Small-caliber artificial blood vessel and preparation method thereof

Abstract

The invention provides a small-caliber artificial blood vessel and a preparation method thereof, and belongs to the technical field of biomedicine. The preparation method comprises the following steps: dissolving a solid polymer material in hexafluoroisopropanol to obtain a spinning solution A; polydeoxyribonucleotide and procyanidine are mixed and dissolved in water, and a spinning solution B is obtained; performing electrostatic spinning on the spinning solution A and the spinning solution B to obtain the small-caliber artificial blood vessel. The PDRN and the PC are used for preparing the small-caliber artificial blood vessel at the same time, the preparation process is simple, use is convenient, the production cost is low, and the prepared artificial blood vessel has good biocompatibility, can promote endothelialization of the blood vessel and has the effect of resisting platelet adhesion.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and in particular relates to a small-caliber artificial blood vessel and a preparation method thereof. Background Art

[0002] In recent years, cardiovascular disease has become one of the leading causes of death, causing 1 / 3 of the deaths worldwide. The occurrence of these diseases is mainly due to vascular stenosis or occlusion, which leads to reduced blood flow, insufficient tissue nutrition supply, and further ischemic tissue damage. At present, the main methods for treating cardiovascular occlusion in clinical practice include drug thrombolysis, percutaneous coronary angioplasty, stent intervention, and vascular replacement. Although drug therapy and minimally invasive surgery have made great progress in recent years, vascular transplantation is still one of the effective methods for treating cardiovascular diseases, and it is even the only treatment for some patients. In this operation, it is sometimes difficult to obtain autologous blood vessels due to the patient's own conditions, so vascular transplantation plays an important role in clinical treatment. Vascular grafts are artificial blood vessels used to repair and replace diseased blood vessels. It is a vascular substitute that does not come from natural organs and tissues, also known as artificial blood vessels. Vascular grafts can be divided into two categories, one is a large-diameter artificial blood vessel with a diameter greater than 6 mm, and the other is a small-diameter artificial blood vessel with a diameter less than 6 mm. At present, large-diameter blood vessels made of materials such as polyester, expanded polytetrafluoroethylene, and polyurethane have been widely used in clinical practice. However, there are no small-caliber artificial blood vessel products in clinical practice.

[0003] The principle of electrospinning is to use high-voltage static electricity to stretch polymer melts or solutions into ultrafine fibers. A typical electrospinning device consists of a high-voltage power supply, a propeller, and a receiving device. Among them, the propeller pushes the solution or polymer melt at a certain speed, and the high-voltage power supply generates an electric field force and acts on the charged droplets. The charged droplets form Taylor under the interaction of electric field force and surface tension, and are then stretched into fibers and fall on the receiver to form a non-woven nanofiber membrane. Electrospinning technology is currently the most widely used method for preparing artificial blood vessels, accounting for about 49%, and is also a common method for preparing small-caliber blood vessels. Synthetic polymer materials are the most commonly used materials for preparing small-caliber vascular grafts due to their controllable sources, high quality stability, and simple processing. Ideal small-caliber blood vessels should have good biocompatibility, good mechanical properties, fast endothelialization speed, low incidence of restenosis and calcification, and more ideally, they can be reshaped as the vascular stent material degrades during vascular healing to form blood vessels with the same mechanical and biological properties as the host's own blood vessels. However, small-caliber blood vessels prepared from simple polymers cannot meet these requirements and often require certain modifications to improve their patency rates for clinical use.

[0004] Procyanidins (PC) are natural plant polyphenols, mainly composed of catechins and epicatechins as monomers, and are most abundant in grape seeds. Procyanidins have the characteristics of good water solubility, high bioavailability and low toxicity. They are natural antioxidants and free radical scavengers. In addition, they have pharmacological effects such as anti-atherosclerosis, anti-inflammatory, anti-tumor, anti-aging and anti-radiation. In the field of biomaterials and medical devices, procyanidins have gradually shown their advantages and have been applied. First of all, procyanidins can be used as cross-linking agents in biomaterials and medical devices. Procyanidins can form hydrogen bonds with aminocarbonyl groups in proteins through their phenolic hydroxyl groups, and have a strong affinity with proline-rich proteins; in addition, procyanidins also have a certain anti-platelet aggregation effect, which may inhibit the activity of NADPH oxidase, thereby affecting the content of platelet NO and superoxide anions, blocking the platelet activation process to a certain extent, and finally achieving the effect of anti-platelet aggregation.

