Dry bioprosthetic valve and preparation method thereof

Abstract

The invention relates to the field of medical materials and medical instruments, and discloses a dry bioprosthetic valve and a preparation method thereof.The preparation method comprises the following steps that S1, pericardium and a cross-linking agent solution are mixed for a cross-linking reaction; s2, carrying out first dehydration treatment on the crosslinked pericardium by adopting a first dehydration solution with gradually increased concentration gradient under an ultrasonic condition; and S3, carrying out second dehydration treatment on the pericardium subjected to the first dehydration treatment by adopting a second dehydration solvent, so as to obtain the dried bioprosthetic valve. According to the preparation method provided by the invention, ethanol, isopropanol and water in a specific proportion are adopted to prepare a dehydration solution with gradient concentration, and two-step dehydration drying is carried out in combination with the physical effect of an ultrasonic technology and glycerol, so that rapid rehydration of the bioprosthetic valve is realized, and the original shape and mechanical property of the bioprosthetic valve can be better recovered after rehydration; and the immediate hemodynamic stability of the valve is improved.

Description

Technical Field

[0001] This invention relates to the field of medical materials and medical devices, specifically to a dried biological valve and its preparation method. Background Technology

[0002] Artificial heart valves are implantable organs that replace natural heart valves and possess their functions. When heart valve disease is severe and valve function cannot be restored or improved through valve separation or repair surgery, artificial heart valve replacement surgery is necessary. In the preservation of bioprosthetic valves, dehydration preservation technology achieves room-temperature storage and transportation of bioprosthetic valves through dehydration. However, current dehydration valve technology still faces the following pressing problems.

[0003] First, there is structural damage. Traditional freeze-drying or vacuum drying can cause the collagen fibers of bioprosthetic valves to collapse. This structural collapse destroys the original fine fiber arrangement and tissue structure of the valve leaflets, reducing their flexibility and fatigue resistance. This makes the valves more susceptible to fatigue damage, breakage, or deformation when facing the complex hemodynamic stresses that the heart valves endure over a long period of time. Consequently, it seriously affects the lifespan and reliability of the valves and fails to meet the clinical needs for long-term stable valve operation.

[0004] Secondly, poor rehydration performance is a significant drawback. Rehydration performance refers to the time required for a bioprosthetic valve to recover its mechanical properties after implantation. Insufficient rehydration performance necessitates a longer preoperative waiting period to ensure the valve performs adequately, increasing both the overall surgical time and complexity. Furthermore, poor valve recovery can negatively impact immediate postoperative hemodynamic stability, posing potential surgical risks and uncertainties to the patient's recovery. Therefore, developing a bioprosthetic valve capable of rapid rehydration and exhibiting stable mechanical and rehydration performance has become a critical technological bottleneck that urgently needs to be overcome in this field. Summary of the Invention

[0005] This invention provides a dried bioprosthetic valve and its preparation method to solve the problems of long preoperative preparation time and hemodynamic instability of valve materials caused by poor mechanical properties and insufficient rehydration performance of dried bioprosthetic valve materials in the prior art.

[0006] In a first aspect, the present invention provides a method for preparing a dried biological valve, comprising the following steps: Step S1: Mix the pericardium with the cross-linking agent solution to carry out the cross-linking reaction; Step S2: Under ultrasound conditions, the cross-linked pericardium is subjected to the first dehydration treatment using a first dehydration solution with an increasing concentration gradient. Step S3: The pericardium after the first dehydration treatment is subjected to a second dehydration treatment using a second dehydration solvent to obtain a dried biological valve.

[0007] The first dehydration solution includes a first dehydration solvent, which includes isopropanol and ethanol in a volume ratio of 1:1-4; the volume fraction of the first dehydration solvent in the first dehydration solution is 30%-100%.

[0008] The second dehydrating solvent is glycerol or an aqueous solution of glycerol with a volume percentage of not less than 50%.

[0009] Furthermore, in step S2, the first dehydration treatment includes immersing the cross-linked pericardium in a first dehydration solution with a volume percentage of 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% for each dehydration solvent, with each immersion time being 1-12 minutes; and / or, the temperature of the ultrasound is 20-40°C and the frequency is 30-50 kHz.

