Multifunctional vascular patch and preparation method and application thereof

Abstract

The present application relates to the technical fields of biomedical materials and medical devices, and discloses a multifunctional vascular patch as well as a preparation method and application thereof, the method comprising: S1: taking healthy mammal pericardial tissue; S2: performing decellularization and antigen-removing treatment on the cleaned mammal pericardial tissue by using a hypotonic solution, a surfactant and a nuclease in combination, to obtain a decellularized pericardial base material; S3: performing first activation on a blood contact surface of an inner layer of the pericardial base material, coating and cross-linking a hydrogel precursor solution containing an anticoagulant recombinant protein and an anti-calcification recombinant protein, to form a hydrogel inner layer carrying double-function proteins in situ; S4: performing second activation on a tissue contact surface of an outer layer of the pericardial base material, to form an outer layer anti-adhesion layer; and S5: performing wet-state shaping, gradient cleaning and sterilization packaging. The multifunctional vascular patch has the advantages of low immunogenicity, anticoagulation, anti-calcification, anti-adhesion, anti-leakage and pro-endothelization, and has high clinical transformation value.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and medical device technology, specifically to a multifunctional vascular patch, its preparation method, and its application. Background Technology

[0002] Vascular repair and reconstruction are core techniques in vascular surgery for treating vascular stenosis, occlusion, injury, and congenital malformations. Vascular patches, as key implant materials, are widely used in clinical scenarios such as carotid endarterectomy, peripheral arteriovenous angioplasty, vascular dilation, arteriovenous access repair, and local reconstruction of large vessels. An ideal vascular patch must simultaneously meet excellent mechanical compliance, blood compatibility, tissue compatibility, anti-calcification ability, anti-adhesion effect, and anti-suture leakage performance to ensure postoperative vascular patency, reduce complications, and achieve long-term stable service. Bovine / porcine pericardium, due to its abundant source, intact natural collagen fiber network, mechanical strength and elasticity matching natural blood vessels, and excellent suture performance, has become an important biological substrate for clinical vascular patches. However, unfunctionalized decellularized bovine / porcine pericardial vascular patches still face multiple technical bottlenecks in clinical application, severely limiting their long-term efficacy and applicability.

[0003] First, the most significant clinical risk is coagulation activation and thrombosis caused by blood contact. After vascular patch implantation, plasma proteins rapidly and non-specifically adsorb onto the material surface, triggering platelet adhesion, aggregation, and a coagulation cascade, leading to early thrombotic occlusion. Traditional anticoagulation strategies often employ heparin immobilization technology, which, while possessing some anticoagulant effect, suffers from drawbacks such as animal-derived contamination, easy activity attenuation, and significant batch-to-batch variability. It may also induce heparin-induced thrombocytopenia, posing significant safety risks. Furthermore, simply relying on physical hydrophilic modification can only reduce protein adsorption and cannot achieve active anticoagulation, making it difficult to meet the needs of high-coagulation-risk scenarios.

[0004] Secondly, long-term post-implantation calcification severely impacts patch lifespan. Decellularized bovine / porcine pericardium retains a large amount of natural collagen structure, with numerous calcium salt binding sites within its molecular chains. After implantation, these sites easily become carriers for hydroxyapatite nucleation and deposition, leading to gradual hardening, loss of elasticity, and decreased compliance of the patch, ultimately resulting in serious consequences such as restenosis and patch rupture. Conventional anti-calcification methods often employ metal ion blocking, small molecule modification, or simple cross-linking treatments, which have limited effectiveness and poor stability, failing to achieve long-term, stable inhibition of calcium salt deposition.

[0005] Third, postoperative tissue adhesion is a long-overlooked but significantly harmful problem in the clinical application of vascular patches. The outer tissue contact surface of the patch lacks an effective insulating layer, making it prone to pathological adhesion to surrounding connective tissue, the adventitia of blood vessels, and muscles. This leads to restricted local tissue movement, nerve compression, chronic pain, and even increases the difficulty of separation and the risk of tissue damage during secondary surgery. Traditional anti-adhesion coatings often use common hydrophilic materials such as hyaluronic acid and chitosan, which have weak protein adsorption inhibition capabilities, poor hydration layer stability, and insufficient abrasion resistance. They are easily degraded and detached in the complex physiological environment of the body, making it difficult to achieve a long-lasting anti-adhesion effect.

[0006] Finally, suture needle leakage is a key issue affecting intraoperative safety and surgical success rate. Bovine / porcine pericardial substrates have a naturally porous structure, making them prone to needle leakage channels after suture puncture, especially under high arterial pressure. This leakage is more pronounced, leading to difficulties in intraoperative hemostasis, prolonged operation time, and increased postoperative hematoma and infection risks. Current technologies rely solely on increasing cross-linking to enhance substrate density, which can result in excessively rigid materials with reduced compliance, failing to balance leak resistance and mechanical compatibility, and lacking an integrated structural and functional design.

[0007] As vascular surgery techniques advance towards minimally invasive, long-lasting, and highly safe approaches, clinical practice demands higher functional standards for vascular patches. Single-function modifications are no longer sufficient for complex applications, necessitating the development of multifunctional composite vascular patches that integrate anticoagulation, anti-calcification, anti-adhesion, and anti-leakage properties. Summary of the Invention

[0008] To address the problems existing in the prior art, this invention provides a multifunctional vascular patch, its preparation method, and its application. The multifunctional vascular patch of this invention is a mammalian (especially bovine / pig) pericardial vascular patch with an inner layer carrying a bifunctional recombinant protein hydrogel and an outer layer of zwitterionic anti-adhesion, which is leak-proof and decellularized. The invention also provides a preparation method to solve the technical defects of existing vascular patches, such as insufficient anticoagulation, easy calcification, tissue adhesion, and suture leakage.

[0009] In a first aspect, the present invention provides a method for preparing a multifunctional vascular patch, comprising the following steps: S1: Take healthy mammalian pericardial tissue, remove fat, fascia and blood clots, cut along the direction of the main collagen fibers, and then wash at low temperature; S2: The cleaned mammalian pericardial tissue was treated with a combination of hypotonic solution, surfactant and nuclease to decellularize and remove antigens, so as to obtain decellularized pericardial substrate. S3: The inner blood contact surface of the pericardial substrate is first activated, coated and crosslinked with a hydrogel precursor fluid containing anticoagulant recombinant protein and anticalcification recombinant protein to form a hydrogel inner layer carrying bifunctional proteins in situ; the anticoagulant recombinant protein includes one or more of SEQ ID NO:1 to SEQ ID NO:5, or a variant having at least 80% sequence identity with it and without significantly altering its anticoagulant compatibility regulatory function; the anticalcification recombinant protein includes one or more of SEQ ID NO:6 to SEQ ID NO:10, or a variant having at least 80% sequence identity with it and without significantly altering its anticalcification function; The mass ratio of the anticoagulation recombinant protein to the anticalcification recombinant protein is (0.5-1):(1-5); The anticoagulant recombinant protein contains 5-15 wt% in the hydrogel precursor solution. The content of anti-calcification recombinant protein in the hydrogel precursor solution is 5-75 wt%; S4: Perform a second activation on the outer tissue contact surface of the pericardial substrate, grafting zwitterionic polymers to form an outer anti-adhesion layer; S5: After wet shaping, gradient cleaning, sterilization and packaging, the target vascular patch is obtained.

[0010] In this invention, mammals include, but are not limited to, pigs, cattle, sheep, horses, or rabbits. In some preferred embodiments, the mammal is a pig or a cattle.

[0011] In step S1 of the present invention, the direction of the main collagen fibers refers to the direction of the head-to-tail longitudinal axis (i.e., cranio-coccygeal direction).

[0012] In some preferred embodiments, in step S1, the temperature range of the low temperature is 2-8°C; In some preferred embodiments, in step S1, the solution used for cleaning includes at least one of PBS, HBSS, HBS, and Tris saline. In some more preferred embodiments, in step S1, the solution used for cleaning is a PBS solution.

[0013] In some preferred embodiments, in step S2, the solution used for hypotonic treatment is hypotonic Hank's solution.

[0014] In step S2 of this invention, the temperature range of the hypotonic treatment is no more than 10°C, so that the low-temperature environment can inhibit the activity of endogenous proteases and collagenases in the tissue, avoid the degradation and breakage of natural collagen fibers, and completely preserve the continuous and orderly collagen fiber network structure of the pericardial substrate. This ensures that the pericardial substrate obtained after treatment can achieve uniform dispersion and firm binding of the two functional proteins, anticoagulation recombinant protein and anticalcification recombinant protein, in the hydrogel network during subsequent treatment, enabling long-term stable release and continuous action, and making the anticoagulation and anticalcification functions highly synergistic. In some preferred embodiments, the temperature range of the hypotonic treatment is 2-4°C.

