Three-layer electrospun fiber-based small-caliber artificial blood vessel and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medical materials, tissue engineering blood vessels and artificial blood vessels, and particularly relates to a three-layer structure electrostatic spinning fiber-based small-diameter artificial blood vessel and a preparation method and application thereof. The artificial blood vessel is composed of an inner nanofiber layer, an intermediate circumferential orientation microfiber layer and an outer random microfiber layer, wherein the inner layer contains a vascular extracellular matrix and a first biological macromolecule, the intermediate layer contains a first degradable polymer material and a second biological macromolecule, and the outer layer contains a second degradable polymer material; the inner layer and the intermediate layer are further grafted or fixed with heparin to improve the antithrombotic performance of the artificial blood vessel and promote vascular tissue regeneration. The small-diameter artificial blood vessel has the functions of antithrombosis, endothelialization promotion, smooth muscle cell circumferential regeneration guidance and degradable reconstruction.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, and in particular to a three-layer electrospun fiber-based small-diameter artificial blood vessel, its preparation method, and its application. Background Technology

[0002] Coronary artery disease, peripheral artery disease, and vascular injury repair often require vascular replacements. Currently, large-diameter artificial blood vessels, such as expanded polytetrafluoroethylene (ePTFE) and polyester vessels, are used clinically. However, for small-diameter artificial blood vessels with an inner diameter of less than 6 mm, there are still problems such as low long-term patency, thrombosis, intimal hyperplasia, insufficient endothelialization, and mismatch between mechanical properties and natural blood vessels.

[0003] After implantation of small-diameter artificial blood vessels, their inner luminal surface is in prolonged contact with blood. If the material surface lacks anticoagulant and endothelialization-promoting properties, it can easily lead to platelet adhesion, activation of the coagulation cascade, and acute thrombosis. Furthermore, the smooth muscle cells in the vascular media exhibit a typical circumferential arrangement, which plays a crucial role in vascular contraction and dilation, mechanical support, and vascular remodeling. Traditional artificial blood vessels are mostly homogeneous or composed of a single random fiber structure, making it difficult to simultaneously meet the requirements of antithrombosis, endothelialization promotion, guiding the circumferential arrangement of smooth muscle cells, and integration with the outer tissue.

[0004] Electrospinning technology can fabricate fibrous scaffolds with high specific surface area, high porosity, and extracellular matrix-like structures, and has been widely used in tissue-engineered vascular research. However, existing electrospun artificial blood vessels still have the following shortcomings: 1. The luminal surface lacks natural vascular matrix signals, and endothelial cell adhesion and functionalization are insufficient; 2. The fibrous structure failed to effectively mimic the layered structure of natural blood vessels; 3. The middle layer fibers lack orientation guidance, making it difficult to induce smooth muscle cells to circumferentially align; 4. The material has insufficient antithrombotic properties; 5. Single-layer or double-layer structures cannot simultaneously achieve endothelialization, middle membrane reconstruction, and outer membrane tissue integration.

[0005] Therefore, it is necessary to develop a small-diameter artificial blood vessel that combines antithrombotic, endothelialization-promoting, smooth muscle cell circumferential regeneration, and degradable remodeling capabilities. Summary of the Invention

[0006] The purpose of this invention is to provide a three-layer electrospun fiber-based small-diameter artificial blood vessel, which achieves the following functions through a biomimetic layered structure design: 1. The inner layer simulates the vascular intima matrix microenvironment, promoting endothelial cell adhesion, migration, and endothelialization; 2. The inner and middle layers fix heparin, improving blood compatibility and inhibiting thrombus formation; 3. The intermediate layer adopts a circumferentially oriented microfiber structure to guide smooth muscle cells to align and regenerate along the circumference of blood vessels; 4. The outer layer adopts a random microfiber structure to improve tissue ingrowth, outer membrane integration, and overall mechanical support; 5. By combining biodegradable polymer materials with bioactive components, the vascular stent can be gradually degraded and remodeled into tissue.

[0007] In a first aspect of the present invention, a three-layer electrospun fiber-based artificial blood vessel is provided. The artificial blood vessel is a hollow cylinder and includes an inner nanofiber layer, a middle circumferentially oriented microfiber layer and an outer random microfiber layer arranged sequentially from the inside to the outside. Heparin is grafted, fixed or loaded on at least one of the inner and middle layers. The inner nanofiber layer is prepared by electrospinning a mixture of vascular extracellular matrix and first biological macromolecule. The intermediate layer is made by electrospinning a mixture of a first polymer material and a second biomacromolecule. The fibers are oriented along the circumferential direction of the blood vessels. The mandrel of the electrospinning is rotated at high speed to receive the fibers, with a rotation speed of 2000-15000 rpm. The outer layer is made of a second polymer material by electrospinning, and the fibers are arranged randomly. Wherein, the first polymer material and the second polymer material are both biodegradable polymer materials, each independently selected from the following group: polycaprolactone PCL, polylactic acid-caprolactone copolymer PLCL, polyglycolic acid PGA, polydioxanone PDO, or combinations thereof; The first and second biomacromolecules are each independently selected from the group consisting of: gelatin, chitosan, oligosaccharides, hyaluronic acid, sodium alginate, collagen, silk fibroin, or combinations thereof.

[0008] In another preferred embodiment, the inner diameter of the artificial blood vessel is 1-20 mm, preferably 1-10 mm, such as 2 mm, 4 mm, 6 mm, or 8 mm.

[0009] In another preferred embodiment, the artificial blood vessel is a small-diameter artificial blood vessel.

[0010] In another preferred embodiment, the first polymer material and the second polymer material may be the same or different, but preferably the same.

