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The mechanical properties of cells, such as leukocytes, in a diseased state differ from those of healthy cells, typically due to their microstructure. The deformability of the cells through a constrictive area is analyzed by the applied stress to the cell. This study investigates the relationship between the sample flow speed and distribution of captured leukocytes based on the cell deformability using a microfluidic device. The device comprises of microfilters that serve as the filtration mechanism. The microfilter gap size gradually decreases from 15 to 3 µm to facilitate the deformability-based separation. Leukocytes have various sizes; hence, they can be separated by microfilters directly from whole blood samples without any cell clogging, and they do not require sample pre-processing such as centrifugation or red blood cell lysis. The distribution of leukocytes captured by the microfilters with respect to the sample flow speed can be analyzed; at higher sample flow speeds of 6 µL/min, small leukocytes with a size of 7 µm could not be captured and they passed through the smallest microfilter gap size of 3 µm. For smaller leukocytes, such as lymphocytes, the distributions are mainly at gap sizes of 4 µm to 8 µm, with most of the lymphocytes captured at the 6 µm microfilter gap size. We conclude that the distribution of the cells captured during the filtration varies depending on the microfilter gap sizes, applied sample flow speed, cell sizes, and the ability of the cells to deform. The deformability imaging profiles of the sample could be developed from the images of the cell distribution, which might be useful for preliminary screening in the clinical applications. This work presents the development of a simple device for the study of cell deformability as the results provide a biophysical marker in high throughput and bulk sample analyses.

We have designed and evaluated a simple and practical system for CD3+ (T-lymphocyte) and CD19+ (B-lymphocyte) cell counting on-chip using a PDMS microfluidic chip which captures these cells using a pillarbased filtration technique. Conventional pillar-based or membrane-based cell separation methods suffer from the problem of cell clogging, thereby reducing the separating and capturing efficiency of the device. To address these issues, we proposed a unique and simple design of a microfluidic chip with an escape route at the pillar-based cell filtration area in the chip which prevents cell clogging. Currently, the diagnosis, monitoring and treatment of immunodeficiency disorder require quantifying the leukocyte population, especially T-lymphocytes and Blymphocytes, in whole blood. We evaluated the performance of our microfluidic chip by obtaining the ratio of T-lymphocytes and Blymphocytes from whole blood and compared the results to those obtained using a flow cytometer. We confirmed that the cell capture area of the microfluidic chip remains clog-free. We achieved 99.8% efficiency in capturing leukocytes using the microfluidic chip, and the ratio of CD3+ and CD19+ had a coefficient of r = 0.9876 and a coefficient of variation (CV) of 2.77%, from 1 μL of whole blood sample.

This paper proposes a 3D assembly method of aligned fiber-structure such as a human skeletal muscle tissue. The innovative point is assembly method of fiber-structure consists of artificial modular micro-fibers. The magnetite particles are included inside of both ends of micro-fibers for “Assembly”, “Positioning” and “Fixation” of micro-fibers, while the capillary force is also used to bundle several fibers tightly. Several different kinds of fibers are fabricated and assembled to form the tissue like structure. In this paper, the muscle fiber and sensor fiber, which has spherical microsensors inside, are used for demonstration of the proof of our concept. After the assembly, the micro-fibers were successfully fused to form a tissue like structure with highly arranged cells, where the microsensors could be used to monitor the inner state of the tissue.

This paper introduces a novel 3D assembly method for mimicking aligned fiber-structure like human skeletal muscle tissue (Fig. 1), and a monitoring system for it. The innovative point is that an artificial single modular micro-fiber comprises magnetic parts in both ends. The magnetic force and capillary force are used to achieve the non-contact manipulation, alignment and assembly of the micro-fibers (Fig. 2). Several different kinds of fibers are fabricated and assembled as a module of the tissue structure. In this paper, the sensor fiber and muscle fiber are used for demonstration of the proof of concept. After the assembly, the micro-fibers were successfully fused to form a tissue structure with highly arranged cells inside. Finally, the tissue would be cultured in the system for realtime monitoring of the internal state. This system has functions consisting of microscope observation, force measuring, fluorescent observation and constant CO2 and temperature control.

