Biological valve material with anti-calcification performance as well as preparation method and application of biological valve material

Abstract

The invention discloses a bioprosthetic valve material with anti-calcification performance and a preparation method and application thereof, and belongs to the technical field of preparation of medical materials and medical instruments. A carbodiimide-based non-aldehyde cross-linking method is adopted, while cross-linking is formed between amino and carboxyl in the valve material, a polyoxazoline compound with the end group being amino or carboxyl is introduced, the polyoxazoline compound is covalently bound with the carboxyl or amino on the valve material, the protein saccharification reaction of the valve material can be weakened, and meanwhile serum protein infiltration is reduced; according to the method, the disordered collagenous fiber surface formed by hydrolysis reaction is removed by using a freezing cutting method, so that the valve material with compactly and uniformly distributed collagenous fibers is obtained, serum protein infiltration is further reduced, the occurrence rate of structural valve decline caused by calcification, saccharification and serum protein infiltration is reduced, and the service life of the valve material is prolonged. And the valve material has long-term stability.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical material and medical device preparation, and particularly relates to a biological valve material with anti-calcification performance, a preparation method and application thereof. Background Art

[0002] Valvular heart disease is a cardiovascular disease with a high incidence rate. According to statistics, the weighted prevalence of valvular heart disease in my country is 3.8%, with approximately 25 million patients. Due to the unique location of the heart valve, there are currently no effective treatments, and heart valve replacement is often necessary. In addition to traditional surgical valve replacement, transcatheter aortic valve replacement (TAVR) has entered clinical routine as an effective alternative for patients with severe aortic stenosis who cannot tolerate surgery or are at high risk for surgical intervention. Recent data have also demonstrated the efficacy and non-inferiority of TAVR in patients with intermediate-risk aortic stenosis over a 2- to 5-year follow-up, and its indications are expected to expand further to low-risk and younger patients.

[0003] The valve materials currently used in clinical TAVR, like surgical bioprosthetic valves, are xenogeneic biomaterials fixed with glutaraldehyde. Bioprosthetic valve materials treated with glutaraldehyde exhibit significantly enhanced mechanical strength and resistance to degradation. However, glutaraldehyde fixation can cause valve calcification, a major cause of structural valve failure. Glutaraldehyde induces cell death and promotes the formation of cell fragments, which subsequently serve as binding sites for calcification. Furthermore, incomplete reaction of glutaraldehyde with tissue proteins produces cytotoxic aldehyde residues, which also induce calcification. In addition to calcification, glycation and serum protein infiltration are also important contributors to structural valve failure. The production of advanced glycation end products (AGEs) leads to collagen degradation, reduced valve compliance, and a proinflammatory response. Serum protein infiltration can cause collagen dislocation and tissue thickening, significantly altering tissue affinity for circulating molecules and synergizing with glycation to contribute to structural valve failure.

[0004] European Patent Application EP1684816A2 discloses a non-glutaraldehyde treatment method for biological tissue. The method first uses a blocking agent to block at least a portion of the tissue collagen's amino groups. After the amino groups are blocked, a non-glutaraldehyde crosslinking agent, such as carbodiimide, is used to activate the collagen's carboxyl groups. A diamino spacer molecule is then added to crosslink the collagen's carboxyl groups with the diamino spacer molecule. Finally, hydrolysis is used to remove at least a portion of the zero-length ester crosslinks in the crosslinked tissue. This method reduces zero-length crosslinks between carboxyl and amino groups, and carboxyl and hydroxyl groups within the tissue, thereby avoiding the increase in tissue hardness caused by zero-length crosslinks. However, this prior art has the following drawbacks: The diamino spacer molecule may only react with carboxyl groups on one end of the amine group within the tissue, resulting in incomplete crosslinking. Furthermore, since the blocking agent blocks the amino groups within the tissue, crosslinks between amino groups and carboxyl groups within the tissue are significantly reduced. The combination of these two factors can reduce the overall crosslinking degree of the tissue. Blocking agents for blocking amino groups often use small aldehydes, which pose toxicity issues. Furthermore, the hydrolytic removal of zero-length ester crosslinks can disrupt the alignment of collagen fibers on the tissue surface, making it more susceptible to infiltration by serum proteins.