[0015] In some preferred embodiments, in step S2, the hypotonic treatment lasts for 10-20 hours to allow the cells to rupture completely and release their intracellular contents. More preferably, the hypotonic treatment lasts for 12 hours.

[0016] In step S2 of this invention, the surfactant is a mild nonionic / amphoteric surfactant such as Triton X-100, Tween series, NP-40, Brij series, or CHAPS, as long as it can dissolve cell membranes and lipid antigens and elute cell debris. In some preferred embodiments, the surfactant is at least one of Triton X-100, Tween-20, and Tween-80.

[0017] In step S2 of this invention, the nuclease can be DNase I, DNase II, RNase A, or RNase T1, as long as it can sufficiently degrade free and bound DNA and RNA in tissues, eliminate nucleic acid immunogens, and reduce the risk of inflammation in the substrate. In some preferred embodiments, the nuclease includes at least one of DNase I, RNase A, and DNase II.

[0018] In some preferred embodiments, in step S2, the decellularization treatment includes: treatment with hypotonic Hank's solution at 4°C for 12 h, followed by 0.1% Triton X. Treatment with 100+0.05% SDS for 2 h, followed by treatment with 0.3% Tween 80 for 8 h; nuclease treatment with 50 U / mL DNase I+20 μg / mL RNase A at 37℃ for 4 h; antigen elution with α-galactosidase.

[0019] Hydrogel materials, due to their advantages such as high water content, high biocompatibility, customizable structure, and ease of loading bioactive components, have become an ideal choice for modifying the interface of blood contact materials. Recombinant protein technology can achieve precise and controllable customization of anticoagulant and anticalcification functional proteins, avoiding the safety risks of natural proteins. Zwitterionic polymers, with their super-strong hydration properties, can construct anti-adhesion interfaces with near-zero protein adsorption. Based on this, this invention organically combines bifunctional recombinant proteins, hydrogel carriers, zwitterionic anti-adhesion layers, and anti-leakage structural design to develop an anti-leakage decellularized mammalian (especially bovine or porcine) pericardial vascular patch with an inner layer carrying a bifunctional recombinant protein hydrogel and an outer zwitterionic anti-adhesion layer. This addresses multiple shortcomings of existing technologies and has significant clinical value and broad application prospects.

[0020] In some preferred embodiments, in step S3, the hydrogel precursor fluid further comprises one or more of the following: hyaluronic acid, methacrylamide gelatin, carboxymethyl chitosan, or alginate.

[0021] In some preferred embodiments, in step S3, the hydrogel precursor material comprises hyaluronic acid and carboxymethyl chitosan in a mass ratio of (1-5):(1-5).

[0022] In some preferred embodiments, in step S3, the amount of hydrogel precursor material in the hydrogel precursor solution is 5%-15% (w / v); more preferably 8%-12% (w / v).

[0023] In step S3 of the present invention, the hydrogel precursor solution uses PBS solution or deionized water as solvent, and the solvent accounts for 85%-95% of the total mass of the hydrogel precursor solution.

[0024] In some preferred embodiments, in step S3, the anticoagulant recombinant humanized collagen includes one or more of SEQ ID NO:1 to SEQ ID NO:5, or a variant having at least 80%, 85%, 90%, 95%, or 98% sequence identity with it and without significantly altering its anticoagulant compatibility regulatory function. SEQ ID NO:1:GPPGPPGPPGERGVEGPPGPPGSEEEED; SEQ ID NO:2:GPPGPPGPRGERGSGDFEEIPEYLQ; SEQ ID NO:3: GPPPGPGPKGDRGDAGSGEDEDE; SEQ ID NO:4: GPPGPPGPDGEAGPPGPPGSDDDSE; SEQ ID NO:5:GPPGPPGPPGQKGEKGSGESESE.

[0025] In this invention, "no significant change" means that the change is not statistically significant.

[0026] In some preferred embodiments, in step S3, the anti-calcification recombinant protein includes an acidic amino acid enrichment region, a calcium ion regulation region, and a collagen / elastin localization region; the anti-calcification recombinant protein includes one or more of SEQ ID NO:6 to SEQ ID NO:10, or variants having at least 80%, 85%, 90%, 95%, or 98% sequence identity with it and without significantly altering its anti-calcification function. SEQ ID NO:6: GDDDDSEEEDDDGSGVGVAPG; SEQ ID NO:7:GSESESEDDDDEEESGPGPKG; SEQ ID NO:8: GDDDDDDGSGESESESGVPGVG; SEQ ID NO:9: GEEEDDDSESDDDGSGFOGER; SEQ ID NO:10: GPGKGDDDEEESSEEDDGG.

[0027] In some preferred embodiments, in step S3, the mass ratio of the anticoagulation recombinant protein to the anticalcification recombinant protein is 1:(1-4).

[0028] In some preferred embodiments, in step S3, the content of the anticoagulant recombinant protein in the hydrogel precursor fluid is 8-12 wt%.

[0029] In some preferred embodiments, in step S3, the content of the anti-calcification recombinant protein in the hydrogel precursor fluid is 10wt%-50wt%.

[0030] In step S3 of this invention, "first activation" refers to modifying the carboxyl groups of the inner layer of pericardial substrate collagen with a carbodiimide reagent to generate succinimide ester active sites, enabling the pericardial substrate to covalently bind to hydrogels and functional proteins, preventing coating erosion and protein loss, and improving interfacial binding strength. This includes three core operational steps: pH adjustment of the buffer system, carboxyl group activation incubation, and rinsing with the free activator. In some preferred embodiments, the activation includes the following steps: preparing an activation solution buffered with MES (0.05-0.1 mol / L EDC + 0.02-0.05 mol / L NHS), adjusting the pH to 5.0-5.5; immersing the inner layer of the pericardial substrate in the activation solution, incubating at room temperature in the dark with low-speed shaking for 30 min; and rinsing 2-3 times with PBS pre-cooled to 0-4℃, 5 min each time, to remove the free activator.

[0031] In step S3 of this invention, the coating conditions include: the substrate is wetted, flattened, and free of water accumulation and wrinkles; blade coating (120-180 μm) or spray coating (0.1 mL / cm). 2 ); 20-25 ℃ away from light, humidity 40%-60%; let stand for 5 min after coating to wet the pores; the coating is uniform, and the cured hydrogel is 20-120 μm thick.

[0032] In step S3 of this invention, the hydrogel precursor solution can be cured by photocrosslinking, enzymatic crosslinking, Schiff base crosslinking, EDC / NHS crosslinking, maleimide-thiol coupling, or dopamine adhesion crosslinking to form a uniform functional layer with a thickness of 20-120 μm. In some preferred embodiments, the hydrogel precursor solution forms a uniform functional layer with a thickness of 20-120 μm by photocrosslinking, EDC / NHS crosslinking, or dopamine adhesion crosslinking. More preferably, curing by EDC / NHS crosslinking includes the following steps: uniformly coating the hydrogel precursor solution onto the inner surface of the activated pericardium substrate, performing a crosslinking reaction at room temperature in the dark for 1-2 h, with a final EDC concentration of 0.01-0.03 mol / L and a final NHS concentration of 0.01-0.02 mol / L in the crosslinking system; after crosslinking, thoroughly rinsing with sterile PBS to remove free crosslinking agent, and curing to form a double-protein-loaded hydrogel inner layer with a thickness of 20-120 μm. More preferably, curing via photocrosslinking includes the following steps: adding 0.5-0.8 wt% of LAP photoinitiator to the hydrogel precursor solution and mixing thoroughly, then uniformly coating it onto the inner layer of the substrate; irradiating with 400-420 nm blue light at an intensity of 4-5 mW / cm². 2In-situ curing is completed by irradiation for 150-180 seconds. More preferably, curing by dopamine adhesion crosslinking includes the following steps: 2-3 wt% dopamine hydrochloride is added to the hydrogel precursor solution, and the pH of the system is adjusted to 8.0-8.5; after being coated on the inner layer of the substrate, it is left to stand at room temperature for 2-3 hours, and crosslinking and curing are achieved by dopamine oxidation self-polymerization.

[0033] In step S4, the zwitterionic polymer must simultaneously meet the following conditions: electrically neutral at physiological pH with a zeta potential of -5 to +5 mV; water contact angle ≤35°; coating equilibrium moisture content ≥70%; grafting density 0.8-2.5 μg / cm³. 2 The wet friction coefficient is ≤0.15; the fibrinogen adsorption inhibition rate is ≥95%; the coating thickness is 50-300 nm (preferably 50-80 nm); the physiological fluid swelling rate is 120%-200%; it is not easily degraded in vivo, and the fibroblast adhesion inhibition rate is ≥90%; its surface can form a dense hydration barrier layer, reducing protein adsorption, cell adhesion, and tissue friction damage, thus giving the pericardial substrate long-lasting anti-adhesion properties. In some preferred embodiments, the zwitterionic polymer is a sulfobetaine (SBA), carboxybetaine (CBA), or phosphorylcholine (MPC) polymer.

[0034] In step S4, "second activation" refers to the selective oxidation of the collagen side chain groups on the outer layer of the pericardial substrate with an oxidant to generate aldehyde active sites, providing covalent binding sites for the zwitterionic polymer without damaging the inner hydrogel layer. This includes oxidative activation and rinsing steps. In some preferred embodiments, the second activation includes the following steps: immersing the outer tissue contact surface of the pericardial substrate in a 0.1-0.2 mol / L sodium periodate aqueous solution at room temperature in the dark for 15-20 min; after activation, thoroughly rinsing with sterile PBS to terminate oxidation and remove residual oxidant.

[0035] Compared with the first activation treatment of the inner layer in step S3: the first activation treatment of the inner layer in step S3 uses EDC / NHS to activate the collagen carboxyl groups for grafting the protein-carrying hydrogel; while the second activation treatment of the outer layer in step S4 uses sodium periodate to oxidize the collagen amino groups to generate aldehyde groups for grafting zwitterionic polymers; the two activation systems have incompatible sites and reagents, and the unilateral stepwise activation can avoid the oxidative inactivation of the recombinant protein in the inner layer, and achieve differentiated and independent functional modification of the inner and outer layers.

[0036] In this invention, activation treatment is equivalent to modification. The first activation treatment of the inner layer in step S3 and the second activation treatment of the outer layer in step S4 can both be understood as modification treatment.

[0037] In step S4, the outer anti-adhesion layer is a dense hydrated layer formed by in-situ polymerization grafting. In some preferred embodiments, the in-situ polymerization grafting includes: preparing a 20-30 wt% zwitterionic monomer solution, adding 0.01-0.02 mol / L APS initiator, coating it on the outside of the activated substrate; isothermal in-situ polymerization at 55-60℃ for 4-5 h to form an anti-adhesion layer with a thickness of 50-300 nm, and rinsing with sterile PBS after polymerization to remove free reactants.

[0038] In step S5, the purpose of wet shaping is to eliminate residual internal stress introduced by the cross-linking and coating processes, regulate the collagen fiber arrangement, and inhibit patch curling and warping. Low-temperature equalization ensures uniform and controllable overall patch thickness, maintains a flat shape, guarantees stable interfacial bonding between the inner and outer coatings and the pericardial substrate, and improves the compatibility with the vascular wound dressing during surgery. In some preferred embodiments, the wet shaping includes the following steps: fully immersing the multifunctional vascular patch, after hydrogel coating and cross-linking, in sterile PBS phosphate buffer (pH 7.2-7.4), laying it flat in a limiting and shaping mold without tensile pre-stress, and performing low-temperature equalization and pressure holding treatment under sealed conditions; the low-temperature equalization and pressure holding treatment conditions are: temperature 4-15℃, pressure holding 0.02-0.12MPa, and holding time 2-12h; more preferably, the temperature is 6-10℃, pressure holding 0.04-0.08MPa, and holding time 4-8h.

[0039] In some preferred embodiments, wet shaping is performed to a total thickness of 0.45-0.55 mm.

[0040] In step S5, gradient washing can smoothly regulate the swelling rate of the hydrogel, thoroughly remove residual polymer impurities, protect the structural integrity of the inner functional protein-carrying hydrogel and the outer SBA anti-adhesion layer, and prevent loss of bioactivity. This allows for more uniform and stable subsequent ethylene oxide sterilization, improving the anti-adhesion and anticoagulation performance of the product after implantation. In some preferred embodiments, the gradient washing includes the following steps: sequentially rinsing with pre-cooled 75% ethanol aqueous solution, 50% ethanol aqueous solution, and 25% ethanol aqueous solution, each with shaking and rinsing 1-2 times (rinsing time 15 min, 12 min, and 10 min respectively), and finally rinsing with sterile PBS with shaking and rinsing 2-3 times (8 min each time), with a shaker speed of 50 r / min, and the entire process is carried out at 4°C in the dark.

[0041] In some preferred embodiments, ethylene oxide sterilization is used in step S5.

[0042] In this invention, the multifunctional vascular patch meets the following requirements: (1) Leakage rate ≤ 0.15 mL / (cm²) at 120 mmHg 2 (min), leakage rate at 250 mmHg ≤ 0.50 mL / (cm) 2 min); (2) Platelet adhesion decreased by ≥60%; (3) The amount of calcium deposition is ≤50% lower than that of the unmodified patch; (4) Endothelial cell coverage ≥60% at 72 h.

[0043] Secondly, the present invention provides a multifunctional vascular patch, which is prepared according to the preparation method described above.

[0044] Thirdly, the present invention provides a medical device comprising the multifunctional vascular patch described above.

[0045] Fourthly, the present invention provides a multifunctional vascular patch prepared according to the above-described preparation method, or the application of the multifunctional vascular patch or the medical device described above in carotid endarterectomy patches, peripheral artery patches, arteriovenous access repair patches, and vascular stent covering materials.

[0046] Compared with the prior art, the present invention has at least the following beneficial technical effects: This invention achieves a synergistic improvement in biocompatibility, functional durability, and clinical safety through an integrated design of a bifunctional recombinant protein-loaded hydrogel inner layer, a zwitterionic anti-adhesion outer layer, and an anti-leakage structure, demonstrating significant technical advantages and application value. The invention constructs a composite hydrogel system loaded with anticoagulant and anti-calcification recombinant proteins in the inner layer. These two functional proteins are uniformly dispersed and firmly bound within the hydrogel network, enabling long-term stable release and sustained action. This allows for highly efficient synergistic effects of anticoagulation and anti-calcification functions, while completely avoiding the safety risks associated with heparin fixation, such as thrombocytopenia, activity attenuation, and uneven source, significantly improving the long-term safety of blood contact.

[0047] The hydrogel layer used in this invention has the characteristics of high water content, low modulus and soft interface, which can significantly improve the hydrophilicity and biocompatibility of the material surface, reduce non-specific adsorption of plasma proteins and platelet adhesion activation, effectively inhibit inflammatory response, and provide a biomimetic microenvironment for endothelial cell adhesion, spreading and migration, accelerate the endothelialization process and reduce the risk of postoperative complications such as thrombosis and intimal hyperplasia.

[0048] This invention constructs an amphoteric polymer anti-adhesion layer on the outer tissue contact surface, which can quickly form a dense, stable, and super-hydrating layer in the physiological environment, significantly reducing protein adsorption and cell adhesion, achieving excellent tissue isolation effect, significantly reducing postoperative tissue adhesion, relieving local pain and tissue traction, reducing the difficulty and trauma risk of secondary surgery, and solving the problems of poor stability, weak anti-adhesion effect, and easy degradation and failure of traditional hydrophilic coatings.

[0049] Meanwhile, this invention employs a triple anti-leakage design of directional collagen fiber arrangement, gradient cross-linking reinforcement, and surface micro-sealing to significantly reduce leakage through suture needle holes without compromising material compliance, thereby improving intraoperative hemostasis efficiency and surgical safety, while taking into account mechanical strength, flexibility, and anti-leakage performance.

[0050] Furthermore, the overall preparation process of this invention is mild and controllable, with low cross-linking agent residue, controllable immunogenicity, and high batch-to-batch stability. It can retain the mechanical advantages and tissue compliance of the natural collagen network of decellularized bovine pericardium to the greatest extent. The production process is simple, highly scalable, and has good potential for clinical translation and large-scale production.

[0051] In summary, the multifunctional vascular patch of this invention uses the pericardium of decellularized mammals (especially bovine or porcine) as the substrate. The inner layer (blood contact surface) features an in-situ constructed hydrogel functional layer loaded with anticoagulant and anticalcification recombinant proteins, achieving efficient, stable, and long-lasting anticoagulation and anticalcification. The outer layer (tissue contact surface) is grafted with zwitterionic polymers to form a dense hydrated layer, significantly reducing tissue adhesion. Simultaneously, fiber orientation, gradient cross-linking, and micro-sealing design solve the problem of leakage through suture needle holes. This multifunctional vascular patch combines advantages such as low immunogenicity, anticoagulation, anticalcification, anti-adhesion, anti-leakage, and endothelialization promotion, making it suitable for vascular repair and reconstruction of the carotid artery, peripheral arteries, and arteriovenous pathways, with high clinical translational value. Attached Figure Description

[0052] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0053] Figure 1 These are schematic diagrams of the layered structure of the multifunctional vascular patch according to Embodiments 1 to 5 of the present invention. Figure 1The patch of this invention has a three-layer differentiated structure, from top to bottom: an outer zwitterionic anti-adhesion layer, a middle decellularized pericardial substrate layer, and an inner layer carrying a bifunctional recombinant protein hydrogel. The illustration clearly shows the positional relationship of each layer, the interface bonding state, and the overall thickness distribution, reflecting the integrated design of the inner layer for anticoagulation and anti-calcification, the outer layer for anti-adhesion, and the substrate providing mechanical support.

[0054] Figure 2 This is a schematic diagram of the preparation process of the multifunctional vascular patch of Examples 1 to 5 of the present invention; the process includes, in sequence: pericardial tissue pretreatment, hypotonic... surfactants The invention presents a complete industrial-scale preparation route, which combines nuclease with decellularization and antigen removal, inner layer activation and in-situ cross-linking of bifunctional protein hydrogels, outer layer zwitterionic polymer grafting, wet shaping, washing, sterilization and aseptic packaging.

[0055] Figure 3 This image shows the cell coverage rate of the multifunctional vascular patch of Example 1, Comparative Example 1, and Comparative Example 2 after 72 h of endothelial cell culture following inoculation.

[0056] Figure 4 This is a SEM image of the surface of the multifunctional vascular patch of Embodiment 1 of the present invention.

[0057] Figure 5 The graph shows the relative calcium deposition content of the multifunctional vascular patch of Example 1 and Comparative Examples 1-3 after 7 days of simulated body fluid calcification.

[0058] Figure 6 The graph shows the quantitative results of the reduction rate of platelet adhesion in vitro of the multifunctional vascular patch in Examples 1, 1, 2 and 4 of this invention.

[0059] Figure 7 This is a graph showing the in vitro coagulation function test results of the multifunctional vascular patch in Test Example 5 of the present invention.

[0060] Figure 8 This is a graph showing the results of testing the anti-leakage performance of the multifunctional vascular patch under a pressure of 120 mmHg in Test Example 7 of the present invention.

[0061] Figure 9 This is a bar chart comparing the maximum tensile strength and elongation at break of the multifunctional vascular patch tested in Test Example 7 of the present invention.

[0062] Figure 10 This is a comparison diagram of the single-line suture traction force of the multifunctional vascular patch tested in Test Example 7 of the present invention.

[0063] Figure 11The results of color Doppler ultrasound follow-up examination of the multifunctional vascular patch implanted in rabbits in the double-layer modified group of Example 1 of this invention 14 days after implantation.

[0064] Figure 12 The results of ELISA detection of serum IgG, IgM, and IgA levels in SD rats 14 days after implantation of different treated patches are presented in Test Example 9 of this invention. Detailed Implementation

[0065] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions mentioned in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used in this application have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0066] In this application, all features defined as numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges. That is, the endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values; these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the ranges, the endpoint values ​​of the ranges and individual point values, and individual point values ​​can be combined to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein. As used herein, when referring to a specific numerical value, it means that the value can vary within a range of ±5%.

[0067] In the description of this application, unless otherwise stated, the terms "multiple / various" and similar terms mean two / more than one. As used in this application, the terms "containing," "including," or "comprising" can be open-ended, semi-closed, or closed-ended. That is, unless otherwise stated, "containing," "including," "containing," "having," or similar terms cover the meaning of "consisting of" and "mainly composed of," for example, "A contains a" covers the meaning of "A contains a and others" and "A contains only a."

[0068] In this application, for the sake of brevity, not all possible combinations of the technical features in each embodiment or example are described. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each embodiment or example can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0069] In the description of this application, it should be noted that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commonly available materials and reagents that can be purchased commercially.

[0070] 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. Furthermore, it should be understood that after reading the contents of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0071] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" refers to weight percentage, "parts" refers to parts by weight, and "ratio" refers to weight proportion.

[0072] Example 1 A multifunctional bovine pericardial vascular patch is prepared according to the following steps: S1: Take healthy bovine pericardial tissue, remove fat, fascia and blood clots, cut it into 30 mm × 50 mm pieces along the main fiber direction, and wash it 3 times with PBS at 4°C. S2: Treat with hypotonic Hank's solution at 4℃ with shaking for 12 h, followed by 0.1% Triton X. Treatment with 100 + 0.05% SDS for 2 h, followed by treatment with 0.3% Tween 80 for 8 h; treatment with 50 U / mL DNase I + 20 μg / mL RNase A at 37℃ for 4 h; α Galactosidase elutes antigen; S3: The inner layer undergoes initial activation, including: preparing a MES buffer (pH=5.5) containing 0.05 mol / L EDC and 0.02 mol / L NHS; immersing decellularized pericardial blood in contact with the inner layer, facing upwards, at room temperature in the dark for 30 min; after activation, rapidly rinsing twice with pre-cooled sterile PBS for 5 min each time to remove free activating agents; then preparing a hyaluronic acid-carboxymethyl chitosan hydrogel precursor solution, wherein the amount of hyaluronic acid is 10% (w / v), the amount of carboxymethyl chitosan is 5% (w / v), and the remainder is deionized water; adding anticoagulation recombinant protein (SEQ ID NO:2) and anticalcification recombinant protein (SEQ ID NO:2). NO:6), mass ratio 1:2 (the content of the anticoagulation recombinant protein in the hydrogel precursor solution is 8%, and the content of the anticalcification recombinant protein in the hydrogel precursor solution is 16wt%); in-situ curing via EDC / NHS crosslinking: the hydrogel precursor solution is uniformly coated on the activated inner layer, and the crosslinking reaction is carried out at room temperature in the dark for 2 h. The final concentration of EDC in the crosslinking system is 0.02 mol / L, and the final concentration of NHS is 0.01 mol / L; after the crosslinking is completed, the unreacted free crosslinking agent is thoroughly rinsed with sterile PBS to remove it, and a double-protein-loaded hydrogel inner layer with a thickness of 30-50 μm is formed. S4: Perform a second activation on the outer tissue contact surface of the pericardial substrate, including immersing the outer contact surface of the pericardial substrate in a 0.1 mol / L sodium periodate aqueous solution at room temperature in the dark for 20 min; after activation, rinse thoroughly with sterile PBS to terminate oxidation and remove residual oxidant; Then, sulfonate betaine (SBA) polymer is grafted onto the outer layer of the pericardial substrate, including preparing a 20 wt% zwitterionic monomer solution, adding 0.01 mol / L APS initiator, and coating it onto the outer side of the activated substrate; in situ polymerization at 60°C for 4 h to form an anti-adhesion layer with a thickness of 50 nm-80 nm; after polymerization, the free reactants are removed by washing with sterile PBS to form an outer anti-adhesion layer, which is a dense hydrated layer formed by in-situ polymerization grafting; S5: Wet shaping: This involves fully immersing the multifunctional vascular patch, after hydrogel coating and cross-linking, in sterile PBS phosphate buffer (pH 7.2-7.4), laying it flat in a limiting shaping mold without tensile pre-stress, and performing low-temperature equalization and pressure holding treatment at 8℃ and 0.06 MPa for 6 hours under sealed conditions to shape to a total thickness of 0.45-0.55 mm. Then, a gradient cleaning process is performed, including: sequentially rinsing twice each with pre-cooled 75% ethanol aqueous solution, 50% ethanol aqueous solution, and 25% ethanol aqueous solution, with rinsing times of 15 min, 12 min, and 10 min respectively; finally, rinsing three times with sterile PBS for 8 min each time, at a shaker speed of 50 r / min, and operating at 4℃ in the dark throughout the process. After the gradient cleaning is completed, the patch is sterilized with ethylene oxide and aseptically packaged to obtain the multifunctional vascular patch.

