JP4273450B2 - Tissue regeneration substrate, transplant material, and production method thereof - Google Patents
Tissue regeneration substrate, transplant material, and production method thereof Download PDFInfo
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- JP4273450B2 JP4273450B2 JP2002547548A JP2002547548A JP4273450B2 JP 4273450 B2 JP4273450 B2 JP 4273450B2 JP 2002547548 A JP2002547548 A JP 2002547548A JP 2002547548 A JP2002547548 A JP 2002547548A JP 4273450 B2 JP4273450 B2 JP 4273450B2
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
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- Animal Behavior & Ethology (AREA)
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- Oral & Maxillofacial Surgery (AREA)
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Description
技術分野
本発明は、医療分野に広く利用可能な組織再生用基材、移植用材料及びそれらの製法に関する。
背景技術
近年、細胞を培養し、組織を再構築する再生組織工学が注目されている。この再生組織工学では、生体親和性の高い種々の材料で形成された組織再生用基材に各種細胞を保持させ、培養することで移植用材料を得ている。
例えば、線維芽細胞等を組み込んだ培養皮膚ではコラーゲンスポンジが組織再生用基材として利用できることが知られており（特開平４−３３２５６１号公報）、最近では、コラーゲンに代えてヒアルロン酸が利用可能であることも示唆されている（特開平１１−３１９０６８号公報）。
ヒアルロン酸は、二糖繰り返し構造を持つムコ多糖の一種であり、主として動物の関節液や眼球硝子体、臍帯や真皮層などの結合組織などに存在する物質である。また、分子量のオーダーは数十万〜数百万であり、非常に多量の水と結合する性質があり、これが例えば関節の低摩擦性や皮膚真皮組織の保水性に関連している。また、損傷を受けた結合組織が修復するとき、ヒアルロン酸の産生が一時的に活発になり、組織再構築における細胞移動を促進する。このようなことから、ヒアルロン酸は創傷被覆材として優れた能力を発揮するものといえる。
しかしながら、ヒアルロン酸は創傷被覆材としては優れた能力を発揮するものの、含水性が非常に高いため、組織再生用基材として利用するには、細胞の接着性が低く、生体外（ｉｎ ｖｉｔｒｏ）での細胞培養が困難であるという問題があった。
本発明は上記問題点を解決することを課題とするものであり、ヒアルロン酸を主体とする組織再生用基材であって生体外での細胞培養と、移植後の組織再生に適したものを提供することを目的とする。また、この組織再生用基材の製法を提供することを別の目的とする。更に、この組織再生用基材を利用した移植用材料及びその製法を提供することを別の目的とする。
発明の開示
上記課題を解決するため、本発明者は鋭意研究の結果、下記の第１〜第４の発明を完成するに至った。
本発明の第１は、ヒアルロン酸及び／又はその誘導体を主体としたヒアルロン酸スポンジと、前記ヒアルロン酸スポンジの少なくとも片面に生体由来の高分子材料からなるスポンジを積層させた細胞接着部とを備えたことを特徴とする。この組織再生用基材の細胞接着部は、生体由来の高分子材料からなるスポンジを積層したものであるため、組織再生用基材に組み込まれる細胞が良好に接着する。また、ヒアルロン酸及び／又はその誘導体を主体としたヒアルロン酸スポンジを備えているため、細胞増殖の促進等の創傷治癒能力にも優れている。したがって、この組織再生用基材によれば、生体外での細胞培養と、移植後の組織再生を良好に行うことができる。また、細胞を高密度で播種した場合、従来のコラーゲンスポンジでは大きな収縮がみられるのに対して、本発明の組織再生用基材ではそのような収縮はほとんどみられない。この組織再生用基材は、細胞を組み込んで移植用材料として使用してもよいが、それ以外に創傷面を被覆する創傷被覆材として使用してもよい。
ここで、ヒアルロン酸の誘導体としては、例えばヒアルロン酸ナトリウムやヒアルロン酸カリウム等のヒアルロン酸金属塩のほか、ヒアルロン酸のヒドロキシル基やカルボキシル基等がエーテル化、エステル化、アミド化、アセタール化、ケタール化されたもの等が挙げられ、中でも、ヒアルロン酸ナトリウムが好ましい。また、ヒアルロン酸スポンジとしては、分子間架橋されたもの（架橋ヒアルロン酸スポンジ）が好ましい。また、生体由来の高分子材料としては、例えばコラーゲン、ゼラチン、フィブリン、アルギン酸等が挙げられるが、このうちコラーゲン特に抗原性の少ないアテロコラーゲンが好ましく、また、コラーゲン、ゼラチンは分子間架橋されたものが好ましい。
この組織再生用基材は、ヒアルロン酸スポンジと細胞接着部との境界付近において、ヒアルロン酸スポンジに生体由来の高分子材料が入り込んだ状態になっていることが好ましい。この場合、ヒアルロン酸スポンジと細胞接着部との膨潤率に差があったとしても、含水時に両者が剥がれたりするおそれがない。
この組織再生用基材は、ヒアルロン酸スポンジが支持体としての織布、不織布又は編物に支持されていることが好ましい。この場合、支持体によって組織再生用基材の強度が増し、ピンセットで摘んで持ち上げたりする際にも欠損するおそれがなく、ハンドリングが良好になる。ここで、支持体の材質としては、ヒアルロン酸スポンジを補強する役割を果たすものであれば特に限定されないが、例えばナイロン、ポリエステル、シリコーンなどの合成高分子材や、絹、木綿、麻などの天然高分子材等が挙げられる。
本発明の第２は、このような組織再生用基材の製法であって、（１）架橋剤を添加したヒアルロン酸及び／又はその誘導体の水溶液を濃縮することによりヒアルロン酸及び／又はその誘導体の分子間架橋物を得る架橋工程と、（２）前記分子間架橋物を真空凍結乾燥することによりヒアルロン酸スポンジを得るスポンジ化工程と、（３）前記ヒアルロン酸スポンジの少なくとも片面から生体由来の高分子材料の水溶液を吸収させたあと真空凍結乾燥することにより前記細胞接着部を形成する積層工程とを含むことを特徴とする。この製法では、ヒアルロン酸及び／又はその誘導体の分子間架橋物を真空凍結乾燥することによりヒアルロン酸スポンジとした上で、その少なくとも片面から生体由来の高分子材料の水溶液を吸収させたあと再度真空凍結乾燥を行う。この製法によれば、本発明の第１の組織再生用基材を比較的容易に作製できる。
ここで、架橋工程において、架橋剤としては、例えば水溶性エポキシ化合物やグルタールアルデヒド等が挙げられるが、そのうち水溶性エポキシ化合物が好ましい。この水溶性エポキシ化合物としては、例えばエチレングリコールジグリシジルエーテル、グリセロールジグリシジルエーテル、グリセロールトリグリシジルエーテル等が挙げられる。架橋剤として水溶性エポキシ化合物を用いた場合、その使用量はヒアルロン酸及び／又はその誘導体に対して、重量比で概ね１／２〜１／１０の割合であることが好ましく、特に１／５〜１／１０程度が好ましい。
この架橋工程においては、ヒアルロン酸及び／又はその誘導体の水溶液を用いるが、この水溶液におけるヒアルロン酸及び／又はその誘導体の濃度は、用いるヒアルロン酸及び／又はその誘導体の種類や分子量等にもよるが、概ね０．５〜１．５重量％である。また、溶媒として用いる水としては、ｐＨ５〜６のイオン交換水が好ましい。
この架橋工程においては、架橋剤を添加したヒアルロン酸及び／又はその誘導体の水溶液を濃縮することによりヒアルロン酸及び／又はその誘導体の分子間架橋反応を行うが、加温して行うのが好ましい。濃縮時の温度は、架橋剤として水溶性エポキシ化合物を用いた場合には、約３０〜６０℃が好ましく、約４０〜６０℃がより好ましく、約５０℃が特に好ましい。６０℃を越えるような高い温度で加熱すると混合液に気泡が生じて、得られるヒアルロン酸スポンジのスポンジ構造の均一性が十分でない場合がある。一方、３０℃より低い温度では、分子間架橋反応速度が小さくなり、所望の濃度の分子間架橋物を得るのに長時間を要することがある。また、ヒアルロン酸及び／又はその誘導体の分子間架橋反応は、中性領域（ｐＨ５〜８）又は酸性領域（ｐＨ３〜５）で行うことが好ましい。また、酸性領域で行うと、中性領域で行う場合に比べて濃縮時間が短くなる傾向にあるため、好ましい。
この架橋工程において、例えばヒアルロン酸及び／又はその誘導体の水溶液の濃度が１〜１０重量％、好ましくは２〜５重量％、特に好ましくは約５重量％になった時点で濃縮を終えることが好ましい。濃縮を十分行わなかった場合には、次のスポンジ化工程後に得られるヒアルロン酸スポンジの膨潤性を十分抑制できないことがあるため、好ましくない。即ち、ヒアルロン酸スポンジが高い膨潤性を有していると、例えば細胞培養時に液体培地等に浸した場合に必要以上に膨潤してしまい、その結果脆弱になると共に形状も大きくなるので、ハンドリングが困難になる。これに対して、上述のように濃縮の終点を制御すれば、ヒアルロン酸スポンジの膨潤性が十分抑制されるため、液体培地等に浸した場合に必要以上に膨潤することがなく、その結果脆弱になったり形状が大きく成りすぎることもないので、ハンドリングが容易になる。なお、濃縮液が完全にフィルム状になるまで架橋工程を行うと、真空凍結乾燥によるスポンジ化工程を行ったとしても、ヒアルロン酸スポンジは形成されないため、濃縮液がフィルム状になる前に濃縮の終点を設定する必要がある。
次に、スポンジ化工程においては、架橋工程後の分子間架橋物を真空凍結乾燥することによりヒアルロン酸スポンジを得るが、真空凍結乾燥時の温度条件は、大きな氷の結晶を形成させないために、約−８５℃〜−３０℃、好ましくは約−８５℃〜−５０℃、より好ましくは約−８５℃であり、真空条件は、３０×１０−３〜１００×１０−３ｍｍｂａｒ（３〜１０Ｐａ）程度が好ましく、より好ましくは３０×１０−３〜５０×１０−３ｍｍｂａｒ（３〜５Ｐａ）である。