[0010] Furthermore, in step S2, the first dehydration treatment involves sequentially immersing the cross-linked pericardium in a first dehydration solution containing 30% by volume of the first dehydrating solvent for 8-12 minutes, 40% by volume of the first dehydrating solvent for 8-12 minutes, 50% by volume of the first dehydrating solvent for 8-12 minutes, 60% by volume of the first dehydrating solvent for 3-7 minutes, 70% by volume of the first dehydrating solvent for 3-7 minutes, 80% by volume of the first dehydrating solvent for 2-4 minutes, 90% by volume of the first dehydrating solvent for 2-4 minutes, and 100% by volume of the first dehydrating solvent for 1-3 minutes.

[0011] Furthermore, in step S1, a decellularization step of the pericardium is included before the pericardium is mixed with the crosslinking agent.

[0012] In some optional embodiments, the decellularizing agent is selected from at least one of Triton X-100, Tween-80, sodium dodecyl sulfate, sodium deoxycholate, sodium lauryl ether sulfate, and trypsin. In some optional embodiments, the decellularization reagent is a phosphate solution containing Triton X-100 and Tween-80; the mass concentration of Triton X-100 in the phosphate solution is 0.4%-0.6%, preferably 0.5%; the mass concentration of Tween-80 in the phosphate solution is 0.4%-0.6%, preferably 0.5%. Furthermore, in step S1, the crosslinking agent is selected from at least one of carbodiimide, N-hydroxysuccinimide, and glutaraldehyde; In some optional embodiments, the solvent for the carbodiimide and N-hydroxysuccinimide is morpholine ethanesulfonic acid buffer, and the solvent for glutaraldehyde is phosphate buffer. In some optional embodiments, the crosslinking agent solution is a morpholine ethanesulfonic acid buffer containing carbodiimide and N-hydroxysuccinimide; the concentration of carbodiimide in the morpholine ethanesulfonic acid buffer is 30-50 mmol / L, preferably 40 mmol / L; and the concentration of N-hydroxysuccinimide in the morpholine ethanesulfonic acid buffer is 10-30 mmol / L, preferably 20 mmol / L.

[0013] Furthermore, the decellularization time is 3-5 hours, and the temperature is 10-25℃; Furthermore, the decellularization is performed under oscillating conditions; In some alternative implementations, the rotational speed of the oscillation is 70-90 rpm.

[0014] Furthermore, in step S1, the crosslinking reaction takes 3-5 hours and is carried out at a temperature of 10-25°C.

[0015] Furthermore, in step S3, the second dehydration treatment is performed 2-3 times, each time for 8-12 minutes, at a temperature of 10-25°C.

[0016] Furthermore, step S3 also includes ventilating and drying the pericardium that has undergone the second dehydration treatment; optionally, the ventilating and drying time is 24-50 hours.

[0017] Secondly, the present invention also provides a dried biological valve, which is prepared by the above-described preparation method.

