Tadashi Kosawada

Mechanics of curved plasma membrane vesicles: resting shapes, membrane curvature, and in-plane shear elasticity.

Highly curved cell membrane structures, such as plasmalemmal vesicles (caveolae) and clathrin-coated pits, facilitate many cell functions, including the clustering of membrane receptors and transport of specific extracellular macromolecules by endothelial cells. These structures are subject to large mechanical deformations when the plasma membrane is stretched and subject to a change of its curvature. To enhance our understanding of plasmalemmal vesicles we need to improve the understanding of the mechanics in regions of high membrane curvatures. We examine here, theoretically, the shapes of plasmalemmal vesicles assuming that they consist of three membrane domains: an inner domain with high curvature, an outer domain with moderate curvature, and an outermost flat domain, all in the unstressed state. We assume the membrane properties are the same in these domains with membrane bending elasticity as well as in-plane shear elasticity. Special emphasis is placed on the effects of membrane curvature and in-plane shear elasticity on the mechanics of vesicle during unfolding by application of membrane tension. The vesicle shapes were computed by minimization of bending and in-plane shear strain energy. Mechanically stable vesicles were identified with characteristic membrane necks. Upon stretch of the membrane, the vesicle necks disappeared relatively abruptly leading to membrane shapes that consist of curved indentations. While the resting shape of vesicles is predominantly affected by the membrane spontaneous curvatures, the membrane shear elasticity (for a range of values recorded in the red cell membrane) makes a significant contribution as the vesicle is subject to stretch and unfolding. The membrane tension required to unfold the vesicle is sensitive with respect to its shape, especially as the vesicle becomes fully unfolded and approaches a relative flat shape.

Characterizing and modulating the mechanical properties of hydrogels from ventricular extracellular matrix

In order to differentiate pluripotent stem cells to cardiomyocytes, the most general method is to expose stem cells to various growth factors related to cardiogenesis. However, a novel method has been reported to induce cardiac differentiation of human ES cells without supplemental growth factors by culturing embryoid body of human ES cells in hybrid gels composed of cardiac extracellular matrix (ECM) and type I collagen. On the other hand, mechanical properties of scaffold is one of the critical cue for differentiation of stem cells. However, it has not been thoroughly investigated the mechanical properties of the scaffold made from cardiac ECM in view of this and other reports about the differentiation of stem cells into cardiomyocytes using cardiac ECM scaffold. In this study, we fabricated bio-hydrogels composed of goat ventricular extracellular matrix, and investigated the mechanical properties by means of uniaxial compression test. It showed that the ECM gels possess viscoelastic property. The elastic modulus K1 in modified non-linear Kelvin model is 9.5 Pa for these gels and K2 is 814.7 Pa. Moreover, we were able to improve the elastic moduli K1 and K2 up to 139.7 Pa and 2023.9 Pa, respectively, by chemical treatment using EDAC.

Analysis of the contraction of fibroblast-collagen gels and the traction force of individual cells by a novel elementary structural model

Based on the experimental data of the contraction ratio of fibroblast-collagen gels with different initial collagen concentrations and cell numbers, we analyzed the traction force exerted by individual cells through a novel elementary structural model. We postulate that the mechanical mechanism of the gel contraction is mainly because that populated cells apply traction force to some of the surrounding collagen fibrils with such proper length potential to be pulled straight so as to be able to sustain the traction force; this traction induce the cells moving closely to each other and consequently compact the fibrillar network; the bending force of the fibrils in turn resists the movement. By employing fiber packing theory for random fibrillar networks and network alteration theory, the bending force of collagen fibrils was deduced. The traction force exerted by individual fibroblasts in the gels was balanced by the bending force and the resistance from interstitial fluid since inertial force can be neglected. The maximum traction force per cell under free floating condition is in the range of 0.27-9.02 nN depending on the initial collagen concentration and populated cell number. The most important outcome of this study is that the traction force of individual cells dynamically varies under different gel conditions, whereas the adhesion force between cell and individual fibrils is relatively converging and stable.

Stress relaxation and stress-strain characteristics of porcine amniotic membrane

BACKGROUND Recently, amniotic membrane (AM) as scaffold is accumulating much more attention in tissue engineering. It is well-known that the mechanical properties of the scaffold inevitably affect the biological process of the incorporated cells. OBJECTIVE This study investigates the stress relaxation and stress-strain characteristics of AM, which have not been sufficiently elucidated before. METHODS Porcine AM samples were prepared at four different AM regions and at three different directions. Ramp-and-hold and stretch-to-rupture tests were conducted on a uniaxial tensile apparatus. A nonlinear viscoelastic model with two relaxation coefficients is proposed to fit the ramp-and-hold data. Rupture strain, rupture stress, and elastic modulus of the linear portion of the stress-strain curve are used to characterize the strength properties of the AM. RESULTS Sample direction has no significant effect on the mechanical properties of the AM. Samples at the ventral region has the maximum rupture strength and elastic modulus, respectively, 2.29±0.99MPa and 6.26±2.69MPa. The average of the relaxation coefficient for the fast and slow relaxation phases are 12.8±4.4s and 37.0±7.7s, respectively. CONCLUSIONS AM is a mechanically isotropic and heterogeneous material. The nonlinear viscoelastic model is suitable to model the AM viscoelasticity and potential for other biological tissues.