このスポンジ化工程においては、架橋工程で十分な濃縮を行えずに得られた分子間架橋物に対しては、凍結し解凍する操作を少なくとも１回以上行ったあと真空凍結乾燥することによりヒアルロン酸スポンジを得ることが好ましい。真空凍結乾燥前に凍結、解凍する操作を行わなかった場合には、含水時に形状を保持しにくいスポンジになることがあるのに対して、真空凍結乾燥前に凍結、解凍する操作を行った場合には、おそらく水素結合のような分子間相互作用が促進された結果と思われるが、含水時に形状を保持しやすいスポンジが得られる。このように真空凍結乾燥前に凍結、解凍する操作は複数回行ってもよいが、製造工程の簡素化を考慮すれば１回行うことが好ましい。また、真空凍結乾燥前に凍結、解凍する操作における凍結の温度条件は、約−８５℃〜−３０℃、好ましくは約−８５℃〜−５０℃、より好ましくは約−８５℃であり、また、解凍の温度条件は約２０℃〜３０℃、好ましくは約３０℃付近である。但し、解凍時に温度を多段階に分けて加温して解凍してもよい。この場合には、分子間架橋物が割れたりしないように注意する。なお、架橋工程において適切な濃縮条件で、十分な濃縮を行って得られた分子間架橋物に対しては、十分な強度を有しているので、真空凍結乾燥前の凍結／解凍操作を必ずしも行う必要はない。
次の積層工程に移る前に、スポンジ化工程で作製したヒアルロン酸スポンジを洗浄する水洗工程を設けることが好ましい。この水洗工程においては、未反応の架橋剤を不活性化させる不活性化剤を洗浄水中に添加しておいてもよい。例えば、架橋剤として水溶性エポキシ化合物を用いた場合には、不活性化剤としては、エポキシ基を開環させる機能を持ったもので生体に害を与えないものが好ましく、例えばグリシンが用いられる。
次に、積層工程においては、ヒアルロン酸スポンジの少なくとも片面から生体由来の高分子材料の水溶液を吸収させたあと真空凍結乾燥することにより細胞接着部を形成するが、生体由来の高分子材料としては既述の通り、コラーゲン、ゼラチン、フィブリン、アルギン酸等が挙げられ、このうちコラーゲン特に抗原性の少ないアテロコラーゲンが好ましい。また、生体由来の高分子材料の水溶液としては、０．２〜１．０重量％の水溶液、好ましくは０．２〜０．５重量％の水溶液、より好ましくは０．５重量％程度の水溶液を用いればよく、また、生体由来の高分子材料とヒアルロン酸との重量比は、１：２〜１０、好ましくは１：２〜４、特に好ましくは１：４程度とすればよい。また、真空凍結乾燥の条件は、スポンジ化工程における真空凍結乾燥の条件と同様である。
また、この積層工程では、ヒアルロン酸スポンジの少なくとも片面から生体由来の高分子材料の水溶液を吸収させているため、得られた組織再生用基材のヒアルロン酸スポンジと細胞接着部との境界付近において、ヒアルロン酸スポンジに生体由来の高分子材料が入り込んだ状態になっている。このため、ヒアルロン酸スポンジと細胞接着部との膨潤率に差があったとしても、組織再生用基材を含水させた時に両者が剥がれたりするおそれがない。
この積層工程において、ヒアルロン酸スポンジの片面に複数の穴を開け、その穴を開けた片面から生体由来の高分子材料の水溶液を吸収させたあと真空凍結乾燥することにより前記細胞接着部を形成してもよい。この場合、生体由来の高分子材料の水溶液は、ヒアルロン酸スポンジの有する多孔に浸透するのに加えて複数の穴にも入り込むため、ヒアルロン酸スポンジとの間にアンカーリングの効果も得られる。このようにして各穴や多孔に生体高分子材料の水溶液を浸透させた後真空凍結乾燥することにより、組織再生用基材におけるヒアルロン酸スポンジと細胞接着部との密着性を一層向上させることができる。なお、ここでいう「穴」には、例えば丸穴や角穴などのような一般的な穴のほか、例えば切れ目や凹凸などのように平面に比べて接触面積が大きくなるようなものも含まれる。
この積層工程において生体由来の高分子材料としてコラーゲン又はゼラチンを用いた場合には、積層工程後に紫外線ランプを照射してコラーゲン又はゼラチンを部分的に分子間架橋することにより含水時にコラーゲン又はゼラチンが流出することを防止するのが好ましく、更にその後にエチレンオキサイドガス等により滅菌するのが好ましい。また、前述の水洗工程を行わなかった場合は、未反応の架橋剤を不活性化させるために、不活性化剤を生体由来の高分子材料の水溶液に添加しておくのが好ましい。不活性化剤としては前述と同様に、例えば、架橋剤として水溶性エポキシ化合物を用いた場合には、グリシン等のエポキシ基を開環させる機能を持ったもので生体に害を与えないものが好ましい。
ところで、前述のスポンジ化工程及び積層工程の代わりに、以下のスポンジ化・積層工程を採用してもよい。即ち、架橋工程後の分子間架橋物を生体由来の高分子材料の水溶液と接触させたあと真空凍結乾燥することにより前記ヒアルロン酸スポンジと共に細胞接着部を形成する工程を採用してもよい。この場合、真空凍結乾燥を一度だけで済ませることができるので、製造プロセスが簡略化される。
このスポンジ化・積層工程では、スポンジ化前の分子間架橋物に生体由来の高分子材料の水溶液を接触させるため、生体由来の高分子材料の水溶液がスポンジ化前の分子間架橋物に浸透しにくく、最終的に得られる組織再生用基材におけるヒアルロン酸スポンジと細胞接着部との密着性が十分でないおそれがある。そこで、ヒアルロン酸及び／又はその誘導体の分子間架橋物の上下方向に複数の穴を設け、生体由来の高分子材料の水溶液との接触面積を大きくしてアンカーリングの効果を得ることが好ましい。このようにして各穴に生体高分子材料の水溶液を浸透させたあと真空凍結乾燥することにより、組織再生用基材におけるヒアルロン酸スポンジと細胞接着部との密着性を向上させることが好ましい。なお、ここでいう「穴」には、例えば丸穴や角穴などのような一般的な穴のほか、例えば切れ目や凹凸などのように平面に比べて接触面積が大きくなるようなものも含まれる。
このスポンジ化・積層工程を採用してヒアルロン酸スポンジが支持体に支持された組織再生用基材を作製するには、例えば以下のような手順で行う。即ち、予め容器の底面に支持体を敷いておき、架橋剤を添加したヒアルロン酸及び／又はその誘導体の水溶液をこの容器内に入れる。その後、この水溶液を加温して濃縮することによりヒアルロン酸及び／又はその誘導体の分子間架橋物を作製する。その後、この容器内で分子間架橋物の上下方向に複数の穴を設け、生体由来の高分子材料の水溶液を加えて分子間架橋物と接触させ、真空凍結乾燥する。なお、必要に応じて、真空凍結乾燥前（例えば穴を設ける前あるいは穴を設けた後）に、分子間架橋物を凍結し解凍する操作を行ってもよい。このような操作を行うことにより、強度が向上し、含水時に形状を保持しやすいスポンジが得られる。
本発明の第３は、移植用材料であって、上述の組織再生用基材と、この組織再生用基材の細胞接着部に保持された細胞とを備えたことを特徴とする。この移植用材料は、細胞が細胞接着部に密着性よく保持されているため生体外で容易に細胞培養が可能であり、しかもヒアルロン酸の細胞移動の促進や保湿効果による創傷治癒能力により組織が適切に再生される。ここで、細胞接着部に保持される細胞はどのような細胞であってもよいが、線維芽細胞、角化細胞、色素産生細胞、血管内皮細胞、内皮細胞、上皮細胞、軟骨細胞、骨芽細胞、筋芽細胞、脂肪細胞、肝細胞、神経細胞、心筋細胞、ランゲルハンス島細胞等が挙げられる。例えば他人由来の線維芽細胞を保持させた移植用材料を使用するには、皮膚創傷部にこの移植用材料を移植し、抗菌剤を含有する膜（例えばポリウレタン膜）で被覆し、更に包帯で被覆する。そのようにすると、他人由来の線維芽細胞から産生される各種サイトカインにより治癒が促進される。
本発明の第４は、このような移植用材料の製法であって、上述の組織再生用基材を作製したあと、この組織再生用基材の細胞接着部に細胞を組み込むことを特徴とする。この製法によれば、本発明の第３の移植用材料を比較的容易に作製できる。ここで、細胞接着部に細胞を組み込む方法としては、例えば、組織再生用基材を培養液に浸漬させると共に細胞を孔内へ取り込ませることにより保持する方法がある。
発明を実施するための最良の形態
以下に、本発明の好適な実施例について説明する。本発明は、下記実施例に何ら限定されるものではない。なお、以下において「％」は特に断りのない限り重量％を表す。
（実施例１）
［１］組織再生用基材の作製（図１参照）
［１−１］ヒアルロン酸スポンジの作製
ヒアルロン酸ナトリウム（分子量約２００万）１０ｇを蒸留水１Ｌに溶解して１％ヒアルロン酸水溶液（ｐＨ６）を作製した。ヒアルロン酸の溶解はメカニカルスターラーで攪拌を行いながら十分な時間をかけて行った。一方、水溶性エポキシ化合物として、デナコールＥＸ３１３（グリセロールジグリシジルエーテル；ナガセ化成工業）１ｇを蒸留水２０ｍｌに希釈し、デナコールＥＸ３１３溶液（以下、ＥＸ３１３溶液という）を作製した。このようにして作製したＥＸ３１３溶液２０ｍｌを、攪拌中のヒアルロン酸水溶液に添加し、さらに攪拌を３０分程度行い、ヒアルロン酸−ＥＸ３１３混合液を作製した。
得られたヒアルロン酸−ＥＸ３１３混合液を底面積が１８０ｃｍ２（１０ｃｍ×１８ｃｍ）のトレーに１８０ｍｌ注入した。このときのヒアルロン酸−ＥＸ３１３混合液の深さは、およそ１ｃｍとなった。トレーに注入されたヒアルロン酸−ＥＸ３１３混合液を室温で２時間静置し、ヒアルロン酸−ＥＸ３１３混合液内の気泡を抜いた後、予めトレーの大きさに応じて切断したベンリーゼ（不織布；旭化成工業）をヒアルロン酸−ＥＸ３１３混合液に上載した。
不織布を上載した状態のヒアルロン酸−ＥＸ３１３混合液を空気循環型乾熱器に入れ、５０℃で１０時間静置した。これにより、ヒアルロン酸の水酸基（ハイドロキシル基）やカルボキシル基とデナコールＥＸ３１３のエポキシ基とが反応してヒアルロン酸の分子間架橋物が生成すると共に、液量が約２／５（つまりヒアルロン酸濃度として約２．５重量％）になるまで濃縮した。このときの濃縮液の深さは、およそ４ｍｍであった。
次に、この濃縮液を−８５℃で凍結した。凍結時間は、フリーザの能力にも左右されるが、およそ５〜６時間程度であり、完全に凍結させた。この凍結後、室温に１〜１．５時間放置して一度解凍し、解凍後、再び５〜６時間程度、−８５℃のフリーザで完全に凍結させた。そして、３０×１０−３〜５０×１０−３ｍｍｂａｒ（３〜５Ｐａ）にて真空凍結乾燥処理を行うことにより、スポンジ構造を有するヒアルロン酸分子間架橋物（以下、架橋ヒアルロン酸スポンジという）を得た。
［１−２］コラーゲンスポンジとの積層化
アテロコラーゲン１ｇを蒸留水５００ｍｌに溶解し、１Ｎ ＨＣｌでｐＨ４に調整し、０．２％アテロコラーゲン水溶液を作製した。作製したアテロコラーゲン水溶液を底面積１８０ｃｍ２のトレーに９０ｍｌ注入した。アテロコラーゲン水溶液は、もとのヒアルロン酸水溶液の液量（１８０ｍｌ）の半分の液量であり、コラーゲンとヒアルロン酸の重量比は１：１０となる。
