JP3687995B2 - Artificial blood vessel and manufacturing method thereof - Google Patents
Artificial blood vessel and manufacturing method thereof Download PDFInfo
- Publication number
- JP3687995B2 JP3687995B2 JP17109594A JP17109594A JP3687995B2 JP 3687995 B2 JP3687995 B2 JP 3687995B2 JP 17109594 A JP17109594 A JP 17109594A JP 17109594 A JP17109594 A JP 17109594A JP 3687995 B2 JP3687995 B2 JP 3687995B2
- Authority
- JP
- Japan
- Prior art keywords
- elastin
- blood vessel
- artificial blood
- water
- soluble
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Materials For Medical Uses (AREA)
- Prostheses (AREA)
Description
【０００１】
【産業上の利用分野】
本発明は、血管疾患の治療に際して、生体血管のバイパス術や置換術に使用される人工血管に関するものである。更に詳しくは、生体血管の内弾性板に類似した構造を人工血管の内腔面に形成することによって、血液の凝固と血漿蛋白の付着を抑制し、小口径でも内膜肥厚を起こさず、高い開存性を有する人工血管及びその製造方法に関するものである。
【０００２】
【従来の技術】
近年、食生活の向上による糖尿病の増加や高齢化などにより、閉塞性動脈硬化症などの血管疾患が増加してきており、代用血管を用いた血行再建術が盛んに行われている。このような状況の中で様々な人工血管が開発されており、胸部大動脈や腹部大動脈、大腿動脈等の再建に使用できる、内径７〜３８ｍｍの大口径人工血管は既に実用化されている。しかしながら、虚血性心疾患の患者の救命の為の冠状動脈の再建や膝下の膝窩動脈や脛骨動脈の再建には、専ら自家内胸動脈や大伏在静脈に頼っており、内径３〜６ｍｍの小口径人工血管の開発が望まれているが、これらの再建に満足して使用できる小口径人工血管は未だ開発されていないのが現状である。
【０００３】
人工血管に必要な性能として、優れた抗血栓性を有し、血栓による閉塞を生じないことと、優れた組織適合性を有し、早期に内膜や外膜が形成され安定化することが求められる。小口径人工血管では、特に血栓閉塞を免れるため優れた抗血栓性が不可欠の特性となるが、抗血栓性の高い人工血管ほど吻合部の組織過形成、即ち吻合部内膜肥厚を高度に形成して閉塞する。即ち抗血栓性と組織適合性は相反する特性であり、抗血栓性の高い材料は組織適合性が悪く、また、抗血栓性の高い材料の組織適合性を高めることは難しい（笹嶋唯博、ＮＩＫＫＥＩ ＭＥＤＩＣＡＬ、１９９２年、２月１０日号）。小口径では内腔が細いことと血流量が少ないことから、僅かな血栓や内膜肥厚の形成が閉塞原因となり、優れた抗血栓性と組織適合性の両立は小口径人工血管の開発における重要な課題である。
【０００４】
例えば、ダクロン人工血管は組織適合性は良好であるが抗血栓性は不十分であり、小口径人工血管には使用できない。一方、fibril length ３０μ以下の eＰＴＦＥ（延伸テフロン）製人工血管は抗血栓性が比較的良好であるが、吻合部内膜肥厚を形成しやすく、有孔度を高めて組織適合性を付与すれば抗血栓性が低下して、低血流量域では早期に血栓閉塞する。また、最近数種の化学修飾生体由来代用血管が開発されており、あるものは極めて良好な抗血栓性を示すが構造的に組織適合性が不良で、やはり何れも移植後数ケ月で吻合部内膜肥厚を生じて、その多くは閉塞に至るという問題を有している。
【０００５】
【発明が解決しようとする課題】
本発明は、従来の人工血管このような問題点を解決しようとするものである。ヒト臍帯静脈は発達したエラスチンを主成分とする内弾性板を有するが、これを化学固定したヒト臍帯静脈グラフトは、採取時や化学固定時に内弾性板が損傷、剥離され、この剥離部では血栓形成が顕著で、これに伴った内膜肥厚を発生するのに対して、内弾性板が温存されている部分は高い抗血栓性を示し、吻合部では内膜肥厚を発生しないことから、組織適合性も優れていることが示された（笹嶋唯博ら、人工臓器，２０（２），４１４−４１９（１９９１））。これらの研究結果を背景として、エラスチン内膜の人工血管としての生物学的適合性に着目するとともに、生体由来のエラスチンを用いることで、人工血管内腔面に均一に内弾性板に匹敵する膜面を構築できることを見出し、鋭意研究して本発明を完成するに至ったものである。
【０００６】
【課題を解決するための手段】
即ち本発明は、合成樹脂を管状にして作製した人工血管基材の内腔面に、水溶性エラスチンを架橋剤によって架橋して得られるエラスチン層を有するか、又は架橋剤によって架橋されたゼラチン層もしくはコラーゲン層を設け、更にその上に水溶性エラスチンを架橋剤によって架橋して形成されたエラスチン層を有する人工血管と、その製造方法に関するものである。
【０００７】
本発明で用いる人工血管基材は、血液の流路としてマクロファージなどの放出する過酸化物分解酵素や加水分解酵素の存在する生体内に長期間留置するため、生体内で酵素などにより分解されず、かつ毒性がなく、また血圧の変動に充分耐えられる材料であることが必要で、その材質としては、ポリウレタン、ポリエステル、ポリテトラフルオロエチレンなどの合成樹脂が好ましい。特にポリウレタンは、生体血管に近いコンプライアンスが得られるため好ましい。また、管状に作製した人工血管基材の内腔面に、ゼラチン、コラーゲン、エラスチン等を強固に固定するためには、内腔面の構造は多孔性、繊維を編んだもの、もしくは繊維が積み重なった構造のものが好ましい。その理由は、このような構造の基材ではゼラチン、コラーゲン、エラスチン等が基材の孔や繊維間に入り込み強いアンカー効果が得られるためである。
