Combined bioprosthetic valve material anti-calcification method and bioprosthetic valve material

Abstract

The invention relates to the field of biomedical materials, and particularly discloses a combined biovalve material anti-calcification method and a biovalve material. A combined bioprosthetic valve material anti-calcification method comprises the following steps: taking an animal-derived biological material, cleaning, removing adipose tissue, cutting a precordial region part to obtain a pericardium material, and storing the pericardium material in ice normal saline for later use; mixing the pericardium material and the decellularized treatment fluid A, oscillating and cleaning; adding the pericardium material into the decellularized treatment solution B, oscillating and cleaning; carrying out crosslinking treatment by utilizing a glutaraldehyde pericardium material; adding the crosslinked pericardium material into a reducing agent solution, oscillating and cleaning; the method comprises the following steps: putting a pericardium material into a glycerol-ethanol mixed solution or a glycerol-isopropanol mixed solution, and soaking for more than 24 hours; according to the anti-calcification method, on the basis that the mechanical property of the valve material is effectively maintained, the anti-calcification performance is improved, and the durability of the valve material is improved.

Description

Technical Field

[0001] The present application relates to the technical field of biomedical materials, and more specifically, to a combined bioprosthetic valve material anti-calcification method and a bioprosthetic valve material. Background Art

[0002] Biological valve materials are mainly derived from bovine pericardium, porcine pericardium, small intestinal submucosa, etc. By chemically cross-linking valve materials derived from animals, the mechanical strength and service life of the materials can be improved. Currently, most of the more mature commercial cross-linking methods are based on glutaraldehyde cross-linking methods. Chemical treatment with glutaraldehyde can not only remove immunogenicity but also stabilize tissue structure. Materials cross-linked with glutaraldehyde from porcine pericardium and bovine pericardium have also been widely put into market use. For example, in the prior art, Chinese invention patent application document with application number CN201410301464.6 discloses a method for chemically treating artificial valves, which includes first treating with an aldehyde solution to cross-link the aldehyde groups with the amino groups in the valve, then treating with EDC / NHS and a diamine solution to activate the carboxyl groups in the material, and then cross-linking the activated carboxyl groups with the amino groups, and then storing the valve in a glutaraldehyde solution.

[0003] The biological valve material made using this method uses glutaraldehyde to cross-link the collagen in the animal-derived valve material. During the degradation process of the pericardial material treated with glutaraldehyde in the body, the parts of the collagen in the material that have been cross-linked by glutaraldehyde will release glutaraldehyde molecules during cleavage, affecting the safety after surgery. The released protein fragments may trigger an immune response of the matrix, which will further lead to calcification. Moreover, glutaraldehyde cross-linking and fixation are performed first to make the tissue structure tight, and cell fragments still remain in the tissue material, affecting the anti-calcification effect. In addition, the final valve material is preserved in glutaraldehyde solution, which will increase the excess aldehyde groups of the valve material and cause calcification of the valve material. Although some biological valve materials require cleaning before surgical implantation, the effect is limited. Summary of the Invention

[0004] In order to make the biological valve material have better anti-calcification effect, the present application provides a combined biological valve material anti-calcification method and biological valve material.

[0005] In a first aspect, the present application provides a method for anti-calcification of a combined bioprosthetic valve material, which adopts the following technical solution: A method for anti-calcification of a combined bioprosthetic valve material comprises the following steps: S1. Obtain animal-derived biological materials, clean and remove adipose tissue, excise the precordial area, obtain pericardial material, and store in ice-cold physiological saline until use; S2. Mix the pericardial material with decellularization solution A, shake at 4-8°C for 3-4 hours, remove the pericardial material, and wash with physiological saline. The decellularization solution A is a buffer solution containing EDTA and PMSF, with a mass ratio of EDTA to PMSF of 2:0.001-0.002. S3. Add the pericardial material prepared in step S2 to decellularization solution B, shake at 4-10° C. for 2-4 hours, remove the pericardial material, and wash with physiological saline. Decellularization solution B comprises decellularization solution A, a nonionic surfactant, and an anionic surfactant. The concentration of the nonionic surfactant is 0.5-2%, and the concentration of the anionic surfactant is 0.5-1.5%. S4, cross-linking the pericardium material obtained in step S3 using glutaraldehyde; S5. Add the cross-linked pericardial material to the reducing agent solution, shake at 4-10°C for 2-4 hours, remove the pericardial material, and wash with normal saline; S6. Place the pericardial material obtained in step S5 in a glycerol-ethanol mixed solution or a glycerol-isopropanol mixed solution with a concentration of 70-75%, and soak for more than 24 hours to complete the anti-calcification treatment.