[0005] Chinese patent application CN115920131A discloses a method for preparing a bioprosthetic valve material with both anticoagulant and anti-calcification properties. The method involves immersing biological tissue in an isocyanoethyl methacrylate solution to produce an in-situ modified bioprosthetic valve with isocyanoethyl methacrylate. The bioprosthetic valve is then immersed and washed, then immersed in a polyethylene glycol diacrylate solution. The polyethylene glycol diacrylate solution and the methacrylate groups on the bioprosthetic valve react in the presence of a free radical polymerization initiator, resulting in a bioprosthetic valve material with both anticoagulant and anti-calcification properties. This technique uses isocyanoethyl methacrylate as an alternative to glutaraldehyde cross-linking, and uses polyethylene glycol derivatives to impart anti-calcification and anti-serum protein infiltration capabilities to the valve material, thereby improving its stability. However, this prior art still has drawbacks: Studies have shown that polyethylene glycol modification can lead to complement activation upon exposure to blood. Furthermore, polyethylene glycol is relatively susceptible to oxidation, leading to loss of function. Polyethylene glycol-modified implants also carry the risk of triggering an immune response.

[0006] Chinese patent CN111494717B - A bioprosthetic valve and its preparation method discloses: by covalently modifying the surface of the bioprosthetic valve with active groups and functional molecules or groups with a high degree of grafting, the anti-thrombotic and anti-calcification functions are improved, and the obtained bioprosthetic valve does not contain aldehyde residues, has good biocompatibility, suitable mechanical properties, and good stability. Among them, the functional molecules or groups are at least one of heparin (15000Mw), hyaluronic acid, polyethylene glycol, argatroban, low molecular weight heparin (4500-5000Mw), hirudin, bivalirudin, fucoidan, ACH11 polypeptide, antithrombin-III, hirudin, APC, sodium citrate, albumin, urokinase, hydroxyl, sulfonic acid, sulfonylphenyl and zwitterion, and the active groups are acrylamide, methacrylamide, acrylate, methacrylate or allyl. On the one hand, the prior art discloses the use of methacrylate groups for non-aldehyde cross-linking through free radical polymerization, and on the other hand, a hydrophilic polyethylene glycol structure is introduced during cross-linking to achieve the purpose of anti-protein adsorption and thus anti-coagulation. However, the chemical stability and antioxidant properties of the artificial bioprosthetic valve prepared therefrom are still relatively poor. At the same time, the polyethylene glycol structure introduced in this patent also has the risk of triggering an immune response.

[0007] Based on the above reasons, the development of a new non-glutaraldehyde treatment system can give artificial valves good anti-calcification properties, reduce the formation of AGE products and resist serum protein accumulation, which is of great significance for improving the long-term stability of the valve. Summary of the Invention

[0008] Based on the above reasons, in response to the problems or defects in the prior art, the purpose of the present invention is to provide a biological valve material with anti-calcification properties and its preparation method and application, so as to solve or at least partially solve the above technical defects in the prior art.

[0009] In order to achieve one of the above purposes of the present invention, the technical solution adopted by the present invention is as follows:

[0010] A method for preparing a biological valve material with anti-calcification properties, the method comprising: decellularizing an animal-derived biological valve material; cross-linking the decellularized valve material using a carbodiimide non-aldehyde cross-linking method; hydrolyzing the cross-linked valve material; and freezing and cutting the hydrolyzed valve material; wherein the cross-linking step comprises modifying the material with a polyoxazoline compound having an amino or carboxyl terminal group.

[0011] Furthermore, in the above technical solution, the cross-linking step also includes a step of further modifying the cross-linked biological valve material with a zwitterionic compound.

[0012] Furthermore, in the above technical solution, the step of modifying the cross-linked valve material with a zwitterionic compound is specifically as follows: soaking the cross-linked biological valve material in an alcohol solution of a zwitterionic compound at 20-40°C for 1-72 hours, preferably at 25°C for 24 hours.

[0013] Preferably, in the above technical solution, the zwitterionic compound includes but is not limited to one or more of 2-[(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl)ammonium hydroxide, methacrylic acid carboxybetaine, and 2-methacryloyloxyethyl phosphorylcholine.

[0014] Preferably, in the above technical solution, the alcohol solution includes but is not limited to one or more of methanol, ethanol, and isopropanol.

[0015] Furthermore, in the above technical solution, the concentration of the alcohol solution of the zwitterionic compound is 0.1 wt%-10 wt%, preferably 5 wt%.

