JP4879404B2 - Porous tissue skeleton-forming material for tissue repair or regeneration - Google Patents

Description

表３ ＰＣＲ最適化サイクル
遺伝子 ｃＤＮＡ（μｌ） サイクル数
アルカリホスファターゼ １ ２５
オステオカルシン １ ３５
ＧＡＰＤＨ １ ２３
【００９８】
結果
生体吸収性ポリマーにおける細胞付着および増殖
種々のポリマーの間およびＴＣＰＳに比較して細胞形態における顕著な差が見られなかった。２４時間の培養後において種々の生体ポリマーフィルムに対する細胞付着はＴＣＰＳと同等であった。３日目において、細胞は４０／６０ＰＣＬ／ＰＬＡを除いて全てのフィルムにおいて良好に増殖した。なお、４０／６０ＰＣＬ／ＰＬＡの場合はＴＣＰＳに比して増殖は６０％であった。さらに、９５／５ＰＬＡ／ＰＧＡおよび９０／１０ＰＧＡ／ＰＬＡフィルムはＴＣＰＳおよび４０／６０ＰＣＬ／ＰＬＡに比べて有意差をもって（ｐ＜０．０５）高い程度の細胞増殖度を支持した（図７）。
【００９９】
分化アッセイ
アルカリホスファターゼ酵素活性
９５／５ＰＬＡ／ＰＧＡ、９０／１０ＰＧＡ／ＰＬＡおよび９５／５ＰＬＡ／ＰＣＬフィルム上で培養した骨芽細胞により発現したアルカリホスファターゼ活性のプロファイルはＴＣＰＳにおいて見られるプロファイルに類似していた。一方、１４日目および２１日目における４０／６０ＰＣＬ／ＰＬＡおよび７５／２５ＰＧＡ／ＰＣＬフィルムにおいて培養した骨芽細胞の場合は、それぞれ、別のフィルムおよびＴＣＰＳに比べてアルカリホスファターゼ比活性が有意差をもって（ｐ＜０．０５）増加した（図８）。
【０１００】
アルカリホスファターゼおよびオステオカルシンのｍＲＮＡの発現
９５／５ＰＬＡ／ＰＧＡ、４０／６０ＰＬＡ／ＰＣＬ、９５／５ＰＬＡ／ＰＣＬフィルムおよびＴＣＰＳにおいて培養した骨芽細胞に対するアルカリホスファターゼ、オステオカルシン、およびＧＡＰＤＨにおけるｍＲＮＡの発現をデンシトメトリーにより評価した。これらの結果を図９に示す。なお、図８におけるデータが最良の半定量分析におけるデータであることに注意されたい。ところが、これらのデータは４０／６０ＰＣＬ／ＰＬＡフィルムがＴＣＰＳに比べて有意差をもって（ｐ＜０．０５）高い程度のオステオカルシンの発現を支持したことを示している。一方、残りのポリマー表面はオステオカルシンおよびアルカリホスファターゼのｍＲＮＡ発現の両方においてＴＣＰＳと同等であった。
【０１０１】
結論
６日間の培養に続いて試験した異なる生体吸収性フィルムまたはメッシュの間に細胞の付着および増殖に関する顕著な差は見られなかった。さらに、これらの結果により、それぞれの分化特性に関してそれぞれの材料の間の差が比較的明瞭であることが分かった。すなわち、４０／６０ＰＣＬ／ＰＬＡフィルムにおける細胞培養は１４日および２１日の培養後の別のフィルムおよびＴＣＰＳにそれぞれ比較して向上したアルカリホスファターゼ活性およびオステオカルシンｍＲＮＡの発現を示した。
【０１０２】
この技法をさらに完全に理解するために、M. A. Aronow、L. C. Gerstenfeld、T. A. Owen、M. S. Tassinari、G. S. SteinおよびJ. B. Lianの「培養した胎児ラット頭蓋冠細胞における骨芽細胞表現型の進行性の発育を助長する因子（Factors that promote progressive development of the osteoblast phenotype in cultured fetal rat calvaria cells）」（Journal of Cellular Physiology, 143:第２１３頁乃至第２２１頁（１９９０年））、およびStein, G. S.、Lian, J. B.およびOwen, T. A.の「骨芽細胞の分化中におけ組織特異性遺伝子発現の調整に対する細胞成長の関係（Relationship of cell growth to the regulation of tissue-specific gene expression during osteoblast differentiation）」(FASEB, 4,第３１１１頁乃至第３１２３頁（１９９０年）)を引用する。
【０１０３】
実施例７
豚皮膚傷治癒モデルにおける発泡体混合物のインビボでの検討
この実施例においては、豚全厚切除傷モデルにおける１ｍｍおよび０．５ｍｍ厚の発泡体骨格形成材料を移植した結果について説明する。発泡体組織骨格形成材料を実施例８において説明するように作成した４０／６０ε−カプロラクトン−コ−ラクチドおよび実施例９において説明するように作成した３５／６５ε−カプロラクトン−コ−グリコリドの混合物により作成した。すなわち、これらのポリマーを混合して実施例３に記載したものとほぼ同じ（冷却速度が２．５℃／分で−５℃に冷却したのみであることを除く）１ｍｍおよび０．５ｍｍ厚の発泡体を形成した。０．５ｍｍ発泡体の走査電子顕微鏡写真を図１１（Ａ），図１１(Ｂ)および図１２に示す。その後、２種類の厚さ（０．５ｍｍおよび１ｍｍ）の発泡体を傷つけた切除モデルにおいてＰＤＧＦの供給有りおよび供給無しの状態で試験して、これらの異なる４種類のサンプルを評価した。
【０１０４】
傷を形成してから８日目に移植した４頭の豚における４８個の全厚切除傷（１頭当たり１２部位）について無作為的な組織学的評価を行った。この評価はＨ＆Ｅ染色したスライドについて行った。この組織学的評価の間に、以下のパラメータを各試料において順位付けおよび／または評価した。すなわち、基質の細胞浸潤についての定性的および定量的評価、（２）基質の接触領域（腹側の表面）内への多形核白血球（ＰＭＮ）の浸潤、（３）基質の（腹側から）下方の肉芽組織床における炎症、（４）基質に対する表皮の反応、および（５）基質の分断の度合いである。
【０１０５】
動物体の管理
豚をそれぞれ個別にケージ（１０平方フィートの最少床面積を有する）内に収容して認識票を付けた。全ての豚に個々の動物体番号を割り当てた。すなわち、動物体番号、種類／血統、手術日、手術技法および実験の継続時間、および安楽死の期日を記載したタグを各動物体ケージに付けた。また、各動物体は永久マーカーによりその首の付け根に動物体番号を印付けした。
【０１０６】
各動物体の部屋を４０％乃至７０％の相対湿度および１５℃乃至２４℃（５９．０乃至７５．２°Ｆ）に維持した。各動物体には１日１回の標準的な豚用の食事を与えたが、麻酔処理を必要とするあらゆる実験処理の前は一晩食事を与えなかった。水は自由に摂ることができるようにした。日常的な明るさ：暗さのサイクルを１２：１２時間とした。
【０１０７】
麻酔処理
実験の最初の日、各評価の日および検死の日に、各動物体を拘束して、Tiletamine HCLプラスZolazepam HCL（Telazol（登録商標）、アイオワ州フォート・ドッジのFort Dodge Animal Health社、４ｍｇ／ｍｌ）およびXylazine（Rompun（登録商標）、カンザス州シャウニー・ミッションのBayer Corporation のアグリカルチャー部、Animal Health 社、４ｍｇ／ｍｌ）または鼻頭を介して投与するIsoflurane(AErrane（登録商標）、アイオワ州フォート・ドッジのFort Dodge Animal Health社）吸入麻酔剤（５％容量）のいずれかにより麻酔処理した。動物体に外科手術を施す場合には、この動物体に鼻頭を介してIsoflurane（AErrane（登録商標））吸入麻酔をかけた状態で維持した。また、各処理から回復した後に食事を与えた。
【０１０８】
外科手術部位の作成
外科処理の１日前に、体重を測定して４頭の豚の背面領域を＃４０外科用シェービングブレードを備える電気クリッパーにより刈り込んだ。その後、毛を刈った皮膚をさらにシェービングクリームと剃刀により綿密に剃って、すすぎ洗いした。この毛を剃った皮膚および動物全体（頭部を除く）をさらにＰＣＭＸ洗浄溶液（Pharmaseal（登録商標）Scrub Care（登録商標）、カリホルニア州バレンシアのBaxter Healthcare社のPharmaseal部）により外科用スクラブ・ブラシスポンジで擦り洗いしてから、HIBICLENS（登録商標）クロルヘキシジングルコネート（イリノイ州シカゴのCOE Laboratories社から入手）によりさらに擦り洗いした。この動物体を滅菌処理したタオルで拭いて乾燥した。その後、各動物体の背面上に滅菌NU-GAUZEガーゼ（テキサス州アーリントンのJohnson & Johnson Medical 社から入手）を置いて、WATERPROOF^★テープ（テキサス州アーリントンのJohnson & Johnson Medical社から入手）により固定した。この動物体の胴部全体をSpandage(商標)弾性伸縮包帯（ニューヨーク州ブルックリンのMediTech International社から入手）で包んで一晩清浄な表面に維持した。
【０１０９】
手術の日に、外科手術部位に動物体を運ぶ直前に、前日に行ったように、その背面の皮膚をＰＣＭＸ洗浄溶液（Pharmaseal（登録商標）、Scrub Care（登録商標））により外科用スクラブ・ブラシスポンジで再び擦り洗いし、すすいで滅菌処理したタオルで拭いて乾燥した。動物体をうつむきに手術台の上に載せて、７０％アルコールで拭いてから滅菌ガーゼで乾かした。滅菌処理した外科用マーカー（マサチューセッツ州レインハムのJohnson & Johnson Professional社の一部門であるCodmanから入手）およびアセテートテンプレートを用いて、各全厚傷の所望の位置に応じて背面の皮膚に標識を作成した。
【０１１０】
外科処理
麻酔をかけてから、滅菌条件下に、メスを用いて左右の背面領域に脊柱に平行に２列に１２個の全厚切除部（１．５ｃｍ×１．５ｃｍ）を各動物体に作成した。さらに、一対の鋏および／またはメスを皮膚および皮下組織の除去のために用いた。出血をスポンジタンポンにより調整した。傷と傷との間に十分な空間を置いて傷同士の干渉を避けた。切除した組織の厚さをデジタルキャリパーにより測定した。
【０１１１】
治療および包帯の供給
各傷を予め準備したコード化した治療手順（各実験対象は全ての治療に対して目隠しした状態であった）で処理した。滅菌した個別の試験物品（１．５ｃｍ×１．５ｃｍの滅菌塩類溶液に２４時間浸漬したもの）から成る一次包帯を所定の計画に従って傷欠損部に配置した。次に、非付着性で、塩類溶液に浸漬した、方形のRELEASE^★包帯（テキサス州アーリントンのJohnson & Johnson Medical社により製造されている）から成る二次包帯を上記の試験物品の上部に配置した。さらに、BIOCLUSIVE^★ 包帯の層（テキサス州アーリントンのJohnson & Johnson Medical社から入手）により傷を密封して傷の湿り気を維持すると共に内部の包帯を保持した。また、Reston(商標)（ミネソタ州セント・ポールの３Ｍ社のメデイカル−サージカル部）ポリウレタン自己付着性発泡材の片状部材を各傷の間に配置して傷の流体漏れによる相互汚染を防ぐと共に傷の損傷および包帯のずれを防いだ。加えて、NU-GAUZE^★ の層をBIOCLUSIVE^★ 包帯およびReston(商標)発泡材の上部に配置し、WATERPROOF^★ テープにより固定して各包帯を保護した。その後、動物体をSpandage(商標)弾性ネットにより包帯して各包帯を保持した。
【０１１２】
上記の二次包帯を毎日除去して、塩類溶液に浸漬したRELEASE^★二次包帯の新しいものと置き換えた。また、一次包帯（試験物品）はこのユニットを置換または傷欠損部から押し出すまで触らなかった。
【０１１３】
術後の医療管理および臨床的観察
麻酔において外科処理を行った後に、動物体を各ケージに戻して回復させた。各動物体に術後すぐおよびその次の日に鎮痛剤（バプレノルフィンヒドロクロライド（Buprenex Injectable、０．０１ｍｇ／ｋｇ)英国ハルのReckitt & Colmanにより販売されている）を与えた。麻酔から回復後に、各豚の深いまたは痛みに対する挙動徴候を観察した。この結果、痛みに対する徴候は全く見られなかった。
【０１１４】
手術日の後に各豚を１日に２回観察して、一般的な挙動、外観、食物消費量、排尿および排便、および異常な排出物の存在を基準にしてその健康状態を決めた。
【０１１５】
安楽死
実験の終了時（傷つけ処理後８日）に、耳周縁部の静脈を介する（（オハイオ州コロンバスのButler社により販売される）Socumb（商標）ペントバルビタールナトリウムおよびフェニトインナトリウム安楽死溶液（１ｍｌ／１０ポンド体重））の静脈注射を伴って各動物体を麻酔により安楽死させた。この安楽死処理の後に、呼吸機能の停止および触診可能な心臓機能が無いことを確認するために動物体を観察した。この際、聴診器が心臓機能の停止の確認を容易にする。
【０１１６】
組織採取
安楽死の直後に、各傷を下層の脂肪および小部分の周囲の皮膚と共に切除した。この組織を１０％中性バッファー化ホルマリンの中に入れた。
【０１１７】
評価
視覚的傷評価
位置ずれ、傷反応および骨格形成材料の物理特性を含む一般的観察を１日目乃至３日目に対して記録した。さらに、詳細な臨床的評価を傷つけ後４日目乃至８日目について行った。以下のパラメータの有り／無し（有り＝１／無し＝０）および／または程度（点数を付ける）について記録した。
【０１１８】
包帯の状態
空気暴露、試験物品の位置ずれ、流通（channeling）、連通communication)およびRELEASE^★ 二次包帯の水分含有量（点数付け基準：４＝湿っている、３＝湿っている／乾燥している、２＝乾燥している／湿っている、１＝乾燥している）。
【０１１９】
傷床状態
試験物品の水分含有量（点数付け基準：４＝湿っている、３＝湿っている／乾燥している、２＝乾燥している／湿っている、１＝乾燥している）、炎症（点数付け基準：３＝重度、２＝中程度、１＝軽度、０＝無し）、再損傷（点数付け基準：３＝重度、２＝中程度、１＝軽度、０＝無し）、凝血、毛嚢炎、感染、試験物品の高さ（点数付け基準：４＝極めて上昇している、３＝上昇している、２＝変化無し、１＝下降している）、フィブリン（点数付け基準：３＝重度、２＝中程度、１＝軽度、０＝無し）、および紅斑。また、試験物品の色も観察した。
【０１２０】
切除した組織サンプルを８日目に採取した。全体の傷を採取して１０％の中性バッファー化したホルマリンに入れた。その後、組織を凍結した部分に調製した。この組織を切り取って、Tissue-Tek（登録商標）OCT 化合物（日本国東京のSakura Finetechnical社により販売されている）を伴う対象物ホルダー上におき、速やかに凍結した。これらの試料を低温槽上で１０μｍに切断して凍結したH&E染色剤により染色した。
【０１２１】
組織学的評価（傷つけ後８日目）
肉芽組織（面積および長さ）および表皮形成の組織学的評価を２０倍乃至４０倍の倍率でH&E染色した試料により評価した。肉芽組織の高さを面積を長さで割ることにより決定した。
【０１２２】
さらに、組織サンプルの組織病理学的評価をH&E染色試料により行い、まず、１００倍乃至４００倍の倍率でこれらのサンプルを評価した。
【０１２３】
結果
全ての部位の大部分における基質の間隙の中に細胞の浸潤が見られた。これらの部位の大部分において、この浸潤は変化する線維芽細胞の集団、マクロファージ、マクロファージ巨細胞、および内皮類似細胞により構成される真の組織の内部成長であり、毛細管の形成も見られたように思える。組織において基質を実質的に埋める基質に対して背面側の密度の高い線維状の接合組織層の実質的な形成がＰＤＧＦを伴うおよび伴わない０．５ｍｍの発泡体における幾つかの部位に見られる。また、１ｍｍの基質は組織の表面にあるか脱落していた。マクロファージ巨細胞の形成は１ｍｍの発泡体骨格形成材に対して０．５ｍｍのものにおいてより多く見られた。１ｍｍの発泡体が下層の肉芽組織から脱落しているか部分的に分離している部位においては、死んだ浸潤細胞が核濃縮の細胞くずの塊を形成していた。
【０１２４】
０．５ｍｍの発泡体骨格形成材料の場合は幾つかの部位において肉芽組織床の中への基質の完全な取り込みが見られた。図１３および図１４はこれらの基質の肉芽組織床の中への取り込みを示している図である。図１３はトリクローム（trichrome)染色した組織サンプルの４０倍の暗視野顕微鏡写真である。また、図１４はＰＤＧＦを含有する発泡体の細胞浸潤を示すトリクローム染色したサンプルの１００倍の複合顕微鏡写真である。これらの両方の写真において肉芽組織床内への基質の完全な取り込みが明らかに分かる。また、これら両方の写真において発泡体骨格形成材料上の高密度の線維組織の存在も明瞭に分かる。これらの存在により、０．５ｍｍの発泡体は表皮組織の成長において適当な基質を構成することが分かった。
【０１２５】
実施例８
ランダムポリ（ε−カプロラクトン−コ−グリコリド）の合成
３５／６５のモル組成を有するε−カプロラクトン−グリコリドのランダムコポリマーを開環重合反応により合成した。この合成法は実施例６における米国特許第５，４６８，２５３号（本明細書に参考文献として含まれる）において記載される方法とほぼ同じである。添加するジエチレングリコール開始剤の量をモノマーの１．１５ミリモル／モルに調節して以下の特性の乾燥状態のポリマーを得た。すなわち、このコポリマーの固有粘度（Ｉ．Ｖ．）は２５℃でヘキサフルオロイソプロパノールにおいて１．５９ｄＬ／ｇであった。ＰＣＬ／ＰＧＡのモル比はプロトンＮＭＲにより３５．５／６４．５であり、０．５％の残留モノマーを含有していた。さらにこのコポリマーのガラス転移点（Ｔｇ）および融点（Ｔｍ）はＤＳＣにより−１℃，６０℃および１２６℃であることが分かった。
【０１２６】
実施例９
連続添加による４０：６０ポリ（ε−カプロラクトン−コ−Ｌ−ラクチド）の合成
グローブボックスにおいて、１００μＬ（３３μモル）の０．３３モルのスズ（ＩＩ）オクトエートのトルエン溶液、１１５μＬ（１．２ミリモル）のジエチレングリコール、２４．６グラム（１７０ミリモル）のＬ−ラクチド、および４５．７グラム（４００ミリモル）のε−カプロラクトンをステンレススチール・メカニカルスターラーおよび窒素ガスブランケットを備えたシラン処理し、火炎乾燥したトゥーネックの２５０ｍＬ丸底フラスコの中に入れた。この反応フラスコを既に１９０℃に設定してあるオイルバス中に入れて保持した。また、このグローブボックス中において、６２．０グラム（４３０ミリモル）のＬ−ラクチドを火炎乾燥して等圧化した滴下ロート中に入れた。このロートをヒートテープで包んで反応フラスコの第２ネックに取り付けた。１９０℃において６時間後に、溶融したＬ−ラクチドを５分かけて反応フラスコ内に添加した。この反応を一晩続けて１９０℃で合計２４時間行った後に、反応物を放置して室温まで冷却した。その後、液体窒素において凍結し、ガラスを破壊してこのコポリマーを単離した。残留したガラス破片はベンチグラインダーによりコポリマーから取り除いた。このコポリマーを液体窒素により再び凍結して、メカニカルスターラーのパドルを取り外した。さらに、Wiley Mill（微粉砕機）によりこのコポリマーを削って風袋を軽量したガラスの広口ビンの中に入れ、真空オーブン内において一晩かけて室温まで加温した。その後、１０３．１３グラムの４０：６０ポリ（ε−カプロラクトン−コ−Ｌ−ラクチド）を風袋を軽量したアルミニウム皿に入れて５４時間かけて１１０℃で真空処理して揮発物質を除去した。この結果、９８．７グラム（９５．７重量％）のコポリマーを脱気処理後に回収した。固有粘度を計測した結果、２５℃でＣＨＣｌ₃ 中（濃度＝０．１ｇ／ｄＬ）において１．１ｄＬ／ｇであった。ＦＴＩＲ（ＫＢｒ窓上におけるＣＨＣｌ₃ からのキャストフィルム、ｃｍ^-1）：２９９３，２９４４，２８６８，１７５９，１４５６，１３８３，１３６２，１１８４，１１３２，１０９４，８７０，および７５６。 ¹Ｈ−ＮＭＲ（４００ＭＨｚ、ＨＦＡＤ／ベンゼン、ｐｐｍ）：δ１．２５、２本の幅広(broad)線（ｅ）；１．３５，２本線（ｆ）；１．４２、３本線；１．５５、２本線；２．２２、３本線；２．３５、４本の幅広線；４．０１、３本線；４．０５、３本線；４．２、四重線；５．０５、３本の幅広線；５．１５、４本線。 ¹Ｈ−ＮＭＲによるポリマー組成：４１．８％ＰＣＬ、５７．５％ＰＬＡ、０．８％Ｌ−ラクチド、＜０．１％ε−カプロラクトン。ＤＳＣ（２０℃／分、第１熱）：Ｔ_m ＝１５４．８℃、ΔＨ_m ＝１８．３Ｊ／ｇ。ＧＰＣ（ポリ（メチルメタクリレート）基準を用いるＴＨＦ中で決定した分子量）：Ｍ_w ＝１６０，０００、Ｍ_n ＝１０１，０００、ＰＤＩ＝１．６。
【０１２７】
実施例１０
９５／５ＰＬＡ／ＰＣＬコポリマーの合成
グローブボックスにおいて、１７０μＬ（１．８ミリモル）のジエチレングリコール、３５０μＬ（１１５μモル）の０．３３モルのスタナスオクトエートのトルエン溶液、１７．１グラム（１５０ミリモル）のε−カプロラクトン、および４１０．４グラム（２．８５モル）のＬ−ラクチドをシラン処理して火炎乾燥した１０００ｍＬ丸底フラスコに入れた。このフラスコはステンレスステンレススチール・メカニカルスターラーおよび乾燥窒素ガスブランケットを保持するための減圧解除用コネクタを備えている。この反応フラスコを既に１８５℃に加熱してあるオイルバスに入れて３時間保持した。その後、フラスコをオイルバスから取り出して室温まで放置冷却した。フラスコをアルミニウムホイルで包んで、これを液体窒素中で凍結することによりポリマーを単離して、ポリマーに付着したガラスを削り取った。その後、このコポリマーをWiley mill（粉砕機）において削り、削ったポリマーを８０℃で２４時間真空乾燥して３０２グラムのコポリマーを回収した。この固有粘度はクロロホルム中（２５℃、濃度＝０．１ｇ／ｄＬ）で２．１ｄＬ／ｇであった。このポリマー組成をプロトンＮＭＲ分光分析により測定して、９７．２モル％のＰＬＡおよび２．８モル％のＰＣＬであることが分かった。残留モノマーは全く検出されなかった。
【０１２８】
実施例１１
６０／４０ＰＬＡ／ＰＣＬハイブリッド発泡体／微小繊維織物構造の合成
工程Ａ．６０／４０ＰＬＡ／ＰＣＬの１４％重量／重量均質トリクロロエタン溶液の作成
１４部の６０／４０ＰＬＡ／ＰＣＬ（Ｉ．Ｖ．＝１．３４ｄｌ／ｇ）を８６部のトリクロロエタン溶媒（ＴＣＥ）で溶解することにより１４％重量／重量ポリマー溶液を調製した。この溶液を磁気攪拌棒を備えたフラスコ内で調製した。小ポリマーを完全に溶解するためには混合物を６０℃で一夜攪拌することが推奨される。
【０１２９】
工程Ｂ． 静電紡糸
この実験には静電スピナーを用いた。スペルマン高電圧直流供給器（Spellman High Voltage DC Supply）（モデル番号：ＲＨＲ３０ＰＮ３０、スペルマン ハイボルテージ エレクトロニクス コーポレーション（Spellman High Voltage Electronics Corporation）, 米国、ニューヨーク州ホッポージ市（Hauppauge, NY, USA））を高圧電源として用いた。印加電圧およびマンドレル速度をラブビューコンピュータソフトにより調節した。紡糸口金との間の距離を機械的に調節した。
【０１３０】
実施例１に記載のように作成した凍結乾燥した６０／４０ＰＬＡ／ＰＣＬ発泡体の平坦なシートをロール掛けして円筒にした。両端部を紡糸において使用した溶媒であるトリクロロエタン数滴で固定した。この発泡体チューブを回転している接地した導電性マンドレルに搭載した。発泡体基体により覆われていないマンドレルの両端部を絶縁テープでマスクして両端部に微小繊維が引きつけられるのを防止した。溶液を紡糸口金内に置き、高電圧をこのポリマー溶液に印加した。
この実験を室温および周囲湿度において行った。
【０１３１】
紡糸時の処理条件は下記の通りであった：
マンドレル電圧 １６，０００Ｖ
マンドレル速度 １００ｒｐｍ
紡糸口金とマンドレルとの距離 １０ ｃｍ
温度 室温
湿度 周囲湿度
【０１３２】
厚さがほぼ２００μｍの層をチューブの他面上に堆積した。
【０１３３】
実施例１２
カプセルに包まれたハイブリッド発泡体／織物構造において培養した軟骨細胞は細胞移動が制限されることを示す
工程Ａ．ハイブリッド６０／４０ＰＬＡ／ＰＣＬ発泡体／微小戦記織物骨格形成材料の作成
実施例１に記載の６０／４０ＰＬＡ／ＰＣＬから作成した凍結乾燥した発泡体を実施例１１に詳細に説明したように、トリクロロエタン中に６０／４０ＰＬＡ／ＰＣＬの静電紡糸の際に、実施例１に記載された６０／４０ＰＬＡ／ＰＣＬから作成された凍結乾燥した発泡体を基体として使用した。
【０１３４】
工程Ｂ．ハイブリッド６０／４０ＰＬＡ／ＰＣＬ発泡体／微小繊維織物骨格形成材料への軟骨細胞の播種
ウシの肩部から単離した軟骨細胞をＤｕｌｂｅｃｃｏの修正Ｅａｇｌｅ培地（高グルコース）に１０％ウシ胎児血清、１０ｍＭ ＨＥＰＥＳ、０．１ｍＭ非必須アミノ酸、２０μｇ／ｍｌのストレプトマイシンおよび０．２５μｇ／ｍｌのアンホテリシンＢを補充したものにおいて培養した。
【０１３５】
３次元移動検定法
この検定法は細胞を移植した収縮されたコラーゲン格子にコラーゲンゲルの中心部に生分解性ポリマー骨格形成材料を埋設したものに基づいている。皮膚の均等物（コラーゲン格子）を下記の混合物から作成した：軟骨細胞培地８ｍｌ、中和培地（０．１Ｎ ＮａＯＨ含有ＤＭＥＭ／１０％ＦＢＳ、使用前に調製）２ｍｌ、コラボラティブ バイオメディカル プロダクツ（Collaborative Biomedical Products）社から入手したラット尾部Ｉ型コラーゲン（３．９９ｍｇ／ｍｌ）４ｍｌ、および軟骨細胞培地に懸濁した軟骨細胞懸濁液２ｍｌ。上記細胞／コラーゲン混合物を２４穴ハイドロゲル塗布プレート（Ｃｏｓｔａｒ、カタログ＃３４７３）の各穴に分配した（２ｍｌ）。最終細胞濃度は６×１０^６個／ｍｌであり、コラーゲン濃度は１ｍｇ／ｍｌであった。コラーゲンゲルを３７℃で１時間重合させた。ゲル重合に続いて、１．５ｍｌの軟骨細胞増殖培地を各穴に適用した。この培地を１日おきに取り替えた。７日間収縮させた後、使い捨て生検用ポンチを使用してコラーゲン格子の厚み全体にわたって直径５ｍｍの傷を作成した。湿った形で供給されたほぼ直径５ｍｍで厚さ２ｍｍの被検骨格形成材料を傷を付けたコラーゲン格子の中心部に移植した。コラーゲン格子が収縮する間に傷床から骨格形成材料押し出されることを確実に防止するために、５０μｌの非細胞性コラーゲンを各構成体の頂部に置いた。これらの構成体を軟骨細胞培地中で培養し、培地は２日間隔で取り替えた。骨格形成材料を含有する収縮したゲルを超低クラスタシャーレにおいて静止状態でまたはバイオリアクタ内で培養し、３週間後に回収した。
【０１３６】
工程Ｃ．骨格形成材料内の細胞の特徴付け
細胞が浸潤した骨格形成材料に組織学を適用した。骨格形成材料を１０％緩衝ホルマリン中で固定し、パラフィンに包埋して切片を作成する（ハイブリッド発泡体／微小繊維織物骨格形成材料）か、または凍結切片を作成した（対照発泡体骨格形成材料）。切片をヘマトキシリン／エオシン（Ｈ／Ｅ、細胞数および形態）、サフラニンＯ（ＳＯ；硫酸化ＧＡＧｓ）およびトリクローム（コラーゲン）で染色した。これらの骨格形成材料への細胞浸潤の定量を画像分析により決定した。データにＴｕｋｅｙ事後テストの分散分析を行った。
【０１３７】
実験結果
３週間培養した構成物のＨ／Ｅ切片の定量的評価により、軟骨細胞は両骨格形成材料に浸潤したことが明らかになった。バイオリアクタ内で培養したハイブリッド発泡体／微小繊維織物骨格形成材料への細胞の浸潤は４０／６０ＰＣＬ／ＰＬＡ発泡体骨格形成材料への浸潤よりも顕著に高かった（Ｐ＜０．０５）。また、静止状態に保った培養においては、ハイブリッド発泡体／微小繊維織物骨格形成材料への細胞の浸潤は４０／６０ＰＣＬ／ＰＬＡ発泡体骨格形成材料への浸潤よりも顕著に高かった（Ｐ＜０．０５）。
【０１３８】
細胞浸潤 ハイブリッド
細胞／ｍｍ^２ ４０／６０ＰＣＬ／ＰＬＡ 発泡体／微小繊維
（３週間） 発泡体骨格形成材料 織物骨格形成材料
静止細胞培養 ５０ ８０
バイオリアクタ １４０ ２００
細胞培養
【０１３９】
ハイブリッド発泡体／微小繊維織物骨格形成材料はもっとも高い細胞密度を示した。本発明者は、細胞−細胞相互作用がハイブリッド発泡体／微小繊維織物骨格形成材料の場合は他の骨格形成材料に比べて高いことが推定されるが、この細胞−細胞相互作用が選択的な軟骨細胞増殖およびハイブリッド発泡体／微小繊維織物骨格形成材料に対する分化された機能に寄与していることがあり得ると仮定する。この細胞−細胞相互作用は軟骨細胞の表現型を維持する重要な役割を果たしていることが仮定される。軟骨細胞は細胞の単一層に展開すると分化することが知られている。単一層で高細胞密度で培養すると、ＩＩ型コラーゲンおよびアグリカンの発現により示されるように軟骨細胞はそれらの表現型を維持する。
【０１４０】
６週間までに４０／６０ＰＣＬ／ＰＬＡ発泡体骨格形成材料は主に構成体の周りのカプセル内に局在する軟骨様形態／性質を示した。これに対して、ハイブリッド発泡体／微小繊維織物骨格形成体においては、カプセルは非常に薄く、かつ構成体内の細胞の形態は軟骨様であり、かつより均一な細胞の分布が観察された。
【０１４１】
本発明の実施態様は以下の通りである。
（Ａ）第１の生体許容性のフィラメント状層とこれに取り付けられた第２の生体許容性の発泡体層を備え、前記生体許容性の発泡体が勾配構造を備える発泡体および流路を備える発泡体からなる群から選ばれる生体許容性の複合体であって、前記勾配構造を備える発泡体は第１の部分および第２の部分を有し、前記第１の部分から前記第２の部分にかけて、組成、剛性、柔軟性、生体吸収速度、および多孔質構造から成る群から選択される少なくとも１個の特性がほぼ連続的に遷移しており、かつ前記流路を備える発泡体は第１の面部と流路を備える第２の面部とを有する生体許容性の複合体。
（１）前記生体許容性の複合体が生体吸収性である実施態様（Ａ）に記載の生体許容性の複合体。
（２）前記生体許容性のフィラメント状層が生体吸収性である実施態様（Ａ）に記載の生体許容性の複合体。
（３）前記生体許容性の複合体が脂肪族ポリエステル、ポリ（アミノ酸）、コポリ（エーテル−エステル）、ポリアルキレンオキサレート、ポリアミド、ポリ（イミノカーボネート）、ポリオルトエステル、ポリオキサエステル、ポリアミドエステル、アミン基を含有するポリオキサエステル、ポリ（酸無水物）、ポリホスファゼン、生体ポリマーおよびこれらの混合物から成る群から選択される生体吸収性のポリマーにより形成されている実施態様（Ａ）に記載の生体許容性の複合体。
（４）前記生体吸収性ポリマーが脂肪族ポリエステルである実施態様（３）に記載の生体許容性の複合体。
