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TEKES/TOKYO TECHNOLOGY SCANNING

Biomaterial Research in Japan


February 22, 2001 Tadaaki (Todd) Toyoshima TEKES, Tokyo

Preface This report lists who does what in the various fields of biomaterial research in Japan. Literature surveys and interviews with researchers are recommended in order to more thoroughly research a specific theme or topic. Tekes Tokyo will be pleased to help you in this respect. Additional information: tadaaki.toyoshima@tekes.fi

Content
1 2 3 4 Strength and Weakness of Biomaterials in Japan.............................................................................................................4 Market ..............................................................................................................................................................................4 Research on Biomaterials.................................................................................................................................................9 3-1 Academic Societies.................................................................................................................................................9 3-2 Major Biomaterial Research Groups ............................................................................................................................10 Ceramics.........................................................................................................................................................................11 4-1 Introduction ..........................................................................................................................................................11 4-2 Artificial Bone Market..........................................................................................................................................12 4-3 Calcium Phosphate Paste......................................................................................................................................14 4-4 Advanced bone materials......................................................................................................................................14 4-5 Bone generation---tissue engineering ...................................................................................................................16 4-6 Drug delivery system using ceramics ...................................................................................................................18 4-7 Ceramics for cancer treatment ..............................................................................................................................18 Metal ..............................................................................................................................................................................18 5-1 Artificial Joints Market.........................................................................................................................................18 5-2 Titanium alloy on the market................................................................................................................................20 5-3 Titanium alloy under research ..............................................................................................................................21 5-4 Metals for dental application ................................................................................................................................21 5-5 Surface modifications ...........................................................................................................................................22 5-6 Other Metals .........................................................................................................................................................23 Polymers and Lipids.......................................................................................................................................................23 6-1 MS Coat................................................................................................................................................................24 6-2 MPC......................................................................................................................................................................24 6-3 Photoreactive polymers ........................................................................................................................................24 6-4 PIPAAm................................................................................................................................................................25 6-5 Polymeric Micelles ...............................................................................................................................................26 6-6 Nanoparticles and Microcapsules .........................................................................................................................26 6-7 Liposomes.............................................................................................................................................................27 6-8 Polysilamine .........................................................................................................................................................27 6-9 Perflourocarbonic acid..........................................................................................................................................28 6-10 Shape Memory Gels .............................................................................................................................................28 6-11 Hollow fibres ........................................................................................................................................................28 Drug Delivery and Targeting .........................................................................................................................................29 7-1 Overall situation ...................................................................................................................................................30 7-2 Active targeting ....................................................................................................................................................32 7-3 Passive Targeting..................................................................................................................................................32 7-4 Stimulus Responsive Materials.............................................................................................................................32 7-5 Electromagnetic Responsive Double Targeting....................................................................................................33 7-6 Biomolecular Design for Biotargeting..................................................................................................................33 7-7 Transdermal & Transmucosal Delivery................................................................................................................34 7-8 Company activities ...............................................................................................................................................35 Gene Delivery ................................................................................................................................................................36 8-1 Gene Therapy Protocols .......................................................................................................................................36 8-2 Research on non-virus vectors..............................................................................................................................37 8-3 Polycations and Liposomes ..................................................................................................................................37 8-4 Polymer Micelles ..................................................................................................................................................38 8-5 Fullerene and Cyclodextrin...................................................................................................................................38 Tissue Engineering.........................................................................................................................................................39 9-1 Regenerative Medical Engineering Programme by the Ministry of Education ....................................................39 9-2 The Yoshizato Group at Hiroshima University ....................................................................................................42 9-3 3D Tissue Module Engineering conducted by NAIR ...........................................................................................43 9-4 Millennium Project ...............................................................................................................................................43

9-5 9-6 9-7 10 11 11-1 11-2 11-3 11-4 11-5 12 13 14

New Institutes .......................................................................................................................................................46 Some topics in Tissue Engineering.......................................................................................................................47 Tissue engineering companies ..............................................................................................................................49 Intelligent materials and others..................................................................................................................................50 Regulations and Guidelines .......................................................................................................................................53 Medical Affairs Law.............................................................................................................................................53 Clone Law.............................................................................................................................................................53 Guideline for research on human embryonic stem cells .......................................................................................54 Guidelines on human cell and tissue utilisation....................................................................................................54 Guideline for Gene Therapy and others................................................................................................................55 List of Universities & Institutes.................................................................................................................................57 List of Companies .....................................................................................................................................................62 Major Sources ...........................................................................................................................................................67

Strength and Weakness of Biomaterials in Japan

Japan is a world leader in dialysis membranes and artificial kidneys, with more than 50% of the world market, owing in part to large fibre manufacturers such as Toray and Asahi Chemical. Furthermore, Japanese companies excel at disposable materials. In contrast, most implant materials are imported, with one exception, ceramic bone. Japan has been historically strong in ceramics, not to mention electronic condensers and fine ceramics in the semiconductor industry. Developments in metal for artificial joints are also catching up with the West. There are many biomaterial researchers in Japan. But, Japanese companies are reluctant to take the risk to be a pioneer in implant applications or prostheses. Japan's strength lies in external rather than implanted components and devices. Most implanted materials have been developed in the West. Components used in interface areas, such as dental materials and contact lenses, are both made in Japan and imported. In Japan, researchers, in close collaboration with clinical doctors, have the advantage in discovering clinical needs and conducting clinical trials. In this respect, bio-material institutes in medical universities have many actual products to bring to market. Most material researchers consider the future of biomaterials to be in tissue engineering or, more narrowly, human cell engineering. With regard to drug delivery systems (DDS), Japan has put successful DDS products on the market, such as Leuprin, polymer nanosphere DDS; and SMANCS, a drug-polymer conjugate suspended in lipiodole. Adhesive transdermal delivery systems, such as pressure sensitive adhesives containing isosorbide dinitrate, are also advanced. Many controlled release type DDS have been developed and the future seems to be in organ, cell, and molecule targeting. In tissue engineering, there are at least three government-supported programmes under way. The government has selected tissue engineering as one of the Millennium Projects, starting two research institutes in the Kobe area: one for basic study and another for application.

Market

Japan imports most of its implant materials or devices. Valves for artificial hearts are 100% imported; artificial blood vessels, 95%; bone bonding materials, 80%; artificial joints, more than 80%; and pacemakers, 90%.

Medical equipment and materials market in Japan Production (million Yen) 1,487,903 298,356 194,395 172,116 Total Heart Valves Pacemakers Blood Tubes Stents Joints and Bones 163,445 7 55 109 384 20,187 8,195 1,234 139 97 63 250 2,937 5,442 44 1,781 Import (million Yen) 834,509 82,872 196,049 46,393 267,879 11,551 44,042 5,941 14,905 84,534 20,167 13,969 358 917 2,995 274 19 41,061 1,789 2,979 Export (million Yen) 365,042 127,215 46,408 95,011 28,232

Grand Total Image diagnostics systems Treatment equipment Measuring and monitoring systems Devices and Equipment for Function Support or Replacement

Joints and Bones total Hip joints Knee joints Shoulder joints Elbow joints Metal bone Resin bone Ceramic bone Bone bonding materials Bone cement Other related

16 10 10

Intraocular Lens Monitors & liquid supply for dialysis Dialysis equipment Pumps & accessories for Artificial Lungs Artificial Lungs Blood Cleaning equipment Blood circuits Home Use Medical equipment X ray equipment Dental materials*

3,936 13,842 73,912 1,429 2,261 14,834 11,962 151,691 103,074 90,752 50,378 6,022 2,825 14,970 5,278 5,071 87,647 85,066 61,843

12,268 421 2,499 3,582 2,922 4,906 15,101 43,511 17,026 4,005 907 165 3,653 1,180 547 49,129 22,329 50,755

1,213 22,094 372 678 1,856 1,072 8,048 16,618 2,142 27 232 211 1,600 17 33 19,796 7,126

Total Metal Crowns Dentures Bonding, Filling materials Impression materials Others

(4/5 are resin) (Almost all are resin) (1/2 are resin)

Eye-related equipment Biopsy inspection equipment Healing & surgery equipment

Dental equipment 35,767 7,616 Equipment for 29,095 2,979 facility Steel equipment 8,080 21,966 Hygiene materials 6,575 Source: Ministry of Health and Welfare Medical Industry Statistics 1999. *Issued a report, in Japanese, on The Market for Dental Equipment, Materials and Chemicals published in October 1999, by the Yano Research Institute, is available (cost - Yen 100,000). Medical equipment and materials market in Japan Grand total growth rate Production Import Export 1995 1.4 % 17.5 % 1996 8.9 20.5 11.3 % 1997 4.0 5.8 21.8 1998 -0.4 11.2 21.7 1999 -1.3 0.0 35.8

The shipment and growth rates of implant materials Shipment in 1999 (100 million Yen) Cardiovascular equipment and materials Artificial bones and joints 710 513

Growth rate in Growth rate 1998/1997 Forecast in (%) 1999/1998 (%) 7.4 15 -17.8 10

Blood cleaning and separation 372 43 No data equipment Dialysis equipment 748 10 10 Total 2,524 7.7 Source: Market for Medical Equipment and Materials, Yano Research Institute.

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3-1

Research on Biomaterials
Academic Societies

According to Dr. Tsuruta, Emeritus Professor, Tokyo University, Japan has a long history in biomaterials. Research on polymers for medical use started in 1958. The Higuchi model for a drug delivery system was reported in 1961. The Toray PMMA hollow fibres for artificial dialysis were put on the market in 1973 and the Kuraray EVA in 1978. The Ministry of Education funded biomedical polymer research programmes from 1972 through 1989 under various programme names. Prof. Tsuruta emphasised, in his keynote speech at the 2000 Biomaterial Conference, that all the development work should have clinical applications as the target. He encouraged researchers to protect themselves against unreasonable lawsuits. Companies should develop their risk management strategies to deal with the biomaterial products and business. He predicted that future biomaterial research would shift to tissue engineering. The following academic societies are focused or have groups focused on biomaterials: Japanese Society for Biomaterials was founded in 1978. It organises an annual conference and publishes a bimonthly journal in Japanese. In addition to individual members, there are about 50 company members. The next annual conference will be held in Kyoto on October 22 and 23, 2001. The Ceramics Society of Japan formed a Bio-related Materials Working Group in October 1998. The group has about 120 members and holds a conference and a Beginners Seminar once a year in November or December. The society publishes two Japanese monthly publications called Journal of Ceramic Society of Japan (for papers) and Ceramics Japan (for reviews). Japan Metal Society has a temporary (March 1998February 2001) group called High Performance Biomaterial Study Committee. The committees aim is to review the appropriate data for the development of biomaterials, and to decide the current status of evaluation methods and standards. There are no industrial standards for biomaterials in Japan. The Society of Polymer Science, Japan has a Research Group on Biomedical Polymers, which was founded in 1972. The research group, 150 members, holds conferences on special themes, and reviewtype seminars. The society publishes an English-language monthly called Polymer Journal. Japan Society of Medical Electronics and Biological Engineering was founded in 1962, and now has 4,000 members. It publishes an English-language quarterly called Frontiers of Medical and Biological Engineering as well as a monthly journal in Japanese. Japan Society of Drug Delivery Systems was founded in 1988 based on the DDS Study Group that began in 1984. It now organises annual conferencesthe next is on July 12, 2001 in Osaka with around 500 researchers expected to attend. The Society publishes a bi-monthly journal in Japanese called Drug Delivery System. The number of members is over 1,000. The Japanese Society of Drug
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Delivery Systems jointly publishes the Journal of Controlled Release (JCR) with the international Controlled Release Society. Japan Society for Tissue Engineering began in 1984 as the Organ Formation Working Group and adopted its current name in 1998. It organises an annual meeting but does not publish a journal. Rather it encourages Japanese members to send papers to Tissue Engineering in the USA.

3-2 Major Biomaterial Research Groups

Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University The oldest biomaterials research institute in Japan has three divisions: the Functional Molecule division; the Materials divisionmetal, inorganic and organic materials; and the Bio Systems division. The institute started dental material research in 1938 and diversified in 1966 to cover other medical fields. It has more than 50 permanent researchers. Tissue Engineering Research Institute at the Institute of Frontier Medical Sciences, Kyoto University This institute is one of the largest Japanese research groups in tissue engineering. The institute was founded in 1978 as the Medical Polymer Research Group and developed into the Research Center for Medical Polymers in 1980, changing its name to Research Center for Biomedical Engineering in 1990, and adopting the current name in 1998. With approximately 80 researchers, it covers all fields related to tissue engineering: biological function, tissue engineering, regeneration control, medical system engineering, and clinical applications. Professor Ikada was former head research director of the institute. Institute of Biomedical Engineering, Tokyo Womens Medical University Prof. Okano, head of the institute, is an expert in temperature responsive polymers and his group is working on applications of the polymers for DDS and gene vectors. Cell Sheet Engineering technology is one of the approaches, promoted by the Institute, to produce complicated organs by accumulating cell sheets one by one using temperature responsive surfaces, similar to the building of large-scale integrated circuits. They recently started collaboration with the Science & Engineering Department of Waseda University. Professor Yoshizato's group, at Hiroshima University. This group is unique in that they are targeting technology development based on tissue engineering to promote local industry, getting support from both the central and local government and from close collaboration with companies. 3D Tissue Module Engineering group in NAIR The 3D Tissue Module Engineering group in the National Institute for Advanced Interdisciplinary Research, under the Ministry of Economy and Industry, was formed in 1998 with more than 40

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researchers, including 11 foreign researchers. This group will likely be a core member in the new laboratory in Osaka called METI Tissue Engineering Center, to be founded in 2002. The International Symposium on Advanced Biomaterials and Tissue Engineering held in 1999 was organised by NAIR to promote industrial applications. National Research Institute for Metal and National Institute for Research in Inorganic Materials, both under the Ministry of Education and Science, are merging in April 2001. Each institute has a biomaterial research group. After the merger, they will have approximately 15-20 researchers.

4 Ceramics
4-1 Introduction1

According to Prof. Nakamura, Kyoto University, there have been three generations of artificial bone: the first generation is sintered hydroxyapatite (HAP) and A-W glass ceramics, the second generation is porous material and Ca-P cement, and the third is the material together with a complex of bone growth factors and bone cells. Three materials for bone replacement have been invented since the 1970s: Bioglass, Sintered hydroxyapatite, and Crystallised glass A-W. Japanese researchers have made a large contribution to the latter two. Dr. H. Aoki, former professor at the Tokyo Medical and Dental University, succeeded in synthesising HAP and manufacturing sintered HAP in 1972, simultaneously but independently from investigators in the USA. He discovered the high affinity of HAP to real bone in 1975. His research theme for developing the manufacturing technology of synthesised HAP for artificial bone and teeth was funded by the Ministry of Education for five years beginning in 1977. Apaceram now sold by Asahi Pentax Co., the top market shareholder of bioceramics in Japan, is based on his research. Prof. Kokubo, from Material Chemistry at the Kyoto University, announced in 1982, and began using in 1991, Crystallised Glass A-W (SiO2-CaO-MgO-P2O5), which is made by the deposit of fine crystals of HAP and wollastonite: CaOSiO2. Nippon Electric Glass Co. has sold A-W as Cerabone since 1991, with annual sales of approximately USD 7 million. It has been used on 45,000 patients. They decided, however, in October 2000 to discontinue the business at the end of 2000, because of slow growth and the poor prospect for future profits.

