Poly (vinyl alcohol) (PVA) hydrogel with high water content is the candidate material for artificial cartilage. PVA hydrogels prepared by freeze-thawing (FT) method (PVA-FT gel), cast-drying (CD) method (PVA-CD gel) and hybrid method of FT and CD (PVA-hybrid gel) were developed and their friction and wear behaviors were evaluated. Sliding pairs of a Cobalt-chromium-molybdenum (Co-Cr-Mo) alloy or alumina ceramic ball and a PVA hydrogel plate were tested in reciprocating friction test. In this study, ultra-pure water and simulated synovial fluid were used as lubricants for friction test. PVA-FT gel showed high friction and severe wear in ultra-pure water. Friction coefficient of PVA-CD gel in ultra-pure water was quite low such as about 0.005, but scratches were observed on the surface of PVA-CD gel. PVA-hybrid gel lubricated in ultra-pure water showed low friction such as 0.004, and intact surface structure of PVA-hybrid gel remained after friction test. Simulated synovial fluid contributed to the improvement of lubrication property of all PVA hydrogels and PVA-hybrid gel showed the lowest friction coefficient such as 0.003 and minimum wear. These results indicated that the hybridization of FT and CD method improved the lubrication property of PVA hydrogel, and PVA-hybrid gel has a great potential as the material for artificial joint.

View full abstract

In joint prostheses where ultra-high molecular weight polyethylene (UHMWPE) is used as bearing material, efficacious treatments such as crosslinking, addition of vitamin E and the grafting of phospholipid polymer are known to have improved wear resistance. However, friction and wear problems have not yet been completely solved under severe conditions, where direct contacts can occur in mixed or boundary lubrication. Accordingly, joint prostheses containing artificial hydrogel cartilage with properties similar to those of articular cartilage are expected to show superior tribological functions. The application of poly(vinyl alcohol) (PVA) hydrogel with high water content as biomimetic compliant artificial cartilage is expected to reproduce similar adaptive multimode lubrication mechanism to natural synovial joints. In this paper, the tribological behaviors of three kinds of PVA hydrogels different in preparation methods were evaluated was evaluated in reciprocating tests lubricated with saline and simulated synovial fluid. The interstitial fluid pressure and friction for PVA hydrogels were estimated by biphasic finite element analysis. The superior lubricating performance of PVA hybrid gel was discussed from biphasic and boundary lubrication.

View full abstract

Traumatic Brain injury (TBI) is one of the most common causes of death or disability and remains an important public health issue. Diffuse Axonal Injury (DAI) is one of the most frequent closed head injuries. This paper presents a reconstruction simulation and evaluation method for prediction of nerve damage by using computational modeling to simulate a realistic falling accident scenario. The motion of the injured people was reconstructed by MADYMO and kinetic data and hit part were generated. In this case the stress of the brain tissue were calculated in detail by the FE human head model and injury risk was predicted.

View full abstract

For the sake of emergency treatment for head injury, and even for applying the appropriate treatment for the patient, to estimate the damage properly has been required. The objective of this study is prediction simulation and visualization of the types of brain injury in head trauma accidents. In this paper, the case of contrecoup contusion of a woman on a bicycle who was contacted by a walker and hit her left side head on the asphalt road was simulated using multibody body dynamics and finite element (1%) methods. The contusion shown on the medical images were compared to the pressure pattern in the FE model and the site and severity of neurological damage were evaluated from von Mises stress. As the result, the prediction simulation matched well the site of brain contusion, and the severity of the predicted neurological injury was consistent with the physician's diagnostic result.

View full abstract

Field accident in American football causes traumatic brain injuries such as concussion and subdural hematoma. It is highly possible that serious brain injury is developed by repeated concussions during a comparatively-short term. Therefore, it is difficult to judge when the injured players are returned to games. We obtained the videotapes in which a head-to-head collision in actual American football game was recorded. A struck player developed concussion without loss of consciousness, with loss of memory and with no apparent abnormalities by CT scan. A reconstruction simulation of player's head collision using mathematical dynamic model (MADYMO) and finite element (FE) head and helmet model was conducted based on the accident videotapes.

View full abstract

Although traumatic brain injuries are caused by rotational impact, the effect of the rotational impact on cell damage is not entirely understood. In previous study, the reconstruction analysis of accident using the head finite element model shows the impact loaded by the rotational acceleration is easier to cause brain injury than by the linear acceleration. However, in vitro experiment taken in consideration of the rotational acceleration that generates biaxial strain with in test specimen is not conducted. This study designs and develops a device that exposes neurons cultured on the gelatinous specimen to biaxial strain generated by the rotational impact. The major components of the device are servo motor, encoder, controller, and dish with the gelatinous specimen. We validate whether the strain field of the gelatinous specimen mimics the deformation induced by head impact and evaluate the tolerance of cultured neuron for biaxial strain.

View full abstract

Diffuse axonal injury (DAI), being the most common type of pathology in Traumatic Brain Injury (TBI), has two distinct types of axonal pathology; swellings that induced by the neurofilament accumulation as a result of the damaged neurofilament structure in the axonal cytoskeleton and focal compaction and/or impaired transport due to the mechanical insult along the axons fallowed by secondary axotomy, and the axonal bulbs which likely represent complete axonal disconnection. These cytoskeletal abnormalities which proceed to the formation of the swellings and secondary axotomy are the morphological indication of DAI. Although previous researches have provided some insight on understanding the cellular mechanism of neuronal injury, and suggested that the degree of electrophysiological impairment and morphological damage of neurons is directly related to the magnitude and rate of axonal stretch, the exact mechanisms that initiate secondary degeneration in DAI are yet to be fully characterized. Our previous studies suggested that the threshold of dysfunction and interruption of axonal transport is strains of 15-22 % at strain rates of 21-27 s^<-1> and both β-APP and Tau are promising candidates for injury analysis. However, quantitative relation between impulsive strain and axonal injury is yet to be discovered thoroughly. Therefore, in this study, axonal injury induced by uniaxial stretch on differentiated mouse neuronal stem cells in order to clarify the relation between the impulsive strain, strain rate and axonal injury. Herein, evaluation is performed by immunohistochemical labeling, with β-APP and tau protein accumulation as biochemical markers of choice.

