Treatment method of biological tissue material and bioprosthetic valve

Abstract

The invention provides a treatment method of a biological tissue material and a biological valve prosthesis. The treatment method comprises the following steps: firstly, fixing and treating the biological tissue material by using a cross-linking agent; then treating the biological tissue material fixed by the cross-linking agent by using a mixed solution of two cryoprotectants; and treating the biological tissue material treated by the mixed solution of the two cryoprotectants by using a freeze-drying technology so as to obtain a freeze-dried product of the biological tissue material. On the basis, the biological tissue material can be stored and transported in a relatively dry state, so that the transportation cost is reduced, and the performance of the biological tissue material can be well maintained.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and particularly relates to a method for treating biological tissue materials and a biological valve prosthesis. Background Art

[0002] The biological materials of artificial biological heart valves mainly come from porcine pericardium or bovine pericardium. The porcine pericardium or bovine pericardium used must be fixed with glutaraldehyde, and it must be stored in glutaraldehyde solution during transportation all the time, which increases the transportation cost and is not conducive to transportation and storage. At present, relevant literature shows that the pericardium stored in glutaraldehyde solution also has problems such as poor biocompatibility, easy calcification, high hemolysis rate, and shortening the service life of biological valves. Moreover, before using the biological valve treated with glutaraldehyde, it is necessary to elute and remove the residual glutaraldehyde on the tissue for a long time to avoid the harm of glutaraldehyde to the human body during use, but too long elution time will affect the use of the valve.

[0003] It should be noted that the information disclosed in the background art part of the present application is only intended to deepen the understanding of the general background art of the present application, and should not be regarded as an admission or any form of suggestion that this information constitutes the prior art known to those skilled in the art. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for treating biological tissue materials and a biological valve prosthesis, aiming to treat biological tissues with cryoprotectants and freeze-drying technology, so that the biological tissue materials can be stored and transported in a relatively dry state.

[0005] To achieve the above purpose, the present invention proposes a method for treating biological tissue materials, which includes:

[0006] Fixing and treating the biological tissue materials with a cross-linking agent;

[0007] Treating the biological tissue materials fixed with the cross-linking agent with a mixed solution of two cryoprotectants. The two cryoprotectants are a shaping agent and a stabilizer. The concentration of the shaping agent is 5% w / v to 20% w / v, and the concentration of the stabilizer is 35% v / v to 50% v / v;

[0008] Treating the biological tissue materials treated with the mixed solution with freeze-drying technology to obtain a freeze-dried product of the biological tissue materials.

[0009] Optionally, the shaping agent is selected from saccharide substances, and the stabilizer is selected from polyol substances and / or amino acid substances.

[0010] Optionally, the saccharide substance is selected from one or a combination of glucose, dextran, sucrose, trehalose, fructose, mannose, and galactose; the polyol substance is selected from one or a combination of mannitol, ethylene glycol, glycerol, sorbitol, n-butanol, and polyethylene glycol; and the amino acid substance is selected from one or a combination of proline, glycine, glutamic acid, arginine, and alanine.

[0011] Optionally, the excipient is trehalose and the stabilizer is polyethylene glycol.

[0012] Optionally, the concentration of the trehalose is one of 5% w / v, 10% w / v, and 15% w / v, and the concentration of the polyethylene glycol is 40% v / v.

[0013] Optionally, the treatment of the biological tissue material treated with the mixed solution by freeze-drying technology includes: an initial freezing treatment, a primary freeze-drying treatment, and a secondary freeze-drying treatment performed in sequence;

[0014] During the initial freezing treatment, the biological tissue material treated with the mixed solution is placed in a freeze-dryer for pre-cooling, cooled to the initial freezing temperature, and after a certain period of time, the initial freezing is completed;

[0015] During the primary freeze-drying treatment, after heating from the initial freezing temperature to the first preset temperature and maintaining for a certain period of time, it is heated from the first preset temperature to the second preset temperature and maintained for a certain period of time, and then the primary freeze-drying is completed;

[0016] During the secondary freeze-drying treatment, after heating from the second preset temperature to the third preset temperature and maintaining for a certain period of time, the secondary freeze-drying is completed;

[0017] Wherein: the initial freezing temperature is lower than the eutectic temperature of the biological tissue material, and the third preset temperature is room temperature.

[0018] Optionally, the initial freezing temperature is 15°C to 20°C lower than the eutectic temperature of the biological tissue material, the initial freezing temperature is -60°C to -40°C, the first preset temperature is -30°C to -20°C, the second preset temperature is -25°C to -10°C, and the third preset temperature is 20°C to 40°C.

[0019] Optionally, the initial freezing temperature is -40°C, the first preset temperature is -30°C, the second preset temperature is -20°C, and the third preset temperature is 25°C.

[0020] Optionally, sterilization is performed after the freeze-drying treatment.

[0021] Optionally, when treating the crosslinker-fixed biological tissue material with the mixed solution of two cryoprotectants, it includes: immersing the crosslinker-fixed biological tissue material in the mixed solution and shaking it at a constant temperature.

