JP5856785B2 - Sheet-like hemostatic material with excellent hemostatic effect - Google Patents
Sheet-like hemostatic material with excellent hemostatic effect Download PDFInfo
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Description
本発明は、フィブリノゲンが固定化された繊維成形体と、トロンビンが固定化された繊維成形体とからなるシート状止血材に関する。 The present invention relates to a sheet-like hemostatic material comprising a fiber molded body on which fibrinogen is immobilized and a fiber molded body on which thrombin is immobilized.
フィブリノゲンは、止血に関与する血液凝固因子のひとつであり、健康人の血漿１００ｍＬ中に２００ｍｇから４００ｍｇ含まれている。フィブリノゲン製剤（フィブリン糊）とは、このフィブリノゲンをヒト血漿中から分離精製して製造される血液製剤の一種（血漿分画製剤）である。医療現場において、止血および組織閉鎖などの組織接着は重要な処置の一つであり、フィブリノゲン製剤は、広く外科手術の領域で使用されている。フィブリノゲン製剤の作用機序は、まず血管が損傷を受けると凝固系の活性化が生じ、最終的に活性化されたトロンビンが可溶性フィブリノゲンを不溶性のフィブリンに変換する。このフィブリンは接着力を有しており、止血や組織接着に有効に働く。
フィブリノゲン製剤は主に２液混合型の液状型製剤（特許文献１）とコラーゲンなどの支持体にフィブリノゲンとトロンビンを固定化したシート型製剤（特許文献２）が存在する。しかしながら、液状型製剤は凍結乾燥されたフィブリノゲンとトロンビンをそれぞれ使用時に溶解して用いる必要があること、さらには完全に溶解させるのに時間がかかり、緊急時に使用できないなどの問題点を有している。
Fibrinogen is one of the blood coagulation factors involved in hemostasis, and is contained in 200 to 400 mg in 100 mL of healthy human plasma. Fibrinogen preparation (fibrin glue) is a kind of blood preparation (plasma fraction preparation) produced by separating and purifying fibrinogen from human plasma. In the medical field, tissue adhesion such as hemostasis and tissue closure is one of the important procedures, and fibrinogen preparations are widely used in the field of surgery. The mechanism of action of fibrinogen preparations is that when blood vessels are damaged, activation of the coagulation system occurs, and finally activated thrombin converts soluble fibrinogen into insoluble fibrin. This fibrin has an adhesive force and works effectively for hemostasis and tissue adhesion.
As for fibrinogen preparations, there are mainly two-component liquid type preparations (Patent Document 1) and sheet type preparations (Patent Document 2) in which fibrinogen and thrombin are immobilized on a support such as collagen. However, liquid-type preparations have problems that it is necessary to dissolve lyophilized fibrinogen and thrombin at the time of use, and that it takes time to completely dissolve and cannot be used in an emergency. Yes.
一方、シート状のフィブリン糊（製品名：タココンブ（登録商標）／ＣＳＬベーリング社）は、支持体であるコラーゲンは厚みがあり、また乾燥状態で臓器閉鎖部位に適用するには支持体自身が硬く、柔軟性を欠くため、閉鎖すべき創傷部位での密着性が低く、効果的な閉鎖は困難である。また、本シート製剤は、支持体が馬コラーゲンであり、ヒト以外の動物成分が使用されているため、適用対象がヒトである場合、異種タンパクに対する抗体の出現やプリオン病等の人畜共通感染症の危険性が存在するため理想的なものとは言い難い。
また、タココンブの場合、同一シート内にフィブリノゲンとトロンビンが共存されており、使用直前に溶液に浸すと同時にフィブリノゲンとトロンビンが溶解して反応が開始するが、その反応はシート内部で起こる。そのため、たとえフィブリンが溶出したとしても組織接着部位へ十分浸透する前に凝固反応が進み、組織表面のみの接着となるため、十分な組織接着効果を示さない。
On the other hand, in the sheet-like fibrin glue (product name: Tacocomb (registered trademark) / CSL Behring Co., Ltd.), the collagen as a support is thick, and the support itself is hard to apply to an organ closure site in a dry state. Due to lack of flexibility, adhesion at the wound site to be closed is low, and effective closure is difficult. In addition, since the support of this sheet preparation is equine collagen and non-human animal components are used, when the application target is a human, the appearance of antibodies against heterologous proteins and zoonotic diseases such as prion diseases It is hard to say that it is ideal because of the dangers.
In the case of an octopus kombu, fibrinogen and thrombin coexist in the same sheet, and the fibrinogen and thrombin start dissolving at the same time as being immersed in the solution immediately before use, but the reaction occurs inside the sheet. For this reason, even if fibrin is eluted, the coagulation reaction proceeds before it sufficiently penetrates into the tissue adhesion site, and only the tissue surface is adhered, so that a sufficient tissue adhesion effect is not exhibited.
したがって、これらの現行製剤により、全ての組織接着、止血が可能となるものではなく、現行製剤では要求される接着力、閉鎖力を示さない場合がある。そのため、医療現場ではより簡易的に使用でき、更に強力な接着力をもった組織接着剤が求められている。
このような問題点を解決するために、生体吸収性材料からなるシート状フィブリン糊接着剤の検討がなされている（特許文献３）。しかしながら、フィブリノゲン固定化シートに非イオン性界面活性剤が必要であること、また、凍結乾燥後のシートは担持させたフィブリノゲンがシートから剥がれ落ちてしまうという問題点を抱えている。
Therefore, these current preparations do not enable all tissue adhesion and hemostasis, and the current preparations may not exhibit the required adhesive force and closing force. Therefore, there is a demand for a tissue adhesive that can be used more easily in the medical field and has a stronger adhesive force.
In order to solve such problems, a sheet-like fibrin glue adhesive made of a bioabsorbable material has been studied (Patent Document 3). However, the non-ionic surfactant is required for the fibrinogen-immobilized sheet, and the lyophilized sheet has problems that the supported fibrinogen is peeled off from the sheet.