[0073] Figure 4 SEM images of the multifunctional vascular patch prepared in Example 1 of this invention are shown. The left image shows the uncrosslinked decellularized bovine pericardium substrate, where collagen fibers are naturally oriented, the surface is dense and smooth, and typical fiber bundle structure is retained. The right image shows the surface of the bilayer modified multifunctional vascular patch, where a uniformly distributed porous hydrogel coating is visible, completely covering the collagen fibers and forming a highly hydrophilic interface. This structure can significantly reduce non-specific protein adsorption and platelet adhesion, endowing the material with excellent anticoagulant and antithrombotic capabilities, confirming that the bifunctional hydrogel layer has been successfully grafted and uniformly distributed on the substrate surface.

[0074] Example 2 A multifunctional bovine pericardial vascular patch is prepared according to the following steps: S1: Take healthy bovine pericardial tissue, remove fat, fascia and blood clots, cut it into 30 mm × 50 mm slices along the main fiber direction, and wash it 3 times with PBS at 4°C. S2: Treat with hypotonic Hank's solution at 4℃ with shaking for 12 h, followed by 0.1% Triton X. Treatment with 100 + 0.05% SDS for 2 h, followed by treatment with 0.3% Tween 80 for 8 h; treatment with 50 U / mL DNase I + 20 μg / mL RNase A at 37℃ for 4 h; α Galactosidase elutes antigen; S3: Perform the first activation on the inner layer, the first activation step is the same as in Example 1; then prepare the methacrylamide gelatin hydrogel precursor solution, wherein the amount of methacrylamide gelatin is 15% (w / v), and the remainder is deionized water; add anticoagulation recombinant protein (SEQ ID NO:5) and anticalcification recombinant protein (SEQ ID NO:7) in a mass ratio of 1:1 (the content of the anticoagulation recombinant protein in the hydrogel precursor solution is 8%, and the content of the anticalcification recombinant protein in the hydrogel precursor solution is 8 wt%); crosslink and cure by blue light: add 0.5 wt% LAP photoinitiator to the hydrogel precursor solution, mix well, and uniformly coat it on the inner layer of the substrate, using 405 nm blue light with a light intensity of 5 mW / cm. 2 Irradiation for 180 s completes in-situ curing, forming a 30-50 μm thick inner layer of double protein-loaded hydrogel; S4: Perform a second activation on the outer tissue contact surface of the pericardial substrate, including immersing the outer contact surface of the outer tissue of the pericardial substrate in a 0.1 mol / L sodium periodate aqueous solution at room temperature in the dark for 20 min; after activation, rinse thoroughly with sterile PBS to terminate oxidation and remove residual oxidant; Then, carboxylate betaine (CBA) polymer is grafted onto the outer layer of the pericardial substrate. This process involves preparing a 20 wt% zwitterionic monomer solution, adding 0.01 mol / L APS initiator, and coating it onto the outer side of the activated substrate. The solution is then in-situ polymerized at 60°C for 4 h to form an anti-adhesion layer with a thickness of 50 nm-80 nm. After polymerization, the free reactants are removed by washing with sterile PBS to form an outer anti-adhesion layer. This anti-adhesion layer is a dense hydrated layer formed by in-situ polymer grafting. S5: The wet-state shaping steps are the same as in Example 1, shaping to a total thickness of 0.45-0.55 mm, followed by gradient cleaning, the gradient cleaning steps are the same as in Example 1, ethylene oxide sterilization, aseptic packaging, to obtain a multifunctional vascular patch.

[0075] Example 3 A multifunctional bovine pericardial vascular patch is prepared according to the following steps: S1: Take healthy bovine pericardium, remove fat, fascia and blood clots, cut it into 30 mm × 50 mm pieces along the main fiber direction, and wash it 3 times with PBS at 4°C. S2: Treat with hypotonic Hank's solution at 4℃ for 12 h with shaking, followed by 0.1% Triton X. Treatment with 100 + 0.05% SDS for 2 h, followed by treatment with 0.3% Tween 80 for 8 h; treatment with 50 U / mL DNase I + 20 μg / mL RNase A at 37℃ for 4 h; α Galactosidase elutes antigen; S3: Perform the first activation on the inner layer, the first activation step is the same as in Example 1; then prepare a dopamine hydrogel precursor solution, using methacrylamide gelatin as the hydrogel precursor material, wherein the amount of methacrylamide gelatin is 15% (w / v), and the remainder is deionized water; add anticoagulation recombinant protein (SEQ ID NO:2) and anticalcification recombinant protein (SEQ ID NO:9) in a mass ratio of 1:4 (the content of the anticoagulation recombinant protein in the hydrogel precursor solution is 8%, and the content of the anticalcification recombinant protein in the hydrogel precursor solution is 32wt%); cross-linking and curing by dopamine adhesion: add 2wt% dopamine hydrochloride to the hydrogel precursor solution, adjust the pH of the system to 8.5, mix well, and then uniformly coat it onto the inner layer of the substrate; let it stand at room temperature for 3 h, and rely on dopamine oxidation self-polymerization to complete in-situ cross-linking and curing, and cure to form a double protein-loaded hydrogel inner layer with a thickness of 30-50 μm. S4: Perform a second activation on the outer tissue contact surface of the pericardial substrate, including immersing the outer contact surface of the outer tissue of the pericardial substrate in a 0.1 mol / L sodium periodate aqueous solution at room temperature in the dark for 20 min; after activation, rinse thoroughly with sterile PBS to terminate oxidation and remove residual oxidant; Then, phosphorylcholine (MPC) polymer is grafted onto the outer layer of the pericardial substrate, including preparing a 20 wt% zwitterionic monomer solution, adding 0.01 mol / L APS initiator, and coating it onto the outer side of the activated substrate; in situ polymerization at 60°C for 4 h to form an anti-adhesion layer with a thickness of 50 nm-80 nm; after polymerization, the free reactants are removed by washing with sterile PBS to form an outer anti-adhesion layer, which is a dense hydrated layer formed by in-situ polymerization grafting; S5: The wet-state shaping steps are the same as in Example 1, shaping to a thickness of 0.45-0.55 mm, followed by gradient cleaning, the gradient cleaning steps are the same as in Example 1, ethylene oxide sterilization, aseptic packaging, to obtain a multifunctional vascular patch.

[0076] Example 4 A multifunctional porcine pericardial vascular patch, the preparation steps are as follows: S1: Take healthy pig pericardium, remove fat, fascia and blood clots, cut it into 30 mm × 50 mm pieces along the main fiber direction, and wash it 3 times with PBS at 4℃. S2: Treat with hypotonic Hank's solution at 4℃ for 12 h with shaking, followed by 0.1% Triton X. Treatment with 100 + 0.05% SDS for 2 h, followed by treatment with 0.3% Tween 80 for 8 h; treatment with 50 U / mL DNase I + 20 μg / mL RNase A at 37℃ for 4 h; α Galactosidase elutes antigen; S3: Perform a first activation on the inner layer, the first activation step being the same as in Example 1; then prepare a dopamine hydrogel precursor solution, wherein the amount of methacrylamide gelatin is 15% (w / v), and the remainder is deionized water; add anticoagulation recombinant protein (SEQ ID NO:2) and anticalcification recombinant protein (SEQ ID NO:9) in a mass ratio of 1:4 (the content of the anticoagulation recombinant protein in the hydrogel precursor solution is 8%, and the content of the anticalcification recombinant protein in the hydrogel precursor solution is 32wt%); cure by dopamine adhesion crosslinking, the dopamine adhesion crosslinking curing step being the same as in Example 3, and cure to form a double-protein-loaded hydrogel inner layer with a thickness of 30-50 μm; S4: Perform a second activation on the outer tissue contact surface of the pericardial substrate, including immersing the outer contact surface of the outer tissue of the pericardial substrate in a 0.1 mol / L sodium periodate aqueous solution at room temperature in the dark for 20 min; after activation, rinse thoroughly with sterile PBS to terminate oxidation and remove residual oxidant; Then, phosphorylcholine (MPC) polymer is grafted onto the outer layer of the pericardial substrate, including preparing a 20 wt% zwitterionic monomer solution, adding 0.01 mol / L APS initiator, and coating it onto the outer side of the activated substrate; in situ polymerization at 60°C for 4 h to form an anti-adhesion layer with a thickness of 50 nm-80 nm; after polymerization, the free reactants are removed by washing with sterile PBS to form an outer anti-adhesion layer, which is a dense hydrated layer formed by in-situ polymerization grafting; S5: The wet-state shaping steps are the same as in Example 1, shaping to a thickness of 0.45-0.55 mm, followed by gradient cleaning, the gradient cleaning steps are the same as in Example 1, ethylene oxide sterilization, aseptic packaging, to obtain a multifunctional vascular patch.

