Toshiro Ohashi

Periodontal tissue activation by vibration: Intermittent stimulation by resonance vibration accelerates experimental tooth movement in rats

INTRODUCTION Accelerating the speed of orthodontic tooth movement should contribute to the shortening of the treatment period. This would be beneficial because long treatment times are a negative aspect of orthodontic treatment. In this study, we evaluated the effects of mechanical stimulation by resonance vibration on tooth movement, and we showed the cellular and molecular mechanisms of periodontal ligament responses. METHODS The maxillary first molars of 6-week-old male Wistar rats were moved to the buccal side by using an expansive spring for 21 days (n = 6, control group), and the amount of tooth movement was measured. Additional vibrational stimulation (60 Hz, 1.0 m/s(2)) was applied to the first molars by using a loading vibration system for 8 minutes on days 0, 7, and 14 during orthodontic tooth movement (n = 6, experimental group). The animals were killed under anesthesia, and each maxilla was dissected. The specimens were fixed, decalcified, and embedded in paraffin. Sections were used for immunohistochemical analysis of receptor activator of NF kappa B ligand (RANKL) expression. The number of osteoclasts in the alveolar bone was counted by using TRAP staining, and the amount of root resorption was measured in sections stained with hematoxylin and eosin. RESULTS The average resonance frequency of the maxillary first molar was 61.02 +/- 8.38 Hz. Tooth movement in the experimental group was significantly greater than in the control group (P <.05). Enhanced RANKL expression was observed at fibroblasts and osteoclasts in the periodontal ligament of the experimental group on day 3. The number of osteoclasts in the experimental group was significantly increased over the control group on day 8 (P <.05). Histologically, there were no pathological findings in either group or significant differences in the amount of root resorption between the 2 groups. CONCLUSIONS The application of resonance vibration might accelerate orthodontic tooth movement via enhanced RANKL expression in the periodontal ligament without additional damage to periodontal tissues such as root resorption.

The pipette aspiration applied to the local stiffness measurement of soft tissues

A simple method of identifying the initial slope of the stress-strain curve (i.e., Youngs modulus of the soft tissue) by introducing the pipette aspiration technique is presented. The tissue was assumed to be isotropic and macroscopically homogeneous. Numerical simulations by the linear finite element analysis were performed for the axisymmetric model to survey the effects of friction at the tissue-pipette contact boundary, pipette cross-sectional geometry, relative size of the specimen to the pipette, and the layered inhomogeneity of the specimen tissue. The friction at the contact region had little effect on the measurement of Youngs modulus. The configuration of the pipette was shown to affect the measurement for small pipette wall thickness. The measurement also depended on the relative size of the specimen to the pipette for relatively small specimens. The extent of the region contributing to the measurement was roughly twice the inside radius of the pipette. In this region, the maximum stress did not exceed the level of the aspiration pressure, with only minor exceptional locations. Calculation of strain energy components indicated that the major contributions to the deformation under pipette aspiration were by the normal extension and shear deformation in pipette axial direction. Experimental verification of the present method for the isotropic, homogeneous artificial material is also presented.

Local elastic modulus of atherosclerotic lesions of rabbit thoracic aortas measured by pipette aspiration method

Changes in mechanical properties of arteries during atherogenesis remain controversial. One of the reasons could be that they have been evaluated with parameters measured in a whole vessel, although the lesions are localized. The local elastic modulus of atherosclerotic lesions was measured by the pipette aspiration method in thoracic aortas of rabbits fed a cholesterol diet for 8, 16, 24 and 28 weeks. The global elastic modulus of the whole aorta was measured by the pressure-diameter test. The local modulus decreased from that of the normal tissue in 8 weeks and then increased during the cholesterol feeding period. The global modulus did not change until 24 weeks and increased by 28 weeks. Histological observation revealed that the initial soft lesion was mainly composed of foam cells, and the stiffening accompanied first the appearance of smooth muscle cells in the top layer of the hyperplastic intima and then calcification in its bottom layer. The global elastic modulus did not change until marked calcification occurred in the tissue. These results suggest that change in mechanical properties of atherosclerotic lesion is not simple and has a close correlation with its histology. Assessment of local mechanical properties is important for studying mechanical properties of atherosclerotic arteries.