View full abstract

Traumatic brain injury caused by traffic accidents, blow, falls and contact sports, is an important public health problem. Diffuse Axonal Injury (DAI) shows a symptom of no consciousness without either cerebral contusion or hematoma. It is thought that DAI is occurred at the damage of the cell level within the brain widely. However, the pathogenesis of DAI is not yet fully clarified, and the effective diagnosis and cure are not established. The ultimate goal in this study is to clarify the threshold for neuronal damage DAI at the cell level of the brain. Therefore, we exposed axons in cultured neuronal cells to the impulsive strain and the effect of the impulsive strain on axonal injury was evaluated at the cell level. Neural stem cells (NSCs) were used as cultured neuronal cells. NSCs can be proliferate and differentiate into neurons and glia. When cultured in medium, NSCs extend the axons towards any direction. However, the in vivo neuronal cells of the human have a characteristic of the orientation. Therefore, the direction of axons growth is controlled by using micro grooves which matched with the size of the cell. Culture surface is used by micro grooves on polydimethylsiloxane (PDMS). It is transcribed from micro groove were generated by using photolithography on resist material (SU8) and the Si substrate. This study has investigated the effects of impulsive strain on axons in cultured neurons differentiated from NSCs.

View full abstract

It is quite important to understand the damage state in brain at head impact, in the view point of the medical treatments and recognition of the brain injury. In past similar studies in which the conventional FEM was used, each part of the brain, such as cerebrum, cerebellum, corpus callosum and brain stem, has been treated as same material, and in most studies the liner elastic properties were chosen to the model instead of the hyper elastic one. Additionally, in some studies some of the brain parts were neglected in the FE model because of the difficulty of the modeling. In this study, to investigate the damage state in the brain at the impact accurately, the head impact simulations were performed using the 3D head model containing all part of the brain as mentioned above, where the material properties of each part of the brain obtained from the tensile test using fresh bovine brain, were applied to each brain part model. Then the validity of the simulation was checked comparing the calculated stress-strain state with medical images. As the result, the minimum principal strain around the corpus callosum was remarkably larger than the one in the case of applying the material properties of cerebrum to the all part of the brain.

View full abstract

Head impact may causes hemorrhage and brain edema. These are thought to be related to impulsive pressure in the skull, but that hasn't been investigated in detail. Therefore, the purpose of this study was to clarify relationship between impulsive pressure and hemorrhage and brain edema. Brain edema is considered to be due to disruption of blood brain barrier (BBB). BBB is a selective permeability barrier. BBB is formed by endothelial cells, pericytes and astrocytes. In this report, we describe about relationship between impulsive pressure and disruption of BBB. Endothelial cells are connected by adhesion proteins. When adhesion protein decrease, blood plasma leak out from BBB and cause brain edema. Impulsive pressure is reproduced using the device loading impulsive pressure. BBB is reproduced using in vitro BBB models. I used two types of models (E00: Endothelial cells, EPA: Endothelial cells, pericytes and astrocytes). I expose impulsive pressure to in vitro BBB models. After that, we measured the transendothelial electrical resistance (TEER) for estimate effect of impulsive pressure. TEER depends on the expression of cell adhesion protein. In the result, TEER decreased after loading impulsive pressure. Decrease of TEER in EPA model is larger than E00 model. These results suggest that impulsive pressure decrease the expression of adhesion protein. The expression of adhesion protein causes brain edema. These results also suggest that pericytes and astrocytes have an effect on permeability of BBB after loading impulsive pressure.

View full abstract

Cancer is one of the most common causes of death in modern society, and metastasis contributes its high mortality. Some types of cancer metastasize to the lymph node, and trigger to the distant metastasis, resulting in poor prognosis. Immigration of tumor cells to the distant organs via lymph node has not been poorly understood and metastasis route in lymphatic vessel has been considered to be different from blood circulation. Our recent study showed that distant metastasis is caused by invasion into vein on the surface of a lymph node after metastasis into the lymph node in a lymph node metastasis mouse model, MXH10/Mo-lpr/lpr (MXH10/Mo/lpr) mouse and suggested that the "lymph node mediated hematogenous metastasis" should be recognized as the final of lymph node metastasis. However, the circulation system around the lymph node that was observed in MXH10/Mo/lpr mice is not be proved in conventional mice. Here we show the similarity of blood vessel network around the subiliac lymph node (SiLN) in MXH10/Mo/lpr mouse C57BL/6 mouse, BALB/cAJcl mouse and NOD/ShiJic-SCID mice. We found that the surficial vein on SiLN is connected to inferior vena cava, and this connection contributes lymph node mediated hematogenous metastasis. We anticipated our investigation to be a start point for advanced research of lymph node mediated hematogenous metastasis.

View full abstract

Cancer is the leading cause of death in Japan, and most of the cancer patients die relation to the metastasis. However, there is still no reliable treatment for metastatic lymph nodes (LNs). In the present study, we propose a drug delivery method via lymph network for metastatic LNs using ultrasound and nano/micro bubbles. We used MXH10/Mo-lpr/lpr mice, which develop systemic swelling of lymph nodes up to 10 mm in diameter, similar in size to human lymph node. The tumor cells were injected into the upper stream LN; the subiliac LN (SiLN) and the metastasis was induced into the downstream LN; the proper axillary LN (PALN). A dye or anticancer agent were injected into another upper stream LN; the accessory axillary LN (AALN). We visualized that the dye can be delivered into the metastatic LN via the lymphatic vessel. In addition, the effectiveness of new lymphatic drug delivery method was examined. We anticipate that the present method has a potential to treat metastatic LNs.