The present study was designed to investigate the local microenvironment of octacalcium phosphate in a granule form upon biomolecules adsorption utilizing an indicator-immobilized-gel-sheet for measuring pH. We previously showed that octacalcium phosphate enhances bone regeneration during its progressive hydrolysis into hydroxyapatite if implanted in bone defects. The gel-sheet was made from a photocrosslinkable prepolymer solution, which can easily immobilize a pH indicator (bromothymol blue; BTB) in the hydrogel. The indicator-immobilized-gel-sheet was mounted on a biochip which was made of polydimethylsiloxane (PDMS) with a flow channel. The pH value was calculated by detecting the color changes in the gel-sheet and displayed as the pH distribution. After pre-adsorption of bovine albumin, β-lactoglobuline or cytochrome C onto octacalcium phosphate granules, the granules with the gel-sheet were further incubated in Tris-HCl buffer solution in the absence or presence of fluoride, known as an accelerator of octacalcium phosphate hydrolysis. pH values of the gel-sheet surrounding octacalcium phosphate granules showed a decrease from pH 7.4 to 6.6 in relation to the proteins adsorbed. Overall, the proposed pH-sensitive gel can be used to detect the pH around octacalcium phosphate granules with a high spatial resolution.

Absolute counting of total leukocytes within small amounts of whole blood is difficult since there is not a good technique to separate the leukocytes from limited volume of whole blood. In this study a microfluidic device with a micropillar array is proposed to filter the leukocytes. Leukocytes are dyed with two different immunofluorescence reagent and using Histogram of Oriented Gradients (HOG) based feature detector, detected by using Support Vector Machines (SVM).

We have proposed a bio-automation system to isolate and recover circulating tumor cells (CTCs) individually from whole blood. An open-channel microfluidic chip-based approach is used to isolate the CTCs. The proposed microfluidic chip design can form a stable air-liquid interface. CTCs are trapped by the gaps in between the pillars of the microfluidic chip due to capillary force associated with the meniscus of the air-liquid interface. We propose a chip design to stabilize air-liquid interface and sample flow speed. We introduce an image analysis algorithm to detect the position of the air-liquid interface. Using the visual feedback from the image analysis algorithm, a control system is proposed to control air-liquid interface position. We succeeded in stabilizing the flow speed, making it feasible for the isolation of 5 mL of whole blood to be completed within 30 min. We achieved an average of position error of air-liquid interface of 4 μm with standard deviation of 7 μm. We have confirmed that air-liquid interface position is a deciding factor for trapping area of CTCs. By controlling air-liquid interface position, we have achieved trapping CTCs in a narrow band with a high concentration.

Training of vitreoretinal surgeries such as peeling of inner limiting membrane (ILM) requires elaborate retina model having contact measurement function since human retina is the delicate tissue. Fabrication of the retina model having measurement function without impairing the physical properties of the human retina is challenging issue. This paper reports the development of a retina model for optical measurement of deformation distribution using digital image correlation (DIC). The retinal model consists of the retina part and the sclera part. Both parts reproduce Young’s modulus of human retina and sclera, respectively. The deformation distribution on the surface of retinal model was measured by the non-contact measurement using DIC. The Young’s modulus of the retina part and sclera part were 30 kPa and 1.9 MPa, respectively. We demonstrated the optical measurement of deformation distribution on the surface of retinal model during the pushing with ILM forceps.

Although there is a great need for suitable vascular replacements in clinical practice, much progress needs to be made toward the development of a fully functional tissue-engineered construct. We propose a fabrication method of engineered tubular tissue for small blood vessels via a layer-by-layer cellular assembly technique using mouse smooth muscle cells, the construction of a poly-(l-lactide-co-e-caprolactone) (PLCL) scaffold, and integration in a microfluidic perfusion culture system. The cylindrical PLCL scaffold is incised, expanded, and its surface is laminated with the cell layers. The construct confirms into tubular structures due to residual stress imposed by the cylindrical PLCL scaffold. The perfusion culture system allows simulation of static, perfusion (laminar flow), and perfusion with pulsatile pressure (Pulsatile flow) conditions in which mimicking the in vivo environments. The aim of this evaluation was to determine whether fabricated tubular tissue models developed their mechanical properties. The cellular response to hemodynamic stimulus imposed by the dynamic culture system is monitored through expression analysis of fibrillin-1 and fibrillin-2, elastin and smooth muscle myosin heavy chains isoforms transcription factors, which play an important role in tissue elastogenesis. Among the available materials for small blood vessel construction, these cellular hybrid vascular scaffolds hold much potential due to controllability of the mechanical properties of synthetic polymers and biocompatibility of integrated cellular components.