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Comparison of physical properties Bioglass HAP

A-W

2.68 3.16 3.07 Density gcm-3 Hardness HV 458 600 680 Compressed strength 500-1000 1080 MPa Bending strength 42 115-200 215 MPa Young modulus 35 80-110 118 GPa Breaking tenacity 1.0 2.0 1/2 MPam Bone invasion speed Highest Low high into ceramics space Containing SiO4 2Yes No Yes (may be toxic) Source: L.L.Hench (1998) J.Am. Ceram. Soc., 81, 1705-28

Dense natural bone 1.6-2.1 100-230 50-150 7-30 2-6

Spongy natural bone

2-12

0.05-0.5

Increasing the tenacity and decreasing the Young Modulus of artificial bone to the level of natural bone is a major challenge. One method of increasing tenacity is to coat titanium alloy with HAP. Another approach is to use collagen-apatite composites. Hybrid materials of organic and inorganic components, bonded at the molecular level to produce material similar to spongy bone, are being investigated. Ceramics that can be moulded in surgery rooms are also required. Mitsubishi Material Co. has developed biodegradable calcium phosphate bioactive paste (major component: a-Tri-Calcium Phosphate) independently from Norian. Mitsubishi started selling the product called BIOPEX in June 2000 through the Welfide Corporation and Taisho Pharmaceutical Co. Ceramics are also used in cancer treatment and drug delivery systems. The major direction of current research is to develop materials to promote bone generation by means of tissue engineering.

4-2

Artificial Bone Market

Market2 In 1998, the market size was USD 35 million, with a 4-5% growth rate expected until the market for osteoporosis treatment takes off. The markets for artificial bones in foreign countries are small because human and animal bones can be easily used. In Japan, such reuse is disliked for social and religious reasons.

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There are two types of artificial bones: ceramics and resin. Sales of artificial bone ceramics totalled USD 30 million and are growing. Artificial bone resin sales were USD 2 million in 1997 and have levelled off. A liquid type resin, which solidifies within the human body, has attracted attention due to potential use in osteoporosis treatment, such as BIOPEX by the Mitsubishi Material Co. Manufacturers Manufacturers sales figures Makers Sales representative Asahi Optical Nippon Special Ceramics Sumitomo Osaka Cement Nippon Electric Glass Mitsubishi Materials Kyocera Asahi Optical Chugai Pharmaceutical Sumitomo Pharmaceutical Discontinued bioglass business Dec. 2000 Welfide Corporation Welfide Sales in 1999 (million Yen) 1,410 600 560 415 331 240 Brand names and product types

APACERAM: HAP CERATITE: Complex of HAP and tri-calcium phosphate (TCP) BONECERAM: HAP Both porous and dense types are available. BIOGLASS A-W BONEFILL and BONETIGHT: HAP BIOPEX: Paste of a-TCP and 4CP BIOCERAM: TCP (Kyocera is an artificial joints manufacturer)

Olympus

Osferion: b-TCP Both porous particles and blocks are available. Source: The Market for Dental Equipment, Materials and Chemicals, October 1999, Yano Research. Direction of technology Hard bone: Increase of bone-forming ability with marrow transplanting and bioactive substances Mitsubishi calcium phosphate paste will be followed by competitors Composites

Olympus

100

Soft bone or cartilage: Replacement of cartilage with artificial bone has not yet succeeded clinically; therefore, joints need to be completely replaced.

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The abrasion of joint sliding surfaces is still a challenge to be solved. Alumina-alumina sliding surfaces and the composites of zirconia and alumina are under study.

4-3

Calcium Phosphate Paste

BIOPEX Mitsubishi Materials Co. launched a calcium phosphate paste in June 2000. The National Health Insurance system listed the material in 1999. At the Japan Biomaterial Conference held in November 2000, Dr Shigeo Miwa from the Aichi Medical University, reported the clinical trial results of Mitsubishi Material BIOPEX. BIOPEX components are Powder: Ca3(PO4)2: 75 % of the weight Ca4(PO4)2O: 18 CaHPO42H2O: 5 Ca10H(PO4)6(OH)2 2 Liquid: 5 % chondroitin sulphate 12 % sodium succinate 83 % distilled water BIOPEX completely changes to apatite in the body in three weeks. Its degree of crystallisation is low and is similar to human bone. Unlike PMMA cement, it does not generate polymerisation heat nor leach non-polymerised monomers to the cardiovascular system. The material is gradually absorbed by the body: 50 % in 3 weeks. Its shortcoming is the weak compressive strength: 6090 M Pa. Therefore, it is not suitable for artificial bone fixing.3 At the Biomaterial Symposium 2000, S. Yamamoto, of the Department of Oral Functional Science, at the Hokkaido University, in co-operation with Asahi Optical Co., presented an application of Calcium Phosphate Cement containing CM-chitin and succinic acid for bone augmentation. In rabbits, he observed new bone formation in a crack in the skull.

4-4

Advanced bone materials4

Many researchers are still trying to develop next generation apatite using composites and surface modification. Nitta Gelatin Inc., together with Dr. Junzo Tanaka and Dr. Masanori Kikuchi, of the National Institute for Research in Inorganic Materials, has developed artificial bone that has a flexural strength of about

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50 MPa, nearly comparable to that of the human bone (40200 MPa). It is a composite consisting of protein-I type collagen and hydroxyapatite. A co-precipitate of a calcium hydroxide solution and an aqueous phosphate solution containing collagen is formed via a cold isostatic pressure process. When the new composite was transplanted onto a rat cranial bone, the composite started to be replaced by natural bone in two weeks. Nitta Gelatin Inc. is also manufacturing collagen for artificial skin in medical treatment. National Industrial Research Institute of Nagoya has found that chitin and chitosan derivatives containing phosphoric acid are effective in forming bone. The surface of chitin or chitosan is modified by phosphoesterification and the derivative put into phosphoric acid and calcium-containing liquid. After 10 days, the same volume of calcium phosphate as the derivative was obtained. Electrically polarised hydroxyapatite is being studied by K. Yamashita, T. Kobayashi, and colleagues, at the Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University. In an oral presentation at the Biomaterial Symposium 2000, they reported that they found that apatite can be electrically polarised and that the stored polarised energy can be measured. They also implanted the polarised apatite in a dog, to evaluate bone formation, and found that new bone started to form on the negative surface in less than a week. This phenomenon can find application in orthopaedic and dental fields. The polarisation can stay in vivo for at least six months. Surface modification of HAP with carboxyl group was reported by Dr. T. Matsumoto, and colleagues, of the Dental School, at Osaka University, together with Dr. M. Okazaki, of Hiroshima University, and Dr. Taira, of the Iwate Medical University. Their idea is based on the fact that bone growth factors such as b-FGF and BMP have affinity to negatively-charged surfaces. They succeeded in producing HAP modified with a carboxyl group and evaluated the material using rats. They found that the weight of the adhered protein and the cell adhesion ability depend on the volume of the carboxyl group on the surface. Zinc-releasing calcium phosphate on collagen is being studied for the promotion of bone generation by Dr. Atsuo Ito, and colleagues, at the National Institute for Advanced Interdisciplinary Research (NAIR), together with T. Hikita, and colleagues, at the Waseda University. They made a NaCl, CaCl22H2O, KCl, ZnCl2 solution, introduced CO2, NaHCO3, Na2HPO412H2O, and put collagen sheet in it. Low crystal apatite containing Zinc was obtained. Dr. H. Kawamura, Kennan Hospital, together with Dr. Ito, at NIAIR, and S. Miyakawa, at the Tsukuba University, conducted a long-term implantation test of Zinc-releasing calcium phosphate ceramics in rabbits. At an oral presentation at the Biomaterial Symposium 2000, they reported that 0.316%ZnTCP/HAP significantly increased the bone-binding ratio but also increased bone absorption. They are currently optimising the zinc content. At a poster presentation at the Biomaterial Symposium 2000, results with magnesium-containing CO3 apatite-collagen composite were reported by Dr. Yasuhiko Yamasaki, of the Dental School of
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Hiroshima University, together with Y. Hamada, and colleagues, of the Dental School, at Osaka University. They tried to synthesise crystals of magnesium-containing CO3 apatite starting from a Ca(CH3COO)2H2O and Mg(CH3COO)24H2O solution. The powder crystal obtained is mixed with 0.5w% collagen to produce cylindrical pellets. The pellets are put in serum containing mouse osteoblasts. Adhesion assays revealed that the Mg composite showed higher adhesion than non-Mg CO3 apatite. Dr. Tanaka, at the Research in Inorganic Materials, and Prof. Miyairi, at the Tokyo Medical and Dental University, are collaborating on creating a composite of polylactic acid and TCP for bone deficiency 20mm and over. They produced the composite with 20-50% polylactic acid and 80-50% TCP to make 0.3mm membranes. With conventional materials, only a 5mm bone deficiency can be rebuilt. Carbonate apatite is being studied by Dr. Masahiro Hasegawa, and colleagues, in Orthopedic Surgery, at the Asahi Memorial Hospital, because human bone contains carbonate ions. They reported at a poster presentation at the Biomaterial Symposium 2000, that they implanted porous apatite with 6 wt% carbonated ions in rabbits. Increased bone forming and the absorption of the material were observed four weeks later. Prof. Masanori Oka, Kyoto University, is developing artificial joint cartilage with titanium fibre mesh injected with PVA hydrogel under high pressure. The properties of lubrication, shock absorption, and the bio-affinity and adhesion to the bone bed are acceptable, except for the antiabrasion. Therefore, the material is suitable for partial hemiarthroplasty for fractures of head of the thigh bone, osteochondritis dissecans, and chondromalacia. This is joint research with China and clinical applications are expected to be appear in a couple of years.

4-5

Bone generation---tissue engineering5

Professor Kunio Takaoka, in orthopaedic surgery, at the Shinshu University, received competitive funding, of 21 million Yen a year, from Ministry of Education for his research on basic theory and operation technique of Bone Morphogenetic Protein (BMP). He used a copolymer of polylactic acid and polyethyleneglycol for the delivery of BMP, as well as for the scaffold. He succeeded in the regeneration of the upper arm bone of rabbits in eight weeks. But the mechanical strength and absorption of the scaffold was unsatisfactory. Prof. K. Shinomiya, of the Tokyo Medical and Dental University, made a block of HAP and collagen composite attached with rhBMP and implanted it in a beagle. Bone generation was observed.

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Prof. Takafumi Yoshikawa, of the Department of Pathology at the Nara Medical University, and colleagues, in co-operation with T. Uemura, and colleagues, at NAIR, have cultured rat marrow cells to make bone. Immediate bone generation right after the transplantation of the cultured bone was observed and osteoblast activity was maintained for a long period. The technology was confirmed with osteoblasts in 29 patients with osteoarthritis and rheumatoid arthritis and injuries. At a presentation at the Biomaterial Symposium 2000, Dr. Ohgushi, in orthopedic surgery, at the Nara Medical University, reported that he is going to culture marrow cells of patients who undergo bone shaving surgery, taking marrow liquid and culturing the cells for three weeks to transplant them to the patients. Professor Kinoshita, of the Kanagawa Dental University, has machined PLLA (poly-L-lactic acid) to meet the shape of the jaw and put sponge marrow taken from waste bone, regenerating jaw bone in six months to one year. He has performed the procedure, clinically, 50 times. The success rate is 80%. The National Institute for Research in Inorganic Materials and Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University have succeeded in developing a complex of tricalcium phosphate (TCP) 70% and polylactic acid 30% to make 0.3mm thin membranes to regenerate lost bone as much as 20mm wide. TCP accelerates bone generation and absorption rate of the materials can be controlled by changing the ratio of the two materials. Moreover, the membranes thermoplasticity makes it easy to form various shapes during surgery. Guoping Chen, and colleagues, of the 3D Tissue Engineering Group, NAIR and T. Sato, at the Graduate School of Medicine, Tsukuba University, have generated cartilage using biodegradable hybrid sponge. At the Biomaterial Symposium 2000, they reported that they introduced collagen sponge into the pores of a copolymer sponge of lactic acid and glycolic acid. They successfully cultured, both in vitro and in mouse, the cartilage cells of the knee joints of cows. Slow release of BMP with gelatin hydrogel was successfully achieved by Dr. Masaya Yamamoto, at the Institute of Frontier Sciences, Kyoto University. Release control was accomplished by changing the water content of the biodegradable hydrogel. The hydrogel was a chemically bridged caustic gelatin with glutalaldehide. BMP easily dissipates in the human body even if it is injected. Dr. N. Saito, and colleagues, at the Shinshu University, are developing an injectable DDS for BMP. They synthesised several blocks of PLA-PEG copolymers, differing in molecular weight, and injected rhBMP-2 and the copolymers at the temperature of 60 degree C into the front of a mouse femur and evaluated the bone generation. It was reported in the Nikkei news that Takeda Chemical Industries Ltd. has developed a new compound contained in slow release polymer capsules, 30 mm in diameter, that promotes the fusion of fractured bone by activating a natural protein in the body for a month. Takeda is now moving to a clinical trial after confirming its effect using mice. The compound, with the development name of
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TAK778, was originally licensed from a Hungarian company and improved by Takeda. It speeds up the healing process of fractured bone by a third. 4-6 Drug delivery system using ceramics

Professor Makoto Otsuka, from the Kobe Pharmaceutical University, is one the most active researchers of drug delivery systems using bone cement. He has studied apatite cement, bioactive glass and complex of bioactive glass, each containing indomethacin. He found that apatite cement suppresses drug release after a week, while bioactive glass allows drug release, even after three weeks. To overcome the brittleness of the bioactive glass, he also tried bisphenol-a-glycidyl methacrylate and triethylene-glycoledimethacrylate, resulting in a decrease in drug release after 100 hours. During tests, he found drug release speed decreases when the calcium concentration in the liquid or the blood is high. He is trying to apply his finding to a drug release system for osteoporosis patients using estradiol. Calcium concentration in blood in osteoporosis rats is lower than normal rats, thus estradiol release in bioactive glass is increased.6

4-7

Ceramics for cancer treatment

It is preferable to irradiate only locally to cancerous tissue. Y2O3-Al2O3-SiO2 glass was reported to be suitable for local irradiation in 1987 by G.J. Ehrhardt and D.E.Day. It was achieved, with neutron radiation of 20-30 mm sphere glass, resulting in conversion of 89Y to 90Y, which radiates b rays. Dr. Kawashita, Kyoto University, has shown that only Y2O3 microspheres with a diameter of 20-30 mm can be easily obtained by high frequency induction heat plasma. Y2O3 shows excellent chemical durability, better than Y2O3-Al2O3-SiO2 glass. Animal trials using the spheres are about to begin. Normal cells can survive up to 48C but cancer cells die at around 43 C. Therefore, it could be effective to heat up cancer tissue in deep parts of bodies, but methods of local heating are not available. Dr. Kawashita has developed fine crystals of magnetite, iron, or iron zinc ferrite deposited in CaO-SiO2 glass matrix and Li ferrite deposited in SiO2-P2O5 glass matrix. The heat generation of these ferromagnetic spheres has been tested using rabbits. Other ferromagnetic spheres with higher heat generating efficiency are under study.

