JP2019508213A - 生体内分解率及び物性の調節が可能な生体適合性豚軟骨来由細胞外基質膜の製造方法及び前記豚軟骨来由細胞外基質を有効成分として含む癒着防止用組成物 - Google Patents
生体内分解率及び物性の調節が可能な生体適合性豚軟骨来由細胞外基質膜の製造方法及び前記豚軟骨来由細胞外基質を有効成分として含む癒着防止用組成物 Download PDFInfo
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Abstract
【解決手段】本発明は、豚軟骨来由細胞外基質を物理化学的方法で処理して多様な剤形の生体素材に製作した。また、物理化学的処理を行ったにもかかわらず前記のような軟骨特異的な機能を維持する特性を確認した。また、豚軟骨来由細胞外基質素材を体内安全性及び癒着防止効能に優れた癒着防止剤としても活用することができる。
【選択図】図1
Description
1.豚軟骨の分離
豚軟骨来由細胞外基質パウダーを製作するために、EN 12442の“Animal tissues and their derivatives utilized in the manufacture of medical devices, part 1; Analysis and management of risk, part 2; controls on sourcing, collection and handling”を参照し、基準に合う施設の豚膝軟骨を購入して使った。
豚軟骨粉末の製造工程は次のようである。
豚軟骨粉末に存在する細胞及び遺伝物質を除去し、純粋な細胞外基質成分のみを得るために次のように脱細胞化過程を遂行した。
脱細胞化した軟骨粉末4g当たり100mlの塩酸水溶液にペプシン(Pepsin;sigma、USA)を処理し、200rpm、4℃で24時間撹拌した。
前記製作した水溶性軟骨粉末1.3gを3次蒸溜水100mlで処理した後、200rpm、常温で1時間撹拌した。
前記製作した細胞外基質膜を銀箔で包装した後、線量5KGy〜100KGyのガンマ線を照射した。
軟骨細胞外基質工程過程による源泉材料の成分分析結果、軟骨細胞外基質に最も大きな比重を占めているコラーゲンと糖蛋白成分の損失なしに維持されていることを確認した。コラーゲンは、酸性溶液とペプシン酵素に源泉材料を溶解し、sirius red assayで測定し、糖蛋白は、パパイン(papain)溶液に溶かし、DMMB assayで測定した(図3)。
1.軟骨細胞外基質膜の架橋前と後かつ厚さによる引張強度分析
癒着防止剤として使われる膜は手術部位を充分に保護することができる物理的性質が重要であるから、食品医薬品安全処ガイドラインに従って製品特性に合う引張強度検査を実施した(図4a)。
癒着防止用膜は手術部位に固定されるときに効果的に癒着防止効果を発揮することができるから、手術時に縫合しても壊れずに耐えることができる強度が必要である。縫合強度は別に食品医薬品安全処に規定されていないから、自ら縫合強度を測定することができるプロトコルを確立して測定した。縫合強度の測定は、下記のようにフィルムを裁断し、フィルムを手術糸で縫合した後、引張強度測定器にそれぞれ手術糸と細胞外基質膜を付着して引張強度を測定する方法で進めた。測定結果、架橋後の細胞外基質膜の縫合強度が架橋前より有意に増加することを確認した(図4b)。
癒着防止用膜は手術部位によって分解期間が違うので、臓器特異的に癒着防止効果を出すためには、各臓器別に分解期間が調節された癒着防止剤を使わなければならない。軟骨細胞外基質膜の放射線照射線量によって分解率調節が可能であるかを確認するために、体外でコラーゲン分解酵素を処理した後、時間による分解挙動を試験した。実験は、互いに異なる放射線照射線量で処理した軟骨細胞外基質膜を1X1cmに切った後、コラーゲン分解酵素で処理したPBSで処理して2週間経過を観察した(図5の左)。また、2週間コラーゲン分解酵素で処理した試料を遠心分離し、上澄み液のヒドロキシプロリン(Hydroxy proline)の量を分析して細胞外基質膜の主要成分であるコラーゲンの分解率を測定した(図5の右)。測定結果、放射線照射線量によってコラーゲン分解酵素処理時の細胞外基質膜の分解率が調節されることを確認した。
細胞外基質膜の放射線照射による体内生分解率の差を確認するために次のように実験を進めた。
図1で製作した軟骨細胞外基質膜を用い、癒着機序で作用する血管内皮細胞の付着差を次のように確認した。
(1)8週齢のC57BL6マウスを用いて癒着モデルを製作した。マウスの腹部の皮膚層と筋肉層をそれぞれ切開した後、筋肉層と皮膚層を縫合することにより、損傷した組職間で癒着が発生するモデルを作って癒着効果を確認した。癒着組職は1週後に発生し、筋肉と皮膚を強く付着させる癒着モデルが形成された(図8a)。
Claims (5)
- 豚軟骨を分離する段階;
前記分離された豚軟骨を凍結乾燥して粉砕する段階;
前記粉砕された豚軟骨粉末を脱細胞化する段階;
前記脱細胞化した豚軟骨粉末を酸性溶液及びペプシン(pepsin)と混合して処理した後、塩基性溶液で中和させて豚軟骨粉末水溶液を製造する段階;
前記豚軟骨粉末水溶液を架橋剤と混合して豚軟骨来由細胞外基質膜を製造する段階;及び
前記豚軟骨来由細胞外基質膜に放射線を照射する段階を含む、生体内分解率及び物性の調節が可能な生体適合性豚軟骨来由細胞外基質膜の製造方法。 - 前記架橋剤はグルタルアルデヒド(Glutaraldehyde)であることを特徴とする、請求項1に記載の生体内分解率及び物性の調節が可能な生体適合性豚軟骨来由細胞外基質膜の製造方法。
- 前記放射線を照射する段階は、5〜100KGyのガンマ線を照射することを特徴とする、請求項1に記載の生体内分解率及び物性の調節が可能な生体適合性豚軟骨来由細胞外基質膜の製造方法。
- 請求項1〜3のいずれか一項に記載の方法によって製造された豚軟骨来由細胞外基質を有効成分として含む、癒着防止用組成物。
- 前記組成物は、軟膏、粉末、ゲル、フィルム、スラブ、ラップ及びスポンジからなる群から選択されたいずれか一形態であることを特徴とする、請求項4に記載の癒着防止用組成物。
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KR1020160029579A KR101772316B1 (ko) | 2016-03-11 | 2016-03-11 | 생체 내 분해속도 및 물성 조절 가능한 생체적합성 돼지연골 유래 세포외기질 막 제조방법 및 상기 돼지연골 유래 세포외기질을 유효성분으로 함유하는 유착방지용 조성물 |
KR10-2016-0029579 | 2016-03-11 | ||
PCT/KR2017/002563 WO2017155328A1 (ko) | 2016-03-11 | 2017-03-09 | 생체 내 분해속도 및 물성 조절 가능한 생체적합성 돼지연골 유래 세포외기질 막 제조방법 및 상기 돼지연골 유래 세포외기질을 유효성분으로 함유하는 유착방지용 조성물 |
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- 2017-03-09 EP EP17763589.3A patent/EP3427765A4/en not_active Withdrawn
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EP3427765A1 (en) | 2019-01-16 |
CN108834404A (zh) | 2018-11-16 |
JP6961629B2 (ja) | 2021-11-05 |
KR101772316B1 (ko) | 2017-08-29 |
EP3427765A4 (en) | 2020-01-15 |
US20190070336A1 (en) | 2019-03-07 |
WO2017155328A1 (ko) | 2017-09-14 |
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