Biomimetic bioprosthesis

Abstract

The present application relates to the field of biomaterials, and particularly relates to a biomimetic biological valve. The biomimetic biological valve is obtained by wrapping a non-crosslinked biological basement membrane around a crosslinked extracellular matrix material to form a sandwich structure. The basement membrane cooperates with the crosslinked extracellular matrix material, fully plays the excellent regenerative repair performance and the characteristics of being rich in bioactive factors of the basement membrane, effectively promotes the growth and adhesion of endothelial cells on the surface of the material, and constructs a tissue structure similar to the natural valve leaflet. The biomimetic biological valve exhibits excellent anti-calcification performance and excellent dynamic mechanical stability and compliance, significantly inhibits the abnormal adhesion of platelets and blood cells, greatly improves the recovery and reconstruction efficiency of the valve function, and can maintain the structural integrity in repeated contraction-expansion cycles, providing an ideal solution for long-term stable application in a physiological environment.

Description

Technical Field

[0001] This invention relates to the field of biomaterials, specifically to a biomimetic bio-valve. Background Technology

[0002] Heart valve replacement surgery is currently the main treatment for valvular heart disease. Heart valves commonly used in valve replacement surgery are mainly divided into mechanical and biological valves. While mechanical valves have high mechanical strength, their poor biocompatibility requires long-term anticoagulation therapy. Biological valves, on the other hand, have good biocompatibility and hemodynamic performance, resulting in better treatment outcomes and are currently the mainstream product. Traditionally, glutaraldehyde-treated pericardial tissue has been used as a biological valve. However, this type of tissue cannot regenerate cells. Without its own repair mechanisms, this non-regenerative tissue cannot cope with wear, fatigue, or calcification damage over long-term use. As its performance gradually declines, micro-cracks widen, accelerating valve failure, thus resulting in a very limited lifespan.

[0003] The patent disclosed in patent number CN115920132A is a bioprosthetic valve with a cross-linked amniotic membrane encapsulating a cross-linked pericardium. The encapsulated structure is further cross-linked as a whole under the initiation of ammonium persulfate and sodium bisulfite to improve the binding force between different membrane layers. The inherent defects of the pericardium are improved by utilizing the regenerative cells of the amniotic membrane.

[0004] However, the amnion's mechanical strength is insufficient. As described in patent CN115920132A, it generally needs to be cross-linked to achieve the mechanical properties required for valves. Secondly, as the surface layer, cross-linking of the amnion also increases the risk of calcification, and the high cost of amnion sources makes mass production difficult. Summary of the Invention

[0005] To overcome the above problems, this invention uses a non-crosslinked biological basement membrane to encapsulate the crosslinked extracellular matrix. This encapsulates the crosslinked extracellular matrix, which has a risk of calcification after crosslinking, so that the bioprosthetic valve has regenerative function. Not only does it not increase the risk of calcification, but it also has a certain anti-calcification ability and has the prospect of mass production.

[0006] The first aspect of the present invention provides a biomimetic biovalve, which is obtained by wrapping a cross-linked extracellular matrix material with a non-cross-linked biological basement membrane to form a sandwich structure.

[0007] The biomimetic bio-valve provided by the first aspect of the present invention comprises a transition layer between a non-crosslinked biological basement membrane and a crosslinked extracellular matrix material, the transition layer comprising a biocompatible colloidal matrix treated with a coupling agent. The biocompatible colloidal matrix has a concentration of at least 8-50 μL / cm³. 2 Biocompatible colloidal matrix dispersions and 1-10 μL / cm 2The coupling agent aqueous solution solidifies to form the coupling agent.

[0008] Examples of biocompatible colloidal matrices include those with a concentration of at least 8-50 μL / cm³. 2 Biocompatible colloidal matrix dispersions and 1-7 μL / cm 2 The coupling agent aqueous solution solidifies to form the coupling agent.

[0009] Examples of biocompatible colloidal matrices include those with a composition of at least 8-45 μL / cm³. 2 Biocompatible colloidal matrix dispersions and 1-10 μL / cm 2 The coupling agent aqueous solution solidifies to form the coupling agent.

[0010] Examples of biocompatible colloidal matrices include those with a composition of at least 8-45 μL / cm³. 2 Biocompatible colloidal matrix dispersions and 1-7 μL / cm 2 The coupling agent aqueous solution solidifies to form the coupling agent.

[0011] Examples of biocompatible colloidal matrices include those with a concentration of at least 8-50 μL / cm³. 2 Biocompatible colloidal matrix dispersions and 1-5 μL / cm 2 The coupling agent aqueous solution solidifies to form the coupling agent.

[0012] Examples of biocompatible colloidal matrices include those with a composition of at least 8-45 μL / cm³. 2 Biocompatible colloidal matrix dispersions and 1-5 μL / cm 2 The coupling agent aqueous solution solidifies to form the coupling agent.

[0013] Examples of biocompatible colloidal matrices include those with a composition of at least 8-44 μL / cm³. 2 Biocompatible colloidal matrix dispersions and 1-5 μL / cm 2 The coupling agent aqueous solution solidifies to form the coupling agent.

[0014] Examples of biocompatible colloidal matrices include those with a composition of at least 8-44 μL / cm³. 2 Biocompatible colloidal matrix dispersions and 1-4 μL / cm 2 The coupling agent aqueous solution solidifies to form the coupling agent.

[0015] The transition layer is formed by spraying a biocompatible colloidal matrix dispersion and a coupling agent aqueous solution onto a non-crosslinked biological basement membrane and / or a crosslinked extracellular matrix material, respectively, and then stacking the layers sequentially and consolidating them to form a whole.

[0016] The preparation method of the biomimetic bio-valve is as follows: spray 8-50 μL / cm onto a non-crosslinked biological basement membrane. 2 Biocompatible colloidal matrix dispersions and 1-7 μL / cm 2A coupling agent aqueous solution was applied, followed by the stacking of cross-linked extracellular matrix materials, and then sprayed at a concentration of 8-50 μL / cm². 2 Biocompatible colloidal matrix dispersions and 1-7 μL / cm 2 After applying the coupling agent aqueous solution, non-crosslinked biological basement membranes are stacked; the membranes are then pressed together under a heating environment (37-50℃) for 8-16 hours to obtain the final product.