[0077] Example 5 A multifunctional porcine pericardial vascular patch, the preparation steps are as follows: S1: Take healthy pig pericardium, remove fat, fascia and blood clots, cut it into 30 mm × 50 mm pieces along the main fiber direction, and wash it 3 times with PBS at 4°C. S2: Treat with 4℃ hypotonic Hank's solution by shaking for 12 h, followed by 0.1% Triton X. Treatment with 100 + 0.05% SDS for 2 h, followed by treatment with 0.3% Tween 80 for 8 h; treatment with 50 U / mL DNase I + 20 μg / mL RNase A at 37℃ for 4 h; α Galactosidase elutes antigen; S3: Perform a first activation on the inner layer, the first activation step being the same as in Example 1; then prepare a hyaluronic acid-carboxymethyl chitosan hydrogel precursor solution, wherein the amount of hyaluronic acid is 10% (w / v), the amount of carboxymethyl chitosan is 5% (w / v), and the remainder is deionized water; add anticoagulation recombinant protein (SEQ ID NO:2) and anticalcification recombinant protein (SEQ ID NO:6) in a mass ratio of 1:2 (the content of the anticoagulation recombinant protein in the hydrogel precursor solution is 8%, and the content of the anticalcification recombinant protein in the hydrogel precursor solution is 16wt%); cure in situ through EDC / NHS crosslinking, the EDC / NHS crosslinking curing step being the same as in Example 1, and cure to form a double-protein-loaded hydrogel inner layer with a thickness of 30-50 μm; S4: Perform a second activation on the outer tissue contact surface of the pericardial substrate, including immersing the outer contact surface of the pericardial substrate in a 0.1 mol / L sodium periodate aqueous solution at room temperature in the dark for 20 min; after activation, rinse thoroughly with sterile PBS to terminate oxidation and remove residual oxidant; Then, sulfonate betaine (SBA) polymer is grafted onto the outer layer of the pericardial substrate, including preparing a 20 wt% zwitterionic monomer solution, adding 0.01 mol / L APS initiator, and coating it onto the outer side of the activated substrate; in situ polymerization at 60°C for 4 h to form an anti-adhesion layer with a thickness of 50 nm-80 nm; after polymerization, the free reactants are removed by washing with sterile PBS to form an outer anti-adhesion layer, which is a dense hydrated layer formed by in-situ polymerization grafting; S5: The wet-state shaping steps are the same as in Example 1, shaping to a thickness of 0.45-0.55 mm, followed by gradient cleaning, the gradient cleaning steps are the same as in Example 1, ethylene oxide sterilization, aseptic packaging, to obtain a multifunctional vascular patch.

[0078] Figure 1 These are schematic diagrams of the layered structure of the multifunctional vascular patch according to Embodiments 1 to 5 of the present invention. Figure 2 This is a schematic diagram of the preparation process of the multifunctional vascular patch of Embodiments 1 to 5 of the present invention.

[0079] Comparative Example 1: Uncrosslinked group The difference from Example 1 is that the EDC / NHS crosslinking in-situ curing step is missing; all other steps are the same as in Example 1.

[0080] Comparative Example 2 Crosslinking Group The difference from Example 1 is that the modification steps of inner layer protein hydrogel grafting in step S3 and outer layer zwitterionic grafting in step S4 are omitted. Only the cross-linking and curing treatment of the substrate is completed. All other steps are the same as in Example 1.

[0081] Comparative Example 3 Inner Layer Modification Group The difference from Example 1 is that only the grafting modification of the inner layer protein hydrogel in step S3 is completed, and the in-situ polymerization grafting of the outer layer zwitterionic polymer in step S4 is not performed. All other steps are the same as in Example 1.

[0082] Comparative Example 4: Outer Layer Modification Group The difference from Example 1 is that only the in-situ polymerization and grafting modification of the outer zwitterionic polymer in step S4 is completed, and the grafting modification of the inner protein-carrying hydrogel in step S3 is not performed. All other steps are the same as in Example 1.

[0083] Test Example 1 This test example measures the cell coverage of the multifunctional vascular patch. The experimental method includes: grouping the samples of the multifunctional vascular patches prepared in Comparative Examples 1, 2, and 1 of this invention into non-crosslinked, crosslinked, and bilayer modified groups, with n=6 in each group; sterilizing and rinsing the multifunctional vascular patches of each group and then laying them flat on a culture plate at a density of 2×10⁻⁶. 4 cells / cm 2 HUVECs were densely seeded and cultured continuously at 37°C and 5% CO2 for 72 h. After cell fixation and staining, the endothelial cell coverage was statistically analyzed by confocal imaging.

[0084] The quantitative statistical results of human umbilical vein endothelial cells (HUVECs) cultured on the surface of the multifunctional vascular patches of Examples 1, 1, and 2 of this invention after 72 h are as follows: Figure 3 As shown in the figure. The results showed that the cell coverage rate of the uncrosslinked group was only about 34.7%, while the crosslinked group showed a slight improvement. The cell coverage rate of the inner layer of the bi-functional recombinant protein hydrogel in the bilayer modified group (Example 1) reached more than 60%, indicating that functional modification can provide a biomimetic hydrophilic microenvironment, significantly promote endothelial cell adhesion, spreading and migration, effectively improve the material's endothelialization ability, reduce the risk of postoperative thrombosis and intimal hyperplasia, and meet the requirements for rapid endothelialization of vascular implants.

[0085] Test Example 2 This test example measures the relative calcium deposition content of a multifunctional vascular patch after 7 days of calcification in a 1.5×SBF simulated body fluid. The experimental method included: grouping the multifunctional vascular patch samples prepared in Comparative Examples 1, 2, 3, and 1 of this invention into non-crosslinked, crosslinked, inner layer modified, and double layer modified groups, with n=6 in each group; preparing a 1.5×SBF simulated body fluid with pH=7.40; the 1L system components were: NaCl 130.67 g, NaHCO3 11.34 g, KCl 4.47 g, K2HPO4 trihydrate 3.61 g, MgCl2 hexahydrate 6.11 g, CaCl2 4.17 g, and Na2SO4 1.34 g; adjusting the pH to 7.40 using a Tris buffer system and 1 mol / L HCl; immersing each group of multifunctional vascular patch samples in the simulated body fluid and incubating at 37°C with shaking for 7 days, followed by incubation every 48 days. Replace with fresh 1.5×SBF; after incubation, rinse, dry, and digest with nitric acid, and determine the calcium content using ICP-OES. The relative calcium deposition content is calculated based on the calcium deposition amount of the uncrosslinked group.

[0086] ICP OES was used to determine the calcium content, and the relative calcification ratio was calculated with the non-crosslinked group as 100%. The results are as follows: Figure 5 As shown in the figure. The results showed that the non-crosslinked group had the most severe calcium deposition, while the crosslinked group showed a slight reduction in calcification. The calcium deposition in the inner layer modified group and the double layer modified group was reduced to less than 44.5% of that in the non-modified group, achieving the design target of ≤50%. This indicates that the anti-calcification recombinant protein can efficiently chelate calcium ions and inhibit hydroxyapatite deposition.

[0087] Test Example 3 This test case examines the in vitro platelet adhesion reduction rate of the multifunctional vascular patch. The experimental method includes: grouping the multifunctional vascular patch samples prepared in Comparative Examples 1, 2, 4, and 1 of this invention into non-crosslinked, crosslinked, outer layer modified, and double layer modified groups, respectively, with n=6 in each group. Then, venous blood from healthy individuals is collected to prepare platelet-rich plasma (PRP), with a platelet concentration of (2.0~3.0)×10⁻⁶. 8 (cells / mL, approximately 2-3 times the concentration of whole blood platelets), the multifunctional vascular patch samples of each group were incubated with PRP at 37°C for 1 h; unbound platelets were removed by PBS washing.

[0088] The number of adherent platelets was quantified using the LDH lysis method, and the platelet adhesion reduction rate was calculated with the non-crosslinked group as a control. The results are as follows: Figure 6As shown in the figure. The results indicate that cross-linking treatment can slightly reduce platelet adhesion, while the outer layer modified group and the bilayer modified group significantly improve the anticoagulant effect, with a platelet adhesion reduction rate of over 60%, confirming that the interface between the bifunctional protein hydrogel and zwitterionic molecules can significantly inhibit platelet adhesion and coagulation activation.

