Preparation method of biogenic vein containing venous valve

Abstract

The invention discloses a preparation method of a biogenic vein containing a venous valve, and belongs to the technical field of biomedical functions.The preparation method of the biogenic vein containing the venous valve comprises the following steps that firstly, a pig or cattle jugular vein pipeline containing a complete valve is cut, cleaned, disinfected and flushed for use; sequentially carrying out cell lysis, cell digestion and nucleic acid digestion treatment; immersing into a 1 mol / L hydroxylamine sulfate solution to carry out heparin covalent cross-linking; immersing into a treating fluid to obtain a biogenic vein containing a venous valve; wherein the treating fluid is a PBS (Phosphate Buffer Solution) containing 4wt% of hydroxyethyl starch and 0.1 wt% of polyhexamethylene biguanide. According to the preparation method of the biogenic vein containing the venous valve, provided by the invention, the treatment liquid containing 4wt% of hydroxyethyl starch and 0.1 wt% of polyhexamethylene biguanide is introduced in the preparation process, so that the technical problem of insufficient dehydration and bacteriostasis in the background technology is remarkably solved, and meanwhile, the valve functionality of the biogenic vein is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering, and specifically relates to a method for preparing a biogenic vein containing a venous valve, and more particularly to a method for preparing a biogenic venous valve that improves anti-dehydration, antibacterial and anticoagulant properties by optimizing the preparation steps. Background Art

[0002] Varicose veins of the lower extremities are a common vascular disease caused by venous valve insufficiency, clinically manifested by venous reflux, vasodilation, and tissue malnutrition. Traditional treatments, such as high ligation and vein stripping, can alleviate symptoms but fail to restore valve function, and postoperative recurrence rates are high and traumatic. Biogenic venous valves, with their biocompatibility and hemodynamic properties similar to those of human tissue, offer a potential cure.

[0003] In existing technologies, bioprosthetic valves prepared through decellularization combined with heparin cross-linking have demonstrated certain anti-thrombotic and anti-calcification properties. However, their long-term preservation and immediate functional maintenance after implantation still face challenges. Existing methods for preserving biogenic venous valves mostly use alcohol or glutaraldehyde solutions, which, while capable of short-term antibacterial effects, have significant drawbacks: (1) Dehydration problem: Alcohol immersion can easily lead to dehydration and shrinkage of collagen fibers, destroying the valve microstructure and affecting the mechanical properties after implantation; (2) Bacteriostatic limitations: Traditional antibacterial agents (such as benzalkonium chloride) may lead to the risk of secondary infection due to concentration decay during long-term storage; These issues result in a short shelf life of the valve and a high risk of thrombosis or infection after implantation, severely restricting its clinical application. Therefore, improved preparation methods are urgently needed to simplify the process while simultaneously addressing the two major challenges of preventing dehydration and achieving long-term antibacterial effects. Summary of the Invention

[0004] This invention improves upon the existing patent CN105770991B and provides a method for preparing a biogenic vein containing venous valves. By optimizing the preparation steps, the dehydration resistance and antibacterial properties of the finished product are significantly improved. The objectives of the invention can be achieved through the following technical solutions: A method for preparing a biogenic vein containing a venous valve comprises the following steps: Step a. Cut a 0.5-1.5 cm diameter, 3-5 cm long pig or bovine jugular vein with an intact valve, clean and disinfect it, and then rinse it with PBS solution for later use; Step b. performing cell lysis, cell digestion and nucleic acid digestion treatment in sequence; Step c. Immerse in 1 mol / L hydroxylamine sulfate solution at room temperature for 12 h, rinse with distilled water, cross-link in heparin-EDC solution at 37 ° C for 48 to 72 h, and then rinse with PBS solution; Step d. The sample was immersed in the treatment solution and treated at room temperature for 5 to 60 minutes, and then rinsed three times with PBS solution to obtain a biogenic vein containing a venous valve; Wherein, the treatment liquid is a PBS solution containing 4 wt % hydroxyethyl starch and 0.1 wt % polyhexamethylene biguanide.

[0005] Furthermore, the disinfection in step a is performed by soaking in a 0.1% chlorhexidine solution for 30 minutes.

[0006] Furthermore, the cell lysis in step b is treated with a PBS solution containing 0.5% Triton X-100 for 24 to 48 hours.

[0007] Furthermore, the cell digestion in step b is: treated with a PBS solution containing 0.025% trypsin and 0.02% EDTA at 37° C. for 30 to 120 minutes.

