A method for preparing a dry biological tissue

Abstract

The application provides a preparation method of dry biological tissue, which comprises the following steps: cleaning the biological tissue; cross-linking the cleaned biological tissue; and dehydrating the cross-linked biological tissue, wherein the dehydrating agent in the dehydrating process is selected from one or more than two of the following substances: alcohol ether with high boiling point and low crystallization point, alcohol amine with high boiling point and low crystallization point, alcohol ether solution with high boiling point and low crystallization point, and alcohol amine solution with high boiling point and low crystallization point. Compared with traditional glycerol dehydration or freeze-drying dehydration, the alcohol ether or alcohol amine dehydrating agent with high boiling point and low crystallization point can remove the calcification sites such as phospholipids in the biological tissue in the dehydration process, further improve the anti-calcification performance of the dry biological tissue, and can also give the dry tissue a wider storage temperature condition.

Description

Technical Field

[0001] This application belongs to the field of biomedicine, specifically relating to a method for preparing dry biological tissues. Background Technology

[0002] To preserve the inherent properties of biological tissue materials, they are typically stored in a liquid environment. Taking bioprosthetic valve materials as an example, these materials are generally stored in solutions of formaldehyde, glutaraldehyde, etc., providing a relatively safe and effective sterile, moist environment for long-term preservation. However, prolonged contact with biological tissues by aldehydes can lead to a large number of free aldehyde groups remaining on the tissue surface. Although products undergo multiple cleanings before use, these aldehyde residues can still cause problems such as toxicity and calcification in the biological tissue.

[0003] To address the problem of residual aldehydes when biological tissues are preserved in liquid form, those skilled in the art have proposed some dry preservation methods. However, traditional dry preservation methods, such as glycerol dehydration or freeze-drying dehydration, can also lead to problems such as physical structural damage, calcification, and denaturation of biological components. Summary of the Invention

[0004] To address the problems encountered in the preservation of the aforementioned biological tissues, this application provides a method for preparing dry biological tissues.

[0005] Specifically, this application relates to the following aspects:

[0006] 1. A method for preparing dry biological tissue, comprising the following steps:

[0007] Cleaning biological tissues;

[0008] Cross-linking of cleaned biological tissues;

[0009] The cross-linked biological tissue was dehydrated.

[0010] The dehydrated biological tissues were dried, packaged, and sterilized to obtain dry biological tissues.

[0011] The dehydrating agent used in the dehydration process is selected from one or more of the following substances:

[0012] High-boiling-point and low-crystallization-point alcohol ethers, high-boiling-point and low-crystallization-point alcohol amines, high-boiling-point and low-crystallization-point alcohol ether solutions, and high-boiling-point and low-crystallization-point alcohol amine solutions.

[0013] 2. The method according to item 1, wherein the alcohol ether is selected from one or more of tetraethylene glycol dimethyl ether, diethylene glycol dimethyl ether, ethylene glycol isooctyl ether, ethylene glycol butyl ether or diethylene glycol monobutyl ether.

[0014] 3. The method according to item 1, wherein the alkanolamine is selected from one or more of N-methyldiethanolamine, N,N-dimethylethanolamine or diethylene glycolamine.

[0015] 4. According to the method described in item 1, wherein,

[0016] The alcohol ether solution comprises an aqueous solution of alcohol ether or a mixed solution of alcohol ether and low molecular weight alcohol;

[0017] The alkanolamine solution comprises an aqueous solution of alkanolamine or a mixed solution of alkanolamine and low molecular weight alcohol.

[0018] The low molecular weight alcohol is selected from ethanol or isopropanol.

[0019] 5. The method according to item 4, wherein the low molecular weight alcohol is ethanol.

[0020] 6. According to the method described in item 4, wherein,

[0021] In the alcohol ether solution or alcohol amine solution, the total volume fraction ratio of alcohol ether or alcohol amine to low molecular weight alcohol is ≥70%, or...

[0022] The volume fraction of water in the alcohol ether solution or alcohol amine solution is ≤30%.

[0023] 7. According to the method described in item 6, wherein,

[0024] In the alcohol ether solution or alcohol amine solution, the total volume fraction ratio of alcohol ether or alcohol amine to low molecular weight alcohol is ≥80%, or...

[0025] In the alcohol ether solution or alcohol amine solution, the volume fraction of water is ≤20%.

[0026] 8. According to the method described in item 4, wherein the volume ratio of alcohol ether or alcohol amine to low molecular weight alcohol in the alcohol ether solution or alcohol amine solution is (7-10):(0-3).

[0027] 9. The method according to item 1, wherein when the dehydrating agent is a mixture of alcohol ether and alcohol amine, the volume ratio of alcohol ether to alcohol amine is (1-4):(4-1).

