Tension in actin filaments plays crucial roles in multiple cellular functions, although little is known about tension dynamics of cells during adhesion to substrates. In this study, we visualized intracellular tension in actin filaments using a newly developed Förster resonance energy transfer (FRET)-based tension sensor (Actinin-sstFRET-GR). Tension dynamics were monitored during adhesion in MC3T3-E1 mouse osteoblastic cells after introduction of the sensor. Whole cell areas continued to increase from 10 to 180 min after plating and tension was monitored in a typical section close to the bottom, where the mean fluorescence was the largest. Tension on the bottom side was negatively correlated with cell area from 10 to 110 min. Thereafter, this correlation was positive until 180 min. We then analyzed central-peripheral differences in tension close to the bottom. In these experiments, tension in the cell periphery increased during expansion of the area and decreased during contraction. As a result, fluctuations of tension in this area were much larger than those in the central area of the cell. Finally, we analyzed upper-lower differences in tension development during initial adhesion and showed continuous decreases in the lower side of the cell from 10 to 110 min after plating, and decreases in the upper side until 70 min, followed by increases. In this study, we successfully visualized dynamic changes in intracellular tension at the sub-cellular level and found that development of tension during initial adhesion processes is time-dependent and has central-peripheral and upper-lower differences. The present FRET tension sensor may become a powerful tool in studies of cell biomechanics.

Keywords

FRET, Tension sensor, Cytoskeleton, Actin filament, α-actinin, MC3T3-E1, Adhesion

Paper information

Junfeng WANG, Masahiro ITO, Wenhao ZHONG, Shukei SUGITA, Tatsuo MICHIUE, Takashi TSUBOI, Tetsuya KITAGUCHI, Takeo MATSUMOTO, “Observations of intracellular tension dynamics of MC3T3-E1 cells during substrate adhesion using a FRET-based actinin tension sensor”, Journal of Biomechanical Science and Engineering, Vol.11, No.4 (2016), p.16-00504. doi:10.1299/jbse.16-00504. Final Version Released on December 16, 2016, Advance Publication Released on October 24, 2016.

In order to determine how cells change their morphology during adhesion process to a substrate, we focused on the actin cytoskeleton and investigated its morphological change along with that of the whole cell during adhesion process. An osteoblastic cell line MC3T3-E1 was used as the test model. We plated cells whose cell cycle had been synchronized by serum starvation on fibronectin-coated glass plate and cultured them for 10 min to 24 h. We then stained their F-actin and nucleus and observed them with a fluorescent microscope for cell area and shape index and 2D parameters for actin morphology, and with a laser scanning microscope for 3D morphology of actin and nucleus. In the beginning of adhesion, the trypsinized cells were round and their nuclei were surrounded uniformly by thick layer of actin. The actin layer in the upper side became actin aggregate (AA) and lower side dense peripheral band (DPB) in 30 min. The upper AA then became smaller and finally to actin filaments (AFs) spanning the cell top. The DPB expanded and finally became AFs on cell bottom by 1 h. The nucleus becomes flattened possibly due to compression by the cell membrane caused by the expansion of the DPB in the early stage of adhesion. In the later stage of adhesion, the number of AFs continuously increased and nucleus became flattened more and more until 12 h. This may be caused by the increase in the top AFs that may compress the nucleus. Cells become more elongated in response to further alignment of AFs until 12 h. These results indicate that change in AFs during adhesion process is complicated not only temporally but also spatially.

Keywords

Cytoskeleton, Actin aggregate, Actin filament, Morphology, MC3T3-E1, Adhesion

Paper information

Junfeng WANG, Shukei SUGITA, Kazuaki NAGAYAMA, Takeo MATSUMOTO, “Dynamics of actin filaments of MC3T3-E1 cells during adhesion process to substrate”, Journal of Biomechanical Science and Engineering, Vol.11, No.2 (2016), p.15-00637. doi:10.1299/jbse.15-00637. Final Version Released on June 24, 2016, Advance Publication Released on January 04, 2016.

Estimation of wall strength of the aortic aneurysms is necessary for the prediction of their rupture risk. We previously found a significant correlation between their tensile strength σ<i>_MAX</i> and a yielding parameter τ_σ, which is the stress when tangent elastic modulus reaches at 63% of the plateau level. This may indicate that the wall strength is estimated from their pressure-diameter relationship. Here we show a possible mechanism of the correlation between τ_σ and σ<i>_MAX</i> by focusing on alignment of collagen fibers. Thin (150-µm) slices of porcine thoracic aortas were uniaxially stretched in circumferential direction until failure under a microscope, and a retardance, a phase shift when polarized light passes through a birefringent material, was measured as the degree of collagen fiber alignment. Strength σ<i>_MAX</i> correlated significantly with τ_σ as obtained previously. The retardance increased with the increase in the stress and reached a plateau at the stress σ<i>_Ret-plateau</i>, indicating that σ<i>_Ret-plateau</i> is the stress at which most of the intramural collagen fibers have aligned. The stress σ<i>_Ret-plateau</i> correlated significantly with τ<i>_σ</i> and both parameters has similar values. This may indicate that the aortic wall yields when all of collagen fibers become straight. Smaller σ<i>_Ret-plateau</i> means that most collagen fibers are stretched and loaded at smaller stress, resulting in failure at smaller stress. This seems to be a reason for the significant correlation between τ_σ and σ<i>_MAX</i>.

