CN113476661A

CN113476661A - Composite self-healing artificial blood vessel with three-layer structure and preparation method thereof

Info

Publication number: CN113476661A
Application number: CN202110756876.9A
Authority: CN
Inventors: 黄平升; 王伟伟; 李双阳; 冯祖建; 董岸杰
Original assignee: Tianjin University; Institute of Biomedical Engineering of CAMS and PUMC
Current assignee: Tianjin University; Institute of Biomedical Engineering of CAMS and PUMC
Priority date: 2021-07-05
Filing date: 2021-07-05
Publication date: 2021-10-08
Anticipated expiration: 2041-07-05
Also published as: CN113476661B

Abstract

The invention discloses a composite self-healing artificial blood vessel with a three-layer structure, which consists of a zwitterionic hydrogel anticoagulant layer, a self-healing polyurethane elastomer layer and a zwitterionic hydrogel anti-inflammatory layer. The zwitterionic hydrogel on the inner layer of the tube body has super-hydrophilicity and super-lubricity, so that the artificial blood vessel has good blood compatibility. The compact self-healing polyurethane elastomer in the middle layer of the tube body can provide mechanical properties required by the artificial blood vessel; because it has the self-healing property, can be quick self-healing after sewing up and puncture, avoid taking place blood leakage and blood vessel damage. The amphoteric ion hydrogel on the outer layer of the tube body has excellent biocompatibility, can reduce rejection reaction stimulation to an implantation position, and is favorable for ensuring the long-term stability and the smoothness of the artificial blood vessel in vivo. The artificial blood vessel has good blood compatibility, permeability resistance, histocompatibility, compliance and mechanical property.

Description

Composite self-healing artificial blood vessel with three-layer structure and preparation method thereof

Technical Field

The invention relates to the field of medical instruments, in particular to a composite self-healing artificial blood vessel with a three-layer structure and a preparation method thereof.

Background

Vascular transplantation and revascularization repair are main or auxiliary treatment means for cardiovascular diseases, and patients with end-stage renal diseases need arteriovenous fistulation for dialysis treatment. Although autologous blood vessels are an ideal substitute for damaged blood vessels, not only are the sources limited, but there are also hazards from secondary trauma and complications. Therefore, how to make artificial blood vessels meeting clinical requirements has been a hot research focus internationally.

At present, large-caliber artificial blood vessels (more than or equal to 6mm) prepared from terylene, expanded polytetrafluoroethylene, real silk, polyurethane and the like are widely applied clinically, particularly polyurethane materials have excellent compliance, wear resistance, elasticity and biocompatibility, overcome the defect of poor compliance of the terylene, the real silk and the expanded polytetrafluoroethylene artificial blood vessels, and have good prospects and great advantages in the fields of artificial blood vessels and artificial hearts. However, the substitution of small-diameter blood vessels (< 6mm) is still extremely undesirable, and because of insufficient anticoagulation, mural thrombus is easily formed in a special state of high tension and low blood flow, and then blood coagulation is promoted, resulting in stenosis and occlusion of the blood vessel, and there is no clinically usable product for small-diameter artificial blood vessels. