Heart valve capable of reducing calcification and improving mechanical property as well as preparation method and application of heart valve

Abstract

The invention provides a heart valve capable of reducing calcification and improving mechanical properties and a preparation method and application of the heart valve. The preparation method comprises the following steps: carrying out first cross-linking treatment on the decellularized pericardial tissue by adopting a procyanidine solution; and carrying out second cross-linking treatment on the decellularized pericardial tissue by adopting a glutaraldehyde solution to prepare the heart valve, wherein the pH (Potential of Hydrogen) value of the procyanidine solution is 6.0 to 8.0; the temperature of the first crosslinking treatment is 21-25 DEG C, and the time is 22-26 hours. According to the present invention, the research results show that the decellularized pericardial tissue is subjected to cross-linking treatment by using the procyanidine solution under the conditions of specific pH value, temperature and time, and then the second cross-linking treatment is performed by using the glutaraldehyde solution, such that the prepared heart valve has excellent tensile fracture resistance and excellent tear resistance; and meanwhile, the viscoelasticity close to that of natural tissues is achieved, so that stress concentration and fatigue damage are reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological valve materials, in particular to a heart valve with reduced calcification and improved mechanical properties, and a preparation method and application thereof. BACKGROUND

[0002] Heart valve disease is a common heart disease worldwide, and its treatment depends on artificial valve replacement. Artificial heart valves mainly include mechanical valves and biological valves. However, mechanical valve replacement requires lifelong anticoagulant therapy, which has a risk of bleeding. Biological valves have hemodynamic performance and do not require long-term anticoagulation, and have become the first choice for artificial valve replacement.

[0003] At present, common xenogeneic heart biological valves are mainly derived from pigs and cows, and their heart valve size, shape and function are similar to human heart valves, and have good biocompatibility. However, before clinical application, the decellularized pericardial tissue from the above sources needs to be cross-linked to meet the clinical requirements of mechanical properties.

[0004] Although the traditional cross-linking treatment method can enhance the mechanical strength of the heart valve, it will irreversibly change the spatial conformation of collagen fibers, accelerate the ectopic deposition of calcium phosphate crystals on the surface of the valve, and ultimately cause the leaflet to harden and tear. Therefore, the traditional treatment method of the heart valve still needs to be improved. SUMMARY

[0005] Therefore, one or more embodiments of the present application provide a heart valve with reduced calcification and improved mechanical properties, and a preparation method and application thereof.

[0006] According to a first aspect of an embodiment of the present application, a preparation method of a heart valve with reduced calcification and improved mechanical properties is provided, comprising the following steps:

[0007] performing first cross-linking treatment on the decellularized pericardial tissue using a procyanidine solution; and

[0008] performing second cross-linking treatment on the decellularized pericardial tissue using a glutaraldehyde solution to prepare the heart valve.

[0009] The pH value of the procyanidine solution is 6.0-8.0, the temperature of the first cross-linking treatment is 21-25°C, and the time is 22-26h.

[0010] In some embodiments, the pH value of the glutaraldehyde solution is 5.0-5.5.

[0011] In some embodiments, after the first cross-linking treatment and before the second cross-linking treatment, the following steps are further included:

[0012] The decellularized pericardial tissue after the first cross-linking treatment is rinsed with a PBS solution.

[0013] Optionally, the rinsing treatment is performed 2-4 times.

[0014] In some embodiments, the concentration of procyanidins in the procyanidin solution is 0.5-8 mg / mL.

[0015] In some embodiments, the rotation speed of the first cross-linking treatment is 60-100 rpm.

[0016] In some embodiments, the volume concentration of glutaraldehyde in the glutaraldehyde solution is 0.3-1%.

[0017] In some embodiments, the temperature of the second cross-linking treatment is 21-25℃; and / or,

[0018] the rotation speed of the second cross-linking treatment is 60-100 rpm; and / or,

[0019] the time of the second cross-linking treatment is 22-26 h.

[0020] In some embodiments, the source of the decellularized pericardial tissue comprises at least one of pig, cow, sheep, horse, and monkey.

