Heart valve stent material and preparation method thereof

Abstract

The invention discloses a heart valve stent material and a preparation method thereof. The preparation method comprises the following steps: performing decellularization treatment on biological tissues; soaking the biological tissue subjected to decellularization treatment in a cross-linking solution, and carrying out a cross-linking reaction; the cross-linking solution is an AMAS cross-linking solution or a BMPS cross-linking solution; and after the cross-linking reaction is completed, cleaning and drying the biological tissue to obtain the heart valve stent material. Through the mode, the preparation method of the heart valve stent material provided by the invention can be used for preparing the heart valve stent material with excellent biocompatibility and excellent mechanical property, effectively reduces the immune reaction risk, can well adapt to the complex mechanical environment of the heart, opens up a brand new path for the field of the heart valve stent material, and has wide application prospects. The heart valve replacement operation effect is expected to be greatly improved, and the prognosis condition of a patient is remarkably improved.

Description

Technical Field

[0001] This application relates to the field of biomedical engineering technology, and in particular to a cardiac valve stent material and its preparation method. Background Technology

[0002] Heart valve disease poses a serious threat to human health worldwide, with its incidence rate increasing year by year. According to statistics from the World Health Organization, a large number of people die from heart valve disease each year. Heart valve replacement surgery, as a primary treatment for moderate to severe heart valve disease, plays a crucial role in improving patients' cardiac function and quality of life. In this type of surgery, the performance of the heart valve stent material, as the supporting structure of the valve, directly affects the long-term success rate of the surgery and the patient's prognosis.

[0003] Over the past few decades, significant progress has been made in the development of biological heart valve stent materials, but many challenges remain to be overcome. Take, for example, the commonly used glutaraldehyde cross-linked biomaterials. Glutaraldehyde reacts chemically with amino groups in the biomaterial to form covalent cross-links between molecules, thereby enhancing the material's mechanical strength. However, this cross-linking process has serious drawbacks. After cross-linking, a large number of aldehyde groups remain on the material's surface and inside. From a cell biology perspective, these residual aldehyde groups have high chemical reactivity and can non-specifically bind to biomolecules such as proteins and nucleic acids on the cell surface, interfering with normal cellular metabolic activities and potentially triggering cytotoxic reactions.

[0004] Meanwhile, residual aldehyde groups can also induce calcification in bio-valve materials. From the perspective of biological calcification mechanisms, aldehyde groups can provide nucleation sites for calcium salt deposition by complexing with calcium ions, thus accelerating the calcification process. Related studies show that more than 50% of glutaraldehyde-crosslinked bio-valve materials exhibit significant calcification 5-10 years after implantation, leading to valve stiffness, restricted leaflet movement, severely affecting the normal opening and closing function of the valve, and greatly shortening its lifespan. Summary of the Invention

[0005] This application primarily provides a cardiac valve stent material and its preparation method. By innovatively introducing AMAS or BMPS as crosslinking agents and precisely controlling the material preparation process, it aims to solve problems such as cytotoxicity and calcification caused by traditional crosslinking agents, while simultaneously achieving a synergistic improvement in biocompatibility and mechanical properties. This provides a safe and long-lasting material solution for the treatment of cardiac valve diseases, ultimately improving patient prognosis and enhancing their quality of life.

[0006] To address the aforementioned technical problems, this application provides a method for preparing a cardiac valve stent material. This method includes: decellularizing biological tissue; immersing the decellularized biological tissue in a crosslinking solution to perform a crosslinking reaction; the crosslinking solution is an AMAS crosslinking solution or a BMPS crosslinking solution; after the crosslinking reaction is completed, cleaning and drying the biological tissue to obtain the cardiac valve stent material.

[0007] In some embodiments, the biological tissues include bovine pericardium, porcine pericardium, heart valves, skin, blood vessels, submucosa of the small intestine, and nerve tissue.

[0008] In some embodiments, prior to decellularization of the biological tissue, the method further includes: The biological tissues were cleaned, disinfected, and separated using physiological saline containing antibiotics or phosphate buffer with a pH of 6.8-8.6 at 0-37°C to remove blood and impurities from the biological tissues.

[0009] In some embodiments, the decellularization of biological tissue includes: The biological tissue was decellularized using a sodium deoxycholate solution with a concentration of 0.1-10% w / v.

[0010] In some embodiments, the decellularization treatment of the biological tissue using sodium deoxycholate solution includes: The biological tissue was placed in the sodium deoxycholate solution and shaken at 100 rpm / min at 37°C for 24 h. The cleaned biological tissue was placed in a nuclease solution and shaken at 0-37°C for 2-8 hours. The nuclease solution was prepared with 0.1-1 mg / mL deoxyribonuclease, 0.1-1 mg / mL ribonuclease and 1% penicillin-antibody sterile PBS buffer.

[0011] In some embodiments, immersing the decellularized biological tissue in a crosslinking solution to perform a crosslinking reaction includes: The biological tissue was immersed in an AMAS crosslinking solution containing 0.1-10% w / v and shaken at 100-200 rpm / min for 24-72 h at 0-37°C and pH 4.0-9.18, allowing the AMAS substance to chemically react with the active groups in the biological tissue to form a crosslinked structure; or The biological tissue was immersed in a BMPS crosslinking solution containing 0.5-10% w / v and shaken at 80-200 rpm / min for 24-48 h at 20-38 °C and pH 6-8, so that the BMPS substance reacted chemically with the active groups in the biological tissue to form a crosslinked structure.

[0012] In some embodiments, the drying process includes either freeze drying or vacuum drying.

