Double-network self-healing acellular bioartificial coronary bypass graft vessel and method of making same

Abstract

The application discloses a kind of double-network self-healing acellular biological type artificial coronary bypass blood vessels, belong to the field of biomedical materials and tissue engineering technology, including each other interpenetrating first network and second network;Wherein the first network is natural animal source small-bore acellular blood vessel matrix;Second network is at least one dynamic reversible bond in hydrazone bond, schiff base bond, hydrogen bond, metal coordination bond, ionic bond, disulfide bond and borate ester bond Crosslinking polymer network.The application further discloses the preparation method of the artificial coronary bypass blood vessel.The application realizes the cooperation of mechanical compliance matching and self-healing function by double-network interpenetrating structure, and the inner surface of lumen can also be provided with functional modification layer to accelerate in-situ endothelialization and inhibit thrombosis.The artificial coronary bypass blood vessel has good mechanical compliance, rapid self-healing ability, excellent antithrombotic and pro-endothelialization performance, can significantly improve the long-term patency rate of small-bore artificial blood vessel, and is suitable for coronary artery bypass grafting.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and tissue engineering technology, specifically relating to a dual-network self-healing decellularized biological small-diameter artificial coronary artery bypass graft and its preparation method. Background Technology

[0002] Coronary artery bypass grafting (CABG) is the primary treatment for severe coronary artery obstruction, but it has long faced challenges in clinical practice, including limited access to grafts and low long-term patency rates. When a patient's own grafts are unavailable, artificial grafts become an alternative.

[0003] Existing synthetic materials (such as ePTFE and PET) suffer from severe compliance mismatch issues in the application of small-diameter artificial blood vessels (<6 mm). Under cardiac pulsation, the elastic difference between rigid artificial blood vessels and flexible natural blood vessels causes shear stress concentration at the anastomosis. This physical stimulation can induce intimal hyperplasia and acute thrombosis, ultimately leading to a significant decrease in long-term patency. Although synthetic materials have achieved some success in large-diameter vascular reconstruction, the more stringent compliance requirements for small-diameter vessels, and the greater susceptibility of smaller diameter vessels to complete occlusion due to intimal hyperplasia, have prevented them from meeting the clinical application requirements of small-diameter vessels such as coronary arteries.

[0004] Ideal coronary artery bypass grafts should possess the following core characteristics: rapid endothelialization capability; allowing the body's own endothelial cells to grow on the inner wall of the artificial blood vessel, forming a natural protective membrane to prevent thrombosis. Mechanical compliance matching; the elasticity of the artificial blood vessel should match that of the natural blood vessel to avoid shear stress concentration at the anastomosis site due to elasticity differences, thereby inhibiting intimal hyperplasia. Self-healing capability; able to automatically repair micro-damage caused by sutures or postoperative fatigue cracks, extending the lifespan of the vessel. Long-term stability; maintaining structural and performance stability under long-term blood flow impact, meeting the clinical needs of patients with a high risk of reoperation.

[0005] In recent years, researchers have attempted to introduce decellularized matrices or natural biomaterials to improve the biocompatibility of artificial blood vessels. For example: CN118286516A discloses an arc-shaped artificial blood vessel that enhances anastomosis adaptability through gradual wall thickness and curved structure design. However, it still uses a mixture of biodegradable polymer and decellularized matrix, which does not solve the problem of material-level compliance matching and does not have self-healing function.

[0006] CN119792647A discloses a pure collagen electrospun artificial blood vessel, which achieves size matching with small coronary arteries (approximately 1.6 mm in diameter) by regulating the fiber structure, and achieves a patency rate of over 82% within one year. However, this approach has the following shortcomings: (1) The mechanical strength of pure collagen raw materials is limited, and there is a risk of structural failure under long-term blood flow impact; (2) It lacks self-healing ability and cannot repair micro-damage caused by sutures or postoperative fatigue cracks; (3) The compliance matching depends entirely on the fiber structure design, and the mechanical compliance of the material itself still differs from that of natural blood vessels; (4) Endothelialization depends on the natural ingrowth of host cells and lacks an active induction mechanism.

[0007] In summary, while the aforementioned existing technologies have improved certain specific performance aspects (e.g., CN118286516A optimizes anastomotic adaptability through structural design, and CN119792647A achieves size matching through a pure collagen system), none of them simultaneously achieve the three core performance characteristics of mechanical compliance matching, rapid in-situ endothelialization induction, and self-healing function. Existing technologies have not yet solved the core technical challenge of multi-performance synergy, and there are currently no ideal products for clinical application.

[0008] Therefore, developing an artificial coronary artery bypass graft that combines mechanical compliance matching, rapid in situ endothelialization induction, and self-healing functions remains a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0009] In response to one or more of the above-mentioned defects or improvement needs of the prior art, the present invention provides a dual-network self-healing decellularized biological artificial coronary artery bypass graft and its preparation method. The bypass graft achieves mechanical compliance matching and self-healing function through the interpenetration of natural decellularized vascular matrix and dynamic cross-linked network, which can significantly improve the long-term patency rate of small-diameter artificial blood vessels and is suitable for coronary artery bypass grafting.