[0021] According to a second aspect of the embodiments of the present application, a heart valve is provided, which is prepared by the method for preparing a heart valve described above.

[0022] According to a third aspect of the embodiments of the present application, the heart valve described above is applied to the preparation of a biological stent material.

[0023] Compared with the conventional technology, the present application has the following beneficial effects:

[0024] The inventors of the present application have found that, by using a procyanidin solution to cross-link the decellularized pericardial tissue under specific pH, temperature, and time conditions, and then using a glutaraldehyde solution for a second cross-linking treatment, the heart valve prepared thereby has excellent tensile fracture resistance and tear resistance, and has viscoelasticity close to that of natural tissue, thereby reducing stress concentration and fatigue damage. DETAILED DESCRIPTION

[0025] For the above purposes, features and advantages of the present application to be more clearly understood, a detailed description of the specific embodiments of the present application is provided. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. However, the present application can be practiced in many different ways from what is described herein, and should not be construed as being limited to the embodiments set forth herein, but should be given the broadest possible interpretation within the scope of the present application.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless otherwise specifically defined, various materials, reagents, instruments and equipment, etc. used in the present application can be obtained from commercial suppliers, such as Sigma-Aldrich, Merck, BioRad, Nalge Nunc International, VWR, and other commercial suppliers.

[0027] The terms "and / or", "or / and", "and / or" used herein are the inclusive or, as understood by persons of ordinary skill in the art. The selection range of the terms "and / or", "or / and", "and / or" includes any one of two or more relevant listed items, and also includes any and all combinations of the relevant listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0028] In the present application, "multiple", "various", "multiple times", "multiple" and the like are used without specific limitation, which means greater than or equal to 2 in number. For example, "one or more" means one or more than two.

[0029] The terms "combination thereof", "any combination thereof", "any combination manner thereof" and the like used herein include all suitable combination manners of any two or more of the listed items.

[0030] In the present application, “suitable”, “suitable manner”, “any suitable manner” and the like are used to describe the manner in which the technical solutions of the present application are implemented, the technical problems of the present application are solved, and the technical effects of the present application are achieved.

[0031] In the present application, “preferably”, “more preferably”, “more preferably”, “preferably” are only used to describe the embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of the present application.

[0032] In the present application, “further”, “more further”, “in particular” and the like are used to describe the purpose, indicating the difference in content, but should not be understood as a limitation on the protection scope of the present application.

[0033] In the present application, “optionally”, “optional” and “optional” mean that it can or can not be present, that is, it means to select from “yes” or “no” in the two parallel schemes. If there are multiple “options” in a technical solution, unless otherwise specified, and there is no contradictory relationship or mutual restriction, each “option” is independent.

[0034] In the present application, the technical features described in an open manner include both closed technical solutions consisting of listed features and open technical solutions containing listed features.

[0035] In the present application, if no special instructions are given, the numerical value distribution in the above numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e. the minimum value and the maximum value) of the numerical range, as well as each numerical value between the two numerical endpoints. If no special instructions are given, when the numerical interval only points to the integers in the numerical interval, including the two endpoint integers of the numerical range and each integer between the two endpoints, in this paper, it is equivalent to directly listing each integer, for example, t is an integer selected from 1-10, which means that t is any one integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe characteristics or properties, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges encompassed therein.

[0036] In the present application, the temperature parameter, unless otherwise specified, allows for constant temperature treatment and allows for variation within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. It is allowed to fluctuate within the range of, for example, ±5℃, ±4℃, ±3℃, ±2℃, ±1℃.

[0037] Some embodiments of the present application provide a method for preparing a heart valve with reduced calcification and improved mechanical properties, comprising S10 and S20:

[0038] S10: performing first cross-linking treatment on the acellular pericardial tissue by using the procyanidin solution; and

[0039] S20: performing second cross-linking treatment on the acellular pericardial tissue by using the glutaraldehyde solution, to prepare the heart valve.

[0040] In the application, the pH value of the procyanidin solution is 6.0-8.0; the temperature of the first cross-linking treatment is 21-25°C, and the time is 22-26h.

