JP5010854B2 - Revascularization material - Google Patents
Revascularization material Download PDFInfo
- Publication number
- JP5010854B2 JP5010854B2 JP2006139069A JP2006139069A JP5010854B2 JP 5010854 B2 JP5010854 B2 JP 5010854B2 JP 2006139069 A JP2006139069 A JP 2006139069A JP 2006139069 A JP2006139069 A JP 2006139069A JP 5010854 B2 JP5010854 B2 JP 5010854B2
- Authority
- JP
- Japan
- Prior art keywords
- vascular
- regeneration material
- layer
- cells
- aliphatic polyester
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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- Materials For Medical Uses (AREA)
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Description
本発明は、血管再生材料およびその製造方法に関する。 The present invention relates to a blood vessel regeneration material and a method for producing the same.
近年、大きく損傷したり失われた生体組織の治療法として、細胞の分化、増殖能を利用し元の生体組織を再構築する再生医療の研究が活発になってきている。血管再生もその１つであり、先天的もしくは病気によるダメージを受けた血管を切除し、欠損部に再生の足場となる材料あるいは足場材料に細胞を含有させた複合材料を架橋し、血管を再生する研究が行われている。
実際の血管組織は血管内面を被覆している内皮細胞を含む内膜、平滑筋細胞からなる中膜、繊維芽細胞からなる外膜の３層から構成されている。内膜は物質の選択的な透過をコントロールするため空間体積の小さい密な構造を有している。また中膜は血管特有の弾性機能を担っている。さらに外膜は血管の外から栄養分を取るため空間体積の大きい疎な構造を有している。血管治療用材料としては、このような血管組織の３層構造を模倣したものが好ましい。
In recent years, research on regenerative medicine that reconstructs an original biological tissue by utilizing cell differentiation and proliferation ability as a treatment method for a biological tissue that has been greatly damaged or lost has become active. Vascular regeneration is one of them, and blood vessels that have been congenital or damaged by disease are excised, and the defective part is regenerated as a scaffold for regeneration or a composite material containing cells in the scaffold is regenerated to regenerate blood vessels. Research has been done.
The actual vascular tissue is composed of three layers: an inner membrane containing endothelial cells covering the inner surface of the blood vessel, a middle membrane made of smooth muscle cells, and an outer membrane made of fibroblasts. The inner membrane has a dense structure with a small space volume to control the selective permeation of substances. The media also has an elastic function peculiar to blood vessels. Furthermore, the outer membrane has a sparse structure with a large space volume in order to take nutrients from outside the blood vessels. As a vascular treatment material, a material that mimics the three-layer structure of such vascular tissue is preferable.
細胞を含有させた複合材料としては、ポリウレタン繊維等からなり血管組織の３層構造を模倣した足場材料に血管内皮細胞および平滑筋細胞を含有させたハイブリッド人工血管が提案されている（特許文献１）。しかし再生医療においては、生分解性材料であることが好ましい。
生分解性材料を用いた足場材料としては、生分解性材料からなる発泡体を生分解性材料からなる補強材によって強化した心血管系組織培養用基材が提案されている（特許文献２）。しかし、このような基材は発泡体であることから実際の血管組織とは構造が大きく相違する。また、生分解性材料からなる織編物およびコラーゲンを構成成分とする足場材料が提案されている（非特許文献１）。しかし、織編物を構成する繊維は繊維径が大きいため比表面積が小さく、高い細胞接着性を得ることは困難であると考えられる。
細胞接着性を向上させた材料として、数ナノメートルから数十マイクロメートルの外径を有するコラーゲン、ポリウレタンなどの医療用高分子の繊維を用いて作製された不織布を含む足場材料が提案されている（特許文献３）。しかし、合成生分解性ポリマーを使用した例は開示されていない。コラーゲン等の動物由来製品は未知のウイルスを有している危険性があるため、生体内に埋め込んで使用する血管治療用材料は、合成生分解性ポリマーからなるものが好ましい。
As a scaffold material using a biodegradable material, a base material for cardiovascular tissue culture in which a foam made of a biodegradable material is reinforced with a reinforcing material made of a biodegradable material has been proposed (Patent Document 2). . However, since such a base material is a foam, the structure is greatly different from that of an actual vascular tissue. Further, a scaffold material comprising a woven or knitted fabric made of a biodegradable material and collagen as constituents has been proposed (Non-patent Document 1). However, since the fiber constituting the woven or knitted fabric has a large fiber diameter, the specific surface area is small, and it is considered difficult to obtain high cell adhesion.
As materials with improved cell adhesion, scaffolding materials including nonwoven fabrics made using fibers of medical polymers such as collagen and polyurethane having an outer diameter of several nanometers to several tens of micrometers have been proposed. (Patent Document 3). However, no examples using synthetic biodegradable polymers are disclosed. Since animal-derived products such as collagen have a risk of having an unknown virus, the material for vascular treatment used by being implanted in a living body is preferably made of a synthetic biodegradable polymer.
本発明は、従来のかかる問題を解決し、生体内に埋め込み可能であり、血管組織に類似した構造を有し、かつ弾性率、弾性回復率に優れた血管再生材料を提供することを目的とする。 An object of the present invention is to solve such conventional problems, and to provide a vascular regeneration material that can be implanted in a living body, has a structure similar to a vascular tissue, and has an excellent elastic modulus and elastic recovery rate. To do.
本発明者は、血管組織に類似した内層、中間層および外層からなる３層構造を有し、かつ同程度の機械的強度を有する血管再生材料について検討した。その結果、内層にポリ乳酸（ＰＬＡ）と、５０〜９０モル％の乳酸由来の繰り返し単位および１０〜５０モル％のカプロラクトン由来の繰り返し単位からなる共重合体（ＰＬＣＡ）とを含有し、前者の含有量が５０〜９０重量％で、後者の含有量が５０〜１０重量％である脂肪族ポリエステルの繊維を用いると、細胞浸潤性を適度に抑制することが出来、表面付近に細胞が局在し、血管内腔に類似の内皮細胞構造を構築できることを見出し、本発明を完成した。 The present inventor examined a vascular regeneration material having a three-layer structure composed of an inner layer, an intermediate layer, and an outer layer similar to a vascular tissue, and having the same mechanical strength. As a result, the inner layer contains polylactic acid (PLA) and a copolymer (PLCA) composed of 50 to 90 mol% lactic acid-derived repeating units and 10 to 50 mol% caprolactone-derived repeating units. in 50 to 90% by weight content, the latter content using fibers of 50-10 wt% der Ru fat aliphatic polyester, it is possible to appropriately inhibit cell invasiveness, cell near the surface The inventors have found that a similar endothelial cell structure can be constructed in the vascular lumen, and the present invention has been completed.
即ち本発明は、同心円状の内層、中間層および外層からなる中空の円筒状であり、各層は、平均繊維径が０．０５〜１０μｍの脂肪族ポリエステルの繊維からなり、内層を構成する脂肪族ポリエステルは、ポリ乳酸（ＰＬＡ）と、５０〜９０モル％の乳酸由来の繰り返し単位および１０〜５０モル％のカプロラクトン由来の繰り返し単位からなる共重合体（ＰＬＣＡ）とを含有し、前者の含有量が５０〜９０重量％で、後者の含有量が５０〜１０重量％である血管再生材料である。該血管再生材料は、細胞を含有することが好ましい。 That is, the present invention is a hollow cylindrical shape comprising a concentric inner layer, an intermediate layer and an outer layer, and each layer is made of an aliphatic polyester fiber having an average fiber diameter of 0.05 to 10 μm, and constitutes an inner layer. The polyester contains polylactic acid (PLA) and a copolymer (PLCA) composed of 50 to 90 mol% of lactic acid-derived repeating units and 10 to 50 mol% of caprolactone-derived repeating units. Is 50 to 90 % by weight, and the content of the latter is 50 to 10 % by weight. The vascular regeneration material preferably contains cells.
また本発明は、同心円状の内層、中間層および外層からなる中空の円筒状であり、各層は、平均繊維径が０．０５〜１０μｍの脂肪族ポリエステルの繊維からなる血管再生材料を製造する方法であって、
(i) ポリ乳酸（ＰＬＡ）と、５０〜９０モル％の乳酸由来の繰り返し単位および１０〜５０モル％のカプロラクトン由来の繰り返し単位からなる共重合体（ＰＬＣＡ）とを含有し、前者の含有量が５０〜９０重量％で、後者の含有量が５０〜１０重量％である脂肪族ポリエステルおよび揮発性溶媒を含有するドープを静電紡糸法にて紡糸し、得られた繊維をコレクタ上に巻き取り内層を形成する工程、
(ii) 脂肪族ポリエステルおよび揮発性溶媒を含有するドープを静電紡糸法にて紡糸し、得られた繊維を内層上に巻き取り中間層を形成する工程、並びに
(iii) 脂肪族ポリエステルおよび揮発性溶媒を含有するドープを静電紡糸法にて紡糸し、得られた繊維を中間層上に巻き取り外層を形成する工程、
を含む血管再生材料の製造方法である。該方法は、さらに細胞を播種する工程を含むことが好ましい。
The present invention also provides a method for producing a vascular regeneration material comprising a hollow cylindrical shape comprising concentric inner layers, intermediate layers and outer layers, each layer comprising an aliphatic polyester fiber having an average fiber diameter of 0.05 to 10 μm. Because
(i) containing polylactic acid (PLA) and a copolymer (PLCA) composed of 50 to 90 mol% of lactic acid-derived repeating units and 10 to 50 mol% of caprolactone-derived repeating units, the former content The dope containing an aliphatic polyester and a volatile solvent having a content of 50 to 90 % by weight and the latter content of 50 to 10 % by weight is spun by an electrostatic spinning method, and the obtained fiber is wound on a collector. Forming a take-up inner layer,
(ii) spinning a dope containing an aliphatic polyester and a volatile solvent by an electrospinning method, winding the obtained fiber on the inner layer to form an intermediate layer, and
(iii) a step of spinning an dope containing an aliphatic polyester and a volatile solvent by an electrostatic spinning method, and winding the obtained fiber on an intermediate layer to form an outer layer;
Is a method for producing a vascular regeneration material. Preferably, the method further comprises the step of seeding the cells.
本発明の血管再生材料は、３層からなり、血管組織に類似した構造を有する。また本発明の血管再生材料は、弾性率、弾性回復率に優れ、血管組織と同程度の弾性率、弾性回復率を有する。よって、本発明の血管再生材料は血管再生用の足場材料に好適である。
また本発明の血管再生材料は、空気透過率の高い外層を有し、空間体積が大きい外膜を有する血管組織の構造に類似している。そのため該血管再生材料は、細胞の浸潤性に優れ血管組織の再生に適している。
また本発明の血管再生材料は、細胞浸潤性の低い内層を有するので、内層に播種した細胞は、血管組織の内皮細胞に類似した密な薄い層に覆われた構造を有する。従って本発明の血管再生材料は再生医療における血管治療用材料として好適である。
The vascular regeneration material of the present invention consists of three layers and has a structure similar to vascular tissue. The vascular regeneration material of the present invention is excellent in elastic modulus and elastic recovery rate, and has an elastic modulus and elastic recovery rate comparable to those of vascular tissue. Therefore, the vascular regeneration material of the present invention is suitable as a scaffold material for vascular regeneration.
The vascular regeneration material of the present invention is similar to the structure of a vascular tissue having an outer layer having a high air permeability and an outer membrane having a large space volume. Therefore, the vascular regeneration material is excellent in cell invasiveness and suitable for regeneration of vascular tissue.
In addition, since the vascular regeneration material of the present invention has an inner layer with low cell invasiveness, cells seeded on the inner layer have a structure covered with a dense thin layer similar to endothelial cells of vascular tissue. Therefore, the vascular regeneration material of the present invention is suitable as a vascular treatment material in regenerative medicine.
以下、本発明について詳述する。
＜血管再生材料＞
本発明の血管再生材料は、同心円状の内層、中間層および外層の３層からなる。各層の厚みは、好ましくは３０〜５００μｍであり、より好ましくは５０〜２５０μｍである。各層は、脂肪族ポリエステル繊維が血管再生材料の軸を中心として、渦巻き状に巻き付けられたものが好ましい。
血管再生材料を構成する脂肪族ポリエステル繊維の平均繊維径は、０．０５〜１０μｍ、好ましくは０．２〜１０μｍである。平均繊維径が、０．０５μｍよりも小さいと血管再生材料の強度が保てないため好ましくない。また平均繊維径が１０μｍを超えると繊維の比表面積が小さく生着する細胞数が少なくなるため好ましくない。平均繊維径は、光学顕微鏡による画像から２０箇所の繊維径の測定値の平均である。
本発明の血管再生材料の断面の一例を図２に示す。図２は説明のための略図であり、本発明の血管再生材料の外面、内面には不規則な凹凸が存在する。本発明の血管再生材料は外径（１１）が、０．５〜５０ｍｍ、好ましくは１〜３０ｍｍである。外径はマイクロメーターにより１０箇所測定を行い、測定値の最小値と最大値の範囲で表す。血管再生材料の内部は中空であり、中空部の内径（１５）は、好ましくは０．１〜４５ｍｍ、より好ましくは０．５〜２５ｍｍである。内径は、測定された外径の値と厚みの値との差である。
Hereinafter, the present invention will be described in detail.
<Vascular regeneration material>
The vascular regeneration material of the present invention comprises three layers of concentric inner layer, intermediate layer and outer layer. The thickness of each layer is preferably 30 to 500 μm, more preferably 50 to 250 μm. Each layer is preferably made of an aliphatic polyester fiber wound in a spiral around the axis of the vascular regeneration material.
The average fiber diameter of the aliphatic polyester fiber constituting the vascular regeneration material is 0.05 to 10 μm, preferably 0.2 to 10 μm. If the average fiber diameter is smaller than 0.05 μm, the strength of the vascular regeneration material cannot be maintained, which is not preferable. An average fiber diameter exceeding 10 μm is not preferable because the specific surface area of the fiber is small and the number of engrafted cells is reduced. The average fiber diameter is an average of measured values of fiber diameters at 20 locations from an image obtained with an optical microscope.
An example of a cross section of the blood vessel regeneration material of the present invention is shown in FIG. FIG. 2 is a schematic diagram for explanation, and irregular irregularities exist on the outer surface and inner surface of the blood vessel regeneration material of the present invention. The vascular regeneration material of the present invention has an outer diameter (11) of 0.5 to 50 mm, preferably 1 to 30 mm. The outer diameter is measured at 10 locations with a micrometer and expressed in the range of the minimum and maximum measured values. The inside of the vascular regeneration material is hollow, and the inner diameter (15) of the hollow portion is preferably 0.1 to 45 mm, more preferably 0.5 to 25 mm. The inner diameter is the difference between the measured outer diameter value and the thickness value.
本発明の血管再生材料は、厚み（１４）が２００〜５，０００μｍ、好ましくは２００〜２，０００μｍである。厚みが２００μｍより薄いと機械強度が低く、血管など負荷の高い組織の再生材料としては好ましくない。厚みは血管再生材料を切り開き、長さ５ｃｍ、幅１ｃｍの試料を調製し、マイクロメーターにより１０箇所測定を行った際の、測定値の最小値と最大値の範囲で表す。
血管再生材料の引張弾性率は、好ましくは０．１〜１０ＭＰａ、より好ましくは０．２〜２．５ＭＰａの範囲である。実際の人体の動脈血管の引張り弾性率は２ＭＰａであるためである（臨床工学ライブラリーシリーズ２，Ｐ５４（秀潤社）、生体物性／医用機械工学 池田研二、嶋津秀照著）。０．１ＭＰａより低いと血液による負荷に耐えられず、破損する場合がある。また引張り弾性率が１０ＭＰａより高いと、移植した際、コンプライアンスミスマッチが起こる場合がある。引張弾性率は、血管再生材料を切り開き、軸方向に５ｃｍ、円周方向に１ｃｍの試料を作製し、軸方向に引張試験を行うことにより求める。
The vascular regeneration material of the present invention has a thickness (14) of 200 to 5,000 μm, preferably 200 to 2,000 μm. If the thickness is less than 200 μm, the mechanical strength is low, which is not preferable as a regenerating material for tissues with high loads such as blood vessels. The thickness is expressed in the range of the minimum value and the maximum value when the blood vessel regeneration material is cut open, a sample having a length of 5 cm and a width of 1 cm is prepared and measured at 10 locations with a micrometer.
The tensile elastic modulus of the vascular regeneration material is preferably in the range of 0.1 to 10 MPa, more preferably 0.2 to 2.5 MPa. This is because the tensile elastic modulus of an arterial blood vessel of an actual human body is 2 MPa (Clinical Engineering Library Series 2, P54 (Shyujunsha), Biophysical / Medical Mechanical Engineering by Kenji Ikeda and Hideteru Shimazu). If it is lower than 0.1 MPa, it may not withstand the load caused by blood and may be damaged. If the tensile modulus is higher than 10 MPa, compliance mismatch may occur when transplanted. The tensile elastic modulus is obtained by cutting a vascular regeneration material, preparing a sample of 5 cm in the axial direction and 1 cm in the circumferential direction, and performing a tensile test in the axial direction.
また本発明の血管再生材料の弾性回復率は、７０〜１００％であることが好ましい。７０％未満であると血液による負荷に耐えられず、破損する可能性がある。よって、引張弾性率が０．１〜１０ＭＰａ、弾性回復率が７０〜１００％である血管再生材料が好ましい。弾性回復率は、以下の式により算出した値である。
［Ｌ０−（Ｌ３０−Ｌ０）］／Ｌ０×１００（％）
Ｌ０：血管再生材料の長さ（ｍｍ）
Ｌ３０：血管再生材料の長さに対し１０％変位の引張り処理を３０回行った後の血管再生材料の長さ（ｍｍ）
The elastic recovery rate of the vascular regeneration material of the present invention is preferably 70 to 100%. If it is less than 70%, it cannot withstand the load of blood and may be damaged. Therefore, a vascular regeneration material having a tensile elastic modulus of 0.1 to 10 MPa and an elastic recovery rate of 70 to 100% is preferable. The elastic recovery rate is a value calculated by the following equation.
[L 0 − (L 30 −L 0 )] / L 0 × 100 (%)
L 0 : Length of blood vessel regeneration material (mm)
L 30 : Length of the revascularized material (mm) after 30 times of tensile treatment with 10% displacement relative to the length of the revascularized material
また血管再生材料は、外層の空気透過率が３０ｃｍ３／ｃｍ２・ｓ以上、好ましくは７０〜２５０ｃｍ３／ｃｍ２・ｓ、より好ましくは７０〜２００ｃｍ３／ｃｍ２・ｓである。外層の空気透過率が３０ｃｍ３／ｃｍ２・ｓ以上であると細胞の浸潤性が高く血管再生用の足場材料として好ましい。外層の空気透過率の上限は実質２５０ｃｍ３／ｃｍ２・ｓである。外層の空気透過率は、差圧１２５Ｐａ、厚み１００μｍに換算した空気透過量で表され、ＪＩＳ−Ｌ１０９６、ＪＩＳ−Ｒ３４２０に従い測定した値である。
血管再生材料は、軸方向に連続する山部（９）および谷部（１０）を有する蛇腹状の血管再生材料であり、山部の間隔（１２）が２ｍｍ以下であり、谷部の深さ（１３）が０．１〜１０ｍｍであることが好ましい。蛇腹状の血管再生材料の断面略図を図３に示す。山部の間隔（１２）が２ｍｍより長いと血管再生材料の伸縮性が十分でない。山部、および谷部の間隔は、光学顕微鏡により１０箇所測定を行い、測定値の最大値と最小値との範囲で表す。図３は説明のための略図である。血管再生材料の蛇腹部の構造は規則的であっても不規則であっても良い。製法によっては血管再生材料の蛇腹部の構造は不規則であり、山部の高さ、谷部の深さ、およびこれらの間隔は一定ではない。
The blood vessel regeneration material has an outer layer air permeability of 30 cm 3 / cm 2 · s or more, preferably 70 to 250 cm 3 / cm 2 · s, more preferably 70 to 200 cm 3 / cm 2 · s. When the outer layer has an air permeability of 30 cm 3 / cm 2 · s or more, the invasiveness of the cells is high, which is preferable as a scaffold for revascularization. The upper limit of the air permeability of the outer layer is substantially 250 cm 3 / cm 2 · s. The air permeability of the outer layer is represented by an air permeation amount converted to a differential pressure of 125 Pa and a thickness of 100 μm, and is a value measured according to JIS-L1096 and JIS-R3420.
