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ABSTRACT: In vitro scaling up of bioengineered tissues is known to be limited by diffusion issues, specifically a lack of vasculature. Here, we report a new strategy for preserving cell viability in three-dimensional tissues using cell sheet technology and a perfusion bioreactor having collagen-based microchannels. When triple-layer cardiac cell sheets are incubated within this bioreactor, endothelial cells in the cell sheets migrate to vascularize in the collagen gel, and finally connect with the microchannels. Medium readily flows into the cell sheets through the microchannels and the newly developed capillaries, while the cardiac construct shows simultaneous beating. When additional triple-layer cell sheets are repeatedly layered, new multi-layer construct spontaneously integrates and the resulting construct becomes a vascularized thick tissue. These results confirmed our method to fabricate in vitro vascularized tissue surrogates that overcomes engineered-tissue thickness limitations. The surrogates promise new therapies for damaged organs as well as new in vitro tissue models.
Scientific Reports 02/2013; 3:1316.
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ABSTRACT: In vitro fabrication of functional vascularized three-dimensional tissues has been a long-standing objective in the field of tissue engineering. Here we report a technique to engineer cardiac tissues with perfusable blood vessels in vitro. Using resected tissue with a connectable artery and vein as a vascular bed, we overlay triple-layer cardiac cell sheets produced from coculture with endothelial cells, and support the tissue construct with media perfused in a bioreactor. We show that endothelial cells connect to capillaries in the vascular bed and form tubular lumens, creating in vitro perfusable blood vessels in the cardiac cell sheets. Thicker engineered tissues can be produced in vitro by overlaying additional triple-layer cell sheets. The vascularized cardiac tissues beat and can be transplanted with blood vessel anastomoses. This technique may create new opportunities for in vitro tissue engineering and has potential therapeutic applications.
Nature Communications 01/2013; 4:1399. · 7.40 Impact Factor
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ABSTRACT: This study experimentally investigated the instability of flow impingement in a cerebral aneurysm, which was speculated to promote the degradation of aneurysmal wall. A patient-specific, full-scale and elastic-wall replica of cerebral artery was fabricated from transparent silicone rubber. The geometry of the aneurysm corresponded to that found at 9 days before rupture. The flow in a replica was analysed by quantitative flow visualization (stereoscopic particle image velocimetry) in a three-dimensional, high-resolution and time-resolved manner. The mid-systolic and late-diastolic flows with a Reynolds number of 450 and 230 were compared. The temporal and spatial variations of near-wall velocity at flow impingement delineated its inherent instability at a low Reynolds number. Wall shear stress (WSS) at that site exhibited a combination of temporal fluctuation and spatial divergence. The frequency range of fluctuation was found to exceed significantly that of the heart rate. The high-frequency-fluctuating WSS appeared only during mid-systole and disappeared during late diastole. These results suggested that the flow impingement induced a transition from a laminar regime. This study demonstrated that the hydrodynamic instability of shear layer could not be neglected even at a low Reynolds number. No assumption was found to justify treating the aneurysmal haemodynamics as a fully viscous laminar flow.
Journal of The Royal Society Interface 01/2013; 10(82):20121031. · 4.40 Impact Factor
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ABSTRACT: The purpose of this study was to propose an innovative approach of setting outlet boundary conditions for the computational fluid dynamics (CFD) simulation of human common carotid arteries (CCAs) bifurcation based on the concept of energy loss minimisation at flow bifurcation. Comparisons between this new approach and previously reported boundary conditions were also made. The results showed that CFD simulation based on the proposed boundary conditions gave an accurate prediction of the critical stenosis ratio of carotid arteries (at around 65%). Other boundary conditions, such as the constant external pressure (P = 0) and constant outflow ratio, either overestimated or underestimated the critical stenosis ratio of carotid arteries. The patient-specific simulation results furthermore indicated that the calculated internal carotid artery flow ratio at CCA bifurcation (61%) coincided with the result obtained by clinical measurements through the use of Colour Doppler ultrasound.