前述のようにして作製した架橋ヒアルロン酸スポンジをアテロコラーゲン水溶液の入ったトレーにベンリーゼが上方に位置するように浸漬し、１時間放置することで架橋ヒアルロン酸スポンジにコラーゲン水溶液をしみ込ませた。そして、このコラーゲン水溶液に浸漬させたヒアルロン酸スポンジを−８５℃で凍結後、真空乾燥した。
真空凍結乾燥後の架橋ヒアルロン酸スポンジにコラーゲンスポンジを積層化したものを、コラーゲンスポンジ側に１５Ｗの紫外線ランプ（２５４ｎｍ）を用いて２５ｃｍの距離から３０分間照射することにより、コラーゲンの分子間架橋を行った。その後、滅菌バッグに詰め、ＥＯＧ（エチレン・オキサイド・ガス）滅菌を６０℃、２０時間行った。これにより、組織再生用基材、つまり架橋コラーゲンスポンジ（細胞接着部）を備えた架橋ヒアルロン酸スポンジであってベンリーゼ（支持体）に支持されたものを得た。
［２］移植用材料の作製
組織再生用基材のベンリーゼとは反対側の面に、１０％ウシ胎児血清（ＦＢＳ）含有ダルベッコ変法イーグル最小必須培地（ＤＭＥＭ＋１０％ＦＢＳ、ギブコ社製）中に浮遊した培養ウサギ線維芽細胞を５×１０４細胞／ｃｍ２の密度で播種したのち、ＣＯ２５％、３７℃のインキュベータ中で７日間培養し、移植用材料としての培養真皮を作製した。培地を除去し、これを凍結保存液（１０％ＤＭＳＯ含有ＤＭＥＭ＋２０％ＦＢＳ）に入れ、−１５２℃の冷凍庫内で保存した。
［３］移植試験
冷凍庫内に保存した培養真皮（移植用材料）を使用時に解凍して凍結保存液を除去し、ハンクス液３０ｍｌで２回洗浄して動物実験に使用した。ウサギの背部に直径７ｃｍの円を描き、全層皮膚を切除し、皮膚欠損創とした。この皮膚欠損創に先の培養真皮を適用し、創傷面の周辺を縫合し、その上にポリウレタンフィルム製創傷被覆材を適用し、更に滅菌パットを載せて、創傷の周辺を縫合し、伸縮性包帯で圧迫固定した。その結果、良好な肉芽組織形成と創面積の顕著な減小が観察された。
（実施例２）
［１］組織再生用基材の作製（図２参照）
［１−１］ヒアルロン酸スポンジの作製
実施例１と同様にして、ヒアルロン酸−ＥＸ３１３混合液を作製し、得られたヒアルロン酸−ＥＸ３１３混合液を底面積が１８０ｃｍ２（１０ｃｍ×１８ｃｍ）のトレーに１８０ｍｌ注入した。トレーにはその大きさに合わせて切断したベンリーゼ（前出）が予め載置されており、少量の蒸留水を加えて含水させてトレー底面に接着させておいた。トレーに注入したヒアルロン酸−ＥＸ３１３混合液の深さは、およそ１ｃｍとなった。
この状態のヒアルロン酸−ＥＸ３１３混合液を空気循環型乾熱器に入れ、５０℃で１５時間静置した。これにより、ヒアルロン酸の水酸基（ハイドロキシル基）やカルボキシル基とデナコールＥＸ３１３のエポキシ基とが反応してヒアルロン酸の分子間架橋物が生成すると共に、液量が約１／５（つまりヒアルロン酸濃度として約５重量％）になるまで濃縮された。このときの濃縮液の深さは、およそ２〜３ｍｍであった。
次に、この濃縮液を−８５℃で凍結した。凍結時間は、フリーザの能力にも左右されるが、およそ５〜６時間程度であり、完全に凍結させた。そして、３０×１０−３〜５０×１０−３ｍｍｂａｒ（３〜５Ｐａ）にて真空凍結乾燥処理を行うことにより、スポンジ構造を有するヒアルロン酸分子間架橋物（以下、架橋ヒアルロン酸スポンジという）を得た。その後、未反応の架橋剤を除去するため、１５Ｌのイオン交換水を入れた容器内に架橋ヒアルロン酸スポンジを一日浸漬させ、水洗した。
［１−２］コラーゲンスポンジとの積層化
アテロコラーゲン２．５ｇを蒸留水５００ｍｌに溶解し、１Ｎ ＨＣｌでｐＨ３．２に調整し、０．５％アテロコラーゲン水溶液を作製した。水洗した架橋ヒアルロン酸スポンジを、不織布を下側にして底面積１８０ｃｍ２のトレーに置き、この上に作製したアテロコラーゲン水溶液を９０ｍｌ注入した。アテロコラーゲン水溶液は、もとのヒアルロン酸水溶液の液量（１８０ｍｌ）の半分の液量であり、コラーゲンとヒアルロン酸の重量比は１：４となる。
架橋ヒアルロン酸スポンジは周囲が若干収縮しており、トレーの側面との間に隙間が生じた。この隙間にアテロコラーゲン水溶液が浸入することにより、ヒアルロン酸スポンジの周囲を包み込むようにコラーゲンが配置され、膨潤時に架橋ヒアルロン酸スポンジとコラーゲンスポンジとが分離するのを抑制した。
アテロコラーゲン水溶液を注入して一昼夜経過後、アテロコラーゲン水溶液を含んだヒアルロン酸スポンジを−８５℃で真空凍結乾燥した。真空凍結乾燥後の架橋ヒアルロン酸スポンジにコラーゲンスポンジが積層化したものを、コラーゲンスポンジ側に１５Ｗの紫外線ランプ（２５４ｎｍ）を用いて２５ｃｍの距離から３０分間照射することにより、コラーゲンの分子間架橋を行った。その後、滅菌バッグに詰め、ＥＯＧ滅菌を６０℃、２０時間行った。これにより、組織再生用基材、つまり架橋コラーゲンスポンジ（細胞接着部）を備えた架橋ヒアルロン酸スポンジであってベンリーゼ（支持体）に支持されたものを得た。
［２］移植用材料の作製
上記方法により作製した１８０ｃｍ２の組織再生用基材を切断し、９０ｃｍ２の大きさにした。切断した組織再生用基材を１８０ｃｍ２のトレーに入れ、ＤＭＥＭ＋１０％ＦＢＳを１００ｍｌ注入し、ｐＨを調整した。ｐＨを調整後（ｐＨ７．４）、培養液を一度廃棄し、ヒト線維芽細胞懸濁液５ｍｌを組織再生用基材に５×１０５細胞／ｃｍ２の密度で播種した。播種後、３７℃で一晩静置し、ヒト線維芽細胞を組織再生用基材に定着させた。その後、上記ＤＭＥＭ＋１０％ＦＢＳ培地を１００ｍｌ加え、３７℃，５％ＣＯ２下で１週間培養を行った。このように作製した培養真皮（移植用材料）は、十分な強度を有しているばかりでなく、周囲が２〜３ｍｍ程度収縮しているのみであるので、ハンドリングが良好であり、欠損するおそれもないものだった。
（実施例３）
［１］組織再生用基材の作製（図３参照）
［１−１］ヒアルロン酸スポンジの作製
ヒアルロン酸ナトリウム（分子量約２００万）２０ｇを蒸留水２Ｌに溶解して１％ヒアルロン酸水溶液（ｐＨ６）を作製した後、１Ｎ ＨＣｌにてｐＨ３．５に調整した。ヒアルロン酸の溶解はメカニカルスターラーで撹拌を行いながら十分な時間をかけて行った。一方、水溶性エポキシ化合物として、デナコールＥＸ８１０（グリセロールジグリシジルエーテル；ナガセ化成工業）４ｇを蒸留水４０ｍｌに希釈し、デナコールＥＸ８１０溶液（以下、ＥＸ８１０溶液という）を作製した。このようにして作製したＥＸ８１０溶液（４０ｍｌ）を、撹拌中のヒアルロン酸水溶液に添加し、さらに撹拌を３０分程度行い、ヒアルロン酸−ＥＸ８１０混合液を作製した。
得られたヒアルロン酸−ＥＸ８１０混合液を底面積が１１０ｃｍ２（１０ｃｍ×１１ｃｍ）のトレーに５０ｇ注入した。トレーにはその大きさに合わせて切断したベンリーゼ（前出）が予め載置されており、少量の蒸留水を加えて含水させてトレー底面に接着させておいた。トレーに注入したヒアルロン酸−ＥＸ８１０混合液の深さは、およそ５ｍｍとなった。
この状態のヒアルロン酸−ＥＸ８１０混合液を空気循環型加熱器に入れ、５０℃で５時間静置した。これにより、ヒアルロン酸の水酸基（ハイドロキシル基）やカルボキシル基とデナコールＥＸ８１０のエポキシ基とが反応してヒアルロン酸の分子間架橋物が生成すると共に、液量が約１／２（つまりヒアルロン酸濃度として約２重量％）になるまで濃縮された。このときの濃縮液の深さは、およそ２〜３ｍｍであった、
次に、この濃縮液を−８５℃で凍結した。凍結時間は、フリーザの能力にも左右されるが、およそ５〜６時間程度であり、完全に凍結させた。そして、３０×１０−３〜５０×１０−３ｍｍｂａｒ（３〜５Ｐａ）にて真空凍結乾燥処理を行った。その後、未反応の架橋剤を除去するため、１Ｌの蒸留水を入れた容器内に架橋ヒアルロン酸スポンジを一晩浸漬させ、水洗した。
水洗後、再び−８５℃のフリーザで完全に凍結させた。そして、３０×１０−３〜５０×１０−３ｍｍｂａｒ（３〜５Ｐａ）にて真空凍結乾燥処理を行うことにより、スポンジ構造を有するヒアルロン酸分子間架橋物（以下、架橋ヒアルロン酸スポンジという）を得た。
［１−２］コラーゲンスポンジとの積層化
コラーゲンスポンジによる積層化に先立ち、剣山を使用して架橋ヒアルロン酸スポンジを穿孔した。このときの孔の間隔は、ほぼ４ｍｍであった。
アテロコラーゲン８ｇを蒸留水１．６Ｌに溶解し、１Ｎ ＨＣｌでｐＨ３．５に調整し、０．５％アテロコラーゲン水溶液を作製した。作製したアテロコラーゲン水溶液を底面積１１０ｃｍ２のトレーに４０ｇ注入した。アテロコラーゲン水溶液は、もとのヒアルロン酸水溶液の液量（５０ｇ）の約４／５の液量であり、コラーゲンとヒアルロン酸の重量比は２：５となる。
前述のようにして作製した架橋ヒアルロン酸スポンジをアテロコラーゲン水溶液の注入したトレーにベンリーゼが上方に位置するように浮かべ、１晩静置することで架橋ヒアルロン酸スポンジにコラーゲン水溶液をしみ込ませた。そして、コラーゲン水溶液をしみ込ませたヒアルロン酸スポンジを−８５℃で完全に凍結した後、真空凍結乾燥した。
真空凍結乾燥後の架橋ヒアルロン酸スポンジにコラーゲンスポンジを積層化したものの両側に、各々１５Ｗの紫外線ランプ（２５４ｎｍ）を用いて２０ｃｍの距離から３０分間照射することにより、コラーゲンの分子間架橋を行った。その後、滅菌バッグに詰め、ＥＯＧ（エチレン・オキサイド・ガス）滅菌を６０℃、２０時間行った。これにより、組織再生用基材、つまり架橋コラーゲンスポンジ（細胞接着部）を備えた架橋ヒアルロン酸スポンジであって、ベンリーゼ（支持体）に支持されたものを得た。
［２］移植用材料の作製
上記方法により作製した組織再生用基材をトレーに入れ、ＤＭＥＭ＋１０％ＦＢＳを５０ｍｌ注入し、ｐＨを調整した。ｐＨを調整後（ｐＨ７．４）、培養液を一度廃棄し、ヒト線維芽細胞懸濁液５ｍｌを組織再生用基材に１×１０５細胞／ｃｍ２の密度で播種した。播種後、３７℃で一晩静置し、ヒト線維芽細胞を組織再生用基材に定着させた。その後、上記ＤＭＥＭ＋１０％ＦＢＳ培地を５０ｍｌ加え、３７℃，５％ＣＯ２下で１週間培養を行った。このように作製した培養真皮（移植用材料）は、培地を除去し、これを凍結保存液（１０％ＤＭＳＯ含有ＤＭＥＭ＋２０％ＦＢＳ）に入れ、−１５２℃の冷凍庫内で保存した。
［３］移植試験
冷凍庫内に保存した培養真皮（移植用材料）を使用時に解凍して凍結保存液を除去し、乳酸リンゲル液３０〜５０ｍｌで３回洗浄してヒト臨床適用した。創傷面は残存壊死組織を除去した後、消毒を行い、生理食塩水で十分に洗浄した。この皮膚欠損創に先の培養真皮を適用し、培養真皮の周囲を縫合固定した。その上に抗生剤含有軟膏を染み込ませたガーゼを適用し、さらに滅菌ガーゼを載せて、創傷の周辺を縫合し、伸縮性包帯で圧迫固定した。その結果、良好な肉芽組織形成と創面積の顕著な減少、創周囲からの上皮化が観察された。
