JP5824108B2 - 多層足場 - Google Patents
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- A—HUMAN NECESSITIES
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—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
- 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
- A61L27/3804—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 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial cells
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/10—Hair or skin implants
- A61F2/105—Skin implants, e.g. artificial skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/58—Materials at least partially resorbable by the body
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/60—Materials for use in artificial skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
- D01F6/00—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof
- D01F6/58—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from homopolycondensation products
- D01F6/62—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from homopolycondensation products from polyesters
- D01F6/625—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from homopolycondensation products from polyesters derived from hydroxy-carboxylic acids, e.g. lactones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction
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- D—TEXTILES; PAPER
- D10—INDEXING SCHEME ASSOCIATED WITH SUBLASSES OF SECTION D, RELATING TO TEXTILES
- D10B—INDEXING SCHEME ASSOCIATED WITH SUBLASSES OF SECTION D, RELATING TO TEXTILES
- D10B2401/00—Physical properties
- D10B2401/10—Physical properties porous
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- D—TEXTILES; PAPER
- D10—INDEXING SCHEME ASSOCIATED WITH SUBLASSES OF SECTION D, RELATING TO TEXTILES
- D10B—INDEXING SCHEME ASSOCIATED WITH SUBLASSES OF SECTION D, RELATING TO TEXTILES
- D10B2401/00—Physical properties
- D10B2401/12—Physical properties biodegradable
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- D—TEXTILES; PAPER
- D10—INDEXING SCHEME ASSOCIATED WITH SUBLASSES OF SECTION D, RELATING TO TEXTILES
- D10B—INDEXING SCHEME ASSOCIATED WITH SUBLASSES OF SECTION D, RELATING TO TEXTILES
- D10B2509/00—Medical; Hygiene
Landscapes