[0028] 10. The method according to item 1, wherein the dehydration treatment comprises the following steps:

[0029] The biological tissue was immersed in a dehydrating agent and shaken to dehydrate it. The dehydration was repeated 1-5 times, each time for 0.5-48 hours, at a temperature of 10-30℃.

[0030] 11. The method according to item 1, wherein the cleaning solution for cleaning biological tissues is selected from an aqueous solution containing a surfactant or a phosphate buffer solution.

[0031] The surfactant is selected from one or more of sodium dodecyl sulfate, amino acid surfactant, Tween 80, glycolipid, lipopeptide or Triton-10;

[0032] The amino acid surfactant is selected from one or more of N-acylglutamate, N-acylsarcosine, N-lauroyl aspartate, cocoyl arginine ethyl ester, or N-acyl lysine.

[0033] The glycolipid is selected from one or more of trehalose, rhamnose, or sophorose.

[0034] The lipopeptide is selected from one or more of surfactants, iturin, spiroin, fentanyl, lichenin, colistin, or octapeptide.

[0035] 12. The method according to claim 1, wherein the crosslinking agent used in the crosslinking is selected from glutaraldehyde, genipin, carbodiimide, N-hydroxysuccinic acid imide, a polycarboxyl compound with at least two carboxyl functional groups, or a polyamino compound with at least two amino functional groups.

[0036] The polycarboxylic compound with at least two carboxyl functional groups is selected from one or more of L-aspartic acid, L-glutamic acid, gluconic acid, malic acid, or succinic acid.

[0037] The polyamino compound with at least two amino functional groups is selected from one or more of 2,4-diaminobutyric acid, 1,3-diamino-2-propanol, chitosan oligosaccharide, L-lysine, arginine, histidine, or propylenediamine.

[0038] 13. The method according to any one of items 1-12, wherein the biological tissue is selected from the pericardium, heart valves, tunica albuginea, pleura, submucosa of the small intestine, dura mater, spinal dura mater, ligaments or skin.

[0039] Invention effects:

[0040] 1. The present application provides a method for preparing dry biological tissue, wherein during the dehydration process, one or more of the following are used as dehydrating agents: alcohol ether with high boiling point and low crystallization point, alcohol amine with high boiling point and low crystallization point, alcohol ether solution with high boiling point and low crystallization point, or alcohol amine solution with high boiling point and low crystallization point, to dehydrate and dry the biological tissue, which has a significant dehydration effect.

[0041] 2. The dehydrating agent used in this application can not only obtain biological tissues that can be preserved in a dry state, but also remove calcification sites such as phospholipids in biological tissues during the dehydration process compared with traditional glycerol dehydration or freeze-drying dehydration, which can further improve the anti-calcification performance of dry biological tissues. At the same time, the high-boiling-point and low-crystallization-point dehydrating agent can give dry tissues more relaxed storage temperature conditions, especially at lower temperatures. Attached Figure Description

[0042] Figure 1 This is a flowchart of the method for preparing dry biological tissues as described in this application. Detailed Implementation

[0043] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0044] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. The terms "comprising" or "including" as used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to". The following descriptions are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0045] To address the issues of calcification and aldehyde residue toxicity associated with liquid preservation of biological tissues, this application provides a method for preparing dry biological tissues, comprising the following steps:

[0046] Cleaning biological tissues;

[0047] Cross-linking of cleaned biological tissues;

[0048] The cross-linked biological tissue was dehydrated.

[0049] The dehydrated biological tissues were dried, packaged, and sterilized to obtain dry biological tissues.

[0050] The dehydrating agent used in the dehydration process is selected from one or more of the following substances:

[0051] High-boiling-point and low-crystallization-point alcohol ethers, high-boiling-point and low-crystallization-point alcohol amines, high-boiling-point and low-crystallization-point alcohol ether solutions, and high-boiling-point and low-crystallization-point alcohol amine solutions.

[0052] In this application, the alcohol ether is selected from one or more of tetraethylene glycol dimethyl ether, diethylene glycol dimethyl ether, ethylene glycol isooctyl ether, ethylene glycol butyl ether, or diethylene glycol monobutyl ether.

[0053] In this application, the alkanolamine is selected from one or more of N-methyldiethanolamine, N,N-dimethylethanolamine, and diethylene glycolamine.

[0054] In this application, the alcohol ether solution comprises an aqueous solution of alcohol ether or a mixed solution of alcohol ether and low molecular weight alcohol; the alcohol amine solution comprises an aqueous solution of alcohol amine or a mixed solution of alcohol amine and low molecular weight alcohol.

[0055] In some embodiments, the low molecular weight alcohol is selected from ethanol or isopropanol; in some preferred embodiments, the low molecular weight alcohol is ethanol.

