A method for preparing a biological vein containing venous valves

Abstract

This invention discloses a method for preparing a biogenic vein containing venous valves, belonging to the field of biomedical functional technology. The method includes the following steps: cutting a porcine or bovine jugular vein containing intact valves, cleaning, disinfecting, and rinsing it for later use; sequentially performing cell lysis, cell digestion, and nucleic acid digestion; then immersing it in a 1 mol / L hydroxylamine sulfate solution for heparin covalent cross-linking; and finally immersing it in a treatment solution to obtain a biogenic vein containing venous valves. The treatment solution is a PBS solution containing 4 wt% hydroxyethyl starch and 0.1 wt% polyhexamethylene biguanide. The method for preparing a biogenic vein containing venous valves provided by this invention significantly solves the technical problems of insufficient dehydration and antibacterial activity mentioned in the background art by introducing a treatment solution containing 4 wt% hydroxyethyl starch and 0.1 wt% polyhexamethylene biguanide during the preparation process. Simultaneously, the valve function of the biogenic vein is significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering, specifically relating to a method for preparing a bio-derived vein containing venous valves, and more particularly to a method for preparing a bio-derived venous valve with improved anti-dehydration, antibacterial, and anticoagulant properties by optimizing the preparation steps. Background Technology

[0002] Varicose veins of the lower extremities are a common vascular disease caused by venous valve insufficiency, clinically manifested as venous blood reflux, vasodilation, and tissue malnutrition. Traditional treatments such as high ligation and vein stripping can relieve symptoms, but they cannot repair valve function and have high recurrence rates and are highly invasive. Bio-derived venous valves, due to their biocompatibility and hemodynamic properties similar to human tissue, have become a potential radical treatment.

[0003] In existing technologies, bioprosthetic valves prepared through decellularization combined with heparin cross-linking have shown certain antithrombotic and anticalcification properties, but their long-term preservation and immediate functional maintenance after implantation still face challenges. Current methods for preserving bio-derived venous valves mostly use alcohol or glutaraldehyde solutions, which, while providing short-term antibacterial effects, have significant drawbacks:

[0004] (1) Dehydration problem: Soaking in alcohol can easily lead to dehydration and shrinkage of collagen fibers, which can damage the microstructure of the valve and affect its mechanical properties after implantation.

[0005] (2) Limitations of antibacterial activity: Traditional antibacterial agents (such as benzalkonium chloride) may lead to secondary infection risk due to concentration decay during long-term storage;

[0006] The aforementioned problems result in a short valve shelf life and a high risk of thrombosis or infection after implantation, severely limiting its clinical application. Therefore, there is an urgent need to improve the preparation method to simultaneously address the two major issues of preventing dehydration and providing long-lasting antibacterial effects while simplifying the procedure. Summary of the Invention

[0007] This invention improves upon existing patent CN105770991B, providing a method for preparing a bio-derived vein containing venous valves. By optimizing the preparation steps, the method significantly enhances the dehydration resistance and antibacterial properties of the finished product. The objective of this invention can be achieved through the following technical solutions:

[0008] A method for preparing a biological vein containing venous valves includes the following steps:

[0009] Step a. Cut a pig or bovine jugular vein with a diameter of 0.5-1.5 cm and a length of 3-5 cm containing an intact valve, clean and disinfect it, and then rinse it with PBS solution for later use;

[0010] Step b. Perform cell lysis, cell digestion, and nucleic acid digestion sequentially;

[0011] Step c. Immerse in 1 mol / L hydroxylamine sulfate solution at room temperature for 12 h, rinse with distilled water, crosslink in heparin-EDC solution at 37 °C for 48–72 h, and then rinse with PBS solution.

[0012] Step d. Immerse in the treatment solution again and treat at room temperature for 5-60 minutes. Then rinse three times with PBS solution to obtain a biological vein containing venous valves.

[0013] The treatment solution is a PBS solution containing 4 wt% hydroxyethyl starch and 0.1 wt% polyhexamethylene biguanide.

