advanced biomedical engineering original paper doi:10
TRANSCRIPT
Optimum Sterilization Methods of Biocompatible Hybrid Material for Arti�cial Organs
Yusuke INOUE,*, **, ***, # Ayaka TASHIRO,** Yukino KAWASE,** Takashi ISOYAMA,** Itsuro SAITO,** Toshiya ONO,** Shintaro HARA,** Kohei ISHII,† Terumi YURIMOTO,†† Yasuyuki SHIRAISHI,*
Akihiro YAMADA,* Tomoyuki YAMBE,* Yusuke ABE**, †††
Abstract We previously reported the development of a new hybrid medical material comprising bio-based materials with high biocompatibility and arti�cial materials with characteristics of excellent strength and pro-cessability. This material shows suf�cient biocompatibility and excellent stability in vivo. Moreover, when ap-plied to the surface of an implantable sensor, the biological reaction on the sensor function surface can be well controlled. For commercialization and widespread use of hybrid materials with such superior properties, steril-ization and storage are critical considerations, given that hybrid materials must be processed outside the body prior to application as medical materials in vivo, thus posing a risk of contamination despite best efforts. There-fore, the aim of the present study was to establish an optimal sterilization method that will not impair the bio-compatibility of the hybrid material. Toward this end, we tested six sterilization methods for the hybrid materi-al: autoclave (121°C, 20 min), dry heat (160°C, 120 min), ethylene oxide gas (37°C, 120 min), hydrogen peroxide plasma (45°C, 45 min), and gamma ray (25 kGy) with and without lyophilization. After sterilization, the material was cultured with vascular endothelial cells to evaluate the engraftment rate, and was observed with light and scanning electron microscopy to determine shape and structure changes. The results demonstrat-ed that gamma sterilization without lyophilization was the best sterilization method for this material, which preserved the collagen network and showed no change in number of adhered vascular endothelial cells com-pared to the pre-sterilized material. These �ndings are useful to promote the commercialization of this hybrid material with combined advantages of synthetic and bio-based materials for widespread clinical application in the engineering of arti�cial organs.
Keywords: biocompatible material, arti�cial heart, sterilization, antithrombotic, in�ammation.
Adv Biomed Eng. 9: pp. 83–92, 2020.
1. Introduction
The main materials currently used for arti�cial organs are synthetic materials and biomaterials. Synthetic mate-
rials have advantageous properties in terms of excellent strength and durability, but suffer from reduced biocom-patibility compared to biomaterials, although this aspect is continuously being improved with the development of new methods [1–3]. By contrast, arti�cial organs based on biomaterials require a chemical processing step of re-moving tissues from the living body, which are then re-turned to the living body [4–8]. This method is associat-ed with a risks of retrovirus and bacterial infection, that are dif�cult to control. Furthermore, arti�cial organs pre-pared with bio-based materials lack strength and durabil-ity and cannot be manipulated easily into an arbitrary desired shape. Therefore, we have focused on the devel-opment of hybrid materials using tissue engineering techniques to create new types of biomaterials with the combined biocompatibility properties of tissues of bio-logical origin and suf�cient strength and durability from synthetic materials, which can be readily formed into in-tended shapes [9–13]. These hybrid materials are pro-duced in vivo by inducing the formation of living tissues
This study was presented at the Symposium on Biomedical Engi-neering 2019, Tokushima, September, 2019. Received on July 22, 2019; revised on October 28, 2019; accepted on December 23, 2019.
* Institute of Development, Aging and Cancer, Tohoku University, Miyagi, Japan.
** Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
*** Advanced Medical Engineering Research Center, Asahikawa Medical University, Hokkaido, Japan.
† National Institute of Technology, Kagawa College, Kagawa, Japan.
†† Central Institute for Experimental Animals, Kawasaki, Japan.
††† Department of Medical and General Sciences, Nihon Institute of Medical Science, Saitama, Japan.
# Seiryo-machi 4–1, Aoba-ku, Sendai, Miyagi 980–8575, Japan. E-mail: [email protected]
Original PaperAdvanced Biomedical Engineering9: 83–92, 2020.
DOI:10.14326/abe.9.83
with arti�cial materials as scaffolds. A core, serving as a scaffold, is inserted into an external form or mold to ef-fectively control the shape. The external mold and cores are then implanted subcutaneously, and are removed once the living tissue is induced. The core organized in the scaffold is removed from the body and decellularized to obtain the �nal material.
This integrated approach for the scaffold makes it possible to obtain biomaterials with good strength and durability (Fig. 1). In addition, we previously demon-strated that these hybrid materials exhibit excellent sta-bility in vivo, and superior ability to control biological reactions when applied to the surface of an implantable sensor [14–17]. To further promote and market these hy-brid materials with superior properties, it is necessary to consider critical aspects of their sterilization and storage. Currently, these hybrid materials have to be processed �rst outside the body for application as medical materi-als in vivo. Although all of these processes are operated under aseptic conditions, a completely aseptic environ-ment cannot be achieved in practice. Thus, establishing a sterilization process is an essential requirement for com-mercial distribution of a medical material. Therefore, in the present study, we sought to establish an optimal ster-ilization method that will not impair the biocompatibility of the hybrid material.
2. Materials and Methods
2.1 Production of hybrid materialsThe core scaffold was a polyester raised �ber that is used for the creation of arti�cial blood vessels, as reported previously [9, 10, 14]. The scaffold was processed into a circle with a diameter of 15 mm and thickness of 1 mm, which was used as the sample for sterilization (Fig. 2A). The scaffold has two surfaces: a surface with raised �-bers and an unprocessed surface. One side of the surface of the scaffold is raised and has a high porosity. To con-trol the shape of the living tissue emerging on the scaf-fold, an acrylic outer mold was prepared, and the scaf-fold was inserted into the mold. The outer mold was composed of an upper and a lower part with a thickness of 2 mm, length of 35 mm, and width of 35 mm. With this design, it is possible to incorporate four scaffolds into a single outer mold. Pores were made in the outer mold to allow cell induction, with �ve pores on each side of the scaffold (Fig. 2B). The prepared core was embed-ded subcutaneously in an adult goat (Japanese-Saanen goat, female, age 4 years, body weight 45 kg), and was left for 3 months to induce living tissue (Fig. 2C). The
Fig. 1 Design concept of the hybrid material. A: Polyester fabric scaffold, B: Acrylic outer mold, C: Implantation on the muscle, D: Removed core from the body, E: Decellularized core, F: Steriliza-tion and storage, G: Clinical use and non-clinical study.
Fig. 2 Arti�cial material as a core of the hybrid material. A1: Scaffold made of polyester velor (raised func-tional surface (front side), A2: Woven back side, B1: External mold to determine the shape of the induced tissue, B2: Outer mold lid with tissue guiding hole, D: Implanted on the muscle (latissimus dorsi).
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Ethics Committee of the University of Tokyo approved the animal experiments (P12-156).
Since the hybrid materials are intended for use in xenotransplantation and allotransplantation, when ap-plied to a recipient, the material was decellularized to prevent immune rejection. Tissue-derived cores were ex-posed for 6 h in a 1.0% aqueous sodium dodecyl sulfate solution at 37°C. In addition, the cores were washed with saline for 3 days to complete preparation of the hybrid material for in vivo application (Fig. 3C). Figure 3B and D shows HE stained images of vertically sliced sec-tions of the material. These images were observed near the front side of the material. The surface of the material has a high clearance rate of the scaffold material and a large proportion of the regenerated tissue. This material is intended for use with the surface in contact with blood or bio-tissue.
2.2 Sterilization methodsSince no sterilization method has yet been established for these novel hybrid materials, we tested several com-mon sterilization methods to determine the most suitable method for this material.
