Biomechanics at Micro- and Nanoscale Levels

The following sections are included:

• PREFACE

• CONTENTS

High sensitivity of human hearing is believed to be achieved by cochlear amplification. The basis of this amplification is thought to be the motility of mammalian outer hair cells (OHCs), i.e., OHCs elongate and contract in response to acoustical stimulation. This motility is thought to be based on voltage-dependent conformational changes of a motor protein embedded in the lateral wall of the OHC. In 2000, this motor protein was identified and termed prestin. Since identification of prestin, intensive research has been done using prestin-expressing cells to elucidate the function of prestin. However, the motor function of prestin at the molecular level is still unclear. To obtain knowledge about the function of prestin, it should be studied using not only prestin in cells, but also prestin in solution. For this purpose, a method of obtaining a large amount of prestin as material is required. In this study, an attempt was therefore made to construct an expression system for prestin. Prestin cDNA was introduced into Escherichia coli (E. coli), insect cells and Chinese hamster ovary (CHO) cells, and the expression of prestin was examined by Western blotting. As CHO cells expressed prestin well, we generated prestin-expressing cell lines using CHO cells by limiting dilution cloning. The stable expression, the N-linked glycosylation and the activity of prestin in generated cell lines were then confirmed.

Actin filaments have been reported to be ∼1000 times stiffer than whole cells. Thus, they must play major roles in mechanical properties of cells. Effects of actin filament distribution and alignment on anisotropy and stiffness were studied on rat aortic smooth muscle cells. Freshly isolated cells (FSMCs), cultured cells (CSMCs), and CSMCs treated with cytochalasin D to disrupt their actin filaments (CSMCs-CYD) were stretched with an originally designed micro tensile tester. FSMCs were stretched in their major and minor axes directions to evaluate their anistropy. The normalized stiffness of FSMCs was significantly higher in the major axis (14.8±4.3kPa, mean±SEM, n=5) than the minor axis (2.8±1.0kPa, n=5). Long and thick fibers of actin filament were found running almost parallel to the major axis of the cells, indicating increased stiffness in this direction. CSMCs and CSMCs-CYD were stained for actin filament after the tensile test while they remained attached to the tester. Relative concentration of the actin filament in the central region of the cell F had significant positive correlation with normalized stiffness in both cells. These results indicate that elastic properties of smooth muscle cells are affected not only by the amount of their actin filaments, but also by their organization and distribution in cells.

Bovine pulmonary microvascular endothelial cells were seeded onto collagen gels with basic fibroblast growth factor (bFGF) to make a micro-vessel formation model. We observed this model in detail using phase contrast microscopy, confocal laser scanning microscopy and electron microscopy. The results show that cells invaded the collagen gel and reconstructed the tubular structures, containing a clearly defined lumen consisting of multiple cells. The model was placed in a parallel-plate flow chamber. A laminar shear stress of 0.3 Pa was applied to the surfaces of the cells for 48 hours. Promotion of micro-vessel network formation was detectable after approximately 10 hours in the flow chamber. After 48 hours, the length of networks exposed to shear stress was 6.17 (± 0.59) times longer than at the initial state, whereas the length of networks not exposed to shear stress was only 3.30 (± 0.41) times longer. The number of bifurcations and endpoints increased for networks exposed to shear stress, whereas the number of bifurcations alone increased for networks not exposed to shear stress. These results demonstrate that shear stress applied to the surfaces of endothelial cells on collagen gel promotes the growth of micro-vessel network formation in the gel and expands the network due to repeated bifurcation and elongation.

The excellent performance of very low friction and long durability of natural synovial joints is maintained with synergistic metabolism of articular cartilage and synovial fluid. To study metabolic mechanism including repair process, it is important to know the stress-strain state of the cartilage and extracellular matrix around chondrocytes during cartilage deformation, where chondrocytes seem to experience similar deformation to extracellular matrix. The articular cartilage of high water content has a biphasic viscoelastic property and shows the time-dependent deformation behavior. In this study, the compressive behaviors in articular cartilage containing chondrocytes were observed in the compressive apparatus located in the stage of confocal laser scanning microscope. The time-dependent behavior of articular cartilage was evaluated by the finite element method for biphasic model.

