JP3966045B2

JP3966045B2 - Collagen nonwoven fabric, its production method, its treatment method and apparatus

Info

Publication number: JP3966045B2
Application number: JP2002101705A
Authority: JP
Inventors: 幸弘守永; 和久松田; 伸年土居; 良輝中野
Original assignee: Nipro Corp
Current assignee: Nipro Corp
Priority date: 2002-04-03
Filing date: 2002-04-03
Publication date: 2007-08-29
Anticipated expiration: 2022-04-03
Also published as: JP2003301362A

Description

【０００１】
【発明の属する技術分野】
本発明は生分解性物質であるコラーゲンからなる不織布に関する。詳細には手術時における止血用メッシュや、各種補填材、補綴材、または再生医療における移植用細胞培養基材、あるいは徐放性ＤＤＳ担体、遺伝子治療用担体などの用途に利用可能な、医療用コラーゲン不織布、その製造方法および装置に関する。
【０００２】
【従来の技術】
種々に処理され調製された形態の医療用コラーゲン材料が、外科的処置並びに外傷の治療に有用である。コラーゲンは生体を構成する主要なタンパク質であり、生体適合性、組織再生、細胞増殖、止血作用等の優れた効果を持ち合わせている為に、特に医療分野において有用な素材である。これらのコラーゲンを用いた医療用材料器具の製造においては、動物や人の組織を直接処理して、組織の形状を維持したまま、主にコラーゲン質のみをそのまま利用したり、さらにこれを後加工する場合もあるが、これらは使い勝手の良い医療用具の形状や剤形として、任意に加工する事が難しい上、コラーゲンの抗原性発現部位がそのまま残された状態である為に問題があった。
そこで、医療用材料器具に使用するコラーゲンは、主として原料である動物から、酸、アルカリ、中性等の条件下で酵素などにより抽出し、粘調なコラーゲン溶液またはこの溶液を乾燥させた固体の状態として得る方法が一般的に用いられるようになった。また更に、ペプシン処理を施すことによって抗原性発現部位を除去し、体内または体表面に移植した際に抗原性が無い、より医療基材に好適なコラーゲン（アテロコラーゲン）を得る方法も用いられている。
【０００３】
このようにして得られたコラーゲン溶液から、医療用基材を製造する方法としては、コラーゲン溶液を凍結乾燥して、スポンジ状の基材を製造する方法や、コラーゲン溶液を湿式または乾式紡糸法で紡糸し、繊維状の基材を製造する方法などが種々の方法が知られている。
特開昭５０−１４１１９号公報には、コラーゲン物質をアミン類、アルカリおよび硫酸ソーダを使用する方法により分子状に水中に可溶化し得たコラーゲン水溶液を紡糸原液として紡糸コラーゲン繊維を生成し、ステープル長に切断して耐水処理するかまたは耐水処理してから切断してコラーゲン繊維ステープルとし、次いで乾式法または湿式法により不織布状に形成することを特徴とする、外科用創傷被覆材の製造方法が記載される。
特公昭５４−３６４４１号公報には、イオン化性、水不溶性のコラーゲンの部分塩である凝血−接着性繊維を、９５〜８０容量部の水混和性有機液体と５〜２０容量部の水からなる混合物中にランダムに縦横に移動させつつエタノール槽底部に沈降させ（スラリー化）し、この繊維を広げてウェッブを製造し、このウェッブを乾燥する、コラーゲン不織布の製造方法が記載される。
特開２０００−９３４９７、特開２０００−２１０３７６及び特開２０００−２７１２０７には、コラーゲン水溶液をエタノール等の親水性有機溶媒中に吐出し、コラーゲンを糸状に成形し、槽底部に沈降しスラリー化したコラーゲンを取り出して、コラーゲン糸の積層構造物を作製する方法が記載されている。
【０００４】
【発明が解決しようとする課題】
従来方法で得られるコラーゲン不織布は、コラーゲン繊維ステープルや親水性有機溶媒中へのコラーゲン吐出物の分散を均一にすることは実質的に不可能であるため、部分的に強度が弱い部分が発生したり、均一な厚みを持った不織布が得られない等の問題があった。また、従来の製造法では、一旦コラーゲンの糸状物をステープル状に切断したり、スラリー化したコラーゲンを取り出す等の煩雑な作業が必要であり、実験室レベルの製造は出来たとしても、工業的生産は困難であった。
【０００５】
【課題を解決するための手段】
本発明はこのような問題に鑑みてなされたものであり、コラーゲンが均一に分散された不織布、その簡便な製造方法および製造装置を提供する。
【０００６】
すなわち、本発明は、
（１）可溶化されたコラーゲン溶液を紡糸原液として紡糸されたコラーゲン糸状物を複数本平行に配列されてなる第１の層と第２の層が、第１の層と第２の層との糸状物の配列方向が角度をなすように積層し、相互に接着していることを特徴とする、コラーゲン不織布、
（２）第１の層または第２の層の上に、さらにコラーゲン糸状物が複数本平行に配列された第３の層が、第３の層の糸状物の配列方向と接する層の糸状物の配列方向とが角度をなすように積層し、相互に接着している、上記（１）のコラーゲン不織布、
（３）コラーゲン糸状物が接着性を有する、上記（１）記載のコラーゲン不織布、
（４）平行に配列したコラーゲン糸状物の糸と糸との間隔が約０〜４０ｍｍである、上記（１）記載のコラーゲン不織布、
（５）平行に配列したコラーゲン糸状物のなす鋭角の角度が約０〜５°である、上記（１）記載のコラーゲン不織布、
（６）コラーゲン糸状物の表面が生分解性物質でコーティングされた、上記（１）記載のコラーゲン不織布、
（７）生分解性物質がコラーゲンである、上記（６）記載のコラーゲン不織布、
（８）上記（１）記載のコラーゲン不織布の層の糸状物が互いに絡みあってなるフェルト状成形物、
（９）一定の回転軸のもとに回転する板状部材に、可溶化されたコラーゲン溶液を紡糸原液として紡糸されたコラーゲン糸状物を平行に巻き取り層（第１の層）を形成させ、該層を形成する糸状物の配列方向と角度をなすようにコラーゲン糸状物を平行に巻き取り、さらに層（第２の層）を形成させることを特徴とする、コラーゲン不織布の製造方法、
（１０）第１の層を形成させ、ついで板状部材の回転軸を変え、さらに第２の層を形成させる、上記（９）記載のコラーゲン不織布の製造方法、
（１１）層を形成する糸状物の配列方向と鋭角の角度が約２０°以下となるようにコラーゲン糸状物を巻き取り、ついで板状部材の回転軸を変え、巻き取った糸状物の配列方向と鋭角の角度が約７０〜９０°となるようにコラーゲン糸状物をさらに巻き取る、上記（９）記載のコラーゲン不織布の製造方法、
（１２）層を形成する糸状物の配列方向が互いに角度をなすように第２の層を形成させた、生分解性物質の溶液に浸漬し、乾燥する、上記（９）記載のコラーゲン不織布の製造方法、
（１３）第２の層を形成させた後、各層の糸状物同士を絡ませ、フェルト状に成形する、上記（９）記載のコラーゲン不織布の製造方法、
（１４）i)可溶化されたコラーゲン溶液を紡糸原液として紡糸されたコラーゲン糸状物が巻き取られる部分である板状部材、ii)該板状部材に連結された内軸、iii)該内軸を収容可能な内腔を有し、先端が斜めの切り口を有する円筒状の外軸、iv)外軸および内軸を回転させる為の駆動機構、およびv)該駆動機構を制御し、外軸および内軸各々の回転を制御する制御機構を有し、板状部材が、内軸との連結部を軸にして板状部材の面に対して水平方向に回動可能であり、かつ外軸の内部に内軸が収容され、外軸の先端の切り口が板状部材の縁部に接した構造を有することによって、自動的に板状部材を方向転換し、コラーゲン糸状物を板状部材の複数方向に巻き取る機能を有することを特徴とする、コラーゲン不織布の製造装置、および
（１５）さらに、コラーゲン糸状物を板状部材の回転軸方向に往復移動させながら送り出す、糸送り機構を有する、上記（１４）記載のコラーゲン不織布の製造装置に関する。
【０００７】
可溶化されたコラーゲンとは、溶媒に溶解できるよう処理が施されたコラーゲンである。例えば、酸可溶化コラーゲン、アルカリ可溶化コラーゲン、酵素可溶化コラーゲン、中性可溶化コラーゲン等の可溶化コラーゲンが挙げられる。特に可溶化処理と同時にコラーゲンの抗原決定基であるテロペプタイドの除去処理が施されている、アテロコラーゲンが好適である。これらコラーゲンの可溶化方法については、特公昭４６−１５００３号公報、特公昭４３−２５９８３９号公報、特公昭４３−２７５１３号公報等に記載されている。またコラーゲンの由来については、ウシ、ブタ、鳥類、魚類、ウサギ、ヒツジ、ネズミ、ヒト等の動物種の皮膚、腱、骨、軟骨、臓器等から抽出されるものである。コラーゲンのタイプとしてはＩ型、III型等の分類可能なタイプのうちいずれかに限定されるものではないが、取り扱い上の観点から、Ｉ型が特に好適である。
【０００８】
可溶化されたコラーゲン溶液の溶媒としてはコラーゲンを可溶化できるものであれば特に限定されない。代表的なものとしては塩酸、酢酸、硝酸等の希酸溶液や、エタノール、メタノール、アセトン等の親水性有機溶媒と水との混合液、水などが挙げられる。これらは単独または２種以上任意の割合で混合して用いても良い。このうち最も好ましくは水である。
また、コラーゲン溶液のコラーゲン濃度は、紡糸可能な濃度であれば特に限定されないが、好ましくは、約４〜１０重量％であり、さらに好ましくは、約５〜７重量％である。
【０００９】
可溶化されたコラーゲン溶液を紡糸原液として紡糸されるとは、コラーゲン溶液を原料として湿式紡糸等の種々公知の紡糸方法（特開平０６−２２８５０５号公報、特開平０６−２２８５０６号公報、特開２０００−９３４９７号公報、特開２０００−２１０３７６号公報及び特開２０００−２７１２０７号公報等）により紡糸されることである。
コラーゲン糸状物は、通常の糸のように柔軟性を有する巻き取り可能なものであればその径は特に限定はされないが、約５μｍ〜１．５ｍｍ程度の外径を有するものが好適で、更に約１０〜２００μｍ程度の外径を有するものが最適である。
コラーゲン糸状物が湿式紡糸法により紡糸される場合、本発明で用いるコラーゲン糸状物は、湿式紡糸法において生成された乾燥前（湿潤状態にある）の糸状物であってもよく、紡糸後に乾燥、架橋処理等を施した糸状物であってもよい。
【００１０】
本発明で用いられるコラーゲン糸状物を作製するための湿式紡糸法としては、親水性有機溶媒を使用する方法、架橋剤を使用する方法など様々な方法が挙げられる。中でも特に親水性有機溶媒を用いて紡糸されたコラーゲン糸状物が好適に用いられる。
親水性有機溶媒を用いて湿式紡糸を行う場合、通常、コラーゲン溶液をノズル等から連続的に親水性有機溶媒等の脱溶媒剤の充填された浴槽中に吐出し、脱水及び凝固させることによりコラーゲン糸状物が得られる。用いる親水性有機溶媒としては、例えば、エタノール、メタノール、イソプロパノールなどの炭素数１から６のアルコール類、アセトン、メチルエチルケトンなどのケトン類等が挙げられる。これらは単独または２種以上を任意の割合で混合して用いても良い。このうち最も好ましい溶媒はエタノールである。親水性有機溶媒の含水率は、通常約５０容量％以下であり、好ましくは約３０容量％以下である。親水性有機溶媒を用いたコラーゲン溶液の紡糸（脱水・凝固）工程は通常、室温ないし４２℃程度で行われ、一連の脱水および凝固による処理時間は約４〜５秒から５時間である。
【００１１】
コラーゲン糸状物が複数本平行に配列された層とは、複数本の糸状物が同一平面上に略均等な間隔をあけて直線的に配置された層であり、同じ層において、配列された糸状物のなす鋭角の角度は約０〜５°であり、好ましくは約０°である。また、同じ層でのコラーゲン糸状物の間隔は、通常、約０〜４０ｍｍであり、好ましくは約０〜１０ｍｍ、さらに好ましくは約０〜１ｍｍである。
【００１２】
第１の層の糸状物の配列方向と第２の層の糸状物の配列方向が角度をなすとは、第１の層に配列された糸状物と第２の層に配列された糸状物との配列方向のなす鋭角の角度が０°ではないことを示す。また、第１の層と第２の層が積層するとは、第１の層と第２の層が互いの面で接触している状態である。本発明のコラーゲン不織布は、少なくともこのような２層からなる積層体を含むコラーゲン不織布である。
本発明のコラーゲン不織布は、第１の層または第２の層の上に、同様のコラーゲン糸状物が複数本平行に配列された第３の層が、第１層または第２層の糸状物の配列方向と第３の層の糸状物の配列方向とが角度をなすように積層し、相互に接着された３層からなる積層体を含むコラーゲン不織布であってもよい。さらに、上記第１の層および第２の層からなる積層体の両面に、同様のコラーゲン糸状物が複数本平行に配列された層が、同様に積層された４層からなる積層体を含むコラーゲン不織布であってもよく、同様に積層された５層以上からなる積層体を含むコラーゲン不織布であってもよい。
３層以上からなる積層体を含む場合、コラーゲン糸状物の配列方向が角度をなすのは、互いに接する層の糸状物配列方向であって、接していない層同士の糸状物配列方向は必ずしも角度をなす必要はなく、なす角度が０°であってもよい。例えば、３層からなる積層体において第３の層が第２の層の上に積層された場合、第１の層と第２の相、ならびに、第２の層と第３の層の糸状物の配列方向は角度をなす必要があるが、第１の層と第３の層の糸状物の配列方向は、角度をなしていてもよく、角度が０°であってもよい。
【００１３】
複数の層からなる積層体としては、積層される糸状物の配列方向のなす角度が一定に保たれた積層体であってもよく、糸状物の配列方向のなす角度がランダムな積層体であってもよい。前者としては、例えば、第１の層の糸状物の配列方向と他の層の糸状物の配列方向がなす鋭角の角度が約２０°以下であるように積層された複数の層からなる積層体が挙げられる。また、そのような積層体が複数積み重ねられることによって形成された積層体であってもよい。この場合、積層される第１の積層体と第２の積層体が接する部分の層の糸状物の配列方向は角度をなしている。３つ以上の積層体を積み重ねる場合、その角度は一定に保たれていてもよく、ランダムであってもよい。前者としては、例えば、その鋭角の角度が約７０〜９０°となるように、複層の積層体が積み重ねられた積層体が挙げられる。
【００１４】
さらに、互いに接する層の糸状物同士がその接触部で接着されることによって、不織布が形成される。例えば、コラーゲン糸状物が、湿式紡糸法において生成された乾燥前（湿潤状態にある）の糸状物である場合は、積層後、乾燥処理を施すことによって、接着がなされる。コラーゲン糸状物が、紡糸後に乾燥、架橋処理等を施した糸状物である場合は、積層後、生分解性物質、例えば、生分解性ポリマーを不織布上に噴霧もしくは含浸し、乾燥処理を施すことによって、接着がなされる。
【００１５】
上記方法で得られるコラーゲン不織布は、必要によりさらに種々公知の物理的または化学的架橋処理を施してもよい。架橋処理を施す段階は問わない。すなわち各種架橋処理を施した糸状物で前記不織布を形成しても良いし、前記不織布を形成した後各種架橋処理を施しても良い。また、２種以上の架橋処理を併用しても良く、その際、処理の順序は問わない。この架橋処理により、生体内に移植された際に分解・吸収される時間を、未架橋の場合に比較して飛躍的に遅延させることが可能となり、また物理的強度も向上する。したがって、コラーゲン不織布を生体の欠損部を補填または補綴する場合に、組織の再生を完了するまでの期間、体内で必要な膜強度を維持することが可能となる。
物理的架橋方法の例としてはγ線照射、紫外線照射、電子線照射、プラズマ照射、熱脱水反応による架橋処理などが挙げられ、化学的架橋方法の例としては、例えばジアルデヒド、ポリアルデヒドなどのアルデヒド類、エポキシ類、カルボジイミド類、イソシアネート類などとの反応、タンニン処理、クロム処理などが挙げられる。
【００１６】
また、上記方法で得られるコラーゲン不織布は、生分解性物質でコーティングを施してもよい。生分解性物質としては、コラーゲン、ヒアルロン酸などが挙げられる。
生分解性物質でコーティングを施す方法の一例としてはバインダー処理が挙げられる。バインダー処理とは、不織布に、溶液状の材料を含浸させた後、適当な乾燥方法で乾燥を行い、不織布中の糸状物同士の結合を補強する処理である。このバインダー処理によりコラーゲン不織布は膜状に成形され、未処理の不織布よりもはるかに物理的強度が向上し、従って縫合強度も格段に向上する。
ただし、バインダー処理を行う際には、コラーゲン不織布に架橋処理が施されていない場合、不織布層自身が含浸させた溶媒に溶解してしまう場合があるため、前述の架橋方法等で、前もって架橋処理を施しておくのが望ましい。これら以外にも、コラーゲン不織布中の糸状物同士の接合を補強する種々の方法を適宜使用することができる。
【００１７】
また、本発明のコラーゲン不織布は、各層の糸状物を絡ませる処理を施してもよい。処理方法としては、例えば、ニードルパンチにより積層されたコラーゲン不織布の各層の糸同士を複雑且つランダムに絡み合わせる処理方法が挙げられる。このような処理によって、フェルト状に成形されたコラーゲン不織布を得ることができる。フェルト状に成形されたコラーゲン不織布は、必要に応じてバインダー処理等を行ってもよい。
【００１８】
コラーゲン不織布およびその２次加工物は、医療用として使用する前に、γ線滅菌、紫外線滅菌等の公知の方法によって、滅菌処理を施す必要がある。熱滅菌はコラーゲンの耐熱性の低さから好ましくない。
また、コラーゲン以外の生分解性物質であるポリグリコール酸、ポリ乳酸、ポリ乳酸ポリグリコール酸共重合体、ポリリン酸などからなる１種もしくは数種の糸状物を用いても、同様な医療用不織布を作製することが可能である。
【００１９】
次に、コラーゲン不織布の製造方法について説明する。
板状部材とは、それ自体が回転等することによって、コラーゲン糸状物を巻き取ることが出来る部材である。板状部材の材質は、コラーゲン糸状物と癒着が生じず、巻き取り状態を維持できる材質であれば特に限定されないが、好ましくは、金属、樹脂等であり、さらに好ましくは、ステンレス、ポリフッ化エチレン系繊維等である。板状部材の形状は、少なくとも２方向にコラーゲン糸状物を巻き取ることが可能な形状であれば特に限定されないが、少なくとも３つの辺を有する板状もしくは枠状であることが好ましく、さらに好ましくは略正方形の板状もしくは枠状である。
【００２０】
板状部材を一定の回転軸のもとに回転するとは、板状部材をその面に対して水平方向に貫通する軸を中心として自転することである。また、板状部材の回転軸を変えるとは、前記回転軸とは別の板状部材を貫通する軸を中心として「前記回転軸と交差する、板状部材の別の１辺と平行な軸を中心として」自転させることをいう。回転軸を変えることによって、コラーゲン糸状物を板状物の別の方向に巻き取り、この操作を繰り返すことによって、本発明のコラーゲン不織布が得られる。
板状部材を回転させる駆動方法は、特に限定されないが、機械的な一定の駆動力によってなされることが好ましい。また、板状部材の回転軸を変える操作は、手動で方向転換させてもよく、自動的に回転軸を変える装置等を用いて行ってもよい。工業的にコラーゲン不織布を製造する場合は、機械的に自動で回転軸を変える装置を用いることが好ましい。
通常、板状部材にコラーゲン糸状物を一定の巻き取り幅で巻き取る場合、板状部材の一辺を複数回往復させるように巻き取った後に、板状部材の回転軸が変えられる。糸状物を往復して巻き取る際の、往きと帰りの糸状物の配列方向のなす鋭角の角度は、通常、約２０°以下であり、好ましくは約１０°以下である。回転軸が変えられた後も同様に巻き取りが行われるが、回転軸が変えられる前の糸状物の配列方向と回転軸が変えられた後の糸状物の配列方向とのなす鋭角の角度は、通常、約７０〜９０°であり、好ましくは、約８０〜９０°である。
さらに、上記のように層を形成する糸状物の配列方向が互いに角度をなすようにコラーゲン糸状物を巻き取った後、生分解性ポリマー溶液に浸漬し、乾燥してもよく、また、各層の糸状物同士を絡ませる処理を施すことによってフェルト状の成型物を得てもよい。
【００２１】
以下に、コラーゲン不織布の製造装置について説明する。
本発明は、上記のように板状部材を自動的に方向変換するための装置に関するものである。
