WO2020057414A1 - Dry biological heart valve capable of rapidly absorbing water and flattening, and preparation method therefor - Google Patents

Abstract

Disclosed are a dry biological heart valve capable of rapidly absorbing water and flattening, and a preparation method therefor. The preparation method comprises the following steps: performing a crosslinking treatment on an animal pericardium using a glutaraldehyde aqueous solution, then immersing the animal pericardium in an extracellular matrix biological enzyme aqueous solution, and finally, drying the animal pericardium to obtain a dry biological heart valve capable of rapidly absorbing water and flattening. The prepared dry biological heart valve has a good performance in terms of rapid water absorption self-flattening, and is capable of rapidly absorbing water and flattening under a simulated fold and pressure test.

Description

Dry biological heart valve capable of quickly absorbing and flattening and preparation method thereof Technical field

The invention relates to the field of medical materials and medical devices, and in particular, to a dry biological heart valve capable of quickly absorbing and flattening, and a preparation method thereof.

Background technique

Heart valve disease is a common valvular disease. Anatomically manifested as narrowed blood passages or valvular insufficiency. Treatment of heart valve disease includes thoracotomy and percutaneous heart valve replacement. Thoracic surgery is very traumatic, high risk, slow recovery, and requires extracorporeal circulation support, which is unacceptable to many patients. Percutaneous heart valve replacement surgery has become the main trend of future valve surgery because it has less trauma to patients and low risk. Biological heart valves are a class of biomedical materials used to replace heart valves in humans. Biological heart valves are generally prepared by cross-linking glutaraldehyde from porcine pericardium and bovine pericardium.

The existing interventional biological valve is generally cross-linked by glutaraldehyde, and then the biological valve is sewn on a metal stent, and then stored in a glutaraldehyde solution. Prior to interventional valve replacement surgery, multiple washing, crimping, and assembly steps are required. This preparation process is cumbersome and easily leads to an increased risk of surgery. The biological valve stored in the glutaraldehyde solution for a long time can easily lead to the residual of glutaraldehyde and increase the calcification and toxicity of the biological valve. Therefore, the development of the biological valve into a dry film stored away from the glutaraldehyde solution and pre-loading in the valve delivery system can better solve the above problems. However, the dry biological valve pre-loaded in the valve delivery system is subject to a longer period of mechanical compression, and because of the higher requirements for the rapid water absorption and self-healing performance of the biological valve in the dry state, the existing Biological valves cannot absorb water quickly and flatten.

Therefore, the existing technology needs to be improved and developed.

Summary of the Invention

In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a dry biological heart valve that can absorb water quickly and flatten, and a preparation method thereof, which aims to solve the problem that existing interventional biological valves need to be stored in glutaraldehyde solution and cannot be used. It is pre-loaded in the conveying system, needs to be temporarily cleaned and pressed, and cannot be quickly absorbed and flattened.

The technical solution of the present invention is as follows:

The invention provides a method for preparing a dry biological heart valve that can absorb water quickly and flatten, comprising the steps of: cross-linking an animal pericardium with an aqueous solution of glutaraldehyde, and then immersing the solution in an extracellular matrix biological enzyme solution , And finally dried to obtain a dry biological heart valve that can absorb and flatten quickly.

The method for preparing a rapidly absorbable and flattened dry biological heart valve, wherein the extracellular matrix biological enzyme aqueous solution is one or more of collagenase, elastase, hyaluronidase, and chondroitin sulfate mixture.

In the method for preparing a rapidly absorbable and flattened dry biological heart valve, the concentration of the extracellular matrix biological enzyme aqueous solution is 0.1-50 U / ml.

The method for preparing a rapidly absorbable and flattened dry biological heart valve, wherein the animal pericardium after cross-linking treatment is immersed in an extracellular matrix biological enzyme aqueous solution under a shaking condition of 25 to 37 ° C for 12 to 72 hours. .

In the method for preparing a quick-absorbing and flattening dry biological heart valve, the volume concentration of the glutaraldehyde aqueous solution is 0.1 to 5%, and the pH is 7 to 8.

In the method for preparing a rapidly absorbable and flattened dry biological heart valve, the animal pericardium is crosslinked and treated with an aqueous solution of glutaraldehyde under shaking conditions of 20 to 45 ° C for 1 to 7 days.

The method for preparing a quick-absorbing and flattening dry biological heart valve, wherein the drying refers to dehydration drying in an alcohol solvent.

In the method for preparing a rapidly-absorbable and flattened dry biological heart valve, the dehydration and drying adopts dehydration and drying of a gradient concentration alcohol mixed solvent.