【０００８】
また、人工血管基材を合成樹脂を繊維状にしたものから作る場合、その繊維径は５〜５０μｍの範囲とするのが適切である。５μｍ以下では平織り又はメリヤス編みとしたとき、繊維間隔が狭くなりすぎて、充分にゼラチンやコラーゲン、エラスチン等が入り込めず、強固に固定できないし、生体内へ植込んだ後も細胞が入り込めず巧く治癒することができない。繊維径５０μｍ以上では繊維間隔が広すぎて、ゼラチンやコラーゲン、エラスチン等を固定する足場が少なく、強固にこれらを固定できない。
【０００９】
また、本発明で用いることのできるエラスチンは特に限定はしないが、ブタ大動脈由来エラスチン、ウシ頚靱帯由来エラスチン、ウシ肺由来エラスチン、ウシ大動脈由来エラスチン、ヒト肺由来エラスチン、ヒト大動脈由来エラスチンなどのエラスチン、又はこれらを熱蓚酸処理によって水溶性にしたα−エラスチンもしくはβ−エラスチン、アルカリエタノール処理によって水溶性にしたκ−エラスチン、ペプシン、エラスターゼなどの酵素で処理し水可溶性にしたエラスチン蛋白質などが挙げられる。中でも組織適合性と抗血栓性の点でヒト大動脈由来エラスチンが望ましく、その理由の詳細は不明であるが、エラスチンはその由来部位と動物の種類によって若干アミノ酸組成が異なり、ヒト大動脈由来エラスチンの有するアミノ酸組成では特に架橋後の裏面を平滑にできるため、血液の凝固活性を引き起こし難い為と考えられる。
【００１０】
また、本発明で用いるゼラチン、およびコラーゲンとしては、動物由来の物が利用できる。エラスチン層を設ける前に予めゼラチンもしくはコラーゲンの層を設ける目的は、主として、前記のように多孔性構造を有する人工血管基材の内表面の孔部を充填して、人工血管内腔面の平滑性を高めるためである。ゼラチン層やコラーゲン層は血液と直接接触し、あるいは作用するものではないが、人工血管内腔面をミクロ的に平滑にすることにより、血液の凝固活性を抑制することが可能になる。
【００１１】
本発明で人工血管の基材内面にゼラチン層又はコラーゲン層を形成する工程において、ゼラチン又はコラーゲンを溶解するｐＨ＝３〜８の緩衝液は特に限定はしないが、クエン酸／水酸化ナトリウム緩衝液、ギ酸／ギ酸ナトリウム緩衝液、クエン酸／クエン酸ナトリウム緩衝液、酢酸／酢酸ナトリウム水溶液、コハク酸／水酸化ナトリウム水溶液、リン酸緩衝溶液、リン酸二水素ナトリウム／水酸化ナトリウム緩衝液などが挙げられる。中でもゼラチンに対してはｐＨ＝７の緩衝液が望ましく、コラーゲンに対してはｐＨ＝３．３の緩衝溶液が好ましい。この理由はゼラチンはｐＨ＝７付近で安定して架橋できるし、またコラーゲンはｐＨ＝４以上では沈澱が生じやすく安定した水溶液と出来ないためである。
【００１２】
また同工程において、ゼラチン及びコラーゲンの濃度は緩衝溶液に対し１〜１０ｗｔ％の範囲が好ましい。この理由は、濃度が低すぎると充分な厚さのゼラチン層又はコラーゲン層が得られず基材が露出してしまうし、濃度が高すぎると溶液の粘度が上昇し、人工血管基材の孔や網目内に入っていくことができないし、ゼラチンやコラーゲンを架橋して得られる人工血管内腔面をミクロ的に平滑にするのが難しいためである。
【００１３】
また、人工血管の基材の内腔面上に直接又はゼラチン層もしくはコラーゲン層を設けた人工血管内腔面上に水溶性エラスチンをコアセルベーション（凝集）させる工程において用いる緩衝溶液も、ｐＨ＝４〜７の範囲のものであればよく特に限定はされない。前記のゼラチン又はコラーゲンを溶解するため緩衝液と同じものが使用できるが、中でもｐＨ＝５で充分な緩衝能を持つクエン酸／水酸化ナトリウム緩衝液、クエン酸／クエン酸ナトリウム緩衝液、酢酸／酢酸ナトリウム水溶液、コハク酸／水酸化ナトリウム水溶液などが、水溶性エラスチンをコアセルベーションさせるのに適している。これは水溶性エラスチンの等電点がこの付近に有り、電気的に中和されたエラスチンが疏水−疏水相互作用を生じやすく、コアセルベーションが安定するためであると考えられる。
【００１４】
また、水溶性エラスチンを緩衝溶液に溶解する量は、ｐＨ＝４〜７の緩衝液に対して１〜３０ｗｔ％の範囲で用いることがでる。この理由はエラスチンの濃度が１ｗｔ％より低すぎると水溶性エラスチンがコアセルベーションし難く、３０ｗｔ％より濃度が高すぎると人工血管の内腔面に形成されるエラスチン層が不均一になってしまう為である。また、温度は７０℃より高い温度ではエラスチンの変性が生じやすいし、３５℃未満の低温では水溶性エラスチンをコアセルベーションさせることができないため３５〜７０℃の範囲が好ましい。
【００１５】
さらに、人工血管の基材の内腔面上に直接又はゼラチン層もしくはコラーゲン層を設けた内腔面上にエラスチン層を形成するには、水溶性エラスチンの溶液を塗布し架橋剤にて架橋したり、予め架橋剤と混合した水溶性エラスチンを塗布し加熱や光によって架橋することも可能であるが、コアセルベーションさせた後で架橋するほうが、形成されたエラスチン層の抗血栓性と組織適合性が良好であるため好ましい。この理由は明確ではないが、エラスチンは生体内でコアセルベート（凝集体）を形成した状態で存在しており、この時の分子の三次構造がエラスチンの生理活性に重要であるためと考えられる。
【００１６】
本発明における各工程において、ゼラチン層、コラーゲン層、あるいはコアセルベーションさせた水溶性エラスチンの層等を架橋させるための架橋剤としては、水溶性の架橋剤であるグルタールアルデヒドやジアルデヒドスターチ、もしくは水溶性のエポキシ化合物等が使用できるが、中でも水溶性エポキシ化合物は、アミノ基とカルボキシル基の両方の官能基と反応でき、また、架橋後は柔らかい蛋白層を与えるため生体血管に近いコンプライアンスを得ることができ、特に好ましい。
【００１７】
また、水溶性エラスチンを人工血管内腔面に均一にコアセルベーションさせるためには、作製する人工血管の内径にもよるが、エラスチン水溶液を充填した人工血管を長手方向に水平に保ちながら、内径２〜６ｍｍの人工血管では円周方向に０．１〜１０ｒｐｍの回転速度で静かに回転させるのが好ましい。この理由は、エラスチンを３５℃以上の温度で静置すると、重力方向にエラスチンがコアセルベーションしてコアセルベート（凝集体）を形成する特性を利用するため、回転速度が速すぎると内腔に充填したエラスチン溶液が攪拌されてしまい、巧くコアセルベーションを内腔面上に形成できないし、回転速度が遅すぎるとエラスチンのコアセルベーション速度より人工血管内腔面の移動速度が遅くなるため、人工血管内腔面に均一にエラスチン層を形成することができないためである。