板状部材に連結された内軸とは、板状部材と連結された部材であり、内軸を回転させることによって板状部材を回転させることができる。外軸は円筒状で、その先端が斜めの切り口を有しており、内軸と外軸は二重構造になっている。内軸と外軸は回転するための駆動機構を有し、その駆動機構を制御する制御機構によって、各々独立して回転、停止させることができ、内軸と外軸共に回転させることもできる。
内軸と板状部材の連結は、内軸先端と板状部材の一頂点が板状部材の面に対して水平方向に回動可能に連結されている。また、板状部材の縁部は外軸先端の斜めの切り口に接している。このような構造において、内軸を固定して外軸のみを回転させることにより、外軸の斜めの切り口の向きが変えられ、板状の巻き取り具の方向を変えることができる。実際にコラーゲン糸状物を巻き取るときの好ましい態様としては内軸と外軸とが共に連動して回転し、それに伴って板状部材が回転する。コラーゲン糸状物を一定回数巻き取り終わった時点で外軸の回転を停止し、内軸だけを回転させ、板状部材の向きを切り替え、再び内軸と外軸を共に回転させて、コラーゲン糸状物を巻き取っていく、といった態様が挙げられる。このようにして、自動的に、板状部材の回転軸が変えられ、複数方向にコラーゲン糸状物を巻き取ることが可能となる。
また、本発明のコラーゲン不織布製造装置は、通常、巻き取りの際、コラーゲン糸状物を板状部材の回転軸方向に往復運動させながら送り出すための糸送り機構を備えている。
【００２２】
本発明により得られるコラーゲン不織布およびその２次加工物は、コラーゲンが元来持ち合わせている、生体内および体表面における分解性および吸収性を有し、毒性もほとんどなく、自体公知の方法に従って、医療用目的等で人間や動物に安全に使用できる。
例えば組織工学分野・再生医療分野における補填および補綴目的で体内に移植される各種膜状物、布状物、袋状物および管状物等（移植用基材）に用いることができる。膜状物としては心膜、胸膜、脳硬膜、漿膜等の代替膜が挙げられ、管状物としては人工血管、ステント、人工神経チャンネル、人工気管、人工食道、人工尿管等が挙げられる。本発明者らが特開２０００−２７１２０７号、特開２０００−２１０３７６にて開示している癒着防止膜に使用することも可能である。
また、接着性細胞等の各種細胞を体外で培養するための基材（細胞培養基材）としても利用できる。上記移植用基材上で、あらかじめ繊維芽細胞、軟骨細胞等の体組織を形成する細胞を常法に従って一定期間培養し、移植用基材の形状に細胞を増殖させて組織を形成した後に、体内へ移植することもできる。
さらに、各種成長因子、薬剤、ベクター等を含浸させ、ドラッグデリバリーシステム担体、徐放性薬剤用担体、遺伝子治療用担体等として利用することもできる。
【００２３】
本発明のコラーゲン不織布の二次加工物としては、コラーゲン製管状物やその他任意の形状を有するコラーゲン製３次元構造物を作製することが可能である。
コラーゲン製管状物を作製する方法としては、コラーゲン不織布を、コラーゲン溶液を接着剤として、ポリフッ化エチレン系繊維製のチューブ等に巻き付けてゆき、乾燥後、チューブを抜く方法が挙げられる。加工されたコラーゲン製管状物に対して、さらに架橋処理を施しても良い。
【００２４】
更に複雑な形状の３次元構造物を作製する方法としては例えば次のような方法が挙げられる。
まず、あらかじめ目的とする３次元構造物の鋳型(雌)を作製しておく。鋳型の素材は特に限定されないが、ポリフッ化エチレン系繊維等の撥水性の高い材料が好ましい。また、鋳型には少なくとも１箇所に穴が開けられていることが好ましく、更に割り型の鋳型が好ましい。
次に、この鋳型にコラーゲン不織布、好ましくはフェルト状に加工された不織布を封入し、穴から生分解性ポリマー溶液を注ぎ込み、各種方法を用いて乾燥させることにより、目的とする複雑な３次元構造物が得られる。
【００２５】
【発明の実施の形態】
以下に、本発明の実施の形態に係るコラーゲン不織布の製造装置について、図を用いて具体的に説明する。なお、本実施の形態は一例であり、本発明は実施の形態に限定されるものではない。
【００２６】
図１にコラーゲン不織布の製造装置の一例を示す。該製造装置は、図に示すように、巻き取り装置１および糸送り装置２から構成されている。
巻き取り装置１は、i)コラーゲン糸状物３が巻き取られる部分である板状もしくは枠状の板状部材１１、ii)該板状部材に連結された内軸１２（図２）、iii)該内軸を収容可能な内腔１３１を有し、先端が斜めの切り口１３２を有する円筒状の外軸１３（図２）、iv)外軸１３および内軸１２を回転させる為の駆動機構１４、およびv)該駆動機構１４を制御し、外軸１３および内軸１２の回転を各々制御する制御機構１５からなるものである。一方、糸送り装置２はローラー２１と往復機構２２とからなるものである。
【００２７】
次に、図２を用いて、板状部材１１と内軸１２との連結部について説明する。板状部材１１は、内軸１２の連結孔１２２と板状部材１１の連結孔１１１が一致するようにして、内軸１２の先端の嵌合部１２１に挟まれた状態で、連結孔１２２および連結孔１１１を貫通するピン４によって連結されている。このため、板状部材１１は、ピン４を軸にして、板状部材の面に対して水平方向に回動可能である。ただし、実施には板状部材１１の回動は外軸１３によって制限される。内軸１２は外軸１３の内部に収容されており、外軸１３先端の切り口１３２が板状部材１１の縁部１１２に接するよう配置される（図３）ためである。
【００２８】
また、図３を用いて、板状部材１１の方向転換を自動的に行う機構を説明する。
図３（ａ）は、一定方向に規定回数の巻き取りを行っている状態を示す。上記したように、板状部材１１は縁部１１２で外軸１３先端の切り口１３２に接しており、切り口１３２は縁部１１２Ｂの方向を向いた状態のままで外軸１３と内軸１２が共に回転するため、板状部材１１の縁部１１２Ａは外軸１３と同一方向に固定されている。この状態のままで、板状部材１１が回転することによって、図面手前側から送り出されるコラーゲン糸状物３が、板状部材１１の縁部１１２Ａに対して垂直方向に巻き取られていく。巻き取り時のコラーゲン糸状物の間隔は一定間隔となるように、糸送り機構によって調整され、巻き取り操作は縁部１１２Ａを一定回数往復し（図３（ｂ））、一定の巻き取り回数が終了するまで行われる。このときの糸状物の巻き取り間隔は通常、約０〜４０ｍｍであり、好ましくは約０〜１０ｍｍ、さらに好ましくは約０〜１ｍｍである。また、糸状物のなす鋭角の角度は、通常、約２０°以下であり、好ましくは、約１０°以下である。
【００２９】
巻き取り終点１１３までコラーゲン糸状物が巻き取られた時点で、内軸の回転を停止し、外軸はそのまま回転させる。板状部材１１は回転を停止し、外軸の切り口１３２が向きを変えることによって、板状部材の面に対して水平方向に方向転換を行う。図３（ｃ）は、板状部材１１が方向変換している途中の状態を示す。外軸１３先端の切り口１３２は板状部材の面に対して垂直方向（手前側）を向いた状態であり、このため板状部材１１は縁部１１２Ａおよび１１２Ｂ共に外軸１３とは異なる方向にある。このような状態を経て、さらに外軸１３のみを回転させると図３（ｄ）に示すように、板状部材１１の方向変換が行われる。
図３（ｄ）の状態では、外軸１３の先端の切り口１３２は板状部材１１の縁部１１２Ａの方向を向いた状態であり、板状部材１１の縁部１１２Ｂが外軸１３と同一方向にある。この状態に達した時点で、再び内軸１２を外軸１３と共に回転させることによって、今度は縁部１１２Ｂに対して垂直方向に、巻き取り始点１１４を開始点として、コラーゲン糸状物の巻き取りが開始される。
【００３０】
【実施例】
次に実施例、実験例を示し本発明を詳細に説明する。
【００３１】
実施例１コラーゲン不織布の作製
ブタ由来Ｉ型、III型混合コラーゲン粉末(日本ハム株式会社製、ＳＯＦＤタイプ、Lot No.０１０２２２６)を注射用蒸留水(大塚製薬社製)に溶解し、７重量%に調製する。そして、この７重量%コラーゲン水溶液を充填したシリンジ(ＥＦＤ社製 Disposable Barrels/Pistons 、５５cc)に充填し、シリンジに装着した針より該コラーゲン水溶液を空気圧により吐出した。この際シリンジに装着の針はＥＦＤ社製 Ultra Dispensing Tips (２７G、ID :φ０．２１ mm)を使用した。吐出した７重量 %コラーゲン水溶液は脱水され糸状になったのち、エタノール槽から引き上げられた。エタノール槽から引き上げられたコラーゲン糸状物を、エタノール槽とは完全に分離独立した第２のエタノール槽に室温で約３０秒間、浸漬し、さらに凝固を施した。続いて、第２のエタノール槽から引き上げられたコラーゲン糸状物を図１と同様の装置を用いて、１辺１５ｃｍ、厚さ５ｍｍの板状部材を１５ｒｐｍで回転させ、コラーゲン糸状物を板状部材に巻き取った。板状部材の直前には、板状部材に均等にコラーゲン糸状物を巻き取っていくためにコラーゲン糸状物の水平位置を周期的に移動させる機構が備え付けられており、その往復速度は１．５ｍｍ／秒とした（糸状物は約６ｍｍの間隔で巻き取られる）。巻き取り装置は、５００回巻き取るたびに板状部材の回転軸を９０度方向転換させるように設定しておき、５００回の巻き取りを６回繰り返し(合計巻き取り数３０００回)、板状部材の両面にコラーゲン糸状物の層を有するコラーゲン巻き取り物を得た。次にこのコラーゲン巻き取り物を、常温で４時間自然乾燥した後、巻き取り物の端部に沿って砕断し、２枚のコラーゲン不織布を得た。
【００３２】
実施例２コラーゲン膜状物への２次加工
実施例１にて作製されたコラーゲン不織布を、バキュームドライオーブン(ＥＹＥＬＡ社製；VOS-３００VD型)と油回転真空ポンプ(ＵＬＶＡＣ社製；GCD１３５-XA型)を用いて１３５℃、減圧下(１Torr以下)で２４時間熱脱水架橋反応を行った。これとは別に、ブタ由来Ｉ型、III型混合コラーゲン粉末(日本ハム株式会社製、ＳＯＦＤタイプ、Lot No.０１０２２６)を注射用蒸留水(大塚製薬社製)に溶解し、１重量%に調製したコラーゲン水溶液を作製した。この１重量％コラーゲン水溶液を、熱脱水架橋反応後のコラーゲン不織布に含浸させ、膜状に成型した後、前記と同様のバキュームドライオーブンを用いて１３５℃、減圧下(１Torr以下)で１２時間熱脱水架橋反応を行い、膜状のコラーゲン不織布を得た。
【００３３】
実施例３フェルト状のコラーゲン不織布の作製
実施例１にて作製されたコラーゲン不織布に対し、ニードルパンチで各層のコラーゲン糸状物をランダムに絡み合わせた後、７０％エタノール溶液(京都光純薬社製)を噴霧し、糸状物同士を接着させ、常温で８時間自然乾燥した。その後バキュームドライオーブン(ＥＹＥＬＡ社製；ＶＯＳ−３００ＶＤ型)と油回転真空ポンプ(ＵＬＶＡＣ社製；ＧＣＤ１３５−ＸＡ型)を用いて１３５℃、減圧下(１Torr以下)で２４時間熱脱水架橋反応を行った。このようして不織布中のコラーゲン糸状物が絡み合った構造をもつ３次元のフェルト状培養基材を作製した。
【００３４】
実験例１コラーゲン不織布を用いた細胞培養実験
実施例１で作製したコラーゲン不織布を用いて、ヒト軟骨細胞、ヒト繊維芽細胞の培養を行った。ヒト繊維芽細胞の培養には、Medium 106S (基礎培地)500ｍＬおよびLSGS(Low Serum Growthfactor Supplement)10ｍＬ（共にCascade Biologics 社製）を混合した混合培地を用いた。ヒト軟骨細胞の培養には、Basal Medium 500ｍＬおよびGrowth Supplement 10ｍＬ（共にCELL APPLICATIONS 社製）を混合した混合培地を用いた。
まず、コラーゲン不織布をシャーレ（ＣＯＲＮＩＮＧ社製、６ウェル）に静置し、不織布上に細胞濃度４．０×１０^５個／ｍＬとなるように細胞を懸濁した上記の混合培地１ｍＬを塗布した。その後シャーレに培地３ｍＬを静かに注入し、その後、３７℃、ＣＯ_２濃度５％の培養条件で静置培養を行った。
両細胞共に培養開始直後に、細胞の基材生着の様子を観察した。
【００３５】
その結果、縦横に配列されたコラーゲン糸状物上に、それぞれの細胞について良好な生着が確認できた。このことから本発明によるコラーゲン不織布が培養基材としての機能を十分に有することが判明した。
【００３６】
実験例２フェルト状コラーゲン不織布での細胞培養実験
実施例３で作製した培養基材に対して、グルタルアルデヒド濃度０．１容量％および０．５容量％の架橋処理を施した。架橋処理後のフェルト状のコラーゲン不織布を用いて、ヒト繊維芽細胞の培養を行った。ヒト繊維芽細胞の培養には、Medium 106S (基礎培地)500ｍＬおよびLSGS(Low Serum Growthfactor Supplement)10ｍＬ（共にCascade Biologics 社製）の混合培地を用いた。
まず、フェルト状コラーゲン不織布をシャーレ（ＣＯＲＮＩＮＧ社製、６ウェル）に静置し、不織布上に細胞濃度４．０×１０^５個／ｍＬとなるように細胞を懸濁した上記の混合培地１ｍＬを塗布した。その後シャーレに培地３ｍＬを静かに注入し、その後、３７℃、ＣＯ_２濃度５％の培養条件で静置培養を行った。
培養開始１４日後における細胞の基材生着の様子を観察した。
【００３７】
その結果、縦横に配列されたコラーゲン糸状物に、細胞の良好な生着が確認できた。このことから本発明によるコラーゲン製３次元培養基材が培養基材としての機能を十分に有することが判明した。
【００３８】
実験例３膜状のコラーゲン不織布を用いた動物埋植実験（補填材としての性能確認）
実施例２で作製したコラーゲン膜状物を用いて、動物への埋植実験を行った。
以下の方法に従ってウサギ腹腔内埋植試験片を作製した。
ウサギ(♂、体重２．６ｋｇ)を正中切開し、腹壁に約１ｃｍ角の欠損部をピンセットを用いて作製した。十分に止血を行った後、実施例２で得られたコラーゲン膜状物を３ｃｍ角に裁断し、先に作製した欠損部に端部４箇所にて縫合固定した。また、コントロールとして、同様に１cm角の欠損を作製し、十分に止血を行った後に放置した部位も作製した。手術後４週間経過した時点で、コラーゲン膜状物を補填した部位、およびコントロール部の様子を観察した。
【００３９】
その結果、コントロール部は作製した欠損の痕跡がはっきりと確認できるのに対し、コラーゲン膜状物を埋植した部位では分解の進行した膜状物が欠損部位に融合し、欠損を補填している様子がうかがえる。周辺には特に顕著な炎症反応も見られず、従って本膜状物が生体適合性が良好で、かつ分解吸収性の補填材料として十分な性能を有することが判明した。
【００４０】
実験例４不織布、組織染色
実施例１にて作製されたコラーゲン不織布を、コラーゲン水溶液(１重量％)を接着剤にして、ポリフッ化エチレン系繊維製チューブに海苔巻き状に巻き付けてゆき、内径２〜３ｍｍ、全長１０ｍｍ程度のコラーゲン製筒状埋植試験片を作製した。埋植試験片形成後、バキュームドライオーブン(ＥＹＥＬＡ社製；ＶＯＳ−３００ＶＤ型）と油回転真空ポンプ（ＵＬＶＡＣ社製；ＧＣＤ１３５−ＸＡ型）を用いて１３５℃、減圧下(１Torr以下)で１２時間熱脱水架橋反応反応を行った。
【００４１】
前記コラーゲン埋植試験片をウサギ(計２羽)背部筋肉に埋植し、同時に他の箇所に対照試験片として同サイズのポリテトラフロロエチレン（ｅＰＴＦＥ）シート（厚さ０．１mm）（商品名ゴアテックスパッチ、Ｇｏｒｅｔｅｘ社製）を筒状に丸めて埋植した。埋植２週間後と４週間後にバイオプシーを採取し、HE染色を施して組織学的評価を行った。
【００４２】
その結果、埋植試験片については、いずれも特に顕著な炎症反応を示すことなく、細胞の浸潤も良好で、また経時的に移植片の分解が進行している様子が確認できた。一方、対照試験片については全く細胞浸潤は見られず、分解吸収の様子も全く確認出来なかった。従って本発明により作製されたコラーゲン単糸がいずれも既存の製品に比べて、生体適合性が良好な分解吸収性材料であることが判明した。
【００４３】
【発明の効果】
本発明のコラーゲン不織布は、コラーゲン製医療用具の材料として、不織布の状態で、通常の環境条件下保存および運搬が容易である。
本発明のコラーゲン不織布製造方法を用いれば、原料となるコラーゲン水溶液を紡糸しながら連続してコラーゲン不織布を得ることができ、工業的生産が簡単かつ容易に行える。また、湿式紡糸等で得られたコラーゲン糸状物は、通常その粘性、相互癒着性等から、織る、編むなどの通常、繊維製造分野で用いられる手法により、織布として製造することは困難であったが、本発明のコラーゲン不織布は、織る、編むといった手法を用いずに、織布と同様な均一性のある布を作製することが可能となる。本発明方法で製造されるコラーゲン不織布を用いれば、複雑な３次元構造をもつコラーゲン製医療用具や、より精密かつ再現性の高いコラーゲン製医療用具の製造も容易にできる。
【００４４】
【図面の簡単な説明】
【図１】本発明のコラーゲン不織布製造装置の一例を示す説明図である。
【図２】本発明のコラーゲン不織布製造装置の巻き取り部分の構造を示す説明図である。
【図３】本発明のコラーゲン不織布製造装置の糸巻き取り機構および板状部材の方向転換機構を示す説明図である。
【符号の説明】
１巻き取り装置
１１板状部材
１１１連結孔
１１２縁部
１２内軸
１２１嵌合部
１２２連結孔
１３外軸
１３１内腔
１３２切り口
２糸送り装置
２１ローラー
２２往復機構
３コラーゲン糸状物
４ピン [0001]
BACKGROUND OF THE INVENTION
  The present invention relates to a nonwoven fabric made of collagen which is a biodegradable substance. Specifically, it can be used for applications such as a hemostatic mesh at the time of surgery, various types of prosthetic materials, prosthetic materials, cell culture substrates for transplantation in regenerative medicine, sustained-release DDS carriers, and carriers for gene therapy. The present invention relates to a collagen nonwoven fabric, a method for manufacturing the same, and an apparatus therefor.
[0002]
[Prior art]
  Variously processed and prepared forms of medical collagen material are useful for surgical procedures as well as for the treatment of trauma. Collagen is a major protein that constitutes a living body, and has excellent effects such as biocompatibility, tissue regeneration, cell proliferation, and hemostasis, and is therefore a useful material particularly in the medical field. In the manufacture of medical materials and equipment using these collagens, animal or human tissues are directly processed to maintain the shape of the tissues while using only collagen as it is, or post-processing However, these are difficult to process arbitrarily as shapes and dosage forms of easy-to-use medical devices, and there are problems because the antigenic expression site of collagen remains as it is.