（５）前記脂肪族ポリエステルがラクチド、グリコリド、グリコール酸、ε−カプロラクトン、ｐ−ジオキサノン（１，４−ジオキサン−２−オン）、トリメチレンカーボネート（１，３−ジオキサン−２−オン）、トリメチレンカーボネートのアルキル誘導体、δ−バレロラクトン、β−ブチロラクトン、γ−ブチロラクトン、ε−デカラクトン、ヒドロキシブチレート、ヒドロキシバレレート、１，４−ジオキセパン−２−オン、１，５，８，１２−テトラオキサシクロテトラデカン−７，１４−ジオン、１，５−ジオキセパン−２−オン、６，６−ジメチル−ジオキセパン−２−オン、およびこれらの混合物のホモポリマーおよびコポリマーからなる群から選ばれる実施態様（４）に記載の生体許容性の複合体。
【０１４２】
（６）前記脂肪族ポリエステルがエラストマーである実施態様（４）に記載の生体許容性の複合体。
（７）前記エラストマーがε−カプロラクトンとグリコリドのコポリマー、ε−カプロラクトンと（Ｌ）ラクチドのコポリマー、ｐ−ジオキサノン（１，４−ジオキサン−２−オン）と（Ｌ）ラクチドのコポリマー、ε−カプロラクトンとｐ−ジオキサノンのコポリマー、ｐ−ジオキサノンとトリメチレンカーボネートのコポリマー、トリメチレンカーボネートとグリコリドとのコポリマー、トリメチレンカーボネートと（Ｌ）ラクチドとのコポリマーおよびこれらの混合物からなる群から選ばれる実施態様（６）に記載の生体許容性複合体。
（８）さらに第２の脂肪族ポリエステルが前記生体許容性の発泡体の構成要素として存在している実施態様（４）に記載の生体許容性の複合体。
（９）さらに第２の脂肪族ポリエステルが前記生体許容性のフィラメント状層の構成要素として存在している実施態様（４）に記載の生体許容性の複合体。
（１０）前記生体許容性の勾配構造を有する発泡体が前記第１の部分から前記第２の部分にかけて、組成においてほぼ連続的に遷移している実施態様（３）に記載の生体許容性の複合体。
【０１４３】
（１１）前記生体許容性の勾配構造を有する発泡体が前記第１の面部から第２の面部にかけて組成において少なくとも２種の異なる脂肪族ポリエステルの第１の比から前記少なくとも２種の異なる脂肪族ポリエステルの第２の比までほぼ連続的に遷移している実施態様（１０）に記載の生体許容性複合体。
（１２）前記生体許容性の勾配構造を有する発泡体が前記第１の部分から前記第２の部分にかけて、剛性においてほぼ連続的に遷移している実施態様（３）に記載の生体許容性の複合体。
（１３）前記生体許容性の勾配構造を有する発泡体が前記第１の部分から前記第２の部分にかけて、生体吸収速度においてほぼ連続的に遷移している実施態様（３）に記載の生体許容性の複合体。
（１４）前記生体許容性の勾配構造を有する発泡体が前記第１の部分から前記第２の部分にかけて、柔軟性においてほぼ連続的に遷移している実施態様（３）に記載の生体許容性の複合体。
（１５）前記生体許容性の勾配構造を有する発泡体が前記第１の部分から前記第２の部分にかけて、構造においてほぼ連続的に遷移している実施態様（３）に記載の生体許容性の複合体。
【０１４４】
（１６）前記生体許容性の勾配構造を有する発泡体が前記第１の部分から前記第２の部分にかけて、構造においてほぼ球状の多孔質形状から柱状の多孔質形状までほぼ連続的に遷移している実施態様（１５）に記載の生体許容性の複合体。
（１７）前記ほぼ球状の気孔の大きさが約３０μｍ乃至約１５０μｍである実施態様（１５）に記載の生体許容性の複合体。
（１８）前記柱状の気孔の直径が約１００μｍ乃至約４００μｍであり、直径に対する長さの比が少なくとも２である実施態様（１５）に記載の生体許容性の複合体。
（１９）さらに、前記生体許容性の勾配構造を有する発泡体の中に治療剤が存在している実施態様（Ａ）に記載の生体許容性の複合体。
（２０）前記生体許容性の発泡体が抗感染剤、ホルモン、鎮痛剤、抗炎症剤、増殖因子、化学療法剤、抗拒絶剤、プロスタグランジン、ＲＤＧペプチド、およびこれらの混合物から成る群から選択される薬剤をさらに含有している実施態様（Ａ）に記載の生体許容性の複合体。
【０１４５】
（２１）前記増殖因子が骨形態発生様タンパク、表皮増殖因子、線維芽細胞増殖因子、血小板由来増殖因子、インシュリン由来増殖因子、トランスホーミング増殖因子、脈管内皮増殖因子およびこれらの混合物である実施態様（２０）に記載の生体許容性複合体。
（２２）前記生体許容性発泡体が生体吸収性の合成ポリマー、生体許容性で生体吸収性の生体ポリマー、生体許容性のセラミック材料およびこれらの混合物から成る群から選択される生体許容性の材料で充填されている実施態様（Ａ）に記載の生体許容性の複合体。
（２３）前記流路を備える発泡体が少なくとも２００μｍの平均長さの流路を有する実施態様（Ａ）に記載の生体許容性複合体。
（２４）前記流路がほぼ前記第１の面部から第２の面部に延在している実施態様（２３）に記載の生体許容性複合体。
（２５）約３０重量％乃至約９９重量％のε−カプロラクトン反復単位を含有する組成により形成されている内部連通気孔を有する発泡体から成る実施態様（Ａ）に記載の生体許容性の複合体。
【０１４６】
（２６）前記ε−カプロラクトン反復単位がラクチド、グリコリド、グリコール酸、ε−カプロラクトン、ｐ−ジオキサノン（１，４−ジオキサン−２−オン）、トリメチレンカーボネート（１，３−ジオキサン−２−オン）、トリメチレンカーボネートのアルキル誘導体、δ−バレロラクトン、β−ブチロラクトン、γ−ブチロラクトン、ε−デカラクトン、ヒドロキシブチレート、ヒドロキシバレレート、１，４−ジオキセパン−２−オン、１，５，８，１２−テトラオキサシクロテトラデカン−７，１４−ジオン、１，５−ジオキセパン−２−オン、６，６−ジメチル−ジオキセパン−２−オン、およびこれらの混合物からなる群から選ばれるコモノマーと共重合している実施態様（２５）に記載の生体許容性の複合体。
（２７）第１の部分および第２の部分を有し、前記生体許容性の発泡体は前記生体許容性の発泡体の前記前記第１の部分から前記第２の部分にかけて、組成、剛性、柔軟性、生体吸収速度、および多孔質構造から成る群から選択される少なくとも１個の特性がほぼ連続的に遷移している実施態様（２５）に記載の生体許容性の複合体。
（２８）前記相互に内部連通している気孔は気孔の大きさが約１０μｍ乃至約２００μｍの範囲内である実施態様（２５）に記載の生体許容性の複合体。
（２９）前記生体許容性の発泡体の多孔質性が約２０％乃至約９８％の範囲内である実施態様（２５）に記載の生体許容性の複合体。
（３０）前記生体許容性発泡体が流路を有する実施態様（２５）に記載の生体許容性の複合体。
【０１４７】
（３１）前記流路の平均長さが少なくとも２００μｍである実施態様（３０）に記載の生体許容性の複合体。
（３２）前記ほぼ球状の気孔の大きさが約３０μｍ乃至約１５０μｍである実施態様（２５）に記載の生体許容性の複合体。
（３３）前記ほぼ球状の気孔の大きさが約３０μｍ乃至約４００μｍであり、直径に対する長さの比が少なくとも２である実施態様（２５）に記載の生体許容性の複合体。
（３４）さらに、前記生体許容性の勾配構造を有する発泡体の中に治療剤が存在している実施態様（２５）に記載の生体許容性の複合体。
（３５）前記生体許容性の発泡体が前記生体許容性の発泡体内の挿入物で形成されている実施態様（Ａ）に記載の生体許容性の複合体。
【０１４８】
（３６）前記挿入物がフィルム、スクリム、織布、編繊維、編組繊維、整形外科用移植片、チューブおよびこれらの混合物からなる群から選ばれる実施態様（３５）に記載の生体許容性の複合体。
（３７）前記生体許容性複合体が３次元形状の構造体に形成されている実施態様（Ａ）に記載の生体許容性の複合体。
（３８）前記３次元形状構造体が管状形、分岐した管状形、球形、半球形、３次元的多角形状、楕円形、トロイド形状、円錐形、円錐台形、ピラミッド形状、これらの中実および中空のもの、並びにこれらの組合せからなる群から選ばれる実施態様（３７）に記載の生体許容性の複合体。
（Ｂ）組織の修復または再生の方法であって、細胞を生体許容性の複合体に接触させる工程から成り、当該生体許容性の複合体が第１の生体許容性のフィラメント状層とこれに取り付けられた第２の生体許容性の発泡体層を備え、前記生体許容性の発泡体が勾配構造を備える発泡体および流路を備える発泡体からなる群から選ばれる生体許容性の複合体であって、前記勾配構造を備える発泡体は第１の部分および第２の部分を有し、前記第１の部分から前記第２の部分にかけて、組成、剛性、柔軟性、生体吸収速度、および多孔質構造から成る群から選択される少なくとも１個の特性がほぼ連続的に遷移しており、かつ前記流路を備える発泡体は第１の面部と流路を備える第２の面部とを有する方法。
（３９）前記生体許容性の複合体が生体吸収性である実施態様（Ｂ）に記載の方法。
（４０）前記生体許容性の複合体が脂肪族ポリエステル、ポリ（アミノ酸）、コポリ（エーテル−エステル）、ポリアルキレンオキサレート、ポリアミド、ポリ（イミノカーボネート）、ポリオルトエステル、ポリオキサエステル、ポリアミドエステル、アミン基を含有するポリオキサエステル、ポリ（酸無水物）、ポリホスファゼン、生体ポリマーおよびこれらの混合物から成る群から選択される生体吸収性のポリマーにより形成されている実施態様（Ｂ）に記載の方法。
【０１４９】
（４１）前記生体吸収性ポリマーが脂肪族ポリエステルである実施態様（４０）に記載の方法。
（４２）前記脂肪族ポリエステルがラクチド、グリコリド、グリコール酸、ε−カプロラクトン、ｐ−ジオキサノン（１，４−ジオキサン−２−オン）、トリメチレンカーボネート（１，３−ジオキサン−２−オン）、トリメチレンカーボネートのアルキル誘導体、δ−バレロラクトン、β−ブチロラクトン、γ−ブチロラクトン、ε−デカラクトン、ヒドロキシブチレート、ヒドロキシバレレート、１，４−ジオキセパン−２−オン、１，５，８，１２−テトラオキサシクロテトラデカン−７，１４−ジオン、１，５−ジオキセパン−２−オン、６，６−ジメチル−ジオキセパン−２−オン、およびこれらの混合物のホモポリマーおよびコポリマーからなる群から選ばれる実施形態（４１）に記載の方法。
（４３）前記脂肪族ポリエステルがエラストマーである実施態様（４２）に記載の方法。
（４４）細胞が前記生体許容性の複合体上に播種される実施態様（Ｂ）に記載の方法。
（４５）細胞が前記生体許容性の複合体上に播種される実施態様（４２）に記載の方法。
【０１５０】
（４６）前記生体許容性の複合体が動物に移植され細胞と接触される実施態様（Ｂ）に記載の方法。
（４７）前記生体許容性の複合体が動物に移植され細胞と接触される実施態様（４２）に記載の方法。
（４８）前記生体許容性の複合体上に細胞を播種し、前記生体許容性の複合体と前記細胞とを細胞培養装置内に置き、前記細胞を前記生体許容性の複合体上で増殖させる実施態様（Ｂ）に記載の方法。
（４９）前記生体許容性の複合体上に細胞を播種し、前記生体許容性の複合体と前記細胞とを細胞培養装置内に置き、前記細胞を前記生体許容性の複合体上で増殖させる実施態様（４２）に記載の方法。
（５０）前記細胞が多能性細胞、幹細胞および前駆体細胞並びにこれらの混合物である実施態様（Ｂ）に記載の方法。
【０１５１】
（５１）前記細胞が筋細胞、脂肪細胞、線維筋芽細胞、外胚葉細胞、筋肉細胞、骨芽細胞、軟骨細胞、内皮細胞、線維芽細胞、膵細胞、肝細胞、胆管細胞、骨髄細胞、神経細胞、尿生殖器細胞およびこれらの混合物である実施態様（Ｂ）に記載の方法。
（５２）前記生体許容性の複合体が抗感染剤、ホルモン、鎮痛剤、抗炎症剤、増殖因子、化学療法剤、抗拒絶剤、プロスタグランジン、ＲＤＧペプチド、およびこれらの混合物から成る群から選択される薬剤を含有している実施態様（Ｂ）に記載の方法。
（５３）前記生体許容性の複合体が約３０重量％乃至約９９重量％のε−カプロラクトン反復単位を含有する組成により形成されている実施態様（Ｂ）に記載の方法。
（５４）前記ε−カプロラクトン反復単位がラクチド、グリコリド、グリコール酸、ε−カプロラクトン、ｐ−ジオキサノン（１，４−ジオキサン−２−オン）、トリメチレンカーボネート（１，３−ジオキサン−２−オン）、トリメチレンカーボネートのアルキル誘導体、δ−バレロラクトン、β−ブチロラクトン、γ−ブチロラクトン、ε−デカラクトン、ヒドロキシブチレート、ヒドロキシバレレート、１，４−ジオキセパン−２−オン、１，５，８，１２−テトラオキサシクロテトラデカン−７，１４−ジオン、１，５−ジオキセパン−２−オン、６，６−ジメチル−ジオキセパン−２−オン、およびこれらの混合物からなる群から選ばれるコモノマーと共重合している実施態様（５３）に記載の生体許容性の複合体。
（５５）細胞が前記生体許容性の複合体上に播種される実施態様（５３）に記載の方法。
【０１５２】
（５６）前記生体許容性の複合体が動物に移植され細胞と接触される実施態様（５３）に記載の方法。
（５７）前記生体許容性の複合体上に細胞を播種し、前記生体許容性の複合体と前記細胞とを細胞培養装置内に置き、前記細胞を前記生体許容性の複合体上で増殖させる実施態様（５３）に記載の方法。
（５８）前記細胞が多能性細胞、幹細胞および前駆体細胞並びにこれらの混合物である実施態様（５７）に記載の方法。
（５９）前記細胞が筋細胞、脂肪細胞、線維筋芽細胞、外胚葉細胞、筋肉細胞、骨芽細胞、軟骨細胞、内皮細胞、線維芽細胞、膵細胞、肝細胞、胆管細胞、骨髄細胞、神経細胞、尿生殖器細胞およびこれらの混合物である実施態様（５７）に記載の方法。
（Ｃ）第１の面部および第２の面部を有する生体許容性の発泡体から成り、前記第１の面部および第２の面部の間において内部連通する気孔および流路を備えている生体許容性の発泡体。
（Ｄ）約３０重量％乃至約９９重量％のε−カプロラクトン反復単位を含有する組成により形成されている内部連通気孔を有する発泡体から成る生体許容性の発泡体。
【０１５３】
【発明の効果】
従って、本発明によれば、組織の修復または再生が有効に行える形態、構造および／または材料組成の連続的な勾配構造を有する生体許容性で生体吸収性の発泡体が提供できる。
【図面の簡単な説明】
【図１】３５／６５ε−カプロラクトン−コ−グリコリドコポリマーの５％溶液により作成した不規則微小構造発泡体の断面の走査電子顕微鏡写真である。
【図２】３５／６５ε−カプロラクトン−コ−グリコリドコポリマーの１０％溶液により作成した垂直方向に開口する流路を有する発泡体の断面の走査電子顕微鏡写真である。
【図３】３５／６５ε−カプロラクトン−コ−グリコリドコポリマーの１０％溶液により作成した構造的な勾配を有する発泡体の断面の走査電子顕微鏡写真である。
【図４】４０／６０ε−カプロラクトン−コ−（Ｌ）ラクチドコポリマーおよび３５／６５ε−カプロラクトン−コ−グリコリドコポリマーの５０／５０混合物により作成した勾配構造を有する発泡体の断面の走査電子顕微鏡写真である。
【図５】４０／６０ε−カプロラクトン−コ−（Ｌ）ラクチドコポリマーおよび３５／６５ε−カプロラクトン−コ−グリコリドコポリマーの５０／５０混合物により作成した勾配構造を有する発泡体の上部の断面の走査電子顕微鏡写真である。
【図６】４０／６０ε−カプロラクトン−コ−（Ｌ）ラクチドコポリマーおよび３５／６５ε−カプロラクトン−コ−グリコリドコポリマーの５０／５０混合物により作成した勾配構造を有する発泡体の底部の断面の走査電子顕微鏡写真である。
【図７】細胞培養における諸データのグラフである。
【図８】細胞培養における諸データのグラフである。
【図９】細胞培養における諸データのグラフである。
【図１０】軟骨組織の解剖構造学的な説明図である。
【図１１】皮膚骨格形成材料としての使用に適する構造を有している３５／６５ε−カプロラクトン−コ−グリコリドコポリマーおよび４０／６０ε−カプロラクトン−コ−（Ｌ）ラクチドコポリマーの５０／５０混合物により作成した０．５ｍｍの発泡体の走査電子顕微鏡写真である。図１１（Ａ）は好ましくは傷床に対向する骨格形成材料の表面の多孔質性を示している走査電子顕微鏡写真であり、図１１（Ｂ）は好ましくは傷床に離れて対向する骨格形成材料の表面の多孔質性を示している走査電子顕微鏡写真である。
【図１２】皮膚骨格形成材料としての使用に適する構造を有している３５／６５ε−カプロラクトン−コ−グリコリドコポリマーおよび４０／６０ε−カプロラクトン−コ−（Ｌ）ラクチドコポリマーの５０／５０混合物により作成した０．５ｍｍの発泡体の走査電子顕微鏡写真である。発泡体の厚さ方向に延在する流路を有する骨格形成材料の断面を示している走査電子顕微鏡写真である。
【図１３】豚モデル内に移植後８日目の図１１（Ａ）乃至図１２に示した発泡体の細胞浸潤を示すトリクローム染色サンプルの４０倍の暗視野顕微鏡写真である。
【図１４】豚モデル内に移植後８日目のさらにＰＤＧＦを含有する図１１（Ａ）乃至図１２に示した発泡体の細胞浸潤を示すトリクローム染色サンプルの１００倍の複合顕微鏡写真である。
【図１５】発泡体基体に融合した静電紡糸繊維層の断面の顕微鏡写真である（両材料は（Ｌ）ラクチドとε−カプロラクトンの６０／４０コポリマーである）。
【図１６】図１５に示す静電紡糸層の表面多孔質構造の顕微鏡写真である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates generally to the field of tissue repair and regeneration. In particular, the present invention provides a template for regeneration, repair or augmentation of porous, bio-acceptable, bioresorbable foams and / or tissues having a constant gradient in composition (gradual change in morphology and function) Relates to a microstructure that acts as
[0002]
[Prior art]
It has been recognized that open cell porous foams are clearly effective for use in tissue repair and regeneration. In tissue repair, the use of amorphous, bio-acceptable foams as a porous plug material for filling bone pores has been intensively studied at an early stage. Brekke et al. (US Pat. No. 4,186,448) describe the use of a porous mesh plug composed of a polyhydroxy acid polymer such as polylactide to heal bone pores. In recent years, for example, US Pat. Nos. 5,522,895 (Mikos) and 5,514,378 (Mikos et al.) Using eluents, US Pat. No. 5,755,792 using vacuum foaming techniques. (Brekke) and 5,133,755 (Brekke), US Pat. Nos. 5,716,413 (Walter et al.) And 5,607,474 (Athanasiou et al.) Using precipitated polymer gel materials, room temperature US Pat. Nos. 5,686,091 (Leong et al.) And 5,677,355 (Shalaby et al.) Using a polymer melt with a labile compound that sublimes at a higher temperature, a base fabric fiber skeleton-forming material TE skeleton forming materials are prepared by different methods such as US Pat. No. 5,770,193 (Vacanti et al.), 5,769,899 (Schwartz et al.), 5,711,960 (Shikinami), etc. Try to It has been. Hinsch et al. (EPA 274,898) also describe a porous open-cell foam of polyhydroxy acid having pores with a size of about 10 μm to about 200 μm for vessel and cell ingrowth. The foam described by this document Hincsh can be reinforced with fibers, yarns, blades, knitted fibers, scrims and the like. The document Hincsh also describes the use of various polyhydroxy acids and copolymers such as poly-L-lactide, poly-DL-lactide, polyglycolide, and polydioxanone. This Hincsh foam has the advantage of having regular pore sizes and shapes that can be adjusted by process conditions, selected solvents, and additives.
[0003]
However, the above technique is limited in producing a skeleton-forming material having a constant gradient structure. Most skeleton-forming materials are isotropic in their form and function and lack the anisotropy characteristics in natural tissues. Furthermore, it has been difficult in the prior art to create a three-dimensional skeleton-forming material that can control the spatial distribution of various pore shapes. The reported method for producing microstructured foams is a low temperature process and has many advantages over other conventional techniques. For example, the method can incorporate thermally sensitive compounds such as proteins, drugs, and other additives including thermally and hydrolytically unstable absorbent polymers.
[0004]
In addition, recently, Athanasiou et al. (US Pat. No. 5,607,474) described a two-layer foam device for repairing osteochondral defects in locations where two dissimilar tissues are present. Suggest use. This Athanasiou device is made up of a first layer and a second layer that are made partially separate and joined together at a later stage. Each skeleton-forming layer is configured to have rigidity and compressibility corresponding to cartilage and bone tissue. Since cartilage and bone often form adjacent layers in the body, this method aims to more clearly resemble the human body structure. However, the interface between cartilage and bone in the human body is not a discontinuous junction of two dissimilar materials with abrupt changes in anatomical features and / or mechanical properties. In other words, chondrocytes have distinctly different cell morphology and orientation depending on the location of the chondrocytes relative to the underlying bone structure. A continuous transition structure to the cartilage interface is formed. Therefore, although Athanasiou's two-tier system is improving, it is not similar to the tissue interface that exists in the human body.
[0005]
Another technique for creating a three-dimensional laminate foam has been proposed by Mikos et al. (US Pat. No. 5,514,378). In this laborious technique, a porous membrane is first created by drying a polymer solution containing eluting salt crystals. Thereafter, a three-dimensional structure is obtained by laminating several membranes together, which are cut into the contour of the desired shape.
[0006]
One of the main drawbacks of the prior art relating to the three-dimensional porous skeleton-forming material used for regeneration of living tissue such as cartilage is that the microstructure of the material is irregular. Such skeletal materials do not change in form or structure, unlike natural tissues. Furthermore, such skeleton-forming materials do not provide nutrient and fluid transport suitable for many applications. In addition, this laminated structure is not fully integrated and is subject to peeling under in vivo conditions.
[0007]
[Problems to be solved by the invention]
Therefore, an object of the present invention is to provide a bio-acceptable and bio-absorbable foam having a continuous gradient of form, structure and / or material. In addition, the foam used in artificial manipulation of tissue, ie repair or regeneration, organizes at the microstructural level and constitutes a template that facilitates cell infiltration, proliferation and differentiation that occurs during functional tissue regeneration. It is preferable to have a certain structure.
[0008]
[Means for Solving the Problems]