（実施例４）
［１］組織再生用基材の作製（図４参照）
［１−１］ヒアルロン酸スポンジの作製
ヒアルロン酸ナトリウム（分子量約２００万）２ｇを蒸留水２００ｍＬに溶解して１％ヒアルロン酸水溶液（ｐＨ６）を作製した後、１Ｎ ＨＣｌにてｐＨ３．５に調整した。ヒアルロン酸の溶解はメカニカルスターラーで撹拌を行いながら十分な時間をかけて行った。一方、水溶性エポキシ化合物として、デナコールＥＸ８１０ ０．２ｇを蒸留水２ｍｌに希釈し、デナコールＥＸ８１０溶液（以下、ＥＸ８１０溶液という）を作製した。このようにして作製したＥＸ８１０溶液（２ｍｌ）を、撹拌中のヒアルロン酸水溶液に添加し、さらに撹拌を３０分程度行い、ヒアルロン酸−ＥＸ８１０混合液を作製した。
得られたヒアルロン酸−ＥＸ８１０混合液を底面積がφ３５ｍｍディッシュに４．８ｇ注入した。ディッシュに注入したヒアルロン酸−ＥＸ８１０混合液の深さは、およそ５ｍｍとなった。
この状態のヒアルロン酸−ＥＸ８１０混合液を空気循環型加熱器に入れ、５０℃で５時間静置した。これにより、ヒアルロン酸の水酸基（ハイドロキシル基）やカルボキシル基とデナコールＥＸ８１０のエポキシ基とが反応してヒアルロン酸の分子間架橋物が生成すると共に、液量が約１／２（つまりヒアルロン酸濃度として約２重量％）になるまで濃縮された。このときの濃縮液の深さは、およそ２〜３ｍｍであった、
次に、この濃縮液を−８５℃で凍結した。凍結時間は、フリーザの能力にも左右されるが、およそ５〜６時間程度であり、完全に凍結させた。そして、３０×１０−３〜５０×１０−３ｍｍｂａｒ（３〜５Ｐａ）にて真空凍結乾燥処理を行った。その後、未反応の架橋剤を除去するため、１Ｌの蒸留水を入れた容器内に一晩浸漬させ、水洗した。水洗後、再び−８５℃のフリーザで完全に凍結させた。そして、３０×１０−３〜５０×１０−３ｍｍｂａｒ（３〜５Ｐａ）にて真空凍結乾燥処理を行うことにより、スポンジ構造を有するヒアルロン酸分子間架橋物（以下、架橋ヒアルロン酸スポンジという）を得た。
［１−２］コラーゲンスポンジとの積層化
コラーゲンスポンジによる積層化に先立ち、剣山を使用して架橋ヒアルロン酸スポンジを穿孔した。このときの孔の間隔は、ほぼ４ｍｍであった。
一方、０．５％コラーゲン水溶液（ＫＯＫＥＮＣＥＬＬＧＥＮ Ｉ−ＰＣ：株式会社高研製）をφ３５ｍｍディッシュに３．８ｇ注入した。コラーゲン水溶液は、もとのヒアルロン酸水溶液の液量（４．８ｇ）の約４／５の液量であり、コラーゲンとヒアルロン酸の重量比はおよそ２：５となる。
前述のようにして作製した架橋ヒアルロン酸スポンジのうち穿孔した面が下になるようにコラーゲン水溶液の注入したディッシュに浮かべ、一晩静置することで架橋ヒアルロン酸スポンジにコラーゲン水溶液をしみ込ませた。そして、コラーゲン水溶液をしみ込ませたヒアルロン酸スポンジを−８５℃で完全に凍結した後、真空凍結乾燥した。
真空凍結乾燥後の架橋ヒアルロン酸スポンジにコラーゲンスポンジを積層化したものの両側に、各々１５Ｗの紫外線ランプ（２５４ｎｍ）を用いて２０ｃｍの距離から３０分間照射することにより、コラーゲンの分子間架橋を行った。その後、滅菌バッグに詰め、ＥＯＧ滅菌を６０℃、２０時間行った。これにより、組織再生用基材、つまり架橋コラーゲンスポンジ（細胞接着部）を備えた架橋ヒアルロン酸スポンジを得た。この組織再生用基材には、ベンリーゼによる強度付加はなされていないが、ピンセット等による取扱いは容易に行うことができた。
本実施例の組織再生用基材は、実施例１〜３と同様、培養真皮として利用することができるほか、創傷被覆材として用いることもできる。創傷被覆材として用いる場合には、生体吸収性を有しているため除去が不要であるし、コラーゲン側を創傷面に当てることによりコラーゲンの細胞保持効果が得られ、創傷適用後にヒアルロン酸が創傷面に溶出して細胞遊走性を発揮し治癒促進効果が得られることが期待される。
なお、実施例２の別例として、ヒアルロン酸−ＥＸ３１３混合液の分子間架橋反応後、真空凍結乾燥することなく架橋反応物の上下方向に穴を開け、その穴を開けた面とアテロコラーゲン水溶液とを接触させたあと真空凍結乾燥することにより、ヒアルロン酸スポンジにすると同時にコラーゲンスポンジにすることもできる。この場合、製造プロセスが簡略化されるし、両スポンジは穴の存在によりアンカーリングの効果が得られるので良好に密着する。
産業上の利用の可能性
本発明は、ヒアルロン酸を主体とする組織再生用基材であって生体外での細胞培養と移植後の組織再生に適しているため、医療分野に広く利用可能である。
【図面の簡単な説明】
図１は実施例１の組織再生用基材の作製手順を表す説明図、図２は実施例２の組織再生用基材の作製手順を表す説明図、図３は実施例３の組織再生用基材の作製手順を表す説明図、図４は実施例４の組織再生用基材の作製手順を表す説明図である。Technical field
The present invention relates to a tissue regeneration substrate, a transplant material, and methods for producing them that can be widely used in the medical field.
Background art
In recent years, regenerative tissue engineering that cultivates cells and reconstructs tissues has attracted attention. In this regenerative tissue engineering, various cells are held on a tissue regeneration base material formed of various materials having high biocompatibility, and cultured to obtain a transplant material.
For example, it is known that collagen sponge can be used as a tissue regeneration substrate in cultured skin incorporating fibroblasts and the like (Japanese Patent Laid-Open No. 4-332561). Recently, hyaluronic acid can be used instead of collagen. This is also suggested (Japanese Patent Laid-Open No. 11-31068).
Hyaluronic acid is a kind of mucopolysaccharide having a disaccharide repeating structure, and is a substance mainly present in animal joint fluid, ocular vitreous, connective tissues such as umbilical cord and dermis layer. Further, the molecular weight is in the order of hundreds of thousands to millions, and has a property of binding to a very large amount of water, which is related to, for example, low friction of joints and water retention of skin dermal tissue. In addition, when damaged connective tissue is repaired, hyaluronic acid production is temporarily activated, facilitating cell migration in tissue remodeling. For these reasons, it can be said that hyaluronic acid exhibits excellent ability as a wound dressing.
However, although hyaluronic acid exhibits an excellent ability as a wound dressing material, it has a very high water content, so that it has low cell adhesiveness for use as a tissue regeneration substrate, and is in vitro. There was a problem that cell culture was difficult.
An object of the present invention is to solve the above-mentioned problems, and is a tissue regeneration substrate mainly composed of hyaluronic acid, which is suitable for cell culture in vitro and tissue regeneration after transplantation. The purpose is to provide. Another object is to provide a method for producing the tissue regeneration substrate. Furthermore, another object is to provide a transplant material using the tissue regeneration substrate and a method for producing the same.
Disclosure of the invention
In order to solve the above problems, the present inventor has completed the following first to fourth inventions as a result of intensive studies.