  Therefore, collagen used in medical material devices is mainly extracted from animals, which are raw materials, using enzymes, etc. under conditions of acid, alkali, neutrality, etc., and a viscous collagen solution or a solid obtained by drying this solution is used. The method of obtaining as a state has come to be generally used. Furthermore, a method of removing antigenic expression sites by performing pepsin treatment and obtaining collagen (atelocollagen) more suitable for a medical base material that is not antigenic when transplanted into the body or body surface is also used. .
[0003]
  As a method for producing a medical base material from the collagen solution thus obtained, a collagen solution is freeze-dried to produce a sponge-like base material, or a collagen solution is prepared by a wet or dry spinning method. Various methods are known such as a method of spinning to produce a fibrous base material.
  Japanese Patent Application Laid-Open No. 50-14119 discloses a method of producing a staple fiber by using a collagen aqueous solution obtained by solubilizing a collagen substance in water by a method using amines, alkali and sodium sulfate as a spinning solution. A method for producing a surgical wound dressing, characterized in that it is cut into lengths and treated with water resistance, or cut into water and then cut into collagen fiber staples, and then formed into a nonwoven fabric by a dry method or a wet method. be written.
  Japanese Patent Publication No. 54-36441 discloses a coagulation-adhesive fiber, which is a partial salt of ionizable, water-insoluble collagen, comprising 95 to 80 parts by volume of a water-miscible organic liquid and 5 to 20 parts by volume of water. A method for producing a collagen non-woven fabric is described in which the mixture is allowed to settle (slurry) at the bottom of an ethanol tank while moving vertically and horizontally in the mixture, and the fibers are spread to produce a web, and the web is dried.
  In JP-A-2000-93497, JP-A-2000-210376, and JP-A-2000-271207, an aqueous collagen solution is discharged into a hydrophilic organic solvent such as ethanol, the collagen is formed into a thread, and settled into a tank bottom to be slurried. A method is described in which collagen is taken out to produce a laminated structure of collagen threads.
[0004]
[Problems to be solved by the invention]
  Since the collagen nonwoven fabric obtained by the conventional method is substantially impossible to uniformly disperse the collagen discharge in collagen fiber staples or hydrophilic organic solvents, a part with weak strength is generated partially. Or a nonwoven fabric having a uniform thickness cannot be obtained. In addition, the conventional manufacturing method requires complicated operations such as once cutting collagen filaments into staples and taking out the slurried collagen, and even if it can be manufactured at the laboratory level, Production was difficult.
[0005]
[Means for Solving the Problems]
  This invention is made | formed in view of such a problem, and provides the nonwoven fabric in which collagen was disperse | distributed uniformly, its simple manufacturing method, and a manufacturing apparatus.
[0006]
  That is, the present invention
(1) A first layer and a second layer, in which a plurality of collagen filaments spun using a solubilized collagen solution as a spinning stock solution are arranged in parallel, are the first layer and the second layer. A non-woven collagen non-woven fabric, characterized by being laminated so that the arrangement direction of the filaments forms an angle and bonded to each other,
(2) The third layer in which a plurality of collagen filaments are arranged in parallel on the first layer or the second layer, and the filamentous layer in contact with the arrangement direction of the third layer filaments The non-woven collagen nonwoven fabric according to (1), wherein the layers are laminated so as to form an angle with each other and bonded to each other,
(3) The collagen nonwoven fabric according to the above (1), wherein the collagen thread has adhesiveness,
(4) The collagen nonwoven fabric according to (1) above, wherein the distance between the threads of collagen filaments arranged in parallel is about 0 to 40 mm,
(5) The collagen nonwoven fabric according to (1), wherein the acute angle formed by the collagen filaments arranged in parallel is about 0 to 5 °,
(6) The collagen nonwoven fabric according to (1) above, wherein the surface of the collagen filamentous material is coated with a biodegradable substance,
(7) The above (the biodegradable substance is collagen)6) Collagen nonwoven fabric as described,
(8) A felt-like molded article in which the filamentous nonwoven fabric layers described in (1) are entangled with each other;
(9) Forming a winding layer (first layer) in parallel on a collagen filament spun using a solubilized collagen solution as a spinning stock solution on a plate-like member that rotates under a fixed rotation axis, A method for producing a collagen nonwoven fabric, comprising: winding collagen filaments in parallel so as to form an angle with the arrangement direction of the filaments forming the layer, and further forming a layer (second layer);
(10) The method for producing a collagen nonwoven fabric according to (9), wherein the first layer is formed, then the rotational axis of the plate-like member is changed, and the second layer is further formed.
(11) The collagen filamentous material is wound up so that the acute angle with the arrangement direction of the filamentous material forming the layer is about 20 ° or less, and then the rotation axis of the plate-like member is changed to arrange the wound filamentous material. And a method for producing a collagen nonwoven fabric according to (9) above, further winding up the collagen filament so that the acute angle is about 70 to 90 °,
(12) The collagen non-woven fabric according to (9), wherein the second layer is formed so that the arrangement directions of the filaments forming the layer form an angle with each other, dipped in a solution of a biodegradable substance, and dried. Production method,
(13) After forming a 2nd layer, the filamentous material of each layer is entangled, and it shape | molds in a felt shape, The manufacturing method of the collagen nonwoven fabric as described in said (9),
(14)i)A plate-like member which is a portion around which a collagen filament spun using a solubilized collagen solution as a spinning dope is wound,ii)An inner shaft connected to the plate-like member,iii)A cylindrical outer shaft having a lumen capable of accommodating the inner shaft and having an oblique cut end;iv)A drive mechanism for rotating the outer shaft and the inner shaft, andv)The drive mechanism is controlled to have a control mechanism for controlling the rotation of each of the outer shaft and the inner shaft, and the plate-like member is in a horizontal direction with respect to the surface of the plate-like member about the connecting portion with the inner shaft. It has a structure that can rotate and the inner shaft is housed inside the outer shaft, and the cut end at the tip of the outer shaft is in contact with the edge of the plate member. An apparatus for producing a collagen nonwoven fabric, which has a function of winding a collagen filamentous material in a plurality of directions of a plate-like member, and
(15) Further, the present invention relates to the apparatus for producing a collagen nonwoven fabric according to (14), further including a yarn feeding mechanism that feeds the collagen filamentous material while reciprocating in the rotational axis direction of the plate member.
[0007]
  Solubilized collagen is collagen that has been treated to be soluble in a solvent. Examples thereof include solubilized collagen such as acid-solubilized collagen, alkali-solubilized collagen, enzyme-solubilized collagen, and neutral-solubilized collagen. In particular, atelocollagen, which has been subjected to the removal treatment of telopeptide, which is an antigenic determinant of collagen, at the same time as the solubilization treatment, is preferred. Methods for solubilizing these collagens are described in Japanese Patent Publication No. 46-15003, Japanese Patent Publication No. 43-259839, Japanese Patent Publication No. 43-27513, and the like. The origin of collagen is extracted from the skin, tendon, bone, cartilage, organ, etc. of animal species such as cattle, pigs, birds, fish, rabbits, sheep, mice and humans. The type of collagen is not limited to any type that can be classified, such as type I and type III, but type I is particularly preferred from the viewpoint of handling.
[0008]
  The solvent of the solubilized collagen solution is not particularly limited as long as it can solubilize collagen. Typical examples include a dilute acid solution such as hydrochloric acid, acetic acid, and nitric acid, a liquid mixture of a hydrophilic organic solvent such as ethanol, methanol, and acetone and water, and water. You may use these individually or in mixture of 2 or more types in arbitrary ratios. Of these, water is most preferred.