[0014] Furthermore, the disinfection in step a involves soaking in a 0.1% benzalkonium chloride solution for 30 minutes.

[0015] Further, in step b, cell lysis involves treatment with PBS solution containing 0.5% Triton X-100 for 24–48 hours.

[0016] Further, in step b, cell digestion involves treating the cells at 37°C for 30–120 min with a PBS solution containing 0.025% trypsin and 0.02% EDTA.

[0017] Further, in step b, the nucleic acid digestion involves treating the sample with a PBS solution containing 30 U / mL DNase I and 0.3 mg / mL RNase A at 37°C for 24–48 hours.

[0018] Further, the heparin-EDC solution in step c is prepared by EDC, sodium heparin, and 0.05 mol / L HCl in a ratio of 1.67 g: 0.835 g: 200 mL; the pH of the heparin-EDC solution is 1.5.

[0019] Furthermore, the processing steps b to d are all carried out on a shaker at 70 to 100 rpm.

[0020] Furthermore, the bio-derived venous seal containing venous valves in step d is preserved in alcohol with a volume fraction of 60% to 80%.

[0021] The beneficial effects of this invention are:

[0022] The method for preparing bio-derived veins with venous valves provided by this invention solves the technical problems of insufficient dehydration and antibacterial activity in the prior art by introducing a treatment solution containing 4 wt% hydroxyethyl starch (HES) and 0.1 wt% polyhexamethylene biguanide (PHMB) in the preparation step. Furthermore, tests have shown that the valve function of the bio-derived vein is significantly improved. The analysis based on the test results is as follows:

[0023] (1) Synergistic antibacterial effect of HES and PHMB:

[0024] A comparison of Comparative Example 1 (containing only PHMB) and Example 2 showed that both achieved antibacterial rates of over 99%, but the combination of... Figure 1 The results showed that the venous valve surface in Example 2 was smoother and showed no signs of bacterial adhesion. This indicates that the hydroxyl groups of HES hinder initial bacterial adhesion (physical barrier) by reducing surface energy, while PHMB disrupts the internal structure of bacteria through chemical penetration (chemical sterilization). The two work synergistically to achieve a "dual blockade" of bacteria. In contrast, the antibacterial rate of Comparative Example 2 (without PHMB) plummeted to 80.4%, demonstrating that PHMB is the core component for long-lasting sterilization, but it cannot completely inhibit bacterial proliferation when acting alone. It relies on HES to reduce bacterial adhesion and lower the subsequent sterilization load. The synergy between the two increases the antibacterial rate from 80%–99.1% for a single component to 99.9%, achieving near-complete sterilization.

[0025] (2) The hydrophilic properties and moisturizing effect of HES:

[0026] The hydroxyl groups of HES are highly hydrophilic and can form a hydration layer to reduce water evaporation. The dehydration rates of Examples 1 and 2 were only 3.2% to 3.5%, while the dehydration rate of Comparative Example 1 (without HES) was as high as 18.7% (Table 1). Figure 2 and Figure 3 Further analysis showed that the HES-treated venous valves maintained a tight tissue structure after alcohol preservation, and the collagen fibers did not shrink due to dehydration, while Comparative Example 1, which did not use HES, showed significant shrinkage and structural damage. Notably, the dehydration rates of Comparative Example 3 (2% HES) and Comparative Example 4 (5% HES) were similar to those of the examples, but the tension improvement rates of Examples 1–2 were higher (82.5%–85.3% vs. 84.4%–85.0%), indicating that 4% HES is the balance point between moisturizing and mechanical properties, effectively locking in moisture while avoiding excessive HES from interfering with valve function.

[0027] (3) Indirect improvement of anticoagulant properties:

[0028] The antibacterial effect of PHMB can reduce the inflammatory response caused by infection, thereby reducing the risk of secondary thrombosis. In addition, the moisturizing effect of HES maintains the integrity of the collagen network and avoids the rough surface caused by dehydration (which easily leads to platelet aggregation). The tension improvement rate of Examples 1-2 (82.5%-85.3%) was significantly higher than that of Comparative Example 1 (62.0%), confirming the positive effect of structural integrity on hemodynamics and indirectly improving anticoagulant performance.