A first aspect of the present invention includes a hyaluronic acid sponge mainly composed of hyaluronic acid and / or a derivative thereof, and a cell adhesion part in which a sponge made of a polymer material derived from a living body is laminated on at least one surface of the hyaluronic acid sponge. It is characterized by that. Since the cell adhesion portion of the tissue regeneration substrate is a laminate of sponges made of a living body-derived polymer material, the cells incorporated in the tissue regeneration substrate adhere well. Moreover, since it has a hyaluronic acid sponge mainly composed of hyaluronic acid and / or a derivative thereof, it is excellent in wound healing ability such as promotion of cell proliferation. Therefore, according to this tissue regeneration substrate, cell culture in vitro and tissue regeneration after transplantation can be performed satisfactorily. Further, when cells are seeded at a high density, the conventional collagen sponge shows a large shrinkage, whereas the tissue regeneration substrate of the present invention hardly shows such a shrinkage. This tissue regeneration substrate may be used as a transplant material incorporating cells, but may also be used as a wound dressing for covering the wound surface.
Here, as the derivative of hyaluronic acid, for example, in addition to hyaluronic acid metal salts such as sodium hyaluronate and potassium hyaluronate, the hydroxyl group and carboxyl group of hyaluronic acid are etherified, esterified, amidated, acetalized, and ketal. Among them, sodium hyaluronate is preferable. The hyaluronic acid sponge is preferably an intermolecularly crosslinked one (crosslinked hyaluronic acid sponge). Examples of biological polymer materials include collagen, gelatin, fibrin, alginic acid and the like. Among these, collagen, particularly atelocollagen with low antigenicity, is preferable, and collagen and gelatin are intermolecularly crosslinked. preferable.
The tissue regeneration substrate is preferably in a state in which a high-molecular material derived from a living body enters the hyaluronic acid sponge in the vicinity of the boundary between the hyaluronic acid sponge and the cell adhesion portion. In this case, even if there is a difference in the swelling rate between the hyaluronic acid sponge and the cell adhesion portion, there is no possibility that the two will be peeled off when containing water.
In the tissue regeneration substrate, hyaluronic acid sponge is preferably supported by a woven fabric, a nonwoven fabric or a knitted fabric as a support. In this case, the strength of the tissue regeneration base material is increased by the support, and there is no fear of loss even when picking up with tweezers and lifting it up, and the handling becomes good. Here, the material of the support is not particularly limited as long as it plays a role of reinforcing the hyaluronic acid sponge. For example, synthetic polymer materials such as nylon, polyester and silicone, and natural materials such as silk, cotton and hemp are used. Examples include polymer materials.
A second aspect of the present invention is a method for producing such a tissue regeneration substrate, wherein (1) hyaluronic acid and / or a derivative thereof is concentrated by concentrating an aqueous solution of hyaluronic acid and / or a derivative added with a crosslinking agent. A cross-linking step for obtaining an intermolecular cross-linked product of (2), (2) a sponge forming step for obtaining a hyaluronic acid sponge by freeze-drying the intermolecular cross-linked product, and (3) a biological origin from at least one side of the hyaluronic acid sponge. And a laminating step of forming the cell adhesion portion by absorbing the aqueous solution of the polymer material and then vacuum lyophilizing it. In this production method, hyaluronic acid and / or a derivative of intermolecular cross-linked product thereof is vacuum-lyophilized to obtain a hyaluronic acid sponge, and an aqueous solution of a biological polymer material is absorbed from at least one side thereof, and then vacuumed again. Freeze-dry. According to this production method, the first tissue regeneration substrate of the present invention can be produced relatively easily.
Here, in the crosslinking step, examples of the crosslinking agent include water-soluble epoxy compounds and glutaraldehyde, and of these, water-soluble epoxy compounds are preferred. Examples of the water-soluble epoxy compound include ethylene glycol diglycidyl ether, glycerol diglycidyl ether, glycerol triglycidyl ether, and the like. When a water-soluble epoxy compound is used as the cross-linking agent, the amount used is preferably about 1/2 to 1/10 by weight with respect to hyaluronic acid and / or a derivative thereof, particularly 1/5. About 1/10 is preferable.
In this crosslinking step, an aqueous solution of hyaluronic acid and / or a derivative thereof is used. The concentration of hyaluronic acid and / or a derivative thereof in this aqueous solution depends on the type and molecular weight of the hyaluronic acid and / or the derivative used. About 0.5 to 1.5% by weight. Moreover, as water used as a solvent, pH 5-6 ion-exchange water is preferable.
In this cross-linking step, an intermolecular cross-linking reaction of hyaluronic acid and / or a derivative thereof is performed by concentrating an aqueous solution of hyaluronic acid and / or a derivative thereof to which a cross-linking agent is added, but it is preferably performed by heating. The temperature at the time of concentration is preferably about 30 to 60 ° C, more preferably about 40 to 60 ° C, and particularly preferably about 50 ° C when a water-soluble epoxy compound is used as a crosslinking agent. When heated at a high temperature exceeding 60 ° C., bubbles are generated in the mixed solution, and the uniformity of the sponge structure of the resulting hyaluronic acid sponge may not be sufficient. On the other hand, at a temperature lower than 30 ° C., the intermolecular cross-linking reaction rate decreases, and it may take a long time to obtain an intermolecular cross-linked product having a desired concentration. Moreover, it is preferable to perform the intermolecular crosslinking reaction of hyaluronic acid and / or a derivative thereof in a neutral region (
In this crosslinking step, for example, the concentration is preferably finished when the concentration of the aqueous solution of hyaluronic acid and / or a derivative thereof becomes 1 to 10% by weight, preferably 2 to 5% by weight, particularly preferably about 5% by weight. . Insufficient concentration is not preferable because the swelling property of the hyaluronic acid sponge obtained after the next sponging step may not be sufficiently suppressed. That is, if the hyaluronic acid sponge has high swellability, for example, when it is immersed in a liquid medium at the time of cell culture, it swells more than necessary, and as a result, it becomes brittle and the shape becomes large. It becomes difficult. On the other hand, if the end point of concentration is controlled as described above, the swelling property of the hyaluronic acid sponge is sufficiently suppressed, so that it does not swell more than necessary when immersed in a liquid medium or the like, and as a result is fragile. And the shape does not become too large, handling becomes easy. Note that if the cross-linking step is performed until the concentrate is completely film-like, the hyaluronic acid sponge is not formed even if the sponge-forming step is performed by vacuum freeze-drying. It is necessary to set the end point.
Next, in the sponge formation step, a hyaluronic acid sponge is obtained by lyophilizing the intermolecular cross-linked product after the crosslinking step, but the temperature condition at the time of vacuum lyophilization is such that large ice crystals are not formed. About −85 ° C. to −30 ° C., preferably about −85 ° C. to −50 ° C., more preferably about −85 ° C. The vacuum condition is 30 × 10 -3 ~ 100 × 10 -3 It is preferably about mmbar (3 to 10 Pa), more preferably 30 × 10. -3 ~ 50x10 -3 mmbar (3-5 Pa).
In this sponge formation step, the intermolecular cross-linked product obtained without sufficient concentration in the cross-linking step is subjected to at least one freezing and thawing operation, followed by vacuum freeze-drying and then hyaluronic acid. It is preferred to obtain a sponge. When the operation of freezing and thawing is not performed before vacuum freeze-drying, it may become a sponge that does not retain its shape when it is wet, whereas when it is frozen and thawed before vacuum freeze-drying This is probably due to the promotion of intermolecular interactions such as hydrogen bonding, but it is possible to obtain a sponge that easily retains its shape when it contains water. As described above, the operation of freezing and thawing before vacuum freeze-drying may be performed a plurality of times, but it is preferably performed once in consideration of simplification of the manufacturing process. In addition, the freezing temperature condition in the operation of freezing and thawing before vacuum freeze-drying is about −85 ° C. to −30 ° C., preferably about −85 ° C. to −50 ° C., more preferably about −85 ° C. The temperature condition for thawing is about 20 ° C. to 30 ° C., preferably about 30 ° C. However, the temperature may be divided in multiple stages at the time of thawing and may be thawed. In this case, care should be taken not to break the intermolecular cross-linked product. In addition, since the intermolecular cross-linked product obtained by sufficient concentration under appropriate concentration conditions in the cross-linking step has sufficient strength, the freeze / thaw operation before vacuum lyophilization is not necessarily performed. There is no need to do it.
It is preferable to provide a water washing step for washing the hyaluronic acid sponge produced in the sponge forming step before proceeding to the next lamination step. In this washing step, an inactivating agent that inactivates the unreacted crosslinking agent may be added to the washing water. For example, when a water-soluble epoxy compound is used as the cross-linking agent, the deactivator preferably has a function of opening the epoxy group and does not harm the living body. For example, glycine is used. .
Next, in the laminating step, a cell adhesion part is formed by absorbing an aqueous solution of a polymer material derived from a living body from at least one surface of a hyaluronic acid sponge and then vacuum lyophilizing it. As described above, collagen, gelatin, fibrin, alginic acid and the like can be mentioned. Among these, collagen, particularly atelocollagen with low antigenicity is preferable. Moreover, as an aqueous solution of the polymer material derived from a living body, an aqueous solution of 0.2 to 1.0% by weight, preferably an aqueous solution of 0.2 to 0.5% by weight, more preferably an aqueous solution of about 0.5% by weight. In addition, the weight ratio of the bio-derived polymer material to hyaluronic acid may be about 1: 2 to 10, preferably 1: 2 to 4, particularly preferably about 1: 4. The conditions for vacuum freeze-drying are the same as the conditions for vacuum freeze-drying in the sponge formation process.
Further, in this laminating process, an aqueous solution of a polymer material derived from a living body is absorbed from at least one side of the hyaluronic acid sponge, and therefore, in the vicinity of the boundary between the hyaluronic acid sponge and the cell adhesion portion of the obtained tissue regeneration substrate. In this state, a high-molecular material derived from a living body enters the hyaluronic acid sponge. For this reason, even if there is a difference in the swelling rate between the hyaluronic acid sponge and the cell adhesion portion, there is no possibility that the two will be peeled off when the tissue regeneration substrate is hydrated.
In this laminating step, a plurality of holes are formed on one side of the hyaluronic acid sponge, an aqueous solution of a polymer material derived from a living body is absorbed from one side of the holes, and then the cell adhesion part is formed by vacuum lyophilization. May be. In this case, the aqueous solution of the polymer material derived from a living body penetrates into the pores of the hyaluronic acid sponge and also enters a plurality of holes, so that an anchoring effect can be obtained between the hyaluronic acid sponge and the hyaluronic acid sponge. By infiltrating an aqueous solution of a biopolymer material into each hole or pore in this way and then vacuum freeze-drying, the adhesion between the hyaluronic acid sponge and the cell adhesion part in the tissue regeneration substrate can be further improved. it can. The “hole” mentioned here includes not only general holes such as round holes and square holes, but also those having a larger contact area than a flat surface such as cuts and irregularities. It is.
When collagen or gelatin is used as a polymer material derived from a living body in this laminating process, collagen or gelatin flows out when water is contained by irradiating an ultraviolet lamp after the laminating process to partially crosslink the collagen or gelatin. It is preferable to sterilize with ethylene oxide gas or the like after that. Moreover, when the above-mentioned water washing process is not performed, in order to inactivate an unreacted crosslinking agent, it is preferable to add the inactivating agent to the aqueous solution of the polymeric material derived from a living body. As described above, for example, when a water-soluble epoxy compound is used as a cross-linking agent, the deactivator has a function of opening an epoxy group such as glycine and does not cause harm to the living body. preferable.