  The collagen concentration of the collagen solution is not particularly limited as long as it is a spinnable concentration, but is preferably about 4 to 10% by weight, and more preferably about 5 to 7% by weight.
[0009]
  Spinning using a solubilized collagen solution as a spinning solution means that various spinning methods such as wet spinning using a collagen solution as a raw material (Japanese Patent Laid-Open Nos. 06-228505, 06-228506, 2000) -93497, JP-A 2000-210376 and JP-A 2000-271207).
  The diameter of the collagen thread is not particularly limited as long as it is flexible and can be wound up like a normal thread, but preferably has an outer diameter of about 5 μm to 1.5 mm. What has an outer diameter of about 10-200 micrometers is optimal.
  When the collagen filamentous material is spun by a wet spinning method, the collagen filamentous material used in the present invention may be a filamentous material before drying (in a wet state) produced in the wet spinning method, and is dried after spinning. It may be a thread-like material subjected to a crosslinking treatment or the like.
[0010]
  Examples of the wet spinning method for producing the collagen thread used in the present invention include various methods such as a method using a hydrophilic organic solvent and a method using a crosslinking agent. Of these, collagen filaments spun using a hydrophilic organic solvent are particularly preferred.
  When performing wet spinning using a hydrophilic organic solvent, collagen is usually discharged by continuously discharging the collagen solution from a nozzle or the like into a bath filled with a desolvent such as a hydrophilic organic solvent, followed by dehydration and solidification. A thread is obtained. Examples of the hydrophilic organic solvent to be used include alcohols having 1 to 6 carbon atoms such as ethanol, methanol and isopropanol, and ketones such as acetone and methyl ethyl ketone. You may use these individually or in mixture of 2 or more types by arbitrary ratios. Of these, the most preferred solvent is ethanol. The water content of the hydrophilic organic solvent is usually about 50% by volume or less, preferably about 30% by volume or less. The spinning (dehydration / coagulation) step of a collagen solution using a hydrophilic organic solvent is usually performed at room temperature to about 42 ° C., and the treatment time by a series of dehydration and coagulation is about 4-5 seconds to 5 hours.
[0011]
  A layer in which a plurality of collagen filaments are arranged in parallel is a layer in which a plurality of filaments are arranged linearly at substantially equal intervals on the same plane, and the filaments arranged in the same layer. The acute angle formed by the object is about 0 to 5 °, preferably about 0 °. The interval between collagen filaments in the same layer is usually about 0 to 40 mm, preferably about 0 to 10 mm, more preferably about 0 to 1 mm.
[0012]
  The arrangement direction of the filaments in the first layer and the arrangement direction of the filaments in the second layer form an angle. The filaments arranged in the first layer and the filaments arranged in the second layer It is shown that the acute angle formed by the arrangement direction is not 0 °. The first layer and the second layer are stacked in a state where the first layer and the second layer are in contact with each other. The collagen nonwoven fabric of the present invention is a collagen nonwoven fabric including a laminate composed of at least such two layers.
  In the collagen nonwoven fabric of the present invention, a third layer in which a plurality of similar collagen filaments are arranged in parallel on the first layer or the second layer is composed of the filaments of the first layer or the second layer. It may be a collagen non-woven fabric including a laminate composed of three layers which are laminated so that the arrangement direction and the arrangement direction of the filaments of the third layer form an angle and are adhered to each other. Furthermore, a collagen comprising a laminate composed of four layers in which a plurality of parallel collagen filaments arranged in parallel on both sides of the laminate composed of the first layer and the second layer are laminated in the same manner. A nonwoven fabric may be sufficient and the collagen nonwoven fabric containing the laminated body which consists of 5 or more layers laminated | stacked similarly may be sufficient.
  In the case of including a laminate composed of three or more layers, the arrangement direction of the collagen filaments forms an angle in the filament arrangement direction of the layers that are in contact with each other, and the filament arrangement direction of the layers that do not contact each other is not necessarily an angle. There is no need to make, and the formed angle may be 0 °. For example, when a third layer is laminated on the second layer in a laminate composed of three layers, the first layer and the second phase, and the filaments of the second layer and the third layer However, the arrangement direction of the filaments of the first layer and the third layer may be an angle, or the angle may be 0 °.
[0013]
  The laminated body composed of a plurality of layers may be a laminated body in which the angle formed by the arrangement direction of the laminated filaments is kept constant, or the laminate formed by random the angle formed by the arrangement direction of the filaments. May be. As the former, for example, a laminate comprising a plurality of layers laminated so that the acute angle formed by the arrangement direction of the filamentous material of the first layer and the arrangement direction of the filamentous material of the other layer is about 20 ° or less. Is mentioned. Moreover, the laminated body formed by stacking two or more such laminated bodies may be sufficient. In this case, the arrangement direction of the filamentous material of the layer of the part which the 1st laminated body and 2nd laminated body which are laminated | stacked contact | connects makes an angle. When three or more laminates are stacked, the angle may be kept constant or may be random. Examples of the former include a laminate in which a multilayer laminate is stacked so that the acute angle is about 70 to 90 °.
[0014]
  Furthermore, the non-woven fabric is formed by bonding the filamentous materials of the layers in contact with each other at the contact portion. For example, when the collagen filamentous material is a filamentous material before drying (in a wet state) generated by a wet spinning method, adhesion is achieved by applying a drying treatment after lamination. When the collagen filamentous material is a filamentous material that has been subjected to drying, cross-linking treatment, etc. after spinning, after lamination, a biodegradable substance, for example, a biodegradable polymer, is sprayed or impregnated onto the nonwoven fabric and then subjected to a drying treatment. By doing so, adhesion is made.
[0015]
  The collagen nonwoven fabric obtained by the above method may be subjected to various known physical or chemical cross-linking treatments as necessary. The stage which performs a crosslinking process is not ask | required. That is, the non-woven fabric may be formed of a filamentous material that has been subjected to various cross-linking treatments, or may be subjected to various cross-linking treatments after the non-woven fabric is formed. Two or more kinds of crosslinking treatments may be used in combination, and the order of treatment is not limited. By this crosslinking treatment, it is possible to dramatically delay the time taken for decomposition and absorption when transplanted into a living body as compared with the case of non-crosslinking, and the physical strength is also improved. Therefore, when a collagen non-woven fabric is filled or prosthetic with a defect in a living body, it is possible to maintain the necessary membrane strength in the body until the tissue regeneration is completed.
  Examples of physical crosslinking methods include γ-ray irradiation, ultraviolet irradiation, electron beam irradiation, plasma irradiation, crosslinking treatment by thermal dehydration reaction, etc. Examples of chemical crosslinking methods include dialdehyde, polyaldehyde and the like. Reaction with aldehydes, epoxies, carbodiimides, isocyanates, tannin treatment, chromium treatment and the like can be mentioned.
[0016]
  Moreover, the collagen nonwoven fabric obtained by the above method may be coated with a biodegradable substance. Examples of biodegradable substances include collagen and hyaluronic acid.