[0011] In another preferred embodiment, both the first polymer material and the second polymer material are PCL or both are PLCL.

[0012] In another preferred embodiment, the polycaprolactone PCL has a molecular weight Mn of 50~100 kDa, preferably 70~90 kDa.

[0013] In another preferred embodiment, the molecular weight Mw of the polylactic acid-caprolactone copolymer PLCL (preferably with a L-lactide:ε-CL copolymer ratio of 50:50) is 50~100 kDa, preferably 60~80 kDa.

[0014] In another preferred embodiment, the ratio of L-lactide monomer to ε-caprolactone in the polylactic acid-caprolactone copolymer PLCL is 10:90 to 90:10, preferably 40:60 to 60:40, for example 50:50.

[0015] In another preferred embodiment, the first biomacromolecule and the second biomacromolecule may be the same or different, but preferably the same.

[0016] In another preferred embodiment, both the first and second biomacromolecules are gelatin.

[0017] In another preferred embodiment, the fibers in the inner nanofiber layer are randomly arranged or poorly oriented. Preferably, the poorly oriented arrangement means that the oriented fibers account for no more than 30% of the total, and more preferably no more than 20%.

[0018] In another preferred embodiment, the fiber diameter of the inner nanofiber layer is 50-800 nm, preferably 50-500 nm, more preferably 80-400 nm, and even more preferably 100-300 nm.

[0019] In another preferred embodiment, the thickness of the inner nanofiber layer is 5-80 μm, preferably 10-50 μm, for example 20 μm or 30 μm.

[0020] In another preferred embodiment, the vascular extracellular matrix in the inner layer is derived from decellularized vascular tissue, including but not limited to decellularized arterial matrix, decellularized venous matrix, decellularized vascular wall matrix, or one or more of their powdered products.

[0021] In another preferred embodiment, the vascular extracellular matrix in the inner layer comprises collagen, elastin, laminin, fibronectin, glycosaminoglycans, and other vascular tissue-related active ingredients to simulate the natural vascular intima and subendothelial matrix microenvironment.

[0022] In another preferred embodiment, in the inner layer, the mass ratio of the extracellular matrix of the vascular cells to the first biomacromolecule is 1:20 to 20:1, preferably 1:9 to 9:1, more preferably 3:7 to 7:3, and even more preferably 5:5.

[0023] In another preferred embodiment, the method for preparing the inner layer includes: (a1) The extracellular matrix of vascular cells is mixed with the first biomacromolecule to prepare an inner spinning solution; (b1) The inner spinning solution is loaded into a syringe and spun onto the surface of a rotating mandrel by electrospinning.

[0024] In another preferred embodiment, in step (a1), the total concentration in the inner spinning solution is 5-30% w / v, preferably 5-20% w / v, for example 10% w / v.

[0025] In another preferred embodiment, in step (a1), the solvent in the inner spinning solution is selected from the group consisting of hexafluoroisopropanol, trifluoroethanol, dichloromethane, trifluoroacetic acid, or combinations thereof.

[0026] In another preferred embodiment, in step (b1), the rotational speed of the mandrel is 100-1800 rpm, preferably 100-1000 rpm, for example 500 rpm.

[0027] In another preferred embodiment, in step (b1), the parameters of the electrospinning include one or more of the following: Spinning voltage: 5-30kV, preferably 8-25 kV, for example 15kV; Receiving distance: 5-25cm, preferably 8-20cm, such as 12cm, 15cm, 18cm; Injection rate: 0.2-2.0 mL / h, preferably 0.5-1.0 mL / h, for example 0.8 mL / h; Temperature: 5-40℃, preferably 15-30℃; Relative humidity: 10-90%, preferably 20-60%.

[0028] In another preferred embodiment, in step (b1), the diameter of the inner fiber and the thickness of the shell are controlled by adjusting the voltage, receiving distance, flow rate, solution concentration and environmental parameters.

[0029] In another preferred embodiment, the fiber diameter of the intermediate layer is 1-20 μm, preferably 2-15 μm, and more preferably 2-10 μm.

[0030] In another preferred embodiment, the thickness of the intermediate layer is 100-800 μm, preferably 100-500 μm, such as 200 μm, 300 μm, or 400 μm.

[0031] In another preferred embodiment, the mass ratio of the first polymeric material to the second biomacromolecule is 95:5-5:95, preferably 95:5 to 50:50, for example 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, or 30:70.

[0032] In another preferred embodiment, when preparing the intermediate layer, the electrospinning speed is 3000-12000 rpm, preferably 5000-10000 rpm, for example 6000 rpm.

[0033] In another preferred embodiment, the method for preparing the intermediate layer includes: (a2) The first polymer material is mixed with the second biomacromolecule to form an intermediate spinning solution; (b2) The intermediate spinning solution is spun onto the surface of a rotating mandrel that has formed an inner nanofiber layer by electrospinning.

[0034] In another preferred embodiment, in step (a2), the total concentration in the intermediate spinning solution is 5-40% w / v, preferably 10-30% w / v, for example 12% w / v, 15% w / v, 18% w / v, or 20% w / v.

[0035] In another preferred embodiment, in step (a2), the solvent in the intermediate spinning solution is selected from the group consisting of hexafluoroisopropanol, trifluoroethanol, dichloromethane, trifluoroacetic acid, or combinations thereof.

[0036] In another preferred embodiment, in step (b2), the rotational speed of the mandrel is 3000-12000 rpm, preferably 5000-12000 rpm, such as 6000 rpm, 7000 rpm, or 8000 rpm.