The blood vessel regeneration material is an accordion-shaped blood vessel regeneration material having a crest (9) and a trough (10) that are continuous in the axial direction, the crest (12) is 2 mm or less, and the depth of the trough (13) is preferably 0.1 to 10 mm. A schematic cross-sectional view of the bellows-like revascularized material is shown in FIG. If the interval (12) between the peaks is longer than 2 mm, the elasticity of the vascular regeneration material is not sufficient. The intervals between the crests and troughs are measured at 10 locations with an optical microscope, and are expressed in the range between the maximum value and the minimum value of the measurement values. FIG. 3 is a schematic diagram for explanation. The structure of the bellows portion of the revascularized material may be regular or irregular. Depending on the manufacturing method, the structure of the bellows part of the vascular regeneration material is irregular, and the height of the peak part, the depth of the valley part, and the interval between them are not constant.
血管再生材料の各層を構成する脂肪族ポリエステルの組成により、細胞浸潤性を制御することが可能である。例えば、グリコール酸由来の繰り返し単位を含有する重合体では細胞との親和性が高く細胞浸潤性が高まる。一方、乳酸単独の重合体では細胞が表面に接着するものの、血管再生材料内部への細胞の浸潤性は低くなる。従って、高い細胞生着が求められる外層ではグリコール酸由来の繰り返し単位を含有するものが好ましく、内層では一層の内皮細胞層を形成することが望まれるため、乳酸由来の繰り返し単位を含有するものが好ましい。従って、内層、中間層、外層の脂肪族ポリエステルは以下のものが好ましい。 Cell invasiveness can be controlled by the composition of the aliphatic polyester constituting each layer of the vascular regeneration material. For example, a polymer containing a repeating unit derived from glycolic acid has high affinity with cells and cell invasiveness. On the other hand, in the case of a polymer of lactic acid alone, cells adhere to the surface, but the invasiveness of the cells into the vascular regeneration material becomes low. Therefore, an outer layer that requires high cell engraftment is preferably one containing a repeating unit derived from glycolic acid, and an inner layer is desired to form one endothelial cell layer, and therefore one containing a repeating unit derived from lactic acid. preferable. Accordingly, the following aliphatic polyesters are preferred for the inner layer, intermediate layer and outer layer.
血管の内膜は、一層の内皮細胞層に覆われた密な構造を有し、物質の選択的な透過をコントロールしている。よって、血管再生材料の内層は、血管組織と同様に一層の内皮細胞層に覆われた構造を有することが好ましい。血管再生材料の最内層にグリコール酸由来の繰り返し単位が含まれると、細胞浸潤性が高まるため一層の内皮細胞構造が構築され難く好ましくない。
そのため、内層を構成する脂肪族ポリエステルは、ポリ乳酸（ＰＬＡ）と、５０〜９０モル％の乳酸由来の繰り返し単位および１０〜５０モル％のカプロラクトン由来の繰り返し単位からなる共重合体（ＰＬＣＡ）とを含有し、前者の含有量が５０〜９０重量％で、後者の含有量が５０〜１０重量％である。ＰＬＣＡは、６０〜９０モル％の乳酸由来の繰り返し単位および１０〜４０モル％のカプロラクトン由来の繰り返し単位からなることが好ましい。
The intima of the blood vessel has a dense structure covered with a layer of endothelial cells and controls the selective permeation of the substance. Therefore, it is preferable that the inner layer of the vascular regeneration material has a structure covered with a single endothelial cell layer like the vascular tissue. When the repeating unit derived from glycolic acid is contained in the innermost layer of the vascular regeneration material, cell invasiveness is increased, and it is difficult to construct a further endothelial cell structure.
Therefore, the aliphatic polyester constituting the inner layer is composed of polylactic acid (PLA), a copolymer (PLCA) composed of 50 to 90 mol% of lactic acid-derived repeating units and 10 to 50 mol% of caprolactone-derived repeating units. It contains the content of the former is 50 to 90 wt%, the latter content is from 50 to 1 0% by weight. PLCA is not preferable that a repeating unit and 10 to 40 mol% of caprolactone-derived repeating units derived from 60 to 90 mole% lactic acid.
中間層を構成する脂肪族ポリエステルは、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、トリメチレンカーボネート、ポリブチレンサクシネート、ポリエチレンサクシネートおよびこれらを構成するモノマーの共重合体などであることが好ましい。これらのうち、乳酸、グリコール酸およびカプロラクトンからなる群より選ばれる少なくとも一種のモノマーの重合体であることが好ましい。血管の中膜は、弾性機能を担っている。そのため血管再生材料の中間層は、弾性を有する５０〜９０モル％の乳酸と１０〜５０モル％のカプロラクトンとの共重合体（ＰＬＣＡ）であることが特に好ましい。 The aliphatic polyester constituting the intermediate layer is preferably polylactic acid, polyglycolic acid, polycaprolactone, polydioxanone, trimethylene carbonate, polybutylene succinate, polyethylene succinate and a copolymer of monomers constituting these. . Among these, a polymer of at least one monomer selected from the group consisting of lactic acid, glycolic acid and caprolactone is preferable. The media of blood vessels has an elastic function. Therefore, the intermediate layer of the vascular regeneration material is particularly preferably an elastic copolymer (PLCA) of 50 to 90 mol% lactic acid and 10 to 50 mol% caprolactone.
外層を構成する脂肪族ポリエステルは、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、ポリトリメチレンカーボネート、ポリブチレンサクシネート、ポリエチレンサクシネートおよびこれらを構成するモノマーの共重合体などであることが好ましい。これらのうち、乳酸、グリコール酸およびカプロラクトンからなる群より選ばれる少なくとも一種のモノマーの重合体であることが好ましい。 The aliphatic polyester constituting the outer layer is preferably polylactic acid, polyglycolic acid, polycaprolactone, polydioxanone, polytrimethylene carbonate, polybutylene succinate, polyethylene succinate, and a copolymer of monomers constituting these. . Among these, a polymer of at least one monomer selected from the group consisting of lactic acid, glycolic acid and caprolactone is preferable.
血管の外膜は、血管の外から栄養分を取るため疎な構造を有し、高い空気透過率を有し、胞浸潤性に優れている。よって、血管再生材料の外層は、細胞の生着性を向上させるため、グリコール酸由来の繰り返し単位を含むことが好ましい。グリコール酸由来の繰り返し単位を含む共重合体としては、乳酸とグリコール酸との共重合体（ＰＬＧＡ）が挙げられる。該共重合体は、好ましくは２０〜８０モル％、より好ましくは４０〜８０モル％の乳酸由来の繰り返し単位および好ましくは２０〜８０モル％、より好ましくは２０〜６０モル％のグリコール酸由来の繰り返し単位からなる。外層には、乳酸とカプロラクトンとの共重合体を含有していてもよい。該共重合体は、５０〜９０モル％の乳酸由来の繰り返し単位および１０〜５０モル％のカプロラクトン由来の繰り返し単位からなることが好ましい。
また外層は、乳酸とグリコール酸との共重合体、および乳酸とカプロラクトンとの共重合体からなる組成物であることが好ましい。組成物中の前者の含有量は好ましくは５０〜９０重量％、後者の含有量は好ましくは１０〜５０重量％である。前者は、２０〜８０モル％の乳酸由来の繰り返し単位および２０〜８０モル％のグリコール酸由来の繰り返し単位からなることが好ましい。後者は、５０〜９０モル％の乳酸由来の繰り返し単位および１０〜５０モル％のカプロラクトン由来の繰り返し単位からなることが好ましい。
The outer membrane of the blood vessel has a sparse structure for removing nutrients from the outside of the blood vessel, has a high air permeability, and is excellent in vesicle infiltration. Therefore, the outer layer of the vascular regeneration material preferably contains a repeating unit derived from glycolic acid in order to improve cell engraftment. Examples of the copolymer containing a repeating unit derived from glycolic acid include a copolymer of lactic acid and glycolic acid (PLGA). The copolymer is preferably derived from 20-80 mol%, more preferably 40-80 mol% of lactic acid-derived repeat units and preferably 20-80 mol%, more preferably 20-60 mol% of glycolic acid. Consists of repeating units. The outer layer may contain a copolymer of lactic acid and caprolactone. The copolymer preferably comprises 50 to 90 mol% of lactic acid-derived repeating units and 10 to 50 mol% of caprolactone-derived repeating units.
The outer layer is preferably a composition comprising a copolymer of lactic acid and glycolic acid and a copolymer of lactic acid and caprolactone. The former content in the composition is preferably 50 to 90% by weight, and the latter content is preferably 10 to 50% by weight. The former is preferably composed of 20 to 80 mol% of repeating units derived from lactic acid and 20 to 80 mol% of repeating units derived from glycolic acid. The latter is preferably composed of 50 to 90 mol% of repeating units derived from lactic acid and 10 to 50 mol% of repeating units derived from caprolactone.
