A method for preventing calcification of a combined bioprosthetic valve material and the bioprosthetic valve material.

Abstract

This application relates to the field of biomedical materials, specifically disclosing a method for anti-calcification of a composite bio-valve material and the bio-valve material itself. The method for anti-calcification of a composite bio-valve material includes the following steps: taking animal-derived biological material, cleaning and removing adipose tissue, cutting the precordial region to obtain pericardial material, and storing it in ice-cold physiological saline for later use; mixing the pericardial material with decellularization solution A, shaking, and washing; adding the pericardial material to decellularization solution B, shaking, and washing; cross-linking the pericardial material using glutaraldehyde; adding the cross-linked pericardial material to a reducing agent solution, shaking, and washing; and immersing the pericardial material in a glycerol-ethanol mixture or a glycerol-isopropanol mixture for at least 24 hours. This anti-calcification method effectively maintains the mechanical properties of the valve material while improving its anti-calcification performance and durability.

Description

Technical Field

[0001] This application relates to the field of biomedical materials technology, and more specifically, it relates to a method for preventing calcification of a combined bioprosthetic valve material and the bioprosthetic valve material. Background Technology

[0002] Biological valve materials are mainly derived from bovine pericardium, porcine pericardium, and the submucosa of the small intestine. Chemical cross-linking of animal-derived valve materials can improve their mechanical strength and lifespan. Currently, most mature commercial cross-linking methods are based on glutaraldehyde. Glutaraldehyde chemical treatment not only removes immunogenicity but also stabilizes tissue structure. Materials from porcine and bovine pericardium cross-linked with glutaraldehyde are already widely used in the market. For example, Chinese invention patent application CN201410301464.6 discloses a method for chemically treating artificial valves. This method includes first treating with an aldehyde solution to cross-link the aldehyde groups with the amino groups in the valve, then treating with an EDC / NHS and diamine solution to activate the carboxyl groups in the material, followed by cross-linking the activated carboxyl groups with the amino groups, and finally storing the valve in a glutaraldehyde solution.

[0003] The bioprosthetic valve material produced using this method involves cross-linking collagen in animal-derived valve materials with glutaraldehyde. During the degradation process of pericardial material treated with glutaraldehyde in vivo, the glutaraldehyde-crosslinked areas of collagen in the material release glutaraldehyde molecules upon lysis, affecting postoperative safety. Furthermore, the released protein fragments may trigger an immune response in the host, further leading to calcification. Moreover, the initial glutaraldehyde cross-linking fixation results in a dense tissue structure, but residual cell debris remains in the tissue material, affecting the anti-calcification effect. Additionally, storing the final valve material in glutaraldehyde solution increases excess aldehyde groups in the valve material, leading to calcification. Although some bioprosthetic valve materials require pre-implantation cleaning, the effect is limited. Summary of the Invention

[0004] In order to improve the anti-calcification effect of bioprosthetic valve materials, this application provides a method for anti-calcification of combined bioprosthetic valve materials and bioprosthetic valve materials.

[0005] In a first aspect, this application provides a method for preventing calcification of a combined bioprosthetic valve material, employing the following technical solution:

[0006] A method for preventing calcification of a composite bioprosthetic valve material includes the following steps:

[0007] S1. Take animal-derived biological materials, clean and remove adipose tissue, cut the precordial region to obtain pericardial material, and store it in ice-cold physiological saline for later use.

[0008] S2. Mix the pericardial material with decellularization solution A, shake at 4-8℃ for 3-4 hours, remove the pericardial material, and wash with physiological saline. The decellularization solution A is a buffer containing EDTA and PMSF, with a mass ratio of EDTA to PMSF of 2:0.001-0.002.

[0009] S3. Add the pericardial material obtained in step S2 to the decellularization solution B, shake at 4-10℃ for 2-4 hours, remove the pericardial material, and wash with physiological saline. The decellularization solution B contains decellularization solution A, nonionic surfactant and anionic surfactant. The concentration of nonionic surfactant is 0.5-2% and the concentration of anionic surfactant is 0.5-1.5%.

[0010] S4. Crosslink the pericardial material obtained in step S3 using glutaraldehyde;

[0011] S5. Add the cross-linked pericardial material to the reducing agent solution, shake at 4-10℃ for 2-4 hours, remove the pericardial material, and wash with physiological saline.

[0012] S6. Place the pericardial material obtained in step S5 into a 70-75% glycerol-ethanol mixed solution or a glycerol-isopropanol mixed solution and soak for more than 24 hours to complete the anti-calcification treatment.