[0013] In some embodiments, the biocompatibility index of the material shall meet the following requirements: relative cell viability ≥ 70%; and the mechanical property index shall meet the following requirements: tensile strength not less than 4 MPa.

[0014] To solve the above-mentioned technical problems, one technical solution adopted in this application is to provide a cardiac valve stent material. The cardiac valve stent material is prepared using the method described above.

[0015] The beneficial effect of this application is that, unlike the prior art, this application discloses a heart valve stent material and its preparation method.

[0016] The AMAS crosslinking agent used in this invention is chosen because of its unique and highly promising application value. Prepared from precisely synthesizable and controllable chemical raw materials, the AMAS crosslinking agent is a functional crosslinking agent with broad application prospects. It possesses strong crosslinking activity, enabling rapid and precise construction of covalent bonds between different biomolecules, thereby achieving efficient crosslinking. It not only degrades gradually in the natural environment without causing long-term harm to the ecosystem, but also exhibits good adaptability when in contact with biological tissues, reducing the probability of adverse effects such as immune responses. Furthermore, it has good solubility, allowing for uniform dispersion in various solution systems, ensuring the crosslinking reaction proceeds stably and uniformly throughout the system. Its mild reaction conditions and performance characteristics mean that it causes almost no damage to the structure and function of the target biomolecules during the crosslinking process. In addition, it exhibits stable chemical properties in aqueous solutions, effectively exerting its crosslinking effect and enhancing the structural stability and durability of heart valve materials. When treating heart valves, the AMAS crosslinking agent can efficiently crosslink the relevant biomolecules in the valve, improving the valve's ability to resist mechanical stress during heartbeats and slowing down the degeneration process of the valve. At the same time, its excellent biodegradability ensures that it will not cause pollution to surrounding tissues and the environment even in the later stages of the material's use.

[0017] Compared with traditional glutaraldehyde, AMAS crosslinking agents have significant advantages in this invention. Glutaraldehyde crosslinking leaves behind cytotoxic aldehyde groups, which can trigger immune responses and material calcification, shortening valve lifespan. AMAS crosslinking does not introduce toxic groups, forms highly stable crosslinked bonds, and produces fewer degradation products. Therefore, the prepared material exhibits excellent biocompatibility, low cytotoxicity, effectively reducing the risk of immune rejection, minimizing inflammation, and extending the in vivo lifespan of the heart valve stent.

[0018] This application uses BMPS as a cross-linking agent primarily because of its high reactivity, good stability, and suitable molecular structure. Specifically, BMPS exhibits good biocompatibility, meaning it can reduce immune and inflammatory responses when in contact with human tissues. When used for heart valve cross-linking, it can reduce the body's rejection response to the valve, improve the valve's tolerance in vivo, and help extend the valve's lifespan. Furthermore, BMPS can undergo efficient cross-linking reactions with proteins and other biomolecules in heart valves, forming a stable three-dimensional network structure. This cross-linking enhances the mechanical properties of the valve tissue, making it better able to withstand the pressure and tension during cardiac circulation, maintaining the valve's morphological and functional stability, and reducing the risk of valve deformation and damage. Moreover, BMPS has high chemical stability in the physiological environment and is not easily degraded by enzymes or other chemicals in the body. This allows the cross-linked heart valve to maintain its structural and performance stability during long-term use, preserving good valve function. Regarding toxicity, under appropriate use conditions, BMPS exhibits low cytotoxicity and has minimal impact on the activity and function of heart valve cells. This helps maintain the normal physiological function of valve cells during the cross-linking process, ensuring the overall health and functional integrity of the valve tissue.

[0019] The advantages of using BMPS compared to pentylene glycol in this application are as follows: Materials cross-linked with glutaraldehyde may have a small amount of residual glutaraldehyde, which has certain cytotoxicity and may trigger inflammatory responses and tissue calcification. BMPS, on the other hand, has good biocompatibility, and residual BMPS has relatively less adverse effects on human tissues and cells, reducing the probability of immune and inflammatory responses. The cross-linking effect is more stable: BMPS cross-links with amino and thiol groups in biomolecules through Michael addition reactions, forming highly stable covalent bonds. Glutaraldehyde cross-linking reactions are more complex and may form multiple cross-linked structures with relatively poor stability. The stable cross-linked structure formed by BMPS helps maintain the performance of biomaterials over the long term; for example, in applications such as heart valves, it better maintains the mechanical properties and morphological stability of the valves. The impact on biomolecular activity is smaller: the cross-linking reaction of BMPS is relatively mild and has less impact on the activity of biomolecules, better preserving their original functions. Glutaraldehyde may react with multiple sites in biomolecules during cross-linking, easily leading to the masking or structural alteration of the active sites of biomolecules, thus affecting their biological activity. High chemical stability: BMPS exhibits high chemical stability in physiological environments and is not easily degraded by enzymes or other chemicals in the body. Glutaraldehyde, on the other hand, has relatively lower chemical stability and may undergo some degradation in vivo, affecting the durability of the cross-linking effect. Low immunogenicity: Due to the good biocompatibility and low residue of BMPS, its immunogenicity is low, resulting in a weaker immune response from the human immune system. In contrast, materials cross-linked with glutaraldehyde may trigger a stronger immune response due to residual glutaraldehyde or its degradation products, affecting the long-term implantation effect of the material. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort, wherein: Figure 1 This is a schematic flowchart of an embodiment of the method for preparing a cardiac valve stent material based on AMAS crosslinking provided in this application; Figure 2 Is it like this? Figure 1 A schematic diagram of the process for step 10 in the preparation method shown; Figure 3 This is a comparison chart of the moisture content test results of the three groups of samples in Example 1; Figure 4 This is a comparison chart of the tensile strength test results of the three groups of samples in Example 1; Figure 5This is a comparison chart of the crosslinking degree detection results of the three groups of samples in Example 1; Figure 6 This is a comparison of the HE staining results of the two groups of samples in Example 1; Figure 7 This is a comparison of the cytotoxicity results and cell live / dead staining fluorescence images of the two groups of samples in Example 1. Figure 8 This is a comparison of the HE staining results of the two groups of samples in Example 2; Figure 9 This is a comparison chart of the moisture content test results of the three groups of samples in Example 2; Figure 10 This is a comparison chart of the tensile strength test results of the three groups of samples in Example 2; Figure 11 This is a comparison chart of the crosslinking degree detection results of the three groups of samples in Example 2; Figure 12 This is a comparison of the cytotoxicity results and cell live / dead staining fluorescence images of the two groups of samples in Example 2. Detailed Implementation