本発明の血管再生材料には、脂肪族ポリエステル中に他の成分をさらに含有しても良い。該成分としては、リン脂質類、糖質類、糖脂質類、ステロイド類、ポリアミノ酸類、タンパク質類、およびポリオキシアルキレン類からなる群から選ばれる少なくとも１種であることが好ましい。具体的な第２成分としては、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のリン脂質類および／またはポリガラクチュロン酸、ヘパリン、コンドロイチン硫酸、ヒアルロン酸、デルマタン硫酸、コンドロイチン、デキストラン硫酸、硫酸化セルロース、アルギン酸、デキストラン、カルボキシメチルキチン、ガラクトマンナン、アラビアガム、トラガントガム、ジェランガム、硫酸化ジェラン、カラヤガム、カラギーナン、寒天、キサンタンガム、カードラン、プルラン、セルロース、デンプン、カルボキシメチルセルロース、メチルセルロース、グルコマンナン、キチン、キトサン、キシログルカン、レンチナンなどの糖質類および／またはガラクトセレブロシド、グルコセレブロシド、グロボシド、ラクトシルセラミド、トリヘキソシルセラミド、パラグロボシド、ガラクトシルジアシルグリセロール、スルホキノボシルジアシルグリセロール、ホスファチジルイノシトール、グリコシルポリプレノールリン酸などの糖脂質類および／またはコレステロール、コール酸、サポゲニン、ジギトキシンなどのステロイド類および／またはポリアスパラギン酸、ポリグルタミン酸、ポリリジンなどのポリアミノ酸類および／またはコラーゲン、ゼラチン、フィブロネクチン、フィブリン、ラミニン、カゼイン、ケラチン、セリシン、トロンビンなどのタンパク質類および／またはポリオキシエチレンアルキルエーテル、ポリオキシエチレンプロピレンアルキルエーテル、ポリオキシエチレンソルビタンエーテルなどのポリオキシアルキレン類、ＦＧＦ（線維芽細胞増殖因子）、ＥＧＦ（上皮増殖因子）、ＰＤＧＦ（血小板由来増殖因子）、ＴＧＦ−β（β型形質転換増殖因子）、ＮＧＦ（神経増殖因子）、ＨＧＦ（肝細胞増殖因子）、ＢＭＰ（骨形成因子）などの細胞増殖因子などが挙げられる。 The blood vessel regeneration material of the present invention may further contain other components in the aliphatic polyester. The component is preferably at least one selected from the group consisting of phospholipids, carbohydrates, glycolipids, steroids, polyamino acids, proteins, and polyoxyalkylenes. Specific examples of the second component include phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, and / or polygalacturonic acid, heparin, chondroitin sulfate, hyaluronic acid, dermatan sulfate, chondroitin, dextran sulfate, Sulfated cellulose, alginic acid, dextran, carboxymethylchitin, galactomannan, gum arabic, tragacanth gum, gellan gum, sulfated gellan, caraya gum, carrageenan, agar, xanthan gum, curdlan, pullulan, cellulose, starch, carboxymethylcellulose, methylcellulose, glucomannan , Carbohydrates such as chitin, chitosan, xyloglucan, lentinan and / or galactocerebroside, Glycolipids such as lucocelebroside, globoside, lactosylceramide, trihexosylceramide, paragloboside, galactosyl diacylglycerol, sulfoquinovosyl diacylglycerol, phosphatidylinositol, glycosyl polyprenol phosphate and / or cholesterol, cholic acid, sapogenin, Steroids such as digitoxin and / or polyamino acids such as polyaspartic acid, polyglutamic acid, polylysine and / or proteins such as collagen, gelatin, fibronectin, fibrin, laminin, casein, keratin, sericin, thrombin and / or polyoxy Ethylene alkyl ether, polyoxyethylene propylene alkyl ether, polyoxyethylene sorbitan ether Which polyoxyalkylenes, FGF (fibroblast growth factor), EGF (epidermal growth factor), PDGF (platelet-derived growth factor), TGF-β (β-type transforming growth factor), NGF (nerve growth factor), HGF Cell growth factors such as (hepatocyte growth factor) and BMP (bone formation factor) can be mentioned.
細胞は、血管細胞、血管前駆細胞および骨髄由来細胞からなる群より選ばれる少なくとも１種であることが好ましい。血管細胞は、血管内皮細胞、平滑筋細胞および線維芽細胞からなる群より選ばれる少なくとも１種であることが好ましい。細胞の継代数は、好ましくは５以下である。継代数が５を越えた細胞は脱分化が進んでいる可能性があるため好ましくない。
細胞は、繊維表面に付着し、血管再生材料中に包含される。播種密度は、好ましくは１０４〜１０７個／ｃｍ２、より好ましくは１０５〜１０６個／ｃｍ２である。本発明の血管再生材料は、内層に含有された細胞の総数を１００％としたとき、少なくとも５０％の細胞が、内層表面から３０μｍまでに存在することが好ましい。
The cell is preferably at least one selected from the group consisting of vascular cells, vascular progenitor cells, and bone marrow-derived cells. The vascular cells are preferably at least one selected from the group consisting of vascular endothelial cells, smooth muscle cells and fibroblasts. The cell passage number is preferably 5 or less. A cell having a passage number exceeding 5 is not preferable because dedifferentiation may have progressed.
Cells adhere to the fiber surface and are included in the vascular regeneration material. The seeding density is preferably 10 4 to 10 7 pieces / cm 2 , more preferably 10 5 to 10 6 pieces / cm 2 . In the vascular regeneration material of the present invention, when the total number of cells contained in the inner layer is 100%, it is preferable that at least 50% of the cells are present up to 30 μm from the inner layer surface.
＜血管再生材料の製造＞
本発明の血管再生材料は、(i) ポリ乳酸（ＰＬＡ）と、５０〜９０モル％の乳酸由来の繰り返し単位および１０〜５０モル％のカプロラクトン由来の繰り返し単位からなる共重合体（ＰＬＣＡ）とを含有し、前者の含有量が５０〜９０重量％で、後者の含有量が５０〜１０重量％である脂肪族ポリエステルおよび揮発性溶媒を含有するドープを静電紡糸法にて紡糸し、得られた繊維をコレクタ上に巻き取り内層を形成する工程、
(ii) 脂肪族ポリエステルおよび揮発性溶媒を含有するドープを静電紡糸法にて紡糸し、得られた繊維を内層上に巻き取り中間層を形成する工程、並びに
(iii) 脂肪族ポリエステルおよび揮発性溶媒を含有するドープを静電紡糸法にて紡糸し、得られた繊維を中間層上に巻き取り外層を形成する工程、
により製造することができる。
<Manufacture of vascular regeneration material>
The blood vessel regeneration material of the present invention comprises (i) polylactic acid (PLA), a copolymer (PLCA) comprising 50 to 90 mol% of lactic acid-derived repeating units and 10 to 50 mol% of caprolactone-derived repeating units. A dope containing an aliphatic polyester having a former content of 50 to 90 % by weight and a latter content of 50 to 10 % by weight and a volatile solvent by electrospinning. Winding the formed fiber on a collector to form an inner layer;
(ii) spinning a dope containing an aliphatic polyester and a volatile solvent by an electrospinning method, winding the obtained fiber on the inner layer to form an intermediate layer, and
(iii) a step of spinning an dope containing an aliphatic polyester and a volatile solvent by an electrostatic spinning method, and winding the obtained fiber on an intermediate layer to form an outer layer;
Can be manufactured.
工程(i)は、脂肪族ポリエステルおよび揮発性溶媒を含有するドープを静電紡糸法にて紡糸し、得られた繊維をコレクタ上に巻き取り内層を形成する工程である。 Step (i) is a step in which a dope containing an aliphatic polyester and a volatile solvent is spun by an electrostatic spinning method, and the obtained fiber is wound on a collector to form an inner layer.
脂肪族ポリエステルは血管再生材料の項で説明した通りである。揮発性溶媒は、脂肪族ポリエステルを溶解し、常圧で沸点が２００℃以下であり、室温で液体である物質である。具体的には、塩化メチレン、クロロホルム、アセトン、メタノール、エタノール、プロパノール、イソプロパノール、トルエン、テトラヒドロフラン、１，１，３，３−ヘキサフルオロイソプロパノール、水、１，４−ジオキサン、四塩化炭素、シクロヘキサン、シクロヘキサノン、Ｎ，Ｎ−ジメチルホルムアミド、アセトニトリルなどが挙げられる。これらのうち、脂肪族ポリエステルの溶解性等から、塩化メチレン、クロロホルム、アセトンが特に好ましい。これらの溶媒は単独で用いても良く、複数の溶媒を組み合わせても良い。また本発明においては、本目的を損なわない範囲で他の溶媒を併用しても良い。 The aliphatic polyester is as described in the section of the blood vessel regeneration material. Volatile solvents are substances that dissolve aliphatic polyester, have a boiling point of 200 ° C. or less at normal pressure, and are liquid at room temperature. Specifically, methylene chloride, chloroform, acetone, methanol, ethanol, propanol, isopropanol, toluene, tetrahydrofuran, 1,1,3,3-hexafluoroisopropanol, water, 1,4-dioxane, carbon tetrachloride, cyclohexane, Examples include cyclohexanone, N, N-dimethylformamide, acetonitrile and the like. Among these, methylene chloride, chloroform, and acetone are particularly preferable in view of the solubility of the aliphatic polyester. These solvents may be used alone, or a plurality of solvents may be combined. Moreover, in this invention, you may use another solvent together in the range which does not impair this objective.
ドープ中の脂肪族ポリエステルの濃度は、好ましくは１〜３０重量％、より好ましくは２〜２０重量％である。脂肪族ポリエステルの濃度が１重量％より低いと、濃度が低すぎるため繊維を形成することが困難となり好ましくない。また、３０重量％より高いと得られる繊維の繊維径が大きくなり好ましくない。 The concentration of the aliphatic polyester in the dope is preferably 1 to 30% by weight, more preferably 2 to 20% by weight. If the concentration of the aliphatic polyester is lower than 1% by weight, it is not preferable because the concentration is too low and it becomes difficult to form fibers. On the other hand, if it is higher than 30% by weight, the fiber diameter of the obtained fiber is undesirably large.