[0037] In another preferred embodiment, in step (b2), the parameters of the electrospinning include one or more of the following: Spinning voltage: 10-30kV, preferably 10-25 kV, preferably 15-25 kV; Receiving distance: 5-25cm, preferably 8-20cm; Injection rate: 1-5 mL / h, preferably 2-5 mL / h.

[0038] In another preferred embodiment, in step (b2), the diameter of the intermediate layer fiber and the shell thickness are controlled by adjusting the spinning solution concentration, receiving distance, injection speed and mandrel rotation speed.

[0039] In another preferred embodiment, the first biomacromolecule and the second biomacromolecule in the inner layer and the middle layer are optionally each independently cross-linked biomacromolecules.

[0040] In another preferred embodiment, the cross-linked biomacromolecules are cross-linked through one or more of chemical cross-linking, photocross-linking, and enzymatic cross-linking.

[0041] In another preferred embodiment, the cross-linked biomacromolecules are cross-linked by one or more of the following methods: genipin, glutaraldehyde vapor, enzymatic cross-linking, carbodiimide coupling, or photocross-linking.

[0042] In another preferred embodiment, both the inner and intermediate layers are grafted, fixed, or loaded with heparin.

[0043] In another preferred embodiment, the heparin is loaded onto the fiber surface by one or more of the following methods: physical adsorption, ion binding, cross-linking fixation, or chemical grafting.

[0044] In another preferred embodiment, the heparin undergoes a stable chemical grafting process by reacting its carboxyl group with an amino group in a first biomolecule, a second biomolecule, or the extracellular matrix of vascular cells via an amidation reaction.

[0045] In another preferred embodiment, the heparin loading rate is at least 30%, preferably at least 50%, and more preferably at least 70%, wherein the loading rate refers to the ratio of heparin loading sites to the total heparin loading sites.

[0046] In another preferred embodiment, the grafting, fixation, or loading of heparin is carried out in the presence of an activator. Preferably, the activator is EDC / NHS or EDC / HOBt.

[0047] In another preferred embodiment, the method of grafting, fixing, or loading heparin includes: (a3) Mix the heparin solution with a double-layered fiber tube consisting of an inner layer and an intermediate layer, such that at least one of the inner layer and the intermediate layer is grafted, fixed or loaded with heparin.

[0048] In another preferred embodiment, in step (a3), the concentration of heparin in the heparin solution is 1-10 mg / mL, preferably 2-5 mg / mL, for example 3 mg / mL.

[0049] In another preferred embodiment, in step (a3), the heparin solution contains an activator, the activator being EDC / NHS.

[0050] In another preferred embodiment, in step (a3), the concentration of EDC in the heparin solution is 5-50 mM, for example 20 mM.

[0051] In another preferred embodiment, in step (a3), the concentration of NHS in the heparin solution is 5-50 mM, for example 20 mM.

[0052] In another preferred embodiment, in step (a3), the grafting, fixing, or loading is performed at 4-40°C.

[0053] In another preferred embodiment, in step (a3), the grafting, fixing or loading time is 2-24 hours, preferably 8-12 hours.

[0054] In another preferred embodiment, step (a3) ​​further includes a post-treatment step of washing with detergent, preferably deionized water, physiological saline, or PBS, each independently.

[0055] In another preferred embodiment, the outer layer has a fiber diameter of 1-20 μm, preferably 2-15 μm, and more preferably 2-10 μm.

[0056] In another preferred embodiment, the outer layer has a thickness of 50-500 μm, preferably 100-300 μm, such as 150 μm, 200 μm, or 300 μm.

[0057] In another preferred embodiment, the method for preparing the outer layer includes: (a4) The second polymer material is made into an outer spinning solution; (b4) The outer spinning solution is spun onto the outer surface of a rotating heparin-treated double-layer fiber tube by electrospinning.

[0058] In another preferred embodiment, in step (a4), the total concentration in the outer spinning solution is 5-40% w / v, preferably 10-30% w / v, for example 12% w / v, 15% w / v, 18% w / v, or 20% w / v.

[0059] In another preferred embodiment, in step (a4), the solvent in the outer spinning solution is selected from the group consisting of hexafluoroisopropanol, trifluoroethanol, dichloromethane, trifluoroacetic acid, or combinations thereof.

[0060] In another preferred embodiment, in step (b4), a heparin-treated double-layer fiber tube is electrospun on a rotating mandrel at a speed of 100-1800 rpm, preferably 100-1000 rpm, for example 500 rpm.

[0061] In another preferred embodiment, in step (b4), the parameters of the electrospinning include one or more of the following: Spinning voltage: 10-30kV, preferably 10-25 kV, preferably 15-25 kV; Receiving distance: 5-25cm, preferably 10-20cm; Injection rate: 1-5 mL / h, preferably 1.5-5 mL / h.

[0062] In a second aspect of the present invention, a method for preparing a three-layer electrospun fiber-based artificial blood vessel as described in the first aspect of the present invention is provided, comprising the following steps: (s1) The inner spinning solution is loaded into a syringe and spun onto the surface of a rotating mandrel by electrospinning; wherein the inner spinning solution contains vascular extracellular matrix and a first biomacromolecule; (s2) The intermediate spinning solution is spun onto the surface of a rotating mandrel with an inner nanofiber layer formed by electrospinning; wherein the intermediate spinning solution contains a first polymer material and a second biomacromolecule; the rotation speed of the mandrel is 2000-15000 rpm; (s3) Mix the heparin solution with a double-layered fiber tube consisting of an inner layer and an intermediate layer, such that at least one of the inner layer and the intermediate layer is grafted, fixed or loaded with heparin. (s4) The outer spinning solution is spun onto the outer surface of a rotating heparin-treated fiber by electrospinning to obtain the three-layer electrospun fiber-based artificial blood vessel; wherein the outer spinning solution contains a second polymer material. Wherein, the first polymer material and the second polymer material are both biodegradable polymer materials, each independently selected from the following group: polycaprolactone PCL, polylactic acid-caprolactone copolymer PLCL, polyglycolic acid PGA, polydioxanone PDO, or combinations thereof; The first and second biomacromolecules are each independently selected from the group consisting of: gelatin, chitosan, oligosaccharides, hyaluronic acid, sodium alginate, collagen, silk fibroin, or combinations thereof.

