Shuichi Mochizuki

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.

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

A new hypothesis on the mechanism of calcification formed on a blood-contacted polymer surface

Calcification on a blood-contacting polymer surface in an artificial heart is one of the most serious problems. Recently, we maintained a goat with a total artificial heart (AH) for 532 days without systemic anticoagulation. Sactype blood pumps coated with segmented polyurethane and incorporating jellyfish valves, thin polymer membrane valves, were used in the experiment. The pump was exchanged for a new one on the 312th day on the left side and the 414th day on the right side. They were analyzed with a scanning electron microscope (SEM) and an X-ray microanalyzer. The valve membrane after 312 days of pumping revealed plastic deformation expanding toward upstream between the spokes by creep fatigue with blood pressure difference when the valve closed. Calcification on the membrane was concentrated in the limited portions that received a strong stretching force: the upstream side of the membrane between the spokes and downstream side of the membrane on the spokes. Slight or no calcification was observed on the opposite side of the membrane that received a compression force, and no calcification was found on nonmoving parts such as the center of the membrane and spokes. A new hypothesis on the mechanism of calcification at the portion that received repeated stretching force was raised. The repeated stretching force would extend the polymer membrane, causing some loosening between polymer molecules and generating microgaps. The blood protein and phospholipid would invade into these microgaps, which would then attract Ca ions followed by phosphate ions to make their complexation. The hypothesis could well explain the calcification phenomena on a blood-contacting polymer surface, and gave a good clue on how to protect from calcification.

Third model of the undulation pump total artificial heart.

The undulation pump is a small, continuous flow displacement type blood pump, and the undulation pump total artificial heart (UPTAH) is a unique, implantable total artificial heart based on this pump. To improve the durability of the UPTAH for investigating long-term pathophysiology with UPTAH, a third model (UPTAH3) has been developed. UPTAH3 was designed to separate the left and right undulation shafts and to be more durable. The undulation pumps were also redesigned. UPTAH3 was implemented with a diameter of 76 mm, width of 78 or 79 mm, total volume of 292 ml, and weight of 620 g. The priming volumes of the left and right pumps are 26 and 21 ml, respectively. The atrial cuffs and outflow cannulae were also redesigned for UPTAH3. The maximum output against an arterial pressure load of 100 mm Hg is about 11 L/min. The maximum pump efficiency is about 15% in the left pump and 18% in the right pump, giving a maximum total efficiency for both of about 11%. To date, UPTAH3 has been tested in 17 goats, and the longest survival period was 46 days. This third model will be useful for investigating pathophysiology with UPTAH.

A step forward for the undulation pump total artificial heart

In the University of Tokyo, various types of total artificial heart (TAH) have been studied. Based on the experiences of TAH research, the development of the undulation pump total artifical heart (UPTAH) started in 1994. The undulation pump is a small-size, continuous-flow, displacement-type blood pump, and the UPTAH is a unique implantable total artificial heart that uses the undulation pump. To date, three models of UPTAH have been developed. The first model, UPTAH1, was developed to investigate the possibility of reducing the size of the device so it could be implanted in small adults, such as Japanese patients, in 1994. The second model, UPTAH2, which was the prototype of the animal experimental model, was developed in 1996 to investigate the possibility of survival with the UPTAH. The third model, UPTAH3, which is the present model, was developed in 1998 to enable long-term survival in animal experiments and to investigate the pathophysiology of the UPTAH. From July 1996 to October 1999, 22 implantations of UPTAH2 or UPTAH3 were performed in goats. In spite of the limitation of their small chest cavity, the UPTAH could be implanted into the chest of all goats. Using UPTAH3, survival of 31 days could be obtained. The research and development of UPTAH are ongoing.