[0056] In this application, the dehydrating agent may be the above-mentioned alcohol ether, alcohol amine, alcohol ether solution or alcohol amine solution;

[0057] It can also be a mixed solution of alcohol ethers, a mixed solution of alcohol ethers and alcoholamines, a mixed solution of alcohol ethers and alcohol ether solutions, or a mixed solution of alcohol ethers and alcoholamines.

[0058] It can also be a mixed solution of one alkanolamine and another alkanolamine, a mixed solution of one alkanolamine and another alkanol ether, or a mixed solution of one alkanolamine and another alkanolamine.

[0059] It can also be a mixed solution of alcohol ether solutions and alcohol ether solutions, or a mixed solution of alcohol ether solutions and alcoholamine solutions;

[0060] It can also be a mixture of one alcoholic amine solution and another alcoholic amine solution.

[0061] Specifically, the dehydrating agent may be tetraethylene glycol dimethyl ether, diethylene glycol dimethyl ether, ethylene glycol isooctyl ether, ethylene glycol butyl ether, diethylene glycol monobutyl ether, N-methyldiethanolamine, N,N-dimethylethanolamine or diethylene glycolamine;

[0062] It can also be a mixed solution of tetraethylene glycol dimethyl ether and diethylene glycol dimethyl ether, a mixed solution of tetraethylene glycol dimethyl ether and ethylene glycol isooctyl ether, a mixed solution of tetraethylene glycol dimethyl ether and ethylene glycol butyl ether, a mixed solution of tetraethylene glycol dimethyl ether and diethylene glycol monobutyl ether, a mixed solution of diethylene glycol dimethyl ether and ethylene glycol isooctyl ether, a mixed solution of diethylene glycol dimethyl ether and ethylene glycol butyl ether, or a mixed solution of diethylene glycol dimethyl ether and diethylene glycol monobutyl ether.

[0063] It can also be a mixed solution of tetraethylene glycol dimethyl ether and N-methyldiethanolamine, a mixed solution of tetraethylene glycol dimethyl ether and N,N-dimethylethanolamine, a mixed solution of tetraethylene glycol dimethyl ether and diethylene glycolamine, a mixed solution of diethylene glycol dimethyl ether and N-methyldiethanolamine, a mixed solution of diethylene glycol dimethyl ether and N,N-dimethylethanolamine, a mixed solution of diethylene glycol dimethyl ether and diethylene glycolamine, a mixed solution of ethylene glycol isooctyl ether and N-methyldiethanolamine, ethylene glycol Mixed solutions of isooctyl ether and N,N-dimethylethanolamine; mixed solutions of ethylene glycol isooctyl ether and diethylene glycolamine; mixed solutions of ethylene glycol butyl ether and N-methyldiethanolamine; mixed solutions of ethylene glycol butyl ether and N,N-dimethylethanolamine; mixed solutions of ethylene glycol butyl ether and diethylene glycolamine; mixed solutions of diethylene glycol monobutyl ether and N-methyldiethanolamine; mixed solutions of diethylene glycol monobutyl ether and N,N-dimethylethanolamine; mixed solutions of diethylene glycol monobutyl ether and diethylene glycolamine.

[0064] It can also be a mixed solution of tetraethylene glycol dimethyl ether and diethylene glycol dimethyl ether solution, a mixed solution of tetraethylene glycol dimethyl ether and ethylene glycol isooctyl ether solution, a mixed solution of tetraethylene glycol dimethyl ether and ethylene glycol butyl ether solution, a mixed solution of tetraethylene glycol dimethyl ether and diethylene glycol monobutyl ether solution, a mixed solution of diethylene glycol dimethyl ether and tetraethylene glycol dimethyl ether solution, a mixed solution of diethylene glycol dimethyl ether and ethylene glycol isooctyl ether solution, a mixed solution of diethylene glycol dimethyl ether and ethylene glycol butyl ether solution, or a solution of diethylene glycol dimethyl ether and diethylene glycol monobutyl ether. Mixed solutions, including: a mixed solution of ethylene glycol isooctyl ether and tetraethylene glycol dimethyl ether; a mixed solution of ethylene glycol isooctyl ether and diethylene glycol dimethyl ether; a mixed solution of ethylene glycol isooctyl ether and ethylene glycol butyl ether; a mixed solution of ethylene glycol isooctyl ether and diethylene glycol monobutyl ether; a mixed solution of diethylene glycol monobutyl ether and tetraethylene glycol dimethyl ether; a mixed solution of diethylene glycol monobutyl ether and diethylene glycol dimethyl ether; a mixed solution of diethylene glycol monobutyl ether and ethylene glycol isooctyl ether; a mixed solution of diethylene glycol monobutyl ether and ethylene glycol butyl ether.