本発明が解決しようとする課題は、フィブリノゲンおよびトロンビンの担持性に優れ、したがって止血性に優れたシート状止血材を提供することである。 The problem to be solved by the present invention is to provide a sheet-like hemostatic material having excellent fibrinogen and thrombin supportability, and thus excellent hemostasis.
発明者らは、フィブリノゲンおよびトロンビンの担持性に優れた繊維成形体について鋭意研究した結果、０．０１ｍｍ２以上の貫通孔を有さずしかも繊維表面の平滑度が１．０７以下である繊維成形体であれば、フィブリノゲンおよびトロンビンの支持体として優れていることを見出し、本発明に到達した。 As a result of diligent research on a fiber molded article excellent in supportability of fibrinogen and thrombin, the inventors have found that there is no through hole of 0.01 mm 2 or more and the fiber surface has a smoothness of 1.07 or less. The present invention has been found to be excellent as a support for fibrinogen and thrombin, and the present invention has been achieved.
すなわち、本発明は繊維成形体（Ａ）にフィブリノゲンが固定化されたシートと、繊維成形体（Ｂ）にトロンビンが固定化されたシートから構成されるシート状止血材であって、
繊維成形体（Ａ）および繊維成形体（Ｂ）はいずれも平均繊維径０．１〜１０μｍの生分解性ポリマー繊維からなり、かつ、
０．０１ｍｍ２以上の貫通孔を有さない特性と、繊維表面の平滑度が１．０７以下である特性とを有する、
ことを特徴とするシート状止血材である。
That is, the present invention is a sheet-like hemostatic material composed of a sheet in which fibrinogen is immobilized on the fiber molded body (A) and a sheet in which thrombin is immobilized on the fiber molded body (B),
The fiber molded body (A) and the fiber molded body (B) are both composed of biodegradable polymer fibers having an average fiber diameter of 0.1 to 10 μm, and
Has a characteristic that no 0.01 mm 2 or more through-holes, and a characteristic smoothness of the fiber surface is 1.07 or less,
This is a sheet-like hemostatic material.
本発明のシート状止血材は、フィブリノゲンおよびトロンビンの担持性に優れ、止血性に優れる。 The sheet-like hemostatic material of the present invention is excellent in fibrinogen and thrombin carrying properties and is excellent in hemostatic properties.
本発明は繊維成形体（Ａ）にフィブリノゲンが固定化されたシートと、繊維成形体（Ｂ）にトロンビンが固定化されたシートから構成されるシート状止血材である。
本発明で用いられるフィブリノゲンやトロンビンは、動物から調製したものでも、遺伝子組換え技術により製造したものでもよい。動物由来であればヒト由来が好ましい。また、アミノ酸配列を改変した蛋白質も使用できる。
なお、シート状止血材はフィブリノゲンおよびトロンビンを含有するため、保存中に一部でフィブリンを生じることがあるが、こうしたフィブリンを含むものも本発明の範囲である。
The present invention is a sheet-like hemostatic material composed of a sheet in which fibrinogen is immobilized on the fiber molded body (A) and a sheet in which thrombin is immobilized on the fiber molded body (B).
The fibrinogen and thrombin used in the present invention may be prepared from an animal or manufactured by a gene recombination technique. Human origin is preferable if it is animal origin. A protein having a modified amino acid sequence can also be used.
In addition, since a sheet-like hemostatic material contains fibrinogen and thrombin, fibrin may be produced in part during storage, but those containing such fibrin are also within the scope of the present invention.
本発明でいう「固定化」とは、フィブリノゲンやトロンビンが繊維成形体の表面だけでなく、繊維成形体の内部にも存在している状態とすることをいうが、フィブリノゲンやトロンビンを繊維の内部にまで存在させることは要しない。
本発明のシート状止血材において、フィブリノゲンやトロンビンを固定化する方法は問わないが、例えばフィブリノゲン等の溶液に予め成形された繊維成形体を浸漬した後、凍結乾燥することにより作製できる。また、フィブリノゲン等をエタノールなどに分散させた溶液をスプレーなどで繊維成形体に噴霧して固定化してもよい。
The term “immobilization” as used in the present invention means that fibrinogen and thrombin are present not only on the surface of the fiber molded body but also inside the fiber molded body. It is not necessary to be present.
The method for immobilizing fibrinogen or thrombin in the sheet-like hemostatic material of the present invention is not limited, but for example, it can be produced by immersing a pre-formed fiber molded body in a solution such as fibrinogen and then freeze-drying. Alternatively, a solution in which fibrinogen or the like is dispersed in ethanol or the like may be sprayed onto the fiber molded body by a spray or the like to be fixed.
フィブリノゲン固定化シート作製時に、フィブリノゲン等に加えて、薬学的に許容しうる添加剤を添加してもよい。そのような添加剤の例として、例えば血液凝固第ＸIII因子（ヒト血液由来または遺伝子組換え技術により得られるものが好ましい）、アルブミン、イソロイシン、グリシン、アルギニン、グルタミン酸、界面活性剤、塩化ナトリウム、糖アルコール（グリセロール、マンニトール等）、およびクエン酸ナトリウムなどがある。これらの添加剤の一つ以上を適宜組み合わせてフィブリノゲン成分の溶解性や、フィブリノゲン固定化シートの柔軟性を向上させることが可能となる。
本発明のシート状止血材において、繊維成形体（Ａ）および繊維成形体（Ｂ）はいずれも生分解性ポリマーからなる。
When preparing the fibrinogen-immobilized sheet, a pharmaceutically acceptable additive may be added in addition to fibrinogen and the like. Examples of such additives include blood coagulation factor XIII (preferably derived from human blood or obtained by genetic recombination technology), albumin, isoleucine, glycine, arginine, glutamic acid, surfactant, sodium chloride, sugar There are alcohols (glycerol, mannitol, etc.) and sodium citrate. One or more of these additives can be combined as appropriate to improve the solubility of the fibrinogen component and the flexibility of the fibrinogen-immobilized sheet.