5 Metal7
5-1 Artificial Joints Market

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Market Units of artificial joints Hip joints Knee Joints Shoulders Knees Others Total Source: Yano Group 1998 (pieces) 55,742 27,527 835 362 508 84,974 Growth rate (%) 2.2 6.9 0.6 16 1.8 3.7 1999 (pieces) 58,932 30,287 865 470 705 91,259 Growth rate (%) 5.7 10.0 3.6 29.8 38.8 7.4

Sales of artificial joints 1998 (million Yen) Hip joints 40,071 Knee Joints 18,028 Shoulders 418 Knees 160 Others 184 Total 84,974 Source: Yano Group

Growth rate (%) 2.6 -0.1 12.9 25 -1.4 1.9

1999 (million Yen) 42,488 19,951 418 217 260 63,335

Growth rate (%) 6.0 10.7 0.0 36.6 41.0 7.6

Titanium alloys are increasingly used instead of SUS316 Co-Cr alloys (approximately 80 % of the market) and have been receiving attention from researchers.

Major manufacturers Companies Stryker Japan BMS Zimmer Debu Japan Kyocera Kobelco Mizuho Medical Nemoto Trading Nakshima Propella The market share of domestic companies is about 14% including Mizuho Medical, Nemoto Trading, and Nakashima Propeller, in addition to Kyocera and Kobelco.
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Market share (%) 26 25 12 11 2

Notes

Kyocera has made 8,700 zirconia-based joints since 1995. The top titanium maker in Japan K-MAX hip joints

Nakashima Propeller Co. Ltd. is expanding their artificial joints business to USD 20 million in five years, three times the current amount. They are competitive in titanium alloys rather than ceramics. Research Direction Metals for orthopedic applications (1) Vanadium-free Titanium alloys to replace the most popular Ti alloy: Ti-6Al-4V, that contains cytotoxic Vanadium. (2) Alloys with low modulusclose to that of real bone. (3) Nickel-free shape memory and ultra-elastic alloysNickel may have adverse physiological effects. Soft bone and cartilage: Replacement of only a part of cartilage with artificial bone has not been clinically successful, therefore, joints are completely replaced. Furthermore, the abrasion of joint sliding surfaces is an issue. (1) Cross-linking of ultra high molecular weight polyethylene (2) Alumina-alumina sliding surface and composites of zirconia and alumina Dental applications: (1) Alloys with low melting point are desired. (2) Alloys easy to machine are desired

5-2

Titanium alloy on the market

A Vanadium-free alloy, Ti-6Al-7Nb, has been developed and put into practical use in Europe. In Japan, a few Vanadium-free alloys are on the market or under research. The Medical Material Division at Kobelco Co. developed Ti-15Mo-5Zr-3Al for cement-type hip joints and Ti-6Al-2Nb-1Ta for cement-less hip joints. The objectives of Kobelcos development were to avoid Vanadium and to achieve high fatigue strength. Ti-15Mo-5Zr-3Al was put into clinical trials in 1991 and has been used since 1995. Because its fatigue strength is 20% higher than that of Ti-6Al-4V, the stem neck out-diameter can be made as small as 9mm.Therefore, the moving area of hip joints is larger, which results in longer life for the joints. Ti-6Al-2Nb-1Ta was put into a clinical trial on 1992 and onto the market in 1996. This alloy is less susceptible to strength deterioration after heat treatment.

20

Kobelco so far has used Ti-15Mo-5Zr-3Al with 3,500 patients and Ti-6Al-2Nb-1Ta with 2,000 patients.

5-3

Titanium alloy under research

Yoshimitsu Okazaki, at the Mechanical Engineering Laboratory under MITI, has developed, in cooperation with Kobelco, Ti-15Zr-4Nb-4Ta-0.2Pd. Zr was added for higher strength; Nb and Ta were added for improved anti-corrosion; Nb and Ta content was kept low for a lower melting point. The composition of the alloy was optimised for a good balance of bio-compatibility, improved anticorrosion, and a low melting point. The alloy is currently being tested on rats. Dr. D. Kuroda and Professor Mitsuo Niinomi, at the Toyohashi University of Technology; Dr. Fukui, and colleagues, of the Dental School, Aichi Gakuin University; T. Kasuga, of the Nagoya Institute of Technology; and Daido Special Steel Co. are developing Ti-29Nb-13Ta-4.6Zr alloy. The goal is to design a new b-type titanium alloy that has a similar Young modulus as bone, but without using cytotoxic materials. Biocompatibility of the alloy, evaluated via cell survival ratio, is the same as titanium and better than Ti-6Al-4V. They have also coated the alloy with calcium phosphate glass: 60CaO30P2O53TiO2 to improve biocompatibility, and found that the adhesion of the glass to the alloy is extremely strong. Professor Hideki Hosoda, of Tsukuba University, an expert in shape memory alloys, is developing Tibased, Ni-free, shape memory. He has so far achieved good mechanical properties and high modulus by adding Nb/Mo for b-phase stability and Al/Sn/Ga for a-phase stability. It seems he can develop shape memory alloys with the specified elements, for medical use. He is looking for commercial collaboration with researchers in the medical field, to study corrosion and bio-compatibility.

5-4

Metals for dental application

Dr Yoneyama, and colleagues, of the Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, are studying Ti-6Al-7Nb alloy produced by Daido Special Steel Co., for dental casting application. They concluded that the alloy is promising because it is much easier to machine than titanium and gives a clear smooth surface. Ti-Ni alloy wire produced by the Furukawa Electric Co. showed ultra elasticity, which is a good for a material from which to develop a new functional device. The titanium had an apatite coating

21

A Ti alloy that is easy to machine is under study by Dr. Takada, and colleagues, at the Tohoku University Dental School. They found that the addition of Ag or Cu improves two or three fold the ability to machine the alloys. Shu, and colleagues, at the Nagoya Institute of Industrial Technology under MITI, have shown that the addition of Si, Ca and B in casting machines for dentists improves mechanical properties, for example, by increasing tensile strength two fold. Prof. Michio Ohta, at Kyushu University, has found from theoretical calculations that the optimum composition for age-hardening alloys at mouth temperature is Au 50%, Cu 44% and Ga 6%. Actually, an alloy with the above composition, plus Ir for finer crystal grains, has been commercialised under the brand name Sofard. Surface treatment of titanium using super critical water is under study by Dr. Ban, and colleagues, at the Aichi Gakuin University Dental School, with the aim of increasing adhesion. The sheer bonding strength of veneer resins to titanium was increased approximately 25% after super critical water treatment.

5-5

Surface modifications

The National Industrial Research Institute of Nagoya has succeeded in coating titanium alloy with hydroxyapatite. They covered the alloy with titanium metal and then added hydroxyapatite particles with a 30 mm diameter and heated up to 800-900C under pressure. They are going to proceed with animal tests. Professor Kokubo, of Kyoto University, in co-operation with Kobelco Co., is developing the formation of amorphous of graded sodium titanate layer on the surface of titanium in order to facilitate apatite formation. It is a three-year study beginning in 1999 with 200 million Yen funding from the Japan Science and Technology Foundation under the Science and Technology Agency. The National Research Institute for Metals has a biometal research group called Center of Cell Engineering headed by Dr. Hanawa. They focus on basic research on the interaction of cell and metal: -Changes in metal surfaces in vivo, -Cell adhesion to an evaporated film of pure metal, -Corrosion behaviour of amorphous alloy in a pseudo-bio environment, -Electrochemical analysis of pure Titanium and SUS316L with culturing cells.

22

Dr. Masao Yoshinari, and colleagues, of the Department of Dental Materials Science, Oral Health Science Center, Tokyo Dental College, is studying surface modifications of titanium to suppress the attachment of oral bacteria. They found that fluorine implantation on titanium polished surfaces decreases the proliferation of bacteria. Dr. Kawabe, and colleagues, of the Okayama University Biomaterial Lab., Faculty of Engineering, are studying the control of apatite-forming ability on the titanium surface using an electrochemical technique. Dr. Shunsuke Fujibayashi, at the Graduate School of Medicine, Kyoto University, is studying the effect of sodium removal treatment on the bone bonding ability of bioactive titanium prepared with alkali and heat treatment.

5-6

Other Metals

(1) Prof. Nakajima, at Osaka University, started the project called Development of advanced medical equipment using porous metal supported by the government Millennium Project. It is a three year project, from 20002002, at a total cost of USD 1.5 million. The project involves Yokohama Medical University, Asahi Intec Co. as well as other science and technical universities. The Lotus-type porous metal so far developed by the professor has uniform straight pores of 1-10 mm and a pore ratio of 80%. They are aiming at making the porous metal with stainless steel, titanium, and shape memory alloy to make guide wires, stents, artificial bone, and dental implants. According to Business & Technology News, the market for guide wire and stents is USD 360 million with an annual growth rate of 23%, and the number of cardiovascular patients is 3.3 million. The market for artificial bone, joints, and dental implants is USD 200 million with 14% annual growth with 3.2 million osteoporosis and arthritis patients. (2) Prof. Shinichi Nittas group, from Tohoku University, and Tokin Co. have jointly developed artificial cardiac muscle with shape memory alloy to be attached on the outside of hearts. The muscle is a Peltier effect element sandwiched by shape memory alloy plates. The size is 1cm*5cm with 0.5 cm of the thickness due to the Ni-Ti alloy. The power is supplied from outside the body by electromagnetic induction. The group tested the effect using a goat.

6 Polymers and Lipids8


There were 40 presentations in the July 2000 Medical Polymer Symposiums. Many researchers are working in this field for various applications such as drug delivery systems, gene delivery systems, and scaffolds. Targeting and gene delivery is one of the most exciting challenges.

23

6-1

MS Coat

MS Coat is the brand name of a desensitiser sold by the SUN MEDICAL CO. to relieve patients with hypersensitive teeth. It was developed by the SUN MEDICAL, a subsidiary of Mitsui Chemical Co., and has been on the US market (Parkell Co. is the sales representative) since 1996 as Pain-free desensitiser. It began sales in Japan in October 1999, through the Morita Co. Its main component is a copolymer of methyl methacrylate and styrenesulfonic acid originally developed by Prof. Nakabayashi, Institute of Biomaterials and Bioengineering Tokyo Medical and Dental University. A clinical trial done by the Tohoku University and the Tokyo Medical and Dental University showed that 87% of patients with hypersensitive teeth achieved immediate relief after just one coating and 82% remained pain-free even after one month. It consists of liquid-A: a MS Coat and liquid-B: calcium compound.

6-2

MPC9

A 2-metacryloyloxyethyl phosphorylcholine (MPC) polymer contains a phospholipid polar group (phosphorylcholine group), the structural component of biomembrane; and a metacryloyl group, which has polymerisation capability with many monomers. MPC polymer reacts extremely weakly with biocomponents such as proteins and blood cells and shows a quite excellent thrombus resistance. Many researchers, both domestic and abroad, are working on using MPC with blood cleaners, implant sensors, blood pumps, blood circuits, artificial blood tubes, and drug carrier nanospheres. MPC polymer was originally synthesised by Professor Nobuo Nakabayashi, at the Tokyo Medical and Dental University, in 1978. Professor Kazuhiko Ishihara of the Department of Materials Engineering, Faculty of Engineering, at the University of Tokyo has established volume production technology of MPC funded by the government. NOF Corporation (Nippon Yushi Co.) has completed a commercial plant for MPC, from monomer through polymer, and anticipates various applications such as contact lens cleaning liquid, catheters, and intraocular lens. The combination of MPC with other materials such as polysulfone membranes and segmented polyurethanes is one of the research themes of the above groups. 6-3 Photoreactive polymers

Dr. Yasuhide Nakayama, an expert in photoreactive polymers, at the National Cardiovascular Disease Center Research Institute, has synthesised, in co-operation with Prof. Takehisa Matsuda, of Kyushu University, photoreactive gelatin by introducing side chains to the benzophenon group, which generates

24

radicals by light radiation.10 The gelatin, dissolved in physiological saline solution together with PEG macromer and hardened using ultraviolet, is useful as a wound-covering reagent or tissue adhesive. Hardening by visible light is also possible in the mixed water solution of gelatin with the styrene group, and water-soluble, camphaquinon. Iniferter polymerisation (iniferters are molecules that have both functions of initiator-transfer and agent-terminator), developed by Dr. Otsu,11 can facilitate the precise structural design of polymer surfaces. This technology can be used for transdermal drug delivery systems and also to bring about blood affinity properties to the inner surface of a device even after moulding and machining. Animal experiments giving blood affinity to artificial hearts are under way in Tokyo University.

6-4

PIPAAm12

Poly N-isopropyle acrylamide (PIPAAm) is well known as a thermally responsive polymer, which drastically changes water affinity with changes in temperature. PIPAAm is highly soluble in water below its lowest critical solution temperature (LSCT), 32 C in water, but becomes water-insoluble at a higher temperature. Dr. Mituo Okano and his group at the Institute of Biomedical Engineering in Tokyo Womens Medical School are developing various applications using the polymer. Some other applications: Cell attachment and detachment to culture plates--Cell Sheet Engineering, explained later in the chapter on Tissue Engineering Chromatography of water solutions without using organic solvents--They are collaborating with Amersham Pharmacia Biotech Co. to develop a protein separation and recovery system by attaching PIPAAm and ligands to a carrier. Copolymer gel of N-isopropylacrylamide (IPAAm) and 2-carboxylisopropylacrylamide (CIPAAm) for DDS Synthesis of block copolymers of PIPAAm chain and hydrophobic polymer chain from polymeric micelles, with a drug in the inner core. With an increase in temperature, the outer shell becomes hydrophobic, and so the drug moves to the outer shellreleasing the drug. Together with the passive targeting technology they have already developed, they are creating an anti-cancer DDS. IPAAm, 2-dimethylaminoethylmetacrylate (DMAEMA) and Butylmethacrylate (BMA) for a gene vector that can control the timing of gene expression Poly N-IPAAm gel with a porous structure has been synthesised through radiation-induced polymerisation by Dr. Kishi, and colleagues at the National Institute of Materials and Chemical Research. Composite capsules of silica and polyN-IPAAm are under development by Prof. Akashi and colleagues, at the Kagoshima University, for the controlled release of drugs. Prof. Akashi's group is also working with various heat and pH responsive materials synthesised from poly(N-vinylamide).