[0013] By employing the above technical solutions, the pericardial material contains a significant amount of attached adipose tissue. If this excess fat is not thoroughly removed, the resulting valve, after implantation, will trigger a severe immune response due to the foreign adipose tissue. EDTA, a strong chelating agent, can form stable chelates with metal ions such as calcium ions, thereby disrupting the connection between cells and the extracellular matrix, making cells easier to separate from the tissue. Furthermore, after removing metal ions, the stability of the cell membrane decreases, and under the force of oscillation, cells are more prone to lysis, releasing intracellular substances and facilitating subsequent washing and removal. PMSF (phenylmethylsulfonyl fluoride) is a serine protease inhibitor that specifically inhibits the activity of serine proteases, preventing the formation of collagen, elastin, and other components in the extracellular matrix. For proteins to be degraded and for tissue structure to remain intact, the buffer solution maintains pH stability and provides a suitable ionic environment, promoting the interaction of EDTA and PMSF with the cell membrane while reducing damage to the extracellular matrix. Shaking increases the contact area and frequency between the buffer solution and bovine pericardial tissue, accelerates the chelation reaction of EDTA and metal ions, promotes cell separation and lysis from the tissue, and also ensures that PMSF is evenly distributed in the buffer solution, increasing its contact opportunities with proteases and more effectively inhibiting protease activity. Therefore, EDTA, PMSF, and buffer solution work synergistically under shaking conditions to fully open tissue cells, remove cells, inhibit protease activity, maintain the structure and function of the extracellular matrix, and facilitate the entry of subsequent reagents into the tissue.

[0014] Next, the pericardial material is further decellularized using decellularization solution A containing both nonionic and anionic surfactants. This process effectively removes cells from the pericardium under relatively gentle conditions, reducing the impact of residual tissue cells on valve calcification while preserving the structure and function of the extracellular matrix to the greatest extent possible. This is beneficial for preparing high-quality bioprosthetic valve materials and improving the biocompatibility and lifespan of bioprosthetic valves. Using a single surfactant not only results in poor decellularization but also requires prolonged treatment time, which can damage the structure of the biological tissue.

[0015] Then, glutaraldehyde is used to cross-link the pericardial material, which cross-links the collagen in the pericardial material, increases its strength, reduces its immunogenicity, increases the stability of the pericardial material, and has a disinfecting effect. Finally, a reducing agent is used to reduce the aldehyde groups remaining after glutaraldehyde cross-linking, directly blocking the residual aldehyde groups and preventing calcium ions from attaching to the residual aldehyde groups to form calcium salt crystals, thereby improving the anti-calcification ability of the valve material and thus improving the service life of the valve.

[0016] Finally, the pericardial material was soaked in a mixture of glycerol and ethanol, which changed the hydration state and surface chemical properties of the pericardial material, reduced calcium ion adsorption, weakened the adsorption capacity of calcification-related proteins, and reduced calcification. In addition, the glycerol solution can penetrate into the collagen molecules, forming hydrogen bonds and stabilizing their triple helix structure. This stabilizing effect can prevent collagen from denaturing in the body environment, thereby maintaining the elasticity and toughness of the valve. At the same time, the glycerol solution can also reduce water loss and maintain the softness of the valve.

[0017] Optionally, the nonionic surfactant is selected from at least one of Triton X-100, Tween-20 / 80, dodecyl polyoxyethylene ether, and ethyl phenyl polyethylene glycol.

[0018] The anionic surfactant is selected from at least one of deoxycholic acid, sodium dodecyl sulfate, and sodium deoxycholate.

[0019] By adopting the above technical solution, Triton X-100, a polyethylene glycol octylphenyl ether, possesses a good hydrophilic-lipophilic balance (HLB value of approximately 13.5). It can form micelles in aqueous solution, effectively dissolving lipid molecules on the cell membrane, disrupting the cell membrane's integrity, and thus releasing cell contents. Furthermore, it causes relatively little damage to the extracellular matrix, preserving its structure and function to a certain extent. Tween-20 has an HLB value of approximately 16.7, and Tween-80 has an HLB value of approximately 15, exhibiting good emulsifying, dispersing, and wetting properties, and exhibiting good protein denaturation properties. The effect is relatively small, which can reduce the damage to the protein components in the extracellular matrix during decellularization, thus helping to maintain the bioactivity and mechanical properties of the bioprosthetic valve; dodecyl polyoxyethylene ether has an HLB value of about 16.9, has good solubility and stability in aqueous solution, can effectively reduce surface tension, and has a mild effect on cell penetration and dissolution, reducing damage to the extracellular matrix while decellularizing, and has little impact on subsequent biocompatibility; ethyl phenyl polyethylene glycol (NP-40) has an effect between Tween and Triton X-100, balancing the decellularization effect and the retention of the extracellular matrix.