[0022] Optionally, the dry moisture content of the freeze-dried product of the biological tissue material is 5% - 15%, and the wet moisture content after rehydration for 10 min is greater than or equal to 70%.

[0023] Optionally, the heat shrinkage temperature of the freeze-dried product of the biological tissue material is greater than 80 °C, and the heat flow is greater than 9.0 J / g.

[0024] Optionally, the breaking force of the freeze-dried product of the biological tissue material is greater than 200 N, the fracture force is greater than 5.6 N, and the fracture strength is greater than 10 MPa.

[0025] Optionally, the thickness difference of the biological tissue material before and after freeze-drying does not exceed 0.1 mm.

[0026] Based on the same inventive concept, the present invention also provides a biological valve prosthesis, which includes the biological tissue material treated by the treatment method of the biological tissue material according to any one of the above.

[0027] In the above treatment method, first, the biological tissue material is fixed with a crosslinker, then, the crosslinker-fixed biological tissue material is treated with the mixed solution of two cryoprotectants, and then the biological tissue material treated with the mixed solution is treated with freeze-drying technology to obtain the freeze-dried product of the biological tissue material. Based on this, the biological tissue material can be stored and transported in a relatively dry state, which not only reduces the transportation cost, but also avoids the long-term cleaning of glutaraldehyde, enabling the valve to be used quickly.

[0028] In the above treatment method, the concentrations of the two cryoprotectants are also particularly considered to ensure that the concentrations of both the excipient and the stabilizer are in a relatively optimal state, and through the synergistic effect of the two cryoprotectants, the cryoprotection effect is better. The present invention uses freeze-drying technology and adds cryoprotectants to perform freeze-drying treatment on the biological tissue material. After obtaining a relatively dry biological tissue material, the biological tissue material can still have a certain degree of flexibility and softness, and its biological properties, physical properties, and chemical properties are well maintained. Moreover, the freeze-dried biological tissue material has a certain anti-calcification ability, low hemolysis rate, and strong protein immunoadsorption ability after being treated with cryoprotectants, improving the anti-fatigue performance of the biological valve.

[0029] Taking bovine pericardium as an example, without adding cryoprotectant, freeze-drying the biological tissue material will cause damage to the tissue, resulting in loose arrangement of the internal fiber structure of the bovine pericardium. However, by adding cryoprotectant to the biological tissue material for freeze-drying treatment in the present invention, the internal fiber structure of the bovine pericardium can be arranged tightly with a small porosity, the cytoplasmic matrix can be stabilized, and the biological tissue material can be stored and transported at room temperature. During the freezing process, due to the addition of cryoprotectant, the collagen, fibrin, etc. of the biological tissue material can also be stabilized without being damaged, so that the biological tissue material maintains good integrity. Description of the Drawings

[0030] Those of ordinary skill in the art will understand that the provided drawings are used to better understand the present invention and do not constitute any limitation to the scope of the present invention. Among them:

[0031] Figure 1 is a flowchart of the method for processing the biological tissue material of the present invention.

[0032] Figure 2 is a tissue cell diagram obtained by directly freeze-drying the biological tissue material fixed with glutaraldehyde (GA) without adding cryoprotectant and performing he staining (i.e., HE staining method) on the freeze-dried sample, wherein the tissue appears red and the cell nucleus appears blue;

[0033] Figure 3 is a tissue cell diagram obtained by performing he staining on the freeze-dried sample of the biological tissue material freeze-dried and protected with 40% v / v polyethylene glycol (PEG), wherein the tissue appears red and the cell nucleus appears blue;

[0034] Figure 4 is a tissue cell diagram obtained by performing he staining on the freeze-dried sample of the biological tissue material freeze-dried and protected with 40% v / v polyethylene glycol (PEG) and 5% w / v trehalose (Tre), wherein the tissue appears red and the cell nucleus appears blue;

[0035] Figure 5 is a tissue cell diagram obtained by performing he staining on the freeze-dried sample of the biological tissue material freeze-dried and protected with 40% v / v polyethylene glycol (PEG) and 10% w / v trehalose (Tre), wherein the tissue appears red and the cell nucleus appears blue;

[0036] Figure 6 is a tissue cell diagram obtained by performing he staining on the freeze-dried sample of the biological tissue material freeze-dried and protected with 40% v / v polyethylene glycol (PEG) and 15% w / v trehalose (Tre), wherein the tissue appears red and the cell nucleus appears blue;

[0037] Figure 7 It is a scanning electron microscope image (i.e., SEM image) of a freeze-dried sample obtained by directly freeze-drying a biological tissue material fixed with glutaraldehyde without adding a cryoprotectant.

[0038] Figure 8 It is a scanning electron microscope image of a freeze-dried sample of a biological tissue material freeze-dried with 40% v / v polyethylene glycol (PEG).

[0039] Figure 9 It is a scanning electron microscope image of a freeze-dried sample of a biological tissue material freeze-dried with 40% v / v polyethylene glycol (PEG) and 5% w / v trehalose (Tre).