[0017] Natural valve tissue consists of three layers: the ventricular layer, the spongy layer, and the fibrous layer, with a total thickness not exceeding 1 mm. The ventricular and fibrous layers provide mechanical integrity, while the spongy layer, located between them, cushions the pressure during cardiac contraction. Interstitial cells exist in the ventricular and fibrous layers near the spongy layer, resulting in indistinct boundaries and smooth transitions between the layers. This invention simulates this natural structure through biomimetic design, exhibiting distinct biomimetic features from the initial implantation stage: the inner layer uses a cross-linked extracellular matrix for shock absorption, while the outer layer uses a non-cross-linked biological basement membrane to simulate the ventricular and fibrous layers, with an intermediate transition layer connecting the two layers. Specifically, the outer non-cross-linked biological basement membrane effectively guides cell attachment and promotes proliferation in the early implantation stage. As the outer non-cross-linked biological basement membrane gradually degrades, the released space is occupied by a network of newly formed cells, ultimately forming a three-dimensional biomimetic structure encapsulating the cross-linked extracellular matrix, achieving a functional simulation closer to natural tissue.

[0018] However, the composite of cross-linked and non-cross-linked membrane materials faces significant technical challenges: insufficient interfacial bonding and mismatched mechanical properties lead to poor immediate composite strength, easily resulting in wrinkles or localized fractures due to uneven stress distribution. Creative research has revealed that, before consolidating the individual membrane layers into a single unit, simultaneously applying a specific area concentration of biocompatible colloidal matrix dispersion and coupling agent between the non-cross-linked biological basement membrane and the cross-linked extracellular matrix material not only significantly improves the problem of poor composite strength but also unexpectedly enhances the biomimetic valve's guiding effect on cell attachment and growth promotion, and helps improve anti-calcification effects. This may be due to the 8-50 μL / cm 2 Biocompatible colloidal matrix dispersions and 1-10 μL / cm 2The coupling agent, in synergy with the aqueous solution, exhibits a certain strain coordination effect and forms an ideal surface energy gradient on the surfaces of both non-crosslinked biological basement membranes and crosslinked extracellular matrix materials. This optimizes intermolecular forces and provides appropriate surface coupling effects to both membranes. This avoids the problem of reduced activity due to interfacial crosslinking caused by the coupling agent and also controls the significant swelling of biocompatible colloidal matrices (such as gelatin, chitosan, and dextran) after water absorption. Therefore, the composite strength and guided-degradation-release effect between the non-crosslinked biological basement membrane and the crosslinked extracellular matrix material are significantly improved. This improvement is most pronounced for gelatin colloidal matrices, which are highly absorbent.

[0019] Furthermore, the area concentration ratio of the biocompatible colloidal matrix dispersion to the coupling agent aqueous solution is (7-12):1.

[0020] Furthermore, the area concentration ratio of the biocompatible colloidal matrix dispersion to the coupling agent aqueous solution is (8-11):1.

[0021] Furthermore, the area concentration ratio of the biocompatible colloidal matrix dispersion to the coupling agent aqueous solution is 10:1.

[0022] In a preferred embodiment, the biocompatible colloidal matrix dispersion includes at least one of gelatin aqueous dispersion, chitosan aqueous dispersion, and dextran aqueous solution:

[0023] Preferably, the biocompatible colloidal matrix dispersion comprises at least one of the following components:

[0024] A gelatin aqueous dispersion of 3wt%-17wt%;

[0025] Chitosan aqueous dispersions ranging from 1.5 wt% to 24.5 wt%;

[0026] A 2.1wt%-20.4wt% aqueous solution of dextran.

[0027] In a preferred embodiment, the biocompatible colloidal matrix dispersion comprises at least one of the following components:

[0028] A gelatin aqueous dispersion of 3wt%-17wt%;

[0029] A 4.1wt%-18.4wt% aqueous solution of dextran.

[0030] Biocompatible colloidal matrix dispersions may include at least one of the following components:

[0031] A gelatin aqueous dispersion of 3wt%-17wt%;

[0032] A 7wt%-10wt% aqueous solution of dextran.

[0033] Biocompatible colloidal matrix dispersions may include at least one of the following components:

[0034] A gelatin aqueous dispersion of 3wt%-17wt%;

[0035] An 8wt%-9wt% aqueous solution of dextran.

[0036] As a preferred embodiment, the biocompatible colloidal matrix dispersion comprises at least 3wt%-17wt% gelatin aqueous dispersion.

[0037] Examples of biocompatible colloidal matrix dispersions include at least 6.2 wt% to 16.7 wt% gelatin aqueous dispersions.

[0038] In a preferred embodiment, the biocompatible colloidal matrix dispersion further includes at least one of determinate collagen and collagen.

[0039] Preferably, the biocompatible colloidal matrix dispersion also includes determinated collagen.

[0040] Further preferably, the biocompatible colloidal matrix dispersion also includes 0.3-0.5 wt% of determinate collagen.

[0041] One example of a method for preparing a biocompatible colloidal matrix dispersion is as follows: gelatin, chitosan, and / or dextran are added to deionized water, dispersed using a magnetic stirrer at 45-70°C, and then determinated collagen and / or collagen are added and dispersed by stirring.

[0042] In a preferred embodiment, the concentration of the coupling agent in the aqueous solution is 0.08-1.4 g / mL.

[0043] As can be listed, the coupling agent is selected from at least one of carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), N-hydroxythiosuccinimide (Slufo-NHS), N,N′-diisopropylcarbodiimide (DIC), and N,N′-dicyclohexylcarbodiimide (DCC).

[0044] Further examples include two of the following coupling agents: carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), N-hydroxythiosuccinimide (Slufo-NHS), N,N′-diisopropylcarbodiimide (DIC), and N,N′-dicyclohexylcarbodiimide (DCC).