[0020] The sulfate ester group in sodium dodecyl sulfate molecules carries a negative charge and can form micelles in aqueous solution, which can effectively disrupt the cell membrane and the lipid bilayer structure inside the cell, resulting in high decellularization efficiency and rapid removal of cells from tissues. Deoxycholic acid is a bile acid whose molecular structure contains a carboxyl group. In aqueous solution, the carboxyl group partially dissociates to release hydrogen ions, giving the deoxycholic acid molecule a negative charge. Its hydrophobic part can insert into the phospholipid bilayer of the cell membrane, while the hydrophilic part is exposed in the aqueous phase. This insertion disrupts the orderly arrangement of phospholipid molecules, increases cell membrane fluidity, and compromises cell membrane integrity. Deoxycholic acid can interact with lipid components in the cell membrane to form mixed micelles, dissolving lipids from the cell membrane. Deoxycholic acid can "wash away" lipids from the cell membrane, thereby causing the cell membrane to lose its barrier function, 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, can destroy the lipid structure of the cell membrane and intracellular cells, and the dissolution 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.

[0021] Optionally, the nonionic surfactant is Triton X-100, and the anionic surfactant is deoxycholic acid.

[0022] By employing the above-mentioned technical solution, Triton X-100 can insert into the phospholipid bilayer of the cell membrane, altering the cell membrane's fluidity and making it more porous. Deoxycholic acid further interacts with lipids in the cell membrane, dissolving lipid components and forming mixed micelles, accelerating cell membrane breakdown. The synergistic effect of these two processes enables faster and more effective disruption of the cell membrane, facilitating the release of cell contents and thus improving decellularization efficiency, shortening processing time, and increasing overall efficiency. Furthermore, this synergistic effect allows for deeper penetration into the cell interior, ensuring more thorough cell removal, reducing the possibility of residual cells, and minimizing the risk of cell recurrence. The presence of residual cell debris and intracellular antigens reduces the immunogenicity of bioprosthetic valves and decreases calcification caused by residual cells. Furthermore, both products cause relatively little damage to the extracellular matrix, preserving its integrity to the greatest extent possible during decellularization. The mechanical properties of the extracellular matrix are crucial for the function of tissue-engineered products such as bioprosthetic valves. Therefore, the combined use of Triton X-100 and deoxycholic acid avoids excessive damage to the fibrous structure of the extracellular matrix, thus maintaining its mechanical properties. Moreover, both have good water solubility, are easy to prepare, require mild processing conditions, and are low in cost, thus improving economic efficiency.

[0023] Optionally, the nonionic surfactant is Triton X-100, and the anionic surfactant is sodium dodecyl sulfate.

[0024] By adopting the above technical solution, Triton X-100 can first destroy the surface structure of the cell membrane, making the cell membrane loose. Then, SDS can further penetrate into the cell and destroy the lipid bilayer structure inside the cell, completely releasing the cell contents. However, it causes greater damage to the extracellular matrix, affecting the mechanical properties and biocompatibility of the bioprosthetic valve material.

[0025] Optionally, the nonionic surfactant is Triton X-100, and the anionic surfactant is sodium deoxycholate.

[0026] By adopting the above technical solution, the mild effect of sodium deoxycholate combined with Triton X-100 can effectively remove cells while reducing damage to the extracellular matrix. Sodium deoxycholate can assist Triton X-100 in destroying cell membranes, and its antibacterial effect helps maintain hygienic conditions during the decellularization process.

[0027] Optionally, the nonionic surfactant is Tween-20 / 80, and the anionic surfactant is sodium dodecyl sulfate.

[0028] 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. Extending the time will lead to a decrease in the mechanical strength of the pericardial material.

[0029] Optionally, the nonionic surfactant is dodecyl polyoxyethylene ether, and the anionic surfactant is sodium deoxycholate.

[0030] By adopting the above technical solution, both dodecyl polyoxyethylene ether and sodium deoxycholate have a mild decellularization effect. The two work synergistically to effectively remove cells while minimizing damage to the extracellular matrix.

[0031] Optionally, the reducing agent solution is one of the following: saturated sodium bisulfite solution, sodium cyanoborohydride in ethanolamine solution, sodium borohydride in ethanol solution, potassium borohydride in ethanol solution, cystine in PBS buffer, glutathione in PBS buffer, and lithium aluminum hydride in anhydrous diethyl ether solution.