Computer Methods in Biomechanics and Biomedical Engineering 01/2012; · 0.85 Impact Factor
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ABSTRACT: The study aim was to develop a novel stentless mitral valve (SMV) and to evaluate its performance, using an original pulsatile simulator developed specifically to analyze the hydrodynamic function of the mitral valve.
The SMV developed at the authors' institution consists of two major components: a large anterior leaflet with commissures, and a small posterior leaflet. The valve is formed by suturing the leaflets (made from bovine pericardium) to a flexible (Duran) ring. The SMV, constructed with a 27 mm flexible ring, was installed into the mitral valve simulator, after which the four papillary flaps of the two leaflets were sutured to artificial papillary muscles. The artificial ventricle was driven pneumatically at a pulse rate of 70 beats/min, with a systolic fraction of 35%. The mean flow, aortic pressure, and atrial pressure were adjusted to 4.5 1/min, 120/80 mmHg, and 10 mmHg, respectively. A 27 mm mechanical valve (MEV; St. Jude Medical Inc.) was employed as a control. The hydrodynamic performance of the SMV and MEV were investigated and compared. An echo-Doppler study was also performed.
The waveforms of the SMV and MEV showed a similar pattern. The mean transvalvular flow was 4.7 +/- 0.4 1/min for the SMV, and 3.55 +/- 0.13 1/min for the MEV (p < 0.001). Mitral regurgitation was 5.07 +/- 1.15 and 3.78 +/- 0.35 ml/beat, respectively (p < 0.05). Echocardiographic data indicated that the regurgitant jet towards the left atrial model was none or trivial for the SMV, and trivial for the MEV.
Within the environment of the mitral valve simulator, the novel SMV prepared from bovine pericardium demonstrated excellent performance characteristics, and may represent a potential future alternative for bioprosthetic stented mitral valves.
The Journal of heart valve disease 01/2012; 21(1):71-5. · 0.81 Impact Factor
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Yuji Haraguchi,
Tatsuya Shimizu,
Tadashi Sasagawa,
Hidekazu Sekine,
Katsuhisa Sakaguchi,
Tetsutaro Kikuchi,
Waki Sekine,
Sachiko Sekiya,
Masayuki Yamato, Mitsuo Umezu,
Teruo Okano
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ABSTRACT: The fabrication of 3D tissues retaining the original functions of tissues/organs in vitro is crucial for optimal tissue engineering and regenerative medicine. The fabrication of 3D tissues also contributes to the establishment of in vitro tissue/organ models for drug screening. Our laboratory has developed a fabrication system for functional 3D tissues by stacking cell sheets of confluent cultured cells detached from a temperature-responsive culture dish. Here we describe the protocols for the fabrication of 3D tissues by cell sheet engineering. Three-dimensional cardiac tissues fabricated by stacking cardiac cell sheets pulsate spontaneously, synchronously and macroscopically. Via this protocol, it is also possible to fabricate other tissues, such as 3D tissue including capillary-like prevascular networks, from endothelial cells sandwiched between layered cell sheets. Cell sheet stacking technology promises to provide in vitro tissue/organ models and more effective therapies for curing tissue/organ failures.