Structural design of a newly developed pediatric circulatory assist device for fontan circulation by using shape memory alloy fiber

Total cavopulmonary connection (TCPC) is commonly applied for the surgical treatment of congenital heart disease such as single ventricle in pediatric patients. Patients with no ventricle in pulmonary circulation are treated along with Fontan algorithm, in which the systemic venous return is diverted directly to the pulmonary artery without passing through subpulmonary ventricle. In order to promote the pulmonary circulation after Fontan procedure, we developed a newly designed pulmonary circulatory assist device by using shape memory alloy fibers. We developed a pulmonary circulatory assist device as a non-blood contacting mechanical support system in pediatric patients with TCPC. The device has been designed to be installed like a cuff around the ePTFE TCPC conduit, which can contract from outside. We employed a covalent type functional anisotropic shape memory alloy fiber (Biometal, Toki Corporation, Tokyo Japan) as a servo actuator of the pulmonary circulatory assist device. The diameter of this fiber was 100 microns, and its contractile frequency was 2–3 Hz. Heat generation with electric current contracts these fibers and the conduit. The maximum contraction ratio of this fiber is about 7% in length. In order to extend its contractile ratio, we fabricated and installed mechanical structural units to control the length of fibers. In this study, we examined basic contractile functions of the device in the mock system. As a result, the internal pressure of the conduit increased to 63 mmHg by the mechanical contraction under the condition of 400 msec-current supply in the mock examination with the overflow tank of 10mmHg loading.

Calcification and Thrombus Formation on Polymer Surfaces of an Artificial Heart

Calcification and thrombus formation are still important problems in artificial heart research. The calcification and thrombus formation generated in artificial heart blood pumps, driven without anticoagulant for 312 days as the left side and 414 days as the right side, were analyzed in this study. A thrombus was observed at the circumference of the sac in the 312-day pump, but it was not associated with calcification. Several phenomena were observed on the polymer membrane valves (jellyfish valves) incorporated into the blood pump: plastic deformation of the valve membrane by creep fatigue; no calcification of stationary parts such as spokes and the center of the membrane; calcification of the particular portion which received repeated stretching stress; and no association of calcification with thrombus. The calcification of the valve area which received repeated stretching force might be explained as follows. Repeated stretching forces extend the polymer membrane, causing some loosening between polymer molecules and generating microgaps. Blood proteins and phospholipids invade these microgaps, which then attract Ca2+ ions followed by phosphate ions(PO4 2-) leading to the formation of calcium phosphate complexes.

Implantable total artificial heart: history and present status at the University of Tokyo

The University of Tokyo has been involved in research and development of the artificial heart since 1959. This paper is a brief review of 40 years of total artificial heart research in the University of Tokyo. Many types of artificial heart and various kinds of materials, blood pumps, valves, drive units, control methods, and pathophysiology have been investigated in our original fashion. The longest survival was 532 days for a goat with a total artificial heart (TAH) placed on the chest wall. These results made us take a step toward the development of an implantable TAH. Two kinds of implantable TAH are now being developed: FTPTAH (flow-transformed pulsatile total artificial heart) and UPTAH (undulation pump total artificial heart). Recently, a goat survived for 31 days with an UPTAH.

Numerical estimation of heat distribution from the implantable battery system of an undulation pump LVAD

We have been developing an implantable battery system using three series-connected lithium ion batteries having an energy capacity of 1800 mAh to drive an undulation pump left ventricular assist device. However, the lithium ion battery undergoes an exothermic reaction during the discharge phase, and the temperature rise of the lithium ion battery is a critical issue for implantation usage. Heat generation in the lithium ion battery depends on the intensity of the discharge current, and we obtained a relationship between the heat flow from the lithium ion battery qc(I) and the intensity of the discharge current I as qc(I) = 0.63 × I (W) in in vitro experiments. The temperature distribution of the implantable battery system was estimated by means of three-dimentional finite-element method (FEM) heat transfer analysis using the heat flow function qc(I), and we also measured the temperature rise of the implantable battery system in in vitro experiments to conduct verification of the estimation. The maximum temperatures of the lithium ion battery and the implantable battery case were measured as 52.2°C and 41.1°C, respectively. The estimated result of temperature distribution of the implantable battery system agreed well with the measured results using thermography. In conclusion, FEM heat transfer analysis is promising as a tool to estimate the temperature of the implantable lithium ion battery system under any pump current without the need for animal experiments, and it is a convenient tool for optimization of heat transfer characteristics of the implantable battery system.