【００１８】
【実施例】
以下に、実施例によって本発明の効果を説明する。
〔実施例１、及び比較例１〕
▲１▼ 溶液の調製及び人工血管の製作
ポリウレタン樹脂（米国エチコン社製バイオマー）を直径１０μｍの繊維状に押出し、直径４ｍｍφ×長さ１ｍのステンレス製マンドレルに巻きとることで、内径４ｍｍφ×長さ１ｍの網目状人工血管基材を作製した。また、牛頸靱帯由来エラスチン（エラスチン・プロダクト社製）を熱蓚酸処理して水溶性にし、コアセルベーションによって凝集させて精製したα−エラスチン４００ｍｇを、２０ｍｌのｐＨ＝４．０酢酸／酢酸ナトリウム緩衝液に４℃によって溶解しエラスチン水溶液を作製した。
【００１９】
次に、上記で得られた人工血管基材を１０ｃｍの長さに切断し、内径４．５ｍｍφ×１２ｃｍのガラス管で、両端に三方コックを取り付け、更に中央部にギヤーを取り付けてモーターによって回転できるようにした治具の中に挿入し装着した。一方の三方コックからエラスチン水溶液を充填し、６０℃に加熱しながらモーターをスタトーさせ、５ｒｐｍで１２時間回転させてエラスチンを人工血管基材の内面にコアセルベーションさせた。ここで、三方コックを開いて管内の溶液を捨て、続いて２０ｍｇの水溶性エポキシ架橋剤（デナコールＥＸ−６１４Ｂ、長瀬産業製）をｐＨ＝７．０のリン酸緩衝液２０ｍｌに溶解した水溶液を人工血管内に充填し、５ｒｐｍの速度で回転させながら６０℃にて２４時間架橋を行い、内腔面に約１００μｍの厚みのエラスチン層を有する人工血管を得た。
【００２０】
▲２▼ 動物実験
体重１１ｋｇのビーグル成犬（雌性）１頭をアトロピンにて前処理し、導入麻酔をフルニトラゼパム０．１ｍｇ／ｋｇ、ケタミン３ｍｇ／ｋｇの静注によって実施した。犬を手術台に固定後、ヘパリン（１００Ｕ／ｋｇ）を静注し、フローセンによる麻酔を維持しながら、腹部を切開して腎動脈下腹部大動脈を５ｃｍ切除し、ここに６−０ポリプロピレン糸を用い端々吻合によって内径４ｍｍφ×長さ５ｃｍの本発明のエラスチン層を有する人工血管を植え込んだ。また、比較例として内径４ｍｍφ×長さ５ｃｍのグルタールアルデヒド処理ヒト臍帯静脈人工血管（Ｄardik Ｂiograft)を、体重１１ｋｇの雑種成犬（雌性）の腎動脈下腹部大動脈に植え込んだ。
【００２１】
術後、抗凝固剤は一切使用せず６ケ月間イヌを飼育した。６ケ月後、イヌをアトロピンにて前処理し、導入麻酔をフルニトラゼパム０．１ｍｇ／ｋｇ、ケタミン３ｍｇ／ｋｇの静注によって実施し、犬を手術台に固定後、ヘパリン（１００ＩＵ／ｋｇ）を静注し、フローセンによる麻酔を維持しながら、腹部を切開し、腎動脈下腹部大動脈に植え込んだ両人工血管を宿主血管と共に摘出した。直ちに、注射器を用いて２５００ＩＵ／５００ｍｌのヘパリンを溶解した生理食塩水にて人工血管の内外面を静かに洗浄し、血液を洗い流し、人工血管を縦に切り開き肉眼的に観察し、本発明のエラスチン層を有する人工血管と比較例のＤardik Ｂiograft との比較を行った。
【００２２】
次に、人工血管を縦に２つ割りにし、一方を４％ホルマリンの中性緩衝溶液に浸漬して固定した後、光学顕微鏡用試料とした。残りの試料は、１％グルタールアルデヒドの中性緩衝溶液及び３％グルタールアルデヒドの中性緩衝溶液に浸して固定し、電子顕微鏡用試料とした。光学顕微鏡用試料は中枢側吻合部、中央部、末梢側吻合部の３つに切断し、ホルマリンを洗浄した後、パラフィン包埋し、各部位から切片を切り出しプレパラートとした。次にヘマトキシリン液に５分浸し、水洗後、１％エオジン液に浸し、水洗、脱水、封入して染色を行った。光学顕微鏡による観察は４０倍と１００倍にて実施し、中枢側吻合部、中央部、末梢側吻合部でそれぞれ新生した内膜の厚みを計測し、本発明のエラスチン層を有する人工血管と比較例のＤardik Ｂiograft との内膜肥厚の程度を比較した。
【００２３】
また、電子顕微鏡用試料はグルタールアルデヒド固定後、０．１Ｍカコジル酸ナトリウム緩衝溶液にて十分洗浄し、試料を中枢側吻合部、中央部、末梢側吻合の３つに分け、１％オスミウム、１％タンニン酸で導電処理を行った後、ｔ−ブタノール凍結乾燥によって乾燥し、試料台に固定してＰｔ−Ｐｄを蒸着した。電子顕微鏡観察は本発明のエラスチン層を有する人工血管と比較例のＤardik Ｂiograft の内腔面の状態を２００倍と１０００倍にて観察し評価した。
【００２４】
尚、光学顕微鏡はニコン社製ＤＩＡＰＨＯＴ−ＴＭＤ型を使用し、走査型電子顕微鏡は日立製Ｓ−２４００型を使用した。
▲３▼ 評価結果
実施例及び比較例の各人工血管の評価結果は、表１に示した通りであった。
【００２５】
【表１】
〔実施例２、及び比較例２〕
▲１▼ 溶液の調製、及びゼラチン層上にエラスチン層を有するシャーレの作製
２％ゼラチンのリン酸緩衝溶液（ｐＨ＝７）を、３５ｍｍφの細胞培養用シャーレ（住友ベークライト（株）製 ＭＳ−１０３５）に２ｍｌ添加して均一に広げた後、４℃にて２時間静置し、ゼラチンをゲル化させた。その後、２％グルタールアルデヒドのリン酸緩衝液（ｐＨ＝７）３ｍｌを添加し、４℃で２４時間静置し、架橋を行った。
【００２７】
次に、牛頸靱帯由来エラスチン（エラスチン・プロダクト社製）を熱蓚酸処理して水溶性にし、コアセルベーションによって凝集させて精製したα−エラスチン４０ｍｇを、２ｍｌのｐＨ＝４．０酢酸／酢酸ナトリウム緩衝液に４℃で溶解した溶液を調製し、上記のゼラチン層を固定したシャーレ内に添加し、５０℃にて１２時間静置し、エラスチンをゼラチン層の上にコアセルベーションさせた。ここでシャーレ内の溶液を捨て、２０ｍｇの水溶性エポキシ架橋剤（デナコールＥＸ−５２１、長瀬産業製）をｐＨ＝７．０のリン酸緩衝液２０ｍｌに溶解した水溶液３ｍｌをシャーレ内に添加し、密栓をして、６０℃にて２４時間架橋を行い、ゼラチン層上にエラスチン層を有する内径３．５ｍｍφのシャーレ作製した。
【００２８】
▲２▼ 細胞培養
