Biological tissue combined crosslinking method

Abstract

The bioprosthetic valve is treated by a first cross-linking agent and a second cross-linking agent, the first cross-linking agent is L-asparaginic acid, L-glutamic acid, glucaric acid, malic acid or succinic acid, and the second cross-linking agent is chitosan oligosaccharide, L-lysine, arginine or histidine. The invention further provides a preparation method of the bioprosthetic valve, according to the bioprosthetic valve and the preparation method of the bioprosthetic valve, almost non-toxic bio-friendly reagents which are widely applied to food and medicine are adopted, cross-linking is carried out twice in sequence, multi-site stable cross-linking of amino and carboxyl of biological tissue is achieved, the physical performance of materials is improved, and the bioprosthetic valve and the preparation method of the bioprosthetic valve are suitable for large-scale popularization and application. Meanwhile, the calcification problem caused by the toxicity of the cross-linking agent is avoided.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological crosslinking, in particular to a method for secondary crosslinking using a biocompatible reagent as a crosslinking agent. BACKGROUND

[0002] Studies have shown that aldehyde-based substances play an important role in the calcification process of biological artificial heart valves, mainly because aldehyde groups can be slowly oxidized to carboxyl groups, becoming potential calcification sites, and aldehyde substances have obvious cytotoxicity, too many aldehyde groups can cause cell apoptosis and cause lipid enrichment, and the phosphate in the lipid is a potential calcification site.

[0003] Most of the currently marketed biological valves rely on aldehyde substances, especially glutaraldehyde, for pericardial crosslinking fixation and even storage, which leads to the need for thorough cleaning of the biological valve before implantation, or even the development of aldehyde-terminated anti-calcification processes. In the past two years, although many non-aldehyde crosslinking fixation processes have emerged, such as genipin, anthocyanin, isothiocyanate, and epoxy compounds, these crosslinking agents still have certain toxicity or insufficient crosslinking strength. SUMMARY

[0004] To solve the above problems, the present application provides a new crosslinking method, which uses a biocompatible reagent with almost no toxicity as a crosslinking agent through secondary crosslinking to achieve stable crosslinking of biological tissue amino and carboxyl groups at multiple sites, improve the physical properties of the material, and avoid the calcification problem caused by the toxicity of the crosslinking agent.

[0005] 1. A biological valve treated with a first crosslinking agent and a second crosslinking agent, wherein the first crosslinking agent is L-aspartic acid, L-glutamic acid, saccharic acid, malic acid or succinic acid, and the second crosslinking agent is chitosan oligosaccharide, L-lysine, arginine or histidine.

[0006] 2. The biological valve according to item 1, wherein the biological valve is a pericardium, preferably a pig pericardium, a cow pericardium, a sheep pericardium or a horse pericardium.

[0007] 3. A method for preparing the biological valve according to item 1, comprising, activating the first crosslinking agent; placing the biological valve in the activated first crosslinking agent for the first crosslinking fixation; activating the biological valve after the first crosslinking fixation, and then adding the second crosslinking agent for the second crosslinking fixation.

[0008] 4. The preparation method according to item 3, wherein the time for the first crosslinking fixation and / or the second crosslinking fixation is 1-72h.

[0009] 5. The method of item 3, wherein the temperature of the first cross-linking fixation and / or the second cross-linking fixation is 1-50°C, preferably 25-45°C.

[0010] 6. The method of item 3, wherein the pH value of the first cross-linking fixation and / or the second cross-linking fixation is 7-11.

[0011] 7. The method of item 3, wherein the activation is performed by an activation solution comprising one or more of N-hydroxysuccinimide, 1-ethyl-3(3-dimethylaminopropyl) carbodiimide, N,N-dicyclohexyl carbodiimide, N,N-diisopropyl carbodiimide, N,N-carbonyldiimidazole, oxalyl chloride and BOP reagent.

[0012] 8. The method of item 3, wherein the activation time is 10-120 min, preferably 30-60 min.

[0013] 9. The method of item 3, wherein the temperature of the activation is 1-50°C, preferably 25-40°C.

[0014] 10. Use of the biological valve of item 1 in the preparation of a biological artificial heart valve.

