Long-acting functionalized double-layer bionic small-caliber vascular prosthesis, and preparation method therefor and use thereof

Abstract

Disclosed in the present invention are a long-acting functionalized double-layer bionic small-caliber vascular prosthesis, and a preparation method therefor and use thereof. The preparation method therefor comprises: separately synthesizing degradable polyurethane elastomers PCHU-DSeSe and PCHU-DTA using a three-step solution polymerization method, and carrying out near-field direct-writing electrospinning of PCHU-DSeSe on a stainless steel mandrel with an axial groove to obtain an axially oriented fibrous vascular inner layer; assembling the axially oriented fibrous vascular inner layer together with the mandrel into a polytetrafluoroethylene mold having radially oriented patterns on the inner wall of the housing; rapidly casting a PCHU-DTA / DMSO solution into the mold for phase separation to obtain a vascular outer layer of an axis-aligned topological microstructure, and carrying out freeze-drying to obtain the long-acting functionalized double-layer bionic small-caliber vascular prosthesis. The long-acting functionalized double-layer bionic small-caliber vascular prosthesis has the functions of continuous release of DSS, NO and H2S, and in-situ dynamic regulation and repair of a vascular tissue microenvironment, can be used for arteriovenous fistula creation, and coronary artery and peripheral vascular bypass therapy, and thus has good application prospects.

Description

Long-acting functionalized double-layer biomimetic small-diameter artificial blood vessel and preparation method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biomaterials for vascular tissue engineering, and particularly relates to a long-acting functionalized double-layer biomimetic small-diameter artificial blood vessel and a preparation method and application thereof. BACKGROUND

[0002] Cardiovascular disease (CVD) has been a major cause of death worldwide, and the World Heart Report 2023 released by the World Heart Federation (WHF) shows that the number of deaths caused by CVD accounted for about one-third of the total number of deaths worldwide in 2021. In China, the prevalence of CVD is also on the rise, and it ranks first in the proportion of disease deaths among urban and rural residents. Clinically, small-diameter vascular injury and disease are very common, and replacing the diseased blood vessel through vascular graft surgery is an important means to treat cardiovascular and orthopedic vascular injury diseases. However, small-diameter artificial blood vessels (inner diameter ≤ 6 mm) have not yet been commercially available due to slow blood flow, easy thrombosis, and low long-term patency rate. Therefore, it is very important and urgent to develop a small-diameter artificial blood vessel with long-term anticoagulant and anti-proliferative properties and a long-term patency rate.

[0003] In the process of vascular tissue regeneration, regulating the physiological microenvironment of damaged or diseased blood vessels is a crucial link to promote vascular tissue regeneration and functional remodeling. Danshensu (DSS) can dynamically regulate oxidative stress, and seleno-compounds can catalyze the continuous release of NO from endogenous donor nitrosothiol (RSNO), thereby inhibiting platelet adhesion / activation and promoting damaged endothelial tissue repair. At the same time, NO can promote the endogenous synthesis of H2S in smooth muscle cells, and H2S can further promote the decomposition and release of NO from RSNO. Currently, there is no report on a biodegradable polyurethane small-diameter artificial blood vessel that has good comprehensive mechanical properties and can dynamically regulate the "full life cycle" release of DSS, NO, or H2S in situ based on "bulk copolymerization".

[0004] Small-diameter artificial blood vessels based on biodegradable polyurethane modification have excellent mechanical compliance and strength, and have significant advantages in terms of process, cost, production conditions, and storage conditions, and can be used for in vivo induction of vascular tissue regeneration. However, the functional biomimetic modification of existing artificial blood vessels cannot achieve controlled release of functional factors, often resulting in early rapid release leading to late functional deficiency, or limited surface grafting sites making it difficult to load more functional factors, and the degradation of non-functionalized surface materials in the early stage leads to the degradation of the early-formed endothelium, making it difficult to achieve long-distance vascular tissue remodeling. Therefore, there is an urgent need for long-acting functional modification of small-diameter artificial blood vessel materials to address the pathological remodeling problems caused by insufficient functionalization.

[0005] In summary, it is of great significance to prepare a long-acting functional double-layer biomimetic small-diameter artificial blood vessel that can dynamically respond to repair the microenvironment in the field of medical devices and biological medicine. Its application and promotion can fill the market gap of current small-diameter artificial blood vessels, alleviate the tense situation of clinical small-diameter blood vessel transplantation, and bring huge economic and social benefits.