ゼラチン層上にエラスチン層を有する内径３．５ｍｍφのシャーレ、及び比較試料として未処理の内径３．５ｍｍφの細胞培養用シャーレにそれぞれ１×１０4個／ｍｌ濃度のヒト子宮頚癌由来Ｈela 細胞を２ｍｌづつ播種し、コウシ血清１０％（大日本製薬製）を含む基本培地ＭＥＭ（大日本製薬製）で４日間３７℃にて培養した。培地を２日毎に新しいものに交換し、培養４日目にセルスクレーパーで細胞を全て剥がし、細胞の浮遊溶液とした。
【００２９】
次に、この細胞浮遊溶液１００μｌに０．３％トリパンブルーのリン酸緩衝溶液１００μｌを加えて染色し、血球計算板上で倒立顕微鏡により対物レンズ１０倍にて観察し、細胞浮遊液１ｍｌ当たりの細胞数及び生死細胞数を計算した。
培養４日目の細胞数から、トリパンブルーにより染色された細胞を死細胞数として生存率を計算し、播種細胞数と培養４日目の細胞数から細胞の増殖率を求め、比較試料の細胞培養用シャーレでの増殖率を１として、ゼラチン層上にエラスチン層を有するシャーレ上での細胞の増殖率比を計算した。尚、倒立顕微鏡はニコン社製ＤＩＡＰＨＯＴ−ＴＭＤ型を使用した。
【００３０】
▲３▼ 評価結果
培養４日目のゼラチン層上にエラスチン層を有するシャーレ、及び比較試料の細胞培養用シャーレ上でのＨela 細胞の生存率、増殖率比を表２に示した。
培養４日目で、ゼラチン層上にエラスチン層を有するシャーレ上でのＨela 細胞の生存率は、比較試料のシャーレと同等であったが、細胞の増殖率比は比較試料を１としたとき、ゼラチン層上にエラスチン層を有するシャーレでは０．６となり、増殖の活発な癌細胞の増殖が抑えられていた。
【００３１】
【表２】
このように、本発明による人工血管の表面は細胞の過激な増殖を抑えることができ、人工血管内腔面での組織や細胞の過剰成長による内膜肥厚を抑えることができる、小口径人工血管に適した材料であることが判明した。
【００３３】
【発明の効果】
以上のように、人工血管基材の内腔面に水溶性エラスチンをコアセルベーション（凝集）させ、架橋剤によって架橋した人工血管は、優れた抗血栓性と組織適合性を併せ持ち、吻合部付近での組織や細胞の過剰成長や未着床を抑え、吻合部内膜肥厚を全く生ずることがないため、従来にない内径４ｍｍ以下といった小口径でも長期間にわたる開存が可能な人工血管であることが明白となった。
本発明は、虚血性心疾患の患者の救命の為の、冠状動脈の再建や膝窩動脈や脛骨動脈の再建に用いることができる小口径でも、長期間開存する有用な材料を提供するものである。[0001]
[Industrial application fields]
The present invention relates to an artificial blood vessel used for a bypass operation or a replacement operation of a biological blood vessel in the treatment of a vascular disease. More specifically, by forming a structure similar to the inner elastic plate of a biological blood vessel on the luminal surface of an artificial blood vessel, it suppresses blood coagulation and plasma protein adhesion, and does not cause intimal thickening even at a small diameter. The present invention relates to an artificial blood vessel having patency and a method for producing the same.
[0002]
[Prior art]
In recent years, vascular diseases such as obstructive arteriosclerosis are increasing due to an increase in diabetes due to an improvement in eating habits and an aging population, and revascularization using a substitute blood vessel is actively performed. Under such circumstances, various artificial blood vessels have been developed, and large-diameter artificial blood vessels having an inner diameter of 7 to 38 mm that can be used for reconstruction of the thoracic aorta, abdominal aorta, femoral artery and the like have already been put into practical use. However, reconstruction of the coronary arteries for lifesaving patients with ischemic heart disease and reconstruction of the popliteal artery and tibial artery below the knee rely exclusively on the autologous internal thoracic artery and great saphenous vein, and the inner diameter is 3 to 6 mm. However, the development of small-diameter artificial blood vessels that can be used satisfactorily for such reconstruction has not yet been developed.