[0063] In another preferred embodiment, step (s1) corresponds to step (b1). In another preferred embodiment, step (s2) corresponds to step (b2). In another preferred embodiment, step (s3) corresponds to step (b3). In another preferred embodiment, step (s4) corresponds to step (b4).

[0064] In another preferred embodiment, the inner layer spinning solution is as described in the first aspect of the present invention, and the method for preparing the inner layer spinning solution is as described in step (a1) of the first aspect of the present invention. In another preferred embodiment, the intermediate layer spinning solution is as described in the first aspect of the present invention, and the method for preparing the intermediate layer spinning solution is as described in step (a2) of the first aspect of the present invention. In another preferred embodiment, the outer layer spinning solution is as described in the first aspect of the present invention, and the method for preparing the outer layer spinning solution is as described in step (a4) of the first aspect of the present invention.

[0065] In another preferred embodiment, step (s3) further includes a step of cross-linking biomacromolecules, including one or more of chemical cross-linking, photocross-linking, and enzymatic cross-linking.

[0066] In another preferred embodiment, the cross-linked biomacromolecules include one or more of the following: genipin cross-linking, glutaraldehyde vapor cross-linking, enzymatic cross-linking, carbodiimide coupling, and photocross-linking.

[0067] In another preferred embodiment, the method is carried out at 4-40°C, preferably 4-37°C.

[0068] In a third aspect of the invention, a medical material is provided, comprising a three-layer electrospun fiber-based artificial blood vessel as described in the first aspect of the invention, and excipients or excipients acceptable in the field of materials.

[0069] In a fourth aspect of the invention, the use of a three-layer electrospun fiber-based artificial blood vessel as described in the first aspect of the invention is provided for the preparation of a medical material as an artificial blood vessel.

[0070] In another preferred embodiment, the medical material is used to treat coronary artery disease, peripheral artery disease, and vascular injury repair.

[0071] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0072] Figure 1 The images shown are (A) optical photographs and cross-sectional SEM images of small-diameter artificial blood vessels; and (B) results of periodic tensile mechanical tests.

[0073] Figure 2 The image shows a stained photograph of endothelial cell adhesion and growth on the inner surface, indicating that the inner layer can promote endothelialization.

[0074] Figure 3 It shows that smooth muscle cells grow along the intermediate layer and penetrate into the interior of the intermediate layer.

[0075] Figure 4 This shows that smooth muscle cells grow without orientation on the outer layer and grow into the interior of the outer layer.

[0076] Figure 5 SEM images before and after heparin grafting are shown, indicating that heparin grafting significantly inhibits platelet adhesion on the material surface.

[0077] Figure 6 A schematic diagram of the preparation process when PCL is selected as the polymer material is shown. Detailed Implementation

[0078] Through extensive and in-depth research, the inventors have developed a three-layer electrospun fiber-based artificial blood vessel based on the natural blood vessel structure and through precise design.

[0079] The inner layer is made by electrospinning vascular extracellular matrix and biomacromolecules, and has a nanofiber structure. It can simulate the microenvironment of natural vascular intima and extracellular matrix, and can provide a biomimetic adhesion microenvironment for endothelial cells, thus accelerating endothelialization. The intermediate layer is made of biodegradable polymer materials and biomacromolecules through high-speed rotational reception of electrospinning. It contains a large number of circumferentially oriented microfibers, which can simulate the ring arrangement characteristics of smooth muscle cells in natural vascular media. It can induce smooth muscle cells to arrange themselves circumferentially, promote the reconstruction of natural vascular media, and provide the main mechanical support. Furthermore, the inner and middle layers are modified with heparin to reduce platelet adhesion and fibrin deposition, thereby reducing the risk of early thrombosis. The outer layer is made of biodegradable polymer material through electrospinning. It has a random micron fiber structure with large pores and high porosity, which is conducive to the ingrowth of host cells and tissues and promotes the integration of artificial blood vessels with surrounding tissues.

[0080] The three-layer electrospun fiber-based artificial blood vessel obtained through precise biomimetic structural design has excellent effects in promoting vascular repair, promoting vascular tissue regeneration, and anti-thrombosis. Based on this, the present invention was completed.

[0081] the term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0082] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0083] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0084] Three-layer electrospun fiber-based small-diameter artificial blood vessel This invention provides a three-layer electrospun fiber-based small-diameter artificial blood vessel, comprising, from the inside out: 1. Inner nanofiber layer The inner layer is in direct contact with blood, and its nanoscale fibrous structure can mimic the natural vascular intima and extracellular matrix microenvironment. The vascular ECM matrix can provide bioactive signals derived from natural blood vessels, while gelatin can enhance hydrophilicity and cell adhesion.

[0085] The inner nanofiber layer is prepared by electrospinning a mixture of vascular extracellular matrix and gelatin, with a fiber diameter of 100-300 nm and a layer thickness of 10-50 μm.