The present invention provides a composite comprising a first layer that is a bio-acceptable filamentous layer and a second layer of a bio-acceptable foam. This composite structure makes it possible to create structures with unique mechanical properties. This fiber layer allows the composite to have mechanical strength that can vary depending on the design, different bioabsorption profiles and different local environments for cell infiltration and inoculation, which are advantageous in a wide range of medical applications. is there. The fibrous layer may be made from various bioacceptable polymers and mixtures of bioacceptable polymers, which are preferably bioabsorbable. This bioacceptable foam may be either a foam with a different gradient structure or a foam with a flow path. The foam with this gradient structure has a generally continuous transition in at least one property selected from the group consisting of composition, stiffness, flexibility, bioabsorption rate, porous structure, and / or microstructure. Yes. Foams having this gradient structure can be made by a mixture of absorbent polymers that form a compositional transition gradient from one polymer material to another. Also, if the single chemical composition is sufficient for application, the present invention can be along one or more directions that can resemble the anatomical features of tissue (eg, cartilage, skin, bone, etc.). A bio-acceptable foam having a microstructural change in structure is provided. The foam with the flow path has a flow path extending through the foam, making it easy for cells to move and nutrients to flow throughout the foam with the flow path.
[0009]
The present invention also provides a method for tissue repair or regeneration, wherein the method adds the first tissue to the complex at a location of the complex having suitable properties to promote tissue growth. It consists of the process of contacting. This complex structure is particularly useful for tissue regeneration between two or more different tissues. In the simplest case of multicellular systems, one cell type may be on one side of the skeletal material and another cell type may be on the other side of the skeleton material. . Examples of such regeneration include: (a) in the case of vascular tissue, smooth muscle on the outside and endothelial cells on the inside to regenerate the vascular structure; (b) in the case of meniscal tissue, By implanting the composite foam inside, and orienting the fibrous surface outward.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses a bioabsorbable porous polymer foam having a novel microstructure. The properties of the foam can be adjusted to suit the desired application by selecting appropriate conditions for forming the foam during lyophilization. The properties of this absorbent polymer have distinct advantages over the prior art where the skeleton-forming material is generally isotropic or irregular in structure. However, foams used in tissue artificial processing (ie, repair or regeneration) organize at the microstructural level, and anatomical, biomechanical and biochemical properties in normal tissue It is preferable to have a certain structure that constitutes a template that facilitates the organization and regeneration of the cells having the. Such foams can be used for the repair or regeneration of tissues (including organs) in animals such as livestock animals, primates and humans.
[0011]
The properties of the foam of the present invention can be adjusted to suit the desired application by selecting appropriate conditions for lyophilization to have one or more of the following properties. (1) interconnected pores with a size in the range of about 10 μm to about 200 μm (or more) forming passages for cell ingrowth and nutrient diffusion; (2) about 20% to Various porosities in the range of about 98%, preferably about 80% to about 95%, (3) gradient in pore size along a certain direction corresponding to selective cell culture, (4) within the foam (5) micropatterning of pores on the surface for cell organization, (6) pore shape and / or orientation ( (E.g., generally spherical, elliptical, cylindrical) selectivity, (7) mechanical property anisotropy, (8) complex foaming with gradients in polymer composition showing or having the advantage of different cellular responses to different materials Body, (9) different speed A mixture of different polymer compositions to form a structure with a portion that breaks at a degree, including (10) RGD, biological factors such as growth factors (PDGF, TGF-β, VEGF, BMP, FGF, etc.) But not limited to these, lyophilized or foamed with a pharmaceutically active compound, (11) ability to create 3D shapes and devices with preferred microstructures, and (12) form composite structures Freeze drying with another part or medical device for. The tuned properties in these absorbent polymers are clearly an advantage over the prior art where the skeleton-forming material is a generally isotropic or irregular structure that does not have a preferred form at the pore level. have. However, the foam used in the tissue skeleton-forming material has a certain structure that forms a template that organizes at the microstructural level and facilitates the organization of cells that can resemble natural tissue. It is preferable. Cells attach, proliferate and differentiate along or through the shape of this structure. This allows for the cultivation of tissue that can resemble the anatomical characteristics of the actual tissue to a significant extent.
[0012]
For example, as shown in FIG. 3, the direction of the main axis of the pores can be changed from the same plane as the foam to a direction perpendicular to the face of the foam. Also, as can be seen from FIG. 3, the pore size can vary from about 30 μm to about 50 μm small pores to about 100 μm to about 200 μm large pores in a foam having a porous gradient structure. Desirably, the foam structure can be configured to facilitate the repair or regeneration of human tissue joints such as cartilage relative to the bone joint present in the joint. The foam is small in length (ie from about 30 μm to about 150 μm) round pores to a relatively large cylindrical shape (ie from about 30 μm to about 400 μm, preferably from about 100 μm to about 400 μm in length in most cases). (The ratio of diameter to diameter is at least 2). It is the figure which has shown the foam provided with the flow path in FIG. 2 and FIG. These flow paths formed by the method of the present invention extend across from the surface on one end side of the foam in the thickness direction of the foam. The length of this channel is generally at least twice the average diameter of the pores, preferably at least 4 times, and most preferably at least 8 times. It should be noted that the flow path for most applications is at least 200 microns long and can extend through the thickness of the foam. The diameter of the channel is at least 1 times the average diameter of the pores, preferably at least 2 to 3 times. Of course, the size and diameter of the channel can be selected according to the desired function of the channel, such as a cell infiltration, nutrient diffusion, or angiogenesis channel.
[0013]
There are many biological tissues that exhibit structures with gradients. That is, examples of tissues in which a skeleton-forming material having a gradient structure can be used include bone, spinal disc, articular cartilage, meniscus, fibrous cartilage, tendon, ligament, dura mater, skin, vascular graft, nerve, liver And pancreas and the like. The following examples show some tissues that can be used for a skeleton-forming material having a gradient structure. The construction of the skeleton-forming material that artificially treats the tissue to facilitate the growth of the organ structure gains significant advantages due to the ability to form a gradient structure in the skeleton-forming material.
[0014]
cartilage
Articular cartilage covers the ends of all bones that form joints in humans and animals. This cartilage acts within the joint as a force distribution mechanism and a support surface between different bones. Without this articular cartilage, stress concentration and friction occur, and the joint cannot move easily. Also, loss of articular cartilage generally results in painful arthritis and reduced joint movement. FIG. 10 schematically shows the morphological characteristics of healthy cartilage.
[0015]
Articular cartilage is a good example of a structure with a natural gradient. That is, articular cartilage is composed of four different regions, including the first 10% to 20% of the surface or peripheral region of the structure (this region includes the joint surface), intermediate It includes an intermediate region that closes 40% to 60% of the structure, a deep region near the reference line, and a transition region between bone and cartilage composed of calcified cartilage. Further, the subchondral bone is present near the reference line, and transitions to the reticular bone. In the superficial or peripheral region, the collagen fibrils are parallel to the surface. These fibers are oriented to resist shear forces that occur during normal articulation. In the middle region, there is a tissue in which collagen fibers having fairly large diameters are irregularly arranged. Further, in the deep region, there are thicker bundles of collagen fibers, and these bundles are perpendicular to the surface and enter the calcified cartilage. These cells tend to collect and arrange in a cylindrical shape in a spheroid form. Furthermore, small cells having a relatively small cytoplasm are present in the calcified cartilage region.
[0016]
A preferred embodiment of the present invention is aimed at forming a foam structure having a gradient that can act as a template corresponding to a number of different regions. These foam structures can be formed into various shapes to regenerate or repair osteochondral defects and cartilage. An example of an effective foam structure can be a cylindrical shape having a rough dimension of 10 mm in diameter and 10 mm in length. Furthermore, the top surface is about 1 mm thick and can be a low porous layer to control fluid passage. By adopting an appropriate treatment method, the porosity of the surface of the foam can be adjusted. That is, the porosity of the skin-like surface can vary from completely impervious to completely porous. The fluid permeability can be adjusted by the porosity of the surface. Under such a coating, the structure can be composed of three types of regions. The upper porous region is close to the cartilage tissue, the lower porous region is close to the bone tissue, and a transition region exists between the upper and lower regions. In the case of articular cartilage, it is presently preferred that the stiffness (elastic modulus) of the upper and lower porous layers has at least the same stiffness as the corresponding adjacent tissue at the time of implantation. That is, in such a case, these porous layers support the environmental load, and can protect the infiltrating cells until they differentiate and merge to proliferate into a tissue that can support the load. For example, the porous structure used for the peripheral region of the surface layer has elongated pores, and the orientation of the structure can be parallel to the surface of the host cartilage. However, the deep region has a porosity of about 80% to about 95% due to pores of about 100 μm (about 80 μm to about 120 μm). That is, it can be expected that chondrocytes infiltrate into this region. Further below this region can be a region having relatively large pores (about 100 μm to about 200 μm) and a porosity in the range of about 50% to about 80%. Such a porous foam of 100 μm to about 200 μm has a structure in which the struts or walls of the pores are relatively large and perpendicular to the load, similar to a structure capable of supporting the load with a natural structure be able to. In addition, there is a need for large pores (about 150 μm to about 300 μm) at the bottom of the structure that have a high stiffness that is structurally comparable to the reticular bone. Therefore, the foam in this part can be reinforced by fibers formed from ceramic particles, calcium phosphate or the like.
[0017]
Recent data obtained from the inventor's work has supported the assumption that cell invasion can be regulated by pore size. In these studies, a skeleton-forming material made of 95/5 mol% poly ((L) lactide-co-ε-caprolactone) with pores of about 80 μm size is (static) About 30 cells / mm)²Chondrocyte infiltration. In addition, the skeleton-forming material made of 40/60 mol% poly (ε-caprolactone-co- (L) lactide) having pores of about 100 μm size is statistically significant (under static conditions). Hold large 50 cells / mm²Cell infiltration. In both of these cases the cells were bovine chondrocytes. Furthermore, a very simple gradient structure having a pore size change of about 80 μm to about 150 μm provided a structure in which chondrocytes easily infiltrate in regions having relatively large pores. On the other hand, chondrocytes are not present in a region having relatively small pores, or are filled with a second cell type (for example, fibroblasts).
[0018]
In a compositionally graded foam, a mixture of two or more elastomeric copolymers or highly elastic semi-crystalline polymers and an additive such as a growth factor or granule may result from the preferred spatial organization of the additive. First, a desired pore gradient can be selected to develop. The compositional gradient is then composed primarily by differences in the polymer-solvent phase separation of each system, using the various techniques referenced in the preferred method of making a foam with a gradient structure. A foam structure having such a gradient exhibits an effective response to chondrocytes or osteoblasts due to spatial arrangement.