25

6-5

Polymeric Micelles

Professor Kataoka, at Tokyo University, studies polymeric micelles and intelligent biomaterials. Block copolymers that have both hydrophilic and hydrophobic polymer chains in a molecule form micelles, in water, with hydrophilic chains outside and hydrophobic chains inside. The micelles are excellent drug carriers, because they can hold drugs firmly in their inner cores and control the drug release by changing the molecular structure of the inner cores. Since polyethyleneglycol (PEG) is used for the hydrophilic chains, the immune system does not recognise the micelles as foreign materials. The micelles also have an enhanced permeability and retention effect, useful for tumour targeting. Micelles containing doxorubicin have been shown to be highly effective against tumours.13 Dr. Kataoka is also developing a glucose-responsive insulin release system using the equilibrium of phenylboronic acid in aqueous solution. He has made a gel by introducing acrylamidephenylboronic acid (AAPBA) to N-isopropylacrylamide (NIPAAm). The gel can release or retain insulin (on and off) in accordance with the glucose concentration.14 He and Prof. T. Aida, from Tokyo University, are applying micelles to DDS for cancer, with a porphyrin dye covered by dendrimer. Porphyrin purges active oxygen in response to light radiation and attacks cancerous cells. The dendrimer is positively-charged, therefore, it tends to attach to the surface of cancerous cells. Thus, the micelles have 28 times more capability of killing cancerous cells than porphyrin alone. Kataoka is planning to cover the micelles with lipid particles, with the diameter of 50 nm, because he already showed that lipid particles gather efficiently around cancerous cells.

6-6

Nanoparticles and Microcapsules

(1) Mucosal peptide delivery systems are under development by Prof. Kawashima, and colleagues, at the Gifu Pharmaceutical University. They have succeeded in developing lactide glycolide copolymer (PLGA) nanospheres containing peptides. They have also succeeded in modifying the surface of the nanosphere with chitosan, for mucosal adhesion. This is because mucosa has a negative charge and chitosan is positively charged, thus the movement in the digestive tract is delayed. They have shown the prolonged effect of calcitonin using rats. They have also made powdered nanospheres for pulmonary delivery that coagulate when inhaled and disperse in the lung and thus function as nanospheres. They have observed that the dextran concentration in blood is extended by using the powdered nanospheres. (2) Prof. Kikuchi, and colleagues, of the Nara Institute of Science and Technology, have succeeded in developing unique spheres with silicon oxide on the surface of a liposome. The diameter of the sphere is 200 nm. The silicon oxide layer is 5 nm. They named the spheres, Cerasome. There were problems with adhesion or deformed shapes due to the soft surface of the liposomes. The siliconised spheres will solve such shortcomings. The researchers are going to add bio-affinity to the surface. (3) Prof. Takayama, and colleagues, of the Hoshi Pharmaceutical University, have developed a neural network system to get the optimum mixing ratio of various materials in order to achieve a micro

26

capsule of a certain diameter. For example, by mixing three materials, such as a component in soybeans, cholesterol and oleic acid, to form a capsule suitable for drug delivery to a cancerous part of the liver, 15 experiments will be enough to get a regression formula in order to determine the ratio for the mixture. At present, as many as 1,000 experiments are needed to determine the optimum ratio. (4) Dr. Ichijou, and colleagues, of the National Institute of Materials and Chemical Research, are working on molecularly imprinted polymers. They have succeeded in synthesising polymer microspheres that can discriminate for oxytocin.

6-7

Liposomes

Prof. Konno's group, at the Research Institute of Advanced Science and Technology, Osaka Prefecture University, are studying functional liposomes modified with polymers. They have succeeded in creating a temperature-sensitive liposome that releases drugs at a temperature slightly higher than body temperature. The temperature response of the liposome copolymer depends on a kind of lipid, for example, egg yellow phosphatidylcholine did not show remarkable drug release in temperatures higher than the lower critical solution temperature (LCST). But adding dioleoilphosphatizylethanolamine (DOPE) gave a very sharp temperature responsive liposome. They have synthesised copolymers, such as NIPAM-AAM (poly-N isopropylacrylamide-Acrylamide) and NIPAM-N-APr (Acryloylpyrolidine), that have LCST around 40 degrees C. A cationic liposome modified with N-APr has also been synthesised. The affinity with negatively charged membranes is controlled by heating. They have also synthesised succinylated polyglycidol, which showed high membrane fusion ability. It can be used for a delivery system into cells.

6-8

Polysilamine

Associate Professor Sachio Nagasaki, at the Science University of Tokyo, is studying polysilamine, a new heterotelechelic oligomer. It can be prepared through an anionic polyaddition reaction between divinyldimethylsilane and 3,6-diazaoctane. It is a gel, with more flexibility than natural gum, but swells and turns solid with a pH change from alkali to acid. Applications for artificial muscle, drug delivery systems, and sensors are anticipated.

27

6-9

Perfluorocarbonic acid

Dr. Azumi, and colleagues, of the Osaka National Research Institute, are studying the displacement characteristics of gold plating on perfluorocarbonic acid membranes, which has been developed by Asahi Glass Corporation. The displacement occurs in response to an electric field. The degree of the displacement depends on the type of counter ions. They have applied the technology to micro catheters that bend in an electric field.

6-10 Shape Memory Gels Dr. Takashi Miyazaki, and colleagues, of the Hokkaido University, are working with shape memory polymer gels prepared from acrylic acid and stearyl acrylate. The shape memory effect takes place at the transition between the amorphous state in the dry condition and the crystalline state, formed with the addition of a certain amount of water. Their aim is to create a reliable valve.

6-11 Hollow fibres Hollow fibres are used for dialysers, and artificial lungs. Materials and the manufacturers of hollow fibres Makers Asahi Kuraray Nikkiso Teijin Medical Regenerate * * Cellulose Cellulose * Acetate Polyacryl* nitril Polymethylmethacrylate Ethylene * vinylalcohol Polysulfone * Polyesterpolymer alloy * Terumo Toray Toyobo

Synthetic polymers are now more used in place of cellulose, with polysulfone as the most popular.

28

Drug Delivery and Targeting

At the Controlled Release Society in Paris in 2000, Prof. Okano, from the Tokyo Women's Medical University, was given the Founders Award for his research on temperature responsive polymers. Dr. Okada, from the Takeda Pharmaceutical Co., and Dr. Toda, a former Takeda employee, now with Ohtsuka Pharmaceutical Co., were also honoured with the Nagai Innovation Award for their invention of Leuprin and its success on the market. It is proof that Japan is world-class in both research and commercialisation of DDS.

29

7-1

Overall situation

Current status of fine particles used in DDS Kinds Materials Size Liposomes Lecithin, Cholesterol, Charged substances 20nma few mm

Sustained drugs Suitable for localising, water-soluble targeting, slow release, & lipidsurface modification, soluble and activation of microphage Lymph targeting, Thermal sterilisation Used mainly for embolisation and absorption enhancement Enhancing absorption in intestines lipid-soluble

Characteristics

Lipid Lecithin, Soya oil, microspheres and Olive oil nanospheres Emulsion Synthetic surfactants, Oily substances

0.2 mm (ave.) Greater than 0.1 mm 5 nm 50200 nm

water-soluble & lipidsoluble lipid-soluble water-soluble & lipidsoluble

Examples under research Topo I inhibitor*, Monoclonal antibody-bonded*, Hemoglobin*, Cationic*, Contrast mediumcontaining* new lipoPGE1*, lipoPGI2*, amphotericin (50nm)* Artificial red cells(fluorocarbon)*, Oral absorption of insulin, calcitonin, & PTH

Commercialised examples Doxil, Daunoxom, Ambisom

Mixed micelles

Synthetic surfactants, Unsaturated fatty acids Polymer micelles Synthetic polymers

Adriamycin, Calcitonin (PEGPAA block copolymer)

30

Micro capsules, Nano capsules

Ion exchange resins Low density lipoprotein

Ethylcellulose, Collagen, Polylactic acid, PLGA, Starch, Geratin, Hemoglobin, Fibrinogen, Albumin Sodium polystyrene sulfonate, Stylene divinylbenzene copolymer Modified LDL

Micro: 5 300 mm, Nano: less than 1000 nm 2045 mm

For slow release, embolisation and imbedding Enhancing mucosa absorption Macropharge-orient

water-soluble & lipidsoluble water-soluble & lipidsoluble water-soluble & lipidsoluble water-soluble

PGLA oral vaccine*, Leuprin by Interferon collagen*, Takeda Pharm. Insulin PLGA Co., PLGA hGH, PLGA LHRH Insulin, calcitonin, theophiline, doxorubicin Thymidine Insulin

20 nm

Red blood cell Red blood cell 7.58.5 ghost membrane mm *Under clinical trials Source: Kikuchi and Igarashi, (2000) Drug Delivery System Vol.15, No.5.

Prof. Abe, at the Science University of Tokyo, has developed an economical production technology for liposomes using supercritical carbon dioxide. Under this method, the packed volume of reagent is increased ten times and the particle size is 20 nm or larger. Prof. M. Higaki, at the St. Marianne Medical University, is working on mucosal immunisation with negative ion exchange resin with sodium polystyrene sulfonate as a carrier.

31

7-2

Active targeting15

Prof. Kitajima, at the Medical School, Keio University, in co-operation with Associate Prof. Imoo, at Okayama University, made ribonuclease combined with EGF by joining the genes ribonuclease and EGF and introducing them into E. coli. Ribonuclease is an enzyme that destroys RNA and inhibits protein synthesis, thus killing cancerous cells. Cancer cells have receptors that bind EGF, a cell growth factor. The combination was effective in a mouse transplanted with cancer cells. In addition to EGF, the transferrin receptors and galactose receptors of the liver cancer cells are under study by Prof. Hashida, at Kyoto University and Prof. Kataoka, at Tokyo University. Dr. Kataoka is also trying to bond recognition molecules to micelles, to provide both passive and active functions. 7-3 Passive Targeting16

The EPR effect (enhanced permeability and retention effect) was discovered by Dr. H. Maeda. Through this effect, polymers are selectively accumulated in cancer tissues compared with normal tissues. SMANCS is a DDS drug utilising this effect. Liposomes modified with polyethyleneglycol can reduce non-specific interactions and suppress uptake into the reticuloendothelial system. Prof. Maruyama, at the Faculty of Pharmaceutical Science, Tokyo University, reported a remarkable increase in residence time in the blood with the modified liposomes. Polymer micelles that have been developed by Prof. Kataoka are now being tested in a DNA delivery application. 7-4 Stimulus Responsive Materials

The following are not necessarily focusing on DDS but on intelligent materials. (1) Prof. S. Aoshima, at Osaka University, is studying living cation polymerisation of vinylethers under an additive base. For example, polyvinylether, with oxyethylene on side chains, is water-soluble at low temperatures but shows phase-separation at a certain temperature (Tps). Tps is changeable by the kind of side chains or random copolymerisation. When the polymer is mixed with polycarbonic acid, it separated at below a certain pH (CpH) to form a complex. (2) The New Energy and Industrial Technology Development Organisation (NEDO) started a five-year research programme called Advanced Stimuli-Responsive Materials in 1996. The programme is focused the intellectual environment responsive functions of biological systems used as industrial

32

materials. The programme has the three targets: 1. Separation materials, 2. Control release materials, 3. Actuating materials. Research on control release materials is conducted at the Osaka National Research Institute in cooperation with Prof. Nagata, of Hokkaido University; Prof. Okano, of the Tokyo Womens Medical University; Prof. Kataoka, of Tokyo University; Prof. Akashi, of Kagoshima University; Prof. Kajiwara, of Kyoto Institute of Technology; and some companies. Under the programme, Dr. Ueno, of the National Institute of Materials and Chemical Research, in cooperation with Tokyo University and Chisso Corporation, is studying magnetic nano particles attached with ligands or nucleonic acid. The particles can be easily recovered using magnets. They copolymerised acryloylglycineamide and biotin monomer, and, just by mixing the copolymer with the magnetic particles, they can get temperature responsive magnetic nano particles that aggregate at less than 20 degrees C and disperse at a higher temperature. This technology has the potential to produce molecular recognition and temperature responsive magnetic particles.

7-5

Electromagnetic Responsive Double Targeting17

Professor Takeshi Kobayashi, in Bioengineering at Nagoya University, is developing Electromagnetic Responsive Double Targeting, in a combination of time targeting, controlled release by physical stimulations from the outside, and space targeting, collecting medicines to a target area by use of monoclonal antibodies and others. Dr. Kobayashi is a developer of magnetic nano-spheres, which heat up in response to an alternating magnetic field. He uses carboxy-methyl cellulose (CMC) and 25nm magnetite in a 4/1 weight ratio to form extrusion needle-shaped magnetite 2-3 mm in diameter. Dr. Wakabayashi of Nagoya University Medical School, has found a use for magnetic nano-spheres in treatment of brain tumours. Liposomes containing magnetic nano-spheres were injected into rat tumours and a 120Hz magnetic field was applied. The tumour disappeared through the heat generated by the particles and the magnetic field. This was also successfully used in rabbits. A monoclonal antibody that binds to tumours has been developed for brain tumours. Magnetic nano-spheres are used for extracting target substances in blood. An antibody for insulin, for example, is attached to the particles, then put into blood. Insulin bind to the particles. Insulin is thus measured at a higher concentration. Glycosylated hemoglobin and fructosamine can also be measured. This way diabetes can be diagnosed in 30-40 minutes with 50 ml of blood. The method is now being marketed by a venture company called M-Bio.

7-6

Biomolecular Design for Biotargeting

33

Biomolecular Design for Biotargeting was accepted by the Ministry of Education and Science from 1998 to 2000 as one of the Grant-in Aid Programmes for the Scientific Research on Priority Areas. The goal of the programme is to review the targeting function of living cells or biomolecules and to reconstruct it from chemical and engineering points of view. The programme consists of the following four research fields involving more than 70 researchers in universities: 1) Biomolecular recognition, represented by Professor Imanaka, Kyoto University -Modifications of the molecular recognition capability of antibodies -Signal transmitting function of antibodies -Modifications of enzymes for molecule recognition analysis 2) Biomolecular architecture, represented by Professor Matusnaga, Tokyo Agriculture and Engineering University -Building a biomolecule architecture on the surface of magnetic nano-spheres and liposomes synthesis of a biomolecular architecture Electromagnetic Responsive Double Targeting, described in the previous chapter, is one of the outcomes of the programme. 3) Cell surface design, represented by Professor Tanaka, Kyoto University -Culturing new functional micro organism cells -Producing biovaccines with antigen presenting cells Modifications of cell function by expressing chimera receptor molecules on the surface of animal cells Analysis of cell surface functions using artificial cells 4) Biotargeting, by Professor Kobayashi, Nagoya University -Tissue and cell targeting of functional materials incorporated with biopolymers Targeting of physiologically functional small molecules with stimulus-responsive polymers Biomolecule targeting with an ultra-micro electrode device

7-7

Transdermal & Transmucosal Delivery

The Suguhara group has conducted a study of how drugs applied on the skin remain locally in dermis or hypodermis or move to the entire body through blood vessels.18 The movement of a drug in skin is rather complicated; therefore, the development of simple evaluation is indispensable.19 The direct transport of a drug from the nasal cavity to the cerebrospinal fluid was reported recently; therefore, targeting the brain is possible.20

34

How to obtain slow release of a drug and adhesion to mucosa is important, but on-off control of drug release is also of interest. Polymer gels that change structure in response to temperature, pH, electric fields, light, ultrasonic, and chemical substances are being studied by Prof. Okana's group.21 Studies on how to enhance the absorption of a drug in skin or mucosa have been carried out by the Josai University group.22 Two Kyoto groups have studied absorption enhancing reagents.23 Electroporation is a relatively new method to enhance absorption.24 The Sugihara group reported that pretreatment of a non-needle syringe increased the transdermal absorption of a drug.25 Phonophoresis has also been used to enhance absorption.26