[0016] Furthermore, in the above technical solution, the animal-derived bio-valve material includes but is not limited to animal-derived heart valve material and / or pericardium material. Specifically, the present invention can use animal tissue to prepare animal-derived bio-valve material. The animal tissue can be mammalian tissue, including animal pericardium (such as porcine pericardium, bovine pericardium, etc.), aortic valve, mitral valve, tricuspid valve, pulmonary valve, etc., which are not specifically limited in the present invention. The animal-derived bio-valve material of the present invention is a bio-valve material obtained by basic processing such as cleaning of fresh biological tissue.

[0017] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the animal-source valve is porcine pericardium, bovine pericardium, equine pericardium or porcine aortic valve.

[0018] Furthermore, in the above technical solution, the decellularization method includes a chemical method, a physical method or an enzymatic digestion method.

[0019] Furthermore, in the above technical solution, the steps of cross-linking the decellularized valve material are as follows: soaking the decellularized biological valve material in an acidic or neutral buffer solution, then adding a polyoxazoline compound with an amino or carboxyl end group, and soaking at 20-40°C for 1-24 hours; then adding a water-soluble carbodiimide cross-linking agent, and gently shaking the reaction for 1-72 hours.

[0020] Specifically, the immersion at 20-40°C for 1-24 hours ensures that the polyoxazoline-modified molecules fully penetrate the valve material's molecular network. The oscillation reaction involves both a cross-linking reaction between amino and carboxyl groups within the material and a covalent bonding of the polyoxazoline-modified molecules to the material, with both reactions occurring simultaneously.

[0021] Furthermore, in the above technical solution, the buffer solution includes but is not limited to 2-(N-morpholino)ethanesulfonic acid (MES) buffer, disodium hydrogen phosphate-citric acid buffer, and disodium hydrogen phosphate-sodium dihydrogen phosphate buffer. In a preferred embodiment of the present invention, the buffer solution is MES buffer.

[0022] Furthermore, in the above technical solution, the pH value of the buffer solution is 3-7, for example, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, and specific points between the above values. In a preferred embodiment of the present invention, the pH value of the buffer solution is acidic, for example, the pH value of the buffer solution is 5.5.

[0023] Furthermore, in the above technical solution, the amount of the bio-valve material after decellularization and the buffer solution is not specifically limited, as long as the valve material can be fully and completely immersed. In a preferred embodiment of the present invention, the amount ratio of the valve material to the buffer solution is 1 cm 2 :(1-10)ml.

[0024] Further, the above technical scheme, the poly-oxazoline compound whose end group is amino or carboxyl includes and is not limited to the poly-(2-methyl-2-oxazoline) of single-ended or double-terminal amino group, the poly-(2-methyl-2-oxazoline) of single-ended or double-terminal carboxyl group, one end is the poly-(2-methyl-2-oxazoline) of carboxyl group and the other end is amino group; the poly-(2-ethyl-2-oxazoline) of single-ended or double-terminal amino group, the poly-(2-ethyl-2-oxazoline) of single-ended or double-terminal carboxyl group, one end is the poly-(2-ethyl-2-oxazoline) of carboxyl group and the other end is amino group. In a preferred embodiment of the present invention, the poly-oxazoline compound whose end group is amino or carboxyl is the poly-(2-methyl-2-oxazoline) of amino or carboxyl group.

[0025] Further, technique scheme, in a preferred embodiment of the present invention, the molecular weight that described end group is the poly-oxazoline compounds of amino or carboxyl is 1000-50000.For example 1000,5000,10000,15000,20000,25000,30000,35000,40000,45000,50000, and the concrete point value between above-mentioned numerical value.In a more preferred embodiment of the present invention, the molecular weight that described end group is the poly-oxazoline compounds of amino or carboxyl is 10000.

[0026] Further, technique scheme, in a preferred embodiment of the present invention, adding end group is after the poly-oxazoline compound of amino or carboxyl, the mass concentration of poly-oxazoline compound is 0.01%-10wt% in the gained solution, for example, 0.01wt%, 0.1wt%, 0.2wt%, 0.5wt%, 1wt%, 2wt%, 3wt%, 5wt%, 10wt%, and the concrete point value between the above-mentioned numerical value. In a more preferred embodiment of the present invention, the mass concentration of poly-oxazoline compound is 1wt% in the described solution.