[0041] The inventors of the application have found that, by using the procyanidin solution to perform cross-linking treatment on the acellular pericardial tissue under specific pH, temperature and time conditions, and then using the glutaraldehyde solution to perform second cross-linking treatment, the heart valve prepared has excellent tensile fracture resistance and tear resistance, and has viscoelasticity close to that of natural tissue, thereby reducing stress concentration and fatigue damage.

[0042] The application analyzes the concentration of the procyanidin solution, the pH value, temperature and time of the first cross-linking, and other factors by gradient experiment and orthogonal design experiment, explores the influence of the factors on the tensile strength, tensile fracture strain and Young's modulus of the heart valve, and selects the preferred conditions for the first cross-linking by using the procyanidin solution; thereby the heart valve has excellent mechanical properties, so that the amount of glutaraldehyde used can be reduced to the greatest extent, and the calcification phenomenon that may occur during glutaraldehyde cross-linking can be reduced.

[0043] It can be understood that the preparation method or acquisition method of the acellular pericardial tissue is not particularly limited in the application, and can be any method commonly used in the art.

[0044] For example, the pH value of the procyanidin solution can be 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, or any value within the range formed by any two of the above-mentioned values. Further, the pH value of the procyanidin solution is 6.5-7.5. Still further, the pH value of the procyanidin solution is 7.0.

[0045] It can be understood that the pH value of the procyanidin solution in the application can be regarded as the environmental pH of the first cross-linking treatment.

[0046] In some embodiments, the preparation method of the heart valve comprises S10 and S20.

[0047] In some embodiments, the size of the decellularized pericardial tissue is 40 mm x 30 mm, the addition amount of the procyanidin solution is 45 mL to 55 mL, and the addition amount of the glutaraldehyde solution is 45 mL to 55 mL. Further, the size of the decellularized pericardial tissue is 40 mm x 30 mm, the addition amount of the procyanidin solution is 50 mL, and the addition amount of the glutaraldehyde solution is 50 mL.

[0048] For example, the temperature of the first cross-linking treatment is 21°C, 22°C, 23°C, 24°C, 25°C, or any value within the range formed by any two of the above values. Further, the temperature of the first cross-linking treatment is 22°C to 24°C. Further, the temperature of the first cross-linking treatment is 23°C.

[0049] In some embodiments, the pH value of the glutaraldehyde solution is 5.0 to 5.5.

[0050] For example, the pH value of the glutaraldehyde solution can be 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, or any value within the range formed by any two of the above values.

[0051] In some embodiments, after the first cross-linking treatment and before the second cross-linking treatment, the method further comprises the step of: rinsing the decellularized pericardial tissue after the first cross-linking treatment with a PBS solution.

[0052] Optionally, the rinsing treatment is performed 2 to 4 times. For example, the rinsing treatment can be performed 2 times, 3 times, 4 times, or any value within the range formed by any two of the above values. By rinsing the decellularized pericardial tissue after the procyanidin solution cross-linking treatment, the procyanidin solution remaining on the surface of the pericardial tissue can be effectively removed.

[0053] In some embodiments, each rinsing treatment is performed for 8 minutes to 12 minutes; further, each rinsing treatment is performed for 10 minutes.

[0054] In some embodiments, the concentration of procyanidin in the procyanidin solution is 0.5 mg / mL to 8 mg / mL.

[0055] For example, the concentration of procyanidins in the procyanidin solution can be 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, or any value within a range defined by any two of the above values. Further, the concentration of procyanidins in the procyanidin solution is 2 mg / mL to 4 mg / mL. Further still, the concentration of procyanidins in the procyanidin solution is 2 mg / mL.

[0056] In some embodiments, the solvent of the procyanidin solution comprises a PBS solution.

[0057] In some embodiments, the rotation speed of the first cross-linking treatment is 60 rpm to 100 rpm.

