Toshihiko Shiraishi

Low-intensity pulsed ultrasound activates the phosphatidylinositol 3 kinase/Akt pathway and stimulates the growth of chondrocytes in three-dimensional cultures: a basic science study

IntroductionThe effect of low-intensity pulsed ultrasound (LIPUS) on cell growth was examined in three-dimensional-cultured chondrocytes with a collagen sponge. To elucidate the mechanisms underlying the mechanical activation of chondrocytes, intracellular signaling pathways through the Ras/mitogen-activated protein kinase (MAPK) and the integrin/phosphatidylinositol 3 kinase (PI3K)/Akt pathways as well as proteins involved in proliferation of chondrocytes were examined in LIPUS-treated chondrocytes.MethodsArticular cartilage tissue was obtained from the metatarso-phalangeal joints of freshly sacrificed pigs. Isolated chondrocytes mixed with collagen gel and culture medium composites were added to type-I collagen honeycomb sponges. Experimental cells were cultured with daily 20-minute exposures to LIPUS. The chondrocytes proliferated and a collagenous matrix was formed on the surface of the sponge. Cell counting, histological examinations, immunohistochemical analyses and western blotting analysis were performed.ResultsThe rate of chondrocyte proliferation was slightly but significantly higher in the LIPUS group in comparison with the control group during the 2-week culture period. Western blot analysis showed intense staining of type-IX collagen, cyclin B1 and cyclin D1, phosphorylated focal adhesion kinase, and phosphorylated Akt in the LIPUS group in comparison with the control group. No differences were detected, however, in the MAPK, phosphorylated MAPK and type-II collagen levels.ConclusionLIPUS promoted the proliferation of cultured chondrocytes and the production of type-IX collagen in a three-dimensional culture using a collagen sponge. In addition, the anabolic LIPUS signal transduction to the nucleus via the integrin/phosphatidylinositol 3-OH kinase/Akt pathway rather than the integrin/MAPK pathway was generally associated with cell proliferation.

Acquisition of Local Neighbor Rules in the Simulation of Pedestrian Flow by Cellular Automata

Cellular Automata is applied to model the pedestrian flow, in which the local neighbor rules implemented to each person in the crowd are determined automatically in the process of simulation. The collision patterns in the visible area settled around each person is introduced, and each person walks around just avoiding the collision patterns. As the simulation proceeds along the time step, various collision patterns are observed. By being informed the collision patterns to all the pedestrians simultaneously, the pedestrian begins to flow quite smoothly and the column formation is observed.

In vitro stability of open wedge high tibial osteotomy with synthetic bone graft.

It has been predicted that significant stress will be applied to the plate and lateral cortical hinge of an osteotomy site when early full weight bearing is commenced after an open wedge high tibial osteotomy. We hypothesized that the stress concentration on the plate or at the lateral cortical hinge would be reduced by inserting bone substitutes into the osteotomy gap. Two different types of tibia model were investigated: Group A, fixation with TomoFix with the osteotomy site left as an open space; and Group B, two beta-TCP wedges are inserted into osteotomy site and fixed with TomoFix. Stress at five points was measured using strain gauges. Specimens were mounted onto a testing machine with an FTA (femoro-tibial angle) of 170 degrees . Cyclic load tests and an ultimate load test were then performed. The mean stress on the plate was measured at 15.5+/-1.8Mpa in Group A. On the other hand, this value in Group B was only 9.52+/-2.1Mpa and this was a significant difference (P<0.01). The mean stress on the lateral hinge in Groups A and B was 3.31+/-0.5 and 2.49+/-0.2, respectively which was also a significant difference (P<0.05). The mean maximum breaking load in Group A was 2500+/-280N and in Group B 4270+/-420N which was a significant difference (P<0.01). Hence, for OWHTO procedures, the use of beta-TCP wedges and TomoFix is thus likely to improve the initial axial and possibly rotational stability at the osteotomy site in comparison with methods that leave the osteotomy gap open.

Estimation of Equivalent Permeability in Magnetorheological Fluid Considering Cluster Formation of Particles

This paper describes a simulation method for the equivalent magnetic permeability of mangetorheological (MR) fluids considering cluster formation of suspended particles. The cluster formation under a magnetic field is simulated by cellular automata (CA). Simulated cluster structures are qualitatively equivalent to those observed experimentally Considering this structure, magnetic permeability analysis is conducted on a representative MR fluid by the finite element method. The equivalent permeability in the MR fluid was obtained from the average magnetic flux density and field. The time evolution of the magnetic characteristics of the MR fluid is shown to correspond to the time evolution of cluster formation.