[0040] Figure 10 It is a scanning electron microscope image of a freeze-dried sample of a biological tissue material freeze-dried with 40% v / v polyethylene glycol (PEG) and 10% w / v trehalose (Tre).

[0041] Figure 11 It is a scanning electron microscope image of a freeze-dried sample of a biological tissue material freeze-dried with 40% v / v polyethylene glycol (PEG) and 15% w / v trehalose (Tre).

[0042] Figure 12 It is the appearance of bovine pericardium after freeze-drying treatment with glutaraldehyde (GA) alone in the comparative example.

[0043] Figure 13 It is the appearance of bovine pericardium after freeze-drying treatment with the addition of alcohol substances and sugar substances in the embodiment of the present invention. Detailed Embodiments

[0044] To make the objectives, advantages and features of the present invention clearer, the following further describes the present invention in detail with reference to the accompanying drawings and specific embodiments. It should be noted that the accompanying drawings are in very simplified forms and not drawn to scale, only for conveniently and clearly assisting in explaining the objectives of the embodiments of the present invention. In addition, the structures shown in the accompanying drawings are often part of the actual structures. In particular, the accompanying drawings need to show different focuses and sometimes use different scales.

[0045] The core of the present invention is to use freeze-drying technology and add a cryoprotectant to perform freeze-drying treatment on biological tissue materials, so that the biological tissue materials can be stored and transported in a relatively dry state, not only reducing the transportation cost, but also enabling the performance of the biological tissue materials to be better maintained, which is beneficial to their use.

[0046] In response to this, the present invention provides a method for treating biological tissue materials. First, the biological tissue materials are fixed with a crosslinking agent, then the biological tissue materials fixed with the crosslinking agent are treated with a mixed solution of two cryoprotectants. After that, the biological tissue materials treated with the mixed solution of two cryoprotectants are treated with freeze-drying technology. During a single freeze-drying process, the moisture of the materials is removed by direct sublimation, thereby reducing the formation of ice crystals during the freeze-drying process, reducing the porosity of collagen fibers, and avoiding damage to collagen fibers caused by the formation of ice crystals. Finally, after obtaining the freeze-dried product of the biological tissue materials, the biological tissue materials can be stored and transported in a relatively dry state, which not only reduces the transportation cost but also avoids long-term cleaning with glutaraldehyde, enabling the biological valve to be used quickly.

[0047] The method for treating biological tissue materials provided by the present invention has the advantages of being non-toxic and simple to operate. Moreover, after adding cryoprotectants, the glass transition temperature of the biological tissue materials can be increased, enabling the biological tissue materials to maintain the basic structure of the cytoplasmic matrix during the freeze-drying process and not causing deformation. During the freeze-drying process, the formation of ice crystals is reduced, avoiding damage to the fiber structure inside the biological tissue materials caused by the formation of ice crystals, and enabling the fiber structure inside the biological tissue materials to be arranged tightly with smaller pores, stabilizing its cytoplasmic matrix. Therefore, the biological tissue materials obtained by freeze-drying still have a certain degree of flexibility and softness, and their biological properties, physical properties, and chemical properties can all be well maintained, and they also have a certain anti-calcification ability, low hemolysis rate, strong protein immunoadsorption ability, and good anti-fatigue performance.

[0048] More specifically, as Figure 1 shown, the method for treating biological tissue materials provided by the present invention includes the following steps:

[0049] Step S10, fixing and treating the biological tissue materials with a crosslinking agent.

[0050] The biological tissue materials in the embodiments of the present invention can be selected from bovine pericardium, porcine pericardium, or other suitable biological tissue materials.

[0051] When fixing and treating the biological tissue materials, the selected crosslinking agent is a common material. In addition to using glutaraldehyde, it can also be other aldehyde-containing reagents well-known to those skilled in the art. In this regard, the present invention is not limited.

[0052] Step S20, treating the biological tissue materials fixed with the crosslinking agent with a mixed solution of two cryoprotectants, and the two cryoprotectants are a shaping agent and a stabilizer.

[0053] During lyophilization treatment, through the combined action of excipients and stabilizers, the lyophilized biological tissue material can not only maintain its original properties, but also have certain anti-calcification ability, good low hemolysis rate and strong protein immunoadsorption ability, and at the same time can improve the anti-fatigue performance.

[0054] A stabilizer refers to a chemical substance that can increase the stability of solutions, colloids, solids, mixtures, etc., and has the functions of slowing down reactions, maintaining chemical equilibrium, reducing surface tension, and preventing photodegradation, thermal decomposition or oxidative decomposition, etc. The stabilizer can be selected from polyol substances, such as but not limited to mannitol, ethylene glycol, glycerol (Gly), sorbitol, xylitol, n-butanol, polyethylene glycol (PEG), etc., and is selected from one or a combination of multiple polyol substances. The stabilizer can also be selected from amino acid substances, such as but not limited to proline, glycine, glutamic acid, arginine, alanine, etc., and is used in one or a combination of multiple amino acid substances. In other embodiments, the stabilizer can be prepared by combining polyol substances and amino acid substances. Using the stabilizer can dehydrate the biological tissue material and stabilize its morphology.