静電紡糸法とは、脂肪族ポリエステルおよび揮発性溶媒を含有するドープを電極間で形成された静電場中に吐出し、ドープを電極に向けて曵糸することにより、繊維状物質をコレクタに巻き付け、血管再生材料を製造する方法である。繊維状物質とは既に溶液の溶媒が留去され、繊維状物質となっている状態のみならず、いまだ溶液の溶媒を含んでいる状態も含む。
静電紡糸法は、例えば図１に示す装置を用いて行うことができる。図１は、ノズル（１）を有する保持槽（３）中に吐出側電極（４）を挿入した装置である。この装置はさらに捕集側電極（５）、高電圧発生器（６）、コレクタ（７）および静電除去器（８）を有する。吐出側電極（４）と捕集側電極（５）との間には、高電圧発生器（６）により所定の電圧が付与される。図１に示す装置において、ドープ（２）を保持槽（３）に充填し、ノズル（１）を通じて静電場中に吐出させ、電界によって曳糸して繊維化させ、コレクタ（７）に集めることにより血管再生材料を得ることができる。静電気除去器（８）を用いることにより外層の空気透過率を高めることができる。
In the electrospinning method, a dope containing an aliphatic polyester and a volatile solvent is discharged into an electrostatic field formed between electrodes, and the dope is spun toward the electrode, whereby the fibrous material is made into the collector. This is a method for producing a wound and revascularized material. The fibrous material includes not only a state in which the solvent of the solution has already been distilled off and becoming a fibrous material, but also a state in which the solvent of the solution is still included.
The electrostatic spinning method can be performed using, for example, the apparatus shown in FIG. FIG. 1 shows an apparatus in which a discharge side electrode (4) is inserted into a holding tank (3) having a nozzle (1). The apparatus further comprises a collection side electrode (5), a high voltage generator (6), a collector (7) and an electrostatic remover (8). A predetermined voltage is applied between the discharge side electrode (4) and the collection side electrode (5) by the high voltage generator (6). In the apparatus shown in FIG. 1, the dope (2) is filled into the holding tank (3), discharged into the electrostatic field through the nozzle (1), threaded by an electric field, fiberized, and collected in the collector (7). Thus, a vascular regeneration material can be obtained. The air permeability of the outer layer can be increased by using the static eliminator (8).
電極は、吐出側電極（４）と捕集側電極（５）とからなる。これらの電極は、金属、無機物または有機物のいかなるものでも導電性を示しさえすれば良い。また、絶縁物上に導電性を示す金属、無機物または有機物の薄膜を持つものであっても良い。静電場は一対または複数の電極間で形成されており、いずれの電極に高電圧を印加しても良い。これは例えば電圧値が異なる高電圧の電極が２つ（例えば１５ｋVと１０ｋV）と、アースにつながった電極の合計３つの電極を用いる場合も含み、または３本を超える数の電極を使う場合も含む。
ドープをコレクタ（７）に向けて曳糸する間に、条件に応じて溶媒が蒸発して繊維状物質が形成される。通常の室温であればコレクタ（７）に捕集されるまでの間に溶媒は完全に蒸発するが、もし溶媒蒸発が不十分な場合は減圧条件下で曳糸しても良い。
コレクタ（７）として鏡面仕上げされていない心棒を用いると、血管再生材料を簡便に製造することが出来る。静電紡糸法により心棒上に血管再生材料を形成する際、心棒を円周方向に回転させることが好ましい。コレクタ（７）を回転させることにより、均質な厚さの円筒体を形成することができる。回転の速度は好ましくは１〜１，０００ｒｐｍ、より好ましくは５〜２００ｒｐｍである。
電極間の距離は、帯電量、ノズル寸法、ドープ吐出量、ドープ濃度等に依存するが、１０ｋＶ程度のときには５〜２０ｃｍの距離が適当である。また、印加される静電気電位は、好ましくは３〜１００ｋＶ、より好ましくは５〜５０ｋＶ、さらに好ましくは５〜３０ｋＶである。
ドープをノズルから静電場中に供給する場合、数個のノズルを用いて繊維状物質の生産速度を上げることもできる。ノズルの内径は好ましくは０．１〜５ｍｍ、より好ましくは０．１〜２ｍｍである。曳糸する温度は溶媒の蒸発挙動や紡糸液の粘度に依存するが、通常は、０〜５０℃である。
An electrode consists of a discharge side electrode (4) and a collection side electrode (5). These electrodes are only required to be conductive with any metal, inorganic or organic material. Further, a metal, inorganic, or organic thin film exhibiting conductivity may be provided over the insulator. The electrostatic field is formed between a pair or a plurality of electrodes, and a high voltage may be applied to any of the electrodes. This includes, for example, the case where two high voltage electrodes having different voltage values (for example, 15 kV and 10 kV) and a total of three electrodes connected to the ground are used, or a case where more than three electrodes are used. Including.
While the dope is drawn toward the collector (7), the solvent evaporates depending on the conditions to form a fibrous material. At normal room temperature, the solvent completely evaporates until it is collected by the collector (7). However, if the solvent evaporation is insufficient, the solvent may be spun under reduced pressure.
When a mandrel that is not mirror-finished is used as the collector (7), a vascular regeneration material can be easily produced. When forming the blood vessel regeneration material on the mandrel by electrostatic spinning, it is preferable to rotate the mandrel in the circumferential direction. By rotating the collector (7), a cylindrical body having a uniform thickness can be formed. The rotation speed is preferably 1 to 1,000 rpm, more preferably 5 to 200 rpm.
The distance between the electrodes depends on the charge amount, nozzle size, dope discharge amount, dope concentration, and the like, but a distance of 5 to 20 cm is appropriate for about 10 kV. The applied electrostatic potential is preferably 3 to 100 kV, more preferably 5 to 50 kV, and still more preferably 5 to 30 kV.
When supplying the dope from the nozzle into the electrostatic field, several nozzles can be used to increase the production rate of the fibrous material. The inner diameter of the nozzle is preferably 0.1 to 5 mm, more preferably 0.1 to 2 mm. The spinning temperature depends on the evaporation behavior of the solvent and the viscosity of the spinning solution, but is usually 0 to 50 ° C.
工程(ii)は、工程（ｉ）で形成された内層の上に、工程（ｉ）と相違する脂肪族ポリエステルを積層する以外は、工程（ｉ）と同じである。工程(iii)は、工程(ii)で形成された中間層の上に、工程(ii)と相違する脂肪族ポリエステルを積層する以外は、工程(ii)と同じである。
外層を形成した後、血管細胞、血管前駆細胞および骨髄由来細胞からなる群より選ばれる少なくとも１種類の細胞を播種することが好ましい。播種密度は、好ましくは１０４〜１０７個／ｃｍ２、より好ましくは１０５〜１０６個／ｃｍ２である。細胞の播種は、細胞を含有する培養液中で、血管再生材料を回転させることにより行うことができる。
Step (ii) is the same as step (i) except that an aliphatic polyester different from step (i) is laminated on the inner layer formed in step (i). Step (iii) is the same as step (ii) except that an aliphatic polyester different from step (ii) is laminated on the intermediate layer formed in step (ii).
After forming the outer layer, it is preferable to seed at least one cell selected from the group consisting of vascular cells, vascular progenitor cells, and bone marrow-derived cells. The seeding density is preferably 10 4 to 10 7 pieces / cm 2 , more preferably 10 5 to 10 6 pieces / cm 2 . Cell seeding can be performed by rotating the vascular regeneration material in a culture solution containing cells.
（蛇腹構造）
本発明によれば、血管再生材料を伸長させることにより弾性回復率を損なわずに蛇腹構造を有する血管再生材料を得ることができる。伸長率としては５０〜３００％が好ましい。ここで５０％伸長とは、１０ｃｍのものを１５ｃｍに伸長させることをいう。十分な弾性回復率を有する血管再生材料を得るために５０％以上伸長させることが好ましい。また３００％を超えて伸長させると血管再生材料自体が破断する場合がある。心棒から血管再生材料を取り外し、血管再生材料の両端を固定し伸長させることで、山部の間隔が２ｍｍ以下であり谷部の深さが０．１〜１０ｍｍの蛇腹構造を有する血管再生材料を得ることができる。蛇腹構造を有する血管再生材料の断面略図を図３に示す。
コレクタ（７）から血管再生材料を取り外すとき、血管再生材料の一端のみに応力をかけ蛇腹構造を得ることができる。即ち、血管再生材料の一端を固定しておき、コレクタ（７）をその固定端の方向に引き抜くことで、一端のみに応力をかけ、蛇腹構造を得ることができる。山部の間隔および谷部の深さは、例えば、デジタルマイクロスコープ(株式会社KEYENCE、VHX DIGITAL MICROSCOPE)により、それぞれ図３の（１２）および（１３）で示す長さを１０箇所程度測定し、範囲を求めることができる。
(Bellows structure)
ADVANTAGE OF THE INVENTION According to this invention, the blood vessel regeneration material which has a bellows structure can be obtained by extending the blood vessel regeneration material, without impairing an elastic recovery rate. The elongation percentage is preferably 50 to 300%. Here, 50% elongation refers to stretching 10 cm to 15 cm. In order to obtain a blood vessel regenerating material having a sufficient elastic recovery rate, it is preferable to stretch 50% or more. Further, when the elongation exceeds 300%, the vascular regeneration material itself may break. By removing the vascular regeneration material from the mandrel, fixing both ends of the vascular regeneration material, and extending the vascular regeneration material, a vascular regeneration material having a bellows structure in which the interval between the peaks is 2 mm or less and the depth of the valley is 0.1 to 10 mm Obtainable. A schematic cross-sectional view of a vascular regeneration material having a bellows structure is shown in FIG.
