Modeling myosin-dependent rearrangement and force generation in an actomyosin network.

Actomyosin contractility is a major force-generating mechanism that drives rearrangement of actomyosin networks; it is fundamental to cellular functions such as cellular reshaping and movement. Thus, to clarify the mechanochemical foundation of the emergence of cellular functions, understanding the relationship between actomyosin contractility and rearrangement of actomyosin networks is crucial. For this purpose, in this study, we present a new particulate-based model for simulating the motions of actin, non-muscle myosin II, and α-actinin. To confirm the model's validity, we successfully simulated sliding and bending motions of actomyosin filaments, which are observed as fundamental behaviors in dynamic rearrangement of actomyosin networks in migrating keratocytes. Next, we simulated the dynamic rearrangement of actomyosin networks. Our simulation results indicate that an increase in the density fraction of myosin induces a higher-order structural transition of actomyosin filaments from networks to bundles, in addition to increasing the force generated by actomyosin filaments in the network. We compare our simulation results with experimental results and confirm that actomyosin bundles bridging focal adhesions and the characteristics of myosin-dependent rearrangement of actomyosin networks agree qualitatively with those observed experimentally.

Direct 3-D morphological measurements of silicone rubber impression using micro-focus X-ray CT.

Three-dimensional computer models of dental arches play a significant role in prosthetic dentistry. The microfocus X-ray CT scanner has the advantage of capturing precise 3D shapes of deep fossa, and we propose a new method of measuring the three-dimensional morphology of a dental impression directly, which will eliminate the conversion process to dental casts. Measurement precision and accuracy were evaluated using a standard gage comprised of steel balls which simulate the dental arch. Measurement accuracy, standard deviation of distance distribution of superimposed models, was determined as +/-0.050 mm in comparison with a CAD model. Impressions and casts of an actual dental arch were scanned by microfocus X-ray CT and three-dimensional models were compared. The impression model had finer morphology, especially around the cervical margins of teeth. Within the limitations of the current study, direct three-dimensional impression modeling was successfully demonstrated using microfocus X-ray CT.

Nanoscale crack initiation and propagation in carbon fiber/epoxy composites using synchrotron: 3D image data.

Crack initiation and propagation in carbon fiber-reinforced plastic (CFRP) was observed in situ under the application of an opening load using nanoscopic synchrotron radiation X-ray computed tomography (nanoscopic SR X-CT) at a high spatial resolution of ∼50 nm. Two datasets of reconstructed and segmented images were produced in typical regions, namely in the thin and thick epoxy regions where the resin thickness between the adjacent carbon fibers was small and large, respectively. This novel study presents the first non-destructive three-dimensional (3D) visualization of resin deformation behavior around crack tips, and provides a valuable and unique insight for the future design of CFRPs.

Dynamic coupling between actin network flow and turnover revealed by flow mapping in the lamella of crawling fragments.

Dynamic turnover and transport of actin filament network is essential for protrusive force generation and traction force development during cell migration. To elucidate the dynamic coupling between actin network flow and turnover, we focused on flow dynamics in the lamella of one of the simplest but elegant motility systems; crawling fragments derived from fish keratocytes. Interestingly, we show that actin network in the lamella of fragments is not stationary as earlier reported, but exhibits a flow dynamics that is strikingly similar to that reported for higher order cells, suggesting that network flow is an intrinsic property of the actin cytoskeleton that is fundamental to cell migration. We also demonstrate that whereas polymerization mediates network assembly at the front, surprisingly, network flow convergence modulates network disassembly toward the rear of the lamella, suggesting that flow and turnover are coupled during migration. These results obtained using simple motility systems are significant to the understanding of actin network dynamics in migrating cells, and they will be found useful for developing biophysical models for elucidating the fundamental mechanisms of cell migration.

Asymmetric intercellular communication between bone cells: propagation of the calcium signaling.