Evaluation of billboards based on pedestrian flow in the concourse of the station

Pedestrian flow in the concourse of the stations was simulated by Cellular Automata, and based on the simulation results, an evaluation index for billboards set along the pathway in the concourse was proposed The practical value of billboards may depend on not only their size or placement, but the number of people who pay attention to them In this paper, some visible area was set to each person walking in the concourse of the station, and the pedestrian flow was simulated by defining various local neighbor rules based on our experience from gates to the door of trains, or door to the gates The score for each billboard was accumulated when a person took a look at the billboard in the view area, and evaluation index was proposed The number of people in the concourse was counted in practice and it showed good agreement with the simulation results.

Control of Apoptosis and Differentiation of Cultured Neural Stem Cells by Mechanical Vibration

In this study, sinusoidal inertia force was applied to cultured neural stem cells and the effects of mechanical vibration on the cells were investigated. Neural stem cells which were obtained from the hippocampus of an adult Fischer rat were seeded in culture plates at the density of 2.5 × 105 cells/ml. After cells were cultured for one day and adhered on the cultured plate, vibration groups of the culture plates were set on the aluminum plate of the experimental setup and cultured under sinusoidal excitation in another CO2 incubator separated from non-vibration groups of the culture plates. Acceleration amplitude was set to 0.25 or 0.5 G and frequency was set to 12.5, 25, or 50 Hz. Time evolution of cell density was obtained by counting the number of cells with a hemocytometer. The expression of Akt, phosphorylated Akt, MAPK, and phosphorylated MAPK was detected by western blotting analysis to understand the mechanism of cell proliferation. Gene expression of MAP-2, neurofilament-H, GFAP, and nestin was detected by a real-time RT-PCR method to obtain a ratio of differentiation of neural stem cells to nerve or glia cells. The results to be obtained are as follows. The mechanical vibration at 25 Hz is most effective on cell proliferation of the present experimental conditions at 0.25 G. The enhancement of cell proliferation is probably caused by the suppression of apoptosis. The differentiation of the neural stem cells depends on acceleration amplitude and the mechanical vibration may maintain some properties of stem cells.Copyright

Effects of Mechanical Vibration on Proliferation and Differentiation of Neural Stem Cells

Neural stem cells have been studied to promote neurogenesis in regenerative therapy. The control of differentiation of neural stem cells to nerve cells and the increase of the number of nerve cells are needed. For the purpose of them, it is important to investigate not only chemical factors but also mechanical factors such as hydrostatic pressure in brain and mechanical vibration in walking. In this study, sinusoidal inertia force was applied to cultured neural stem cells and the effects of mechanical vibration on the cells were investigated. After the cells were cultured in culture plates for one day and adhered on the cultured plane, vibrating group of the culture plates was set on an aluminum plate attached to an exciter and cultured under sinusoidal excitation for 24 hours a day during 26 days. The amplitude of the acceleration on the culture plate was set to 0.25 G and the frequency was set to 25 Hz. The time evolution of cell density was obtained by counting the number of cells at every 3 or 4 days. The expression of Akt, phosphorylated Akt (p-Akt), MAPK, and phosphorylated MAPK (p-MAPK) was detected by western blotting analysis at 7 days of culture to understand the mechanism of cell proliferation. Akt and MAPK are part of signaling pathways in relation to cell proliferation. The phosphorylation of Akt suppresses apoptosis and the phosphorylation of MAPK activates cell division. The gene expression of MAP-2, NFH, GFAP, and nestin was detected by real-time RT-PCR analysis at 7 days of culture to obtain a ratio of differentiation of neural stem cells to nerve or glia cells. MAP-2 and NFH are nerve cell markers, GFAP is a glia cell marker, and nestin is a stem cell marker. The results obtained are as follows. The cell density of the vibrating group was three times higher than that of the non-vibrating group at 26 days of culture. p-Akt was enhanced by the mechanical vibration while p-MAPK was not. There is no significant difference of the gene expression level of MAP-2, NFH, GFAP, and nestin between the vibrating and non-vibrating groups. These results suggest that the mechanical vibration promotes the proliferation of neural stem cells and its cause is likely the suppression of apoptosis but not the activation of cell division, and that the mechanical vibration at the experimental condition does not affect the differentiation of neural stem cells to nerve or glia cells.© 2008 ASME

Effects of Acceleration Amplitude and Frequency of Mechanical Vibration on Osteoblast-Like Cells