[0021] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0022] The terms "first," "second," and "third" used in the embodiments of this application are for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first," "second," or "third" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device that includes a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to these processes, methods, products, or devices.

[0023] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a mutually exclusive, independent, or alternative embodiment. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0024] See Figure 1 , Figure 1 This is a schematic flowchart of an embodiment of the method for preparing a cardiac valve stent material provided in this application. The method for preparing the cardiac valve stent material includes: Step 10: Decellularize the biological tissue.

[0025] Before step 10, the procedure also includes: pretreatment of the biological tissue.

[0026] Biological tissues can be derived from xenogenes, allogeneic organisms, autologous tissues, or autologous organs. These biological tissues include bovine pericardium, porcine pericardium, heart valves, skin, blood vessels, submucosal layer of small intestine, and nerve tissue. Among these, heart valves, skin, blood vessels, submucosal layer of small intestine, and nerve tissue can be from the patient's own body or from their relatives or volunteers.

[0027] Biological tissues used to prepare valve stent materials can be one of the following: bovine pericardium, porcine pericardium, heart valves, skin, blood vessels, submucosa of the small intestine, and nerve tissue. Biological tissues such as bovine pericardium, porcine pericardium, heart valves, skin, blood vessels, submucosa of the small intestine, and nerve tissue have good biocompatibility and mechanical properties, which can meet the requirements for making heart valve stent materials.

[0028] Pretreatment of biological tissues includes cleaning, disinfection, and removal of excess impurities to ensure sterility, initially reduce immunogenicity, and improve biocompatibility and mechanical strength.

[0029] Specifically, biological tissues are cleaned, disinfected, and separated using physiological saline containing antibiotics or phosphate buffer with a pH of 6.8-8.6 at 0-37°C to remove blood and impurities from the biological tissues.

[0030] In this embodiment, bovine pericardium is used as the source of biological tissue. Fresh biological tissue is placed in physiological saline containing antibiotics or phosphate buffer with a pH of 6.8-8.6 and soaked and washed at 0-37°C. Excess fat and other tissues are separated from the biological tissue. During the washing process, the biological tissue is disinfected and most of the residual blood and impurities in the biological tissue are removed.

[0031] After pretreatment, select biological tissues with no surface damage and uniform thickness for trimming to the required size, ensuring smooth edges without burrs.

[0032] The size of the biological tissue obtained after trimming can range from 1x1cm to 10x10cm, and it can be trimmed according to specific needs.

[0033] Subsequently, the obtained biological tissue is washed again, for example, by using saline or phosphate buffer containing antibiotics. After washing, it can be stored in saline or sterile phosphate buffer containing antibiotics at 1℃-8℃.

[0034] After pretreatment, the biological tissues are decellularized.

[0035] Step 10 specifically includes: decellularizing biological tissues with sodium deoxycholate solution, wherein the sodium deoxycholate content in the sodium deoxycholate solution is 0.1-10% w / v.

[0036] The pruned biological tissue is soaked in a sodium deoxycholate solution for a certain period of time to remove cellular components while preserving the extracellular matrix, ensuring the structural integrity and functional stability of the tissue. The soaking time can be adjusted according to the thickness and cell density of the biological tissue, typically between 24 and 48 hours. The treated biological tissue needs to be rinsed again to thoroughly remove any residual sodium deoxycholate solution and ensure no cell residue remains.

[0037] The sodium deoxycholate solution contains 0.1%-10% w / v sodium deoxycholate, with concentrations of 0.1%, 0.3%, 0.5%, 0.8%, 1%, 3%, 5%, 8%, or 10% w / v. The specific concentration should be determined based on the characteristics of the biological tissue and the experimental results to ensure optimal decellularization. The treated biological tissue needs to be washed multiple times with sterile physiological saline or phosphate buffer until no sodium deoxycholate residue remains, ensuring the purity and safety of the biological tissue.

[0038] See Figure 2 , Figure 2 Is it like this? Figure 1 The schematic diagram of step 10 in the preparation method shown indicates that the biological tissue is decellularized using sodium deoxycholate solution, specifically including: Step 11: Place the biological tissue in sodium deoxycholate solution and shake at 100-200 rpm / min at 0-37℃ for 24-48 hours.

[0039] After the shaking is complete, the biological tissue is washed again with sterile saline or phosphate buffer for 10 minutes each time, for a total of 3 times to ensure that sodium deoxycholate residue is completely removed.

[0040] Step 12: Place the cleaned biological tissue into a nuclease solution and shake at 0-37℃ for 2-8 hours. The nuclease solution is prepared with 0.1-1 mg / mL deoxyribonuclease, 0.1-1 mg / mL ribonuclease and sterile PBS buffer containing 1% penicillin and antibiotics.