[0003]
The required performance of the artificial blood vessel has excellent antithrombogenicity, does not cause clogging due to thrombus, and has excellent tissue compatibility, and the intima and outer membranes are formed and stabilized at an early stage. Desired. In small-diameter artificial blood vessels, excellent antithrombogenicity is indispensable especially for avoiding thrombus obstruction, but an artificial blood vessel with higher antithrombogenicity forms more anastomotic tissue hyperplasia, that is, anastomotic intimal thickening Then block. In other words, antithrombogenicity and histocompatibility are contradictory properties, and materials with high antithrombogenicity have poor tissue compatibility, and it is difficult to improve the tissue compatibility of materials with high antithrombotic properties (Yukihiro Takashima, NIKKEI MEDICAL, February 10, 1992). Small caliber has a narrow lumen and low blood flow, so the formation of slight thrombus and intimal thickening causes occlusion, and it is important for the development of small-diameter artificial blood vessels to have both excellent antithrombogenicity and tissue compatibility. It is a difficult task.
[0004]
For example, Dacron artificial blood vessels have good tissue compatibility but insufficient antithrombotic properties and cannot be used for small-diameter artificial blood vessels. On the other hand, an artificial blood vessel made of ePTFE (stretched Teflon) with a fibril length of 30 μm or less has relatively good antithrombogenicity, but it is easy to form an intimal thickening of the anastomosis, and increases the porosity and provides tissue compatibility. The antithrombogenicity is reduced, and thrombosis occurs early in the low blood flow area. In addition, several types of chemically modified living blood substitutes have been developed recently, and some of them show extremely good antithrombotic properties but structurally have poor tissue compatibility. Intimal thickening occurs, many of which have the problem of leading to obstruction.
[0005]
[Problems to be solved by the invention]
The present invention is intended to solve such problems of conventional artificial blood vessels. The human umbilical vein has a developed internal elastic plate mainly composed of elastin. However, the human umbilical vein graft, which is chemically fixed, is damaged and peeled during collection and chemical fixation. The formation is conspicuous, and intimal thickening associated with this occurs, whereas the part where the inner elastic plate is preserved shows high antithrombogenicity, and intimal thickening does not occur in the anastomosis part. It was also shown that the compatibility was excellent (Yukihiro Tsujishima et al., Artificial Organ, 20 (2), 414-419 (1991)). Based on these research results, we focused on the biocompatibility of the elastin intima as an artificial blood vessel, and by using elastin derived from a living body, a membrane that is uniformly comparable to the inner elastic plate on the surface of the artificial blood vessel The inventors have found that a surface can be constructed, and have intensively researched to complete the present invention.
[0006]
[Means for Solving the Problems]
That is, the present invention has a gelatin layer that has an elastin layer obtained by crosslinking water-soluble elastin with a crosslinking agent on the inner surface of an artificial blood vessel substrate made of synthetic resin in the form of a tube, or is crosslinked with a crosslinking agent. Alternatively, the present invention relates to an artificial blood vessel having an elastin layer formed by providing a collagen layer and further cross-linking water-soluble elastin with a crosslinking agent thereon, and a method for producing the same.
[0007]
Since the artificial blood vessel substrate used in the present invention is placed in a living body in the presence of a peroxide-degrading enzyme or hydrolase released from macrophages as a blood flow path for a long period of time, it is not decomposed by an enzyme or the like in the living body. In addition, it is necessary that the material is non-toxic and can sufficiently withstand fluctuations in blood pressure, and the material is preferably a synthetic resin such as polyurethane, polyester, or polytetrafluoroethylene. In particular, polyurethane is preferable because compliance close to that of living blood vessels can be obtained. In addition, in order to firmly fix gelatin, collagen, elastin, etc. to the lumen surface of a tubular artificial blood vessel substrate, the structure of the lumen surface is porous, fiber knitted, or fibers stacked. Those having a different structure are preferred. The reason for this is that gelatin, collagen, elastin and the like enter between the pores and fibers of the base material and have a strong anchor effect in the base material having such a structure.
[0008]
Moreover, when making an artificial blood vessel base material from what made synthetic resin a fiber form, it is suitable that the fiber diameter shall be the range of 5-50 micrometers. Below 5μm, when weaving plain or knitted, the fiber spacing becomes too narrow, and gelatin, collagen, elastin, etc. cannot enter sufficiently and cannot be firmly fixed, and cells can enter even after implantation in the living body. Can't heal well. When the fiber diameter is 50 μm or more, the fiber interval is too wide, and there are few scaffolds for fixing gelatin, collagen, elastin, etc., and these cannot be firmly fixed.
[0009]
The elastin that can be used in the present invention is not particularly limited, but elastin such as porcine aorta-derived elastin, bovine cervical ligament-derived elastin, bovine lung-derived elastin, bovine aorta-derived elastin, human lung-derived elastin, human aorta-derived elastin, etc. Or α-elastin or β-elastin made water-soluble by treatment with hot oxalic acid, elastin protein treated with an enzyme such as κ-elastin, pepsin, or elastase made water-soluble by alkaline ethanol treatment to make it water-soluble It is done. Among them, elastin derived from human aorta is desirable in terms of histocompatibility and antithrombogenicity, and the details of the reason are unclear, but elastin has a slightly different amino acid composition depending on the site of origin and the type of animal, and has elastin derived from human aorta. In the amino acid composition, the back surface after cross-linking can be smoothed.
[0010]
In addition, animal-derived products can be used as gelatin and collagen used in the present invention. The purpose of providing the gelatin or collagen layer in advance before providing the elastin layer is to fill the pores on the inner surface of the artificial blood vessel substrate having a porous structure as described above, thereby smoothing the surface of the artificial blood vessel lumen. This is to increase sex. The gelatin layer and the collagen layer do not directly contact or act on blood, but it is possible to suppress blood coagulation activity by microscopically smoothing the artificial blood vessel lumen surface.
