A method of reducing calcification of a bioprosthesis tissue and a bioprosthesis tissue

Abstract

The application provides a method for reducing calcification of a biological prosthesis tissue and the biological prosthesis tissue, and the method comprises the following steps: placing at least partially cross-linked fixed biological prosthesis tissue into a combined anti-calcification solution for anti-calcification treatment, wherein the combined anti-calcification solution comprises at least two kinds of amino compounds with different molecular weights. The method for reducing calcification of the biological prosthesis tissue provided by the application can solve the problems of the prior art, such as the unsatisfactory anti-calcification effect of the anti-calcification method and the performance reduction of the tissue after the anti-calcification treatment.

Description

Technical Field

[0001] This invention relates to the field of medical device technology, and in particular to a method for reducing calcification of biological prosthesis tissue and the biological prosthesis tissue itself. Background Technology

[0002] Currently, commonly used bioprosthetic valves and patches in clinical practice, such as bovine pericardium and small intestinal submucosa, typically use animal-derived collagen fiber tissue materials that are stored or cross-linked and fixed with chemical reagents such as glutaraldehyde. This type of fixation method often results in residual aldehyde groups in the bioprosthetic tissue, and numerous studies have shown that bioprosthetic tissue fixed with glutaraldehyde can cause calcification in vivo after implantation.

[0003] Existing methods for treating bioprosthetic tissues to prevent calcification include decellularization, alcohol treatment, surfactant treatment, and metal ion competition. While these methods have some anti-calcification effects, they are not ideal in practical applications. Decellularization can remove most of the antigens and DNA from the tissue, but it makes the tissue porous, leading to a decrease in the mechanical properties of the bioprosthetic tissue. Alcohol treatment shows good short-term anti-calcification effects, but it cannot remove residual aldehyde groups on the bioprosthetic tissue. Due to the presence of residual aldehyde groups, the long-term risk of calcification in the bioprosthetic tissue remains high. Surfactant treatment can remove phospholipids from the bioprosthetic tissue, achieving some anti-calcification effects, but aldehyde groups, as calcification inducing factors, still exist, resulting in poor anti-calcification effects. Metal ion competition can bind to existing binding sites in the bioprosthetic tissue, achieving some anti-calcification effects, but it also cannot completely eliminate the long-term calcification-inducing effect of aldehyde groups.

[0004] US Patent No. 4976733A describes a method for treating bioprosthetic tissue with 2-aminooleic acid to prevent bioprosthetic tissue calcification. In this method, bovine pericardium fixed with glutaraldehyde is placed in a sodium borate buffer solution (pH 11) containing sodium 2-aminooleate and incubated overnight at standard temperature. In vivo implantation showed that the treated tissue significantly improved calcification compared to untreated tissue. However, studies using a lamb model for valve replacement by Gott JP et al. found that while this method improves valve calcification, it can easily lead to tissue loosening and pericardial hairline hematoma.

[0005] Existing methods for preventing calcification in biological prostheses have limitations, including unsatisfactory anti-calcification effects and the risk of reduced prosthesis tissue performance after treatment. There is significant room for improvement in anti-calcification treatment processes to better meet the requirements for the performance and biocompatibility of biological prostheses. Summary of the Invention

[0006] The purpose of this invention is to provide a method and a biological prosthesis tissue for reducing calcification, so as to solve the problems of unsatisfactory anti-calcification effect and tissue performance decline after anti-calcification treatment in the prior art.

[0007] To address the aforementioned technical problems, the present invention provides a method for reducing calcification of biological prosthesis tissue, comprising: placing at least partially cross-linked and fixed biological prosthesis tissue into a combined anti-calcification solution for anti-calcification treatment, wherein the combined anti-calcification solution comprises at least two amino compounds of different molecular weights.

[0008] Furthermore, the combined anti-calcification solution comprises a first amino compound and a second amino compound, wherein the molecular weight of the first amino compound is 181-400 and the molecular weight of the second amino compound is 45-180.