Bone functional adaptation by remodeling is achieved by harmonized activities of bone cells in which osteocytes in the bone matrix are believed to play critical roles in sensing mechanical stimuli and transmitting signals to osteoclasts/osteoblasts on the bone surface in order to regulate their bone remodeling activities through the lacuno-canalicular network with many slender osteocytic processes. In this study, we investigated the intercellular communication between bone cells, particularly focusing on its directionality, through in vitro observations of the calcium signaling response to mechanical stimulus and its propagation to neighboring cells (NCs). Direct mechanical stimulus was applied to isolated bone cells from chick calvariae, osteocytes (Ocys) and bone surface cells (BSCs) mainly containing osteoblasts, and the percentage of calcium signaling propagation from the stimulated cell to NCs was analyzed. The results revealed that, regardless of the type of stimulated cell, the signaling propagated to BSCs with a significantly higher percentage, implying that calcium signaling propagation between bone cells strongly depends on the type of receiver cell and not the transmitter cell. In addition, in terms of mutual communication between Ocys and BSCs, the percentage of propagation from Ocys to BSCs is significantly higher than that in the opposite direction, suggesting that the calcium signaling mainly propagates asymmetrically with a bias from Ocys in bone matrix to BSCs on bone surfaces. This asymmetric communication between Ocys and BSCs suggests that osteocytic mechanosensing and cellular communications, which significantly affect bone surface remodeling activities to achieve functional adaptation, seem to be well coordinated and active at the location of biologically suitable and mechanically sensitive regions close to the bone surfaces.

Strain field in actin filament network in lamellipodia of migrating cells: implication for network reorganization.

Cell motility is spatiotemporally regulated by interactions among mechanical and biochemical factors involved in the regulation of cytoskeletal actin structure reorganization. Although the molecular mechanisms underlying cell motility have been well investigated, the contributions of mechanical factors such as strain in the network reorganization remain unclear. In this study, we have quantitatively evaluated the strain field in the actin filament network forming the lamellipodia of migrating fish keratocytes to elucidate the mechanism by which actin filament network reorganization is regulated by biomechanical factors. The results highlight the existence of a negative (compressive) strain in the lamellipodia whose direction is parallel to that of cell movement. A close correlation was found between the distributions of the strain and the actin filament density in the lamellipodia, suggesting that negative strain may be involved in filament depolymerization. Based on this result, we propose a selective depolymerization model which suggests that negative strain may couple with biomechanical factors such as ADF/cofilin to promote selective depolymerization of filaments oriented in the direction of the deformation because such filaments experience relatively higher levels of the deformation. This model, in conjunction with others, may explain the observed reduction in filament density and the reorganization of actin filament network at the back of the lamellipodia of migrating fish keratocytes. Thus, we suggest that by coupling with biochemical factors, mechanical factors are involved in the regulation of actin filament depolymerization, thereby contributing to the regulation of cell motility.

Wall boundary model for primitive chain network simulations.

In condensed polymeric liquids confined in slit channels, the movement of chains is constrained by two factors: entanglement among the chains and the excluded volume between the chains and the wall. In this study, we propose a wall boundary (WB) model for the primitive chain network (PCN) model, which describes the dynamics of polymer chains in bulk based on coarse graining upon the characteristic molecular weight of the entanglement. The proposed WB model is based on the assumptions that (i) polymers are not stuck but simply reflected randomly by the wall, and (ii) subchains below the entanglement length scale behave like those in bulk even near the wall. Using the WB model, we simulate the dynamics of entangled polymer chains confined in slit channels. The results show that as the slit narrows, the chains are compressed in the direction normal to the wall, while they are expanded in the parallel direction. In addition, the relaxation time of the end-to-end vector increases, and the diffusivity of the center of mass decreases. The compression in the normal direction is a natural effect of confinement, while the expansion is introduced by a hooking process near the wall. The trends revealed that the relaxation time and diffusivity depend on the increase in friction due to an increased number of entanglements near the wall, which is also associated with the hooking process in the PCN model. These results are expected within the assumptions of the PCN model. Thus, the proposed WB model can successfully reproduce the effects of wall confinement on chains.

Measurement of local strain on cell membrane at initiation point of calcium signaling response to applied mechanical stimulus in osteoblastic cells.