[0086] Controlling the inner layer fiber diameter to 100-300 nm is beneficial for increasing the specific surface area; improving endothelial cell adhesion; promoting endothelial cell spreading and migration; accelerating the formation of a continuous endothelial layer; and reducing the risk of thrombosis caused by direct contact between exposed artificial materials and blood.

[0087] 2. Intermediate circumferentially oriented microfiber layer The middle layer serves as the primary mechanical support layer for the artificial blood vessel, with its fibers oriented circumferentially along the vessel wall. This structure mimics the circular arrangement of smooth muscle cells in the tunica media of natural blood vessels.

[0088] The functions of the intermediate layer include: providing radial compressive and burst-resistant mechanical support; guiding smooth muscle cells to circumferentially align; promoting the regeneration of medial structures; improving the mechanical fit of artificial blood vessels; and inhibiting intimal hyperplasia caused by disordered cell migration.

[0089] The intermediate layer is made by electrospinning a mixture of biodegradable polymer material and gelatin at high speed. The fibers are oriented circumferentially along the blood vessels, with a fiber diameter of 2-10 μm and a layer thickness of 200-400 μm.

[0090] 3. Heparinized inner and intermediate layers Grafting or fixing heparin onto the inner and middle layers can significantly improve blood compatibility, reduce platelet adhesion and fibrin deposition, and lower the risk of early thrombosis.

[0091] Intermediate layer heparinization can also provide continuous anticoagulation during the local degradation or remodeling of the inner layer, thereby improving the early and intermediate patency of small-diameter artificial blood vessels.

[0092] 4. Outer random microfiber layer The outer layer adopts a random microfiber structure with large pores and high porosity, which is conducive to the ingrowth of host cells and tissues and promotes the integration of artificial blood vessels with surrounding tissues.

[0093] The outer layer functions include: providing adventitia-like support; promoting the ingrowth of fibroblasts and perivascular cells; enhancing post-implantation stability; improving the overall toughness and tear resistance of the scaffold; and providing space for the formation of new microvessels.

[0094] The outer layer is made of biodegradable polymer material by electrospinning, with fibers arranged randomly, fiber diameter of 2-10 μm, and layer thickness of 100-200 μm.

[0095] The inner and middle layers are grafted, fixed, or loaded with heparin to improve the anticoagulant properties of the artificial blood vessel lumen and the region near the lumen.

[0096] Preferably, the inner diameter of the small-diameter artificial blood vessel is 2-6 mm.

[0097] The main constituent materials of each layer of the small-diameter artificial blood vessel include: 1. Vascular extracellular matrix The extracellular matrix of the blood vessels may be derived from decellularized vascular tissue, including but not limited to decellularized arterial matrix, decellularized venous matrix, decellularized vascular wall matrix or their powdered products.

[0098] The extracellular matrix of blood vessels may contain collagen, elastin, laminin, fibronectin, glycosaminoglycans and other vascular tissue-related active ingredients to mimic the natural microenvironment of the vascular intima and subendothelial matrix.

[0099] 2. Gelatin The gelatin is used to improve the hydrophilicity, cell adhesion and cross-linkability of the fiber layer, and to provide amino and other reaction sites for heparin grafting or fixation.

[0100] 3. Biodegradable polymer materials The biodegradable polymeric materials include, but are not limited to: polycaprolactone (PCL); polylactic acid-caprolactone copolymer (PLCL); polyglycolic acid (PGA); polydioxanone (PDO); or one or more combinations of the above materials.

[0101] The biodegradable polymer material is used to provide mechanical support, degradation and reconstruction capabilities, and fiber structure stability for artificial blood vessels.

[0102] 4. Heparin The heparin is used to endow artificial blood vessels with anticoagulant and antithrombotic functions, reduce platelet adhesion and coagulation reactions, and improve the compatibility of blood in the lumen.

[0103] Heparin can be loaded onto the surface of inner and middle layer fibers through physical adsorption, ion binding, cross-linking fixation, or chemical grafting. Preferably, an EDC / NHS activation system is used to amidate the carboxyl groups of heparin with the amino groups in gelatin or the extracellular matrix of vascular cells, thereby achieving stable grafting of heparin.

[0104] Preparation method The method for preparing the three-layer electrospun fiber-based small-diameter artificial blood vessel of the present invention includes the following steps: Step 1: Preparation of inner layer spinning solution Take the extracellular matrix of vascular cells and gelatin, mix them according to a predetermined mass ratio, and dissolve them in trifluoroethanol or hexafluoroisopropanol to prepare an inner layer spinning solution with a mass-volume concentration of 8-15% w / v.

[0105] The mass ratio of vascular extracellular matrix to gelatin can be 1:9-9:1, preferably 3:7-7:3.

[0106] Step 2: Electrospinning to prepare the inner nanofiber layer The inner spinning solution is loaded into a syringe and spun onto the surface of a rotating mandrel by electrospinning.

[0107] The inner diameter of the mandrel corresponds to the inner diameter of the artificial blood vessel, and the mandrel diameter is 2-6 mm. The mandrel rotation speed is 100-1000 rpm, used to obtain a random or low-orientation nanofiber inner layer.

[0108] By adjusting the voltage, receiving distance, flow rate, solution concentration, and environmental parameters, the inner fiber diameter is controlled to be 100-300 nm, and the inner layer thickness is controlled to be 10-50 μm.

[0109] The preferred spinning parameters are: spinning voltage: 8-25 kV; receiving distance: 8-20 cm; injection speed: 0.2-2.0 mL / h; temperature: 15-30℃; relative humidity: 20-60%.

[0110] Step 3: Preparation of intermediate layer spinning solution A biodegradable polymer material was mixed with gelatin and dissolved in hexafluoroisopropanol to prepare an intermediate spinning solution.