The method for preparing a rapidly absorbable and flattened dry biological heart valve, wherein the alcohol solvents include methanol, ethanol, n-propanol, isopropanol, glycerol, n-butanol, n-pentanol, n- One or more of undecanol, n-dodecanol, 2-propanol, 2-butanol, 2-hexanol, cyclohexanol, and tert-butanol.

The method for preparing a fast-absorbing and flattening dry biological heart valve, wherein an animal pericardium is sequentially immersed in 25% ethanol / 25% isopropanol / 50% glycerol at 25-45 ° C in a range of 20- 30h, 45% ethanol / 45% isopropanol / 10% glycerol for 20-30 hours, complete gradient dehydration and drying.

The method for preparing a fast-absorbing and flattening dry biological heart valve, wherein before the animal pericardium is cross-linked with a glutaraldehyde aqueous solution, the method further includes decellularizing the animal pericardium.

The method for preparing a fast-absorbing and flattening dry biological heart valve, wherein, before the animal pericardium is cross-linked with an aqueous solution of glutaraldehyde, the fresh animal pericardium is heated at 3 to 5 ° C and 80 to 80 ° C. Rinse with distilled water at 120RPM for 1.5 ~ 3h under shaking conditions.

The present invention also provides a dry biological heart valve that can quickly absorb and flatten, and is prepared by using the preparation method described in any one of the above.

Beneficial effect: The present invention provides a dry biological heart valve that can absorb water quickly and flatten, and a preparation method thereof. The dry biological heart valve prepared by using the preparation method of the invention has good performance of rapid water absorption and self-healing. Can quickly absorb water and flatten under pressure test.

detailed description

The present invention provides a dry biological heart valve that can quickly absorb and flatten, and a method for preparing the same. In order to make the purpose, technical solution, and effect of the present invention clearer and clearer, the present invention is described in further detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

A method for preparing a dry biological heart valve capable of quickly absorbing and flattening provided by a preferred embodiment of the present invention, that is, an improved method for quickly absorbing and recovering water from a pre-installed dry biological heart valve, which includes the steps of: The animal pericardium is cross-linked with an aqueous glutaraldehyde solution, then immersed in an extracellular matrix bioenzyme aqueous solution, and finally dried to obtain a dry biological heart valve that can absorb and flatten quickly. Among them, preferably, the drying means dehydration drying in an alcohol solvent.

The present invention focuses on improving the fast water-absorbing and self-healing performance of a dry biological valve. In order to achieve this purpose, the present invention creatively adds biological enzymes to the pericardium of an animal glutaraldehyde cross-linked treatment, so as to make artificial biological valve tissue The effect of rapid water repelling, so as to obtain a pre-installed dry biological heart valve with fast water absorption and self-healing performance.

In general, the preparation method of the present invention includes: 1. cross-linking of a biological valve; 2. water absorption improvement of the biological valve to improve biochemical treatment; 3. dehydration and drying of a gradient concentration of an alcohol solvent. Among them, the water absorption improvement of the biovalve to improve the biochemical treatment and the dehydration and drying of the gradient concentration alcohol solvent are the key steps of the present invention.

Further, the animal pericardium according to the present invention includes, but is not limited to, a pig pericardial valve, a bovine pericardial valve, and the like. In this embodiment, before the animal pericardium is cross-linked with an aqueous solution of glutaraldehyde, the method further includes the steps of decellularizing the animal pericardium and cleaning the animal pericardium. During specific implementation, fresh pericardial tissues of pigs or cattle were collected and stored under low temperature and humid conditions at 4 ° C. The pericardial tissue was washed with distilled water under gentle vibration and fluid pressure under oscillating conditions to remove adhered non-pericardial and non-collagenous tissue. The washing according to the present invention achieves effective decellularization of the pericardial tissue through osmotic shock, and preferably the washing is continued until there is no visible adhesion of non-pericardial or non-collagenous tissue. In specific implementation, preferably, the step of cleaning the animal pericardium cleanly comprises: washing the fresh animal pericardium with distilled water at a temperature of 3 to 5 ° C. and a rotation speed of 80 to 120 RPM for 1.5 to 3 hours. For example, the pericardium of fresh animal (pig or cow) can be immersed in glutaraldehyde solution for cross-linking treatment under the condition of 4 ℃ 100RPM rotating speed and shaking for 2h.