[0006] By adopting the above technical solution, there is a lot of attached fat tissue in the pericardial material. If the material is not completely cleared of excess fat, the foreign fat tissue will cause a serious immune response after the valve is implanted into the human body. EDTA is a strong chelating agent that can form stable chelates with metal ions such as calcium ions, thereby destroying the connection between cells and extracellular matrix, making it easier for cells to separate from the tissue. Moreover, after the metal ions are removed, the stability of the cell membrane decreases. Under the force of the oscillation treatment, the cells are more likely to lyse, thereby releasing substances in the cells, which is convenient for subsequent cleaning and removal; PMSF (phenylmethylsulfonyl fluoride) is a serine protease inhibitor that can specifically inhibit the activity of serine proteases, preventing the degradation of collagen, elastin and other heavy proteins in the extracellular matrix. To ensure protein degradation and maintain the integrity of tissue structure; the buffer solution can maintain pH stability and provide a suitable ionic environment, promote the interaction between EDTA and PMSF and cell membranes, and reduce damage to the extracellular matrix; oscillation can increase the contact area and frequency between the buffer solution and bovine pericardial tissue, accelerate the chelation reaction between EDTA and metal ions, promote the separation and lysis of cells from tissues, and also make PMSF evenly distributed in the buffer solution, increase its contact opportunity with proteases, and more effectively inhibit the activity of proteases. Therefore, EDTA, PMSF and buffer solution work synergistically under oscillation treatment conditions to fully open tissue cells, which can not only remove cells but also inhibit protease activity, while maintaining the structure and function of the extracellular matrix and facilitating the entry of subsequent reagents into the tissue.

[0007] The pericardial material is then further decellularized using a decellularization solution A containing nonionic and anionic surfactants. This effectively removes pericardial cells under relatively mild conditions, minimizing the impact of residual tissue cells on valve calcification while maximally preserving the structure and function of the extracellular matrix. This facilitates the preparation of high-quality bioprosthetic valve materials and improves their biocompatibility and longevity. Using a single surfactant for treatment results in poor decellularization and requires extended treatment time, which can damage the biological tissue structure.

[0008] The pericardial material is then cross-linked with glutaraldehyde to cross-link the collagen in the pericardial material, thereby increasing its strength, reducing its immunogenicity, increasing its stability, and achieving a disinfecting effect. Finally, a reducing agent is used to reduce the aldehyde groups remaining after glutaraldehyde cross-linking, directly sealing the remaining aldehyde groups to prevent calcium ions from adhering to the remaining aldehyde groups and forming calcium salt crystals, thereby improving the anti-calcification ability of the valve material and thereby increasing the service life of the valve.

[0009] Finally, the pericardial material is soaked in a mixture of propylene glycol and ethanol, thereby changing the hydration state and surface chemical properties of the pericardial material surface, reducing the adsorption of calcium ions, weakening the adsorption capacity of calcification-related proteins, and reducing the occurrence of calcification; in addition, the propylene glycol solution can penetrate into the collagen molecules, forming hydrogen bonds with the collagen molecules and stabilizing their triple helix structure. This stabilizing effect can prevent the collagen from denaturing in the in vivo environment, thereby maintaining the elasticity and toughness of the valve. At the same time, the propylene glycol solution can also reduce water loss and maintain the softness of the valve.

[0010] Optionally, the nonionic surfactant is selected from at least one of Triton X-100, Tween-20 / 80, dodecyl polyoxyethylene ether, and ethylphenyl polyethylene glycol. The anionic surfactant is selected from at least one of deoxycholic acid, sodium lauryl sulfate, and sodium deoxycholate.

[0011] By adopting the above technical solution, Triton X-100 is polyethylene glycol octylphenyl ether, which has a good hydrophile-lipophile balance value (HLB value is about 13.5), can form micelles in aqueous solution, effectively dissolve lipid molecules on the cell membrane, destroy the integrity of the cell membrane, thereby releasing the cell contents, and has relatively little damage to the extracellular matrix, and can retain the structure and function of the extracellular matrix to a certain extent. The HLB value of Tween-20 is about 16.7, and the HLB value of Tween-80 is about 15. It has good emulsification, dispersion and wetting properties, and is effective in preventing protein deformation. The effect is relatively small, and it can reduce the damage to the protein components in the extracellular matrix during the decellularization process, which is beneficial to maintaining the biological activity and mechanical properties of the biological valve; the HLB value of dodecyl polyoxyethylene ether is about 16.9, and it has good solubility and stability in aqueous solution, can effectively reduce surface tension, and has a mild effect on cell penetration and dissolution. It reduces damage to the extracellular matrix while decellularizing and has little effect on subsequent biocompatibility; ethylphenyl polyethylene glycol (NP-40) has an effect between Tween and Triton X-100, balancing the decellularization effect and the retention of the extracellular matrix.

