MSA  Vol.4 No.8 A , August 2013
Usefulness of Agarose Mold as a Storage Container for Three-Dimensional Tissue-Engineered Cartilage

The efficiency of substance exchange may be decreased when the thickness and volume of such a tissue-engineered cartilage that is composed of cultured cells and porous scaffold increase. Moreover, during the transport of this construct with complicated shapes, excessive and focal mechanical loading may cause deformation. The establishment of incubation and transport methods is necessary for the three-dimensional tissue-engineered cartilage. Therefore, we investigated the preparation of an agarose mold with a concavity similar to the shape of 3-dimensional tissue-engineered cartilage to prevent excessive and focal concentration of stress, while avoiding interference with substance exchange as much as possible. Firstly, we investigated the preparation at 1% - 4% agarose concentrations. Since the mechanical strength was insufficient at 1%, 2% was regarded as appropriate. Using 2% agarose, we prepared a mold with a 5 × 5 × 5 mm concavity to accommodate tissue-engineered cartilage (5 × 5 × 5 mm mixture of 1.5 × 107 cells and collagen gel), and stored the regenerative cartilage in it for 2 and 24 hours. On comparison with storage in a plastic mold with the same shape in which substance exchanged from side and bottom was impossible, although no significant differences were noted in the number or viability of cells after 2 hours, these were markedly reduced in the plastic mold after 24 hours. It was confirmed that favorable cell numbers and viability were maintained by immediately retaining the regenerative cartilage in the culture medium in the agarose mold and keeping the temperature at 37°C. Since this agarose mold also buffers against mechanical forces loaded on the three-dimensional regenerative tissue, it may be useful as a container for storage and transport of large-sized three-dimensional regenerative tissue.

Cite this paper: Y. Mori, S. Kanazawa, M. Watanabe, H. Suenaga, K. Okubo, S. Nagata, Y. Fujihara, T. Takato and K. Hoshi, "Usefulness of Agarose Mold as a Storage Container for Three-Dimensional Tissue-Engineered Cartilage," Materials Sciences and Applications, Vol. 4 No. 8, 2013, pp. 73-78. doi: 10.4236/msa.2013.48A010.

[1]   J. A. Baddour, K. Sousounis and P. A. Tsonis, “Organ Repair and Regeneration: An Overview,” Birth Defects Research C: Embryo Today, Vol. 96, No. 1, 2012, pp. 1-29. doi:10.1002/bdrc.21006

[2]   M. Ochi, Y. Uchio, K. Kawasaki, S. Wakitani and J. Iwasa, “Transplantation of Cartilage-Like Tissue Made by Tissue Engineering in the Treatment of Cartilage Defects of the Knee,” The Journal of Bone & Joint Surgery British, Vol. 84, No. 4, 2002, pp. 571-578. doi:10.1302/0301-620X.84B4.11947

[3]   H. Yanaga, K. Yanaga, K. Imai, M. Koga, C. Soejima and K. Ohmori, “Clinical Application of Cultured Autologous Human Auricular Chondrocytes with Autologous Serum for Craniofacial or Nasal Augmentation and Repair,” Plastic and Reconstructive Surgery, Vol. 117, 2006, pp. 2019-2030.

[4]   H. Yamaoka, Y. Tanaka, S. Nishizawa, Y. Asawa, T. Takato and K. Hoshi, “The Application of Atelocollagen Gel in Combination with Porous Scaffolds for Cartilage Tissue Engineering and Its Suitable Conditions,” Journal of Biomedical Materials Research Part A, Vol. 93, 2010, pp. 123-132.

[5]   Y. Tanaka, H. Yamaoka, S. Nishizawa, S. Nagata, T. Ogasawara, Y. Asawa, Y. Fujihara, T. Takato and K. Hoshi, “The Optimization of Porous Polymeric Scaffolds for Chondrocyte/Atelocollagen Based Tissue-Engineered Cartilage,” Biomaterials, Vol. 31, No. 16, 2010, pp. 4506-4516. doi:10.1016/j.biomaterials.2010.02.028

[6]   K. Hoshi, Y. Fujihara, Y. Asawa, S. Nishizawa, S. Kanazawa, T. Sakamoto, M. Watanabe, T. Ogasawara, H. Saijo and T. Takato, “Recent Trends of Cartilage Regenerative Medicine and Its Application to the Oral and Maxillofacial Surgery,” Oral Science International, Vol. 10, No. 1, 2013, pp. 15-19. doi:10.1016/S1348-8643(12)00049-3

[7]   S. Sekiya, T. Shimizu and T. Okano, “Vascularization in 3D Tissue Using Cell Sheet Technology,” Regenerative Medicine, Vol. 8, No. 3, 2013, pp. 371-377. doi:10.2217/rme.13.16

