JP5763790B2 - 哺乳類の軟骨組織由来の生体移植材 - Google Patents
哺乳類の軟骨組織由来の生体移植材 Download PDFInfo
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- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
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Description
(実施例1.最適ガンマ線量の決定)
豚の耳から抽出した新鮮な軟骨を9%のNaCl溶液に5時間浸漬し、超音波を照射した。これにより、軟骨細胞が除去されるようにした。その後、ウイルス不活化溶液として1%の過酸化水素溶液に上記組織を浸漬し、45分間撹拌した。この過程を経りながらウイルス及びその他の病原体が不活化されるようにした。また、アルカリ溶液として0.5Mの水酸化ナトリウム(NaOH)溶液に30分間浸漬した。各工程別の溶液で処理する前に、洗浄溶液を用いて前の工程の処理溶液が残留しないように完全に洗浄した。その後、組織の損傷を最小化するために、50%、80%及び100%のアセトンに順次浸漬し、脱水した後、包装容器に密封してガンマ線を照射した。最適のガンマ線量を決めるために、上記の工程により製造された軟骨移植材に対し、5〜30kGy範囲で、5kGy単位でガンマ線を照射した。ガンマ線照射による活性軟骨細胞の程度及びその他の軟骨を構成する物質の量を数値化して最適のガンマ線量を選択し、その結果を下記の表2に示す。
製造された軟骨組織は、アセトン処理またはグリセロールを用いて脱水することができる。グリセロールを用いた処理方法は、抗生剤を約0.11%含むグリセロールに組織を浸漬した後、これを再び99.9%以上のグリセロールに移して浸漬することにより、組織内の水分をグリセロールで置換する方法である。
(実施例3.工程別のECM因子の確認)
図5及び図6に示すように、新鮮な豚の耳介軟骨と最終的に製造された軟骨移植材に対して、ケラタン硫酸、コラーゲンII、フィブロネクチン、コンドロイチン硫酸の含量を調査した。その結果、ケラタン硫酸は、基質全般にわたって観察され、コンドロイチン硫酸は、軟骨膜の周辺で主に観察された。特に、コンドロイチン硫酸は、軟骨膜の一部の周辺に偏重して現われる傾向にあることが観察された。ケラタン硫酸、コラーゲンII、フィブロネクチン、コンドロイチン硫酸は、最終的に製造された軟骨移植材においては残留量が多少低減する傾向にあったが、依然として維持及び保存されていることが確認された。
上記の処理を通じて処理された最終軟骨移植材に、抗原性を誘発する軟骨細胞が存在するか、その他の細胞外基質はどのくらい維持されるのかを検証するために、それぞれの要素に特異的な染料で組織検査を実施した。
最終産物の3次元的構造を電子顕微鏡(SEM)で観察した。その結果は、図8に示すように、元の軟骨組織の構造と比べて、多孔性構造及び3次元的構造が損傷されずに保存されていることが分かる。
工程に投入されていない軟骨組織(赤い線)、及び工程処理中の軟骨組織(青い線)及び最終産物(紫線)の化学的含量を調査した。図9に示すように、それぞれのサンプルから同一のピークが現われており、加工工程及び滅菌により原料の特性が変わらないことを示している。
7週齢のSprague Dawley rat(SD−rat、male)を入手して実験個体に、動物の一般健康状態の観察及び順化のために実験前の1週間の期間を飼育した。その後、平均体重に到逹する個体を選別して実験を実施した。Zoletile(20mg/kg)とXylazine(5mg/kg)を筋肉注射して麻酔を誘導し、その後、脊椎を基準にして左/右に、それぞれ長さ方向に2cmを切開した。試験物質を移植するために、筋膜と真皮を分離してポケットを作り、予め用意した0.5cmの大きさの軟骨移植材を筋肉内に移植した。移植材の移植後には、4−0ナイロンを用いて単純結節縫合で欠損部位を縫合した。移植実験3ヶ月及び4ヶ月後に実験個体を犠牲させ、正常組織を含む移植部位を摘出して組織検査を実施した。
Claims (7)
- 哺乳類の軟骨組織を[アセトンまたはメタノール]:クロロホルム=1:1(v/v)の割合で混合した溶液に24〜72時間浸漬して脱脂させる工程と、
前記脱脂した軟骨組織に超音波を印加しながら6〜12%の塩化ナトリウム高張液に4〜6時間浸漬する工程と、
前記高張液で浸漬した軟骨組織を0.5〜1.5%の過酸化水素、0.1〜1.0%の過酢酸、70〜99%のアルコール、及び0.001〜3%の次亜塩素酸ナトリウムを含むウイルス不活化溶液に、30分〜1時間浸漬する工程と、
前記ウイルス不活化溶液で浸漬した軟骨組織を0.25〜0.75Mの水酸化ナトリウム溶液に15〜45分間浸漬する工程と、
前記水酸化ナトリウム溶液に浸漬した軟骨組織を脱水処理または冷凍処理する工程と、
前記脱水処理または冷凍処理された軟骨組織にガンマ線を照射する工程と、
を含む軟骨移植材の製造方法。 - 前記哺乳類は、豚、人、馬、牛、犬、鼠からなる群より選択される1種以上であることを特徴とする請求項1に記載の軟骨移植材の製造方法。
- 前記軟骨組織は、硝子軟骨、弾性軟骨及び繊維軟骨からなる群より選択される1種以上であることを特徴とする請求項1に記載の軟骨移植材の製造方法。
- 前記哺乳類の軟骨組織は、豚の軟骨組織であることを特徴とする請求項1に記載の軟骨移植材の製造方法。
- 前記豚の軟骨組織は、耳介軟骨組織または肋軟骨組織であることを特徴とする請求項1に記載の軟骨移植材の製造方法。
- 前記脱水処理は、50%、80%、及び100%濃度のアセトンに順次浸漬する方法、無水アルコールに浸漬する方法、50%、80%濃度のアセトンに順次浸漬した後に無水アルコールに浸漬する方法、または0.1〜1%の抗生剤及び90〜99.9%のグリセロールを含む溶液に軟骨移植材を1次浸漬した後に100%のグリセロール溶液に移して2次浸漬する方法からなる群より選択される1つ以上であることを特徴とする請求項1に記載の軟骨移植材の製造方法。
- 前記冷凍処理は、前記アルカリ溶液で処理した軟骨組織を−70℃で1〜2時間冷凍させた後、48〜72時間凍結乾燥させることである請求項1に記載の軟骨移植材の製造方法。
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