A rapid construction process is necessary to build up numerous cell modules into three-dimensional (3D) tissues that retain the tissue geometries and initial conditions of the cells. We propose a new 3D assembly technique using water transfer printing to fabricate a hollow tubular tissue structure. Utilizing this assembly technique, we discuss the relationship between the 3D transcriptional body of a gel matrix and the developed shape of transferred tissue. We then fabricate hollow tubular tissue. Simulation of the 3D environment in which tissues normally develop and function is crucial for the engineering of in vitro models that can be used for the formation of complex tissues. These artificial hollow tubular tissues could be used as in vitro simulators for drug efficiency evaluation and operative training.

We have developed a microfluidic chip that can separate gastric cancer cells from peritoneal washes. Gastric cancer cells tend to be agglomerated in the peritoneal wash, and are larger than the other cells and the soft tissue fragments. This paper presents a novel microfluidic chip to enrich low-concentrated cancer cells from the peritoneal wash by size-dependent cell filtration. This microfluidic chip has the advantage that it can separate agglomerated cells in high speed because larger objects are filtered by a gap in the microchannel. Two-step exposure was performed to fabricate a precise uneven channel for cell filtration. The main and shallow channels have heights of 100 μm and 8 μm, respectively. Furthermore, inserting fins in the microchannel that assist sheath flow enhanced continuous and robust separation against flow fluctuation in the microchannel. The microfluidic channel was designed to have high filtration efficiency and a high throughput while having a compact profile. We used patient's peritoneal washes to isolate cancer cells. As a result, we were able to enrich the cancer cells in peritoneal wash.

Rapid and effective injection of functional nanobeads into a specific living cell with high cell viability is one of the most important issues for single cell analysis. This paper reports a novel method of injecting single magnetic nanobeads into a living Madin-Darby canine kidney (MDCK) cell using a continuous-wave near infrared laser. Experimental results demonstrated the successful injection of magnetic nanobeads into living cells with high injection rates reaching 100 %, maximal cell survival rates of 90 % at 14 mW laser power. Injection times was achieved about 1.4 sec.

Techniques that allow cells to self-assemble into three-dimensional (3D) spheroid microtissues provide powerful in vitro models that are becoming increasingly popular in fields such as stem cell research, tissue engineering, and cancer biology. Appropriate simulation of the 3D environment in which tissues normally develop and function is crucial for the engineering of in vitro models that can be used for the formation of complex tissues. We have developed a unique multicellular aggregate formation platform that utilizes a maskless gray-scale photolithography. The cellular aggregate formed using this platform has a toroidal-like geometry and includes a micro lumen that facilitates the supply of oxygen and growth factors and the expulsion of waste products. As a result, this platform was capable of rapidly producing hundreds of multicellular aggregates at a time, and of regulating the diameter of aggregates with complex design. These toroidal multicellular aggregates can grow as long-term culture. In addition, the micro lumen can be used as a continuous channel and for the insertion of a vascular system or a nerve system into the assembled tissue. These platform characteristics highlight its potential to be used in a wide variety of applications, e.g. as a bioactuator, as a micro-machine component or in drug screening and tissue engineering.