[0018] The technical solution of this invention has the following advantages: 1. The present invention provides a method for preparing a dried bioprosthetic valve, comprising the following steps: S1 step: mixing pericardium with a cross-linking agent solution to carry out a cross-linking reaction; S2 step: under ultrasound conditions, performing a first dehydration treatment on the cross-linked pericardium using a first dehydration solution with an increasing concentration gradient; S3 step: performing a second dehydration treatment on the pericardium after the first dehydration treatment using a second dehydration solvent to obtain a dried bioprosthetic valve; the first dehydration solution comprises a first dehydration solvent, which comprises isopropanol and ethanol in a volume ratio of 1:1-4; the volume fraction of the first dehydration solvent in the first dehydration solution is 30%-100%; the second dehydration solvent is glycerol or a glycerol aqueous solution with a volume percentage of not less than 50%. The preparation method provided by this invention uses a specific ratio of ethanol and isopropanol with water to prepare a dehydration solution in step S2. This solution can replace water in biological tissues and gradually remove free water by forming hydrogen bonds with water molecules. After dehydration with alcohol reagents, the stability of the cross-linked valve tissue can be enhanced, residual phospholipids can be effectively removed, the tendency of biological valves to calcify can be reduced, and the anti-calcification performance can be improved. Secondly, the concentration of ethanol and isopropanol is gradually increased during the dehydration process. The gradual concentration adjustment achieves gentle dehydration. Combined with the physical action of ultrasound technology, this not only avoids shrinkage or deformation caused by rapid changes in osmotic pressure due to rapid dehydration of biological tissues, but also improves dehydration efficiency and shortens the processing cycle. Furthermore, in step S3, the polyhydroxy structure of glycerol forms hydrogen bonds with water molecules and macromolecules such as collagen in biological tissues, which can also replace free water in biological tissues. The hydroxyl groups of glycerol form hydrogen bonds with groups on pericardial collagen to maintain the hydration state of the material, support the cavities between fibers, and ensure the microstructure of the material in an anhydrous environment, thereby maintaining its mechanical properties. In addition, using glycerin as the final dehydration and drying solution can shorten the rehydration time of the material, simplify the preoperative preparation steps and shorten the preparation time. After rehydration, it can better restore its original shape and mechanical properties, and improve the immediate hemodynamic stability of the valve.

[0019] 2. The present invention provides a dried bioprosthetic valve, prepared by the above-described method. This material is stable and can be stored for a long time, improving storage convenience. Furthermore, the dry preservation method effectively avoids aldehyde residue caused by traditional glutaraldehyde solution preservation, reducing the potential risk of triggering immune responses. It also improves anti-calcification properties, thus delaying the calcification process after valve implantation and extending its service life. In addition, the material can better restore its original shape and mechanical properties after rehydration, ensuring a stable hemodynamic state immediately after valve implantation, providing a reliable guarantee for surgical outcomes. Therefore, the dried bioprosthetic valve provided by the present invention has the advantages of rapid rehydration, stable mechanical properties, long-term storage, and superior durability. Detailed Implementation

[0020] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0021] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0022] Example 1 This embodiment provides a dried biological valve and its preparation method, the specific steps of which are as follows: (1) At 25°C, fresh bovine pericardium was soaked in 0.5% Triton X-100 and 0.5% Tween-80 phosphate (PBS) buffer (the decellularization reagent was prepared using PBS buffer at pH 7.4 as the solvent) and placed on a shaker at 80 rpm for 4 h. After rinsing with 0.9% sodium chloride solution 3 times for 15 min each time, the bovine pericardium was soaked in morpholine ethanesulfonic acid (MES) buffer containing 40 mmol / L EDC and 20 mmol / L NHS (the cross-linking agent solution was prepared using MES buffer at pH 5.5 as the solvent) for cross-linking for 4 h at 25°C. After the cross-linking was completed, it was rinsed with 0.9% sodium chloride solution 3 times for 15 min each time. (2) Prepare a mixed solvent with a volume ratio of isopropanol and anhydrous ethanol of 1:2, and then prepare a dehydration solution with water at a volume fraction of 30%-100%; under ultrasonic conditions, immerse the material obtained in the previous step in the dehydration solution with a volume percentage of 30% for 10 min, 40% for 10 min, 50% for 10 min, 60% for 5 min, 70% for 5 min, 80% for 3 min, 90% for 3 min, and 100% for 2 min, respectively. The ultrasonic temperature is 40℃ and the frequency is 40KHz. (3) Soak the material obtained in the previous step in pure glycerin twice, for 10 minutes each time, at a temperature of 25°C. After the soaking, wipe the surface of the glycerin as dry as possible and place it in a fume hood for ventilation and drying for 48 hours.

