S34 Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S29–S40

The focus of this study is 1,3-bis(p-carboxyphenoxy)propane(CPP) and sebacic acid (SA) copolymers. They are made via melt–condensation into homopolymers and copolymers to investigate thedifferent degradation patterns and drug releasemechanisms of matrixdevices andmicrospheres. By using double emulsion and other micro-encapsulation methods, the solid microspheres and double-wallmicrospheres (DWMS) of polymers are fabricated. During thefabricating processes, drugs can be loaded into these microspheres.A drug release implant device could be used after operation for slowrelease of drug, and such device can be produced by moldingcompression of polyanhydride microspheres. These microspherescan be used as parts to form release devices by molding method. Byraising the temperature approach to the melting points (~70–80 °C),these microspheres could be compressed into a microchamberpolyanhydride device. And the desirable release profiles could beobtained by adjusting the polymer components and structure ofdevices.

doi:10.1016/j.jbiosc.2009.08.075

BM-P6

Effect of carboxylates on triglyceride accumulation and a TNFαproduction in mouse cells

Maeda Miwako, Mimura Minori, and Shiomi Naofumi

Kobe College, Nishinomiya, Hyogo, Japan

When a person has an obese condition, insulin resistance andmetabolic syndrome are often induced by abnormal secretions of theadipocytokines, such as TNFα and leptin. Here, we tried to clarify theeffects of carboxylates on the triglyceride accumulation and the TNFαproduction in mouse cell lines.

The adipocyte 3T3-L24, hepatocyte NMuLi, and myoblast C2C12were cultured in DMEM containing 10% FBS to examine thecharacteristics of triglyceride accumulation. When 0.28 mM long-chain fatty acids were added in the medium, triglyceride wasunusually accumulated in those cells. The result suggested thatlong-chain fatty acid was a key factor of triglyceride accumulation.Next carboxylates were added to inhibit the triglyceride synthesis.Sodium citrate or acetate added in the medium effectively inhibitedtriglyceride accumulation in those cells. The mechanism of inhibitionwas also examined. In case of the addition of sodium citrate, the rate ofglucose consumption was enhanced but the rate of sodium oleic wasthe same as that of control. Thus, the inhibition of triglycerideaccumulation by citrate was caused by both the inhibition of thesynthesis of triglyceride and the enhancement of degradation of oleicacid. Additionally, some other compounds containing carboxylic bondwere also examined. Carnitine, which was a transporter of fatty acidsinto the inner membrane of mitochondria, was strongly inhibited theaccumulation.

Finally the effect of the carboxylates on TNFα production in theadipocyte was examined. Production of TNFα was increased by thedifferentiation from pre-adipocyte to adipocyte. On the contrary,TNFα production was depressed a little by the addition of sodiumcitrate. Therefore, TNFα production did not closely relate to thetriglyceride accumulation and was supported by another transcrip-tional factor.

doi:10.1016/j.jbiosc.2009.08.076

BM-P7

Quality assessment of collagen substrate by morphologicalresponse of chondrocytes

Masrina Mohd Nadzir,1 Nao Maruyama,1 Masahiro Kino-oka,2

and Masahito Taya1

Divisions of Chemical Engineering, Graduate School of Engineering Science,Osaka University, Toyonaka, Japan1 and Department of Biotechnology,Graduate School of Engineering, Osaka University, Suita, Japan2

Difficulty in non-invasive evaluation of chondrocyte behaviors inthree-dimensional collagen-embedded cultures has brought forwardthe requirement of a two-dimensional evaluation system. Previouswork reported that the collagen substrate could estimate the extent ofdedifferentiating the state of chondrocytes by observation of cellularmorphology (1). In this study, the morphological changes of rabbitchondrocytes were investigated in relation to structural variation ofcollagen surface. Culture surfaces coated with high density collagentype I (CL substrate) were prepared under a conditionwith or withoutexposure to air. By the scanning electron microscopy of substrate, lessfibril formation and non-fibrous collagen in spaces among collagenfibrils were observed on the air-exposed substrate. Yunoki et al. (2)suggested the inhibitory effect on fibril formation by predominantcross-linking of monomeric collagen. One day incubation of cells on CLsubstrate prepared through exposure to air was conducted to estimatethe frequency of round shape cells (fR). The decrease in fR value wasobserved on CL substrate with exposure to air compared to that on theCL substrate without exposure. This suggested that the collagenbinding sites to cell integrin receptors decreased with decreasing fibrilformation. Modulation of fibril properties is normally achievedthrough chemical cross-linking of collagen (3). The current studydemonstrated the possibility of using oxygen for regulating collagenfibril formation for the regulation of cell signaling.