[0032] By employing the above technical solutions, sodium bisulfite can undergo a nucleophilic addition reaction with residual aldehyde groups to generate stable aldehyde adducts, thereby blocking the aldehyde groups. The reaction conditions and temperature do not destroy the already formed cross-linked structure, and the cost is low and the operation is relatively simple. However, it cannot reduce the Schiff base formed by glutaraldehyde and protein amino groups, only blocking the free aldehyde groups, and 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 the cross-linking of glutaraldehyde and protein amino groups to stable secondary amino bonds. While blocking the free aldehyde groups, it stabilizes the Schiff base, making the cross-linked network more stable. However, it requires strict cleaning to avoid cyano residue.

[0033] Optionally, the concentration of the sodium cyanoborohydride ethanolamine solution is 0.2-0.5M.

[0034] By adopting the above technical solution, sodium cyanoborohydride can provide sufficient reducing power at the lower concentration to complete the aldehyde group blocking within a reasonable time, and the reaction process is relatively easy to control with few side reactions.

[0035] Optionally, the buffer solution is one of 0.01M Tris-HCl buffer, 0.01M PBS buffer, and 0.01M D-Hanks buffer.

[0036] By employing the above-mentioned technical solution, the Tris-HCl buffer has a wide pH range, can resist certain acid-base changes, and maintains relative pH stability of the solution. The PBS buffer contains sodium, potassium, and chloride ions, which are similar to the ionic composition of extracellular fluid, providing an ionic atmosphere similar to the physiological environment for the decellularization process. In the suitable environment provided by the buffer, nonionic and anionic surfactants can better bind to the cell membrane, exerting their decellularization effect, while the structure and function of the extracellular matrix are better preserved. Furthermore, the buffer can dilute harmful substances generated during the decellularization process, reducing their toxic effects on the extracellular matrix.

[0037] Optionally, the weight ratio of the pericardial material to the volume of the decellularization treatment solution A is 1:4-10;

[0038] The weight ratio of the pericardial material obtained in step S2 to the volume of the decellularization treatment solution B is 1:4-10.

[0039] By adopting the above technical solution, a good decellularization effect can be achieved within a certain range of the amount of pericardial material and decellularization solution used. If the volume of the decellularization solution is small and the concentration of the effective components in the solution is relatively high, but the solution is insufficient to fully wet the pericardial material, it will lead to incomplete decellularization, more residual cells, calcium ion deposition, accelerated calcification process, and local high concentration of decellularization reagent may excessively degrade the extracellular matrix, resulting in a decrease in the mechanical properties of the pericardial material. Moreover, cell debris cannot be removed in time and accumulates on the surface or inside of the pericardial material, affecting the structural integrity of the material. If the volume of the decellularization solution is large, although it can fully wet the pericardial material, with high decellularization efficiency and less cell residue, it will increase the processing time and cost.

[0040] Optionally, the glutaraldehyde concentration is 0.4-0.65%, the crosslinking pH value is 6.5-7.4, the crosslinking temperature is 20-40℃, and the crosslinking time is 6-7 days.

[0041] By adopting the above technical solution, with appropriate concentration, pH value and reaction temperature, glutaraldehyde can be completely cross-linked with collagen and other components in pericardial materials, thereby improving the mechanical properties of the cross-linked pericardial materials. If the cross-linking is incomplete, the degree of cross-linking will be 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, losing its flexibility and elasticity.

[0042] Optionally, the oscillation frequency in steps S2, S3 and S5 is 100-160 rpm.

[0043] By adopting the above technical solutions, an appropriate oscillation frequency can improve decellularization efficiency, reduce cell residue, protect the integrity of the material structure and the uniformity of processing, and shorten the processing time.

[0044] Optionally, the pericardial material is pig pericardium or bovine pericardium.

[0045] Secondly, this application provides a biological valve material, which adopts the following technical solution:

[0046] A bioprosthetic valve material is prepared by the aforementioned anti-calcification method for composite bioprosthetic valve materials.

[0047] By adopting the above technical solution, a biological valve material is prepared by first decellularizing in two steps, and then cross-linking with glutaraldehyde, blocking aldehyde groups, and soaking treatment. The material has fewer cell debris and excess aldehyde group residues, avoids calcification caused by the introduction of aldehyde groups during glutaraldehyde soaking, and has a good anti-calcification effect.

[0048] In summary, this application has the following beneficial effects:

[0049] 1. The method of this application first performs two-step decellularization on the biological material. Using EDTA, PMSF and buffer, the tissue structure of the pericardium is first opened to facilitate the subsequent penetration of surfactants. Then, nonionic surfactants and anionic surfactants are used in combination to further remove cell debris, remove highly antigenic epithelial cells and fibroblasts, reduce antigenicity, reduce important sites for calcification of biological tissue, and retain the extracellular matrix, thereby enhancing the anti-calcification performance of the biological material. Then, glutaraldehyde is 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 a glycerol ethanol solution to avoid the introduction of aldehyde groups by soaking in glutaraldehyde, while giving the pericardial material good flexibility and elasticity.

