JP4669662B2 - 生物活性材料、生物活性材料の製法およびその使用方法 - Google Patents
生物活性材料、生物活性材料の製法およびその使用方法 Download PDFInfo
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Description
)まで低下させることができる酸である。好ましい酸はクエン酸、アスコルビン酸、乳酸、上記の酸の組合せ等である。添加する酸の量は、溶液のpHを所望のpHまで低下させるのに十分な量であり、それは当業者によって容易に決定される。
当該分野で公知の技術を用い、モリカイコの繭をまず精錬した。膠様セリシン蛋白質を、以下のように、炭酸ナトリウム(Na2CO3)の水溶液中での反復洗浄によって抽出した。繭を、まず、98℃の1.1g/l(1リットル当たりのグラム)のNa2CO3の水溶液中で1時間洗浄し、次いで、98℃にて0.4g/lのNa2CO3を含有する水溶液中で1時間すすいだ。次いで、繭を98℃から室温に低下させる温度にて蒸留水中で反復洗浄し、セリシンを含まないフィブロインを得た。水中の生のフィブロインの量は10g/lであった。次いで、精錬したフィブロインを、65℃で、100gフィブロイン/l溶液の初期組成にて、臭化リチウム(LiBr)の9.3モル水溶液に3時間で溶解させた。溶解が完了すると、水溶液を蒸留水の添加によって5%重量/容量フィブロインまで希釈した。残存している不純物および未溶解繊維を、孔隙サイズn.1を持つフィルターを用いる濾過によって除去した。次いで、3,500の分子量カットオフの再生したセルロース膜を用い、72時間の蒸留水に対する透析によって塩をフィブロイン混合物から除去した。透析したフィブロインの最終濃度は1から2%純度の絹フィブロインであった。
フィブロイン水溶液(20ml(ミリリットル))を、−20℃から50℃の温度で2から24時間インキュベートしたポリスチレンカプセルに注いだ。−20℃でインキュベートした溶液を加熱し、ゲル化が起こるまで室温に維持した。4℃から50℃の一定温度に保持された他のフィブロイン溶液は、ゲル化が起こるのに2から24時間のインキュベーションが必要であった。熱重力分析によって測定したゲルの含水量は95から98重量%までで変化した。懸濁液の粘度は、それをインキュベートする時間および温度に依存した。従って、フィブロインのレオロジー特性は熱処理によって変化することが証明された。
水性フィブロイン混合物を、特定のアミノ酸の結合部位との反応に特異的な種々の蛋白質分解酵素で処理した。目標は平均分子量を低下させ、生分解速度を増加させ、および細胞接着因子、成長因子および薬理学的分子との媒介に特異的なペプチドとの最終的な相互作用のための特異的活性部位を露出させることであった。以下の酵素を調べた:
クエン酸またはグリセロールをフィブロイン溶液に添加することによって、ゲル化を誘導した。
ポリマーでコーティングしたヒドロキシアパタイト粒子のコア/シェル粒子の粉末を作成した。ほぼ6,000の分子量を持つ(ポリ)ラクチド(PLA)を、生分解性ポリマーのシェルとして用いた。2グラムのシラン処理ヒドロキシアパタイトを50mlのブタノール中の6グラムのポリラクチドに添加した。溶液を2時間で70℃まで加熱し、室温まで冷却し、50mlのポリエチレングリコールの2%重量/容量水溶液と混合した。コーティングした粒子の沈降速度は、コーティングプロセスの相対的均一性を維持するのに十分なほどに遅かった。コア/シェル混合物を攪拌し、ガラスペトリ皿に注ぎ、50℃にて乾燥し、軽く粉砕して粉末とした。得られた物質はむしろ広いサイズ分布の球状ポリマーコーティング凝集体であった。
グリセロールをフィブロイン水溶液に添加し、4℃にてフィブロイン水溶液を処理することによって作成したフィブロインゲル上で、ヒト骨芽細胞様細胞(MG63)を72時間培養した。テストした物質上で、および対照として用いた空のポリスチレンウェルで増殖させたMG63の生化学的および免疫酵素パラメータを評価して、細胞増殖および活性を測定した。
クエン酸由来のフィブロインゲルを、ウサギの大腿顆にドリルで開けたキャビティー(6mm直径、10mm深さ)に移植した。インプラントを含まないキャビティーを対照として用いた。移植から1ヶ月後にウサギを犠牲とし、インプラント部位の組織学的結果をインプラントを含まない部位のそれと比較した。
Claims (14)
- フィブロイン溶液を、熱処理、酸、蛋白質分解酵素、生体適合性ポリマー、のうち一つかそれ以上の因子の組み合わせで処置されたフィブロイン溶液と、シェル/コア粒子を含む孔隙形成粒状材料を含むことを特徴とする組成物。
- 該コア/シェル粒子が、カルシウム化合物、リン酸塩源、からなる群から選択された少なくとも一つの成分からなる、請求項1に記載の組成物。
- 該シェルが、生分解性ポリマーからなる、請求項1又は2に記載の組成物。
- オクルージョン、ゲル、クリーム又はペーストのいずれかの形態である、請求項1−3のいずれかに記載の組成物。
- 該フィブロインが、モリカイコフィブロインからなる請求項1−4のいずれかに記載の組成物。
- 注射可能又は注入可能である請求項1−5のいずれかに記載の組成物。
- 該孔隙形成粒状材料が、ヒドロキシアパタイト、リン酸三カルシウム、セラミック、サンゴのうち一つかそれ以上の前記材料の組み合わせからなる、請求項1−6のいずれかに記載の組成物。
- 該コアが、ヒドロキシアパタイト、リン酸カルシウム化合物、再吸着可能なガラス充填剤、のうち一つかそれ以上の前記材料の組み合わせからなり、該シェルが、ポリ乳酸、ポリグリコール酸、ポリカプロラクトン、ポリトリメチル炭酸塩、ポリエチレングリコールジアクリレート、ポリアンヒドリド、ポリオルトエステル、ポリホスファジン、ポリアセタール、ポリエステル、ポリ尿素、ポリカーボネート、ポリウレタン、ポリアルファーヒロドキシ酸、ポリアミド、ポリアミノ酸、のうち一つかそれ以上の前記生分解性ポリマーからなる請求項1−7に記載の組成物。
- 細胞、抗生物質、骨形成蛋白質、骨再生を促進する化合物、創傷修復を促進する化合物、のうち一つかそれ以上の因子の組み合わせ、のグループから選択される生理活性剤を含む、請求項1−8のいずれかに記載の組成物。