[0089] Test Example 4 This test case is used to verify the in vitro anticoagulant function of the anticoagulant protein sequence of the present invention. The inhibitory effect of the protein on the coagulation process is determined by detecting coagulation indicators. The specific steps are as follows: S1: The anticoagulated recombinant protein of SEQ ID NO: 1-5 was prepared into protein solutions with gradient concentrations of 0.1 μg / mL, 1 μg / mL, 10 μg / mL and 100 μg / mL using PBS buffer. S2: Take fresh citrate-anticoagulated plasma from healthy individuals, mix it evenly with protein solution at a volume ratio of 1:9, incubate at 37°C for 10 min, and set up pure PBS-treated plasma as a blank control group; S3: The activated partial thromboplastin time (APTT) and prothrombin time (PT) of plasma in each group were measured using a fully automated coagulation analyzer, with 3 parallel samples set up for each group; S4: Statistically analyze and compare the changes in clotting time in each group, and analyze the coagulation inhibitory activity of anticoagulant proteins.

[0090] Experimental results: All anticoagulant proteins exhibited concentration-dependent anticoagulant effects. At a concentration of 100 μg / mL, proteins SEQ ID NO:1-5 all prolonged plasma APTT by more than 30% compared to the control group. Among them, SEQ ID NO:2 and SEQ ID NO:4 showed the best anticoagulant effects, with APTT prolongation exceeding 40%, and also slightly prolonged PT. This confirms that the anticoagulant proteins of the present invention possess excellent intrinsic coagulation inhibitory activity, effectively delaying blood coagulation, and forming the core functional basis for the overall anticoagulant performance of the multifunctional vascular patch.

[0091] Test Example 5 This test example examines the in vitro coagulation function of the multifunctional vascular patch. The experimental method includes: grouping the multifunctional vascular patch samples prepared in Comparative Examples 1, 2, 3 and 1 of this invention into non-crosslinked group, crosslinked group, monolayer modified group and bilayer modified group, respectively; the blank control group was treated with pure PBS; n=6 in each group; and then co-incubating each group of multifunctional vascular patches with anticoagulated plasma from healthy individuals. The APTT and PT coagulation indicators were detected by a fully automated coagulation analyzer.

[0092] Figure 7This paper presents the in vitro coagulation function test results of the multifunctional vascular patch of Example 1, Comparative Examples 1-3, and the blank control group. The results show that the PT clotting time in the non-crosslinked group was not significantly different from that in the blank plasma. The crosslinked group slightly prolonged both APTT and PT clotting times. The inner layer modified group showed a significantly improved anticoagulant effect, and the double-layer modified group showed significantly prolonged APTT and PT with the best effect. P <0.05, P <0.01 (compared to the blank control group), demonstrating that the bifunctional composite modified structure can effectively inhibit the coagulation cascade reaction and endow the patch with excellent anticoagulant properties.

[0093] Figure 7 The significance markers (asterisks) were determined based on the statistical analysis results of the raw data from this batch of in vitro coagulation tests: one-way ANOVA was used, combined with Bonferroni multiple comparison tests; the significance markers were defined as follows: P <0.05, P <0.01, the marked group is compared with the blank control group.

[0094] Test Example 6 This test case is used to verify the calcium ion chelating ability and hydroxyapatite inhibition ability of the anti-calcification protein sequence of the present invention. The specific steps are as follows: S1: Prepare 100 μmol / L solutions of each anticalcification protein (SEQ ID NO:6-10) and 10 mmol / L CaCl2 titrant. Titrate at 25℃ using isothermal titration calorimetry to determine the binding constant Ka of each protein to calcium ions and the number of binding sites. S2: Prepare a solution containing 8 mmol / L Ca 2+ 4 mmol / L HPO4 2- High-calcium simulated body fluid SBF was prepared by adding each anti-calcification protein to a final concentration of 100 μg / mL, and a protein-free blank SBF control group was set up. S3: Incubate at 37℃ for 72 h, and use a spectrophotometer to detect the turbidity of the system at 600 nm. Combine with ICP-OES to detect the residual calcium ion concentration in the supernatant. Three parallel samples were set up for each group. S4: Compare the changes in turbidity and the rate of calcium ion consumption in each group to evaluate the protein's anti-calcification activity.

[0095] Experimental results: The binding constant Ka of the anticalcification protein (SEQ ID NO:6-10) to calcium ions is ≥10. 4 M -1SEQ ID NO:7 and SEQ ID NO:9 exhibit the best binding performance, with a Ka value reaching 10. 5 M -1 In conclusion, compared to the control group, the protein experimental group showed a significantly reduced rate of turbidity increase and a more than 50% reduction in calcium ion consumption, effectively delaying the formation of hydroxyapatite precipitation. This confirms that the anti-calcification protein of this invention possesses excellent calcium ion chelation and calcium salt crystal growth inhibition capabilities, providing core support for the long-term anti-calcification performance of the patch.

[0096] Test Example 7 The multifunctional bovine pericardial vascular patch prepared in Example 1 was cut into standard samples of 10 mm × 50 mm. Mechanical properties were tested along the main direction of collagen fibers. Six parallel samples were set up in each group, and the average value of the results was taken.

[0097] 1. Using an electronic universal testing machine, rectangular strips of specimens were cut with an initial gauge length of 20 mm. Uniaxial tensile tests were conducted at a tensile rate of 20 mm / min. The maximum tensile strength and elongation at break of the multifunctional vascular patches prepared in Example 1, Comparative Example 1, and Comparative Example 2 were measured. Figure 9 This is a bar chart comparing the maximum tensile strength and elongation at break of the multifunctional vascular patches obtained in Test Example 7 of this invention, with n=6 for each group. The results show that the uncrosslinked group has lower strength and higher flexibility; the tensile strength is significantly improved and the elongation at break is reasonably reduced after gradient crosslinking; the single-layer and double-layer modified groups have further improved mechanical strength while maintaining good elastic compliance, and the curve shape is highly matched with natural blood vessels, which can meet the mechanical support requirements during vascular reconstruction surgery and the requirements for long-term dynamic service.

[0098] 2. Maximum tensile elongation and elastic deformation rate test: The specimen is clamped in an electronic universal testing machine with a clamping distance of 30 mm, and subjected to uniaxial tension at a rate of 100 mm / min until fracture. The load is recorded. Displacement curve. The elastic deformation rate was calculated based on the linear recoverable interval in the curve, and the maximum tensile elongation was calculated at fracture. The results show that the maximum tensile elongation of the patch of the present invention is 26.3%, the elastic deformation rate is 20.1%, and the elastic deformation ratio reaches 76.4%, indicating that it has good elastic recovery ability and can adapt to vascular pulsation deformation.

[0099] 3. The single-line suture tensile strength test uses 6 A polypropylene suture was inserted through a single needle 2 mm from the edge of the sample and pulled at a constant speed of 100 mm / min. The maximum tensile force at which the suture tore or the material broke was recorded. Figure 10 To compare the single-line suture traction force of the multifunctional vascular patch samples prepared in Comparative Examples 1, 2, and 1 of this invention, they were grouped into an uncrosslinked group, a crosslinked group, and a double-layer modified group, with n=6 in each group. Six... A single-point suture with polypropylene suture was performed, and the suture was pulled at a rate of 100 mm / min until failure, with the maximum tensile force recorded. Results showed that the single-line suture tensile force of the patch of this invention was 32.6 N, meeting the clinical requirements for vascular suture tear resistance. The uncrosslinked group showed weaker tear resistance. After gradient crosslinking, the suture strength was significantly improved. The double-layer modified group consistently achieved a tensile force of approximately 32.0 N, far exceeding the clinical safety threshold, effectively reducing the risks of intraoperative suture tearing and pinhole dehiscence, and improving surgical safety and reliability.

[0100] 4. The bursting strength test was conducted using a 5 mm diameter spherical indenter, which was vertically applied to the center of the sample at a rate of 100 mm / min. The maximum force at which the patch burst was recorded. The results showed that the bursting strength of the patch of this invention was 64.8 N, demonstrating good resistance to localized high pressure and damage from instrument handling.

[0101] 5. The anti-leakage performance test was conducted using a simulated vascular pressurization device, and the permeation volume per unit area per unit time was measured at pressures of 120 mmHg and 250 mmHg, respectively. Figure 8 The results show the leakage of the multifunctional vascular patch samples prepared in Comparative Examples 1, 2, and 1 of this invention, divided into uncrosslinked, crosslinked, and bilayer modified groups, under a pressure of 120 mmHg, with n=6 for each group. The fluid permeation per unit area per unit time was measured using a simulated vascular pressurization device. The results showed that the uncrosslinked group had the highest leakage. After gradient crosslinking and surface modification, the leakage significantly decreased, with the bilayer modified group showing a leakage as low as 0.11 mL / (cm²) at 120 mmHg. 2 •min), satisfying ≤0.15 mL / (cm 2 The clinical requirement is 0.38 mL / (cm³) at 250 mmHg. 2 All of them (·min) meet the clinical standards for anti-leakage of vascular patches, indicating that the fiber orientation, gradient cross-linking and inner and outer layer micro-sealing design of the multifunctional vascular patch can effectively solve the problem of leakage through suture needle holes.