In the sheet-like hemostatic material of the present invention, both the fiber molded body (A) and the fiber molded body (B) are made of a biodegradable polymer.
本発明で用いられる生分解性ポリマーとしては、具体的にはポリ乳酸、ポリグリコール酸、ポリカプロラクトン、ポリジオキサノン、乳酸−グリコール酸共重合体や乳酸−カプロラクトン共重合体の如き乳酸の共重合体、ポリグリセロールセバシン酸、ポリヒドロキシアルカン酸、ポリブチレンサクシネートなどの脂肪族ポリエステルが挙げられる。さらに好ましくは、ポリ乳酸、ポリグリコール酸、乳酸−グリコール酸共重合体などの脂肪族ポリエステルであり、最も好ましいのはポリ乳酸、乳酸−グリコール酸共重合体である。
このとき、乳酸の共重合体は、伸縮性を付与するモノマー成分が少ないほうが好ましい。ここで伸縮性を付与するモノマー成分としては、カプロラクトンモノマーや、エチレングリコール、１，２−プロピレングリコール、１，３−プロピレングリコール、１，２−ブタンジオール、１，４−ブタンジオール、ポリカプロラクトンジオール、ポリアルキレンカーボネートジオール、ポリエチレングリコールユニットなどの軟質成分が例示できる。これらの軟質成分はポリマー重量比で２０％未満であることが好ましい。これよりも軟質成分が多いと自己支持性を失いやすく、柔らかすぎて取り扱いにくい繊維成形体になる。
Specific examples of the biodegradable polymer used in the present invention include polylactic acid, polyglycolic acid, polycaprolactone, polydioxanone, a lactic acid copolymer such as a lactic acid-glycolic acid copolymer and a lactic acid-caprolactone copolymer, Aliphatic polyesters such as polyglycerol sebacic acid, polyhydroxyalkanoic acid, polybutylene succinate and the like can be mentioned. More preferred are aliphatic polyesters such as polylactic acid, polyglycolic acid and lactic acid-glycolic acid copolymer, and most preferred are polylactic acid and lactic acid-glycolic acid copolymer.
At this time, it is preferable that the copolymer of lactic acid has few monomer components which impart stretchability. Here, as the monomer component imparting stretchability, caprolactone monomer, ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, 1,2-butanediol, 1,4-butanediol, polycaprolactone diol Examples thereof include soft components such as polyalkylene carbonate diol and polyethylene glycol unit. These soft components are preferably less than 20% by weight of the polymer. When there are more soft components than this, it becomes easy to lose a self-supporting property, and it becomes a fiber molded object which is too soft and difficult to handle.
生分解性ポリマーとしてポリ乳酸、乳酸の共重合体を用いる場合、モノマーにはＬ−乳酸およびＤ−乳酸があるが、特に制限はない。また、ポリ乳酸の光学純度や分子量、Ｌ体とＤ体の組成比、配列には特に制限はないが、好ましくはＬ体の多いポリ乳酸である。ポリＬ乳酸とポリＤ乳酸のステレオコンプレックスを用いてもよい。
また、生分解性ポリマーの重量平均分子量としては、１×１０３〜５×１０６であり、好ましくは１×１０４〜１×１０６、より好ましくは５×１０４〜５×１０５である。また、生分解性ポリマーの末端構造やポリマーを重合する触媒は任意に選択できる。
When a polylactic acid or lactic acid copolymer is used as the biodegradable polymer, the monomers include L-lactic acid and D-lactic acid, but there is no particular limitation. Further, the optical purity and molecular weight of polylactic acid, the composition ratio of L-form and D-form, and the arrangement are not particularly limited, but polylactic acid having many L-forms is preferred. A stereocomplex of poly L lactic acid and poly D lactic acid may be used.
The weight average molecular weight of the biodegradable polymer is 1 × 10 3 to 5 × 10 6 , preferably 1 × 10 4 to 1 × 10 6 , more preferably 5 × 10 4 to 5 × 10 5 . is there. The terminal structure of the biodegradable polymer and the catalyst for polymerizing the polymer can be arbitrarily selected.
本発明で用いる繊維成形体においては、その目的を損なわない範囲で、他のポリマーや他の化合物を併用してもよい。例えば、ポリマー共重合、ポリマーブレンド、化合物混合である。
本発明で用いる生分解性ポリマーは高純度であることが好ましく、とりわけ生分解性ポリマー中に含まれる添加剤や可塑剤、残存触媒、残存モノマー、成型加工や後加工に用いた残留溶媒などの残留物は少ないほうが好ましい。特に医療に用いる場合は、安全性の基準値未満に抑える必要がある。
In the fiber molded body used in the present invention, other polymers and other compounds may be used in combination as long as the purpose is not impaired. For example, polymer copolymerization, polymer blending, compound mixing.
The biodegradable polymer used in the present invention preferably has a high purity, and in particular, additives, plasticizers, residual catalysts, residual monomers, residual solvents used for molding processing and post-processing included in the biodegradable polymer, etc. Less residue is preferred. In particular, when used for medical treatment, it is necessary to keep it below the safety standard value.
本発明における繊維成形体の繊維径は０．１〜１０μｍである。ここで平均繊維径が０．１μｍよりも小さい、または１０μｍよりも大きいと、所望のフィブリノゲンおよびトロンビンの担持性が得られない。好ましい平均繊維径は１．０〜８．０μｍであり、さらに好ましくは２．０〜７．０μｍである。なお、繊維径とは繊維断面の直径をいう。繊維断面の形状は円形に限らず、楕円形や異形になることもありうる。この場合の繊維径は、該楕円形の長軸方向の長さと短軸方向の長さの平均値とする。また、繊維断面が円形でも楕円形でもないときには円または楕円に近似して繊維径を算出する。 The fiber diameter of the fiber molded body in the present invention is 0.1 to 10 μm. Here, if the average fiber diameter is smaller than 0.1 μm or larger than 10 μm, desired fibrinogen and thrombin supportability cannot be obtained. A preferable average fiber diameter is 1.0 to 8.0 μm, and more preferably 2.0 to 7.0 μm. In addition, a fiber diameter means the diameter of a fiber cross section. The shape of the fiber cross section is not limited to a circle, and may be an ellipse or an irregular shape. The fiber diameter in this case is the average value of the length of the ellipse in the major axis direction and the length in the minor axis direction. When the fiber cross section is neither circular nor elliptical, the fiber diameter is calculated by approximating a circle or ellipse.