View full abstract

Cancer is one of the major diseases that cause death all over the world. One of the first steps of metastasis is lymphatic metastasis. Systemic drug administration, which is major chemotherapeutic approach is of minor benefit for the metastatic lymph nodes and causes strong side effects. In our laboratory, we have studied on a novel drug delivery system, combining nano/microbubbles with ultrasound (US), which shows considerable potential for the delivery of exogenous polymeric molecules into lymph node cells through the lymphatic sinus. In this study, we selected some fluorescent molecules with different molecular weight (MW) as exogenous molecules, and evaluated delivery efficiencies into tumor cells in the metastatic lymph node depending MW. We found that delivery efficacy into lymph node cells has difference between MW.

View full abstract

Intracellular delivery of genes and macromolecules is an essential technique in biological researches and clinical application. A unique method of intracellular delivery using rapid squeeze of cells in a microfluidic device has recently been established. In this method, rapid deformation of the cell membrane increases the permeability of the membrane and makes it possible to introduce various macromolecules into the cells. This method realized high efficiency as well as high cell viability; however, kinetics of the membrane perforation is not elucidated. Therefore, in this study, we developed a new apparatus for microscopic observation of intracellular delivery under compressive deformation. PC-3 cells were compressed between a pair of slide glasses with a gap of 5 μm and stained with propidium iodide and fluorescent dextran with three different molecular weight: 3, 40, and 70 kDa. Our methods successfully showed that intracellular delivery increased by compressive deformation of the cells and its efficacy changed depending on time after the compression and molecular weight of macromolecules.

View full abstract

Understanding the time when molecular delivery occurs during sonoporation is important to improve the efficiency of successful sonoporation and fraction of viable cells. Here, we report how ultrasound conditions affect the time for sonoporation. We employed real-time observations of propidium iodide (PI) delivery to 3T3-Swiss albino cells under 5 seconds 1 MHz continuous ultrasound waves treatment with fluorescence (PI) and microbubbles SONAZOID^[○!R]. Sonoporation timing was evaluated by analyzing the time courses of the altering fluorescence intensity of the cells. Three ultrasound intensity conditions, 0.15, 0.20 and 0.30 W/cm^2, were examined. Fluorescence intensity increased for 0.20 and .30 W/cm^2-sonication, indicating PI delivery into the cells, while no apparent increase was observed for 0.15 W/cm^2-sonication. Fluorescence intensity clearly started to increase earlier for 0.30 W/cm^2 than for 0.20 W/cm^2-sonication. Sonoporation timing ranged from 2 to 6 seconds for 0.30 W/cm^2-sonication. The most delayed timing was obtained for cells that had the largest adhesion area. As a conclusion, different microbubble responses stimulated by various ultrasound conditions may cause different sonoporation timing.

View full abstract

This paper describes development of regenerative medical system using microcapsules including gas by shock waves. The newly proposed system has two features. One is culture mode by pressure wave, the other is rupture mode by interactions between shock waves and bubbles in microcapsules. Especially, the disintegration mechanism of polymer capsules including gas bubbles by shock wave was investigated and analysis of deformation process of a bubble inside was done in this paper. Deformation process of a capsule and a bubble were observed by microscopy. It was found that there are two modes such as oscillation, disintegration (damage) after working pressure wave (414kHz frequency) on the microcapsules. It was also found that these modes depend on gas ratio in the liquid capsule, membrane thickness, pressure amplitude and frequency. And deformation process of a bubble in the capsule was mathematically analyzed to compare the experimental result, such as effects of initial internal pressure of a bubble and gas ratio on the deformation process.

View full abstract

Currently, Alzheimer's treatment and medication is become to popularity topic in the world. Scientist and Researchers are looking for new treatments to alter the course of the disease and improve the quality of life for a person who was with Alzheimer's disease (AD). The potential of Low level light therapy (LLLT) has a lot of benefits in the areas of physical medicine and rehabilitation. In this studies bring the basic knowledge between optics and cell culture. Neuron cells were mimicked liked Alzheimer's disease by using P-amyloid solution Then, Alzheimer's cell were treated by light emitting diode (LED)-called phototherapy at 630 nm for proving in the cellular level that light from LED can inhibited P-amyloid peptides.

View full abstract

The use of magnetic nanoparticles is advantageous in manipulating targets remotely. Magnetic labeling of target cells with magnetic nanoparticle enables us to manipulate and control cells by applying an external magnetic field. We have developed functional magnetite (Fe_3O_4) nanoparticles to label target cell magnetically, and applied the functional magnetite nanoparticles to tissue engineering process including cell isolation (magnetic cell sorting), gene transfer (magnetofection), magnetic cell patterning and three-dimensional tissue construction. We have thus developed a novel methodology designated "magnetic force-based tissue engineering (Mag-TE)". In particular, forming three-dimensional tissue constructs is important in tissue engineering. In the Mag-TE method, target cells are labeled with magnetite cationic liposomes (MCLs) and magnetically accumulated to form three-dimensional tissue-like structures. In this talk, I am going to focus on resent results of skeletal muscle tissue engineering using MCLs. We have succeeded to fabricate electrically excitable tissue-engineered skeletal muscle constructs in vitro by using the Mag-TE technique.