The hybrid material was freeze-dried since the sam-ple must be in a dry state for practical application of most sterilization methods. For this purpose, lyophilization was performed because it is desirable to maintain a sta-ble long-term sterile condition while the sample is main-tained in a dry state. The sample was cut into quarters, and each section was �ash-frozen in liquid nitrogen and then lyophilized for 24 h using a lyophilizer (FDU-1200, EYELA, Japan). The lyophilized samples were then ster-ilized by �ve methods: autoclave, dry heat, ethylene ox-ide gas, hydrogen peroxide gas plasma, and gamma ray. In addition, another sample was prepared by gamma-ray sterilization of the material in physiological saline with-out freeze-drying. Thus, a total of six types of samples were produced. Each sterilization method was performed using the standard protocol, and the conditions are sum-
marized in Table 1.
2.2.1 Veri�cation of sterilizationTo verify the effects of various methods of sterilization, the samples were checked by viable count methods [18]. Sterilized samples were homogenized in sterilized water, diluted 10-fold, and applied to an agar medium. The number of colonies was counted after culturing in an in-
Fig. 3 Removed core and decellularized material. A: Enriched blood vessels and tissues were induced into the scaffold, B: Tissue image of the core with many nuclei (HE staining), C: Completed hybrid material after decellularization, D: Hybrid material with only collagen and remaining scaffold (HE staining).
Table 1 Methods and conditions of sterilization.
Method Abbreviation Mechanism Conditions Equipment
Autoclave AC High-pressure steam 121°C, 20 min TA33E, TOHO, Japan
Dry heat DH Heat 160°C, 120 min SH62, Yamato Scienti�c, Japan
Ethylene oxide gas EO Chemical 37°C, 11 h SA-N540, Canon, Japan
Hydrogen peroxide gas plasma GP Chemical 45°C, 45 min STERRAD50, APS Japan, Japan
Gamma ray with freeze-drying GD Ionizing radiation 25 kGyKoga isotope(Outsourcing)
Gamma ray without freeze-drying GW Ionizing radiation 25 kGy, SalineKoga isotope(Outsourcing)
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cubator (35°C) for 48 hours.
2.3 Evaluation of the negative effects of sterilizationTo compare the effects of the various sterilization meth-ods, we assessed the adverse effects of sterilization in terms of changes in shape, structure, and cell adhesion of the hybrid material before and after sterilization.
2.3.1 Evaluation of structural changes by light and electron microscopy
The sterilized samples were observed with a light stereo-microscope (SZ61, Olympus, Japan) and scanning elec-tron microscope (SEM; S-2250N, Hitachi, Japan). The sample was coated with platinum Pt (Pt + Pd) using ion sputtering for SEM observation at an acceleration volt-age of 20 kV and a current of 120 μA.
2.3.2 Adhesion of vascular endothelial cellsWhen a hybrid material is applied to the blood contact surface, it is recellularized by recipient cells and exhibits high antithrombotic properties. Therefore, the cell via-bility of the material was assessed by culturing with bo-vine-derived vascular endothelial cells (BAE-1) to assess the effects of sterilization on cell adhesion. After steril-ization, 150 μL of a cell suspension (2.5 × 106 cells/mL) was added to the sample and placed in a 5% CO2 incuba-tor (BNA-11, ESPEC, Japan) at 37°C for 5 days. After culture, tissue staining was performed to count the num-ber of adhering cells.
2.4 Histological evaluationTo con�rm the cell adhesion ability, hematoxylin and eo-sin (HE) staining was performed to observe the process of cellularization and decellularization. The samples were �xed in 10% buffered formalin solution for 24 h.