[0055] The characteristics of excipients are that they are stable in nature, do not produce side effects, do not affect the curative effect, are not prone to deformation, cracking or being eaten by insects at room temperature, are harmless to the human body, have no physiological effects, do not chemically or physically interact with the main drug, and do not affect the content determination of the main drug, etc. The excipient can form hydrogen bonds with the polar groups of protein groups in the biological tissue material, replace water molecules, maintain the cytoplasmic matrix, and prevent protein denaturation, etc. Using the excipient aims to increase the glass transition temperature of the biological tissue material and stabilize the protein structure. The excipient is mainly selected from some saccharide substances, such as one or a combination of multiple ones selected from glucose, dextran, sucrose, fructose, trehalose (Tre), mannose, galactose, etc.

[0056] It has been found through research that when the concentration of the stabilizer and the excipient exceeds a certain level, crystals (especially saccharide substances) will precipitate during the mixing process, reducing the concentration of the solution and affecting the preparation effect of the solution. In addition, if the concentration of the stabilizer is too low, the biological tissue material is prone to deformation (such as curling, etc.) during lyophilization, and if the concentration of the excipient is too low, its use effect will also be affected, and it cannot fully stabilize proteins and increase the glass transition temperature. To balance these problems, in the processing method of the biological tissue material of the present invention, the concentration of the excipient is optimized to 5% w / v - 20% w / v, and the concentration of the stabilizer is optimized to 35% v / v - 50% v / v; preparing a mixed solution of the stabilizer and the excipient according to these concentrations not only avoids crystal precipitation, but also can achieve a good cryoprotective effect, so that the biological tissue material will not be damaged during lyophilization.

[0057] The concentration of the stabilizer can be 35% v / v or 40% v / v or 50% v / v, and a more suitable concentration is 40% v / v, with better effects.

[0058] The concentration of the excipient can be 5% w / v or 10% w / v or 15% w / v or 20% w / v, and more appropriate concentrations are 5% w / v or 10% w / v or 15% w / v.

[0059] Step S30: After the mixed solution of the two cryoprotectants fully reacts with the biological tissue material, the biological tissue material treated with the mixed solution is then processed using freeze-drying technology to obtain a freeze-dried product of the biological tissue material.

[0060] Generally, the biological tissue material is placed in a freeze-dryer for freeze-drying treatment. After the freeze-drying treatment, a freeze-dried product of the biological tissue material can be obtained. It should be understood that by using a freeze-dryer for freeze-drying the biological valve material, free water and part of the bound water can be removed from the biological tissue material during the drying process, enabling the biological tissue material to maintain a relatively dry state after the freeze-drying is completed, so that it can be stored at room temperature.

[0061] Optionally, after the freeze-drying treatment, sterilization is performed. For example, sterilization is carried out using ethylene oxide.

[0062] Specifically, in this embodiment, when using freeze-drying technology to process the biological tissue material treated with the mixed solution of the two cryoprotectants, it includes three basic processes: initial freezing treatment (i.e., pre-cooling stage), primary freeze-drying treatment, and secondary freeze-drying treatment.

[0063] During the initial freezing treatment, the biological tissue material treated with the mixed solution is directly placed in a freeze-dryer for pre-cooling, and the temperature is lowered to the initial freezing temperature and maintained for a certain period of time to complete the initial freezing.

[0064] During the primary freeze-drying treatment, after raising the temperature from the initial freezing temperature to the first preset temperature and maintaining for a certain period of time, the temperature is raised from the first preset temperature to the second preset temperature and maintained for a certain period of time to complete the primary freeze-drying.

[0065] During the secondary freeze-drying treatment, after raising the temperature from the second preset temperature to the third preset temperature and maintaining for a certain period of time, the secondary freeze-drying is completed.

[0066] After the secondary freeze-drying is completed, the sample is directly taken out of the freeze-dryer, and the biological tissue material can be stored and transported at room temperature.

[0067] Thus, when freeze-drying is required, the cryoprotectant-treated biological tissue material is directly placed in a freeze-dryer. First, it is pre-cooled, directly cooled to the initial freezing temperature (i.e., the pre-cooling stage). After pre-freezing for a period of time, the biological tissue material can be rapidly frozen. Then, it is heated to the first preset temperature, evacuated, and kept frozen for a period of time. After that, it is further heated to the second preset temperature, evacuated, and freeze-dried for a period of time, thus completing one cycle of freeze-drying. After one cycle of freeze-drying, it enters the secondary freeze-drying stage. At this time, it is heated to the third preset temperature, evacuated, and freeze-dried until the end.

[0068] The eutectic temperature of the biological tissue material is the critical temperature for obtaining the best freeze-drying effect. Therefore, the initial freezing temperature should be lower than the eutectic temperature of the biological tissue material. Preferably, the initial freezing temperature is 15°C - 20°C lower than the eutectic temperature of the biological tissue material to obtain a better freeze-drying effect. In addition, the third preset temperature is room temperature, that is, normal temperature or ambient temperature, which can be defined as 20°C - 40°C, preferably 25°C.