[0019]
In addition, the purpose of the functional gradient is to reduce the stress concentration effects at the abruptly changing interface by more evenly distributing the stress in regions where the mechanical and / or physical properties are changing. This condition is more similar to real biological tissues and structures in which the structural transition between different tissues such as cartilage and bone is gradual. The object of the present invention is therefore to provide an implant in which there is a functional gradient between the material phases. That is, the present invention provides a bioresorbable implant having multi-phase functional stages with attachment means for use in the surgical repair of osteochondral defects or osteoarthritis. In several patents, systems have been proposed for repairing cartilage that can be used with the porous scaffolding material of the present invention. For example, US Pat. No. 5,769,899 describes a device for repairing a cartilage defect, and US Pat. No. 5,713,374 describes a method for fixing a cartilage repair device with a bone anchor. (Both of which are hereby incorporated by reference).
[0020]
Bone
Gradient structures are naturally present in the bone / cartilage boundary region. In the inventor's research, it was found that differences in materials significantly affect cell function. That is, polymer films of primary osteoblasts (95/5 (L) -lactide-co-glycolide copolymer, 90/10 glycolide-co- (L) lactide copolymer, 95/5 (L) -lactide-co-ε- Caprolactone copolymer, 75/25 glycolide-co- (L) lactide copolymer and 40 / 60ε-caprolactone-co- (L) lactide copolymer) and knitted mesh (95/5 (L) lactide-co-glycolide and 90/10 glycolide) The initial and long term responses to (co- (L) lactide copolymers) were evaluated in vitro. From this result, it was found that after 6 days of culture, osteoblasts adhered and proliferated well on all biodegradable polymer films and meshes. With the exception of 40 / 60ε-caprolactone-co- (L) lactide copolymer film, none of the polymer films tested showed significant enhancement of primary rat osteoblast differentiation compared to tissue culture polystyrene (control). . On the other hand, films made with 40/60 ε-caprolactone-co- (L) lactide copolymer promoted cultured osteoblasts as shown by increased alkaline phosphatase activity and osteocalcin mRNA expression compared to other films and TCPS. Showed differentiation. Therefore, it is clear that different absorptive materials significantly change cell function and differentiation. Therefore, by selecting a material that is optimal for cell growth and differentiation, it is possible to optimize tissue regeneration by different cell types in the same skeleton-forming material using a composite material having a gradient structure of composition.
[0021]
Therefore, in a device or skeleton-forming material for bone repair or regeneration, a homopolymer or copolymer of linear, branched or star structure (random, block, segmented block, tapered block, graft, containing ε-caprolactone , Triblock, etc.) are particularly preferred. Note that aliphatic polyester copolymers containing ε-caprolactone in the range of about 30% to about 99% by weight are currently considered preferred. Suitable repeating units that can be copolymerized with ε-caprolactone are well known in the art. For example, suitable comonomers that can be copolymerized with ε-caprolactone include lactic acid, lactide (including L-, D-, meso and D, L mixtures), glycolic acid, glycolide, p-dioxanone (1,4-dioxane- 2-one), trimethylene carbonate (1,3-dioxane-2-one), δ-valerolactone, β-butyrolactone, ε-decalactone, 2,5-diketomorpholine, pivalolactone, α, α-diethylpropio Lactone, ethylene carbonate, ethylene oxalate, 3-methyl-1,4-dioxane-2,5-dione, 3,3-diethyl-1,4-dioxane-2,5-dione, γ-butyrolactone, 1,4 -Dioxepane-2-one, 1,5-dioxepane-2-one, 6,6-dimethyl-dioxepane-2-one, 6, - dioxabicycloctane rust cyclooctane-7-one, and combinations thereof without limitation.
[0022]
In addition, as a preferable medical device or tissue skeleton-forming material for bone tissue repair and / or regeneration containing a bioabsorbable polymer made of ε-caprolactone, a porous foam skeleton-forming material (described in the present application) is used. Etc.), fibrous three-dimensional drawn filaments, non-woven fabrics, woven fabrics, knitted fabrics, or braided tissue skeleton-forming materials, composite materials containing reinforcing fibers, substrates, and mixtures thereof.
[0023]
Skin
Furthermore, skin is another example of tissue having a gradient structure. The basic structure of the skin has two distinct but strongly integrated layers, and the thickness of each layer varies in different parts of the body. For example, the outer layer or epidermis is avascular and is mainly composed of keratinocytes with a small number of immune cells (Langerhans cells) and pigment cells (melanocytes). This keratinocyte produces keratin fibers and corneocyte envelopes, which impart durability and protective functionality to the epidermis. The development of these structures is completely dependent on the differentiation state of the epidermis. That is, the epidermis forms a stratified epithelium with different expression patterns as the cells further migrate from the basement membrane. This layered layer of differentially expressed cells needs to be formed in order to maintain the function of the epidermis. Underneath this epidermis is the dermis, which is a dense, irregularly connected tissue with many blood vessels. This layer is highly dense with collagenous and elastic fibers that impart exceptional elasticity and strength to this layer. Fibroblasts are the main cell type in this layer. In addition, there is a basement membrane between these two layers, which acts as an attachment site for epidermal cells and acts to regulate the function and differentiation of these cells. The layer of keratinocytes directly attached to the basement membrane has a cubic shape and is highly aligned. This adhesion state and structure becomes an important condition for producing a highly scaly structure in the epidermis. That is, this basal layer provides a source of precursor cells for epidermal repair and replacement. The scaly layer also provides strength and resistance to injury and infection.
[0024]
Any material used for skin replacement should be considered capable of inducing infiltration of cells, such as fibroblasts, necessary to produce skin components in the healed tissue. Furthermore, it should be considered that this material should preferably improve rather than inhibit the rate of re-epithelialization in such a way that another basal layer of the epidermis is formed. Materials that allow invasion into the skeletal material by moving keratinocytes can produce partially differentiated cells.
[0025]
For this reason, it can have functional advantages due to the porous configuration facilitating the regulation of access of specific cell types and the regeneration of natural tissues. FIG. 11A, FIG. 11B, and FIG. 12 are diagrams each showing a microstructure of the foam skeleton-forming material of the present invention. Further, FIG. 13 (100 × magnification) and FIG. 14 (40 × composite photo) show photographic evidence of infiltration of fibroblasts, macrophages, macrophage giant cells and endothelium-like cells into 0.5 mm foam. . The foam tissue skeleton-forming material 101 shown in both these photographs is a 50:50 mixture of ε-caprolactone-co-glycolide copolymer and ε-caprolactone-co-lactide copolymer (made as described in Example 7). Met. These photographs were taken on the eighth day after transplantation in a 1.5 cm × 1.5 cm × 0.2 cm excision wound model in a Yorkshire pig model. In both photographs, complete uptake of the substrate into the granulation tissue bed is clearly seen. It also appears that the dense fibrous tissue above the foam tissue skeleton-forming material provides a suitable matrix for excessive growth of the epidermis. PDGF is further incorporated into the foam tissue skeleton-forming material shown in FIG. That is, it is clearly understood that a growth factor such as PDGF is necessary in a wound healing model in which a wound is formed.
[0026]
According to the inventors' initial research, a foam tissue skeleton-forming material having a thickness of about 150 μm to about 3 mm, preferably about 300 μm to about 1500 μm, most preferably about 500 μm to about 1000 μm is used as the skin skeleton-forming material. It seemed desirable to do. Clearly different skin injuries (ie diabetic ulcers, venous stasis ulcers, decubitus ulcers, burns, etc.) require different foam thicknesses. In addition, the patient's condition requires the incorporation of growth factors, antibiotics and antibacterial agents to facilitate wound healing.
[0027]
Vascular graft
The formation of a tubular structure with a gradient is also relevant to the present invention. A vascular graft with a relatively large pore in the outer diameter portion and a small transition in size as it reaches the inner diameter portion, or the tube in the opposite state is used for vascular tissue culture. It is useful in the culture of endothelial cells and smooth muscle cells.
[0028]
The multi-layered tubular structure allows tissue regeneration similar to the mechanical and / or biological properties of blood vessels and can be used as a vascular graft. That is, the desired mechanical properties, bioabsorbability, and tissue ingrowth rate can be obtained with a plurality of concentric layers formed with different compositions under different processing conditions. The innermost luminal layer is optimized for endothelialization by adjusting the porosity of its surface and possible additions in the surface treatment. On the other hand, the outermost outer membrane layer of the vascular graft optimizes its porosity (porosity, pore size, pore shape and pore size distribution) to optimize bioactivity, drug, or It is adjusted to induce tissue ingrowth by incorporating cells. In this case, a barrier layer with low porosity may or may not be provided between these two types of porous layers in order to increase strength and reduce leakage.
[0029]
Foam composition
A variety of absorbent polymers can be used to make the foam. Examples of bioacceptable and bioabsorbable polymers suitable for use include aliphatic polyesters, poly (amino acids), copoly (ether-esters), polyalkylene oxalates, polyamides, poly (iminocarbonates), polyorthoesters, Included are polymers selected from the group consisting of polyoxaesters, polyamide esters, polyoxaesters containing amine groups, poly (anhydrides), polyphosphazenes, biomolecules, and mixtures thereof. Further, as aliphatic polyesters for the purpose of the present invention, lactide (including lactic acid, D-, L-, and meso-lactide), glycolide (including glycolic acid), ε-caprolactone, p-dioxanone (1,4-dioxane) -2-one), trimethylene carbonate (1,3-dioxan-2-one), alkyl derivatives of trimethylene carbonate, δ-valerolactone, β-butyrolactone, γ-butyrolactone, ε-decalactone, hydroxybutyrate (repeated) Unit), hydroxyvalerate (repeating unit), 1,4-dioxepan-2-one (1,5,8,12-tetraoxacyclotetradecane-7,14-dione of the dimer), 1,5- Dioxepane-2-one, 6,6-dimethyl-1,4-dioxan-2-one, 2,5-diketomol Holin, pivalolactone, α, α-diethylpropiolactone, ethylene carbonate, ethylene oxalate, 3-methyl-1,4-dioxane-2,5-dione, 3,3-diethyl-1,4-dioxane-2, These include, but are not limited to, homopolymers and copolymers of 5-dione, 6,8-dioxabicyclooctane-7-one, and mixtures of these polymers. Moreover, as poly (imino carbonate) which meets the object of the present invention,Handbook of Biodegradable Polymers(Edited by Domb, Kost and Wisemen, Hardwood Academic Press, 1997, pp. 251 to 272), and those described in Kemnitzer and Kohn. Copoly (ether-esters) for the purposes of the present invention include Cohn and Younes ("Journal of Biomaterials Research", Vol. 22, pages 993 to 1009, 1988) and Cohn (Polymer Preprints). (ACS Division of Polymer Chemistry) Vol. 30 (1), page 498, 1989), and copolyester-ethers (for example, PEO / PLA). Examples of polyalkylene oxalates that meet the object of the present invention include U.S. Pat. Nos. 4,208,511, 4,141,087, 4,130,639, which are incorporated herein by reference. Nos. 4,140,678, 4,105,034 and 4,205,399 are included. Furthermore, polyphosphazenes, and copolymers, terpolymers and higher order mixed monomers made from L-lactide, D, L-lactide, lactic acid, glycolide, glycolic acid, para-dioxanone, trimethylene carbonate and ε-caprolactone, etc. The literature Allcock (in which references are incorporated herein by reference are polymers based onThe Encyclopedia of Polymer Science, Vol. 13, pp. 31-41, Wiley Intersciences, John Wiley & Sons, 1988), and Vandorpe, Schacht, Dejardin and Lemmouchi (Handbook of Biodegradable Polymersedited by Domb, Kost and Wisemen, Hardwook Academic Press, 1977, pages 161 to 182). In addition, poly (acid anhydride) has a chemical formula HOOC-C in which m is an integer in the range of 2 to 8.₆H_Four-O- (CH₂)_m-O-C₆H_FourIncluded are -COOH diacids and those made by copolymers of this compound with aliphatic alpha-omega diacids of up to 12 carbon atoms. In addition, polyoxaesters, polyoxaamides and polyoxaesters containing amine groups and / or amide groups are incorporated herein by reference, US Pat. Nos. 5,464,929, 5,595,751. No. 5,597,579, No. 5,607,687, No. 5,618,552, No. 5,620,698, No. 5,645,850, No. 5 648,088, US Pat. No. 5,698,213, US Pat. No. 5,700,583, and US Pat. No. 5,859,150. Polyorthoesters are described in the document Heller (included herein by reference).Handbook of Biodegradable Polymers, edited by Domb, Kost and Wisemen, Hardwook Academic Press, 1977, pages 99 to 118).
[0030]
In general, aliphatic polyesters are absorbable polymers and are suitable for making foams having a gradient structure. The aliphatic polyester can be a homopolymer or copolymer (random, block, segmented block, tapered block, graft, triblock, etc.) having a linear, branched or star structure, preferable. Suitable monomers for making such aliphatic homopolymers and copolymers are lactic acid, lactide (including D-, L-, meso, D, L mixtures), glycolic acid, glycolide, ε-caprolactone, p-dioxanone ( 1,4-dioxane-2-one), trimethylene carbonate (1,3-dioxan-2-one), δ-valerolactone, β-butyrolactone, ε-decalactone, 2,5-diketomorpholine, pivalolactone, α , Α-diethylpropiolactone, ethylene carbonate, ethylene oxalate, 3-methyl-1,4-dioxane-2,5-dione, 3,3-diethyl-1,4-dioxane-2,5-dione, γ -Butyrolactone, 1,4-dioxepan-2-one, 1,5-dioxepan-2-one, 6,6-dimethyl-dio Sepang-2-one, 6,8-dioxabicycloctane rust cyclooctane-7-one, and can be selected from the group consisting of combinations is not limited thereto.
[0031]
Elastomeric copolymers are also particularly useful in the present invention. Suitable bioabsorbable and bioacceptable elastomers include ε-caprolactone and glycolide elastomeric copolymers (preferably about 35:65 to about 65:35, more preferably 45:55 to 35:65 ε-caprolactone). Having a molar ratio of glycolide), ε-caprolactone and L-lactide, a D-lactide mixture of L-lactide or an elastomeric copolymer of lactide including lactic acid copolymers (preferably from about 35:65 to about 65:35). And more preferably having a molar ratio of ε-caprolactone to lactide of 45:55 to 30:70 or about 95: 5 to about 85:15), p-dioxanone (1,4-dioxane-2-one) ) And L-lactide, D-lactide and lactide-containing elastomer copolymers Remer (preferably having a molar ratio of p-dioxanone to lactide of about 40:60 to about 60:40), an elastomeric copolymer of ε-caprolactone and p-dioxanone (preferably about 30:70 to about An elastomeric copolymer of p-dioxanone and trimethylene carbonate (preferably from about 30:70 to about 70:30 p-dioxanone to tri) having a molar ratio of ε-caprolactone to p-dioxanone of 70:30. An elastomeric copolymer of trimethylene carbonate and glycolide (preferably having a molar ratio of trimethylene carbonate to glycolide of from about 30:70 to about 70:30), Methylene carbonate and L-lactide, D-La Lactide elastomeric copolymers (preferably having a molar ratio of trimethylene carbonate to lactide of about 30:70 to about 70:30), and mixtures thereof, including tides, mixtures thereof and lactic acid copolymers Is selected from, but is not limited to.
[0032]
Examples of suitable bioabsorbable elastomers are also described in US Pat. Nos. 4,045,418, 4,057,537, and 5,468,253, which are incorporated herein by reference. ing. These elastomeric polymers have from about 1.2 dL / g to about 4 dL / g, preferably about 1 when measured at 25 ° C. in a 0.1 gram / deciliter (g / dL) solution in hexafluoroisopropanol (HFIP). It has an intrinsic viscosity of from 2 dL / g to about 2 dL / g, most preferably from about 1.4 dL / g to about 2 dL / g.
[0033]
Preferably, these elastomers exhibit high stretch and low modulus, as well as good tensile strength and good recovery properties. In a preferred embodiment of the present invention, the elastomer forming the foam exhibits a stretch ratio of about 200% or more, preferably about 500% or more. These properties indicate the degree of elasticity of the bioabsorbable elastomer described above and are approximately 500 psi (35.1 kgf / cm).²) Or more, preferably about 1000 psi (70.3 kgf / cm)²) And a tensile strength of about 50 lbs / inch (8.9 kg weight / cm) or more, preferably about 80 lbs / inch (14 kg weight / cm) or more.
[0034]
A polymer or copolymer suitable for forming a foam having a gradient structure for tissue regeneration depends on several factors. That is, chemical composition, spatial distribution of components, polymer molecular weight, and degree of crystallization all determine the polymer's in vitro and in vivo behavior to some extent. However, the choice of polymer to create a foam having a gradient structure for tissue regeneration is highly dependent on (but not limited to) the following factors. (A) bioresorption (or biodegradation) rate, (b) in vivo mechanical properties, (c) cell responsiveness to materials related to cell attachment, proliferation, migration and differentiation, and (d) biotolerability. .
[0035]
The properties of the material matrix that are timely absorbed by the body environment are important. However, differentiation in absorption time under in vivo conditions is also a criterion when mixing two different copolymers. For example, a 35:65 ε-caprolactone and glycolide copolymer (relatively fast absorbing polymer) is mixed with 40:60 ε-caprolactone and (L) lactide copolymer (relatively slow absorbing polymer) to form a foam. Can be formed. Such foams can have several different physical structures depending on the processing technique used. That is, these two types of components are two continuous layers irregularly interconnected, have a constant gradient structure in the thickness direction, or an integral boundary between the layers of these two types of components. A laminate-type composite having portions can be constructed. In addition, these foam microstructures can be optimized to regenerate or repair the desired anatomy of the tissue undergoing artificial processing.
[0036]
A preferred embodiment of the present invention aims to use a polymer mixture to form a structure that transitions from one composition to another in a gradient fashion. Foams with such a gradient structure are used to repair or regenerate the structure of natural tissues such as cartilage (joints, meniscus, septum, trachea, etc.), esophagus, skin, bone and vascular tissue This is particularly advantageous in the artificial processing of tissues. For example, an epsilon-caprolactone-co-glycolide elastomer is mixed with ε-caprolactone-co-lactide (in a molar ratio of about 5:95) to provide a soft sponge-like foam that resembles the cartilage-to-bone transition. The foam can be formed to have a transition from a hard foam to a hard foam. Of course, different polymer blends can be used to construct different gradient structures such as similar gradient effects or different absorbency profiles, stress response profiles, or different degrees of elasticity. In addition, these foams can use the specific skeletal material of the present invention including, but not limited to, spinal disc, dura mater, nerve tissue, liver, pancreas, kidney, bladder, tendon, ligament and breast tissue. Can be used for organ replacement or regeneration.
[0037]
The above elastomeric polymers can be lyophilized, supercritical solvent foaming (ie, methods as described in EP 464,163B1), gas injection extrusion, gas injection molding or extractable materials (ie salts, sugars, etc.). Can be foamed by a casting process with any means known to those skilled in the art. At present, it is considered preferable to produce bioabsorbable and bioacceptable elastomeric foams by lyophilization. A suitable method for lyophilizing elastomeric polymers to form foams is assigned to Ethicon, filed June 30, 1999, which is hereby incorporated by reference in the following examples and specification. No. ETH-1352, described in a co-pending patent application entitled “Process for Manufacturing Bopmedical Foams”.
[0038]
The foam produced in the present invention can be produced by a polymer-solvent phase separation technique that is a modification of the prior art that unexpectedly formed a gradient structure in the foam structure. In general, polymer solutions are (a) thermally induced gelation / crystallization, (b) solvent-free induced separation of solvent and polymer phases, (c) chemically induced phase separation, and (d) thermally induced cusps. It can be separated into two phases by any of four methods of spinodal decomposition. The polymer solution is separated in a prepared manner and splits into two separate phases or two continuous phases. Subsequent removal of the solvent phase results in a porous structure with pores in the micrometer range that are less dense than the original polymer (AT Young's “Microcellular foams via phase separation”). (See J. Vac. Sci. Technol. A4 (3), May / June 1986)). The processes related to the production of these foams include a process of selecting an appropriate solvent for a polymer that needs to be freeze-dried and a process of creating a uniform solution. The polymer solution is then frozen and subjected to a vacuum drying process. In this freezing step, phase separation of the polymer solution is performed, and in the vacuum drying step, the solvent is sublimated and removed, and the remaining porous polymer structure, that is, a porous foam having internal open cells is dried.
[0039]
Solvents generally suitable as a starting point for selecting a solvent for the preferred absorbent aliphatic polyester are formic acid, ethyl formate, acetic acid, hexafluoroisopropanol (HFIP), cyclic ethers (ie, THF, DMF, and PDO), acetone, C2 to C5 alcohol acetate (such as ethyl acetate and t-butyl acetate), glyme (ie monoglyme, ethylglyme, diglyme, ethyldiglyme, triglyme, butyldiglyme and tetraglyme), methyl ethyl ketone, dipropylene Glycol methyl ether, lactone (γ-valerolactone, δ-valerolactone, β-butyrolactone, γ-butyrolactone, etc.), 1,4-dioxane, 1,3-dioxolane, 1,3-dioxolan-2-one Ethylene carbonate), dimethyl carbonate, benzene, toluene, benzyl alcohol, p-xylene, naphthalene, tetrahydrofuran, N-methylpyrrolidone, dimethylformamide, chloroform, 1,2-dichloromethane, morpholine, dimethyl sulfoxide, hexafluoroacetone / sesquihydrate (HFAS), anisole, and a solvent selected from the group consisting of mixtures thereof include, but are not limited to. Among these solvents, 1,4-dioxane is a preferred solvent. Note that a uniform solution of the polymer in this solution can be made by standard techniques.
[0040]
Thus, it can be appreciated that the applicable polymer concentration or the amount of solvent that can be used varies with each system. Suitable phase diagram curves for some systems are already known. However, if a suitable curve is not available, this curve can be generated by known techniques. For example, examples of such suitable techniques are described in the documents Smolders, Van Aartsen and Steenbergen (Kolloid-Z, u. Z. Polymere, 243, 14 (1971)). As a general usage, the amount of polymer in solution can vary between about 0.5% to about 90% by weight, preferably 0.5% to about 30% by weight, and the polymer in any solvent Depends greatly on the solubility of the resin and the final properties of the desired foam.
[0041]
In addition, solid materials can be added to the polymer-solvent system. The solid material added to the polymer-solvent system preferably does not react with the polymer or solvent. Suitable solid materials include materials that promote tissue regeneration or regrowth, buffers, reinforcements, porous modifiers, and the like. Preferred solid materials include demineralized bone particles, calcium phosphate particles, or calcium carbonate particles for bone repair, an elution solid material for pore formation, and a bioabsorbable material that does not dissolve in the solvent system as a reinforcement. Examples include, but are not limited to, polymer particles or polymer particles that form pores when absorbed.
