JP5179459B2 - 組織工学による靱帯、腱、および他の組織を産生するためのマトリックス - Google Patents
組織工学による靱帯、腱、および他の組織を産生するためのマトリックス Download PDFInfo
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Description
本発明は、生物工学による組織、特に靱帯および腱を産生するために用いられるマトリックスまたは足場構造に向けられる。より詳しく述べると、本発明は、それを必要とするレシピエントに移植するために、多能性細胞をエクスビボでその上に播種してもよく、細胞が増殖して前十字靱帯に分化する新規絹骨格マトリックスに関する。
毎年、何十万人のアメリカ人が、膝、肘、手、肩、手首、および顎の靱帯および腱を捻挫、断裂、または破断する(Langerら、Science 260:920〜926(1993)(非特許文献1))。特に重要なものは膝の前十字靱帯である。アメリカだけでも毎年200,000人を超える人々が、前十字靱帯(ACL)を断裂または破断すると予想される(Albrightら、1999、第42章、「Knee and Leg:Soft-Tissue Trauma」、「Orthopaedic Knowledge Update 6」、アメリカ整形外科学会(非特許文献2))。ACLは、前脛骨並進の一次安定化剤として、および外反内反膝屈曲角形成の第二の安定化剤として作用し、スポーツの損傷および交通事故に関連した屈曲-回転-外反力に起因する破断または断裂を多くの場合受けやすい。破断または断裂は多くの場合、重度の運動制限、疼痛および不快感に至り、スポーツおよび運動に参加できなくなる。ACLの障害は、三つの範疇に分類される:(1)靱帯性(引っ張り応力による靱帯線維の断裂)、(2)骨折を伴わない骨-靱帯界面での障害、ならびに(3)骨および靱帯の結合部位での骨折を伴う骨-靱帯界面の障害。最も一般的なタイプのACL障害は、第一の範疇、すなわち靱帯性である。
[A]類似の細胞およびそれを取り巻く細胞内物質の集合体。体内には基本的な四つの組織が存在する:1)上皮;2)血液、骨、および軟骨を含む結合組織;3)筋組織;ならびに4)神経組織。
本発明は、その上に多能性細胞を播種して、細胞が増殖して靱帯および腱線維芽細胞に分化し、それによって前十字靱帯(ACL)、または他の靱帯、腱、もしくは組織の形成が得られる、新規絹繊維骨格のマトリックスに向けられる。新規絹繊維骨格のマトリックスは、ワイヤロープ(ねじれたまたは編まれたような)形態の繊維を有するように設計され、これは天然の前十字靱帯(下記の表1を参照されたい)と同一の機械特性を示し、マトリックスの構造および形態に単純な変化を加えることによって如何なる所望の靱帯または腱組織(下記の表2を参照されたい)も形成することができる。
a.絹被膜の調製
図1Aに示す未加工のカイコガの繊維を抽出して、天然の絹フィブロインをコーディングする膠様タンパク質であるセリシンを除去した(図1A〜Cを参照されたい)。1群あたり適当数の繊維を平行に配置して、0.02 M Na2CO3水溶液および0.3%(w/v)アイボリー石けん溶液において90℃で60分間抽出した後、水で十分にすすいで膠様のセリシンタンパク質を抽出した。
絹繊維骨格のマトリックスの機械的特性を予測するために、鎖3本のワイヤロープに関するコステロの等式を導出した。導出されたモデルは、マトリックスの全体的な強度および剛性を所定のレベルの幾何学的階層に関してピッチ角の関数として計算するために、抽出された絹繊維材料の特性および所望のマトリックス幾何学的階層を組み合わせて考慮に入れる一連の等式である。
SEMによって決定したように、90℃で60分後に完全なセリシンの除去を認めた(図1A〜Cを参照されたい)。絹繊維からのセリシンの除去は、繊維の超構造を変化させ、それによってよりなめらかな繊維表面が得られ、その下に存在する絹フィブロインが出現して(図1A〜Cに示す)、平均直径は20〜40 μmの範囲であった。フィブロインは、極限の引っ張り強さの有意な15.2%減少を示した(1.033±0.042 N/繊維〜0.876±0.1 N/繊維)(p<0.05、対応のあるスチューデントt-検定)(図1Dを参照されたい)。最適にした絹マトリックスの機械的特性(図2を参照されたい)を、上記の表1、図3A(マトリックス1)および図3C(マトリックス2に関して)に概要する。最適にした絹マトリックスは、天然のACLと同等の値を示したことは、これらの結果から明らかであり、これらの値は平均で極限の引っ張り強さ(UTS)〜2100 N、剛性〜250 N/mm、降伏点〜2100 Nおよび破断時の33%伸長を有することが報告されている(Wooら、「The Tensile Properties of Human Anterior Cruciate Ligament(ACL)and ACL graft tissue.」、「Knee Ligaments:Structure, Function, Injury and Repair」D. Danielら編、レイブン出版社、279〜289頁(1990))。