[0111] The mass ratio of the biodegradable polymer material to gelatin can be 95:5-50:50, preferably 90:10-60:40. The concentration of the intermediate layer spinning solution can be 10-30% w / v, preferably 12-25% w / v.

[0112] Step 4: Electrospinning to prepare a centrally circumferentially oriented microfiber layer The intermediate spinning solution is electrospun onto the surface of a mandrel that has formed an inner nanofiber layer. The mandrel is then rotated at high speed to receive the fibers, with a rotation speed of 4000-8000 rpm, so that the fibers are oriented and aligned along the circumferential direction of the blood vessels.

[0113] By adjusting the concentration of the spinning solution, the receiving distance, the injection speed, and the mandrel rotation speed, the diameter of the intermediate layer fiber is controlled to be 2-10 μm, and the layer thickness is controlled to be 200-400 μm.

[0114] This circumferentially oriented microfiber layer is used to mimic the circumferential arrangement of smooth muscle cells in the tunica media of natural blood vessels and guides smooth muscle cells to adhere, migrate, and regenerate circumferentially.

[0115] Step 5: Heparin grafting or fixation treatment The composite tubular fiber membrane, consisting of an inner layer and an intermediate layer, is immersed in a heparin solution, which fixes the heparin in the fiber structure of the inner and intermediate layers.

[0116] The concentration of the heparin solution is 2-5 mg / mL.

[0117] Preferably, EDC and NHS are added to the heparin solution to activate the heparin carboxyl group and react with the amino groups in gelatin and vascular extracellular matrix, thereby achieving heparin chemical grafting. The concentration of EDC can be 5-50 mM, the concentration of NHS can be 5-50 mM, the reaction time is 2-24 h, and the reaction temperature is 4-37℃.

[0118] After heparin fixation, the product can be thoroughly washed with deionized water, PBS, or physiological saline to remove unreacted heparin and residual cross-linking agents.

[0119] In another embodiment, the gelatin-containing fiber layer may be cross-linked using genipin, glutaraldehyde vapor, enzymatic cross-linking, carbodiimide coupling, or photocross-linking, and heparin fixation may be performed simultaneously or subsequently.

[0120] Step Six: Preparation of Outer Spinning Solution A biodegradable polymer material was dissolved in hexafluoroisopropanol to prepare an outer spinning solution.

[0121] The outer biodegradable polymer material can be the same as or different from the material used in the middle layer, and is preferably one or more of PCL, PLCL, PGA, and PDO.

[0122] The concentration of the outer spinning solution can be 10-30% w / v, preferably 12-25% w / v.

[0123] Step 7: Electrospinning to prepare the outer random microfiber layer The outer spinning solution is electrospun onto the outer surface of the heparin-treated intermediate layer, and the mandrel is rotated at 100-1000 rpm to receive the outer layer of random microfibers.

[0124] By controlling the spinning parameters, the outer fiber diameter is made to be 2-10 μm and the outer layer thickness is 100-200 μm.

[0125] The outer random microfiber layer has high porosity and a three-dimensional interconnected structure, which is conducive to the ingrowth of fibroblasts, smooth muscle cells and surrounding tissue cells, and improves the integration ability of artificial blood vessels with host tissues.

[0126] Step 8: Post-processing After spinning, the tubular support is removed from the mandrel and then dried, residual solvent is removed, washed, sterilized, and stored.

[0127] The drying method can be vacuum drying, freeze drying, or room temperature ventilation drying.

[0128] The sterilization method may be ethylene oxide sterilization, gamma ray sterilization, electron beam sterilization, low-temperature plasma sterilization, or ultraviolet sterilization.

[0129] Preferably, the finished artificial blood vessels are subjected to residual solvent testing, mechanical property testing, suture strength testing, burst pressure testing, water leakage testing, heparin release testing, and blood compatibility testing.

[0130] Compared with the prior art, the present invention has at least the following advantages: 1. Bionic layered structure By using a three-layer structure of inner, middle and outer layers to simulate the intima, media and adventitia of blood vessels respectively, it is beneficial to achieve layered regeneration of vascular tissue.

[0131] 2. Excellent endothelialization-promoting properties The inner layer is composed of vascular ECMs matrix and gelatin, and has a 100-300 nm nanofiber structure, which can provide a biomimetic adhesion microenvironment for endothelial cells and accelerate endothelialization.

[0132] 3. Enhanced antithrombotic properties Grafting or fixing heparin into the inner and middle layers can reduce platelet adhesion and coagulation reactions, and improve the blood compatibility of small-diameter artificial blood vessels.

[0133] 4. Guides the circumferential regeneration of smooth muscle cells The intermediate layer uses a high-speed rotating receiver at 4000-8000 rpm to prepare circumferentially oriented microfibers, which can induce smooth muscle cells to align circumferentially and promote the reconstruction of the vascular media that resembles natural blood vessels.

[0134] 5. Balancing mechanical properties and organizational integration The middle layer provides the main mechanical support, while the outer random microfiber layer allows host cells and tissues to grow in, improving implantation stability and tissue integration.

[0135] 6. The material is biodegradable and reconfigurable. Biodegradable polymers such as PCL, PLCL, PGA, and PDO can be gradually degraded in vivo, providing conditions for the replacement of artificial materials with new blood vessel tissue.

[0136] 7. Suitable for small-diameter blood vessel replacement The artificial blood vessel has an inner diameter of 2-6 mm, and is particularly suitable for small-diameter artery replacement, peripheral vascular repair, coronary artery bypass grafting material research, and tissue-engineered vascular construction.