Further, in this embodiment, the volume concentration of the glutaraldehyde aqueous solution is preferably 0.1 to 5%, for example, it can be 0.1%, 1%, 5%, etc., and the pH of the glutaraldehyde aqueous solution is preferably 7 to 8 , For example, 7, 7.4, 8 and so on. Preferably, the animal pericardium is cross-linked with an aqueous solution of glutaraldehyde under shaking conditions at 20 to 45 ° C. for 1 to 7 days. This step is the cross-linking of the biological valve, which will achieve the stable cross-linking of most collagen tissues, improve the structural stability of the entire pericardial tissue, and reduce or even eliminate immunogenicity.

Further, in this embodiment, the extracellular matrix biological enzyme aqueous solution is one or more mixed solutions of collagenase, elastase, hyaluronidase, and chondroitin sulfate. The concentration of the extracellular matrix biological enzyme aqueous solution is preferably 0.1 to 50 U / ml, and may be, for example, 0.1 U / ml, 1 U / ml, 20 U / ml, 50 U / ml, or the like. Preferably, the animal pericardium after the cross-linking treatment is immersed in an extracellular matrix bioenzyme aqueous solution under shaking conditions at 25 to 37 ° C. for 12 to 72 hours. This step is to improve the biochemical treatment of the water absorption of the biological valve, and it is necessary to ensure that the extracellular matrix bioenzyme reaches a near-saturated physical penetration, so as to introduce as much extracellular matrix bioenzyme as possible.

Further, in this embodiment, the dehydration and drying in an alcohol solvent according to the present invention is preferably a dehydration and drying with a gradient concentration alcohol mixed solvent. The alcohol solvents include, but are not limited to, methanol, ethanol, n-propanol, isopropanol, glycerol, n-butanol, n-pentanol, n-undecanol, n-dodecanol, 2-propanol, One or more of 2-butanol, 2-hexanol, cyclohexanol, tert-butanol, and the like are mixed. In specific implementation, preferably, the animal pericardium is immersed in order of 25% ethanol / 25% isopropanol / 50% glycerol at 25 to 45 ° C for 20 to 30 hours (for example, 24 hours), 45% ethanol / 45% Isopropanol / 10% glycerol for 20 to 30 hours (for example, 24 hours), to complete the gradient dehydration and drying of the animal pericardium.

The embodiment of the present invention also provides a dry biological heart valve that can quickly absorb and flatten, and is prepared by using the preparation method described in any one of the above. The dried biological heart valve prepared by using the preparation method of the present invention has good fast water absorption and self-healing performance, and can quickly absorb water and flatten under the simulated folding test.

The invention adopts a unique pre-installed dry biological heart valve to quickly absorb water and self-recovery and improve technology, so that the prepared dry biological heart valve has good water-absorbing and self-healing performance after being implanted into a human body, and can quickly absorb water and flatten under a simulated folding test. . Pre-packed dry biovalves stored without glutaraldehyde solution. It can reduce the residue of glutaraldehyde, reduce the calcification problem and toxicity caused by glutaraldehyde, simplify the preoperative installation of the valve system, and reduce the additional risk of surgery.

The following describes the present invention in detail with specific embodiments:

Example 1

Fresh pig pericardium comes from the local slaughterhouse. Glutaraldehyde is from Chengdu Best Reagent Company. Collagenase is from Sigma-Aldrich. The cleaned pig pericardium was crosslinked in a 1% volume concentration glutaraldehyde aqueous solution (pH 7.4, 25 ° C) for 48h. Wash with distilled water. Then, it was immersed in a 10 U / ml aqueous collagenase solution at 37 ° C for 24 hours. Then immerse in 25% ethanol / 25% isopropanol / 50% glycerol for 24h, 45% ethanol / 45% isopropanol / 10% glycerol for 24h at 25 ° C to realize the gradient dehydration of the biological valve . A pre-loaded dry biological heart valve with good water absorption and self-healing properties is prepared.

After testing, it was found that the dried biological heart valve prepared in this embodiment can quickly flatten out within 3 minutes of immersion in water under simulated bending test conditions (10kg weight folding after two folds) without obvious folding damage. Under the uniaxial tensile test (1 * 5cm sample size), the tensile breaking stress is greater than 15N.

Example 2

Fresh pig pericardium comes from the local slaughterhouse. Glutaraldehyde is from Chengdu Best Reagent Company. Elastase is from Shanghai Aladdin Biochemical Technology Co., Ltd. The cleaned pig pericardium was crosslinked in a 1% volume concentration glutaraldehyde aqueous solution (pH 7.4, 25 ° C) for 48h. Wash with distilled water. Then immerse it in a 5U / ml elastase aqueous solution at 37 ° C for 24h. Then immerse in 25% ethanol / 25% isopropanol / 50% glycerol for 24h, 45% ethanol / 45% isopropanol / 10% glycerol for 24h at 25 ° C to realize the gradient dehydration of the biological valve . A pre-loaded dry biological heart valve with good water absorption and self-healing properties is prepared.