When the vascular regeneration material is removed from the collector (7), stress can be applied to only one end of the vascular regeneration material to obtain a bellows structure. That is, by fixing one end of the vascular regeneration material and pulling out the collector (7) in the direction of the fixed end, stress is applied to only one end, and an accordion structure can be obtained. The distance between the peaks and the depth of the valleys are measured, for example, by using a digital microscope (KEYENCE, VHX DIGITAL MICROSCOPE) about 10 locations at lengths indicated by (12) and (13) in FIG. A range can be determined.
以下の実施例により本発明を具体的に説明する。しかし、本発明はこれら実施例に限定されるものではない。
（１）実施例に使用した材料は以下の通りである。
The following examples illustrate the invention. However, the present invention is not limited to these examples.
(1) The materials used in the examples are as follows.
(i)ＰＬＧＡ（５０／５０、ＩＶ＝０．６）：乳酸（５０モル％）−グリコール酸（５０モル％）共重合体、固有粘度（以下ＩＶ）＝０．６ｄＬ／ｇ（ＨＦＩＰ中、３０℃）、Birmingham Polymers社製、
(ii)ＰＬＧＡ（５０／５０、ＩＶ＝１．０８）：乳酸（５０モル％）−グリコール酸（５０モル％）共重合体、ＩＶ＝１．０８ｄＬ／ｇ（ＨＦＩＰ中、３０℃）、Absorbable Polymers International社製、
(iii)ＰＬＡ：乳酸重合体、島津製作所（株）製、
(iv)ＰＬＣＡ（７７／２３）：乳酸（７７モル％）−カプロラクトン（２３モル％）共重合体、Ｍｗ＝２．５×１０５、多木化学（株）製、
(v)塩化メチレン、エタノール、マイヤーヘマトキシリン溶液：和光純薬工業（株）製、
(vi)正常ブタ大動脈内皮細胞：APPLICATIONS社製、
(vii)ＦＢＳ（ウシ胎児血清）：HYCLONE社製、
(viii)DMEM、PBS:Invitrogen社製、
(ix)Tissue-Tek OCT compound、エオジン溶液：サクラファインテックジャパン（株）製、
(x)７．５％中性ホルムアルデヒド溶液(pH7.2)：シグマアルドリッチジャパン（株）製、
(xi)AQUATEX:Merck社製
(i) PLGA (50/50, IV = 0.6): lactic acid (50 mol%)-glycolic acid (50 mol%) copolymer, intrinsic viscosity (hereinafter IV) = 0.6 dL / g (in HFIP, 30 ℃), Birmingham Polymers,
(ii) PLGA (50/50, IV = 1.08): lactic acid (50 mol%)-glycolic acid (50 mol%) copolymer, IV = 1.08 dL / g (in HFIP, 30 ° C.), Absorbable Made by Polymers International,
(iii) PLA: lactic acid polymer, manufactured by Shimadzu Corporation
(iv) PLCA (77/23): lactic acid (77 mol%)-caprolactone (23 mol%) copolymer, Mw = 2.5 × 10 5 , manufactured by Taki Chemical Co., Ltd.
(v) Methylene chloride, ethanol, Meyer's hematoxylin solution: manufactured by Wako Pure Chemical Industries,
(vi) Normal porcine aortic endothelial cells: manufactured by APPLICATIONS,
(vii) FBS (fetal bovine serum): manufactured by HYCLONE
(viii) DMEM, PBS: manufactured by Invitrogen,
(ix) Tissue-Tek OCT compound, eosin solution: manufactured by Sakura Finetech Japan Co., Ltd.
(x) 7.5% neutral formaldehyde solution (pH 7.2): manufactured by Sigma Aldrich Japan Co., Ltd.
(xi) AQUATEX: Merck
（２）血管再生材料の物性は以下の方法により測定した。
(i)平均繊維径：デジタルマイクロスコープ（株式会社KEYENCE、VHX DIGITAL MICROSCOPE)により２０箇所測定を行い、その平均値を平均繊維径とした。
(ii)外径：マイクロメーター（株式会社ミツトヨ）により１０箇所測定を行い、測定値の最小値と最大値との範囲を外径とした。
(iii)厚み：血管再生材料を切り開き、長さ５ｃｍ、幅１ｃｍの試料を調製し、マイクロメーターにより１０箇所測定を行い、測定値の最小値と最大値との範囲を厚みとした。
(iv)内径：測定された外径の値と厚みの値との差より求めた。
(v)引張弾性率：血管再生材料を切り開き、血管再生材料の軸方向に５ｃｍ、円周方向に１ｃｍの試料を作製し、血管再生材料の軸方向に張力をかけテンシロン（EZ TEST：島津製作所）により引張試験を行った。
(vi)弾性回復率：血管再生材料を切り開き、血管再生材料の軸方向に５ｃｍ、円周方向に１ｃｍの試料を作製し、血管再生材料の軸方向に張力をかけ、Ｌ０およびＬ３０を求め、以下の式により算出した。
［Ｌ０−（Ｌ３０−Ｌ０）］／Ｌ０×１００（％）
Ｌ０：血管再生材料の長さ（ｍｍ）
Ｌ３０：血管再生材料の長さに対し１０％変位の引張り処理を３０回行った後の血管再生材料の長さ（ｍｍ）
(2) The physical properties of the vascular regeneration material were measured by the following methods.
(i) Average fiber diameter: 20 positions were measured with a digital microscope (KEYENCE, VHX DIGITAL MICROSCOPE), and the average value was defined as the average fiber diameter.
(ii) Outer diameter: Ten locations were measured with a micrometer (Mitutoyo Co., Ltd.), and the range between the minimum value and the maximum value of the measured values was defined as the outer diameter.
(iii) Thickness: A blood vessel regenerating material was cut open, a sample having a length of 5 cm and a width of 1 cm was prepared, and measurement was performed at 10 points with a micrometer, and the range between the minimum value and the maximum value of the measured value was defined as the thickness.
(iv) Inner diameter: It was determined from the difference between the measured outer diameter value and thickness value.
(v) Tensile elastic modulus: Opening the vascular regeneration material, preparing a sample of 5cm in the axial direction of the vascular regeneration material and 1cm in the circumferential direction, applying tension to the axial direction of the vascular regeneration material (EZ TEST: Shimadzu Corporation) ) To perform a tensile test.
(vi) Elastic recovery rate: Cut the vascular regeneration material, prepare a sample of 5 cm in the axial direction of the vascular regeneration material and 1 cm in the circumferential direction, apply tension in the axial direction of the vascular regeneration material, and set L 0 and L 30 And calculated by the following formula.
[L 0 − (L 30 −L 0 )] / L 0 × 100 (%)
L 0 : Length of blood vessel regeneration material (mm)
L 30 : Length of the revascularized material (mm) after 30 times of tensile treatment with 10% displacement relative to the length of the revascularized material
(vii)外層の空気透過率：血管再生材料を切り開き外層のみを剥がし、５ｃｍ×５ｃｍになるように形状を整え、フラジール・パーミヤメータ（（株）東洋精機製作所）を用いて、JIS-L1096、JIS-R3420に従い空気透過率（ｃｍ３／ｃｍ２・ｓ）を測定し、厚み１００μｍにおける空気透過率に換算した。 (vii) Outer layer air permeability: Open the revascularized material, peel off only the outer layer, adjust the shape to 5cm x 5cm, and use fragile perimeter (Toyo Seiki Co., Ltd.), JIS-L1096, JIS The air permeability (cm 3 / cm 2 · s) was measured according to -R3420 and converted to an air permeability at a thickness of 100 μm.
＜参考例１＞（ドープの調製）
（ドープＡの調製）
ＰＬＧＡ（５０／５０、ＩＶ＝０．６）０．８ｇ、ＰＬＣＡ（７７／２３）０．２ｇ、塩化メチレン／エタノール＝８／１（重量部／重量部）９ｇを室温（２５℃）で混合し濃度１０重量％のドープＡを調製した。
（ドープＢの調製）
ＰＬＣＡ（７７／２３）１ｇ、塩化メチレン／エタノール＝８／１（重量部／重量部）９ｇを室温（２５℃）で混合し濃度１０重量％のドープＢを調製した。
（ドープＣの調製）
ＰＬＧＡ（５０／５０、ＩＶ＝１．０８）０．８ｇ、ＰＬＣＡ（７７／２３）０．２ｇ、塩化メチレン／エタノール＝８／１（重量部／重量部）９ｇを室温（２５℃）で混合し濃度１０重量％のドープＣを調製した。
（ドープＤの調製）
ＰＬＡ０．８ｇ、ＰＬＣＡ（７７／２３）０．２ｇ、塩化メチレン／エタノール＝８／１（重量部／重量部）９ｇを室温（２５℃）で混合し濃度１０重量％のドープＤを調製した。
（ドープＥの調製）
ＰＬＡ０．５ｇ、ＰＬＣＡ（７７／２３）０．５ｇ、塩化メチレン／エタノール＝８／１（重量部／重量部）９ｇを室温（２５℃）で混合し濃度１０重量％のドープＥを調製した。表１にドープＡ〜Ｅに含有された脂肪族ポリエステルの組成を示す。
<Reference Example 1> (Preparation of dope)
(Preparation of dope A)
PLGA (50/50, IV = 0.6) 0.8 g, PLCA (77/23) 0.2 g, methylene chloride / ethanol = 8/1 (parts by weight / parts) 9 g were mixed at room temperature (25 ° C.). A dope A having a concentration of 10% by weight was prepared.
(Preparation of dope B)
1 g of PLCA (77/23) and 9 g of methylene chloride / ethanol = 8/1 (parts by weight / parts by weight) were mixed at room temperature (25 ° C.) to prepare a dope B having a concentration of 10% by weight.
(Preparation of dope C)
PLGA (50/50, IV = 1.08) 0.8 g, PLCA (77/23) 0.2 g, methylene chloride / ethanol = 8/1 (parts by weight) 9 g was mixed at room temperature (25 ° C.). A dope C having a concentration of 10% by weight was prepared.