In adaptive bone remodeling, it is believed that bone cells such as osteoblasts, osteocytes and osteoclasts can sense mechanical stimuli and modulate their remodeling activities. However, the mechanosensing mechanism by which these cells sense mechanical stimuli and transduce mechanical signals into intracellular biochemical signals is still not clearly understood. From the viewpoint of cell biomechanics, it is important to clarify the mechanical conditions under which the cellular mechanosensing mechanism is activated. The aims of this study were to evaluate a mechanical condition, that is, the local strain on the cell membrane, at the initiation point of the intracellular calcium signaling response to the applied mechanical stimulus in osteoblast-like MC3T3-E1 cells, and to investigate the effect of deformation velocity on the characteristics of the cellular response. To apply a local deformation to a single cell, a glass microneedle was directly indented to the cell and moved horizontally on the cell membrane. To observe the cellular response and the deformation of the cell membrane, intracellular calcium ions and the cell membrane were labeled using fluorescent dyes and simultaneously observed by confocal laser scanning microscopy. The strain distribution on the cell membrane attributable to the applied local deformation and the strain magnitude at the initiation point of the calcium signaling responses were analyzed using obtained fluorescence images. From two-dimensionally projected images, it was found that there is a local compressive strain at the initiation point of calcium signaling. Moreover, the cellular response revealed velocity dependence, that is, the cells seemed to respond with a higher sensitivity to a higher deformation velocity. From the viewpoint of cell biomechanics, these results provide us a fundamental understanding of the mechanosensing mechanism of osteoblast-like cells.

Effects of post-training lesions in the hippocampus and the parietal cortex on idiothetic information processing in the rat.

Dead reckoning can be defined as the ability to navigate using idiothetic information based on self-movement cues without using allothetic information such as environmental cues. In the present study, we investigated the effects of hippocampal and parietal cortex lesions on homing behavior using dead reckoning in rats. Experimentally naive Wistar rats were trained with a homing task in which rats were required to take a food pellet from a cup in the arena and to return home with the pellet. After training, rats were divided into a control (CONT) group (n = 16), hippocampal lesioned (HIPP) group (n = 16), and parietal cortex lesioned (PARC) group (n = 16), and rats in the lesioned groups underwent surgery. After surgery, Test 1 (with four cups) and Test 2 (with one cup but the outgoing path was diverted by a barrier) were conducted. The HIPP group showed severe impairment in homing, but the performance of the PARC group did not differ from that of the CONT group. HIPP rats either approached wrong doors or ate the pellet in the arena. Circular statistics showed that homing directions of CONT and PARC rats showed concentration towards home, whereas those of HIPP rats did not. Our results exhibiting HIPP rats' failure in homing agree with many previous studies, but the results obtained from PARC rats were different from previous studies. These results indicate that the intact hippocampus is important for dead reckoning, but the role of the parietal cortex in dead reckoning is still not clear.

Framework for optimal design of porous scaffold microstructure by computational simulation of bone regeneration.

In bone tissue engineering using a biodegradable scaffold, geometry of the porous scaffold microstructure is a key factor for controlling mechanical function of the bone-scaffold system in the regeneration process as well as after the regeneration. In this study, we propose a framework for the optimal design of the porous scaffold microstructure by three-dimensional computational simulation of bone tissue regeneration that consists of scaffold degradation and new bone formation. The rate of scaffold degradation due to hydrolysis, that leads to decrease in mechanical properties, was simply assumed to relate to the water content diffused from the surface to the bulk material. For the new bone formation on both bone and scaffold surfaces, the rate equation of trabecular surface remodeling driven by mechanical stimulation was applied. Solving these two phenomena in the same time frame, the bone regeneration process in the bone-scaffold system was predicted by computational simulation using a voxel finite element method. The change in the mechanical function of the bone-scaffold system during the regeneration process was quantitatively evaluated by measuring the change in total strain energy, and this was used for the evaluation function to optimize the scaffold microstructure that provides the desired mechanical function during and after the bone regeneration process. A case study conducted for the scaffold with a simple microstructure demonstrated that the proposed simulation method could be applied to the design of a porous scaffold microstructure. In addition, the regeneration process was found to be very complex even though the simple rate equations for scaffold regeneration and new bone formation were used because of the coupling effects of these phenomena.