[0008] Furthermore, the nucleic acid digestion in step b is carried out by treating the cells with a PBS solution containing 30 U / mL DNase I and 0.3 mg / mL RNase A at 37° C. for 24 to 48 hours.

[0009] Furthermore, the heparin-EDC solution in step c is prepared by mixing EDC, heparin sodium and 0.05 mol / L HCl in a dosage ratio of 1.67 g:0.835 g:200 mL; the pH of the heparin-EDC solution is 1.5.

[0010] Furthermore, the treatment processes in steps b to d are all carried out in a shaking table at 70 to 100 rpm.

[0011] Furthermore, the biogenic venous seal containing the venous valve in step d is stored in alcohol with a volume fraction of 60% to 80%.

[0012] Beneficial effects of the present invention: The method for preparing a biogenic vein containing a venous valve provided by the present invention solves the technical problems of dehydration and insufficient antibacterial properties proposed in the prior art by introducing a treatment solution containing 4 wt% hydroxyethyl starch (HES) and 0.1 wt% polyhexamethylene biguanide (PHMB) into the preparation step. Furthermore, testing has shown that the valve function of the biogenic vein is significantly improved. The test results are analyzed as follows: (1) Synergistic antibacterial effect of HES and PHMB: The comparison between Comparative Example 1 (containing only PHMB) and Example 2 shows that the antibacterial rates of both are above 99%, but the combination of Figure 1 From the appearance results, it can be seen that the surface of the venous valve in Example 2 is smoother and has no signs of bacterial attachment. This shows that the hydroxyl groups of HES hinder the initial adhesion of bacteria by reducing the surface energy (physical barrier), while PHMB destroys the internal structure of bacteria through chemical penetration (chemical sterilization), and the two work together to achieve a "double blockade" of bacteria. In contrast, the antibacterial rate of Comparative Example 2 (without PHMB) dropped sharply to 80.4%, proving that PHMB is the core component of long-term bactericidal effect, but it cannot completely inhibit bacterial proliferation when acting alone, and it needs to rely on HES to reduce bacterial attachment to reduce the subsequent bactericidal load. The synergy of the two has increased the antibacterial rate from 80% to 99.1% of a single component to 99.9%, achieving a nearly complete antibacterial effect.

[0013] (2)HES’s hydrophilic properties and moisturizing properties: 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 (no HES) was as high as 18.7% (Table 1). Figure 2 and Figure 3 Further results showed that HES-treated venous valves maintained a tight structure after alcohol storage, with collagen fibers showing no shrinkage due to dehydration, whereas Comparative Example 1, which did not use HES, exhibited significant shrinkage and structural damage. Notably, while the dehydration rates of Comparative Examples 3 (2% HES) and 4 (5% HES) were similar to those of the Examples, the tension improvement rates of Examples 1-2 were higher (82.5%-85.3% vs. 84.4%-85.0%). This suggests that 4% HES strikes a balance between moisture retention and mechanical properties, effectively locking in moisture while preventing excessive HES from interfering with valve function.

[0014] (3) Indirect improvement of anticoagulant performance: 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, avoiding the rough surface caused by dehydration (which easily triggers platelet aggregation). The tension improvement rate of Examples 1-2 (82.5%-85.3%) is significantly higher than that of Comparative Example 1 (62.0%), confirming the positive impact of structural integrity on hemodynamics and indirectly improving anticoagulant performance.

[0015] (4) Process compatibility and stability: 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 while the dehydration rate varied slightly within the 2% to 5% HES concentration range, Example 2, with 4% HES, achieved the best results in both inhibition (99.9%) and tension improvement (85.3%), indicating that at this concentration, hydrophilicity and antimicrobial permeability achieved optimal synergy. Furthermore, PHMB's chemical stability reduces its risk of concentration decay during long-term alcohol storage compared to traditional antimicrobial agents.

[0016] (5) The valve function of the biogenic vein is significantly improved: Figure 1 It can be seen that the valve of the venous valve of the biogenic vein is transformed from a curled state before treatment with the treatment fluid to a stretched state after treatment, and the closing function of the valve is significantly improved. At the same time, it can be seen from the reflux percentage data in Table 1 that the anti-reflux performance of the valve after treatment with the treatment fluid is improved, and the significant improvement in the functionality of the overall valve has been verified.

[0017] Conclusion: The present invention breaks through the limitations of a single function 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 together to achieve long-term 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 biogenic venous valves treated with HES is significantly improved. The above technical solution provides a reliable improvement strategy for the clinical transformation of biogenic venous valves. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described below with reference to the accompanying drawings.