Nature Protocol 01/2012; 7(5):850-8. · 8.36 Impact Factor
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Ichiro Suzuki,
Yasuyuki Shiraishi,
Shota Yabe,
Yusuke Tsuboko,
Telma Keiko Sugai,
Ken Matsue,
Takeyoshi Kameyama,
Yoshifumi Saijo,
Takashi Tanaka,
Yoshihiro Okamoto,
Zhonggang Feng,
Takako Miyazaki,
Masaaki Yamagishi,
Makoto Yoshizawa, Mitsuo Umezu,
Tomoyuki Yambe
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ABSTRACT: The purpose of this study was to examine the hemodynamic characteristics of expanded polytetrafluoroethylene (ePTFE) pulmonary valves with bulging sinuses quantitatively in a pediatric pulmonary mechanical circulatory system designed by us, in order to propose the optimal design for clinical applications. In this study, we developed a pediatric pulmonary mock circulation system, which consisted of a pneumatic right ventricular model, a pulmonary heart valve chamber, and a pulmonary elastic compliance tubing with resistive units. The hemodynamic characteristics of four different types of ePTFE valves and a monoleaflet mechanical heart valve were examined. Relationships between the leaflet movements and fluid characteristics were evaluated based on engineering analyses using echocardiography and a high-speed video camera under the pediatric circulatory conditions of the mock system. We successfully performed hemodynamic simulations in our pediatric pulmonary circulatory system that could be useful for quantitatively evaluating the pediatric heart valves. In the simulation study, the ePTFE valve with bulging sinuses exhibited a large eddy in the vicinity of the leaflets, whereas the straight tubing exhibited turbulent flow. The Reynolds number obtained in the valve with bulging sinuses was calculated to be 1667, which was smaller than that in the straight tubing (R (e) = 2454).The hemodynamic characteristics of ePTFE pediatric pulmonary heart valves were examined in our mock circulatory system. The presence of the bulging sinuses in the pulmonary heart valve decreased the hydrodynamic energy loss and increased the systolic opening area. Based on an in vitro experiment, we were able to propose an optimal selection of pulmonary valve design parameters that could yield a more sophisticated pediatric ePTFE valve shape.
Journal of Artificial Organs 09/2011; 15(1):49-56. · 1.59 Impact Factor
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ABSTRACT: The study objective was to evaluate various types of Norwood arch reconstruction methods and to show the factors that affect the cardiac workload of the single ventricle. The Norwood procedure is one of the most challenging congenital heart surgeries. Several aortic arch reconstruction techniques have been reported to avoid recoarctation, ensure coronary perfusion, and improve long-term outcomes. Inside the arch, complicated turbulent flow is generated; however, little is known about the cause of the disadvantageous inefficient flow and the surgical techniques to avoid it.
We created patient-specific computational hemodynamic models of 9 patients who underwent different types of arch reconstruction methods. Four patients had aortic atresia, and 5 patients had aortic stenosis. Flow profiles were defined by echocardiography data corrected with body surface area. Turbulent pulsatile flow was analyzed with the finite volume method. Flow energy loss was calculated to estimate cardiac workload, and wall shear stress was calculated to estimate vessel wall stiffness increase.
Recoarctation and acute arch angles increased wall shear stress and energy loss. In the patients with aortic atresia, a longitudinal incision toward the descending aorta was effective in creating a smooth arch angle. In the patients with aortic stenosis, arch repair with the Damus-Kaye-Stansel procedure in a single anastomotic site was effective in creating sufficient anastomosis space and a smooth arch angle.
Creation of a large anastomotic space and a smooth aortic arch angle reduced wall shear stress and energy loss, and should improve long-term cardiac performance after the Norwood procedure.
The Journal of thoracic and cardiovascular surgery 09/2011; 144(1):130-8. · 3.41 Impact Factor
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ABSTRACT: Rectangular microchannels 50 μm high and 30, 40, 50, 60, or 70 μm wide were fabricated by adjusting the width of a gap cut in a polyethylene sheet 50 μm thick and sandwiching the sheet between an acrylic plate and a glass plate. Flux in the microchannels was measured under three different inner surface conditions: uncoated, albumin-coated, and confluent growth of rat fibroblasts on the bottom of the microchannels. The normalized flux in microchannels with cultured fibroblasts or albumin coating was significantly larger than that in the uncoated channels. The experimental data for all microchannels deviated from that predicted by classical hydrodynamic theory. At small aspect ratio the flux in the microchannels was larger than that predicted theoretically, whereas it became smaller at large aspect ratio. The aspect ratio rather than Reynolds number is the correct property for predicting the variation of the normalized friction factor. We postulate that two counteracting effects, rotation of large molecules and slip velocity at the corners of the microchannels, are responsible for the deviation. From these results we conclude that albumin coating should be carried out in the same way as when fabricating our integrating cell-culture system. The outcomes of this study are not only important for the design of our culture system, but also quite informative for general microfluidics.