After testing, it was found that the dried biological heart valve prepared in this embodiment can quickly flatten out within 3 minutes of immersion in water under simulated bending test conditions (10kg weight folding after two folds) without obvious folding damage. Under the uniaxial tensile test (1 * 5cm sample size), the tensile breaking stress is greater than 15N.

Example 3

Fresh pig pericardium comes from the local slaughterhouse. Glutaraldehyde is from Chengdu Best Reagent Company. Elastase and hyaluronidase are from Shanghai Aladdin Biochemical Technology Co., Ltd. The cleaned pig pericardium was cross-linked in a 1% volume glutaraldehyde aqueous solution (pH 7.4, 25 ° C) for 48 hours. Wash with distilled water. Then immerse it in a mixed solution of 1U / ml elastase and 5U / ml hyaluronidase at 37 ° C for 24h. Then immerse in 25% ethanol / 25% isopropanol / 50% glycerol for 24h, 45% ethanol / 45% isopropanol / 10% glycerol for 24h at 25 ° C to realize the gradient dehydration of the biological valve . A pre-loaded dry biological heart valve with good water absorption and self-healing properties is prepared.

After testing, it was found that the dried biological heart valve prepared in this embodiment can quickly flatten out within 3 minutes of immersion in water under simulated bending test conditions (10kg weight folding after two folds) without obvious folding damage. Under the uniaxial tensile test (1 * 5cm sample size), the tensile breaking stress is greater than 15N.

Example 4

Fresh pig pericardium comes from the local slaughterhouse. Glutaraldehyde is from Chengdu Best Reagent Company. Elastase and hyaluronidase are from Shanghai Aladdin Biochemical Technology Co., Ltd. The cleaned pig pericardium was crosslinked in a 1% volume concentration glutaraldehyde aqueous solution (pH 7.4, 25 ° C) for 48h. Wash with distilled water. Then, it was immersed in a 50U / ml chondroitin sulfate mixed aqueous solution at 37 ° C for 24 hours. Then immerse in 25% ethanol / 25% isopropanol / 50% glycerol for 24h, 45% ethanol / 45% isopropanol / 10% glycerol for 24h at 25 ° C to realize the gradient dehydration of the biological valve . A pre-loaded dry biological heart valve with good water absorption and self-healing properties is prepared.

After testing, it was found that the dried biological heart valve prepared in this embodiment can quickly flatten out within 3 minutes of immersion in water under simulated bending test conditions (10kg weight folding after two folds) without obvious folding damage. Under the uniaxial tensile test (1 * 5cm sample size), the tensile breaking stress is greater than 15N.

Example 5

Fresh pig pericardium comes from the local slaughterhouse. Glutaraldehyde is from Chengdu Best Reagent Company. Elastase and hyaluronidase are from Shanghai Aladdin Biochemical Technology Co., Ltd. The cleaned pig pericardium was crosslinked in a 1% volume concentration glutaraldehyde aqueous solution (pH 7.4, 25 ° C) for 48h. Wash with distilled water. Then immerse in a 50U / ml collagenase and chondroitin sulfate aqueous solution at 37 ° C for 24h. Then immerse in 25% ethanol / 25% isopropanol / 50% glycerol for 24h, 45% ethanol / 45% isopropanol / 10% glycerol for 24h at 25 ° C to realize the gradient dehydration of the biological valve . A pre-loaded dry biological heart valve with good water absorption and self-healing properties is prepared.

After testing, it was found that the dried biological heart valve prepared in this embodiment can quickly flatten out within 3 minutes of immersion in water under simulated bending test conditions (10kg weight folding after two folds) without obvious folding damage. Under the uniaxial tensile test (1 * 5cm sample size), the tensile breaking stress is greater than 15N.

Example 6

Fresh pig pericardium comes from the local slaughterhouse. Glutaraldehyde is from Chengdu Best Reagent Company. Elastase and hyaluronidase are from Shanghai Aladdin Biochemical Technology Co., Ltd. The cleaned pig pericardium was crosslinked in a 1% volume concentration glutaraldehyde aqueous solution (pH 7.4, 25 ° C) for 48h. Wash with distilled water. Then immerse in a mixed aqueous solution of collagenase and chondroitin sulfate at 0.1 U / ml for 24 hours at 37 ° C. Then immerse in 25% ethanol / 25% isopropanol / 50% glycerol for 24h, 45% ethanol / 45% isopropanol / 10% glycerol for 24h at 25 ° C to realize the gradient dehydration of the biological valve . A pre-loaded dry biological heart valve with good water absorption and self-healing properties is prepared.