  An example of a method for coating with a biodegradable substance is binder treatment. The binder treatment is a treatment in which a nonwoven fabric is impregnated with a solution-like material and then dried by an appropriate drying method to reinforce the bonding between the filaments in the nonwoven fabric. By this binder treatment, the collagen non-woven fabric is formed into a film shape, and the physical strength is much improved as compared with the untreated non-woven fabric, and therefore the suturing strength is remarkably improved.
  However, when the binder treatment is performed, if the collagen nonwoven fabric is not crosslinked, it may be dissolved in the solvent impregnated by the nonwoven fabric layer itself. It is desirable to give. In addition to these, various methods for reinforcing the bonding between the filamentous materials in the collagen nonwoven fabric can be used as appropriate.
[0017]
  Moreover, the collagen nonwoven fabric of this invention may give the process which makes the filamentous material of each layer get entangled. Examples of the treatment method include a treatment method in which the yarns of the respective layers of the collagen nonwoven fabric laminated by needle punching are entangled in a complicated and random manner. By such treatment, a non-woven collagen nonwoven fabric formed into a felt shape can be obtained. The collagen nonwoven fabric formed into a felt shape may be subjected to a binder treatment or the like as necessary.
[0018]
  The collagen nonwoven fabric and its secondary processed product must be sterilized by a known method such as γ-ray sterilization or ultraviolet sterilization before being used for medical purposes. Thermal sterilization is not preferred because of the low heat resistance of collagen.
  The same non-woven fabric for medical use can be obtained by using one or several kinds of filamentous materials composed of polyglycolic acid, polylactic acid, polylactic acid polyglycolic acid copolymer, polyphosphoric acid and the like which are biodegradable substances other than collagen. Can be produced.
[0019]
  Next, the manufacturing method of a collagen nonwoven fabric is demonstrated.
  The plate-like member is a member that can wind up the collagen filamentous material by rotating itself. The material of the plate-like member is not particularly limited as long as it is a material that does not adhere to the collagen thread and can maintain the wound state, but is preferably a metal, a resin, or the like, and more preferably stainless steel, polyfluorinated ethylene. Fiber. The shape of the plate member is not particularly limited as long as it is a shape capable of winding the collagen thread in at least two directions, but is preferably a plate shape or a frame shape having at least three sides, more preferably It has a substantially square plate shape or frame shape.
[0020]
  Rotating the plate-like member around a fixed rotation axis means that the plate-like member rotates around an axis that penetrates the plate-like member in the horizontal direction with respect to the surface. Further, changing the rotation axis of the plate-like member means that “the axis parallel to another side of the plate-like member intersecting the rotation axis is centered on an axis passing through a plate-like member different from the rotation axis. Rotating around the center. By changing the rotation axis, the collagen thread is wound in another direction of the plate and the operation is repeated to obtain the collagen nonwoven fabric of the present invention.
  The driving method for rotating the plate-like member is not particularly limited, but it is preferable that the driving method is performed with a constant mechanical driving force. Further, the operation of changing the rotation axis of the plate-like member may be manually changed in direction, or may be performed using a device that automatically changes the rotation axis. When manufacturing a collagen nonwoven fabric industrially, it is preferable to use a device that automatically changes the rotation axis.
  Usually, when a collagen thread is wound around a plate-like member with a constant winding width, the axis of rotation of the plate-like member is changed after winding the plate-like member so as to reciprocate a plurality of times. When the filamentous material is reciprocally wound, the acute angle formed by the arrangement direction of the forward and return filamentous materials is usually about 20 ° or less, and preferably about 10 ° or less. Winding is performed in the same way after the rotation axis is changed, but the acute angle between the arrangement direction of the filamentous material before the rotation axis is changed and the arrangement direction of the filamentous material after the rotation axis is changed is In general, the angle is about 70 to 90 °, and preferably about 80 to 90 °.
  Furthermore, after winding the collagen filament so that the arrangement direction of the filaments forming the layers forms an angle as described above, the collagen filament may be immersed in a biodegradable polymer solution and dried. A felt-like molded product may be obtained by performing a process of entwining the filaments.
[0021]
  Below, the manufacturing apparatus of a collagen nonwoven fabric is demonstrated.
  The present invention relates to an apparatus for automatically changing the direction of a plate-shaped member as described above.
  The inner shaft connected to the plate member is a member connected to the plate member, and the plate member can be rotated by rotating the inner shaft. The outer shaft has a cylindrical shape, the tip of which has an oblique cut, and the inner shaft and the outer shaft have a double structure. The inner shaft and the outer shaft have a drive mechanism for rotating, and can be rotated and stopped independently by a control mechanism that controls the drive mechanism, and both the inner shaft and the outer shaft can be rotated.
  The connection between the inner shaft and the plate-like member is such that the tip of the inner shaft and one vertex of the plate-like member are turnable in the horizontal direction with respect to the surface of the plate-like member. The edge of the plate member is in contact with an oblique cut at the tip of the outer shaft. In such a structure, by rotating only the outer shaft while fixing the inner shaft, the direction of the oblique cut of the outer shaft can be changed, and the direction of the plate-shaped winder can be changed. As a preferred mode for actually winding the collagen filamentous material, the inner shaft and the outer shaft rotate in conjunction with each other, and the plate member rotates accordingly. When the collagen thread has been wound up a certain number of times, the rotation of the outer shaft is stopped, only the inner shaft is rotated, the direction of the plate-like member is switched, and both the inner shaft and the outer shaft are rotated again, so that the collagen thread is rotated. The mode of winding up is mentioned. In this way, the rotation axis of the plate-like member is automatically changed, and the collagen thread can be wound up in a plurality of directions.
  Moreover, the collagen nonwoven fabric manufacturing apparatus of this invention is normally equipped with the thread | yarn feed mechanism for sending out while reciprocating the collagen thread-like material in the rotating shaft direction of a plate-shaped member in the case of winding.
[0022]
  The collagen non-woven fabric obtained by the present invention and the secondary processed product thereof are degradable and absorbable in vivo and on the surface of the body that collagen originally possesses, have almost no toxicity, and are in accordance with a method known per se. It can be used safely on humans and animals for purposes.
  For example, it can be used for various membranes, cloths, bags, tubulars and the like (transplant base materials) transplanted into the body for the purpose of filling and prosthesis in the field of tissue engineering and regenerative medicine. Examples of the membranous material include alternative membranes such as pericardium, pleura, cerebral dura mater, and serosa. Examples of the tubular material include artificial blood vessels, stents, artificial nerve channels, artificial trachea, artificial esophagus, and artificial ureter. It can also be used for the anti-adhesion film disclosed by the present inventors in JP-A Nos. 2000-271207 and 2000-210376.
  Moreover, it can utilize also as a base material (cell culture base material) for culturing various cells, such as adhesive cells, outside the body. On the base material for transplantation, cells that previously form body tissues such as fibroblasts and chondrocytes are cultured for a certain period of time according to a conventional method, and after the cells are grown in the shape of the base material for transplantation, the tissue is formed. It can also be transplanted into the body.
  Furthermore, it can be impregnated with various growth factors, drugs, vectors and the like, and can be used as a drug delivery system carrier, a sustained-release drug carrier, a gene therapy carrier and the like.
