) cells stably transfected with gerbil prestin. First, since it is unclear which detergents are suitable for solubilization of prestin, the best detergent for solubilization was selected from among 8 kinds of detergent commonly used for membrane protein isolation. The optimum concentration of the detergent was also determined. As a result, it was clarified that 10 mM n-nonyl-β-D-thiomaltopyranoside efficiently solubilizes prestin. Next, using this detergent, purification of prestin by anti-FLAG affinity chromatography was performed, and 84 ± 23 μg of purified prestin was obtained from 2×109 3×FLAG-tagged prestin-expressing CHO cells.
    Download PDF (1221K)
  • Ryo SUDO, Toshihiro MITAKA, Mariko IKEDA, Kazuo TANISHITA
    2008 Volume 3 Issue 2 Pages 235-248
    Published: 2008
    Released on J-STAGE: August 22, 2008
    JOURNAL FREE ACCESS
    Vertical cell-cell contact is an important difference between 2D- and 3D-culture models. We hypothesized that this difference in cell-cell contacts is critical in maintaining hepatocyte functions. Here we developed a simple culture technique to investigate the effect of the vertical cell-cell contacts on morphogenesis, proliferation, and differentiation of hepatocytes. Two separate monolayers of primary rat hepatocytes were first cultured on a microporous membrane and a culture dish and one was then inverted and placed on top of the other to create a 3D-configuration. Imaging techniques revealed that hepatocytes recovered cell polarity and formed bile canaliculi in response to the addition of the vertical cell-cell contacts. Quantitative analysis revealed that the cells exhibited cuboidal cell shape with vertical cell-cell contacts and maintained differentiated functions while their proliferation activity was inhibited. In contrast, hepatocytes in a monolayer gradually flattened due to the lack of vertical cell-cell contacts, resulting in cells losing differentiated functions which correlated to the deformation of cell shape. Controlling 2D- and 3D-configurations is important in switching growth and differentiation of hepatocytes. These results implicate how we apply 2D- and 3D-cultures for tissue engineering of the liver.
    Download PDF (2198K)
  • Ryo SUDO, Norio TAKAHASHI, Toshihiro MITAKA, Mariko IKEDA, Kazuo TANIS ...
    2008 Volume 3 Issue 2 Pages 249-262
    Published: 2008
    Released on J-STAGE: August 22, 2008
    JOURNAL FREE ACCESS
    Cell response on microporous membranes is important since the membranes are often used for the applications of tissue engineering. We previously developed the 3D stacked-up culture of hepatic progenitor cells, which are small hepatocytes (SHs), where the cells were cultured on microporous membranes for reconstructing layered tissues in vitro. Since membranes with randomly distributed micropores were used in the culture, the size and distribution of the micropores remained to be optimized. Here we prepared microporous membranes with different pore sizes and spacing to investigate the SH morphogenesis on the membranes. Laser-ablation technique was used to fabricate micropores in desired patterns. Phase-contrast microscopy revealed that the formation of SH colonies was correlative to the micropores. Furthermore, SH colonies exhibited collective cell movements along the micropatterned pores. The correlation enhanced with increasing sizes (>10 μm) and decreasing spacing (<40 μm) of micropores. Scanning electron microscope images showed that SHs located at the colony edge extended protrusions invading into the micropores. Furthermore, immunofluorescent staining revealed that the focal complexes localized intensively around the micropores, suggesting that the SH morphogenesis was regulated by the focal complexes. This study demonstrated that the surface topography of microporous membranes plays an important role in the formation and movement of SH colonies.
    Download PDF (3587K)
  • Atsutaka TAMURA, Sadayuki HAYASHI, Kazuaki NAGAYAMA, Takeo MATSUMOTO
    2008 Volume 3 Issue 2 Pages 263-274
    Published: 2008
    Released on J-STAGE: August 22, 2008
    JOURNAL FREE ACCESS
    Mechanical properties of brain tissue characterized in high-rate loading regime are indispensable for the analysis of traumatic brain injury (TBI). However, data on such properties are very limited. In this study, we measured transient response of brain tissue subjected to high-rate extension. A series of uniaxial extension tests at strain rates ranging from 0.9 to 25 s-1 and stress relaxation tests following a step-like displacement to different strain levels (15-50%) were conducted in cylindrical specimens obtained from fresh porcine brains. A strong rate sensitivity was found in the brain tissue, i.e., initial elastic modulus was 4.2 ± 1.6, 7.7 ± 4.0, and 18.6 ± 3.6 kPa (mean ± SD) for a strain rate of 0.9, 4.3, and 25 s-1, respectively. In addition, the relaxation function was successfully approximated to be strain-time separable, i.e., material response can be expressed as a product of time-dependent and strain-dependent components as:K(t) = G(te(ε), where G(t) is a reduced relaxation function, G(t) = 0.416e-t/0.0096+0.327e-t/0.0138+0.256e-t/1.508, and σe(ε) is the peak stress following a step input of ε. Results of the present study will improve biofidelity of computational models of a human head and provide useful information for the analysis of TBI under injurious environments with strain rates greater than 10 s-1.
