JP5570425B2 - 組織工学用多孔質足場の調製方法 - Google Patents
組織工学用多孔質足場の調製方法 Download PDFInfo
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- JP5570425B2 JP5570425B2 JP2010528420A JP2010528420A JP5570425B2 JP 5570425 B2 JP5570425 B2 JP 5570425B2 JP 2010528420 A JP2010528420 A JP 2010528420A JP 2010528420 A JP2010528420 A JP 2010528420A JP 5570425 B2 JP5570425 B2 JP 5570425B2
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Description
a)ある量の少なくとも1種の多糖および1種の架橋剤を含むアルカリ性水溶液を調製するステップと、
b)ステップa)の水溶液を凍結させるステップと、
c)ステップb)の凍結溶液を昇華させるステップと
からなるステップを含む、多孔質足場の調製方法であって、ステップa)の溶液中で多糖の架橋結合が起こる前にステップb)を行うことを特徴とする方法を提供することである。
本明細書で使用する「多糖」という用語は、2つ以上の単糖ユニットを含む分子を指す。
本発明の第1の目的は、
a)ある量の少なくとも1種の多糖および1種の架橋剤を含むアルカリ性水溶液を調製するステップと、
b)ステップa)の水溶液を凍結させるステップと、
c)ステップb)の凍結溶液を昇華させるステップと
からなるステップを含む多孔質足場の調製方法であって、ステップa)の溶液中で多糖の架橋結合が起こる前にステップb)を行うことを特徴とする方法に関する。
本発明の足場は、特に組織工学、組織修復、または組織再生に適している。多孔度の違いにより、足場の適当な領域への異なる細胞型の移入が促進されうる。他の実施形態において、多孔度の違いにより、組織の発生/修復/再生の適当な構造化に必要な、足場を構成する細胞型間における適当な細胞間連結の発生が促進されうる。例えば、細胞突起の伸長は、足場材料の多様な多孔度を介してより適当になるよう適応されうる。したがって、足場は任意の組織の細胞を含んでよい。
多糖ベースの足場の調製
プルラン/デキストラン75:25、水中総濃度24.5%(w/v)の混合物(プルラン、MW200,000、Hayashibara Inc.Okayama,Japan;デキストラン、MW500,000、Pharmacia)を調製した。アルカリ性条件下で、架橋剤のトリメタリン酸ナトリウム(STMP)(11%(w/v)、Sigma)を用いて多糖を化学的に架橋結合させた。簡単に言えば、多糖溶液9mLを、10MのNaOH1mLと混合し、次いでその混合物に水1mL中STMP300mgの水溶液を加えた。その溶液をすぐに60mmのペトリ皿中に注ぎ込み、次いで−80℃で保管した。Lyovac freeze−dryer (GT2,STERIS Rotary vane pump,BOC EDWARDS)内での凍結乾燥プロセス中に、凍結した混合物を架橋結合させた。水分を完全に除去できるように足場を24時間凍結乾燥させた。凍結乾燥プロセス中に架橋結合した足場は不透明で若干もろいものであった。その足場は所望のサイズと形状に容易に切断でき、再含水させることができるものであった(図1)。
凍結乾燥条件の影響
実施例1に従って多糖足場の調製を行った。凍結乾燥ステップでは、制御漏出量によって異なる真空度を調整した(0.1mbar、0.75mbar、3mbar、1.5mbar、および6.5mbar)。
膨張の割合
実施例2に従って多糖足場の調製を行った。凍結乾燥させた足場を、かみそりで切断して長方形の足場(2.5cm×2cm、厚さ3mm)を得た。足場を脱イオン水で洗浄して緩衝塩を全て除去し、次いで50℃で36時間、脱水した。試料の乾燥重量(乾燥W)と、脱イオン水で24時間再含水させた後の膨張状態の重量(膨張W)を、電子てんびん(AG 204 Deltarange(登録商標)mettler Toledo;最大81g/210g;d=0.1mg/1mg)を用いて測定した。計量前に、膨張した足場を柔らかい紙の上に注意深く置いて過剰な水分を除去した。それぞれの実験を3連で行った。膨張の割合を次式に従って計算した。膨張の割合=(膨張W−乾燥W)/乾燥W)×100。