References

1. Kino-oka, M., Maeda, Y., Sato, Y., Maruyama, N., Takezawa, Y., Khoshfetrat, A.B.,Sugawara, K., and Taya, M.: Morphological evaluation of chondrogenic potency inpassaged cell populations, J. Biosci. Bioeng., 107, 544-551 (2009).

2. Yunoki, S., and Matsuda, T.: Simultaneous processing of fibril formation and cross-linking improves mechanical properties of collagen, Biomacromolecules, 9, 879-885(2008).

3. Friess, W.: Collagen – biomaterial for drug delivery, Eur. J. Pharm. Biopharm., 45,113-136 (1998).

doi:10.1016/j.jbiosc.2009.08.077

BM-P9

Optical properties of the FITC–AMP nanoparticles

Taeho Lee, and Hyun-Jae Shin

Chosun University, Gwangju, Republic of Korea

The FITC (fluorescein isothiocyanate) is the fluorescence imagingprobe in vivo. We investigated the morphology, optical properties andphoto-stability of the AMP (aminopropyl-Mg-pyrosilicate) and FITC–AMP nanoparticle. FITC–APTS conjugate was synthesized by thethiourea reaction of the FITC and APTS (3-aminopropyltriethoxysi-lane) in ethanol. To this solution was added MgCl2 and then stirredmagnetically for 24 h. The FITC–AMP nanoparticles were then

S35Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S29–S40

centrifuged and subsequently washed three times with ethanol. TheFITC–APTS conjugate solution and FITC–AMP nanoparticles areprotected from light during reaction and storage to prevent photo-bleaching. The particle size of the AMP nanoparticles were about50 nm, and the terrace structure of the nanosheet was observed bySEM, and the interlayer d-spacing of the AMP was 1.4 nm by XRD. Inthe FT-IR study, we observed the Mg-O (559 cm−1), Si-O-C(937 cm−1), Si-O-Si (1030 cm−1), \OH (3390 cm−1), and \NH3

+

(3005 cm−1) vibrational modes of the AMP nanoparticle. But the\NH3

+ vibrational mode of the FITC–AMP nanoparticle was notobserved caused by the thiourea reaction of the FITC and primaryamine group of APTS. The fluorescence band of the FITC–AMPnanoparticles (fluorescence peak 543 nm) was observed in the redshift and decreased bandwidth in the green region when comparedwith the fluorescence band of the FITC (fluorescence peak 521 nm).The photo-degradation of the FITC was observed in the fluorescencequenching as exposed by 300 W Xe light. However, the FITC–AMPnanoparticles were observed the photoisomerization with light suchas quenching and recovery of fluorescence with light-on and light-offconditions, respectively. Therefore we will be discussing the photo-isomerization of the FITC–AMP nanoparticle in this presentation.

Reference

1. Swadeshmukul, S., Bernd, L., Chiara, B., Debamitra, D., Zehui, C., Weihong, T.,Brij, M. M., and Robert, A. M.: Fluorescence lifetime measurements to determinethe core-shell nanostructure of FITC-doped silica nanoparticles: An optical approachto evaluate nanoparticle photostability, J. Luminescence, 117, 75-82 (2006).

doi:10.1016/j.jbiosc.2009.08.078

BM-P10

Construction of skeletal muscular tissue-like structures by amagnetic force-based tissue engineering technique

Yasunori Yamamoto,1 Akira Ito,1 Masahiro Kato,1 Yoshinori Kawabe,1

Kazunori Shimizu,2 Hideaki Fujita,2 Eiji Nagamori,2

and Masamichi Kamihira1

Kyushu University, Fukuoka, Japan1 and Toyota Central R&D LaboratoriesInc., Aichi, Japan2