[0011]
In the step of forming the gelatin layer or collagen layer on the inner surface of the artificial blood vessel in the present invention, the buffer solution of pH = 3 to 8 for dissolving gelatin or collagen is not particularly limited, but a citrate / sodium hydroxide buffer solution. , Formic acid / sodium formate buffer, citric acid / sodium citrate buffer, acetic acid / sodium acetate aqueous solution, succinic acid / sodium hydroxide aqueous solution, phosphate buffer solution, sodium dihydrogen phosphate / sodium hydroxide buffer solution, etc. It is done. Among them, a buffer solution with pH = 7 is desirable for gelatin, and a buffer solution with pH = 3.3 is preferable for collagen. This is because gelatin can be stably cross-linked around pH = 7, and collagen is likely to precipitate at pH = 4 or more and cannot form a stable aqueous solution.
[0012]
In this step, the gelatin and collagen concentrations are preferably in the range of 1 to 10 wt% with respect to the buffer solution. The reason is that if the concentration is too low, a sufficiently thick gelatin layer or collagen layer cannot be obtained, and the substrate is exposed. If the concentration is too high, the viscosity of the solution increases, and the pores of the artificial blood vessel substrate are increased. This is because it cannot enter the mesh or the mesh, and it is difficult to make the luminal surface of the artificial blood vessel obtained by crosslinking gelatin or collagen microscopically smooth.
[0013]
Further, the buffer solution used in the step of coacervating (aggregating) water-soluble elastin directly on the lumen surface of the artificial blood vessel substrate or on the artificial blood vessel lumen surface provided with the gelatin layer or the collagen layer is also pH = There is no particular limitation as long as it is in the range of 4-7. In order to dissolve the gelatin or collagen, the same buffer solution can be used, but among them, citric acid / sodium hydroxide buffer solution having sufficient buffer capacity at pH = 5, citric acid / sodium citrate buffer solution, acetic acid / Sodium acetate aqueous solution, succinic acid / sodium hydroxide aqueous solution and the like are suitable for coacervation of water-soluble elastin. This is considered to be because the isoelectric point of water-soluble elastin is in the vicinity, and the electrically neutralized elastin is likely to cause a water-water-water interaction, thereby stabilizing the coacervation.
[0014]
Moreover, the amount which dissolves water-soluble elastin in a buffer solution can be used in the range of 1-30 wt% with respect to the buffer solution of pH = 4-7. The reason is that if the concentration of elastin is too low, the water-soluble elastin is difficult to coacervate, and if the concentration is too high, the elastin layer formed on the luminal surface of the artificial blood vessel becomes uneven. Because of that. The temperature is preferably in the range of 35 to 70 ° C. because elastin is easily denatured at temperatures higher than 70 ° C., and water-soluble elastin cannot be coacervated at temperatures lower than 35 ° C.
[0015]
Furthermore, in order to form an elastin layer directly on the lumen surface of the artificial blood vessel substrate or on the lumen surface provided with the gelatin layer or collagen layer, a water-soluble elastin solution is applied and crosslinked with a crosslinking agent. It is also possible to apply water-soluble elastin mixed with a crosslinking agent in advance and crosslink by heating or light, but it is better to crosslink after coacervation and the antithrombogenicity and tissue compatibility of the formed elastin layer It is preferable because of its good properties. The reason for this is not clear, but it is thought that elastin exists in the form of coacervates (aggregates) in vivo, and the tertiary structure of the molecule at this time is important for the physiological activity of elastin.
[0016]
In each step in the present invention, as a crosslinking agent for crosslinking a gelatin layer, a collagen layer, or a coacervated water-soluble elastin layer, water-soluble crosslinking agents such as glutaraldehyde and dialdehyde starch, Alternatively, water-soluble epoxy compounds can be used, but water-soluble epoxy compounds can react with both functional groups of amino groups and carboxyl groups, and provide a soft protein layer after cross-linking so that compliance close to that of living blood vessels can be achieved. It can be obtained and is particularly preferred.
[0017]
In order to uniformly coacervate water-soluble elastin on the luminal surface of the artificial blood vessel, the inner diameter of the artificial blood vessel filled with the elastin aqueous solution is kept horizontal in the longitudinal direction, depending on the inner diameter of the artificial blood vessel to be produced. In an artificial blood vessel of 2 to 6 mm, it is preferable to rotate gently in the circumferential direction at a rotation speed of 0.1 to 10 rpm. The reason for this is that when elastin is allowed to stand at a temperature of 35 ° C. or higher, elastin coacervates in the direction of gravity to form a coacervate (aggregate). As the elastin solution is stirred, coacervation cannot be skillfully formed on the lumen surface, and if the rotation speed is too slow, the moving speed of the artificial blood vessel lumen surface is slower than the coacervation speed of elastin, This is because the elastin layer cannot be uniformly formed on the artificial blood vessel lumen surface.
[0018]
【Example】
The effects of the present invention will be described below with reference to examples.
[Example 1 and Comparative Example 1]
(1) Preparation of solution and production of artificial blood vessel Polyurethane resin (Biomer manufactured by Ethicon, USA) is extruded into a fiber with a diameter of 10 μm, and wound on a stainless steel mandrel with a diameter of 4 mmφ × length of 1 m, whereby an inner diameter of 4 mmφ × length A 1 m reticulated artificial blood vessel substrate was prepared. In addition, 400 mg of α-elastin purified from bovine cervical ligament-derived elastin (manufactured by Elastin Products), treated with hot oxalic acid to make it water-soluble, and coagulated for purification, was added to 20 ml of pH = 4.0 acetic acid / sodium acetate buffer. An elastin aqueous solution was prepared by dissolving in the liquid at 4 ° C.
[0019]
Next, the artificial blood vessel substrate obtained above is cut to a length of 10 cm, and a three-way cock is attached to both ends with a glass tube with an inner diameter of 4.5 mmφ × 12 cm, and a gear is attached to the center, and is rotated by a motor. It was inserted into a jig that was made available. An elastin aqueous solution was filled from one of the three-way cocks, and the motor was started while heating to 60 ° C., and the elastin was coacervated on the inner surface of the artificial blood vessel substrate by rotating at 5 rpm for 12 hours. Here, the three-way cock is opened and the solution in the tube is discarded. Subsequently, an aqueous solution in which 20 mg of a water-soluble epoxy crosslinking agent (Denacol EX-614B, manufactured by Nagase Sangyo) is dissolved in 20 ml of a phosphate buffer solution having a pH of 7.0 is prepared. The artificial blood vessel was filled and crosslinked at 60 ° C. for 24 hours while rotating at a speed of 5 rpm to obtain an artificial blood vessel having an elastin layer having a thickness of about 100 μm on the inner surface.