[0015] The present application uses L-aspartic acid and chitooligosaccharide as cross-linking agents to jointly cross-link biological tissues by a secondary cross-linking method. The method uses a widely used biological friendly reagent in food and medicine which is almost non-toxic, and performs two cross-linking operations in succession to realize stable cross-linking of multiple sites of amino and carboxyl groups in biological tissues, improve the physical properties of the material, and avoid the calcification problem caused by the toxicity of the cross-linking agent. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a flow chart of the cross-linking method described in the present application. DETAILED DESCRIPTION

[0017] The specific embodiments of the present application will be described in more detail below. Although the following describes specific embodiments of the present application, it should be understood that the present application can be implemented in various forms and should not be limited by the specific embodiments or examples described herein. On the contrary, these specific embodiments or examples are provided so that the present application can be more thoroughly understood and the scope of the present application can be fully conveyed to those skilled in the art.

[0018] The present application provides a biological valve treated by a first cross-linking agent and a second cross-linking agent, wherein the first cross-linking agent is L-aspartic acid, L-glutamic acid, glucose diacid, malic acid or succinic acid, and the second cross-linking agent is chitooligosaccharide, L-lysine, arginine or histidine.

[0019] In some embodiments, the first cross-linking agent is L-aspartic acid and the second cross-linking agent is chitooligosaccharide.

[0020] In some embodiments, the biological valve is pericardium, in particular, porcine pericardium, bovine pericardium, sheep pericardium or horse pericardium.

[0021] In one aspect, the application also provides a method for joint cross-linking of biological tissue, wherein a first cross-linking agent containing carboxyl groups at a concentration of 0.1-5 wt% is placed in an activation solution at a concentration of 0.1-5 wt%, and first activation is performed at a temperature of 0-50°C for 10-120 min. After the first activation, the biological tissue is placed in the first cross-linking agent after activation at a concentration of 0.1-5 wt%, and first cross-linking fixation is performed at a temperature of 0-50°C and a pH of 7-11 for 1-72 h. After the first cross-linking fixation, the biological tissue after the first cross-linking fixation is placed in an activation solution at a concentration of 0.1-5 wt%, and second activation is performed at a temperature of 0-50°C for 10-120 min, and then a second cross-linking agent containing amino groups at a concentration of 0.1-5 wt% is added, and second cross-linking fixation is performed at a temperature of 0-50°C and a pH of 7-11 for 1-72 h.

[0022] In some embodiments, the biological tissue is pericardium tissue.

[0023] In some embodiments, the biological tissue is porcine pericardium, bovine pericardium, sheep pericardium or horse pericardium.

[0024] In some embodiments, the biological tissue is bovine pericardium.

[0025] In some embodiments, the first cross-linking agent is L-aspartic acid, L-glutamic acid, saccharic acid, malic acid or succinic acid.

[0026] In some embodiments, the first cross-linking agent is L-aspartic acid.

[0027] In some embodiments, the concentration of the first crosslinking agent is 0.1-5 wt%, specifically, it can be 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2.0 wt%, 2.1 wt%, 2.2 wt%, 2.3 wt%, 2.4 wt%, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%.

[0028] In some embodiments, the activation solution comprises one or more of N-hydroxysuccinimide (NHS), 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC), N,N- dicyclohexylcarbodiimide (DCC), N,N-diisopropylcarbodiimide (DIC), N,N-carbonyldiimidazole (CDI), oxalyl chloride, BOP reagent.

[0029] In some embodiments, the concentration of the activation solution is 0.1-5 wt%, in particular, it can be 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2.0 wt%, 2.1 wt%, 2.2 wt%, 2.3 wt%, 2.4 wt%, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.0 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%.

[0030] In some embodiments, the activation solution is N-hydroxysuccinimide and 1-ethyl-3(3-dimethylaminopropyl)carbodiimide.

[0031] In some embodiments, the temperature of the first activation and / or the second activation is 1-50°C, in particular, it can be 1°C, 2°C, 4°C, 6°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C.

[0032] In some embodiments, the time of the first activation and / or the second activation is 10-120 min, in particular, it can be 10 min, 20 min, 40 min, 60 min, 80 min, 100 min, 120 min.

[0033] In some embodiments, the temperature of the first cross-linking fixation is 0-50°C, in particular, it can be 1°C, 2°C, 4°C, 6°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C.

[0034] In some embodiments, the temperature of the first cross-linking fixation is 25-35°C.

[0035] In some implementation schemes, the first crosslinking fixation time is 1-72 hours, specifically, it can be 1 hour, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 42 hours, 48 ​​hours, 54 hours, 60 hours, 66 hours, or 72 hours.

[0036] In some implementations, the first crosslinking fixation time is 36-72 hours.

[0037] In some implementations, the pH value for the first crosslinking fixation is 7-11, specifically, it can be 7, 8, 9, 10, or 11.