[8]   D. A. Rees, “Strucutre, Conformation, and Mechanism in the Formation of Polysaccaride Gels and Networks,” Advances in Carbohydrate Chemistry & Biochemistry, Vol. 24, 1969, pp. 267-332. doi:10.1016/S0065-2318(08)60352-2

[9]   S. Arnott, A. Fulmer, W. E. Scott, I. C. Dea, R. Moorhouse and D. A. Rees, “The Agarose Double Helix and Its Function in Agarose Gel Structure,” Journal of Molecular Biology, Vol. 90, No. 2, 1974, pp. 269-284. doi:10.1016/0022-2836(74)90372-6

[10]   K. Yonenaga, S. Nishizawa, Y. Fujihara, Y. Asawa, S. Kanazawa, S. Nagata, T. Takato and K. Hoshi, “The Optimal Conditions of Chondrocyte Isolation and Its Seeding in the Preparation for Cartilage Tissue Engineering,” Tissue Engineering Part C: Methods, Vol. 16, No. 6, 2010, pp. 1461-1469. doi:10.1089/ten.tec.2009.0597

[11]   Y. Tanaka, T. Ogasawara, Y. Asawa, H. Yamaoka, S. Nishizawa, Y. Mori, T. Takato and K. Hoshi, “Growth Factor Contents of Autologous Human Sera Prepared by Different Production Methods and Their Biological Effects on Chondrocytes,” Cell Biology International, Vol. 32, 2008, pp. 505-514. doi:10.1016/j.cellbi.2007.12.012

[12]   T. Takahashi, T. Ogasawara, J. Kishimoto, G. Liu, H. Asato, T. Nakatsuka, E. Uchinuma, K. Nakamura, H. Kawaguchi, T. Takato and K. Hoshi, “Synergistic Effects of FGF-2 with Insulin or IGF-I on the Proliferation of Human Auricular Chondrocytes,” Cell Transplant, Vol. 14, No. 9, 2005, pp. 683-693. doi:10.3727/000000005783982675

[13]   H. Yamaoka, H. Asato, T. Ogasawara, S. Nishizawa, T. Takahashi, T. Nakatsuka, I. Koshima, K. Nakamura, H. Kawaguchi, U. I. Chung, T. Takato and K. Hoshi, “Cartilage Tissue Engineering Using Human Auricular Chondrocytes Embedded in Different Hydrogel Materials,” Journal of Biomedical Materials Research Part A, Vol. 78, No. 1, 2006, pp. 1-11. doi:10.1002/jbm.a.30655

[14]   R. Aoyagi and T. Yoshida, “Freuqency Equations of an Ultrasonic Vibrator for the Elastic Sensor Using a Contact Impedance Method,” Japanese Journal of Applied Physics, Vol. 5B, 2004, pp. 3204-3209. doi:10.1143/JJAP.43.3204

[15]   Y. Kang, J. Yang, S. Khan, L. Anissian and G. A. Ameer, “ A New Biodegradable Polyester Elastomer for Cartilage Tissue Engineering,” Journal of Biomedical Materials Research Part A, Vol. 77, No. 2, 2006, pp. 331-339. doi:10.1002/jbm.a.30607

[16]   K. Yonenaga, S. Nishizawa, M. Akizawa, Y. Asawa, Y. Fujihara, T. Takato and K. Hoshi, “Utility of NucleoCounter for the Chondrocyte Count in the Collagenase Digest of Human Native Cartilage,” Cytotechnology, Vol. 62, 2010, pp. 539-545. doi:10.1007/s10616-010-9304-y

[17]   Y. Ohyabu, N. Kida, H. Kojima, T. Taguchi, J. Tanaka and T. Umemura, “Cartilaginous Tissue Formation from Bone Marrow Cells Using Rotating Wall Vessel (RWV) Bioreactor,” Biotechnology and Bioengineering, Vol. 95, No. 5, 2006, pp. 1003-1008. doi:10.1002/bit.20892

[18]   S. Sakai, H. Mishima, T. Ishii, H. Akaogi, T. Yoshioka, Y. Ohyabu, F. Chang, N. Ochiai and T. Umemura, “Rotating Three-Dimensional Dynamic Culture of Adult Human Bone Marrow-Derived Cells for Tissue Engineering of Hyaline Cartilage,” Journal of Orthopaedic Research, Vol. 27, No. 4, 2009, pp. 517-521. doi:10.1002/jor.20566

[19]   K. S. Furukawa, H. Suenaga, K. Toita, A. Numata, J. Tanaka, T. Ushida, Y. Sakai and T. Tateishi, “Rapid and Large-Scale Formation of Chondrocyte Aggregates by Rotational Culture,” Cell Transplant, Vol. 12, 2003, pp. 475-479.

[20]   C. Cohen, “Optical Rotation and Helical Polypeptide Chain Configuration in Collagen and Gelatin,” Journal of Biophysical and Biochemical Cytology, Vol. 1, No. 1, 1955, pp. 203-214. doi:10.1083/jcb.1.3.203