[0050] 2. In this application, Triton X-100 is preferably used as a nonionic surfactant and deoxycholic acid is used as an anionic surfactant. The combination of the two can more effectively destroy the cell membrane, with higher decellularization efficiency, reducing residual cells, reducing the possibility of calcification, and the two have relatively less damage to the extracellular matrix, preserving the integrity of the extracellular matrix, thereby maintaining the mechanical properties of the pericardial material.

[0051] 3. The method in this application first performs decellularization, then uses two surfactants for treatment, blocks the aldehyde groups after cross-linking with glutaraldehyde, and then soaks in a glycerol mixed solution. It can be used directly afterward. The processing time is short and the execution efficiency is high. It avoids the calcification effect caused by glutaraldehyde soaking. The combination of two surfactants and multiple methods results in valve materials with less damage, fewer residual cells and aldehyde groups, excellent anti-calcification effect, and good durability. Attached Figure Description

[0052] Figure 1 The image in the middle shows the calcification of the valve material of Example 1 and Comparative Example 7 8 weeks after subcutaneous implantation (Experimental group: Example 1; Control group: Comparative Example 7).

[0053] Figure 2 The diagram in the middle shows the calcium content detection after subcutaneous implantation of the valve materials prepared in Example 1 and Comparative Example 7 (Experimental group is Example 1, control group is Comparative Example 7). Detailed Implementation

[0054] The following embodiments provide a further detailed description of this application.

[0055] Example

[0056] Example 1: A method for preventing calcification of a combined bioprosthetic valve material, comprising the following steps:

[0057] S1. Take fresh bovine pericardium material, wash it with clean water, remove excess fat tissue on the surface, cut a relatively uniform part of the precordial area to obtain pericardium material, and store it in ice-cold physiological saline for later use.

[0058] S2. Add the pericardial material to decellularization solution A and shake at 4°C for 4 hours at a shaking frequency of 120 rpm. Discard the liquid and retain the pericardial material. Wash three times with physiological 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).

[0059] S3. Add the pericardial material obtained in step S2 to the decellularization solution B, and shake at 4°C for 4 hours at a shaking frequency of 120 rpm. Remove the pericardial material and wash it 3 times with physiological saline. The ratio of the pericardial material obtained in step S2 to the decellularization solution B is 1:4 (weight:volume). The decellularization solution B contains a nonionic surfactant, an anionic surfactant, and decellularization solution A. The nonionic surfactant is Triton X-100 with a concentration of 0.5%, and the anionic surfactant is deoxycholic acid with a concentration of 1%.

[0060] S4. The pericardial material obtained in step S3 is placed in glutaraldehyde for cross-linking treatment. The concentration of glutaraldehyde is 0.65%, the pH value during cross-linking treatment is 7.4, the cross-linking temperature is 40℃, and the cross-linking time is 7 days.

[0061] S5. Add the cross-linked pericardial material to the reducing agent solution, which is saturated sodium sulfite. The ratio of the cross-linked pericardial material to the reducing agent solution is 1:3 (weight:volume). Shake at 4°C for 4 hours at a shaking frequency of 120 rpm. Remove the pericardial material and wash it 3 times with physiological saline.

[0062] S6. Place the pericardial material obtained in step S5 into a 75% glycerol-ethanol mixed solution and soak it at 4°C for more than 24 hours to complete the anti-calcification treatment.

[0063] Example 2: A method for preventing calcification of a combined bioprosthetic valve material, comprising the following steps:

[0064] S1. Take fresh bovine pericardium material, wash it with clean water, remove excess fat tissue on the surface, cut a relatively uniform part of the precordial area to obtain pericardium material, and store it in ice-cold physiological saline for later use.

[0065] S2. Add the pericardial material to decellularization solution A, shake at 8°C for 3 hours at a shaking frequency of 160 rpm, discard the liquid and retain the pericardial material, wash 3 times with physiological 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).

[0066] S3. Add the pericardial material obtained in step S2 to the decellularization solution B, and shake at 10°C for 2 hours at a shaking frequency of 160 rpm. Remove the pericardial material and wash it 3 times with physiological saline. The ratio of the pericardial material obtained in step S2 to the decellularization solution B is 1:10 (weight:volume). The decellularization solution B contains a nonionic surfactant, an anionic surfactant, and decellularization solution A. The nonionic surfactant is Triton X-100 with a concentration of 2%, and the anionic surfactant is deoxycholic acid with a concentration of 1.5%.

