JP4451658B2 - 組織および器官のリモデリング - Google Patents
組織および器官のリモデリング Download PDFInfo
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Description
本発明は、組織エンジニアリング、より詳しくは組織のリモデリングに関する。
この最初の実施例では、再増殖、再血管形成、および一体化を含む初期のリモデリング現象を実証するために、移植後の初期(1週間)の修復現象を検討した。さらに、異なる構成の無細胞マトリクス材料を試験した。Xenoderm(無細胞ブタ皮膚マトリクスシート)および微粒子状Xenoderm(凍結破砕無細胞ブタ皮膚マトリクス)が関節の軟骨および骨の損傷を修復する有効性を評価するために、ユカタンミニブタモデルを使用した。動物の飼育および手術は、所内動物実験委員会(IACUC)の必要条件に従って実施した。一般的に、動物をテラゾール(8mg/kg)、ケタミン(4mg/kg)およびキシラジン(4mg/kg)を用いて筋肉内(IM)麻酔した。動物は挿管し、2〜3%イソフルランおよび1〜2L/分のO2で維持した。術前投薬にはセファゾリン約40mg/kgを静脈内投与(IV)、ブプレノルフィン0.007mg/kg IM、およびグリコピロレート0.01mgkg IMが含まれた。術後の抗菌物質はセファゾリン3.0〜3.5g IVであった。術後の鎮痛としては、ブプレノルフィン0.007mg/kg IM、および必要に応じて1〜3日毎に貼付したフェンタニル50、75μg/hour(経皮)パッチが含まれた。
手術が関節腔に入るものと比較して傷害性が低いと考えられたため、両後肢を処置した。大腿骨の外側面から脛骨粗面へ伸びる皮膚切開を伴う、後膝関節(膝の関節)への側面からのアプローチを用いた。皮下組織を切開し、骨膜起子を用いて筋膜の遠位大腿骨および近位脛骨への結合を除去した。
微粉末化XenoDermを充填した損傷では、XenoDermシートは外れていたが鋲止めの一ヶ所で定位置に保たれていた。顆と膝蓋骨関節表面の間で出血が明らかであった。損傷は外観上僅かに凹(表面下1〜1.5mm)、赤みがかって、出血性であった。鋲止めの穴は明らかに視認され、色は黒であった。軟骨−骨ブロックを摘出して10%ホルマリン固定液中に4日間、4℃にて置いた。XenoDermシートは別に同条件下で10%ホルマリン中で固定した。
XenoDermの覆いはそのままであって軟骨表面によく固定されていた。縫合を切断し覆いは上記の通り10%ホルマリン中に置いた。縫合糸は損傷に癒着していた。損傷は軟骨表面と連続的であり触れると固く、いくらか血があった。軟骨表面全体は綺麗に見えた。軟骨−骨を摘出して10%ホルマリン中で上記の通り固定した。
(a)右肢、遠位大腿骨
損傷を覆うXenoDermシートは定位置にあったが、手術部位周辺の血腫が明らかであった。覆いの中央に僅かな窪みがあった(約1mm);移植片全体と周囲の骨を骨のこを用いて摘出し、ブロックを10%ホルマリン中に置いた。ホルマリン中で4日後、ブロックを2分割し移植片の肉眼的外観を観察した。
損傷の周縁は識別が容易でなく、顕著な血腫が明らかであった。移植片の表面は不規則で、しかしプローブを用いた貫通に対しては固かった。移植片全体と周囲の骨を骨のこを用いて摘出し、ブロックを10%ホルマリン中に置いた。ホルマリン中で4日後、ブロックを2分割し移植片の肉眼的外観を観察した。
PLLA鋲を用いて固定したXenoDermシートはそのままで、目立たず、周囲の骨膜に癒着したように見えた。シートの下の移植材料はプロービングに対して固かった。移植片全体と周囲の骨を骨のこを用いて摘出し、ブロックを10%ホルマリン中に置いた。ホルマリン中で4日後、ブロックを2分割し移植片の肉眼的外観を観察した。
自家骨移植片は突出した骨性の断片を有する粗い表面を示した。移植片はプロービングに抵抗性であった。移植片全体と周囲の骨を骨のこを用いて摘出し、ブロックを10%ホルマリン中に置いた。ホルマリン中で4日後、ブロックを2分割し移植片の肉眼的外観を観察した。
