Development of the robust yet practical tissue culture systems for vascular grafting is one of the major challenges for biomedical engineering. The following questions should be solved in priority: (1) flexibility of the tissue reservoir allowing dynamic stretching, (2) seeding of cells and extraction of tissues should be easy and safe, (3) system must allow morphological observation in real time, (4) maintenance of metabolic activity of tissues should be performed automatically. In our study, we attempted to solve these problems designing in vivo-like culture chamber made of PDMS, and developed an integrated system with a perfusion bioreactor and a small digital microscope. We developed disposable cell chamber with following qualities: transparent, autoclave-sterilizable, non cell-adhesive, and having low autofluorescence. The polyacetal mold made it possible to prepare a chamber hosting the tissue of the desired shape and size. In our case, a porous tube made from PTFE was fixed inside the chamber and tubular cell culture space was prepared for loading of preformed cell spheroids. Perfusion of the media within the porous tube continuously supplied the nutrients and oxygen to the spheroids. Growth and fusion of the cell spheroids inside the chamber can be observed real time by the small digital microscope and analyzed retrospectively by time-laps movies. We loaded batches up to 900 goat fibroblast spheroids into the system; growth, development and morphological fusion of the spheroids were followed out for 10 days. Removal of the tissue without disturbing its structural integrity was possible, and histological analysis revealed reasonable fusion degree between spheroids, and 72.5 % cell survival rate as estimated by TUNEL staining. In conclusion, our system has the basic performance necessary for culturing cell spheroids for tissue-engineered vascular graft.
A fall can result in fractures, sequelae, or even death in the worst-case scenario. Falls often occur because people misjudge step height and do not lift their feet sufficiently. One strategy to combat this issue is the use of vertical stripes to make stairs appear taller than they really are, which can thus prevent underestimation of the real height. However, the appropriate size of the space between the stripes is different for everyone. This study demonstrates how the intensity of an illusion can influence the height of foot clearance when ascending stairs. We validate the feasibility of using a model for determining the striping that can be used to adapt the pattern to individual differences. We constructed the model based on a contrast-sensitivity function using a Gaussian model. In an experiment, we measured the location of the toe via motion capture as participants climbed stairs. As a result, the relationship between foot-clearance and the spatial frequency approximated the function described by the CSF, Contrast Sensitive Function. The coefficients of determination exceeded 0.9 for one participant, 0.8 for two participant and 0.6 for other two participants. We concluded that the model presented CSF would fit the foot clearance with striped stairs for any individual.
Skeletal fragility is an important orthopedic concern. In this study, the effect of the recovery period after damage under different mode of damage loading on the human cortical bone was investigated. Human cortical bone samples were damaged with one of three different loading modes (tension, compression and torsion) and the change of mechanical properties (stiffness, viscous relaxation and energy dissipation) before and after the damage with respect to different recovery time (1, 10, 30, 60 and 100 minutes) were measured. Results showed that measures of mechanical properties after damage varied significantly with recovery time and load modes. Typically, significant changes in property ratios occurred in the first 10 minutes for loading modulus measures in all loading modes. In addition, results showed that, with respect to loading mode effects, all torsional modulus changes were largest followed by tensile and then compressive modulus changes. We concluded that a minimum 10 minutes recovery period is required for all three loading modes while tension and torsion would require 30 minutes recovery time to obtain an accurate measure of the damage state and recovery effect because more than 90% of strain recovery and modulus recovery were observed during this period.
Poly(lactic acid) (PLA) has attracted much attention as a material for bioabsorbable bone fixation devices, however degradation rate of PLA is very low. Surface treatment of PLA has been investigated to increase degradation rate due to improvement in hydrophilicity. In this study, effects of ion-implantation on degradation rate and mechanical properties were investigated. Argon gas which is chemically stable was used as ion source, and argon ion was implanted in the surface of PLA. The contact angle and the surface roughness of the ion-implanted PLA were measured to evaluate hydrophilicity, and tensile tests and micro-indentation tests were conducted to evaluate mechanical properties. The difference in surface morphology after in vitro degradation test between ion-implanted and un-implanted region was observed to investigate degradation properties of ion-implanted PLA. As a result, tensile strength increased by 8.2 % compared to un-implanted specimen and Vickers hardness increased by 9.5%. The ion-implantation might affect mechanical properties in the overall specimen rather than those in the surface. The result of in vitro degradation test showed that an initial degradation rate was accelerated by ion-implantation. This result is consistent with the increasing hydrophilicity of ion-implanted PLA. Those results suggested that ion-implantation accelerates degradation of PLA without decreasing mechanical properties.