[0029] (4) Process compatibility and stability:

[0030] The combination of HES and PHMB was introduced after heparin cross-linking (step d), without interfering with the previous decellularization and cross-linking steps. Comparative Examples 3 and 4 showed that the dehydration rate varied little within the HES concentration range of 2% to 5%, but Example 2 with 4% HES achieved the best results in both antibacterial rate (99.9%) and tension improvement rate (85.3%), indicating that the hydrophilicity and antibacterial permeability reached optimal synergy at this concentration. Furthermore, the chemical stability of PHMB reduces its risk of concentration decay during long-term alcohol storage compared to traditional antibacterial agents.

[0031] (5) Valve function of biological veins was significantly improved:

[0032] Figure 1 It can be seen that the venous valves of the biologically derived veins changed from a curled state before treatment to a relaxed state after treatment, and the valve closing function was significantly improved. At the same time, the reflux percentage data in Table 1 shows that the anti-reflux performance of the valves after treatment was improved, and the overall valve function was significantly improved, which was verified.

[0033] Conclusion: This invention overcomes the limitations of single-function approaches through the synergistic effect of HES and PHMB: the hydroxyl groups of HES reduce bacterial adhesion and provide moisturizing, while PHMB penetrates and kills bacteria and inhibits infection; the two work synergistically to achieve long-lasting antibacterial effect (99.9%). The hydrophilic properties of HES reduce collagen dehydration (dehydration rate ≤3.5%) and maintain valve mechanical properties (tension improvement rate ≥82.5%). At the same time, the function of bio-derived venous valves treated with HES is significantly improved. The above technical solutions provide a reliable improvement strategy for the clinical translation of bio-derived venous valves. Attached Figure Description

[0034] The present invention will now be further described with reference to the accompanying drawings.

[0035] Figure 1 The images show the appearance of the biogenic vein containing venous valves after treatment with the treatment solution for 2 hours in step e of this invention (the left image is Comparative Example 1 without hydroxyethyl starch treatment; the right image is Example 2 after hydroxyethyl starch treatment).

[0036] Figure 2 The image shows the appearance of the biological vein containing venous valves after being sealed and stored in alcohol for 24 hours and then dried at room temperature for 1 hour in step e of this invention (the upper image is Comparative Example 1 without hydroxyethyl starch treatment; the lower image is Example 2 with hydroxyethyl starch treatment).

[0037] Figure 3 The image shows an HE-stained photograph of a biological vein containing venous valves after being sealed and stored in alcohol for 24 hours in step e of this invention and then dried at room temperature for 1 hour. (The left image is Comparative Example 1 without hydroxyethyl starch treatment; the right image is Example 2 with hydroxyethyl starch treatment.) Detailed Implementation

[0038] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1

[0040] A method for preparing a biological vein containing venous valves includes the following steps:

[0041] Step a. Material Acquisition and Pretreatment:

[0042] Take a fresh pig jugular vein with a diameter of 0.5 cm and containing a valve from a daily slaughter at the slaughterhouse. Turn it over to find the valve part, cut a 3 cm long section containing the intact valve, turn it back to the right side, trim away the peripheral fat tissue and excess connective tissue, then irrigate with physiological saline to remove blood clots, then immerse in 0.1% benzalkonium chloride solution for disinfection for 30 minutes, and rinse with PBS solution for later use.

[0043] Step b. Decellularization:

[0044] Cell lysis: The material obtained in step a was treated with PBS containing 0.5% Triton X-100 for 24 hours to lyse the cells, and then rinsed with PBS solution.

[0045] Cell digestion: The cells were then immersed in PBS solution containing 0.025% trypsin and 0.02% EDTA (0.025g trypsin and 0.02g EDTA dissolved in 100mL PBS solution) for 30min to digest the cells, and then rinsed with PBS solution.