[0012] The sulfate groups in the sodium dodecyl sulfate molecule carry a negative charge and can form micelles in aqueous solution, which can effectively destroy the cell membrane and the lipid bilayer structure within the cell. It has a high decellularization efficiency and can quickly remove cells from tissues. Deoxycholic acid is a bile acid that contains carboxyl groups in its molecular structure. In aqueous solution, the carboxyl groups will partially dissociate into hydrogen ions, making the deoxycholic acid molecule negatively charged. Its hydrophobic portion can be inserted into the phospholipid bilayer of the cell membrane, while the hydrophilic portion is exposed to the aqueous phase. This insertion will destroy the orderly arrangement of phospholipid molecules, increase the fluidity of the cell membrane, and cause the integrity of the cell membrane to be damaged. Deoxycholic acid can interact with the lipid components in the cell membrane to form mixed micelles, dissolving the lipids from the cell membrane. Deoxycholic acid can "wash off" the lipids on the cell membrane, thereby causing the cell membrane to lose its barrier function and effectively removing cells from the pericardial tissue while relatively well preserving the structure and function of the extracellular matrix. Sodium deoxycholate has a good ability to dissolve lipids and can destroy the lipid structure of the cell membrane and the cell. Moreover, its dissolving effect is relatively mild, causing less damage to the extracellular matrix. It also has a certain antibacterial effect, reducing the risk of contamination during the decellularization process.

[0013] Optionally, the nonionic surfactant is Triton X-100, and the anionic surfactant is deoxycholic acid.

[0014] By adopting the above technical solution, Triton X-100 can be inserted into the phospholipid bilayer of the cell membrane, changing the fluidity of the cell membrane and making the cell membrane loose. Deoxycholic acid further interacts with the lipids in the cell membrane, dissolving the lipid components, forming mixed micelles, and accelerating the disintegration of the cell membrane. The two work synergistically to destroy the cell membrane more quickly and effectively, making it easier to release the cell contents, thereby improving the decellularization efficiency, shortening the processing time, and improving the processing efficiency. Moreover, this synergistic effect can penetrate deeper into the cells, ensuring that the cells can be removed more thoroughly, reducing the possibility of residual cells, and reducing cell The two products can remove residual cell fragments and intracellular antigens, reduce the immunogenicity of biological valves, and reduce calcification caused by residual cells; in addition, the two products have relatively little damage to the extracellular matrix, and can retain the integrity of the extracellular matrix to the greatest extent while decellularizing. The mechanical properties of the extracellular matrix are crucial to the function of tissue engineering products such as biological valves. Therefore, the combined use of Triton X-100 and deoxycholic acid can avoid excessive damage to the fiber structure in the extracellular matrix, thereby maintaining its mechanical properties. Moreover, the two products have good water solubility, simple preparation, mild processing conditions, low cost, and improved economic benefits.

[0015] Optionally, the nonionic surfactant is Triton X-100, and the anionic surfactant is sodium lauryl sulfate.

[0016] By adopting the above technical solution, Triton X-100 can first destroy the surface structure of the cell membrane, making the cell membrane loose, and then SDS can further penetrate into the cell, destroying the lipid bilayer structure in the cell and completely releasing the cell contents. However, it causes greater damage to the extracellular matrix, affecting the mechanical properties and biocompatibility of the biological valve material.

[0017] Optionally, the nonionic surfactant is Triton X-100, and the anionic surfactant is sodium deoxycholate.

[0018] By adopting the above technical solution, the mild effect of sodium deoxycholate is combined with Triton X-100, which can not only effectively remove cells but also reduce damage to the extracellular matrix. Sodium deoxycholate can assist Triton X-100 in destroying cell membranes, and its antibacterial effect helps maintain the sanitary conditions of the decellularization process.

[0019] Optionally, the nonionic surfactant is Tween-20 / 80, and the anionic surfactant is sodium lauryl sulfate.

[0020] By adopting the above technical solution, the mild properties of Tween-20 / 80 can reduce the damage of SDS to the extracellular matrix. Tween-20 / 80 first dissolves and disperses the cell membrane, and then SDS further exerts its effect to improve the decellularization efficiency. However, the decellularization efficiency is not high, and the processing time needs to be extended. Prolonging the time will lead to a decrease in the mechanical strength of the pericardial material.

[0021] Optionally, the nonionic surfactant is lauryl polyoxyethylene ether, and the anionic surfactant is sodium deoxycholate.

[0022] By adopting the above technical solution, both dodecyl polyoxyethylene ether and sodium deoxycholate have a mild decellularization effect, and the two can work synergistically to effectively remove cells while minimizing damage to the extracellular matrix.

[0023] Optionally, the reducing agent solution is one of saturated sodium bisulfite solution, ethanolamine solution of sodium cyanoborohydride, ethanol solution of sodium borohydride, ethanol solution of potassium borohydride, PBS buffer solution of dry cystine, PBS buffer solution of glutathione, and anhydrous ether solution of lithium aluminum hydride.

[0024] By adopting the above technical solution, sodium bisulfite can undergo a nucleophilic addition reaction with the residual aldehyde groups to generate a stable aldehyde adduct, thereby blocking the aldehyde groups. The reaction conditions and temperature will not destroy the formed cross-linked structure, and it is low cost and simple to operate. However, it cannot reduce the Schiff base formed by glutaraldehyde and protein amino groups, and only blocks free aldehyde groups, which cannot inhibit the hydrolysis of the Schiff base. Sodium cyanoborohydride is a selective reducing agent that can reduce free aldehyde groups and can also reduce the easily hydrolyzed Schiff base formed by cross-linking glutaraldehyde and protein amino groups to a stable secondary amino bond. While blocking the free aldehyde groups, it stabilizes the Schiff base and makes the cross-linked network more stable. However, it requires strict cleaning to avoid residual cyanide groups.