効果的な組織修復および再生には、移植材料が再血管形成される必要がある。再血管形成は、リモデリング過程を推進する修復細胞の再増殖を促進する。微粉末化XenoDermで充填した、個体1に形成した骨軟骨性損傷の組織学的分析は、すべての無細胞移植片構成において生じるこれらの過程を代表する。移植後7日の骨軟骨性損傷のヘマトキシリン・エオジン(HE)染色は、下にある骨の軟骨下骨にはっきりと侵入している直径6mmの穴で、最初の損傷のおよその大きさが明瞭に明確化されることを示している。周囲のホスト関節軟骨、および下にある小柱骨および骨髄因子もまた同定された。このような早い時点でさえ、表面での軟骨の内殖、および損傷の両壁面および底部に沿った新しい骨形成の証拠があった。移植片と周囲のホスト軟骨との間の効果的な一体化、移植片全体の多数の血管によって証明された高度な再血管形成、および移植片の底部から生じる、新しい骨形成を代表する小柱の伸長が観察された。これらの現象はすべての無細胞移植材料において、移植片の構成に依存してさまざまな程度でみられた。微粉末化XenoDermはこの早い時点では、シート状XenoDermと比較してより高度な再血管形成および細胞の再増殖を示した。しかし、一般的な結論は、7日目に、軟骨および骨の修復を促進する適当なリモデリング現象が生じているということであった。
関節の軟骨損傷の基礎となる骨の損傷の修復について無細胞皮膚マトリクス足場の有効性を実証するために、ユカタンミニブタ骨軟骨性プラグ損傷モデルを使用した試験を実施した。移植片の3種類の処方を評価し、どの処方でも充填されていない損傷と比較した。試験した処方は:(1)表面をフィブリン糊で密封した微粉末化XenoDermパテ(〜600mg/mL)(2)フィブリノゲンおよびトロンビンと組み合わせてゲルを作成し、表面をフィブリン糊で密封した、微粉末化XenoDerm(〜330mg/mL)、または(3)軟骨損傷の周囲に縫合したAlloDermのシートで定位置に保った微粉末化XenoDermペースト(〜330mg/mL)、であった。従って、処方(1)および(3)の無細胞マトリクス成分は、微粉末化(粒子状)XenoDermの濃度に関してのみ異なった。
採取した損傷のそれぞれを肉眼的外観について検査した。この主観的分析は、関節内障害の形成、関節表面の回復、軟骨の侵食および外観について得点を分配する。肉眼的評価尺度を下記に示す:
評価
関節内癒着
無し= 2
ごく小さい/細い遊離した線維状組織= 1
大きい/太い線維状組織= 0
関節軟骨の回復
完全= 2
部分的= 1
無し= 0
軟骨の侵食
無し= 2
損傷部位および境界部= 1
損傷部位および隣接する正常軟骨= 0
軟骨の外観
半透明= 2
不透明= 1
変色または不規則= 0
理論上の最高得点= 8
3種類の移植材料および空の対照損傷の肉眼的評価点は以下の通りである:
移植材料 得点合計
空の損傷 3
Cymetra 5
Cymetra+フィブリン 6
Cymetraパテ 4
これらの視覚的観察は、Cymetra/フィブリンで充填した損傷について、無処置対照と比較して軟骨表面の修復の顕著な改善を示している。
本物の関節軟骨の同定は、超微細構造および/または生化学パラメータを試験することによって達成することができる。関節軟骨は、同定可能な領域を有する軟骨組織の連続的な層を形成する。表層は、平らな形態を持つ軟骨細胞、およびトルイジンブルーで染色されにくい、硫酸化グリコサミノグリカン(主にアグリカン)の相対的な不在を示す細胞外ネットワークによって特徴づけられる。中層および深層の軟骨細胞は球状の外観を持ち、マトリクスはトルイジンブルーでの染色によって証明されるように豊富な硫酸化プロテオグリカンを含む。
移植片 得点合計
空の損傷 7
Cymetra 7
Cymetra+フィブリン 17
Cymetraパテ 18
すべての移植材料は無処置対照より顕著に良好に得点した。しかし、Cymetra/フィブリンの組み合わせは軟骨修復の指標についてより良好に得点し、またCymetraパテは小柱骨修復についてより良好に得点したことが注目された。にもかかわらず、無処置損傷と比較して、無細胞マトリクス移植片の組み合わせは骨誘導性であり(すなわち、骨修復を可能にする)、そして軟骨修復の足場として作用する。