[0023] Example 2 This embodiment provides a dried biological valve and its preparation method, the specific steps of which are as follows: (1) At 18°C, fresh bovine pericardium was soaked in 0.5% Triton X-100 and 0.5% Tween-80 phosphate (PBS) buffer (the decellularization reagent was prepared using PBS buffer at pH 7.4 as the solvent) and placed on a shaker at 90 rpm for 5 h. After rinsing with 0.9% sodium chloride solution 3 times for 15 min each time, the bovine pericardium was soaked in morpholine ethanesulfonic acid (MES) buffer containing 40 mmol / L EDC and 20 mmol / L NHS (the cross-linking agent solution was prepared using MES buffer at pH 5.5 as the solvent) for cross-linking for 3 h at 18°C. After the cross-linking was completed, it was rinsed with 0.9% sodium chloride solution 3 times for 15 min each time. (2) Prepare a mixed solvent with a volume ratio of isopropanol and anhydrous ethanol of 1:2. Then, prepare a dehydration solution with water at a volume fraction of 30%-100%. Under ultrasonic conditions, immerse the material obtained in the previous step in the dehydration solution with a volume percentage of 30% for 12 min, 40% for 12 min, 50% for 12 min, 60% for 7 min, 70% for 7 min, 80% for 4 min, 90% for 4 min, and 100% for 3 min. The ultrasonic temperature is 20℃ and the frequency is 50KHz. (3) Soak the material obtained in the previous step in pure glycerin twice, for 12 minutes each time, at a temperature of 18°C. After the soaking, wipe the surface of the glycerin as dry as possible and place it in a fume hood for ventilation and drying for 50 hours.

[0024] Example 3 This embodiment provides a dried biological valve and its preparation method, the specific steps of which are as follows: (1) At 10°C, fresh bovine pericardium was soaked in 0.5% Triton X-100 and 0.5% Tween-80 phosphate (PBS) buffer (the decellularization reagent was prepared using PBS buffer at pH 7.4 as the solvent) and placed on a shaker at 70 rpm for 3 h. After rinsing with 0.9% sodium chloride solution 3 times for 15 min each time, the bovine pericardium was soaked in morpholine ethanesulfonic acid (MES) buffer containing 40 mmol / L EDC and 20 mmol / L NHS (the cross-linking agent solution was prepared using MES buffer at pH 5.5 as the solvent) for cross-linking for 5 h at 10°C. After the cross-linking was completed, it was rinsed with 0.9% sodium chloride solution 3 times for 15 min each time. (2) Prepare a mixed solvent with a volume ratio of isopropanol and anhydrous ethanol of 1:3. Then, prepare a dehydration solution with water at a volume fraction of 30%-100%. Under ultrasonic conditions, immerse the material obtained in the previous step in dehydration solutions with volume percentages of 30% for 8 min, 40% for 8 min, 50% for 8 min, 60% for 3 min, 70% for 3 min, 80% for 2 min, 90% for 2 min, and 100% for 1 min. The ultrasonic temperature is 30℃ and the frequency is 30KHz. (3) Soak the material obtained in the previous step in pure glycerin three times, each time for 8 minutes at a temperature of 10°C; after the soaking, wipe the surface of the glycerin as dry as possible and place it in a fume hood for ventilation and drying for 24 hours.

[0025] Example 4 The dried biological valve and its preparation method provided in this embodiment are basically the same as those in Example 1, except that the pure glycerol in step (3) is replaced with a glycerol aqueous solution with a volume fraction of 70%.

[0026] Comparative Example 1 The dry bioprosthetic valve and its preparation method provided in this comparative example are basically the same as those in Example 1, except that isopropanol in step (2) is replaced with methanol.

[0027] Comparative Example 2 The dry bioprosthetic valve and its preparation method provided in this comparative example are basically the same as those in Example 1, except that the mixed solution of isopropanol and anhydrous ethanol in step (2) is replaced with anhydrous ethanol alone.

[0028] Comparative Example 3 The dry biological valve and its preparation method provided in this comparative example are basically the same as those in Example 1, except that step (3) is omitted.

[0029] Comparative Example 4 The dry bioprosthetic valve and its preparation method provided in this comparative example are basically the same as those in Example 1, except that the ultrasonic conditions in step (2) are omitted, that is, the dehydration treatment in step (2) is carried out in a static state.

[0030] Experimental Example 1 The dried bioprosthetic valves prepared in Examples 1-4 and Comparative Examples 1-4 were subjected to rehydration treatment. Specifically, the dried bioprosthetic valves were immersed in 500 mL of water at 25°C for 2 minutes. Then, the mechanical properties of the rehydrated valves were tested. The specific method is as follows: Cut the sample into 10×50mm strips, set the parameters of the tensile testing machine, set the speed of the tensile testing machine to 100mm / min, the clamping distance to 25mm, clamp the sample with the clamp, start the tensile testing machine, and record the tensile strength and elongation at break data when the sample breaks. The results are shown in Table 1.