In order to improve bioactivity of hydroxyapatite (HA), the surface of HA was modified with calcium or magnesium ion irradiation. Calcium ion irradiation improved bioactivity of HA up to ion dose 1014 ions/cm2, whereas magnesium ion irradiation did not affect bone-like apatite formation. It is found that this different phenomena were attributed to surface electrical potential. Ion irradiation generally induces surface nano-scopic damage, however the strength of HA was not significantly affected by ion irradiation. From cell proliferation, no toxicity of HA irradiated with calcium ions was confirmed. From these results, the effectiveness of calcium ion irradiation on the treatment of bone defect using HA is confirmed.
Bone tissue is subjected to multiple forms of mechanical stress. Even in the absence of external loads, however, residual stress is measured, although the underlying mechanisms remain unknown. This study measured the changes in residual stresses, diaphyseal size, and the micro- and nanostructures of bone during growth and maturation, periods associated with different in vivo mechanical loads due to increasing body weight. Mid-diaphyses from bovine femurs in the following three age groups were examined: 1) less than one month old, 2) two years old, and 3) 8−9 years old. Residual stresses along the bone axis at anterior, posterior, lateral, and medial positions on the diaphyseal surface were measured by X-ray diffraction and averaged. Diaphyseal size, porosity, mineral contents, and degree of hydroxyapatite crystal orientation of transverse cross-sections were investigated for relations with residual stress. Residual stress increased significantly from less than one month old (83.7 ± 53.3 MPa) to two years old (125.5 ± 61.9 MPa) in parallel with expanding diaphyseal width and cortical thickness. Residual stress plateaued until 8−9 years old (114.6 ± 42.2 MPa) and was correlated with local cortical thickness (p < 0.05). At the stage, diaphyseal width was only slightly greater than at 2 years and cortical thickness was not significantly different. For all measurements across groups, residual stress statistically correlated with porosity (p < 0.05), mineral contents (p < 0.01), and degree of crystal orientation (p < 0.01). These observations suggest that residual stresses are generated due to bone formation and reconstruction under changing in vivo mechanical loads with age. In conclusion, residual stresses in bone are generated during development and maintained in maturation, and are indirectly related to diaphyseal size and both bone micro- and nanostructure.
Deformability of epithelial tissues plays a crucial role in embryogenesis, homeostasis, wound healing, and disease. The deformability is determined by the mechanical balance between active force generation and passive response of cells. However, little is known about how multiple cells in epithelial tissues passively respond to external forces. Using a 3D vertex model, we performed computational simulations of longitudinal tension and compression tests of an epithelial tube. Under tension, the tube extended with necking as exhibiting cell rearrangements that play a role in reducing local stiffness of the tube. On the other hand, under compression, the tube buckled with kinking without cell rearrangements. The cell rearrangements occurred when apical and basal cell surfaces stored elastic deformation energies. These results illustrate the variance of deformation modes of epithelial tissues in the single cell level as well as the importance of cell rearrangements in regulating epithelial deformability.
Human thumb and fingers are usually subjected to an external loading during daily activity. The information of how muscles in the forearm cooperate with each other in order to response to the external loading is still unknown. Such information may be helpful in understanding muscle function pathology and motor disorder. A novel method called electromyography computed tomography (EMG-CT) was developed to visualize muscle activity within a whole cross-section of the forearm by measuring surface EMG signals around the forearm. The current study aimed to extend the previous work by using the EMG-CT to investigate muscle cooperative activity under loading application to thumb or each finger. Loads of 0.98-9.8 N were applied to the thumb or each finger of four subjects in eight loading directions. The loading directions on thumb and index, middle, and little fingers were inner, outer, and upper directions. EMG signals around the subject’s forearm were recorded during the loading by using EMG band consisting of 40 pairs of bipolar electrodes. The results show different muscle cooperative activity pattern between loading conditions. During load was applied to thumb, muscle in lower region in pronation cross-section were highly active. When load was applied to a finger, muscles in lateral-lower region were highly active. In all subjects, total muscle activity in the whole cross-section and the maximum value of muscle activity increased in proportion to loading. This study demonstrates effectiveness of EMG-CT method by showing that the muscle cooperative activity of an individual is specific to force application conditions.