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- Chemical & Material Sciences (AREA)
- Transplantation (AREA)
- Public Health (AREA)
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Description
本出願は、いずれもその全体が参照により本明細書に組み込まれている、2007年1月25日に出願した「Multilayer Scaffold」という表題の英国出願第0801405.2号および2007年2月15日に出願した「Multilayer Scaffold」という表題の英国特許出願第0802767.4号の優先権を主張するものである。
「最大検出孔径」とは、キャピラリーフローポロメーターが試料中に検出できる最大孔径である;
「最大孔径分布での直径」とは、分布のピークでの孔径(すなわち、モード孔径(modal pore size))を示す;
「平均流孔径」とは、平均孔径を示す。
・エレクトロスピニングポリマー溶液濃度
・エレクトロスピニング溶媒
・エレクトロスピニング電圧
・エレクトロスピニング持続時間
・繊維コレクターの種類、形状または構造材料
・円筒状コレクターの直径、回転速度または長さ
・針の横断距離、横断回数または横断速度
・針の直径、長さ、断面形状または構造材料
・針の本数または針の配置
・針からコレクターまでの分離距離
・高圧構造
・添加剤(例えば塩)による溶媒導電性
・繊維コレクターを覆うために使用する基材(非剥離紙(no release paper)の使用を含む)
・環境大気組成、環境大気圧、環境大気温度または環境大気湿度
・溶媒が完全またはほぼ完全に繊維から蒸発し、繊維がコレクターに衝突する際に一緒に結合しないことを確実にするための、層の1つまたは複数に対する上記条件のいずれかの変更
・溶媒には、実質的に蒸発するのに十分な時間が与えられず、それによりコレクター上で他の繊維と部分的に結合し、高度に相互結合した多孔質メッシュを形成する部分的に溶媒和された繊維になることを確実にするための、層の1つまたは複数に対する上記条件のいずれかの変更
・繊維が、足場の強度および構造保持力を改善するために、足場の繊維性を実質的に変えずにコレクター上の他の繊維と一緒に結合することを可能にするのに十分な溶媒を保持する、中間体の状態に対する上記条件のいずれかの変更。
(i)約1μmから100μmの間の直径を有する孔を含む第1の繊維材料を創傷内に配置する工程であり、前記第1の繊維材料が、皮膚線維芽細胞によりコロニー形成することができ、それにより真皮の再生を促進する工程と、
(ii)第2の繊維材料を第1の繊維材料の上に配置する工程であり、第2の繊維材料が、約50nmから20μmの間の直径を有する孔を含み、第2の繊維材料が、ケラチン生成細胞によりコロニー形成することができ、それにより真皮の再生を促進する工程と
を含む。
1. Chong E.J et al (2007) “Evaluation of electrospun PCL/gelatine nanofibrous scaffold for wound healing and layered dermal reconstruction.” Acta Biomaterialia 3, 321 to 330
2. Ma, PX (2004) “Scaffolds for tissue fabrication”, Materials Today, 2004, 30 to 40
3. Desmouliere A et al (2005) “Tissue repair, contraction and the myofibroblast” Wound Rep Regen 13 (1) 7 to 12
4. Werner, S et al (2007) “Keratinocyte-fibroblast interactions in wound healing” J Invest Dermatol 127 (5) 998 to 1008
不織布単層足場を、1,1,1,3,3,3-ヘキサフルオロプロパン-2-オール(ヘキサフルオロイソプロパノール、HFIP)中のポリ(グリコール酸)(PGA)の溶液をエレクトロスピニングすることにより調製した。
PURAC Biomaterialsから供給されたPGA(概算重量平均分子量130000)を、Rondol Linear 18一軸スクリュー押出機を用いて260〜274℃で溶融押し出しし、次いで5〜10℃にて水中ですぐに失活させた。この押し出したPGAを使用して、Apollo Scientific Ltdから供給された分光光度等級のHFIP中の7w/w%溶液(約0.35Pa.sの溶液粘度に対応)を調製した。この溶液を、溶解するまで21℃で終夜回転した。エレクトロスピニングする前に、HFIP中のPGA溶液を10.0μmのWhatman Polydisc HDフィルター(ポリプロピレンフィルター、直径50mm)を介して濾過し、20mLのシリンジ(ポリプロピレン、潤滑剤不使用、内径20.0mm)に直接入れた。得られたポリマー溶液は、視認できる微粒子を含んでいなかった。