[0027] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the temperature at which the decellularized biological valve material is immersed in an acidic or neutral buffer solution is room temperature, and the room temperature refers to the natural room temperature conditions in all seasons without additional cooling or heating treatment. Generally, the room temperature is controlled at 10-30°C, for example, it can be 10°C, 12°C, 15°C, 20°C, 25°C, 30°C, and specific point values between the above values.

[0028] Furthermore, in the above technical solution, the time for soaking the decellularized bioprosthetic valve material in the acidic or neutral buffer solution can be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or 24 hours, as well as specific points between the above values. In a more preferred embodiment of the present invention, the soaking time is 12 hours.

[0029] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the water-soluble carbodiimide crosslinking agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

[0030] Furthermore, in the above technical solution, after the EDC and NHS are added to the buffer solution, the concentration of EDC is 50-500mM, and the concentration of NHS is 10-200mM. In a more preferred embodiment of the present invention, the concentration of EDC is 100mM, and the concentration of NHS is 60mM.

[0031] Furthermore, in the above technical solution, the temperature of the oscillation reaction is 10-40° C. In a preferred embodiment of the present invention, the temperature of the oscillation reaction is room temperature, generally controlled at 10-30° C., for example, 25° C.

[0032] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the oscillation reaction time is 12-36 hours, for example, the oscillation reaction time is preferably 24 hours.

[0033] Furthermore, in the above technical solution, the step of hydrolyzing the cross-linked valve material is specifically as follows: placing the cross-linked valve material in an alkaline buffer solution and immersing it under shaking at 20-40°C.

[0034] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the pH value of the buffer solution is 8-10. More preferably, the pH value of the buffer solution is 9.5.

[0035] Furthermore, in the above technical solution, the buffer solution includes but is not limited to sodium carbonate-sodium bicarbonate buffer, Tris-hydrochloric acid buffer, borate buffer, etc. Preferably, the buffer solution is borate buffer.

[0036] Furthermore, in the above technical solution, the oscillation temperature is room temperature, which is generally controlled at 10-30°C, for example, 25°C.

[0037] Furthermore, in the preferred embodiment of the present invention, the oscillation process is as follows: oscillating at a speed of 30-80 rpm for 24-120 hours. More preferably, the oscillation is oscillating at 50 rpm for 72 hours.

[0038] Furthermore, in the above technical solution, the steps of freezing and cutting the hydrolyzed valve material are specifically as follows: freezing the hydrolyzed valve material, then cutting the front and back surfaces of the obtained frozen valve material, and finally thawing it.

[0039] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the specific process adopted for freezing is as follows: freezing at -50 to -20°C for 1-10 minutes.

[0040] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the cutting amplitude is 1-50% of the thickness of the frozen valve material.

[0041] In the present invention, unless otherwise specified, "cutting width" refers to the percentage of the thickness of the frozen valve material cut off to the overall thickness of the frozen valve material.

[0042] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the temperature used for thawing is 20-40°C.

[0043] The second object of the present invention is to provide a biological valve material with anti-calcification properties prepared by the above method.

[0044] The above-mentioned biological valve has long-term stability. This is because the method of the present invention can reduce the incidence of structural valve failure caused by calcification, glycation and serum protein infiltration, thereby making the valve material have long-term stability.

[0045] The third object of the present invention is to provide a bio-valve material with anti-calcification properties prepared by the above-mentioned method for use as a valve material required for replacement surgery of the pericardium, aortic valve, pulmonary valve, venous valve, mitral valve, tricuspid valve, etc.

[0046] The main raw materials used in the present invention play the following roles in the present invention:

[0047] The polyoxazoline compounds with amino or carboxyl end groups used in the present invention are macromolecular compounds generated by ring-opening polymerization, and their repeating units do not contain cyclic structures. The tertiary amide in the structure has a strong ability to form hydrogen bonds with water molecules, has good hydrophilicity, can form a hydration layer to resist protein adsorption, has excellent anti-fouling ability, and the structural unit is not easy to form peroxides, has excellent chemical stability, antioxidant properties, and is non-immunogenic. After the polyoxazoline compounds with amino or carboxyl end groups are modified to biological valve materials, their excellent anti-fouling ability can inhibit the uptake of serum proteins and the formation of advanced glycation end products of the implanted biological valve materials, thereby avoiding the pro-inflammatory reaction caused by sending signals to product receptors, and giving the implant materials anti-inflammatory and antioxidant properties. At the same time, since fibrinogen adsorption is a necessary condition for thrombosis, the anti-thrombotic ability of the implant material can be improved by reducing protein adsorption. In addition, when the polyoxazoline compounds of the present invention are combined with the biological valve materials, the amino and carboxyl groups of the biological valve materials themselves can undergo internal cross-linking, thereby having a stable cross-linking degree.