By the way, instead of the above-described sponge formation process and lamination process, the following sponge formation and lamination processes may be adopted. That is, a step of forming a cell adhesion portion together with the hyaluronic acid sponge by bringing the intermolecular cross-linked product after the cross-linking step into contact with an aqueous solution of a polymer material derived from a living body and then vacuum freeze-drying may be employed. In this case, since the vacuum freeze-drying can be completed only once, the manufacturing process is simplified.
In this sponge / laminate process, the aqueous solution of the polymer material derived from the living body is brought into contact with the intermolecular crosslinked product before the sponge formation, so that the aqueous solution of the polymer material derived from the living body penetrates into the intermolecular crosslinked product before the sponge formation. It is difficult, and the adhesion between the hyaluronic acid sponge and the cell adhesion portion in the finally obtained tissue regeneration substrate may be insufficient. Therefore, it is preferable to provide a plurality of holes in the vertical direction of the intermolecular cross-linked product of hyaluronic acid and / or a derivative thereof to increase the contact area with the aqueous solution of the polymer material derived from the living body to obtain the anchoring effect. Thus, it is preferable to improve the adhesion between the hyaluronic acid sponge and the cell adhesion part in the tissue regeneration substrate by infiltrating each hole with an aqueous solution of a biopolymer material and then freeze-drying it in vacuo. The “hole” mentioned here includes not only general holes such as round holes and square holes, but also those having a larger contact area than a flat surface such as cuts and irregularities. It is.
In order to produce a tissue regeneration base material in which a hyaluronic acid sponge is supported on a support by adopting this sponge-forming / laminating step, for example, the following procedure is performed. That is, a support is laid in advance on the bottom of the container, and an aqueous solution of hyaluronic acid and / or a derivative thereof to which a crosslinking agent has been added is placed in the container. Thereafter, this aqueous solution is heated and concentrated to prepare an intermolecular cross-linked product of hyaluronic acid and / or a derivative thereof. Thereafter, a plurality of holes are provided in the container in the vertical direction of the intermolecular cross-linked product, an aqueous solution of a polymer material derived from a living body is added to contact the intermolecular cross-linked product, and vacuum freeze-dried. If necessary, an operation for freezing and thawing the intermolecular cross-linked product may be performed before vacuum freeze-drying (for example, before or after providing holes). By performing such an operation, a sponge is obtained that has improved strength and can easily retain its shape when it is wet.
According to a third aspect of the present invention, there is provided a transplant material, comprising the above-described tissue regeneration base material and cells held in the cell adhesion portion of the tissue regeneration base material. This transplant material allows cells to be easily cultured in vitro because the cells are held in the cell adhesion part with good adhesion, and the tissue is stimulated by the ability of hyaluronic acid to migrate and wound healing due to its moisturizing effect. Play properly. Here, the cells held in the cell adhesion part may be any cells, but fibroblasts, keratinocytes, pigment producing cells, vascular endothelial cells, endothelial cells, epithelial cells, chondrocytes, osteoblasts. Examples include cells, myoblasts, fat cells, hepatocytes, nerve cells, cardiomyocytes, and Langerhans islet cells. For example, in order to use a transplant material that holds fibroblasts derived from another person, the transplant material is transplanted into a skin wound, covered with a film containing an antibacterial agent (for example, a polyurethane film), and further dressed. Cover. By doing so, healing is promoted by various cytokines produced from fibroblasts derived from others.
A fourth aspect of the present invention is a method for producing such a transplant material, characterized in that after the above-described tissue regeneration base material is produced, cells are incorporated into the cell adhesion portion of the tissue regeneration base material. . According to this production method, the third transplanting material of the present invention can be produced relatively easily. Here, as a method for incorporating cells into the cell adhesion part, for example, there is a method of holding a tissue regeneration substrate by immersing the tissue regeneration substrate in a culture solution and taking the cells into the pores.
BEST MODE FOR CARRYING OUT THE INVENTION
In the following, preferred embodiments of the present invention will be described. The present invention is not limited to the following examples. In the following, “%” represents “% by weight” unless otherwise specified.
(Example 1)
[1] Fabrication of tissue regeneration substrate (see FIG. 1)
[1-1] Preparation of hyaluronic acid sponge
10 g of sodium hyaluronate (molecular weight of about 2 million) was dissolved in 1 L of distilled water to prepare a 1% hyaluronic acid aqueous solution (pH 6). The dissolution of hyaluronic acid was carried out with sufficient time while stirring with a mechanical stirrer. On the other hand, 1 g of Denacol EX313 (glycerol diglycidyl ether; Nagase Kasei Kogyo) as a water-soluble epoxy compound was diluted in 20 ml of distilled water to prepare a Denacol EX313 solution (hereinafter referred to as EX313 solution). 20 ml of the EX313 solution thus prepared was added to the stirring hyaluronic acid aqueous solution, and further stirred for about 30 minutes to prepare a hyaluronic acid-EX313 mixed liquid.
The obtained hyaluronic acid-EX313 mixed solution has a bottom area of 180 cm. 2 180 ml was injected into a (10 cm × 18 cm) tray. The depth of the hyaluronic acid-EX313 mixed solution at this time was about 1 cm. The hyaluronic acid-EX313 mixed solution injected into the tray was allowed to stand at room temperature for 2 hours, air bubbles were removed from the hyaluronic acid-EX313 mixed solution, and then cut in advance according to the size of the tray. ) Was added to the hyaluronic acid-EX313 mixed solution.
The hyaluronic acid-EX313 mixed solution on which the nonwoven fabric was placed was placed in an air circulation type dry heat oven and allowed to stand at 50 ° C. for 10 hours. As a result, the hydroxyl group (hydroxyl group) or carboxyl group of hyaluronic acid reacts with the epoxy group of Denacol EX313 to form an intermolecular crosslinked product of hyaluronic acid, and the liquid volume is about 2/5 (that is, the concentration of hyaluronic acid To about 2.5% by weight). The depth of the concentrated liquid at this time was about 4 mm.
The concentrate was then frozen at -85 ° C. Although the freezing time depends on the ability of the freezer, it is about 5 to 6 hours and was completely frozen. After this freezing, it was allowed to stand at room temperature for 1 to 1.5 hours and thawed once. After thawing, it was completely frozen again in a freezer at -85 ° C for about 5 to 6 hours. And 30x10 -3 ~ 50x10 -3 By performing vacuum freeze-drying treatment at mmbar (3 to 5 Pa), a hyaluronic acid intermolecular cross-linked product having a sponge structure (hereinafter referred to as cross-linked hyaluronic acid sponge) was obtained.
[1-2] Lamination with collagen sponge
1 g of atelocollagen was dissolved in 500 ml of distilled water and adjusted to
The crosslinked hyaluronic acid sponge produced as described above was immersed in a tray containing an aqueous solution of atelocollagen so that Benlyse was positioned above, and left for 1 hour to impregnate the crosslinked hyaluronic acid sponge with the collagen aqueous solution. And the hyaluronic acid sponge immersed in this collagen aqueous solution was frozen at -85 degreeC, and was vacuum-dried.
The collagen sponge layered on the crosslinked hyaluronic acid sponge after vacuum freeze-drying is irradiated to the collagen sponge side for 30 minutes from a distance of 25 cm using a 15 W UV lamp (254 nm), thereby cross-linking the collagen molecules. went. Then, it filled in the sterilization bag and performed EOG (ethylene oxide gas) sterilization at 60 degreeC for 20 hours. As a result, a tissue regeneration base material, that is, a crosslinked hyaluronic acid sponge provided with a crosslinked collagen sponge (cell adhesion part) and supported by a Benize (support) was obtained.
[2] Production of transplant material
Cultured rabbit fibroblasts suspended in Dulbecco's modified Eagle's minimum essential medium (DMEM + 10% FBS, manufactured by Gibco) containing 10% fetal bovine serum (FBS) on the opposite side of the tissue regeneration substrate from
[3] Transplant test
The cultured dermis (transplant material) stored in a freezer was thawed at the time of use to remove the cryopreservation solution, washed twice with 30 ml of Hanks' solution, and used for animal experiments. A 7 cm diameter circle was drawn on the back of the rabbit, and the skin of all layers was excised to form a skin defect wound. Apply the previous cultured dermis to this skin defect wound, suture the periphery of the wound surface, apply the polyurethane film wound dressing on it, place a sterilized pad, suture the wound periphery, stretch It was pressed and fixed with a bandage. As a result, good granulation tissue formation and a marked reduction in wound area were observed.
(Example 2)
[1] Fabrication of tissue regeneration substrate (see FIG. 2)
[1-1] Preparation of hyaluronic acid sponge
In the same manner as in Example 1, a hyaluronic acid-EX313 mixed liquid was prepared, and the obtained hyaluronic acid-EX313 mixed liquid had a bottom area of 180 cm. 2 180 ml was injected into a (10 cm × 18 cm) tray. Benlyse (previously) cut in accordance with the size of the tray was placed in advance on the tray, and a small amount of distilled water was added to hydrate and adhered to the bottom of the tray. The depth of the hyaluronic acid-EX313 mixed solution injected into the tray was about 1 cm.
The hyaluronic acid-EX313 mixed solution in this state was put into an air circulation type dry heat oven and allowed to stand at 50 ° C. for 15 hours. As a result, the hydroxyl group (hydroxyl group) or carboxyl group of hyaluronic acid reacts with the epoxy group of Denacol EX313 to produce an intermolecular crosslinked product of hyaluronic acid, and the liquid volume is about 1/5 (that is, the hyaluronic acid concentration). To about 5% by weight). The depth of the concentrated liquid at this time was about 2 to 3 mm.
The concentrate was then frozen at -85 ° C. Although the freezing time depends on the ability of the freezer, it is about 5 to 6 hours and was completely frozen. And 30x10 -3 ~ 50x10 -3 By performing vacuum freeze-drying treatment at mmbar (3 to 5 Pa), a hyaluronic acid intermolecular cross-linked product having a sponge structure (hereinafter referred to as cross-linked hyaluronic acid sponge) was obtained. Thereafter, in order to remove the unreacted crosslinking agent, the crosslinked hyaluronic acid sponge was immersed in a container containing 15 L of ion exchange water for one day and washed with water.
[1-2] Lamination with collagen sponge
2.5 g of atelocollagen was dissolved in 500 ml of distilled water and adjusted to pH 3.2 with 1N HCl to prepare a 0.5% atelocollagen aqueous solution. Cross-linked hyaluronic acid sponge washed with water, bottom area 180cm with the nonwoven fabric facing down 2 And 90 ml of the prepared atelocollagen aqueous solution was injected thereon. The atelocollagen aqueous solution is half the amount of the original hyaluronic acid aqueous solution (180 ml), and the weight ratio of collagen to hyaluronic acid is 1: 4.