[0009] Furthermore, the first amino compound is selected from one or more mixtures of amino acid alkyl alcohol esters, saturated alkylamines, polyetheramines, aminoalkyl acids, aminoalkyl esters, aminoalkylamides, aminoalkyl diacids, aminoalkyl diacids, and amino surfactants.

[0010] Furthermore, the second amino compound is selected from one or more of amino acids, amino alcohols, diamines, and glucosamine.

[0011] Furthermore, the mass ratio of the first amino compound to the second amino compound in the anti-calcification solution is 1:5 to 1:50.

[0012] Furthermore, the combined anti-calcification solution is a phosphate buffer containing an amino compound.

[0013] Furthermore, in the combined anti-calcification solution, the mass-volume ratio of the first amino compound is 0.01%-5%.

[0014] Furthermore, in the combined anti-calcification solution, the mass-volume ratio of the second amino compound is 0.02%-10%.

[0015] Furthermore, the pH value of the combined anti-calcification solution is 5-10.

[0016] Furthermore, the pH value of the combined anti-calcification solution is 5.8-9.6.

[0017] Furthermore, the concentration of phosphate in the combined anti-calcification solution is 0.02-1 mol / L.

[0018] This invention provides a method for reducing calcification in biological prosthetic tissues, specifically comprising:

[0019] S1: Select biological prosthetic tissue that has been at least partially cross-linked and fixed for cleaning;

[0020] S2: The cleaned biological prosthesis tissue from step S1 is placed in a combined anti-calcification solution for anti-calcification treatment;

[0021] S3: Clean the bioprosthetic tissue after the anti-calcification treatment in step S2.

[0022] Furthermore, in S1 and S3, when cleaning the biological prosthesis tissue, sterile physiological saline, phosphate buffer solution or alcohol-containing solution is used for cleaning.

[0023] Preferably, the concentration of the phosphate buffer solution is 0.05-0.2 mol / L, and the pH value is 6.8-8.6; in the alcohol-containing solution, the volume fraction of alcohol is greater than 0 and less than 50%.

[0024] The present invention also provides a biological prosthesis tissue, which is prepared by processing with the method described above to reduce calcification of the biological prosthesis tissue.

[0025] In summary, compared with the prior art, the method for reducing calcification of biological prosthesis tissue provided by the present invention has the following advantages:

[0026] This invention employs a combined anti-calcification solution to treat bioprosthetic tissues against calcification. The anti-calcification agent contains at least one amino group, which chemically bonds with residual aldehyde groups on the bioprosthetic tissue, blocking free aldehyde groups and generating a Schiff base. This chemical bond is stable, resulting in better anti-calcification performance. Furthermore, this invention innovatively uses a combination of macromolecular and small-molecule anti-calcification agents. The steric hindrance of the macromolecules and the good permeability of the small molecules complement each other, leading to a better anti-calcification effect on animal-derived materials. Bioprosthetic tissues treated with this anti-calcification process can effectively reduce the residual aldehyde content of the biomaterial, thereby improving its anti-calcification performance.

[0027] Furthermore, in the technical solution of the present invention, when performing anti-calcification treatment, a high concentration of PBS buffer can neutralize part of the alkalinity of the amino group, and the anti-calcification treatment is carried out by using a suitable temperature combined with shaking, which improves the reaction rate between the amino group and the aldehyde group.

[0028] Furthermore, in the combined anti-calcification process of this invention, the anti-calcification reagents used are mostly water-soluble, easy to penetrate, and easy to wash off, which has the advantage of causing little damage to tissues. The treated animal-derived materials have good biocompatibility.

[0029] Furthermore, the anti-calcification method provided by this invention has strong process adaptability, has little impact on the performance of biological prosthetic tissues, and the resulting biological prosthetic tissues have a wider range of applications. Biological prosthetic tissues treated by the method of this invention can be dehydrated and dried, sterilized with EO, or stored in liquid and sterilized in liquid, and can be used to manufacture dry materials. Attached Figure Description

[0030] Figure 1 This is a flowchart illustrating a method for reducing calcification in biological prosthetic tissues provided by the present invention. Detailed Implementation

[0031] The following detailed description, in conjunction with the accompanying drawings and specific embodiments, provides a method for reducing calcification in biological prostheses and the biological prosthesis tissue proposed in this invention. The advantages and features of this invention will become clearer from the following description.