[0019] Figure 1 This is a picture of the appearance of a biogenic vein containing a venous valve after being treated with the treatment solution in step e of the present invention for 2 hours (the left picture is Comparative Example 1 without hydroxyethyl starch treatment; the right picture is Example 2 after hydroxyethyl starch treatment); Figure 2 This is a picture of the appearance of a biogenic vein containing a venous valve after being sealed and stored in alcohol for 24 hours in step e of the present invention and then dried at room temperature for 1 hour (the upper picture is Comparative Example 1 without hydroxyethyl starch treatment; the lower picture is Example 2 after hydroxyethyl starch treatment); Figure 3 This is a HE-stained photograph of a biogenic vein containing a venous valve after being sealed and stored in alcohol for 24 hours in step e of the present invention and then dried at room temperature for 1 hour (the left picture is Comparative Example 1 without hydroxyethyl starch treatment; the right picture is Example 2 after hydroxyethyl starch treatment). DETAILED DESCRIPTION

[0020] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0021] Example 1

[0022] A method for preparing a biogenic vein containing a venous valve comprises the following steps: Step a. Material acquisition and pretreatment: Take a fresh pig jugular vein tube with a diameter of 0.5 cm and a valve from a slaughterhouse. Turn it over to find the valve part, cut a 3 cm long segment containing a complete valve, turn it back to the front and trim off the peripheral fat tissue and excess connective tissue of the blood vessel. Then use normal saline to irrigate it in the antegrade direction to remove blood clots, then immerse it in 0.1% chlorhexidine solution for disinfection for 30 minutes, and then rinse it with PBS solution for use.

[0023] Step b. Decellularization: Cell lysis: Treat the material obtained in step a with PBS solution containing 0.5% Triton X-100 for 24 h to lyse the cells, and then rinse with PBS solution.

[0024] Cell digestion: Immerse in a PBS solution containing 0.025% trypsin and 0.02% EDTA (0.025 g trypsin, 0.02 g EDTA dissolved in 100 mL PBS solution) for 30 min to digest the cells, and then rinse with PBS solution.

[0025] Nucleic acid digestion: Finally, immerse the tube in a 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.

[0026] Cell lysis, cell digestion, and nucleic acid digestion were all performed at 37°C in a shaker at 70 rpm.

[0027] Step c. Heparin covalent cross-linking: The material treated in step b was immersed in a 1 mol / L hydroxylamine sulfate solution and shaken at 70 rpm at room temperature for 12 h. After that, it was rinsed with distilled water three times, each time for 10 min. 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 shaken at 37 ° C and 70 rpm for cross-linking for 48 h. After that, it was rinsed with PBS solution three times.

[0028] Step d. Improvement process: The material treated in step c was immersed in a treatment solution (a PBS solution containing 4 wt% hydroxyethyl starch and 0.1 wt% polyhexamethylene biguanide), treated at room temperature for 5 minutes, and then rinsed with PBS solution three times to obtain a biogenic vein containing a venous valve. Finally, the obtained biogenic vein containing a venous valve was sealed and stored in 60% alcohol by volume and sealed at room temperature.

[0029] Example 2

[0030] A method for preparing a biogenic vein containing a venous valve comprises the following steps: Step a. Material acquisition and pretreatment: Take a fresh bovine jugular vein tube with a diameter of 1.5 cm and a valve from a slaughterhouse, turn it over to find the valve part, cut a 5 cm long segment containing a complete valve, turn it back to the front and trim off the peripheral fat tissue and excess connective tissue of the blood vessel, then use normal saline to irrigate it in the antegrade direction to remove blood clots, then immerse it in 0.1% chlorhexidine solution for disinfection for 30 minutes, and then rinse it with PBS solution for use.

[0031] Step b. Decellularization: Cell lysis: Treat the material obtained in step a with PBS solution containing 0.5% Triton X-100 for 48 h to lyse the cells, and then rinse with PBS solution.

[0032] Cell digestion: Immerse in a PBS solution containing 0.025% trypsin and 0.02% EDTA (0.025 g trypsin, 0.02 g EDTA dissolved in 100 mL PBS solution) for 120 min to digest the cells, and then rinse with PBS solution.

[0033] Nucleic acid digestion: Finally, immerse the tube in a PBS solution containing 30 U / mL DNase I and 0.3 mg / mL RNase A for 48 hours to digest the nucleic acid, and then rinse with PBS solution.

[0034] Cell lysis, cell digestion, and nucleic acid digestion were all performed at 37°C in a shaker at 100 rpm.