[0137] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0138] Example 1: Three-layer small-diameter artificial blood vessel with PCL / gelatin middle layer Synthesis diagram as shown Figure 6 As shown.

[0139] Decellularized vascular ECMs powder and gelatin were mixed at a mass ratio of 5:5 and dissolved in hexafluoroisopropanol to prepare an inner layer spinning solution with a total concentration of 10% w / v. This spinning solution was electrospun onto the surface of a rotating mandrel with a diameter of 4 mm at a mandrel speed of 500 rpm, a spinning voltage of 15 kV, a receiving distance of 12 cm, and a feeding rate of 0.8 mL / h to prepare a random nanofiber inner layer. The resulting inner layer fibers had a diameter of approximately 100-300 nm and a thickness of approximately 30 μm.

[0140] PCL and gelatin were mixed at a mass ratio of 80:20 and dissolved in hexafluoroisopropanol to prepare an intermediate layer spinning solution with a total concentration of 18% w / v. This solution was then spun onto the inner layer surface at a mandrel speed of 6000 rpm to form a circumferentially oriented microfiber intermediate layer. The resulting intermediate layer fibers had a diameter of approximately 2-10 μm and a thickness of approximately 300 μm.

[0141] The inner and middle layer composite tubes were immersed in a solution containing 3 mg / mL heparin, 20 mM EDC, and 20 mM NHS, and reacted at room temperature for 12 h to graft heparin onto the fiber layer containing gelatin and ECMs. After the reaction, the tubes were thoroughly washed with PBS and deionized water.

[0142] PCL was dissolved in hexafluoroisopropanol to prepare an outer layer spinning solution with a concentration of 18% w / v. The outer layer spinning solution was spun onto the outer surface of the intermediate layer at a mandrel rotation speed of 500 rpm to form a random microfiber outer layer. The resulting outer layer fibers had a diameter of 2-10 μm and a thickness of approximately 150 μm.

[0143] Finally, the tubular stent was vacuum dried to remove residual solvent, resulting in a three-layer electrospun fiber-based small-diameter artificial blood vessel with an inner diameter of 4 mm.

[0144] Example 2: Three-layer small-diameter artificial blood vessel with PLCL / gelatin middle layer The process was essentially the same as in Example 1, except that the biodegradable polymer material used in the intermediate layer was PLCL, with a PLCL to gelatin mass ratio of 70:30, and the intermediate layer spinning solution concentration was 20% w / v. The mandrel rotation speed was 7000 rpm, and the resulting intermediate layer fibers exhibited a distinct circumferential orientation.

[0145] The outer layer is made of random microfibers fabricated using PLCL. This artificial blood vessel exhibits good elasticity and compliance, making it suitable for small-diameter vascular stents that require high flexibility and elasticity matching.

[0146] Example 3 Based on the method of Example 1, by changing the diameter of the mandrel, a small-diameter artificial blood vessel with an inner diameter of 6 mm was obtained using electrospun fiber-based material. The image was captured and recorded using a stereomicroscope. Figure 1 As shown in the left figure of Figure A, the artificial blood vessel is a hollow tubular structure.

[0147] The sample was then immersed in liquid nitrogen for 3-5 minutes to freeze, and then removed and fractured. A flat cross-section sample was sputter-coated with gold and characterized by SEM. The results are as follows: Figure 1 As shown in the right figure of A, a clear three-layer structure can be seen. The inner layer is 10-50 μm thick, with randomly oriented fibers and is relatively dense. The middle layer is 200-400 μm thick and contains a large number of circumferentially oriented fibers with diameters in the micrometer range. The outer layer is 100-200 μm thick, with fiber thickness also in the micrometer range, and the fibers are mainly randomly arranged.

[0148] Next, a 10mm long artificial blood vessel sample was taken, and the upper and lower clamps were passed through the lumen and fixed to the upper and lower clamps of the strength tester. The sample was stretched circumferentially at a stretching rate of 5mm / min and repeated 100 times. It can be seen that the artificial blood vessel sample has excellent circulatory performance.

[0149] Example 4 Fiber membranes were prepared using the set inner layer spinning parameters, cut into 10 mm diameter discs, and placed in 24-well plates, followed by sterilization. A 1 × 10⁻⁶ stencil was then applied to the surface of the fiber membrane. 4 Endothelial cells were seeded in each well and cultured for 3 days. The samples were then fixed in 4% paraformaldehyde solution at 4 °C for 24 h. The fixed cells were then co-stained with Oregon Green 488 Phalloidin and DAPI and observed and characterized using a fluorescence microscope.

[0150] The results are as follows Figure 2 As shown, a large number of endothelial cells have adhered and grown on the inner surface.

[0151] Subsequently, a fiber membrane was prepared using the set intermediate layer spinning parameters. Smooth muscle cells were then seeded and stained using the same method described above. The results are as follows: Figure 3 As shown, a large number of smooth muscle cells adhered and grew in the intermediate layer. The smooth muscle cells also grew in a circumferential orientation, and some cells grew into the interior of the intermediate layer.

[0152] Then, a fiber membrane was prepared using the set outer spinning parameters. Smooth muscle cells were then seeded and stained using the same method described above. The results are as follows: Figure 4 As shown, a large number of smooth muscle cells adhered and grew in the outer layer. The smooth muscle cells also grew randomly, and some cells grew into the interior of the outer layer.