    Download PDF (579K)
  • Chwei-Goong TSENG, Yuan-Shou JIANG, Kan-Shan SHIH
    2008 Volume 3 Issue 2 Pages 275-286
    Published: 2008
    Released on J-STAGE: August 27, 2008
    JOURNAL FREE ACCESS
    The purpose of this study was to compare the effects of various designs of implants' placement on stress distribution in bone around the implants supporting one-unit fixed complete dentures. A computer tomography image was redrawn to reconstruct a digital three-dimensional solid model of a mandible including the cortical bone and cancellous bone. Moreover, the reverse engineering method and computer-aided design were employed to construct a digital three-dimensional solid model of fixed complete dentures as well as implant and abutment. All of the digital three-dimensional solid models were combined and transformed to the FE models, and they were classified into 7 configurations according to the number and location of the implants. The MSC/Patran software was used to develop the FE mesh comprising of 1101954 elements with 252693 nodes. The MSC/Nastran software was utilized as pre and post-processor for all mathematical calculations necessary to evaluate dental and mandibular biomechanics. One set of multiple vertical loads was used to simulate the possibility of occlusion status. And the von Mises stress values in the cortical bone, cancellous bone and implants were evaluated. The simulated results indicated that the stress distributions for FE models were largely affected by the number and location of implants. In the bone, similar to the single-tooth case, the von Mises stresses were all concentrated toward the cortical bone around the collar of the implants for FE models. By increasing the thickness of each connector of complete dentures, the stress distribution for cortical bone around implants could be decreased.
    Download PDF (4047K)
  • Takashi TADENUMA, Koji IIDA, Michio MURAKOSHI, Shun KUMANO, Kouhei TSU ...
    2008 Volume 3 Issue 2 Pages 287-298
    Published: 2008
    Released on J-STAGE: September 05, 2008
    JOURNAL FREE ACCESS
    A source of the electromotility of the outer hair cells (OHCs), which enables the high sensitivity of mammalian hearing, is believed to be voltage-dependent conformational change of the motor protein prestin embedded in the lateral wall of the OHCs. As a result of extensive studies on this unique motor protein using prestin-expressing cells, knowledge about its structure and function has been gradually accumulated. To obtain further knowledge about prestin, research using purified prestin molecules is necessary, and thus a method for efficiently obtaining prestin molecules is required. In the present study, construction of an expression system for prestin using the baculovirus/Sf9 insect cell system was attempted. The expression and localization in the plasma membrane of prestin in infected Sf9 cells were confirmed by Western blotting and immunofluorescence analysis, and prestin's functional activity was determined by patch-clamp measurements. Furthermore, to obtain prestin from the cells efficiently, culture conditions of the cells were examined, and it was clarified that cells should be harvested around 72 hours after infection. Quantitative Western blotting revealed that the maximal amount of prestin per liter of culture medium was around 800 μg. The results of this study indicate that the expression system which was constructed is potentially useful for purification of prestin due to the rapidity of its construction and its high yield.
    Download PDF (782K)
  • Akinori UEDA, Ryo SUDO, Mariko IKEDA, Ken-ichi KOKUBO, Susumu KUDO, Hi ...
    2008 Volume 3 Issue 2 Pages 299-310
    Published: 2008
    Released on J-STAGE: September 24, 2008
    JOURNAL FREE ACCESS
    Three-dimensional (3-D) control of microvessel formation is critical for regeneration medicine and tissue engineering. Formation and maintenance of organ functions require deep and extensive microvessel networks that can supply O2 and nutrients to tissue-forming cells. In this study, we examined the effects of hypoxia on 3-D network formation using an in vitro model system in which the effects of hypoxia were isolated from those of other factors affecting network formation, such as growth factors. When we quantified network formation by endothelial cells (ECs) cultured on collagen gel under hypoxic (5% O2) and normoxic (21% O2) conditions, we found that hypoxia caused ECs to penetrate into the underlying collagen gel and to form 3-D, capillary-like networks. We also examined the detailed 3-D morphology of the networks using confocal laser-scanning microscopy. The networks promoted by hypoxia were more extensive and penetrated more deeply into the underlying collagen gel than did those formed under normoxic conditions. Our results demonstrate that hypoxia can induce 3-D network formation by ECs in vitro in the absence of other factors. In examining the mechanism by which hypoxia induces network formation, we found that hypoxia promotes expression of many EC genes, causes EC to secrete network-inducing factors, and increases the sensitivity of ECs to growth factors.
    Download PDF (1307K)
feedback
Top