Keywords

Collagen Fibers, Failure Stress, Porcine Thoracic Aorta, Stiffness, Birefringence

Paper information

Shukei SUGITA and Takeo MATSUMOTO, “Yielding Phenomena of Aortic Wall and Intramural Collagen Fiber Alignment: Possible Link to Rupture Mechanism of Aortic Aneurysms”, Journal of Biomechanical Science and Engineering, Vol. 8, No. 2 (2013), pp. 104-113. doi:10.1299/jbse.8.104

In order to determine how cells change their traction forces at focal adhesions (FAs) under macroscopic deformation conditions, we investigated the dynamic changes in traction force at FAs by culturing porcine aortic smooth muscle cells (SMCs) on elastic micropillar substrates and giving them macroscopic deformation by stretching the substrates. We patterned adhesion region on the top surface of a polydimethylsiloxane-based micropillar array using our original micropatterning technique to align the cells on the pillar array parallel to the stretch direction. SMCs plated on the micropillars successfully spread in the adhesion region and their actin stress fibers (SFs) aligned in the direction to be stretched. Cells were then stretched and released cyclically with strain rates of 0.3%/15s up to 3--6% strain, and deflection of micropillars at both side regions of cells were measured simultaneously to obtain the traction force at each FA <i>in situ</i>. SMCs aligned in the stretch direction showed two types of responses: almost a half of the SMCs changed their force in phase with the applied strain, and showed gradual active contraction with the stretch cycles (synchronous group); and the rest tended to keep their force constant and became elongated with the cycles (asynchronous group). In the asynchronous group, the force sometimes changed in antiphase with the cell strain as if the cells maintain intracellular traction force at a constant level. These results may indicate that SMCs sometimes exhibit active homeostatic responses to keep their pretension constant during macroscopic stretching, and such tensional homeostatic responses may occur concurrently with cell elongation.

Keywords

Cell Biomechanics, Mechanical Properties, Stress Fibers, Force Transmission, Cytoskeleton

Paper information

Kazuaki NAGAYAMA, Akifumi ADACHI and Takeo MATSUMOTO, “Dynamic Changes of Traction Force at Focal Adhesions during Macroscopic Cell Stretching Using an Elastic Micropillar Substrate: Tensional Homeostasis of Aortic Smooth Muscle Cells”, Journal of Biomechanical Science and Engineering, Vol. 7, No. 2 (2012), pp.130-140 . doi:10.1299/jbse.7.130

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

Paper information

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

The effects of three-dimensional (3-D) culture and cyclic stretch stimulation on the expression of contractile proteins were investigated in freshly isolated rat aortic smooth muscle cells (FSMC). Primary cells were cultured statically on cell culture dishes (two-dimensional (2-D) culture) or in type I collagen gel matrix (3-D culture). Changes in their expression level of actin filaments (AFs) and smooth muscle myosin heavy chain (SM-MHC) were measured quantitatively using an accurately-calibrated fluorescent microscopy. The expression of AFs and SM-MHC decreased in both cultures in their early stages. Cell morphology was quite different between the two cultures: the cells had a flattened and irregular shape in the 2-D culture, while they had a fusiform shape with a well-defined long axis in the 3-D. Nineteen-day culture in the gel significantly increased the expression levels of AFs and SM-MHC while the expression levels remained low in the 2-D. Further and early increase in the expression levels was observed in the cells cultured in the gel with cyclic stretch of ?8% amplitude and 1 Hz frequency. The cyclic stretch also induced alignment of FSMCs in the gel parallel to the stretch direction, and the cell alignment was observed earlier than the increase in their contractile proteins. These results indicate that the 3-D culture in collagen gel may increase the expression level of contractile proteins in FSMCs while maintaining their fusiform morphology, and cyclic stretch may efficiently increase the expression levels when the cells aligned in the stretch direction.