[0046] Nucleic acid digestion: Finally, immerse the sample in PBS solution containing 30 U / mL DNase I and 0.3 mg / mL RNase A for 24 hours to digest the nucleic acid, and then rinse with PBS solution.

[0047] Cell lysis, cell digestion, and nucleic acid digestion were all performed at 37°C in a shaker at 70 rpm.

[0048] Step c. Heparin covalent crosslinking:

[0049] The material treated in step b was immersed in a 1 mol / L hydroxylamine sulfate solution and treated at room temperature with shaking at 70 rpm for 12 h. After rinsing with distilled water three times for 10 min each time, it was immersed in heparin-EDC solution (1.67 g EDC + 0.835 g heparin sodium + 0.05 mol / L HCl 200 mL, pH 1.5) and crosslinked at 37 °C with shaking at 70 rpm for 48 h. After rinsing with PBS solution three times.

[0050] Step d. Improved processing:

[0051] The material treated in step c was immersed in a treatment solution (PBS solution containing 4 wt% hydroxyethyl starch and 0.1 wt% polyhexamethylene biguanide) and treated at room temperature for 5 min. After rinsing three times with PBS solution, a biological vein containing a venous valve was obtained. Finally, the obtained biological vein containing a venous valve was sealed and stored in 60% alcohol at room temperature.

[0052] Example 2

[0053] A method for preparing a biological vein containing venous valves includes the following steps:

[0054] Step a. Material Acquisition and Pretreatment:

[0055] Take a fresh bovine jugular vein with a diameter of 1.5 cm and containing a valve from a daily slaughterhouse. Turn it over to find the valve part, cut a 5 cm long section containing the intact valve, turn it back to the right side, trim away the peripheral fat tissue and excess connective tissue, then irrigate with physiological saline to remove blood clots, then immerse in 0.1% benzalkonium chloride solution for disinfection for 30 minutes, and rinse with PBS solution for later use.

[0056] Step b. Decellularization:

[0057] Cell lysis: The material obtained in step a was treated with PBS containing 0.5% Triton X-100 for 48 hours to lyse the cells, and then rinsed with PBS solution.

[0058] Cell digestion: The cells were then immersed in PBS solution containing 0.025% trypsin and 0.02% EDTA (0.025g trypsin and 0.02g EDTA dissolved in 100mL PBS solution) for 120min to digest the cells, and then rinsed with PBS solution.

[0059] Nucleic acid digestion: Finally, immerse the sample in PBS solution containing 30 U / mL DNase I and 0.3 mg / mL RNase A for 48 h to digest the nucleic acid, and then rinse with PBS solution.

[0060] Cell lysis, cell digestion, and nucleic acid digestion were all performed at 37°C in a shaker at 100 rpm.

[0061] Step c. Heparin covalent crosslinking:

[0062] The material treated in step b was immersed in a 1 mol / L hydroxylamine sulfate solution and treated at room temperature with shaking at 100 rpm for 12 h. After rinsing with distilled water three times for 10 min each time, it was immersed in heparin-EDC solution (1.67 g EDC + 0.835 g heparin sodium + 0.05 mol / L HCl 200 mL, pH 1.5) and crosslinked at 37 °C with shaking at 100 rpm for 72 h. After rinsing with PBS solution three times.

[0063] Step d. Improved processing:

[0064] The material treated in step c was immersed in a treatment solution (PBS solution containing 4 wt% hydroxyethyl starch and 0.1 wt% polyhexamethylene biguanide) and treated at room temperature for 60 min. After rinsing three times with PBS solution, a biological vein containing a venous valve was obtained. Finally, the obtained biological vein containing a venous valve was sealed and stored in 80% alcohol at room temperature.

[0065] Comparative Example 1

[0066] Comparative Example 1 served as the control group for Example 2. 4 wt% hydroxyethyl starch was removed from the improved solution in step e of Example 2, while the remaining raw materials, raw material amounts, and preparation methods remained consistent with those in Example 2. A biogenic vein containing venous valves was obtained. Finally, the obtained biogenic vein containing venous valves was sealed and stored in 80% alcohol by volume at room temperature.