[0025] Optionally, the concentration of the sodium cyanoborohydride solution in ethanolamine is 0.2-0.5M.

[0026] By adopting the above technical solution, at the same concentration, sodium cyanoborohydride can provide sufficient reducing power to complete the aldehyde group blocking within a reasonable time, and the reaction process is relatively easy to control with few side reactions.

[0027] Optionally, the buffer is one of 0.01M Tris-HCl buffer, 0.01M PBS buffer and 0.01M D-Hanks buffer.

[0028] By adopting the above technical solution, Tris-HCl buffer has a wide pH range, can withstand a certain degree of acid-base changes, and maintain a relatively stable solution pH. PBS buffer contains sodium ions, potassium ions, chloride ions, and other ions. These ions are similar to the ionic composition of extracellular fluid, which can provide an ionic atmosphere similar to the physiological environment during the decellularization process. In the suitable environment provided by the buffer, nonionic surfactants and anionic surfactants can better bind to cell membranes, exerting the decellularization effect, while effectively preserving the structure and function of the extracellular matrix. In addition, the buffer can dilute harmful substances produced during the decellularization process, reducing their toxic effects on the extracellular matrix.

[0029] Optionally, the ratio of the weight of the pericardial material to the volume of the decellularization treatment solution A is 1:4-10; The ratio of the weight of the pericardial material obtained in step S2 to the volume of the decellularization solution B is 1:4-10.

[0030] By adopting the above technical solution, the amount of pericardial material and decellularization treatment fluid is within a certain range, which can achieve a better decellularization effect. If the volume of the decellularization treatment fluid is small, the concentration of the effective ingredients in the treatment fluid is relatively high, but the treatment fluid is not enough to fully infiltrate the pericardial material, it will lead to incomplete decellularization, a large number of residual cells, calcium ion deposition, accelerated calcification process, and local high concentration of decellularization reagents may excessively degrade the extracellular matrix, resulting in a decrease in the mechanical properties of the pericardial material, and cell fragments cannot be removed in time, and accumulate on the surface or inside of the pericardial material, affecting the structural integrity of the material. If the volume of the decellularization treatment fluid is large, although it can fully infiltrate the pericardial material, the decellularization efficiency is high, and there are few residual cells, it will increase the processing time and cost.

[0031] Optionally, the glutaraldehyde concentration is 0.4-0.65%, the cross-linking pH value is 6.5-7.4, the cross-linking temperature is 20-40° C., and the cross-linking time is 6-7 days.

[0032] By adopting the above technical solution, appropriate concentration, pH value and reaction temperature can completely cross-link glutaraldehyde with collagen and other components in the pericardial material, thereby improving the mechanical properties of the cross-linked pericardial material. If the cross-linking is incomplete, the degree of cross-linking is low, and the mechanical properties and biological stability of the material cannot be effectively improved. If the cross-linking is excessive, the material will become hard and brittle, and lose its flexibility and elasticity.

[0033] Optionally, the oscillation frequency in step S2, step S3 and step S5 is 100-160 rpm.

[0034] By adopting the above technical solutions, appropriate oscillation frequency can improve decellularization efficiency, reduce cell residues, protect the structural integrity of the material and the uniformity of the treatment, and shorten the treatment time.

[0035] Optionally, the pericardium material is porcine pericardium or bovine pericardium.

[0036] In a second aspect, the present application provides a bioprosthetic valve material, which adopts the following technical solution: A biological valve material is prepared by the anti-calcification method of the combined biological valve material.

[0037] By adopting the above technical solution, a two-step decellularization process is first performed, followed by glutaraldehyde cross-linking, aldehyde group blocking and soaking treatment to prepare a biological valve material. Less cell debris and excess aldehyde groups remain, avoiding calcification caused by the introduction of aldehyde groups by glutaraldehyde soaking, and having a good anti-calcification effect.

[0038] In summary, this application has the following beneficial effects: 1. The method of the present application first performs a two-step decellularization on the biomaterial. EDTA, PMSF and buffer are used to first open the tissue structure of the pericardium to facilitate the subsequent penetration of surfactants. Then, nonionic surfactants and anionic surfactants are used in combination to further remove cell debris and remove epithelial cells and fibroblasts with strong antigenicity, thereby reducing antigenicity and reducing important sites of calcification of biological tissues. The extracellular matrix is ​​retained, and the anti-calcification properties of the biomaterial are enhanced. Glutaraldehyde is then used for cross-linking to increase the structural stability of the pericardial material. A reducing agent is used to block residual aldehyde groups to reduce the calcification reaction caused by residual aldehyde groups. Finally, the pericardial material is soaked in an ethanol solution of glycerol to avoid the introduction of aldehyde groups by soaking in glutaraldehyde, while giving the pericardial material better flexibility and elasticity.