[0032] In this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0033] Existing technologies involve treating biomaterials with anti-calcification solutions, but the reaction conditions are complex, affecting the performance of the resulting biomaterials. The purpose of this invention is to provide a method for reducing calcification in bioprosthetic tissues, addressing the problems of unsatisfactory anti-calcification effects and decreased tissue performance after anti-calcification treatment in existing technologies. The anti-calcification treatment method employed in this invention can protect residual aldehyde groups in biomaterials and is easily implemented.

[0034] To achieve the above-mentioned idea, the present invention provides a method for reducing calcification of biological prosthesis tissue, comprising placing at least partially cross-linked and fixed biological prosthesis tissue into a combined anti-calcification solution for anti-calcification treatment, wherein the combined anti-calcification solution comprises at least two amino compounds of different molecular weights.

[0035] like Figure 1 As shown, specifically, in embodiments of the present invention, the method for reducing calcification of biological prosthesis tissue may further include the following steps:

[0036] S1: Select biological prosthetic tissue that has been at least partially cross-linked and fixed for cleaning;

[0037] S2: The cleaned biological prosthesis tissue from step S1 is placed in a combined anti-calcification solution for anti-calcification treatment;

[0038] S3: Clean the bioprosthetic tissue after the anti-calcification treatment in step S2.

[0039] In step S1, the tissue source for the bioprosthetic tissue can be bovine pericardium, porcine pericardium, porcine tissue valves, blood vessels, skin, submucosa of the small intestine, meninges, bladder lining, ligaments, and tendons, etc. Cross-linking and fixing of the bioprosthetic tissue can be performed using glutaraldehyde, a commonly used material in the industry. After fixing, the bioprosthetic tissue is cleaned with a cleaning solution to thoroughly remove free glutaraldehyde within the tissue. During cleaning, sterile physiological saline, PBS buffer (phosphate buffer) with a pH of 6.8-8.6 and a concentration of 0.05-0.2 mol / L, or an alcohol-containing solution (wherein the alcohol concentration is greater than 0 and less than or equal to 50%) can be used. Cleaning can be repeated 3-5 times at room temperature, with each cleanse lasting 3-5 minutes, to ensure effective cleaning.

[0040] In step S2 of this embodiment of the invention, immersing the cleaned bioprosthetic tissue in a combined anti-calcification solution for anti-calcification treatment is one of the key steps of the invention. The combined anti-calcification solution refers to a solution containing at least two amino compounds with different molecular weights. The amino groups of the amino compounds can react with the free aldehyde groups in the bioprosthetic tissue to generate Schiff bases, which can block and protect the aldehyde groups, thereby reducing the problem of bioprosthetic tissue calcification caused by free aldehyde groups.

[0041] Furthermore, the combined anti-calcification solution comprises a first amino compound and a second amino compound, wherein the molecular weight of the first amino compound is 181-400 and the molecular weight of the second amino compound is 45-180. This invention employs at least two amino compounds of different molecular weights in combination to treat biological prosthetic tissues against calcification. The first amino compound is a long-chain macromolecular amino compound, which may include one or more of the following: amino acid alkyl alcohol esters (e.g., glycine lauryl ester hydrochloride, alanine hexadecyl ester hydrochloride, etc.), saturated alkylamines (e.g., dodecylamine, octadecylamine, etc.), polyetheramines, aminoalkyl acids (e.g., 12-aminododecanoic acid, 14-aminotetradecanoic acid, 2-aminohexadecanoic acid, etc.), aminoalkyl esters (e.g., methyl 8-aminooctanoate hydrochloride, methyl 12-aminolaurate, etc.), aminoalkylamides (e.g., 2-(methylamino)-N-phenylacetamide hydrochloride, 4-aminobenzenesulfonamide hydrochloride, etc.), aminoalkyl diacids (e.g., 2-aminooctanoic acid, 5-aminoisophthalic acid, etc.), aminoalkyl diacids (e.g., diethyl aminomalonate hydrochloride, L-2-aminohexanoic acid 6-methyl ester hydrochloride, etc.), and amino surfactants (e.g., 2-aminooleyl alcohol, TEA salt of cocoyl glutamate, etc.). The second amino compound is a short-chain, small-molecule amino compound, which can be selected from one or more of amino acids (e.g., glutamic acid, glycine, alanine, etc.), amino alcohols (e.g., ethanolamine, isopropanolamine, etc.), diamines (e.g., ethylenediamine, propylenediamine, butylenediamine, etc.), and glucosamine. Large-molecule amino compounds can react with free aldehyde groups in biological prostheses to form Schiff bases, and can also modify and alter the surface of biological prostheses, enhancing their mechanical properties. Small-molecule amino compounds, on the other hand, can more easily penetrate into the interior of biological prostheses, reacting with internal free aldehyde groups. This allows for the sealing and protection of free aldehyde groups both on the surface and inside the prosthesis, more thoroughly reducing the impact of free aldehyde groups on calcification of the biological prosthesis.