本発明で用いられる繊維成形体は長繊維よりなる。長繊維とは、紡糸から繊維成形体への加工にいたるプロセスの中で、繊維を切断する工程を加えずに形成される繊維成形体をいい、エレクトロスピニング法、スパンボンド法、メルトブロー法などで形成することができる。これらのうち、エレクトロスピニング法（静電紡糸法、エレクトロスプレー法）が好ましく用いられる。エレクトロスピニング法は、ポリマーを溶媒に溶解させた溶液に高電圧を印加することで、電極上に繊維成形体を得る方法である。工程としては、ポリマーを溶媒に溶解させて溶液を製造する工程と、該溶液に高電圧を印加する工程と、該溶液を噴出させる工程と、噴出させた溶液から溶媒を蒸発させて繊維成形体を形成させる工程と、任意に実施しうる工程として形成された繊維成形体の電荷を消失させる工程と、電荷消失によって繊維成形体を累積させる工程を含む。 The fiber molded body used in the present invention is composed of long fibers. Long fiber refers to a fiber molded body that is formed without adding a fiber cutting step in the process from spinning to fiber molded body. Electrospinning, spunbonding, melt blowing, etc. Can be formed. Of these, the electrospinning method (electrostatic spinning method, electrospray method) is preferably used. The electrospinning method is a method of obtaining a fiber molded body on an electrode by applying a high voltage to a solution in which a polymer is dissolved in a solvent. The steps include a step of producing a solution by dissolving a polymer in a solvent, a step of applying a high voltage to the solution, a step of ejecting the solution, and evaporating the solvent from the ejected solution to form a fiber molded body. A step of forming the fiber molded body, a step of eliminating the charge of the fiber molded body formed as an optional process, and a step of accumulating the fiber molded body by the charge loss.
本発明のシート状止血材において、繊維成形体（Ａ）は、０．０１ｍｍ２以上の貫通孔を有さないという特性と、繊維表面の平滑度が１．０７以下であるという特性との両方の特性をさらに有する。また、繊維成形体（Ｂ）も、０．０１ｍｍ２以上の貫通孔を有さないという特性と、繊維表面の平滑度が１．０７以下であるという特性との両方の特性をさらに有する。
Both the sheet-like hemostatic material of the present invention, fiber molded body (A) is a characteristic that no 0.01 mm 2 or more through-holes, the characteristic that the smoothness of the fiber surface is 1.07 or less It has the following characteristics. The fiber molded article (B) also, that further Yusuke and property that no 0.01 mm 2 or more through-holes, the characteristics of both the characteristics of smoothness of the fiber surface is 1.07 or less .
従来、生体吸収性繊維として使用されているウェブ、ニット、およびメッシュ等は、単繊維を束ねたサブミリオーダーの繊維束が網目構造における網を形成し、サブミリオーダーの貫通孔が存在し、そのような貫通孔を消失することは困難であった。となると、フィブリノゲンまたはトロンビンを複合させる際、網部と貫通孔部とで疎と密な構造を取らざるをえない。よって、止血効果に不均一性が生じることは容易に想像できる。さらには、貫通孔を有するため突き刺しのような刺激に対して非常に弱いという欠点を有していた。
すなわち、０．０１ｍｍ２以上の貫通孔を有する場合、凍結乾燥後のフィブリノゲンまたはトロンビンの保持性が低下することがある。また、０．０１ｍｍ２以上の貫通孔を有する場合、フィブリノゲンまたはトロンビンは容易にシートから脱落するため、シート内で担持量に斑が生じ、止血効果にばらつきが生じることがある。
Conventionally, in webs, knits, meshes, etc. used as bioabsorbable fibers, submillimeter-order fiber bundles in which single fibers are bundled form a net in a mesh structure, and submillimeter-order through holes exist. It was difficult to eliminate a through hole. Then, when fibrinogen or thrombin is compounded, a sparse and dense structure is unavoidable between the net part and the through hole part. Therefore, it can be easily imagined that non-uniformity occurs in the hemostatic effect. Furthermore, since it has a through-hole, it has a drawback that it is very weak against irritation such as piercing.
That is, when it has a through-hole of 0.01 mm 2 or more, the retainability of fibrinogen or thrombin after lyophilization may decrease. Moreover, when it has a through-hole of 0.01 mm < 2 > or more, fibrinogen or thrombin easily drops off from the sheet, so that the amount carried is uneven within the sheet, and the hemostatic effect may vary.
一方、繊維表面の平滑度が１．０７以下であるとは、繊維表面を電子走査線顕微鏡などで観察した際に、表面に凹凸などが見られないものをいう。例えば国際公開ＷＯ２００６／０２２４３０号には、繊維表面が凹凸の繊維成形体が示されているが、このような凹凸を一定限度しかもたない繊維をいう。具体的には、繊維の表面形態が、原子間力顕微鏡ＡＦＭを用いて、ＡＦＭ観察視野範囲１×１μｍ２における表面積率により評価したものが１．０７以下である繊維をいう。好ましくはこの値が１．０５以下であり、より好ましくは１．０３以下である。かかる繊維成形体はフィブリン糊との親和性に優れる。 On the other hand, when the fiber surface has a smoothness of 1.07 or less, when the fiber surface is observed with an electronic scanning line microscope or the like, the surface has no irregularities. For example, International Publication No. WO2006 / 022430 shows a fiber molded body having a concavo-convex fiber surface, and refers to a fiber having such a concavo-convex limit. Specifically, a fiber whose surface morphology is evaluated by a surface area ratio in an AFM observation visual field range of 1 × 1 μm 2 using an atomic force microscope AFM is 1.07 or less. This value is preferably 1.05 or less, more preferably 1.03 or less. Such a fiber molded body is excellent in affinity with the fibrin glue.