[0067] Among them, see Figure 1 As shown, Figure 1Images of the appearance of the bio-derived veins containing venous valves after treatment with the treatment solution in step e for 2 hours (left image is Comparative Example 1 without hydroxyethyl starch treatment, right image is Example 2 with hydroxyethyl starch treatment). Conclusion: The venous valves treated with hydroxyethyl starch have open valves, significantly reduced wrinkles, and a smoother and more moist surface.

[0068] Then, see Figure 2 As shown, Figure 2 After being sealed and stored in alcohol for 24 hours in step e, the appearance of the bio-derived vein containing venous valves was taken out and dried at room temperature for 1 hour (the upper image is Comparative Example 1 without hydroxyethyl starch treatment, and the lower image is Example 2 with hydroxyethyl starch treatment). Conclusion: The bio-derived vein treated with hydroxyethyl starch maintained its moisture and did not dehydrate.

[0069] Finally, see Figure 3 As shown, Figure 3 HE-stained images of biological veins containing venous valves after being sealed and stored in alcohol for 24 hours in step e and then dried at room temperature for 1 hour are shown (left image: Comparative Example 1 without hydroxyethyl starch treatment; right image: Example 2 with hydroxyethyl starch treatment). The right image shows a significantly denser tissue structure. Conclusion: Hydroxyethyl starch treatment has an anti-ethanol dehydration effect.

[0070] Comparative Example 2

[0071] Comparative Example 2 served as the control group for Example 2. The 0.1 wt% polyhexamethylene biguanide PBS solution in the improved solution in step e of Example 2 was removed, while the remaining raw materials, raw material amounts, and preparation methods remained consistent with those in Example 2. A biogenic vein containing a venous valve was obtained. Finally, the obtained biogenic vein containing a venous valve was sealed and stored in 80% alcohol by volume at room temperature.

[0072] Comparative Example 3

[0073] Comparative Example 3 served as the control group for Example 2. The concentration of 4 wt% hydroxyethyl starch in the improved solution in step e of Example 2 was replaced with 2 wt%, while the remaining raw materials, raw material amounts, and preparation methods remained consistent with those in Example 2. A biogenic vein containing a venous valve was obtained. Finally, the obtained biogenic vein containing a venous valve was sealed and stored in 80% alcohol by volume at room temperature.

[0074] Comparative Example 4

[0075] Comparative Example 4 served as the control group for Example 2. The concentration of 4 wt% hydroxyethyl starch in the improved solution in step e of Example 2 was replaced with 5 wt%, while the remaining raw materials, raw material amounts, and preparation methods remained consistent with those in Example 2. A biogenic vein containing a venous valve was obtained. Finally, the obtained biogenic vein containing a venous valve was sealed and stored in 80% alcohol by volume at room temperature.

[0076] Test Example 1

[0077] The performance of the bio-derived veins containing venous valves prepared in Examples 1-2 and Comparative Examples 1-4 was tested. The testing process is as follows, and the test results are shown in Table 1:

[0078] 1. Tissue moisture content:

[0079] Biological veins containing venous valves were collected, and their weight before drying, mass after drying, and percentage of weight loss were determined according to the method for determining loss on drying in Part III of the 2020 edition of the Chinese Pharmacopoeia. The percentage of weight loss can refer to the water content.

[0080] 2. Anti-dehydration performance test:

[0081] Methods: After being sealed and stored in alcohol for 24 hours, the biological vein containing venous valves was removed and dried at room temperature for 1 hour and weighed (W0). It was then stored in an environment of 25℃ / 50% humidity for 30 days and weighed again (W1). At least 6 parallel samples were prepared for each group, and the dehydration rate (%) was calculated.

[0082] Dehydration rate (%) = (W0-W1) / W0×100%.