[0042] Through the inventors' experiments, to ensure that the combination of at least two amino compounds effectively seals and protects the free aldehyde groups in the biological prosthesis tissue while also modifying the tissue to maximize its mechanical properties, the mass ratio of the first amino compound to the second amino compound in the anti-calcification solution can be 1:5 to 1:50. This allows as many small-molecule amino compounds as possible to penetrate the interior of the biological prosthesis tissue, sealing the free aldehyde groups and improving the anti-calcification effect.

[0043] The reaction of amino compounds with free aldehyde groups in biological prosthetic tissue to form Schiff bases requires a suitable reaction environment. Preferably, the combined anti-calcification solution of the present invention is a phosphate buffer solution containing amino compounds. The amino compound in the combined anti-calcification solution has a mass-volume percentage of 0.01%-10%, preferably 0.02%-5%. The pH of the combined anti-calcification solution is 5-10, preferably 5.8-9.6. The phosphate concentration is 0.02-1 mol / L, preferably 0.02-0.5 mol / L. Immersing the biological prosthetic tissue in this slightly alkaline combined anti-calcification solution provides optimal reaction conditions to promote the reaction of amino and aldehyde groups to form Schiff bases. Furthermore, during anti-calcification treatment, the cleaned biological prosthetic tissue can be immersed in the combined anti-calcification solution, and the reaction vessel can be placed in a constant-temperature shaking incubator at 15-50°C and 50-200 rpm for 2-96 hours to ensure sufficient reaction between the amino and aldehyde groups.

[0044] After step S2 is completed, the anti-calcification treated biological prosthesis tissue is removed and cleaned to remove residual anti-calcification reagent. During cleaning, sterile physiological saline, PBS buffer (phosphate buffer solution) with a pH of 6.8-8.6 and a concentration of 0.05-0.2 mol / L, or an alcohol-containing solution (wherein the alcohol concentration in the solution is greater than 0 and less than or equal to 50%) can be used for cleaning. The cleaning can be repeated 3-5 times at room temperature, with each cleaning lasting 3-5 minutes, to fully remove residual anti-calcification reagent from the biological prosthesis tissue.

[0045] The present invention also provides a biological prosthesis tissue, which is prepared by processing with the method described above to reduce calcification of the biological prosthesis tissue.

[0046] To further understand the present invention, preferred embodiments will be described below in more detailed and specific terms to highlight the features and characteristics of the method for reducing calcification of biological prosthetic tissue provided by the present invention. These descriptions are merely illustrative of the features and advantages of the method of the present invention and are not intended to limit the scope of protection of the present invention.

[0047] Example 1

[0048] The method for reducing calcification of biological prosthesis tissue provided in Example 1 includes the following steps:

[0049] Biological prosthesis tissue cleaning: Bovine pericardial tissue fixed with 0.625% glutaraldehyde was removed from the fixation solution and cleaned in physiological saline three times, 5 minutes each time.