Effect of spatial gradient in fluid shear stress on morphological changes in endothelial cells in response to flow

Arterial bifurcations are common sites for development of cerebral aneurysms. Although this localization of aneurysms suggests that high shear stress (SS) and high spatial SS gradient (SSG) occurring at the bifurcations may be crucial factors for endothelial dysfunction involved in aneurysm formation, the details of the relationship between the hemodynamic environment and endothelial cell (EC) responses remain unclear. In the present study, we sought morphological responses of ECs under high-SS and high-SSG conditions using a T-shaped flow chamber. Confluent ECs were exposed to SS of 2-10Pa with SSG of up to 34Pa/mm for 24 and 72h. ECs exposed to SS without spatial gradient elongated and oriented to the direction of flow at 72h through different processes depending on the magnitude of SS. In contrast, cells did not exhibit preferred orientation and elongation under the combination of SS and SSG. Unlike cells aligned to the flow by exposure to only SS, development of actin stress fibers was not observed in ECs exposed to SS with SSG. These results indicate that SSG suppresses morphological changes of ECs in response to flow.

A microwell array device with integrated microfluidic components for enhanced single-cell analysis

Increasing awareness of the importance of cell heterogeneity in many biological and medical contexts is prompting increasing interest in systems that allow single‐cell analysis rather than conventional bulk analysis (which provides average values for variables of interest from large numbers of cells). Recently, we presented a microwell chip for long‐term, high‐throughput single‐cell analysis. The chip has proved to be useful for purposes such as screening individual cancer and stem cells for protein/gene markers. However, liquids in the wells can only be added or changed by manually rinsing the chip, or parts of it. This procedure has several well‐known drawbacks – including risks of cross‐contamination, large dead volumes and laboriousness – but there have been few previous attempts to integrate liquid rinsing/switching channels in “ready‐to‐use” systems for single‐cell analysis. Here we present a microwell system designed (using flow simulations) for single‐cell analysis with integrated microfluidic components (microchannels, magnetically driven micropumps and reservoirs) for supplying the cell culture wells with reagents, or rinsing, thus facilitating controlled, directed liquid handling. It can be used totally independently, since tubing is not essential. The practical utility of the integrated system has been demonstrated by culturing endothelial cells in the microwells, and successfully applying live‐cell Calcein AM staining. Systems such as this combining high‐density microwell chips with microfluidic components have great potential in numerous screening applications, such as exploring the important, but frequently undetected, heterogeneity in drug responses among individual cells.

Application of scanning acoustic microscopy for assessing stress distribution in atherosclerotic plaque.

AbstractScanning acoustic microscopy (SAM) was equipped to assess the acoustic properties of normal and atherosclerotic coronary arteries. The SAM image in the atherosclerotic lesion clearly demonstrated that the sound speed was higher than that in the normal intima, and that the variation of elasticity was found within the fibrous cap of the plaque. Youngs elastic modulus of each region was calculated and the finite element analysis was applied to derive the stress distribution in these arterial walls. In a case of normal coronary artery, the stress was dominant in the intima and the distribution was rather homogeneous and in a case of atherosclerosis, high stress was concentrated to the relatively soft lesion in the fibrous cap overlying lipid pool. SAM provides information on the physical properties, which cannot be obtained by the optical microscope. The results would help in understanding the pathological features of atherosclerosis.

Three-dimensional imaging of biological cells with picosecond ultrasonics

We use picosecond ultrasonics to image animal cells in vitro—a bovine aortic endothelial cell and a mouse adipose cell—fixed to Ti-coated sapphire. Tightly focused ultrashort laser pulses generate and detect GHz acoustic pulses, allowing three-dimensional imaging (x, y, and t) of the ultrasonic propagation in the cells with ∼1 μm lateral and ∼150 nm depth resolutions. Time-frequency representations of the continuous-wavelet-transform amplitude of the optical reflectivity variations inside and outside the cells show GHz Brillouin oscillations, allowing the average sound velocities of the cells and their ultrasonic attenuation to be obtained as well as the average bulk moduli.