Artificial muscular tissues composed of mouse myoblast C2C12cells were fabricated using a magnetic force-based tissue engineeringtechnique (1). Magnetite cationic liposomes, in which magnetitenanoparticles were encapsulated by cationic liposomes, were used formagnetic labeling of C2C12 cells. C2C12 cells labeled with magnetitecationic liposomes were seeded into a well of ultra-low cellattachment culture plates. When a magnet was positioned under-neath the well, cells accumulated evenly onto the culture surface andformed a multilayered cell sheet. When a silicone plug was positionedat the center of the well during fabrication of the cell sheets, the cellsheets drastically shrank and formed a ring-like assembly around theplug. Histological study revealed that cells in the cellular ring werehighly oriented in the direction of circumference by the tensiongenerated within the structure. The cellular ring was hooked aroundtwo pins separated by 10 mm and could be cultured for 6 d in adifferentiation medium. The C2C12 cells were differentiated to formmyogenin-positive multinucleated myotubes. After a 7-d culture inthe differentiation medium, C2C12 cellular rings were successfullycontracted in response to an electric stimulation (15 V, 10-ms widepulse) and the contraction force was 20.1 μN. These results suggest

that this procedure can provide a novel strategy for muscular tissueengineering.

Reference

1. Yamamoto, Y., Ito, A., Kato, M., Kawabe Y., Shimizu, K., Fujita, H., Nagamori, E., andKamihira, M.: Preparation of artificial skeletal muscle tissues by a magnetic force-based tissue engineering technique, J. Biosci. Bioeng., (in press).

doi:10.1016/j.jbiosc.2009.08.079

BM-P11

Genetically modified angiogenic cell sheets fabricated bymagnetic force-based tissue engineering techniques

Hirokazu Akiyama, Akira Ito, Yoshinori Kawabe, andMasamichi Kamihira

Kyushu University, Fukuoka, Nishi-ku, Japan

In tissue engineering, an angiogenic potential of transplants isconsidered to be important for the long-term maintenance of cellsurvival and tissue functions particularly in the regeneration of highmetabolic tissues including skeletal muscle tissue. Insufficientvascularization induces the improper cell integration or cell death inthe grafts, leading to nonfunctional tissues with low cell density. Inthis report, we combined two magnetic force-based techniquesmediated by magnetite cationic liposomes (MCLs), magnetofection,and magnetic cell accumulation, to create three-dimensional (3D)tissue constructs genetically-engineered to express vascular endothe-lial growth factor (VEGF), a potent angiogenic stimulator. For genedelivery, a retroviral vector encoding an expression cassette of VEGFwas labeledwithMCLs so that the viral particles could bemagneticallyattracted onto a monolayer of C2C12 cells, that is, the skeletal musclemyoblast cell line. By the MCL-mediated infection, the transductionefficiency increased up to 6.7-fold compared with the conventionalmethod. For fabrication of tissue constructs, the magnetic force-basedtissue engineering (Mag-TE) technique, whereMCL-labeled cells wereaccumulated in the presence of a magnetic field, was applied topromote the spontaneous formation of a multilayered cell sheet. VEGFgene-engineered C2C12 (C2C12/VEGF) cell sheets constructed usingthe two magnetic force-based techniques were transplanted sub-cutaneously into nude mice. Histological analyses revealed that theC2C12/VEGF cell sheet grafts produced thick tissues in the mice andmaintained high-cell density with promoted vascularization on day14. Moreover, the cells formed multinucleated myotubes in both coreand peripheral regions of the C2C12/VEGF cell sheet-derived tissues.Taken together, these results suggest that the method combiningmagnetofection and Mag-TE techniques represents a powerfulstrategy in tissue engineering involving skeletal muscle repair.

doi:10.1016/j.jbiosc.2009.08.082

BM-P12

BMP-2 immobilized gelatin-β-chitosan scaffold for enhancingbone regeneration

Il Keun Kwon, Sung Eun Kim, Min Sun Bae, Do Wan Kim,and Kyoung Kyu Choi

Kyung Hee University, Seoul, Republic of Korea