[0023]
  As the secondary processed product of the collagen nonwoven fabric of the present invention, it is possible to produce a collagen tubular product or a collagen three-dimensional structure having any other shape.
  Examples of a method for producing a collagen tubular material include a method in which a collagen nonwoven fabric is wound around a tube made of a polyfluorinated ethylene fiber using a collagen solution as an adhesive, and the tube is pulled out after drying. The processed collagenous tubular product may be further subjected to a crosslinking treatment.
[0024]
  As a method for producing a three-dimensional structure having a more complicated shape, for example, the following method can be cited.
  First, a target three-dimensional structure mold (female) is prepared in advance. The material of the mold is not particularly limited, but a material having high water repellency such as polyfluorinated ethylene fiber is preferable. Moreover, it is preferable that the mold has a hole in at least one place, and a split mold is more preferable.
  Next, a collagen non-woven fabric, preferably a non-woven fabric processed into a felt shape, is enclosed in this mold, a biodegradable polymer solution is poured from the hole, and dried using various methods, so that the desired complex three-dimensional structure is obtained. Things are obtained.
[0025]
DETAILED DESCRIPTION OF THE INVENTION
  Below, the manufacturing apparatus of the collagen nonwoven fabric which concerns on embodiment of this invention is demonstrated concretely using figures. The present embodiment is an example, and the present invention is not limited to the embodiment.
[0026]
  An example of the manufacturing apparatus of a collagen nonwoven fabric is shown in FIG. The manufacturing apparatus includes a winding device 1 and a yarn feeding device 2 as shown in the figure.
  The winding device 1i)A plate-like or frame-like plate-like member 11 which is a portion around which the collagen thread 3 is wound,ii)An inner shaft 12 (FIG. 2) connected to the plate-like member;iii)A cylindrical outer shaft 13 (FIG. 2) having a lumen 131 capable of accommodating the inner shaft and having a cut end 132 having a slanted tip.iv)A drive mechanism 14 for rotating the outer shaft 13 and the inner shaft 12, andv)The driving mechanism 14 is controlled and a control mechanism 15 for controlling the rotation of the outer shaft 13 and the inner shaft 12 is provided. On the other hand, the yarn feeding device 2 includes a roller 21 and a reciprocating mechanism 22.
[0027]
  Next, the connection part of the plate-shaped member 11 and the inner shaft 12 is demonstrated using FIG. The plate-like member 11 is sandwiched between the fitting hole 121 at the tip of the inner shaft 12 so that the connection hole 122 of the inner shaft 12 and the connection hole 111 of the plate-like member 11 coincide with each other. The pins 4 that pass through the connecting holes 111 are connected. For this reason, the plate-like member 11 can be rotated in the horizontal direction with respect to the surface of the plate-like member around the pin 4. However, in practice, the rotation of the plate-like member 11 is limited by the outer shaft 13. This is because the inner shaft 12 is accommodated inside the outer shaft 13 and the cut end 132 at the tip of the outer shaft 13 is disposed so as to contact the edge 112 of the plate-like member 11 (FIG. 3).
[0028]
  A mechanism for automatically changing the direction of the plate member 11 will be described with reference to FIG.
  FIG. 3A shows a state in which a specified number of windings are performed in a certain direction. As described above, the plate-like member 11 is in contact with the cut end 132 at the tip of the outer shaft 13 at the edge 112, and the cut 132 is the edge 1.1Since the outer shaft 13 and the inner shaft 12 rotate together while facing the direction of 2B, the edge 112A of the plate-like member 11 is fixed in the same direction as the outer shaft 13. When the plate-like member 11 is rotated in this state, the collagen thread 3 delivered from the front side of the drawing is wound up in the vertical direction with respect to the edge 112A of the plate-like member 11. The yarn threading mechanism is adjusted so that the interval between the collagen filaments during winding is constant, and the winding operation reciprocates the edge 112A a certain number of times (FIG. 3B). It is done until it ends. The winding interval of the filamentous material at this time is usually about 0 to 40 mm, preferably about 0 to 10 mm, and more preferably about 0 to 1 mm. Further, the acute angle formed by the filamentous material is usually about 20 ° or less, and preferably about 10 ° or less.
[0029]
  When the collagen thread is wound up to the winding end point 113, the rotation of the inner shaft is stopped and the outer shaft is rotated as it is. The plate-like member 11 stops rotating, and the direction of the cut surface 132 of the outer shaft is changed, so that the direction is changed in the horizontal direction with respect to the surface of the plate-like member. FIG.3 (c) shows the state in the middle of the plate-shaped member 11 changing the direction. The cut end 132 at the distal end of the outer shaft 13 is in a state in which it is oriented in a direction perpendicular to the surface of the plate-like member (front side). is there. When only the outer shaft 13 is further rotated through such a state, the direction of the plate member 11 is changed as shown in FIG.
  In the state shown in FIG. 3D, the cut end of the outer shaft 13 is cut.132Is the state of facing the edge 112 </ b> A of the plate-like member 11, and the edge 112 </ b> B of the plate-like member 11 is in the same direction as the outer shaft 13. When this state is reached, the inner shaft 12 is rotated together with the outer shaft 13 again, so that the collagen filamentous material is wound up in the direction perpendicular to the edge 112B, starting from the winding start point 114. Be started.
[0030]
【Example】
  Next, the present invention will be described in detail with reference to examples and experimental examples.
[0031]
Example 1 Preparation of collagen nonwoven fabric
  Porcine-derived type I and type III mixed collagen powder (manufactured by Nippon Ham Co., Ltd., SOFD type, Lot No. 0102226) is dissolved in distilled water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) to prepare 7% by weight. Then, this 7 wt% collagen aqueous solution was filled in a syringe (Disposable Barrels / Pistons, 55 cc manufactured by EFD), and the collagen aqueous solution was discharged from the needle attached to the syringe by air pressure. At this time, Ultra Dispensing Tips (27G, ID: φ0.21 mm) manufactured by EFD was used as a needle attached to the syringe. The discharged 7 wt% collagen aqueous solution was dehydrated and turned into a string, and then pulled up from the ethanol tank. The collagen filaments pulled up from the ethanol tank were immersed in a second ethanol tank completely separated from the ethanol tank for about 30 seconds at room temperature, and further solidified. Subsequently, the collagen filaments pulled up from the second ethanol tank are rotated at 15 rpm on a plate member having a side of 15 cm and a thickness of 5 mm using the same apparatus as in FIG. Rolled up. Immediately before the plate-like member, a mechanism for periodically moving the horizontal position of the collagen filamentous material is provided to wind up the collagen filamentous material evenly around the plate-like member, and the reciprocating speed is 1.5 mm. / Second (the filamentous material is wound at an interval of about 6 mm). The winding device is set so that the rotation axis of the plate-shaped member is turned 90 degrees every time 500 windings are performed, and 500 windings are repeated 6 times (total winding number is 3000 times). A collagen wound product having layers of collagen thread on both sides of the member was obtained. Next, this collagen wound product was naturally dried at room temperature for 4 hours, and then crushed along the end of the wound product to obtain two collagen nonwoven fabrics.
[0032]
Example 2 Secondary processing to collagen membrane
  The collagen non-woven fabric produced in Example 1 was vacuum-reduced (1 Torr) at 135 ° C. using a vacuum dry oven (manufactured by EYELA; VOS-300VD) and an oil rotary vacuum pump (manufactured by ULVAC; GCD135-XA). In the following, a thermal dehydration crosslinking reaction was carried out for 24 hours. Separately, porcine-derived type I and type III mixed collagen powder (manufactured by Nippon Ham Co., Ltd., SOFD type, Lot No. 010226) is dissolved in distilled water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) to prepare 1% by weight. An aqueous collagen solution was prepared. This 1 wt% collagen aqueous solution is applied to the collagen nonwoven fabric after the thermal dehydration crosslinking reaction.ImpregnateAfter forming into a membrane, thermal dehydration crosslinking reaction was performed for 12 hours at 135 ° C. under reduced pressure (1 Torr or less) using a vacuum dry oven similar to the above to obtain a membrane-like collagen nonwoven fabric.