[0039] 2. In this application, Triton X-100 is preferably used as a non-ionic surfactant and deoxycholic acid is used as an anionic surfactant. The combination of the two can more effectively destroy the cell membrane, has a higher decellularization efficiency, reduces residual cells, and reduces the possibility of calcification. Moreover, the two have relatively little damage to the extracellular matrix, retaining the integrity of the extracellular matrix, thereby maintaining the mechanical properties of the pericardial material.

[0040] 3. The method in this application first performs decellularization, then uses two surfactants for treatment, seals the aldehyde groups after cross-linking with glutaraldehyde, and soaks in a propylene glycol mixed solution before it can be used directly. The processing time is short and the execution efficiency is high, avoiding the calcification effect caused by glutaraldehyde soaking. The two surfactants are used in combination with multiple means, and the valve material obtained has less damage, less residual cells and aldehyde groups, excellent anti-calcification effect, and good durability. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 8 shows the calcification images of the valve materials of Example 1 and Comparative Example 7 after subcutaneous implantation for 8 weeks (the experimental group is Example 1, and the control group is Comparative Example 7).

[0042] Figure 2 Schematic diagram of the detection of calcium content after subcutaneous implantation of valve materials prepared in Example 1 and Comparative Example 7 (the experimental group is Example 1, and the control group is Comparative Example 7). DETAILED DESCRIPTION

[0043] The following examples further illustrate the present application in detail. Example

[0044] Example 1: A method for anti-calcification of a combined bioprosthetic valve material, comprising the following steps: S1. Take fresh bovine pericardium, wash it with clean water, remove excess fat tissue on the surface, cut a relatively uniform portion of the precordial area, obtain the pericardium material, and store it in ice-cold physiological saline until used; S2. Add the pericardial material to decellularization solution A and shake at 4°C for 4 hours at 120 rpm. Discard the liquid and retain the pericardial material. Rinse three times with normal saline. The ratio of pericardial material to decellularization solution A is 1:4 (weight:volume). Decellularization solution A is prepared by mixing 0.2 g EDTA, 100 μg PMSF, and 0.01 M Tris-HCl buffer (pH 8). S3. Add the pericardial material prepared in step S2 to decellularization solution B, shake at 4° C. for 4 h at a frequency of 120 rpm, remove the pericardial material, and wash it three times with normal saline. The ratio of the pericardial material prepared in step S2 to the decellularization solution B is 1:4 (weight:volume). The decellularization solution B comprises a nonionic surfactant, an anionic surfactant, and decellularization solution A. The nonionic surfactant is Triton X-100 at a concentration of 0.5%, and the anionic surfactant is deoxycholic acid at a concentration of 1%; S4, placing the pericardium material obtained in step S3 into glutaraldehyde for cross-linking treatment, wherein the glutaraldehyde concentration is 0.65%, the pH value during cross-linking treatment is 7.4, the cross-linking temperature is 40° C., and the cross-linking time is 7 days; S5. Add the cross-linked pericardial material to a reducing agent solution of saturated sodium sulfite in a ratio of 1:3 (weight:volume). Oscillate at 4°C for 4 hours at a frequency of 120 rpm. Remove the pericardial material and wash it three times with normal saline. S6. Place the pericardial material obtained in step S5 in a 75% glycerol-ethanol mixed solution and soak it at 4° C. for more than 24 hours to complete the anti-calcification treatment.

[0045] Example 2: A method for anti-calcification of a combined bioprosthetic valve material, comprising the following steps: S1. Take fresh bovine pericardium, wash it with clean water, remove excess fat tissue on the surface, cut a relatively uniform portion of the precordial area, obtain the pericardium material, and store it in ice-cold physiological saline until used; S2. Add the pericardial material to decellularization solution A and shake at 8°C for 3 h at 160 rpm. Discard the liquid and retain the pericardial material. Rinse three times with normal saline. The ratio of pericardial material to decellularization solution A is 1:10 (weight:volume). Decellularization solution A is prepared by mixing 0.2 g EDTA, 200 μg PMSF, and 0.01 M Tris-HCl buffer (pH 8). S3. Add the pericardial material prepared in step S2 to decellularization solution B, shake at 10° C. for 2 h at a frequency of 160 rpm, remove the pericardial material, and wash it three times with normal saline. The ratio of the pericardial material prepared in step S2 to the decellularization solution B is 1:10 (weight:volume). The decellularization solution B comprises a nonionic surfactant, an anionic surfactant, and decellularization solution A. The nonionic surfactant is Triton X-100 at a concentration of 2%, and the anionic surfactant is deoxycholic acid at a concentration of 1.5%; S4, placing the pericardium material obtained in step S3 into glutaraldehyde for cross-linking treatment, wherein the glutaraldehyde concentration is 0.4%, the pH value during cross-linking treatment is 6.5, the cross-linking temperature is 20° C., and the cross-linking time is 7 days; S5. Add the cross-linked pericardial material to a reducing agent solution of saturated sodium sulfite in a ratio of 1:4 (weight:volume). Oscillate at 10°C for 2 hours at a frequency of 160 rpm. Remove the pericardial material and wash it three times with normal saline. S6. Place the pericardial material obtained in step S5 in a 70% glycerol-ethanol mixed solution and soak it at 8° C. for more than 24 hours to complete the anti-calcification treatment.