[0020]
(2) Animal experiment One beagle adult dog (female) having a body weight of 11 kg was pretreated with atropine, and induction anesthesia was carried out by intravenous injection of flunitrazepam 0.1 mg / kg and ketamine 3 mg / kg. After fixing the dog on the operating table, heparin (100 U / kg) was intravenously injected, and while maintaining anesthesia with Frocene, the abdomen was incised and 5 mm of the inferior abdominal aorta was excised. An artificial blood vessel having an elastin layer of the present invention having an inner diameter of 4 mmφ × length of 5 cm was implanted by end-to-end anastomosis. Further, as a comparative example, a glutaraldehyde-treated human umbilical vein artificial blood vessel (Dardik Biograft) having an inner diameter of 4 mmφ × length of 5 cm was implanted in the renal artery lower abdominal aorta of a hybrid adult dog (female) weighing 11 kg.
[0021]
After the operation, dogs were raised for 6 months without using any anticoagulant. Six months later, the dog was pretreated with atropine, and induction anesthesia was performed by intravenous injection of flunitrazepam 0.1 mg / kg and ketamine 3 mg / kg. After fixing the dog to the operating table, heparin (100 IU / kg) was statically administered. In addition, while maintaining anesthesia with Frocene, the abdomen was incised, and both artificial blood vessels implanted in the lower abdominal aorta of the renal artery were removed together with the host blood vessels. Immediately, the inner and outer surfaces of the artificial blood vessel were gently washed with a physiological saline in which 2500 IU / 500 ml of heparin was dissolved using a syringe, the blood was washed away, the artificial blood vessel was cut vertically, and visually observed. A comparison was made between the artificial blood vessel having a layer and the Dardik Biograft of the comparative example.
[0022]
Next, the artificial blood vessel was divided into two vertically, and one was immersed and fixed in a neutral buffer solution of 4% formalin, and then used as a sample for an optical microscope. The remaining sample was immersed and fixed in a neutral buffer solution of 1% glutaraldehyde and a neutral buffer solution of 3% glutaraldehyde to prepare a sample for an electron microscope. The sample for an optical microscope was cut into three parts, a central anastomosis part, a central part, and a peripheral anastomosis part, washed with formalin, embedded in paraffin, cut out from each part, and used as a preparation. Next, it was immersed in a hematoxylin solution for 5 minutes, washed with water, then immersed in a 1% eosin solution, washed with water, dehydrated and sealed for dyeing. Observation with an optical microscope is performed at 40 times and 100 times, and the thickness of the intima newly formed at the central anastomosis part, the central part, and the peripheral anastomosis part is measured, and compared with the artificial blood vessel having the elastin layer of the present invention. The degree of intimal thickening was compared with the example Dardik Biograft.
[0023]
In addition, after fixing the glutaraldehyde with an electron microscope sample, the sample was sufficiently washed with a 0.1 M sodium cacodylate buffer solution, and the sample was divided into a central anastomosis part, a central part, and a peripheral anastomosis, 1% osmium, After conducting a conductive treatment with 1% tannic acid, it was dried by t-butanol lyophilization, fixed to a sample stage, and Pt-Pd was deposited. The observation under an electron microscope was evaluated by observing the state of the luminal surface of the artificial blood vessel having the elastin layer of the present invention and the Dardik Biograft of the comparative example at 200 and 1000 times.
[0024]
The optical microscope used was a DIAPHOT-TMD type manufactured by Nikon Corporation, and the scanning electron microscope used was an S-2400 type manufactured by Hitachi.
(3) Evaluation results The evaluation results of the respective artificial blood vessels in Examples and Comparative Examples are as shown in Table 1.
[0025]
[Table 1]
[Example 2 and Comparative Example 2]
(1) Preparation of solution and preparation of petri dish having elastin layer on gelatin layer A phosphate buffer solution (pH = 7) of 2% gelatin was mixed with a 35 mmφ cell culture petri dish (Sumitomo Bakelite Co., Ltd. MS-1035) 2 ml) was added to spread uniformly and allowed to stand at 4 ° C. for 2 hours to gelatinize the gelatin. Thereafter, 3 ml of 2% glutaraldehyde phosphate buffer (pH = 7) was added, and the mixture was allowed to stand at 4 ° C. for 24 hours for crosslinking.
[0027]
Next, elastin derived from bovine cervical ligament (manufactured by Elastin Products Co., Ltd.) was treated with hot succinic acid to make it water-soluble, and 40 mg of α-elastin purified by coagulation was purified with 2 ml of pH = 4.0 acetic acid / sodium acetate. A solution dissolved in a buffer solution at 4 ° C. was prepared, added to the petri dish where the gelatin layer was fixed, and allowed to stand at 50 ° C. for 12 hours, so that elastin was coacervated on the gelatin layer. Here, the solution in the petri dish is discarded, and 3 ml of an aqueous solution in which 20 mg of a water-soluble epoxy crosslinking agent (Denacol EX-521, manufactured by Nagase Sangyo) is dissolved in 20 ml of a phosphate buffer having a pH of 7.0 is added to the petri dish. Sealed and crosslinked at 60 ° C. for 24 hours to prepare a petri dish having an inner diameter of 3.5 mmφ having an elastin layer on the gelatin layer.
[0028]
(2) A human cervix having a concentration of 1 × 10 4 cells / ml in a petri dish with an inner diameter of 3.5 mmφ having an elastin layer on a cell culture gelatin layer and an untreated cell culture petri dish with an inner diameter of 3.5 mmφ as a comparative sample. 2 ml each of cancer-derived Hela cells were seeded and cultured in a basic medium MEM (Dainippon Pharmaceutical Co., Ltd.) containing 10% calf serum (Dainippon Pharmaceutical Co., Ltd.) for 4 days at 37 ° C. The medium was replaced with a new one every 2 days, and all cells were peeled off with a cell scraper on the 4th day of culture to obtain a suspended solution of cells.