- 注射可能で生物活性フィブロインゲルを形成する方法であって、フィブロイン溶液を、熱処理、蛋白質分解酵素、生体適合性ポリマー、のうち一つかそれ以上の因子の組み合わせで処置することと、コア/シェル粒子を含む孔隙形成粒状材料を添加することからなる、注射可能で生活性フィブロインゲルを形成する方法。
- 前記因子の熱処理が、約−20℃から約50℃の温度まで加熱することを含む請求項10に記載の方法。
- 該生体適合性ポリマーが、ポリエチレングリコール、ポリエチレンオキシド、ポリビニルピロリドン、のうち一つかそれ以上の前記生体適合性ポリマーの組み合わせからなり、該蛋白質分解酵素が、ストレプトマイセス・グリセウス由来のプロテアーゼ、パパイン、キモトリプシン、のうち一つかそれ以上の前記蛋白質分解酵素の組み合わせからなる、請求項10に記載の方法。
- 組成物を調整する方法であって、フィブロイン溶液を、熱処理、酸、蛋白質分解酵素、生体適合性ポリマー、のうち一つかそれ以上の因子の組み合わせで処置することと、シェル/コア粒子を含む孔隙形成粒状材料を添加することからなる、請求項1−9のいずれかに記載の組成物を調整する方法。
- 前記因子がクエン酸、アスコルビン酸、乳酸、からなるグループから一つかそれ以上選択される、請求項13に記載の方法。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US34300601P | 2001-10-25 | 2001-10-25 | |
PCT/US2002/034252 WO2003035124A2 (en) | 2001-10-25 | 2002-10-25 | Fibroin compositions and methods of making the same |
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JP4669662B2 true JP4669662B2 (ja) | 2011-04-13 |
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JP2003537688A Expired - Fee Related JP4669662B2 (ja) | 2001-10-25 | 2002-10-25 | 生物活性材料、生物活性材料の製法およびその使用方法 |
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US (3) | US7727542B2 (ja) |
EP (1) | EP1446169B1 (ja) |
JP (1) | JP4669662B2 (ja) |
CN (1) | CN1277584C (ja) |
AT (1) | ATE420671T1 (ja) |
AU (1) | AU2002348072A1 (ja) |
CA (1) | CA2466001C (ja) |
DE (1) | DE60230907D1 (ja) |
ES (1) | ES2321915T3 (ja) |
WO (1) | WO2003035124A2 (ja) |
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- 2002-10-25 CA CA2466001A patent/CA2466001C/en not_active Expired - Fee Related
- 2002-10-25 DE DE60230907T patent/DE60230907D1/de not_active Expired - Lifetime
- 2002-10-25 AT AT02784287T patent/ATE420671T1/de not_active IP Right Cessation
- 2002-10-25 EP EP02784287A patent/EP1446169B1/en not_active Expired - Lifetime
- 2002-10-25 ES ES02784287T patent/ES2321915T3/es not_active Expired - Lifetime
- 2002-10-25 CN CNB028212819A patent/CN1277584C/zh not_active Expired - Fee Related
- 2002-10-25 US US10/281,096 patent/US7727542B2/en not_active Expired - Fee Related
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ATE420671T1 (de) | 2009-01-15 |
JP2005510268A (ja) | 2005-04-21 |
US8071118B2 (en) | 2011-12-06 |
CA2466001C (en) | 2012-01-03 |
US7727542B2 (en) | 2010-06-01 |
US20030099630A1 (en) | 2003-05-29 |
WO2003035124A3 (en) | 2003-09-12 |
AU2002348072A1 (en) | 2003-05-06 |
WO2003035124A2 (en) | 2003-05-01 |
CN1575188A (zh) | 2005-02-02 |
EP1446169B1 (en) | 2009-01-14 |
ES2321915T3 (es) | 2009-06-15 |
CN1277584C (zh) | 2006-10-04 |
CA2466001A1 (en) | 2003-05-01 |
EP1446169A2 (en) | 2004-08-18 |
US20120040907A1 (en) | 2012-02-16 |
DE60230907D1 (de) | 2009-03-05 |
US20070190099A1 (en) | 2007-08-16 |
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