View full abstract

We have been proposing a label-free cell separation device called "cell rolling column" in which cells selectively roll on antibody-immobilized column surfaces. The aim of this study was to investigate the possibilities of separation of undifferentiated induced pluripotent stem (iPS) cells from heterogeneous cells. Three types of phospholipid polymer, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-w-butyl methacrylate (BMA)-co-p-nitrophenyl oxycarbonyl poly (ethylene glycol) methacrylate (MEONP)], were synthesized with monomer feed ratios of MPC:BMA:MEOPN of 30:60:10 (PMBN 10), 33:66:1 (PMBN 1) and 33:67:0 (PMBN 0). A microfluidic channel was coated with PMBNs and immobilized with antibodies to stage-specific embryonic antigen 1 (SSEA-1), known as a pluripotency marker for murine cells. Suspended iPS cells were infused into the channels, and cell rolling speed and SSEA-1 expression were investigated. The results indicate that PMBN 10 coated channel showed highest density of anti-SSEA-1 antibody immobilization, and SSEA-1 positive cells were rolled on the surface. It is concluded that the cell rolling column can be a promising tool for a cell separation system for undifferentiated iPS cells.

View full abstract

The uterus is referred to as a dynamic organ due to chemical stimuli such as menstrual hormone change, but it is also affected by a spontaneous contractile activity by its myometrium. We hypothesized that mechanical loading leads to physiological change in human endometrial stromal cells (hESCs). In this study, we applied 15% of uniaxial cyclic strain for 7days in hESCs and reported that the differentiation of smooth muscle cells was enhanced by cyclic strain. After loading cyclic strain into hESCs, it also higly up-regulated the mRNA expressions of HB-EGF and ITGB3 that are known as key markers for bloastocyst attachment and endometrial receptivity, respectively. Moreover, we firstly found out that cyclic strain also elevated the production level of cAMP in hESCs, so that inhibitor tests using SQ22536 and H-89 were carried out to identify the involvement of cAMP signaling pathway in the promotion of differentiation for smooth muscle cells under mechanical loading. Therefore, we suggested the possibility of using mechanical stimuli to regenerate uterin tissue with smooth muscle cells.

View full abstract

To recover serious uterine damages which cause infertility, tissue engineering is a promising method. However, there is a lack of effective models of the regenerative tissues which can be utilized in clinical application. In such a situation, we have noted the effectiveness of decellularized tissues, which are produced from native tissues by removing cells which cause an immune reaction. Moreover, it is assumed the decellularized tissues are useful also in their mechanical characteristic because they remain the structure of native tissues. We have reported that the tissue decellularized with hyper hydrostatic pressure and then implanted in a defective part of native rat uterus encourages the regeneration of the tissues, and this study is performed to make more progress in the uterine regeneration with the decellularized tissues. For the purpose of rapid migration of the uterine cells into transplanted tissue and vascularization needed for nourishment to the tissue, we removed proteoglycan, a kind of protein in the uterus, from the decellularized tissues with chemical treatment and seeded mesenchymal stem cells into the tissues with the perfusion culture device. We then evaluated them by staining and quantification of DNA and proteoglycan, and confirmed that they has more space into which uterine cells can migrate and distribution of the seeded MSCs.

View full abstract

In 3D tissue engineering, scaffold has very important role in holding 3D structure, cell adhesion, cell differentiation and mechanical property. Previous studies show that mesenchymal stem cells differentiate smooth muscle cells or vascular endothelial cells by mechanical stress. Other studies show that the microstructure of cell adhesion surface influence to the stem cell differentiation. We set up the hypothesis that 3D scaffold with microstructure and mechanical stress can control the cell differentiation. Poly L-lactic acid (PLLA) is biodegradable polymer widely used as scaffold. In our research, two types of PLLA scaffold with microstructure is used. Porous scaffolds are made by mixing PLLA and NH_4HCO_3. Fibrous scaffolds are made by using electrospinning method, which makes polymer nanofiber. SEM images of scaffold show that pore size of porous scaffold is about 50〜100um and that of fibrous scaffold is about 5〜10um. Seeding hMSCs on porous/fibrous 3D scaffold and culture 3 days in the dynamic culture devise. After cell culture, the differentiation rate is analyzed by PCR. Results of PCR show that fibered structure inhibits the differentiation to endothelial cells and that the dynamic culture promotes the differentiation to smooth muscle cells. These results show a part of the relation between microstructure and cell differentiation. For blood vessel regeneration, further study about microstructure and mechanical stress is need.

View full abstract

It has been well recognized that the heart transplant is one of the most effective ways to cure severe cardiac diseases. However, this treatment has severe limitation, for example, it is very difficult to prepare fresh hearts for transplantation. Recently, tissue engineering approaches such as the cell sheet engineering have been utilized for partial heart diseases such as myocardial infraction. Furthermore, cardiomyocytes derived from iPS cells have been considered to be used to construct engineered cardiac walls. The aim of this study was to understand the fundamental pulsation behavior and mechanism of cardiomyocyte colonies derived from human iPS cells. Effects of co-culture with MSCs on the pulsation were also investigated.

View full abstract

The aim of this study is to develop hybrid tubular structure of PLCL porous tube and mesenchymal stem cells (MSCs). Such graft was constructed by layering MSC sheets on porous PLCL tubular graft. Cell proliferation assay confirmed that the number of cells increased steadily during 11 days of in vitro study. Moreover, it was also found that co-culture cell sheet of MSCs and endothelial cells (ECs) exhibited higher proliferation rate than that of MSCs' monoculture cell sheet, indicating that the co-culture cell sheet can modulate higher proliferation of cells. Tensile mechanical testing of the hybrid tubes was also conducted and it was found that the mechanical properties of the hybrid tube with co-culture cell sheets tended to be larger than those of the tube with monoculture sheets.