[0006] SUMMARY

[0007] In order to solve the above problems, the main purpose of the present application is to provide a preparation method of long-acting functional double-layer biomimetic small-diameter artificial blood vessel, which comprises synthesizing a mechanically matched degradable polyurethane artificial blood vessel material capable of long-acting release of active components by a three-step solution polymerization method based on the principle of "bulk copolymerization", spinning an axially oriented fiber blood vessel inner layer by near-field direct writing, and preparing an axially oriented topological microstructure blood vessel outer layer by a special mold combined with a thermal phase separation technique.

[0008] Another purpose of the present application is to provide a long-acting functional double-layer biomimetic small-diameter artificial blood vessel prepared by the preparation method of the long-acting functional double-layer biomimetic small-diameter artificial blood vessel, which has the functions of continuously releasing DSS, NO and H2S with interactive strengthening effect and dynamically regulating and repairing the microenvironment of blood vessel tissue in the "whole life cycle".

[0009] Still another purpose of the present application is to provide the application of the long-acting functional double-layer biomimetic small-diameter artificial blood vessel as or in the preparation of medical products that can dynamically regulate and repair the microenvironment.

[0010] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0011] The present application provides a preparation method of long-acting functional double-layer biomimetic small-diameter artificial blood vessel, comprising the following steps:

[0012] (1) Based on bulk copolymerization, degradable polyurethane elastomers PCHU-DSeSe and PCHU-DTA are synthesized by a three-step solution polymerization method, respectively. The monomers include soft segment diol and diisocyanate, and also include chain extender, so that the weight average molecular weight of PCHU-DSeSe and PCHU-DTA elastomers is kept between 8-10 million, and the viscosity coefficient is kept between 1.2-1.8;

[0013] (2) PCHU-DSeSe in step (1) is obtained by near-field direct writing electrospinning on a stainless steel shaft core with axial grooves in the mold, to obtain PCHU-DSeSe oriented fibers as the axially oriented fiber blood vessel inner layer, the fiber diameter is controlled between 0.1-80 μm, and the fiber and shaft core are placed in a vacuum drying box for drying for at least 2 days;

[0014] (3) Assembling the composite of step (2) together with the axial oriented fiber vascular inner layer to the polytetrafluoroethylene mold with radially oriented texture on the inner wall of the shell;

[0015] (4) Weighing PCHU-DTA, dissolving in dimethyl sulfoxide (DMSO) at 50-60℃, stirring to dissolve, obtaining a clear and uniform solution of 8-16% (mass volume ratio, w / v, g / mL), which is quickly cast into the polytetrafluoroethylene mold with oriented fiber layer in step (3), and quickly placed at-80℃ to cause thermally induced phase separation, with a placement time of 48h or more, to obtain an axially oriented topological microstructure vascular outer layer;

[0016] (5) Taking out the mold, soaking in a 0℃ ultrapure ice water mixture for 10min after removing the shell of the mold, obtaining an artificial blood vessel, and taking it off from the axis of the mold, continuing to soak in a 0℃ ultrapure ice water mixture, changing the water every 4h, a total of 6-10 times; taking out the artificial blood vessel from the deionized water and freeze-drying for 3 days or more, obtaining a long-acting functionalized double-layer biomimetic small-diameter artificial blood vessel.

[0017] Preferably, in step (1), the molar ratio of the soft segment diol, diisocyanate and chain extender in the synthesis of the degradable polyurethane elastomer PCHU-DSeSe and PCHU-DTA is 1:2:1.

[0018] Preferably, in step (1), the soft segment diol is selected from one or more of polycaprolactone diol (HO-PCL-OH), polycarbonate diol (HO-PC-OH), polylactic acid diol (HO-PLLA-OH), poly(lactic acid-glycolic acid) diol (HO-PLGA-OH), poly(lactic acid-caprolactone) diol (HO-PLCL-OH), poly(glycolic acid-caprolactone) diol (HO-PGCL-OH).

[0019] Preferably, in step (1), the diisocyanate is selected from one or more of toluene diisocyanate (TDI), isophorone diisocyanate (IPDI), diphenylmethane diisocyanate (MDI), dicyclohexylmethane diisocyanate (HMDI), hexamethylene diisocyanate (HDI), lysine diisocyanate (LDI).