[0058] For example, the rotation speed of the first cross-linking treatment can be 60 rpm, 61 rpm, 62 rpm, 63 rpm, 64 rpm, 65 rpm, 66 rpm, 67 rpm, 68 rpm, 69 rpm, 70 rpm, 71 rpm, 72 rpm, 73 rpm, 74 rpm, 75 rpm, 76 rpm, 77 rpm, 78 rpm, 79 rpm, 80 rpm, 81 rpm, 82 rpm, 83 rpm, 84 rpm, 85 rpm, 86 rpm, 87 rpm, 88 rpm, 89 rpm, 90 rpm, 91 rpm, 92 rpm, 93 rpm, 94 rpm, 95 rpm, 96 rpm, 97 rpm, 98 rpm, 99 rpm, 100 rpm, or any value within a range defined by any two of the above values. Further, the rotation speed of the first cross-linking treatment is 70 rpm to 90 rpm. Further still, the rotation speed of the first cross-linking treatment is 80 rpm.

[0059] It can be understood that, under the condition of shaking at a specific rotation speed, the procyanidin solution can more uniformly cross-link the pericardial tissue.

[0060] In some embodiments, the volume concentration of glutaraldehyde in the glutaraldehyde solution is 0.3% to 1%.

[0061] For example, the volume concentration of glutaraldehyde in the glutaraldehyde solution can be 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, or any value within a range defined by any two of the above values. Further, the volume concentration of glutaraldehyde in the glutaraldehyde solution is 0.3% to 0.6%. Further still, the volume concentration of glutaraldehyde in the glutaraldehyde solution is 0.3%.

[0062] In some embodiments, the solvent of the glutaraldehyde solution comprises water.

[0063] It can be understood that the concentration of glutaraldehyde in the glutaraldehyde solution is close to the concentration of glutaraldehyde in the wet valve preservation method of the valve, and therefore the second cross-linking treatment using the glutaraldehyde solution with the specific volume concentration can also have the effect of material preservation.

[0064] In some embodiments, the temperature of the second cross-linking treatment is 21℃-25℃.

[0065] For example, the temperature of the second cross-linking treatment is 21℃, 22℃, 23℃, 24℃, 25℃, or any value within the range formed by any two of the above values. Further, the temperature of the second cross-linking treatment is 23℃.

[0066] In some embodiments, the rotation speed of the second cross-linking treatment is 60rpm-100rpm.

[0067] For example, the rotation speed of the first cross-linking treatment can be 60rpm, 61rpm, 62rpm, 63rpm, 64rpm, 65rpm, 66rpm, 67rpm, 68rpm, 69rpm, 70rpm, 71rpm, 72rpm, 73rpm, 74rpm, 75rpm, 76rpm, 77rpm, 78rpm, 79rpm, 80rpm, 81rpm, 82rpm, 83rpm, 84rpm, 85rpm, 86rpm, 87rpm, 88rpm, 89rpm, 90rpm, 91rpm, 92rpm, 93rpm, 94rpm, 95rpm, 96rpm, 97rpm, 98rpm, 99rpm, 100rpm, or any value within the range formed by any two of the above values.

[0068] In some embodiments, the time of the second cross-linking treatment is 22h-26h.

[0069] For example, the time of the second cross-linking treatment can be 22h, 23h, 24h, 25h, 26h, or any value within the range formed by any two of the above values. Further, the time of the second cross-linking treatment is 24h.

[0070] In some embodiments, the source of the decellularized pericardial tissue comprises at least one of pig, cow, sheep, horse, and monkey. It can be understood that the method for preparing the heart valve of the present application is not limited to the decellularized pericardial tissue of the above specific sources, but can also be applied to pericardial tissue of other sources.

[0071] The heart valve prepared by the method has excellent tensile fracture resistance and tear resistance, and has elasticity similar to that of a natural heart valve, so that stress concentration and fatigue damage can be reduced, and the anti-calcification ability and mechanical properties of the heart valve can be improved.

[0072] Some embodiments of the present application also provide a heart valve prepared by the above method.

[0073] The heart valve has excellent mechanical properties, is not prone to calcification, and has viscoelasticity similar to that of a natural biological valve, and is suitable as an artificial biological valve material.

[0074] Some embodiments of the present application also provide the use of the above heart valve in the preparation of a biological stent material.

