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“Preface”, Journal of Biomechanical Science and Engineering, Vol.11, No.2 (2016), p.16preface2. doi:10.1299/jbse.16preface02. Released on J-STAGE June 24, 2016

The endothelial cells lining our cardiovascular system are constantly affected by shear stress, which can alter both the morphology and biological activity of the cells. Methods to study the basic shear stress response by creating stable flow profiles on the macro scale are well established, but they do not allow the generation of controlled high precision flow profiles. The emergence of microfluidic devices has enabled well-defined individual cellular response studies on endothelial cells in scale-relevant tools. However, so far, no shear stress studies on clonal heterogeneity have been published. We have developed a novel bioassay system to study several shear stress conditions in parallel on clonal expanded single cells. The device consists of a silicon/glass microwell slide with integrated polydimethylsiloxane microchannels, which delivers shear stress to cells in a well-controlled manner using micropumps. The flow behavior of the device was numerically characterized by computational fluid dynamics analysis, which confirmed that the desired fluid-imposed shear stress was obtained. Bovine aortic endothelial cells were cultured in the microwells for 24 hours and then subjected to a fluid shear stress of up to 2.0 Pa for 6 hours. The results showed that alignment and elongation of the endothelial cells along the flow direction were dependent on the level of shear stress applied. It was demonstrated that multiple experimental conditions can be examined simultaneously within a single device and the compartmentalized structure of the microwell slide can be used to ensure physical separation of cells in individual wells. Moreover, it was shown that the device could reduce consumption of expensive reagents and enable screening of rare samples.

Keywords

Endothelial cells, Bioassay system, MEMS, Microfluidics, Fluid shear stress, Microwells

Paper information

Emilie WEIBULL, Shunsuke MATSUI, Helene ANDERSSON SVAHN and Toshiro OHASHI, “A microfluidic device towards shear stress analysis of clonal expanded endothelial cells”, Journal of Biomechanical Science and Engineering, Vol.9, No.1 (2014), p.JBSE0006. doi:10.1299/jbse.2014jbse0006

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Shinsuk PARK, Kyehan RHEE and Toshiro OHASHI, “Preface”, Journal of Biomechanical Science and Engineering, Vol. 7, No. 4 (2012), pp.335 . doi:10.1299/jbse.7.335

Cellular traction forces were measured by using a microfabricated substrate, particularly exploring how cytoskeletal structures such as actin filaments and microtubules contribute to traction forces. Smooth muscle cells isolated from bovine aortas were cultured and transfected with fluorescence proteins to visualize cell microstructures and then plated on a micropatterned elastomer substrate with arrays of micropillars. Cell spreading on the substrates produced deflection of micropillars which was used for estimation of cellular traction forces, and was closely associated with organization of stress fibers of actin filaments. Traction forces varied considerably among cells, showing the order of several 10 nN. After disruption of microtubules with nocodazole, traction forces significantly increased and there was no detectable change in formation of stress fibers. To inhibit the ROCK pathway, a signaling pathway of myosin light chain phosphorylation, possibly being induced by disruption of microtubules, significantly depressed the increase in traction forces after the disruption of microtubules. This result indicates that microtubules disassembly may regulate the actomyosin-based contractile system mainly through the ROCK pathway. The present study suggests that formation of stress fibers are mainly involved in cellular traction forces and a contribution of microtubules should include not only a force balance but also rather a modulator of the actomyosin contractile system in actin stress fibers.

Keywords

Traction Forces, Stress Fibers, Microtubules, Actomyosin Force Generation, Intracellular Stress Balance

Paper information

Toshiro OHASHI, Norifumi KAMEDA, Shouji NAKAMURA and Masaaki SATO, “Biomechanical Contribution of Cytoskeletal Structures to Traction Forces in Cultured Smooth Muscle Cells”, Journal of Biomechanical Science and Engineering, Vol. 5, No. 3 (2010), pp.262-271 . doi:10.1299/jbse.5.262