The cross-linked hyaluronic acid sponge was slightly shrunk around, and a gap was formed between the side surfaces of the tray. When the atelocollagen aqueous solution entered the gap, the collagen was disposed so as to wrap around the hyaluronic acid sponge, and the separation of the crosslinked hyaluronic acid sponge and the collagen sponge during swelling was suppressed.
After a day and night after injecting the aqueous solution of atelocollagen, the hyaluronic acid sponge containing the aqueous solution of atelocollagen was lyophilized at −85 ° C. under vacuum. Collagen intermolecular cross-linking is achieved by irradiating a collagen sponge layered on a cross-linked hyaluronic acid sponge after vacuum freeze-drying using a 15 W UV lamp (254 nm) for 30 minutes from a distance of 25 cm. went. Then, it filled in the sterilization bag and performed EOG sterilization at 60 degreeC for 20 hours. As a result, a tissue regeneration base material, that is, a crosslinked hyaluronic acid sponge provided with a crosslinked collagen sponge (cell adhesion part) and supported by a Benize (support) was obtained.
[2] Production of transplant material
180cm produced by the above method 2 90 cm of the tissue regeneration substrate 2 The size of. 180cm of the cut tissue regeneration substrate 2 And 100 ml of DMEM + 10% FBS was injected to adjust the pH. After adjusting the pH (pH 7.4), the culture solution is discarded once, and 5 ml of human fibroblast suspension is used as a tissue regeneration substrate at 5 × 10 5. 5 Cells / cm 2 Seeded at a density of After seeding, the mixture was allowed to stand overnight at 37 ° C. to fix human fibroblasts on the tissue regeneration substrate. Thereafter, 100 ml of the above DMEM + 10% FBS medium was added, and the temperature was 37 ° C., 5% CO2. 2 Incubation was carried out for 1 week. The cultured dermis (transplant material) produced in this way has not only sufficient strength, but also the surroundings are only shrunk by about 2 to 3 mm, so that handling is good and there is a risk of loss. There was nothing.
(Example 3)
[1] Fabrication of tissue regeneration substrate (see FIG. 3)
[1-1] Preparation of hyaluronic acid sponge
20 g of sodium hyaluronate (molecular weight of about 2 million) was dissolved in 2 L of distilled water to prepare a 1% aqueous solution of hyaluronic acid (pH 6), and then adjusted to pH 3.5 with 1N HCl. The dissolution of hyaluronic acid was carried out with sufficient time while stirring with a mechanical stirrer. On the other hand, 4 g of Denacol EX810 (glycerol diglycidyl ether; Nagase Kasei Kogyo) as a water-soluble epoxy compound was diluted in 40 ml of distilled water to prepare a Denacol EX810 solution (hereinafter referred to as EX810 solution). The EX810 solution (40 ml) thus prepared was added to the stirring hyaluronic acid aqueous solution, and further stirred for about 30 minutes to prepare a hyaluronic acid-EX810 mixed solution.
The obtained hyaluronic acid-EX810 mixed solution has a bottom area of 110 cm 2 50 g was injected into a (10 cm × 11 cm) tray. Benlyse (previously) cut in accordance with the size of the tray was placed in advance on the tray, and a small amount of distilled water was added to hydrate and adhered to the bottom of the tray. The depth of the hyaluronic acid-EX810 mixed solution injected into the tray was about 5 mm.
The hyaluronic acid-EX810 mixed solution in this state was placed in an air circulation heater and allowed to stand at 50 ° C. for 5 hours. As a result, the hydroxyl group (hydroxyl group) or carboxyl group of hyaluronic acid reacts with the epoxy group of Denacol EX810 to produce an intermolecular cross-linked product of hyaluronic acid, and the liquid volume is about 1/2 (that is, the concentration of hyaluronic acid To about 2% by weight). The depth of the concentrate at this time was approximately 2 to 3 mm.
The concentrate was then frozen at -85 ° C. Although the freezing time depends on the ability of the freezer, it is about 5 to 6 hours and was completely frozen. And 30x10 -3 ~ 50x10 -3 The vacuum freeze-drying process was performed at mmbar (3-5 Pa). Thereafter, in order to remove the unreacted crosslinking agent, the crosslinked hyaluronic acid sponge was immersed overnight in a container containing 1 L of distilled water and washed with water.
After washing with water, it was completely frozen again with a freezer at -85 ° C. And 30x10 -3 ~ 50x10 -3 By performing vacuum freeze-drying treatment at mmbar (3 to 5 Pa), a hyaluronic acid intermolecular cross-linked product having a sponge structure (hereinafter referred to as cross-linked hyaluronic acid sponge) was obtained.
[1-2] Lamination with collagen sponge
Prior to lamination with a collagen sponge, a cross-linked hyaluronic acid sponge was drilled using Kenzan. The distance between the holes at this time was approximately 4 mm.
8 g of atelocollagen was dissolved in 1.6 L of distilled water, adjusted to pH 3.5 with 1N HCl, and a 0.5% atelocollagen aqueous solution was prepared. The prepared atelocollagen aqueous solution is bottom area 110cm 2 40 g was injected into the tray. The atelocollagen aqueous solution is about 4/5 of the amount of the original hyaluronic acid aqueous solution (50 g), and the weight ratio of collagen to hyaluronic acid is 2: 5.
The cross-linked hyaluronic acid sponge prepared as described above was floated on a tray into which the atelocollagen aqueous solution was injected so that Benlyse was positioned above, and allowed to stand overnight, so that the cross-linked hyaluronic acid sponge was soaked in the cross-linked hyaluronic acid sponge. Then, the hyaluronic acid sponge soaked with an aqueous collagen solution was completely frozen at -85 ° C. and then vacuum freeze-dried.
Intermolecular cross-linking of collagen was performed by irradiating both sides of a collagen sponge layered on a cross-linked hyaluronic acid sponge after vacuum freeze-drying using a 15 W ultraviolet lamp (254 nm) for 30 minutes from a distance of 20 cm. . Then, it filled in the sterilization bag and performed EOG (ethylene oxide gas) sterilization at 60 degreeC for 20 hours. As a result, a tissue regeneration base material, that is, a crosslinked hyaluronic acid sponge provided with a crosslinked collagen sponge (cell adhesion part), which was supported by Benlyse (support), was obtained.
[2] Production of transplant material
The tissue regeneration substrate produced by the above method was placed in a tray, and 50 ml of DMEM + 10% FBS was injected to adjust the pH. After adjusting the pH (pH 7.4), the culture solution is discarded once, and 5 ml of human fibroblast suspension is used as a tissue regeneration substrate at 1 × 10 5 Cells / cm 2 Seeded at a density of After seeding, the mixture was allowed to stand overnight at 37 ° C. to fix human fibroblasts on the tissue regeneration substrate. Then, 50 ml of the above DMEM + 10% FBS medium was added, and the temperature was 37 ° C., 5% CO2. 2 Incubation was carried out for 1 week. The cultured dermis (transplant material) thus prepared was removed from the medium, placed in a cryopreservation solution (10% DMSO-containing DMEM + 20% FBS), and stored in a −152 ° C. freezer.
[3] Transplant test
The cultured dermis (transplant material) stored in a freezer was thawed at the time of use to remove the cryopreservation solution, washed 3 times with 30-50 ml of lactated Ringer's solution and applied to human clinical practice. The wound surface was disinfected after removing the remaining necrotic tissue and thoroughly washed with physiological saline. The previous cultured dermis was applied to the skin defect wound, and the periphery of the cultured dermis was sutured and fixed. A gauze impregnated with an antibiotic-containing ointment was applied thereon, a sterilized gauze was placed thereon, the periphery of the wound was sutured, and it was fixed by pressing with an elastic bandage. As a result, good granulation tissue formation, marked reduction in wound area, and epithelialization from around the wound were observed.
(Example 4)
[1] Fabrication of tissue regeneration substrate (see FIG. 4)
[1-1] Preparation of hyaluronic acid sponge
2 g of sodium hyaluronate (molecular weight of about 2 million) was dissolved in 200 mL of distilled water to prepare a 1% hyaluronic acid aqueous solution (pH 6), and then adjusted to pH 3.5 with 1N HCl. The dissolution of hyaluronic acid was carried out with sufficient time while stirring with a mechanical stirrer. On the other hand, as a water-soluble epoxy compound, 0.2 g of Denacol EX810 was diluted in 2 ml of distilled water to prepare a Denacol EX810 solution (hereinafter referred to as EX810 solution). The EX810 solution thus prepared (2 ml) was added to the stirring hyaluronic acid aqueous solution and further stirred for about 30 minutes to prepare a hyaluronic acid-EX810 mixed solution.
4.8 g of the obtained hyaluronic acid-EX810 mixed solution was poured into a dish having a bottom area of φ35 mm. The depth of the hyaluronic acid-EX810 mixed solution injected into the dish was about 5 mm.
The hyaluronic acid-EX810 mixed solution in this state was placed in an air circulation heater and allowed to stand at 50 ° C. for 5 hours. As a result, the hydroxyl group (hydroxyl group) or carboxyl group of hyaluronic acid reacts with the epoxy group of Denacol EX810 to produce an intermolecular cross-linked product of hyaluronic acid, and the liquid volume is about 1/2 (that is, the concentration of hyaluronic acid To about 2% by weight). The depth of the concentrate at this time was approximately 2 to 3 mm.
The concentrate was then frozen at -85 ° C. Although the freezing time depends on the ability of the freezer, it is about 5 to 6 hours and was completely frozen. And 30x10 -3 ~ 50x10 -3 The vacuum freeze-drying process was performed at mmbar (3-5 Pa). Then, in order to remove an unreacted crosslinking agent, it was immersed in the container containing 1 L distilled water overnight, and washed with water. After washing with water, it was completely frozen again with a freezer at -85 ° C. And 30x10 -3 ~ 50x10 -3 By performing vacuum freeze-drying treatment at mmbar (3 to 5 Pa), a hyaluronic acid intermolecular cross-linked product having a sponge structure (hereinafter referred to as cross-linked hyaluronic acid sponge) was obtained.
[1-2] Lamination with collagen sponge
Prior to lamination with a collagen sponge, a cross-linked hyaluronic acid sponge was drilled using Kenzan. The distance between the holes at this time was approximately 4 mm.
On the other hand, 0.5% collagen aqueous solution (KOKENCELLGEN I-PC: manufactured by Koken Co., Ltd.) was injected into a φ35 mm dish. The collagen aqueous solution is about 4/5 the amount of the original hyaluronic acid aqueous solution (4.8 g), and the weight ratio of collagen to hyaluronic acid is about 2: 5.
The cross-linked hyaluronic acid sponge produced as described above was floated on a dish into which a collagen aqueous solution had been injected so that the perforated surface was down, and allowed to stand overnight, so that the collagen aqueous solution was soaked into the cross-linked hyaluronic acid sponge. Then, the hyaluronic acid sponge soaked with an aqueous collagen solution was completely frozen at -85 ° C. and then vacuum freeze-dried.