View full abstract

The brain consists of neurons, glial cells, blood vessels, extracellular matrix and other components. The neurovascular unit system serves as a model to study interactions between neurons and other kinds of cells. In this study, we investigated culture conditions to construct this unit in a microfluidic cell culture system. Our microfluidic device has three microchannels; the central channel contains hydrogel, which is sandwiched by the other two channels filled with medium. Neural stem cells or human brain microvascular endothelial cells were seeded into the medium channel and cultured on the surface of the gel. In the field of tissue engineering, some kinds of gels, such as collagen I, fibrin, Matrigel, have been used for cell culture. Therefore, we tested several kinds of gel materials to clarify which gel material facilitates construction of neural networks or microvascular networks. The culture of neural stem cells was analyzed by measuring the length of axons of neurons, while the culture of endothelial cells was investigated in terms of the ability of angiogenesis. We found that neural stem cells differentiated into neurons and constructed neural network in fibrin-Matrigel mixed gel. This gel material also allowed endothelial cells to construct microvascular networks.

View full abstract

Tissue engineered cell sheets composed of oriented muscle fibers differentiated from myoblasts can be regarded as more suitable for the regeneration of skeletal muscles. A novel method for fabricating monolayer cell sheets by culturing cells on a suspended mesh patterned structure consisting of narrow mesh lines (< 5 μm) and large apertures (> 100 μm) was developed in our laboratory. In this study, we investigated the influence of mesh shape on the orientation of myoblast and myotubes derived from them. To do this, we cultured C2C12 (mouse myogenic cell line) on fibronectin-coated mesh patterned structure and differentiated them to myotubes after confluency was reached. Using a diamond shaped mesh with a 100 nm long minor axis and a minor angle of 70°, we obtained myoblast cell sheets with cells oriented in the longest axis, in accordance with our previous studies. Importantly, myotubes derived from the oriented myoblasts were also similarly oriented, suggesting that lengthwise fusion as opposed to sidewise fusion occurs predominantly in orientated myoblasts. Using Fluo-4 indicator, we monitored calcium dynamics in the oriented myotubes and found that some myotubes displayed a unique calcium wave that travelled from end-to-end at a speed of 10-30 μm/s. This calcium dynamics was clearly distinct from the typical calcium spike displayed by a majority of myotubes. In our future studies, we will investigate the relationship between this calcium dynamics and the beating motion also observed in some oriented myoblast.

View full abstract

During wound healing process, dermal fibroblasts infiltrate into wound site and secrete collagen to repair the wound. The fibroblasts are usually subjected to tensional force that stimulates cell infiltration and proliferation. Therefore, it is important to measure the shrinkage force of skin and to evaluate the effect of force on dermal fibroblasts. It is possible to simulate the dermal layer of skin by embedding the fibroblasts in three-dimensional collagen gel in vitro. Using this culture model, the collagen gel shrinkage by the fibroblasts could also simulate the wound shrinkage of healing process. In this study, we developed the culturing and monitoring device for skin fibroblast-seeded collagen gel to measure shrinking force. The fibroblast-seeded collagen gels were cultured in the culture medium with or without ascorbic acid. As the results, the shrinking force under both culturing conditions increased with increase in the culture time. The force under the ascorbic acid dosing condition increased rapidly compared to that under the no-dosing condition.

View full abstract

Stem cell-based self-assembled tissues (scSATs) bio-synthesized from synovium-derived mesenchymal stem cell have a great potential for the repair and regeneration of biological soft tissues. However, the mechanical properties of the scSAT were insufficient for clinical application to a relatively large tissue defect. A candidate solution to the problem is to promote the generation of the extracellular matrix in the scSAT via a cell/tissue cultivation under cyclic strain. In the present study, MSCs were cultured in the presence of ascorbic acid for 7 days on the elastic tissue culture plates, then subsequently cultured under cyclic tensile strain for 7days, with 10 % at 1 Hz, for either 1 hour (1 h group), 3 hours (3 h group) or 5 hours (5 h group) a day. Specimens cultured for 14 days under static condition on the elastic tissue culture plate were defined as static group. Morphological observation of the surface structure of 1 h group was performed using a scanning electron microscope. The scSAT specimens of 3 h group, 5 h group and static group were subjected to tensile testing. Results revealed that the load at failure and tensile strength of the scSAT were higher in 3 h and 5 h groups than in static group. One-way analysis of variance demonstrated a significant effect of duration of strain application on the tensile strength and modulus of the scSATs. Morphological observation indicated that collagen fiber-like structure aligned parallel with the direction of strain application in 1 h group. It is suggested that the structural alignment due to the cell/tissue cultivation under cyclic strain resulted in the improvement of mechanical properties of the scSATs.

View full abstract

The aim of this research project is to develop a novel engineered cartilage tissue that has mechanical properties comparable with natural articular tissue. As a part of the fundamental work, culture condition were mainly examined to optimize the proliferation and differentiation of MSCs. As a new novel scaffold for cartilage regeneration, collagen gel/sponge two-phase scaffold was developed by placing sponge structure in gel phase. Effects of combined medium and the number of seeding cells on the biological and mechanical properties of the engineered cartilage were examined. Two kinds of combined culture condition were found to effectively activate the proliferation and differentiation of MSCs, resulting in increase of the compressive modulus. It was also found that increased cell number can activate the proliferation and result in the increase of compressive mechanical property.