[0033]
Example 3 Production of felt-like collagen nonwoven fabric
  The collagen nonwoven fabric produced in Example 1 was randomly entangled with each layer of collagen filaments with a needle punch, and then sprayed with a 70% ethanol solution (manufactured by Kyoto Hikari Pharmaceutical Co., Ltd.) to adhere the filaments together. It was naturally dried at room temperature for 8 hours. Thereafter, a thermal dehydration crosslinking reaction is performed at 135 ° C. under reduced pressure (1 Torr or less) for 24 hours using a vacuum dry oven (EYELA; VOS-300VD) and an oil rotary vacuum pump (ULVAC; GCD135-XA). It was. In this way, a three-dimensional felt-shaped culture substrate having a structure in which the collagen filaments in the nonwoven fabric are intertwined was produced.
[0034]
Experimental Example 1 Cell culture experiment using collagen non-woven fabric
  Human chondrocytes and human fibroblasts were cultured using the collagen nonwoven fabric produced in Example 1. For culture of human fibroblasts, a mixed medium in which 500 mL of Medium 106S (basal medium) and 10 mL of LSGS (Low Serum Growth factor Supplement) (both manufactured by Cascade Biologics) was used. For the culture of human chondrocytes, a mixed medium containing 500 mL of Basal Medium and 10 mL of Growth Supplement (both manufactured by CELL APPLICATIONS) was used.
  First, the collagen non-woven fabric is allowed to stand in a petri dish (CORNING, 6 well), and the cell concentration is 4.0 × 10 on the non-woven fabric.⁵1 mL of the above mixed medium in which the cells were suspended so as to be the number of cells / mL was applied. Thereafter, 3 mL of the medium was gently poured into the petri dish, and then 37 ° C, CO₂Static culture was performed under a culture condition of 5% concentration.
  Immediately after the start of culture for both cells, the state of cell substrate engraftment was observed.
[0035]
  As a result, good engraftment of each cell could be confirmed on the collagen filaments arranged vertically and horizontally. From this, it was found that the collagen nonwoven fabric according to the present invention has a sufficient function as a culture substrate.
[0036]
Experimental Example 2 Cell culture experiment with felt-like collagen nonwoven fabric
  The culture substrate prepared in Example 3 was subjected to crosslinking treatment with a glutaraldehyde concentration of 0.1% by volume and 0.5% by volume. Human fibroblasts were cultured using the felt-like non-woven collagen fabric after the crosslinking treatment. For culture of human fibroblasts, a mixed medium of 500 mL Medium 106S (basal medium) and 10 mL LSGS (Low Serum Growthfactor Supplement) (both manufactured by Cascade Biologics) was used.
  First, a felt-like collagen nonwoven fabric is allowed to stand in a petri dish (CORNING, 6 wells), and the cell concentration is 4.0 × 10 on the nonwoven fabric.⁵1 mL of the above mixed medium in which the cells were suspended so as to be the number of cells / mL was applied. Thereafter, 3 mL of the medium was gently poured into the petri dish, and then 37 ° C, CO₂Static culture was performed under a culture condition of 5% concentration.
  The state of cell substrate engraftment 14 days after the start of culture was observed.
[0037]
  As a result, good engraftment of cells could be confirmed on the collagen filaments arranged vertically and horizontally. From this, it was found that the collagen three-dimensional culture substrate according to the present invention has a sufficient function as a culture substrate.
[0038]
Experimental example 3 Animal implantation experiment using membrane-like collagen nonwoven fabric (Performance confirmation as filling material)
  Using the collagen membrane produced in Example 2, an animal implantation experiment was conducted.
  A rabbit intraperitoneal implant specimen was prepared according to the following method.
  A rabbit (sputum, weight 2.6 kg) was cut through a midline, and a 1 cm square defect was created in the abdominal wall using tweezers. After sufficient hemostasis, the collagen membrane obtained in Example 2 was cut into 3 cm square, and sutured and fixed to the previously prepared defect at four locations. In addition, as a control, a 1 cm square defect was similarly prepared, and a site that was allowed to stand after sufficient hemostasis was also prepared. At the time when 4 weeks had passed after the operation, the site supplemented with the collagen membrane and the state of the control part were observed.
[0039]
  As a result, the control part can clearly confirm the trace of the created defect, but the membrane that has been decomposed is fused to the defect site to compensate for the defect at the site where the collagen film is implanted. You can see the situation. No particularly prominent inflammatory reaction was observed in the periphery, and thus it was found that this membrane-like product has good biocompatibility and has sufficient performance as a degradable and absorbable supplementary material.
[0040]
Experimental Example 4 Nonwoven fabric, tissue staining
  The collagen non-woven fabric produced in Example 1 was wound around a polyfluorinated ethylene fiber tube in a laver shape with a collagen aqueous solution (1% by weight) as an adhesive, and had an inner diameter of 2-3 mm and a total length of about 10 mm. A collagen tubular implant specimen was prepared. After the formation of the embedded specimen, 12 hours at 135 ° C. under reduced pressure (1 Torr or less) using a vacuum dry oven (manufactured by EYELA; VOS-300VD type) and an oil rotary vacuum pump (manufactured by ULVAC; GCD135-XA type). Thermal dehydration crosslinking reaction was performed.
[0041]
  Polyethylene fluoroethylene (ePTFE) sheet (thickness: 0.1 mm) of the same size as a test specimen is implanted in the back muscle of a rabbit (total of 2) with the collagen-implanted specimens at the same time. A Gore-Tex patch (manufactured by Goretex) was rolled into a cylindrical shape and embedded. Biopsies were collected 2 weeks and 4 weeks after implantation and subjected to HE staining for histological evaluation.
[0042]
  As a result, none of the implant specimens showed a particularly remarkable inflammatory reaction, the cell infiltration was good, and it was confirmed that the degradation of the graft progressed over time. On the other hand, no cell infiltration was observed in the control test piece, and the state of degradation and absorption could not be confirmed at all. Accordingly, it has been found that any single collagen yarn produced according to the present invention is a biodegradable and absorbable material compared to existing products.
[0043]
【The invention's effect】
  The collagen nonwoven fabric of the present invention can be easily stored and transported under normal environmental conditions as a material for a collagen medical device in a nonwoven fabric state.
  If the collagen nonwoven fabric manufacturing method of this invention is used, a collagen nonwoven fabric can be obtained continuously, spinning the collagen aqueous solution used as a raw material, and industrial production can be performed simply and easily. In addition, collagen filaments obtained by wet spinning or the like are usually difficult to manufacture as woven fabrics by techniques commonly used in the fiber manufacturing field such as weaving and knitting because of their viscosity and mutual adhesion. However, the collagen non-woven fabric of the present invention can produce a uniform cloth similar to a woven cloth without using a method of weaving or knitting. By using the collagen nonwoven fabric produced by the method of the present invention, it is possible to easily produce a collagen medical device having a complicated three-dimensional structure and a collagen medical device having higher precision and reproducibility.
[0044]
[Brief description of the drawings]
FIG. 1 is an explanatory view showing an example of a collagen nonwoven fabric manufacturing apparatus of the present invention.
FIG. 2 is an explanatory view showing a structure of a winding portion of the collagen nonwoven fabric manufacturing apparatus of the present invention.
FIG. 3 is an explanatory view showing a yarn winding mechanism and a plate-like member direction changing mechanism of the collagen nonwoven fabric manufacturing apparatus of the present invention.
[Explanation of symbols]
  1 Winding device
  11 Plate member
  111 connecting hole
  112 Edge
  12 Inner shaft
  121 Fitting part
  122 Connecting hole
  13 Outer shaft
  131 Lumen
  132 Cut
  2 Yarn feeder
  21 Roller
  22 Reciprocating mechanism
  3Collagen thread
  4      pin