(Preparation of dope D)
A dope D having a concentration of 10% by weight was prepared by mixing 0.8 g of PLA, 0.2 g of PLCA (77/23), and 9 g of methylene chloride / ethanol = 8/1 (parts by weight / parts by weight) at room temperature (25 ° C.).
(Preparation of dope E)
0.5 g of PLA, 0.5 g of PLCA (77/23) and 9 g of methylene chloride / ethanol = 8/1 (parts by weight / parts by weight) were mixed at room temperature (25 ° C.) to prepare a dope E having a concentration of 10% by weight. Table 1 shows the composition of the aliphatic polyester contained in the dopes A to E.
＜実施例１＞（Ｄ／Ｂ／Ａ）
（血管再生材料の製造）
図１に示す装置を組み立てた。ノズル（１）の内径は０．８ｍｍとした。ノズル（１）から捕集電極（５）までの距離は１０ｃｍに設定した。捕集側電極（５）として、外径４ｍｍ、長さ２０ｃｍのステンレス棒を用いた。ノズル（１）と捕集側電極（５）との間に、コレクタ（７）および静電除去器（８）を設置した。
（内層の形成）
ドープＤを保持槽（３）に入れ、ドープＤを捕集側電極（５）に向けて５分間吐出し、内層を形成した。吐出側電極（４）と捕集側電極（５）との電圧は１４ｋＶに設定した。コレクタ（７）は１００ｒｐｍで回転させながら捕集した。このとき、静電除去器（８）は使用しなかった。内層の厚みは６０〜８０μｍであった。内層を構成する繊維の平均繊維径は６μｍであった。
（中間層の形成）
さらに、ドープＤの代わりにドープＢを保持槽（３）に入れ、５分間吐出し、中間層を形成した。このとき、静電除去器（８）は使用しなかった。中間層の厚みは６０〜８０μｍであった。中間層を構成する繊維の平均繊維径は４μｍであった。
（外層の形成）
次に、ドープＢの代わりにドープＡを保持槽（３）に入れ、５分間吐出し、外層を形成し血管再生材料を得た。このとき、静電除去器（８）を使用した。外層の厚みは６０〜８０μｍであった。外層を構成する繊維の平均繊維径は６μｍであった。得られた血管再生材料は、長さ１０ｃｍ、外径４．４〜４．５ｍｍ、厚み２００〜２４０μｍであった。血管再生材料の引張弾性率は、１．１７ＭＰａ、弾性回復率は９７．３％、空気透過率は１４９cm3/cm2・sであった。
<Example 1> (D / B / A)
(Manufacture of revascularized materials)
The apparatus shown in FIG. 1 was assembled. The inner diameter of the nozzle (1) was 0.8 mm. The distance from the nozzle (1) to the collecting electrode (5) was set to 10 cm. A stainless steel rod having an outer diameter of 4 mm and a length of 20 cm was used as the collection side electrode (5). A collector (7) and an electrostatic remover (8) were installed between the nozzle (1) and the collection side electrode (5).
(Formation of inner layer)
The dope D was put in the holding tank (3), and the dope D was discharged toward the collecting electrode (5) for 5 minutes to form an inner layer. The voltage of the discharge side electrode (4) and the collection side electrode (5) was set to 14 kV. The collector (7) was collected while rotating at 100 rpm. At this time, the electrostatic remover (8) was not used. The thickness of the inner layer was 60 to 80 μm. The average fiber diameter of the fibers constituting the inner layer was 6 μm.
(Formation of intermediate layer)
Furthermore, dope B was put in the holding tank (3) instead of dope D and discharged for 5 minutes to form an intermediate layer. At this time, the electrostatic remover (8) was not used. The thickness of the intermediate layer was 60 to 80 μm. The average fiber diameter of the fibers constituting the intermediate layer was 4 μm.
(Formation of outer layer)
Next, in place of the dope B, the dope A was put in the holding tank (3) and discharged for 5 minutes to form an outer layer to obtain a vascular regeneration material. At this time, an electrostatic remover (8) was used. The thickness of the outer layer was 60 to 80 μm. The average fiber diameter of the fibers constituting the outer layer was 6 μm. The obtained vascular regeneration material had a length of 10 cm, an outer diameter of 4.4 to 4.5 mm, and a thickness of 200 to 240 μm. The blood vessel regeneration material had a tensile elastic modulus of 1.17 MPa, an elastic recovery rate of 97.3%, and an air permeability of 149 cm 3 / cm 2 · s.
（ヒト臍帯静脈内皮細胞の播種）
得られた円柱状の血管再生材料の内部に一方の端部から細胞懸濁液を注入し、細胞を播種した。細胞懸濁液は、血管再生材料の内径と長さから播種密度が１×１０６ｃｅｌｌｓ／ｃｍ２となるように細胞数を調整しておいた。注入は、血管再生材料を垂直にし、下から上に向かって注入することにより行った。上側まで細胞懸濁液面が上がってきた時点で注入を止めた。
注入後、血管再生材料の両端（注入側、反対側）に一方向弁付きの栓を取り付けた後、培地入りの円筒状の培養容器内に固定した。培養容器をウェーブローター（株式会社サーモニクス）に置き３７℃で約３−６回／分で、約４−６時間回転させながら培養を行った。その後、栓は開放し静地で培養を継続した。培養後の担体断面のＨＥ染色像を図４に示す。なお、ＨＥ染色法は実施例３に示す通りである。図４によれば、血管再生材料の内層の中空部側に細胞層が形成されているのが分かる。
(Seeding of human umbilical vein endothelial cells)
A cell suspension was injected from one end into the obtained cylindrical vascular regeneration material, and cells were seeded. The number of cells in the cell suspension was adjusted so that the seeding density was 1 × 10 6 cells / cm 2 from the inner diameter and length of the vascular regeneration material. The injection was performed by injecting the revascularized material vertically and injecting it from the bottom to the top. The injection was stopped when the cell suspension surface reached the upper side.
After the injection, stoppers with one-way valves were attached to both ends (injection side, opposite side) of the vascular regeneration material, and then fixed in a cylindrical culture container containing a medium. The culture vessel was placed on a wave rotor (Thermonics Co., Ltd.) and cultured at 37 ° C. at about 3-6 times / minute for about 4-6 hours. Thereafter, the stopper was opened and the culture was continued in a static place. FIG. 4 shows an HE-stained image of the carrier cross-section after culture. The HE staining method is as shown in Example 3. FIG. 4 shows that a cell layer is formed on the hollow portion side of the inner layer of the blood vessel regeneration material.
＜実施例２＞（Ｅ／Ｂ／Ａ）
内層をドープＥを用いて形成する以外は実施例１と同じ方法で血管再生材料を得た。血管再生材料の物性を表２に示す。その後、実施例１と同じ方法で細胞を播種した。
<Example 2> (E / B / A)
A blood vessel regeneration material was obtained in the same manner as in Example 1 except that the inner layer was formed using Dope E. Table 2 shows the physical properties of the revascularized material. Thereafter, the cells were seeded in the same manner as in Example 1.
＜比較例１＞（Ａ／Ｂ／Ａ）
内層をドープＡを用いて形成する以外は実施例１と同じ方法で血管再生材料を得た。血管再生材料の物性を表２に示す。その後、実施例１と同じ方法で細胞を播種した。
<Comparative Example 1> (A / B / A)
A blood vessel regeneration material was obtained in the same manner as in Example 1 except that the inner layer was formed using Dope A. Table 2 shows the physical properties of the revascularized material. Thereafter, the cells were seeded in the same manner as in Example 1.
＜比較例２＞（Ｃ／Ｂ／Ａ）
内層をドープＣを用いて形成する以外は実施例１と同じ方法で血管再生材料を得た。血管再生材料の物性を表２に示す。その後、実施例１と同じ方法で細胞を播種した。
<Comparative Example 2> (C / B / A)
A blood vessel regeneration material was obtained in the same manner as in Example 1 except that the inner layer was formed using Dope C. Table 2 shows the physical properties of the revascularized material. Thereafter, the cells were seeded in the same manner as in Example 1.
＜実施例３＞（細胞浸潤性の評価）
実施例１の血管再生材料の細胞浸潤性を以下の方法で評価した。
（血管内皮細胞培養）
実施例１で外層を形成する前の内層（ａ）および中間層（ｂ）からなる血管再生材料をシート状に切り開き、直径１７ｍｍの円形に切り取った。内層上に正常ブタ大動脈内皮細胞を１×１０６個／ｃｍ２の密度で播種し、１０％ＦＢＳを含む培養液（ＤＭＥＭ）を用いて５％ＣＯ２、３７℃の環境下で７日間培養した。
（凍結切片作製）
培養後のシートをＰＢＳを用いて洗浄後、Tissue-Tek OCT compound内に包埋し、液体窒素を用いて凍結した。この試料からクライオスタット（ライカマイクロシステムズ（株）社製、ＣＭ１８００）を用いて厚さ５μｍの凍結縦切片を作製し、スライドグラス(Polyscience社製、Tissue Tack Microscope Slides)に貼り付け、室温で一晩静置することにより自然乾燥させた。
（ＨＥ染色）
上記手順により作製した標本を７．５％中性ホルムアルデヒド溶液に１０分間浸すことにより固定した。水道水で洗浄後、マイヤーヘマトキシリン溶液に５分間浸すことにより核を染色した。水道水に１５分間浸した後、エオジン溶液に３分間浸すことにより細胞質を染色した。水道水で洗浄後、余分な水分を取り除き乾燥後、封入剤ＡＱＵＡＴＥＸを用いてカバーグラス（松浪硝子工業（株）製）内に封入した。
（評価）
染色後の標本を光学顕微鏡（ＯＬＹＭＰＵＳ（株）製、ＢＸ５１）を用いて観察し、細胞が内層表面に接着している様子および内層内部に浸潤している様子を確認した。その結果、内層（ａ）の無作為に抽出した幅１ｍｍ範囲内に存在する全細胞のうち、６７％の細胞が内層表面（ｃ）から３０μｍまでに分布していた。その顕微鏡写真を図５に示す。
<Example 3> (Evaluation of cell invasiveness)
The cell invasiveness of the vascular regeneration material of Example 1 was evaluated by the following method.