[0050] Combined anti-calcification treatment: Prepare 1L of PBS solution with a concentration of 0.1mol / L containing 0.1% glycine lauryl hydrochloride and 1.0% glutamic acid, with a pH value between 7.40±0.1. Place the washed and cross-linked fixed bovine pericardial tissue into the prepared combined anti-calcification solution and place it in a constant temperature shaker at 25℃ for 24h with a rotation speed of 60rpm. After the treatment, wash 5 times with 0.1mol / L sterile PBS solution, 5 min each time.

[0051] Example 2

[0052] The method for reducing calcification of biological prosthesis tissue provided in Example 2 includes the following steps:

[0053] Biological prosthesis tissue cleaning: Bovine pericardial tissue fixed with 0.625% glutaraldehyde was removed from the fixation solution and cleaned three times in physiological saline for 5 minutes each time.

[0054] Combined anti-calcification treatment: Prepare 1L of PBS solution with a concentration of 0.1mol / L containing 0.05% dodecylamine and 1.0% isopropanolamine, with a pH value between 8.0±0.1. Place the washed and cross-linked fixed bovine pericardial tissue into the prepared combined anti-calcification solution and place it in a constant temperature shaker at 60rpm and 25℃ for 72h. After the treatment, wash 5 times with 0.1mol / L sterile PBS solution, 5 min each time.

[0055] Example 3

[0056] The method for reducing calcification of biological prosthesis tissue provided in Example 3 includes the following steps:

[0057] Biological prosthesis tissue washing: Bovine pericardial tissue fixed with 0.625% glutaraldehyde was removed from the fixation solution and washed three times with 0.1 mol / L PBS buffer at pH 7.4 for 5 min each time.

[0058] Combined anti-calcification treatment: Prepare 1L of PBS solution with a concentration of 0.1mol / L containing 0.1% 12-aminododecanoic acid and 2.0% glucosamine, with a pH value between 8.0±0.1. Place the washed and cross-linked fixed bovine pericardial tissue into the prepared combined anti-calcification solution and place it in a constant temperature shaker at 30℃ for 48h with a rotation speed of 60rpm. After the treatment, wash 5 times with 0.1mol / L sterile PBS solution, 5 min each time.

[0059] Example 4

[0060] The method for reducing calcification of biological prosthesis tissue provided in Example 4 includes the following steps:

[0061] Cleaning of biological prosthesis tissue: Bovine pericardial tissue fixed with 0.625% glutaraldehyde was removed from the fixation solution and cleaned three times in 20% ethanol solution for 5 minutes each time.

[0062] Combined anti-calcification treatment: Prepare 1L of PBS solution with a concentration of 0.1mol / L containing 0.1% methyl 12-aminolaurate and 2.0% glycine, with a pH value between 7.40±0.1. Place the washed and cross-linked fixed bovine pericardial tissue into the prepared combined anti-calcification solution and place it in a constant temperature shaker at 100rpm and 25℃ for 72h. After treatment, wash with sterile physiological saline 5 times, 5min each time.

[0063] Example 5

[0064] The method for reducing calcification of biological prosthesis tissue provided in Example 5 includes the following steps:

[0065] Biological prosthesis tissue cleaning: Bovine pericardial tissue fixed with 0.625% glutaraldehyde was removed from the fixation solution and cleaned three times in physiological saline for 5 minutes each time.

[0066] Combined anti-calcification treatment: Prepare 1L of PBS solution with a concentration of 0.1mol / L containing 0.05% polyetheramine and 0.8% ethylenediamine, with a pH value between 9.0±0.1. Place the washed and cross-linked fixed bovine pericardial tissue into the prepared combined anti-calcification solution and place it in a constant temperature shaker at 48℃ for 24 hours with a rotation speed of 60 rpm. After the treatment, wash 5 times with 0.1mol / L sterile PBS solution, 5 minutes each time.

[0067] Example 6

[0068] The method for reducing calcification of biological prosthesis tissue provided in Example 6 includes the following steps:

[0069] Biological prosthesis tissue cleaning: Bovine pericardial tissue fixed with 0.625% glutaraldehyde was removed from the fixation solution and cleaned three times in physiological saline for 5 minutes each time.