[0005] Polydeoxyribonucleotide (PDRN) is an active polynucleotide mixture with a molecular weight range of 50 to 2000 kDa. Studies have shown that it has the effects of promoting cell proliferation and anti-apoptosis, inhibiting the expression of inflammatory factors, and promoting blood flow recovery. It has been used in a variety of tissue repairs, including skin wound repair, corneal injury repair, gastrointestinal mucosal injury repair, nerve damage repair, bone defect repair, etc.

[0006] At present, PDRN has not been used to prepare small-caliber artificial blood vessels, and PC and PDRN have not been used simultaneously to prepare small-caliber artificial blood vessels. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide a small-caliber artificial blood vessel and a preparation method thereof. Using PDRN and PC simultaneously for the preparation of small-caliber artificial blood vessels can improve the endothelialization efficiency of vascular endothelial cells and improve their blood compatibility.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for preparing a small-caliber artificial blood vessel, comprising the following steps:

[0010] 1) dissolving a solid polymer material in hexafluoroisopropanol to obtain a spinning solution A;

[0011] 2) dissolving the polydeoxyribonucleotide and proanthocyanidin in water to obtain a spinning solution B;

[0012] 3) The spinning solution A and the spinning solution B are subjected to electrospinning to obtain a small-caliber artificial blood vessel.

[0013] Preferably, the solid polymer material includes polycaprolactone, poly-L-lactide-caprolactone, polylactic acid, polylactic acid-glycolic acid copolymer, polyglycolic acid or polytrimethylene carbonate.

[0014] Preferably, the final concentration of the solid polymer material in the spinning solution A in step 1) is 5 to 15 wt %.

[0015] Preferably, in step 2), the final concentration of the polydeoxyribonucleotide in the spinning solution B is 0.2-3 mg / mL, and the final concentration of the proanthocyanidins in the spinning solution B is 2-5 mg / mL.

[0016] Preferably, the specific steps of the electrospinning in step 3) are:

[0017] Use two syringes to absorb spinning solution A and spinning solution B respectively, fix the two syringes on the propulsion pumps respectively, connect the two syringes to the double-channel section of the nozzle with the same nozzle with a silicone tube, connect the positive electrode of the high-voltage power supply to the single-channel section of the nozzle with the same nozzle, and connect the negative electrode to the receiving shaft, turn on the power to spin, and collect the spun fibers on the receiving shaft; dry the receiving shaft with the spun fibers collected, demould, and obtain a small-caliber artificial blood vessel.

[0018] Preferably, the propulsion speed of the propulsion pump is 5 to 15 μL / min; the voltage of the high-voltage power supply is 8 to 30 kV.

[0019] Preferably, the length of the single-channel needle is 5 to 10 cm; and the distance between the syringe and the receiving axis is 10 to 15 cm.

[0020] Preferably, the spinning environment temperature is 20-26° C., and the humidity is 10%-70%.

[0021] Preferably, the drying temperature is 30-40° C., and the drying time is 5-9 days.

[0022] The present invention also provides a small-caliber artificial blood vessel prepared by the preparation method, wherein the diameter of the small-caliber artificial blood vessel is less than 6 mm.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention uses PDRN and PC simultaneously for the preparation of small-caliber artificial blood vessels, has a simple preparation process, is easy to use, and has a low production cost. The prepared artificial blood vessels have good biocompatibility, can promote vascular endothelialization, and have an anti-platelet adhesion effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of a method for preparing a small-caliber artificial blood vessel;

[0026] Figure 2 It is the appearance morphology and electron microscope image of small-caliber artificial blood vessels (wherein, A is the appearance morphology of PLCL small-caliber artificial blood vessels loaded with PDRN and PC obtained by spinning, and B is the scanning electron microscope observation result of PLCL small-caliber artificial blood vessels loaded with PDRN and PC (PC concentration 4 mg / mL, PDRN concentration 1.0 mg / mL));

[0027] Figure 3 The results of the platelet adhesion test of small-caliber artificial blood vessels (A is the PLCL artificial blood vessel group, and B is the PLCL artificial blood vessel group loaded with PDRN and PC);

[0028] Figure 4 These are the results of a vascular endothelial cell proliferation test on small-caliber artificial blood vessels. DETAILED DESCRIPTION

[0029] The present invention provides a method for preparing a small-caliber artificial blood vessel, comprising the following steps:

[0030] 1) dissolving a solid polymer material in hexafluoroisopropanol to obtain a spinning solution A;

[0031] 2) dissolving the polydeoxyribonucleotide and proanthocyanidin in water to obtain a spinning solution B;

[0032] 3) Electrospinning the spinning solution A and the spinning solution B to obtain a small-caliber artificial blood vessel. Figure 1 shown.