[0020] Preferably, in step (1), the chain extender used in the synthesis of the degradable polyurethane elastomer PCHU-DSeSe and PCHU-DTA includes chain extender 1 and chain extender 2; wherein the chain extender 1 used in the synthesis of PCHU-DSeSe is Danshensu (DSS), and the chain extender 2 is selected from one or more of 2-(2-hydroxyethyl diselenide) ethanol (SeDO), selenocystamine hydrochloride (SeCA), 3,3'-diselenodipropionic acid (SeDPA), 4,4'-dithio-bis(4,1-phenylene) dimethanol, bis(4-carboxyphenyl) diselenide, bis(4-aminophenyl) diselenide); the chain extender 1 used in the synthesis of PCHU-DTA is Danshensu (DSS), and the chain extender 2 is O-hydroxymethyl-2-hydroxythioacetate (TA); the order of addition of the chain extenders is: first add chain extender 1 and then add chain extender 2.

[0021] More preferably, in step (1), the molar ratio of chain extender 1 to chain extender 2 used in the synthesis of PCHU-DSeSe is 1:99 to 99:1.

[0022] More preferably, in step (1), the molar ratio of chain extender 1 to chain extender 2 used in the synthesis of PCHU-DTA is 1:99 to 99:1.

[0023] Preferably, in step (1), during the synthesis of PCHU-DSeSe and PCHU-DTA, the reaction temperature of the soft segment diol and diisocyanate is 50 to 80°C, the stirring rate is 80 to 250 rpm, and the reaction time is 2 to 3 h.

[0024] Preferably, in step (1), during the synthesis of PCHU-DSeSe and PCHU-DTA, DSS is added dropwise after being dissolved in DMSO, the concentration of the DSS / DMSO solution is controlled at 0.02 to 0.01 g / mL, and the optimal concentration is 0.04 g / mL; the dropwise addition rate is 8 to 12 mL / h, and the optimal rate is 10 mL / h.

[0025] Preferably, in step (1), during the synthesis of PCHU-DSeSe and PCHU-DTA, the stirring rate after adding the chain extender is controlled at 500 to 800 rpm, the reaction time after adding DSS is controlled at 3 to 5 h, triethylamine (TEA) is added as an acid-binding agent after the chain extender 2 is dissolved in DMAc, then the chain extender 2 / DMAc / TEA is added dropwise into the prepolymer, 0.1 mL of TEA is added per milliliter of the chain extender 2 / DMAc solution, the concentration of the chain extender 2 / DMAc solution is 0.02 to 0.01 g / mL, the dropwise addition rate is 8 to 12 mL / h, and the reaction time in each reaction is 8 to 24 h.

[0026] Preferably, in step (2), when the near-field direct writing electrospinning is used to prepare the axially oriented fiber intravascular layer, the rotational speed of the core is 10-18 rpm, and the moving speed of the needle is 800-1200 mm / min.

[0027] Preferably, the size of the inner and outer layers of the long-acting functionalized double-layer biomimetic small-diameter artificial blood vessel is controlled and adjusted through electrospinning and a mold, and is controlled to be: an inner diameter (d1) of 1.2-6 mm, a total wall thickness (T) of 0.1-3 mm, an inner layer PCHU-DSeSe oriented fiber thickness (t1) of 0.05-1.5 mm, an outer layer oriented topological microstructure PCHU-DTA porous material thickness (t2) of 0.05-2 mm, and an artificial blood vessel length (L) controlled to be 0.5-25 cm; the outer layer thread form is triangular or trapezoidal, the ratio of the thread height (h) to the total wall thickness (T) of the artificial blood vessel is 1:5-10, the thread number is 1-2, the pitch is 0.1-2 mm, and the lead is 0.1-4 mm.

[0028] The application also provides a long-acting functionalized double-layer biomimetic small-diameter artificial blood vessel prepared by the preparation method of the long-acting functionalized double-layer biomimetic small-diameter artificial blood vessel.

[0029] The application also provides application of the long-acting functionalized double-layer biomimetic small-diameter artificial blood vessel as or in the preparation of a medical product for arteriovenous fistula, heart coronary and peripheral vascular bypass treatment.

[0030] Preferably, the long-acting functionalized double-layer biomimetic small-diameter artificial blood vessel has the functions of continuously releasing DSS, NO and H2S in the "whole life cycle" and dynamically regulating and repairing the vascular tissue microenvironment in situ.

[0031] Compared with the prior art, the application has at least the following beneficial effects:

[0032] 1. The application is based on the principle of bulk copolymerization, and two kinds of degradable polyurethane elastomers PCHU-DSeSe and PCHU-DTA are prepared by a three-step solution polymerization method, which has good biocompatibility, processability and mechanical elasticity, and the preparation cost is low, the process is simple and the performance is controllable.