(Vascular endothelial cell culture)
The vascular regeneration material composed of the inner layer (a) and the intermediate layer (b) before forming the outer layer in Example 1 was cut into a sheet shape and cut into a circle having a diameter of 17 mm. Normal porcine aortic endothelial cells are seeded on the inner layer at a density of 1 × 10 6 cells / cm 2 , and cultured for 7 days in an environment of 5% CO 2 and 37 ° C. using a culture solution (DMEM) containing 10% FBS. did.
(Frozen section preparation)
The cultured sheet was washed with PBS, embedded in Tissue-Tek OCT compound, and frozen with liquid nitrogen. From this sample, a frozen longitudinal section having a thickness of 5 μm was prepared using a cryostat (manufactured by Leica Microsystems Co., Ltd., CM1800), attached to a slide glass (Polyscience, Tissue Tack Microscope Slides), and overnight at room temperature. It naturally dried by standing.
(HE staining)
The specimen prepared by the above procedure was fixed by immersing it in a 7.5% neutral formaldehyde solution for 10 minutes. After washing with tap water, nuclei were stained by soaking in Mayer's hematoxylin solution for 5 minutes. After soaking in tap water for 15 minutes, the cytoplasm was stained by soaking in eosin solution for 3 minutes. After washing with tap water, excess water was removed and dried, and then encapsulated in a cover glass (manufactured by Matsunami Glass Industry Co., Ltd.) using an encapsulant AQUATEX.
(Evaluation)
The specimen after staining was observed using an optical microscope (BX51, manufactured by OLYMPUS Co., Ltd.), and it was confirmed that cells were adhered to the inner layer surface and infiltrated into the inner layer. As a result, 67% of the cells present in the range of 1 mm width randomly extracted from the inner layer (a) were distributed from the inner layer surface (c) to 30 μm. The micrograph is shown in FIG.
＜実施例４＞（細胞浸潤性の評価）
実施例２の血管再生材料の細胞浸潤性を実施例３と同じ方法で評価した。その結果、内層の（ａ）無作為に抽出した幅１ｍｍ範囲内に存在する全細胞のうち、６８％の細胞が内層表面（ｃ）から３０μｍまでに分布していた。
<Example 4> (Evaluation of cell invasiveness)
The cell invasiveness of the vascular regeneration material of Example 2 was evaluated by the same method as in Example 3. As a result, 68% of the cells present in the inner layer (a) within a randomly extracted 1 mm width range were distributed from the inner layer surface (c) to 30 μm.
＜比較例３＞（細胞浸潤性の評価）
比較例１の血管再生材料の細胞浸潤性を実施例３と同じ方法で評価した。その結果、内層（ａ）の無作為に抽出した幅１ｍｍ範囲内に存在する全細胞のうち、３９％の細胞が内層表面（ｃ）から３０μｍまでに分布していた。その顕微鏡写真を図６に示す。
<Comparative Example 3> (Evaluation of cell invasiveness)
The cell invasiveness of the vascular regeneration material of Comparative Example 1 was evaluated by the same method as in Example 3. As a result, 39% of cells were distributed from the inner layer surface (c) to 30 μm among all the cells present in the range of 1 mm width randomly extracted from the inner layer (a). The micrograph is shown in FIG.
＜比較例４＞（細胞浸潤性の評価）
比較例２の血管再生材料の細胞浸潤性を実施例３と同じ方法で評価した。その結果、内層（ａ）の無作為に抽出した幅１ｍｍ範囲内に存在する全細胞のうち、４１％の細胞が内層表面（ｃ）から３０μｍまでに分布していた。
<Comparative Example 4> (Evaluation of cell invasiveness)
The cell invasiveness of the vascular regeneration material of Comparative Example 2 was evaluated by the same method as in Example 3. As a result, 41% of the cells present in the 1 mm width range randomly extracted from the inner layer (a) were distributed from the inner layer surface (c) to 30 μm.
細胞浸潤性の評価から分かるように、内層（ａ）の組成により細胞浸潤性に差が認められる。即ち、グリコール酸由来の繰り返し単位を含有しない内層（ａ）を有する実施例１および２の血管再生材料は、グリコール酸由来の繰り返し単位を含有する内層（ａ）を有する比較例１および２の血管再生材料に比べ、細胞浸潤性が有意に抑えられ、表面付近に細胞が局在し、血管内腔に類似の内皮細胞構造を構築することができる。 As can be seen from the evaluation of cell invasiveness, there is a difference in cell invasiveness depending on the composition of the inner layer (a). That is, the blood vessel regeneration material of Examples 1 and 2 having the inner layer (a) not containing the repeating unit derived from glycolic acid is the blood vessel of Comparative Examples 1 and 2 having the inner layer (a) containing the repeating unit derived from glycolic acid. Compared with regenerated materials, cell infiltration is significantly suppressed, cells are localized near the surface, and a similar endothelial cell structure can be constructed in the blood vessel lumen.
本発明の血管再生材料は血管組織に類似した構造および物理特性を示すため、再生医療における血管治療用材料として有用である。 The vascular regeneration material of the present invention exhibits a structure and physical properties similar to vascular tissue, and is useful as a vascular treatment material in regenerative medicine.
１． ノズル
２． ドープ
３． 保持槽
４． 吐出側電極
５． 捕集側電極
６． 高電圧発生器
７． コレクタ
８． 静電除去器
９． 山部
１０． 谷部
１１． 外径
１２． 山部の間隔
１３． 谷部の深さ
１４． 厚み
１５． 内径
ａ． 内層
ｂ． 中間層
ｃ． 内層表面
1. Nozzle 2. Dope Holding tank 4. 4. Discharge side electrode Collection side electrode 6. High voltage generator 7. Collector 8. Static eliminator 9. Yamabe 10. Tanibe 11. Outer diameter 12. Mountain spacing 13. Valley depth 14. Thickness 15. Inner diameter a. Inner layer b. Intermediate layer c. Inner layer surface
Claims (11)
(i) ポリ乳酸（ＰＬＡ）と、５０〜９０モル％の乳酸由来の繰り返し単位および１０〜５０モル％のカプロラクトン由来の繰り返し単位からなる共重合体（ＰＬＣＡ）とを含有し、前者の含有量が５０〜９０重量％で、後者の含有量が５０〜１０重量％である脂肪族ポリエステルおよび揮発性溶媒を含有するドープを静電紡糸法にて紡糸し、得られた繊維をコレクタ上に巻き取り内層を形成する工程、
(ii) 脂肪族ポリエステルおよび揮発性溶媒を含有するドープを静電紡糸法にて紡糸し、得られた繊維を内層上に巻き取り中間層を形成する工程、並びに
(iii) 脂肪族ポリエステルおよび揮発性溶媒を含有するドープを静電紡糸法にて紡糸し、得られた繊維を中間層上に巻き取り外層を形成する工程、
を含む血管再生材料の製造方法。 It is a hollow cylindrical shape composed of concentric inner layer, intermediate layer and outer layer, and each layer is a method for producing a blood vessel regeneration material composed of aliphatic polyester fibers having an average fiber diameter of 0.05 to 10 μm,
(i) containing polylactic acid (PLA) and a copolymer (PLCA) composed of 50 to 90 mol% of lactic acid-derived repeating units and 10 to 50 mol% of caprolactone-derived repeating units, the former content The dope containing an aliphatic polyester and a volatile solvent having a content of 50 to 90 % by weight and the latter content of 50 to 10 % by weight is spun by an electrostatic spinning method, and the obtained fiber is wound on a collector. Forming a take-up inner layer,
(ii) spinning a dope containing an aliphatic polyester and a volatile solvent by an electrospinning method, winding the obtained fiber on the inner layer to form an intermediate layer, and
(iii) a step of spinning an dope containing an aliphatic polyester and a volatile solvent by an electrostatic spinning method, and winding the obtained fiber on an intermediate layer to form an outer layer;
A method for producing a vascular regeneration material comprising:
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| US8840660B2 (en) | 2006-01-05 | 2014-09-23 | Boston Scientific Scimed, Inc. | Bioerodible endoprostheses and methods of making the same |
| EP2081616B1 (en) | 2006-09-15 | 2017-11-01 | Boston Scientific Scimed, Inc. | Bioerodible endoprostheses and methods of making the same |
| JP5541846B2 (en) * | 2008-04-22 | 2014-07-09 | 帝人株式会社 | Cotton-like structure for cardiovascular treatment and method for producing the same |
| WO2009134991A2 (en) | 2008-04-30 | 2009-11-05 | Ethicon, Inc. | Tissue engineered blood vessel |
| US7998192B2 (en) * | 2008-05-09 | 2011-08-16 | Boston Scientific Scimed, Inc. | Endoprostheses |
| US8668732B2 (en) | 2010-03-23 | 2014-03-11 | Boston Scientific Scimed, Inc. | Surface treated bioerodible metal endoprostheses |
| WO2015008152A2 (en) * | 2013-05-31 | 2015-01-22 | M-S Medical Sagl | Methods and compositions for blood vessel tissue repair and engineering |
| CA2948171A1 (en) | 2014-05-08 | 2015-11-12 | Secant Medical, Inc. | Composite lumen with reinforcing textile and matrix |
| CN106075596B (en) * | 2016-07-21 | 2021-02-09 | 南开大学 | Preparation technology of three-layer artificial blood vessel |
| CN109172073B (en) * | 2018-09-03 | 2023-11-21 | 上海市第一人民医院 | Electrostatic spinning nanofiber membrane for controlling release of growth factors and esophageal tectorial membrane memory stent |
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