7-8

Company activities

(1) Leuprin, launched by the Takeda Pharmaceutical Co., is the most successful polymer nano-sphere DDS. It is sold in more than 10 countries, including the USA, with the annual sales of more than USD 1 billion. In 1989, Drs. Ogawa, Okada, Toda, and colleagues, at Takeda, succeeded in putting leuprolerin acetate, an antagonist of the luteinising hormone-releasing hormone, in microspheres. The microshperes, made of a copolymer of polylactic acid and glycol acid, to keep released leuprolerin for more than a month after a one-time injection. It is currently used for the treatment of prostate cancer and breast cancer. (2) SMANCS, developed by Prof. Maeda, at the Medical School, Kumamoto University, and launched by the Yamanouchi Pharmaceutical Co. in 1996, is the first and only polymer anticancer drug approved by the Ministry of Health and Welfare. It is a styrene-maleic acid conjugate of neocarcinostatin. The molecular weight of SMANCS itself is 16,000, but it bonds with albumin in the human body to behave like a polymer with 80,000 MW. It gathers and accumulates around cancerous tissue, a phenomena Prof. Maeda discovered in 1986 and called the EPR effect. SMANCS can be oil-philic by the butylesterification of the carboxyl group of the styrene-maleic acid copolymer, thus, increasing the affinity to lipiodol. Therefore, both the targeting and controlled release of lipiodol are achieved. (3) A venture company called NanoCarrier Co., located in Chiba prefecture, has developed micelletype DDS that accumulate drugs near tumour tissues. The materials are molecules of hydrophilic polyethyleneglycol (PEG) bonded with hydrophobic polyaminoacid. The particles have a diameter of 20200 nm. In an experiment using mice, the anticancer drug contained in the micelle accumulated near the tumour tissue at a concentration seven times higher than the drug alone. They explain that micelles normally cannot pass through blood vessels around healthy cells, but that passages in blood vessels around cancerous cells are large enough for the micelles to go through. (4) The DDS annual conference was held in July 2000 in Akita, Japan. The following information related to biomaterial DDS was reported at the proceedings. Nippon Shinyaku Co. is developing new drugs using lipid ultra fine particles (LNSTM) that contain soybean oil and egg yellow lecithin and have a diameter of several dozen nano-metres. LNS does not go to the liver or the spleen and has passive targeting capability.

35

Welfide Co. is developing bromperidole, a drug for mental illness, contained in slow release polylactic acid microspheres. NOF Corporation (Nippon Yushi Co.) is ready to sell highly pure liposome for medical applications. Ohtsuka Pharmaceutical Co. is studying liposome triggering cytokine production in human peripheral blood cells.

8 8-1

Gene Delivery
Gene Therapy Protocols

Protocols for gene therapy that have been approved by the Advanced Medical Technology Evaluation Committee in the Ministry of Health and Welfare by the end of 2000. No Date of Diseases Vectors/Gene Hospitals Number of approval patients treated 1 1995 Feb. ADA Retro virus Hokkaido Univ. 1 (1995) deficiency 2 1997 May HIV infection Retro virus Kumamoto Univ. 3 1998 Aug. Kidney cancer Retro virus Tokyo Univ. 3 Medical Institute 4 1998 Oct. Lung cancer Adenovirus Okayama Univ. 8 5 6 7 8 9 10 11 12 13 2000 May 2000 Feb. With-drawn 2000 Jan. 2000 Jan. 2000 Jan. 2000 Jun. 2000 Jan Esophagus cancer Breast cancer Liver cancer Malignant glioma Lung cancer Lung cancer Prostate cancer Lung cancer Adenovirus/ P53 Retrovirus Adenovirus Cationic Liposome Adenovirus Adenovirus Adenovirus Adenovirus DNA alone Chiba Univ. Japan Foundation for Cancer Research Tokyo Univ. Medical Institute Nagoya Univ. Tokyo Jikei Medical Univ. Tohoku Univ. Okayama Univ. Tokyo Univ. Medical Institute Osaka Univ. 1 (19/12/2000)

1 1 1

Under examin- Chronic ation obstructive arteriosclerosis

36

As seen in the above table, Nagoya University is the only group that used non-virus vectors for clinical studies of gene therapy. Dr. Yoshida, and colleagues, have been developing gene therapy for malignant glioma with the IFNb gene, using a cationic liposome as the vector. Liposomes are synthetic lipid vesicles that can entrap drugs or genes within their aqueous compartment or lipid bilayer. Dr. Yoshida's group conducted the first therapy in April 2000. They have also worked on entrapping recombinant adenoviral vector in multi-layer liposomes to improve transduction efficiency and reduce virus antigenicity. They have also succeeded, in co-operation with Aichi Medical University, in producing a single chain antibody for type III mutant EGFR, which expressed in malignant glioma and not in normal cells.

8-2

Research on non-virus vectors

Cationic liposomes and polycations are the two major materials used for gene vectors. Prof. Akaike's group in the Department of Biomolecular Engineering in Tokyo Institute of Technology and Prof. Hashida, in the Pharmaceutical Department in Kyoto University, are some of the major research groups in the field. Dr. Prof. Akaike's group, including Dr. Maruyama and Dr. Ishihara, has synthesised a graft copolymer (comb type copolymer) with poly(L-lysine) as the backbone and hyaluronic acid side chains. This polymer formed a complex with plasmid DNA. They showed with in-vivo experiments that the complex is specifically taken into the liver. They also made graft copolymer of poly(L-lysine) and arabinogalactan, a kind of polysaccharide, and showed a similar targeting effect to the liver. They are also studying the expression of graft copolymers in cells. Prof. Akaike is one of the leading researchers of hybrid livers. (1) Prof. Hashida, of Kyoto University, and Dr. Kawakami at Nagasaki University, have synthesised a sugar-modified cationic liposome that created a complex with plasmid DNA. They used a cholesterol derivative modified with galactose or mannose, both showing high gene expression in the liver. (2) Prof. Ogihara, Osaka University Hospital, has applied for permission to provide gene therapy using catheters, in-stead of vectors, to bring hepatocyte growth factors to the target part of the inner wall of the heart. Another group in Osaka University has already applied for permission to inject HGF in cardiac muscle.

8-3

Polycations and Liposomes

(1) Prof. Mayumi and Prof. Nakagawa, of the Department of Pharmeutical Science at Osaka

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University, are studying membrane fusion liposomes, a kind of hybrid vector, with the membrane fusion capability of a virus. They have also worked on the expression system in cytoplasm. (2) Prof. Konno's group in Osaka Prefecture University, made a complex of succynilated polyglycidol bonded with transferrin and cationic liposome-plasmid conjugate. It was tested as a gene delivery system for tumour cells and proved to have high expression efficiency, because plasmid DNA was introduced into target cells through transferrin receptors. (3) Increasing the gene-introducing efficiency of cationic liposomes to the level of viral vectors is being studied by various research groups at the Tokyo University, Tokushima University, and Shizuoka Prefecture University. (4) Aomori Prefecture Green Bio Center has applied for permission to use a new gene vector, positively charged polyornithine fused with plasmid, developed by Agriculture Research Center, to create herbicide-resistant rice.

8-4

Polymer Micelles

Prof. Kataoka and his group are also applying micelles to non-viral vectors. Polyethylene glycol-poly a, b-aspartic acid as anion block copolymer mixed with polyethylene glycol-poly L-lysine (PEG-PLL) as cation block copolymer generates PIC with a diameter of dozens of nano-metres in a sharp diameter distribution. Micelles 80 nm in diameter were formed with plasmid DNA and PEG-PLL. Higher expression of luciferase in cultured cells was obtained with the micelles, compared with conventional lipofectin. He has also reported an environment-responsive micelle vector by reversible cross linking of the core through disulfide bonds.

8-5

Fullerene and Cyclodextrin

(1) Prof. Nakamura, of the Science Faculty and Prof. Okayama, of the Medical Faculty in Tokyo University have been successful in applying for permission to use fullerene (C60) as a gene vector. C60 is a cubic molecule with a 1 nano-metre diameter. Profs. Nakamura and Okayama bound DNA, containing the luciferase gene on an expression plasmid, to positively charged C60, and added them to a culture of monkeys kidney cells. Expression was achieved in 25% of the cells. Nakamura and Okayama claim that C60 gradually enters into the nucleus. Thus, C60 can be a DDS. Together with a pharmaceutical company, they have applied for a patent.

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(2) Prof. Kamigama, Pharmaceutical Faculty, Kumamoto University, is trying to use a conjugate of cyclodextrin and dendrimer as a gene vector. The conjugate showed higher activity than conventional LipofectinTM.

Tissue Engineering

Tissue engineering has been strongly supported by the government in the past several years and was chosen as one of the focus fields of the Millennium Project supported by the Prime Minister. A national centre of tissue engineering, from basic research to clinical development, is being built in Kobe. Among the significant number of researchers in the field, Kyoto University is a pioneer in this field and its research is considered world-class. Tissue engineering research in Japan is not lagging behind the USA, but commercialisation or business applications are clearly behind. Only recently have some venture companies been created. According to J-TEC, a venture company for skin culture, the Japanese market potential for tissue engineering will be USD 8 billion and the world market, USD 45 billion . Three government-supported research programmes in the tissue engineering field are of note: The Regenerative Medical Engineering Programme, supported by the Ministry of Education and conducted by hundreds of university researchers---1996--2000 Hiroshima prefecture Tissue Engineering Project, organised by Professor Katsuoshi Yoshizato, of Hiroshima University 3D Tissue Module Engineering, conducted by NAIR (National Institute for Advanced Interdisciplinary Research), under the Ministry of Economics, Trade and Industry (A part of the Millennium Project supported by the Prime Minister)

9-1

Regenerative Medical Engineering Programme by the Ministry of Education

The programme started in 1996 as a five-year programme with about USD 1 million annually. The programme consists seven groups: Tissue Engineering for Hard Tissues; Tissue Engineering for Soft Tissues; Biomaterials for Tissue Engineering; Bioprocess Engineering of Functional Regeneration of Culture; Tissue Engineering for Organ Regeneration, started in 1996; and, Function Regeneration, and Bio Tissue Engineering, started in 1997 and 1998, respectively.

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(1) Tissue Engineering for Hard Tissues The project leader is Prof. Shoji Enomoto, of the Dental Faculty, Tokyo Medical and Dental University. About 20 researchers are involved in the project at the university. The objective is to analyse the molecular mechanism of the differentiation and function control of osteoblasts and osteoclasts in hard tissue generation and regeneration. Study on ADDI, HESI, and scleraxis, transcription factors of osteoblast Study on factors of periodontium Regeneration of jaw bone Study of the regeneration of heavy-load-bearing bone (2) Biomaterials for Tissue Engineering The project leader is Prof. Mitsuo Okano, of the Institute of Biomedical Engineering, Tokyo Womens Medical University, and the researchers involved are mostly from the same university. Masayuki Yamato is a major researcher in Cell Sheet Engineering. They have proposed a concept of Cell Sheet Engineering, which is to put cell sheets one upon another 3-dimensionally to create tissues and organs applying Temperature Responsive Culture Plates with poly N-isopropyle acrylamide(PIPAAm), which is known to drastically change water affinity with changes in temperature. PIPAAm is highly soluble in water below a lowest critical solution temperature (LSCT), 32 C in water, but becomes water-insoluble at higher temperatures. Electron Beam (EB) radiation or specific chemical reactions are used for the adhesion of cells to the plates. At 37 C, for example, various cells adhere to the plates but cells detach when the temperature is less than LSCT. With the plates, they have succeeded in culturing various cells such as fibroblasts, hemangioendotherial cells, macrophage, cells in a pigmented layer of retina, and microglia cells. Cell sheets recovered with this method are more functional than those recovered with trypsin. When EB is irradiated through a patterned mask, various shapes of cell sheets are made, and, by repeating the process, complicated layered tissues are made systematically.

The following is a summary of Okanos presentation at the Biomaterials for Tissue Reconstruction27 Here we demonstrate two novel cell co-culture methods that exploit intelligent surfaces grafted with a thermally responsive polymer (poly(N-isopropylacrylamide. PIPAAm)) to alter cell-surface interactions. Many cell types adhere on the grafted surface as on tissue culture polystyrene dishes at 37'C. Only by reducing the temperature below 32 'C can the spread cells detach from the surfaces without trypsin. Because cells are recovered maintaining intact cell-cell junctions together with formerly deposited extracellular matrix, harvested cell sheets can be transferred to other surfaces and

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readily adhere to them. Therefore, we transferred harvested cell sheets onto another cell type monolayer and co-cultured them. The other method is a patterned co-culture utilising patterned graft of PIPAAm by masked electron beam irradiation. One cell type is then cultured to confluency at 37'C. Reducing the temperature to below 32 'C., the cells are only detached from these grafted patterns. Another cell type is then seeded over the same surface at 37'C. These subsequently seeded cells only adhere to the now-exposed polymer-grafted domains. Initially, seeded cells remaining adherent on nonpatterned surfaces and cells added in the second seeding are then co-cultured at 37'C together in wellordered patterns. These two methods enabled long-term co-culture of primary hepatocytes with other cell types. Hepatocytes co-cultured with endothelial cells maintained the differentiated functions such as albumin synthesis for more than two months. These findings might be related to the morphological resemblance to liver tubule structure in terms of the adjacent lining of hepatocytes and endothelial cells.

(3) Bioprocess engineering of functional regeneration of culture Prof. Ohshima, at Tsukuba University Medical School, is the project leader and the objective is to develop an artificial liver. They have developed the volume culture of liver cells: a packed-bed reactor using polyvinylformal as a scaffold. Based on various other studies, they are currently trying to scale up reactors, create model animals, and a mixed culture of foetal liver cells. Recovery and purification of hepatic stem cells is also under study. (4) Tissue Engineering for Soft Tissues The leader is Prof. Shimizu, of the Advanced Medical Research Institute, Kyoto University and many researchers in the institute are working on the project. They have made progress in especially recovering the following tissues: Bronch made three-dimensional collagen, Ischemic myocardium, Membranes like pleura and duramater, and nerves. (5) Tissue Engineering for Organ Regeneration Prof. Yamaoka, at Kyoto University Medical School, is the leader, and many researchers in the university are focusing on liver regeneration in vitro liver supporting device using a pig liver, application of human liver cells for an artificial liver, and the promotion of liver cell proliferation using gene manipulation. (6) Functional Regeneration by Medical Bioengineering Prof. Tomomitsu Hotta, Tokai University Medical School, and his group in co-operation with Kyoto

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University, are tackling the in vitro proliferation of hematopoetic stem cells, especially in cord blood, and lymph cells. They are also working on gene introduction to hematopoetic stem cells. Other activities include a drug delivery system for the adrenal steroid hormone using poly-D, L-lactic acid (PDLLA) microcapsules (7) Biological Tissue Engineering Prof. Umakoshi, in the Engineering Department, at Osaka University, is the leader in co-operation with Osaka University Dental School and Kyoto University. The Osaka University group is working on the relationship between structures or tissues and the property of biomaterials, using such technologies as electron beam radiation introducing lattice defects and laser radiation for infusion of foreign elements in very small quantities. The dental group is working on a composite of apatite and collagen. The Kyoto University group is evaluating regenerated tissues and developing evaluation methods.

