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.

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

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

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

Diffuse axonal injury (DAI) is a specific type of closed head injury often seen in automobile accidents, that directly leads to the morbidity and mortality, however, the injury mechanism of DAI has yet to be clarified. DAI is characterized by structural and functional damage in nerve fibers in the white matter, which may be caused by excessive tensile strain. While the white matter has a network-like structure of nerve fibers embedded in neuroglia and the extracellular matrix, the nerve fibers are undulated and the mechanical properties of these components are not necessarily equal. Thus, the strain in the white matter can be different from that in the fibers. In this study, we have measured stretch ratios of the nerve fibers running in various directions in porcine brain tissue subjected to uniaxial stretch and compared them with global strain. It was found that the fiber direction positively correlated with neural fiber strain whilst the fiber strain was not equal to global strain. Particularly, the maximum neural fiber strain was ?25% of its surrounding tissue strain, indicating that the local strain in the neural fibers is not equal to global strain in the brain tissue. Consideration of neural fiber alignment in the white matter is important in studying the mechanical aspects of pathogenesis of DAI.

Keywords

Nerve Fiber, Brain Tissue, Stretch Ratio, Uniaxial Tension, Diffuse Axonal Injury

Paper information

Atsutaka TAMURA, Kazuaki NAGAYAMA and Takeo MATSUMOTO, “Measurement of Nerve Fiber Strain in Brain Tissue Subjected to Uniaxial Stretch (Comparison Between Local Strain of Nerve Fiber and Global Strain of Brain Tissue)”, Journal of Biomechanical Science and Engineering, Vol. 1, No. 2 (2006), pp.304-315 . doi:10.1299/jbse.1.304

We established a quasi-in situ tensile test to measure the tensile properties of smooth muscle cells (SMCs) cultured on substrate maintaining their shape and cytoskeletal integrity. SMCs were cultured on a substrate coated with thermoresponsive gelatin (PNIPAAm-gelatin) and were held with a pair of micropipettes coated with an adhesive. Cells were detached from the substrate by lowering ambient temperature to dissolve the PNIPAAm-gelatin. Tensile tests for fusiform SMCs up to ?15% strain performed 3 times in normal and Ca2+-free Hank's balanced salt solution (HBSS(+) and HBSS(-), respectively) in order to investigate the effects of Ca2+ on the change in their tensile properties during loading/unloading cycles. The stiffness of the fusiform SMCs obtained by the first loading process in HBSS(-) and in HBSS(+) was 0.041±0.024 N/m (n=6, mean±SEM) and 0.031±0.008 N/m (n=6), respectively, and was significantly lower than that of spherical cells detached from the substrate by trypsinization (?0.09 N/m), indicating that cell stiffness is overestimated when cells are trypsinized. Cell stiffness increased from the first cycle to the second and then stabilized in HBSS(-), while it increased continuously with the number of the cycles in HBSS(+). These results suggest that the mechanical properties of SMCs change with stretching and that extracellular Ca2+ has a significant effect on their response to stretch.

Keywords

Cellular Biomechanics, Micromanipulation, Internal Tension, Cyclic Tensile Properties, Actin Filaments

Paper information

Kazuaki NAGAYAMA, Akira TSUGAWA and Takeo MATSUMOTO, “Tensile Properties of Cultured Aortic Smooth Muscle Cells Obtained in a quasi-in situ Tensile Test with Thermoresponsive Gelatin”, Journal of Biomechanical Science and Engineering, Vol. 1, No. 1 (2006), pp.256-267 . doi:10.1299/jbse.1.256