[0046] Example 3: A method for anti-calcification of a combined bioprosthetic valve material, comprising the following steps: S1. Take fresh bovine pericardium, wash it with clean water, remove excess fat tissue on the surface, cut a relatively uniform portion of the precordial area, obtain the pericardium material, and store it in ice-cold physiological saline until used; S2. Add the pericardial material to decellularization solution A and shake at 6°C for 4 h at 140 rpm. Discard the liquid and retain the pericardial material. Rinse three times with normal saline. The ratio of pericardial material to decellularization solution A is 1:8 (weight:volume). Decellularization solution A is prepared by mixing 0.2 g EDTA, 100 μg PMSF, and 0.01 M Tris-HCl buffer (pH 8). S3. Add the pericardial material prepared in step S2 to decellularization solution B, shake at 8° C. for 3 h at a frequency of 140 rpm, remove the pericardial material, and wash it three times with normal saline, wherein the ratio of the pericardial material prepared in step S2 to the decellularization solution B is 1:7 (weight:volume), and the decellularization solution B comprises a nonionic surfactant, an anionic surfactant, and decellularization solution A, wherein the nonionic surfactant is Triton X-100 at a concentration of 1%, and the anionic surfactant is deoxycholic acid at a concentration of 1%; S4, placing the pericardium material obtained in step S3 into glutaraldehyde for cross-linking treatment, wherein the glutaraldehyde concentration is 0.5%, the pH value during cross-linking treatment is 7, the cross-linking temperature is 30° C., and the cross-linking time is 7 days; S5. Add the cross-linked pericardial material to a reducing agent solution of saturated sodium sulfite at a ratio of 1:5 (weight:volume). Oscillate at 8°C for 3 hours at a frequency of 140 rpm. Remove the pericardial material and wash it three times with normal saline. S6. Place the pericardial material obtained in step S5 in a 70% glycerol-isopropyl alcohol mixed solution and soak it at 6° C. for more than 24 hours to complete the anti-calcification treatment.

[0047] Example 4: A method for anti-calcification of a combined biological valve material, which differs from Example 1 in that the nonionic surfactant is Triton X-100 and the anionic surfactant is sodium lauryl sulfate.

[0048] Example 5: A combined bioprosthetic valve material anti-calcification method, which differs from Example 1 in that the nonionic surfactant is Triton X-100 and the anionic surfactant is sodium deoxycholate.

[0049] Example 6: A method for anti-calcification of a combined biological valve material, which differs from Example 1 in that the nonionic surfactant is Tween-20 and the anionic surfactant is sodium lauryl sulfate.

[0050] Example 7: A method for preventing calcification of a combined biological valve material. The method differs from Example 1 in that the nonionic surfactant is dodecyl polyoxyethylene ether and the anionic surfactant is sodium deoxycholate.

[0051] Example 8: A combined bioprosthetic valve material anti-calcification method, which differs from Example 1 in that the reducing agent is a 0.5 M sodium cyanoborohydride solution in ethanolamine.

[0052] Comparative Example Comparative Example 1: A combined bioprosthetic valve material anti-calcification method, which differs from Example 1 in that step S3 is not provided, and the pericardial material obtained in step S2 is directly subjected to glutaraldehyde cross-linking treatment, and the remaining steps and parameters are the same as those in Example 1.

[0053] Comparative Example 2: A combined anti-calcification method for biological valve materials, which differs from Example 1 in that step S2 is not provided, the pericardial material obtained in step S1 is directly added to the decellularization treatment fluid B, and an equal amount of physiological saline is used in the decellularization treatment fluid B instead of the decellularization treatment fluid A. The remaining steps and parameters are the same as those in Example 1.

[0054] Comparative Example 3: A combined bioprosthetic valve material anti-calcification method, which differs from Example 1 in that step S2 is not provided, and the pericardial material obtained in step S1 is directly added to the decellularization treatment solution B. The remaining steps and parameters are the same as those in Example 1.

[0055] Comparative Example 4: A combined anti-calcification method for biological valve materials, which differs from Example 1 in that, in step S3, the decellularization treatment liquid B contains only the decellularization treatment liquid A and the non-ionic surfactant Triton X-100, the concentration of the non-ionic surfactant is 0.5%, and the other parameters are the same as those in Example 1.

[0056] Comparative Example 5: A combined anti-calcification method for biological valve materials, which differs from Example 1 in that, in step S3, the decellularization treatment liquid B contains only the decellularization treatment liquid A and the anionic surfactant deoxycholic acid, the concentration of the anionic surfactant is 1%, and the other parameters are the same as those in Example 1.