[0083] 3. Antibacterial performance test:

[0084] Method: A bio-derived vein containing venous valves was immersed in a solution containing 1×10 6 CFU / mL E. coli / PBS suspension was incubated at 37℃ for 24 h, and the soaking solution was serially diluted, spread on agar plates, and counted to obtain the colony count of the experimental group. The control group did not add biologically derived veins containing venous valves, and then the inhibition rate (%) was calculated.

[0085] Inhibition rate (%) = [(number of colonies in control group - number of colonies in experimental group) / number of colonies in control group] × 100%.

[0086] 4. Venous tension measurement:

[0087] Adult beagle dogs (weighing 10±1 kg, fasted for 24 hours preoperatively) were selected and anesthetized with intravenous sodium pentobarbital (30 mg / kg). The neck was shaved and disinfected. An incision was made along the lateral border of the sternocleidomastoid muscle, and the external jugular vein was freed 5 cm. A pressure sensor probe was vertically fixed to the vein wall, and venous tension (P0) was recorded in a head-down position (30°). A treated valve (length matched to the vessel diameter) was implanted into the vein incision, and the incision was sutured continuously with 7-0 Prolene sutures. Seven days after recovery, venous tension was recorded again in a head-down position (P1). The tension improvement rate (%) was calculated.

[0088] Tension improvement rate (%) = [(P0-P1) / P0] × 100%.

[0089] 5. Reflux percentage:

[0090] Referring to GB12279-2008 "Cardiovascular Implants - Artificial Heart Valves", the percentage (%) of reflux in the bio-derived veins containing venous valves prepared in Examples 1-2 and Comparative Examples 1-4 was measured.

[0091] Table 1 Test Results

[0092]

[0093] It should be noted that, in this document, terms such as “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus.

[0094] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a biologically derived vein containing venous valves, characterized in that, Includes the following steps: Step a. Cut a pig or bovine jugular vein with a diameter of 0.5-1.5 cm and a length of 3-5 cm containing an intact valve, clean and disinfect it, and then rinse it with PBS solution for later use; Step b. Perform cell lysis, cell digestion, and nucleic acid digestion sequentially; Step c. Immerse in 1 mol / L hydroxylamine sulfate solution at room temperature for 12 h, rinse with distilled water, crosslink in heparin-EDC solution at 37 °C for 48–72 h, and then rinse with PBS solution. Step d. Immerse in the treatment solution again and treat at room temperature for 5-60 minutes. Then rinse three times with PBS solution to obtain a biological vein containing venous valves. The treatment solution is a PBS solution containing 4 wt% hydroxyethyl starch and 0.1 wt% polyhexamethylene biguanide.

2. The method for preparing a biologically derived vein containing venous valves according to claim 1, characterized in that, The disinfection in step a is to soak in 0.1% benzalkonium chloride solution for 30 minutes.

3. The method for preparing a biologically derived vein containing venous valves according to claim 1, characterized in that, Cell lysis in step b: treatment with PBS solution containing 0.5% Triton X-100 for 24–48 h.

4. The method for preparing a biologically derived vein containing venous valves according to claim 1, characterized in that, Cell digestion in step b: treat with PBS solution containing 0.025% trypsin and 0.02% EDTA at 37°C for 30–120 min.

5. The method for preparing a biologically derived vein containing venous valves according to claim 1, characterized in that, Nucleic acid digestion in step b: treatment with PBS solution containing 30 U / mL DNase I and 0.3 mg / mL RNase A at 37°C for 24–48 h.

6. The method for preparing a biologically derived vein containing venous valves according to claim 1, characterized in that, The heparin-EDC solution in step c is prepared by EDC, sodium heparin, and 0.05 mol / L HCl in a ratio of 1.67 g: 0.835 g: 200 mL; the pH of the heparin-EDC solution is 1.

5.

7. The method for preparing a biologically derived vein containing venous valves according to claim 1, characterized in that, The processing steps b to d are all carried out on a shaker at 70 to 100 rpm.

8. The method for preparing a biogenic vein containing venous valves according to claim 1, characterized in that, In step d, the bio-derived vein containing venous valves is sealed and preserved in alcohol with a volume fraction of 60%–80%.

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