[0153] Example 5: Evaluation of platelet adhesion properties Samples before and after grafting were placed in 24-well cell culture plates and co-incubated with 500 μL of platelet-rich plasma (PRP). The PRP was prepared by centrifuging porcine whole blood at 1500 rpm for 15 min. After incubation at 37 °C for 2 h, the samples were washed with phosphate-buffered saline (PBS) to remove unattached free platelets. The samples were then fixed in 2.5% glutaraldehyde solution at 4 °C for 48 h. After fixation, the samples were dehydrated stepwise using a gradient of 50%, 70%, 80%, 90%, 95%, and 100% ethanol solutions, dried at room temperature, sputter-coated with gold, and characterized by scanning electron microscopy (SEM).

[0154] The results are as follows Figure 5 As shown, the left image is the sample without heparin grafting, and it can be seen that a large number of platelets adhere to the surface, while the right image is the sample with heparin grafting, and it can be seen that heparin grafting can inhibit platelet adhesion.

[0155] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A three-layer electrospun fiber-based artificial blood vessel, characterized in that, The artificial blood vessel is a hollow cylinder, comprising an inner nanofiber layer, a middle circumferentially oriented microfiber layer and an outer random microfiber layer arranged sequentially from the inside to the outside, wherein at least one of the inner and middle layers is grafted, fixed or loaded with heparin. The inner nanofiber layer is prepared by electrospinning a mixture of vascular extracellular matrix and first biological macromolecule; the fiber diameter of the inner nanofiber layer is 50-800 nm. The intermediate layer is made by electrospinning a mixture of a first polymer material and a second biomacromolecule. The fibers are oriented along the circumferential direction of the blood vessels. The mandrel of the electrospinning is rotated at high speed to receive the fibers, with a rotation speed of 2000-15000 rpm. The outer layer is made of a second polymer material by electrospinning, and the fibers are arranged randomly. Wherein, the first polymer material and the second polymer material are both biodegradable polymer materials, each independently selected from the following group: polycaprolactone PCL, polylactic acid-caprolactone copolymer PLCL, polyglycolic acid PGA, polydioxanone PDO, or combinations thereof; The first and second biomacromolecules are each independently selected from the group consisting of: gelatin, chitosan, oligosaccharides, hyaluronic acid, sodium alginate, collagen, silk fibroin, or combinations thereof.

2. The three-layer electrospun fiber-based artificial blood vessel as described in claim 1, characterized in that, The inner diameter of the artificial blood vessel is 1-20 mm, preferably 1-10 mm.

3. The three-layer electrospun fiber-based artificial blood vessel as described in claim 1, characterized in that, Both the first polymer material and the second polymer material are PCL, or both are PLCL; Both the first and second biomacromolecules are gelatin.

4. The three-layer electrospun fiber-based artificial blood vessel as described in claim 1, characterized in that, The inner nanofiber layer has a fiber diameter of 50-500 nm, preferably 80-400 nm, and more preferably 100-300 nm; The thickness of the inner nanofiber layer is 5-80 μm, preferably 10-50 μm.

5. The three-layer electrospun fiber-based artificial blood vessel as described in claim 1, characterized in that, The fiber diameter of the intermediate layer is 1-20 μm, preferably 2-15 μm, and more preferably 2-10 μm; The thickness of the intermediate layer is 100-800μm, preferably 100-500μm.

6. The three-layer electrospun fiber-based artificial blood vessel as described in claim 1, characterized in that, The outer layer has a fiber diameter of 1-20 μm, preferably 2-15 μm, and more preferably 2-10 μm; The outer layer has a thickness of 50-500 μm, preferably 100-300 μm.

7. A method for preparing a three-layer electrospun fiber-based artificial blood vessel as described in any one of claims 1-6, characterized in that, Includes the following steps: (s1) The inner spinning solution is loaded into a syringe and spun onto the surface of a rotating mandrel by electrospinning; wherein the inner spinning solution contains vascular extracellular matrix and a first biomacromolecule; (s2) The intermediate spinning solution is spun onto the surface of a rotating mandrel with an inner nanofiber layer formed by electrospinning; wherein the intermediate spinning solution contains a first polymer material and a second biomacromolecule; the rotation speed of the mandrel is 2000-15000 rpm; (s3) Mix the heparin solution with a double-layered fiber tube consisting of an inner layer and an intermediate layer, such that at least one of the inner layer and the intermediate layer is grafted, fixed or loaded with heparin. (s4) The outer spinning solution is spun onto the outer surface of a rotating heparin-treated fiber by electrospinning to obtain the three-layer electrospun fiber-based artificial blood vessel; wherein the outer spinning solution contains a second polymer material. Wherein, the first polymer material and the second polymer material are both biodegradable polymer materials, each independently selected from the following group: polycaprolactone PCL, polylactic acid-caprolactone copolymer PLCL, polyglycolic acid PGA, polydioxanone PDO, or combinations thereof; The first and second biomacromolecules are each independently selected from the group consisting of: gelatin, chitosan, oligosaccharides, hyaluronic acid, sodium alginate, collagen, silk fibroin, or combinations thereof.

8. The preparation method according to claim 7, characterized in that, The method has one or more features selected from the group consisting of: (a) In step (s1), the rotational speed of the mandrel is 100-1800 rpm, preferably 100-1000 rpm; (b) In step (s2), the rotational speed of the mandrel is 3000-12000 rpm, preferably 5000-12000 rpm; (c) In step (s4), the heparin-treated double-layer fiber tube is electrospun on a rotating mandrel at a speed of 100-1800 rpm, preferably 100-1000 rpm.

9. A medical material comprising a three-layer electrospun fiber-based artificial blood vessel as described in any one of claims 1-6, and excipients or excipients acceptable in the field of materials.

10. The use of the three-layer electrospun fiber-based artificial blood vessel according to any one of claims 1-6, characterized in that, Used to prepare medical materials for use as artificial blood vessels.

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