[0065] It can also be a mixed solution of tetraethylene glycol dimethyl ether and N-methyldiethanolamine, a mixed solution of tetraethylene glycol dimethyl ether and N,N-dimethylethanolamine, a mixed solution of tetraethylene glycol dimethyl ether and diethylene glycolamine, a mixed solution of diethylene glycol dimethyl ether and N-methyldiethanolamine, a mixed solution of diethylene glycol dimethyl ether and N,N-dimethylethanolamine, a mixed solution of diethylene glycol dimethyl ether and diethylene glycolamine, a mixed solution of ethylene glycol isooctyl ether and N-methyldiethanol ... Mixed solutions of octyl ether and N,N-dimethylethanolamine; mixed solutions of ethylene glycol isooctyl ether and diethylene glycolamine; mixed solutions of ethylene glycol butyl ether and N-methyldiethanolamine; mixed solutions of ethylene glycol butyl ether and N,N-dimethylethanolamine; mixed solutions of ethylene glycol butyl ether and diethylene glycolamine; mixed solutions of diethylene glycol monobutyl ether and N-methyldiethanolamine; mixed solutions of diethylene glycol monobutyl ether and N,N-dimethylethanolamine; mixed solutions of diethylene glycol monobutyl ether and diethylene glycolamine.

[0066] It can also be a mixed solution of N-methyldiethanolamine and N,N-dimethylethanolamine, a mixed solution of N-methyldiethanolamine and diethylene glycolamine, or a mixed solution of N,N-dimethylethanolamine and diethylene glycolamine.

[0067] It can also be a mixed solution of N-methyldiethanolamine and tetraethylene glycol dimethyl ether, a mixed solution of N-methyldiethanolamine and diethylene glycol dimethyl ether, a mixed solution of N-methyldiethanolamine and ethylene glycol isooctyl ether, a mixed solution of N-methyldiethanolamine and ethylene glycol butyl ether, a mixed solution of N-methyldiethanolamine and diethylene glycol monobutyl ether, a mixed solution of N,N-dimethylethanolamine and tetraethylene glycol dimethyl ether, or a mixed solution of N,N-dimethylethanolamine and diethylene glycol dimethyl ether. Mixed solutions of N,N-dimethylethanolamine and ethylene glycol isooctyl ether, N,N-dimethylethanolamine and ethylene glycol butyl ether, N,N-dimethylethanolamine and diethylene glycol monobutyl ether; mixed solutions of diethylene glycolamine and tetraethylene glycol dimethyl ether, diethylene glycol dimethyl ether, diethylene glycol isooctyl ether, diethylene glycol butyl ether, and diethylene glycol monobutyl ether.

[0068] It can also be a mixed solution of N-methyldiethanolamine and N,N-dimethylethanolamine solution, a mixed solution of N-methyldiethanolamine and diethylene glycolamine solution, a mixed solution of N,N-dimethylethanolamine and N-methyldiethanolamine solution, a mixed solution of N,N-dimethylethanolamine and diethylene glycolamine solution, a mixed solution of diethylene glycolamine and N-methyldiethanolamine solution, or a mixed solution of diethylene glycolamine and N,N-dimethylethanolamine solution.

[0069] It can also be a mixed solution of tetraethylene glycol dimethyl ether solution and diethylene glycol dimethyl ether solution, a mixed solution of tetraethylene glycol dimethyl ether solution and ethylene glycol isooctyl ether solution, a mixed solution of tetraethylene glycol dimethyl ether solution and ethylene glycol butyl ether solution, a mixed solution of tetraethylene glycol dimethyl ether solution and diethylene glycol monobutyl ether solution, a mixed solution of diethylene glycol dimethyl ether solution and ethylene glycol isooctyl ether solution, a mixed solution of diethylene glycol dimethyl ether solution and ethylene glycol butyl ether solution, or a mixed solution of diethylene glycol dimethyl ether solution and diethylene glycol monobutyl ether solution;

[0070] It can also be a mixed solution of tetraethylene glycol dimethyl ether and N-methyldiethanolamine, a mixed solution of tetraethylene glycol dimethyl ether and N,N-dimethylethanolamine, a mixed solution of tetraethylene glycol dimethyl ether and diethylene glycolamine, a mixed solution of diethylene glycol dimethyl ether and N-methyldiethanolamine, a mixed solution of diethylene glycol dimethyl ether and N,N-dimethylethanolamine, a mixed solution of diethylene glycol dimethyl ether and diethylene glycolamine, a mixed solution of ethylene glycol isooctyl ether and N-methyldiethanolamine, or a mixed solution of ethylene glycol isooctyl ether. Mixed solutions of ether and N,N-dimethylethanolamine; mixed solutions of ethylene glycol isooctyl ether and diethylene glycolamine; mixed solutions of ethylene glycol butyl ether and N-methyldiethanolamine; mixed solutions of ethylene glycol butyl ether and N,N-dimethylethanolamine; mixed solutions of ethylene glycol butyl ether and diethylene glycolamine; mixed solutions of diethylene glycol monobutyl ether and N-methyldiethanolamine; mixed solutions of diethylene glycol monobutyl ether and N,N-dimethylethanolamine; mixed solutions of diethylene glycol monobutyl ether and diethylene glycolamine.