Fluorescent staining was performed to visualize the living cells. For this purpose, we added 1 μL of 4’,6-di-amidino-2-phenylindole dihydrochloride (DAPI) solu-tion (Dojin Chemical, Japan) to 2 mL of 0.3% TritonTM X-100 (Sigma-Aldrich, Japan) solution. The samples were in�ltrated with this solution for 5 min. The samples were then observed with a �uorescence microscope (BZ-9000, KEYENCE, Japan), and the numbers of viable cells were counted.
2.5 Statistical analysisDifferences in number of cells among the seven samples were assessed using one-way analysis of variance (ANOVA) and Tukey’s multiple comparisons (p < 0.01). All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University), which is a graphical user interface for R (the R Founda-tion for Statistical Computing) [19].
3. Results
3.1 Veri�cation of sterilizationThe results of veri�cation of sterilization are shown in Fig. 5. Many colonies were found in the non-sterile hy-brid material used as a control sample. No colonies were found in all samples sterilized by the six methods.
3.2 Effects of sterilization on structural changesThe changes in shape of the materials after sterilization are shown in Fig. 4. With autoclaving, the material sur-face turned brown and shrank. In particular, extensive contraction was observed on the raised surface side, where many living tissues are present. SEM images showed some portions with a clear network structure, and the collagen �ber diameter of the tissue increased markedly. After dry heat sterilization, the sample surface turned slightly brown. SEM observation demonstrated a network structure and a uniform surface structure with-out gaps. Moreover, the �ber diameter of the pores was essentially maintained. Ethylene oxide gas sterilization did not result in any discoloration or deformation of the sample, and SEM observation showed a network struc-ture with the same thin �ber diameter as that observed before sterilization. After hydrogen peroxide gas plasma sterilization, the raised surface with many biological tis-sues became rounded and shrunk, and the raised surface side was very fragile. SEM observation showed only a few portions in which the network structure could be con�rmed, and the �ber diameter increased. No discolor-ation or deformation was observed after gamma steriliza-tion with freeze-drying and without freeze-drying. More-over, SEM observation showed a network structure with the same �ne �ber diameter as that before gamma steril-ization with freeze-drying.
Fig. 5 Veri�cation of sterilization. Control, No sterilization; AC, autoclave steriliza-tion; DH, dry heat sterilization; EO, ethylene oxide gas sterilization; GP, hydrogen peroxide gas plasma sterilization; GD, gamma-ray sterilization with freeze-drying; GW, gamma-ray sterilization without freeze-drying.
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Figure 6 shows the results of culturing vascular en-dothelial cells on the samples after sterilization. The HE-stained vertical sections in the left column show the structural changes of the samples. Compared to Control and GW, the other samples were stained with eosin that binds to positively charged proteins. In particular, AC had a thick eosin area and was stained strongly, showing strong protein aggregation. In DH, the result of HE stain-ing (Fig. 6) and SEM (Fig. 4) showed a greatly enlarged spaces, and the structure changed to an unstable state. GP changed to a rough surface with a fuzzy collagen sur-face. When GD and GW were compared, GD had larger pores and larger �ber diameter. GW showed no signi�-cant difference from Control.
3.3 Cell adhesion test by culture of vascular endo-thelial cells
The results after culturing the cells for 5 days on the ma-terials after sterilization are shown in Fig. 6. The left col-umn shows HE-stained vertical sections of the samples, with the nuclei of the cultured cells stained deep purple. The right column shows the DAPI-stain viable cells (white dots) observed under �uorescence microscopy. The number of cells per unit area is shown in Fig. 7.
In both the control (non-sterilized hybrid material) and gamma ray-sterilized samples, cells were found on the entire surface of the material, with no signi�cant dif-ference in cell numbers (p > 0.9). For all other steriliza-tion methods, the cells were sparsely distributed and al-most no nuclei were observed in the vertical sections (Figs. 6, 7). There was a signi�cant difference in cell
numbers among AC, DH, EO, GP, GD (methods with lyophilization) and GW (gamma-ray without lyophiliza-tion) groups (ANOVA, p < 0.01). There was no signi�-cant difference among AC, DH, EO, GP, and GD groups (p > 0.01).