9-2

The Yoshizato Group at Hiroshima University

This six-year project, started in 1997, is supported by the Science and Technology Agency, which merged with the Ministry of Education and Science in January 2001. This is an application-oriented research programme to promote local industry. It will be funded to a total of USD 20 million. 40 researchers will be involved. The following are themes related to biomaterials. (1) Search for hair growing factors The stem cells of skin are already known. Yoshizatos group is now trying to clarify the differentiation process of skin stem cells in order to use them for generating skin that forms hair follicles. The Institute of Immunology Co., a diagnostic reagent maker, and Molten Co., a sporting goods company located in Hiroshima, are co-operating on the project (2) Insulin secreting cells implanted in skin To modify the genes of non-pancreatic cells to secrete insulin and place them into skin fibroblasts to cure diabetes-derived skin tumours (3) Establish a therapy for acute liver insufficiency using the hepatocytes of the patients themselves Yoshizatos group has proved the existence of hepatic stem cells and developed in vitro culture of human liver cells. (4) Production of human collagen via gene modification of silk worms

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This project, supported by Termo, leading catheter maker, is to make silk worms produce collagen. It is the most difficult project, according to Prof. Yoshizato.

9-3

3D Tissue Module Engineering conducted by NAIR

This project, started in 1998, is a pre-research project, a kind of a survey before an extensive project starts. More than 40 researchers are working on the project. The major results: (1) A prototype of a culture reactor for liver cells using porous non-woven Teflon has been developed. (2) A system for three-dimensional culturing of cartilage cells under the static pressure has been developed. Proteoglycans, and other proteins secreted by cartilage, have been produced at a pressure of 50 atm. NAIR held an International Symposium on Advanced Biomaterial & Tissue Engineering in 1999 in Tokyo, covering Scaffolds for hard and soft tissue, Cell growth factors & Cell culture technology, Hybrid biomaterials, as well as Tissue engineered devices and Artificial organs. The Ministry of Economics, Trade and Industry has decided to continue the pre-project and convert it into a leading research project in 2001.

9-4

Millennium Project

The Millennium Project, starting in 2000, has a total budget of USD 640 million for biotechnology in 2000. Out of that budget, USD 108 million has been allocated to Tissue Engineering. (1) Targets

Tissue engineering in the fields of bone & cartilage, blood vessels, nerves, skin, blood, and transplanting technology in the five years from 2000 through 2004. Blood vein regeneration, skin transplanting, quick detection of a virus in cells, and large volume tissue culturing will be in clinical use by 2002.

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(2) Fields

Major Research Organisations Major Research Organisations Nagoya University. Kitasato University., and St. Marianne University. Tokyo University. Nagoya University, Nagoya First Red Cross Hospital, and Autonomy Medical School National Center of Neurology and Psychiatry Tokyo Medical & Dental Univ., Tsukuba Univ., and Nagoya Univ. National Institute of Health Sciences Represented by Prof. Ueda, Nagoya University. Prof. Kuroyanagi, Kitazato University. Prof. B Nagai, Tokyo University. Prof. Saito, Nagoya University. Dr. Takasaka, National Center of Neurology and Psychiatry Dr. Nomoto, Japan Transplanting Society Dr. Hayakawa, National Institute of Health Sciences

Bone & Cartilage Skin & Cornea Blood vessels Blood cells Nerve Transplanting technology Development, Differentiation & Regeneration (3) Research strategy Fields Bone & Cartilage

Skin & Cornea

Blood vessels

Blood cells Nerve

Research Strategy Establishing large volume culture technology of osteoblasts and chondroblasts, improving scaffold materials, and storage and packaging methods for providing hospitals with cultured bone and cartilage for transplantation and regeneration Development and clinical application of 1 Outer skin cultured from patients keratinised cells (Prof. Kumagai, St Marianne University) 2 Inner skin cultured from own or others' fibroblasts (Prof. Kuroyanagi, Kitasato University) 1 Promotion and suppression of blood vessel generation with gene introduction. Human HGF, bFGF for promotion and receptor for VEGF for suppression 2 Blood vessel generation with cell transplantation 3 Prevention of re-arctation with transcription factor from activation of smooth muscle or modification of signals inducing apoptosis 1 Hemopoietic cells for new therapies 2 Blood in umbilical cords for transplantation and regeneration 3 Curing hematopoietic insufficiency with gene modification of patients To make use of foetus or human nerve stem cells for Parkinson's disease therapy

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Transplanting technology

Quality assurance of cells, tissue, and materials and device

1 Introducing immune tolerance 2 Immune suppression methods using monoclonal antibodies, decoy molecules, and antisense genes 3 Avoiding chronic rejection with antisense genes and decoy genes 4 Development of a transplanting system for the proliferation of organ transplantation 1 Assurance of infection-free cells 2 Cell evaluation with multi-labelled FISH 3 Analysis with cell-derived proteins as index 4 Forecast technology of carcinogenesis of cells and tissue 5 Metabolism of physiologically active proteins

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9-5

New Institutes

The government has just decided to set up two large institutes dedicated to tissue engineering: (1) RIKEN Center for Development Biology

The centre will conduct basic research in embryology. About two hundred researchers will be employed in seven large groups and in more than 20 small groups lead by young researchers in their early 30s. The seven large groups are 1. Stem cells, lead by Prof. Nishikaw, at Kyoto University; 2. Cell & tissue differentiation, lead by Prof. Sasai, at Kyoto University - who established an efficient technology for the generation of nerve cells from mice ES cells; 3. Vertebrate body plan; 4. Morphogenetic signals; 5. Non-symmetric segmentation; 6. Higher order structure building; and 7. Development and evolution. The centre is currently physically located in Tsukuba but will move to Kobe in January 2002. Dr. Masatoshi Takeichi is director general. Approximately USD 50 million from the Millennium Project budget has been allocated for construction. For pre-clinical study and clinical tests, Kobe is building the Advanced Medical Center next to the RIKEN Center and bringing in the Kobe City Hospital as well. The Ministry of Education, Science and Technology has decided to annually grant to Kobe USD 4 million for five years to develop the technology basis from the Regional S & T Promotion portfolio. (2) METI Tissue Engineering Center

The centre will open with about 150 researchers in 2002 near Osaka, 25 km from the RIKEN Center for Development Biology. It will act as a bridge between basic technology and industry. Its aim is to develop devices based on cell culture technology that function as replacements for tissues or organs. The following themes will be studied: Three-dimensional cell and tissue production technology to induce specific functions in cells using physical, chemical or biochemical stimulus. At the same time, the development of biodegradable scaffolds that contain functional factors. Search for stem cells from the differentiated tissues to form functional specific tissues. Application of embryonic stem cells in co-operation with other institutions in the Osaka-Kyoto area. Search for genes related to tissue formation, and control and application of the genes. Development of devices that can replace animal tests It will have a large scale cell culture line (Cell Processing Center)

(3) Tokai University will start a donation course called Tissue Engineering, in April 2001, with the help of BCS Corporation. It plans to culture the skin of burned patients and transplant it back to the patients. They are developing a two layer structure of inner and outer skin. The Tokai University Hospital has already succeeded in transplanting regenerated skin for more than 10 patients.

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9-6

Some topics in Tissue Engineering

Kidneys Dialysis equipment with hollow fibres is quite successful in prolonging the lives of 200,000 chronic kidney patients who receive dialysis regularly: three times a week each for 4 hours. The dialysis can control water and electrolyte in the blood properly but cannot fully discharge waste materials in the body, nor can it produce hormones. But the development of an artificial kidney is not an immediate aim. Livers Liver disease patients total about 610,000 . Organ donors are very rare in Japan. Many researchers are working on the development of an artificial liver. Prof. Ikai and Prof. Iwata, at Kyoto University, are working on different approaches: culturing pig liver cells packed with dextran (glucose polymer) microcarriers; separating and culturing human foetus precursor liver cells, cells in bioreactors or hollow fibres, as well as whole pig liver out-of-the-body circulating systems. Prof. Akaike and the Watanabe group, in the Bioengineering Department, at Tokyo Institute of Technology, is working on the mechanism of formation of the liver, and drug delivery systems for hepatitis. They have introduced VEGF, a blood vessel generating factor, into hepatocytes and showed blood vessels developing in the body. To concentrate caspase inhibitor (Z-ASP) around hepatocytes, they used PVLA nano-particles in polylactic acid matrix, to bind to asialoglycoprotein receptors,. Prof. Funatsu, and his group, in Chemical Engineering, at Kyushu University, are developing a module of spherical multicellular aggregates (spheroids) of primary porcine hepatocytes packed in multicapillary polyurethane foam. They have succeeded in maintaining function for two weeks and have applied for approval for a clinical test under Prof. Sugimachi, of the Medical Department, at Kyushu University. The Hiroshima group is also working on an artificial liver as described above. Pancreas A wearable glucose sensor, incorporated with an insulin feeder, has been developed successfully by Prof. Sakakida, of the Medical Department, at Kumamoto University. He has developed a glucose sensor from a ferrocene-added minute needle type covered with MPC film (copolymer of MPC, or 2methacryloyloxyethyl phosphorylcholine and BMA, or n-butyl methacrylate). Continuous measurement for two weeks was possible. An implant system using a telemeter system is under development. Prof. Hiroo Iwata, at Kyoto University, has developed membranes for immuno-isolation. In a hybrid pancreas, islets of Langerhans are transplanted after they are sealed with immuno-isolation membranes.
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Iwata is focusing on membranes that suppress the activity of complements. One of the membranes is agarose-polystylene sulfonic acid. The Nara Medical University used the membranes in a diabetic dog and normalised the blood glucose level for more than 100 days. Prof. Miyamoto, and his group, at Kyoto University, have used bFGF together with hialuronic acid packed in PVA for subcutaneous implantation of islets with enhanced generation of blood vessels and controlled release. They have maintained normal glucose levels, in diabetic rats, for more than three months. Blood Vessels Cardiovascular surgery was performed on 41,016 patients in 1998 in 440 hospitals in Japan: 16,822 bypass operations for ischemic heart diseases; congenital disease, 9,254; valvular disorder, 8,788; and, Great vessel operation, 4,605. The total number doubled compared with 1986 while the latter two levelled off, but ischemic diseases increased. Dr. Yasuko Tomisawa, of the Tokyo Womens Medical University, has proposed the use of hybrid artificial blood vessels using the patients own tissue portion. Porous cloth is used for the scaffold and the tissue portion taken during an operation is cut in pieces and put on the scaffold. This idea was shown to be as being technically feasible not only in animal tests but also in a human trial.28 Dr. Sinoka, at Tokyo Womens Medical University, has succeeded in producing tissue-engineered blood vessels. Seed cells from a cultured thigh vein of a child patient were placed in a woven mesh of polyglactin, sandwiched by polyglycol acid meshes. After 30-60 minutes of in-vitro culturing, it was implanted in the patient. A blood vessel was successfully produced. This technology can now be used only for vessels under a medium to low pressure. It is indispensable to develop a biodegradable elastomer for this application. Hearts A total of 450 ventricular assist systems are clinically used in Japan. They were developed by National Cardiovascular Center Research Institute (NCCRI), commercialised by Toyobo Co., developed by Tokyo University, and manufactured by Nippon Zeon. Totally implantable artificial hearts are under development by Tokyo University, NCCRI, Tohoku University, METI Industrial Technology Research Institute, and some other universities. Lungs More than 95% of the artificial lungs in Japan are made from hollow fibres with heparin treatment. Matrix materials According to Prof. Yoshihiro Ito, at Tokushima University, the progress of matrix and scaffold materials owes a great deal to Japanese researchers. First, micro-machining methods of materials have
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been devised, such as photolithography, to precisely control cell function and bring cells to the tissue. Prof. Matsuda, at Kyushu University, in co-operation with Dr. Yasuhide Nakayama, for example, has succeeded in controlling hydrophilic and hydrophobic areas and fixing the area of cell adhesive peptides in micrometre scale. Second is the application of temperature responsive polymers by Prof. Okano, Tokyo Womens Medical University, as described above. Third, advanced functions of cells such as growth, differentiation, and apoptosis can now be controlled by artificial materials. Studies have revealed that growth factors fixed in materials can be taken into cells and control cell functions.

9-7

Tissue engineering companies

(1) Japan Tissue Engineering Co. (J-TEC), Japans first tissue engineering venture company founded in 1999 by Professor Minoru Ueda, from Nagoya University, a pioneer in skin regeneration, to manufacture and market cultured outer skin grafts produced from the patients own oral mucous membranes. J-TEC, located near Nagoya , plans to put the skin product on the market in 2002, as well as develop other skin products such as allograft outer skin, inner skin, auto-cartilage, and cultured bone. It expects USD 3 million sales in 2004 and USD 40 million in 2009. It will receive loans from the Ministry of Health and Welfare over the next five years. (2) Menicon Co., a leading contact lens company with USD 300 million annual sales, announced last August it was entering the skin-culture business in 2003. They have developed the technology in cooperation with Kitasato University and are going to conduct clinical tests at the Aichi Medical University. They will start volume production of allograft outer skin, produced from cells or tissues of donors, in 2003, and inner skin in 2005. In the immediate future, they are targeting cornea regeneration to help visually-handicapped people. (3) Foreign companies are also entering the Japanese market. Smith & Nephew, approved by the Ministry of Health and Welfare in July 1999, is undertaking clinical trials of cultured skin products from donor tissue, using the technology developed by Advanced Tissue Sciences Inc., US. (4) Genzyme Tissue Repair, the US tissue-engineering division of Genzyme Corp., founded in 1999, is providing Japanese doctors with cultured cartilage grafts from patients own tissue. The first treatment of a joint using chondrocyte cultured in the US took place in Chiba University in June 1999.29 (5) Asahi Medical Co., a leading manufacturer of hollow polysulfon fibre and membranes for artificial kidneys, has developed filters that can separate stem cells from blood in the umbilical cord. They have applied for FDA, as well as Japanese Ministry of Health and Welfare, approval. The company has opened a donation class in Tokyo University Medical Science Institute for co-operative development. They had already developed polyester filters with a diameter of 1-2 micrometres for the separation of white blood cells, among which stem cells are scattered. They are going to put the filters on the European market in autumn 2001.