[0033] In the present invention, a solid polymer material and hexafluoroisopropanol are mixed and dissolved to obtain a spinning solution A. The solid polymer material includes polycaprolactone (PCL), poly-L-lactide-caprolactone (PLCL), polylactic acid (PLA), polylactic acid-co-glycolic acid (PLGA), polyglycolic acid (PGA) or polytrimethylene carbonate (PTMC); the final concentration of the solid polymer material in the spinning solution A is preferably 5 to 15 wt%, and more preferably 7 to 14 wt%.

[0034] In the present invention, polydeoxyribonucleotides and proanthocyanidins are mixed and dissolved in water to obtain a spinning solution B. The final concentration of proanthocyanidins in the spinning solution B is preferably 2.0 to 5.0 mg / mL, more preferably 3.0 to 4.0 mg / mL; the final concentration of polydeoxyribonucleotides in the spinning solution B is preferably 0.2 to 3 mg / mL, more preferably 0.2 to 2.0 mg / mL.

[0035] In the present invention, spinning solution A and spinning solution B are electrospun to obtain a small-caliber artificial blood vessel. The specific steps of the electrospinning are: using two syringes to absorb the spinning solution A and the spinning solution B respectively, fixing the two syringes on the propulsion pumps respectively, connecting the two syringes with the double-channel section of the nozzle with the same nozzle by a silicone tube, connecting the positive electrode of the high-voltage power supply to the single-channel section of the nozzle with the same nozzle, and connecting the negative electrode to the receiving shaft, turning on the power supply to spin, and collecting the spun fibers on the receiving shaft; drying the receiving shaft on which the spun fibers are collected, demoulding, and obtaining a small-caliber artificial blood vessel. The propulsion speed of the propulsion pump is preferably 5-15μL / min, and more preferably 7-13μL / min; the length of the single-channel segment needle is preferably 5-10cm, and more preferably 6-9cm; the distance between the syringe and the receiving axis is preferably 10-15cm, and more preferably 11-14cm; the voltage of the high-voltage power supply is preferably 8-30kV, and more preferably 10-28kV; the ambient temperature during spinning is preferably 20-26°C, and more preferably 22-25°C, and the humidity is preferably 10%-70%, and more preferably 20%-60%; the drying temperature is preferably 30-40°C, and more preferably 32-38°C; the drying time is preferably 5-9 days, and more preferably 6-8 days.

[0036] The present invention also provides a small-caliber artificial blood vessel prepared by the preparation method, wherein the diameter of the small-caliber artificial blood vessel is less than 6 mm.

[0037] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0038] Example 1

[0039] Weigh PLCL, stir and dissolve it in hexafluoroisopropanol at room temperature to obtain a 10wt% PLCL solution (spinning solution A); weigh PC and PDRN, dissolve them in 60℃ water for injection, and the final concentrations of PC and PDRN are 4mg / mL and 1.0mg / mL respectively (spinning solution B). Use two syringes to absorb spinning solutions A and B, and then fix the two syringes on the propulsion pump respectively, connect the two syringes to the double-channel section of the nozzle with a silicone tube, connect the positive electrode of the high-voltage power supply to the single-channel section of the nozzle with the nozzle, adjust the needle length of the single-channel section to 5cm, and connect the negative electrode to the receiving shaft. Set the spinning parameters, the voltage is 17.0kV, the distance between the syringe and the receiving shaft is 13cm, and the propulsion speed of the propulsion pump is 15μL / min. At the same time, control the ambient temperature to 20-26℃, the humidity to 40%-60%, then turn on the power, and collect the electrospun fibers on the receiving shaft. After spinning, the receiving shaft that receives the spun fibers is placed in a vacuum drying oven at 37℃ and dried for 1 week. The prepared blood vessel material is demoulded from the receiving shaft to obtain a small-caliber artificial blood vessel.

[0040] Example 2

[0041] Weigh PTMC, stir and dissolve it in hexafluoroisopropanol at room temperature to obtain a 10wt% PTMC solution (spinning solution A); weigh PC and PDRN, dissolve them in 60℃ water for injection, and the final concentrations of PC and PDRN are 3mg / mL and 1.5mg / mL respectively (spinning solution B). Use two syringes to absorb spinning solutions A and B, and then fix the two syringes on the propulsion pump respectively, connect the two syringes to the double-channel section of the nozzle with a silicone tube, connect the positive electrode of the high-voltage power supply to the single-channel section of the nozzle with the nozzle, adjust the needle length of the single-channel section to 5cm, and connect the negative electrode to the receiving shaft. Set the spinning parameters, the voltage is 17.0kV, the distance between the syringe and the receiving shaft is 10cm, and the propulsion speed of the propulsion pump is 8μL / min. At the same time, control the ambient temperature to 20-26℃, the humidity to 50%-70%, then turn on the power, and collect the electrospun fibers on the receiving shaft. After spinning, the receiving shaft that receives the spun fibers is placed in a vacuum drying oven at 37℃ and dried for 1 week. The prepared blood vessel material is demoulded from the receiving shaft to obtain a small-caliber artificial blood vessel.