[0069] Furthermore, the primary freezing temperature can be -60°C - -40°C, preferably -40°C, and the initial freezing time can be 5h - 10h, preferably 5h (i.e., 300min). In the initial freezing stage, the cooling rate can be -10°C / min - -0.1°C / min, such as -0.1°C / min.

[0070] The first preset temperature can be -30°C - -20°C, preferably -30°C, and the freeze-drying time at the first preset temperature can be 6h (360min) - 10h (600min), preferably 8h (480min).

[0071] The second preset temperature can be -25°C - -10°C, preferably -20°C, and the freeze-drying time at the second preset temperature can be 4h (240min) - 8h (480min), such as 5h (i.e., 300min). In the first cycle of freeze-drying stage, the heating rate can be 1°C / min - 10°C / min, such as 1°C / min.

[0072] The freeze-drying time at the third preset temperature can be 4h (240min) - 8h (480min), such as 5h (i.e., 300min). In the secondary freeze-drying stage, the heating rate can be 1°C / min - 10°C / min, such as 1°C / min.

[0073] It should also be noted that freeze-drying needs to be completed in a vacuum environment to effectively remove the moisture of the biological valve. Therefore, during the freeze-drying process, it is necessary to evacuate the air and maintain a certain pressure for freeze-drying. For example, a certain pressure needs to be set during both the primary drying and the secondary drying processes, and the pressure range is 5 Pa to 50 Pa, such as 15 Pa. In this way, the moisture can directly sublimate to achieve the purpose of freeze-drying.

[0074] In addition, in step S20, when treating the crosslinker-fixed biological tissue material with a mixed solution of two cryoprotectants, the specific operation can be as follows: Immerse the crosslinker-fixed biological tissue material in the mixed solution and shake it at a constant temperature so that the two cryoprotectants in the mixed solution can fully react with the biological tissue material.

[0075] The conditions for the constant-temperature shaking treatment are preferably: the shaking temperature is 20°C to 50°C, a more suitable temperature is 37°C, the shaking speed (rotation speed) is 50 r / min to 100 r / min, a more suitable speed is 70 r / min, and the shaking time is 4 h to 24 h, preferably 24 h. During the cryoprotection treatment, any suitable device can be used for shaking treatment, including but not limited to a constant-temperature shaker.

[0076] Furthermore, this embodiment also provides a biological valve prosthesis, which includes the biological tissue material processed by the processing method of the biological tissue material described in any one of the above.

[0077] Specifically in this embodiment, when treating the crosslinker-fixed biological tissue material with a mixed solution of polyethylene glycol (PEG) and trehalose (Tre), polyethylene glycol (PEG) can keep the sample in its original form during the freeze-drying process, and trehalose (Tre) can form hydrogen bonds with the polar groups of protein groups in the biological valve tissue, replace water molecules, maintain the cytoplasmic matrix, and prevent protein denaturation, so that the biological tissue material will not be damaged during freeze-drying.

[0078] Specifically in this embodiment, when treating the crosslinker-fixed biological tissue material with a mixed solution of polyethylene glycol (PEG) and trehalose (Tre), polyethylene glycol (PEG) can keep the sample in its original form during the freeze-drying process, and trehalose (Tre) can form hydrogen bonds with the polar groups of protein groups in the biological valve tissue, replace water molecules, maintain the cytoplasmic matrix, and prevent protein denaturation, so that the biological tissue material will not be damaged during freeze-drying.

[0079] When preparing a mixed solution of trehalose (Tre) and polyethylene glycol (PEG), considering that when the concentrations of trehalose (Tre) and polyethylene glycol (PEG) are too high, there will be a problem of trehalose (Tre) crystallization, which cannot meet the concentration requirements and is difficult to meet the required requirements. When the concentrations of trehalose (Tre) and polyethylene glycol (PEG) are too low, they cannot play their due roles and affect the cryoprotective effect. Therefore, the concentration of polyethylene glycol (PEG) is 35% v / v to 50% v / v, and the concentration of trehalose (Tre) is 5% w / v to 20% w / v. Especially when the concentration of polyethylene glycol (PEG) is 40% v / v, the performance in all aspects is optimal. Therefore, the concentration of polyethylene glycol (PEG) is preferably 40% v / v, and the concentration of trehalose (Tre) is preferably one of 5% w / v, 10% w / v, and 15% w / v. Mixing 40% v / v of polyethylene glycol (PEG) with trehalose (Tre) at any one of the concentrations of 5% w / v, 10% w / v, and 15% w / v can make the freeze-dried biological tissue material reach a better state.