[0010] To achieve the above objectives, according to one aspect of the present invention, a dual-network self-healing decellularized biological artificial coronary artery graft is provided, comprising a first network and a second network intercalated within the first network; The first network consists of naturally derived small-diameter decellularized blood vessels that retain the extracellular matrix, serving as a mechanical framework support layer; The second network is a dynamic cross-linked network with self-healing function, containing at least one dynamic reversible bond selected from hydrazone bonds, Schiff base bonds, hydrogen bonds, metal coordination bonds, ionic bonds, disulfide bonds, and borate ester bonds.

[0011] As a further improvement of the present invention, the naturally derived small-diameter decellularized blood vessels are derived from the coronary arteries, carotid arteries, or ureters of pigs or sheep.

[0012] As a further improvement of the present invention The intravascular diameter of the first network is 0.8~5 mm; and / or, The vessel wall thickness of the first network is 200~800μm; and / or, The collagen content of the blood vessels in the first network is 40% to 80%.

[0013] As a further improvement of the present invention, the second network includes hydrogen bonds; The hydrogen bonds are formed by the hydrogen bond interactions between UPy units and between UPy units and polyether segments after the UPy functionalized natural polymer and polyether segments are bridged by diisocyanate. The UPy-functionalized natural polymer is selected from one or more of UPy-functionalized gelatin, UPy-functionalized chitosan, or UPy-functionalized hyaluronic acid; the polyether segment is one or more of polytetramethylene ether glycol and polyethylene glycol.

[0014] As a further improvement of the present invention, the inner surface of the lumen of the artificial coronary artery graft is further provided with a functionalized modification layer, the functionalized modification layer comprising endothelial progenitor cell capturing peptides and nitric oxide sustained-release units covalently grafted onto the inner surface of the lumen of the artificial coronary artery graft.

[0015] According to another aspect of the present invention, a method for preparing a dual-network self-healing decellularized biological artificial coronary artery graft is provided, comprising the following steps: (1) Decellularization: Decellularization is performed on blood vessels of natural origin to remove cellular components while retaining the fibrous network structure of the extracellular matrix, thereby obtaining the first network framework; (2) Preparation of the second network precursor solution: Prepare a solution containing a polymer or monomer that can form dynamic reversible bonds as the second network precursor solution; (3) Interpenetrating composite: The first network skeleton is placed in the second network precursor liquid, so that the second network precursor liquid penetrates into the fiber pores of the first network skeleton; (4) Induced crosslinking: The second network precursor liquid is induced to undergo a dynamic crosslinking reaction to form dynamic reversible bonds, thereby forming a second network in situ within the pores of the first network skeleton.

[0016] As a further improvement of the present invention, in step (1), the decellularization process includes: treating the blood vessels of natural origin with a surfactant to remove cellular components, then treating them with a DNA enzyme to remove residual nucleic acids, and finally rinsing them with a buffer solution; wherein the surfactant is selected from one or more of SDS and Triton X-100.

[0017] As a further improvement of the present invention, in step (3), vacuum infusion is used to allow the second network precursor liquid to penetrate into the fiber pores of the first network skeleton. The vacuum conditions are -0.05MPa to -0.095MPa, and the processing time is 10 to 60 minutes.

[0018] As a further improvement of the present invention, in step (4), the induced crosslinking is carried out in situ in the second network precursor solution when the first network skeleton is placed in the second network precursor solution, or after the permeated first network skeleton is removed from the second network precursor solution, depending on the type of the selected dynamic reversible bond.

[0019] As a further improvement of the present invention, it also includes a step of endothelialization modification of the artificial coronary graft: Inject a functionalized solution containing S-nitrosothiol and endothelial progenitor cell capture peptide into the inner wall of the artificial coronary artery graft prepared in step (4), so that the S-nitrosothiol and endothelial progenitor cell capture peptide are fixed on the inner surface of the lumen by covalent bonding; the endothelial progenitor cell capture peptide is selected from one or more of REDV, TPS, and YIGSR.

[0020] In summary, the technical solutions conceived by this invention have the following beneficial effects compared with the prior art: (1) This invention successfully simulates the stress-strain characteristics of natural blood vessels by using the microscopic interpenetrating structure of the first network (natural decellularized vascular matrix) and the second network (dynamic polymer cross-linked network). The first network provides mechanical support and biocompatibility, while the second network provides self-healing ability and compliance regulation. The two work together to effectively solve the problem of shear stress concentration caused by elasticity differences at the anastomosis site, and significantly reduce the risk of intimal hyperplasia.

[0021] (2) The dynamic reversible bonds (selected from hydrazone bonds, Schiff base bonds, hydrogen bonds, disulfide bonds, etc.) introduced in the second network of this invention can spontaneously heal at the broken points of the graft vessels under physiological conditions through molecular chain diffusion and recombination when the graft vessels are mechanically damaged during suturing or postoperatively, without the need for external intervention. This function can significantly reduce the risk of bleeding from suture needle holes, promptly repair microcracks caused by long-term pulsating fatigue, extend the service life of graft vessels, and reduce the need for secondary surgical intervention.

[0022] (3) This invention covalently grafts endothelial progenitor cell-capturing peptides and nitric oxide donor molecules onto the luminal surface. The capture peptides specifically capture circulating endothelial progenitor cells, accelerating the in situ endothelialization process; the nitric oxide donor continuously releases nitric oxide, inhibiting platelet adhesion and smooth muscle proliferation. The two work synergistically to construct an active interface for active capture and antithrombosis on the luminal surface, forming a stable functional endothelial layer and significantly improving the long-term patency rate of bypass grafts.