Claims

A plurality of collagen filaments spun using a solubilized collagen solution as a spinning dope are arranged in parallel. The first layer and the second layer are formed of the filaments of the first layer and the second layer. A collagen non-woven fabric, which is laminated so that the arrangement direction forms an angle and is adhered to each other.

The third layer in which a plurality of collagen filaments are arranged in parallel on the first layer or the second layer, and the arrangement direction of the filaments in the layer in contact with the arrangement direction of the filaments in the third layer The collagen nonwoven fabric according to claim 1, wherein the layers are laminated so as to form an angle and are adhered to each other.

The collagen nonwoven fabric according to claim 1, wherein the collagen filamentous material has adhesiveness.

The collagen nonwoven fabric according to claim 1, wherein a distance between the threads of the collagen filaments arranged in parallel is 0 to 40 mm.

The collagen nonwoven fabric according to claim 1, wherein the surface of the collagen thread is coated with a biodegradable substance.

The collagen nonwoven fabric according to claim 5 , wherein the biodegradable substance is collagen.

A spiral wound layer (first layer) is formed on a plate-like member rotating around a fixed rotation axis in parallel with a collagen filament spun using a solubilized collagen solution as a spinning stock solution. A method for producing a collagen nonwoven fabric, comprising: winding a collagen filament in parallel so as to form an angle with an arrangement direction of the filament to be formed, and further forming a layer (second layer).

The method for producing a collagen nonwoven fabric according to claim 7 , wherein the first layer is formed, the rotation axis of the plate member is changed, and the second layer is further formed.

The collagen filamentous material is wound up so that the acute angle with the arrangement direction of the filamentous material forming the layer is 20 ° or less, then the rotation axis of the plate-like member is changed, and the arrangement direction of the wound filamentous material and the acute angle angle The method for producing a collagen nonwoven fabric according to claim 7 , wherein the collagen filamentous material is further wound so as to be 70 to 90 °.

The method for producing a collagen nonwoven fabric according to claim 7 , wherein the second layer is formed so that the arrangement directions of the filaments forming the layers form an angle with each other, immersed in a solution of the biodegradable substance, and dried.

A layer (first layer) is formed by winding a collagen filament spun using a solubilized collagen solution as a spinning stock solution along one side of a plate-like member that rotates around a fixed rotation axis along one side of the plate-like member. And the collagen filamentous material is wound along one side of the plate-like member so as to form an angle with the arrangement direction of the filamentous material forming the first layer, and further overlapped on the first layer. Collagen non-woven fabric produced by forming a layer (second layer).

A plurality of collagen filaments spun using a solubilized collagen solution as a spinning dope are arranged in parallel. The first layer and the second layer are formed of the filaments of the first layer and the second layer. A method for treating a collagen nonwoven fabric, in which the filaments of each layer of the collagen nonwoven fabric are entangled and formed into a felt shape, wherein the layers are laminated so that the arrangement direction forms an angle and are adhered to each other.

A spirally wound collagen layer (first layer) is formed in parallel on a plate-like member rotating around a fixed rotation axis using a solubilized collagen solution as a spinning stock solution, and the first layer is formed. Collagen filaments are wound in parallel so as to form an angle with the arrangement direction of the filaments forming the layers of layers, and the filaments of each layer of the collagen nonwoven fabric formed with the layer (second layer) are entangled to form a felt A method for treating a collagen non-woven fabric to be molded.

A plate-like member which is a portion around which a collagen filament spun using a solubilized collagen solution as a spinning dope is wound,
An inner shaft connected to the plate-like member,
A cylindrical outer shaft having a lumen capable of accommodating the inner shaft and having an oblique cut end;
A drive mechanism for rotating the outer shaft and the inner shaft,
And a control mechanism for controlling the drive mechanism and controlling the rotation of each of the outer shaft and the inner shaft,
The plate-like member is rotatable in the horizontal direction with respect to the surface of the plate-like member about the connecting portion with the inner shaft, and the inner shaft is accommodated inside the outer shaft, and a cut end at the tip of the outer shaft Having a structure that is in contact with the edge portion of the plate-like member, thereby automatically changing the direction of the plate-like member and having a function of winding the collagen filament in a plurality of directions of the plate-like member. Nonwoven manufacturing equipment.

The collagen nonwoven fabric manufacturing apparatus according to claim 14, further comprising a yarn feeding mechanism that feeds the collagen filamentous material while reciprocating in parallel with the rotation axis direction of the plate-like member.