[0042] Example 3

[0043] Weigh PLGA, stir and dissolve it in hexafluoroisopropanol at room temperature to obtain a 5wt% PLGA solution (spinning solution A); weigh PC and PDRN, dissolve them in 60℃ water for injection, and the final concentrations of PC and PDRN are 2mg / mL and 0.2mg / mL (spinning solution B), respectively. Use two syringes to absorb spinning solutions A and B, and then fix the two syringes on the propulsion pump respectively, connect the two syringes to the double-channel section of the same nozzle with a silicone tube, connect the positive electrode of the high-voltage power supply to the single-channel section of the same nozzle, adjust the needle length of the single-channel section to 5cm, and connect the negative electrode to the receiving shaft. Set the spinning parameters, the voltage is 8.0kV, the distance between the syringe and the receiving shaft is 10cm, and the propulsion speed of the propulsion pump is 5μL / min. At the same time, control the ambient temperature to 20-26℃, the humidity to 50%-70%, then turn on the power, and collect the electrospun fibers on the receiving shaft. After spinning, the receiving shaft that receives the spun fibers is placed in a vacuum drying oven at 30℃ and dried for 5 days. The prepared blood vessel material is demoulded from the receiving shaft to obtain a small-caliber artificial blood vessel.

[0044] Example 4

[0045] Weigh PCL, stir and dissolve it in hexafluoroisopropanol at room temperature to obtain a 15wt% PCL solution (spinning solution A); weigh PC and PDRN, dissolve them in 60℃ water for injection, and the final concentrations of PC and PDRN are 5mg / mL and 3mg / mL respectively (spinning solution B). Use two syringes to absorb spinning solutions A and B, and then fix the two syringes on the propulsion pump respectively, connect the two syringes to the double-channel section of the same nozzle with a silicone tube, connect the positive electrode of the high-voltage power supply to the single-channel section of the same nozzle, adjust the needle length of the single-channel section to 10cm, and connect the negative electrode to the receiving shaft. Set the spinning parameters, the voltage is 30.0kV, the distance between the syringe and the receiving shaft is 15cm, and the propulsion speed of the propulsion pump is 15μL / min. At the same time, control the ambient temperature to 20-26℃, the humidity to 30%-60%, then turn on the power, and collect the electrospun fibers on the receiving shaft. After spinning, the receiving shaft that receives the spun fibers is placed in a vacuum drying oven and dried at 40℃ for 9 days. The prepared blood vessel material is demoulded from the receiving shaft to obtain a small-caliber artificial blood vessel.

[0046] Experimental Example 1

[0047] The surface structure of the small-caliber artificial blood vessel prepared in Example 1 was observed using a scanning electron microscope (SEM, Hitachi S-4800, Japan).

[0048] Experimental results: Figure 2 As shown, this small-caliber artificial blood vessel has a structure in which thick and thin fibers coexist.

[0049] Experimental Example 2

[0050] (1) Preparation of anticoagulated rabbit blood: Collect blood from the ear artery of New Zealand rabbits and add 3.8% sodium citrate solution at a ratio of 9:1 for anticoagulation. Then take 8 mL of fresh anticoagulated rabbit blood and mix it with 10 mL of 0.9% sodium chloride injection to obtain diluted anticoagulated rabbit blood;

[0051] (2) Centrifugation: Add 12 mL of anticoagulated rabbit blood to each 15 mL centrifuge tube and centrifuge twice. The first centrifugation speed is 1000 rpm, and 2 mL of supernatant is discarded. The second centrifugation speed is 2000 rpm to obtain platelet-rich plasma (PRP), and 5 mL is collected for later use;

[0052] (3) Platelet adhesion experiment: The PLCL artificial blood vessels without PDRN and PC loading and pure PLCL artificial blood vessels prepared in Example 1 were cut into discs with a diameter of 6 mm, respectively, and placed in a 96-well plate. 0.5 mL of PRP covering material was added to each well. After incubation at 37°C for 1 hour, the plates were rinsed 3 times with PBS buffer to remove unattached platelets. The plates were then fixed with Gluta fixative at 4°C for 12 hours, dehydrated with gradient alcohol, and finally the treated blood vessel samples were placed in a fume hood to dry naturally. The results were observed by SEM electron microscopy.