[0045] For example, when the coupling agent is selected from two of the following: carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), N-hydroxythiosuccinimide (Slufo-NHS), N,N′-diisopropylcarbodiimide (DIC), and N,N′-dicyclohexylcarbodiimide (DCC), the coupling agent includes a first coupling agent and a second coupling agent, and the concentration ratio of the first coupling agent to the second coupling agent is (1.5-4):(0.9-1.2).

[0046] For example, when the coupling agents are EDC and NHS, the concentration ratio of EDC to NHS is (1.5-4):(0.9-1.2).

[0047] As a preferred embodiment, the non-crosslinked biological basement membrane is derived from mammalian tissues such as skin, gastrointestinal tract, blood vessels, and bladder.

[0048] Examples of non-crosslinked biological basement membranes originate from the gastrointestinal tract and / or bladder of mammals.

[0049] Examples of non-crosslinked biological basement membranes originate from the small intestine and / or bladder of mammals.

[0050] For example, the biomimetic biomimetic valve consists of, from top to bottom, a non-crosslinked biological basement membrane derived from bladder tissue, a transition layer, a crosslinked extracellular matrix, a transition layer, and a non-crosslinked biological basement membrane derived from the small intestine; for example, the biomimetic biomimetic valve consists of, from top to bottom, a non-crosslinked biological basement membrane derived from bladder tissue, a transition layer, a crosslinked extracellular matrix, a transition layer, and a non-crosslinked biological basement membrane derived from bladder tissue.

[0051] Examples of non-crosslinked biological basement membranes include, at a minimum, the bladder.

[0052] One example of a non-crosslinked biological basement membrane is the non-crosslinked bladder basement membrane.

[0053] Examples of cross-linked extracellular matrix materials include those derived from mammalian vascular valves, aortic valves, heart valves, pericardium, and dermis.

[0054] Examples of cross-linked extracellular matrix materials include those derived from one or more of mammalian vascular valves, aortic valves, heart valves, and pericardium.

[0055] Examples of cross-linked extracellular matrix materials are derived from mammalian aortic valves, heart valves, and / or pericardium.

[0056] Preferably, the cross-linked extracellular matrix material is derived from the pericardium of mammals.

[0057] Preferably, non-crosslinked biological basement membranes and crosslinked extracellular matrix materials have different sources.

[0058] Especially when the cross-linked extracellular matrix material is derived from the pericardium of mammals, applying 8-50 μL / cm 2 Biocompatible colloidal matrix dispersions and 1-10 μL / cm 2 The aqueous solution of the coupling agent significantly improved the interlayer bonding strength and cell growth promotion effect, and the overall stability of the biomimetic valve's contraction and expansion behavior was particularly outstanding. The applicant hypothesizes that at this point, the coupling agent and the pericardial interface, as well as the molecular chains of gelatin, chitosan, or dextran, can enhance their binding through electrostatic attraction or osmosis, forming a tighter mixed structure. Simultaneously, the cross-linking points are more evenly distributed, thus affecting the binding between the cross-linked pericardium and the non-cross-linked biological basement membrane.

[0059] Non-crosslinked biological basement membranes can be prepared using the Abraham method. That is, non-crosslinked biological basement membranes can be obtained using conventional decellularization methods such as detergent washing, acid washing, and alkali washing, or they can be obtained using improved decellularization methods, such as those described in patent applications 201610531693.6 or 201710860898.3.

[0060] As a preferred embodiment, the preparation of cross-linked extracellular matrix materials includes at least decellularization and cross-linking.

[0061] For example, cross-linked extracellular matrix can be prepared by the Abraham method during decellularization. That is, it can be decellularized by the general method of washing with detergent, acid washing, and alkali washing in sequence, or it can be decellularized through a specific decellularization process to achieve the effect of over-decellularization, so as to remove impurities on the material more thoroughly and reduce the immunogenicity of the material to the greatest extent.

[0062] For example, cross-linked extracellular matrix undergoes a specific decellularization process during decellularization to achieve excessive decellularization, thus more thoroughly removing impurities from the material and minimizing its immunogenicity.

[0063] For example, cross-linked extracellular matrix materials undergo at least two discontinuous peroxide treatments during decellularization, and are pretreated with enzymes and alcohol solutions prior to the peroxide treatment.

[0064] Preferably, the decellularization of the cross-linked extracellular matrix material involves a sequential process of at least a first peroxide treatment, a surfactant treatment, and a second peroxide treatment.

[0065] The peroxide is selected from one or more of peracetic acid, perpropionic acid, hydrogen peroxide, and sodium peroxide.

[0066] The first peroxide that can be listed is hydrogen peroxide.

[0067] Examples of second peroxides include peracetic acid and / or perpropionic acid.

[0068] Generally, excessive decellularization leads to decreased material compliance and insufficient adhesion to other membrane materials, making interlayer separation easy. However, in this technology, the cross-linked extracellular matrix material that has undergone excessive decellularization does not easily separate from the non-cross-linked biological basement membrane, exhibiting good compliance. After several expansion and contraction behaviors following hydration, the material still does not shrink back. This may be related to the specific gelatin / chitosan / dextran matrix-coupling agent network, which affects and regulates the material's surface energy, charge distribution, and physiological adaptability.

[0069] For example, the surfactant is selected from one or more of the following: glyceryl monoladate (GML), polysorbate-20 (Tween-20), polysorbate-80 (Tween-80), octylphenyl polyoxyethylene ether (TritonX-100), and sodium dodecyl sulfonate (SDS).

[0070] In a preferred embodiment, the cross-linked extracellular matrix material has undergone pretreatment with enzymes and / or alcohol solutions before peroxide treatment during the decellularization process.

[0071] For example, in the decellularization process of cross-linked extracellular matrix materials, they have undergone pretreatment with proteases, nucleases, and / or alcohol solutions before peroxide treatment.

[0072] Further examples include trypsin.

[0073] In the crosslinking process of preparing crosslinked extracellular matrix materials, a homologous bifunctional crosslinking agent is used for crosslinking. After crosslinking and cleaning, the materials are immersed in a capping agent solution.