[0031] Table 1 Mechanical property test results

[0032] As shown in Table 1, the dried bioprosthetic valves prepared in Examples 1-4 are significantly better than those in Comparative Examples 1-4 in terms of tensile strength and / or elongation at break, indicating that the bioprosthetic valves prepared by the method of the present invention have more stable mechanical properties.

[0033] Experiment Example 2 The dehydration rate and rehydration water content of the dried bioprosthetic valves prepared in Examples 1-4 and Comparative Examples 1-4 were tested respectively, and the specific methods are as follows: Method for detecting dehydration rate: Take the material obtained after step (1) and cut it into slices of 20mm×20mm, absorb the surface moisture, weigh it and record it as W_wet; take the final valve product and cut it into slices of 20mm×20mm, absorb the surface moisture, weigh it and record it as W_dehydrated; dry the final valve in an oven at 105℃ for 5h, take it out and weigh it and record it as W_dry; the formula for calculating the dehydration rate is: (W_wet - W_dehydrated) / (W_wet - W_dry) × 100%, and the results are shown in Table 2.

[0034] Method for detecting the water content after 30 seconds of rehydration: The final obtained valve was cut into 20mm×20mm slices, placed in 500mL of water, and after rehydration for 30 seconds, it was taken out, the surface moisture was absorbed, and the weight was recorded as Wf; the rehydrated valve was dried in an oven at 105℃ for 5 hours, and the weight was recorded as Wd; the formula for calculating the water content after 30 seconds of rehydration is: water content after 30 seconds of rehydration = (Wf-Wd) / Wd×100%, and the results are shown in Table 2.

[0035] The final moisture content was determined as follows: The obtained valve was cut into 20mm×20mm slices, placed in 500mL of water, fully rehydrated, removed and dried, and weighed. The valve was then placed in 500mL of water again for 10 minutes, removed and dried, and weighed again until the weight was equal to the previous weight. This weight was recorded as Wf. The rehydrated valve was then dried in an oven at 105℃ for 5 hours, and weighed again, which was recorded as Wd. The final moisture content was calculated using the formula: Final moisture content = (Wf - Wd) / Wd × 100%. The results are shown in Table 2.

[0036] Table 2 Results of Dehydration Rate and Rehydrated Moisture Content Tests

[0037] As shown in Table 2, the dried bioprosthetic valves prepared in Examples 1-4 are significantly better than those in Comparative Examples 1-4 in terms of dehydration rate and water content after rehydration. The method for preparing the dried bioprosthetic valve provided by this invention maintains the final dehydration rate of the valve at 50%-60%, which can provide better rehydration while maintaining good mechanical and hemodynamic properties, and has the advantage of rapid rehydration, thereby shortening the preoperative preparation time and improving the convenience of clinical use.

[0038] Experimental Example 3 Hemodynamic tests were performed on the dried bioprosthetic valves prepared in Examples 1-2 and Comparative Example 1 after rehydration, respectively. The specific methods are as follows: The prepared valve was immersed in 500 mL of water at 25 °C for 2 min to rehydrate, and then tested using a pulsating flow tester. During the test, the valve movement should be fully open and fully closed. The transvalvular pressure difference, total regurgitation percentage, and opening area of ​​the valve were recorded. The results are shown in Table 3.

[0039] Table 3. Hemodynamic monitoring results after rehydration

[0040] As shown in Table 3, the hemodynamic parameters of the dried bioprosthetic valves prepared in Examples 1 and 2 are all better than those in Comparative Example 1, indicating that the bioprosthetic valves prepared by the method of the present invention have a stable hemodynamic state.