[0071] It can also be a mixed solution of N-methyldiethanolamine and N,N-dimethylethanolamine, a mixed solution of N-methyldiethanolamine and diethylene glycolamine, or a mixed solution of N,N-dimethylethanolamine and diethylene glycolamine.

[0072] In some preferred embodiments, the dehydrating agent is a mixed solution of ethylene glycol butyl ether and ethanol, wherein the volume ratio of ethylene glycol butyl ether to ethanol is (7-10):(0-3). The value of 7-10 can be any value between 7 and 10, for example, 7, 8, 9, or 10; the value of 0-3 can be any value between 0 and 3, for example, 0, 1, 2, or 3.

[0073] In some preferred embodiments, the dehydrating agent is a mixed solution of N-methyldiethanolamine and ethanol, wherein the volume ratio of N-methyldiethanolamine to ethanol is (7-10):(0-3). The value of 7-10 can be any value between 7 and 10, for example, 7, 8, 9, or 10; the value of 0-3 can be any value between 0 and 3, for example, 0, 1, 2, or 3.

[0074] In some preferred embodiments, the dehydrating agent is an aqueous solution of a mixture of ethylene glycol butyl ether and ethanol, wherein the total volume fraction of ethylene glycol butyl ether and ethanol is ≥70%, for example, the total volume fraction of ethylene glycol butyl ether and ethanol can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0075] In some preferred embodiments, the dehydrating agent is a mixed solution of ethylene glycol butyl ether and N-methyldiethanolamine, wherein the volume ratio of ethylene glycol butyl ether to N-methyldiethanolamine is (1-4):(1-4), and the value of 1-4 can be any value between 1 and 4, for example, 1, 2, 3, or 4.

[0076] In some preferred embodiments, the dehydrating agent is tetraethylene glycol dimethyl ether, diethylene glycol dimethyl ether, ethylene glycol butyl ether, N,N-dimethylethanolamine, or diethylene glycolamine.

[0077] In some preferred embodiments, the dehydrating agent is an aqueous solution of ethylene glycol butyl ether or an aqueous solution of N-methyldiethanolamine, wherein the volume fraction of water is ≤30%, for example, the volume fraction of water can be 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%.

[0078] In this application, the dehydration treatment step includes the following steps: immersing biological tissue in a dehydrating agent and shaking to dehydrate, dehydrating 1-5 times, each time for 0.5-48 hours, at a dehydration temperature of 10-30℃.

[0079] For example, the number of dehydration cycles can be 1, 2, 3, 4, or 5; the time for each dehydration cycle can be 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, or 24h; and the dehydration temperature can be 10℃, 11℃, 12℃, 13℃, 14℃, 15℃, 16℃, 17℃, 18℃, 19℃, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, or 30℃.

[0080] In this application, the cleaning solution for cleaning biological tissues is selected from aqueous solutions containing surfactants or phosphate buffer solutions.

[0081] In this application, the surfactant is selected from one or more of sodium dodecyl sulfate, amino acid surfactant, Tween 80, glycolipid, lipopeptide, or Triton-10; the amino acid surfactant is selected from one or more of N-acylglutamate, N-acylsarcosinate, N-lauroyl aspartate, cocoyl arginine ethyl ester, or N-acyl lysine; the glycolipid is selected from one or more of trehalose, rhamnolipid, or sophorolipid; and the lipopeptide is selected from one or more of surfactants, iturin, spirochetin, fentanyl, lichenin, colistin, or octapeptide.

[0082] In this application, the crosslinking agent used for crosslinking is selected from glutaraldehyde, genipin, carbodiimide, N-hydroxysuccinic acid imide, a polycarboxylic acid compound with at least two carboxyl functional groups, or a polyamino acid compound with at least two amino functional groups. The polycarboxylic acid compound with at least two carboxyl functional groups is selected from one or more of L-aspartic acid, L-glutamic acid, gluconic acid, malic acid, or succinic acid. The polyamino acid compound with at least two amino functional groups is selected from one or more of 2,4-diaminobutyric acid, 1,3-diamino-2-propanol, chitosan oligosaccharide, L-lysine, arginine, histidine, or propylenediamine.

[0083] In this application, the biological tissue is selected from the pericardium, heart valves, tunica albuginea, pleura, submucosa of the small intestine, dura mater, spinal dura mater, ligaments, or skin.