[0038] In some implementations, the pH value for the first crosslinking fixation is 8-9.

[0039] In some embodiments, the second crosslinking agent is chitosan oligosaccharide, L-lysine, arginine, or histidine.

[0040] In some embodiments, the second cross-linking agent is chitosan oligosaccharide. Carboxyl groups can become potential calcification sites. During the second cross-linking reaction, the amino groups of the second cross-linking agent, chitosan oligosaccharide, react with the carboxyl groups on the bioprosthetic valve and the carboxyl groups remaining after the first cross-linking reaction, removing these carboxyl groups and thus eliminating potential calcification sites. Simultaneously, chitosan oligosaccharide does not contain carboxyl groups, so the cross-linking reaction does not introduce new carboxyl calcification sites. Therefore, using chitosan oligosaccharide as the second cross-linking agent can improve the anti-calcification performance of the bioprosthetic valve.

[0041] In some embodiments, the concentration of the second crosslinking agent is 0.1-5 wt%, specifically, it can be 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2.0 wt%, 2.1 wt%, 2.2 wt%, 2.3 wt%, 2.4 wt%, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt%, 3.8 wt%, 3.9 wt%. wt%, 4.1wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%.

[0042] In the process of cross-linking fixation of biological tissues, the cross-linking agent is usually in excess to ensure that the amino and hydroxyl groups in the tissue react fully, thereby improving the fixation effect and material stability. In this application, L-aspartic acid and chitosan oligosaccharide are used as cross-linking agents to achieve stable cross-linking of amino and carboxyl groups at multiple sites in biological tissues, thereby improving the physical properties of the material and avoiding calcification problems caused by the toxicity of cross-linking agents.

[0043] In some implementations, the temperature for the second crosslinking fixation is 1-50°C, specifically, it can be 1°C, 2°C, 4°C, 6°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 42°C, 44°C, 46°C, 48°C, or 50°C.

[0044] In some implementations, the temperature for the second crosslinking fixation is 35-45°C.

[0045] In some implementation schemes, the second crosslinking fixation time is 1-72 hours, specifically, it can be 1 hour, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 42 hours, 48 ​​hours, 54 hours, 60 hours, 66 hours, or 72 hours.

[0046] In some implementations, the second crosslinking fixation time is 36-72 hours.

[0047] In some implementations, the pH value for the second crosslinking fixation is 7-11, specifically, it can be 7, 8, 9, 10, or 11.

[0048] In some implementations, the pH value for the second crosslinking fixation is 8-9.

[0049] Example Example 1 18 g of EDC (1.8 wt%) and 12 g of NHS (1.2 wt%) were added to 1000 ml of L-aspartic acid solution (concentration 1 wt%), and the solution was activated at 35 °C for 60 min. After activation, a piece of bovine heart was placed in the activated L-aspartic acid solution and subjected to the first cross-linking fixation at 35 °C and pH 8 for 48 h. After the first cross-linking fixation, the bovine heart was placed in 1000 ml of activation solution with EDC and NHS concentrations of 9 g (0.9 wt%) and 6 g (0.6 wt%), respectively, and activated at 35 °C for 60 min. Then, 13 g of chitosan oligosaccharide (1.3 wt%) was added, and the solution was subjected to the second cross-linking fixation at 35 °C and pH 8 for 48 h.

[0050] For cross-linked bovine pericardium, the heat shrinkage temperature and tensile strength of the pericardium were determined according to the method described in QB / T 2710-2018 "Determination of tensile strength and elongation of leather physical and mechanical tests".

[0051] Example 2-18 The pericardium was processed according to the method described in Example 1, wherein: The only difference between Examples 2-3 and Example 1 is the temperature at which the first crosslinking fixation is performed. The only difference between Examples 4-6 and Example 1 is the duration of the first crosslinking fixation. The only difference between Examples 7-9 and Example 1 is that the pH of the first crosslinking fixation is different; The only difference between Examples 10-12 and Example 1 is that the temperature for the second crosslinking fixation is different; The only difference between Examples 13-15 and Example 1 is the time taken for the second crosslinking fixation. The only difference between Examples 16-18 and Example 1 is that the pH of the second crosslinking fixation is different; The bovine pericardium treated in Examples 2-18 was tested to determine its heat shrinkage temperature and tensile strength. The treatment conditions and test data are shown in Table 1.

[0052] Table 1

[0053] Examples 19-21 The only difference between Examples 19-21 and Example 1 is that the crosslinking agent used is different, in accordance with the method described in Example 1.