[0070] Combined anti-calcification treatment: Prepare 1L of PBS solution with a concentration of 0.1mol / L containing 0.1% 4-aminobenzenesulfonamide hydrochloride and 1.0% glutamic acid, with a pH value between 7.4±0.1. Place the washed and cross-linked fixed bovine pericardial tissue into the prepared combined anti-calcification solution and place it in a constant temperature shaker at 48℃ for 24 hours with a rotation speed of 60 rpm. After the treatment, wash five times with 0.1mol / L sterile PBS solution, each time for 5 minutes.

[0071] Example 7

[0072] The method for reducing calcification of biological prosthesis tissue provided in Example 7 includes the following steps:

[0073] Biological prosthesis tissue cleaning: Bovine pericardial tissue fixed with 0.625% glutaraldehyde was removed from the fixation solution and cleaned in physiological saline three times, 5 minutes each time.

[0074] Combined anti-calcification treatment: Prepare 1L of PBS solution with a concentration of 0.1mol / L containing 0.5% diethyl aminomalonate hydrochloride and 4.0% glucosamine, with a pH value between 7.4±0.1. Place the washed and cross-linked fixed bovine pericardial tissue into the prepared combined anti-calcification solution and place it in a constant temperature shaker at 48℃ for 24 hours with a rotation speed of 60 rpm. After the treatment, wash five times with 0.1mol / L sterile PBS solution, each time for 5 minutes.

[0075] Example 8

[0076] The method for reducing calcification of biological prosthesis tissue provided in Example 8 includes the following steps:

[0077] Biological prosthesis tissue cleaning: Bovine pericardial tissue fixed with 0.625% glutaraldehyde was removed from the fixation solution and cleaned in physiological saline three times, 5 minutes each time.

[0078] Combined anti-calcification treatment: Prepare 1L of PBS solution with a concentration of 0.1mol / L containing 0.1% 2-aminooleyl alcohol and 1.0% ethylenediamine, with a pH value between 7.4±0.1. Place the washed and cross-linked fixed bovine pericardial tissue into the prepared combined anti-calcification solution and place it in a constant temperature shaker at 48℃ for 24 hours with a rotation speed of 60 rpm. After the treatment, wash five times with 0.1mol / L sterile PBS solution, each time for 5 minutes.

[0079] Example 9

[0080] The method for reducing calcification of biological prosthesis tissue provided in Example 9 includes the following steps:

[0081] Biological prosthesis tissue cleaning: Bovine pericardial tissue fixed with 0.625% glutaraldehyde was removed from the fixation solution and cleaned three times in 30% ethylene glycol solution for 5 minutes each time.

[0082] Combined anti-calcification treatment: Prepare 1L of PBS solution with a concentration of 0.1mol / L containing 0.1% methyl 12-aminolaurate and 2.0% glycine, with a pH value between 7.40±0.1. Place the washed and cross-linked fixed bovine pericardial tissue into the prepared combined anti-calcification solution and place it in a constant temperature shaker at 100rpm and 25℃ for 72h. After treatment, wash with sterile physiological saline 5 times, 5min each time.

[0083] To evaluate the performance of the biological prosthetic tissues prepared in Examples 1-9 of this invention, three control group experiments were also set up. The performance indicators of the control groups were tested together with the biological prosthetic tissues obtained from the embodiments of this invention. The specific technical solutions for the three control groups are as follows.

[0084] Control group 1

[0085] The biological prosthesis tissue in control group 1, after cross-linking, only underwent cleaning without any other anti-calcification treatment. The specific steps were as follows:

[0086] Biological prosthesis tissue cleaning: Bovine pericardial tissue fixed with 0.625% glutaraldehyde was removed from the fixation solution and cleaned in physiological saline three times, 5 minutes each time.

[0087] Control group 2

[0088] The anti-calcification treatment regimen for the biological prosthesis tissue in control group 2 is as follows:

[0089] Biological prosthesis tissue cleaning: Bovine pericardial tissue fixed with 0.625% glutaraldehyde was removed from the fixation solution and cleaned in physiological saline 3 times, 5 minutes each time;

[0090] Anti-calcification treatment: Prepare 1L of 0.1mol / L PBS solution containing 2.0% glycine, with a pH value between 7.40±0.1. Place the cleaned and fixed bovine heart into the prepared anti-calcification solution and place it on a constant temperature shaker at 60rpm and 48℃ for 24h. After completion, wash 5 times with 0.1mol / L sterile PBS solution, each time for 5min.