[0074] Examples of capping agents include amino acids and / or amino acid derivatives.

[0075] Further examples include amino acids selected from glutamic acid, alanine, arginine, glycine, aspartic acid, lysine, etc.

[0076] Further examples include amino acid derivatives selected from polyamino acids such as polylysine or polyaspartic acid.

[0077] Examples of homologous bifunctional crosslinking agents include one or more of glutaraldehyde, bis(succinimide) glutarate (DSG), dimethyl adipic acid (DMA), 3,3′-dithiobis(propenyline) dimethyl ester dihydrochloride (DTBP), and ethylene glycol bis(sulfosuccinimide) succinate (Sulfo-EGS).

[0078] Furthermore, the homologous bifunctional crosslinking agent is glutaraldehyde.

[0079] Specifically, to achieve excessive decellularization during the decellularization process of the cross-linked extracellular matrix, the preparation methods for cross-linked extracellular matrix materials are as follows:

[0080] S1. Decellularization: Cut mammalian vascular valves, aortic valves, heart valves and / or pericardium, wash them, soak them in a protease aqueous solution, rinse them, soak them in an alcohol aqueous solution; rinse them, soak them in an aqueous solution of the first peroxide, rinse them, soak them in an aqueous solution of the surfactant, rinse them, soak them in an aqueous solution of the second peroxide, and rinse them.

[0081] S2. Crosslinking: Decellularized mammalian vascular valves, aortic valves, heart valves and / or pericardium are immersed in an aqueous solution of a homologous bifunctional crosslinking agent for crosslinking, washed, immersed in a capping agent solution, and then removed to obtain the product.

[0082] Specifically, to achieve excessive decellularization during the decellularization process of the cross-linked extracellular matrix, the preparation methods for cross-linked extracellular matrix materials are as follows:

[0083] S1. Decellularization: Cut mammalian vascular valves, aortic valves, heart valves and / or pericardium, wash them, soak them in a 0.1-0.5 wt% protease aqueous solution for 4-8 hours, rinse them, soak them in a 60-80 wt% alcohol aqueous solution for 8-14 hours; rinse them, soak them in an aqueous solution of the first peroxide, rinse them, soak them in an aqueous solution of the surfactant, rinse them, soak them in an aqueous solution of the second peroxide, and rinse them.

[0084] S2. Crosslinking: Decellularized mammalian vascular valves, aortic valves, heart valves and / or pericardium are immersed in an aqueous solution of a homologous bifunctional crosslinking agent for 12-24 hours for crosslinking. After washing, they are immersed in a capping agent solution for 7-12 hours and then removed to obtain the product.

[0085] The second aspect of the present invention also provides an application of a biomimetic bio-valve in heart valves and vascular valves.

[0086] Beneficial effects:

[0087] (1) In this invention, the basement membrane works synergistically with cross-linked vascular valves, aortic valves, heart valves, and / or pericardial extracellular matrix materials. The non-cross-linked biological basement membrane and the cross-linked extracellular matrix materials have different sources, giving full play to the excellent regenerative and repair properties of the basement membrane and its rich bioactive factors, effectively promoting the growth and adhesion of endothelial cells on the material surface, thereby constructing a tissue structure that mimics the leaflets of a natural valve. The resulting biomimetic valve exhibits excellent anti-calcification properties, significantly inhibits abnormal adhesion of platelets and blood cells, and greatly improves the efficiency of valve function recovery and reconstruction.

[0088] (2) The biomimetic valve material has good blood compatibility as a whole, which can minimize the phenomenon of red blood cell rupture and dissolution, and has a very low damaging effect on red blood cells, thus fundamentally reducing the risk of coagulation. At the same time, the surface energy, charge distribution and hydrophilicity / hydrophobicity of the material are closer to the physiological environment, reducing the non-specific interaction between the red blood cell membrane and the material.

[0089] (3) Before consolidating each membrane layer into a whole, a specific area concentration of biocompatible colloidal matrix dispersion and coupling agent are simultaneously applied between the non-crosslinked biological basement membrane and the crosslinked extracellular matrix material membrane layers. This not only significantly improves the problem of poor composite strength, but also unexpectedly enhances the guiding and growth-promoting effect of the biomimetic valve on cell attachment, and helps to inhibit calcium deposition, block calcium ion binding sites, and significantly delay the calcification process.

[0090] (4) The biomimetic bio-valve exhibits excellent dynamic mechanical stability and compliance, maintaining structural integrity during repeated contraction-expansion cycles. After hydration, the material maintains its initial dimensions without significant shrinkage after multiple deformations, ensuring its reliable performance under long-term dynamic loads. The material achieves uniform stress distribution through a unique molecular network design, possessing both the ability to adapt to complex deformations and the ability to effectively resist plastic deformation, providing an ideal solution for its long-term stable application in physiological environments. Attached Figure Description

[0091] Figure 1 This is a stained image of a calcification experimental section, in which... Figure 1 (a) is a staining diagram of Example 1. Figure 1 (b) is the staining diagram of Example 5. Figure 1 (c) is the staining diagram of control example 1. Figure 1 (d) is the coloring diagram of Comparative Example 5. Figure 1 (e) is the coloring diagram of Comparative Example 6. Detailed Implementation

[0092] Compare with Example 1

[0093] This example provides a valve composed of only one layer of cross-linked extracellular matrix material. The preparation of the cross-linked extracellular matrix material includes decellularization and cross-linking, wherein the decellularization is excessive decellularization.

[0094] The cross-linked extracellular matrix material was prepared as follows:

[0095] S1. Decellularization: Cut the bovine pericardium, wash it, soak it in a 0.2 wt% protease aqueous solution for 6 hours, rinse it, soak it in a 65 wt% ethanol aqueous solution; rinse it with water, soak it in a 2.6 wt% first peroxide (specifically hydrogen peroxide) aqueous solution, rinse it with water, soak it in a 1 wt% surfactant (specifically Triton X-100) aqueous solution, rinse it with water, soak it in a 0.2 wt% second peroxide (specifically peracetic acid) aqueous solution, and rinse it with water.