[0042]
Suitable eluting solid materials include salts (ie, sodium chloride, potassium chloride, calcium chloride, sodium tartrate, sodium citrate, etc.), bioacceptable mono- and disaccharides (ie, glucose, fructose, dextrose, maltose). , Lactose and sucrose), polysaccharides (ie starch, alginate), water-soluble proteins (ie gelatin and agarose). In general, these materials all have an average diameter of about 1 mm or less, preferably from about 50 μm to 500 μm. These particles also constitute from about 1% to about 50% by volume of the total volume of the particles and polymer-solvent mixture (this total volume% is equal to 100% by volume). This eluting material is immersed in a solvent that dissolves the foam in a time amount that dissolves the foam together with the eluting material but dissolves the foam and does not adversely change the foam. Can be removed. The preferred extraction solvent is water, most preferably distilled deionized water. This process is described in US Pat. No. 5,514,378, which is included herein as a reference. Preferably, unless the accelerated absorbency of the foam is desired, the foam is dried at low temperature and / or vacuum after completion of the elution process to minimize its hydrolysis.
[0043]
After forming the polymer-solvent mixture, the mixture is solidified. For a particular polymer-solvent system, the freezing point, melting temperature, and apparent glass transition point of the polymer-solvent system can be determined by standard differential calorimetry (DSC) techniques. Theoretically, although not meant to limit the scope of the invention, it is believed that when the polymer-solvent system cools, initial solidification occurs at temperatures near or below the freezing point of the solvent. This corresponds to the solidification of most of the solvent in the system. This initial solidification appears as a first exothermic peak. Thereafter, a second freezing point occurs when the solvent remaining with the polymer solidifies. This second freezing point is shown as the second exothermic peak. Furthermore, a clear Tg is exhibited at a temperature at which the fully frozen system exhibits a first exothermic shift upon reheating.
[0044]
An important parameter to adjust is the freezing rate of the polymer-solvent system. That is, the type of pore morphology that is fixed during the freezing process is a function of the thermodynamics of the solution, the freezing rate, the temperature to be cooled, the concentration of the solution, and homogeneous or heterogeneous nucleation. A detailed description of such a phase separation phenomenon is included in this specification as a reference (AT Young's “Microbubble Foam by Phase Separation” (J. Vac. Sci. Technol. A4 (3), May). / June 1986) and S. Matsuda “Thermodynamics of Formation of Porous Polymeric Membrane from Solutions” (Polymer J. Vol. 23, No. 5, 435) Pp. 444, 1991).
[0045]
Such previously reported polymer solutions can be solidified in various ways, such as placing or pouring the solution into a mold and cooling the mold in a suitable bath or on a freezer shelf. Alternatively, the polymer solution can be solidified layer by layer by spraying the polymer solution onto a cooled surface by spraying with a sprayer. The cooled surface can be a medical device or a component or film thereof. The shape of the solidified spray film is similar to the shape of the sprayed surface. Alternatively, the solidified mixture can be formed into a fixed shape during cutting or freezing. Using these and other methods, foams can be made in various shapes and dimensions (ie, tubular, branched, spherical, hemispherical, three-dimensional polygonal, oval (ie, kidney shape), Toroidal shapes, conical shapes, frustoconical shapes, pyramid shapes, solid and hollow shapes, and combinations thereof).
[0046]
Alternatively, another method for making foamed parts is the use of cold fingers (a metal part whose surface shows the inside of the part to be manufactured). The cold finger is removed after being immersed in a polymer in a suitable solvent. This process is very similar to the process of immersing an ice cream cream stick in warm chocolate to solidify it and hardening it, or immersing the compact in rubber latex to form gloves or condoms. At this time, the thickness and morphology of the foam produced is a function of temperature, residence time, and drawing speed of the cold finger in the mixture. That is, the longer the residence time and the slower the drawing speed, the thicker the coating. After withdrawal, the cold finger is placed on a high heat capacity instrument in contact with the lyophilized freezing tray. From this point, the primary and secondary drying processes as described above begin. This method is suitable for making tubes, branched tubular structures or sleeves that can be formed into a shape that fits a portion of the device or animal anatomy.
[0047]
In addition, the polymer solutions described above can be solidified by various inserts incorporated with solutions such as films, scrims, wovens, nonwovens, knitted fabrics or braided fiber structures. In addition, this solution can be separated from plastic surgical implants (eg screws, pins, nails and plates) or tubular or branched tubular structures (skeletal forming materials for vessels or for tubular organs). It can be created along with the structure. These inserts can be formed from at least one bio-acceptable material and may be non-absorbable, absorbable, or a combination thereof.
[0048]
The polymer solution in the mold is cooled in a certain direction through the mold wall in contact with the freeze-drying shelf and processed in a constant thermal cycle. The mold and its surface can be formed of any material that does not affect the polymer-solvent system, but is preferably formed of a material with high thermal conductivity. That is, heat transfer is transferred from the freeze dryer shelf through the mold wall and into the polymer solution. Thereafter, when the temperature of the mixture falls below its gel point and / or freezing point, the mixture phase separates.
[0049]
This phase-separated system configuration is fixed during the solidification process of the lyophilization process, and the formation of continuous pores begins with the start of the vacuum drying process and the solvent sublimes. However, the mixture in the container or mold cooled by the cooling radiator will solidify before freezing. That is, even though the mixture appears to be in a solid state, there is initially some residual solvent associated with the uncrystallized polymer. Theoretically, this is not meant to limit the present invention, but the solidification reaction is completed after the freezing point is moved through the mixture by the cooling radiator and the mixture is apparently solidified. Therefore, at any time, the material in front of the front of the freezing point is not as cold as the material in the back of the front and is not completely frozen.
[0050]
When the inventor supplies a vacuum to this apparently solid polymer solvent mixture immediately after it appears in a solidified state, the foam can be formed with a gradient structure having various pore sizes and structures and channels. I found Therefore, the start time of the sublimation process (by depressurization, ie vacuum drying) is an important step in the method for forming a transition state in the structure. The start time of this sublimation process is affected by the thickness of the foam produced, the concentration of the solution, the heat transfer rate, and the direction of heat transfer. It should be noted that those skilled in the art can handle specific polymer-solvent systems by monitoring the thermal transition rate of the foam at various depths and locations within the apparatus to be foamed using a thermocouple. It can be seen that this process can be monitored and characterized. That is, by controlling this sublimation treatment, a structure having gradient and anisotropy in the pore form can be formed. Furthermore, this method makes it possible to form microstructures similar to tissues such as cartilage, bone and skin. For example, in general, the flow path can be formed by supplying a vacuum immediately after the solution is apparently solidified. However, when the same solution is further solidified, the foam has relatively large pores near the surface to which the vacuum is supplied (on the side opposite to the cooling radiator), and on the side closer to the cooling radiator. Has relatively small pores in the region.
[0051]
This treatment method is in contrast to prior art treatment methods aimed at completely freezing the solution to form a foam having a uniform microstructure (irregularly interconnected pores). In such prior art, the vacuum drying process was performed only after a considerable amount of time to complete the desired phase separation (US Pat. No. 5,755,792 (Brekke), US Pat. No. 755 (Brekke), No. 5,716,413 (Walter et al.), No. 5,607,474 (Athanasiou et al.), No. 5,686,091 (Leong et al.), No. 5,677 , 355 (Shalaby et al.), And European Patent Publications E0274898 (Hinsch) and EPA 594148 (Totakura)).
[0052]
2, 3 and 4 show foams having various structures. For example, as shown in FIG. 3, the orientation of the main axis of the pores can vary from the same plane as the foam until it is perpendicular to the plane of the foam. Theoretically, although not meant to limit the scope of the present invention, in a conventional foaming process, when the solvent crystallizes, the freezing front moves within the solution, causing the solution to be parallel to the freezing front and the crystalline layer. It is thought to solidify within. However, if a vacuum is applied before the solution is completely frozen, the foam morphology is formed such that the pores are arranged generally parallel to the vacuum source, as shown in FIG.
[0053]
As can be seen in FIG. 3, the pore size can vary from a small pore of about 10 μm to about 60 μm to a porous gradient foam from a relatively large pore of about 60 μm to about 200 μm. In this case as well, this state can be obtained by supplying a vacuum to the solution in an apparently solidified state before the solution is completely solidified. When adjusting the bubble size, the polymer concentration in the solution and the cooling rate are also important parameters. Preferably, the foam structure is formed so that it can act as a template for restoring a human tissue joint, such as cartilage, to the bone joint present in the joint. This foaming progresses from small spherical pores to relatively large cylindrical (ie, having a diameter to length ratio of at least 2: 1). Furthermore, the stiffness of the foam can be adjusted by mixing two different polymers with different foam structures or different Young's modulus.
[0054]
Further, the foam can have a flow path as shown in FIG. The channel formed by this method can traverse the thickness of the foam and is generally about 30 to about 200 μm in diameter. Generally, the channel has a length that is at least twice, preferably at least four times, and most preferably at least eight times the average diameter of the channel. Of course, the size and diameter of the channel can be selected based on the desired functionality of the channel, such as cell infiltration, nutrient diffusion or angiogenesis channels.
[0055]
A person skilled in the art can easily set the above-mentioned directionality in an arbitrary three-dimensional state by setting the appropriate mold configuration and, if necessary, the mold wall at different temperatures. Can be seen visually. The following types of gradient structures can be formed by changes in pore size and / or shape through a thickness having a uniform composition. That is, there is one type and size of pores for a specific thickness, followed by another type and size of pores, composition gradient mainly due to one type of composition on one side There is a composition gradient due to another type of composition on the other side, a structure in which the composition transitions from one side to the other, and a low-porosity thick film with a small pore layer This is followed by a structure with a large pore area, followed by a foam with vertical pores with spatial organization, and these vertical pores can act as a nutrient diffusion channel These are the structures in which these pores are created in a three-dimensional mold to form a three-dimensional foam having a desired microstructure combination of compositional and structural gradients. Generally, the foam formed in the container or mold has a thickness in the range of about 0.25 mm to about 100 mm, preferably about 0.5 mm to about 50 mm. Thicker foams can be formed, but the time of the lyophilization process becomes very long, making it difficult to control the final foam structure and increasing the residual solvent content.
[0056]
As already explained, the process of the invention for producing a bio-acceptable foam is performed under temperature conditions above the apparent glass transition temperature and below the solidification temperature of the mixture (preferably just below the solidification temperature). It can be significantly reduced by performing a sublimation process. That is, the time of the combined processing steps (freezing process + primary drying process + secondary drying process) is much shorter than the time described in the prior art. For example, the combined processing time for aliphatic polyesters with volatile solvents is generally 72 hours or less, preferably 48 hours or less, more preferably 24 hours or less, and most preferably 10 hours or less. In practice, this combined processing time is less than 3 hours for foams less than 1 mm thick with specific aliphatic polyesters, less than 6 hours for foams about 2 mm thick, and about 3 mm thick foam. It is 9 hours or less with the body. On the other hand, in the prior art, this processing time is generally 72 hours or more. Further, the residual solvent concentration in the foam prepared by the method of the present invention is also extremely low. The residual 1,4-dioxane concentration is 10 ppm (parts per million) or less, preferably 1 ppm or less, most preferably, as described for aliphatic polyester foams made using 1,4-dioxane as the solvent. 100 ppb (parts pervillion) or less.
[0057]
In addition, if it is an expert in the said technical field, said directionality can be set to arbitrary three-dimensional states by making the temperature of the wall part of the said shaping | molding die into a different temperature as needed as needed. Is easily seen visually. The following types of gradient structures can be created by the present invention. That is,
1. A structure in which the size and / or shape of the pores varies through a certain thickness with a uniform composition,
2. A structure with one type and size of pores for a particular thickness, followed by another type and size of pores,
3. A structure in which there is a gradient mainly from one type of composition on one side, a gradient from another type of composition on the other side, and the composition transitions from one side to the other,
4). A structure with a small porous layer with a low porosity, followed by a large pore region,
5. A foam comprising vertical pores with spatial organization, wherein these vertical pores can act as channels for nutrient diffusion,
6. a structure for producing these foams in a three-dimensional mold to form a three-dimensional foam having a desired microstructure; and
7). It is a combined structure of composition and structural gradient.
[0058]
In addition, various proteins (including short peptides), proliferative agents, chemotactic agents and therapeutic agents (antibiotics, analgesics, anti-inflammatory agents, anti-rejection agents (eg immunosuppressive agents) and anti-cancer agents), or ceramics The particles can be added to the foam during the preparation process, absorbed on the surface or back of the foam after the foam is created, and filled into the foam. For example, synthetic polymers or biopolymers (such as collagen and elastin) or biocompatible ceramic materials (such as hydroxyapatite) that can absorb foam pores with bioacceptability, and combinations thereof (such as tissue growth in the device). Can be partially or completely filled with or without a prompting material). Suitable materials include autograft, allograft or xenograft bone, bone marrow, morphogenetic protein (BMP), epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet derived growth factor ( PDGF), insulin-derived growth factors (IGF-I and IGF-II), transforming growth factor (TGF-β), vascular endothelial growth factor (VEGF) or other osteoinductive known in the art or This includes, but is not limited to, osteoconductive materials. The biopolymer can also be used as a delivery vehicle for conductive or chemotactic materials or growth factors. Examples include recombinant or animal-derived collagen or elastin or hyaluronic acid. Bioactive coatings or surface treatments can also be applied to the surface of the material. For example, biologically active peptide sequences (RDG) can be attached to facilitate protein absorption and subsequent attachment of cellular tissue. In addition, therapeutic agents can be supplied by these foams.
[0059]
In another embodiment of the present invention, the polymers and mixtures used to form the foams described above can contain a therapeutic agent. To form such a foam, the therapeutic agent can be mixed with the polymer before forming the foam, or the therapeutic agent can be loaded after forming the foam. The variety of different therapeutic agents that can be used in the foams of the present invention is numerous. In general, therapeutic agents that can be administered via the pharmaceutical composition of the present invention include anti-infective agents such as antibiotics and antiviral agents, chemotherapeutic agents (ie, anticancer agents), anti-rejection agents, analgesics and analgesic combinations. Drugs, anti-inflammatory agents, hormones such as steroids, growth factors (bone morphogenetic proteins (ie BMP1-7), bone morphogenesis-like proteins (ie GFD-5, GFD-7 and GFD-8), epidermal growth) Factor (EGF), fibroblast growth factor (ie FGF1-9), platelet derived growth factor (PDGF), insulin-like growth factors (IGF-I and IGF-II), transforming growth factor (ie TGF-βI) -III), vascular endothelial growth factor (VEGF)), and other natural or genetically engineered proteins, polysaccharides, glycoproteins or Including but Potanpaku such as but not limited to these. These growth factors are described in the literature of Vicki Rosen and R. Scott Thies, which are included herein as references (The Cellular and Molecular Basis of Bone Formation and Repairpublished by R. G. Landes Company).
[0060]
Foams containing bioactive materials can be blended and foamed by mixing one or more therapeutic agents with the polymer or solvent or polymer-solvent mixture for making the foam. Alternatively, the therapeutic agent can be coated on the foam, preferably with a pharmaceutically acceptable carrier. Any drug carrier that does not dissolve the foam can be used. The therapeutic agent can be present as a liquid, a finely divided solid, or other suitable physical form. Generally or alternatively, the substrate includes one or more additives such as diluents, carriers, excipients, stabilizers, and the like.
[0061]
The amount of therapeutic agent depends on the particular drug used and the medical condition being treated. In general, the amount of drug is from about 0.001% to about 70%, preferably from about 0.001% to about 50%, most preferably from about 0.001% to about 20% by weight of the substrate. %. Furthermore, the amount and type of polymer incorporated into the drug delivery substrate will vary depending on the desired release profile and the amount of drug used.
[0062]
Upon contact with body fluids, the drug is released. When the drug is incorporated into the foam, the drug is released as the foam gradually disintegrates (mainly by hydrolysis). In this way, an effective amount (for example, 0.0001 mg / kg / hour to 10 mg / kg / hour) of a drug is supplied for a long time (for example, 1 hour to 5000 hours, preferably 2 hours to 800 hours). Can be done. Such dosage form can be determined according to the condition of the subject to be treated, the degree of pain, the judgment of the prescribing doctor, and the like. Various formulations can be made by those skilled in the art according to the above or similar methods.
[0063]
The foam can also act as a skeleton-forming material amount for artificial processing of tissue. That is, the porous gradient structure induces cell growth. Cells are harvested from the patient (before or during the surgery to repair the tissue) as described in an existing patent (Vacanti US Pat. No. 5,770,417) and the cells are subjected to sterile processing conditions. Processing below can yield specific cell types (ie, pluripotent cells, stem cells and progenitor cells such as the mesenchymal stem cells described in US Pat. No. 5,486,359). Suitable cells that can be contacted or inoculated with the foam skeleton-forming material of the present invention include myocytes, fat cells, fibromyoblasts, ectoderm cells, muscle cells, osteoblasts (ie, bone cells), chondrocytes ( Ie, osteochondrocytes), endothelial cells, fibroblasts, pancreatic cells, hepatocytes, bile duct cells, bone marrow cells, neurons, genitourinary cells (including nephritic cells), and combinations thereof. Absent. The skeleton-forming material of the present invention can treat various cells (that is, autogenous, allogeneic and xenogeneic cells, etc.). These cells may also contain an inserted DNA encoding a protein capable of stimulating tissue attachment, proliferation or differentiation. The foam is placed in a cell culture environment and cells are seeded on or within the structure. The foam is maintained in a sterile environment and then implanted into a donor patient that has infiltrated the cells with the microstructure of the device. This in vitro inoculation of cells allows for faster tissue growth and differentiation. The ability of this cell to differentiate and form a specific extracellular matrix of the tissue is important in the artificial processing of the tissue with a functional graft.
[0064]
The user can choose to inoculate different cell types into different porous structures. Schaufer et al. Report that different cell types (ie, stromal cells and chondrocytes) can be cultured on different structures. These structures are then combined and a biphasic tissue structure can be generated for transplantation by bringing the entire structure back into the cell culture environment. Gradient structures can also be generated for co-cultured tissue scaffolding materials (Schaefer, D et al.). In addition, radiopaque markers can be added to the foam to perform image processing after implantation. After a predetermined in vitro growth period (eg, 3 weeks), the tissue treated graft is removed and transplanted into the patient. In the case of cell-free treatment, a sterilized cell-free skeleton-forming material can be used to replace damaged or damaged tissue.
[0065]
The foam skeleton-forming material of the present invention can be sterilized by conventional techniques including sterilization based on radiation (ie gamma radiation), sterilization based on chemical treatment (ethylene oxide treatment) or other suitable treatment. This sterilization treatment is preferably treatment with ethylene oxide at 52 ° C. to 55 ° C. within 8 hours. After this sterilization treatment, the foam skeleton-forming material is packaged in a suitable sterilization and moisture resistant package that can be used for shipping and in health care facilities such as hospitals.
[0066]
In another embodiment of the present invention, the firing pair may have a fibrous fabric fused to the top or bottom surface. In this way, the surface properties such as porosity, permeability, disintegration rate and mechanical properties of the structure can be adjusted. This fibrous fabric can be produced by an electrospinning method, in which a fiber layer can be formed on a freeze-dried foam surface. Electrospinning is a method that has been used to make fibers. Electrospinning of many polymeric materials is described in the following patents and literature: US Pat. Nos. 4,522,709, 5,024,789, 5,311,884, No. 5,522,879, “Nanometre diameter fibers of polymer, produced by electrospinning” (Nanotechnology, 1 (1996) pp. 216 to 233), “ Structure and morphology of small diameter electrospun aramid fibers ”(Polymer Inter., 36 (1995) pp. 195 to 201), and incorporated herein by reference. “Carbon nanofiber from polyacrylonitrile and mesophase pitch” (Journal of Advanced Materials, Vol. 3, No. 1) 1999) pages 36 to 41).
[0067]
In this method, a polymer jet is applied with an electric power that overcomes the surface tension of the solution to form a charged jet. A jet of this solution is jetted onto a grounded substrate, dried and solidified. By adjusting the spinning conditions, the resulting fiber can be in the range of about 0.1 μm to about 20 μm, and preferably in the range of about 0.3 μm to about 5.0 μm. In this case, it is a freeze-dried foam. Two examples of such structures are shown in FIGS. These layer structures can be made of at least one bio-acceptable material and can be non-absorbable, absorbable or a combination thereof.
[0068]
The composition, thickness and porosity of the fiber layer can be adjusted to provide the desired mechanical and biological properties. For example, the bioabsorption rate of the fiber layer can be selected to provide a bioabsorbable structure that is longer or shorter than the underlying foam layer. In addition, the fiber layer may have greater integrity to the composite and mechanical force may be applied to the fiber side of the structure. For example, thanks to the fibrous layer, sutures, staples or other fastening devices can be used to hold the composite in place. As a general guide, but not limiting the scope of the invention in any way, it is generally preferred that the fiber layer be composed of layers having a thickness of about 1 to 1000 microns.
[0069]
One effective application of a foam structure having a fibrous surface layer is its use as a vascular graft. The layers in such a structure will form a plurality of concentric cylinders and the fiber layer will reduce permeability. The lyophilized foam structure can be optimized for cell infiltration by smooth muscle cells or fibroblasts on the outer membrane surface and endothelial cells on the luminal surface. The fibrous fabric layer acts as a barrier to blood leakage and improves the mechanical properties of this structure. In addition, the fiber layer acts as a barrier to smooth muscle cell proliferation and prevents intimal hyperplasia.
[0070]
Another effective application of such a foam / woven hybrid structure is the use as a skeletal material for breast regeneration. The fiber layer enhances mechanical integrity and maintains the original shape of the skeleton-forming material. When performing a biopsy or removing a tumor, the cavity can be filled with a foam skeletal material coated with such fibers, and the skeletal material can be poured with cells or bioactive agents. If not.
[0071]
Another useful application of such a foam / fiber structure is the use as a skeletal material for artificial processing of meniscal tissue. In this case, a wedge-shaped graft can be made with a fibrous covering that wraps the foam core that has been lyophilized. Cells can be injected into the center of the graft through this fiber layer. This textile surface layer allows diffusion of nutrients and waste products, but restricts the migration of cells from the skeleton-forming material.
[0072]
The following examples are intended to illustrate the principles and practice of the present invention, but are not limited thereto. That is, many additional embodiments within the spirit and scope of the present invention can be readily envisioned by those skilled in the art.
