Koki Takiura

Development of a three-dimensional bioprinter: construction of cell supporting structures using hydrogel and state-of-the-art inkjet technology.

We have developed a new technology for producing three-dimensional (3D) biological structures composed of living cells and hydrogel in vitro, via the direct and accurate printing of cells with an inkjet printing system. Various hydrogel structures were constructed with our custom-made inkjet printer, which we termed 3D bioprinter. In the present study, we used an alginate hydrogel that was obtained through the reaction of a sodium alginate solution with a calcium chloride solution. For the construction of the gel structure, sodium alginate solution was ejected from the inkjet nozzle (SEA-Jet, Seiko Epson Corp., Suwa, Japan) and was mixed with a substrate composed of a calcium chloride solution. In our 3D bioprinter, the nozzle head can be moved in three dimensions. Owing to the development of the 3D bioprinter, an innovative fabrication method that enables the gentle and precise fixation of 3D gel structures was established using living cells as a material. To date, several 3D structures that include living cells have been fabricated, including lines, planes, laminated structures, and tubes, and now, experiments to construct various hydrogel structures are being carried out in our laboratory.

Microcirculation of the bulbar conjunctiva in the goat implanted with a total artificial heart: effects of pulsatile and nonpulsatile flow.

A new system to observe the microcirculation on the bulbar conjunctiva was developed using a digital high definition microscope to investigate the influence of the flow patterns on the microcirculation in a goat with a total artificial heart (TAH). The undulation pump TAH was implanted into the goat. When the whole body condition became stable, the flow pattern was modulated between the pulsatile and the nonpulsatile mode, and the changes in the microcirculation were observed. When the flow pattern was changed from pulsatile to nonpulsatile mode, the erythrocyte velocity in capillaries dropped from 526 ± 83 to 132 ± 41 μm/s and remained at a low level. The number of perfused capillaries decreased as well. Then the nonpulsatile flow mode was maintained for 20 minutes. After the flow pattern was returned to the pulsatile mode again, the erythrocyte velocity recovered to the initial level (433 ± 71 μm/s). In many cases, the flow of the nonperfused capillaries in the nonpulsatile mode recovered to the initial level after the flow pattern was changed to the pulsatile mode again. The perfused capillary density in the nonpulsatile mode (19.7 ± 4.1 number of capillaries/mm ) was significantly lower than that in the pulsatile mode (34.7 ± 6.3 number of capillaries/mm ).It is thought that the basal and flow stimulated endothelium derived nitric oxide release in the microvessels decreased because of the disappearance of pulsatility and that the nitric oxide induced the constriction of arterioles after the flow pattern was changed to the nonpulsatile mode. At the same time, the baroceptors might sense the decrease in the arterial peak pressure or dp/dt, and the sympathetic nerve increases activities and induce the constriction of arterioles. Then, the erythrocyte velocity in capillaries would decrease. Because of the flow pattern further in the chronic phase, it is important to follow the change in the microcirculation.

Development of mechanical circulatory support devices at the University of Tokyo.

The development of mechanical circulatory support devices at the University of Tokyo has focused on developing a small total artificial heart (TAH) since achieving 532 days of survival of an animal with a paracorporial pneumatically driven TAH. The undulation pump was invented to meet this purpose. The undulation pump total artificial heart (UPTAH) is an implantable TAH that uses an undulation pump. To date, the UPTAH has been implanted in 71 goats weighting from 39 to 72 kg. The control methods are very important in animal experiments, and sucking control was developed to prevent atrial sucking. Rapid left–right balance control was performed by monitoring left atrial pressure to prevent acute lung edema caused by the rapid increase in both arterial pressure and venous return associated with the animal becoming agitated. Additionally, 1/R control was applied to stabilize the right atrial pressure. By applying these control methods, seven goats survived more than 1 month. The maximum survival period was 63 days. We are expecting to carry out longer term animal experiments with a recent model of TAH. In addition to the TAH, an undulation pump ventricular assist device (UPVAD), which is an implantable ventricular assist device (VAD), has been in development since 2002, based on the technology of the UPTAH. The UPVAD was implanted in six goats; three goats survived for more than 1 month. While further research and development is required to complete the the UPVAD system, the UPVAD has good potential to be realized as an implantable pulsatile-flow VAD.