[0048] The key steps in the preparation method of the present invention play the following roles in the present invention:

[0049] (1) The purpose of the step of cross-linking the animal-derived biological valve material of the present invention is that the biological valve material presents a porous fibrous tissue arrangement, which contains groups such as carboxyl, amino and hydroxyl groups. When the biological valve material is combined with a polyoxazoline compound having an amino or carboxyl end group, the amino and carboxyl groups of the material itself can undergo internal cross-linking, thereby having a stable cross-linking degree.

[0050] (2) The purpose of the present invention to perform zwitterion modification treatment on the cross-linked valve material is that the zwitterion binding can further enhance the material's ability to resist protein adsorption, while the alcohol solution soaking has a degreasing effect, which can reduce the internal calcification sites of the material and further enhance the anti-calcification ability.

[0051] (3) The purpose of the step of hydrolyzing the cross-linked valve material in the present invention is to remove the ester bonds and improve the softness of the obtained valve material.

[0052] (4) The purpose of the step of freeze-cutting the hydrolyzed valve material in the present invention is to remove the surface of disordered collagen fibers.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] (1) The present invention adopts a non-aldehyde cross-linking method based on carbodiimide, and at the same time as the amino and carboxyl groups inside the valve material form cross-links, a polyoxazoline compound with an amino or carboxyl end group is introduced, so that it is covalently bonded to the carboxyl or amino group on the valve material, which can weaken the protein glycation reaction of the valve material and reduce serum protein infiltration; a freezing cutting method is used to remove the disordered surface of the collagen fibers formed by the hydrolysis reaction, thereby obtaining a valve material with dense and uniform collagen fiber distribution, further reducing serum protein infiltration, thereby reducing the incidence of structural valve failure caused by calcification, glycation and serum protein infiltration, and making the valve material have long-term stability.

[0055] Compared to European patent application EP1684816A2, the present invention does not block the amino groups in the valve material during the cross-linking process, thus avoiding the toxicity issues associated with the use of small molecule aldehydes. While the valve material is bonded to the polyoxazoline, the amino and carboxyl groups of the material itself can undergo internal cross-linking, resulting in a stable degree of cross-linking. Furthermore, following the hydrolysis treatment, the present invention uses a freeze-cutting method to remove the disordered surface of the collagen fibers formed by the hydrolysis reaction. Compared to EP1684816A2, the present invention has a valve material with a more uniform surface structure, further reducing serum protein infiltration.

[0056] The polyoxazoline compounds used in this invention have excellent anti-protein adsorption properties, better chemical stability and antioxidant properties than polyethylene glycol compounds, and are non-immunogenic. Modification with polyoxazoline compounds in this invention can inhibit serum protein uptake and the formation of advanced glycation end products in implant materials, impart anti-inflammatory and antioxidant properties to the implant materials, and enhance their anti-thrombotic capabilities.

[0057] Compared to Chinese patent application CN115920131A, the present invention utilizes polyoxazoline compounds instead of polyethylene glycol compounds to modify valve materials. Polyoxazoline compounds not only exhibit excellent resistance to protein adsorption but also possess improved chemical stability and antioxidant properties compared to polyethylene glycol compounds. They are non-immunogenic and can inhibit serum protein uptake and the formation of advanced glycation end products (AGEs) in the implant material, while also imparting anti-inflammatory and antioxidant properties to the implant material.