[0096] S2. Crosslinking: The decellularized bovine pericardium was soaked in an aqueous solution of 0.7wt% homologous bifunctional crosslinking agent (specifically glutaraldehyde) for 24 hours for crosslinking. After washing, it was soaked in an aqueous solution of 0.4wt% capping agent (specifically lysine) for 7 hours and then removed to obtain the product.

[0097] Compare with Example 2

[0098] This example provides a valve composed of only one layer of cross-linked extracellular matrix material. The preparation of the cross-linked extracellular matrix material includes decellularization and cross-linking. The decellularization is not excessive decellularization, but rather follows the traditional decellularization method. Specifically, after decellularization according to the Abraham method, it is immersed in a 0.7wt% glutaraldehyde aqueous solution for cross-linking for 24 hours. After washing, it is immersed in a 0.4wt% lysine aqueous solution for 7 hours and then removed to obtain the valve.

[0099] Example 1

[0100] This example provides a biomimetic bio-valve, which is obtained by wrapping a non-crosslinked biological basement membrane derived from the bladder with a crosslinked extracellular matrix material derived from the pericardium (the preparation of the crosslinked extracellular matrix material is the same as in Control Example 1), forming a sandwich structure. A transition layer is provided between the non-crosslinked biological basement membrane and the crosslinked extracellular matrix material, and the transition layer consists of 20 μL / cm 2 Biocompatible colloidal matrix dispersion and 2 μL / cm 2 After the coupling agent aqueous solution is sprayed onto the non-crosslinked biological basement membrane and / or crosslinked extracellular matrix material, the layers are stacked in sequence and then consolidated to form a whole.

[0101] Preparation of biomimetic bio-valve

[0102] Spray 20 μL / cm onto 3 layers of non-crosslinked biological basement membrane. 2 Biocompatible colloidal matrix dispersion and 2 μL / cm 2 A coupling agent aqueous solution was applied, followed by a layer of cross-linked extracellular matrix material, and then sprayed at a concentration of 20 μL / cm². 2 Biocompatible colloidal matrix dispersion and 2 μL / cm 2 After applying the coupling agent aqueous solution, three layers of non-crosslinked biological substrate membrane were stacked; the membrane was then pressed at 40°C for 12 hours to obtain the final product.

[0103] Biocompatible colloidal matrix dispersion

[0104] The biocompatible colloidal matrix dispersion comprises 11.9 wt% gelatin aqueous dispersion and 0.34 wt% determinated collagen. The preparation method of the biocompatible colloidal matrix dispersion is as follows: 3.5 g of gelatin (purchased from Beyotime Biotechnology Co., Ltd., acid-processed gelatin, model ST1339) is added to 26 mL of deionized water, dispersed at 50 °C using a magnetic stirrer, and then 0.1 g of determinated collagen (purchased from Cosmobio Japan, product number KOU-CLP-01) is added and stirred to disperse.

[0105] Coupling agent aqueous solution

[0106] The coupling agent aqueous solution is a 0.10 g / mL EDC aqueous solution. Specifically, it is obtained by dissolving 0.2 g of EDC powder in 2 mL of deionized water.

[0107] Non-crosslinked biological basement membrane

[0108] Non-crosslinked biological basement membranes were prepared from porcine bladder tissue using the Abraham method.

[0109] The preparation of the cross-linked extracellular matrix material was the same as in Control Example 1.

[0110] Example 2

[0111] This example provides a biomimetic valve, which differs from Example 1 in that the transition layer consists of 8 μL / cm 2 Biocompatible colloidal matrix dispersion and 1 μL / cm 2 After the coupling agent aqueous solution is sprayed onto the non-crosslinked biological basement membrane and / or crosslinked extracellular matrix material, the layers are stacked in sequence and then consolidated to form a whole.

[0112] Preparation of biomimetic bio-valve

[0113] Spray 8 μL / cm onto 3 layers of non-crosslinked biological basement membrane. 2 Biocompatible colloidal matrix dispersion and 1 μL / cm 2 A coupling agent aqueous solution was applied, followed by a layer of cross-linked extracellular matrix material, and then sprayed at a concentration of 8 μL / cm². 2 Biocompatible colloidal matrix dispersion and 1 μL / cm 2 After applying the coupling agent aqueous solution, three layers of non-crosslinked biological substrate membrane were stacked; the membrane was then pressed at 40°C for 8 hours to obtain the final product.

[0114] Coupling agent aqueous solution

[0115] The coupling agent aqueous solution is a 0.10 g / mL EDC-NHS aqueous solution. Specifically, it is obtained by dissolving 0.15 g of EDC powder and 0.04 g of NHS powder in 2 mL of deionized water. The first coupling agent is EDC and the second coupling agent is NHS, with a concentration ratio of 3.75:1.

[0116] Example 3

[0117] This example provides a biomimetic bio-valve, which differs from Example 2 in that the non-crosslinked biological basement membrane is derived from the bladder and small intestine, and the transition layer consists of 44 μL / cm². 2 Biocompatible colloidal matrix dispersion and 4 μL / cm 2 After the coupling agent aqueous solution is sprayed onto the non-crosslinked biological basement membrane and / or crosslinked extracellular matrix material, the layers are stacked in sequence and then consolidated to form a whole.

[0118] The preparation method of the biomimetic bio-valve is as follows: spray 44 μL / cm onto a 3-layer non-crosslinked biological basement membrane derived from the bladder. 2 Biocompatible colloidal matrix dispersion and 4 μL / cm 2 A coupling agent aqueous solution was applied, followed by a layer of cross-linked extracellular matrix material, and then sprayed at a concentration of 44 μL / cm². 2 Biocompatible colloidal matrix dispersion and 4 μL / cm 2 After applying the coupling agent aqueous solution, three layers of non-crosslinked biological basement membranes derived from the small intestine (the non-crosslinked biological basement membranes derived from the small intestine were prepared from small intestine tissue according to the Abraham method) were stacked; the whole membrane was then pressed at 40°C for 14 hours to obtain the final product.