[0084] The application provides a method for preparing dry biological tissue, wherein the dehydrating agent used in the process not only dehydrates and dries the biological tissue, but also removes calcification sites such as phospholipids in the biological tissue, thereby further improving the anti-calcification performance of the dry biological tissue. At the same time, the dehydrating agent with a high boiling point and low crystallization point can provide the dry tissue with more relaxed storage temperature conditions, especially at lower temperatures.

[0085] Figure 1 This is a flowchart of the method for preparing dry biological tissues as described in this application.

[0086] Example 1

[0087] 1. Cleaning: Bovine pericardium (obtained from local slaughterhouse) was cleaned by shaking with 2% N-acylglutamate PBS solution at 4℃. The shaking frequency was 100 RPM, the pH was 7.4, the feed-to-solution ratio was 100 ml / pcs, and the shaking was repeated 3 times for 30 min each time.

[0088] 2. Cross-linking: 18g EDC (1.8wt%) and 12g NHS (1.2wt%) were added to 1000ml of L-aspartic acid solution (concentration 1wt%), and activated at 35℃ for 60min. After activation, a bovine heart slice was placed in the activated L-aspartic acid solution, and the first cross-linking fixation was performed at 35℃ and pH 8 for 48h. After the first cross-linking fixation, the bovine heart slice with the first cross-linking fixation was placed in 1000ml of activation solution with EDC and NHS concentrations of 9g (0.9wt%) and 6g (0.6wt%), respectively, and activated at 35℃ for 60min. Then, 3.5g of 1,3-diamino-2-propanol (3.5wt%) was added, and the second cross-linking fixation was performed at 35℃ and pH 8 for 48h.

[0089] 3. Dehydration: The anti-calcification biological tissue was dehydrated three times in sequence using ethanol:ethylene glycol butyl ether at volume ratios of 20%:80%, 10%:90%, and 0%:100%. The first two dehydration times were 2 hours each, and the last dehydration time was 24 hours. The temperature was room temperature, the material-to-liquid ratio was 100 ml / pcs, and the shaking frequency was 100 RPM.

[0090] 4. Drying: After dehydration, the biological tissue is dried in a clean environment using a centrifuge at 1500 rpm for 2 minutes to remove excess dehydrating agent.

[0091] 5. Packaging and sterilization: The dried biological tissue is placed in a dialysis bag for packaging and sterilized using EO. After sterilization, the dialysis bag is wrapped in a foil bag, vacuum-sealed, and stored.

[0092] Example 2

[0093] The dehydration step used 100% tetraethylene glycol dimethyl ether solution as the dehydrating agent and was carried out once for 24 hours. The remaining steps were the same as in Example 1.

[0094] Example 3

[0095] The dehydration step used a 100% diethylene glycol dimethyl ether solution as the dehydrating agent, and the remaining steps were the same as in Example 2.

[0096] Example 4

[0097] The dehydration step used ethanol:N-methyldiethanolamine in volume ratios of 20%:80%, 10%:90%, and 0%:100% as the dehydrating agent, and the remaining steps were the same as in Example 1.

[0098] Example 5

[0099] The dehydration step used 100% N,N-dimethylethanolamine as the dehydrating agent, and the remaining steps were the same as in Example 2.

[0100] Example 6

[0101] The dehydration step used a 100% diethylene glycolamine solution as the dehydrating agent, and the remaining steps were the same as in Example 2.

[0102] Example 7

[0103] The dehydration step uses a 100% ethanol solution as the dehydrating agent, and the remaining steps are the same as in Example 2.

[0104] Example 8

[0105] The dehydration step used 100% ethylene glycol butyl ether solution as the dehydrating agent, and the remaining steps were the same as in Example 2.

[0106] Example 9

[0107] The dehydration step uses ethanol:ethylene glycol butyl ether:water in volume ratios of 30%:50%:20%, 15%:75%:10%, and 5%:95%:0% as the dehydrating agent, and the remaining steps are the same as in Example 1.

[0108] Example 10

[0109] The dehydration step uses ethanol:ethylene glycol butyl ether:water in volume ratios of 20%:60%:20%, 10%:80%:10%, and 0%:100%:0% as the dehydrating agent, and the remaining steps are the same as in Example 1.

[0110] Example 11

[0111] The dehydration step uses ethanol:ethylene glycol butyl ether:water in volume ratios of 10%:10%:80%, 20%:30%:50%, and 30%:50%:20% as the dehydrating agent, and the remaining steps are the same as in Example 1.

[0112] Example 12

[0113] The dehydration step used ethylene glycol butyl ether and N-methyldiethanolamine in a volume ratio of 20%:80% as the dehydrating agent, and the remaining steps were the same as in Example 2.

[0114] Example 13

[0115] The dehydration step uses ethylene glycol butyl ether and N-methyldiethanolamine in a volume ratio of 50%:50% as the dehydrating agent, and the remaining steps are the same as in Example 2.