The present study was performed to establish a novel ocular surgery simulator for training in peeling of the inner limited membrane (ILM). This simulator included a next-generation artificial ILM with mechanical properties similar to the natural ILM that could be peeled underwater in the same manner as in actual surgery. An artificial eye consisting of a fundus and eyeball parts was fabricated. The artificial eye was installed in the eye surgery simulator. The fundus part was mounted in the eyeball, which consisted of an artificial sclera, retina, and ILM. To measure the thickness of the fabricated ILM on the artificial retina, we calculated the distance of the step height as the thickness of the artificial ILM. Two experienced ophthalmologists then assessed the fabricated ILM by sensory evaluation. The minimum thickness of the artificial ILM was 1.9 ± 0.3 μm (n = 3). We were able to perform the peeling task with the ILM in water. Based on the sensory evaluation, an ILM with a minimum thickness and 1000 degrees of polymerization was suitable for training. We installed the eye model on an ocular surgery simulator, which allowed for the performance of a sequence of operations similar to ILM peeling. In conclusion, we developed a novel ocular surgery simulator for ILM peeling. The artificial ILM was peeled underwater in the same manner as in an actual operation.

Muscloskeletal tissues, bone, cartilage, muscles and tendons regulate and support the body's actions and are differentiated from mesenchymal stem cells. These organs generate and/or respond to mechanical stress, which is inevitable in daily life. Among these tissues, cartilages play roles in articular function in joints where shear stress is loaded in combination with cyclical or intermittent compressive force by joint action, and provides a template for bone growth under compressive stress directed along the long axis of long bones. Along with soluble factors, such as cytokines and growth factors, mechanical stress has been recognized as one of the epigenetic factors that regulates the gene expression of various types of cells. Thus, the molecular mechanisms of the mechano-sensing, mechano-transduction, and mechano-response of cells have become a focus of connective tissue biology. Here, we will discuss the mechanisms through which differentiated chondrocytes and mesenchymal cells that are differentiating into chondrocytes respond to various types of mechanical stress by altering their phenotype and how these phenotypic changes are molecularly regulated. We will focus on the roles of cell-extracellular matrix interactions through integrins and downstream signaling pathways involving mitogen-activated protein kinases.

Octacalcium phosphate (OCP) has been advocated to be a precursor of biological apatite crystals in bones and teeth. Our previous studies showed that synthetic OCP stimulates bone regeneration, followed by the progressive conversion of OCP into hydroxyapatite (HA), when implanted in bone defects. However, the precise mechanism to induce the osteogenic phenotype in osteoblasts by OCP has not been identified. The present study was designed to investigate whether the physicochemical aspect, specific to and derived from the structural properties of OCP, influences the function of an osteoblastic cell line, mouse bone marrow stromal ST-2 cells. Different amounts of synthetic OCP and synthetic sintered ceramic HA were coated onto 48-well tissue culture plates. The amounts of OCP and HA were controlled to strengthen their intrinsic physicochemical properties, in which the milieu around the crystals will be modified during the culture. The roughness of the OCP coatings was independent of the amount of coating. Chemical analyses of the supernatants of the OCP coatings revealed that the concentration of Ca2+ decreased with increasing amounts of OCP, while the concentration of inorganic phosphate increased markedly, most probably through OCP--apatite conversion. ST-2 cells were cultured on the OCP or HA coatings up to day 21. The OCP coating caused a significant decrease in cell attachment and in the initial stage of proliferation, dependent upon the amount of coating. On the other hand, OCP enhanced the expression of osteogenic markers, including type I collagen, alkaline phosphatase, and osterix. However, HA did not alter the expression of these markers in ST-2 cells cultured on different amounts of HA coating. These results demonstrated that OCP is capable of inducing the differentiation of stromal cells into osteoblastic cells, especially differentiation into early stage osteoblastic cells, prior to reaching the stage of mature osteoblastic cell lineage.

We succeeded in isolating circulating tumor cells from whole blood of Mouse-CTC model with using controlled air-liquid interface on an open-chip. Also, we could pick-up isolated CTC from the open-chip and obtain enough high viability for downstream analysis. Furthermore, we are developing fully automated platform for this whole process.

This paper reports temperature indicator for surgical simulator and temperature calculation method using image processing for visualization of temperature increase and distribution in the arterial biological model. The indicator consists of polydimethylsiloxane (PDMS) and thermochromic pigment presenting non-reversible color change according to temperature. The indicator has transparency to observe the catheter inside the model during simulation. Temperature of color indicator is calculated from the color change using several color spaces such as YCrCb color space. The range of the temperature measurement is from 50 to 110 degree of C. The accuracy of the temperature measurement is approximately ±2.0 °C. Temperature distribution of ablated model was measured (Ablation temperature: 90 °C.). Finally, temperature change measurement of ablated renal vessel model was demonstrated.