[0041] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a dried biological valve, characterized in that, Includes the following steps: Step S1: Mix the pericardium with the cross-linking agent solution to carry out the cross-linking reaction; Step S2: Under ultrasound conditions, the cross-linked pericardium is subjected to the first dehydration treatment using a first dehydration solution with an increasing concentration gradient. Step S3: The pericardium after the first dehydration treatment is subjected to a second dehydration treatment using a second dehydrating solvent to obtain a dried bioprosthetic valve; The first dehydration solution comprises a first dehydration solvent, which includes isopropanol and ethanol in a volume ratio of 1:1-4; the volume fraction of the first dehydration solvent in the first dehydration solution is 30%-100%. The second dehydrating solvent is glycerol or an aqueous solution of glycerol with a volume percentage of not less than 50%.

2. The method for preparing a dried biological valve according to claim 1, characterized in that, In step S2, the first dehydration treatment includes immersing the cross-linked pericardium in a first dehydration solution with a volume percentage of 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% for each dehydration solvent, with each immersion time being 1-12 minutes; and / or, the temperature of the ultrasound is 20-40°C and the frequency is 30-50 kHz.

3. The method for preparing a dried biological valve according to claim 2, characterized in that, In step S2, the first dehydration treatment involves sequentially immersing the cross-linked pericardium in a first dehydration solution containing 30% by volume of the first dehydrating solvent for 8-12 minutes, 40% by volume of the first dehydrating solvent for 8-12 minutes, 50% by volume of the first dehydrating solvent for 8-12 minutes, 60% by volume of the first dehydrating solvent for 3-7 minutes, 70% by volume of the first dehydrating solvent for 3-7 minutes, 80% by volume of the first dehydrating solvent for 2-4 minutes, 90% by volume of the first dehydrating solvent for 2-4 minutes, and 100% by volume of the first dehydrating solvent for 1-3 minutes.

4. The method for preparing a dried biological valve according to claim 1, characterized in that, In step S1, a decellularization step of the pericardium is also included before the pericardium is mixed with the crosslinking agent; Optionally, the decellularizing agent is selected from at least one of Triton X-100, Tween-80, sodium dodecyl sulfate, sodium deoxycholate, sodium lauryl ether sulfate, and trypsin. Alternatively, the decellularization reagent is a phosphate solution containing Triton X-100 and Tween-80; the mass concentration of Triton X-100 in the phosphate solution is 0.4%-0.6%, preferably 0.5%; the mass concentration of Tween-80 in the phosphate solution is 0.4%-0.6%, preferably 0.5%.

5. The method for preparing a dried biological valve according to claim 1, characterized in that, In step S1, the crosslinking agent is selected from at least one of carbodiimide, N-hydroxysuccinimide, and glutaraldehyde; Optionally, the solvent for the carbodiimide and N-hydroxysuccinimide is morpholine ethanesulfonic acid buffer, and the solvent for glutaraldehyde is phosphate buffer. Alternatively, the crosslinking agent solution is a morpholine ethanesulfonic acid buffer containing carbodiimide and N-hydroxysuccinimide; the concentration of carbodiimide in the morpholine ethanesulfonic acid buffer is 30-50 mmol / L, preferably 40 mmol / L; and the concentration of N-hydroxysuccinimide in the morpholine ethanesulfonic acid buffer is 10-30 mmol / L, preferably 20 mmol / L.

6. The method for preparing a dried biological valve according to claim 4, characterized in that, The decellularization time is 3-5 hours, and the temperature is 10-25℃; And / or, the decellularization is performed under oscillating conditions; Optionally, the rotational speed of the oscillation is 70-90 rpm.

7. The method for preparing a dried biological valve according to claim 1, characterized in that, In step S1, the crosslinking reaction takes 3-5 hours and is carried out at a temperature of 10-25°C.

8. The method for preparing a dried biological valve according to claim 1, characterized in that, In step S3, the second dehydration treatment is performed 2-3 times, each time for 8-12 minutes, at a temperature of 10-25°C.

9. The method for preparing a dried biological valve according to any one of claims 1-8, characterized in that, Step S3 further includes ventilating and drying the pericardium that has undergone the second dehydration treatment; optionally, the ventilating and drying time is 24-50 hours.

10. A dried biological valve, characterized in that, It is prepared by the preparation method according to any one of claims 1-9.

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