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Paper information

Toshiro OHASHI and Helene ANDERSSON-SVAHN, “Preface”, Journal of Biomechanical Science and Engineering, Vol. 5, No. 3 (2010), pp.185-185 . doi:10.1299/jbse.5.185

Mechanical properties of human aortic aneurysm tissues were measured with a biaxial tensile tester. Fifteen-mm-square specimens were obtained from thoracic aortic aneurysms of various origins and from undilated aortas adjacent to the aneurysms during aneurysmectomy, and were stored frozen until the measurement. Each specimen was stretched biaxially in physiological saline at room temperature at the rate of ?0.2 mm/sec. Although the ordered displacement was set equal for both directions, real strain applied to the specimens was not equibiaxial. The stress-strain curves under equibiaxial stretch were obtained by fitting measured curves with a strain energy function considering material anisotropy. Effects of freezing and ambient temperature on the mechanical properties were evaluated with porcine thoracic aortas. The mechanical properties of the frozen-stored specimens at 23°C were almost similar to those of the fresh specimens at 37 °C. Elastic modulus at zero load averaged for both directions Hmi = (Hxi+Hyi)/2 was higher (P &lt; 0.01) in the aneurysm tissues (1450 ± 250 kPa, mean ± SEM, n = 26) than in the undilated tissues (650 ± 140 kPa, n = 10). Anisotropy index K = |Hxi-Hyi|/Hmi was not significantly different between the aneurysm (20 ± 3%) and the undilated tissues (12 ± 3%) for all specimens. For the specimens whose elastic modulus Hmi was smaller than 1 MPa, however, the index K was significantly higher (P &lt; 0.05) in the aneurysm specimens (23.1 ± 5.3%, n = 14) than the undilated tissues (9.5 ± 2.5%, n = 8). These results indicate aneurysm tissues are not only stiffer but also more anisotropic than the nonaneurysmal tissues.

Keywords

True Aneurysm, Dissecting Aneurysm, Annulo Aortic Ectasia, Post-Stenotic Dilatation, Marfan's Syndrome, Mechanical Properties, Tensile Test, Anisotropy, Elastic Modulus

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Takeo MATSUMOTO, Tomohiro FUKUI, Toshihiro TANAKA, Naoko IKUTA, Toshiro OHASHI, Kiichiro KUMAGAI, Hiroji AKIMOTO, Koichi TABAYASHI and Masaaki SATO, “Biaxial Tensile Properties of Thoracic Aortic Aneurysm Tissues”, Journal of Biomechanical Science and Engineering, Vol. 4, No. 4 (2009), pp.518-529 . doi:10.1299/jbse.4.518

Control of the gliding directions of kinesin-driven microtubules (MTs) in vitro has good feasibility for the development of nano-scale transport systems. A requirement for the control of transporters in these systems includes detecting the positions of gliding MTs; however, no studies have reported on the monitoring of the positions of gliding MTs. Here, we suggest an algorithm to detect tip coordinates of gliding MTs by binarization, skeletonization, and filtration of fluorescent images of MTs. The algorithm was first applied to artificially drawn segments with given lengths (10-80 pixels), widths (1-10 pixels), and curvature radii (20-120 pixels) to verify the effect of the sizes of MTs on accuracy of tip coordinates extracted by the algorithm, and error was estimated by referring to the true coordinates. The estimated errors were as small as 2 pixels in the width and were not affected by the length and the curvature radius, indicating that our algorithm is useful to extract the tips of MTs. The algorithm was subsequently applied to images of gliding MTs. Since distances from the trajectories of the MTs to the centers of gravity of the MTs (3.7 ± 2.1 pixels) were significantly larger than those to the tips (1.9 ± 0.5 pixels), the use of the tips as representative points of gliding MTs was verified. A detection method using tips of MTs, as suggested in this study, may be a useful technique for monitoring each MT in nanoscale transport systems.