After testing, it was found that the dried biological heart valve prepared in this embodiment can quickly flatten out within 3 minutes of immersion in water under simulated bending test conditions (10kg weight folding after two folds) without obvious folding damage. Under the uniaxial tensile test (1 * 5cm sample size), the tensile breaking stress is greater than 15N.

In summary, the present invention provides a dry biological heart valve that can absorb water quickly and flatten, and a preparation method thereof. The dry biological heart valve prepared by using the preparation method of the present invention has good rapid water absorption and self-healing properties. It can quickly absorb water and flatten under the simulated folding test. The pre-installed dry biological valve stored without glutaraldehyde solution can reduce the residual glutaraldehyde, reduce the calcification problem and toxicity caused by glutaraldehyde, simplify the preoperative installation of the valve system, and reduce the additional risk of surgery.

It should be understood that the application of the present invention is not limited to the above-mentioned examples. For those of ordinary skill in the art, improvements or changes can be made according to the above description. All these improvements and changes should fall into the protection scope of the appended claims of the present invention.

Claims (13)

1. A method for preparing a fast-absorbing and flattening dry biological heart valve, which is characterized by comprising the steps:

   The animal pericardium was cross-linked with an glutaraldehyde aqueous solution, then immersed in an extracellular matrix bioenzyme aqueous solution, and finally dried to obtain a dry biological heart valve that can quickly absorb and flatten.
2. The method of claim 1, wherein the extracellular matrix biological enzyme aqueous solution is collagenase, elastase, hyaluronidase, and chondroitin sulfate. One or more mixed solutions.
3. The method of claim 1, wherein the concentration of the aqueous extracellular matrix biological enzyme solution is 0.1-50 U / ml.
4. The method for preparing a rapidly absorbable and flattened dry biological heart valve according to claim 3, characterized in that the animal pericardium after the cross-linking treatment is shaken in an extracellular matrix biological enzyme aqueous solution at 25 to 37 ° C Soak for 12 to 72 hours under conditions.
5. The method of claim 1, wherein the volume concentration of the glutaraldehyde aqueous solution is 0.1 to 5%, and the pH is 7 to 8.
6. The method for preparing a rapidly absorbable and flattened dry biological heart valve according to claim 5, characterized in that the animal pericardium is cross-linked with an aqueous solution of glutaraldehyde under shaking conditions of 20 to 45 ° C for 1 to 7 day.
7. The method of claim 1, wherein the drying refers to dehydration and drying in an alcohol solvent.
8. The method for preparing a rapidly-absorbable and flattened dry biological heart valve according to claim 7, wherein the dehydration and drying adopts dehydration and drying of a gradient concentration alcohol mixed solvent.
9. The method for preparing a rapidly absorbable and flattened dry biological heart valve according to claim 7, wherein the alcohol solvents include methanol, ethanol, n-propanol, isopropanol, glycerol, and n-butanol , One or more of n-pentanol, n-undecanol, n-dodecanol, 2-propanol, 2-butanol, 2-hexanol, cyclohexanol, tert-butanol.
10. The method for preparing a rapidly absorbable and flattened dry biological heart valve according to claim 8 or 9, wherein the animal pericardium is sequentially immersed in 25% ethanol / 25% isopropanol at 25-45 ° C / 50% glycerol for 20-30 hours, 45% ethanol / 45% isopropanol / 10% glycerol for 20-30 hours, and the gradient dehydration and drying are completed.
11. The method for preparing a rapidly absorbable and flattened dry biological heart valve according to claim 1, characterized in that before the animal pericardium is crosslinked with a glutaraldehyde aqueous solution, the method further comprises removing the animal pericardium. Cell processing.
12. The method for preparing a fast-absorbing and flattening dry biological heart valve according to claim 1, characterized in that, before the animal pericardium is cross-linked with a glutaraldehyde aqueous solution, the fresh animal pericardium is dried at 3 Rinse with distilled water for 1.5 to 3 hours under oscillating conditions at ~ 5 ° C and 80 to 120 RPM.
13. A dry biological heart valve capable of quickly absorbing and flattening, which is prepared by using the preparation method according to any one of claims 1 to 12.