[0119] Biocompatible colloidal matrix dispersion

[0120] The biocompatible colloidal matrix dispersion comprises 16.7 wt% gelatin aqueous dispersion and 0.50 wt% determinated collagen. The preparation method of the biocompatible colloidal matrix dispersion is as follows: 5 g of gelatin (purchased from Beyotime Biotechnology Co., Ltd., acid-processed gelatin, model ST1339) is added to 25 mL of deionized water, dispersed at 50 °C using a magnetic stirrer, and then 0.15 g of determinated collagen (purchased from Cosmobio Japan, product number KOU-CLP-01) is added and stirred to disperse.

[0121] Example 4

[0122] This example provides a biomimetic bio-valve, which differs from Example 2 in that the biocompatible colloidal matrix dispersion includes 6.2 wt% gelatin aqueous dispersion, 8.5 wt% dextran aqueous solution and 0.46 wt% determinated collagen.

[0123] The preparation method of the biocompatible colloidal matrix dispersion is as follows: 1.9g of gelatin and 2.6g of dextran are added to 26mL of deionized water, dispersed with a magnetic stirrer at 55℃, and then 0.14g of determinate collagen is added and stirred to disperse.

[0124] Example 5

[0125] This example provides a biomimetic bio-valve, which differs from Example 2 in that the preparation of the pericardial-derived cross-linked extracellular matrix material is the same as in Control Example 2.

[0126] Comparative Example 1

[0127] This example provides a biomimetic valve, which differs from Example 1 in that the transition layer consists of a 70 μL / cm² structure. 2 Biocompatible colloidal matrix dispersion and 7 μL / cm 2 After the coupling agent aqueous solution is sprayed onto the non-crosslinked biological basement membrane and / or crosslinked extracellular matrix material, the layers are stacked in sequence and then consolidated to form a whole.

[0128] Preparation of biomimetic bio-valve

[0129] Spray 70 μL / cm onto 3 layers of non-crosslinked biological basement membrane. 2 Biocompatible colloidal matrix dispersion and 7 μL / cm 2 A coupling agent aqueous solution was applied, followed by a layer of cross-linked extracellular matrix material, and then sprayed at a concentration of 70 μL / cm². 2 Biocompatible colloidal matrix dispersion and 7 μL / cm 2 After applying the coupling agent aqueous solution, three layers of non-crosslinked biological substrate membrane were stacked; the membrane was then pressed at 45°C for 16 hours to obtain the final product.

[0130] Comparative Example 2

[0131] This example provides a biomimetic valve, which differs from Example 1 in that the transition layer consists of 7 μL / cm 2 Biocompatible colloidal matrix dispersion and 1 μL / cm 2 After the coupling agent aqueous solution is sprayed onto the non-crosslinked biological basement membrane and / or crosslinked extracellular matrix material, the layers are stacked in sequence and then consolidated to form a whole.

[0132] Preparation of biomimetic bio-valve

[0133] Spray 7 μL / cm onto 3 layers of non-crosslinked biological basement membrane. 2 Biocompatible colloidal matrix dispersion and 1 μL / cm 2 A coupling agent aqueous solution was applied, followed by a layer of cross-linked extracellular matrix material, and then sprayed at a concentration of 7 μL / cm². 2Biocompatible colloidal matrix dispersion and 1 μL / cm 2 After applying the coupling agent aqueous solution, three layers of non-crosslinked biological substrate membrane were stacked; the membrane was then pressed at 40°C for 8 hours to obtain the final product.

[0134] Comparative Example 3

[0135] This example provides a biomimetic valve, which differs from Example 1 in that the transition layer consists of 30 μL / cm 2 Biocompatible colloidal matrix dispersion and 6 μL / cm 2 After the coupling agent aqueous solution is sprayed onto the non-crosslinked biological basement membrane and / or crosslinked extracellular matrix material, the layers are stacked in sequence and then consolidated to form a whole.

[0136] Preparation of biomimetic bio-valve

[0137] Spray 30 μL / cm onto 3 layers of non-crosslinked biological basement membrane. 2 Biocompatible colloidal matrix dispersion and 6 μL / cm 2 A coupling agent aqueous solution was applied, followed by a layer of cross-linked extracellular matrix material, and then sprayed at a concentration of 30 μL / cm². 2 Biocompatible colloidal matrix dispersion and 6 μL / cm 2 After applying the coupling agent aqueous solution, three layers of non-crosslinked biological substrate membrane were stacked; the membrane was then pressed at 40°C for 10 hours to obtain the final product.

[0138] Comparative Example 4

[0139] This example provides a biomimetic bio-valve, which differs from Example 1 in that the non-crosslinked bio-basement membrane is derived solely from the small intestine.

[0140] Comparative Example 5

[0141] This example provides a biomimetic valve, which differs from Example 1 in that the transition layer consists of a 20 μL / cm² structure. 2 After the biocompatible colloidal matrix dispersion is sprayed onto non-crosslinked biological basement membranes and / or crosslinked extracellular matrix materials, the layers are stacked sequentially and solidified to form a whole.

[0142] Spray 20 μL / cm onto non-crosslinked biological basement membranes 2 A biocompatible colloidal matrix dispersion was then layered with cross-linked extracellular matrix material, and sprayed at a concentration of 20 μL / cm². 2 A biocompatible colloidal matrix dispersion was prepared by stacking non-crosslinked biological basement membranes and then pressing the entire membrane at 40°C for 10 hours.

[0143] Comparative Example 6

[0144] This example provides a biomimetic bio-valve, which differs from Example 1 in that the biocompatible colloidal matrix dispersion includes 21.1 wt% chitosan aqueous dispersion.

[0145] The preparation method of the biocompatible colloidal matrix dispersion is as follows: 8g of chitosan is added to 30mL of deionized water and dispersed with a magnetic stirrer at 60℃.