本発明のシート状止血材は、フィブリノゲンが固定化されたシートと、トロンビンが固定化されたシートから構成される。好ましくはこれらの二層構造であるが、フィブリノゲンが固定化されたシートおよびトロンビンが固定化されたシートは、それぞれ二層以上あってもよく、また各シートを積層する順序も問わない。さらに、使用時にそれぞれのシートを重ねて使用するようになっているシート状止血材キットであってもよい。
また、本発明のシート状止血材において、繊維成形体（Ａ）および繊維成形体（Ｂ）の厚みは５０μｍから３００μｍが好ましい。５０μｍよりも薄いと必要量のフィブリノゲンおよびトロンビンが担持できず好ましくない。３００μｍよりも厚いと必要となるフィブリノゲンおよびトロンビンの量が過剰量必要となり好ましくない。
The sheet-like hemostatic material of the present invention is composed of a sheet on which fibrinogen is immobilized and a sheet on which thrombin is immobilized. These two-layer structures are preferable, but the sheet on which fibrinogen is immobilized and the sheet on which thrombin is immobilized may each have two or more layers, and the order in which the sheets are laminated is not limited. Furthermore, it may be a sheet-like hemostatic material kit that is used by overlapping each sheet when used.
In the sheet hemostatic material of the present invention, the thickness of the fiber molded body (A) and the fiber molded body (B) is preferably 50 μm to 300 μm. If it is thinner than 50 μm, the necessary amounts of fibrinogen and thrombin cannot be supported, which is not preferable. If it is thicker than 300 μm, an excessive amount of fibrinogen and thrombin is required, which is not preferable.
本発明で用いる繊維成形体は、リン脂質を生分解性ポリマーに対して０．１〜１０重量％含有することが好ましい。リン脂質の含有量が０．１重量％より少ないと、フィブリノゲンまたはトロンビン溶液との親和性に効果を示し難く、１０重量％よりも多いと、繊維成形体自体の耐久性が低下し易くなるため好ましくない。好ましい含有量は０．２〜５重量％であり、さらに好ましくは０．３〜３重量％である。
本発明で用いるリン脂質は、動物組織から抽出したものでも人工的に合成したものでもよい。かかるリン脂質としては、例えばホスファチジルコリン、ホスファチジルエタノールアミン、ホスファチジルセリン、ホスファチジルグリセロールなどが挙げられる。これらは１種類を選択してもよいし、２種類以上の混合物を用いてもよい。好ましくはホスファチジルコリンまたはホスファチジルエタノールアミンであり、さらに好ましくはジラウロイルホスファチジルコリンまたはジオレオイルホスファチジルエタノールアミンである。
The fiber molded body used in the present invention preferably contains 0.1 to 10% by weight of phospholipid with respect to the biodegradable polymer. If the phospholipid content is less than 0.1% by weight, it is difficult to show an effect on the affinity with the fibrinogen or thrombin solution, and if it exceeds 10% by weight, the durability of the fiber molded body tends to be lowered. It is not preferable. The preferred content is 0.2 to 5% by weight, more preferably 0.3 to 3% by weight.
The phospholipid used in the present invention may be extracted from animal tissue or artificially synthesized. Examples of such phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol. One of these may be selected, or a mixture of two or more may be used. Preferred is phosphatidylcholine or phosphatidylethanolamine, and more preferred is dilauroylphosphatidylcholine or dioleoylphosphatidylethanolamine.
以下、実施例により本発明の実施の形態を説明するが、これらは本発明の範囲を制限するものではない。 Hereinafter, although an example explains an embodiment of the present invention, these do not limit the range of the present invention.
１．平均繊維径：
得られた繊維成形体の表面を走査型電子顕微鏡（キーエンス株式会社：商品名「ＶＥ８８００」）により、倍率２０００倍で撮影して得た写真から無作為に２０箇所を選んで繊維の径を測定し、すべての繊維径の平均値を求めて平均繊維径とした。ｎ＝２０である。
２．平均厚：
高精度デジタル測長機（株式会社ミツトヨ：商品名「ライトマチックＶＬ−５０」）を用いて測長力０．０１Ｎによりｎ＝１０にて繊維成形体の膜厚を測定し、平均値を算出した。なお、本測定においては測定機器が使用可能な最小の測定力で測定を行った。
３．貫通孔の有無および平均表面開孔面積
得られた繊維成形体の表面を走査型電子顕微鏡（キーエンス株式会社：商品名「ＶＥ８８００」）で撮影して得た写真から、貫通孔の有無を確認した。
４．繊維表面の平滑度：
ＡＦＭとして、デジタルインスツルメント社製Ｎａｎｏ Ｓｃｏｐｅ ＩＩＩａを使用した。カンチレバーはＡＣ−２４０ＴＳ（シリコン製：ばね定数２Ｎ／ｍ）を使用した。観察に際して分解能低下を防ぐため、カンチレバーは探針の汚染、磨耗がない新品を使用した。また、探針−試料表面間に働く力は必要最小限の力に設定し、走査中の試料の破損、探針の磨耗を防いだ。観察は、探針が繊維中心部にコンタクトするようにし、探針走査方向が繊維軸と一致するように観察視野１×１μｍ２で行った。走査速度は０．７Ｈｚとした。分解能は２５６×２５６ｐｉｘｅｌｓ以上とした。繊維は曲率をもっているため、ＡＦＭ観察後、装置に付属している傾き補正ソフト等を用いて繊維が有する曲率、マクロな形態上のうねりをキャンセルした。傾き補正は、探針が完全に繊維中心部にコンタクトし、走査方向と繊維軸方向が一致した場合は二次傾き補正を行ったが、そうでない場合は三次傾き補正処理、フラット処理を行った。傾き補正後、装置付属の表面粗さ解析を行い、表面積率を算出した。表面積率とは、観察面が理想的にフラットであると仮定したときの面積Ｓ０に対する実際の表面積Ｓの比率Ｓｒａｔｉｏであり、Ｓｒａｔｉｏ＝Ｓ／Ｓ０で表される。評価はランダムに行った観察視野２０点の平均値をもって行った。
５．フィブリノゲン保持量（重量変化確認）
１×１ｃｍ２に切り出したシートの重量（Ａ）を測定し、シートの両端をピンセットで挟み、１０回折りまげを実施した後、再度シートの重量（Ｂ）を測定する。
重量変化（％）＝Ｂ／Ａ×１００
1. Average fiber diameter:
Measure the diameter of the fiber by randomly selecting 20 points from the photograph obtained by photographing the surface of the obtained fiber molded body with a scanning electron microscope (Keyence Corporation: trade name “VE8800”) at a magnification of 2000 times. And the average value of all the fiber diameters was calculated | required, and it was set as the average fiber diameter. n = 20.