We succeeded in isolating pre-counted small amounts of circulating tumor cells from whole blood. And we also confirmed isolation of CTCs from mouse-CTC model with same method. On our open microfluidic chip, CTCs are isolated by their size-difference from other blood cells. Therefore, we arranged micropillars on the microfluidic chip. Blood sample is injected between chip and inclined coverglass. And then, by suction of blood sample, CTCs are drifted and collected. Near 90% of CTCs were isolated without any pretreatments. We also operated animal testing with same method.

We proposed a fabrication technique of tubular tissues as an artery and the circulatory culture system for evaluating the development mechanisms of them. The tubular tissues were 2–6 mm in diameter, using a poly (L-lactide-co-ipusironn-caprolactone) (PLCL) scaffold. They consisted of the cell membranes and the PLCL scaffolds. Moreover, these tubular tissues had a mechanical property of artery because of the PLCL scaffolds. We successfully integrated them to the circulatory culture system. After that, we can evaluate the elastic fiber development of the tubular tissues with the mechanical stimulus utilizing the developed circulatory culture system.

Computer topography (CT) imaging is one of the powerful-tools for nondestructive observation technique. In this paper, we present observation of bone from mice by scanning electron microscope (SEM)-CT system using environmental- SEM (E-SEM). The advantage of this SEM-CT system is able to image in high-resolution with less than 400 nm resolution. We investigated the effects of X-ray wavelengths and intensities to improve the imaging contrast for SEM-CT observation. From experimental results, a porous structures were observed inside the bone sample with approximately 10 μm pores.

We developed an active virus filter (AVF) that enables virus enrichment for single virus infection, by using insulator-based dielectrophoresis (iDEP). A 3D-constricted flow channel design enabled the production of an iDEP force in the microfluidic chip. iDEP using a chip with multiple active virus filters (AVFs) was more accurate and faster than using a chip with a single AVF, and improved the efficiency of virus trapping. We utilized maskless photolithography to achieve the precise 3D gray-scale exposure required for fabrication of constricted flow channel. Influenza virus (A PR/8) was enriched by a negative DEP force when sinusoidal wave was applied to the electrodes within an amplitude range of 20 Vp-p and a frequency of 10 MHz. AVF-mediated virus enrichment can be repeated simply by turning the current ON or OFF. Furthermore, the negative AVF can inhibit virus adhesion onto the glass substrate. We then trapped and transported one of the enriched viruses by using optical tweezers. This microfluidic chip facilitated the effective transport of a single virus from AVFs towards the cell-containing chamber without crossing an electrode. We successfully transported the virus to the cell chamber (v = 10 µm/s) and brought it infected with a selected single H292 cell.

We proposed a 3D assembly technique and a circulatory culture system. The technique gives multilayer-structured tubular tissues for blood vessel models. By the circulatory culture system, this study was aimed to determine whether the tissue-engineered tubular models maintain the normally mechanical property as the development and function. In this work, we were able to fabricate tubular tissues from multilayered tissues rapidly in the suitable conditions and circularly culture the fabricated multilayer-structured tubular tissues.

We proposed a 3D assembly technique of small-diameter blood vessels using a PLCL (poly (L-lactide-co-e-caprolactone)) scaffold. The technique uses a residual stress of PLCL scaffolds to fabricate a multilayer structured tubular tissue, and gives a tissue mechanical property which blood vessels originally have. In the future, we try to test the circulatory culture system in order to investigate whether the tissue-engineered structure maintains the equivalent mechanical property as the human blood vessel. In this work, we demonstrated that fabricated tissues could attach on the inside of tubular PLCL scaffold in the appropriate conditions.

The CTCs (Circulating Tumor Cells) isolation method enables to sort very rare single CTC from the whole blood. We utilized the technique of convective self-assembly, which is a technique for depositing cells onto the microfluidic channel, to isolate CTCs. Through the convective self-assembly processes, the cells including CTCs and blood cells are deposited in the channels of microfluidic chip. After giving the sheath flow, only the CTCs remain in the channels and the other cells are removed. As the result, it was indicated that a specific design of microchannel has the highest collection rate of about 90%.