[0146] Comparative Example 7

[0147] This example provides a biomimetic bio-valve. The preparation of the non-crosslinked biological basement membrane and the crosslinked extracellular matrix material are the same as in Example 1. The non-crosslinked biological basement membrane and the crosslinked extracellular matrix material are connected only through a crosslinking agent and lamination. Specifically, a concentration of 25 μL / cm² is sprayed onto the non-crosslinked biological basement membrane. 2 After preparing the coupling agent aqueous solution, a layer of cross-linked extracellular matrix material is stacked on top, wherein the preparation method of the coupling agent aqueous solution is the same as in Example 1; and an area concentration of 25 μL / cm² is sprayed onto the cross-linked extracellular matrix material. 2 After applying the coupling agent aqueous solution, another layer of non-crosslinked biological base membrane is stacked on top, and the membrane is pressed at 45°C for 14 hours to obtain the final product.

[0148] Test characterization

[0149] 1. Degradation experiment: During the preparation of the biomimetic bio-valve, after obtaining the non-crosslinked biological basement membrane and the crosslinked extracellular matrix material, the mass m1 of the non-crosslinked biological basement membrane and the mass m2 of the crosslinked extracellular matrix material were weighed respectively. It can be seen that the mass ratio of the non-crosslinked biological basement membrane in the final bio-valve obtained in Example 1 is A = 2 × m1 / (2 × m1 + m2) × 100% (the mass of the solute in the first mixture and the second mixture can be ignored).

[0150] The biomimetic biomimetic valve sample was cut into 1x3cm pieces, and its initial mass m0 was recorded. It was then degraded in vitro under an ultra-high concentration degradation environment (degradation environment: 10mg collagenase: 100ml 1XPBS enzymatic hydrolysate). Degradation time points were set at 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h, 216h, and 240h, and the mass m of the sample was recorded at each degradation time point. The average value of three parallel samples at each time point was taken. The degradation rate B was calculated by the change in mass of the sample before and after degradation: B = (m0 - m) / m0 × 100%.

[0151] In Example 1, the degradation rate of the sample stabilized on day 5, and a comparison of the degradation rates A and B on day 5 showed that A ≈ B. This suggests that the non-crosslinked biological basement membrane of the biomimetic valve had essentially degraded completely by this point. This indicates that on day 5, the biomimetic biomimetic valve of this invention, consisting of a non-crosslinked biological basement membrane, a biocompatible colloidal matrix treated with a coupling agent, and a crosslinked extracellular matrix material, had completed the guidance of regenerated cell adhesion and released sufficient space for the regenerated cells to encapsulate the inner crosslinked extracellular matrix. By day 5, the inner crosslinked extracellular matrix material was encapsulated within the regenerated cell layer. It also demonstrates that the biomimetic valve obtained by this technology reaches material stability relatively quickly after implantation.

[0152] The degradation time points when the sample degradation rate began to stabilize and no longer changed were recorded. The results are shown in Table 1:

[0153] Table 1

[0154]

[0155] The samples in Examples 1-5 all tended to stabilize starting on day 5, which also shows that the preparation method of the present invention has good stability, that is, the rate of steady-state equilibrium of the material is stable, ensuring the stable performance of the material function.

[0156] 2. Mechanical performance testing:

[0157] The biomimetic bio-valve sample was cut into 1x3cm size and hydrated (soaked in 20×PBS for 20min). The length, width and thickness of the hydrated sample material were tested. Five points were randomly selected on the sample surface for each thickness test. The average thickness d0 was taken and the initial hydrated sample area was calculated.

[0158] The bonding strength between the non-crosslinked biological basement membrane layer and the crosslinked extracellular matrix material layer was tested according to YY / T 0729.2-2009 Test Method for Adhesive Bonding Properties of Tissue Adhesives Part 2: Test for T-Peel Tensile Bearing Strength. The tensile strength of the hydrated samples was measured according to GB / T1040 Determination of Tensile Properties of Plastics Part 3: Test Conditions for Films and Sheets. The results are shown in Table 2.

[0159] After conducting 3 million fatigue tensile tests in the laboratory on the hydrated samples of Examples 1-5 and Comparative Examples 1-5, the thickness was measured again (see Table 2). The sample area was calculated by measuring the length and width and compared with the initial hydrated sample area to calculate whether the sample size had shrunk or expanded (here, shrinkage and expansion refer to the possible deformation of the membrane material after the tensile fatigue test, resulting in an increase in surface area and a decrease in thickness). The presence of delamination in the samples was also observed. The results are shown in Table 2.

[0160] Table 2

[0161]

[0162] In the repeated experiments on connection strength and tensile strength of Comparative Example 7, the inventors found that the bioprosthetic valve prepared according to the method of Comparative Example 7 was not stable. Sometimes the material became obviously hard and sometimes it was difficult to form a whole.

[0163] The samples in Examples 1-5 exhibit excellent dynamic mechanical stability and compliance, maintaining structural integrity during repeated shrinkage-expansion cycles (i.e., tensile fatigue testing). The hydrated material retains its initial dimensions after multiple deformations, showing no significant shrinkage or expansion, ensuring reliable performance under long-term dynamic loads. This material achieves uniform stress distribution through a unique molecular network design, possessing both the ability to adapt to complex deformations and resistance to delamination, while also effectively resisting plastic deformation.

[0164] Compared to Example 1, Comparative Example 1 contained too much biocompatible colloidal matrix dispersion. After tensile fatigue testing, the membrane material of Comparative Example 1 did not exhibit significant shrinkage or expansion, but severe delamination occurred, leading to a significant increase in the overall thickness of the membrane material. Although the instantaneous bonding strength (2.1 N / cm) of Comparative Example 1 was similar to that of Example 1, the severe delamination after tensile fatigue testing reflects the difficulty in maintaining good interlayer bonding strength in Comparative Example 1, which is not conducive to practical applications. Compared to Example 1, Comparative Example 2 contained less biocompatible colloidal matrix dispersion. After tensile fatigue testing, the membrane material of Comparative Example 2 did not exhibit significant shrinkage or expansion, but delamination occurred, leading to an increase in the overall thickness of the membrane material. The inventors speculate that, in the presence of a coupling agent, too little or too much biocompatible colloidal matrix dispersion negatively impacts the flexibility and interfacial stress between interfaces (i.e., the transition layer), causing decreased interfacial flexibility and interfacial stress mismatch.