[0080] For example: To prepare 1 L of the mixed solution, including 40% v / v of polyethylene glycol (PEG) and 10% w / v of trehalose (Tre), it can be mixed as follows: First, measure 400 mL of the polyethylene glycol (PEG) solution with a measuring cylinder and pour it into a beaker. Then, add the 10% w / v trehalose (Tre) solution to the beaker and stir continuously until it is evenly mixed until 1 L of the solution is obtained, that is, a mixed solution of 40% v / v polyethylene glycol (PEG) and 10% w / v trehalose (Tre) is obtained. When mixing the two cryoprotectants, it is best to stir to make the two solutions fully mixed to ensure the prepared concentration of the solution. For example, the mixed solution of the two cryoprotectants can be prepared on a magnetic stirrer. During the preparation process, it can be magnetically stirred until the solution is clear and transparent to make the solution fully mixed. The mixing treatment of the two cryoprotectants generally refers to mixing two different cryoprotectants according to different types of mixing methods such as volume ratio, mass ratio, molar concentration ratio, etc., so that the two cryoprotectants can fully exert their effects and achieve the effects required by the present invention.

[0081] The following takes the freeze-drying treatment experiment of bovine pericardium to further illustrate the advantages of the treatment method of the biological tissue material of the present invention. However, considering that the samples treated only with trehalose (Tre) have obvious deformation after the freeze-drying step and do not meet the actual requirements, the following tests and evaluations are no longer carried out for the simple trehalose (Tre) treatment.

[0082] I. Morphology and appearance

[0083] From Figure 12It can be seen that if glutaraldehyde is used alone for freeze-drying treatment of bovine pericardium materials, it will cause deformation of the appearance of the bovine pericardium, which is not conducive to use. However, from Figure 13 it can be seen that after freeze-drying treatment of bovine pericardium with a cryoprotectant solution composed of alcohols and sugars, the bovine pericardium material hardly undergoes deformation. This shows that using a combination of two cryoprotectants for freeze-drying treatment is beneficial to the freeze-drying of bovine pericardium materials and can achieve a good cryoprotective effect.

[0084] II. Water content test

[0085] Refer to Table 1, which is the measurement result of the water content of the freeze-dried sample of bovine pericardium after rehydration for 10 min. In Table 1: GA refers to the freeze-dried sample that has been fixed with glutaraldehyde but not subjected to freeze-drying protection treatment; 40% v / v PEG (polyethylene glycol) + 5% w / v Tre (trehalose) refers to the freeze-dried sample that has been fixed with glutaraldehyde and then subjected to freeze-drying protection treatment with a concentration of 40% v / v PEG and 5% w / v Tre; 40% v / v PEG + 10% w / v Tre refers to the freeze-dried sample that has been fixed with glutaraldehyde and then subjected to freeze-drying protection treatment with a concentration of 40% v / v PEG and 10% w / v Tre; 40% v / v PEG + 15% w / v Tre refers to the freeze-dried sample that has been fixed with glutaraldehyde and then subjected to freeze-drying protection treatment with a concentration of 40% v / v PEG and 15% w / v Tre. Other bovine pericardium samples treated with different concentrations are as described above and will not be elaborated one by one. Freeze-drying rehydration means adding water to the freeze-dried bovine pericardium and observing the degree to which it returns to its original state. The wet-state water content refers to the water content of the freeze-dried bovine pericardium after rehydration, and the dry-state water content refers to the water content of the freeze-dried bovine pericardium.

[0086] Table 1: Wet-state and dry-state water contents of bovine pericardium after rehydration for 10 min

[0087]

[0088] It can be seen from Table 1 that after the freeze-dried samples of bovine pericardium added with cryoprotectant are rehydrated for 10 min, measured by the Karl Fischer method, the wet-state water content (water content after rehydration) after rehydration is greater than or equal to 70%, and the dry-state water content (i.e., the water content after freeze-drying without rehydration) is between 5% and 15%. This shows that after adding cryoprotectant, the bovine pericardium can be quickly rehydrated after freeze-drying, which is conducive to use. The above results show that the freeze-dried bovine pericardium material can be quickly rehydrated and can maintain its performance.

[0089] III. Thermal stability test

[0090] Refer to Table 2, which is the result of DSC (differential scanning calorimetry) test on the freeze-dried bovine pericardium.

[0091] Table 2: Thermal shrinkage temperature

[0092] Grouping Unit (°C) GA 83.14 40% v / v PEG + 5% w / v Tre 84.96 40% v / v PEG + 10% w / v Tre 84.88 40% v / v PEG + 15% w / v Tre 84.86 50% v / v PEG and 5% w / v Tre 84.31 50% v / v PEG and 10% w / v Tre 83.25 50% v / v PEG and 15% w / v Tre 84.18 40% v / v PEG and 5% w / v Dextran 83.15 40% v / v PEG and 10% w / v Dextran 82.75 40% v / v PEG and 15% w / v Dextran 83.49 30% v / v Gly and 5% w / v Tre 82.34 30% v / v Gly and 10% w / v Tre 81.24 30% v / v Gly and 15% w / v Tre 80.14 40% v / v Gly and 5% w / v Dextran 82.54 40% v / v Gly and 10% w / v Dextran 81.65 40% v / v Gly and 15% w / v Dextran 81.65