[0091] Control group 3

[0092] The anti-calcification treatment regimen for the control group 3 pairs of biological prosthesis tissues is as follows:

[0093] Biological prosthesis tissue cleaning: Bovine pericardial tissue fixed with 0.625% glutaraldehyde was removed from the fixation solution and cleaned in physiological saline 3 times, 5 minutes each time;

[0094] Anti-calcification treatment: Prepare 1L of 0.1mol / L PBS solution containing 0.1% 12-aminododecanoic acid, with a pH value between 8.0±0.1. Place the cleaned and fixed bovine heart into the prepared anti-calcification solution and place it on a constant temperature shaker at 60 rpm and 48℃ for 24 hours. After completion, wash 5 times with 0.1mol / L sterile PBS solution, each time for 5 minutes.

[0095] The performance indicators of the biological prosthesis tissues obtained in Examples 1-9 and Control Groups 1-3 of the present invention were tested, and the comparison was carried out from two aspects: cytotoxicity and aldehyde content.

[0096] Cytotoxicity comparison experiment

[0097] The cytotoxicity of the samples from Examples 1-9 and Control Groups 1-3 of this invention was evaluated using the MTT assay, and the test results are shown in Table 1 below.

[0098] Table 1 Comparison of cytotoxicity

[0099]

[0100] As can be seen from the results in Table 1, the biological prosthesis tissues obtained by the technical solutions of Examples 1-9 of the present invention have lower cytotoxicity and better biocompatibility.

[0101] Aldehyde content comparison experiment

[0102] In the comparative experiment of aldehyde content, biological prosthetic tissue samples were taken, and the aldehyde content was tested with a probe. The experimental results were recorded. The aldehyde residue of control group 1 was used as 100% baseline. The relative aldehyde residue of control groups 2-3 and biological prosthetic tissues of Examples 1-9 of the present invention were evaluated, as shown in Table 2 below.

[0103] Table 2. Relative Residual Amounts of Aldehydes in Beef Heart

[0104]

[0105] As can be seen, the biological prosthesis tissues treated with the technical solutions of Examples 1-9 of the present invention have less aldehyde residue compared with the control group, and their anti-calcification effect is better. In particular, after the biological prosthesis tissues are treated with the solution in Example 3 of the present invention for anti-calcification, the biological tissues have the least toxicity and the least aldehyde content, and the anti-calcification effect is significantly better than the control group and other experimental groups.

[0106] In summary, compared with the prior art, the method for reducing calcification of biological prosthesis tissue provided by the present invention has the following advantages:

[0107] This invention employs a combined anti-calcification solution to treat bioprosthetic tissues against calcification. The anti-calcification agent contains at least one amino group, which chemically bonds with residual aldehyde groups on the bioprosthetic tissue, blocking free aldehyde groups and generating a Schiff base. This chemical bond is stable, resulting in better anti-calcification performance. Furthermore, this invention innovatively uses a combination of macromolecular and small-molecule anti-calcification agents. The steric hindrance of the macromolecules and the good permeability of the small molecules complement each other, leading to a better anti-calcification effect on animal-derived materials. Bioprosthetic tissues treated with this anti-calcification process can effectively reduce the residual aldehyde content of the biomaterial, thereby improving its anti-calcification performance.

[0108] Furthermore, in the technical solution of the present invention, when performing anti-calcification treatment, a high concentration of PBS buffer can neutralize part of the alkalinity of the amino group, and the anti-calcification treatment is carried out by using a suitable temperature combined with shaking, which improves the reaction rate between the amino group and the aldehyde group.

[0109] Furthermore, in the combined anti-calcification process of this invention, the anti-calcification reagents used are mostly water-soluble, easy to penetrate, and easy to wash off, which has the advantage of causing little damage to tissues. The treated animal-derived materials have good biocompatibility.