2. Average thickness:
Using a high-precision digital length measuring machine (Mitutoyo Co., Ltd .: trade name “Lightmatic VL-50”), the film thickness of the fiber molded body is measured at a length measuring power of 0.01 N and n = 10, and the average value is calculated. did. In this measurement, the measurement was performed with the minimum measuring force that can be used by the measuring device.
3. Presence or absence of through-holes and average surface open area The presence or absence of through-holes was confirmed from a photograph obtained by photographing the surface of the obtained fiber molded body with a scanning electron microscope (Keyence Corporation: trade name “VE8800”). .
4). Fiber surface smoothness:
As the AFM, Nano Scope IIIa manufactured by Digital Instruments was used. The cantilever used was AC-240TS (made of silicon: spring constant 2 N / m). In order to prevent resolution degradation during observation, a new cantilever with no contamination or wear of the probe was used. In addition, the force acting between the probe and the sample surface was set to the minimum necessary force to prevent sample breakage and probe wear during scanning. The observation was performed with an observation field of view of 1 × 1 μm 2 so that the probe was in contact with the center of the fiber and the scanning direction of the probe coincided with the fiber axis. The scanning speed was 0.7 Hz. The resolution was 256 × 256 pixels or higher. Since the fiber has a curvature, after the AFM observation, the curvature of the fiber and the undulation on the macro form were canceled using inclination correction software attached to the apparatus. In the tilt correction, when the probe contacted the fiber center completely and the scanning direction coincided with the fiber axis direction, the secondary tilt correction was performed. Otherwise, the tertiary tilt correction processing and flat processing were performed. . After the inclination correction, the surface roughness analysis attached to the apparatus was performed to calculate the surface area ratio. The surface area ratio is a ratio Sratio of the actual surface area S to the area S0 when it is assumed that the observation surface is ideally flat, and is represented by Sratio = S / S0. The evaluation was performed with an average value of 20 observation visual fields performed at random.
5. Fibrinogen retention (weight change confirmation)
The weight (A) of the sheet cut out to 1 × 1 cm 2 is measured, both ends of the sheet are sandwiched with tweezers, 10-folding is performed, and then the weight (B) of the sheet is measured again.
Weight change (%) = B / A × 100
実施例１
０．４％のジラウロイルホスファチジルコリンを含有したポリ乳酸（重量平均分子量２６万２千、Ｐｕｒａｃ社製）１１重量部をジクロロメタン溶液９０重量部で溶解し、均一な溶液を調製した。エレクトロスピニング法により紡糸を行い、シート状の繊維成形体を得た。噴出ノズルの内径は０．８ｍｍ、電圧は８ｋＶ、噴出ノズルから平板までの距離は２５ｃｍであった。上記平板は、紡糸時は陰極として用いた。得られた繊維成形体に、７０℃１０分間熱処理を実施した。得られた繊維成形体の平均繊維径は４．１μｍ、厚さは７９μｍであった。０．０１ｍｍ２以上の貫通孔は観察されなかった。繊維表面の平滑度は、１．００４であった。
市販の生体組織接着剤である「ボルヒール」（登録商標、一般財団法人化学及血清療法研究所製、以下同じ）のキットに含まれるフィブリノゲン溶液１．２５ｍＬを、上記で作製した繊維成形体（５×５ｃｍ２）上に染み込ませた。フィブリノゲン溶液は、容易にシート内に染み込んだ。この検体を凍結後、２４時間凍結乾燥させたものをフィブリノゲン固定化シートとした。フィブリノゲンは、シート内部全面に均一に担持されていた。そのＳＥＭ像を図１に示す。シートの両端をピンセットで挟み、１０回折りまげを実施し、重量変化を確認した。本シートを取り扱っても担持したフィブリノゲンが崩壊することはなく、重量変化はなかった（１００％保持）。
次に、ボルヒールのキットに含まれるトロンビンを付属の溶解液により溶解し、１８７５単位／ｍＬのトロンビン溶液を調製した。このトロンビン溶液３３．７５μＬを、上記で作製した繊維成形体（２×２．５ｃｍ２）上に均一にしみ込ませた。トロンビン溶液は、容易にシート内に染み込んだ。この検体を凍結後、２４時間凍結乾燥させたものをトロンビン固定化シートとした。トロンビンは、シート内部全面に均一に担持されていた。
Example 1
11 parts by weight of polylactic acid (weight average molecular weight 262,000, manufactured by Purac) containing 0.4% dilauroylphosphatidylcholine was dissolved in 90 parts by weight of a dichloromethane solution to prepare a uniform solution. Spinning was performed by electrospinning to obtain a sheet-like fiber molded body. The inner diameter of the ejection nozzle was 0.8 mm, the voltage was 8 kV, and the distance from the ejection nozzle to the flat plate was 25 cm. The flat plate was used as a cathode during spinning. The obtained fiber molded body was heat-treated at 70 ° C. for 10 minutes. The obtained fiber molded body had an average fiber diameter of 4.1 μm and a thickness of 79 μm. A through-hole of 0.01 mm 2 or more was not observed. The smoothness of the fiber surface was 1.004.