4. Discussion
This exploratory study demonstrated the most suitable method for sterilizing hybrid materials (composed of bio-based and synthetic materials) for ultimate applica-tion as arti�cial organs, by comparing several commonly used sterilization methods. Since many of these conven-tional methods were originally developed for sterilizing metal-based medical instruments, adverse effects of pro-tein denaturation were observed in the hybrid materials after sterilization. In particular, deformation of the auto-claved and dry heat-sterilized samples was caused by the effects of pressure and heat. This occurred because most of the hybrid materials are composed of collagen pro-teins that readily deform upon heating [20].
In this study, the samples were lyophilized as a pre-treatment for sterilization. When protein is in a dry state, the energy is relatively thermodynamically low, and heat would have less effect compared with a saline-based ma-terial [21–24]. Since the extent of denaturation of a pro-tein in the dry state is unclear for these hybrid materials, heating was carried out at 120°C in an autoclave and at 160°C in dry heat sterilization. As a result, the sample shrank and deformation of the collagen network was ob-served. Light microscope and SEM observations showed that the autoclaved sample was more deformed than the
Fig. 4 Comparison of structural changes induced by various sterilization methods. Upper row: Optical microscope images; Bottom row: Structural comparison by scanning electron microscopy. AC, auto-clave sterilization; DH, dry heat sterilization; EO, ethylene oxide gas sterilization; GP, hydrogen peroxide gas plasma sterilization; GD, gamma-ray sterilization with freeze-drying.
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dry heat-sterilized sample. This result is considered to re�ect the effect of high pressure.
In ethylene oxide gas sterilization, an irreversible re-action is caused by substituting the hydrogen atoms of amino groups (-NH2), hydroxyl groups (-OH), and thiol groups (-SH) in microorganisms with alkyl groups. Since the hybrid material is also considered to contain a hy-droxyl group, it is likely that the structure would be changed by performing ethylene oxide gas steriliza-tion [25–27]. Although we did not perform chemical analysis, no apparent structural destruction or change was observed when comparing the pre- and post-steril-ized samples, at least by SEM observation.
The structural change of the sample subjected to hy-drogen oxide gas plasma sterilization is probably due to the action of free radicals. Hydrogen peroxide low-tem-perature plasma sterilization has a combined bactericidal effect from the vaporized hydrogen peroxide itself along with a killing effect from the various active free radical substances that are generated by bringing the hydrogen peroxide into a plasma state at low temperature. Indeed, this is a common sterilization method that kills microor-ganisms. By irradiating with high-frequency energy, hy-drogen peroxide is plasmi�ed, and the released electrons produce hydroxyl and hydroperoxyl radicals, which are highly reactive free radicals and oxidizing agents; the former react with all biological substances such as car-bohydrates, proteins, and lipids. Consequently, DNA and
Fig. 6 Results of cell adhesion test by culturing with vascu-lar endothelial cells. Left column: HE-stained vertical sections; Right column: DAPI-stained viable cells.
Fig. 7 Number of surviving vascular endothelial cells per square millimeter. AC, autoclave sterilization; DH, dry heat steriliza-tion; EO, ethylene oxide gas sterilization; GP, hy-drogen peroxide gas plasma sterilization; GD, gam-ma-ray sterilization with freeze-drying; GW, gamma-ray sterilization without freeze-drying.
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RNA of microorganisms are damaged to achieve a steril-ized state [28–30]. However, almost 50% of the compo-sition of the hybrid materials are proteins that will also be damaged as part of the mechanism of microbial steril-ization, which explains why the sample shrank and be-came very fragile. In addition, the hybrid material had a porous structure, facilitating adsorption of hydrogen per-oxide that penetrated the material. Collectively, these phenomena resulted in a change in shape of the material following sterilization.