[0023] (4) The first network of the present invention is processed by decellularization process, which completely removes the cellular components that may cause immune rejection, while completely preserving the three-dimensional ultrastructure and bioactive components of the natural extracellular matrix, providing a natural bioactive interface for rapid in situ endothelialization.

[0024] (5) The raw materials for the preparation method of the present invention are widely available, including natural blood vessels such as coronary arteries, carotid arteries, or ureters from pigs and sheep. The cost is controllable and the raw materials are easy to obtain. The preparation process is standardized, simple to operate, and suitable for industrial production. By adjusting the composition and cross-linking conditions of the second network precursor solution, bridge vessels with different performance specifications can be prepared to meet diverse clinical needs. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of a dual-network self-healing decellularized biological artificial coronary artery graft according to an embodiment of the present invention; Figure 2 This is a diagram illustrating the self-healing effect of artificial coronary artery graft puncture according to an embodiment of the present invention. Figure 3 The results of in vitro endothelial cell experiments for endothelialization functionalization are from embodiments of the present invention. Figure 4 This is an animal experimental surgery for the dual-network self-healing decellularized biological artificial coronary artery graft of the present invention. Figure 5 The DSA patency rate of the dual-network self-healing decellularized biological artificial coronary graft in animal experiments according to embodiments of the present invention. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0027] Please see Figure 1This invention provides a dual-network self-healing decellularized bio-artificial coronary artery graft, comprising a first network and a second network. The first network consists of naturally derived small-diameter decellularized blood vessels retaining extracellular matrix, serving as a mechanical framework support layer. The second network is a dynamically cross-linked network with self-healing function, containing at least one dynamically reversible bond selected from hydrazone bonds, Schiff base bonds, hydrogen bonds, metal coordination bonds, ionic bonds, disulfide bonds, and borate ester bonds. The second network interpenetrates within the first network at the microscale, forming an interpenetrating network structure. The first network provides mechanical support and a biocompatibility basis, while the second network endows the graft with compliance matching and self-healing function. The synergistic effect of the two networks makes the graft approximate natural coronary arteries in both mechanical properties and biological function.

[0028] In a preferred embodiment, the naturally derived decellularized blood vessels are derived from the coronary arteries, carotid arteries, or ureters of pigs or sheep. These animal blood vessels are widely available and cost-effective, and their mechanical properties are highly similar to those of human blood vessels. After decellularization, they retain ECM active components rich in RGD sequences, which can actively induce endothelial cell adhesion and proliferation, providing a natural bioactive interface for rapid in-situ endothelialization. The ureter, in particular, has a thick wall and high collagen content (60%–80%), with a burst pressure exceeding 3000 mmHg, making it suitable for high-mechanical-strength applications.

[0029] In a preferred embodiment, the inner diameter of the first network (decellularized vascular matrix) is 0.8-5 mm, the wall thickness is 200-800 μm, and the collagen content is 40%-80%.

[0030] Among them, an inner diameter of 0.8–5 mm can precisely meet the clinical needs of coronary artery bypass surgery. Coronary arteries, carotid arteries, and ureters from pigs and sheep can all obtain vascular matrix within this diameter range through decellularization, without the need for complex sizing modifications. A wall thickness of 200–800 μm balances mechanical strength and compliance, matching natural coronary arteries. A collagen content of 40%–80% achieves a dynamic balance between mechanical support and tissue remodeling.

[0031] In this invention, the dynamically reversible bonds can be formed in ways known in the art: hydrazone bonds and Schiff base bonds are formed through the condensation reaction of aldehyde groups with acylhydrazides or amino groups; disulfide bonds are formed through the oxidative coupling reaction of thiol groups; borate ester bonds are formed through the esterification reaction of boric acid with vicinal diols; metal coordination bonds are formed through the coordination of polymers containing coordinating groups with metal ions; ionic bonds are formed through the electrostatic interaction between positively charged and negatively charged polymer chain segments; and hydrogen bonds are formed through the self-assembly of hydrogen bond donors and acceptors on the polymer chain. All of the above formation methods are conventional techniques in the art, and those skilled in the art can choose appropriate methods to construct the second network as needed.

[0032] In a preferred embodiment, the dynamic reversible bonds contain hydrogen bonds. The second network is constructed by bridging UPy-functionalized natural polymers and polyether segments with diisocyanate. UPy units on the UPy-functionalized natural polymers self-assemble through quadruple hydrogen bonding to form hydrogen-linked crosslinks. Simultaneously, UPy units interact with hydroxyl groups on the polyether segments through hydrogen bonding to form auxiliary crosslinks. Together, they constitute a multi-layered hydrogen-bonded network, endowing the material with excellent self-healing ability, mechanical toughness, and fatigue resistance. This system, with a natural polymer framework, exhibits excellent biocompatibility and a simple preparation process.

[0033] More preferably, the UPy-functionalized natural polymer is selected from one or more of UPy-functionalized gelatin, UPy-functionalized chitosan, or UPy-functionalized hyaluronic acid; the polyether segment is selected from one or more of polytetramethylene ether glycol and polyethylene glycol.

[0034] More preferably, the second network comprises a synergistic combination of hydrogen bonds and hydrazone bonds, or a synergistic combination of hydrogen bonds and disulfide bonds.