[0102] In summary, the vascular patch of the present invention has stable mechanical properties, and its tensile, suture, puncture, and anti-leakage indices are all within the ideal clinical window. At the same time, it maintains good elasticity and compliance, and can meet the usage requirements of various vascular repair scenarios such as carotid artery, peripheral artery, and arteriovenous access.

[0103] Test Example 8 This test case examines the ultrasound imaging of the multifunctional vascular patch prepared in Example 1 of this invention implanted in an adult rabbit. The experimental method included: selecting healthy adult rabbits and constructing a carotid artery defect model; after general anesthesia, the rabbit's neck was prepared and disinfected, an incision was made along the midline of the neck, and the carotid artery on one side was exposed layer by layer. The vessel segment was freed, blood flow to the proximal and distal ends of the vessel was blocked, and approximately 8 mm of vessel wall tissue was removed to form a lateral wall defect; the multifunctional vascular patch of Example 1 was implanted for vascular patch formation, and the patch was continuously sutured to the vessel edge using 60 absorbable sutures; after suturing, the vascular occlusion was released, and after confirming no significant bleeding, the surgical incision was closed layer by layer; the rabbit was fed normally for 14 days post-surgery, and high-frequency ultrasound (frequency 12-15MHz) was used to scan and image the implanted area to observe the patch morphology, lumen patency, vessel wall hyperplasia, and complications.

[0104] Figure 11 This is the color Doppler ultrasound follow-up result of the multifunctional vascular patch of Example 1 of the present invention (i.e., the double-layer modified group) implanted in rabbits 14 days after surgery. The ultrasound imaging results showed that 14 days after surgery, the patch vascular morphology was intact, the lumen remained patent, the patch adhered well to the host blood vessels, the degree of vessel wall hyperplasia was low, and no complications such as thrombosis or vascular occlusion occurred.

[0105] Test Example 9 This test case examines the immune effects of the multifunctional vascular patch prepared in Example 1 and Comparative Examples 1-3 of this invention implanted into rats.

[0106] The experimental methods included: SD rats were randomly divided into a blank control group, a non-crosslinked group, a crosslinked group, a monolayer modified group, and a bilayer modified group. Except for the blank group (normal rats without implanted patches), the multifunctional vascular patches of Comparative Example 1, Comparative Example 2, and Example 1 were implanted subcutaneously in the other groups of rats, respectively. Peripheral blood was collected and serum was separated after 14 days of feeding. The serum IgG, IgM, and IgA concentrations were quantitatively detected using an ELISA kit.

[0107] Figure 12 The serum IgG, IgM, and IgA levels in SD rats were detected by ELISA 14 days after implantation of different treated patches, with a sample size of n=6 for each group. Compared with the blank control group, implantation of the non-crosslinked multifunctional vascular patch significantly induced humoral immune responses in rats, with the highest immunoglobulin concentration; the immunological indicators of the multifunctional vascular patch implanted in the crosslinked group were lower than those in the non-crosslinked group; and the immunosuppressive effect was most significant in the bilayer modified group. This demonstrates that crosslinking treatment can reduce the basic immunogenicity of the patch, and zwitterionic grafting can further inhibit immune activation. The multifunctional vascular patch in Example 1 is the best bilayer modified patch with low immunogenicity.

[0108] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a multifunctional vascular patch, characterized in that, Includes the following steps: S1: Take healthy mammalian pericardial tissue, remove fat, fascia and blood clots, cut along the direction of the main collagen fibers, and then wash with PBS at low temperature; S2: The cleaned mammalian pericardial tissue was treated with a combination of hypotonic solution, surfactant and nuclease to decellularize and remove antigens, so as to obtain decellularized pericardial substrate. S3: The inner blood contact surface of the pericardial substrate is first activated, coated and crosslinked with a hydrogel precursor fluid containing anticoagulant recombinant protein and anticalcification recombinant protein to form a hydrogel inner layer carrying bifunctional proteins in situ; the anticoagulant recombinant protein includes one or more of SEQ ID NO:1 to SEQ ID NO:5, or a variant having at least 80% sequence identity with it and without significantly altering its anticoagulant compatibility regulatory function; the anticalcification recombinant protein includes one or more of SEQ ID NO:6 to SEQ ID NO:10, or a variant having at least 80% sequence identity with it and without significantly altering its anticalcification function; The mass ratio of the anticoagulation recombinant protein to the anticalcification recombinant protein is (0.5-1):(1-5); The anticoagulant recombinant protein contains 5-15 wt% in the hydrogel precursor solution. The content of anti-calcification recombinant protein in the hydrogel precursor solution is 5-75 wt%; S4: Perform a second activation on the outer tissue contact surface of the pericardial substrate, grafting zwitterionic polymers to form an outer anti-adhesion layer; S5: After wet shaping, gradient cleaning, sterilization and packaging, the target vascular patch is obtained.

2. The method for preparing the multifunctional vascular patch according to claim 1, characterized in that, In step S1, the mammal is a pig or a cow; and / or In step S1, the temperature range of the low temperature is 2-8°C; and / or In step S1, the solution used for the cleaning includes at least one of PBS, HBSS, HBS, and Tris saline.

3. The method for preparing the multifunctional vascular patch according to claim 1, characterized in that, In step S2, the hypotonic treatment lasts for 10-20 hours; and / or In step S2, the temperature range of the hypotonic treatment is no more than 10°C; and / or In step S2, the surfactant includes at least one of Triton X-100, Tween-20, and Tween-80; and / or In S2, the nuclease includes at least one of DNase I, RNase A, and DNase II.

4. The method for preparing the multifunctional vascular patch according to claim 1, characterized in that, In step S3, the mass ratio of the anticoagulation recombinant protein to the anticalcification recombinant protein is 1:(1-4); and / or In step S3, the content of the anticoagulant recombinant protein in the hydrogel precursor solution is 8-12 wt%; and / or In step S3, the content of the anti-calcification recombinant protein in the hydrogel precursor solution is 10-50 wt%; and / or In step S3, the hydrogel precursor solution further comprises one or more of the following: hyaluronic acid, methacrylamide gelatin, carboxymethyl chitosan, or alginate; and / or In step S3, the hydrogel precursor solution forms a uniform functional layer with a thickness of 20-120 μm through photocrosslinking, enzymatic crosslinking, Schiff base crosslinking, EDC / NHS crosslinking, maleimide-thiol coupling, or dopamine adhesion crosslinking.

5. The method for preparing the multifunctional vascular patch according to claim 1, characterized in that, In step S4, the zwitterionic polymer is a sulfobetaine (SBA), carboxybetaine (CBA), or phosphorylcholine (MPC) polymer; and / or In step S4, the outer anti-adhesion layer is a dense hydrated layer formed by in-situ polymerization grafting.

6. The method for preparing the multifunctional vascular patch according to claim 1, characterized in that, In step S2, the decellularization treatment includes: treatment with hypotonic Hank's solution at 4°C for 12 h, followed by 0.1% Triton X. Treatment with 100+0.05% SDS for 2 h, followed by treatment with 0.3% Tween 80 for 8 h; nuclease treatment with 50 U / mL DNase I+20 μg / mL RNase A at 37℃ for 4 h; antigen elution with α-galactosidase.

7. A multifunctional vascular patch, characterized in that, It is prepared by any one of claims 1-6.

8. A medical device, characterized in that, It includes the multifunctional vascular patch as described in claim 7.

9. The multifunctional vascular patch according to claim 7, characterized in that, The multifunctional vascular patch meets the following requirements: (1) Leakage rate ≤ 0.15 mL / (cm²) at 120 mmHg 2 (min), leakage rate at 250 mmHg ≤ 0.50 mL / (cm) 2 min); (2) Platelet adhesion decreased by ≥60%; (3) The amount of calcium deposition is ≤50% lower than that of the unmodified patch; (4) Endothelial cell coverage ≥60% at 72 h.

10. The application of the multifunctional vascular patch prepared by any one of claims 1-6, or the multifunctional vascular patch according to claim 7 or 9, or the medical device according to claim 8, in carotid endarterectomy patches, peripheral artery patches, arteriovenous access repair patches, and vascular stent covering materials.

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