Fabrication of 3D Cell Supporting Structures With Multi-Materials Using the Bio-Printer

We produced three-dimensional (3-D) cell supporting structures for use in engineering regenerative living tissue. The structures were formed by alginate gel, a type of hydro-gel, and our originally developed printer, termed a 3-D bio-printer. A droplet ejected from an inkjet printer nozzle has the same size as several cells. Thus, we considered that such a printer nozzle would be able to eject cultured living cells, along with growth factor, protein, and other materials. If a 3-D gel structure could be formed with such cells, and the materials and cells self-assembled, a variety of living tissues could be obtained. In this report, our 3-D bio-printer and the method of fabrication of multiple material gel structures is presented. Our 3-D bio-printer has a printing mechanism that operates in 3 directions with a positioning resolution of 0.5 micrometers, which is adequate for precise positioning of the ejected cells. Further, a piezoelectric inkjet nozzle head that does not became heated during operation is used and cells can be ejected without heating. The head has 4 nozzles and is able to eject 4 different kinds of materials simultaneously. We used a sodium alginate solution and ejected it from the inkjet nozzle into a calcium chloride solution as a substrate, thus alginate gel beads were obtained. Several types of gel structures could be constructed by distributing the beads precisely and the resolution of the gel structures was as small as the size of an individual bead, about 10–60 μm in diameter. By continuous operation of the inkjet nozzle, gel lines were able to be formed. The substrate was a 10% calcium chloride solution on a slide glass and the ejected-droplets contained a 0.8% sodium alginate solution. In addition, gel sheets were formed by parallel gel lines. In that case, a 10% calcium chloride solution was also used as the substrate. It is possible that such 3-D gel sheet structures could be constructed by lamination in a high viscosity substrate. We also formed gel rings, which were stacked and allowed to sink into the substrate, thus obtaining gel tubes. By utilizing gel tubes with inner and outer walls formed using different types of gels with vascular endothelial cells and smooth muscle cells, blood vessel structures could be fabricated with the present system.Copyright

in Vitro And in Vivo Heat Dissipation Of An Electrohydraulic Totally Implantable Artificial Heart

The authors evaluated the heat transfer characteristics of an electrohydraulic totally implantable artificial heart (EH-TAH) developed at our institute. In three in vitro experiments, the heat dissipation of the EH-TAH was investigated. First, the EH-TAH was connected to a closed mock circuit filled with 1 L of saline, and driven at an input power of 20 W. The estimated heat conducted to the blood was ∼10.3 W, which was almost half of the input power. Second, we simulated heat transfer with the circulation of a calf by using a heat exchanger. The amount of heat dissipating directly from the EH-TAH surface was calculated to be 10 W. Third, the temperature of the actuator examined with thermography was found to be almost uniform, and no prominent high temperature area was observed. In an in vivo study, the EH-TAH was implanted for 10 days in a calf weighing 62 kg. The input power was 18 ± 2 W, the temperature of the actuator-tissue contacting surface was 39.4 ± 0.8°C, and that of the pump blood chamber was 39.8 ± 0.4°C. This slight temperature elevation was thought to be attributable to heat dissipation to the blood. On histologic study of the chest wall and the lung in contact with the actuator, vascularized connective tissue envelopes were observed, but unfavorable side effects, such as tissue necrosis, were not observed. These results suggest that the thermal effect of this system is acceptable at the input power used. ASAIO Journal 1997; 43:M592-M597.

In vitro and in vivo evaluation of a left-right balancing capacity of an interatrial shunt in an electrohydraulic total artificial heart system

The authors evaluated the basic performance of an interatrial shunt (IAS) made by punching a hole in the atrial septum, in accommodating the left-right imbalance in our electrohy-draulic total artificial heart (EHTAH) system. In an in vitro study conducted in a closed mock circuit connected with the EHTAH, the interatrial pressure gradient changed in compliance with the amount of bronchial flow and the size of the IAS. The IAS of 4.4 mm diameter or larger maintained the interatrial pressure gradient within physiologically permissible limits when the amount of bronchial flow was 5% of cardiac output or less. A left-to-right one-way valve made of a piece of pericardium, a possible option in this IAS method, successfully prevented right-to-left reverse shunt flow through the IAS. In a chronic in vivo study using a calf implanted with the EHTAH for 10 days, a 4.5 mm IAS without the one-way valve demonstrated satisfactory dynamic left-right balancing capacity with a stable interatrial pressure gradient of 4 ± 1 mmHg over a wide range of atrial pressures. No thrombus was found in or around the IAS at autopsy. The authors conciude that the IAS is a simple and promising means

Early changes in circulating blood volume and volume regulating humoral factors after implantation of an electrohydraulic total artificial heart.