細胞浸潤
ウィスターラット由来の大腿骨骨髄間葉系幹細胞(MSC)を実施例1の通りに調製した足場で培養した。円形の穿孔器を用いて、直径6mm、厚さ1mmの円形の多孔質足場を切り出した。細胞を播種する前に、37℃で24時間、24ウェルプレートの培養培地中で足場を平衡化させた。培養培地は、10%のウシ胎児血清と1%のペニシリン/ストレプトマイシン(Sigma)を含む低グルコースDMEM(Gibco,Life Technology,New York)からなるものを使用した。細胞を足場の上部に播種した(足場あたり細胞106個の細胞密度)。アルコルビン酸(50μg/ml)を補充した培養培地を2〜3日ごとに取り替えた。試料を培養物中で最大1週間維持した。培養培地中でインキュベートした非播種の多孔質足場を対照として用いた。動物由来およびヒト由来の一次血管平滑筋細胞および内皮細胞などの他の細胞型を用いて同様の実験を首尾よく行った。
足場にMSCを浸潤させながら、多孔質足場の表面に細胞を2時間未満付着させた。
播種ステップの前に蛍光染料PKH26(Sigma)を用い、製造者の取扱説明書に従って細胞標識することにより、細胞追跡を行った。細胞を非標識足場とFITC−足場の両方に播種した。次いで細胞播種した足場を4%のパラホルムアルデヒド/PBSで固定した後、共焦点顕微鏡(Zeiss LSM 510)で分析した。
生存細胞によって加水分解され、それによって強度な均一の緑色蛍光(波長485〜535nm)を生成するポリアニオン系染料である、カルセインAM(Calbiochem,San Diego CA)を用いて、細胞の生存率をアッセイした。この染料を、1日目、5日目、および7日目に、製造者の取扱説明書に従って、多孔質の非標識足場およびFITC−足場に加えた。次いでこの播種された足場を4%のパラホルムアルデヒド/PBSで固定した後、共焦点顕微鏡(Zeiss LSM 510)で分析して、足場およびFITC−足場内の細胞分布を可視化した。
足場内へのタンパク質の組み込み
実施例1に従い、ゼラチンおよびI型コラーゲンなどの接着タンパク質を組み込むために以下の改変を加えて、多糖足場を調製した。ゼラチンに関しては、多糖溶液9mLを10MのNaOH1mLと混合し、次いでその混合物にゼラチン500μgを含む水1mL中STMP300mgの水溶液(0.1%のゼラチン溶液500μL)を加えた。I型コラーゲンの組み込みに関しては、多糖溶液に0.4%のコラーゲン溶液(Upstate #08115)500μLを加えた後に架橋剤(500μL中300mg)を加えた。足場の厚い切片のクーマシーブルー染色およびシリウスレッド染色により、足場内のタンパク質分布が確認された。平均タンパク質含量は、足場直径6mmにつきゼラチン約1μg、および足場直径6mmにつきコラーゲン4μgであると見積もられた。
代用血管としての管状足場
実施例1に記載の通り調製した多糖ベースの管状足場は、代用血管として使用できうる。
足場内への薬物の組み込み
実施例1に従い、ノルフロキサシンなどの薬物を組み込むために以下の改変を加えて、多糖足場を調製した。ノルフロキサシンは、フルオロキノロン系カルボン酸であり、広く使用されている抗菌剤である。現在ノルフロキサシンは、主にその水溶性の低さに起因して、生体利用率が低いモデル化合物とされている。固体状態のノルフロキサシン(Sigma)(60mg)を多糖溶液(10g)に加え、その混合物を、均一になるまで撹拌した。次いで、結果生じた混合物を10MのNaOH1mLと混合し、次いでその混合物に水1mL中STMP300mgの水溶液を加えた。次いで、実施例1に従って架橋結合プロセスを行った。
Claims (20)
- a)ある量の少なくとも1種の多糖および1種の架橋剤を含むアルカリ性水溶液を調製するステップと、
b)ステップa)で得られた水溶液を凍結させるステップと、
c)ステップb)で得られた凍結溶液を昇華させるステップと
からなるステップを含む、多孔質足場の調製方法であって、
ステップa)において、前記水溶液は37℃以下の温度で調製され、