[0067] S4. The pericardial material obtained in step S3 is placed in glutaraldehyde for cross-linking treatment. The concentration of glutaraldehyde is 0.4%, the pH value during cross-linking treatment is 6.5, the cross-linking temperature is 20℃, and the cross-linking time is 7 days.

[0068] S5. Add the cross-linked pericardial material to the reducing agent solution, which is saturated sodium sulfite. The ratio of the cross-linked pericardial material to the reducing agent solution is 1:4 (weight:volume). Shake at 10°C for 2 hours at a shaking frequency of 160 rpm. Remove the pericardial material and wash it 3 times with physiological saline.

[0069] S6. Place the pericardial material obtained in step S5 into a 70% glycerol-ethanol mixed solution and soak it at 8°C for more than 24 hours to complete the anti-calcification treatment.

[0070] Example 3: A method for preventing calcification of a combined bioprosthetic valve material, comprising the following steps:

[0071] S1. Take fresh bovine pericardium material, wash it with clean water, remove excess fat tissue on the surface, cut a relatively uniform part of the precordial area to obtain pericardium material, and store it in ice-cold physiological saline for later use.

[0072] S2. Add the pericardial material to decellularization solution A and shake at 6°C for 4 hours at a shaking frequency of 140 rpm. Discard the liquid and retain the pericardial material. Wash three times with physiological 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).

[0073] S3. Add the pericardial material obtained in step S2 to the decellularization solution B, and shake at 8°C for 3 hours at a shaking frequency of 140 rpm. Remove the pericardial material and wash it 3 times with physiological saline. The ratio of the pericardial material obtained in step S2 to the decellularization solution B is 1:7 (weight:volume). The decellularization solution B contains a nonionic surfactant, an anionic surfactant, and decellularization solution A. The nonionic surfactant is Triton X-100 with a concentration of 1%, and the anionic surfactant is deoxycholic acid with a concentration of 1%.

[0074] S4. The pericardial material obtained in step S3 is placed in glutaraldehyde for cross-linking treatment. The concentration of glutaraldehyde is 0.5%, the pH value during cross-linking treatment is 7, the cross-linking temperature is 30℃, and the cross-linking time is 7 days.

[0075] S5. Add the cross-linked pericardial material to the reducing agent solution, which is saturated sodium sulfite. The ratio of the cross-linked pericardial material to the reducing agent solution is 1:5 (weight:volume). Shake at 8°C for 3 hours at a shaking frequency of 140 rpm. Remove the pericardial material and wash it 3 times with physiological saline.

[0076] S6. Place the pericardial material obtained in step S5 into a 70% glycerol-isopropanol mixed solution and soak it at 6°C for more than 24 hours to complete the anti-calcification treatment.

[0077] Example 4: A method for preventing calcification of a combined bioprosthetic valve material, which differs from Example 1 in that the nonionic surfactant is Triton X-100 and the anionic surfactant is sodium dodecyl sulfate.

[0078] Example 5: A method for preventing calcification of a combined bioprosthetic valve material, which differs from Example 1 in that the nonionic surfactant is Triton X-100 and the anionic surfactant is sodium deoxycholate.

[0079] Example 6: A method for preventing calcification of a combined bioprosthetic valve material, which differs from Example 1 in that the nonionic surfactant is Tween-20 and the anionic surfactant is sodium dodecyl sulfate.

[0080] Example 7: A method for preventing calcification of a combined bioprosthetic valve material, which differs from Example 1 in that the nonionic surfactant is dodecyl polyoxyethylene ether and the anionic surfactant is sodium deoxycholate.

[0081] Example 8: A method for preventing calcification of a combined bioprosthetic valve material, which differs from Example 1 in that the reducing agent is an ethanolamine solution of sodium cyanoborohydride at a concentration of 0.5M.

[0082] Comparative Example

[0083] Comparative Example 1: A method for preventing calcification of a combined bioprosthetic valve material. The difference from Example 1 is that step S3 is not set, and the pericardial material obtained in step S2 is directly subjected to glutaraldehyde crosslinking treatment. The remaining steps and parameters are the same as in Example 1.

[0084] Comparative Example 2: A method for preventing calcification of a combined bioprosthetic valve material, which differs from Example 1 in that step S2 is omitted, and the pericardial material obtained in step S1 is directly added to the decellularization treatment solution B. In addition, an equal amount of physiological saline is used in the decellularization treatment solution B instead of the decellularization treatment solution A. The remaining steps and parameters are the same as in Example 1.

[0085] Comparative Example 3: A method for preventing calcification of a combined bioprosthetic valve material, which differs from Example 1 in that step S2 is omitted, 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 in Example 1.