View full abstract

Although articular cartilage plays an important role in joint lubrication, it has a limited healing potential due to the lack of vascularity. We have been performing research on cartilage repair by the use of a mesenchymal stem cell-derived tissue engineered construct (TEC). Recently, we developed a novel TEC (gTEC) through a bovine serum-free medium in order to avoid infection due to viral pathogens such as prion protein. The purpose of the present study is to determine the frictional and compressive properties of repaired cartilage treated by gTEC in comparison with that treated by normal TEC. Porcine synovial MSCs were cultured at a high cell density in bovine serum-free medium and extracellular matrix produced by the MSCs were detached from the substratum. The cell-matrix complex spontaneously contracted to develop a gTEC. For compression, a normal TEC was produced in bovine serum medium. The gTEC and TEC were implanted in vivo into chondral defects in the medial femoral condyle of 3-month-old pigs, and repair tissues were obtained after 6 months. Indentation and friction tests were conducted by the use of a friction tester to determine the stiffness and friction coefficient of repaired tissues. Stiffness of gTEC- and TEC-treated cartilage had a tendency to decrease as compared with that of normal cartilage. Coefficient of start-up friction of gTEC- and TEC-treated cartilage were higher than that of normal cartilage, but there was no significant difference between the gTEC-treated and TEC-treated repair tissues. Coefficient of dynamic friction of gTEC- and TEC-treated cartilage were almost identical to that of normal cartilage. gTEC-treated repair cartilage have equivalent frictional and compressive properties with TEC-treated repair cartilage although it is softer than normal cartilage.

View full abstract

In this study, the porous PLLA microfiber scaffold was developed by electrospinning method to maintain the nutrient supply into cultured cartilage. Porous fiber scaffold and solid fiber scaffold were fabricated by spinning poly-L-lactic acid (PLLA)/polyethylene oxide (PEO) solution and PLLA solution respectively with an electrospinning unit. According to SEM observation, porous structure was confirmed in the porous fiber and fiber diameter of both types of fibers were about 5.5 μm Porcine chondrocytes mixed in collagen type I gel were seeded in the both scaffolds. Live cells and dead cells were observed in the both fiber scaffolds as cell viability observation by using a multiphoton microscopy. After 28 days of cultivation, dead cells for the porous fiber scaffold was less than those for the solid fiber scaffold, and synthesized glycosaminoglycan (GAG) for the porous fiber scaffold was higher than that for the solid fiber scaffold. According to fluorescent observation of penetration of rhodamine B solution through the porous fiber scaffold embedded in agarose gel, mesh-like fluorescent image was observed and luminance value of the image increased with diffusion of rhodamine B solution through the porous fiber scaffold. These results showed that the developed porous fiber scaffold can maintain the nutrient supply into the cultured cartilage and inhibit cell death during cultivation.

View full abstract

As an alternative therapeutic approach to treating arthropathy such as osteoarthritis (OA), tissue engineering may be promising. To recreate the articular cartilage in vitro, researchers have used scaffold-free approaches such as pellets, cell aggregates and self-assembled tissues, while applying chemical and mechanical stimuli to replicate the in vivo environment. However, there are still many problems to be overcome. To synthesize thick cartilage tissues with adequate mechanical properties, it is crucial to supply nutrients and oxygen to the center of the tissues. In this study, we have developed a new method, the multilayer technique to solve this problem. Compared to conventional techniques constructing tissues in one single block, our proposed layering technique is able to create thicker viable tissues. By optimizing parameters such seeding density and number of layers, we were able to create tissues with better mechanical properties. Further studies are needed to elucidate the underlying mechanism of such technique.

View full abstract

We aimed to investigate mechanical properties of Achilles' tendon autograft after ovine anterior cruciate ligament reconstruction. Mature three female ovine were used in this study. In each ovine, the right knee underwent anterior cruciate ligament reconstruction. They were sacrificed at 13 weeks after surgery. In each ovine, both the knees were harvested, and the left knee was used to obtain the data of the native anterior cruciate ligament. Drawer test for stability evaluation and tensile test for evaluation of mechanical properties were performed. The knee was mounted on a jig with 5 degrees of freedom for tensile testing, four cycles of anterior-posterior shear loads (50 N) were applied to the knee at 30°, 60° and 90° of the flexion with a cross head speed set at 20 mm/min. The displacement of the femur relative to tibia was defined as the anterior-posterior translation of the knee. (1) Stress relaxation test (5 N, 10 min), (2) cyclic load test (0-50 N, 10 cycles, 10 mm/min), and (3) tensile test (300 mm/min) were performed in turn. Before each testing, tissue cross-sectional area was measured with the device using a laser displacement sensor. At 13 weeks after the implantation, the anterior-posterior translation of the reconstructed knee was greater than native anterior cruciate ligament. Ultimate strength and maximum Young's modulus of the femur-graft-tibia complex were smaller than native anterior cruciate ligament. These testing methods presented here is effective to gain insight into in vivo biomechanical remodeling of reconstructed tissues.

View full abstract

In the treatment of anterior cruciate ligament rupture, anterior cruciate ligament reconstruction is performed using autologous tendon. However, there are problems in the invasiveness and limitation in the source of autologous tendon. In order to solve these problems, a decellularized and sterilized bovine tendon has been developed. The aim of this study is to investigate cell infiltration into the decellularized and sterilized bovine tendon in rat anterior cruciate ligament reconstruction. In 4 and 26 weeks after the implantation, the tissues were explanted and subjected to hematoxylin-eosin staining. In this study, rat autologous tendon were also implanted to compare cell infiltration into the reconstructed tissues between the decellularized and sterilized bovine tendon and rat autologous tendon In the implantation period of 4 weeks, the number of cells in the decellularized and sterilized bovine tendon was higher than that in the rat autologous tendon In the implantation period of 26 weeks, the number of cells in the decellularized and sterilized bovine tendon and that in the rat autologous tendon became comparable. In the implantation period of 26 weeks, the number of cells in the decellularized and sterilized bovine tendon and that in rat anterior cruciate ligament also became comparable. These data indicated that the decellularized and sterilized bovine tendon has a promise as a prosthesis for anterior cruciate ligament reconstruction.