昇華ステップ(ステップc))中に多糖の架橋結合が起こることを特徴とする方法。 - 前記ステップa)において、前記水溶液を4℃〜25℃の範囲の温度で調製する、請求項1に記載の方法。
- 前記多糖が、デキストラン、寒天、アルギン酸、ヒアルロン酸、イヌリン、プルラン、ヘパリン、キトサン、およびフコイダンからなる群から選択される、請求項1または2に記載の方法。
- 前記架橋剤が、トリメタリン酸三ナトリウム(STMP)、オキシ塩化リン(POCl3)、エピクロロヒドリン、ホルムアルデヒド、水溶性カルボジイミド、およびグルタルアルデヒドからなる群から選択される、請求項1から3のいずれか一項に記載の方法。
- 前記架橋剤がトリメタリン酸三ナトリウム(STMP)である、請求項4に記載の方法。
- ステップa)の水溶液を凍結乾燥する、請求項1から5のいずれか一項に記載の方法。
- ステップa)の水溶液を、0.1mBar〜6.5mBarの圧力下で凍結乾燥する、請求項6に記載の方法。
- ステップa)の水溶液を、ステップb)の前に型に注ぎ込む、請求項1から7のいずれか一項に記載の方法。
- 前記足場を造形する、請求項1から8のいずれか一項に記載の方法。
- ステップc)で得られた足場に再度含水させることからなるステップをさらに含む、請求項1から9のいずれか一項に記載の方法。
- 請求項1から10のいずれか一項に記載の方法によって得られる多孔質足場。
- 孔のサイズが1μm〜500μmで構成されている、請求項11に記載の多孔質足場。
- 多孔度が4%〜50%で構成されている、請求項11または12に記載の多孔質足場。
- ある量の細胞を担持していることを特徴とする、請求項11から13のいずれか一項に記載の多孔質足場。
- 細胞が、酵母細胞、哺乳動物細胞、昆虫細胞、および植物細胞からなる群から選択される、請求項14に記載の多孔質足場。
- 哺乳動物細胞が、軟骨細胞;線維軟骨細胞;骨細胞;骨芽細胞;破骨細胞;筋細胞;滑膜細胞;骨髄細胞;間葉細胞;上皮細胞、肝細胞、間質細胞;幹細胞;胚性幹細胞;脂肪組織由来の前駆体細胞;末梢血前駆細胞;成体組織から単離された幹細胞;および遺伝子形質転換細胞からなる群から選択される、請求項15に記載の多孔質足場。
- 組織工学、三次元細胞培養、または治療上での細胞送達のための、請求項11から16のいずれか一項に記載の多孔質足場。
- 請求項11から14のいずれか一項に記載の足場を用いて作製された代用血管。
- 請求項11から14のいずれか一項に記載の足場を用いて作製された、軟骨または骨のインプラント。
- 請求項11から14のいずれか一項に記載の足場を用いて作製された、活性薬剤の放出制御システム。
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US9522218B2 (en) * | 2007-10-11 | 2016-12-20 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method for preparing porous scaffold for tissue engineering, cell culture and cell delivery |
US20110300203A1 (en) * | 2008-10-22 | 2011-12-08 | Trustees Of Columbia University In The City Of New York | Cartilage regeneration without cell transplantation |
TWI421339B (zh) * | 2009-10-16 | 2014-01-01 | Academia Sinica | 三維支架之製備方法及其裝置 |
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KR101333381B1 (ko) * | 2012-05-07 | 2013-11-28 | 영남대학교 산학협력단 | 단일공정에 의한 이중층 스캐폴드의 제조방법 및 상기 제조방법에 의해 얻어진 이중층 스캐폴드를 이용한 조직 재생방법 |
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