Keywords

Cellular Biomechanics, Smooth Muscle Phenotype, Contractile Properties, Extracellular Matrix, Actin Stress Fibers

Paper information

Kazuaki NAGAYAMA, Naoki MORISHIMA and Takeo MATSUMOTO, “Effects of Three-Dimensional Culture and Cyclic Stretch Stimulation on Expression of Contractile Proteins in Freshly Isolated Rat Aortic Smooth Muscle Cells”, Journal of Biomechanical Science and Engineering, Vol. 4, No. 2 (2009), pp.286-297 . doi:10.1299/jbse.4.286

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Keywords

Paper information

Takeo MATSUMOTO and Marie OSHIMA, “Preface”, Journal of Biomechanical Science and Engineering, Vol. 3, No. 3 (2008), pp.311-311 . doi:10.1299/jbse.3.311

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(t)σe(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.

Keywords

Brain Tissue, Visocoelasticity, Stress, Strain, High-Rate Extension, Relaxation, Traumatic Brain Injury (TBI)

Paper information

Atsutaka TAMURA, Sadayuki HAYASHI, Kazuaki NAGAYAMA and Takeo MATSUMOTO, “Mechanical Characterization of Brain Tissue in High-Rate Extension”, Journal of Biomechanical Science and Engineering, Vol. 3, No. 2 (2008), pp.263-274 . doi:10.1299/jbse.3.263

Mechanical properties of brain tissue in high strain region are indispensable for the analysis of brain damage during traffic accidents. However, accurate data on the mechanical behavior of brain tissue under impact loading condition are sparse. In this study, mechanical properties of porcine brain tissues were characterized in their cylindrical samples cored out from their surface. The samples were compressed in their axial direction at strain rates ranging from 1 to 50 s-1. Stress relaxation test was also conducted following rapid compression with a rise time of ?30 ms to different strain levels (20-70%). Brain tissue exhibited stiffer responses under higher impact rates: initial elastic modulus was 5.7±1.6, 11.9±3.3, 23.8±10.5 kPa (mean±SD) for strain rate of 1, 10, 50 s-1, respectively. We found that stress relaxation K(t,ε) could be analysed in time and strain domains separately. The relaxation response could be expressed as the product of two mutually independent functions of time and strain as:<br />K(t,ε)=G(t)σe(ε), where σe(ε) is an elastic response, i.e., the peak stress in response to a step input of strain ε, and G(t) is a reduced relaxation function:<br />G(t)=0.642e-t/0.0207+0.142e-t/0.482+0.216e-t/18.9, i.e., the time-dependent stress response normalized by the peak stress. The reduced relaxation function obtained here will serve as a useful tool to predict mechanical behavior of brain tissue in compression with strain rate greater than 10 s-1.

Keywords

Brain Tissue, Viscoelasticity, Stress, Strain, High-Rate Compression, Relaxation

Paper information

Atsutaka TAMURA, Sadayuki HAYASHI, Isao WATANABE, Kazuaki NAGAYAMA and Takeo MATSUMOTO, “Mechanical Characterization of Brain Tissue in High-Rate Compression”, Journal of Biomechanical Science and Engineering, Vol. 2, No. 3 (2007), pp.115-126 . doi:10.1299/jbse.2.115

The stress relaxation test was performed for cultured rat aortic smooth muscle cells (SMCs) to investigate the effect of actin filaments (AFs) on viscoelastic properties of the cells. Untreated cells and cells treated with cytochalasin D to disrupt their AFs were stretched by 70-85%, and their length was kept constant with a laboratory-made micro tensile tester with feed-back control to obtain their stress relaxation curve. Viscoelastic analysis with 4-parameter Maxwell model showed that the stress relaxation process of the cells could be divided into two phases with different time constants: a fast phase with a time constant in the order of minutes, and a slow phase with a time constant in the order of hours. Elastic parameters in the two phases decreased similarly by about a half with AF disruption. Viscous parameters also decreased by ?1/3 and ?1/4 in the fast and the slow phase, respectively, with AF disruption. No difference was observed for the relaxation time constant in the fast phase in response to AF disruption, while the time constant in the slow phase decreased significantly by about a half. Fluctuation in tension was observed in the stress relaxation curve of the untreated cells. Such fluctuation disappeared in cells treated with cytochalasin D. These results indicates that AFs have significant effects on viscosity of SMCs in the slow phase and on the fluctuation in tension, both of which may be caused by the dynamic change of AFs.

Keywords

Cellular Biomechanics, Mechanical Properties, Micromanipulation, Normalized Tension, Actin Filament Reorganization

Paper information

Kazuaki NAGAYAMA, Shinichiro YANAGIHARA and Takeo MATSUMOTO, “Actin Filaments Affect on Not Only Elasticity But Also Late Viscous Response in Stress Relaxation of Single Isolated Aortic Smooth Muscle Cells”, Journal of Biomechanical Science and Engineering, Vol. 2, No. 3 (2007), pp.93-104 . doi:10.1299/jbse.2.93