[0058] (2) The present invention adopts a non-aldehyde cross-linking system (EDC / NHS cross-linking), and introduces a modified molecule polyoxazoline compound during cross-linking. The advantage is that polyoxazoline compounds have better chemical stability and antioxidant properties than polyethylene glycol compounds, and are non-immunogenic. They can also inhibit serum protein uptake and the formation of advanced glycation end products, giving the modified valve anti-inflammatory and antioxidant properties, thereby improving the long-term stability of the valve. In addition, the cross-linking, hydrolysis, and freeze-cutting of the present invention are a complete set of interrelated valve material processing technologies. Since EDC / NHS non-aldehyde cross-linking is adopted, which belongs to zero-length cross-linking, it is necessary to use a hydrolysis method to increase the softness of the EDC / NHS cross-linked valve material, thereby improving its durability; and the freeze-cutting method, on the one hand, removes the disordered surface of the collagen fibers after hydrolysis, reduces the surface roughness of the valve material, thereby reducing serum protein adsorption, and on the other hand, it can also reduce the thickness of the valve material, so that the leaflet is easy to compress into the stent to achieve its interventional implantation.

[0059] (3) The polyoxazoline compound used in the present invention is a macromolecular compound generated by ring-opening polymerization, and its repeating unit does not contain a ring structure. The bioprosthetic valve material modified with the polyoxazoline compound has the ability to inhibit serum protein uptake and the formation of advanced glycation end products. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0061] Figure 1To quantitatively analyze the AGE deposited on the bioprosthetic valve material using immunohistochemistry;

[0062] Figure 2 To quantitatively analyze the serum proteins deposited on bioprosthetic valve materials using immunohistochemistry;

[0063] Figure 3 This is an SEM image of the bioprosthetic valve material before cryosectioning in Example 2;

[0064] Figure 4 This is an SEM image of the biological valve material after cryosectioning in Example 2. DETAILED DESCRIPTION

[0065] The present invention discloses a biological valve material with anti-calcification properties, as well as its preparation method and application. The key point of the present invention is that polyoxazoline compounds are used to covalently modify the valve material on the basis of non-aldehyde cross-linking. While resisting calcification, the serum protein infiltration and the formation of glycation end products of the valve material are inhibited, and the protein glycation reaction of the valve material is weakened; the use of zwitterionic compounds for modification further improves the anti-protein adsorption capacity; on the other hand, the use of a freeze-cutting method removes the disordered surface of the collagen fibers formed by the hydrolysis reaction, further reducing serum protein infiltration. The synergistic effect of multiple aspects reduces the possibility of structural valve failure caused by calcification, glycation and serum protein infiltration, and makes the valve material have long-term stability.

[0066] The present invention is further described in detail below through an implementation case. This implementation case is implemented based on the technology of the present invention. Detailed implementation methods and specific operating procedures are now given to illustrate the creativity of the present invention, but the protection scope of the present invention is not limited to the following implementation case.

[0067] Based on the information contained in this application, it will be readily apparent to those skilled in the art that various changes can be made to the precise description of the present invention. It should be understood that the scope of the present invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are provided only to illustrate specific aspects of the present invention.

[0068] In order to better understand the present invention and not to limit the scope of the present invention, all numbers used in this application to express amounts, percentages, and other numerical values should be understood to be modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification are approximate values, which may be changed according to the different ideal properties to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods.

[0069] The equipment and raw materials used in the present invention can be purchased from the market or are commonly used in the art. The methods in the following examples are all conventional methods in the art unless otherwise specified.

[0070] Example 1

[0071] The preparation method of a bioprosthetic valve (polyoxazoline-modified bioprosthetic valve) material with anti-calcification performance in this embodiment includes the following steps:

[0072] 1. Decellularize the cleaned porcine pericardium using chemical extraction method;

[0073] 2. The porcine pericardium after decellularization in step 1 was cleaned with PBS buffer, soaked in phosphate buffer at pH 5, and added with 0.5 wt% of amino-terminated poly(2-ethyl-2-oxazoline) with a molecular weight of 5000. The mixture was soaked at 25°C for 4 hours. Then, a 150 mM / 30 mM EDC / NHS solution was added and the mixture was reacted at 37°C with gentle shaking for 4 hours. The mixture was then washed with PBS three times for 15 minutes each.

[0074] 3. Soak the cross-linked bovine pericardium in a 5 wt% isopropanol solution of carboxybetaine methacrylate at 20°C for 12 hours;

[0075] 4. Soak the porcine pericardium modified in step 3 in Tris-HCl buffer (pH 8.8) at 25°C and 50 rpm for 120 hours, and then wash with PBS three times for 15 minutes each time.

[0076] 5. Freeze the porcine pericardium hydrolyzed in step 4 at -50°C for 1 minute, cut the front and back surfaces of the material using a freezing microtome, with the cutting width being 3% of the sample thickness, and then thaw at 20°C.