Expression of microRNA-1, microRNA-133a and Hand2 protein in cultured embryonic rat cardiomyocytes

In this study, we investigated the expression of the pathway, SRF–microRNA-1/microRNA-133a–Hand2, in the Wistar rat embryonic ventricular cardiomyocytes under conventional monolayer culture. The morphological observation of the cultured cardiomyocytes and the mRNA expression levels of three vital constituent proteins, MLC-2v, N-cadherin, and connexin43, demonstrated the immaturity of these cultured cells, which was featured by less myofibril density, immature sarcomeric structure, and significantly lower mRNA expression of the three constituent proteins than those in neonatal ventricular samples. More importantly, results in this study suggest that the change of SRF–microRNA-1/microRNA-133a–Hand2 pathway results into the attenuation of the Hand2 repression in cultured cardiomyocytes. These outcomes are valuable to understand the cellular state as embryonic cardiomyocytes to be in vitro model and might be useful for the assessment of engineered cardiac tissue and cardiac differentiation of stem cells.

Non-invasive Stiffness Detection Method for Living Cell Nucleus by Using Piezoelectric Micro Sensor

Cell nucleus is a body including various components and it has complicated and heterogeneous structure. Though most functions of nuclear domains have been well studied, mechanical properties of the nuclear domains has not been studied. In this study, the experimental system was developed to measure the stiffness of living cell nucleus in order to verify the inhomogeneity of the cell nucleus. The system is composed of two main devices: a sensor which can measure stiffness of micro cell nucleus and a rotatable device which can rotate nucleus horizontally. The devices are assembled on the stage of an inverted phase contrast microscope. Experimental studies have been carried out by using normal human osteoblast. The method has shown capability to detect difference of stiffness on cell nucleus. There is significant difference between nucleolus and other nuclear domains.

Dynamic Analysis of Circular Engineered Cardiac Tissue to Evaluate the Active Contractile Force

Circular engineered cardiac tissue was fabricated by embedding rat embryonic cardiomyocytes into collagen (type I) gels. The engineered tissue was set to a specific configuration and the spontaneous beat displacement at one site of it was measured. The active contractile force of the embedded cardiomyocytes was derived from the displacement data. In this process, the engineered tissue was constitutively modeled as three components in parallel: i.e., an active contractile component representing the cardiomyocyte contraction, a pre-force component representing the effects of gel compaction during the tissue fabrication, and a Kelvin model for the passive properties of the tissue. Dynamic analysis of the beat displacement allowed solving out the active contractile force. In addition, energy coefficient was defined to evaluate the pump function of the engineered tissue. It demonstrated that this approach can detect the active contractile force as small as ~0.01 mN and can sensitively reveal the change of the active contractile force under different culture conditions. Besides being an assay to evaluate the mechanical performance of engineered cardiac tissue, this novel method is particularly suitable to be used in pharmacological response testing of stem cell-derived cardiomyocytes under three-dimensional culture attributed to its high sensitivity and feasibility for continuous and in situ measurement.

Novel Three-dimensional Micro Vibration Stage and Its Ability to Influence in Cellular Senescence

Although most cells are facing to mechanical stimuli, environment of cultured cells in an ordinary CO2 incubator may be inappropriate, because these cultured cells are normally free from mechanical stimuli. In this study, a novel three-dimensional micro vibration stage is developed to stimulate cultured cells mechanically. The simple two times twisted v-shaped steric structure of the stage makes possible to excite a culture dish three-dimensionally. By using the stage, the effect of vibration stimulation to adhesive cells was morphologically investigated. The experimental result using normal human osteoblast shows that the vibration stimulation decreases the projected area and increases the slenderness ratio of the cells. It suggests that the dynamic stimulation inhibits the morphological change according to progression of cellular senescence. A newly defined parameter, anti-aging effect, is proposed to describe the effect of the dynamic stimulation. It is found that the most effective vibration direction and frequency is unidirectional, horizontal and 10 Hz.

Three-Dimensional Micro Vibration Stage and Its Application to Cell Culture

One of the important roles of cytoskeleton is to maintain global mechanical strength of the cell. It is expected the effect of environmental mechanical stimulations appears in cell morphology. An environment of cultured cells in an ordinary incubator might be inappropriate, because those are normally free from mechanical stimulations such as fluid flow shear stress, tensile strain, compression and vibration. In this study, a three-dimensional micro vibration stage, to exert vibration stimulation non-invasively and three-dimensionally onto cultured cells, is developed. This vibration stage is assumed to be installed and operated in a CO2 incubator, so it has a compact and simple cantilever structure. In order to excite vibrations in each direction of orthogonal coordinate system, it has made from one stainless steel strip into a V-shaped vibrator, which is kinetically designed. Three piezoelectric ceramics were bonded on the vibrator, so that it is able to apply the three-dimensional vibration stimulation onto the cells on the culture dish. By using the developed stage, the effect of vibration stimulation was investigated morphologically. The experimental result shows that the vibration stimulation decreases the projected area and increases the slenderness ratio. In other words, the dynamic stimulation inhibits the progression of morphological change according to cell senescence. This might suggest that the stimulation affects gene expression pattern. Also, the most effective condition of vibration was horizontal direction of 10 Hz. It is found that the developed micro vibration stage is quite useful as a control device for cell culture.

Mechanics of Plasma Membrane Vesicles in Cells

The mechanical characteristics of biological membranes are of fundamental importance in cellular biology. Many cellular processes, such as endocytosis, exocytosis and cell fusion, are strongly involved with large mechanical deformations of the membrane accompanied by changes in curvature [1]. One of the remarkable features in this respect are local membrane regions with high curvature, such as the clathrin-coated pits, vesicles, chained vesicles and channels which facilitate the cell to transport specific macromolecules. In this study, mechanics of plasma membrane vesicles in cells have theoretically been investigated based on minimization of bending and in-plane shear strain energy of the membrane. Effects of outer surrounding cytoplasmic flat membrane upon mechanically stable shapes of the vesicles were revealed as well as the effects of the in-plane shear elasticity.