The fiber molded body (5) prepared by using 1.25 mL of the fibrinogen solution contained in the kit of “Bolheel” (registered trademark, manufactured by Chemical and Serum Therapy Laboratory, the same below), which is a commercially available biological tissue adhesive, as described above. X5 cm 2 ). The fibrinogen solution soaked easily into the sheet. This specimen was freeze-dried for 24 hours and used as a fibrinogen-immobilized sheet. Fibrinogen was uniformly supported on the entire surface inside the sheet. The SEM image is shown in FIG. The both ends of the sheet were sandwiched with tweezers, 10-folding was performed, and the weight change was confirmed. Even when the sheet was handled, the supported fibrinogen was not disintegrated, and the weight was not changed (100% retention).
Next, thrombin contained in the Bolheel kit was dissolved with the attached dissolution solution to prepare a 1875 unit / mL thrombin solution. 33.75 μL of this thrombin solution was uniformly soaked on the fiber molded body (2 × 2.5 cm 2 ) prepared above. The thrombin solution soaked easily into the sheet. This specimen was frozen and then lyophilized for 24 hours to obtain a thrombin-immobilized sheet. Thrombin was uniformly supported on the entire surface inside the sheet.
実施例２
０．７％のジラウロイルホスファチジルコリンを含有したポリ乳酸（重量平均分子量２６万２千、Ｐｕｒａｃ社製）１１重量部をジクロロメタン溶液９０重量部で溶解し、均一な溶液を調製した。エレクトロスピニング法により紡糸を行い、シート状の繊維成形体を得た。噴出ノズルの内径は０．８ｍｍ、電圧は８ｋＶ、噴出ノズルから平板までの距離は２５ｃｍであった。上記平板は、紡糸時は陰極として用いた。得られた繊維成形体に、７０℃１０分間熱処理を実施した。得られた繊維成形体の平均繊維径は３．８μｍ、厚さは８５μｍであった。０．０１ｍｍ２以上の貫通孔は観察されなかった。繊維表面の平滑度は、１．００６であった。
ボルヒールのキットに含まれるフィブリノゲン溶液１．２５ｍＬを、上記で作製した繊維成形体（５×５ｃｍ２）上に染み込ませた。フィブリノゲン溶液は、容易にシート内に染み込んだ。この検体を凍結後、２４時間凍結乾燥させたものをフィブリノゲン固定化シートとした。フィブリノゲンは、シート内部全面に均一に担持されていた。そのＳＥＭ像を図２に示す。シートの両端をピンセットで挟み、１０回折りまげを実施し、重量変化を確認した。本シートを取り扱っても担持したフィブリノゲンが崩壊することはなく、重量変化はなかった（１００％保持）。
次に、ボルヒールのキットに含まれるトロンビンを付属の溶解液により溶解し、１８７５単位／ｍＬのトロンビン溶液を調製した。このトロンビン溶液３３．７５ｍＬを、上記で作製した繊維成形体（２×２．５ｃｍ２）上に均一にしみ込ませた。トロンビン溶液は、容易にシート内に染み込んだ。この検体を凍結後、２４時間凍結乾燥させたものをトロンビン固定化シートとした。トロンビンは、シート内部全面に均一に担持されていた。
Example 2
11 parts by weight of polylactic acid (weight average molecular weight 262,000, manufactured by Purac) containing 0.7% dilauroylphosphatidylcholine was dissolved in 90 parts by weight of a dichloromethane solution to prepare a uniform solution. Spinning was performed by electrospinning to obtain a sheet-like fiber molded body. The inner diameter of the ejection nozzle was 0.8 mm, the voltage was 8 kV, and the distance from the ejection nozzle to the flat plate was 25 cm. The flat plate was used as a cathode during spinning. The obtained fiber molded body was heat-treated at 70 ° C. for 10 minutes. The obtained fiber molded body had an average fiber diameter of 3.8 μm and a thickness of 85 μm. A through-hole of 0.01 mm 2 or more was not observed. The smoothness of the fiber surface was 1.006.
1.25 mL of the fibrinogen solution contained in the Borheel kit was soaked on the fiber molded body (5 × 5 cm 2 ) prepared above. The fibrinogen solution soaked easily into the sheet. This specimen was freeze-dried for 24 hours and used as a fibrinogen-immobilized sheet. Fibrinogen was uniformly supported on the entire surface inside the sheet. The SEM image is shown in FIG. The both ends of the sheet were sandwiched with tweezers, 10-folding was performed, and the weight change was confirmed. Even when the sheet was handled, the supported fibrinogen was not disintegrated, and the weight was not changed (100% retention).
Next, thrombin contained in the Bolheel kit was dissolved with the attached dissolution solution to prepare a 1875 unit / mL thrombin solution. 33.75 mL of this thrombin solution was uniformly soaked on the fiber molded body (2 × 2.5 cm 2 ) prepared above. The thrombin solution soaked easily into the sheet. This specimen was frozen and then lyophilized for 24 hours to obtain a thrombin-immobilized sheet. Thrombin was uniformly supported on the entire surface inside the sheet.