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They have also successfully applied their hollow fibre filters in a filtration process for the removal of prion, a protein causing Creutzfeldt-Jacobi disease, in co-operation with Professor Kitamoto, at Tohoku University. They are testing the efficacy of the process in the US and plan to launch the product by the year-end, if positive results are obtained.30 (6) Gunze Co., an underwear company, has various biomaterials such as collagen sheets, bonebonding materials made of polylactic acid, as well as sewing materials made of polyglycolic acid. (7) Kyowa Hakko Co. announced on October 25, 2000 that they have succeeded in producing cerebral neurocytes from embryonic stem cells taken from a mouse embryo, in co-operation with Prof. Sasai, at Kyoto University. They cultured the ES cells in specific marrow cells of mice and almost all the ES cells were converted to neurocytes in a week, 30% of which generate dopamine and continued to work even after being returned to a mouse brain. It will possibly be used for the treatment of Parkinson disease in a few years. The patent is pending. (8) Sumitomo Bakelite Co. announced that they have developed a culture system for rat liver cells on collagen gel. Compared with conventional systems, synthesised glycogen, a marker of liver function, is 6-7 times higher in levels, and the life of cultured hepatocytes is more than doubled to two weeks. They are planning to sell the system to pharmaceutical companies. 31 (9) Tanabe Pharmaceutical Co. has filed a patent on the efficient cultivation of ES cells from invitro fertilised monkeys cells, based on the research done in co-operation with the Institute of Frontier Medical Sciences at Kyoto University; Prof. Nakatsuji, of Shiga Medical University; and Kinki University. This is the second reported success producing monkey stem cells, following the University of Wisconsin in the US. They want to provide the cultured stem cells to researchers in universities and enterprises upon a request. 32 (10) Kirin Brewery Co. is building a facility dedicated to dendritic cell culture. They are planning to take dendritic cells from patients and grow them with a cancer antigen and inject them back in the patient. (11) Medogene Bioscience, a venture company, is aiming at commercialisation of HGF (hepatocyte growth factor) in co-operation with Prof. Toshiichi Nakamura, from Osaka University, discoverer of the protein.

10

Intelligent materials and others

Intelligent Materials Forum The Society of Non-traditional Technology organises the Intelligent Materials Forum, for the exchange of information on intelligent material research. In addition to the annual symposium and bi-annual workshops, it organises international conferences. The following are the titles of presentations on Biorelated polymers and molecular synchronisation copied from the proceedings of the International

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Conference on Intelligent Materials held in October 1998 in Japan. The author will continue to monitor this forum. 1 Cellular and Molecular Responses to Fluid Shear Stress in Vascular Endothelial Cells by J.Ando R.Korenaga, K.Kosaki and A.Kamiya (University of Tokyo) 2 Control of 'Hepatocyte Differentiation by Manipulation of Cell Environments by H.Oda and A.Kakinulna PVagoya (University of Tokyo) 3 Reconstruction of Tissue Architectures from Cell Sheets Recovered from Temperature-responsive Culture Surfaces by M.Yamato, A.Kushida, C.Konno, M.Okuhara, A.Kikuchi, F.Karikusa, Y.Sakurai and T.Okano (Tokyo Women 's Medical University.) 4 Reconstruction of Pulmonary Epithelia Tissues on the Basement Membrane Produced in Vitro by the Cooperation of Alveolar Epithelial Cells and Pulmonary Fibroblasts by K.Mochitate and A.Furuyama (National Institute for Environmental Studies) 5 Characterisation of Collagenous Proteins as Major Components in the Animal Tissues by T.Hayashi (University of Tokyo) 6 Regulation of Cellular Function by Electric Stimulation System by Y.Yanagida, A.Mizuno, K.Kimura, T.Motegi, T.Haruyama, E.Kobatake and M.Aizawa (Tokyo Institute of Technology.) 7 Photolithographic Synthesis of Intelligent Microgels by Y.Ito (NAIST) 8 Cell Response tc Stimulus Given by Cell Adhesive Peptide-cairying Thermosensitive Microspheres by K.Fujinoto, K.Kisara and H.Kawaguchi (Keio University.) 9 Creation of High-performance Surface Modified by Reactive Polymeric Micelle by M. Iijima, Y.Nagasaki, M.Kato and K.Kataoka* (Science University of Tokyo, * University of Tokyo). 10 Functionalization of Polylactide Surface Using Aldehyde-ended Poly (Ethylene Glycol) and the Surface Characterization by H.Otsuka, Y.Nagasaki*, T.Okano**, Y.Sakurai** and K. Kataoka (Univ of Tokyo, * Science University. of Tokyo **Tokyo Women 's Medical University.) 11 Interaction of Poly(ethylene glycol)-Poly(D. L-lactic acid) Micelles with Surfaces by K.Enroto, Y.Nagasaki and K.Kataoka* (Science University of Tokyo, *University of Tokyo) 12 Design of an Intelligent MRI Contrast Agent Depicting Microenvironmental Differences. by M.Mikawa*,**, N.Miwa** T.Akaike and A. Maruyama* (Tokyo Institute of Technology **Njhon Scherliag KK) 13 Stimu-Responsive Behavior of a Polyrotaxane Consisting of B - Cyclodextrins and a Poly(ethylene glycol)-Poly(propylene glycol) Triblock-Copolymer by H.Fujita, T.Ooya and N.Yui fJapap. (Advanced Institute of Science and Technology.) 14 Biodegradable Polyrotaxanes for Advanced Drug Delivery Systems by T.Ooya and N.Yui (Japan Advanced Institute of Science and Technology) 15 Design of Artificial Redox Proteins with Specific Binding Activity and Its Application for Biosensor Fabrication by H.Shinohara E Yokota K Yoshitani and M Sisido (Okayama University.) 16 pH-Responsive Hydrogels for Protein Delivery by K.Kato, M.Nishida and K.Nakamae (Kobe University.) 17 A Novel Polypyrrole Film Actuator by H.Okuzaki and K.Funasaka (Yamanashi University.) 18 Regulation of Complexation Between Cyclodextrins and Azobenzene-terminated Polymer by T.Ikeda, T.Ooya and N.Yui (JAIST) 19 The Fusion Degree of Ceramic Components on Dental Casting Alloys by G.Hristea, M.Ignat and I.Patrascu (R&D. Institute for Electrical Engineering) 20 A Method for Direct Harvest of Bacterial Cellulose Filaments During Continuous Cultivation of Acetobacter Xylinum by Nakayama, H.Tamura and S.Tokura (Kansai University)

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21 Establishment of Brain Capillary Endothelial Cell Line: Application of Poly-Nisopropylacrylamide for the Trypsin Free Passage by T.Terasaki, K.Hosoya, K.Nagase, K.Tetsuka, S.Saeki, M.Tomi, N.Yanai, M Obinat M Ueda A Kikuchi** and T Okano** (Tohoku University., * YS New Tech. Lab., ** Tokyo Women 's Medical University.) 22 Characterization of Thermo-responsive Polymer-modified Surfaces Used as Intelligent Chromatography Stationary Phase by T.Yakushiji, K.Sakai, A.Kikuchi*, T.Aoyagi*, Y.Sakurai* and T.Okano* (Waseda University., *Tokyo Women 's Medical University.) 23 Cisplatin-loaded Polymeric Micelle for Tumor-directed Cancer Therapy by N.Nishiyama, M.Yokoyama*, TAoyagi*, T.Okano*, Y.Sakurai* and K.Kataoka (University. of Tokyo. *Tokyo Women 's Medical University.) 24 On-off Control of Enzymatic Activity Synchronized with Reversible Micellization of Lysozyme with Anionic Block Copolymer by A. Harada and K. Kataoka (University. of Tokyo)

Micromachines A micromachine technology development programme has been supported by METI for the past ten years with the total budget of USD 250 million with the Micromachine Center as the administrator. Medicine is one of the three application fields, as well as Microfactory, and Maintenance of electric power generation stations. Development of the necessary elements for diagnostics and treatment of cerebral blood vessels has been selected as the theme for the medical application field. The development of micro laser catheters, for example, is the target of Terumo Corporation, the major manufacturer of catheters in Japan. On the other hand, Prof. Kataoka, at Tokyo University has made a survey of the fusion of micromachine technology with gene therapy vectors. He thought non-virus vectors could be called micromachines and studied the design basis for micromachines for gene vectors, starting injection, accumulation at a target organ, and transmission into cells and nucleus. Prof. Ito, of Tokushima University, is trying to build a micro lens array on a stimuli-responsive gel, for compound eyes. This report does not cover the micromachine research from the material point of view, but the micromachine development that would give material researchers a new prospect. The micromachine programme ends this year but its concept will be continued in a new national programme probably to be called Nanotechnology Manufacturing, supported by METI.

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Regulations and Guidelines

11-1 Medical Affairs Law Medical materials and equipment are governed by the Medical Affairs Law, from government approval, manufacturing, distribution, through evaluation after sales. The materials and equipment are listed in the Medical Affairs Enforcement Ordinance issued by the Ministry of Health and Welfare. Manufacturers or importers of medical materials and equipment have to get approval from the local government before starting business. Foreign manufacturers can apply for the approval without using Japanese entities. In this case, importers are not required to obtain additional approval. The medical materials and equipment are classified into four categories: Class 1, 2, 3 and 4. Materials and equipment in Class 1 do not need approval for each kind of material or equipment. Products in this category, for example, are diagnostic equipment used outside the human body, as well as dental tools. Class 2, such as electronic blood pressure monitors and image diagnostic equipment, do not require approval if they meet the standards. Class 3, including bone, contact lenses, and artificial kidneys require approval and a clinical test in some cases. Class 4, such as pacemakers, animal-derived tools or materials need approval for each product and a clinical test in principle. There are no complete lists of products classified in each of the four categories. The government must be consulted in advance in order to ascertain in which category the product will be classified. Clinical test results conducted in foreign countries will be accepted if the tests are conducted by specified foreign organisations SETI in Finland, for example and satisfy the other criteria set by the government. The detailed information necessary for the application is described in the book (in Japanese and English) Pharmaceutical Affairs Law, Enforcement Ordinance and Enforcement Regulation. Even minor modifications of the product sometimes require a new application. It is highly recommended to discuss the necessity of an application or re-application with the government in advance.

11-2 Clone Law The Clone Law, or Law concerning Restriction of Clone Technology of Humans, was passed the Diet in December 2000. The basic points of the law are: 1 The law forbids, under penalty, transplanting, to human beings or animals, human clone embryos, human-animal hybrid embryos1) and human-animal chimera embryos2). Note 1. Fertilisation of a human generative cell and an animal generative cell or transplantation of the nucleus of a human cell to animal cell without the nucleus, Note 2. Binding a human embryo with an animal embryo or animal cells

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2 3

The Minister of Education and Science shall decide the guidelines on creation, distribution, and handling of the embryos. Such guidelines are obligatory. People handling the embryos must inform the Minister of such handling. The Minister may order changes if the plan does not conform to the guidelines.

The following shall be forbidden by the guidelines based on the law. Transplantation of the nucleus of human embryo cells to nucleus-removed human eggs Separation of early human embryos Transplantation of the nucleus of animal cells to nucleus-removed human eggs Binding animal embryos with human cell Binding human embryos with human cells This means these cases may likely be reconsidered in the near future. 11-3 Guideline for research on human embryonic stem cells The Ministry of Education and Science announced a draft of the guidelines for research with human ES cells in February 2001, requesting public opinions. It will be formalised by the summer following examination in the Integrated Science and Technology Committee of the Cabinet. The draft restricts the ES cell source only to spare embryos that are obtained in infertility treatments and would be otherwise discarded as waste. The generation of human embryos by fertilisation of sperm and eggs, in order to obtain ES cells, is forbidden. The embryos should be frozen and should be used within 14 days following fertilisation. The embryos should be free of charge except for appropriate transportation costs. Appropriate informed consents should be obtained. The use of ES cells should be limited to the basic study of life and medical science for the development of new diagnostics, treatment, or drugs. Commercial applications of ES cells are forbidden for the time being until other safety guidelines are in place. Injecting ES cells in human embryos or producing individuals from ES cells or animal embryos injected with human ES cells, is strictly forbidden. Organisations extracting ES cells will be limited to those approved by the government.

11-4 Guidelines on human cell and tissue utilisation There are no guidelines enacted yet. However, the Advanced Medical Technology Evaluation Committee in the Ministry of Health and Welfare completed a draft of A Guideline of Utilisation of Human Tissue for Transplantation and Others in April 2000. The same committee made a report of Requirements for Providing Human Tissues and Using Them for Research and Development in November 1999.

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Also, The Japan Human Cell Society will produce guidelines for the handling of human live cells as research material, by autumn 2001. The guidelines will handle collection, preservation, provision, and informed consent. JHCS is a society of doctors in the fields of surgery and obstetrics. A study made by many researchers headed by Dr. Nakamura, of the National Institute of Health Sciences, from 19961998 has provided the basis for the above drafts and reports.

11-5 Guideline for Gene Therapy and others The Ministry of Education issued the Guideline for Clinical Study on Gene Therapy in June 1994. It requires the director general of the body carrying out the gene therapy to submit a plan to the Minister of Education. The laws regarding Infertility treatments will be prepared within the next three years. The public has been informed concerning the guidelines. The ethical guidelines for human genome research will be decided by spring 2001.

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List of Universities & Institutes


Major researchers Prof. Kojima, Institute of Molecular and Cellar Regulation Prof. Katsutoshi Yoshizato, leader of Hiroshima Tissue Regeneration Project Prof. Osada Prof. Suzuki Prof. M. Akashi, Polymer Science Prof. Keiichi Fukuda Prof. Osoya, plastic surgery Prof. Kuroyanagi, Human Skin Development Center Prof. H. Maeda Fields Location Succeeded in the culture of Maebashi pancreatic stem cells to secrete insulin by finding a substance that increases b cells. Liver regeneration, Volume Hiroshima production of collagen, Insulin secreting skin Polymer gels Develops complex skin: derma and epidermis, began clinical trials at the end of 2000 Polymer nanoshperes Creates cardiovascular muscle from stem cells of rat marrow Cultures fingers Skin regeneration and repair Polymeric materials for DDS Hokkaido Kagawa, Sgikoku Kagoshima ( Kyushu) Tokyo Osaka Kanagawa Kumamoto English-language websites

Names Gunma University

Hiroshima University

http://devbiol.bio.sci.hiroshimau.ac.jp/index.html

Hokkaido University Hishi Kagawa Medical School Kagoshima University Keio University Kinki University Kitasato University Kumamoto

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University Kurume University Kyoto University Kyoto Institute of Technology Kyushu University Mechanical Engineering Laboratory Nagoya University Institute of Advanced Medical Sciences Prof. Kajiwara, Material Science Prof. Takehisa Matsuda, Medical Engineering Dr. Takashi Yamane Prof. Minoru Ueda, Department of Oral and Maxillofacial Surgery Prof. Takafumi Yoshikawa, Department of Pathology and Orthopedic Surgery Dr. Yoshiyuki Taenaka Dr. Y. Nakayama

Regeneration in animals of blood veins from umbilical cord One of the most advanced Biomaterials and Tissue Engineering research Photo-responsive materials

(Kyushu) Kyushu Kyoto Kyoto www.frontier.kyoto-u.ac.jp

Polymers for medical Kyushu applications, cardiovascular tissue engineering Centrifugal blood pumps for Tsukuba artificial hearts Skin, Mucosa, Bone, Cartilage, Secretory gland Hydroxyapatite Nagoya

www.aist.go.jp/MEL/soshiki/kiso/biome ch/biohome.htm

Nara Medical University

Nara

National Cardiovascular Center

Artificial Heart development, both pulsatile implantable type and centrifugal pump type Photoreactive polymers

Osaka Osaka

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National Institute for Advanced Interdisciplinary Research National Institute for Research in Inorganic Materials National Research Institute for Metals