In reorganization process of the actin stress fibers, a change in its mechanical condition has been suggested as one of the key mediators. Some experimental studies have clarified that tension release in the stress fibers induces the fiber depolymerization that is considered as the initial phase of its reorganization. However, quantitative mechanical values such as strain or stress that induce the depolymerization still have not been evaluated. This study tried to quantify the fiber strain that induces the depolymeridzaion of the actin stress fibers to gain a basic understanding of the reorganization phenomenon from a mechanical viewpoint. Osteoblastic cells were cultured on prestretched silicone rubber substrate. Compressive deformation was applied to the cells by uniaxially releasing the prestretched substrate strain and the change of the stress fiber structure was observed. The results indicated that the compressive strain, not in the whole cell body but in the stress fiber itself, is important to induce the disassembly of the fiber structure. In addition, the existence of a threshold strain for initiating fiber disassembly was suggested.

Morphological responses of cultured bovine endothelial cells (ECs) exposed to hydrostatic pressure were investigated. ECs were exposed to physiological blood pressure under a hydrostatic head of culture medium for 24 h. Pressured ECs exhibited marked elongation and orientation with the random direction, together with development of centrally located, thick stress fibers. Pressured ECs also exhibited multilayered structure unlike under control conditions. The area and the shape index value significantly decreased after exposure to hydrostatic pressure, which was in good agreement with the results from conventional flow-imposed experiments. In contrast, a tortuosity index, which was newly introduced to represent cell shape tortuosity, significantly increased for pressured ECs, while sheared ECs had no difference in turtuosity index from control. In addition, pressured ECs aligned with no predominant direction, while sheared ECs aligned in the flow direction. These results indicate that ECs can respond very specifically to the type of imposed mechanical stimuli such as hydrostatic pressure and fluid shear stress.

Uptake of food, water and nutrients may cause mechanical stress on the intestinal villi that regulates villous and intestinal motilities and digestive functions. However, the mechanism of mechanosensing in intestine is not yet clear. We pay attention a cellular system, subepithelial fibroblasts, that form a cellular network just under the epithelium of the gastrointestinal tract. Using primary cultured cells isolated from rat duodenal villi, we previously found that subepithelial fibroblasts had many kinds of receptors for vaso- and neuro-active substances, such as endothelins (ETs), ATP, serotonin, and that they reversibly changed cell morphology between flat and stellate-shape depending on intracellular cAMP levels. Recently we found that subepithelial fibroblasts were sensitive to mechanical stress such as “touching” and “stretching”. Mechanical stimulations evoked Ca²⁺-increase in the cells and ATP-release from the cells. The released ATP activated P2Y1 type ATP receptors on the surrounding cells and propagated Ca²⁺-waves through the network. Concomitant with Ca²⁺-waves, a transient contraction of the network was observed. ATP-release and Ca²⁺ signaling were cell-shape dependent, i.e., they were abolished in stellate-shaped cells treated with dBcAMP, and recovered or further enhanced in re-flattened cells treated with ETs. From these unique properties, we propose that subepithelial fibroblasts work as a mechano-sensor in the intestine, and as a regulator of mechanical property and movement of the villi.

Progress in both cell biology and biomaterial technology has led to the possibility of therapeutic applications of tissue engineering for the repair of cartilage defects. The regulation of cell differentiation and the cell source have been a big challenge for this tissue engineering approach. The re-aggregate approach is an attempt to achieve a more or less complete regeneration of tissues from dispersed cells of a particular origin, under controlled culture conditions. Mesenchymal stem cells (MSCs) are expected to be a useful cell souse for cartilage tissue engineering. In order to explore the feasibility of applying these cells, MSC aggregates mixed with chondrocytes were examined. For regulating cell differentiation and forming micro-tissue element of cartilage, rotational culture system was innovated. Rapid and large-scale “microelements of cartilage” were formed in this system.

It is suspected that due to the presence of a filtration flow of water at the vessel wall, concentration polarization of low-density lipoproteins (LDL) occurs at the luminal surface of an implanted graft, leading to an increase in the uptake of LDL by the cells forming a pseudointima, rapid proliferation of the cells, and eventual development of intimal hyperplasia in the graft. Hence we have studied the interrelationship between water filtration velocity at the vessel wall measured before and after implantation of the graft and the thickness of a pseudointima formed in the graft by implanting three kinds of artificial grafts having an approximately the same diameter (3.0 mm) but very different water permeability at the vessel wall into the common carotid artery of the dog and harvesting them at different times postoperatively. It was found that the greater the water filtration velocity measured before and after implantation of the graft, the greater the thickness of the pseudointima formed in the graft, suggesting the impotance of the filtration flow of water which cause concentration polarization of LDL at the luminal surface of the implanted graft in the development of intimal hyperplasia.

