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Evaluation of <i>in vivo</i>migration of chondrocytes from tissue-engineered cartilage that was subcutaneously transplanted in mouse model

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Open Journal of Regenerative Medicine 2013年第4期2卷 93-98页

作者： Mariko Matsuyama yuko fujihara Ryoko Inaki Satoru Nishizawa Satoru Nagata Tsuyoshi Takato Kazuto Hoshi Department of Cartilage & Bone Regeneration (Fujisoft) Graduate School of Medicine The University of Tokyo Tokyo Japan Department of Sensory and Motor System Medicine The University of Tokyo Hospital Tokyo Japan NAGATA Microtia and Reconstructive Plastic Surgery Clinic Toda Saitama Japan Translational Research Center The University of Tokyo Hospital Tokyo Japan

For regenerative medicine, clarification of in vivo migration of transplanted cells is an important task to secure the safety of transplanted tissue. We had prepared tissue-engineered cartilage consisting of cultured chondrocytes with collagen hydrogel and a biodegradable porous polymer, and we clinically applied it for treatment of craniofacial anomaly. To verify the safety of this tissue-engineered cartilage, we had syngenically transplanted the tissue-engineered cartilage using chondrocytes harvested from EGFP-transgenic mice into subcutaneous pocket of wild type mice, and investigated localizations of transplanted chondrocytes in various organs including cerebrum, lung, liver, spleen, kidney, auricle, gastrocnemius, and femur. After 8 to 24 weeks of the transplantation, accumulation of cartilaginous matrices was observed in tissue-engineered cartilage, while EGFP-positive transplanted chondrocytes were localized in this area. Otherwise, no EGFP was immunohistochemically detected in each organ, suggesting that subcutaneously-transplanted chondrocytes do not migrate to other organs through the circulation. In cartilage tissue engineering using cultured chondrocytes, risk for migration and circulation of transplanted cells seemed negligible, and that ectopic growth of the cells was unlikely to occur, showing that this is safe technique with regard to the in vivo migration of transplanted cells.

关键词： Cartilage Tissue Engineering Chondrocyte Migration

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Usefulness of Agarose Mold as a Storage Container for Three-Dimensional Tissue-Engineered Cartilage

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Materials Sciences and Applications 2013年第8期4卷 73-78页

作者： Yoshiyuki Mori Sanshiro Kanazawa Makoto Watanabe Hideyuki Suenaga Kazumi Okubo Satoru Nagata yuko fujihara Tsuyoshi Takato Kazuto Hoshi Department of Cartilage & Bone Regeneration (Fujisoft) Graduate School of Medicine The University of Tokyo Tokyo Japan Division of Tissue Engineering The University of Tokyo Hospital Tokyo Japan Department of Sensory and Motor System Medicine Graduate School of Medicine The University of Tokyo Tokyo Japan Department of Sensory and Motor System Medicine Graduate School of Medicine The University of Tokyo Tokyo Japan Department of Cartilage & Bone Regeneration (Fujisoft) Graduate School of Medicine The University of Tokyo Tokyo Japan Department of Sensory and Motor System Medicine Graduate School of Medicine The University of Tokyo Tokyo Japan Department of Cartilage & Bone Regeneration (Fujisoft) Graduate School of Medicine The University of Tokyo Tokyo Japan Division of Tissue Engineering The University of Tokyo Hospital Tokyo Japan NAGATA Microtia and Reconstructive Plastic Surgery Clinic Saitama Japan

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.

关键词： Tissue Engineering Cartilage Container Storage Transport

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Hollow Fiber Module Applied for Effective Proliferation and Harvest of Cultured Chondrocytes

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Materials Sciences and Applications 2013年第8期4卷 62-67页

作者： Yoshiyuki Mori Makoto Watanabe Sakae Nakagawa Yukiyo Asawa Satoru Nishizawa Kazumi Okubo Hideto Saijo Satoru Nagata yuko fujihara Tsuyoshi Takato Kazuto Hoshi Department of Cartilage & Bone Regeneration (Fujisoft) Graduate School of Medicine The University of Tokyo Tokyo Japan Department of Cartilage & Bone Regeneration (Fujisoft) Graduate School of Medicine The University of Tokyo Tokyo Japan Division of Tissue Engineering The University of Tokyo Hospital Tokyo Japan Department of Sensory and Motor System Medicine Graduate School of Medicine The University of Tokyo Tokyo Japan Department of Sensory and Motor System Medicine Graduate School of Medicine The University of Tokyo Tokyo Japan Department of Cartilage & Bone Regeneration (Fujisoft) Graduate School of Medicine The University of Tokyo Tokyo Japan Division of Tissue Engineering The University of Tokyo Hospital Tokyo Japan Department of Sensory and Motor System Medicine Graduate School of Medicine The University of Tokyo Tokyo Japan Division of Tissue Engineering The University of Tokyo Hospital Tokyo Japan NAGATA Microtia and Reconstructive Plastic Surgery Clinic Saitama Japan Nomura Unison Co. Ltd Nagano Japan

Steady and useful culture for chondrocytes is essential for cartilage regenerative medicine. However, in conventional plate culture, the chondrocytes become dedifferentiated and lose their ability to make cartilage matrices. Three-dimensional culture mimicking the physiological environment in native chondrocytes is useful to maintain the chondrocyte properties during the proliferation culture. However, the three-dimensional culture is practically a hard task due to difficult harvest of the cells. Thus, we attempted to apply porous materials, hollow fibers for the three-dimensional culture, and developed their module to realize the effective harvest of the cells. Polyethersulfone-based hollow fibers, whose safety and cell affinity were confirmed by the experiment of the coculture with human chondrocytes, were collected to fabricate a module. The hollow fiber module was installed with screw ends, and enabled the easy removal of chondrocytes from the inner unit. Cultured human chondrocytes embedded within collagen hydrogel were put into the outer lumen of the hollow fiber module, while chondrocyte prolfieration medium was perfused through the inner lumen at 0 to 30 mL/min. After 2 weeks’ culture, the flow rate of 3 to 10 mL/min effectively supported the chondrocyte proliferation. Then, long-term culture using the hollow fiber module at flow rate of 5 mL/min was performed, revealing that the cell growth in this module at 3 weeks was approximately twice larger than that in static culture. The numbers of viable cells could be maintained by week 7. The hollow fiber module installed with screw ends can effectively culture and harvest the chondrocytes.

关键词： Hollow Fiber Module Proliferation Cell Culture Chondrocyte Tissue Engineering

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