[0035] When the second network includes hydrogen bonds and hydrazone bonds, the hydrogen bonds are formed by the self-assembly of UPy units through quadruple hydrogen bonds between UPy units, after UPy-functionalized natural polymers and polyether segments are bridged by diisocyanates. The hydrazone bonds are formed by the condensation reaction between aldehyde-containing components (such as oxidized dextran and aldehyde-modified hyaluronic acid) and hydrazide-containing components (such as natural polymers modified with adipic acid dihydrazide). Hydrogen bonds provide rapid self-healing capabilities, while hydrazone bonds, as dynamic covalent bonds, provide additional mechanical stability. Together, they construct a hierarchical energy dissipation network.

[0036] When the second network contains both hydrogen bonds and disulfide bonds, the hydrogen bonds are formed in the same way as above; the disulfide bonds are formed by oxidative coupling reactions of thiol-containing components (such as thiolized gelatin) in the presence of oxygen. The synergistic effect of hydrogen bonds and disulfide bonds endows the material with both rapid self-healing ability and high mechanical strength.

[0037] In a preferred embodiment, the inner surface of the artificial coronary artery graft is further provided with a functionalized modification layer, which includes endothelial progenitor cell capturing peptides and nitric oxide sustained-release units covalently grafted onto the inner surface of the artificial coronary artery graft.

[0038] This invention further incorporates a functionalized modification layer on the inner surface of the vessel lumen. Through an EDC / NHS activation reaction, endothelial progenitor cell capturing peptides (such as the REDV sequence) and nitric oxide donor molecules (such as S-nitrosothiol or diethylenetriamine / nitric oxide adduct) are covalently grafted onto the active groups of the decellularized vascular matrix and the second network. The capturing peptides specifically capture endothelial progenitor cells in circulating blood, accelerating their adhesion and endothelial differentiation on the vessel lumen surface. The nitric oxide donor molecules continuously release nitric oxide, inhibiting platelet adhesion and smooth muscle proliferation. Synergistically, the capturing peptides initiate rapid endothelialization, while nitric oxide provides an antithrombotic microenvironment, forming a stable and functionalized endothelial layer on the vessel lumen surface, thereby improving the long-term patency of bypass grafts.

[0039] Furthermore, this embodiment of the invention also provides a method for preparing a dual-network self-healing decellularized biological artificial coronary artery graft, comprising the following steps: (1) Decellularization: Natural animal-derived blood vessels are decellularized to remove cellular components while retaining the fibrous network structure of the extracellular matrix, thus obtaining the first network framework; Preferably, in step (1), the first network is made from animal-derived blood vessels (such as the coronary artery, carotid artery or ureter of pigs or sheep) through a decellularization process to remove immunogenic cellular components and retain the extracellular matrix (ECM).

[0040] More preferably, the decellularization process includes: treating the naturally derived blood vessels with a surfactant to remove cellular components, followed by treatment with a DNase to remove residual nucleic acids, and finally rinsing with a buffer solution; wherein the surfactant is selected from one or more of SDS and Triton X-100.

[0041] Specifically, it includes the following steps: Naturally derived blood vessels were placed in a solution containing surfactant and shaken at 35-40°C for 12-24 hours. Then add DNase and continue to treat with shaking at 35~40℃ for 2~6 hours; Finally, rinse with PBS buffer to remove any residual reagents; Preferably, the surfactant is selected from one or more of sodium dodecyl sulfate (SDS) and Triton X-100, with the concentration of SDS being 0.1~0.25 wt%, the concentration of Triton X-100 being 0.5~1.0 vol%, and the concentration of DNase being 40~80 U / mL.

[0042] (2) Preparation of precursor solution: Prepare a solution containing a polymer or monomer that can form dynamic reversible bonds as a second network precursor solution; Those skilled in the art will understand that the preparation method of the second network precursor depends on the type of dynamic reversible bond selected. For example, when the dynamic reversible bond is a hydrogen bond, it can be prepared by bridging UPy functionalized natural polymers and polyether segments with diisocyanate; when the dynamic reversible bond is a hydrazone bond, it can be prepared by mixing aldehyde-containing components and hydrazide-containing components under suitable conditions; when the dynamic reversible bond is a disulfide bond, it can be prepared by oxidative coupling using mercapto-containing polymers. When preparing a combination of multiple dynamic bonds, the preparation methods of multi-dynamic bond network systems known in the art can be referred to. The above preparation methods are all conventional techniques in the art, and those skilled in the art can choose appropriate preparation methods as needed.

[0043] (3) Interpenetration and composite: The first network skeleton is placed in the second network precursor liquid, and the second network precursor liquid is fully penetrated into the fiber pores of the first network by vacuum infusion; Preferably, the vacuum conditions for vacuum infusion are -0.05 MPa to -0.095 MPa, and the treatment time is 10 to 60 minutes.

[0044] (4) Induced crosslinking: Inducing the polymer monomers or polymers in the second network precursor liquid to undergo dynamic crosslinking reaction, so that dynamic reversible bonds are formed, thereby forming the second network in situ within the pores of the first network skeleton, and obtaining a double network structure.

[0045] Those skilled in the art will understand that the specific method of inducing crosslinking depends on the type of dynamic reversible bond selected. Depending on the type of dynamic reversible bond selected, conventional techniques in the art are used to adjust environmental conditions, including but not limited to adjusting temperature, humidity, pH, introducing metal ions, or physical dehydration.