Keywords

Image Analysis, Tip Detection, Microtubule, Kinesin, Nano-Scale Transport System

Paper information

Shukei SUGITA, Naoya SAKAMOTO, Toshiro OHASHI and Masaaki SATO, “Novel Image Analysis for Trajectory of Microtubules Gliding on Kinesins with Tip Detection”, Journal of Biomechanical Science and Engineering, Vol. 4, No. 3 (2009), pp.404-414 . doi:10.1299/jbse.4.404

Kinesins, biomolecular motors moving along microtubules (MTs) in cells, can potentially be utilized as nano-scale transport systems with an inverted gliding assay, in which the MTs glide on a kinesin-coated surface. Although the key requirements include controls of gliding direction and velocity of MTs, the details of motility properties of MTs have not been well known. In this study, we quantitatively measured angular and gliding velocities, particularly focusing on the effects of MT length and kinesin density. The gliding assay of MTs of up to 20 μm in length was performed on a substrate coated with the kinesin density of 7.5, 38, and 75 μg/ml that resulted in the kinesin spacing of 7.8, 4.2, and 3.1 μm, respectively. The angular velocity for MTs shorter than kinesin spacings significantly decreased with increasing their length, and that for MTs longer than kinesin spacings was not affected by their length. Moreover, the angular velocity was substantially higher at lower kinesin density. These results suggest that both the number of associated kinesins with MTs and the kinesin spacings may contribute to the gliding direction. In contrast, the gliding velocity was independent of the MT length, ranging from 0.3 to 0.5 μm/s with decreasing the kinesin density. This may potentially imply the existence of an underlying mechanism with respect to the number of kinesins per the unit length of MTs. Towards development of high throughput nano-scale transport systems, long MTs and low kinesin densities would be effective for high directionality and high velocity, respectively.

Keywords

Nanoscale Transport System, Motor Protein, Microtubule, Angular Velocity, Gliding Velocity

Paper information

Shukei SUGITA, Naoya SAKAMOTO, Toshiro OHASHI and Masaaki SATO, “Characterization of Motility Properties of Kinesin-Driven Microtubules Towards Nano-Scale Transporter: Focusing on Length of Microtubules and Kinesin Density”, Journal of Biomechanical Science and Engineering, Vol. 3, No. 4 (2008), pp.510-519 . doi:10.1299/jbse.3.510

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.

Keywords

Endothelial Cells, Nucleus Remodeling, Shear Stress, Cyclic Stretching, Hydrostatic Pressure, Cytoskeletal Meshworks

Paper information

Toshiro OHASHI, Kazuhiko HANAMURA, Daisaku AZUMA, Naoya SAKAMOTO and Masaaki SATO, “Remodeling of Endothelial Cell Nucleus Exposed to Three Different Mechanical Stimuli”, Journal of Biomechanical Science and Engineering, Vol. 3, No. 2 (2008), pp.63-74 . doi:10.1299/jbse.3.63

This study investigated nitric oxide (NO) production and phenotype changes of smooth muscle cells (SMC) in a cocultured model (CM) exposed to fluid shear stress. The CM was composed of human umbilical endothelial cells (EC) and SMC, a collagen layer, and a porous membrane. After exposing the CM to shear stress of 1.5 Pa for 24 hours, α-smooth muscle actin (α-SMA) expression of SMC and NO production in culture media were examined. Under static conditions, α-SMA expression in the CM was significantly lower than that of a SMC monocultured model (SMC model). After exposure to shear stress, NO production in the CM increased compared to that in the static CM, and there was no significant difference in α-SMA expression between the CM and SMC model. These results suggest that EC may regulate phenotype changes of cocultured SMC, and NO may be one of the factors which induce dedifferentiation of SMC.

Keywords

Coculture, Endothelial Cell, Smooth Muscle Cell, Shear Stress, Smooth Muscle Phenotype, Nitric Oxide

Paper information

Naoya SAKAMOTO, Masaki OI, Toshiro OHASHI and Masaaki SATO, “Nitric Oxide Production and Smooth Muscle Cell Phenotype of Endothelial Cell-Smooth Muscle Cell Cocultured Model”, Journal of Biomechanical Science and Engineering, Vol. 1, No. 1 (2006), pp.224-233 . doi:10.1299/jbse.1.224