JP4486347B2 - 軟骨組織再生用基材および軟骨細胞との複合体とその製造方法 - Google Patents
軟骨組織再生用基材および軟骨細胞との複合体とその製造方法 Download PDFInfo
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Images
Description
[実施例1]
ポリ乳酸(分子量100000)のクロロホルム溶液(5g/L)に両親媒性ポリマーとしてポリアクリルアミド共重合体(重量平均分子量:85000)を10:1の割合で混合し、ガラス基板上にキャストし室温、湿度70%の条件下に静置し、溶媒を徐々に飛ばすことでハニカム構造を有するフィルムを調製した。得られたフィルムの光学顕微鏡写真を図3に示す。
実施例1で作製したハニカム構造フィルムを70%エタノールで滅菌し、滅菌済みのボイデンチャンバー内にフィルムを挿んだ。一方、ウサギ膝関節の軟骨から薄い軟骨片をメスで剃りおろし、細かく刻んだ後、0.15(w/v)%のトリプシンを含有するPBS(−)中で1時間酵素処理し、さらに0.15(w/v)%のコラーゲナーゼを含有するPBS(−)中で37℃で2時間30分インキュベートした。そして、ポアサイズが70μmのナイロンフィルターで濾過した濾液を1500rpmで3分間遠心し、抗生物質と10%ウシ胎児血清を含有するα−MEM血清培地で2回洗浄した後、ウサギ膝軟骨細胞を得た。得られた軟骨細胞をα−MEM血清培地で37℃、5%CO2インキュベータ内で培養した。2回継代培養した軟骨細胞を0. 25%トリプシン/1mmol EDTA/PBS(−)で剥離・採集し、2×105cells/ml細胞液を調製した。ハニカム構造フィルムを1mlの培地で濡らしてから、上記の細胞液を1ml播種した。次いで6ウェルプレート内に収め、5%CO2、37℃インキュベーター内で培養をした。次の日培地をすべて吸引し25μg/mlのAscorbic Acid入りの培地でボイデンチャンバー内に2ml加えた培地交換を行い、その後、2日おきに培地交換を行い、3週間まで静置して培養した。培養後、容器から移植体を外し、GAG測定を行った。その結果を表1に示す。
また得られた軟骨細胞と軟骨組織再生用基材との複合体の光学顕微鏡写真を図4に示す。
実施例2と同様の大きさのシャーレ上で同様に軟骨細胞を培養し(単層培養)、3週間培養後GAG測定を行った。その結果を表1に示す。
実施例2と同様にウサギ軟骨細胞を取り出し、得られた軟骨細胞を培養液と混合して軟骨細胞浮遊液を作製し、この軟骨細胞浮遊液と同量のコラーゲン(アテロコラーゲン:高研(株))内に2×105cells/mlの密度になるように包埋した後、培養を開始した。このときコラーゲンの最終濃度は2.4重量%となった。次いで6ウェルプレート内に収め、5%CO2、37℃インキュベーター内で25μg/mlのAscorbic Acid入りの培地を用い培養をし、3週間培養後GAG測定を行った。その結果を表1に示す。
2 軟骨組織再生用基材
3 軟骨細胞
4 軟骨組織再生用基材
Claims (4)
- 生分解性脂肪族ポリエステルまたは生分解性脂肪族ポリカーボネートと、アクリルアミドポリマーを主鎖骨格とし、疎水性側鎖としてのドデシル基と親水性側鎖としてのラクトース基もしくはカルボキシル基とを併せもつ両親媒性ポリマーとからなり、生分解性脂肪族ポリエステルまたは生分解性脂肪族ポリカーボネートと両親媒性ポリマーの組成比が99:1から50:50(wt/wt)であるハニカム構造を有する生分解性フィルムからなる軟骨組織再生用基材。
- 該ハニカム構造の平均空隙内径が20μm以下であることを特徴とする請求項1に記載された軟骨組織再生用基材。
- 請求項1〜2のいずれかに記載された軟骨組織再生用基材と該軟骨組織再生用基材に担持された軟骨細胞からなる軟骨組織再生用複合体。
- 請求項1〜2のいずれかに記載された軟骨組織再生用基材上で軟骨細胞を培養し、軟骨組織再生用基材上に軟骨細胞が担持された軟骨組織再生用複合体を製造する方法。
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Families Citing this family (8)
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EP1693075B1 (en) * | 2003-11-21 | 2012-10-03 | Teijin Limited | Substrate for tissue regeneration |
WO2006093207A1 (ja) * | 2005-03-02 | 2006-09-08 | National University Corporation Hokkaido University | 細胞の分化/増殖を制御するための基材 |
WO2007086421A1 (ja) * | 2006-01-26 | 2007-08-02 | National University Corporation Hokkaido University | 硬組織再生治療に用い得るハニカム状多孔質体 |
JP6467493B2 (ja) * | 2015-03-18 | 2019-02-13 | 富士フイルム株式会社 | 軟骨再生材料及びその製造方法 |
EP3272370B1 (en) | 2015-03-18 | 2022-04-27 | FUJIFILM Corporation | Cartilage-regenerating material |
CN110087698B (zh) | 2016-10-17 | 2022-06-24 | 国立大学法人九州大学 | 医疗用蜂窝结构体 |
JP2021037281A (ja) | 2019-08-27 | 2021-03-11 | 邦夫 石川 | 医療用炭酸カルシウム組成物、および関連医療用組成物、ならびにこれらの製造方法 |
JP2021137577A (ja) | 2020-03-05 | 2021-09-16 | 邦夫 石川 | 医用ハニカム構造体およびその製造方法、医用組織再建袋、成形型 |
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