We proposed a 3D assembly technique using water transfer printing to fabricate a multilayer structured tubular tissue. This study was aimed to determine whether the tissue-engineered tubular structure maintains the normally mechanical property as the development and function by the artificial circulatory system. In this work, we demonstrated that fabricated tissues could rapidly assemble into aligned tubular tissue in the appropriate geometrical conditions using engineering approaches. This technique does not require a solid biodegradable scaffold. Therefore, this approach presents the simple and rapid method to create through the exploitation of the intrinsic potential of cells to assemble fabricated tissues into functional 3D tissues in a suitable tubular tissue environment. The described technique is applicable to many different cell types and can be used to engineer tissue constructs of user-defined size and shape with micro-scale control of the cellular organization, which could form the basis for constructing 3D engineered tissues with a hollow tubular tissue in vitro.

We propose a concurrent microvascular connection method called suction-induced vascular fixation (SVF) method for the achievement of Organ-Explant-Chip which is a biologically-designed simulator having biological materials such as cells, tissues, and organs. The advantages of proposed method with using a microfluidic device are as follows: (1) operation of flexible objects (blood vessels), (2) alignment the blood vessels concurrently, and (3) reduction of the DOFs of the blood vessels. From the experimental results, we confirmed that four cardiovascular of the explanted embryonic chick heart can be induced into the fabricated microfluidic device concurrently. We have also succeeded in construction of hybrid circulatory system between artifacts and embryonic chick heart, and monitoring the response of the heart of chick embryo by supplying the culture medium.

We newly propose a microfluidic device with multiple functions for simple microvascular connection. By using this microfluidic device with suction mechanism, we can operate the blood vessels in a single device for assisting the microvascular connection procedure such as fixing the position of the blood vessels, expanding the diameter of the blood vessels, and pouring the adhesion and drugs. We have succeeded in connecting the microcardiovascular of the extracted chicken embryonic heart to artificial mictotubes in a shorter time than the manual operation such as suturing. We have also succeeded in constructing the biological simulator that can observe a living organ continuously while circulating the culture medium through the inside of the heart. Finally, we showed the prototype platform of the evaluating the migration of the cells at the connection part between the extracted tissue from living organism and the assembled tissue structure.

In this paper, we newly propose an organ-explanted bionic simulator (OBiS) using an isolated organ tissue. We use a heart isolated from the chick embryo for proposed simulator. To achieve the OBiS, we perform the concurrent microvascular anastomosis by using suction-induced vascular fixation (SVF) method for easily connection to artificial tubes with blood vessels led to the explanted chick's heart. Through the experiment with a simulated cardiovascular, it is confirmed that vessels can be aligned by using the proposed method. In addition, we have succeeded in connecting the Alg tubes with the blood vessels led to the heart of chick embryo. OBiS is available for the evaluation of new drugs immune response not only without using large animals but also as a response of organs beyond cellular level.

We propose a simple and rapid microcardiovascular connection method called suction-induced vascular fixation (SVF) method for the achievement of bionic simulator with using chicken embryonic heart. The advantages of proposed method with using a microfluidic device are as follows: (1) operation of flexible objects (blood vessels), (2) alignment the blood vessels concurrently, and (3) reduction the DOF of the blood vessels. From the experimental results, we confirmed that four cardiovascular can be induced into the fabricated device concurrently. Furthermore, we have succeeded in connecting to a heart of chick embryo with microtubes. We have also succeeded in construction of hybrid circulatory system between artificial tubes and blood vessels led to the heart of chick embryo.