View full abstract

This paper describes visualization of thrombus formation process on wall surface in Couette flows. The aim is to investigate the effect of shear stress or shear rate, especially constant shear rate in Couette flows, on the thrombus formation. In this investigation, by visualization of the thrombus formation in blood plasma flow, it was found that the flow rate and shear rate have strong effects on the thrombus formation rate. The thrombus formation rates in the Couette flows was obtained by special double cylindrical test rig. Effect of the different of coating and shear rate on thrombus formation rate was investigated. From these results, even if coating surface is changed, the relationship between the shear rate and Rs is same.

View full abstract

To analysis platelet deposition on high shear flows, reaction rate of platelet activation is redefined to be the product of agonists and shear rate factors instead of the original only agonist activation model developed by Sorensen et al.. The new model is then applied to orifice flow. It is found that platelet deposition is mainly caused by resting platelets in the flow field and fraction of surface available for platelet-surface adhesion drops rapidly when platelet activation begins.

View full abstract

Arterial pulsation in arteries is deeply related to cardiovascular diseases. Although experimental studies under the pulsation are important, experiments in vivo have various problem including high-cost, invasiveness, and individual difference. Then the instrument that can reproduce the behavior of pulsation in human body have developed in this study. Compared to previous studies, the instrument is developed by modifying input system and adding fluidic resistance. Therefore, the developed instrument can be a new tool to explore vascular mechanobiology.

View full abstract

The aim of this study is to develop an accelerated fatigue tester for the Normo valve. Because the Normo valve has a unique structure similar to natural mitral valve, a novel accelerated fatigue tester which can mount the Normo valve and test in the condition recommended by the International Organization for Standardization (IS05840) was developed. We succeeded in producing the targeted closed duration percentage over the defined trans-valvular pressure gradient by adjusting the systolic and the drive frequency. The results showed that the drive condition of 10 Hz in drive frequency and 50% in systolic fraction met the requirement noted in ISO 5840.

View full abstract

The purpose of this study is to investigate influences of multi-loads and stenosis in femoral-popliteal artery on fracture of self-expandable stent using finite element analysis. A nitinol alloy stent was selected. The three-dimensional CAD models of the stent and vessel were constructed using SolidWorks. First, the stent was compressed by four cylinder models. Second, the stent was deployed in 50%, 60% and 75% stenotic artery models. Third, cyclic multi-loads were applied to the artery model. Multi-loads were two types. One is shortening of 12.7% and torsion of 0.28 degree/mm (Average). Another is shortening of 6.9% and torsion of 0.14 degree/mm (Half of average). When average loads were applied, strain amplitude of stent were 0.61%, 0.75%, and 0.87% in 50%, 60% and 75% stenotic model. When half of average loads were applied, those were 0.36%, 0.41%, and 0.54%. According to constant-life diagrams of average loads, when the stent was deployed in over 60% stenotic artery model, risk of fracture was high. This study indicated that risk of fracture is increased by multi-loads and stenosis.

View full abstract

Almost all pumps for presently available blood purification systems are roller pumps. We are developing a system of a small centrifugal blood pump with a vascular access to peripheral vein in order to downsize the hemofiltration systems for easy transportation and to make its use safer. We have developed small centrifugal pumps: NY1, NY2 and NY3 whose impeller diameters are 30 mm. NY4 and NY5 whose impeller diameters are 34 mm. Their impellers are supported with double pivot bearing. Another difference of NY pumps is pivot contact areas. Their contact angle of prospect are 30° for NY1 and NY5, 45° for NY2, and 60° for NY3 and NY4. These pumps provides stable blood flow of 50 mL/min. Hemolysis tests were conducted fir all pumps. The total quantity of plasma free hemoglobin of all pumps were found to be under half of that of a commercial centrifugal pump for extracorporeal circulation. Especially it was found that NY5 pump showed only 10 percent hemolysis compared to a commercial centrifugal pump.

View full abstract

Hemofilters are medical devices for assisting deteriorated renal functions, which purify the blood by removing pathogen and wastes. For continuous hemofilters, antithronbogenecity is one of the most important requirements. The aim of this study is to investigate the key parameters for the establishment of a reliable in vitro thrombogenecity testing methodology for continuous hemofilters using human blood from healthy volunteers. This study used adjusted activated clotting time of human blood drawn from healthy volunteers to 150-200 sec using heparin. The blood was circulated in the air-contactless circuit with flow and pressure in a clinical situation was reproduces. The tests were terminated when the inlet pressure elevated to 150 mmHg from the initiation pressure of 70 mmHg at the inlet. The average circulation duration was 6.5±1.7 hours(n=4). The circulation duration using human blood from healthy volunteers were shorter than using porcine blood which was previously reported. Hematocrit of porcine blood was lower than the human blood from healthy volunteers, whereas the hematocrit of porcine blood was comparable to dialysis patients. These data indicated the importance of hematocrit of blood in the assessment of the thrombogenecity of continuous hemofilters.

View full abstract

Though several kinds of implantable ventricular assist devices (VAD) are available at present, their application is limited to transplantation candidates and other patients, who need partial circulatory assist, cannot use VADs in Japan. We are developing a small portable axial-flow pump whose blood contacting parts are made of polymer. Though axial flow pump models, KAP2, KAP3, and KAP4 were developed with different pivot size, their hemolytic properties have not changed so much as 5 times of a conventional centrifugal pump. Though KAP5 changed its fluid dynamic design and found an almost the same level hemolysis at 10000 rpm, it attained very low level of hemolysis at 5000 rpm. We will investigate the suitable ranges of use for next models partial circulatory assist.