[0110] Furthermore, the anti-calcification method provided by this invention has strong process adaptability, has little impact on the performance of biological prosthetic tissues, and the resulting biological prosthetic tissues have a wider range of applications. Biological prosthetic tissues treated by the method of this invention can be dehydrated and dried, sterilized with EO, or stored in liquid and sterilized in liquid, and can be used to manufacture dry materials.

[0111] The above description is merely a description of preferred embodiments of the present invention and is not intended to limit the scope of the invention in any way. Any changes or modifications made by those skilled in the art based on the above disclosure are within the scope of the claims. Obviously, those skilled in the art can make various modifications and variations to the invention without departing from the spirit and scope of the invention. Therefore, if these modifications and variations of the invention fall within the scope of the claims and their equivalents, the invention also intends to include these modifications and variations.

Claims

1. A method for reducing calcification in biological prosthetic tissue, characterized in that, include: Biological prosthesis tissue that has been at least partially cross-linked and fixed is placed in a combined anti-calcification solution for anti-calcification treatment, the combined anti-calcification solution comprising at least two amino compounds of different molecular weights; The combined anti-calcification solution comprises a first amino compound and a second amino compound, wherein the molecular weight of the first amino compound is 181-400 and the molecular weight of the second amino compound is 45-180.

2. The method for reducing calcification of biological prosthesis tissue according to claim 1, characterized in that, The first amino compound is selected from one or more of the following: amino alkyl alcohol esters, saturated alkylamines, polyetheramines, aminoalkyl acids, aminoalkyl esters, aminoalkylamides, aminoalkyl diacids, and aminoalkyl diacids.

3. The method for reducing calcification of biological prosthetic tissue according to claim 1, characterized in that, The second amino compound is selected from one or more of amino acids, amino alcohols, diamines, and glucosamine.

4. The method for reducing calcification of biological prosthesis tissue according to claim 1, characterized in that, The mass ratio of the first amino compound to the second amino compound in the combined anti-calcification solution is 1:5 to 1:

50.

5. The method for reducing calcification of biological prosthetic tissue according to claim 1, characterized in that, The combined anti-calcification solution is a phosphate buffer containing an amino compound.

6. The method for reducing calcification of biological prosthetic tissue according to claim 5, characterized in that, In the combined anti-calcification solution, the mass-volume ratio of the first amino compound is 0.01%-5%.

7. The method for reducing calcification of biological prosthetic tissue according to claim 5, characterized in that, In the combined anti-calcification solution, the mass-volume ratio of the second amino compound is 0.02%-10%.

8. The method for reducing calcification of biological prosthetic tissue according to claim 5, characterized in that, The pH value of the combined anti-calcification solution is 5-10.

9. The method for reducing calcification of biological prosthetic tissue according to claim 8, characterized in that, The pH value of the combined anti-calcification solution is 5.8-9.

6.

10. The method for reducing calcification of biological prosthesis tissue according to claim 5, characterized in that, The concentration of phosphate in the combined anti-calcification solution is 0.02-1 mol / L.

11. The method for reducing calcification of biological prosthetic tissue according to claim 1, characterized in that, The method includes: S1: Select biological prosthetic tissue that has been at least partially cross-linked and fixed for cleaning; S2: The cleaned biological prosthesis tissue from step S1 is placed in a combined anti-calcification solution for anti-calcification treatment; S3: Clean the bioprosthetic tissue after the anti-calcification treatment in step S2.

12. The method for reducing calcification of biological prosthesis tissue according to claim 11, characterized in that, In S1 and S3, when cleaning the biological prosthesis tissue, sterile physiological saline, phosphate buffer solution or alcohol-containing solution is used for cleaning.

13. The method for reducing calcification of biological prosthesis tissue according to claim 12, characterized in that, The concentration of the phosphate buffer solution is 0.05-0.2 mol / L, and the pH value is 6.8-8.6; In the alcohol-containing solution, the volume fraction of alcohol is greater than 0 and less than 50%.

14. A biological prosthetic tissue, characterized in that, It was prepared by treating the tissue with the method for reducing calcification of biological prostheses as described in any one of claims 1-13.

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