[0116] Example 14

[0117] The dehydration step used ethylene glycol butyl ether and N-methyldiethanolamine in a volume ratio of 80%:20% as the dehydrating agent, and the remaining steps were the same as in Example 2.

[0118] Example 15

[0119] The dehydration step uses ethanol:ethylene glycol butyl ether in volume ratios of 30%:70%, 20%:80%, and 10%:90% as the dehydrating agent, and the remaining steps are the same as in Example 1.

[0120] Example 16

[0121] The dehydration step used ethanol:ethylene glycol butyl ether in volume ratios of 10%:90%, 5%:95%, and 0%:100% as the dehydrating agent, and the remaining steps were the same as in Example 1.

[0122] Example 17

[0123] The dehydration step used ethanol:N-methyldiethanolamine in volume ratios of 30%:70%, 20%:80%, and 10%:90% as the dehydrating agent, and the remaining steps were the same as in Example 1.

[0124] Example 18

[0125] The dehydration step used ethanol:N-methyldiethanolamine in volume ratios of 10%:90%, 5%:95%, and 0%:100% as the dehydrating agent, and the remaining steps were the same as in Example 1.

[0126] Example 19

[0127] The dehydration step uses ethylene glycol butyl ether at a volume ratio of 20%:80% as the dehydrating agent, and the remaining steps are the same as in Example 2.

[0128] Example 20

[0129] The dehydration step uses ethylene glycol butyl ether at a volume ratio of 50%:50% as the dehydrating agent, and the remaining steps are the same as in Example 2.

[0130] Example 21

[0131] The dehydration step uses ethylene glycol butyl ether at a volume ratio of 80%:20% as the dehydrating agent, and the remaining steps are the same as in Example 2.

[0132] Example 22

[0133] The dehydration step used N-methyldiethanolamine:water at a volume ratio of 80%:20% as the dehydrating agent, and the remaining steps were the same as in Example 2.

[0134] Example 23

[0135] The dehydration step uses ethylene glycol butyl ether at a volume ratio of 80%:20% as the dehydrating agent, and the remaining steps are the same as in Example 2.

[0136] Comparative Example

[0137] The dehydration step uses 100% glycerol dehydrating agent, and the remaining steps are the same as in Example 1.

[0138] The dehydration effect of the dried biological tissues prepared in some of the above examples and comparative examples was evaluated by testing their water content. The experimental method referred to the national standard GB 5009.3-2016, "Determination of Moisture in Food," and the results are shown in Table 1 below.

[0139] The anti-calcification properties of the dried biological tissues prepared in Examples 1-23 and the comparative examples were evaluated. The experimental method followed the standard YYT1859-2022, Evaluation of Anti-calcification of Animal-Derived Cardiovascular Implants: Subcutaneous Implantation Test in Rats. The results are shown in Table 2 below.

[0140] Table 1 Evaluation results of dehydration effect in some examples and comparative examples

[0141]

[0142] As can be seen from the dehydration effects of the various embodiments on biological tissues in Table 1, when tetraethylene glycol dimethyl ether, N,N-dimethylethanolamine, and ethylene glycol butyl ether are used as dehydrating agents in this application, they have obvious dehydration effects on biological tissues, and the dehydration effect is better than that of glycerol; when ethylene glycol butyl ether, N-methyldiethanolamine and ethanol are used in combination in a certain proportion, the dehydration effect is more significant compared with that of a single agent.

[0143] Table 2 Evaluation results of the anti-calcification performance of the examples and comparative examples

[0144]

[0145]

[0146] As can be seen from the anti-calcification performance results of each embodiment in Table 2, the dry biological tissues prepared in the embodiments of this application, under certain conditions of dehydrating agent treatment, have significantly improved anti-calcification performance compared with glycerol dehydration.

[0147] Examples 1, 4, and 15-18 show that the dehydrating agent of ethylene glycol butyl ether or N-methyldiethanolamine combined with ethanol has the most significant effect on the anti-calcification performance of biological tissues. When the volume ratio of ethanol to ethylene glycol butyl ether is (0-3):(7-10), its anti-calcification performance reaches 1.25-1.97 ug / mg. Further examples 9-11 show that when the dehydrating agent of ethylene glycol butyl ether combined with ethanol is prepared into an aqueous solution, the total volume fraction of ethylene glycol butyl ether and ethanol is between 80% and 10%. When used in combination, the anti-calcification performance remains good even at 0% concentration. However, when the total volume fraction of ethylene glycol butyl ether and ethanol decreases to 20-50%, its anti-calcification performance declines to 10.92 ug / mg. This means that when the total volume fraction of ethylene glycol butyl ether and ethanol is ≥70%, it exhibits good anti-calcification performance. As shown in Example 23, when ethylene glycol butyl ether and ethanol are used in combination, even with a single dehydration method, their anti-calcification performance is significantly higher than other dehydrating agents, reaching 1.69 ug / mg. Therefore, when ethylene glycol butyl ether and ethanol are used together as dehydrating agents, whether using a three-gradient dehydration method or a single dehydration method, they exhibit significant anti-calcification effects on biological tissues at certain concentrations and ratios.