Effects of loading frequency on the functional adaptation of trabeculae predicted by bone remodeling simulation.

The process of bone remodeling is regulated by metabolic activities of many bone cells. While osteoclasts and osteoblasts are responsible for bone resorption and formation, respectively, activities of these cells are believed to be controlled by a mechanosensory system of osteocytes embedded in the extracellular bone matrix. Several experimental and theoretical studies have suggested that the strain-derived interstitial fluid flow in lacuno-canalicular porosity serves as the prime mover for bone remodeling. Previously, we constructed a mathematical model for trabecular bone remodeling that interconnects the microscopic cellular activities with the macroscopic morphological changes in trabeculae through the mechanical hierarchy. This model assumes that fluid-induced shear stress acting on osteocyte processes is a driving force for bone remodeling. The validity of this model has been demonstrated with a remodeling simulation using a two-dimensional trabecular model. In this study, to investigate the effects of loading frequency, which is thought to be a significant mechanical factor in bone remodeling, we simulated morphological changes of a three-dimensional single trabecula under cyclic uniaxial loading with various frequencies. The results of the simulation show the trabecula reoriented to the loading direction with the progress of bone remodeling. Furthermore, as the imposed loading frequency increased, the diameter of the trabecula in the equilibrium state was enlarged by remodeling. These results indicate that our simulation model can successfully evaluate the relationship between loading frequency and trabecular bone remodeling.

Effect of tensile force on the mechanical behavior of actin filaments.

Actin filaments are the most abundant components of the cellular cytoskeleton, and play critical roles in various cellular functions such as migration, division and shape control. In these activities, mechanical tension causes structural changes in the double-helical structure of the actin filament, which is a key modulator of cytoskeletal reorganization. This study performed large-scale molecular dynamics (MD) and steered MD simulations to quantitatively analyze the effects of tensile force on the mechanical behavior of actin filaments. The results revealed that when a tensile force of 200pN was applied to a filament consisting of 14 actin subunits, the twist angle of the filament decreased by approximately 20°, corresponding to a rotation of approximately -2° per subunit, representing a critical structural change in actin filaments. Based on these structural changes, the variance in filament length and twist angle was found to decrease, leading to increases in extensional and torsional stiffness. Torsional stiffness increased significantly under the tensile condition, and the ratio of filament stiffness under tensile force to that under no external force increased significantly on longer temporal scales. The results obtained from this study contribute to the understanding of mechano-chemical interactions concerning actin dynamics, showing that increased tensile force in the filament prevents actin regulatory proteins from binding to the filament.

Trabecular bone remodelling simulation considering osteocytic response to fluid-induced shear stress.

In bone functional adaptation by remodelling, osteocytes in the lacuno-canalicular system are believed to play important roles in the mechanosensory system. Under dynamic loading, bone matrix deformation generates an interstitial fluid flow in the lacuno-canalicular system; this flow induces shear stress on the osteocytic process membrane that is known to stimulate the osteocytes. In this sense, the osteocytes behave as mechanosensors and deliver mechanical information to neighbouring cells through the intercellular communication network. In this study, bone remodelling is assumed to be regulated by the mechanical signals collected by the osteocytes. From the viewpoint of multi-scale biomechanics, we propose a mathematical model of trabecular bone remodelling that takes into account the osteocytic mechanosensory network system. Based on this model, a computational simulation of trabecular bone remodelling was conducted for a single trabecula under cyclic uniaxial loading, demonstrating functional adaptation to the applied mechanical loading as a load-bearing construct.

Estimation of bone permeability considering the morphology of lacuno-canalicular porosity.