Journal of Artificial Organs 04/2011; 14(3):238-44. · 1.59 Impact Factor
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Takashi Yamane,
Shunei Kyo,
Hikaru Matsuda,
Yusuke Abe,
Kou Imachi,
Toru Masuzawa,
Takeshi Nakatani,
Kazuhiro Sase,
Koichi Tabayashi,
Setsuo Takatani,
Eisuke Tatsumi, Mitsuo Umezu,
Toshie Tsuchiya
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ABSTRACT: To facilitate research and development (R&D) and to expedite the review processes of medical devices, the Ministry of Health, Labor and Welfare (MHLW) and the Ministry of Economy, Trade and Industry (METI) founded a joint committee to establish guidance for newly emerging technology. From 2005 to 2007, two working groups held discussions on ventricular assist devices and total artificial hearts, including out-of-hospital programs, based on previous guidance documents and standards. Based on this discussion, the METI published the R&D Guidelines for innovative artificial hearts in 2007, and in 2008 the MHLW published a Notification by Director regarding the evaluation criteria for emerging technology.
Artificial Organs 09/2010; 34(9):699-702. · 2.00 Impact Factor
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ABSTRACT: The viscoelastic characteristics of contracted collagen gels populated with rat fibroblasts or cardiomyocytes were investigated by uniaxial tensile testing. Rat type I collagen-Dulbecco's modified Eagle's medium solution (each 2 ml in volume, 0.5 mg/ml collagen concentration) containing 2.0 million rat fibroblasts or cardiomyocytes were cast in a circular shape. After gelation and culture for 10 days the contracted gels were first stretched to a tensile strain of approximately 0.20 at 4.6 × 10(-3)/s strain rate, and then the strain was kept unchanged for 3 min. The tensile stress in the gels was recorded. The results were regressed against the equations of the Kelvin viscoelastic model. It was found that the two elastic coefficients in the model were 6.5 ± 1.7 and 10.2 ± 3.2 kPa, respectively, for gels with cardiomyocytes and 5.1 ± 1.6 and 4.5 ± 0.9 kPa for those with fibroblasts; the values for gels with cardiomyocytes were significantly higher than those for gels with fibroblasts. The viscous coefficient was 169.6 ± 60.7 kPa s for the cardiomyocytes and 143.6 ± 44.7 kPa s for the fibroblasts. The relaxation time constant for gels with cardiomyocytes was 19.6 ± 10.6 s, significantly smaller than for gels with fibroblasts (36.4 ± 13.3 s). This study is the first to obtain viscoelastic data for living cell-contracted collagen gels. These data show that the viscous effect has a vital effect on the mechanical behavior of the gels and cannot be neglected in the culture and function of artificial substitutes based on contracted collagen gels. Furthermore, the data may imply that viscous coefficient of the gels might be closely related to collagen density rather than to cross linking among collagen fibrils.
Journal of Artificial Organs 09/2010; 13(3):139-44. · 1.59 Impact Factor
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ABSTRACT: Modern imaging technologies, such as computed tomography (CT) angiography, magnetic resonance (MR) angiography, and digital subtraction (DS) angiography are widely used for pretreatment evaluation of cerebral aneurysms, but the relative accuracies of these modalities are unclear. This study compared the measurements of aneurysm neck and dome height and width on CT angiography, time-of-flight (TOF)-MR angiography, and DS angiography using a three-dimensional workstation. An elastic model of a side-wall aneurysm was connected to an artificial heart pulsatile circuit system. The aneurysm model was prepared using a silicone membrane of 0.6-mm thickness under normal physiological circulation parameters. Using this aneurysm model, three-dimensional TOF-MR angiography, contrast-enhanced CT angiography, and DS angiography were performed. Source images were post-processed on a dedicated workstation to calculate the aneurysm size. DS angiography measurements were found to be the most accurate. In contrast, aneurysm neck sizes measured on CT angiography were significantly wider than actual values (p < 0.05) and aneurysm heights measured using TOF-MR angiography were significantly lower than actual values (p < 0.01). In this in-vitro model, at least one aneurysm dimension measured with CT angiography and with TOF-MR angiography differed significantly from actual values. Aneurysm neck width markedly affects therapeutic planning, as a wide neck requires craniotomy or endovascular treatment using an adjunctive device, so inaccuracies should be considered when aneurysm treatment is planned using modern methods of visualization.