Cytoskeletal tension modulates MMP-1 gene expression from tenocytes on micropillar substrates

Actin cytoskeletons, aggregated with myosin II, generate intracellular cytoskeletal tension, which is induced to cell attaching substrate as cell traction forces. It is thought that cytoskeletal tension links closely to cell functions. The present study examined quantitative relationships between cytoskeleton tension and the balance of cell metabolism of tenocytes. Using micromachining techniques, micropillar substrates were prepared with polydimethylsiloxane, having three different values of substrate elasticity (6, 18 and 33 kPa) by changing the micropillar height. After 24h incubation of bovine tenocytes on these micropillar substrates, cell traction forces were determined. Gene expressions for type I collagen (anabolic marker) and MMP-1 (catabolic marker) from tenocytes on micropillars were also analyzed with qPCR. In addition, effects of an inhibition of myosin II activity on tenocyte cytoskeletal tension and metabolism were examined using the inhibitor, blebbistatin. It was exhibited that cell traction forces were significantly larger in tenocytes on 33 kPa substrates compared to those on 6 kPa substrates. This was associated with significant lower expression of MMP-1 mRNA on 33 kPa substrates. Cell traction forces were decreased significantly by the supplementation of blebbistatin in a dose-dependent manner. Indeed, there were significant smaller traction forces and higher expression for MMP-1 mRNA from tenocytes treated with 10 μM blebbistatin compared to their corresponding controls. Accordingly, tenocyte responses to substrate stiffness are associated with alterations in intracellular tension and catabolic gene expression. On the other hand, tenocyte anabolism, as measured by type I collagen mRNA expression, was not altered with substrate stiffness.

A new experimental system for simultaneous application of cyclic tensile strain and fluid shear stress to tenocytes in vitro

Tenocyte mechanotransduction has been of great interest to researchers in tendon mechanobiology and biomechanics. In vivo, tenocytes are subjected to tensile strain and fluid shear stress, but most studies of tenocyte mechanobiology have been to understand how tenocytes regulate their functions in response to tensile strain. Thus, there is still much to know about tenocyte responses to fluid shear stress, partly due to the difficulty of devising a suitable experimental set-up and understanding the exact magnitude of imposed fluid shear stress. Therefore, this study was performed to test a new experimental system, which is suitable for the application of tensile strain and fluid shear stress to tenocytes in vitro. It was experimentally and numerically confirmed that tenocytes could maintain their in situ morphology within microfabricated microgrooves; also, physiological tensile strain and a wide range of fluid shear stress magnitudes can be applied to these cells. Indeed, it was demonstrated that the combined stimulation of cyclic tensile strain and oscillatory fluid shear stress induced a greater synergetic effect on tenocyte calcium response and significantly increased the percentage of tenocyte exhibiting increases in intracellular Ca2+ concentration compared to the solo applications of these two modes of mechanical stimulation. The experimental system presented here is suitable for research of tenocyte mechanobiology, particularly mechanotransduction events, which were difficult to study using previous experimental models like explants and cell monolayers.

A versatile micro-mechanical tester for actin stress fibers isolated from cells

Conventional atomic force microscopy is one of the major techniques to evaluate mechanical properties of cells and subcellular components. The use of a cantilever probe for sample manipulation within the vertical plane often makes absolute positioning of the probe, subject to thermal drift, difficult. In addition, the vertical test is unable to observe changes in the sample structure responsible for mechanical behavior detected by the probe. In the present study, an alternative mechanical tester was developed that incorporated a pair of micro-needles to manipulate a sample in a project plane, allowing acquisition of the accurate probe position and entire sample image. Using a vision-based feedback control, a micro-needle driven by a piezo actuator is moved to give user-defined displacements or forces to sample. To show its usefulness and versatility, three types of viscoelastic measurements on actin stress fibers isolated from smooth muscle cells were demonstrated: strain rate-controlled tensile tests, relaxation tests and creep tests. Fluorescence imaging of the stress fibers using Qdots over the course of the measurements, obtained through multiple image detectors, was also carried out. The technique described here is useful for examining the quantitative relationship between mechanical behavior and related structural changes of biomaterials.