シリンジ出口を、HFIP耐性軟質プラスチック管に接続し、次いでそれを2本の管に分けた。これらの管は、モーター(図2、アイテム6)により横断させることが可能である、針アーム(図2、アイテム2)で支持された2本の先端が平らな21ゲージのスチール針(図2、アイテム3)につながっていた。針を、接地された直径50mm、長さ200mmのスチールマンドレル(図2、アイテム4)の回転軸(図2、アイテム7)に対して垂直に配向し、針の先端からマンドレルまでの分離距離(図2、アイテム5)を150mmに設定した。針を、18.5秒ごとに1横断の速度で、全長200mmのマンドレルに沿って横断するように設定した(横断は、横断距離の長さに沿った単一の前進運動または後進運動として定義される)。
作製した単一の足場層の厚さを、Mitutoyo Absolute Digimaticデジタルキャリパーを用いてその長さに沿って(すなわち、マンドレルの回転軸に平行)いくつかの地点で測定した。
足場試料を、いずれかの端部に2つの小片の両面接着剤を用いて12mmのアルミニウムSEMスタブに付着させ、中心帯は接着剤なしで残した。試料を、足場の上面が視認できるように付着させた(すなわち、表面には実験が終わる頃に付着)。次いで、試料を金/パラジウム合金で、約30nmの推定深さにスパッタ被覆した。次いで、被覆した試料を、FEI Quanta 3.1.1ソフトウェアとともに電圧5.0kVおよびスポット直径2.5nmを用いて高圧モードでFEI-Quanta Inspect SEMで撮像した。12,000倍率で取得したSEM画像例を図3に示す。
エレクトロスピニングした各繊維足場の試料1つに対して、適切な倍率の3枚のSEM画像を記録および印刷し、これらを用いて平均繊維径を計算した。各画像に対して、ランダムに選択した直線に沿った、最初の20本の明確に視認できる繊維の直径を、定規を用いて測定した。3枚の画像から総計60の測定値を使用して、平均繊維径および標準偏差を計算した。
円形試料(直径26mm)を、テンプレートおよび外科用メスを用いて、足場の厚さが均一な部分から切断した。これらの試料に対して、PMI Capillary Flow Porometer CFP-1100-AEXLを用いてキャピラリーフローポロメトリー分析を行った。使用した湿潤液は、Galwic(表面張力15.9dyn.cm-1)であり、使用した試験方法は、8psiまたは12psiの最大圧力を用いたドライアップ/ウェットアップ(Dry Up/Wet Up)であった。
厚さ=中心60%の足場長さに沿って100〜120μm
平均繊維径=0.44μm±0.20μm
最大検出孔径=1.98μm
平均流孔径(平均孔径)=1.11μm
最大孔径分布での直径=0.93μm
HFIP中のPGAの8w/w%溶液を調製し使用して、実施例1に記載されているのと同じ一般的方法を用いて不織布単層足場材料を調製した。このHFIP中のPGAの濃度は、約0.55Pa.sの溶液粘度に対応している。図4は、10000倍率で取得した足場のSEM画像を示している。
厚さ=中心65%の足場長さに沿って120〜140μm
平均繊維径=0.51μm±0.12μm
最大検出孔径=2.29μm
平均流孔径(平均孔径)=1.15μm
最大孔径分布での直径=0.94μm
HFIP中のPGAの9w/w%溶液を調製し使用して、実施例1に記載されているのと同じ一般的方法を用いて不織布単層足場材料を調製したが、水性塩化ナトリウムは、HFIP中のPGA溶液に加えなかった。HFIP中のPGAの濃度は、約0.85Pa.sの溶液粘度に対応している。さらに、エレクトロスピニング持続時間を68分に増加した。図5は、6000倍率で取得した足場のSEM画像を示している。
厚さ=中心70%の足場長さに沿って100〜110μm
平均繊維径=0.81μm±0.38μm
最大検出孔径=3.44μm
平均流孔径(平均孔径)=1.87μm
最大孔径分布での直径=1.58μm
異なる構造の2層を含む不織布の二重層足場を、各々1.7Pa.sおよび0.55Pa.sの溶液粘度に対応するHFIP中のPGAの11w/w%溶液および8w/w%溶液を用いて調製した。
第1の層
厚さ=中心75%の足場長さに沿って60〜70μm
平均繊維径=2.58μm±0.44μm
厚さ=中心60%の足場長さに沿って120〜130μm
平均繊維径=0.68μm±0.37μm
ケラチン生成細胞の移動および増殖を支持する第2の繊維材料層の能力を示すために、実施例1〜3で調製した足場上でのヒトケラチン生成細胞のインビトロ移動挙動を評価した。これらの足場を、2つの陽性対照:Thermanoxカバースリップ(Nunc GmbHから供給);および実施例1に記載されているのと同じ一般的方法(ただし、HFID中のPGAの11w/w%を使用)を用いて調製した、より大きな平均繊維径2.46μm(S.D.0.50μm)を有する100〜110μmの厚さのエレクトロスピニングしたPGA足場と比較した。エレクトロスピニングしたPGA足場は、国際公開第07/132186号(Smith and Nephew)に記載されているものと同様に、線維芽細胞の移動および増殖を支持することが示された。