Neurologia medico-chirurgica 01/2010; 50(10):893-9. · 0.61 Impact Factor
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ABSTRACT: Porcine aortic valve (AoF) tissues cross-linked with glutaraldehyde and epoxy compounds were reported to have high anticalcification properties, but their hydrodynamic characteristics have not been evaluated. The aim of the present study was to investigate the hydrodynamic differences between porcine AoFs, cross-linked with concomitant use of an epoxy compound and glutaraldehyde, at different fixation periods. The valves were mounted on a pulsatile flow circulation mimicking a left heart. The left atrial and left ventricular pressures and mitral and aortic flows were measured at every 0.002 seconds, and the hydrodynamic factor of the valves mounted on the mitral position was estimated. Effective orifice area and the regurgitation volume, which are used as indicators of valve efficiency, failed to detect significant differences due to glutaraldehyde fixation time. In addition, the pressure gradient across the bioprosthetic valve and the variation of mitral flow also had no significant differences. The flow circuit model of the present study was mimicking of a left heart. The evaluation of the mitral valvular function with different glutaraldehyde fixation times was accomplished by relating the pressure with the flow, and by estimating the time lag between valve motion and transvalvular flow.
ASAIO journal (American Society for Artificial Internal Organs: 1992) 01/2009; 55(1):13-8. · 1.39 Impact Factor
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ABSTRACT: There is an essential demand for tissue engineered autologous small-diameter vascular graft, which can function in arterial high pressure and flow circulation. We investigated the potential to engineer a three-layered robust and elastic artery using a novel hemodynamically-equivalent pulsatile bioreactor.
Endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts were harvested from bovine aorta. A polyglycolic acid (PGA) sheet and a polycaprolactone sheet seeded with SMCs, and a PGA sheet seeded with fibroblast, were wrapped in turn on a 6-mm diameter silicone tube and incubated in culture medium for 30 days. The supporting tube was removed, and the lumen was seeded with ECs and incubated for another 2 days. The pulsatile bioreactor culture, under regulated gradual increase in flow and pressure from 0.2 (0.5/0) L/min and 20 (40/15) mm Hg to 0.6 (1.4/0.2) L/min and 100 (120/80) mm Hg, was performed for an additional 2 weeks (n=10). The engineered vessels acquired distinctly similar appearance and elasticity as native arteries. Scanning electron microscopic examination and Von Willebrand factor staining demonstrated the presence of ECs spread over the lumen. Elastica Van Gieson and Masson Tricrome Stain revealed ample production of elastin and collagen in the engineered grafts. Alpha-SMA and calponin staining showed the presence of SMCs. Tensile tests demonstrated that engineered vessels acquired equivalent ultimate strength and similar elastic characteristics as native arteries (Ultimate Strength of Native: 882+/-133 kPa, Engineered: 827+/-155 kPa, each n=8).
A robust and elastic small-diameter artery was engineered from three types of vascular cells using the physiological pulsatile bioreactor.
Circulation 10/2008; 118(14 Suppl):S52-7. · 14.74 Impact Factor
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ABSTRACT: The weak contractile force exerted by engineered cardiac muscle is a big problem in cardiac muscle tissue engineering, even though the field has made great progress over the past decade. We believe that one major reason for the weak contractile force is that the expression of genes regulating cardiomyocyte differentiation and cardiac tissue syncytium may be different for in vivo and cultured cells. In the present study, we investigated the difference of mRNA expression under in vivo and culture conditions in order to seek a target for further gene transfer treatment in the process of cardiac tissue construction. To this end, mRNA expression of four major transcriptional factors (SRF, p300, Nkx2.5, and myocardin) and two intercalated disk constituent proteins (N-cadherin and connexin43) in rat cardiomyocytes was measured by means of ratiometric reverse-transcription polymerase chain reaction. Cardiomyocytes were harvested from the hearts of 18-day (about 3 days before birth) Wistar-rat embryos (embryonic cells), 12-day neonatal rat hearts (neonatal cells), or 14-day successive dish culture of the embryonic cells harvested from 18-day embryos (cultured cells). The results indicated that, except for SRF, the mRNAs had a lower expression tendency in cultured cells than in embryonic and in neonatal cells; in particular, the mRNA expression of myocardin, N-cadherin, and connexin43 of cultured cardiomyocytes was significantly lower than that of neonatal cells. Therefore, myocardin is a candidate for forced gene up-expression during the construction of engineered cardiac tissue; in addition, a plausible reason for the weak contractile force of engineered cardiac tissue is the weak constitution of intercalated disk, because it was elucidated that mRNA expression of proteins related to intercalated disk were lower in culture.