実施例３
ポリ乳酸（重量平均分子量２６万２千、Ｐｕｒａｃ社製）１１重量部をジクロロメタン溶液９０重量部で溶解し、均一な溶液を調製した。エレクトロスピニング法により紡糸を行い、シート状の繊維成形体を得た。噴出ノズルの内径は０．８ｍｍ、電圧は８ｋＶ、噴出ノズルから平板までの距離は２５ｃｍであった。上記平板は、紡糸時は陰極として用いた。得られた繊維成形体に、７０℃１０分間熱処理を実施した。得られた繊維成形体の平均繊維径は３．６μｍ、厚さは８１μｍであった。０．０１ｍｍ２以上の貫通孔は観察されなかった。繊維表面の平滑度は、１．００５であった。
ボルヒールのキットに含まれるフィブリノゲン溶液１．２５ｍＬを、上記で作製した繊維成形体（５×１０ｃｍ２）上に染み込ませた。フィブリノゲン溶液はシートへ染み込みにくかった。この検体を凍結後、２４時間凍結乾燥させたものをフィブリノゲン固定化シートとした。フィブリノゲンは、シート内部全面に均一に担持されていた。そのＳＥＭ像を図３に示す。シートの両端をピンセットで挟み、１０回折りまげを実施し、重量変化を確認した。本シートを取り扱っても担持したフィブリノゲンが崩壊することはなく、重量変化はなかった（１００％保持）。
次に、ボルヒールのキットに含まれるトロンビンを付属の溶解液により溶解し、１８７５単位／ｍＬのトロンビン溶液を調製した。このトロンビン溶液３３．７５μＬを、上記で作製した繊維成形体（５×１０ｃｍ２）上に均一にしみ込ませた。トロンビン溶液はシートへ染み込みにくかった。この検体を凍結後、２４時間凍結乾燥させたものをトロンビン固定化シートとした。トロンビンは、シート内部全面に均一に担持されていた。
Example 3
11 parts by weight of polylactic acid (weight average molecular weight 262,000, manufactured by Purac) was dissolved in 90 parts by weight of a dichloromethane solution to prepare a uniform solution. Spinning was performed by electrospinning to obtain a sheet-like fiber molded body. The inner diameter of the ejection nozzle was 0.8 mm, the voltage was 8 kV, and the distance from the ejection nozzle to the flat plate was 25 cm. The flat plate was used as a cathode during spinning. The obtained fiber molded body was heat-treated at 70 ° C. for 10 minutes. The obtained fiber molded body had an average fiber diameter of 3.6 μm and a thickness of 81 μm. A through-hole of 0.01 mm 2 or more was not observed. The smoothness of the fiber surface was 1.005.
1.25 mL of the fibrinogen solution contained in the Borheel kit was soaked on the fiber molded body (5 × 10 cm 2 ) prepared above. The fibrinogen solution was difficult to penetrate into the sheet. This specimen was freeze-dried for 24 hours and used as a fibrinogen-immobilized sheet. Fibrinogen was uniformly supported on the entire surface inside the sheet. The SEM image is shown in FIG. The both ends of the sheet were sandwiched with tweezers, 10-folding was performed, and the weight change was confirmed. Even when the sheet was handled, the supported fibrinogen was not disintegrated, and the weight was not changed (100% retention).
Next, thrombin contained in the Bolheel kit was dissolved with the attached dissolution solution to prepare a 1875 unit / mL thrombin solution. 33.75 μL of this thrombin solution was uniformly soaked on the fiber molded body (5 × 10 cm 2 ) prepared above. The thrombin solution was difficult to penetrate into the sheet. This specimen was frozen and then lyophilized for 24 hours to obtain a thrombin-immobilized sheet. Thrombin was uniformly supported on the entire surface inside the sheet.
比較例１
繊維成形体として、ポリグリコール酸系不織布であるネオベール（登録商標、グンゼ株式会社製、平均繊維径２０μｍ、厚さ１５０μｍ）を用いた以外は、実施例１と同様に、フィブリノゲン固定化シートおよびトロンビン固定化シートを作製した。ＳＥＭ観察により０．０１ｍｍ２以上の貫通孔が観察された。繊維表面の平滑度は、１．０６９であった。
上で調製したフィブリノゲン溶液はシートへ染み込みにくかった。この検体を凍結後、２４時間凍結乾燥させたものをフィブリノゲン固定化シートとした。シートの両端をピンセットで挟み、１０回折りまげを実施し、重量変化を確認した。フィブリノゲンは、シート内部全面に均一に担持されておらず、シートはピンセットで取り扱うと、担持されていたフィブリノゲンが剥落した（重量変化８９％）。ＳＥＭ像を図４に示す。
同様に、上で調製したトロンビン溶液もシートへの染み込みが悪かった。この検体を凍結後、２４時間凍結乾燥させたものをトロンビン固定化シートとした。トロンビンは、シート内部全面に均一に担持されていた。
Comparative Example 1
The fibrinogen-immobilized sheet and thrombin were the same as in Example 1 except that Neobale (registered trademark, manufactured by Gunze Co., Ltd., average fiber diameter: 20 μm, thickness: 150 μm), which is a polyglycolic acid-based nonwoven fabric, was used as the fiber molded body. An immobilization sheet was produced. Through holes of 0.01 mm 2 or more were observed by SEM observation. The smoothness of the fiber surface was 1.069.
The fibrinogen solution prepared above was difficult to penetrate into the sheet. This specimen was freeze-dried for 24 hours and used as a fibrinogen-immobilized sheet. The both ends of the sheet were sandwiched with tweezers, 10-folding was performed, and the weight change was confirmed. Fibrinogen was not uniformly supported on the entire inner surface of the sheet, and when the sheet was handled with tweezers, the supported fibrinogen peeled off (weight change 89%). An SEM image is shown in FIG.
Similarly, the thrombin solution prepared above had poor penetration into the sheet. This specimen was frozen and then lyophilized for 24 hours to obtain a thrombin-immobilized sheet. Thrombin was uniformly supported on the entire surface inside the sheet.
本発明のシート状止血材は高い止血効果を有しており、外科手術や外傷の止血材として使用できるので、例えば医療品製造業で利用される。
Since the sheet-like hemostatic material of the present invention has a high hemostatic effect and can be used as a hemostatic material for surgery and trauma, it is used, for example, in the medical product manufacturing industry.
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