[0035] Step c. Heparin covalent cross-linking: The material treated in step b was immersed in a 1 mol / L hydroxylamine sulfate solution and shaken at 100 rpm at room temperature for 12 h. After that, it was rinsed with distilled water three times, each time for 10 min. 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 shaken at 37 ° C and 100 rpm for cross-linking for 72 h. After that, it was rinsed with PBS solution three times.

[0036] Step d. Improvement process: The material treated in step c is immersed in a treatment solution (a PBS solution containing 4 wt% hydroxyethyl starch and 0.1 wt% polyhexamethylene biguanide) and treated at room temperature for 60 minutes. It is then rinsed three times with a PBS solution to obtain a biogenic vein containing a venous valve. Finally, the obtained biogenic vein containing a venous valve is sealed and stored in 80% alcohol by volume and sealed at room temperature.

[0037] Comparative Example 1 Comparative Example 1 is the control group of Example 2. 4 wt% hydroxyethyl starch in the improved solution in step e of Example 2 is removed, and the remaining raw materials, raw material amounts and preparation methods remain the same as in Example 2 to obtain a biogenic vein containing a venous valve. Finally, the obtained biogenic vein containing a venous valve is sealed and stored in 80% alcohol by volume and sealed at room temperature.

[0038] Among them, see Figure 1 As shown, Figure 1 These are pictures of the appearance of the biogenic vein containing venous valves after being treated with the treatment liquid in step e for 2 hours (the left picture is Comparative Example 1 without hydroxyethyl starch treatment, and the right picture is Example 2 after treatment with hydroxyethyl starch). Conclusion: The venous valves treated with hydroxyethyl starch are open, the wrinkles are significantly reduced, and the surface is smoother and moister.

[0039] Then, see Figure 2 As shown, Figure 2 The following are pictures of the appearance of the biogenic vein 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 (the upper picture is comparative example 1 without hydroxyethyl starch treatment, and the lower picture is example 2 after hydroxyethyl starch treatment). Conclusion: The biogenic vein after hydroxyethyl starch treatment maintains moisture and is not dehydrated.

[0040] Finally, see Figure 3 As shown, Figure 3This HE-stained photograph shows a biogenic vein containing venous valves after 24 hours of alcohol-sealed storage in step e and then drying at room temperature for 1 hour. (The left image shows Comparative Example 1, untreated with hydroxyethyl starch; the right image shows Example 2, treated with hydroxyethyl starch.) The right image clearly shows a more compact tissue structure. Conclusion: Treatment with hydroxyethyl starch provides resistance to ethanol dehydration.

[0041] Comparative Example 2 Comparative Example 2 is the control group of Example 2. The 0.1wt% polyhexamethylene biguanide PBS solution in the improved solution in step e of Example 2 is removed, and the remaining raw materials, raw material amounts and preparation methods remain the same as in Example 2 to obtain a biogenic vein containing a venous valve. Finally, the obtained biogenic vein containing a venous valve is sealed and stored in 80% alcohol by volume and sealed at room temperature.

[0042] Comparative Example 3 Comparative Example 3 is the control group of Example 2, except that the concentration of 4 wt% hydroxyethyl starch in the improved solution in step e of Example 2 is replaced with 2 wt%, and the remaining raw materials, raw material amounts and preparation methods remain the same as in Example 2 to obtain a biogenic vein containing a venous valve. Finally, the obtained biogenic vein containing a venous valve is sealed and stored in 80% alcohol by volume and sealed at room temperature.

[0043] Comparative Example 4 Comparative Example 4 is the control group of Example 2, except that the concentration of 4 wt% hydroxyethyl starch in the improved solution in step e of Example 2 is replaced with 5 wt%, and the remaining raw materials, raw material amounts and preparation methods remain the same as in Example 2 to obtain a biogenic vein containing a venous valve. Finally, the obtained biogenic vein containing a venous valve is sealed and stored in 80% alcohol by volume and sealed at room temperature.

[0044] Test Example 1 The performance of the biogenic veins containing venous valves prepared in Examples 1 to 2 and Comparative Examples 1 to 4 was tested. The test process is as follows, and the test results are shown in Table 1: 1. Tissue water content: Take a biological vein containing a venous valve, and determine the weight before drying, mass after drying and weight loss percentage according to the drying loss determination method in Part III of the 2020 edition of the "Chinese Pharmacopoeia", where the weight loss percentage can refer to the water content.