[0033] 2. The long-acting functionalized double-layer biomimetic small-diameter artificial blood vessel in the application has the functions of continuously releasing DSS and NO in the inner layer and continuously releasing DSS and H2S in the outer layer, and the active molecules released in the inner and outer layers can interact to enhance the effect, realize mutual corresponding linkage and synergistic effect, and dynamically regulate and repair the vascular tissue microenvironment in situ, so as to quickly promote vascular tissue remodeling.

[0034] 3、The long-acting functional double-layer biomimetic small-caliber artificial blood vessel in the application realizes the recruitment of endothelial cells, promotes the migration and proliferation of endothelial cells by the axial orientation of the inner surface of the fibers obtained by electrospinning; the oriented porous structure of the outer surface obtained by thermal phase separation not only improves the bending resistance of the blood vessel, but also dynamically regulates the migration and proliferation of smooth muscle cells; the inner and outer layers of the artificial blood vessel can continuously release DSS, respond to inflammatory and oxidative stress microenvironment, effectively inhibit related adverse reactions, and protect endothelial cells and smooth muscle cells from damage.

[0035] 4、The long-acting functional double-layer biomimetic small-caliber artificial blood vessel in the application can be used for arteriovenous fistula, heart coronary and peripheral vascular bypass treatment, the oriented fibers of the inner layer PCHU-DSeSe of the blood vessel can catalyze the continuous release of NO from the endogenous NO source in situ, and the oriented topological microstructure PCHU-DTA of the outer layer can controllably degrade the porous polyurethane material to release H2S, NO and H2S, which can interactively promote and regulate the function of the blood vessel.

[0036] 5、The long-acting functional double-layer biomimetic small-caliber artificial blood vessel in the application has adjustable mechanical compliance, burst strength and biocompatibility, has no hypersensitivity reaction to the human body during transplantation surgery, has affinity with blood and surrounding tissues in the human body, can realize the regulation of active small molecule release in the whole degradation cycle after transplantation, can quickly and stably remodel the endothelial cell layer to realize long-acting anticoagulation, dynamically regulate the repair microenvironment, quickly realize the regeneration and functional remodeling of long-distance small-caliber vascular tissues, and has good application prospect in cardiovascular surgery, hemodialysis center, nephrology department and orthopedic repair surgery. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is a synthesis reaction schematic diagram of the degradable polyurethane elastomer PCHU-DSeSe for preparing the oriented fibers of the inner layer of the artificial blood vessel in Example 1 by the "three-step solution polymerization method".

[0038] Figure 2 is a synthesis reaction schematic diagram of the degradable polyurethane elastomer PCHU-DTA for preparing the oriented topological microstructure porous material of the outer layer of the artificial blood vessel in Example 1 by the "three-step solution polymerization method".

[0039] Figure 3 is a preparation process schematic diagram of the long-acting functional double-layer biomimetic small-caliber artificial blood vessel in the example.

[0040] Figure 4 is a biological regulation mechanism schematic diagram of the long-acting functional double-layer biomimetic small-caliber artificial blood vessel in the example.

[0041] Figure 5 is the long-acting functional double-layer biomimetic small-caliber artificial blood vessel prepared in the example and its X-ray diffraction electron microscope diagram, wherein Figure 5A is a physical diagram of the long-acting functional double-layer biomimetic small-caliber artificial blood vessel prepared, Figure 5B is a cross-sectional view, Figure 5C is a longitudinal section view, 5D is a cross-sectional view of the lumen, and Figure 5E is an outer surface scanning view. DETAILED DESCRIPTION

[0042] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Example 1

[0044] In this example, a long-lasting functionalized double-layer biomimetic small-caliber artificial blood vessel is prepared. The monomer soft segment diol used in the synthesis of the degradable polyurethane elastomer PCHU-DSeSe is HO-PCL-OH, the diisocyanate is HDI, and the chain extender 2 is SeDO. The molar ratio of chain extender 1 to chain extender 2 is 1:3. The monomer soft segment diol used in the synthesis of the degradable polyurethane elastomer PCHU-DTA is HO-PCL-OH, the diisocyanate is HDI, and the molar ratio of DSS to TA is 1:3. The steps are as follows:

[0045] (1) The specific operation of preparing degradable polyurethane elastomers PCHU-DSeSe and PCHU-DTA by three-step solution polymerization based on the principle of bulk copolymerization is as follows: weigh 5g of polycaprolactone diol (PCL diol, Mn is 2000) in a 250mL three-necked flask, stir at 180rpm at 60℃ for 10min; then heat to 120℃ and remove water in vacuum for 30min, then pass nitrogen protection and cool to 60℃; take 0.81mL HDI and add it to the three-necked flask to react for 2.5h; then cool to 45℃, adjust the speed to 560rpm, weigh 0.12g DSS and dissolve it completely with 5mL DMSO and add it drop by drop into the three-necked flask, add it in about 30min, continue stirring and react for 3h, and add about 10mL DMSO during the reaction according to the change of solution viscosity; weigh 0.47g of SeDO and dissolve it with 10mL DMSO and add it drop by drop into the three-necked flask to continue reacting for 12h, and add about 15mL After stopping the reaction, the polymer was precipitated with deionized water and solvent exchanged for 48 hours, with the deionized water replaced every 6 hours for a total of eight times to thoroughly remove the DMSO. The washed material was then frozen at -80°C for 3 hours and freeze-dried to obtain PCHU-DSeSe. The preparation process for PCHU-DTA was similar to that for PCHU-DSeSe, except that 0.47g of SeDO was replaced with 0.23g of TA, DMAc was used as the reaction solvent, and TEA was added to adjust the reaction pH. PCHU-DTA was finally prepared.

[0046] (2) Take 0.35 g PEO and dissolve in 10 mL deionized water, completely dissolved, to obtain a release layer spinning solution, and obtain a uniform PEO release layer by near-field direct writing on a shaft core with a diameter of 3 mm, and place overnight to volatilize the water; take 0.8 g PCHU-DSeSe and dissolve in 10 mL hexafluoroisopropanol (HFIP), completely dissolved, and then electrospun under the conditions of a shaft core rotating speed of 10 rpm and a needle moving speed of 1000 mm / min, and after vacuum drying, an axially oriented artificial blood vessel inner layer with a thickness of 0.4 mm is obtained.

[0047] (3) The axially oriented fiber tube in step (2) is assembled into a mold shell with an inner diameter of 5 mm, and a polytetrafluoroethylene mold with a radially oriented texture (triangular thread, thread count 1, pitch 1 mm, thread height 0.2 mm) is prepared; then take 0.8 g of PCHU-DTA on an analytical balance and dissolve in 10 mL of DMSO at 60°C, stir to dissolve, and obtain a clear and uniform solution. Then, the mixed solution is quickly cast into the specially designed polytetrafluoroethylene mold with the oriented fiber layer, and quickly placed at -80°C to phase separate, and the placement time is 48 h or more. Then take out the mold, remove the mold shell and soak in a 0°C ultrapure ice water mixture, after 10 min, the artificial blood vessel is removed from the shaft core of the mold, and continues to soak in the 0°C ultrapure ice water mixture, and the water is exchanged every 4 h, a total of 6-10 times. After taking the artificial blood vessel out of the deionized water, freeze-drying for 3 days or more, a double-layer biomimetic small-diameter artificial blood vessel with an inner diameter of about 3 mm and a thickness of about 1 mm is obtained.

[0048] Example 2

[0049] This example prepares a long-acting functionalized double-layer biomimetic small-diameter artificial blood vessel, and the degradable polyurethane elastomer PCHU-DSeSe is synthesized by using HO-PC-OH as the soft segment monomer, HDI as the diisocyanate, and SeDO as the chain extender 2, and the molar ratio of chain extender 1 to chain extender 2 is 1:1; PCHU-DTA is synthesized by using HO-PCL-OH as the soft segment monomer, HDI as the diisocyanate, and the molar ratio of DSS to TA is 1:1, and the operation steps are as follows:

[0050] (1) The specific operation of preparing PCHU-DSeSe and PCHU-DTA based on the principle of bulk copolymerization three-step solution polymerization is as follows: weigh 5 g of polycarbonate diol (PC diol, Mn ~ 2000) in a 250 mL three-necked flask, stir at 60°C and 180 rpm for 10 min; then heat to 120°C and vacuum for 30 min, then protect with nitrogen and cool to 60°C; take 0.81 mL of HDI and add it to the three-necked flask and react for 2.5 h; then cool to 45°C, adjust the stirring speed to 560 rpm, weigh 0.24 g of DSS, completely dissolve it in 10 mL of DMSO and add it dropwise into the three-necked flask, about 50 min for dropwise addition, continue to stir for 3 h, and add about 10 mL of DMSO according to the change of solution viscosity during the reaction; then weigh 0.11 g of SeDO, dissolve it in 6 mL of DMSO and add it dropwise into the three-necked flask, continue to react for 12 h, and add about 15 mL of DMSO according to the change of viscosity during the reaction; stop the reaction, precipitate the polymer with deionized water and perform solvent exchange for 48 h, replace the deionized water every 6 h, a total of 8 times to completely wash away the DMSO; then take out the washed material, freeze it at -80°C for 3 h, and obtain PCHU-DSeSe by freeze-drying. The preparation process of PCHU-DTA is the same as that of PCHU-DSeSe, except that 0.47 g of SeDO is changed to 0.15 g of TA, the reaction solvent is DMAc, and the pH value of the reaction needs to be adjusted by adding TEA, and finally PCHU-DTA is prepared.