足場および対照を、Samco SB-25液圧プレスを用いて直径13mmのディスクに切断し、Minucellクリップ(部品番号1300、Minucell and Minutissue Vertriebs, GmbH)に配置し、Amersham UVクロスリンカーを用いて紫外線下で20分滅菌した。正常なヒトケラチン生成細胞(NHEK、Promocell GmbHから供給)を、1ディスク当たり100000細胞の密度で、ケラチン生成細胞増殖培地(KGM-2; Promocell GmbH)100μl中のディスク上に播種し、空気95%およびCO25%の混合物中で37℃にて1時間接着させた。1時間後、ディスクを、滅菌リン酸緩衝液(PBS)に浸し、付着しなかった全細胞を除去し、KGM-2培地2mlを含有する24ウェルプレートのウェルに配置した。得られたディスクを、空気95%およびCO25%の混合物中で37℃にて24時間インキュベートした。
ケラチン生成細胞が1つの平面上でコロニーとして移動するため、移動は、細胞数を定量するのではなく視覚的に評価された。インキュベーション後、ディスクをPBSで2回洗浄し、4',6-ジアミジノ-2-フェニリンドール(DAPI; Vector Laboratories Ltdから供給)を含有するマウンティング培地を用いてガラススライド上に載置した。次いで、スライドをLeica DMLB蛍光顕微鏡を用いて視覚化した。
・NHEK細胞は、エレクトロスピニングした足場すべてに接着し、24時間のインキュベーション後足場表面上に視認できる。
・NHEK細胞は、96時間のインキュベーション内で全足場の端部に移動する。
・実施例1および2で調製した2つの足場は、NHEK細胞の移動を、ケラチン生成細胞シートの移動端で覆われた距離により明示された足場対照よりも良好に支持した。これは、異なる構造(実施例1および2は、より小さい平均繊維径および孔径を有していた)によるものである。
・実施例3で調製した足場は、実施例1および2と比較してより大きい平均繊維径および孔径を有していたため、足場対照と同じ態様で挙動した。
2 針アーム
3 スチール針
4 スチールマンドレル
5 針の先端からマンドレルまでの分離距離
6 モーター
7 回転軸
8 モーター
9高電圧発生器
Claims (9)
- 少なくとも2種の繊維材料を含む生体吸収性合成足場であって、第1の繊維材料が4μmから9μmの間の直径を有する孔を含み、第2の繊維材料が0.1μmから3.5μmの間の直径を有する孔を含み、第2の繊維材料の繊維は、0.1μmから1.2μmの直径を有する、足場。
- 第1の繊維材料および第2の繊維材料が、異なる組成から成る、請求項1に記載の足場。
- 少なくとも2種の繊維材料が足場内で層を形成する、請求項1または2に記載の足場。
- 前記層が実質的に平面である、請求項3に記載の足場。
- 前記層が互いに隣接している、請求項3または4に記載の足場。
- 前記足場が、第2の繊維材料層に結合した第1の繊維材料層を含む積層体であり、前記第1および第2の材料が、異なる組成から成る、請求項2から5のいずれか一項に記載の足場。
- 前記繊維材料が不織布である、請求項1から6のいずれか一項に記載の足場。
- 第1の繊維材料および第2の繊維材料の少なくとも1つがエレクトロスピニングされている、請求項1から7のいずれか一項に記載の足場。
- 4μmから9μmの間の直径を有する孔を含む第1の繊維材料、および0.1μmから3.5μmの間の直径を有する孔を含む第2の繊維材料を備え、第2の繊維材料の繊維は、0.1μmから1.2μmの直径を有する、真皮および表皮の再生を促進するための、キット。
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GB0801405.2 | 2008-01-25 | ||
GB0801405A GB0801405D0 (en) | 2008-01-25 | 2008-01-25 | Multilayer scaffold |
GB0802767.4 | 2008-02-15 | ||
GB0802767A GB0802767D0 (en) | 2008-02-15 | 2008-02-15 | Multilayer scaffold |
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AU2009207489A1 (en) | 2009-07-30 |
AU2009207489B2 (en) | 2014-09-11 |
EP2244754A2 (en) | 2010-11-03 |
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