[0075] The present application will be further described in conjunction with specific examples and comparative examples, but should not be understood as limiting the scope of protection of the present application. The raw materials involved in the following specific examples, if not specifically stated, can be sourced from the market, the instruments used, if not specifically stated, can be sourced from the market, and the processes involved, if not specifically stated, are commonly selected by those skilled in the art.

[0076] Example 1

[0077] (1) Effect of different concentrations of procyanidins on the first crosslinking treatment

[0078] (1.1) 0.5 mg, 1 mg, 2 mg, 4 mg and 8 mg of procyanidins (PC) were dissolved in 1 mL of PBS solution, respectively, to prepare five different concentrations (0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL and 8 mg / mL) of PC crosslinking solution.

[0079] (1.2) The decellularized bovine pericardium with a length of 40 mm and a width of 30 mm was immersed in the above-mentioned five different concentrations of crosslinking solution, and crosslinked at 23°C for 24 h under the condition of continuous gentle shaking at 80 rpm.

[0080] Performance test: after clamping the sample between the two end clamps, slowly stretch the two end clamps manually until the sensor receives a signal of 0.1 N of tensile force, which is the sign that the sample can start the tensile test. Along the long axis of the pericardium sheet, set the constant tensile rate of the tensile machine to 0.01 mm / s, and perform a quasi-static uniaxial tensile test, and record the load and displacement in real time until the material is pulled apart. After the test, calculate the tensile strength, tensile fracture strain and Young's modulus; the results are shown in Table 1.

[0081] Table 1

[0082]

[0083] From the above table, it can be seen that the tensile fracture strain of the heart valve increases with the increase of the concentration of procyanidins, and significantly increases at 8 mg / mL; the tensile strength and Young's modulus increase with the increase of the concentration between 0.5 mg / mL and 2 mg / mL, and tend to be stable or even slightly decrease after 2 mg / mL.

[0084] For the heart valve, the deformation when it is opened and closed under the action of blood flow usually does not exceed 10%, and the tensile fracture strain of all experimental groups is greater than 10%, so the concentration that makes the tensile strength and Young's modulus larger is selected as the crosslinking concentration of procyanidins; that is, the concentration of procyanidins is preferably 2 mg / mL.

[0085] (2) Effect of different crosslinking conditions on the first crosslinking treatment

[0086] The procyanidin concentration (2 mg / mL) determined in (1) is used as the crosslinking concentration, and the pH, temperature and crosslinking time are used as factors affecting the crosslinking effect of procyanidins, and each factor is set to have three different levels (as shown in Table 2).

[0087] Table 2

[0088]

[0089] Further, a three-factor three-level orthogonal experiment is designed, and nine experimental groups are obtained (as shown in Table 3). According to the method described in (1.2), uniaxial tensile experiments are respectively carried out on the nine experimental groups, the tensile strength, tensile fracture strain and Young's modulus are calculated, and the results are subjected to range analysis, and the results are shown in Table 4.

[0090] Table 3

[0091]

[0092] Table 4

[0093]

[0094] It should be noted that "k1", "k2" and "k3" in Table 4 respectively represent the sum of the test indexes corresponding to the first level, the second level and the third level of a certain factor; "r" represents the range.

[0095] From the range r results in Table 4, the degree of influence of each factor on the tensile strength is in the order of temperature > time > pH, and the maximum tensile strength is obtained at a temperature of 23°C; the degree of influence on the tensile strain at break is in the order of pH > temperature > time, and the influence of pH on the tensile strain at break is much greater than that of the other two factors, and the maximum tensile strain at break is obtained at a pH of 7.0. The degree of influence on the Young's modulus is in the order of pH > temperature > time, and the maximum Young's modulus is obtained at a pH of 7.0.

[0096] In order to obtain better tensile strength and Young's modulus, the preferred conditions for the first cross-linking treatment are pH = 7.0, 23°C and 24h; in order to obtain better tensile strain at break, the preferred conditions for the first cross-linking treatment are pH = 7.0, 37°C and 12h. However, according to the range values, the influence of temperature and time on the tensile strain at break is very small and can be ignored, and the tensile strain at break meets the requirement of being greater than 10%, so the combination with better mechanical strength is preferred as the cross-linking scheme.