[0045] 2. Anti-dehydration performance test: Methods: After 24 hours of sealed storage in alcohol, the biogenic vein containing venous valves was taken out and dried at room temperature for 1 hour, and then weighed (W0). It was stored in a 25℃ / 50% humidity environment for 30 days and weighed again (W1). At least 6 parallel samples were prepared for each group, and the dehydration rate (%) was calculated.

[0046] Dehydration rate (%) = (W0-W1) / W0×100%.

[0047] 3. Antibacterial performance test: Methods: A biogenic vein containing a venous valve was immersed in a solution containing 1×10 6 The suspension of Escherichia coli (CFU / mL) in PBS was incubated at 37°C for 24 hours, and then the immersion solution was diluted in gradients and spread on agar plates for culture and counting to obtain the number of colonies in the experimental group. The control group did not add the biological source vein containing venous valves, and then calculated the inhibition rate (%).

[0048] Inhibition rate (%) = [(number of colonies in the control group - number of colonies in the experimental group) / number of colonies in the control group] × 100%.

[0049] 4. Venous tension test: Adult beagle dogs (weighing 10 ± 1 kg, fasted for 24 hours before surgery) were anesthetized with intravenous sodium pentobarbital (30 mg / kg), and the neck was shaved and disinfected. A skin incision was made along the lateral edge of the sternocleidomastoid muscle, and the external jugular vein was freed 5 cm. A pressure transducer probe was fixed perpendicularly to the venous wall, and venous tension (P0) was recorded with the head in a low position (30°). The venous incision was implanted with a treated valve (length matching the vessel diameter), and sutured continuously with 7-0 Prolene sutures. After 7 days of recovery, venous tension (P1) was recorded again with the head in a low position. The tension improvement rate (%) was calculated.

[0050] Tension improvement rate (%) = [(P0-P1) / P0] × 100%.

[0051] 5. Reflux percentage: With reference to GB12279-2008 “Cardiovascular Implants - Artificial Heart Valves”, the reflux percentages (%) of the biogenic veins containing venous valves prepared in Examples 1 to 2 and Comparative Examples 1 to 4 were measured respectively.

[0052] Table 1 Test results

[0053] It should be noted that, in this document, terms such as "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or elements that are inherent to such process, method, article or apparatus.

[0054] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may 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 biogenic vein containing a venous valve, characterized in that: The following steps are involved: Step a. Cut a 0.5-1.5 cm diameter, 3-5 cm long pig or bovine jugular vein with an intact valve, clean and disinfect it, and then rinse it with PBS solution for later use; Step b. performing cell lysis, cell digestion and nucleic acid digestion treatment in sequence; Step c. Immerse in 1 mol / L hydroxylamine sulfate solution at room temperature for 12 h, rinse with distilled water, cross-link in heparin-EDC solution at 37 ° C for 48 to 72 h, and then rinse with PBS solution; Step d. The sample was immersed in the treatment solution and treated at room temperature for 5 to 60 minutes, and then rinsed three times with PBS solution to obtain a biogenic vein containing a venous valve; Wherein, the treatment liquid is a PBS solution containing 4 wt % hydroxyethyl starch and 0.1 wt % polyhexamethylene biguanide.

2. The method for preparing a biogenic vein containing a venous valve according to claim 1, characterized in that: The disinfection in step a is performed by soaking in a 0.1% chlorhexidine solution for 30 minutes.

3. The method for preparing a biogenic vein containing a venous valve according to claim 1, characterized in that: The cell lysis in step b: treatment with PBS solution containing 0.5% Triton X-100 for 24 to 48 hours.

4. The method for preparing a biogenic vein containing a venous valve according to claim 1, characterized in that: The cell digestion in step b is carried out by treating the cells with a PBS solution containing 0.025% trypsin and 0.02% EDTA at 37° C. for 30 to 120 minutes.

5. The method for preparing a biogenic vein containing a venous valve according to claim 1, characterized in that: The nucleic acid digestion in step b was performed by treating the cells with a PBS solution containing 30 U / mL DNase I and 0.3 mg / mL RNase A at 37° C. for 24 to 48 hours.

6. The method for preparing a biogenic vein containing a venous valve according to claim 1, characterized in that: The heparin-EDC solution in step c is prepared by mixing EDC, heparin sodium, 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 biogenic vein containing a venous valve according to claim 1, characterized in that: The treatment processes in steps b to d are all carried out in a shaking table at 70 to 100 rpm.

8. The method for preparing a biogenic vein containing a venous valve according to claim 1, characterized in that: The biogenic venous seal containing the venous valve in step d is stored in alcohol with a volume fraction of 60% to 80%.

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