[0041] The residual nucleic acid components are further broken down by shaking the nuclease solution again, ensuring the purity of the biological tissue.

[0042] Step 20: Immerse the decellularized biological tissue in a cross-linking solution to carry out the cross-linking reaction. The cross-linking solution is either an AMAS cross-linking solution or a BMPS cross-linking solution.

[0043] When the crosslinking solution is an AMAS crosslinking solution, the biological tissue is immersed in a crosslinking solution containing 0.1-10% w / v AMAS (maleimide succinimide ester), and shaken at 100-200 rpm / min for 24-72 h at 0-37℃ and pH 4.0-9.18, so that AMAS reacts chemically with the active groups in the biological tissue to form a crosslinked structure.

[0044] Alternatively, the crosslinking solution is a BMPS crosslinking solution. Biological tissue is immersed in a BMPS crosslinking solution containing 0.5-10% w / v and shaken at 80-200 rpm / min for 24-48 hours at 20-38°C and pH 6-8, allowing the BMPS to chemically react with the active groups in the biological tissue to form a crosslinked structure. The cross-linking solution also contains a cross-linking solution buffer and an antioxidant. The cross-linking solution buffer includes phosphate buffer and Tris buffer, and the cross-linking solution buffer can be either phosphate buffer or Tris buffer. The antioxidant includes EDTA and ascorbic acid, and the antioxidant can be either EDTA or ascorbic acid. The concentration of the antioxidant is 0.1%-5% w / v, which plays a role in maintaining the stability of the cross-linking solution and preventing tissue browning.

[0045] Step 30: After the cross-linking reaction is completed, the biological tissue is cleaned and dried to obtain the heart valve stent material.

[0046] After the cross-linking reaction is completed, the biological tissue is thoroughly cleaned with deionized water or physiological saline for 3-5 times, with each cleaning time being 3-5 minutes, to remove unreacted AMAS or BMPS substances and other impurities. Then, the biological tissue is dried to obtain the heart valve stent material.

[0047] The drying process includes either freeze drying or vacuum drying.

[0048] Among them, the biocompatibility indicators of the obtained heart valve stent material must meet the following requirements: no obvious cell toxicity, and relative cell survival rate ≥70%; the mechanical performance indicators must meet the following requirements: tensile strength not less than 4 MPa.

[0049] Finally, the obtained cardiac valve stent material was tested and evaluated, including water content, tensile strength, residual amino groups, matrix fiber structure, and cytotoxicity. In this embodiment, the original biological tissue used was bovine pericardium, and the cardiac valve stent material was obtained by cross-linking bovine pericardium.

[0050] Example 1: Cardiac valve stent material obtained based on AMAS crosslinking.

[0051] Determination of water content in cross-linked cardiac valve stents: The heart valve stent material removed from the preservation solution was first rinsed three times with deionized water, and then cut into 2×2 cm² pieces. After carefully absorbing the water from the surface of the heart valve stent material with filter paper, the weight of the cut heart valve stent material was measured using an electronic balance and recorded as W0. It was then placed in a freeze dryer and freeze-dried under negative pressure for 24 hours, and weighed again, recorded as W1. The water content was calculated based on the change in mass before and after the freeze-drying process.

[0052]

[0053] The results show that the water content of cardiac valve stent materials can reflect the degree of looseness and compactness within the biomaterial. For example... Figure 3 As shown, compared with the fresh group (93.7±0.4%), the water content of all groups of materials after cross-linking was significantly reduced.

[0054] The NATIVE group had the highest water content (approximately 93.7 ± 0.4%), indicating that the fresh, uncrosslinked natural valve scaffold material has extremely strong hydrophilicity, which is consistent with the natural high water content characteristic of biological tissues.

[0055] GA group: The water content was significantly reduced to about 80%, indicating that glutaraldehyde (GA) crosslinking leads to the densification of the internal structure of the heart valve stent material and significantly reduces the hydration capacity of the heart valve stent material.

[0056] AMAS group: The water content is similar to that of NATIVE group, indicating that AMAS retains the hydrophilic structure of biological tissues well during the cross-linking process, and can achieve a balance between mechanical strength and biomimetic properties.

[0057] Compared to the GA group, the heart valve stent material obtained after AMAS crosslinking can achieve effective crosslinking while maintaining high water content, making it more suitable for stent designs that require biomimetic natural valve characteristics, and it has a stronger anti-calcification effect.

[0058] Determination of tensile strength of cross-linked cardiac valve stents: Decellularized bovine pericardium and various control group test samples were cut into 5×1 cm rectangular membranes and immersed in PBS to equalize osmotic pressure for 2 hours. The thickness of the membranes at three random locations on the samples was measured using vernier calipers, and the average value was calculated. The ends of the rectangular membranes were wrapped with appropriately sized sandpaper and mounted on the biaxial testing apparatus fixture. The initial length of the membranes between the fixtures was recorded, and a tension of 0.1 N was pre-loaded on the membranes. The samples were stretched at a speed of 12 mm / min until fracture, and the stress-strain curves were recorded. The Cauchy stress and Green's strain of the material were calculated, and the tangential modulus and fracture stress of the material were further analyzed using the following formulas:

[0059]

[0060] Where L is the instantaneous length of the sample, L0 is the initial length of the sample, F is the instantaneous tension on the sample, T0 is the initial thickness of the sample, and W0 is the initial width of the sample.