Although the effi cacy of the in vivo osteogenic capabilities of synthetic octacalcium phosphate (OCP) crystal implantation can be explained through its stimulatory capacity for the differentiation of the host osteoblastic cell lineage, direct evidence that OCP supports bone regeneration by osteogenic cells in vivo has not been shown. Mesenchymal stem cells (MSCs) isolated from 4-week-old male Wistar rat long bones were pre-incubated in osteogenic or maintenance medium in the presence or absence of basic fi broblast growth factor (bFGF). OCP/Collagen (OCP/Col) or collagen disks were seeded with MSCs that had been pre-incubated in osteogenic medium containing bFGF, which exhibited the highest differentiation induction, and then incubated for an additional day. The disks were implanted in critical-sized calvaria defects of 12-weekold male Wistar rats and the specimens were analysed radiographically, histologically, histomorphometrically, and by micro-computed tomography (CT) imaging at 4 and 8 weeks after the implantation. The OCP/Col·MSCs group rapidly induced more bone regeneration, even within 4 weeks, compared to the OCP/Col group without MSCs. The bone mineral density of the OCP/Col·MSCs group was also greater than the OCP/Col group. The Col·MSCs group did not exhibit prominent osteogenicity. These results indicate that OCP crystals in a collagen matrix effi ciently promote exogenously introduced osteogenic cells to initiate bone regeneration if the cells are pre-treated in a suitable differentiation condition.

This paper presents an innovative driving method for an on-chip robot actuated by permanent magnets in a microfluidic chip. A piezoelectric ceramic is applied to induce ultrasonic vibration to the microfluidic chip and the high-frequency vibration reduces the effective friction on the MMT significantly. As a result, we achieved 1.1 micrometre positioning accuracy of the microrobot, which is 100 times higher accuracy than without vibration. The response speed is also improved and the microrobot can be actuated with a speed of 5.5 mm s(-1) in 3 degrees of freedom. The novelty of the ultrasonic vibration appears in the output force as well. Contrary to the reduction of friction on the microrobot, the output force increased twice as much by the ultrasonic vibration. Using this high accuracy, high speed, and high power microrobot, swine oocyte manipulations are presented in a microfluidic chip.

The present study was designed to investigate whether mechanical testing in conjunction with microcomputed tomography (microCT) analysis can be used to evaluate the quality of regenerated bone enhanced by the implantation of a composite composed of granular octacalcium phosphate and collagen (OCP/Col) matrix. Previous studies confirmed that the granules of OCP alone or OCP in Col matrix tend to mature into bone-like hydroxyapatite and enhance bone regeneration coupled with its own biodegradation, if implanted in various bony sites. OCP/Col was implanted in rat calvaria critical-sized bone defect for 4 to 12 weeks for microindentation, mechanical testing, microCT imaging, and histological examinations. The microindentation testing of the regenerated bone revealed a progressive increase of the Vickers hardness showing the highest value in 12 weeks. The Vickers hardness was in good agreement with both the parameters of microCT and the mechanical property; the quality of regenerated bone increased progressively with the implantation periods. The regenerated bone exhibited a mature bone-like matrix structure with osteocytes histologically. The quality of the regenerated bone was compatible to that of normal control calvaria bone regarding the mechanical properties. The results indicate that microindentation testing can be used to evaluate the quality of regenerated bone in the initial regeneration and the development of bone enhanced by OCP granules within Col matrix. It appears that the implantation of OCP/Col could be a model study to determine the quality of the regenerated bone.

Previous studies have suggested that the biodegradability of octacalcium phosphate-collagen (OCP/Col) composite by osteoclasts is accelerated in association with mechanical stress suffered by the host tissue around the implant. The present study was designed to investigate whether alleviation of mechanical stress restores the bone regenerative properties of OCP/Col, as previously shown in nonload-bearing sites. OCP/Col discs supported with a polytetrafluoroethylene (PTFE) ring, which has a higher modulus than OCP/Col, were implanted in a rat subperiosteal pocket for up to 12 weeks. The structural features of the implant and biological responses were analyzed by X-ray diffraction, Fourier transform infrared spectroscopy, histomorphometry, histochemistry, and tissue mRNA expression around the implants. The effect of compression was analyzed using mouse stromal ST-2 cells by deforming the cell-seeded OCP/Col discs in vitro with or without a PTFE ring. The results clearly indicated the restoration of bone formation by the alleviation of mechanical stress and the upregulation of osteoblast-related genes, such as osterix on the other hand, the implantation of OCP/Col on calvaria or in an in vitro test without PTFE support resulted in the upregulation of osteoclast-related genes, such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K, in the tissues or receptor activator of the nuclear factor-kappaB ligand (RANKL) in ST-2 cells. The results confirmed that calvaria augmentation is enhanced by implanting OCP/Col if suitable conditions regarding mechanical stress are provided.