Approximately one in three people over 65 years of age fall each year. The resulting physiological and psychological trauma can lead to physical deconditioning, social isolation and early mortality. Recent research has reported balance recovery can be trained in a single session resulting in dramatic reductions in fall rates. However, most previous research has used repeated exposures to a single hazard in a fixed location and not controlled for reductions in walking speed. It follows, that the biomechanical mechanisms important for reactive balance recovery (in the absence of anticipatory adjustments) are probably not well understood. Here, we investigated the biomechanics of successful reactive balance recovery following the first exposures to unexpected trip and slip hazards in different locations. Ten healthy adults (29.1±5.6 years) completed 32 walks at fixed speed, cadence and step length over a custom 10-meter walkway while being exposed to randomly presented and located slip and trip hazards. Balance recovery kinematics were assessed using a VICON motion analysis system. Repeated exposures to unexpected hazards induced significant reductions (p≤0.05) in anteroposterior (AP) trunk sway following the trips (26.7° to 14.3°; Cohen’s d -1.24) and slips (32.7° to 19.0°; Cohen’s d -0.93). During recovery from unexpected trips, reduced AP trunk sway was strongly correlated with a more posterior centre-of-mass position relative to the stepping foot (r=0.91) and a longer step length (r=-0.71). During recovery from unexpected slips, reduced AP trunk sway was moderately correlated with slower slipping speed (r=0.54) and a less posterior centre-of-mass position relative to the stance (slipping) foot (r=-0.39). The biomechanical mechanisms required for the successful reactive balance recovery from trips and slips were different. Future experimental protocols to optimize reactive balance recovery for fall prevention should therefore use progressive exposures to both slip and trip hazards using specialized equipment and determine if similar biomechanical mechanisms are observed in young and elderly people at risk of falls.
Osteoblasts change their intracellular calcium ion concentration in response to mechanical stimuli. Although it has been reported that osteoblasts sense and respond to stretching of a substrate on which osteoblastic cells have adhered, the details of the dynamic characteristics of their calcium signaling response remain unclear. Motion artifacts such as loss of focus during stretch application make it difficult to conduct precise time-course observations of calcium signaling responses. Therefore, in this study, we observed intracellular calcium signaling responses to stretch in a single osteoblastic cell by video rate temporal resolution. Our originally developed cell-stretching microdevice enables in situ observation of a stretched cell without excessive motion artifacts such as focus drift. Residual minor effects of motion artifacts were corrected by the fluorescence ratiometric method with fluorescent calcium indicator Fluo 8H and fluorescent cytoplasm dye calcein red-orange. We succeeded to detect the intracellular calcium signaling response to stretch by video rate temporal resolution. The results revealed a time lag from stretch application to initiation of the intracellular calcium signaling response. We compared two time lags measured at two different cell areas: central and peripheral regions of the cell. The time lag in the central region of the cell was shorter than that in the peripheral region. This result suggests that the osteoblastic calcium signaling response to stretching stimuli initiates around the central region of the cell.
Cells in our body utilize a variety of adaptor proteins for transmitting context specific signals that arise from the cellular microenvironment. Adaptor proteins lack enzymatic activity and typically perform their function by acting as scaffolds that bind other signaling proteins. While most adaptor proteins are functionally modulated by biochemical alterations such as phosphorylation, a subset of adaptor proteins are functionally modulated by a mechanical alteration in their structure that makes cryptic sites available for binding to downstream signaling proteins. α-catenin is one such adaptor protein that localizes to cadherin-based cell adhesions by binding the membrane-localized cadherin-β-catenin complex at one side and the cytosolic F-actin on the other side. An increase in actomyosin tension is directly relayed to α-catenin resulting in a change in its conformation making cryptic binding sites accessible to its interacting partners. Here, I describe an updated view of the mechanical regulation of α-catenin in the context of cellular adhesion, including the role of cadherin clustering in its activation.
Determination of left-right asymmetry of the body plan is achieved in the early embryo. At the 4-6 somite stage, a cavity structure, called a node, is observed in the ventral midline surface, in which hundreds of cilia rotate. Nodal cilia are typically tilted toward the posterior and rotate in the clockwise direction, resulting in the generation of leftward flow in the node. Such leftward flow acts as a trigger of left-specific gene expression, and fluid mechanics plays a role in left-right symmetry breaking. To understand the cilia-driven nodal flow, it is necessary to determine the hydrodynamic interactions among rotating cilia, as ciliary motions interact with each other through fluid motion. In this study, we numerically investigated the elastohydrodynamic synchronization of two rotating cilia, as well as the flow field. The ciliary motion was determined by the balance of cytoskeletal elastic force, motor protein-induced active force, and fluid viscous force. According to the geometric clutch hypothesis, the frequency of rotating cilia is controlled by the bending curvature. Owing to hydrodynamic interactions, bending deformations of two cilia become time-dependent, and the rotation is finally locked in anti-phase regardless of the relative position and initial phase difference. By locking in the reverse phase, the average propulsion flow rate becomes 2-3 times larger than in-phase beating. The results of this study form a basis for understanding cilium-driven nodal flow.