[0046] For example, for hydrogen-bonded networks, the first network framework after permeation needs to be removed from the second network precursor solution, and self-assembly is induced by adjusting the ambient temperature or humidity. For dynamic covalent networks (such as hydrazone bonds, Schiff base bonds, disulfide bonds, etc.), the cross-linking reaction can be promoted by adjusting the pH or temperature of the precursor solution, and cross-linking can take place in the precursor solution. For coordination-bonded networks, metal ions can be pre-added to the second network precursor solution, or the framework can be removed after permeation and immersed in a solution containing metal ions for cross-linking. The above-mentioned methods of inducing cross-linking are all conventional techniques in this field, and those skilled in the art can choose appropriate methods as needed.

[0047] (5) Post-processing: Remove residual reagents, sterilize and seal for storage.

[0048] In a preferred embodiment, after the induction of crosslinking in step (4), the procedure further includes an endothelialization modification of the artificial coronary graft: The inner wall of the induced cross-linked dual-network artificial blood vessel is perfused with a functionalized solution containing S-nitrosothiol (concentration of 0.5~5 mg / mL) and endothelial progenitor cell capturing peptide (concentration of 0.1~1.0 mg / mL), and the solution is subjected to a shaking reaction at 35~40℃ for 2~6 hours, so that the S-nitrosothiol and endothelial progenitor cell capturing peptide are covalently fixed to the inner surface of the lumen; wherein the endothelial progenitor cell capturing peptide is selected from one or more of REDV, TPS, and YIGSR.

[0049] After the reaction, the vessel was washed multiple times with phosphate-buffered saline (PBS, pH 7.4) for 5-10 minutes each time to remove unbound functionalized molecules and residual reaction reagents, thus obtaining endothelialized functionalized artificial coronary artery grafts.

[0050] It should be noted that, in this invention, physiological conditions refer to conditions simulating the human body's internal environment, including a temperature of 35-40°C, a pH of 7.2-7.6, and an osmotic pressure of 280-320 mOsm / L. In the following specific embodiments of this invention, physiological conditions refer to a phosphate buffer environment at 37°C and pH 7.4.

[0051] The following are specific embodiments and comparative examples: Example 1 The method for preparing the dual-network self-healing decellularized biological artificial coronary artery graft in this embodiment includes the following steps: Preparation of the first network (decellularized scaffold): Fresh porcine coronary arteries (approximately 3 mm in diameter and 400 μm in wall thickness) were placed in a mixture of 1 vol% Triton X-100 and 0.1 wt% SDS and treated with shaking at 37°C for 16 hours. Then, 50 U / mL of DNase I was added, and the mixture was further treated with shaking at 37°C for 4 hours to remove residual nucleic acids. Finally, the mixture was washed with sterile PBS buffer for 24 hours to obtain the decellularized first network scaffold.

[0052] Preparation of the second network precursor solution: UPy functionalized gelatin (grafting rate 25%) and PTMEG (molecular weight 2000 Da) with isocyanate-terminated groups were mixed at a mass ratio of 1:1. Stannous octoate catalyst with a mass fraction of 0.05% was added and stirred evenly to prepare the second network precursor solution.

[0053] Intercalation and in-situ crosslinking: The decellularized scaffold was placed in the above-mentioned precursor solution and perfused under vacuum (0.08 MPa) for 30 minutes to allow the precursor solution to fully penetrate into the vascular fiber pores. After removal, it was placed in a constant temperature and humidity chamber (37°C, 85%±5% relative humidity) for 24 hours. Humidity induces the reaction between isocyanate groups and amino groups and water on the gelatin, while UPy units spontaneously assemble through quadruple hydrogen bonds, forming a dynamic and reversible crosslinked network.

[0054] Post-processing: The prepared double-network blood vessels were immersed in 75% ethanol for 12 hours to wash away residual monomers, and then rinsed three times with sterile PBS buffer (pH 7.4) for 15 minutes each time. The vessels were then sealed and stored at 4°C.

[0055] Example 2 The method for preparing the dual-network self-healing decellularized bio-type artificial coronary artery graft in this embodiment enhances the endothelial progenitor cell capture and NO sustained release functions, and also demonstrates the synergistic effect of two dynamic reversible bonds: hydrogen bonds and disulfide bonds. The method includes the following steps: Preparation of the first network (decellularized scaffold): The first network was prepared by using a porcine carotid artery (with an inner diameter of about 4 mm and a wall thickness of about 500 μm) and processing it according to the decellularization process in Example 1 to obtain the decellularized first network scaffold.

[0056] Preparation of the second network precursor solution: UPy-functionalized chitosan (grafting rate 20%) and isocyanate-terminated PTMEG (molecular weight 1000 Da) were mixed at a mass ratio of 1:2. 0.05% stannous octoate catalyst was added, and the mixture was stirred until homogeneous. Subsequently, thiolated gelatin (thiol grafting rate 15%) was added at a mass ratio of 1:1 (UPy-functionalized chitosan to thiolized gelatin), and the mixture was stirred until homogeneous to obtain the second network precursor solution containing UPy side groups and free thiol groups.