[0073]
Example
In the following examples, each polymer and monomer has its chemical composition and purity (NMR, FT-IR), thermal analysis (DSC), molecular weight (intrinsic viscosity), and baseline andIn vitroIt is characterized for its mechanical properties (Instron stress / strain).
[0074]
¹H-NMR measurement at 300 MHz NMR with CDCl_ThreeAlternatively, HFAD (hexafluoroacetone sesqua deuterium oxide) was used as a solvent. In addition, thermal analysis of the polymer and monomer was performed using a DuPont 912 differential calorimetry (DSC) apparatus. Furthermore, the intrinsic viscosity (IV dL / g) of each polymer and copolymer was 50 boa soaked in a constant temperature water bath at 25 ° C. with chloroform or hexafluoroisopropanol (HFIP) as a solvent at a concentration of 0.1 g / dL. Measured with a Cannon-Ubbelhode dilution viscometer.
[0075]
Further, in the following examples, specific abbreviations are used such that ε-caprolactone polymer is shown as PCL, glycolide polymer as PGA, and (L) lactide polymer as PLA. Furthermore, the% values listed before each copolymer indicate the mole% value of each component, respectively.
[0076]
Example 1
Creation of foams with irregular microstructures (unfavorable structures)
Step A. Creation of a 5% weight / weight uniform solution of 35/65 PCL / PGA in 1,4-dioxane
1 part by weight of 35/65 PCL / PGA was dissolved in 19 parts by weight of solvent 1,4-dioxane to make a polymer volume of 5% weight / weight. The 35/65 PCL / PGA copolymer was formed generally as described in Example 8. This solution was made in a flask with a magnetic stir bar. In order to completely dissolve the copolymer, it is preferred that the mixture is gently heated to 60 ± 5 ° C. and continuously stirred for a minimum of 4 hours and no more than 8 hours. Subsequently, a transparent and homogeneous solution was obtained by filtering using a dry filter with ultra-coarse porous filter (Pyrex extraction filter paper with frit disk) while filtering this viscous solution.
[0077]
Step B. freeze drying
Freezemobile 6 from VIRTIS, a laboratory scale freeze dryer, was used for this experiment. The lyophilizer was started and the shelf chamber was maintained at 20 ° C. under dry nitrogen for about 30 minutes. A thermocouple for monitoring the shelf temperature was installed and monitored. Before actually starting the processing step, the mold was carefully filled with the uniform polymer solution prepared in step A. In this experiment, a glass mold was used, which was inert to 1,4-dioxane, had good heat transfer properties, and had a surface that could easily remove the foam. If there is, a mold formed of any material can be used. The glass mold or pan used in this example weighed 620 grams, was 5.5 mm thick optical glass, and was cylindrical with an outer diameter of 21 cm and an inner diameter of 19.5 cm. Moreover, the height of the mouth of this dish was 2.5 cm. Next, the following process was performed to form a 2 mm thick foam. That is,
(I) The glass dish containing the solution was placed carefully (not tilted) on the freeze-dryer shelf maintained at 20 ° C. The treatment process was started and the shelf temperature was maintained at 20 ° C. for 30 minutes for thermal conditioning.
(Ii) The solution was then cooled to -5 ° C by cooling the shelf to -5 ° C.
(Iii) Sixty minutes after freezing at −5 ° C., vacuum was supplied to initiate primary drying of dioxane by sublimation. In order to remove most of the solvent, a primary drying treatment at -5 ° C. under vacuum under reduced pressure is required for 1 hour. At the end of this drying phase, the vacuum level is generally below about 50 millitorr (6.7 Pa).
(Iv) Next, a secondary drying process was performed in two stages under a reduced pressure of 50 mTorr (6.7 Pa) or less to remove the absorbed dioxane. In the first stage, the shelf temperature was raised to 5 ° C. and maintained at this temperature for 1 hour. The stage of the second drying process starts from the end of the first stage. In this second drying stage, the shelf temperature was raised to 20 ° C. and maintained at this temperature for about 1 hour.
(V) At the end of the second stage, the lyophilizer temperature was returned to room temperature and nitrogen was supplied to release the vacuum. Thereafter, the chamber was filled with dry nitrogen for about 30 minutes, and then the door was opened.
[0078]
The above process is suitable for making a foam having a thickness of about 2 mm or less. In addition, if it is an expert of the said technical field, various conditions described in this specification are specific, Comprising: Each operating condition is, for example, solution concentration, polymer molecular weight and composition, solution volume, molding. It will be appreciated that it will vary over a certain range due to several factors such as mold parameters, mechanical variables such as cooling rate and heating rate. FIG. 1 shows an SEM of a cross section of a foam formed according to the method described in this example. Note the irregular microstructure (not the preferred structure) in this foam.
[0079]
Example 2
Making foam with vertical flow path
In this example, the creation of a 35/65 PCL / PGA with a vertical flow path that can constitute a pathway for nutrient transfer and directed tissue regeneration is described.
[0080]
The inventor made this foam using an FTS Dura lyophilizer equipped with a computer control and data monitoring system. The first step in creating the foam is creating a uniform solution. That is, a 10% weight / weight 35/65 PCL / PGA uniform solution was prepared in the same manner as described in Step A of Example 1. The polymer solution was then carefully filled into a dish immediately before actually starting the processing step. This dish weighed 620 grams, was 5.5 mm thick optical glass, and was cylindrical with an outer diameter of 21 cm and an inner diameter of 19.5 cm. Moreover, the height of the mouth of this dish was 2.5 cm. Next, the following process was performed to form a foam having a desired structure with a thickness of 2 mm. That is,
(I) The dish containing the solution was placed on a lyophilizer shelf precooled to -17 ° C. The treatment process was started and the shelf temperature was maintained at -17 ° C for 15 minutes to quench the polymer solution.
(Ii) After 15 minutes of rapid cooling to −17 ° C., vacuum was applied to initiate primary drying of dioxane by sublimation and maintained at 100 millitorr (13.3 Pa) for 1 hour.
(Iii) Next, secondary drying was performed at 5 ° C. for 1 hour and at 20 ° C. for 1 hour. At each temperature, the vacuum level was maintained at 20 millitorr (2.7 Pa).
(Iv) At the end of the second stage, the temperature of the lyophilizer was returned to room temperature and nitrogen was supplied to release the reduced pressure. Thereafter, the chamber was filled with dry nitrogen for about 30 minutes, and then the door was opened.
[0081]
FIG. 2 is an SEM photograph showing a cross section of a foam having a vertical flow path. These channels extend through the thickness of the foam.
[0082]
Example 3
Foam with structural gradient
In this example, the preparation of a foam having a gradient in the form of foam as shown in FIG. 3 with a 10% solution of 35 / 65ε-caprolactone-co-glycolide will be described. The method for making such a foam is similar to the method described in Example 2 with one difference. That is, in the freeze-drying process (ii), the time for maintaining the solution in the freezing process is 30 minutes.
[0083]
FIG. 3 is an electron micrograph of a cross section of the foam of this example. Note the size of the pores and the shape of the pores through the thickness of the foam.
[0084]
Example 4
Composition gradient foam
In this example, a method for producing a foam having a composition gradient and not necessarily a morphological gradient will be described. Such foams can be made with polymer solutions made from a physical mixture of two or more polymers. This example illustrates a foam having a composition gradient made with 35/65 PCL / PGA and 40/60 PCL / PLA.
[0085]
Step A. Preparation of a solution mixture of 35/65 PCL / PGA and 40/60 PCL / PLA in 1,4-dioxane
In a preferred method, two separate solutions were first prepared: (a) a 10% weight / weight polymer solution of 35/65 PCL / PGA and (b) a 10% weight / weight polymer solution of 40/60 PCL / PLA. . After these solutions were made as described in Example 1, equal parts of each solution was poured into the mixing flask. In addition, about each polymer used in order to make these solutions, it describes in Example 8 and Example 9. A homogeneous solution of this physical mixture was obtained by gentle heating to 60 ± 5 ° C. and continuous stirring with a magnetic stir bar for about 2 hours.
[0086]
Step B. freeze drying
The inventor made this foam using an FTS Dura lyophilizer equipped with a computer control and data monitoring system. The first step in creating the foam is the creation of the uniform solution described in step A above. The solution was then carefully filled into a dish immediately before actually starting the processing step. This cylindrical glass dish weighed 117 grams, was made of 2.5 mm thick optical glass, and had a cylindrical shape with an outer diameter of 100 mm and an inner diameter of 95 mm. Moreover, the height of the mouth of this dish was 50 mm. Next, the following process was performed to form a 25 mm thick foam having a compositional gradient. That is,
(I) The glass dish containing this solution was placed on a shelf of a freeze dryer maintained at 20 ° C., and the condition of the solution was adjusted at 20 ° C. for 30 minutes. Next, the process step was started and the shelf temperature was set to -5 [deg.] C. by a cooling rate program of 0.5 [deg.] C./min.
(Ii) The solution was then maintained for 5 hours under freezing conditions (−5 ° C.).
(Iii) Next, vacuum was supplied to initiate primary drying of dioxane by sublimation, and maintained at 100 mTorr (13.3 Pa) for 5 hours.
(Iv) Thereafter, a secondary drying treatment was performed at 5 ° C. for 5 hours and at 20 ° C. for 10 hours. The vacuum level was maintained at 20 millitorr (2.7 Pa) at each temperature.
(V) At the end of the second stage, the lyophilizer temperature was returned to room temperature and nitrogen was supplied to release the vacuum. Thereafter, the chamber was filled with dry nitrogen for about 30 minutes, and then the door was opened. The obtained foam clearly has a gradient in chemical composition by examining the form of the foam wall as shown in FIGS. 4, 5 and 6. This gradient in chemical composition was further confirmed by NMR data as described below.
[0087]
The foam sample made by the above method had a thickness of about 25 mm and was characterized in mole percent composition. This foam sample is composed of a mixture of PCL / PLA and PCL / PGA. First, it was confirmed that the material had a gradient in composition by preparing and analyzing a slice of the foam sample. That is, these sample slices are (1) foam IA (top slice), (2) foam IB (top middle slice), (3) foam IC (bottom middle slice), and (4) foam. ID (bottom slice) was defined. Thereafter, 5 mg of each material was dissolved in 300 μL of hexafluorocesquaterium oxide (HFAD) and then 300 μL of C₆D₆NMR samples were prepared by diluting with
Table 1¹H-NMR result: mol% composition
Sample ID PLA PGA PCL
Foam IA 47.2 12.4 40.5
Foam IB 12.3 51.3 36.5
Foam IC 7.7 56.5 35.8
Foam ID 7.8 56.3 35.8
[0088]
From the above NMR results, it can be seen that these foam samples have a gradient in composition. That is, the foam top layer has a high PLA concentration (47 mol%), while the foam bottom layer has a high PGA concentration (56 mol%). From these results, it is considered that during the freezing process, the PCL / PGA copolymer and the PCL / PLA copolymer have different phase separation characteristics, so that a foam having a specific composition gradient was formed.
[0089]
Example 5
Foam having a structural gradient
In this example, a method of making a foam having a compositional and structural gradient and not necessarily a morphological gradient will be described. Such foams can be made with polymer solutions made from a physical mixture of two or more polymers. This example was made with 35/65 PCL / PLA (as described in Example 9) and 95/5 PLA / PCL (a random copolymer having an IV of 1.8 in the HFIP measurement as described herein). The foam having the composition gradient will be described. 35 / 65PCL / PLA is a soft elastomer copolymer, and 95 / 5PLA / PCL is a relatively rigid copolymer. Compositional and structural gradients can be constructed by combining these two types of copolymers. This foam can be made by the steps described in Example 4; first, a 10% weight / weight solution of 35/65 PCL / PLA in 1,4-dioxane and 10% weight / weight of 95/5 PLA / PCL. We began with the creation of a uniform 50/50 physical mixture of heavy solutions. Foams having such compositional gradients can provide a good template for tissue junctions such as bone-cartilage interfaces.
[0090]
Example 6
Cell culture and differentiation data
Films made with 95/5 PLA / PGA, 90/10 PGA / PLA, 95/5 PLA / PCL, 75/25 PGA / PCL and 40/60 PCL / PLA were tested. Tissue culture polystyrene (TCPS) was used as a positive control in all assays. Prior to testing, each polymer disc was pre-humidified for 20 minutes at the bottom of a 24-well ultra low cluster dish.
[0091]
The 95/5 PLA / PGA copolymer used in this example is a random copolymer having an IV of 1.76 when measured in HFIP at 25 ° C. and used in the Panacryl ™ suture (Ethicon, Somerville, NJ). Has been. The 90/10 PGA / PLA copolymer is also a random copolymer with an IV of 1.74 when measured in HFIP at 25 ° C. and is used in the Vicyl ™ suture (Ethicon, Somerville, NJ). The 95/5 PLA / PCL polymer was made as described in Example 10 and has an IV of 2.1 when measured in HFIP at 25 ° C. Also, 75/25 PGA / PCL copolymer is a segment block copolymer having an IV of 1.85 and is described in US Pat. No. 5,133,739. This copolymer is used in the Monocril ™ suture (Ethicon, Somerville, NJ). In addition, a 40/60 PCL / PLA copolymer was made as described in Example 9 and has an IV of 1.44.
[0092]
Cell attachment and proliferation
Cells were seeded at 40,000 cells / well in 24-well ultra low cluster dishes (Corning) containing each polymer. The ultra-low cluster dish is coated with a layer of hydrogel polymer that retards protein and cell attachment to the well. Thus, the adherence of cells to each biopolymer was determined after 24 hours of culture (N = 3 for each polymer). The attached cells were released by trypsinization, and the number of the cells was determined by a hemocytometer. Furthermore, cell proliferation was evaluated by counting the number of cells on the 3rd and 6th day after inoculation.
[0093]
Proliferation assay
Alkaline phosphatase activity
Alkaline phosphatase activity was determined by colorimetric assay with p-nitrophenol phosphate substrate (Sigma 104) and manufacturer's instructions. That is, cells were seeded on a film or mesh at a density of 40,000 cells / well and cultured for 1, 6, 14, and 21 days. On the sixth day, when the cells were confluent, they were fed mineralization medium (culture medium supplemented with 10 mM β-glycerophosphate, 50 μg / ml ascorbic acid). Alkaline phosphatase activity was then determined in cell homogenates (0.05%, Triton X-100) at each of the above times. The amount of protein in the cell extract was determined with Pierce's micro BCA reagent. In addition, primary rat osteoblasts cultured on films and meshes were stained for membrane-bound alkaline phosphatase using a histological staining kit (Sigma). Three samples for each group were tested for all films and meshes.
[0094]
Osteocalcin ELISA
Osteocalcin secreted into the medium by osteoblasts cultured on various films was quantified by ELISA (Osteocalcin ELISA kit, Biomedical Technologies, Boston). Prior to measuring this protein by ELISA, aliquots of media from the wells containing each polymer film were lyophilized. Three samples were tested for each polymer and the ELISA was repeated twice.
[0095]
Von Kossa ( Von Kossa) staining
Three samples for each polymer were stained on tissue mineralized by von Kossa silver nitrate staining.
[0096]
Expression of alkaline phosphatase and osteocalcin mRNA
Intracellular expression of alkaline phosphatase and osteocalcin mRNA was evaluated by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) using RNA extracted from cells cultured for 21 days on film. Seven days after inoculation, the culture medium was replaced with mineralized medium (3 mM β-glycerophosphate and 50 μg / ml ascorbic acid were added). The cells were further cultured for 2 weeks for a total of 3 weeks. Total RNA was extracted from 4 samples per group using the RNeasy mini kit provided by Qiagen. The nature and amount of total RNA was measured for each polymer group. First, reverse transcription was performed to reverse-transcribe the entire RNA (Superscript II, Gibco) to obtain cDNA. Next, the cDNA corresponding to osteocalcin, alkaline phosphatase, and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was amplified by the PCR protocol already described (GIBCO BRL manufacturer's instructions). Primer sequences (Table 2) corresponding to osteocalcin, alkaline phosphatase, and GAPDH were obtained by the FASTA program (Genetic Computer Group, Madison, Wis.). Preliminary studies were also performed to optimize the number of PCR cycles for each primer (Table 3) to determine the range of RNAs that showed contrast to the cDNA. These PCR products were electrophoresed on a 1% (weight) agarose gel containing ethidium bromide. These gels were then photographed under UV light and the expression of osteocalcin and alkaline phosphatase mRNA against GAPDH was evaluated by densitometry.
[0097]
Statistical analysis
The degree of significant difference was evaluated for all quantitative tests using analysis of variance (ANOVA) with post hoc post-hoc comparison (post hoc).

Table 3 PCR optimization cycle
Gene cDNA (μl) Number of cycles
Alkaline phosphatase 125
Osteocalcin 1 35
GAPDH 1 23
[0098]
result
Cell attachment and proliferation in bioabsorbable polymers
There was no significant difference in cell morphology between the various polymers and compared to TCPS. Cell adhesion to various biopolymer films after 24 hours of culture was equivalent to TCPS. On day 3, the cells grew well in all films except 40/60 PCL / PLA. In the case of 40/60 PCL / PLA, the proliferation was 60% compared to TCPS. Furthermore, 95/5 PLA / PGA and 90/10 PGA / PLA films supported a higher degree of cell proliferation with a significant difference (p <0.05) compared to TCPS and 40/60 PCL / PLA (FIG. 7).
[0099]
Differentiation assay
Alkaline phosphatase enzyme activity
The profile of alkaline phosphatase activity expressed by osteoblasts cultured on 95/5 PLA / PGA, 90/10 PGA / PLA and 95/5 PLA / PCL films was similar to that seen in TCPS. On the other hand, in the case of osteoblasts cultured in the 40/60 PCL / PLA and 75/25 PGA / PCL films on the 14th and 21st days, the alkaline phosphatase specific activity is significantly different from that of the other films and TCPS, respectively. (P <0.05) increased (FIG. 8).
[0100]
Expression of alkaline phosphatase and osteocalcin mRNA
Expression of mRNA in alkaline phosphatase, osteocalcin, and GAPDH on osteoblasts cultured in 95/5 PLA / PGA, 40/60 PLA / PCL, 95/5 PLA / PCL film and TCPS was assessed by densitometry. These results are shown in FIG. Note that the data in FIG. 8 is the data in the best semi-quantitative analysis. However, these data indicate that the 40/60 PCL / PLA film supported a higher degree of osteocalcin expression with a significant difference (p <0.05) compared to TCPS. On the other hand, the remaining polymer surface was comparable to TCPS in both osteocalcin and alkaline phosphatase mRNA expression.
[0101]
Conclusion
There was no significant difference in cell attachment and proliferation between the different bioabsorbable films or meshes tested following 6 days of culture. Furthermore, these results indicated that the differences between each material with respect to each differentiation property were relatively clear. That is, cell culture on 40/60 PCL / PLA film showed improved alkaline phosphatase activity and osteocalcin mRNA expression compared to another film after 14 days and 21 days of culture and TCPS, respectively.
[0102]
To further understand this technique, MA Aronow, LC Gerstenfeld, TA Owen, MS Tassinari, GS Stein, and JB Lian “facilitating the progressive development of the osteoblast phenotype in cultured fetal rat calvarial cells. Factors that promote progressive development of the osteoblast phenotype in cultured fetal rat calvaria cells (Journal of Cellular Physiology, 143: 213 to 221 (1990)), and Stein, GS, Lian, JB and Owen, TA, “Relationship of cell growth to the regulation of tissue-specific gene expression during osteoblast differentiation” (FASEB, 4, No. 4) 3111 to 3123 (1990)) are cited.
[0103]
Example 7
In vivo study of foam blends in a porcine skin wound healing model
In this example, the results of implanting 1 mm and 0.5 mm thick foam skeleton-forming materials in a porcine full thickness excision wound model will be described. A foam tissue skeleton-forming material was made with a mixture of 40 / 60ε-caprolactone-co-lactide prepared as described in Example 8 and 35 / 65ε-caprolactone-co-glycolide prepared as described in Example 9. did. That is, these polymers were mixed and almost the same as those described in Example 3 (except that the cooling rate was only 2.5 ° C./min and −5 ° C.) 1 mm and 0.5 mm thick A foam was formed. Scanning electron micrographs of the 0.5 mm foam are shown in FIGS. 11 (A), 11 (B), and 12. FIG. These four different samples were then evaluated in an ablation model in which two thicknesses (0.5 mm and 1 mm) of foam were injured, with and without the supply of PDGF.
[0104]
Random histological evaluation was performed on 48 full thickness excision wounds (12 sites per animal) in 4 pigs transplanted 8 days after formation of the wound. This evaluation was performed on slides stained with H & E. During this histological evaluation, the following parameters were ranked and / or evaluated in each sample. Qualitative and quantitative evaluation of substrate cell infiltration, (2) Infiltration of polymorphonuclear leukocytes (PMN) into the contact area (ventral surface) of the substrate, (3) Substrate (from the ventral side) ) Inflammation in the lower granulation tissue bed, (4) epidermal response to substrate, and (5) degree of substrate fragmentation.
[0105]
Animal body management
Each pig was individually housed in a cage (having a minimum floor area of 10 square feet) and was tagged. All pigs were assigned individual animal body numbers. That is, a tag describing the animal body number, type / lineage, date of surgery, surgical technique and duration of experiment, and date of euthanasia was attached to each animal body cage. Each animal body was marked with an animal number at the base of its neck with a permanent marker.
[0106]
Each animal room was maintained at 40% to 70% relative humidity and 15 ° C to 24 ° C (59.0 to 75.2 ° F). Each animal received a standard pig meal once a day, but no overnight meal prior to any experimental treatment requiring anesthesia treatment. Water was made available freely. The daily brightness: dark cycle was 12:12 hours.
[0107]
Anesthesia treatment
Tiedamine HCL plus Zolazepam HCL (Telazol®, Fort Dodge Animal Health, Fort Dodge, Iowa, 4 mg / day on the first day of the experiment, on the day of each evaluation and on the day of necropsy ml) and Xylazine (Rompun®, Agriculture Department, Bayer Corporation, Shawnee Mission, Kansas, Animal Health, 4 mg / ml) or Isoflurane (AErrane®, Fort Iowa) administered via the nasal head Anesthesia treatment with one of the inhalation anesthetics (5% volume) by Dodge Fort Dodge Animal Health. When a surgical operation was performed on an animal body, the animal body was maintained under an inhalation anisoflurane (AErrane (registered trademark)) inhalation anesthesia via the nasal head. A meal was also given after recovery from each treatment.