[0061] Tangential modulus is a mechanical property indicator that reflects collagen strength, while tensile stress at break is one of the important indicators that reflects the biomechanical strength of tissue.

[0062] The results showed that the tangential modulus of the AMAS sample was 29.90±3.883 MPa, which was higher than that of the GA sample (glutaraldehyde cross-linked valve material) (23.108±2.339 MPa), demonstrating that the collagen fibers of the AMAS-cross-linked decellularized bovine pericardium have higher mechanical strength. Figure 4 B); Compared with glutaraldehyde cross-linked valve material (8.818±0.686 MPa), AMAS cross-linked material (10.13±1.183 MPa) showed an increase in tensile stress at break. The above test results prove that AMAS cross-linked material with the same cross-sectional area can withstand higher mechanical loads than GA cross-linked material. Figure 4 C).

[0063] Detection and determination of residual amino groups in cross-linked cardiac valve stents: This application aims to determine the degree of crosslinking of samples using the ninhydrin reaction. The principle is that ninhydrin can undergo oxidative deamination with α-amino acids, proteins, and free polypeptide amino groups, producing a purple compound with a significant absorption peak at 570 nm. The reaction intensity is directly proportional to the amount of amino acid. Before the experiment, reagents need to be prepared as follows: First, accurately weigh 0.1 g of glycine powder using a benchtop balance, dissolve it in distilled water, and bring the volume to 100 ml. Transfer the solution to a reagent bottle for storage to obtain a 1.0 mg / ml glycine standard solution. Second, weigh 2 g of ninhydrin powder using a benchtop balance, add 100 ml of distilled water, and prepare a 2% (w / w) ninhydrin colorimetric solution. Third, measure 20 ml of isopropanol solution and distilled water using a graduated cylinder, and mix them to prepare a 50% (w / w) isopropanol solution. In the experimental procedure, five samples weighing a fixed 30 mg each were placed in EP tubes. 1 ml of 2% ninhydrin colorimetric solution was added to each tube, and the tubes were heated at 95°C for 30 minutes, then cooled to room temperature. Next, 250 μl of 50% isopropanol solution was added to each tube, and after mixing, 100 μl of the supernatant was transferred to a 96-well plate. The absorbance at 570 nm was measured using a spectrophotometer, and the molar number of free amino acids in each sample was calculated based on the glycine solution standard curve. Finally, with M0 representing the molar number of free amino acids in the uncrosslinked sample and M1 representing the molar number of free amino acids in the crosslinked sample, the degree of crosslinking was calculated using the following formula:

[0064] Note: All reactions should be performed in the dark.

[0065] The results showed that the residual amino content in the sample, detected by the ninhydrin colorimetric method, could indirectly quantify the degree of cross-linking of the sample. Figure 5 It can be seen that the cross-linking degree of the heart valve material after decellularization was significantly reduced (28.03±1.82%), while the cross-linking degree of the tissue after cross-linking was significantly increased. The cross-linking degree of the AMAS group was (80.85±0.87%), and that of the GA group was (79.69±0.70%), with the AMAS group being higher than the GA group.

[0066] Matrix fiber structure: Decellularized scaffolds of heart valves were fixed with 4% paraformaldehyde, embedded in paraffin, cut into 5-7 micrometer thick slices, dewaxed with xylene, dehydrated with a series of alcohols, and stained with hematoxylin and eosin (HE) to observe the distribution and structural changes of collagen fibers, elastic fibers, and muscle fibers in the matrix.

[0067] The results showed that collagen and elastin were intact and neatly arranged, indicating that the fiber distribution was well preserved (see [reference]). Figure 6 ).

[0068] Cytotoxicity assay of cross-linked cardiac valve stents: Decellularized samples were thoroughly washed with deionized water and then sterilized by immersion in 75% ethanol for 24 hours. Subsequently, they were washed three times with sterile PBS buffer for 15 minutes each time in a sterile operating room. Complete culture medium was prepared by adding 1% penicillin-dextrose antibody and 15% fetal bovine serum to DMEM medium. Then, 1 cm... 2 (n=20) Samples were soaked in 10 mL of complete culture medium and incubated at 37°C for 3 days to prepare sample extract. Human umbilical vein endothelial cells (HUVECs) were seeded at a density of 5000 per well in 96-well plates. After culturing for 24 hours, the old culture medium was replaced with the extract, and untreated complete culture medium was used as a control. After 1 and 3 days of incubation at 37°C, the culture medium was aspirated, and 100 μL of complete culture medium supplemented with 10% Cell Counting Kit-8 (CCK-8) reagent was added to each well. The cells were incubated at 37°C for 2 hours. The culture medium was aspirated and transferred to new 96-well plates, and the absorbance of the solution at 450 nm was measured using a microplate reader. The remaining cells were washed with PBS, and 100 μL of staining solution containing 2 μM calcein and 8 μM propidium iodide was added to each well. The cells were incubated at 37°C for 30 minutes for live / dead cell staining and observed using a fluorescence microscope. The relative cell viability was calculated using the following formula:

[0069] The OD value is the absorbance of the sample solution at 450 nm.

[0070] The results showed that the cytotoxicity of the AMAS group was significantly lower than that of the GA group, indicating that the AMAS group had higher biosafety. Fluorescence images further confirmed this result (see [link to relevant documentation]). Figure 7 ).