[0054] The treated bovine pericardium was tested to determine its heat shrinkage temperature and tensile strength. The specific crosslinking agents used in Examples 19-21 and the test data are shown in Table 2.

[0055] Table 2

[0056] In Example 20, chitosan was used as the second crosslinking agent. Due to the large molecular weight and poor permeability of chitosan, its crosslinking effect was slightly poor. Furthermore, chitosan has the risk of hydrolysis in the blood over a long period of time. Therefore, in this application, chitosan oligosaccharide is more suitable as a crosslinking agent for preparing biological valves.

[0057] Comparative Example Comparative Example 1 18 g EDC (1.8 wt%) and 12 g NHS (1.2 wt%) were added to 1000 ml of L-aspartic acid solution (concentration 1 wt%), and the solution was activated at 35 °C for 60 min. After activation, a bovine pericardium slice was placed in the activated L-aspartic acid solution, and the first cross-linking fixation was performed at 35 °C and pH 8 for 48 h.

[0058] Comparative Example 2 The difference between Comparative Example 2 and Comparative Example 1 is that the cross-linking agent is chitosan oligosaccharide.

[0059] Comparative Example 3 One slice of ox heart was placed in 1000 ml of 0.625 wt% glutaraldehyde solution and cross-linked and fixed at 35°C for 48 h.

[0060] The veal hearts treated in Comparative Examples 1-3 were tested according to the method described in Example 1, and the results are shown in Table 3.

[0061] Table 3

[0062] The difference between Comparative Examples 1-3 and Example 1 is that Comparative Examples 1-3 only underwent one crosslinking process, Comparative Examples 1-2 used L-aspartic acid or chitosan oligosaccharide as crosslinking agents, and Comparative Example 3 used glutaraldehyde as a crosslinking agent. Experimental data show that only one crosslinking process cannot simultaneously cover the effective crosslinking functional groups amino and carboxyl groups in the pericardium. Therefore, the heat shrinkage temperature and tensile strength of the pericardium with only one crosslinking process are significantly weaker than those with two crosslinking processes. The commonly used crosslinking agent glutaraldehyde in Comparative Example 3 only crosslinks with the amino groups in the pericardium, while L-aspartic acid and chitosan oligosaccharide used in this application crosslink with the amino and carboxyl groups in the pericardium, respectively. After treatment, the heat shrinkage temperature of the pericardium is significantly increased, indicating that two crosslinking processes can significantly improve the crosslinking strength and enhance the physical properties of the pericardium. At the same time, compared with glutaraldehyde, the new crosslinking agent has significantly reduced toxicity, which can effectively reduce the risk of calcification caused by reagent residue toxicity.

[0063] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0064] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A biological valve, treated with a first cross-linking agent and a second cross-linking agent, wherein the first cross-linking agent is L-aspartic acid, L-glutamic acid, gluconic acid, malic acid or succinic acid, and the second cross-linking agent is chitosan oligosaccharide, L-lysine, arginine or histidine.

2. The biological valve according to claim 1, wherein, The bioprosthetic valve is a pericardium, preferably a porcine pericardium, bovine pericardium, sheep pericardium, or equine pericardium.

3. The method for preparing the biological valve according to claim 1, comprising: The first crosslinking agent is activated; The bioprosthetic valve was placed in the activated first cross-linking agent for the first cross-linking fixation; The bioprosthetic valve, after the first cross-linking fixation, was activated, and then a second cross-linking agent was added for a second cross-linking fixation.

4. The preparation method according to claim 3, wherein, The time for the first crosslinking fixation and / or the second crosslinking fixation is 1-72 hours.

5. The preparation method according to claim 3, wherein, The temperature for the first crosslinking fixation and / or the second crosslinking fixation is 1-50℃, preferably 25-45℃.

6. The preparation method according to claim 3, wherein, The pH value for the first crosslinking fixation and / or the second crosslinking fixation is 7-11.

7. The preparation method according to claim 3, wherein, Activation is performed using an activation solution comprising one or more of N-hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N-dicyclohexylcarbodiimide, N,N-diisopropylcarbodiimide, N,N-carbonyldiimidazole, oxalyl chloride, and BOP reagent.

8. The preparation method according to claim 3, wherein, The activation time is 10-120 min, preferably 30-60 min.

9. The preparation method according to claim 3, wherein, The activation temperature is 1-50℃, preferably 25-40℃.

10. The use of the bioprosthetic valve according to claim 1 in the preparation of bioartificial heart valves.

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