[0097] Taking all factors into consideration, the preferred conditions for the first cross-linking treatment are pH = 7.0, a temperature of 23°C and a time of 24h.

[0098] (3) Performance of the heart valve after co-crosslinking of procyanidins and pentanediol

[0099] (a) A 0.3% glutaraldehyde (GA) solution was prepared; a decellularized bovine pericardium with a length of 40mm and a width of 30mm was freely immersed in the glutaraldehyde solution, and cross-linked at 23°C under gentle shaking at 80rpm for 24h to obtain a glutaraldehyde-crosslinked decellularized bovine pericardium, which was named as the GA group.

[0100] (b) The decellularized bovine pericardium was freely immersed in a 2mg / mL procyanidin (PC) solution, and the pH of the procyanidin solution was adjusted to 7.0 using HCl and NaOH solutions, and cross-linked at 23°C under gentle shaking at 80rpm for 24h, which was named as the PC group.

[0101] (c) A 0.3% glutaraldehyde solution was prepared, and the PC group was placed in the 0.3% glutaraldehyde solution for second cross-linking, and a procyanidin and glutaraldehyde co-crosslinked decellularized bovine pericardium was obtained after cross-linking at 23°C under gentle shaking at 80rpm for 24h, which was named as the PG group.

[0102] (d) Performance test

[0103] Uniaxial tensile fracture test: Fresh bovine pericardium tissue, decellularized bovine pericardium tissue without crosslinking treatment, GA group, PC group and PG group samples were cut into 2mm x 35mm uniaxial tensile standards with a dumbbell-shaped mold. After clamping the samples at both ends, the two end clamps were slowly stretched manually until the sensor received a signal of 0.1N tensile force, which was the sign that the sample was stretched flat and could start the tensile test. Along the long axis of the pericardium sheet, the constant tensile rate of the tensile machine was set to 0.01mm / s, and the quasi-static uniaxial tensile test was carried out, and the load and displacement were recorded in real time until the material was pulled apart. After completing the test, the tensile strength, tensile fracture strain, secant modulus, Young's modulus and fracture energy were calculated; the results are shown in Table 5.

[0104] Table 5

[0105]

[0106] From the above table, it can be seen that the tensile strength and secant modulus of the PG group are between the GA group and the PC group, the tensile fracture strain and fracture energy are greater than the other two groups, and the Young's modulus is less than the other two groups; it shows that the co-crosslinking method of procyanidine and glutaraldehyde is beneficial to improve the strength and stiffness of the heart valve, which may be due to the simultaneous action of procyanidine and glutaraldehyde on the crosslinking sites of the fibers, which plays a synergistic crosslinking role.

[0107] Tear test: The above bovine pericardium samples were cut into 15mm x 45mm rectangles, and a 22mm cut was made in the middle of the short side along the parallel direction of the long side to form a trouser-shaped sample. Adjust the initial distance between the two clamps to 22mm, and install the two legs of the sample so that the main axis of the sample coincides with the center line of the clamp. Apply an external force parallel to the cut plane direction to the specified trouser-shaped sample at a stretching speed of 200mm / min; calculate the tear strength according to the test curve, and the results are shown in Table 6.

[0108] Table 6

[0109]

[0110] From the above table, it can be seen that the tear strength of the PG group is greater than that of the GA group and the PC group, which shows that the co-crosslinking method of procyanidine and glutaraldehyde is beneficial to improve the tear resistance of the heart valve.

[0111] Stress relaxation test: The bovine pericardium samples of each group were cut into 10 mm x 40 mm rectangular strips, and the two ends of the sample were clamped on the experimental machine. The two end clamps were manually operated slowly until the sensor received a signal of 0.1 N tensile force, which was the sign that the sample was stretched flat and could start the formal experiment. The sample was stretched to 20% strain at a speed of 0.01 mm / s, and the clamp position was kept fixed. The force (F) change over time was recorded within 10 min. The stress relaxation curve was fitted using a two-order generalized Maxwell model, and the viscoelastic mechanical parameters were extracted. The results are shown in Table 7.