[0051] (2) Weigh 0.35 g of PEO and dissolve it in 10 mL of deionized water to obtain a demolding layer spinning solution, obtain a uniform PEO demolding layer on a 3 mm diameter shaft core by near-field direct writing, and place it overnight to volatilize the water; weigh 1 g of PCHU-DSeSe and dissolve it in 10 mL of hexafluoroisopropanol (HFIP), completely dissolve it, and then spin the PCHU-DSeSe / HFIP solution under the conditions of a shaft core speed of 12 rpm and a needle moving speed of 1200 mm / min to obtain an axially oriented artificial blood vessel inner layer with a thickness of 0.4 mm after vacuum drying.

[0052] (3) The step (2) of the composite together with the axial orientation of the core of the fiber tube is assembled into the inner diameter of 5 mm mold shell, the inner wall has a radial orientation of the polytetrafluoroethylene mold with the texturing (triangular thread, line number is 1, pitch is 1 mm, thread height 0.2 mm) ready; Then 1g of PCHU-DTA is weighed on an analytical balance, dissolved in 10 mL of DMSO at 60 DEG C, and stirred to obtain a clear and uniform solution. Subsequently, the mixed solution is quickly cast into the specific model of the polytetrafluoroethylene mold with the oriented fiber layer, quickly placed at -80 DEG C to make it phase separation, and placed for more than 48 h. Then the mold is taken out, the shell of the mold is removed, and then soaked in a 0 DEG C ultrapure ice water mixture. After 10 min, the artificial blood vessel is removed from the core of the mold and continues to be soaked in a 0 DEG C ultrapure ice water mixture. The solvent is replaced every 4 h, a total of 6-10 times. The artificial blood vessel is taken out of the deionized water and freeze-dried for more than 3 days to obtain a double-layer biomimetic small-diameter artificial blood vessel with an inner diameter of about 3 mm and a thickness of about 1 mm.

[0053] Comparative Example 1

[0054] The difference between this comparative example and Example 1 is that the molar ratio of chain extender 1 to chain extender 2 in the synthesis of PCHU-DSeSe is 15:85; and the molar ratio of DSS to TA in the synthesis of PCHU-DTA is 15:85.

[0055] Comparative Example 2

[0056] The difference between this comparative example and Example 1 is that the molar ratio of chain extender 1 to chain extender 2 in the synthesis of PCHU-DSeSe is 35:65; and the molar ratio of DSS to TA in the synthesis of PCHU-DTA is 35:65.

[0057] Comparative Example 3

[0058] The difference between this comparative example and Example 1 is that the molar ratio of chain extender 1 to chain extender 2 in the synthesis of PCHU-DSeSe is 1:1; and the molar ratio of DSS to TA in the synthesis of PCHU-DTA is 1:1.

[0059] Comparative Example 4

[0060] The difference between this comparative example and Example 1 is that the molar ratio of chain extender 1 to chain extender 2 in the synthesis of PCHU-DSeSe is 65:35; and the molar ratio of DSS to TA in the synthesis of PCHU-DTA is 65:35.

[0061] Comparative Example 5

[0062] The present comparative example differs from Example 1 in that the molar ratio of chain extender 1 to chain extender 2 in the synthesis of PCHU-DSeSe is 75:25; the molar ratio of DSS to TA in the synthesis of PCHU-DTA is 75:25.

[0063] Comparative Example 6

[0064] The present comparative example differs from Example 1 in that the molar ratio of chain extender 1 to chain extender 2 in the synthesis of PCHU-DSeSe is 85:15; the molar ratio of DSS to TA in the synthesis of PCHU-DTA is 85:15.

[0065] Performance test

[0066] The long-acting functionalized double-layer biomimetic small-diameter artificial blood vessels prepared in the above examples and comparative examples were respectively subjected to radial mechanical tensile test and endothelial coverage rate statistics, and the fiber orientation of the inner layer was observed by scanning electron microscope (SEM).

[0067] Radial mechanical tensile test

[0068] All samples of the examples and comparative examples were cut into the same length (5 mm) for radial mechanical tensile test. Before the tensile test, all samples were respectively immersed in 0.01 M phosphate buffer solution (PBS, pH = 6.8) and soaked at 37°C for 24 h. A tensile testing machine (HY-940FS) was used, radial uniaxial tension test was adopted, the sensor load range was 0-200 N, and the tensile speed was 1 mm / min.