[0057] Intercalation and in-situ crosslinking: The decellularized scaffold was placed in the aforementioned second network precursor solution and maintained under vacuum perfusion technology (vacuum degree -0.09 MPa) for 45 minutes to allow the precursor solution to fully penetrate into the vascular fiber pores. After removal, it was placed in a constant temperature and humidity chamber (temperature 37℃, relative humidity 85%±5%) for 24 hours of static reaction. During this process, UPy units spontaneously assembled to form a dynamic hydrogen-bonded network through quadruple hydrogen bonds; simultaneously, in the presence of oxygen, free thiol groups (-SH) underwent oxidative coupling reactions to form a disulfide-bonded dynamic network. The synergistic effect of hydrogen bonds and disulfide bonds endows the material with both rapid self-healing ability and high mechanical strength.

[0058] Endothelialization modification (REDV / SNAP grafting): A mixed solution containing endothelial progenitor cell capture peptide REDV (concentration 0.5 mg / mL) and nitric oxide donor SNAP (concentration 2 mg / mL) was perfused into the luminal wall of the blood vessel and reacted with gentle shaking at 37°C for 4 hours, so that REDV and SNAP were covalently grafted onto the luminal surface.

[0059] Post-processing: After the reaction, the vessel was washed three times (15 minutes each time) with PBS buffer (pH 7.4) containing 0.5 IU / mL heparin to remove physically adsorbed free molecules and obtain an artificial coronary artery graft with both self-healing and highly efficient endothelialization-promoting functions.

[0060] Comparative Example 1 In this comparative example, decellularized porcine coronary arteries were prepared according to the method of Example 1, but without performing the second network interpenetration and cross-linking steps, the decellularized blood vessels were directly cleaned and sterilized to obtain single-network decellularized blood vessels.

[0061] Comparative Example 2 In this comparative example, decellularized vascular skeletons were not used. The second network precursor liquid from Example 1 was injected into a cylindrical mold and cured according to the crosslinking conditions of Example 1 (37°C, relative humidity 85%±5%, 24 hours) to obtain a dynamic crosslinked network pipe of pure synthetic material.

[0062] Comparative Example 3 This comparative example provides an irreversible covalently cross-linked dual-network blood vessel, prepared using a method essentially the same as in Example 1, except that the second network uses ordinary gelatin without UPy functionalization. The preparation method is as follows: Preparation of the first network backbone: Same as in Example 1.

[0063] Preparation of the second network precursor solution: Non-UPy functionalized ordinary gelatin and isocyanate-terminated PTMEG are mixed at a mass ratio of 1:1, 0.05% catalyst stannous octoate is added, and the mixture is stirred evenly to prepare the second network precursor solution.

[0064] Intercalation and cross-linking: Same as in Example 1 (after vacuum infusion, stand at 37°C and 85% humidity for 24 hours). During this process, the isocyanate groups react with the amino groups and moisture on the gelatin to form an irreversible covalent cross-linked network of urea bonds and urethane bonds.

[0065] Post-processing: Same as in Example 1.

[0066] Comparative Example 4 This comparative example provides an inert polyurethane interpenetrating network blood vessel, wherein the second network is made of conventional polyurethane, and the preparation method is as follows: Preparation of the first network backbone: Same as in Example 1.

[0067] Preparation of inert polyurethane prepolymer: Using diphenylmethane diisocyanate (MDI) and polycarbonate diol (PCDL) as polyurethane monomers and small molecule butanediol (BDO) as chain extender, the three in a molar ratio of 3:1:2, polyurethane prepolymer is prepared by conventional methods, dissolved in N,N-dimethylacetamide (DMAc), and prepared into a 2-5% solution.

[0068] Intercalation and compounding: The decellularized vascular skeleton is fully impregnated with the above polyurethane prepolymer solution and allowed to act at room temperature for 2 hours.

[0069] Curing and elution: The above intermediate product is fully immersed in water for curing, causing the polyurethane prepolymer to undergo an irreversible cross-linking reaction to form an inert polyurethane network. After elution to remove residual solvent, an interpenetrating network structure of decellularized blood vessels and inert polyurethane is obtained.

[0070] Post-processing: Same as in Example 1.

[0071] Comparative Example 5 This comparative example uses natural human coronary artery mechanical performance data as a theoretical reference. Specific data are as follows: static compliance approximately 3.0 ± 7.0% / 100 mmHg, burst pressure approximately 2000~3000 mmHg. These data are used to compare with the test results of the embodiments of this invention.

[0072] Experimental Example 1: In vitro performance testing The following performance tests were performed on Examples 1, 2, and Comparative Examples 1-5: Self-healing efficiency test: The blood vessel was completely cut and aligned, and left to stand in a humid environment at 37°C for 10-30 minutes. The tensile strength recovery rate was recorded.

[0073] Compliance test: In a simulated blood circulation system (pulsating pressure 80-120 mmHg), the rate of change of blood vessel diameter with pressure was measured.

[0074] Burst pressure test: Continuous pressure is applied to the inside of the blood vessel until the vessel wall ruptures, and the maximum pressure that can be withstood is recorded.

[0075] In situ endothelialization assay (cell experiment): Endothelial cells were seeded and cultured with blood vessels to observe the efficiency of REDV in capturing endothelial cells.

[0076] Table 1. Experimental results of artificial blood vessel materials in examples and comparative cases.