[0112] Table 7

[0113]

[0114] From the above table, it can be seen that E1 and E2 of the PG group are smaller than those of the GA group and the PC group, and τ1 and τ2 have no significant difference from the other two groups. This indicates that the co-crosslinking of procyanidins and pentanediol can further enhance the stability of the crosslinking network through synergistic effect, thereby significantly improving the elastic modulus and making it have excellent rigidity, so as to effectively resist the high stress of the heart valve in the systolic-diastolic cycle.

[0115] Dynamic mechanical analysis: The bovine pericardium samples of each group were cut into 3 mm x 10 mm rectangular strips. Before the test started, the thickness of the sample at three different positions was measured using a vernier caliper and the arithmetic mean was taken, and the thickness was recorded as t. After the sample was clamped stably, the experimental test mode was set to periodic actuation, and the strain control mode was selected to maintain a constant strain amplitude. The sample was subjected to 500 cycles of motion. At a frequency of 1 Hz, a sinusoidal strain waveform was applied, and the stress response of the sample was recorded. The stress and strain curves over time were Fourier fitted to obtain the dynamic mechanical parameters, and the results are shown in Table 8.

[0116] Table 8

[0117]

[0118] An ideal biological valve material needs high elasticity to maintain shape, proper viscosity to buffer stress, and tan δ should not be too low to avoid excessive energy dissipation leading to fatigue. From the above table, it can be seen that although the E' of the GA group is high, the tan δ is also the highest, which may be too rigid; the E' and E'' of the PG group perform excellently, and the tan δ is moderate, which is closer to the performance of natural tissues and is suitable for long-term use.

[0119] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described, but as long as the combinations of the technical features do not contradict, they should be considered within the scope of the present disclosure.

[0120] The above-described embodiments are merely illustrative of several embodiments of the present application, which are described in more detail and in a specific manner, but should not be construed as limiting the scope of the patent. It should be noted that for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

Claims

1. A method for preparing a heart valve that reduces calcification and improves mechanical properties, characterized in that: The steps include: The decellularized pericardial tissue was first cross-linked using a proanthocyanidin solution; and, performing a second cross-linking treatment on the decellularized pericardial tissue using a glutaraldehyde solution to prepare the heart valve; The pH value of the proanthocyanidin solution is 6.0-8.0; the temperature of the first cross-linking treatment is 21° C.-25° C., and the time is 22 h-26 h.

2. The method for preparing a heart valve according to claim 1, wherein: The pH value of the glutaraldehyde solution is 5.0-5.

5.

3. The method for preparing a heart valve according to claim 1, wherein: After the first cross-linking treatment and before the second cross-linking treatment, the method further includes the following steps: The decellularized pericardial tissue after the first cross-linking treatment was rinsed with PBS solution; Optionally, the rinsing process is performed 2 to 4 times.

4. The method for preparing a heart valve according to any one of claims 1 to 3, characterized in that: The concentration of proanthocyanidins in the proanthocyanidin solution is 0.5 mg / mL to 8 mg / mL.

5. The method for preparing a heart valve according to any one of claims 1 to 3, characterized in that: The rotation speed of the first cross-linking treatment is 60 rpm to 100 rpm.

6. The method for preparing a heart valve according to any one of claims 1 to 3, characterized in that: The volume concentration of glutaraldehyde in the glutaraldehyde solution is 0.3% to 1%.

7. The method for preparing a heart valve according to any one of claims 1 to 3, characterized in that: The temperature of the second cross-linking treatment is 21°C to 25°C; and / or, The rotation speed of the second cross-linking treatment is 60 rpm to 100 rpm; and / or, The second cross-linking treatment is performed for 22 to 26 hours.

8. The method for preparing a heart valve according to any one of claims 1 to 3, characterized in that: The decellularized pericardial tissue may be derived from at least one of pigs, cows, sheep, horses and monkeys.

9. A heart valve, characterized in that: The heart valve is prepared by the preparation method of any one of claims 1 to 8.

10. Use of the heart valve according to claim 9 in preparing a biological scaffold material.

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