The effects of mechanical stimuli to the fibrous tissues were investigated utilizing the collagen gel culture method. A specimen of thin collagen gel membrane, in which fibroblasts were proliferating and was supported by stainless steel wire mesh, was cultured in medium and subjected to static or repeated dynamic uniaxial tensile/compressive load in an incubator. The cell alignment and the shrinkage of collagen gel matrix were observed. Furthermore, mechanical tests were carried out under microscope and changes in mechanical properties of the specimens were measured. We found that both of static and dynamic loads strengthened the collagen matrix, however, the dynamic stimuli were concluded to be more effective in matrix strengthening than the static stimuli.

Monochromatic synchrotron radiation (SR) allows ultrahigh-resolution computed tomogtaphy (CT) and accurate density measurements. The purpose of this study was to demonstrate the utility of monochromatic SRCT in microstructural analysis of cortical bone. Tibial diaphyses of growing rat (14 weeks, n=8) undergoing unilateral sciatic neurectomy 8 weeks ago were imaged with 5.83–μm voxel resolution by 20-keV SRCT at the synchrotron radiation facility (SPring-8). Reconstructed image data were translated into local mineral densities using a calibrated linear relationship between linear absorption coefficients and concentrations of homogeneous K₂HPO₄ solution. After bone segmentation by a simple thresholding, pure bone 3D images were analyzed for macro- and microscopic structural properties. In neurectomized hindlimbs, the cortical canal network rarefaction as well as the bone atrophy were found. The former was characterized by 30% smaller porosity, 11% smaller canal density in transverse section, and 38% smaller canal link density than those in the contralateral bone, implying the reduced bone microcirculation in the disuse-induced atrophic tibia. On the other hand, no difference was found in the bone mineral density between neurectomized and intact hindlimbs. In conclusion, the SRCT is a promising method for the 3D analysis of cortical microstructure.

To study the microcirculation for the analysis of thrombosis and haemolytic anaemia, fluid (plasma) flow and the motion of cellular components such as the red blood cell (RBC) and platelets must be included in the model. Vascular wall should also be included when the mechanical and biological interactions between the wall and cells are of concern. In the microvascular region, the cells and the plasma must be separately modeled because the length scale of the cellular components is comparable, sometimes larger, than the vascular diameter. To model the mechanical interactions between the cell and the wall, including their collisions, highly deformable nature of the cellular membrane must be represented in the model. In this study, a new computer simulation using particle method was proposed to analyze microscopic behavior of blood flow. A simulation region including plasma, red blood cells (RBCs) and platelets was modeled by an assembly of discrete particles. The proposed method was applied to the motion and deformation of single RBC and multiple RBCs, and the thrombogenesis process due to platelet aggregation.

Skeletal muscle models for computer simulations are reviewed, and special emphasis is located on models taking into account a three-dimensional architecture and microscopic structure of skeletal muscle. Then a model describing damage as well as deformation is proposed by taking into account a three-dimensional architecture and microscopic structure and by modifing the model in literature. It is ascertained that the proposed model describes damage properties of muscle qualitatively.

We made theoretical predictions of the orientation of actin stress fibers (SFs) in a substrate-attached endothelial cell subjected to cyclic stretch within a constant range of strains. Taking account of polymerization/depolymerization of actin monomers and binding of SFs to adjacent actin filaments (AFs), we simulated the formation of SFs at the basal membrane. The numerical simulations predicted that AFs would be bundled but remain crossed with other AFs, which suggested that an additional mechanism was needed for the model to reproduce the actual distribution of AFs. Next, we validated two working hypotheses that defined the maximum strain in a SF during deformation as either the strain measured at maximal substrate deformation or the strain measured at maximal compression or stretching. Nonuniform cell deformation was also incorporated into the model. Under certain conditions of cyclic stretching of SFs for which one end of the fiber was attached to the apical membrane, the different measures of maximum strain produced clear differences in the position of the other end of the fiber.

The following sections are included:

• SUBJECT INDEX