[0071] The AMAS crosslinking agent used in this invention is chosen because of its unique and highly promising application value. Prepared from precisely synthesizable and controllable chemical raw materials, the AMAS crosslinking agent is a functional crosslinking agent with broad application prospects. It possesses strong crosslinking activity, enabling rapid and precise construction of covalent bonds between different biomolecules, thereby achieving efficient crosslinking. It not only degrades gradually in the natural environment without causing long-term harm to the ecosystem, but also exhibits good adaptability when in contact with biological tissues, reducing the probability of adverse effects such as immune responses. Furthermore, it has good solubility, allowing for uniform dispersion in various solution systems, ensuring the crosslinking reaction proceeds stably and uniformly throughout the system. Its mild reaction conditions and performance characteristics mean that it causes almost no damage to the structure and function of the target biomolecules during the crosslinking process. In addition, it exhibits stable chemical properties in aqueous solutions, effectively exerting its crosslinking effect and enhancing the structural stability and durability of heart valve materials. When treating heart valves, the AMAS crosslinking agent can efficiently crosslink the relevant biomolecules in the valve, improving the valve's ability to resist mechanical stress during heartbeats and slowing down the degeneration process of the valve. At the same time, its excellent biodegradability ensures that it will not cause pollution to surrounding tissues and the environment even in the later stages of the material's use.

[0072] Compared with traditional glutaraldehyde, AMAS crosslinking agents have significant advantages in this invention. Glutaraldehyde crosslinking leaves behind cytotoxic aldehyde groups, which can trigger immune responses and material calcification, shortening valve lifespan. AMAS crosslinking does not introduce toxic groups, forms highly stable crosslinked bonds, and produces fewer degradation products. Therefore, the prepared material exhibits excellent biocompatibility, low cytotoxicity, effectively reducing the risk of immune rejection, minimizing inflammation, and extending the in vivo lifespan of the heart valve stent.

[0073] In this application, the method for preparing cardiac valve stent materials innovatively uses maleimide succinimide (AMAS), a chemically active compound, as a crosslinking agent to crosslink the biomaterial. This process enables the preparation of cardiac valve stent materials with excellent biocompatibility and superior mechanical properties, effectively reducing the risk of immune reactions and allowing them to adapt well to the complex mechanical environment of the heart. The preparation process encompasses the fine pretreatment of biomaterials, the precisely controlled AMAS crosslinking reaction, and comprehensive and meticulous post-processing steps. This opens up a new path in the field of cardiac valve stent materials and is expected to greatly improve the outcome of cardiac valve replacement surgery and significantly improve patient prognosis.

[0074] Example 2: Cardiac valve stent material obtained based on BMPS crosslinking.

[0075] Matrix fiber structure: Two types of bioprosthetic valves, obtained by crosslinking with glutaraldehyde and BMPS respectively, were fixed with 4% paraformaldehyde, embedded in paraffin, cut into 5-7 micrometer thick slices, dewaxed with xylene, dehydrated with a series of alcohols, and stained with HE to observe the distribution and structural changes of collagen fibers, elastic fibers and muscle fibers in the matrix.

[0076] The results showed that, compared with the GA group (glutaraldehyde crosslinking), the collagen and elastin of the bioprosthetic valve in the BMPS group were intact and neatly arranged, indicating that the fiber distribution was well preserved (see...). Figure 8 ).

[0077] Determination of water content in cross-linked cardiac valve stents: Pericardial tissue material taken from the preservation solution was first rinsed three times with deionized water, then cut into 2×2 cm² slices. After carefully absorbing the surface water with filter paper, the pericardial material was weighed using an electronic balance and recorded as W0. It was then placed in a freeze dryer and freeze-dried under negative pressure for 24 hours, and weighed again, recorded as W1. The water content was calculated based on the change in mass before and after the freeze-drying process.

[0078]

[0079] The results showed that the water content of bovine pericardium could also reflect the degree of looseness and compactness within the biological material. Figure 9 Compared with the fresh group (water content 93.7±0.4%), the water content of the bioprosthetic valve materials in both the GA group and the BMPS group was significantly reduced after cross-linking. The water content of the bioprosthetic valve after BMPS cross-linking (78.7±0.6%) was significantly lower than that of the GA group (81.9±0.8%), indicating that BMPS cross-linking made the material structure more compact.

[0080] Determination of tensile strength of cross-linked cardiac valve stents: Decellularized bovine pericardium and control group test samples were cut into 5×1 cm rectangular sheets and immersed in PBS to equalize osmotic pressure for 2 hours. The thickness at three random locations on the sample was measured using vernier calipers, and the average value was calculated. The ends of the rectangular sheet were wrapped with appropriately sized sandpaper and mounted on a biaxial testing apparatus fixture. The initial length of the sheet between the fixtures was recorded, and a tension of 0.1 N was pre-loaded on the sheet. The sample was stretched at a speed of 12 mm / min until fracture. The stress-strain curve was recorded, and the Cauchy stress and Green's strain of the material were calculated. Further analysis of the tangential modulus and fracture stress of the material was conducted. Figure 10 AC), the calculation formula is as follows:

[0081]

[0082] Where L is the instantaneous length of the sample, L0 is the initial length of the sample, F is the instantaneous tension on the sample, T0 is the initial thickness of the sample, and W0 is the initial width of the sample.

[0083] The results showed that tangential modulus is a mechanical property indicator reflecting collagen strength. The tangential modulus of the BMPS sample was 34.288±2.504 MPa, which was higher than that of the GA sample (23.108±2.339 MPa), demonstrating that the collagen fibers of the BMPS-crosslinked bioprosthetic valve have higher mechanical strength. Figure 10 B). Tensile stress at break is one of the important indicators reflecting the biomechanical strength of tissue. Compared with glutaraldehyde cross-linked valve material (tensile stress at break of 8.818±0.686 MPa), BMPS cross-linked material (15.803±1.119 MPa) showed an increased tensile stress at break. The above experimental results prove that BMPS cross-linked material with the same cross-sectional area can withstand higher mechanical loads than GA cross-linked material. Figure 10 C).

