Grant-in-Aid for Scientific Research (B)
|Allocation Type||Single-year Grants |
|Research Institution||Kyoto Prefectural University of Medicine (2003)|
Kyoto University (2001-2002)
KAIHARA Satoshi Kyoto Prefectural University of Medicine, Graduate School of Medical Science Transplantation and Regenerative Surgery, Associate Professor, 医学研究科, 助教授 (70324647)
TABATA Yasuhiko Kyoto University, Institute for Frontier Medical Sciences, Professor, 再生医科学研究所, 教授 (50211371)
UEMOTO Sinji Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Professor, 医学研究科, 教授 (40252449)
TANAKA Kouichi Kyoto University, Graduate School of Medical Science, Professor, 医学研究科, 教授 (20115877)
OKAMOTO Masahiko Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Assistant Professor, 医学研究科, 講師 (90295650)
YOSHIMURA Norio Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Professor, 医学研究科, 教授 (00191643)
木内 哲也 京都大学, 医学研究科, 助教授 (40303820)
|Project Period (FY)
2001 – 2003
Completed(Fiscal Year 2003)
|Budget Amount *help
¥14,300,000 (Direct Cost : ¥14,300,000)
Fiscal Year 2003 : ¥2,600,000 (Direct Cost : ¥2,600,000)
Fiscal Year 2002 : ¥3,300,000 (Direct Cost : ¥3,300,000)
Fiscal Year 2001 : ¥8,400,000 (Direct Cost : ¥8,400,000)
|Keywords||regeneration of small intestine / tissue engineering / extracellular matrix / microcapsule|
1)Isolation and implantation of cells from small intestine
Mixtures of mucosal and mesenchymal cells, so called 'intestinal epithelial organoid units', were isolated from neonatal rats. We modified this method with changing the concentration of collagenase suspension and the duration of digestion, which improved the number of isolated organoid units 20% more then the conventional method.
The isolated organoid units were seeded to polymer tubes created from polyglycolic acid (PGA) and implanted to the syngenic rat. Three weeks after the implantation, the unit-polymer constructs grew up to create cyst, which was lined with well-developed neomucosa. The neointestine was then anastomosed to native jejunum. Twelve weeks after the anastomosis, the neointestine grew up to 4cm in average length and showed the same level of glucose absorptive function as normal intestine.
2)Development of polymer scaffolds
The polymer scaffold made from PGA fiber had a disadvantage in cell attachment since the seed
ed cells only attached at the crossing of the fibers. We assumed that the PGA fiber was not suitable for cell attachment due to its round surface and created membranes between each fiber with 7% poly-lactic acid (PLLA) solution. Moreover, the surface of the PLLA membrane was hydrolyzed with NaOH solution because PLLA itself was poor in cell affinity. With histological study of the polymer after seeding, these modifications improved the attachment of the seeded cells to the polymer scaffolds dramatically.
The modified PGA polymer was implanted into rats after cell seeding ; however, we missed to induce neomorphogenesis of small intestine in this approach. To clarify the reason for this disappointed result, the polymer scaffolds were harvested a few days after implantation and sent to histological study, which showed that most of the seeded cells, especially the cells close to the center of the polymer scaffold, died within a couple of days after implantation. From these findings, we assumed that the modification of the PGA polymer increased the number of implanted cells, which amplified consumption of oxygen and nutrients for their survival. However, the PLLA membrane disturbed their transport and most of the implanted cells died due to the lack of oxygen and nutrients for their survival.
3)Evolution of microcapsule
Microcapsule made of gelatin could be used for controlled release of epidermal growth factor (EGF). Also the concentration and duration of EGF release could be managed with changing the water density of the gelatin. However, the microcapsule was not applied to in vivo experiment because of the failure in creating new polymer scaffolds. Less