By contrast, gamma-ray sterilization breaks the chemical bonds in a molecule by the ionizing energy generated from applying radiation to a substance. The active species generated outside the cell then invade the cell and combine with the active species generated inside the cell. By damaging the DNA molecules, they stop the cell proliferation, leading to cell death [31–33]. While damaging the bacteria on the hybrid material, it is possi-ble that the gamma rays also cause a change in the struc-ture of the hybrid material itself. Despite the lack of chemical analysis, no such apparent structural destruc-tion or change was observed in the pre- and post-steril-ized samples, at least by SEM observation.
Overall, for long-term storage, it is more desirable to store hybrid materials in a dry state than using storage methods that require liquids such as saline. However, comparing the results of gamma ray sterilization with and without lyophilization, we found that lyophilization should be avoided when sterilizing this material.
This paper focuses on the biocompatibility of hybrid materials by sterilization, but the changes in mechanical strength of the hybrid materials by sterilization are not clear. Gamma sterilization (25 kGy) has been reported not to change the structure of polyester [34]. Hara et al. [35] and Bessho et al. [36] reported that the degrada-tion and cross-linking of collagen and gelatin occurred simultaneously by irradiation with gamma rays. They also reported that collagen was degraded when heat treatment was performed before gamma irradiation [35, 37]. Depending on the pH of the liquid to be stored, cross-linking of collagen changes after gamma steriliza-tion [38]. The effect of sterilization on polyester fabric is considered to be suf�ciently small compared to living tissue [39]. On the other hand, heat and gas produced by general sterilization methods have an effect on polymer materials [40]. Sterilization obviously affects macromol-ecules and living tissues, but the magnitude of negative effects depends on conditions such as dose, temperature, and pH. Therefore, the change in mechanical strength by sterilization requires further study. In addition, to deter-mine speci�c sterilization conditions, it is necessary to evaluate effectiveness by animal experiments.
None of the sterilized samples showed culture
growth on an agar medium, demonstrating the overall success of each of the sterilization methods tested.
5. Conclusion
We explored the optimal sterilization method that does not compromise the biocompatibility of a hybrid materi-al by investigating morphological changes and cell adhe-sion. Although lyophilization is generally considered to be effective for sterilization and storage, this treatment negatively affected cell adhesion to the hybrid material. Based on these results, gamma sterilization without lyo-philization appears to be the most suitable sterilization method for hybrid materials, which can promote their clinical application and marketability.
Acknowledgement
This work was supported by JSPS KAKENHI Grant Numbers JP25670105, JP15K12503, JP19K12772. And this work was supported by the cooperation program of research institutes in Tohoku University.
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Advanced Biomedical Engineering. Vol. 9, 2020.(90)
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Yusuke INOUE
Yusuke INOUE received his Ph.D. degree in Medi-
cine from The University of Tokyo, Japan in 2011.
From 2011 to 2015 he was postdoctoral researcher
at The University of Tokyo. He moved to Tohoku
University in 2015 and right now he is an Assistant
Professor of Department of Medical Engineering
and Cardiology, Institute of Development, Aging and Cancer. His cur-
rent research �eld is biomedical engineering and biomaterials for arti-
�cial heart. He is a member of the Japanese Society for Medical and
Biological Engineering, the Japanese Society for Arti�cial Organs, the
Society of Life Support Engineering, Research Conference for Arti�-
cial Heart and Assisted Circulation of Japan.
Ayaka TASHIRO
Ayaka TASHIRO received her master’s degree from
Kitasato University, Japan in 2016. From 2016 and
right now she is a Clinical Engineer at Ikegami
General Hospital, Medical Corporation Showakai,
Japan. Her current research �eld is biomedical en-
gineering and biomaterials for arti�cial heart. She
was a member of the Japanese Society for Medical and Biological En-
gineering, the Japanese Society for Arti�cial Organs, Research Confer-
ence for Arti�cial Heart and Assisted Circulation of Japan.