[0084] Analysis of the degree of cross-linking of heart valves after cross-linking: First, accurately weigh 0.1g of glycine powder using a benchtop balance, dissolve it in distilled water, and bring the volume to 100ml. Transfer the solution to a reagent bottle for storage, thus preparing a 1.0mg / ml glycine standard solution. Second, weigh 2g of ninhydrin powder using a benchtop balance and add it to 100ml of distilled water to prepare a 2% (w / w) ninhydrin colorimetric solution. Third, measure 20ml of isopropanol solution and distilled water into each well using a graduated cylinder, mix thoroughly, and prepare a 50% (w / w) isopropanol solution. Select 5 samples from each group, fix the weight of each sample to 30mg, and place them in EP tubes. Finally, add 1ml of 2% ninhydrin colorimetric solution to each tube, heat at 95℃ for 30 minutes, and remove and cool to room temperature. Add 250ul of 50% isopropanol solution to each tube, gently shake well, and take 100μl of the supernatant from each tube into a 96-well plate. Measure the absorbance at 570nm using a spectrophotometer. Based on the glycine solution standard curve, the molar number of free amino acids in each sample was calculated. Let M0 represent the molar number of free amino acids in the uncrosslinked sample and M1 represent the molar number of free amino acids in the crosslinked sample. The degree of crosslinking was calculated using the following formula: (Light protection required)

[0085] The results showed that the residual amino content in the sample, detected by the ninhydrin colorimetric method, could indirectly quantify the degree of cross-linking of the sample. Figure 11 It can be seen that the degree of crosslinking of the bioprosthetic valve after BMPS crosslinking is no different from that of GA, but the degree of crosslinking is significantly increased compared with DBP.

[0086] In vitro cytotoxicity assay of cross-linked cardiac valve stents: Decellularized samples were thoroughly washed with deionized water and then sterilized by immersion in 75% ethanol for 24 hours. Subsequently, they were washed three times with sterile PBS buffer for 15 minutes each time in a sterile operating room. Complete culture medium was prepared by adding 1% penicillin-dextrose antibody and 15% fetal bovine serum to DMEM medium. 1 cm² (n=20) samples were then immersed in 10 mL of complete culture medium and incubated at 37°C for 3 days to prepare the sample extract. Human umbilical vein endothelial cells (HUVECs) were seeded at a density of 5000 cells per well in 96-well plates. After 24 hours of culture, the extract was used instead of the old culture medium, with untreated complete culture medium used as a control. After 1 and 3 days of incubation at 37°C, the culture medium was aspirated, and 100 μL of complete culture medium supplemented with 10% Cell Counting Kit-8 (CCK-8) reagent was added to each well of the cells, and the cells were incubated at 37°C for 2 hours. The culture medium was aspirated and transferred to new 96-well plates. The absorbance of the solution at 450 nm was measured using a microplate reader. The remaining cells were washed with PBS, and 100 μL of staining solution containing 2 μM calcein and 8 μM propidium iodide was added to each well. The cells were incubated at 37°C for 30 minutes for live / dead cell staining, and observed using a fluorescence microscope. The relative cell viability was calculated using the following formula:

[0087] The OD value is the absorbance of the sample solution at 450 nm.

[0088] The results showed that the CCK8 assay indicated that the GA group samples exhibited significant cytotoxicity, while the BMPS group samples showed no cytotoxicity. Fluorescence images further confirmed this result (see [link to CCK8 assay]). Figure 12 ).

[0089] This application uses BMPS as a cross-linking agent primarily because of its high reactivity, good stability, and suitable molecular structure. Specifically, BMPS exhibits good biocompatibility, meaning it can reduce immune and inflammatory responses when in contact with human tissues. When used for heart valve cross-linking, it can reduce the body's rejection response to the valve, improve the valve's tolerance in vivo, and help extend the valve's lifespan. Furthermore, BMPS can undergo efficient cross-linking reactions with proteins and other biomolecules in heart valves, forming a stable three-dimensional network structure. This cross-linking enhances the mechanical properties of the valve tissue, making it better able to withstand the pressure and tension during cardiac circulation, maintaining the valve's morphological and functional stability, and reducing the risk of valve deformation and damage. Moreover, BMPS has high chemical stability in the physiological environment and is not easily degraded by enzymes or other chemicals in the body. This allows the cross-linked heart valve to maintain its structural and performance stability during long-term use, preserving good valve function. Regarding toxicity, under appropriate use conditions, BMPS exhibits low cytotoxicity and has minimal impact on the activity and function of heart valve cells. This helps maintain the normal physiological function of valve cells during the cross-linking process, ensuring the overall health and functional integrity of the valve tissue.