[0077] The results showed that the burst pressures of Examples 1 and 2 were significantly higher than those of Comparative Example 2 (pure synthetic material), comparable to those of Comparative Example 3 (ordinary gelatin + PTMEG) and Comparative Example 4 (inert polyurethane), and close to the reference range of Comparative Example 5 (natural human coronary artery), indicating that the dual-network structure of the present invention possesses sufficient mechanical strength. The burst pressure of Example 2 was slightly higher than that of Example 1, attributed to the enhanced material strength resulting from the introduction of the disulfide bond dynamic network. The burst pressure of Comparative Example 2 was far below the physiological arterial pressure, failing to meet clinical requirements.

[0078] The static compliance of Examples 1 and 2 is within the reference range of Comparative Example 5 (natural human coronary artery), showing a high degree of matching with natural blood vessels, indicating that the present invention can effectively reduce anastomotic shear stress. Comparative Example 1 has significantly low compliance (too stiff), and Comparative Example 2 has significantly high compliance (too soft), neither of which can achieve mechanical matching. Although the compliance of Comparative Examples 3 and 4 is within the standard deviation range of natural blood vessels, it is lower than that of Examples 1 and 2, and the compliance matching effect is not as good as that of the present invention. The above results show that the dual-network structure of the present invention achieves precise control of compliance through the first network providing a mechanical framework and the second network providing elastic regulation, and is superior to the irreversible covalent cross-linked systems of Comparative Examples 3 and 4.

[0079] Examples 1 and 2 both exhibited excellent rapid self-healing capabilities, with tensile strength recovery rates exceeding 85% after 30 minutes. Comparative Examples 1, 3, and 4 showed no self-healing ability (recovery rate of 0%). Although Comparative Example 2 had a relatively high self-healing efficiency, its mechanical strength was severely insufficient. The self-healing efficiency of Example 2 was slightly better than that of Example 1, possibly related to the synergistic effect of hydrogen bonds and disulfide bonds. The above results indicate that the present invention, by introducing a dynamic cross-linking network, endows the material with rapid self-healing capability while maintaining mechanical strength, while Comparative Examples 3 and 4, due to the irreversible covalent cross-linking of the second network, completely lack self-healing ability.

[0080] Figure 2 The self-healing process of the artificial coronary artery bypass graft in Example 1 of this invention is shown. Immediately after puncture, a clear puncture hole was visible on the vessel surface; 30 minutes after puncture, the puncture hole had essentially healed, and the surface was restored to its original integrity. The results indicate that the bypass graft of this invention possesses excellent rapid self-healing ability, achieving effective closure of the damaged interface within 30 minutes. This is attributed to the dynamic reversible recombination mechanism of the UPy quadruple hydrogen bonds in the second network. Furthermore, Example 1 retains the complete ECM components and exhibits good tissue compatibility. Example 2 further grafts REDV capturing peptide and SNAP nitric oxide donor, possessing the function of actively capturing endothelial progenitor cells and inhibiting platelet aggregation, with excellent endothelialization tendency. Comparative Example 1, while retaining ECM components, lacks self-healing ability; Comparative Example 2 is a purely synthetic material and lacks bioinducibility.

[0081] In addition, from Figure 3 It can be seen that the endothelial-functionalized coating has a good ability to promote endothelial cell adhesion. Compared with Example 1, Example 2 showed a greater number of endothelial cells adhering, a larger spreading area, and a significantly reduced platelet adhesion density. This is attributed to the specific capture effect of REDV on circulating endothelial progenitor cells and the inhibitory effect of SNAP-catalyzed NO release on platelet aggregation. The above results demonstrate that the endothelial-functionalized coating described in this invention can effectively accelerate the in situ endothelialization process, providing a biological guarantee for the long-term patency of bypass grafts.

[0082] In summary, Examples 1 and 2 are significantly superior to Comparative Examples 1-4 in terms of burst pressure, compliance, self-healing efficiency, and tissue compatibility. While Comparative Examples 3 and 4 possess a certain mechanical strength, their second network is irreversibly covalently cross-linked, resulting in a lack of self-healing ability and poor compliance. Comparative Example 2 has high self-healing efficiency but severely insufficient mechanical strength. Comparative Example 1 exhibits poor compliance and no self-healing. In contrast, Example 2, through the synergistic effect of a dual dynamic network of hydrogen and disulfide bonds and endothelial functionalization modification, achieves the best overall performance. The dual-network self-healing decellularized biological artificial coronary artery bypass graft described in this invention meets the clinical requirements for coronary artery bypass surgery in both mechanical properties and biological function.

[0083] Experimental Example 2: In vivo animal experiments The dual-network self-healing decellularized biological artificial coronary artery graft prepared in Example 2 was implanted into the coronary artery of an adult sheep (e.g., Figure 4 Animal experiments were conducted for in vivo evaluation, and the patency of the bypass graft was monitored three months after implantation using digital subtraction angiography (DSA). The results were as follows. Figure 5 As shown.