[0069] Endothelial coverage test

[0070] Test method: The PCHU-DSeSe of the examples and comparative examples was prepared into an oriented fiber membrane of 24-well size, sterilized and laid on the bottom of a 24-well plate, a special sterile 316L stainless steel strip was overlaid on the fiber membrane in each well of the 24-well plate, and the well was divided into two parts. Then, HUVECs primary cells were evenly inoculated on the sample membrane in the 24-well plate (1 x 10 5 After 12 h of culture, the steel strip was removed, and the culture was continued for 48 h. After 48 h, the cells were fixed with 4% paraformaldehyde for more than 2 h, washed with PBS for 3-5 times; rhodamine-labeled phalloidin was added and incubated at room temperature for 1.5 h, washed with PBS for 3-5 times; DAPI staining solution was added and incubated in the dark for 5 min, washed with PBS for 3-5 times. Finally, the cell migration morphology and the migration distance of the stained cell nucleus compared with the original state were observed under an inverted fluorescence microscope, and the percentage of cell coverage was calculated by Image J software, the endothelial coverage percentage (%) = cell coverage area / well plate reserved original area x 100%.

[0071] Table 1: Example and comparative example performance test results

[0072] As shown in Table 1, only by controlling the ratio of DSS and chain extender 2 to TA and the processing parameters, can the long-acting functionalized double-layer biomimetic small-diameter artificial blood vessels capable of effectively exerting the synergistic effect of NO and H2S and matching in mechanical properties be prepared by the three-step solution polymerization method, electrospinning and thermally induced phase separation technology, which can stably and long-actingly regulate the vascular transplantation microenvironment in situ and dynamically, realize the rapid endothelialization effect, shorten the vascular remodeling period and realize long-distance vascular tissue regeneration.

[0073] The above describes the preferred embodiment of the present application, but the present application should not be limited to the content disclosed in the embodiment. Therefore, any equivalent or modification completed without departing from the disclosed spirit of the present application falls within the scope of protection of the present application.

Claims

1. A method for preparing a long-acting functionalized double-layer biomimetic small-caliber artificial blood vessel, characterized in that, The method comprises the following steps: (1) synthesizing degradable polyurethane elastomers PCHU-DSeSe and PCHU-DTA by a three-step solution polymerization method based on bulk copolymerization, the monomers including soft segment diol and diisocyanate, and further including chain extender; The weight average molecular weight of PCHU-DSeSe and PCHU-DTA is 80-100 thousand, and the viscosity coefficient is 1.2-1.8; (2) obtaining PCHU-DSeSe oriented fibers as axial oriented fiber inner layer of blood vessel by near-field direct writing electrospinning of PCHU-DSeSe in step (1) on a stainless steel shaft core with axial grooves of a mold, the fiber diameter being 0.1-80 μm, and the fiber and shaft core being vacuum dried as a whole for at least 2 days; (3) assembling the axial oriented fiber inner layer of blood vessel in step (2) together with the shaft core into a polytetrafluoroethylene mold with radially oriented grooves on the inner wall of the shell; (4) weighing PCHU-DTA, dissolving it in DMSO at 50-60 ℃, stirring to obtain a clear and uniform solution with a concentration of 8-16 w / v %, and rapidly casting the solution into the polytetrafluoroethylene mold assembled in step (3), and rapidly placing it at-80 ℃ to cause thermal induced phase separation, and placing it for not less than 48 h to obtain an axially oriented topological microstructure outer layer of blood vessel; (5) taking out the mold, immersing it in a 0 ℃ ultrapure ice water mixture for 10 min after removing the mold shell to obtain a preformed artificial blood vessel, taking it off the shaft core of the mold, and continuing to immerse it in the 0 ℃ ultrapure ice water mixture, changing the water every 4 h, a total of 6-10 times, and taking out the artificial blood vessel from the 0 ℃ ultrapure ice water, and freeze-drying it for not less than 3 days to obtain a long-acting functionalized double-layer biomimetic small-diameter artificial blood vessel.

2. The method for preparing a long-lasting functionalized double-layer bionic small-caliber artificial blood vessel according to claim 1, characterized in that: In step (1), the molar ratio of the soft segment diol, diisocyanate and chain extender is 1:2:

1.