Yukino KAWASE
Yukino KAWASE received her master’s degree from
Kitasato University, Japan in 2015. Her current re-
search �eld is biomedical engineering and bioma-
terials for arti�cial heart. She was a member of the
Japanese Society for Medical and Biological Engi-
neering, the Japanese Society for Arti�cial Organs,
Research Conference for Arti�cial Heart and Assisted Circulation of
Japan.
Takashi ISOYAMA
Takashi ISOYAMA received Ph.D. degree from The
University of Tokyo, Japan in 1995. From 1986 to
1992 he was Scientist at AISIN SEIKI Co., Ltd..
From 1992 to 1998 he was a Scientist at AISIN
COSMOS R&D CO.,LTD.. From 1998 to 2002 he
was Research Associate at Research Center for
Advanced Science and Technology, The University of Tokyo, Japan.
From 2002 and right now he is a Lecturer at Department of Biomedical
Engineering, Graduate School of Medicine, The University of Tokyo,
Japan. His current research �eld is biomedical engineering. He is a
member of the Japanese Society for Medical, Biological Engineering,
the Japanese Society for Arti�cial Organs, Research Conference for
Arti�cial Heart and Assisted Circulation of Japan, Japanese Society of
Autologous Blood Transfusion, The Japan Society for Aeronautical
and Space Sciences.
Itsuro SAITO
Itsuro SAITO received his Ph.D. degree in Engineer-
ing from The University of Tokyo, Japan in 2000.
From 2000 to 2007 he was Research Associate at
The University of Tokyo. From 2007 to 2009 he
was a Project Assistant Professor of The University
of Tokyo. From 2009 and right now he is a Project
Researcher of Department of Biomedical Engineering, Graduate
School of Medicine, The University of Tokyo, Japan. And he is Presi-
dent & Division Director of iMed Japan Inc. His current research �eld
is physiological control method for total arti�cial heart and ultrasonic
generator for ultrasonic metal welding machine. He is a member of the
Japanese Society for Medical and Biological Engineering, the Japanese
Society for Arti�cial Organs, the Society of Life Support Engineering,
Research Conference for Arti�cial Heart and Assisted Circulation of
Japan.
Toshiya ONO
Toshiya ONO received his bachelor’s degree from
Senshu University, Japan in 1983. From 1978 to
1998 he was Technical Of�cial at Ministry of Edu-
cation. From 1997 to 2004 he was a Technical
Staff, from 2004 to 2012 he was Technical Special-
ist, from 2012 to 2014 he was a Senior Technical
Specialist, and from 2014 and right now he is a Head of Technical Staff
at Department of Biomedical Engineering, Graduate School of Medi-
cine, The University of Tokyo, Japan. His current research �eld is bio-
medical engineering. He is a member of the Japanese Society for Med-
ical, Biological Engineering, the Japanese Society for Arti�cial Organs,
Japan Society for Laser Surgery and Medicine, The Japanese Society
of Thermology, Research Conference for Arti�cial Heart and Assisted
Circulation of Japan.
Shintaro HARA
Shintaro HARA received his Ph.D. degree from The
University of Tokyo, Japan in 2016. From 2016 to
2017, he was postdoctoral researcher at The Uni-
versity of Tokyo. He moved to The University of
Tokyo Hospital and was postdoctoral researcher
from 2017 to 2019. Now, he is a Specially Ap-
pointed Assistant Professor of Department of Bioengineering, The Uni-
versity of Tokyo, Japan. His current research �led is computational
�ow dynamics of extra-coporeal membrane (ECMO). He is member of
Japanese Society for Medical and Biological Engineering, Japanese
Society for Arti�cial Organs and Japanese Society of Mechanical Engi-
neering.