[0086] Comparative Example 4: A method for preventing calcification of a combined bioprosthetic valve material, which differs from Example 1 in that, in step S3, the decellularization treatment solution B contains only decellularization treatment solution A and nonionic surfactant Triton X-100, with a concentration of 0.5% for the nonionic surfactant. The remaining parameters are the same as in Example 1.

[0087] Comparative Example 5: A method for preventing calcification of a combined bioprosthetic valve material, which differs from Example 1 in that, in step S3, the decellularization treatment solution B contains only decellularization treatment solution A and the anionic surfactant deoxycholic acid, with the concentration of the anionic surfactant being 1%, and the remaining parameters being the same as in Example 1.

[0088] Comparative Example 6: A method for preventing calcification of a combined bioprosthetic valve material, which differs from Example 1 in that, in step S5, it is preserved in a 0.2 wt% glutaraldehyde solution.

[0089] Comparative Example 7: A method for preventing calcification of a biological valve material, comprising the following steps: freshly collected bovine pericardium is washed with distilled water for 4 hours under oscillation conditions at 4°C and 120 rpm, then soaked in a 0.625% glutaraldehyde solution for 24 hours. After the reaction is completed, it is taken out and soaked in a 0.2% glutaraldehyde solution for storage.

[0090] Performance testing

[0091] Biological valve materials were prepared according to the methods in the examples and comparative examples, and their performance was tested according to the following methods. The test results are recorded in Table 1.

[0092] 1. Calcium deposition: Referring to the method in YY / T1859-2022 "Evaluation of Anti-calcification 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 cm². 2 For each 10mm×10mm specimen, 3-week-old SPF-grade Wistar rats weighing 45-55g were selected. After weighing and anesthesia, one anti-calcification specimen was implanted in each of four areas: the upper and lower subcutaneous regions of the left and right back, for a total of four specimens per rat. The skin was then sutured. At the specified time endpoint (8 weeks in this test), the rats were weighed, and the specimens were carefully removed. The transplanted surface was rinsed with physiological saline and smoothed. The specimens were then dried in a constant-temperature oven to constant weight. After digestion, the calcium deposition was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES), expressed as micrograms per milligram of dry weight. Five rats were tested in each example, and the results were averaged.

[0093] 2. Uniaxial tensile test: Each group of materials was cut into strips of 1cm×5cm (n=20). The tensile test was performed using an electronic tensile testing machine (Instron, USA) at a tensile speed of 10mm / min. The stress-strain curves were analyzed to obtain the maximum tensile strength and elastic modulus.

[0094] Table 1 Performance test results of valve materials

[0095]

[0096]

[0097] As can be seen from Table 1 and the anti-calcification methods in Examples 1-3, the valve materials obtained by the anti-calcification methods in Examples 1-3 have a low amount of calcium deposits after implantation, and have a large maximum tensile strength and elastic modulus, exhibiting good stability and anti-calcification ability.

[0098] In Example 4, Triton X-100 was used as a nonionic surfactant and sodium dodecyl sulfate as an anionic surfactant. The two were used together to further decellularize the pericardial material. The amount of calcium attached to the valve material increased not significantly, but its maximum tensile strength and elastic modulus decreased. This indicates that although the combination of Triton X-100 and sodium dodecyl sulfate can remove cell debris, it increases the damage to the extracellular matrix.

[0099] 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 as in Example 1.

[0100] Compared with Example 1, Example 6 uses Tween-20 as the nonionic surfactant and sodium dodecyl sulfate as the cationic surfactant. Its decellularization effect is not as good as that of Example 1, but the amount of calcium adsorbed is increased.

[0101] In Example 7, dodecyl polyoxyethylene ether and sodium deoxycholate were used in synergy. Compared with Example 1, the anti-calcification ability was slightly reduced, but the mechanical properties were similar.

[0102] Compared with Example 1, Example 8 uses a low-concentration sodium cyanoborohydride ethanolamine solution as a reducing agent, which shows that the blocking effect on aldehyde groups is good, and it can also reduce Schiff bases, making the structure stable.

[0103] Compared with Example 1, Comparative Example 1 did not use decellularization solution B, but only decellularization solution A. In Comparative Example 2, only decellularization solution B was used, and decellularization solution B did not contain decellularization solution A. In Comparative Example 3, only decellularization solution B was used. It can be seen that Comparative Example 3 had the best decellularization effect, while Comparative Example 2 had the worst effect and the weakest anti-calcification ability.

[0104] Compared with Example 1, Comparative Examples 4 and 5 contain only a single surfactant in the decellularization treatment solution B. That is, the decellularization and re-treatment is carried out using a single surfactant, which shows that the anti-calcification ability of the prepared valve material is weakened.