[0093] As can be seen from Table 2, the thermal shrinkage temperatures of the freeze-dried samples with and without cryoprotectant added are both greater than 80 °C. The thermal shrinkage temperature can illustrate the thermal stability of proteins. By measuring the thermal shrinkage temperature of bovine pericardium with different treatments using DSC, it can be known that different combinations of alcohols and saccharides also meet the requirements for the thermal stability of bovine pericardium after freeze-drying. However, when using the combination of polyethylene glycol (PEG) and trehalose (Tre), compared with other reagent combinations, the treatment of bovine pericardium material is better and the thermal shrinkage temperature is higher. In any case, when different concentration combinations of polyethylene glycol (PEG) and trehalose (Tre) are formed, the thermal shrinkage temperatures of bovine pericardium treated with these two cryoprotectants are both higher than that of the bovine pericardium treated with glutaraldehyde (GA) fixation (P < 0.05). This shows that the combination of polyethylene glycol (PEG) and trehalose (Tre) is better, and the thermal stability of the collagen in the freeze-dried bovine pericardium is better than that of the bovine pericardium fixed with GA.

[0094] IV. Mechanical Property Testing

[0095] The thickness of the freeze-dried biological tissue usually becomes thinner. However, through the mechanical property testing with a universal tensile testing machine, it is found that the mechanical properties of the freeze-dried samples of bovine pericardium added with cryoprotectant are basically the same as those of the freeze-dried samples of bovine pericardium fixed with glutaraldehyde (GA). For details, please refer to Table 3.

[0096] Table 3: Results of Mechanical Property Determination

[0097]

[0098] Note: P > 0.05, no significant difference. The breaking force is the maximum force when pulled to break. The rupture force is the maximum force when the sample is stressed and ruptured. Generally, after freeze-drying treatment, the acceptance criteria for the freeze-dried products of biological tissue materials are as follows: the rupture force is greater than 200 N, the breaking force is greater than 5.6 N, and the breaking strength is greater than 10 MPa.

[0099] As can be seen from Table 3, the mechanical properties of bovine pericardium treated with different combinations all meet the acceptance criteria after freeze-drying, but the combination of polyethylene glycol (PEG) and trehalose (Tre) is superior to other reagent combinations. Moreover, by comparing polyethylene glycol at concentrations of 40% v / v and 50% v / v, it can be seen that when the combination of 40% v / v PEG and 10% w / v Tre is used, the mechanical properties of the bovine pericardium are better. Generally speaking, the mechanical properties of the 40% v / v PEG + 10% w / v Tre combination are superior to those of the other two reagent combinations and different combinations of 50% v / v PEG and Tre, which indicates that the 40% v / v PEG + 10% w / v Tre combination is more suitable for the freeze-drying preservation of bovine pericardium. In short, the bovine pericardium added with cryoprotectant is suitable to replace the GA solution as the preservation and transportation material of biological tissue materials after freeze-drying.

[0100] V. Thickness measurement before and after freeze-drying

[0101] The thickness of the bovine pericardium was measured using a thickness gauge as shown in Table 4, and Table 4 shows the thickness changes of the bovine pericardium treated under different experimental conditions. As can be seen from Table 4, the thickness difference of the bovine pericardium after freeze-drying before and after freeze-drying basically does not exceed 0.1 mm, which indicates that the bovine pericardium can be quickly rehydrated and maintain its original performance.

[0102] Table 4: Thickness changes before and after freeze-drying

[0103]

[0104]

[0105] Note: P < 0.05, there is a significant difference.

[0106] VI. Staining treatment and scanning electron microscopy observation

[0107] To observe the fiber structure of the freeze-dried samples, the freeze-dried samples of bovine pericardium treated under different experimental conditions were also subjected to he staining and scanning electron microscopy observation.

[0108] The results showed that, as Figure 2 and Figure 7 shown, in the freeze-dried samples of bovine pericardium fixed with GA but without cryoprotectant, the fiber structure ( Figure 2 the red part in

[0109] was arranged disorderly, and there were relatively large voids inside, which indicates that when directly freeze-dried after GA fixation, the pores between the collagen fibrils of the freeze-dried samples increased. Therefore, ice crystals were generated during the freeze-drying process, resulting in pores. Figures 3 to 6 , and Figures 8 to 11As shown, after being treated with cryoprotectant and then freeze-dried, the fiber structures of the freeze-dried samples can be arranged tightly and orderly, and the pores between collagen fibers are significantly reduced. This indicates that the generation of ice crystals is reduced during the freeze-drying process, which is conducive to maintaining the structural stability between collagens. Therefore, the internal fiber arrangement of the freeze-dried bovine pericardium treated by adding cryoprotectant is tighter, which is beneficial to the freeze-drying preservation of bovine pericardium without affecting its physical properties.

[0110] In summary, in the method for treating biological tissue materials provided by the present invention, by adding two cryoprotectants, the biological valve can be stored in a dry state at room temperature after being freeze-dried, solving the problem of difficult storage and transportation of biological valves at normal temperature. After freeze-drying, the biological valve can be stored in a dry state at room temperature. The freeze-dried samples of the biological valve obtained after freeze-drying treatment have the following characteristics: (1) having a certain flexibility and good biological properties; (2) being convenient for storage and transportation, capable of being transported at room temperature, and effectively reducing the transportation and storage costs; (3) during the freezing process, due to the addition of cryoprotectant, the collagen, fibrin, etc. of the biological valve can be stabilized without being damaged, thus stabilizing the cytoplasmic matrix and protein structure of the biological valve, enabling the biological valve to maintain good integrity. In particular, the biological valve can also have certain anti-calcification ability, low hemolysis rate, strong protein immunoadsorption ability and good anti-fatigue performance.