3. The method for preparing a long-lasting functionalized double-layer bionic small-caliber artificial blood vessel according to claim 1, characterized in that: In step (1), the soft segment diol is selected from one or more of HO-PCL-OH, HO-PC-OH, HO-PLLA-OH, HO-PLGA-OH, HO-PLCL-OH and HO-PGCL-OH; and / or the diisocyanate is selected from one or more of TDI, IPDI, MDI, HMDI, HDI and LDI; and / or the chain extender used in the synthesis of PCHU-DSeSe and PCHU-DTA includes chain extender 1 and chain extender 2; the chain extender 1 used in the synthesis of PCHU-DSeSe is danshensu molecule (DSS), and the chain extender 2 is selected from one or more of 2-(2-hydroxyethyl diselenide) ethanol, selenocystamine hydrochloride, 3,3'-bis(seleno) dipropionic acid, 4,4'-dithio-bis(4,1-phenylene) dimethanol, bis(4-carboxyphenyl) diselenide and bis(4-aminophenyl) diselenide; the chain extender 1 used in the synthesis of PCHU-DTA is DSS, and the chain extender 2 is TA; The addition sequence of the chain extender is: first adding the chain extender 1 and then adding the chain extender 2.

4. The method for preparing a long-lasting functionalized double-layer bionic small-caliber artificial blood vessel according to claim 3, characterized in that: In step (1), the molar ratio of chain extender 1 to chain extender 2 used in the synthesis of the PCHU-DSeSe is 1:99-99:1; and / or the chain extender used in the synthesis of the PCHU-DTA is DSS and the molar ratio of TA is 1:99-99:

1.

5. The method for preparing a long-lasting functionalized double-layer bionic small-caliber artificial blood vessel according to claim 1, characterized in that: In step (1), the reaction temperature of the soft segment diol and diisocyanate is 50-80°C, the stirring rate is 80-250 rpm, and the reaction time is 2-3 h; and / or the chain extender DSS is dissolved in DMSO and added dropwise, and the concentration of the DSS / DMSO solution is 0.02-0.01 g / mL, and the dropwise addition rate is 8-12 mL / h; and / or the stirring rate after the addition of the chain extender is 500-800 rpm, the reaction time after the addition of DSS is 3-5 h, and the chain extender 2 is dissolved in DMAc and then triethylamine (TEA) is added as an acid-binding agent, and then the chain extender 2 / DMAc / TEA is added dropwise into the prepolymer, 0.1 mL of TEA is added per milliliter of the chain extender 2 / DMAc solution, the concentration of the chain extender 2 / DMAc solution is 0.02-0.01 g / mL, and the dropwise addition rate is 8-12 mL / h, and the reaction time in each reaction is 8-24 h.

6. The method for preparing a long-lasting functionalized double-layer bionic small-caliber artificial blood vessel according to claim 1, characterized in that: In step (2), when the near-field direct writing electrospinning is used to prepare the axially oriented fiber vascular inner layer, the core rotation speed is 10-18 rpm, and the needle moving speed is 800-1200 mm / min.

7. The method for preparing a long-lasting functionalized double-layer bionic small-caliber artificial blood vessel according to claim 1, characterized in that: The inner and outer layer sizes of the long-acting functional double-layer biomimetic small-diameter artificial blood vessel are controlled and adjusted through electrospinning and a mold, and are controlled to be: an inner diameter of 1.2-6 mm, a total wall thickness of 0.1-3 mm, an inner layer PCHU-DSeSe oriented fiber thickness of 0.05-1.5 mm; an outer layer oriented topological microstructure PCHU-DTA porous material thickness of 0.05-2 mm, and an artificial blood vessel length of 0.5-25 cm; an outer layer thread type is triangular or trapezoidal, a ratio of thread height to artificial blood vessel total wall thickness is 1:5-10, a thread number is 1-2, a pitch is 0.1-2 mm, and a lead is 0.1-4 mm.

8. A long-term functionalized double-layer biomimetic small-diameter artificial blood vessel, characterized in that, The long-acting functional double-layer biomimetic small-diameter artificial blood vessel is prepared by the preparation method of any one of claims 1-7.

9. Use of the long-acting functional double-layer biomimetic small-diameter artificial blood vessel of claim 8 in the preparation of a medical product for treating arteriovenous fistula, limb arterial vessel injury repair, heart coronary and peripheral vessel bypass.

10. Use according to claim 9, characterized in that, The long-acting functional double-layer biomimetic small-diameter artificial blood vessel has the functions of continuously releasing DSS, NO in the "full life cycle", continuously releasing DSS, H2S in the outer layer, and the active molecules released by the inner and outer layers can interact to enhance the effect, and can dynamically regulate and repair the vascular tissue microenvironment in situ.

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