[0090] The advantages of using BMPS compared to pentylene glycol in this application are as follows: Materials cross-linked with glutaraldehyde may have a small amount of residual glutaraldehyde, which has certain cytotoxicity and may trigger inflammatory responses and tissue calcification. BMPS, on the other hand, has good biocompatibility, and residual BMPS has relatively less adverse effects on human tissues and cells, reducing the probability of immune and inflammatory responses. The cross-linking effect is more stable: BMPS cross-links with amino and thiol groups in biomolecules through Michael addition reactions, forming highly stable covalent bonds. Glutaraldehyde cross-linking reactions are more complex and may form multiple cross-linked structures with relatively poor stability. The stable cross-linked structure formed by BMPS helps maintain the performance of biomaterials over the long term; for example, in applications such as heart valves, it better maintains the mechanical properties and morphological stability of the valves. The impact on biomolecular activity is smaller: the cross-linking reaction of BMPS is relatively mild and has less impact on the activity of biomolecules, better preserving their original functions. Glutaraldehyde may react with multiple sites in biomolecules during cross-linking, easily leading to the masking or structural alteration of the active sites of biomolecules, thus affecting their biological activity. High chemical stability: BMPS exhibits high chemical stability in physiological environments and is not easily degraded by enzymes or other chemicals in the body. Glutaraldehyde, on the other hand, has relatively lower chemical stability and may undergo some degradation in vivo, affecting the durability of the cross-linking effect. Low immunogenicity: Due to the good biocompatibility and low residue of BMPS, its immunogenicity is low, resulting in a weaker immune response from the human immune system. In contrast, materials cross-linked with glutaraldehyde may trigger a stronger immune response due to residual glutaraldehyde or its degradation products, affecting the long-term implantation effect of the material.

[0091] This method leverages the unique chemical structure and reactivity of BMPS (3-maleimide propionic acid-N-hydroxysuccinimide ester) to crosslink with proteins in bioprosthetic valve tissue under mild reaction conditions. By precisely controlling the temperature, pH, concentration, and reaction time of the reaction system, effective reinforcement and stabilization of the collagen fiber network in the valve tissue are achieved. The detailed preparation process includes pretreatment of the bioprosthetic valve raw materials to remove cells and impurities, thereby reducing tissue immunogenicity; subsequently, the BMPS crosslinking agent is introduced into an optimized reaction environment for crosslinking; finally, the crosslinked valve undergoes post-treatment to improve its biocompatibility and mechanical properties. Experimental results show that the bioprosthetic valve prepared using this method exhibits excellent mechanical stability and low cytotoxicity in testing. Compared with bioprosthetic valves prepared by traditional glutaraldehyde crosslinking, the bioprosthetic valve prepared by the method of this invention has significant advantages in durability and biosafety, and is expected to provide a more reliable and long-lasting bioprosthetic valve product for heart valve replacement surgery, with broad application prospects in the field of cardiovascular disease treatment.

[0092] The above description is merely an embodiment of this application and does not limit the patent scope of this application. Any equivalent structural or procedural transformations made using the content of this application's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of this application.

Claims

1. A method for preparing a cardiac valve stent material, characterized in that, include: Decellularization of biological tissues; The decellularized biological tissue was immersed in a cross-linking solution to carry out a cross-linking reaction; The crosslinking solution is an AMAS crosslinking solution or a BMPS crosslinking solution; After the cross-linking reaction is completed, the biological tissue is cleaned and dried to obtain a heart valve stent material.

2. The preparation method according to claim 1, characterized in that, The biological tissues include bovine pericardium, porcine pericardium, heart valves, skin, blood vessels, submucosa of the small intestine, and nerve tissue.

3. The preparation method according to claim 1 or 2, characterized in that, Before decellularizing biological tissues, the following steps are also included: The biological tissues were cleaned, disinfected, and separated using physiological saline containing antibiotics or phosphate buffer with a pH of 6.8-8.6 at 0-37°C to remove blood and impurities from the biological tissues.

4. The preparation method according to claim 1, characterized in that, The decellularization process of the biological tissue includes: The biological tissue was decellularized using a sodium deoxycholate solution with a concentration of 0.1-10% w / v.

5. The preparation method according to claim 4, characterized in that, The decellularization treatment of the biological tissue using sodium deoxycholate solution includes: The biological tissue was placed in the sodium deoxycholate solution and shaken at 100-200 rpm / min at 0-37°C for 24-48 hours. The cleaned biological tissue was placed in a nuclease solution and shaken at 0-37°C for 2-8 hours. The nuclease solution was prepared with 0.1-1 mg / mL deoxyribonuclease, 0.1-1 mg / mL ribonuclease and 1% penicillin-antibody sterile PBS buffer.

6. The preparation method according to claim 1, characterized in that, The step of immersing the decellularized biological tissue in a crosslinking solution to carry out a crosslinking reaction includes: The biological tissue was immersed in an AMAS crosslinking solution containing 0.1-10% w / v and shaken at 100-200 rpm / min for 24-72 h at 0-37°C and pH 4.0-9.18, allowing the AMAS substance to chemically react with the active groups in the biological tissue to form a crosslinked structure; or The biological tissue was immersed in a BMPS crosslinking solution containing 0.5-10% w / v and shaken at 80-200 rpm / min for 24-48 h at 20-38 °C and pH 6-8, so that the BMPS substance reacted chemically with the active groups in the biological tissue to form a crosslinked structure.

7. The preparation method according to claim 6, characterized in that, The crosslinking solution also contains a crosslinking solution buffer and an antioxidant, wherein the crosslinking solution buffer is a phosphate buffer and a Tris buffer, and the antioxidant includes EDTA and ascorbic acid, and the concentration of the antioxidant is 0.1%-5% w / v.

8. The preparation method according to claim 1, characterized in that, The drying process includes either freeze drying or vacuum drying.

9. The preparation method according to claim 1, characterized in that, The biocompatibility of the heart valve stent material must meet the following requirements: no significant toxicity to cells, and a relative cell survival rate of ≥70%; the mechanical performance must meet the following requirements: tensile strength of not less than 4 MPa.

10. A cardiac valve stent material, characterized in that, The heart valve stent material is prepared using the method for preparing heart valve stent material as described in any one of claims 1 to 9.

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