[0084] Three months post-implantation DSA angiography showed that the graft vessel prepared in Example 2 had a patent lumen, with smooth contrast agent passage and no obvious stenosis, occlusion, or thrombosis. No stenosis or aneurysmal dilatation was observed at the anastomosis site, the vessel wall remained intact, and there was no leakage or pseudoaneurysm. This indicates that the graft vessel of the present invention maintained good structural stability and antithrombotic ability under in vivo blood flow impact. Its radial compliance highly matches that of the natural coronary artery, effectively eliminating anastomotic shear stress concentration and inhibiting intimal hyperplasia. Simultaneously, the mechanical strength and self-healing function provided by the dual-network structure ensured the integrity of the lumen. These results confirm that the dual-network self-healing acellular biological coronary graft of the present invention has good patency and biocompatibility in vivo, providing strong experimental evidence for the clinical application of coronary artery bypass grafting.

[0085] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A dual-network self-healing decellularized biological artificial coronary artery graft, characterized in that, This includes a first network and a second network interspersed within the first network; The first network consists of naturally derived small-diameter decellularized blood vessels that retain the extracellular matrix, serving as a mechanical framework support layer; The second network is a dynamic cross-linked network with self-healing function, containing UPy quadruple hydrogen bonds; The UPy quadruple hydrogen bond is formed by the hydrogen bond interaction between UPy units and the hydrogen bond interaction between UPy units and polyether segments after the UPy functionalized natural polymer and polyether segments are bridged by diisocyanate. The UPy-functionalized natural polymer is selected from one or more of UPy-functionalized gelatin, UPy-functionalized chitosan, or UPy-functionalized hyaluronic acid; the polyether segment is one or more of polytetramethylene ether glycol and polyethylene glycol. The method for preparing the artificial coronary artery bypass graft includes the following steps: (1) Decellularization: Decellularization is performed on blood vessels of natural origin to remove cellular components while retaining the fibrous network structure of the extracellular matrix, thereby obtaining the first network framework; (2) Preparation of the second network precursor solution: Prepare a solution containing a polymer or monomer that can form dynamic reversible bonds as the second network precursor solution; (3) Interpenetrating composite: The first network skeleton is placed in the second network precursor liquid, so that the second network precursor liquid penetrates into the fiber pores of the first network skeleton; (4) Induced crosslinking: The second network precursor liquid is induced to undergo a dynamic crosslinking reaction to form dynamic reversible bonds, thereby forming a second network in situ within the pores of the first network skeleton.

2. The dual-network self-healing decellularized biological artificial coronary artery graft according to claim 1, characterized in that, The naturally derived small-diameter decellularized blood vessels are derived from the coronary arteries or carotid arteries of pigs or sheep; and / or, The intravascular diameter of the first network is 0.8~5 mm; and / or, The vessel wall thickness of the first network is 200~800μm; and / or, The collagen content of the blood vessels in the first network is 40% to 80%.

3. The dual-network self-healing decellularized biological artificial coronary artery graft according to claim 1 or 2, characterized in that, The inner surface of the artificial coronary artery graft is further provided with a functionalized modification layer, which includes S-nitrosothiol and endothelial progenitor cell capturing peptides covalently grafted onto the inner surface of the artificial coronary artery graft; the endothelial progenitor cell capturing peptides are selected from one or more of REDV, TPS, and YIGSR.

4. A method for preparing a dual-network self-healing decellularized biological artificial coronary artery graft according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Decellularization: Decellularization is performed on blood vessels of natural origin to remove cellular components while retaining the fibrous network structure of the extracellular matrix, thereby obtaining the first network framework; (2) Preparation of the second network precursor solution: Prepare a solution containing a polymer or monomer that can form dynamic reversible bonds as the second network precursor solution; (3) Interpenetrating composite: The first network skeleton is placed in the second network precursor liquid, so that the second network precursor liquid penetrates into the fiber pores of the first network skeleton; (4) Induced crosslinking: The second network precursor liquid is induced to undergo a dynamic crosslinking reaction to form dynamic reversible bonds, thereby forming a second network in situ within the pores of the first network skeleton.

5. The method for preparing a dual-network self-healing decellularized biological artificial coronary artery graft according to claim 4, characterized in that, In step (1), the decellularization process includes: treating the blood vessels of natural origin with a surfactant to remove cellular components, then treating them with a DNA enzyme to remove residual nucleic acids, and finally rinsing them with a buffer solution; the surfactant is selected from one or more of SDS and Triton X-100.

6. The method for preparing a dual-network self-healing decellularized biological artificial coronary artery graft according to claim 4, characterized in that, In step (3), vacuum infusion is used to allow the second network precursor liquid to penetrate into the fiber pores of the first network skeleton. The vacuum conditions are -0.05MPa to -0.095MPa, and the treatment time is 10 to 60 minutes.

7. The method for preparing a dual-network self-healing decellularized biological artificial coronary artery graft according to claim 4, characterized in that, In step (4), the induced crosslinking is performed either in situ in the second network precursor solution after the first network skeleton is placed in the second network precursor solution, or after the permeated first network skeleton is removed from the second network precursor solution, depending on the type of the selected dynamic reversible bond.

8. The method for preparing the dual-network self-healing decellularized biological artificial coronary artery graft according to any one of claims 4-7, characterized in that, It also includes the step of endothelialization of the artificial coronary grafts: Inject a functionalized solution containing S-nitrosothiol and endothelial progenitor cell capture peptide into the inner wall of the artificial coronary artery graft prepared in step (4), so that the S-nitrosothiol and endothelial progenitor cell capture peptide are fixed on the inner surface of the lumen by covalent bonding; the endothelial progenitor cell capture peptide is selected from one or more of REDV, TPS, and YIGSR.

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