[0108]
Creating a surgical site
One day prior to the surgical procedure, weighed and trimmed the dorsal area of four pigs with an electric clipper equipped with a # 40 surgical shaving blade. Thereafter, the cut skin was further shaved with a shaving cream and a razor and rinsed. This shaved skin and whole animal (except the head) is further treated with a PCMX cleaning solution (Pharmaseal® Scrub Care®, Pharmaseal Department, Baxter Healthcare, Valencia, Calif.) With a surgical scrub brush After scrubbing with a sponge, it was further scrubbed with HIBICLENS® chlorhexidine gluconate (obtained from COE Laboratories, Chicago, Ill.). The animal body was wiped dry with a sterilized towel. Then place a sterile NU-GAUZE gauze (obtained from Johnson & Johnson Medical, Arlington, Texas) on the back of each animal, and WATERPROOF^★Fixed with tape (obtained from Johnson & Johnson Medical, Arlington, Texas). The entire torso of the animal body was wrapped with a Spandage ™ elastic stretch bandage (obtained from MediTech International, Brooklyn, NY) and kept on a clean surface overnight.
[0109]
On the day of surgery, just prior to moving the animal to the surgical site, the skin on the dorsal surface of the dorsal skin was treated with PCMX lavage solution (Pharmaseal®, Scrub Care®) as was done the previous day. It was rubbed again with a brush sponge, rinsed and wiped dry with a sterilized towel. The animal body was placed on the operating table, and wiped with 70% alcohol, and then dried with sterile gauze. Marking the dorsal skin with a sterile surgical marker (obtained from Codman, a division of Johnson & Johnson Professional, Raynham, Mass.) And acetate template depending on the desired location of each full thickness wound did.
[0110]
Surgical treatment
After anesthesia, 12 full-thickness resections (1.5 cm × 1.5 cm) were created in each animal body in two rows parallel to the spinal column on the left and right dorsal regions using a scalpel under sterile conditions. . In addition, a pair of scissors and / or scalpels were used for skin and subcutaneous tissue removal. Bleeding was adjusted with a sponge tampon. A sufficient space was placed between the scratches to avoid interference between the scratches. The thickness of the excised tissue was measured with a digital caliper.
[0111]
Treatment and bandage supply
Each wound was treated with a pre-coded coded treatment procedure (each experimental subject was blinded to all treatments). A primary bandage consisting of sterilized individual test articles (soaked in 1.5 cm x 1.5 cm sterile saline solution for 24 hours) was placed in the wound defect according to a predetermined plan. Next, non-adhesive, square RELEASE soaked in saline solution^★A secondary bandage consisting of a bandage (manufactured by Johnson & Johnson Medical, Arlington, Texas) was placed on top of the test article. In addition, BIOCLUSIVE^★ The wound was sealed with a bandage layer (obtained from Johnson & Johnson Medical, Arlington, Texas) to maintain the wound's moisture and retain the internal bandage. In addition, Reston ™ (3M Medical-Surgical Department of St. Paul, Minn.) Polyurethane self-adhesive foam strips are placed between each wound to prevent cross-contamination due to wound fluid leakage. Prevented wound damage and bandage slippage. In addition, NU-GAUZE^★ Layer of BIOCLUSIVE^★ Place on top of bandages and Reston (TM) foam, WATERPROOF^★ Each bandage was protected with tape. Thereafter, the animal body was bandaged with a Spandage ™ elastic net to hold each bandage.
[0112]
RELEASE soaked in saline solution with daily removal of the above secondary dressing^★Replaced with a new secondary bandage. The primary bandage (test article) was not touched until the unit was replaced or pushed out of the wound defect.
[0113]
Postoperative medical management and clinical observation
After surgical treatment in anesthesia, the animals were returned to their respective cages for recovery. Each animal was given analgesic (baprenorphine hydrochloride (Buprenex Injectable, 0.01 mg / kg) sold by Reckitt & Colman, Hull, UK) immediately after surgery and the next day. After recovery from anesthesia, each pig was observed for behavioral signs of deep or pain. As a result, there were no signs of pain.
[0114]
Each pig was observed twice daily after the day of surgery to determine its health based on general behavior, appearance, food consumption, urination and defecation, and the presence of abnormal discharge.
[0115]
Euthanasia
At the end of the experiment (8 days after wounding), Socumb ™ pentobarbital sodium and phenytoin sodium euthanasia solution (sold by the company Butler of Columbus, Ohio) (1 ml / 10) Each animal body was euthanized by anesthesia with an intravenous injection of pound body weight)). Following this euthanasia treatment, the animals were observed to confirm that there was no respiratory function and no palpable heart function. At this time, the stethoscope facilitates confirmation of cardiac function stoppage.
[0116]
Tissue collection
Immediately following euthanasia, each wound was excised with underlying fat and skin surrounding a small portion. The tissue was placed in 10% neutral buffered formalin.
[0117]
Evaluation
Visual wound assessment
General observations were recorded for days 1-3, including misalignment, wound response and physical properties of the skeleton-forming material. In addition, a detailed clinical evaluation was performed on days 4-8 after injury. The following parameters were recorded for the presence / absence (presence = 1 / non-existence = 0) and / or degree (score).
[0118]
Bandage condition
Air exposure, misalignment of test article, channeling, communication) and RELEASE^★ Moisture content of the secondary dressing (scoring criteria: 4 = wet, 3 = wet / dry, 2 = dry / wet, 1 = dry).
[0119]
Wound bed condition
Moisture content of test article (scoring criteria: 4 = moist, 3 = wet / dry, 2 = dry / moist, 1 = dry), inflammation (score) Rating criteria: 3 = severe, 2 = moderate, 1 = mild, 0 = none), re-injury (scoring criteria: 3 = severe, 2 = moderate, 1 = mild, 0 = none), blood clot, folliculitis , Infection, test article height (scoring criteria: 4 = extremely rising, 3 = rising, 2 = no change, 1 = falling), fibrin (scoring criteria: 3 = severe 2 = moderate, 1 = mild, 0 = none), and erythema. The color of the test article was also observed.
[0120]
Excised tissue samples were taken on day 8. Whole wounds were collected and placed in 10% neutral buffered formalin. Thereafter, the tissue was prepared in a frozen part. The tissue was cut and placed on an object holder with Tissue-Tek® OCT compound (sold by Sakura Finetechnical, Tokyo, Japan) and quickly frozen. These samples were stained with a frozen H & E stain cut to 10 μm on a cryostat.
[0121]
Histological evaluation (8 days after injury)
The histological evaluation of granulation tissue (area and length) and epidermis formation was evaluated by H & E stained samples at 20 to 40 times magnification. The granulation tissue height was determined by dividing the area by the length.
[0122]
Furthermore, histopathological evaluation of tissue samples was performed with H & E stained samples, and these samples were first evaluated at a magnification of 100 to 400 times.
[0123]
result
Cell infiltration was seen in the interstices of the matrix in most of all sites. In most of these sites, this invasion is a true tissue ingrowth composed of a changing population of fibroblasts, macrophages, macrophage giant cells, and endothelial-like cells, and appears to have formed capillaries. It seems to be. Substantial formation of a dense fibrillar connective tissue layer on the dorsal side with respect to the substrate that substantially fills the substrate in the tissue is seen at several sites in the 0.5 mm foam with and without PDGF . Also, the 1 mm substrate was on the surface of the tissue or was missing. Macrophage giant cell formation was seen more in 0.5 mm vs. 1 mm foam scaffold. At the site where the 1 mm foam had fallen off or partially separated from the underlying granulation tissue, dead infiltrating cells formed a nucleus-enriched cell lump mass.
[0124]
In the case of the 0.5 mm foam skeleton-forming material, complete incorporation of the substrate into the granulation tissue bed was seen at several sites. Figures 13 and 14 show the uptake of these substrates into the granulation tissue bed. FIG. 13 is a 40 × dark field photomicrograph of a tissue sample stained with trichrome. FIG. 14 is a 100-fold composite photomicrograph of a trichrome-stained sample showing cell infiltration of a foam containing PDGF. In both these photographs, complete uptake of the substrate into the granulation tissue bed is clearly visible. Moreover, the presence of high-density fibrous tissue on the foam skeleton-forming material is clearly seen in both these photographs. Due to their presence, 0.5 mm foam was found to constitute a suitable substrate in the growth of epidermal tissue.
[0125]
Example 8
Synthesis of random poly (ε-caprolactone-co-glycolide)
A random copolymer of ε-caprolactone-glycolide having a molar composition of 35/65 was synthesized by a ring-opening polymerization reaction. This synthesis is similar to that described in US Pat. No. 5,468,253 in Example 6 (included herein by reference). The amount of diethylene glycol initiator added was adjusted to 1.15 mmol / mol of monomer to give a dry polymer with the following characteristics. That is, the intrinsic viscosity (IV) of this copolymer was 1.59 dL / g in hexafluoroisopropanol at 25 ° C. The PCL / PGA molar ratio was 35.5 / 64.5 by proton NMR and contained 0.5% residual monomer. Further, the glass transition point (Tg) and melting point (Tm) of this copolymer were found to be -1 ° C, 60 ° C and 126 ° C by DSC.
[0126]
Example 9
Synthesis of 40:60 poly (ε-caprolactone-co-L-lactide) by continuous addition
In a glove box, 100 μL (33 μmol) of 0.33 mol of tin (II) octoate in toluene, 115 μL (1.2 mmol) of diethylene glycol, 24.6 grams (170 mmol) of L-lactide, and 45. Seven grams (400 mmol) of ε-caprolactone was placed in a silane-treated flame-dried two-necked 250 mL round bottom flask equipped with a stainless steel mechanical stirrer and a nitrogen gas blanket. The reaction flask was placed and held in an oil bath already set at 190 ° C. Further, in this glove box, 62.0 g (430 mmol) of L-lactide was placed in a dropping funnel which was flame-dried and made equal pressure. The funnel was wrapped with heat tape and attached to the second neck of the reaction flask. After 6 hours at 190 ° C., molten L-lactide was added into the reaction flask over 5 minutes. The reaction was continued overnight at 190 ° C. for a total of 24 hours, after which the reaction was allowed to cool to room temperature. The copolymer was then isolated by freezing in liquid nitrogen and breaking the glass. Residual glass fragments were removed from the copolymer with a bench grinder. The copolymer was frozen again with liquid nitrogen and the mechanical stirrer paddle was removed. Further, the copolymer was shaved with a Wiley Mill (fine pulverizer), and the tare was put into a light glass wide-mouth bottle, and heated to room temperature overnight in a vacuum oven. Thereafter, 103.13 grams of 40:60 poly (ε-caprolactone-co-L-lactide) was placed in a lightly tared aluminum pan and vacuum treated at 110 ° C. for 54 hours to remove volatiles. As a result, 98.7 grams (95.7% by weight) of copolymer was recovered after the deaeration treatment. As a result of measuring the intrinsic viscosity, CHCl at 25 ° C._ThreeIt was 1.1 dL / g in the inside (concentration = 0.1 g / dL). FTIR (CHCl over KBr window_ThreeCast film from, cm^-1): 2993, 2944, 2868, 1759, 1456, 1383, 1362, 1184, 1132, 1094, 870, and 756.¹H-NMR (400 MHz, HFAD / benzene, ppm): δ1.25, two broad lines (e); 1.35, two lines (f); 1.42, three lines; 1.55, 2 lines; 2.22, 3 lines; 2.35, 4 wide lines; 4.01, 3 lines; 4.05, 3 lines; 4.2, quadruple lines; 5.05, 3 wide lines Line; 5.15, 4 lines.¹Polymer composition by H-NMR: 41.8% PCL, 57.5% PLA, 0.8% L-lactide, <0.1% ε-caprolactone. DSC (20 ° C./min, first heat): T_m= 154.8 ° C., ΔH_m= 18.3 J / g. GPC (molecular weight determined in THF using poly (methyl methacrylate) standard): M_w= 160,000, M_n= 101,000, PDI = 1.6.
[0127]
Example 10
Synthesis of 95/5 PLA / PCL copolymer
In the glove box, 170 [mu] L (1.8 mmol) diethylene glycol, 350 [mu] L (115 [mu] mol) 0.33 mol stannous octoate in toluene, 17.1 grams (150 mmol) [epsilon] -caprolactone, and 410.4 Gram (2.85 moles) of L-lactide was placed in a 1000 mL round bottom flask that had been silanized and flame dried. The flask is equipped with a stainless steel mechanical stirrer and a decompression release connector for holding a dry nitrogen gas blanket. The reaction flask was placed in an oil bath already heated to 185 ° C. and held for 3 hours. Thereafter, the flask was removed from the oil bath and allowed to cool to room temperature. The polymer was isolated by wrapping the flask with aluminum foil and freezing it in liquid nitrogen, and the glass adhering to the polymer was scraped off. The copolymer was then shaved in a Wiley mill and the shaved polymer was vacuum dried at 80 ° C. for 24 hours to recover 302 grams of copolymer. This intrinsic viscosity was 2.1 dL / g in chloroform (25 ° C., concentration = 0.1 g / dL). The polymer composition was measured by proton NMR spectroscopy and found to be 97.2 mol% PLA and 2.8 mol% PCL. No residual monomer was detected.
[0128]
Example 11
Synthesis of 60/40 PLA / PCL hybrid foam / microfiber fabric structure
Step A. Preparation of a 14% weight / weight homogeneous trichloroethane solution of 60/40 PLA / PCL
A 14% weight / weight polymer solution was prepared by dissolving 14 parts 60/40 PLA / PCL (IV = 1.34 dl / g) in 86 parts trichloroethane solvent (TCE). This solution was prepared in a flask equipped with a magnetic stir bar. It is recommended to stir the mixture overnight at 60 ° C. to completely dissolve the small polymer.
[0129]
Step B. Electrostatic spinning
An electrostatic spinner was used for this experiment. Spellman High Voltage DC Supply (Model Number: RHR30PN30, Spellman High Voltage Electronics Corporation, USA, Hauppauge, NY, USA) Using. The applied voltage and mandrel speed were adjusted with Love View computer software. The distance between the spinneret was adjusted mechanically.
[0130]
A flat sheet of lyophilized 60/40 PLA / PCL foam prepared as described in Example 1 was rolled into a cylinder. Both ends were fixed with a few drops of trichloroethane, the solvent used in spinning. The foam tube was mounted on a rotating grounded conductive mandrel. Both ends of the mandrel not covered with the foam substrate were masked with an insulating tape to prevent the microfibers from being attracted to both ends. The solution was placed in a spinneret and a high voltage was applied to the polymer solution.
This experiment was performed at room temperature and ambient humidity.
[0131]
The processing conditions during spinning were as follows:
Mandrel voltage 16,000V
Mandrel speed 100rpm
Distance between spinneret and mandrel 10 cm
Temperature room temperature
Humidity Ambient humidity
[0132]
A layer approximately 200 μm thick was deposited on the other side of the tube.
[0133]
Example 12
Chondrocytes cultured in encapsulated hybrid foam / textile structure show limited cell migration
Step A. Preparation of hybrid 60/40 PLA / PCL foam / micro warfare fabric skeleton forming material
The lyophilized foam made from 60/40 PLA / PCL described in Example 1 was subjected to Example 1 during electrospinning of 60/40 PLA / PCL in trichloroethane as described in detail in Example 11. A freeze-dried foam made from 60/40 PLA / PCL described in 1 was used as the substrate.
[0134]
Step B. Seeding of chondrocytes into hybrid 60/40 PLA / PCL foam / microfiber fabric skeleton-forming material
Chondrocytes isolated from bovine shoulders were transferred to Dulbecco's modified Eagle medium (high glucose) with 10% fetal bovine serum, 10 mM HEPES, 0.1 mM non-essential amino acids, 20 μg / ml streptomycin and 0.25 μg / ml amphotericin Cultured in supplemented with B.
[0135]
3D movement test
This assay is based on a contracted collagen lattice implanted with cells with a biodegradable polymer skeleton-forming material embedded in the center of the collagen gel. A skin equivalent (collagen lattice) was made from the following mixture: 8 ml of chondrocyte medium, 2 ml of neutralization medium (DMEM / 10% FBS containing 0.1 N NaOH, prepared before use), Collaborative Biomedical Products (Collaborative Biomedical) Products) 4 ml of rat tail type I collagen (3.99 mg / ml) obtained from the company, and 2 ml of chondrocyte suspension suspended in chondrocyte medium. The cell / collagen mixture was dispensed (2 ml) into each well of a 24-well hydrogel-coated plate (Costar, catalog # 3473). Final cell concentration is 6 x 10⁶Pieces / ml, and the collagen concentration was 1 mg / ml. The collagen gel was polymerized at 37 ° C. for 1 hour. Following gel polymerization, 1.5 ml of chondrocyte growth medium was applied to each well. This medium was changed every other day. After shrinking for 7 days, a disposable biopsy punch was used to create a 5 mm diameter wound throughout the collagen lattice thickness. The test skeleton-forming material, approximately 5 mm in diameter and 2 mm thick, supplied in wet form, was implanted in the center of the damaged collagen lattice. 50 μl of non-cellular collagen was placed on top of each construct to ensure that the scaffolding material was not pushed out of the wound bed while the collagen lattice contracted. These constructs were cultured in chondrocyte medium, and the medium was changed every two days. Shrinked gels containing skeletal material were cultured in an ultra-low cluster petri dish at rest or in a bioreactor and harvested after 3 weeks.
[0136]
Step C. Characterization of cells in skeletal materials
Histology was applied to the skeleton-forming material infiltrated with cells. Skeletal material is fixed in 10% buffered formalin and embedded in paraffin to create sections (hybrid foam / microfiber woven skeleton material) or frozen sections (control foam skeleton material) ). Sections were stained with hematoxylin / eosin (H / E, cell number and morphology), safranin O (SO; sulfated GAGs) and trichrome (collagen). Quantification of cell infiltration into these skeletal materials was determined by image analysis. Tukey post-test ANOVA was performed on the data.
[0137]
Experimental result
Quantitative evaluation of H / E sections of constructs cultured for 3 weeks revealed that chondrocytes infiltrated both skeleton-forming materials. Cell infiltration into the hybrid foam / microfiber fabric skeleton-forming material cultured in the bioreactor was significantly higher than that into the 40/60 PCL / PLA foam skeleton-forming material (P <0.05). Further, in the culture kept stationary, the infiltration of cells into the hybrid foam / microfiber fabric skeleton-forming material was significantly higher than the infiltration into the 40/60 PCL / PLA foam skeleton-forming material (P <0). .05).
[0138]
Cell infiltration hybrid
Cells / mm²      40 / 60PCL / PLA Foam / Microfiber
(3 weeks) Foam skeleton forming material Woven skeleton forming material
Resting cell culture 50 80
Bioreactor 140 200
Cell culture
[0139]
The hybrid foam / microfiber fabric skeleton-forming material showed the highest cell density. The present inventor presumes that the cell-cell interaction is higher in the case of the hybrid foam / microfiber fabric skeleton-forming material than the other skeleton-forming material, but this cell-cell interaction is selective. Suppose that it may be possible to contribute to chondrocyte proliferation and differentiated function for hybrid foam / microfibre woven scaffolding material. It is hypothesized that this cell-cell interaction plays an important role in maintaining the chondrocyte phenotype. Chondrocytes are known to differentiate when deployed into a single layer of cells. When cultured at a high cell density in a monolayer, chondrocytes maintain their phenotype as shown by the expression of type II collagen and aggrecan.
[0140]
By 6 weeks, the 40/60 PCL / PLA foam skeleton-forming material exhibited a cartilage-like morphology / property localized primarily in capsules around the construct. In contrast, in the hybrid foam / microfiber woven fabric skeleton, the capsule was very thin, the cell morphology in the construct was cartilage-like, and a more uniform cell distribution was observed.
[0141]
  Embodiments of the present invention are as follows.
(A) a first bio-acceptable filamentous layer and a second bio-acceptable foam layer attached thereto, the bio-acceptable foam having a gradient structure and a flow path; A bio-acceptable composite selected from the group consisting of foams, the foam comprising the gradient structure having a first part and a second part, from the first part to the second At least one property selected from the group consisting of composition, rigidity, flexibility, bioabsorption rate, and porous structure is substantially continuously changed over the portion, and the foam including the flow path is the first one. A bio-acceptable composite having one surface portion and a second surface portion having a flow path.
  (1) The biologically acceptable complex is bioabsorbableEmbodiment (A)The biotolerable complex according to 1.
  (2) The bioacceptable filamentous layer is bioabsorbableEmbodiment (A)The biotolerable complex according to 1.
  (3) The bioacceptable complex is an aliphatic polyester, poly (amino acid), copoly (ether-ester), polyalkylene oxalate, polyamide, poly (imino carbonate), polyorthoester, polyoxaester, polyamide ester Formed by a bioabsorbable polymer selected from the group consisting of: polyoxaesters containing amine groups, poly (anhydrides), polyphosphazenes, biopolymers and mixtures thereofEmbodiment (A)The biotolerable complex according to 1.
  (4) The bioacceptable complex according to embodiment (3), wherein the bioabsorbable polymer is an aliphatic polyester.
  (5) The aliphatic polyester is lactide, glycolide, glycolic acid, ε-caprolactone, p-dioxanone (1,4-dioxane-2-one), trimethylene carbonate (1,3-dioxane-2-one), tri Alkyl derivatives of methylene carbonate, δ-valerolactone, β-butyrolactone, γ-butyrolactone, ε-decalactone, hydroxybutyrate, hydroxyvalerate, 1,4-dioxepan-2-one, 1,5,8,12-tetraOxaEmbodiments selected from the group consisting of homopolymers and copolymers of cyclotetradecane-7,14-dione, 1,5-dioxepan-2-one, 6,6-dimethyl-dioxepan-2-one, and mixtures thereof (4 ).
[0142]
(6) The bioacceptable composite according to embodiment (4), wherein the aliphatic polyester is an elastomer.
(7) The elastomer is a copolymer of ε-caprolactone and glycolide, a copolymer of ε-caprolactone and (L) lactide, a copolymer of p-dioxanone (1,4-dioxane-2-one) and (L) lactide, ε-caprolactone And an embodiment selected from the group consisting of copolymers of p-dioxanone, copolymers of p-dioxanone and trimethylene carbonate, copolymers of trimethylene carbonate and glycolide, copolymers of trimethylene carbonate and (L) lactide, and mixtures thereof ( The biotolerable complex according to 6).
(8) The bioacceptable composite according to embodiment (4), wherein a second aliphatic polyester is present as a component of the bioacceptable foam.
(9) The bioacceptable composite according to embodiment (4), wherein a second aliphatic polyester is present as a constituent of the bioacceptable filamentous layer.
(10) The biocompatible composition according to the embodiment (3), wherein the foam having the biocompatible gradient structure transitions substantially continuously in composition from the first portion to the second portion. Complex.