The present study was designed to investigate whether the amount of octacalcium phosphate (OCP) affects the characteristics of alginate (Alg)/OCP scaffolds regarding the pore formation and its distribution, and the thermodynamic stability from OCP to hydroxyapatite (HA) in an in vitro physiological environment. Alg/OCP composites with weight ratios of 100/0, 75/25, 50/50, and 25/75 were prepared through mixing the ground synthesized OCP crystals with an Alg solution and applying lyophilization. Analysis of X-ray diffraction, Fourier transform infrared (FTIR) spectroscopy, and mercury intrusion porosimetry verified that the crystalline phase of OCP and the porosities were retained regardless of the OCP amount. On the other hand, the elastic modulus, determined by mechanical testing, and, interestingly, the pore size increased with increasing the OCP amount. The immersion of the composites in a simulated body fluid up to 14 days revealed that OCP in Alg matrices tends to convert to HA with enhancing the calcium consumption depending on the OCP amount. The results indicated that the inclusion of OCP crystals in the Alg matrix by the mixing process controls the character of the pore distribution in Alg/OCP composites while maintaining the transitory nature of OCP.

The present study was designed to investigate whether compressive mechanical strain affects osteoblastic cell differentiation. We developed a culture system with the multi channel that induces the mechanical stimuli with quantitative gradient strain on seeded cells during the culture. This culture system was a computer-driven device that the various mode of mechanical strains can be applied, using the pressurization or depressurization, to deform cells cultured on PDMS membrane. The mRNA expression levels of osteoblast-related genes, such as osterix, collagen type I, and osteopontin, determined by real-time PCR, decreased by the compressive strain. On the contrary, the expression of RANKL, a factor related to the induction of osteoclasts, increased in the compressive strain, while the expression of osteoprotegrin, a molecule that antagonizes the function of the RANKL, decreased in the same strain. It was suggested that the function of osteoblastic cells may be influenced by the compressive strain.

The musculoskeletal system is a unique combination of soft and hard tissues in the body, consisted of bone, cartilage, muscle and tendon. These skeletal tissue formation are in essence organized by the tissue specific cells, including osteoblasts, osteocytes, chondrocytes, myocytes and fibroblasts. Mechanical stress has a variety of effects on the structure and function of their cells. However, little is known about the effect of mechanical stress on cell differentiation regarding the quality and quantity of the stress and the timing applied. In the present study, we developed a culture device that enables to load the mechanical stress quantitatively with ease to evaluate proliferation and differentiation of cells.

We described an assembly technique and perfusion culture system for constructing artery tissue models. This technique differed from previous studies in that it does not require a solid biodegradable scaffold; therefore, using sheet-like tissues, this technique allowed the facile fabrication of tubular tissues can be used as model. The fabricated artery tissue models had a multilayer structure. The assembly technique and perfusion culture system were applicable to many different sizes of fabricated arteries. The shape of the fabricated artery tissue models was maintained by the perfusion culture system; furthermore, the system reproduced the in vivo environment and allowed mechanical stimulation of the arteries. The multilayer structure of the artery tissue model was observed using fluorescent dyes. The equivalent Young's modulus was measured by applying internal pressure to the multilayer tubular tissues. The aim of this study was to determine whether fabricated artery tissue models maintained their mechanical properties with developing. We demonstrated both the rapid fabrication of multilayer tubular tissues that can be used as model arteries and the measurement of their equivalent Young's modulus in a suitable perfusion culture environment.

A rapid construction process is necessary for building up numerous cell modules into 3D tissues maintaining the tissue geometries and initial conditions of cells. We proposed new 3D assembly technique to fabricate a hollow tubular tissue structure using by water transfer printing. By utilizing this assembly technique, we discuss the relation between 3D transcriptional body of gel matrix and a developed figure of transfer tissue, and perform fabricating on a hollow tubular tissue. Appropriately simulation of the 3D environment in which tissues normally develop and function is crucial for the engineering in vitro models that can be used for the formation of complex tissues. These artificial hollow tubular tissues would be used for drug efficiency evaluation and operative training as in vitro simulators.