Bone formation is subject in vivo to mechanical stimulation. Although many researches for bone cells of osteoblastic lineage sensing and responding to mechanical stimulation have been reported mainly in the biochemical field, effects of mechanical stimulation on bone cells are not well understood. In this study, in order to clarify effects of acceleration amplitude and frequency of mechanical stimulation on MC3T3-E1, which is an osteoblast-like cell line derived from mouse calvaria, in the sense of mechanical vibrations, their cell proliferation, cell morphology, bone matrix generation and gene expression of alkaline phosphatase (ALP) were investigated when sinusoidal inertia force was applied to the cells. After the cells were cultured in culture plates in a CO2 incubator for one day and adhered on the cultured plane, vibrating groups of the culture plates were set on an aluminum plate attached to a exciter and cultured under sinusoidal excitation in another incubator separated from non-vibrating groups of the culture plates. Acceleration amplitude and frequency were set to several kinds of conditions. The time evolution of cell density was obtained by counting the number of cells with a hemocytometer. The cell morphology was observed with a phase contrast microscope. Calcium salts generated by the cells were observed by being stained with alizarin red S solution and their images were captured with a CCD camera. The vibrating groups for the cell proliferation and the calcium salts staining were sinusoidally excited for 24 hours a day during 28-day cultivation. Gene expression of ALP was measured by a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) method. After the vibrating groups for the PCR were excited for 7 days, the total RNAs were extracted. After reverse transcription, real-time RT-PCR was performed. Gene expression for ALP and a housekeeping gene were determined simultaneously for each sample. ALP gene level in each sample was normalized to the measured housekeeping gene level. The results to be obtained are as follows. In the range from 12.5 to 200 Hz, saturation cell density for the cell proliferation shows tendency of increase as frequency decreases and ALP gene expression shows a peak to frequency at 50 Hz. Among 0, 0.25 and 0.5 G, saturation cell density and ALP gene expression show tendency of increase as acceleration amplitude increases.© 2007 ASME

Effects of Amplitude and Frequency of Vibration Stimulation on Cultured Osteoblasts

Mechanical stimulation to bones affects osteogenesis such as decrease of bone mass of astronauts under zero gravity, walking rehabilitation to bone fracture and fracture repair with ultrasound devices. Bone cells have been reported to sense and response to mechanical stimulation at cellular level morphologically and metabolically. In the view of mechanical vibrations, bone cells are deformed according to mechanical stimulation and their mechanical characteristics. Recently, it was reported that viscoelasticity of cells was measured using tensile and creep tests and that there was likely natural frequency and nonlinearity of cells in the sense of structural dynamics. It suggests that there is effective frequency and amplitude of mechanical stimulation on osteogenesis by bone cells. In this study, sinusoidal inertia force was applied to cultured osteoblasts, MC3T3-E1, and effects of frequency and acceleration amplitude of mechanical vibration on the cells were investigated in respect of cell proliferation, cell morphology, bone matrix generation and alkaline phosphatase (ALP) gene expression. After the cells were cultured in culture plates in a CO2 incubator for one day and adhered on the cultured plane, vibrating groups of the culture plates were set on an aluminum plate attached to a exciter and cultured under sinusoidal excitation in another incubator separated from non-vibrating groups of the culture plates. Acceleration amplitude and frequency were set to several kinds of conditions. The time evolution of cell density was obtained by counting the number of cells with a hemocytometer. The cell morphology was observed with a phase contrast microscope. Calcium salts generated by the cells were observed by being stained with alizarin red S solution and their images were captured with a CCD camera. The vibrating groups for the cell proliferation and the calcium salts staining were sinusoidally excited for 24 hours a day during 28-day cultivation. Gene expression of ALP was measured by a real-time RT-PCR method. After the vibrating groups for the PCR were excited for 6 hours, the total RNAs were extracted. After reverse transcription, real-time RT-PCR was performed. Gene expression for ALP and a housekeeping gene were determined simultaneously for each sample. Gene levels in each sample were normalized to the measured housekeeping gene levels. As a result, it is shown that saturate cell density becomes high and bone matrix generation is promoted by applying mechanical vibration and that there may be some peaks to frequency and a certain threshold value to acceleration amplitude of mechanical vibration for saturation cell density and bone matrix generation.Copyright

A flow modeling of lubricating greases under shear deformation by cellular automata

A Cellular Automata modeling of the lubricating grease flow under the shear deformation is proposed Lubricating greases are composed of thickening agent, liquid lubricant and various kinds of additives The thickening agent forms fibrous microstructures in liquid lubricant, and lubricating greases present their special feature due to this microstructure Though they are widely used in mechanical components, there is little understanding on the lubrication mechanism including the contribution of the fibrous microstructure There have been proposed no other theoretical model except for the average flow model based on the fluid lubrication theory in tribology field In the present paper, the flow modeling of lubricating greases under shear deformation was proposed by Cellular Automata, where aggregated thickening agent and liquid lubricant were represented by virtual particles movable on the two-dimensional cell space It was assumed that the fibrous microstructure was composed of multiple particles, and external stress induces the flow of grease where the particles interacts one another.