[0057] Comparative Example 6: A combined bioprosthetic valve material anti-calcification method, which differs from Example 1 in that, in step S5, the material is preserved in a glutaraldehyde solution with a concentration of 0.2 wt%.

[0058] Comparative Example 7: A method for anti-calcification of biological valve materials, comprising the following steps: washing freshly collected bovine pericardium with distilled water at 4°C and 120 rpm for 4 hours, then soaking it in a glutaraldehyde solution with a mass concentration of 0.625% for 24 hours, and after the reaction is completed, taking it out and soaking it in a glutaraldehyde solution with a mass fraction of 0.2% for preservation.

[0059] Performance testing The bioprosthetic valve material was prepared according to the methods in the examples and comparative examples, and the performance was tested according to the following methods. The test results are recorded in Table 1.

[0060] 1. Calcium content: Referring to the method in YY / T1859-2022 "Anti-calcification evaluation of animal-derived cardiovascular implants in rats: subcutaneous implantation test", the prepared bioprosthetic valve material was cut into single pieces with an area of ​​1 cm 2 Three-week-old, SPF-grade Wistar rats weighing 45-55g were selected for the test. The rats were weighed and anesthetized, and one anti-calcification-treated specimen was implanted subcutaneously in the upper and lower regions of the left and right backs, for a total of four specimens per rat. The skin was then sutured. At the prescribed time point (8 weeks in this test), the rats were weighed and the specimens removed. The host tissue on the graft surface was carefully removed, and the specimens were rinsed with physiological saline and smoothed. The specimens were then dried in a constant-temperature oven to a constant weight. After digestion, the amount of calcium present was determined using inductively coupled plasma emission spectrometry (ICP-AES) and expressed as μg / mg dry weight. Five rats were tested for each example, and the experimental results were averaged.

[0061] 2. Mechanical uniaxial tensile test: Each group of materials was cut into 1 cm × 5 cm strips (n = 20) and stretched at a speed of 10 mm / min using an electronic tensile testing machine (Instron, USA). The stress-strain curve was analyzed to obtain the maximum tensile strength and elastic modulus.

[0062] Table 1 Performance test results of valve materials Combining Table 1 with the anti-calcification methods in Examples 1-3, it can be seen that the valve material obtained by the anti-calcification methods in Examples 1-3 has a low amount of calcium after implantation, and has a high maximum tensile strength and elastic modulus, and has good stability and anti-calcification ability.

[0063] In Example 4, Triton X-100 was used as a nonionic surfactant and sodium lauryl sulfate was used as an anionic surfactant. The two were combined to further decellularize the pericardial material. The amount of calcium attached to the valve material produced did not increase significantly, but its maximum tensile strength and elastic modulus decreased. This shows that although Triton X-100 and sodium lauryl sulfate can remove cell debris, they increase damage to the extracellular matrix.

[0064] In Example 5, Triton X-100 was used as a nonionic surfactant and sodium deoxycholate was used as an anionic surfactant. The valve material prepared had similar anti-calcification effects and mechanical properties to those in Example 1.

[0065] Compared with Example 1, Example 6 uses Tween-20 as the nonionic surfactant and sodium lauryl sulfate as the cationic surfactant. The decellularization effect is not as good as that of Example 1, and the amount of calcium hanging is increased.

[0066] In Example 7, dodecyl polyoxyethylene ether and sodium deoxycholate were used in synergy. Compared with Example 1, the anti-calcification ability was slightly reduced, and the mechanical properties were similar.

[0067] Compared with Example 1, Example 8 uses a low concentration of sodium cyanoborohydride ethanolamine solution as the reducing agent solution. It can be seen that the aldehyde group is blocked effectively and the Schiff base can be reduced to stabilize the structure.

[0068] Compared with Example 1, Comparative Example 1 did not use decellularization treatment liquid B for treatment, but only used decellularization treatment liquid A for treatment, while in Comparative Example 2, only decellularization treatment liquid B was used for treatment, and the decellularization treatment liquid B did not contain decellularization treatment liquid A, while in Comparative Document 3, only decellularization treatment liquid B was used for treatment. It can be seen that the decellularization effect in Comparative Example 3 is the best, while the effect in Comparative Example 2 is the worst and the anti-calcification ability is the weakest.

[0069] Compared with Example 1, in Comparative Examples 4 and 5, the decellularization treatment solution B only contains a single surfactant, that is, a single surfactant is used for decellularization reprocessing. It can be seen that the anti-calcification ability of the valve materials prepared is weakened.

[0070] Comparative Example 6 uses glutaraldehyde solution to soak and store the valve material. It can be seen that the valve material has poor anti-calcification ability after implantation; Comparative Example 7 is a valve material made by only using glutaraldehyde cross-linking and soaking in glutaraldehyde solution. The data in Table 1 show that compared with Example 1, the amount of calcium hanging on the valve materials prepared in Comparative Example 6 and Comparative Example 7 is increased, and the anti-calcification ability is weakened.