[0143]
(11) The foam having the bio-acceptable gradient structure is composed of the at least two different aliphatics from the first ratio of at least two different aliphatic polyesters in the composition from the first surface part to the second surface part. The bio-acceptable composite according to embodiment (10), which transitions substantially continuously to a second ratio of polyester.
(12) The bio-acceptable body according to the embodiment (3), wherein the foam having the bio-acceptable gradient structure is substantially continuously changed in rigidity from the first part to the second part. Complex.
(13) The bio-acceptable according to the embodiment (3), wherein the foam having the bio-acceptable gradient structure transitions substantially continuously in the bioabsorption rate from the first part to the second part. Sex complex.
(14) The bio-acceptability according to the embodiment (3), wherein the foam having the bio-acceptable gradient structure is substantially continuously transitioned in flexibility from the first part to the second part. Complex.
(15) The bio-acceptable body according to the embodiment (3), wherein the foam having the bio-acceptable gradient structure transitions substantially continuously in the structure from the first part to the second part. Complex.
[0144]
  (16) The foam having the bio-acceptable gradient structure has a substantially spherical porous shape in the structure from the first part to the second part.ColumnarThe bio-acceptable complex according to embodiment (15), wherein the composite is substantially continuously transitioned to the porous shape.
  (17) The bio-acceptable complex according to embodiment (15), wherein the size of the substantially spherical pore is about 30 μm to about 150 μm.
  (18) The bio-acceptable complex according to embodiment (15), wherein the columnar pores have a diameter of about 100 μm to about 400 μm, and a ratio of length to diameter is at least 2.
  (19) Furthermore, a therapeutic agent is present in the foam having the biotolerable gradient structure.Embodiment (A)The biotolerable complex according to 1.
  (20) The bioacceptable foam is from the group consisting of an anti-infective agent, hormone, analgesic agent, anti-inflammatory agent, growth factor, chemotherapeutic agent, anti-rejection agent, prostaglandin, RDG peptide, and mixtures thereof. Contains further selected drugEmbodiment (A)Described inLiving bodyTolerable complex.
[0145]
  (21) The growth factor is a bone morphogenesis-like protein, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-derived growth factor, transforming growth factor, vascular endothelial growth factor and a mixture thereof The bio-acceptable complex according to aspect (20).
  (22) The biocompatible material wherein the biocompatible foam is selected from the group consisting of a bioabsorbable synthetic polymer, a biocompatible bioabsorbable biopolymer, a biocompatible ceramic material, and a mixture thereof. Filled withEmbodiment (A)The biotolerable complex according to 1.
  (23) The foam including the flow path has a flow path having an average length of at least 200 μm.Embodiment (A)The biotolerable complex according to 1.
  (24) The bio-acceptable complex according to embodiment (23), wherein the flow path substantially extends from the first surface portion to the second surface portion.
  (25) comprising a foam having internally connected vents formed of a composition containing from about 30% to about 99% by weight of ε-caprolactone repeating unitsEmbodiment (A)The biotolerable complex according to 1.
[0146]
  (26) The ε-caprolactone repeating unit is lactide, glycolide, glycolic acid, ε-caprolactone, p-dioxanone (1,4-dioxane-2-one), trimethylene carbonate (1,3-dioxan-2-one) , Alkyl derivatives of trimethylene carbonate, δ-valerolactone, β-butyrolactone, γ-butyrolactone, ε-decalactone, hydroxybutyrate, hydroxyvalerate, 1,4-dioxepan-2-one, 1,5,8,12 -TetraOxaEmbodiments copolymerized with a comonomer selected from the group consisting of cyclotetradecane-7,14-dione, 1,5-dioxepan-2-one, 6,6-dimethyl-dioxepan-2-one, and mixtures thereof The biotolerable complex according to (25).
  (27) having a first part and a second part, wherein the bioacceptable foam has a composition, rigidity, from the first part to the second part of the bioacceptable foam; The bio-acceptable composite according to embodiment (25), wherein at least one property selected from the group consisting of flexibility, bioabsorption rate, and porous structure transitions substantially continuously.
  (28) The bio-acceptable complex according to embodiment (25), wherein the pores interconnected with each other have a pore size in the range of about 10 μm to about 200 μm.
  (29) The bio-acceptable composite according to embodiment (25), wherein the bio-acceptable foam has a porosity in the range of about 20% to about 98%.
  (30) The bio-acceptable composite according to embodiment (25), wherein the bio-acceptable foam has a flow path.
[0147]
  (31) The bio-acceptable complex according to embodiment (30), wherein the average length of the flow path is at least 200 μm.
  (32) The bio-acceptable complex according to embodiment (25), wherein the size of the substantially spherical pore is about 30 μm to about 150 μm.
  (33) The bio-acceptable complex according to embodiment (25), wherein the size of the substantially spherical pore is about 30 μm to about 400 μm, and the ratio of length to diameter is at least 2.
  (34) The biocompatible composite according to embodiment (25), wherein a therapeutic agent is present in the foam having the biocompatible gradient structure.
  (35) The bio-acceptable foam is formed of an insert in the bio-acceptable foam.Embodiment (A)The biotolerable complex according to 1.
[0148]
  (36) The insert is a film, scrim, woven fabric, knitted fiber,BraidingThe bio-acceptable composite according to embodiment (35), selected from the group consisting of fibers, orthopedic implants, tubes and mixtures thereof.
  (37) The bioacceptable complex is formed in a three-dimensional structure.Embodiment (A)The biotolerable complex according to 1.
  (38) The three-dimensional shape structure is tubular, branched, spherical, hemispherical, three-dimensional polygonal, elliptical, toroidal, conical, frustoconical, pyramidal, solid and hollow And a bio-acceptable complex according to embodiment (37) selected from the group consisting of these and combinations thereof.
(B) A method for tissue repair or regeneration, comprising the step of contacting cells with a bio-acceptable complex, wherein the bio-acceptable complex includes a first bio-acceptable filamentous layer and A bio-acceptable composite comprising a second bio-acceptable foam layer attached, wherein the bio-acceptable foam is selected from the group consisting of a foam having a gradient structure and a foam having a flow path; The foam having the gradient structure has a first part and a second part, and the composition, rigidity, flexibility, bioabsorption rate, and porosity are from the first part to the second part. A method in which at least one characteristic selected from the group consisting of a textured structure transitions substantially continuously, and the foam comprising the flow path has a first face and a second face comprising the flow path .
  (39) The bioacceptable complex is bioabsorbableEmbodiment (B)The method described in 1.
  (40) The bioacceptable complex is an aliphatic polyester, poly (amino acid), copoly (ether-ester), polyalkylene oxalate, polyamide, poly (iminocarbonate), polyorthoester, polyoxaester, polyamide ester Formed by a bioabsorbable polymer selected from the group consisting of: a polyoxaester containing an amine group, a poly (anhydride), a polyphosphazene, a biopolymer and mixtures thereofEmbodiment (B)The method described in 1.
[0149]
  (41) The method according to embodiment (40), wherein the bioabsorbable polymer is an aliphatic polyester.
  (42) The aliphatic polyester is lactide, glycolide, glycolic acid, ε-caprolactone, p-dioxanone (1,4-dioxane-2-one), trimethylene carbonate (1,3-dioxane-2-one), tri Alkyl derivatives of methylene carbonate, δ-valerolactone, β-butyrolactone, γ-butyrolactone, ε-decalactone, hydroxybutyrate, hydroxyvalerate, 1,4-dioxepan-2-one, 1,5,8,12-tetraOxaEmbodiments selected from the group consisting of homopolymers and copolymers of cyclotetradecane-7,14-dione, 1,5-dioxepan-2-one, 6,6-dimethyl-dioxepan-2-one, and mixtures thereof (41 ) Method.
  (43) The method according to embodiment (42), wherein the aliphatic polyester is an elastomer.
  (44) Cells are seeded on the biotolerable complexEmbodiment (B)The method described in 1.
  (45) The method according to embodiment (42), wherein the cells are seeded on the biocompatible complex.
[0150]
  (46) The biotolerable complex is transplanted into an animal and contacted with a cellEmbodiment (B)The method described in 1.
  (47) The method according to embodiment (42), wherein the biotolerable complex is transplanted into an animal and contacted with a cell.
  (48) Cells are seeded on the bio-acceptable complex, the bio-acceptable complex and the cell are placed in a cell culture device, and the cells are grown on the bio-acceptable complex.Embodiment (B)The method described in 1.
  (49) Cells are seeded on the bio-acceptable complex, the bio-acceptable complex and the cell are placed in a cell culture device, and the cells are grown on the bio-acceptable complex. The method according to embodiment (42).
  (50) The cells are pluripotent cells, stem cells and precursor cells, and mixtures thereofEmbodiment (B)The method described in 1.
[0151]
  (51) the cells are muscle cells, fat cells, fibromyoblasts, ectoderm cells, muscle cells, osteoblasts, chondrocytes, endothelial cells, fibroblasts, pancreatic cells, hepatocytes, bile duct cells, bone marrow cells, Are nerve cells, genitourinary cells and mixtures thereofEmbodiment (B)The method described in 1.
  (52) The bioacceptable complex is selected from the group consisting of anti-infective agents, hormones, analgesics, anti-inflammatory agents, growth factors, chemotherapeutic agents, anti-rejection agents, prostaglandins, RDG peptides, and mixtures thereof. Contains the selected drugEmbodiment (B)The method described in 1.
  (53) The bio-acceptable complex is formed with a composition containing about 30% to about 99% by weight of ε-caprolactone repeating units.Embodiment (B)The method described in 1.
  (54) The ε-caprolactone repeating unit is lactide, glycolide, glycolic acid, ε-caprolactone, p-dioxanone (1,4-dioxane-2-one), trimethylene carbonate (1,3-dioxan-2-one) , Alkyl derivatives of trimethylene carbonate, δ-valerolactone, β-butyrolactone, γ-butyrolactone, ε-decalactone, hydroxybutyrate, hydroxyvalerate, 1,4-dioxepan-2-one, 1,5,8,12 -TetraOxaEmbodiments copolymerized with a comonomer selected from the group consisting of cyclotetradecane-7,14-dione, 1,5-dioxepan-2-one, 6,6-dimethyl-dioxepan-2-one, and mixtures thereof (53) The biotolerable complex according to (53).
  (55) The method according to embodiment (53), wherein the cells are seeded on the biotolerable complex.
[0152]
  (56) The method according to embodiment (53), wherein the biotolerable complex is transplanted into an animal and contacted with a cell.
  (57) Cells are seeded on the bio-acceptable complex, the bio-acceptable complex and the cell are placed in a cell culture device, and the cells are grown on the bio-acceptable complex. The method of embodiment (53).
  (58) The method according to embodiment (57), wherein the cells are pluripotent cells, stem cells and precursor cells, and a mixture thereof.
  (59) the cells are muscle cells, fat cells, fibromyoblasts, ectoderm cells, muscle cells, osteoblasts, chondrocytes, endothelial cells, fibroblasts, pancreatic cells, hepatocytes, bile duct cells, bone marrow cells, The method according to embodiment (57), which is a nerve cell, urogenital cell and a mixture thereof.
(C) A bio-acceptable body comprising a bio-acceptable foam having a first surface portion and a second surface portion, and having pores and flow paths that communicate with each other between the first surface portion and the second surface portion. Foam.
(D) A bio-acceptable foam comprising a foam having internal open pores formed with a composition containing from about 30 wt% to about 99 wt% ε-caprolactone repeating units.
[0153]
【The invention's effect】
Therefore, according to the present invention, a bio-acceptable and bio-absorbable foam having a continuous gradient structure with a form, structure and / or material composition capable of effectively repairing or regenerating tissue can be provided.
[Brief description of the drawings]
FIG. 1 is a scanning electron micrograph of a cross section of an irregular microstructured foam made with a 5% solution of 35 / 65ε-caprolactone-co-glycolide copolymer.
FIG. 2 is a scanning electron micrograph of a cross-section of a foam having a vertically open channel made with a 10% solution of 35 / 65ε-caprolactone-co-glycolide copolymer.
FIG. 3 is a scanning electron micrograph of a cross-section of a foam having a structural gradient made with a 10% solution of 35 / 65ε-caprolactone-co-glycolide copolymer.
FIG. 4 is a scanning electron micrograph of a cross-section of a foam having a gradient structure made with a 50/50 mixture of 40 / 60ε-caprolactone-co- (L) lactide copolymer and 35 / 65ε-caprolactone-co-glycolide copolymer. is there.
FIG. 5: Scanning electron microscope of the top cross-section of a foam having a gradient structure made with a 50/50 mixture of 40 / 60ε-caprolactone-co- (L) lactide copolymer and 35 / 65ε-caprolactone-co-glycolide copolymer. It is a photograph.
6 is a scanning electron microscope of the bottom cross section of a foam having a gradient structure made with a 50/50 mixture of 40 / 60ε-caprolactone-co- (L) lactide copolymer and 35 / 65ε-caprolactone-co-glycolide copolymer. It is a photograph.
FIG. 7 is a graph of various data in cell culture.
FIG. 8 is a graph of various data in cell culture.
FIG. 9 is a graph of various data in cell culture.
FIG. 10 is an explanatory diagram of the anatomical structure of cartilage tissue.
FIG. 11 made with a 50/50 mixture of 35 / 65ε-caprolactone-co-glycolide copolymer and 40 / 60ε-caprolactone-co- (L) lactide copolymer having a structure suitable for use as a skin skeleton-forming material. It is the scanning electron micrograph of the 0.5 mm foam. FIG. 11A is a scanning electron micrograph showing the porosity of the surface of the skeleton-forming material, preferably facing the wound bed, and FIG. 11B is preferably the skeleton formation facing away from the wound bed. It is a scanning electron micrograph which shows the porosity of the surface of material.
FIG. 12 made with a 50/50 mixture of 35 / 65ε-caprolactone-co-glycolide copolymer and 40 / 60ε-caprolactone-co- (L) lactide copolymer having a structure suitable for use as a skin skeleton-forming material. It is the scanning electron micrograph of the 0.5 mm foam. It is a scanning electron micrograph which shows the cross section of the frame | skeleton formation material which has the flow path extended in the thickness direction of a foam.
FIG. 13 is a 40 × dark field photomicrograph of a trichrome-stained sample showing cell infiltration of the foam shown in FIGS. 11 (A) to 12 on the 8th day after transplantation in a pig model.
14 is a 100-fold composite photomicrograph of a trichrome-stained sample showing cell infiltration of the foam shown in FIGS. 11 (A) to 12 containing further PDGF 8 days after transplantation in a pig model. .
FIG. 15 is a photomicrograph of a cross section of an electrospun fiber layer fused to a foam substrate (both materials are 60/40 copolymers of (L) lactide and ε-caprolactone).
16 is a photomicrograph of the surface porous structure of the electrospun layer shown in FIG.

Claims (31)

A foam having a first bio-acceptable filamentous layer and a second bio-acceptable foam layer attached thereto, the bio-acceptable foam having a gradient structure, and a flow path A bio-acceptable composite selected from the group consisting of: a foam comprising the gradient structure has a first part and a second part, from the first part to the second part; At least one property selected from the group consisting of composition, stiffness, flexibility, bioabsorption rate, and porous structure is substantially continuously transitioned, and the foam comprising the flow path has a first a surface portion and a second surface, and a flow path between them possess,
The bio-acceptable filament of the first bio-acceptable filamentous layer and the bio-acceptable foam are aliphatic polyester, poly (amino acid), copoly (ether-ester), and polyalkylene oxalate, respectively. , Polyamides, poly (iminocarbonates), polyorthoesters, polyoxaesters, polyamide esters, polyoxaesters containing amine groups, poly (anhydrides), polyphosphazenes, biopolymers and mixtures thereof Formed from a bioabsorbable polymer,
The foam having the bio-acceptable gradient structure has a composition of the at least two different aliphatic polyesters from a first ratio of at least two different aliphatic polyesters in composition from the first surface portion to the second surface portion. Transitions almost continuously to the second ratio,
Furthermore, a second aliphatic polyester is present as a component of the bioacceptable foam,
And a second aliphatic polyester is present as a component of the bioacceptable filamentous layer,
A biotolerable complex.   The bioacceptable complex according to claim 1, wherein the aliphatic polyester is lactide, lactic acid, glycolide, glycolic acid, ε-caprolactone, p-dioxanone (1,4-dioxane-2-one), trimethylene carbonate ( 1,3-dioxan-2-one), alkyl derivatives of trimethylene carbonate, δ-valerolactone, β-butyrolactone, γ-butyrolactone, ε-decalactone, hydroxybutyrate, hydroxyvalerate, 1,4-dioxepane-2 -One, 1,5,8,12-tetraoxacyclotetradecane-7,14-dione, 1,5-dioxepan-2-one, 6,6-dimethyl-1,4-dioxane-2-one, and these A bio-acceptable composite selected from the group consisting of homopolymers and copolymers of body.   The biocompatible composite according to claim 1, wherein the aliphatic polyester is an elastomer.   The bioacceptable complex according to claim 3, wherein the elastomer is a copolymer of ε-caprolactone and glycolide, a copolymer of ε-caprolactone and (L) lactide, p-dioxanone (1,4-dioxane-2-one), and (L) lactide copolymer, ε-caprolactone and p-dioxanone copolymer, p-dioxanone and trimethylene carbonate copolymer, trimethylene carbonate and glycolide copolymer, trimethylene carbonate and (L) lactide copolymer and these A bio-acceptable complex selected from the group consisting of mixtures.   The bioacceptable composite according to claim 1, wherein the foam having the bioacceptable gradient structure transitions substantially continuously in rigidity from the first portion to the second portion. A biotolerable complex.   2. The biocompatible composite according to claim 1, wherein the foam having the biocompatible gradient structure transitions substantially continuously in the bioabsorption rate from the first portion to the second portion. A bio-acceptable complex.   The biocompatible composite according to claim 1, wherein the foam having the biocompatible gradient structure transitions substantially continuously in flexibility from the first portion to the second portion. A bio-acceptable complex.   The biocompatible composite according to claim 1, wherein the foam having the biocompatible gradient structure transitions substantially continuously in the structure from the first portion to the second portion. A biotolerable complex.   9. The bio-acceptable composite according to claim 8, wherein the foam having the bio-acceptable gradient structure has a structure from a substantially spherical porous shape to a columnar shape from the first portion to the second portion. A bio-acceptable complex that transitions almost continuously to a porous shape.   The bio-acceptable composite according to claim 9, wherein the size of the substantially spherical pore is 30 to 150 µm.   The bio-acceptable composite according to claim 9, wherein the diameter of the columnar pores is 100 µm to 400 µm, and the ratio of the length to the diameter is at least 2.   The biocompatible complex according to claim 1, further comprising a therapeutic agent in the biocompatible complex.   2. The biotolerable complex of claim 1, wherein the anti-infective agent, hormone, analgesic agent, anti-inflammatory agent, growth factor, chemotherapeutic agent, anti-rejection agent, prostaglandin, RDG peptide, and mixtures thereof. A bio-acceptable complex, further comprising a drug selected from the group consisting of:   14. The biotolerable complex of claim 13, wherein the growth factor is bone morphogenetic protein, bone morphogenesis-like protein, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor, trans A bio-acceptable complex that is selected from homing growth factor, vascular endothelial growth factor and mixtures thereof.   The biocompatible composite according to claim 1, wherein the biocompatible foam is from a bioabsorbable synthetic polymer, a biocompatible bioabsorbable biopolymer, a biocompatible ceramic material, and mixtures thereof. A bio-acceptable complex filled with a bio-acceptable material selected from the group consisting of:   The bioacceptable composite according to claim 1, wherein the foam comprising the flow path has a flow path with an average length of at least 200 µm.   The biotolerable complex according to claim 16, wherein the flow path extends substantially from the first surface portion to the second surface portion.   The bioacceptable composite according to claim 1, wherein the bioacceptable foam has internal open pores formed of a composition containing 30 wt% to 99 wt% of ε-caprolactone repeating units. A biotolerable complex.   19. The bioacceptable complex according to claim 18, wherein the ε-caprolactone repeating unit is lactide, lactic acid, glycolide, glycolic acid, p-dioxanone (1,4-dioxane-2-one), trimethylene carbonate (1 , 3-dioxan-2-one), alkyl derivatives of trimethylene carbonate, δ-valerolactone, β-butyrolactone, γ-butyrolactone, ε-decalactone, hydroxybutyrate, hydroxyvalerate, 1,4-dioxepane-2- ON, 1,5,8,12-tetraoxacyclotetradecane-7,14-dione, 1,5-dioxepan-2-one, 6,6-dimethyl-1,4-dioxane-2-one, and these A biotolerable complex copolymerized with a comonomer selected from the group consisting of a mixture.   19. The bioacceptable composite according to claim 18, comprising a first part and a second part, wherein the bioacceptable foam is from the first part of the bioacceptable foam. A bio-acceptable composite in which at least one property selected from the group consisting of composition, rigidity, flexibility, bioabsorption rate, and porous structure transitions substantially continuously over the second portion. .   19. The biotolerable complex according to claim 18, wherein the pores interconnected with each other have a pore size within a range of 10 μm to 200 μm.   The biocompatible composite according to claim 18, wherein the porosity of the biocompatible foam is in the range of 20% to 98%.   19. The biocompatible composite according to claim 18, wherein the biocompatible foam has a flow path.   24. The biocompatible composite of claim 23, wherein the flow path has an average length of at least 200 [mu] m.   19. The bio-acceptable complex according to claim 18, wherein the substantially spherical pore size is 30 μm to 150 μm.   19. The bio-acceptable complex according to claim 18, wherein the substantially columnar pore has a diameter of 30 μm to 400 μm and a ratio of length to diameter is at least 2.   19. The bioacceptable complex according to claim 18, further comprising a therapeutic agent in the bioacceptable foam.   The biocompatible composite according to claim 1, wherein the biocompatible foam is formed of an insert in the biocompatible foam.   29. The biocompatible composite of claim 28, wherein the insert is selected from the group consisting of films, scrims, woven fabrics, knitted fibers, braided fibers, orthopedic grafts, tubes and mixtures thereof. Tolerable complex.   The bio-acceptable complex according to claim 1, wherein the bio-acceptable complex is formed into a three-dimensional structure.   32. The bioacceptable complex according to claim 30, wherein the three-dimensional shape structure is tubular, branched, spherical, hemispherical, three-dimensional polygonal, elliptical, toroidal, conical, conical. A bio-acceptable complex selected from the group consisting of trapezoids, pyramids, solid and hollow, and combinations thereof.

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