骨の直径の2倍に達する骨幹中部の分断損傷(約3cm)を、ブタ2個体のそれぞれの一つの大腿骨に一則性に、外科的に作成した。損傷はこのように自然には治癒しない臨界サイズ損傷であった。骨にネジ止めした金属製の骨板を損傷上にわたって適用し、それによって骨切除された骨を正しい解剖学的位置に固定した。Xenodermのシートは、外科的適用の前に約10分間生理食塩水に浸漬することによって再構成した。XenoDermシートを円筒形の骨損傷の周りに巻き付け、チューブを形成した。シートは隙間の両側の骨の近位および遠位端に約5mm重ね、縫合で骨膜に固定した。XenoDermのチューブを閉じる前に、XenoDermシートによって定義されるチューブを、OSTE0SET(登録商標)(硫酸カルシウム)ペレットと、レシピエントブタの近位上腕骨から得た海綿状自家骨との1:1混合物で充填した。チューブを移植材料で充填した後、XenoDermシートを長さに沿って縫合糸を用いて連続パターン縫合で閉じた。
2種類のラット異所性心移植モデルを試験した。一方は虚血心室異常のモデル(「虚血モデル」)であり、もう一方は先天性左心肥厚(「形成不全左心モデル」)である。虚血モデルでは、左主冠状動脈は結紮され、虚血範囲は摘出され、そして心筋の適当な部分が摘出部分と同一の比率を有するマトリクスで置換されている。その操作は全体的な心室の形および構成を保存する。形成不全左心モデルは、動脈結紮または摘出は含まないが、心室の空洞の全体的な大きさを拡大する必要に応じて、左心室壁の切開および継ぎ当て拡大を含んだ。さらに、これらのモデルの両方で、ドナー心臓の吻合接続を適当に操作することによって(非特許文献6;非特許文献7)、機能する(すなわち、正常な心室充満)または無負荷(心室バイパス)左心室のいずれかを作成することができる。心室に移植されたマトリクスは、強度と支持のためのゴアテックス(登録商標)(ポリテトラフルオロエチレン;PTFE)(W.L. Gore & Associates, Inc、アリゾナ州Flagstaff)の1mmの層と、組織の再生を誘導する無細胞マトリクス(たとえば、AlloDerm、XenoDerm、または無細胞血管マトリクス)の内層との2層の形に構成されている。別の方法として、粒子状マトリクスをシートの間に挟んだ、無細胞マトリクスの2枚のシートを用いることができる。
Claims (14)
- 哺乳動物の骨膜の欠陥を有する骨を修復または改善するための組成物であって、非粒子状無細胞皮膚マトリクスを含有し、前記骨膜の欠陥を有する骨の中または表面に配置されることを特徴とする組成物。
- 哺乳動物の長骨を修復または改善するための組成物であって、粒子状無細胞皮膚マトリクスを含有し、前記長骨の中または表面に配置されることを特徴とする組成物。
- 前記哺乳動物がヒトであることを特徴とする請求項1または2記載の組成物。
- 前記哺乳動物と組織適合する生存細胞をさらに含むことを特徴とする請求項1または2記載の組成物。
- 前記細胞が前記哺乳動物に由来することを特徴とする請求項4記載の組成物。
- 前記細胞が、表皮細胞、ケラチノサイト、内皮細胞、線維芽細胞、胚性幹細胞、成体または胚性間葉系幹細胞、臍帯幹細胞、前軟骨芽細胞、軟骨芽細胞、軟骨細胞、軟骨細胞、前骨芽細胞、骨細胞および破骨細胞から選択されることを特徴とする請求項4記載の組成物。
- 細胞成長因子、血管新生因子、分化因子、サイトカイン、ホルモン、ケモカインより成る群から選択される一または複数の物質と組み合わせて用いられることを特徴とする請求項1または2記載の組成物。
- 前記一または複数の物質が、前記組成物に含まれることを特徴とする請求項7記載の組成物。
- 前記一または複数の物質を、前記組成物とは別に前記哺乳動物に注射または注入することを特徴とする請求項7記載の組成物。
- 転写または翻訳調節因子に調節可能に連結している細胞成長因子、血管新生因子、分化因子、サイトカイン、ホルモン、ケモカインより成る群から選択される一または複数の物質をコードする一または複数の核酸配列を含む一または複数の発現ベクターと組み合わせて用いられることを特徴とする請求項1または2記載の組成物。
- 前記一または複数の発現ベクターが、前記哺乳動物に投与される一または複数の細胞に含まれることを特徴とする請求項10記載の組成物。
- 前記一または複数の細胞が、前記組成物に含まれることを特徴とする請求項11記載の組成物。
- 重篤な骨の隙間異常に関連する骨膜を修復するためのものである請求項1記載の組成物。
- 脱灰骨粉末をさらに含むことを特徴とする請求項2記載の組成物。
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