[0077] Example 2

[0078] The preparation method of a bioprosthetic valve (polyoxazoline-modified bioprosthetic valve) material with anti-calcification performance in this embodiment includes the following steps:

[0079] 1. Decellularize the cleaned bovine pericardium using the hypertonic and hypotonic methods;

[0080] 2. The decellularized bovine pericardium from step 1 was cleaned with PBS buffer, soaked in MES buffer (pH 5.5), and added with 10,000 molecular weight, 1 wt% amino-terminated poly(2-methyl-2-oxazoline) at 25°C for 12 hours. Then, a 150 mM / 60 mM EDC / NHS solution was added and the mixture was incubated at 37°C with gentle shaking for 12 hours. The mixture was then washed with saline three times for 15 minutes each.

[0081] 3. Soak the cross-linked bovine pericardium in a 2 wt% ethanol solution of 2-[(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl)ammonium hydroxide at 25° C. for 24 h.

[0082] 4. Soak the modified bovine pericardium in pH 8.4 boric acid buffer at 37°C and shake at 60 rpm for 72 hours, and then wash with saline three times for 15 minutes each time.

[0083] 5. Freeze the bovine pericardium hydrolyzed in step 4 at -30°C for 3 minutes, cut the front and back surfaces of the material using a freezing microtome, with the cutting width being 5% of the sample thickness, and then thaw at 37°C.

[0084] Example 3

[0085] A bioprosthetic valve (polyoxazoline-modified bioprosthetic valve) material with anti-calcification properties, the preparation method of which comprises the following steps:

[0086] 1. Decellularize the cleaned porcine aortic valve using enzymatic digestion;

[0087] 2. The porcine aortic valve decellularized in step 1 was cleaned with 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer, soaked in MES buffer at pH 4.5, and poly(2-ethyl-2-oxazoline) with a molecular weight of 50,000 and a concentration of 5 wt% carboxyl end groups was added. The valve was soaked at 37°C for 24 hours. 200 mM / 100 mM EDC / NHS was then added and the reaction was carried out at 20°C with gentle shaking for 72 hours. The valve was then washed with HEPES buffer three times for 15 minutes each time.

[0088] 3. Soak the cross-linked bovine pericardium in a 1 wt% 2-methacryloyloxyethyl phosphorylcholine ethanol solution at 30° C. for 48 h;

[0089] 4. Soak the porcine aortic valve modified in step 3 in a sodium carbonate-sodium bicarbonate buffer solution with a pH value of 9.6 at 37°C and shaken at 80 rpm for 24 hours. Wash with HEPES buffer three times for 15 minutes each time.

[0090] 5. Freeze the porcine aortic valve after hydrolysis in step 4 at -20°C for 5 minutes, cut the front and back surfaces of the material using a freezing microtome, with the cutting width being 10% of the sample thickness, and then thaw at 20°C.

[0091] Comparative Example 1

[0092] This comparative example is a bioprosthetic valve material modified with polyethylene glycol having an amino terminal group, and its preparation method comprises the following steps:

[0093] 1. Decellularize the cleaned bovine pericardium using the hypertonic and hypotonic methods;

[0094] 2. The decellularized bovine pericardium from step 1 was cleaned with PBS buffer, soaked in MES buffer (pH 5.5), and added with 10,000 molecular weight, 1 wt% amino-terminated polyethylene glycol. The mixture was soaked at 25°C for 12 hours. A 150 mM / 60 mM EDC / NHS solution was then added and the mixture was incubated at 37°C with gentle shaking for 12 hours. The mixture was then washed with saline three times for 15 minutes each.

[0095] 3. Soak the cross-linked bovine pericardium in a 2 wt% ethanol solution of 2-[(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl)ammonium hydroxide at 25° C. for 24 h.

[0096] 4. Soak the cross-linked bovine pericardium modified in step 3 in a pH 8.4 boric acid buffer solution at 37°C and shake at 60 rpm for 72 hours, and then wash with saline three times for 15 minutes each time.

[0097] 5. Freeze the bovine pericardium hydrolyzed in step 4 at -30°C for 3 minutes, cut the front and back surfaces of the material using a freezing microtome, with the cutting width being 5% of the sample thickness, and then thaw at 37°C.