View full abstract

Thrombus formation at the interface between left ventricular wall and the inflow cannula of left ventricular assist device (LVAD) remains to be an unsolved problem that induces complications in clinical practice. The aim of this study is to develop an in vitro airless circuit which produces flow and pressure environments in patients with a LVAD, and to investigate an influence of MPC coating of the inflow cannula of EVAHEART^[○!R] LVAD (Sun Medical Technology Research Co., Japan) on thrombus detachment from the inflow cannula. In the thrombogenicity test, two identical circuits except for the cannula were prepared to form a comparative study. Heparinized blood from a single porcine with activated clotting time ranged within 130-210 sec was circulated for 4.5 hours. The tests were performed under the combinations of the total flow rate of 4 L/min or 5 L/min, and the pump rotational speed of 1800, 1900 or 2000 rpm, respectively. It was elucidated that higher amount of thrombus formed on the MPC non-coated cannula compared with MPC coated cannula. However, there was no difference in the amount of thrombus detached between the two cannula (Student's t-test, p=0.91). From these results, it was elucidated that the MPC non-coated cannula yielded significantly lower proportion of thrombus detachment to thrombus formation than the MPC coated cannula (Student's t-test, p<0.05).

View full abstract

We have developed in vitro test methodologies modeling in vivo practical-use situations, to quantitatively assess safety and effectiveness of innovative medical devices. We believe that expediting development of these in vitro methodology not only reinforce preclinical evaluation and enhance timely access to innovative medical devices for patients but also reduce potential risk when used in real-world clinical practice, because controlled clinical trials have certain limitation in fully demonstrating benefit and risk. With the grant from Ministry of Health, Labour and Welfare intended for "Promotional Project for Practical Application of Innovative Pharmaceutical, Medical Device, and Tissue Engineering Products", we have developed drafts on in vitro test guideline for "Durability testing methods for coronary artery stent", "Durability testing methods for vascular stent intended for Superficial femoral - Popliteal artery", and "Method for in vitro thrombogenicity testing for inflow cannula of left ventricular assist system". Further 5 guideline drafts are under preparation. Here, we introduce impacts of in vitro test methods modeling in vivo intended-use situations on expediting availability of new medical devices and timely assessment and management of risk.

View full abstract

For the wheelchair users, pressure distribution cushion is essential to prevent pressure ulcer. On the other hand, cushion needs to have the function of keeping sitting posture, especially for spinal cord injury, cervical cord injury or elderly persons. For this issue, we propose the cushion with a layered structure of the silicon rubber block cube. This contrivance provides both of the scattering pressure and maintaining sitting posture. The block cubes are constructed by wrapping soft silicone rubber up in the hard silicone rubber. And because of the layered structure, the cushion can be custom-made to fit the shape of the individual users' hip. In this paper, we have tried to develop custom-made silicone rubber cushion and we compared the pressure distributions when users use the silicon rubber cushion and same shape urethane cushion. Both of silicon rubber and urethane cushion have excellent enough performances of the pressure prevention. The pressure distributions by using the silicone rubber cushion haven't showed big difference according to the wheelchair tilting. These results suggest that the silicone rubber cushion has the good performance of keeping users' body posture on their wheelchair.

View full abstract

Excessive hip joint loads during daily activities could be a risk factor for initiation and progression of hip osteoarthritis (Hip-OA). In the daily activities, walking is one of the highest frequent opportunities that make loads on hip joint. A gait modification could a noninvasive treatment option that reduces the hip joint force to care and cure Hip-OA. However, direct measurements of hip joint load during walking are difficult. It has also been reported that there is a positive correlation between maximum hip joint load and maximum hip adduction moment during walking. In this study, we are aiming to find a suitable "step width" to reduce the hip adduction moment during walking. Ten subjects walked varying "step width" from 0 m to 0.4 m by 0.1 m increments. As results, all ten subjects showed the maximum adduction moment decreases with larger step width than normal step width. Given those results, we believe that a gait modification by varying step width could be one of the noninvasive treatment options to care and cure Hip-OA.

View full abstract

For the old people over their ages of 65 years old, the frequency of injuries from fall accidents is very high enough that one of every 3 persons experiences some injuries resulting from a fall accident every year, and the number of old people dying or suffering from the fall injuries is gradually increased. This study aimed to investigate an fall's influence on the human body's injury from the impact aspect by realizing a 3D musculoskeletal motion dynamic model. In the falling simulations, the maximum contact force was primarily calculated when the hip was collided with the ground (1.90 times of subject's weight). And the maximum contact force was secondarily calculated (0.57 times of subject's weight) when the wrist was touched onto the ground. While the experiment targeting real human bodies showed similar pattern of findings to those of simulation results, although the former showed somewhat higher values (2.21 times of the subject's weight at the hip, and 0.63 times of the subject's weight at the wrist). It is considered the 3D musculoskeletal dynamic simulation model will be utilized as a useful tool to acquire basic information on the falling-injury researches according to human body's various changes and environmental changes in future.

View full abstract

At the present time, a safety criterion for human support robot has not been established in spite of the fact that it will be doubtless widespread due to decreasing birthrate and aging of the population in Japan, Under this situation, an attempt is made to estimate the strength and fracture in the human metatarsals by conducting the simulation of human-robot interactions and modeling the human foot on the basis of CT images. This result will contribute to the realization of high accuracy in the prediction of injury risk due to human and robot interactions.

View full abstract

In order to know the breathing at the time of the crowd accident, the chest and abdomen of female subjects was compression in experiment. The results, vital capacity and tidal volume can not resist the load, and decreased with time, minute ventilation was continued keeping the ventilation of more than normal value by respiratory rate increases. It is expected that be difficult to increase the ventilation of the intended to one cycle of breaths during crowd accident. Minute ventilation is maintained by increasing the respiratory rate, and ventilation rate is exacerbated with increasing death space ventilation.

View full abstract