[0105] Comparative Example 6 used glutaraldehyde solution to soak and store the valve material, and it can be seen that the valve material had poor anti-calcification ability after implantation. Comparative Example 7 is a valve material made by cross-linking with glutaraldehyde and soaking in glutaraldehyde solution. As can be seen from the data in Table 1, compared with Example 1, the amount of calcium attached to the valve materials prepared by Comparative Example 6 and Comparative Example 7 is increased and the anti-calcification ability is weakened.

[0106] Following the method in YY / T1859-2022 "Evaluation of Anti-calcification 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 cm². 2 (10mm×10mm / single piece) specimen slides were prepared from 3-week-old SPF-grade Wistar rats weighing 45-55g. The rats were weighed, anesthetized, and the skin was sutured subcutaneously in the upper left back. At the specified time endpoint (8 weeks in this test), the rats were weighed and the specimens removed. The host tissue on the transplanted surface was carefully removed, the specimens were rinsed with physiological saline, smoothed, and air-dried. A small portion was taken for histochemical examination. Figure 1 As shown in the figure, observations reveal that after Von Kossa calcium salt staining, the experimental group remained very smooth with no obvious calcification spots, demonstrating good anti-calcification properties. In contrast, the control group prepared in Comparative Example 5 showed obvious calcification spot deposition. The amount of calcium deposited in the experimental group of Example 1 and the control group of Comparative Example 5 is as follows: Figure 2 As shown (experimental group 0.6±0.3μg / mg, control group 135.1±13.8μg / mg), it can be seen that two steps of removing cell debris are performed before glutaraldehyde crosslinking and fixation. In addition, the use of surfactant combination during cell debris removal can produce valve material with strong anti-calcification ability.

[0107] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A combined method of bioprosthetic valve material calcification resistance, characterized by, It comprises the following steps: S1, taking animal-derived biomaterials, washing, removing adipose tissue, cutting the precordial part, obtaining pericardial material, and storing in ice physiological saline for standby; S2, adding the pericardial material to the decellularization treatment liquid A, oscillating at 4-8℃ for 3-4h, taking out the pericardial material, and washing with physiological saline, wherein the decellularization treatment liquid A is a buffer containing EDTA and PMSF, and the mass ratio of EDTA and PMSF is 2:0.001-0.002; S3, adding the pericardial material prepared in step S2 to the decellularization treatment liquid B, oscillating at 4-10℃ for 2-4h, taking out the pericardial material, and washing with physiological saline, wherein the decellularization treatment liquid B contains the decellularization treatment liquid A, non-ionic surfactant and anionic surfactant, the concentration of non-ionic surfactant is 0.5-2%, and the concentration of anionic surfactant is 0.5-1.5%; S4, cross-linking the pericardial material obtained in step S3 by using glutaraldehyde; S5, adding the cross-linked pericardial material to the reducing agent solution, oscillating at 4-10℃ for 2-4h, taking out the pericardial material, and washing with physiological saline; S6, placing the pericardial material obtained in step S5 in a 70-75% concentration of glycerol-ethanol mixed solution or glycerol-isopropanol mixed solution, and immersing at 4-8℃ for 24h or more to complete the anti-calcification treatment; The non-ionic surfactant is triton X-100, and the anionic surfactant is deoxycholic acid; The reducing agent solution is saturated sodium bisulfite solution or sodium cyanoborohydride ethanolamine solution.

2. The combination biovalve material anti-calcification method of claim 1, wherein: The buffer is one of 0.01M Tris-HCl buffer, 0.01M PBS buffer and 0.01M D-Hanks buffer.

3. The combination biovalve material anti-calcification method of claim 1, wherein: The ratio of the weight of the pericardial material to the volume of the decellularization treatment liquid A is 1:4-10; The ratio of the weight of the pericardial material prepared in step S2 to the volume of the decellularization treatment liquid B is 1:4-10; The ratio of the weight of the cross-linked pericardial material in step S5 to the volume of the reducing agent solution is 1:3-5.

4. The combination biovalve material anti-calcification method of claim 1, wherein: The concentration of glutaraldehyde is 0.4-0.65%, the cross-linking pH value is 6.5-7.4, the cross-linking temperature is 20-40℃, and the cross-linking time is 6-7 days.

5. The combination biovalve material anti-calcification method of claim 1, wherein: The oscillation frequency in steps S2, S3 and S5 is 120-160rpm.

6. The combination biovalve material anti-calcification method of claim 1, wherein: The pericardial material is pig pericardium or bovine pericardium.

7. A biological valve material, characterized by, Prepared by the method of claim 1-6. Prepared by the method of claim 1-6.

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