[0071] According to the method in YY / T1859-2022 "Anti-calcification evaluation of animal-derived cardiovascular implants - subcutaneous implantation test in rats", the prepared bioprosthetic valve material was cut into single pieces with an area of ​​1 cm 2 Specimen pieces (10 mm × 10 mm / single piece) were selected from 3-week-old SPF Wistar rats weighing 45-55 g. They were weighed and anesthetized, and the skin was sutured on the upper part of the left back subcutaneously. At the specified time end point (8 weeks in this test), the rats were weighed and the specimens were removed. The host tissue on the transplanted surface was carefully removed, the specimens were rinsed with physiological saline, flattened, and dried. A small portion was taken for histochemical examination, such as Figure 1 As shown, it can be seen that after VonKossa calcium salt staining, the experimental group is still very smooth, without obvious calcification spots, and has good anti-calcification performance, while the control group prepared in Comparative Example 5 has obvious calcification spots deposited. The amount of calcium in the experimental group of Example 1 and the control group of Comparative Example 5 is as follows Figure 2 As shown in the results (0.6±0.3μg / mg in the experimental group and 135.1±13.8μg / mg in the control group), it can be seen that the two-step removal of cell debris before glutaraldehyde cross-linking and fixation, and the use of a surfactant combination when removing cell debris, can produce a valve material with strong anti-calcification ability.

[0072] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for anti-calcification of a combined bioprosthetic valve material, characterized in that: The following steps are involved: S1. Obtain animal-derived biological materials, clean and remove adipose tissue, excise the precordial area, obtain pericardial material, and store in ice-cold physiological saline until use; S2. Add the pericardial material to decellularization solution A, shake at 4-8°C for 3-4 hours, remove the pericardial material, and wash with physiological saline. The decellularization solution A is a buffer solution containing EDTA and PMSF, and the mass ratio of EDTA to PMSF is 2:0.001-0.002; S3, adding the pericardial material prepared in step S2 to decellularization solution B, shaking at 4-10° C. for 2-4 hours, removing the pericardial material, and washing with physiological saline, wherein the decellularization solution B comprises the decellularization solution A, a nonionic surfactant, and an anionic surfactant, wherein the concentration of the nonionic surfactant is 0.5-2%, and the concentration of the anionic surfactant is 0.5-1.5%; S4, cross-linking the pericardium material obtained in step S3 using glutaraldehyde; S5. Add the cross-linked pericardial material to the reducing agent solution, shake at 4-10°C for 2-4 hours, remove the pericardial material, and wash with normal saline; S6. Place the pericardial material obtained in step S5 in a 70-75% glycerol-ethanol mixed solution or a glycerol-isopropanol mixed solution, and soak at 4-8° C. for more than 24 hours to complete the anti-calcification treatment.

2. The method for anti-calcification of a combined bioprosthetic valve material according to claim 1, characterized in that: The nonionic surfactant is selected from at least one of Triton X-100, Tween-20 / 80, dodecyl polyoxyethylene ether, and ethylphenyl polyethylene glycol; The anionic surfactant is selected from at least one of deoxycholic acid, sodium lauryl sulfate, and sodium deoxycholate.

3. The method for anti-calcification of a combined bioprosthetic valve material according to claim 2, characterized in that: The nonionic surfactant is Triton X-100, and the anionic surfactant is deoxycholic acid.

4. The method for anti-calcification of a combined bioprosthetic valve material according to claim 1, characterized in that: The reducing agent solution is one of saturated sodium bisulfite solution, ethanolamine solution of sodium cyanoborohydride, ethanol solution of sodium borohydride, ethanol solution of potassium borohydride, PBS buffer solution of dry cystine, PBS buffer solution of glutathione, and anhydrous ether solution of lithium aluminum hydride.

5. The method for anti-calcification of a combined bioprosthetic valve material according to claim 1, characterized in that: The buffer solution is one of 0.01M Tris-HCl buffer solution, 0.01M PBS buffer solution and 0.01M D-Hanks buffer solution.

6. The method for anti-calcification of a combined bioprosthetic valve material according to claim 1, characterized in that: The ratio of the weight of the pericardial material to the volume of the decellularization solution A is 1:4-10; The ratio of the weight of the pericardial material obtained in step S2 to the volume of the decellularization solution B is 1:4-10; In step S5, the ratio of the weight of the cross-linked pericardium material to the volume of the reducing agent solution is 1:3-5.

7. The method for anti-calcification of a combined bioprosthetic valve material according to claim 1, characterized in that: The glutaraldehyde concentration is 0.4-0.65%, the cross-linking pH value is 6.5-7.4, the cross-linking temperature is 20-40° C., and the cross-linking time is 6-7 days.

8. The method for anti-calcification of a combined bioprosthetic valve material according to claim 1, characterized in that: The oscillation frequency in steps S2, S3 and S5 is 120-160 rpm.

9. The method for anti-calcification of a combined bioprosthetic valve material according to claim 1, characterized in that: The pericardium material is porcine pericardium or bovine pericardium.

10. A biological valve material, characterized in that: The combined bioprosthetic valve material is prepared by the anti-calcification method according to any one of claims 1 to 9.

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