[0148] As can be seen from Examples 12-14, the combined effect of ethylene glycol butyl ether and N-methyldiethanolamine is the second best. When the volume ratio of ethylene glycol butyl ether to N-methyldiethanolamine is (1-4):(1-4), its anti-calcification performance reaches 2.26-2.59ug / mg.

[0149] Examples 2, 3, and 5-8 show that when alcohol ethers or alcohol amines are used alone as dehydrating agents, their anti-calcification performance reaches 2.03-4.08 ug / mg. Ethanol also has some anti-calcification properties; however, in specific operations, ethanol easily evaporates during subsequent storage, while alcohol ethers and alcohol amines are non-volatile, allowing for lower storage temperatures. Examples 19-22 show that when alcohol ethers or alcohol amines are prepared into aqueous solutions, their anti-calcification performance is inferior to that of the comparative example glycerol when the volume fraction is below 50%.

[0150] The description in this disclosure is provided for illustrative and descriptive purposes only and is not intended to be exhaustive or to limit the disclosure to its forms. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of this disclosure and to enable those skilled in the art to understand this disclosure and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A method for preparing dry biological tissue, comprising the following steps: Cleaning biological tissues; Cross-linking of cleaned biological tissues; The cross-linked biological tissue was dehydrated. The dehydrated biological tissues were dried, packaged, and sterilized to obtain dry biological tissues. The dehydrating agent used in the dehydration process is selected from one of the following substances: High-boiling-point and low-crystallization-point alcohol ethers, solutions of high-boiling-point and low-crystallization-point alcohol ethers, and mixtures of high-boiling-point and low-crystallization-point alcohol ethers and high-boiling-point and low-crystallization-point alkanolamines. The alcohol ether solution is an aqueous solution of alcohol ether, or the alcohol ether solution is a mixed solution of alcohol ether and low molecular weight alcohol; The low molecular weight alcohol is ethanol; In the aqueous solution of the alcohol ether, the total volume fraction ratio of the alcohol ether to the low molecular weight alcohol is ≥80%; In the mixed solution of alcohol ether and low molecular weight alcohol, the volume ratio of alcohol ether to low molecular weight alcohol is (7-10):(0-3) and the volume ratio is not (7-10):0; In the mixture, the volume ratio of alcohol ether to alcohol amine is (1-4):(1-4). The alcohol ether is selected from one of tetraethylene glycol dimethyl ether, diethylene glycol dimethyl ether, or ethylene glycol butyl ether; The alkanolamine is selected from one of N-methyldiethanolamine, N,N-dimethylethanolamine, or diethylene glycolamine.

2. The method according to claim 1, wherein, The dehydration process includes the following steps: The biological tissue was immersed in a dehydrating agent and shaken to dehydrate it. The dehydration was repeated 1-5 times, each time for 0.5-48 hours, at a temperature of 10-30℃.

3. The method according to claim 1, wherein, The cleaning solution for cleaning biological tissues is selected from aqueous solutions containing surfactants or phosphate buffer solutions. The surfactant is selected from one or more of sodium dodecyl sulfate, amino acid surfactant, Tween 80, glycolipid, lipopeptide or Triton-10; The amino acid surfactant is selected from one or more of N-acylglutamate, N-acylsarcosine, N-lauroyl aspartate, cocoyl arginine ethyl ester, or N-acyl lysine. The glycolipid is selected from one or more of trehalose, rhamnose, or sophorose. The lipopeptide is selected from one or more of surfactants, iturin, spiroin, fentanyl, lichenin, colistin, or octapeptide.

4. The method according to claim 1, wherein, The crosslinking agent used in the crosslinking process is selected from glutaraldehyde, genipin, carbodiimide, N-hydroxysuccinic acid imide, polycarboxylic acid compounds with at least two carboxyl functional groups, or polyamino compounds with at least two amino functional groups. The polycarboxylic compound with at least two carboxyl functional groups is selected from one or more of L-aspartic acid, L-glutamic acid, gluconic acid, malic acid, or succinic acid. The polyamino compound with at least two amino functional groups is selected from one or more of 2,4-diaminobutyric acid, 1,3-diamino-2-propanol, chitosan oligosaccharide, L-lysine, arginine, histidine, or propylenediamine.

5. The method according to any one of claims 1-4, wherein, The biological tissues are selected from the pericardium, heart valves, lamina, pleura, submucosa of the small intestine, dura mater, spinal dura mater, ligaments, or skin.

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