Load-induced interstitial fluid flow in lacuno-canalicular porosity is believed to play an important role in cellular activities regulating adaptive bone remodeling. To investigate interstitial fluid behavior based on poroelasticity, it is important to determine the anisotropic permeability tensor reflecting the morphological features of the lacuno-canalicular porosity as fluid channels. In this study, we presented an estimation method of trabecular permeability by describing the analytical relationship between the volume orientation (VO) fabric tensor, which represents the canalicular orientation, and the permeability tensor. The relationship showed that the trabecular permeability tensor is proportional to the product of the volume fraction of the interstitial fluid and the VO fabric tensor of the canaliculi. We applied the proposed method to a two-dimensional fluorescent image of a trabecular cross section to quantify the canalicular anisotropy and the trabecular permeability tensor. The results indicated that the canaliculi are predominantly oriented in the radial direction of the trabecula, and the permeability depends strongly on the canalicular morphology.

Evaluation of extensional and torsional stiffness of single actin filaments by molecular dynamics analysis.

It is essential to investigate the mechanical behaviour of cytoskeletal actin filaments in order to understand their critical role as mechanical components in various cellular functional activities. These actin filaments consisting of monomeric molecules function in the thermal fluctuations. Hence, it is important to understand their mechanical behaviour on the microscopic scale by comparing the stiffness based on thermal fluctuations with the one experimentally measured on the macroscopic scale. In this study, we perform a large-scale molecular dynamics (MD) simulation for a half-turn structure of an actin filament. We analyse its longitudinal and twisting Brownian motions in equilibrium and evaluated its apparent extensional and torsional stiffness on the nanosecond scale. Upon increasing the sampling-window durations for analysis, the apparent stiffness gradually decreases and exhibits a trend to converge to a value that is close to the experimental value. This suggests that by extrapolating the data obtained in the MD analysis, we can estimate the experimentally determined stiffness on the microsecond to millisecond scales. For shorter temporal scales, the apparent stiffness is larger than experimental values, indicating that fast, local motions of the molecular structure are dominant. To quantify the local structural changes within the filament on the nanosecond scale and investigate the molecular mechanisms, such as the binding of the actin-regulatory proteins to the filaments, it is preferable to analyse the mechanical behaviour on the nanometre and nanosecond scales using MD simulation.

Simulations of dynamics of actin filaments by remodeling them in shear flows.

A mechanochemical relationship between intracellular flow fields of actin and remodeling of actin filaments is important to the realization of cell motility. In this study, to investigate this mechanochemical relationship, we present a mathematical model describing motions of actin filament and remodeling of the filaments in shear flows. We perform simulations of the dynamics of actin filaments remodeled by polymerization, depolymerization, and severing, and investigate contributions of these remodeling processes to effective shear viscosities of the system.

Effectiveness of scaffolds with pre-seeded mesenchymal stem cells in bone regeneration--assessment of osteogenic ability of scaffolds implanted under the periosteum of the cranial bone of rats.

To date, there has been no study on the development of novel regimens based on the following tissue engineering principles: seeding and culturing mesenchymal stem cells (MSCs) on a scaffold before surgery or injecting cultured MSCs into a scaffold during surgery. The purpose of this study was to assess the in vivo osteogenic ability of scaffold/MSCs implanted beneath the periosteum of the cranial bone of rats in three different sample groups: one in which MSCs were pre-seeded and cultured on a scaffold to produce the 3-D woven fabric scaffold/MSC composite using osteo-lineage induction medium, one in which cultured MSCs produced by osteo-lineage induction in cell cultivation flasks were injected into a scaffold during surgery and a control group, in which only the 3-D woven fabric scaffold was implanted. The results indicate that pre-seeding MSCs on a scaffold leads to a higher osteogenic ability than injecting cultured MSCs into a scaffold during surgery.

Coarse-grained modeling and simulation of actin filament behavior based on Brownian dynamics method.