Intermolecular cross-linking of collagen was performed by irradiating both sides of a collagen sponge layered on a cross-linked hyaluronic acid sponge after vacuum freeze-drying using a 15 W ultraviolet lamp (254 nm) for 30 minutes from a distance of 20 cm. . Then, it filled in the sterilization bag and performed EOG sterilization at 60 degreeC for 20 hours. As a result, a tissue regeneration substrate, that is, a crosslinked hyaluronic acid sponge provided with a crosslinked collagen sponge (cell adhesion part) was obtained. The tissue regeneration substrate was not added with strength by Benize, but could be easily handled with tweezers or the like.
The tissue regeneration substrate of this example can be used as a cultured dermis as well as Examples 1 to 3, and can also be used as a wound dressing. When used as a wound dressing, removal is unnecessary because it is bioabsorbable, and the collagen cell retention effect can be obtained by applying the collagen side to the wound surface. It is expected that it will elute on the surface and exhibit cell migration and can promote healing.
As another example of Example 2, after intermolecular cross-linking reaction of the hyaluronic acid-EX313 mixed liquid, a hole was formed in the vertical direction of the cross-linked reaction product without vacuum freeze-drying, and the surface having the hole and atelocollagen aqueous solution Can be made into a hyaluronic acid sponge and a collagen sponge at the same time. In this case, the manufacturing process is simplified, and both sponges adhere well because the anchoring effect is obtained by the presence of holes.
Industrial applicability
The present invention is a tissue regeneration substrate mainly composed of hyaluronic acid, and is suitable for cell culture in vitro and tissue regeneration after transplantation, and thus can be widely used in the medical field.
[Brief description of the drawings]
FIG. 1 is an explanatory diagram showing the procedure for producing the tissue regeneration substrate of Example 1, FIG. 2 is an explanatory diagram showing the procedure for producing the tissue regeneration substrate of Example 2, and FIG. 3 is for tissue regeneration of Example 3. FIG. 4 is an explanatory diagram showing the procedure for producing the tissue regeneration substrate of Example 4. FIG.
Claims (10)
（２）該ヒアルロン酸架橋工程で得られた分子間架橋物を真空凍結乾燥することによりヒアルロン酸スポンジを得るスポンジ化工程と、(2) a sponge forming step of obtaining a hyaluronic acid sponge by vacuum lyophilization of the intermolecular crosslinked product obtained in the hyaluronic acid crosslinking step;
（３）該スポンジ化工程で得られたヒアルロン酸スポンジの少なくとも片面にアテロコラーゲン水溶液を吸収させたあと真空凍結乾燥することにより細胞接着部としてコラーゲンスポンジを形成する積層工程と、(3) a laminating step in which a collagen sponge is formed as a cell adhesion part by absorbing a solution of atelocollagen on at least one side of the hyaluronic acid sponge obtained in the sponge step and then vacuum lyophilizing;
（４）該積層工程によって形成されたコラーゲンスポンジに分子間架橋を施すコラーゲン架橋工程とを含むことを特徴とする組織再生用基材の製法。(4) A method for producing a tissue regeneration base material, comprising a collagen crosslinking step of performing intermolecular crosslinking on the collagen sponge formed by the lamination step.
（２）該ヒアルロン酸架橋工程で得られた分子間架橋物を真空凍結乾燥することによりヒアルロン酸スポンジを得るスポンジ化工程と、(2) a sponge forming step of obtaining a hyaluronic acid sponge by vacuum lyophilization of the intermolecular crosslinked product obtained in the hyaluronic acid crosslinking step;
（３）該スポンジ工程で得られたヒアルロン酸スポンジの少なくとも片面からアテロコラーゲン水溶液を吸収させたあと真空凍結乾燥することにより細胞接着部としてのコラーゲンスポンジを形成する積層工程と、(3) a laminating step of forming a collagen sponge as a cell adhesion part by absorbing an atelocollagen aqueous solution from at least one side of the hyaluronic acid sponge obtained in the sponge step and then vacuum-freezing and drying;
（４）該積層工程によって形成されたコラーゲンスポンジに分子間架橋を施すコラーゲン架橋工程とを含むことを特徴とする組織再生用基材の製法。(4) A method for producing a tissue regeneration base material, comprising a collagen crosslinking step of performing intermolecular crosslinking on the collagen sponge formed by the lamination step.
前記第１の架橋工程によって得られた分子間架橋物を生体由来の高分子材料の水溶液と接触させたあと真空凍結乾燥することにより、前記ヒアルロン酸スポンジと該高分子材料からなる前記細胞接着部とを同時に形成するスポンジ化・積層工程であることを特徴とする請求項１〜５のいずれか一つに記載の組織再生用基材の製法。 The cell adhesion part consisting of the hyaluronic acid sponge and the polymer material by bringing the intermolecular crosslinked product obtained by the first crosslinking step into contact with an aqueous solution of a polymer material derived from a living body and then vacuum lyophilization The method for producing a tissue regeneration substrate according to any one of claims 1 to 5, which is a sponge forming and laminating step in which the steps are simultaneously formed.
前記組織再生用基材の細胞接着部に保持された細胞とCells held in the cell adhesion portion of the tissue regeneration substrate;
を備えたことを特徴とする移植用材料。A transplant material characterized by comprising:
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2000373116 | 2000-12-07 | ||
| JP2000373116 | 2000-12-07 | ||
| PCT/JP2001/010751 WO2002045767A1 (en) | 2000-12-07 | 2001-12-07 | Substrate for tissue regeneration, material for transplantation, and processes for producing these |
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| JPWO2002045767A1 JPWO2002045767A1 (en) | 2004-04-08 |
| JP4273450B2 true JP4273450B2 (en) | 2009-06-03 |
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| JP2002547548A Expired - Fee Related JP4273450B2 (en) | 2000-12-07 | 2001-12-07 | Tissue regeneration substrate, transplant material, and production method thereof |
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| JP (1) | JP4273450B2 (en) |
| KR (1) | KR20030061378A (en) |
| CN (1) | CN1774272A (en) |
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| WO (1) | WO2002045767A1 (en) |
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| JPS6358531A (en) * | 1986-08-29 | 1988-03-14 | Hitachi Ltd | Detector for display position |
| AU2003298519A1 (en) * | 2002-06-27 | 2004-06-23 | Roberto Beretta | Methods for preparing a solid-fibrin web |
| WO2004103427A1 (en) * | 2003-05-23 | 2004-12-02 | M D Bio Inc | Filling material and manipulation method |
| US20100136082A1 (en) | 2006-12-22 | 2010-06-03 | Laboratoire Medidom S.A. | In situ system for intra-articular chondral and osseous tissue repair |
| CN101724164B (en) * | 2008-10-31 | 2011-12-14 | 科妍生物科技股份有限公司 | Method for preparing cross-linked hyaluronic acid |
| CN101912633A (en) * | 2010-08-03 | 2010-12-15 | 孙伟庆 | Hyaluronic acid sponge and preparation method thereof |
| EP2606828B1 (en) * | 2011-12-20 | 2018-04-11 | Angioclinic AG | Hyaluronic acid and its use for treating venous insufficiency and varicose veins |
| CN102558600A (en) * | 2011-12-01 | 2012-07-11 | 上海白衣缘生物工程有限公司 | Cross-linked hyaluronan sponge and preparation method for same |
| KR101240518B1 (en) * | 2012-03-26 | 2013-03-11 | 주식회사 제네웰 | Raw materials for transplantation using biocompatible polymers |
| EP2910255A1 (en) * | 2014-02-19 | 2015-08-26 | MedSkin Solutions Dr. Suwelack AG | Methods for the production of biopolymers with defined average molecular weight |
| CN106110367A (en) * | 2016-07-29 | 2016-11-16 | 北京化工大学常州先进材料研究院 | Based on natural polymer MULTILAYER COMPOSITE medical dressing and preparation method thereof |
| WO2019180454A1 (en) * | 2018-03-22 | 2019-09-26 | Queen Mary University Of London | Implantable cell dressing for treatment of disease |
| KR102113778B1 (en) * | 2018-09-27 | 2020-05-21 | 한국화학연구원 | Myocardial-like structures prepared using porous supports method for evaluating drug toxicity using the same |
| CN109998775A (en) * | 2019-03-21 | 2019-07-12 | 中国人民解放军军事科学院军事医学研究院 | A kind of compressed tampon dressing and the preparation method and application thereof |
| CN114681654B (en) * | 2020-12-31 | 2023-04-18 | 广州迈普再生医学科技股份有限公司 | Absorbable sponge dressing with wound repair function and preparation method thereof |
| CN115671365B (en) * | 2022-11-04 | 2024-02-13 | 浙江诸暨聚源生物技术有限公司 | Crosslinked recombinant collagen sponge and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| AU632273B2 (en) * | 1988-03-09 | 1992-12-24 | Terumo Kabushiki Kaisha | Medical material permitting cells to enter thereinto and artificial skin |
| JPH04332561A (en) * | 1991-05-09 | 1992-11-19 | Koken Co Ltd | Matrix for culture skin |
| IT1263144B (en) * | 1993-02-04 | 1996-08-01 | Lanfranco Callegaro | PHARMACEUTICAL COMPOSITIONS INCLUDING SPONGY MATERIAL CONSTITUTED FROM FOREIGN DERIVATIVES OF HYALURONIC ACID IN ASSOCIATION WITH OTHER PHARMACOLOGICALLY ACTIVE SUBSTANCES |
| JPH06292716A (en) * | 1993-04-09 | 1994-10-21 | Shimizu Yoshihiko | Medical material |
| JPH11322807A (en) * | 1998-05-11 | 1999-11-26 | Mitsubishi Chemical Corp | Production of crosslinked hyaluronic acid sponge |
| JPH11319066A (en) * | 1998-05-11 | 1999-11-24 | Mitsubishi Chemical Corp | Wound coating material |
| EP1022031B1 (en) * | 1999-01-21 | 2005-03-23 | Nipro Corporation | Suturable adhesion-preventing membrane |
| JP2000237294A (en) * | 1999-02-18 | 2000-09-05 | Denki Kagaku Kogyo Kk | Medical material containing hyaluronic acid gel |
| JP2001212224A (en) * | 2000-02-04 | 2001-08-07 | Toyobo Co Ltd | Wound coating material |
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2001
- 2001-12-07 JP JP2002547548A patent/JP4273450B2/en not_active Expired - Fee Related
- 2001-12-07 KR KR10-2003-7005483A patent/KR20030061378A/en not_active Application Discontinuation
- 2001-12-07 WO PCT/JP2001/010751 patent/WO2002045767A1/en active Application Filing
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| AU2002221089A1 (en) | 2002-06-18 |
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| KR20030061378A (en) | 2003-07-18 |
| JPWO2002045767A1 (en) | 2004-04-08 |
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