[0053] Experimental results: Figure 3 As shown, the results showed that the PLCL group had more platelet adhesion on the surface of the artificial blood vessels, but the PLCL artificial blood vessels loaded with PDRN and PC had less platelet adhesion on the surface.

[0054] Experimental Example 3

[0055] The experiment was divided into 3 groups, namely, pure PLCL artificial blood vessel group, PLCL artificial blood vessel group loaded with 4 mg / mL PC, and PLCL artificial blood vessel group loaded with 1.0 mg / mL PDRN and 4 mg / mL PC. The preparation methods were the same as those in Example 1. Before cell culture, the prepared vascular materials were cut into tubes with a length of 0.5 cm / root. After ultraviolet irradiation for 24 hours, the tubes were cut into 6 cm long tubes. 2 / mL ratio, and the cells were extracted with DMEM medium for 24 hours. EA.hy926 cells were cultured at 1×10 5 Cells were seeded at a density of 100 μL / mL in a 96-well plate. After cell seeding, the samples were incubated at 37°C, 5% CO2. Cell proliferation was assessed at 24 hours using the CCK-8 assay.

[0056] Experimental results: Figure 4As shown, the results showed that there was no significant difference in cell survival rate between the PLCL artificial blood vessel group loaded with PC and the PLCL group artificial blood vessels (P>0.05), while the cell survival rate of the PLCL artificial blood vessel group loaded with PDRN and PC was significantly increased compared with the pure PLCL group artificial blood vessels (P<0.05), showing the effect of promoting endothelialization.

[0057] It can be seen from the above embodiments and experimental examples that the small-caliber artificial blood vessel prepared by the present invention can improve the endothelialization efficiency of vascular endothelial cells, improve its blood compatibility, and has the effect of resisting platelet adhesion.

[0058] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a small-caliber artificial blood vessel, characterized in that: The following steps are involved: 1) dissolving a solid polymer material in hexafluoroisopropanol to obtain a spinning solution A; 2) dissolving the polydeoxyribonucleotide and proanthocyanidin in water to obtain a spinning solution B; 3) The spinning solution A and the spinning solution B are subjected to electrospinning to obtain a small-caliber artificial blood vessel.

2. The preparation method according to claim 1, characterized in that: The solid polymer material includes polycaprolactone, poly-L-lactide-caprolactone, polylactic acid, polylactic acid-glycolic acid copolymer, polyglycolic acid or polytrimethylene carbonate.

3. The preparation method according to claim 1, characterized in that: Step 1) The final concentration of the solid polymer material in the spinning solution A is 5 to 15 wt%.

4. The preparation method according to claim 1, characterized in that: Step 2) The final concentration of polydeoxyribonucleotides in the spinning solution B is 0.2-3 mg / mL, and the final concentration of proanthocyanidins in the spinning solution B is 2-5 mg / mL.

5. The preparation method according to claim 1, characterized in that: Step 3) The specific steps of the electrospinning are: Use two syringes to absorb spinning solution A and spinning solution B respectively, fix the two syringes on the propulsion pumps respectively, connect the two syringes to the double-channel section of the nozzle with the same nozzle with a silicone tube, connect the positive electrode of the high-voltage power supply to the single-channel section of the nozzle with the same nozzle, and connect the negative electrode to the receiving shaft, turn on the power to spin, and collect the spun fibers on the receiving shaft; dry the receiving shaft with the spun fibers collected, demould, and obtain a small-caliber artificial blood vessel.

6. The preparation method according to claim 5, characterized in that: The propulsion speed of the propulsion pump is 5 to 15 μL / min; the voltage of the high-voltage power supply is 8 to 30 kV.

7. The preparation method according to claim 5, characterized in that: The length of the single-channel needle is 5 to 10 cm; the distance between the syringe and the receiving axis is 10 to 15 cm.

8. The preparation method according to claim 5, characterized in that: The spinning environment temperature is 20-26° C., and the humidity is 10%-70%.

9. The preparation method according to claim 5, characterized in that: The drying temperature is 30-40° C., and the drying time is 5-9 days.

10. The small-caliber artificial blood vessel prepared by the preparation method according to any one of claims 1 to 9, characterized in that: The diameter of the small-caliber artificial blood vessel is less than 6 mm.

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