[0111] The above description is only a description of the preferred embodiments of the present invention, and does not limit the scope of the present invention in any way. Any changes and modifications made by those of ordinary skill in the art of the present invention according to the above disclosure belong to the protection scope of the technical solution of the present invention.

Claims

1. A method for treating biological tissue materials, characterized in that, Comprising: Fixing and treating biological tissue materials with a crosslinking agent; Treating the biological tissue materials fixed with the crosslinking agent with a mixed solution of two cryoprotectants, the two cryoprotectants being a shaping agent and a stabilizer, the concentration of the shaping agent being 5% w / v to 20% w / v, and the concentration of the stabilizer being 35% v / v to 50% v / v; Treating the biological tissue materials treated with the mixed solution with freeze-drying technology to obtain freeze-dried products of the biological tissue materials.

2. The processing method of the biological tissue material according to claim 1, characterized in that, The shaping agent is selected from saccharide substances, and the stabilizer is selected from polyol substances and / or amino acid substances.

3. The processing method of the biological tissue material according to claim 2, characterized in that, The saccharide substances are selected from one or a combination of glucose, dextran, sucrose, trehalose, fructose, mannose, and galactose, the polyol substances are selected from one or a combination of mannitol, ethylene glycol, glycerol, sorbitol, xylitol, n-butanol, and polyethylene glycol, and the amino acid substances are selected from one or a combination of proline, glycine, glutamic acid, arginine, and alanine.

4. The processing method of the biological tissue material according to claim 1, characterized in that The shaping agent is trehalose, and the stabilizer is polyethylene glycol.

5. The processing method of the biological tissue material according to claim 4, characterized in that, The concentration of the trehalose is one of 5% w / v, 10% w / v, and 15% w / v, and the concentration of the polyethylene glycol is 40% v / v.

6. The processing method of the biological tissue material according to claim 1, wherein The treating the biological tissue materials treated with the mixed solution with freeze-drying technology includes: an initial freezing treatment, a primary freeze-drying treatment, and a secondary freeze-drying treatment performed in sequence; During the initial freezing treatment, the biological tissue materials treated with the mixed solution are placed in a freeze-dryer for precooling, cooled to the initial freezing temperature and maintained for a certain time to complete the initial freezing; During the primary freeze-drying treatment, after heating from the initial freezing temperature to a first preset temperature and maintaining for a certain time, heating from the first preset temperature to a second preset temperature and maintaining for a certain time to complete the primary freeze-drying; During the secondary freeze-drying treatment, heating from the second preset temperature to a third preset temperature and maintaining for a certain time to complete the secondary freeze-drying; Wherein: the initial freezing temperature is lower than the eutectic temperature of the biological tissue materials, and the third preset temperature is room temperature.

7. The processing method of the biological tissue material according to claim 6, characterized in that, The initial freezing temperature is 15°C to 20°C lower than the eutectic temperature of the biological tissue materials, the initial freezing temperature is -60°C to -40°C, the first preset temperature is -30°C to -20°C, the second preset temperature is -25°C to -10°C, and the third preset temperature is 20°C to 40°C.

8. The processing method of the biological tissue material according to claim 7, wherein The initial freezing temperature is -40°C, the first preset temperature is -30°C, the second preset temperature is -20°C, and the third preset temperature is 25°C.

9. The processing method of the biological tissue material according to claim 6, characterized in that, Sterilization is performed after the freeze-drying treatment.

10. The processing method of the biological tissue material according to claim 1, characterized in that, When treating the biological tissue materials fixed with the crosslinking agent with the mixed solution of two cryoprotectants, it includes: immersing the biological tissue materials fixed with the crosslinking agent in the mixed solution and oscillating at a constant temperature.

11. The processing method of the biological tissue material according to claim 1, characterized in that, The dry-state water content of the freeze-dried products of the biological tissue materials is 5% to 15%, and the wet-state water content after rehydration for 10 min is greater than or equal to 70%.

12. The processing method of the biological tissue material according to claim 1, wherein The heat shrinkage temperature of the freeze-dried product of the biological tissue material is greater than 80 °C, and the heat flow is greater than 9.0 J / g.

13. The processing method of the biological tissue material according to claim 1, characterized in that, The breaking force of the freeze-dried product of the biological tissue material is greater than 200 N, the fracture force is greater than 5.6 N, and the fracture strength is greater than 10 MPa.

14. The processing method of the biological tissue material according to claim 1, characterized in that, The thickness difference of the biological tissue material before and after freeze-drying does not exceed 0.1 mm.

15. A biological valve prosthesis, characterized in that, It includes a biological tissue material processed by the processing method of the biological tissue material according to any one of claims 1-14.

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