The actin filament, which is the most abundant component of the cytoskeleton, plays important roles in fundamental cellular activities such as shape determination, cell motility, and mechanosensing. In each activity, the actin filament dynamically changes its structure by polymerization, depolymerization, and severing. These phenomena occur on the scales ranging from the dynamics of actin molecules to filament structural changes with its deformation due to the various forces, for example, by the membrane and solvent. To better understand the actin filament dynamics, it is important to focus on these scales and develop its mathematical model. Thus, the objectives of this study were to model and simulate actin filament polymerization, depolymerization, and severing based on the Brownian dynamics method. In the model, the actin monomers and the solvent were considered as globular particles and a continuum, respectively. The motion of the actin molecules was assumed to follow the Langevin equation. The polymerization, which increases the filament length, was determined by the distance between the center of the actin particle at the barbed end and actin particles in the solvent. The depolymerization, which decreases the filament length, was modeled such that the number of dissociation particles from the filament end per unit time was constant. In addition, the filament severing, in which one filament divides into two, was modeled to occur at an equal rate along the filament. Then, we simulated the actin filament dynamics using the developed model, and analyzed the filament elongation rate, its turnover, and the effects of filament severing on the polymerization and depolymerization. Results indicated that the model reproduced the linear dependence of the filament elongation on time, filament turnover process by polymerization and depolymerization, and acceleration of the polymerization and depolymerization by severing, which qualitatively agreed with those observed in experiments.

Actomyosin contractility spatiotemporally regulates actin network dynamics in migrating cells.

Coupling interactions among mechanical and biochemical factors are important for the realization of various cellular processes that determine cell migration. Although F-actin network dynamics has been the focus of many studies, it is not yet clear how mechanical forces generated by actomyosin contractility spatiotemporally regulate this fundamental aspect of cell migration. In this study, using a combination of fluorescent speckle microscopy and particle imaging velocimetry techniques, we perturbed the actomyosin system and examined quantitatively the consequence of actomyosin contractility on F-actin network flow and deformation in the lamellipodia of actively migrating fish keratocytes. F-actin flow fields were characterized by retrograde flow at the front and anterograde flow at the back of the lamellipodia, and the two flows merged to form a convergence zone of reduced flow intensity. Interestingly, activating or inhibiting actomyosin contractility altered network flow intensity and convergence, suggesting that network dynamics is directly regulated by actomyosin contractility. Moreover, quantitative analysis of F-actin network deformation revealed that the deformation was significantly negative and predominant in the direction of cell migration. Furthermore, perturbation experiments revealed that the deformation was a function of actomyosin contractility. Based on these results, we suggest that the actin cytoskeletal structure is a mechanically self-regulating system, and we propose an elaborate pathway for the spatiotemporal self-regulation of the actin cytoskeletal structure during cell migration. In the proposed pathway, mechanical forces generated by actomyosin interactions are considered central to the realization of the various mechanochemical processes that determine cell motility.

Computer simulation of trabecular remodeling in human proximal femur using large-scale voxel FE models: Approach to understanding Wolff's law.

Ever since Julius Wolff proposed the law of bone transformation in the 19th century, it has been widely known that the trabecular structure of cancellous bone adapts functionally to the loading environment. To understand the mechanism of Wolff's law, a three-dimensional (3D) computer simulation of trabecular structural changes due to surface remodeling was performed for a human proximal femur. A large-scale voxel finite element model was constructed to simulate the structural changes of individual trabeculae over the entire cancellous region. As a simple remodeling model that considers bone cellular activities regulated by the local mechanical environment, nonuniformity of local stress was assumed to drive the trabecular surface remodeling to seek a uniform stress state. Simulation results demonstrated that cell-scale ( approximately 10microm) remodeling in response to mechanical stimulation created complex 3D trabecular structures of the entire bone-scale ( approximately 10cm), as illustrated in the reference of Wolff. The bone remodeling reproduced the characteristic anisotropic structure in the coronal cross section and the isotropic structures in other cross sections. The principal values and axes of a structure characterized by fabric ellipsoids corresponded to those of the apparent stress of the structure. The proposed large-scale computer simulation indicates that in a complex mechanical environment of a hierarchical bone structure of over 10(4) length scale (from approximately 10microm to approximately 10cm), a simple remodeling at the cellular/trabecular levels creates a highly complex and functional trabecular structure, as characterized by bone density and orientation.