細胞の単離と培養
適当な条件を形成すれば、所望の靱帯線維芽細胞系列への分化を指示できることから、骨髄間質細胞(BMSC)、骨原性、軟骨原性、腱原性、脂肪細胞原性、および筋原性系列に分化することができる多能性細胞を選択した(Markolfら、J. Bone Joint Surg., 71A:887〜893(1989);Caplanら、「Mesenchymal stem cells and tissue repair」、「The Anterior Cruciate Ligament:Current and Future Concepts」、D.W. Jacksonら編、レイブン出版社、ニューヨーク(1993);Youngら、J. Orthopaedic Res. 16:406〜413(1998))。
凍結したP1 hBMSCsを解凍して、5×103個/cm2で再度播種して(P2)、ほぼコンフルエントに達してからトリプシン処理して、マトリックス播種のために用いた。滅菌した(エチレンオキサイド)絹マトリックス(特にマトリックス1および2のコード1本、平行な抽出絹繊維の束30本およびコラーゲン繊維のワイヤロープ)に、細胞容積を最少にして、細胞-マトリックス接触を増加するように、テフロンブロックに機械処理した特注の播種チャンバー(全量1 ml)に細胞を播種した。細胞のスラリー(3.3×106個BMSCs/ml)と共に4時間インキュベーション後の播種したマトリックスを、実験期間のあいだ適当量の細胞培養用培地を含むペトリ皿に移した。
絹マトリックスに対する骨髄間質細胞の反応も同様に調べた。BMSCsは、培養1日後、絹およびコラーゲンマトリックスに容易に接着してその上で増殖し(図4A〜Cおよび図10Aを参照されたい)、隣接する繊維を架橋するように細胞伸長部を形成した。図4Dおよび図10Bに示すように、構築物を覆う均一な細胞シートを培養14日目および21日目でそれぞれ認めた。MTT分析によって、播種したBMSCsによる完全なマトリックスの被覆が培養14日後に確認された(図5を参照されたい)。マトリックス1(図6Aを参照されたい)およびマトリックス2(図6Bを参照されたい)において増殖させた細胞の総DNA定量から、BMSCsが絹構築物上で増殖して成長し、培養21日後および14日後にそれぞれ測定したDNAが最高量であったことを確認した。
バイオリアクター系における骨髄間質細胞からの靱帯形成に及ぼす定方向の多次元機械刺激の影響に洞察を与える研究を行う。バイオリアクターは、発達中の靱帯に対して独立したしかし同時の周期的な多次元のひずみ(例えば、並進、回転)を適用することができる。7〜14日間の静的な休息期間の後(播種後時間)、回転的および並進的ひずみ率ならびに直線および回転変形は、1〜4週間のあいだ一定である。並進的ひずみ(3.3%〜10%、1〜3 mm)および回転的ひずみ(25%、90°)を、周波数0.0167 HzでBMSCsを播種した絹骨格のマトリックスに同時に適用し(1分間当たりの応力および弛緩の完全な1サイクル);機械的負荷を行わない播種したマトリックスを有するそれ以外の同一のセットのバイオリアクターを対照とした。実験期間において靱帯を一定の周期的ひずみに曝露した。
Claims (3)
- ワイヤーロープ形態に構築されたセリシン抽出された絹フィブロンのみを含む、絹繊維骨格のマトリックスであって、
マトリックスが、2000Nより大きい極限の引っ張り強さを有し、
マトリックスが、100N/mm〜600N/mmの範囲の直線剛性を有し、および、
マトリックスが、ヒトの体における移植用である、該マトリックス。 - 絹が、カイコからの絹である、請求項1に記載のマトリックス。
- マトリックスが、破断時の%伸長が少なくとも29%である、請求項2に記載のマトリックス。
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