[0029]
Next, 100 μl of a 0.3% trypan blue phosphate buffer solution was added to 100 μl of this cell suspension solution, and the cells were stained and observed with an inverted microscope on a hemocytometer with a 10 × objective lens. Cell counts and viable cell counts were calculated.
From the number of cells on the 4th day of culture, the survival rate was calculated using the cells stained with trypan blue as the number of dead cells, and the cell growth rate was calculated from the number of seeded cells and the number of cells on the 4th day of culture. Assuming that the growth rate in the culture petri dish was 1, the cell growth rate ratio on the petri dish having an elastin layer on the gelatin layer was calculated. The inverted microscope used was a DIAPHOT-TMD type manufactured by Nikon Corporation.
[0030]
(3) Evaluation results Table 2 shows the survival rate and proliferation rate ratio of the Hela cells on the petri dish having an elastin layer on the gelatin layer on the 4th day of culture and the cell culture petri dish of the comparative sample.
On the 4th day of culture, the survival rate of Hela cells on a petri dish having an elastin layer on the gelatin layer was equivalent to that of the petri dish of the comparative sample. In the petri dish having an elastin layer on the gelatin layer, the value was 0.6, and the proliferation of actively proliferating cancer cells was suppressed.
[0031]
[Table 2]
As described above, the surface of the artificial blood vessel according to the present invention can suppress the rapid proliferation of cells, and can suppress intimal thickening due to excessive growth of tissue and cells on the surface of the artificial blood vessel. It was found that the material is suitable for the above.
[0033]
【The invention's effect】
As mentioned above, artificial blood vessels coagulated (aggregated) with water-soluble elastin on the luminal surface of the artificial blood vessel base material and cross-linked with a cross-linking agent have both excellent antithrombogenicity and tissue compatibility, and near the anastomosis It is an artificial blood vessel that can be maintained for a long period of time even with a small diameter of 4 mm or less, which is unconventional, because it suppresses overgrowth and non-implantation of tissues and cells in the body and does not cause any intimal intimal thickening. It became clear.
The present invention provides a useful material that can be preserved for a long time even with a small diameter that can be used for coronary artery reconstruction, popliteal artery and tibial artery reconstruction for the lifesaving of patients with ischemic heart disease. is there.
Claims (11)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP17109594A JP3687995B2 (en) | 1994-07-22 | 1994-07-22 | Artificial blood vessel and manufacturing method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP17109594A JP3687995B2 (en) | 1994-07-22 | 1994-07-22 | Artificial blood vessel and manufacturing method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0833661A JPH0833661A (en) | 1996-02-06 |
| JP3687995B2 true JP3687995B2 (en) | 2005-08-24 |
Family
ID=15916901
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP17109594A Expired - Fee Related JP3687995B2 (en) | 1994-07-22 | 1994-07-22 | Artificial blood vessel and manufacturing method thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3687995B2 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1403304B1 (en) * | 2001-05-30 | 2008-08-27 | Keiichi Miyamoto | Crosslinked elastin and processes for its production |
| EP1618856B1 (en) | 2003-03-31 | 2011-06-08 | Teijin Limited | Composite of support substrate and collagen, and process for producing support substrate and composite |
| JP2006068401A (en) * | 2004-09-03 | 2006-03-16 | Kyushu Institute Of Technology | Artificial blood vessel |
| JP4078431B2 (en) | 2004-10-29 | 2008-04-23 | 国立大学法人九州工業大学 | Water-soluble elastin, method for producing the same, food and medicine containing the same |
| JP2007045722A (en) * | 2005-08-08 | 2007-02-22 | Kyushu Institute Of Technology | Water-soluble elastin, and food and pharmaceutical each containing the same |
| JP5935094B2 (en) | 2010-02-19 | 2016-06-15 | 国立大学法人九州工業大学 | Chemically modified water-soluble elastin, mixed gel of chemically modified water-soluble elastin and collagen, and production method thereof |
-
1994
- 1994-07-22 JP JP17109594A patent/JP3687995B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0833661A (en) | 1996-02-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4167045A (en) | Cardiac and vascular prostheses | |
| EP0457430B1 (en) | Collagen constructs | |
| JP3784798B2 (en) | Implantable tubular prosthesis made of polytetrafluoroethylene | |
| AU2001255741B2 (en) | Decellularized vascular prostheses | |
| US5628781A (en) | Implant materials, methods of treating the surface of implants with microvascular endothelial cells, and the treated implants themselves | |
| Hanel et al. | Current PTFE grafts: a biomechanical, scanning electron, and light microscopic evaluation. | |
| Ichikawa et al. | Use of a bovine jugular vein graft with natural valve for right ventricular outflow tract reconstruction: a one-year animal study | |
| WO1992009312A1 (en) | Implant material | |
| AU2001255741A1 (en) | Decellularized vascular prostheses | |
| JP2002527191A (en) | Cardiovascular component for transplantation and method of manufacturing the same | |
| JPH07250887A (en) | Artificial blood vessel and its production | |
| JP3687995B2 (en) | Artificial blood vessel and manufacturing method thereof | |
| Noishiki et al. | Healing pattern of collagen-impregnated and preclotted vascular grafts in dogs | |
| JP2003126125A (en) | Artificial blood vessel and method of preparing it | |
| JP3573554B2 (en) | Artificial blood vessel and method for producing the same | |
| JPH05269196A (en) | Multilayered artificial blood vessel | |
| JP2005507755A (en) | Synthetic vascular prosthesis | |
| JPH0576588A (en) | Composite artificial blood vessel | |
| JPH0240341B2 (en) | ||
| JPS62152468A (en) | Tubular organ prosthetic article having composite structure | |
| JP4277939B2 (en) | Cell growth factor-producing cell-embedded medical material | |
| JPH0889569A (en) | Artificial blood vessel and its production | |
| CN114225115B (en) | Nondestructive modified blood vessel substitute containing living cells and preparation method thereof | |
| JPH04187154A (en) | Artificial blood vessel and patch | |
| JPS602254A (en) | Artificial blood vessel and production thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20040430 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20040728 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20040927 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20050607 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20050607 |
|
| R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20080617 Year of fee payment: 3 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20090617 Year of fee payment: 4 |
|
| LAPS | Cancellation because of no payment of annual fees |