Yusuke INOUE, et al: Sterilization Methods of Hybrid Materials (91)
Kohei ISHII
He received a Ph.D. degree from the University of
Tokyo in 2013. And he is presently a lecturer at
Department of Electro-Mechanical Systems Engi-
neering, National Institute of Technology, Kagawa
College. His research �eld is biomedical engineer-
ing. He is a member of the Japanese Society for
Medical and Biological Engineering.
Yasuyuki SHIRAISHI
Yasuyuki SHIRAISHI received the D.Eng. degree in
Mechanical Engineering from Waseda University,
Tokyo, Japan in 2002, and Ph.D. in Medical Sci-
ence from Tohoku University, Sendai, Japan, in
2008. He is currently an Associate Professor of the
Department of PreClinical Evaluation, PreClinical
Research Center, Institute of Developing, Aging and Cancer, Tohoku
University. His research interests include mechanical arti�cial internal
organs, modeling and simulation of cardiovascular systems. He is a
member of the Japanese Society for Medical and Biological Engineer-
ing, the Japanese Society for Arti-cial Organs, the Japan Society of
Mechanical Engineers, and IEEE.
Akihiro YAMADA
Akihiro YAMADA received Ph.D. degrees in Bio-
medical Engineering from Tohoku University in
2015. From 2015 to present, he worked at Institute
of Development, Aging and Cancer, Tohoku Uni-
versity as an Assistant Professor. His research in-
terests include arti�cial organs, ventricular assist
device, shape memory alloy, congenital heart disease, and electrical
engineering for medical devices, etc. He is a member of the Japanese
Society for Medical and Biological Engineering, Japanese Society for
Arti�cial Organs, IEEE, and Japan Society for Simulation Technology.
Tomoyuki YAMBE
He received the M.D. and Ph.D. degrees in Medi-
cine from Tohoku University in 1985 and in 1989,
respectively. He was a Research Associate from
1992 at the Division of Medical Engineering and
clinical investigation and Department of Medical
Engineering and Cardiology, Institute of Develop-
ment, Aging and Cancer, Tohoku University. He has been a Professor
in the same department from 2004. From 2015 to present, he became
the director of the PreClinical Research Center, Institute of Develop-
ing, Aging and Cancer, Tohoku University. He engages in arti�cial in-
ternal organs, autonomic nervous system analysis and telemedicine. He
is a member of Japanese College of Cardiology, the Japanese Society
for Medical and Biological Engineering, Japanese Society for Arti�cial
Organs, Japan Society of Circulation Control in Medicine, Japanese
Society of Clinical Physiology and Japanese Society of Neurovegeta-
tive Research.
Yusuke ABE
He received the M.D. degree from Hirosaki Uni-
versity in 1984 and the Ph.D. degree in Medicine
from the University of Tokyo in 1994. He was a
Research Associate from 1987 to 1998 at Institute
of Medical Electronics, Faculty of Medicine, the
University of Tokyo, Japan. He moved to Graduate
School of Medicine, the University of Tokyo and he was a Research
Associate from 1998 to 1999. From 1999 to 2018 he was an Associate
Professor at Graduate School of Medicine, the University of Tokyo.
From 2019 and right now he is a Professor at Department of Medical
and General Sciences, Nihon Institute of Medical Science, Japan. His
research �eld is Medical Engineering and General Surgery. He is a
member of the Japanese Society for Medical and Biological Engineer-
ing, the Japanese Society for Arti�cial Organs and Japan Society for
Laser Surgery and Medicine.
Terumi YURIMOTO
Terumi YURIMOTO received his Ph.D. degree from
The University of Tokyo, Japan in 2017. And he
received DVM degree at Asabu University in 2013.
From 2017 to now he is a researcher at Central In-
stitute of Experimental Animals. His current re-
search �eld is laboratory animal science and veter-
inary medicine. He is a member of the Japanese Society of Veterinary
Science and Society for Neuroscience.
Advanced Biomedical Engineering. Vol. 9, 2020.(92)