The early changes in circulating blood volume (CBV) and volume regulating humoral factors after implantation of an electrohydraulic total artificial heart (EH-TAH) were investigated in a calf and compared with results in a sham operated control calf. CBV was measured by the dye dilution method using indocyanine green. CBV and humoral factors were periodically investigated. In the EH-TAH implanted calf, the cardiac output was estimated at 6–7 L/min (94–109 ml/kg/min), and the aortic pressure and aerobic metabolic condition were favorable. Nevertheless, the CBV was increased to 132 and 168% of the pre-operative value (range in the control calf, 83–103%) on post operative days 4 and 8, respectively. The atrial natriuretic peptide level on days 2, 5, and 7 was 23, 170, and 240 (in the control calf, 19–61) pg/ml, respectively, and the anti-diuretic hormone level was 7.3, 2.0, and 1.3 (0.5–1.3) pg/ml, respectively. The plasma renin activity was 3.2, 3.7, and 3.1 (0.5–0.3) ng/ml/hr, respectively. The angiotensin-I and angiotensin-II levels were also increased in the EH-TAH implanted calf. It is concluded that significant water retention occurs even at sufficient cardiac output early after EH-TAH implantation. The changes in humoral factors are suggested to arise secondary to the increased CBV or other unknown factors.

A new approach to detection of the cavitation on mechanical heart valves

The cavitation on the mechanical heart valves (MHVs) is thought to be a cause of the mechanical failure of the occluder; also, the free radicals that would be generated when the cavitation bubbles implode might affect the patients chemically. These cavitation effects are attributed to the bubble collapse. Therefore, it is important to detect the bubble implosion behavior to analyze the cavitation on MHVs. The cavitation bubbles induce the generation of free radicals at their implosion, and the excited hydroxyl radicals emit the faint light. Based on this fact, we have tried to observe the faint light emission from a MHV to specifically capture the implosion of the cavitation bubbles. A highly sensitive CCD (charge coupled device) camera (C2400–35 VIM camera, Hamamatsu Photonics, Hamamatsu, Japan) was adopted in this study. This camera can observe low light down to the single photon counting range, and it gives two-dimensional mapping of the light. A 20 mm Björk-Shiley valve was submerged in the water tank of 10 L deionized water with luminol as a light enhancer, and then the pressure difference of 150 mm Hg was exerted on the valve at a rate of 60 bpm with a pulse duplicator. The camera and the water tank were settled in the lightproof configuration. After 2 hours of exposure, faint light images have been obtained successfully. The light emits mostly from the edge of the occluder on the inflow side in the major orifice of the valve. Therefore the results suggest that the bubbles would implode around this region and that free radicals caused by cavitation might be produced on MHV, which has coincided with our preliminary result by an electron spin resonance spectrometry.

Development of a novel intrafascicular nerve electrode

Artificial organs could be controlled using autonomic neural signals, because they exhibit rapid responses to physical needs similar to those of natural organs. A nerve electrode must satisfy many requirements to measure autonomous neural signals such as a long lifetime, high signal-to-noise ratio, multichannel recording, simple installation into a nerve fascicle, and good manufacturing productivity. The purpose of our study is to propose and evaluate a novel nerve electrode that satisfies these conditions, which to date has not been developed. A novel intrafascicular nerve electrode was designed, fabricated, and evaluated on autonomic nerves. Conventional extrafascicular and intrafascicular nerve electrodes were fabricated and tested for comparison to our novel intrafascicular nerve electrode. The novel intrafascicular nerve electrode had a 3-week lifetime, whereas the conventional extrafascicular nerve electrode had a 2-week lifetime. The signal-to-noise ratio was improved from 1.6 to 2.0 compared with the conventional extrafascicular nerve electrode. The novel intrafascicular nerve electrode was easier to install into a nerve fascicle and had better manufacturing productivity than the conventional intrafascicular nerve electrode. We succeeded in demonstrating the feasibility of our novel intrafascicular nerve electrode.

Preliminary study of a new type of energy transmission system for artificial hearts

Abstract A transcutaneous energy transmission (TET) system is the most common way to power artificial hearts and ventricular assist devices. However, an external battery used with a TET system poses several problems, such as its heavy mass, small charge capacity, and long recharging time. The battery is indispensable when patients want to be ambulatory. This article proposes a new type of TET system that does not require an external battery because electrical energy is supplied remotely by using electromagnetic waves. For this system to operate, multiple transmitting antennas have to be mounted in a room or facility that has been shielded from electromagnetic waves, and a receiving antenna is attached to the patient. Electromagnetic waves transmit electrical power from the transmitting antennas to the receiving antenna. The received electrical power is sent to an implanted device through the TET system. The total power efficiency was plotted against the transmitter–receiver distance by measuring the power that was input to the transmitting antennas, and the final direct current (DC) power that was received by the receiving antenna. A 430-MHz frequency was applied in the experiments. The obtained efficiency was around 10% within a transmitter–receiver distance of 1 m when Yagi-Uda antennas were used for the transmitting antennas and two other types of antenna were used for the receiving antennas: a folded dipole with a reflector and a single loop with a reflector. The results suggested that the proposed system is worth considering. The proposed system would go a long way toward enhancing the patients quality of life compared with the currently used conventional TET system.