[0098] Figure 1 and Figure 2 Immunohistochemistry was used to quantitatively analyze the AGE and serum protein deposits on the polyoxazoline-modified bioprosthetic valve materials one month after they were implanted into rabbit muscles. The polyoxazoline-modified bioprosthetic valve material was prepared using the method described in Example 2. As controls, an unmodified bioprosthetic valve material and a bioprosthetic valve material modified with polyethylene glycol (PEG) with a molecular weight of 10,000 and amino groups as prepared in Comparative Example 1 were also implanted. The AGE and serum protein deposits on the unmodified bioprosthetic valve material were set as 100%. Both polyoxazoline and PEG modifications significantly reduced AGE and serum protein deposits on the bioprosthetic valve material (P < 0.001). Polyoxazoline modification significantly reduced AGE (P < 0.001) and serum protein deposits (P < 0.005) compared to the bioprosthetic valve material modified with polyethylene glycol (PEG) with a molecular weight of 10,000 and amino groups as prepared in Comparative Example 1.

[0099] Figure 3 and Figure 4 These are the biological valve materials before and after cryosectioning in Example 2. It can be seen that the fiber distribution after cryosectioning is significantly denser and more uniform.

[0100] Table 1 shows the calcium content of the bioprosthetic valve material prepared in Example 2 (non-aldehyde cross-linking) after 60 days of rabbit muscle implantation, as measured by ICP. Compared to commercial valve materials cross-linked with glutaraldehyde, the bioprosthetic valve material prepared in Example 2 showed significantly lower calcium content.

[0101] Table 1 Calcium content test results (unit: g / kg dry weight tissue)

[0102] Grouping Non-aldehyde cross-linking Glutaraldehyde cross-linking average value 44.96 72.44 deviation 49.17 72.31 median 25.02 43.11

Claims

1. A method for preparing a bioprosthetic valve material with anti-calcification properties, characterized by: The method comprises: decellularizing an animal-derived biological valve material; cross-linking the decellularized valve material using a carbodiimide non-aldehyde cross-linking method; hydrolyzing the cross-linked valve material; and freezing and cutting the hydrolyzed valve material; wherein the cross-linking step is modified by using a polyoxazoline compound having an amino or carboxyl terminal group.

2. The method according to claim 1, wherein: The cross-linking step further includes the step of further modifying the cross-linked biological valve material with a zwitterionic compound.

3. The method according to claim 2, wherein: The zwitterionic compound includes one or more of 2-[(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl)ammonium hydroxide, methacrylic acid carboxybetaine, and 2-methacryloyloxyethyl phosphorylcholine.

4. The method according to claim 1, wherein: The cross-linking treatment of the decellularized valve material includes the following steps: soaking the decellularized biological valve material in an acidic or neutral buffer solution, then adding a polyoxazoline compound with an amino or carboxyl end group, and soaking at 20-40°C for 1-24 hours; then adding a water-soluble carbodiimide cross-linking agent and gently shaking the reaction for 1-72 hours.

5. The method according to claim 1, wherein: The polyoxazoline compounds with amino or carboxyl terminal groups include poly(2-methyl-2-oxazoline) with a single or double amino terminal, poly(2-methyl-2-oxazoline) with a single or double carboxyl terminal, poly(2-methyl-2-oxazoline) with a carboxyl terminal at one end and an amino terminal at the other end, poly(2-ethyl-2-oxazoline) with an amino terminal at one end or a double amino terminal, poly(2-ethyl-2-oxazoline) with a single or double carboxyl terminal, and poly(2-ethyl-2-oxazoline) with a carboxyl terminal at one end and an amino terminal at the other end.

6. The method according to claim 4, characterized in that: The molecular weight of the polyoxazoline compound with amino or carboxyl terminal groups is 1,000-50,000.

7. The method according to claim 4, characterized in that: The water-soluble carbodiimide cross-linking agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS.

8. The method according to claim 7, wherein: After the EDC and NHS are added to the buffer solution, the concentration of EDC is 50-500 mM, and the concentration of NHS is 10-200 mM.

9. A biological valve material with anti-calcification properties prepared by the method according to any one of claims 1 to 8.

10. Use of the biovalve material with anti-calcification properties prepared by the method according to any one of claims 1 to 8 as valve material required for pericardium, aortic valve, pulmonary valve, venous valve, mitral valve, and tricuspid valve replacement.

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