[0165] The membrane material in Comparative Example 3 underwent deformation, resulting in a certain expansion of its surface area. Although slight delamination was observed after tensile fatigue testing, the thickness (compared to before the tensile fatigue test) actually decreased. This indicates that when the biocompatible colloidal matrix changes from 5-50 μL / cm², the surface area expands. 2 Biocompatible colloidal matrix dispersions and 1-10 μL / cm 2Under the premise of the solidification of the coupling agent aqueous solution, the area concentration ratio of the biocompatible colloidal matrix dispersion and the coupling agent aqueous solution will significantly affect the deformation of the membrane material after tensile fatigue testing, and this deformation effect is significantly greater than the delamination effect. This is equivalent to the fact that, in this scheme, the area concentration ratio of the biocompatible colloidal matrix dispersion and the coupling agent aqueous solution will significantly affect the anti-plastic deformation cumulative performance under cyclic loading, that is, it will have a key impact on the material's ability to maintain geometric stability during long-term mechanical fatigue.

[0166] Compared to Example 1, the non-crosslinked biological basement membrane of Comparative Example 4 did not originate from the bladder, but only from the submucosa of the small intestine. The sample expanded after tensile fatigue testing, which indicates that the origin of the non-crosslinked biological basement membrane has a certain impact on the material's ability to maintain dimensional stability during long-term mechanical fatigue.

[0167] In Comparative Example 5, no coupling agent aqueous solution was added to the transition layer. The sample underwent significant deformation after tensile fatigue testing, and this deformation was uneven. Wrinkles appeared on the material surface, and the edges curled to a certain extent. Therefore, the thickness was not measured after tensile fatigue testing.

[0168] 3. Cell viability test: The cell viability of the samples was tested according to GB / T 16886.5-2017 Biological evaluation of medical devices - Part 5: In vitro cytotoxicity test. A cell viability rate >70% indicates that the material has low toxicity and meets the requirements for biological evaluation of medical devices. The higher the cell viability rate, the better the effect of promoting cell adhesion and guiding cell growth. The sample results are shown in Table 3.

[0169] Table 3

[0170]

[0171] Whether from Example 1 and Comparative Example 1, or from Example 5 and Comparative Example 2, the biomimetic bio-valve with a specific area concentration transition layer and a non-crosslinked biological basement membrane layer obtained by the present invention effectively blocks the cytotoxicity brought about by the crosslinked decellularized matrix.

[0172] 4. In vivo calcification test: Materials from the pouch implantation experimental group (Examples 1, 5, Comparative Examples 5 and 6) and the control group (Control Example 1) were obtained from the back of rats. Samples were removed at the 2-month time point and the tissue sections were stained with Alizarin Red. The results are as follows: Figure 1 The results showed Figure 1 (a) and Figure 1 No obvious calcium deposition was observed in (b). Figure 1 (c) contains a clear and abundant area of ​​calcium salt deposition. Figure 1 (d) and Figure 1(e) shows areas of varying degrees of calcium salt deposition, indicating that no calcification occurred in the samples of Example 1 and Example 5, and that the present invention has a significant anti-calcification effect.

[0173] 5. Blood compatibility test: The hemolysis rate was tested according to GB / T 16886.5-2017 Biological evaluation of medical devices - Part 4. The final hemolysis rate of the material in Example 1 was <5% in the in vitro blood test.

Claims

1. A biomimetic bio-valve, wherein the biomimetic bio-valve is obtained by encapsulating a cross-linked extracellular matrix material with a non-cross-linked biological basement membrane to form a sandwich structure; characterized in that, The sources of non-crosslinked biological basement membranes include at least the bladder; A transition layer is provided between the non-crosslinked biological basement membrane and the crosslinked extracellular matrix material, the transition layer comprising a biocompatible colloidal matrix treated with a coupling agent; The biocompatible colloidal matrix has a concentration of at least 8-44 μL / cm³. 2 Biocompatible colloidal matrix dispersions and 1-4 μL / cm 2 The coupling agent is solidified to form a dispersion, wherein the biocompatible colloidal matrix dispersion comprises at least determinate collagen and 3wt%-17wt% gelatin aqueous dispersion.

2. The biomimetic valve according to claim 1, characterized in that, The area concentration ratio of the biocompatible colloidal matrix dispersion to the coupling agent aqueous solution is (7-12):

1.

3. The biomimetic bio-valve according to any one of claims 1-2, characterized in that, The non-crosslinked biological basement membrane and the crosslinked extracellular matrix material have different sources.

4. The biomimetic valve according to claim 1, characterized in that, The preparation of the cross-linked extracellular matrix material includes at least decellularization and cross-linking.

5. The biomimetic valve according to claim 4, characterized in that, Cross-linked extracellular matrix materials undergo at least two discontinuous peroxide treatments during decellularization.

6. The biomimetic valve according to claim 4, characterized in that, The decellularization of cross-linked extracellular matrix materials involves a sequential process of at least a first peroxide treatment, a surfactant treatment, and a second peroxide treatment.

7. The biomimetic valve according to claim 5, characterized in that, The peroxide is selected from one or more of peracetic acid, perpropionic acid, hydrogen peroxide, and sodium peroxide.

8. The biomimetic valve according to claim 6, characterized in that, The first peroxide is hydrogen peroxide; the second peroxide is peracetic acid and / or perpropionic acid.

9. The biomimetic bio-valve according to any one of claims 5-8, characterized in that, In the decellularization process of cross-linked extracellular matrix materials, the materials have undergone initial treatment with enzymes and alcohol-water solutions before peroxide treatment.

10. The biomimetic valve according to claim 4, characterized in that, In the crosslinking process of preparing crosslinked extracellular matrix materials, a homologous bifunctional crosslinking agent is used for crosslinking.

11. The biomimetic bio-valve according to claim 4 or 10, characterized in that, The preparation of the cross-linked extracellular matrix material also includes cross-linking cleaning followed by immersion in a capping agent solution.

12. The application of the biomimetic valve according to claim 1 in the preparation of heart valve and vascular valve materials.

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