Prof. Tetsuya Tateishi

3D Tissue Module Engineering, Zn-releasing calcium phosphate ceramics

Tsukuba

www.aist.go.jp/NAIR/nair_e.html

HAP and collagen nanocomposite Tsukuba Metallic biomaterials, especially surface modification Research on Planarian Tsukuba Okayama Okazaki (near Nagoya) Osaka Kobe (in the future) Develops epidermis Succeeded in making 3D liver tissue using mouse stem cells, with artificial marrow, applying gene recovery technology Uses laser for separation of stem cells, creating cardiovascular muscle Tokyo Hokkaido

www.nirim.go.jp/nirim/indexe.html www.nrim.go.jp:8080/public/kikaku/engl ish/index.html

Dr. Takao Hanawa, Biomaterials Research Team Okayama University Prof. K. Agata, Biology Faculty Okazaki National Integrated Research Institute Bioscience Center Osaka University RIKEN Saint Marianne University Sapporo Medical School Tokai University Institute of Molecular and Cellular Biology RIKEN Center for Development Biology Prof. Kumagai Prof. Toshihro Mitaka Prof. Kiyoshi Ando

www.orion.ac.jp/index.html www.imcb.osaka-u.ac.jp/imcbE.html www.riken.go.jp/engn/rworld/riken/form/cdb.html

Kanagawa

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Tokushima University Tokushima University Tokyo Dental College

Prof. Yoshihiro Ito Bioengineering Prof. Asaoka, Dental School Prof. Tsubota Dr. Yoshinari, Oral Health Science Center Prof. Toshihiro Akaike, Biomolecular Engineering Institute of Biomaterials and Bioemgineering Prof. Kataoka, Materials Department Prof. Ishihara, Materials Department Prof. Yokota and Dr. Maekawa, Medical Science Research Institute

Polymers for medical applications Shikoku Dental biomaterials Transplantation of stem cells from normal ectocornea to abnormal cornea Surface modification of titanium for dental implants Shikoku Tokyo Tokyo

Tokyo Institute of Technology Tokyo Medical and Dental University Tokyo University

Artificial liver, Tokyo DDS, Physically directed cell differentiation Covering all fields of biomaterials Tokyo Micelles, Glucose-responsive gel, Intelligent materials MPC ES cells and Hemopoietic stem cells (A centre for gene research in Japan) Tokyo Tokyo Tokyo

www.i-mde.tmd.ac.jp/index-E.html

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Tokyo Womens Medical University Tottori Medical School Toyohashi University of Technology

Prof. Teruo Okano, Institute of Biomedical Engineering Prof. Ochi Prof. Niinomi, Medical and Welfare Engineering

Temperature-responsive polymer, Cell sheet engineering, Intelligent materials Cartilage of the knee with atero collagen as scaffold Titanium for medical use

Tokyo

Tottori Toyohashi (near Nagoya)

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List of Companies

Supporting members of the Japan Biomaterials Society Company names Annual Biomaterial Products (Major Turnover* products) in million USD 3M Health Care Akita Sumitomo Subsidiary Catheters Bake of Sumitomo Bake Co. (1,100) Alcare 100 Products for orthopedic surgery Asahi Medical Subsidiary Blood dialysis equipment, of Asahi Plasma purifiers, White cell Chemical separators, ES cell separators Co. (11,000) Asahi Optical (700) Bone ceramics (Camera) (Asahi Pentax) Bacster Belinger Ingelheim Chemicals Chugai (1700) Bone ceramics (Drugs) Pharmaceutical GC 300 Dental material and equipment Screw implant SETiO

Location

English-language websites

Akita

Tokyo

Tokyo

Tokyo Tokyo

www.chugai-pharm.co.jp/ceidn02/index.html www.gcdental.co.jp/english/

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Gunze

(1,600)

Polyglycol acid (PGA) fibres, sheets and tubes for sutures, Polylactic acid (PLA) for bonebonding, Collagen (Clothing) (Drugs) Intraocular lens (Glass) Ion implantation Non-ferrous metals (Petroleum)

Osaka

Hemonetics Japan Hisamitsu Pharmaceutical HOYA Ion Engineering Institute Japan Energy Japan Gore Tex JMS Johnson & Johnson Medical Kao Kawasumi Laboratory Inc. Koken Kuraray Kyocera Medikitt Mitsubishi Materials Nippi Collagen Nikkiso Nippon Electric Glass

(450) (2,000) (18,000)

Kyushu Tokyo Osaka Tokyo www.hoya.co.jp/index-e.html www.J-energy.co.jp/eng/index.html

(6,000) 300 (250) (2,300) (5,000) (9,000) 60 (400) (2,000)

(Detergent, Food oil) Dialyses Atero collagen (Silicone) Dental resins, Artificial Kidneys (Fibres) Artificial Joints (Ceramics) Bone paste (Cement) Collagen Dialysis (Glass)

Tokyo Tokyo Osaka Kyoto Tokyo Tokyo Tokyo Ohtsu (near Kyoto)

www.kao.co.jp/e/ www.kawasumi.co.jp/e_top.html www.kuraray.co.jp/en/index.html www.kyocera.co.jp/english/index1.html www.mmc.co.jp/english/top_e.html

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Medotronics Japan Sherwood Japan NOF Corporation Nippon Zeon Nissho Nitta Gelatin Pfizer Pahrmaceutical Pristol Myer PURAC BIOCHEM BD Sata Trading Shigematsu Trading Smith & Nephew Sumitomo 3M Sumitomo Electric Sumitomo Osaka Cement Sun Medical

(1,500) (600) (180)

MPC polymer (Unsaturated fatty acids) (Rubber) Artificial kidneys, disposal products, Cord blood banking system (Supermarket) Gelatin

Tokyo Tokyo Osaka Osaka www.nissho.co.jp/english/index.html

Importer of medical materials and equipment (Importer of chemicals and foods) (7,000) (1,500) 20 (Subsidiar y of Mitsui Chemical Co.) (Cables) Bone material (Cement) Import and export of medical equipment, Dental bonding materials, MS coat

Tokyo Osaka

Osaka Osaka Shiga (near Kyoto)

www.sei.co.jp/ www5.mediagalaxy.co.jp/soc./sumitomo_e/in dex.html

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Takiron

PLLA bone bonding material, Osaka www.takiron.co.jp/english/english/index.html Gels for ultrasonic tests (Housing materials) Teijin (5,500) oxygen therapy, pharmaceuticals Osaka www.teijin.co.jp/japanese/indexJ.html (Fibres, Drugs) Terumo (1,400) Catheters & disposal products Tokyo www.terumo.co.jp/English/index.html Tokai Medical Catheters, Balloon catheters with Kasugai (near Products drug delivery function Nagoya) Toray (9,000) Artificial Kidney, Dialysis Tokyo www.toray.co.jp/e/index.html (Fibres) Toyo Boseki (2,500) Licensee of the Apligraf double Osaka www.toyobo.co.jp/e/index.html layer skin from Organogenesis, US (Fibres) Ube Kosan Artificial blood vessels Ube www.ube-ind.co.jp/english/index_e.html Yunichika (2,000) (Fibres) Osaka www.unitika.co.jp/home-e.html Sources: Japan Biomaterials Society (in part) *Annual turnover: figures without brackets are bio-related products; those in the brackets are for all products.

(500)

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Biomaterial companies, not members of the Japan Biomaterials Society Company names Annual Biomaterial Products (Major Location English-language websites Turnover* products) in million USD J-Tec Venture company for artificial Gamagouri skin (near Nagoya) Kaneka (2,300) Blood Purifier Lixell Osaka www.kaneka.co.jp/kaneka-e/index.html Kirin Beer Co. (13,000) Blood related drugs (Beer) Tokyo www.kirin.co.jp/english/ Menicon 300 Hard Contact Lens Nagoya www.menicon.co.jp/english/index.html Mizuho Medical 80 Grips for Titan aneurysm Tokyo Industry implants Takara (1,800) Enzymes, DNA arrays Tokyo www.takara.co.jp/english/index.html *Annual turnover: figures without brackets are bio-related products; those in the brackets are for all products.

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(1) (2) (3) (4)

Major Sources

Japan Biomaterial Symposium, November 2000 High Performance Biomaterials Symposium (Biometals), November 2000 Medical Polymer Symposium, Feb. 2001 The third symposium on Advanced Functional Materials by Chemical Technology Strategic Promotion Organisation, November 2000 (5) Research Plan presentation on Disease Genome and Regenerative Medical Treatment by the Ministry of Health and Welfare, May 2000 (6) International Symposium on Advanced Biomaterial & Tissue Engineering, February 1999 (7) Medical Polymer Symposium Proceedings, July 2000 (8) Proceedings of annual meeting of DDS, July 2000 (9) Proceedings of the third annual meeting of the Japanese Society for Tissue Engineering, June 2000 (10) Symposium proceedings on Industrialisation of Tissue Engineering, March 2000 (11) Proposal for Impending Key Issues in Medical Bio Engineering by Japan Society of Science, May 2000 (12) Biomaterials Journal (13) Ceramics Journal (14) Drug Delivery System (15) NEDO brochures (16) JSPS Internet report on Tissue Engineering

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References: Sources: Biomaterial Symposium, November 2000; Cermaics (1999) 34:7 JAFCO report, January 2000; The Market for Dental Equipment, Materials and Chemicals, October 1999, Yano Research. 3 Shigeo Miwa (2000) Biomaterial Symposium (oral presentation) 4 Oka, M., Yura, S., and Toguchida, J. (1999) The development of artificial osteochodral composite material. Advances in Science and Technology 28:385-397 5 Yoshikawa, T., Ohgushi H ,et al., J. (1998) Biomed. Mater. Res., 41, 568-73; Yoshikawa T., et al., (2000) Transplantation 69, 128-134; Yoshikawa T., et al., (2000) J Bone Miner Res. 15, 1147-1157 6 Otsuka, M Yoneoka K., et al., (1999) J. Pharm. Pharmacol., 51, 475-81 7 High Performance Biomaterial Symposium, November 2000; Biomaterial Symposium, November 2000
1 2

Medical Polymer Symposiums, July 2000; Medical Polymer Symposiums, February 2001; Biomaterial Symposium, November 2000. 9 Ishihara, H. et al., (1992) J. Biomed. Mater. Res., 26, 1543-1552; Ishihara, K. et al., (1999) Modification of polysulfone with phospholipid polymer for improvement of the blood compatibility. Part 2. Protein adsorption and platelet adhesion-, Biomaterials, 20(17), 1553-1559 10 Nakayama, Y., Matsuda T. (1999) J. Biomed. Mater. Res. 48, 511 11 2Otsu, T., Matsumoto A. (1998) Adv. Polym. Sci., 136, 75 12 Matsumura, Y. et al. J. J. (1999) Cancer Research, 90 (1), 122-128; Chung, J.E. et al. (1998) J. Controlled Release, 53, 119-130 13 Matsumura, Y. et al. J. J. (1999) Cancer Res., 90, 122-128 14 Kataoka, K. et al. (1998) J. Amer. Chem. Soc., 120, 12694-12695 15 Takakura Y, Hashida M (1996) Macromolecular carrier systems for targeted drug delivery, Pharmacokinetic considerations on biodistribution. Pharm Res 13: 820-831; Koyama Y, Miyagawa T, Kawaide A, Kataoka K(1996) Receptor-mediated absorption of high molecular weight dextran from intestinal tract. J Contro Rel 41:171-176 16 Maeda H, Matsumoto T, Konno T, Iwai K, Ueda M (1984) Tailor-making of protein drugs by polymer conjugation for tumour targeting: a brief review of SMANCS. J Protein Chem 3: 181-193; Maruyama K, et al.(1992) Prolonged circulation time in vivo of large unilamellar liposomes composed of distearoyl phosphatidylcholine and cholesterol containing amphiphathic poly(ethylene glycol), Biochim Biophys Acta 1128: 44-49; Katayose S, Kataoka K (1999) Remarkable increase in nuclease resistance of plasmid DNA through supramolecular assembly with poly(ethylene glycol)-poly(Llysine)block copolymer, J Pharm Sci 87: 160-163 17 Masashige Shinkai, Mitsugu Yanase, Masataka Suzuki, Hiroyuki Honda, Toshihiko Wakabayashi, Jun Yoshida and Takeshi Kobayashi (1999) Intracelluar hyperthermia for cancer using magnetic cationic liposomes, Journal of Magnetism and Magnetic Materials, 194(1), 176-184 18 Sugibayashi K, et al. (1999) Analysis of skin disposition of flurbiprofen after topical application in hairless rats. Chem Pharm Bull 47: 749-754 19 Yanagimoto G. et al. (1998) A new in-situ experimental method to evaluate the cutaneous disposition of flurbiprofen after topical application in hairless rats. Pharm. Pharmcol. Commun. 4: 261-265 20 Sakane T., et al. (1995) Direct drug transport from the rat nasal cavity to the cerebrospinal fluid: the relation to the molecular weight of drugs. J Pharm Pharmacol 47: 379-381 21 Okano T. et al. (1993) Intelligent polymeric materials for drug delivery. Biomedical Applications of Polymeric Materials. 407-428 (CRC Press, Inc. Boca Raton)
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Hatanaka T et al. (1994) An application of the hydrodynamic pore theory to percutaneous absorption of drugs. Pharm Res 11: 654-658 23 Yamashita F. et al. (1993) Analysis of skin penetration enhancement based on a two-layer skin diffusion model with polar and nonpolar routes in the stratum corneum: dose-dependent effects of 1geranylazacycloheptan-2-one on drugs with different lipophilicities. Bio Pharm Bull 16: 690-697; Yamamoto A. et al. (1994) Absorption enhancement of intrapulmonary administered insulin by various absorption enhancers and protease inhibitors in rats. J Pharmcol 46: 14-18 24 Yoshida M. et al. (2000) Effects of application voltage and cathode and anode positions at electroporation on the in vitro permeation of benzoic acid through hairless rat skin Chem. Pharm. Bull. 39 25 Inoue N. et al (1996) Fundamental investigation of a novel drug delivery system-A transdermal delivery system with jet injection. Int J Pharmaceut 137: 75-84 26 Ueda H. et al. (1996) Combined effect of ultrasound and chemical enhancers on the skin permeation of aminopyrine. Int J Pharmaceut 143: 37-45 27 Okano, M (2000) 7th Symposium on Interface, Tokyo; Yamato, M., Konno, C., Kushida, A., Hirose. M., Utsumi. M., Kikuchi. A., Okano. T. (2000) Biomaterials, 21, 981-986; Kushida, A., Yamato. M., Konno, C., Kikuchi, A., Sakurai, Y., Okano. T. (2000) J. Biomed. Mater. Res., 51. 226-233; Hirose. M., Kwon, O.H., Yamato, M., Kikuchi. A., Okano, T. (2000) Biomacrocolecules, 1, 377-381 28 Matsumoto, A., et al. (2000) J Thoroac Cardiovasc Surg., 48, 153-160 29 Nikkei Weekly, Sept. 4, 2000; Nikkei, Jan. 15, 2001 30 Business and Technology News, Oct. 5, 2000; Nikkei, Jan. 15; Nikkei Feb 8 2001 31 Nikkei Sangyo News October 30, 2000 32 Nikkei Sangyo Sept. 25, 2000
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