Journal of Artificial Organs 02/2008; 11(3):134-40. · 1.59 Impact Factor
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ABSTRACT: A series of guidelines for development and assessment of next-generation medical devices has been drafted under an interagency collaborative project by the Ministry of Health, Labor and Welfare and the Ministry of Economy, Trade and Industry. The working group for assessment guidelines of next-generation artificial hearts reviewed the trend in the prevalence of heart failure and examined the potential usefulness of such devices in Japan and in other countries as a fundamental part of the process of establishing appropriate guidelines. At present, more than 23 million people suffer from heart failure in developed countries, including Japan. Although Japan currently has the lowest mortality from heart failure among those countries, the number of patients is gradually increasing as our lifestyle becomes more Westernized; the associated medical expenses are rapidly growing. The number of heart transplantations, however, is limited due to the overwhelming shortage of donor hearts, not only in Japan but worldwide. Meanwhile, clinical studies and surveys have revealed that the major causes of death in patients undergoing long-term use of ventricular assist devices (VADs) were infection, thrombosis, and mechanical failure, all of which are typical of VADs. It is therefore of urgent and universal necessity to develop next-generation artificial hearts that have excellent durability to provide at least 2 years of event-free operation with a superior quality of life and that can be used for destination therapy to save patients with irreversible heart failure. It is also very important to ensure that an environment that facilitates the development, testing, and approval evaluation processes of next-generation artificial hearts be established as soon as possible.
Journal of Artificial Organs 02/2007; 10(4):187-94. · 1.59 Impact Factor
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ABSTRACT: This study focuses on the development of self-training system for surgical operation and quantitative evaluation of the surgical skills. Our group has developed a self-training system for anastomotic technique in Coronary Artery Bypass Grafting (CABG) to contribute the education of cardiovascular surgery without a risk to patients. The self-training system consists of following portions, 1) "YOUCAN", coronary and graft vascular silicone model, 2) "BEAT", a device, simulating stabilized myocardial surface, and 3) Quantitative evaluation system based on in vitro mock circulatory system. The coronary and graft model has been anastomosed by expert and trainee cardiac surgeon. The anastomosed model was mounted onto test section of the in vitro mock circulatory system then identical waveforms of coronary artery was applied into the inlet of an anastomosis. The energy loss was quantified as a pressure difference between proximal and distal ends of anastomosis. The energy loss was obtained as 67.3+/-1.75 mJ (trainee) and 41.3+/-3.08 mJ (registered surgeon). It was founded that average energy loss by expert surgeon was lower by 38.6% than that by trainee surgeon. The major difference among the models of expert and trainee was the Effective Orifice Area (EOA) of the anastomosis. Through the experiment, EOA was confirmed by image analysis as 2.73 mm2 for an expert against 0.534 mm2 for a trainee. In conclusion, it was suggested that the anastomotic skill among expert and trainee surgeons could be hydrodynamically differentiated by using in vitro mock circulatory system.
Conference proceedings: ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference 02/2007; 2007:2705-8.
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Life System Modeling and Simulation, International Conference, LSMS 2007, Shanghai, China, September 14-17, 2007, Proceedings; 01/2007
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Life System Modeling and Simulation, International Conference, LSMS 2007, Shanghai, China, September 14-17, 2007, Proceedings; 01/2007
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Life System Modeling and Simulation, International Conference, LSMS 2007, Shanghai, China, September 14-17, 2007, Proceedings; 01/2007
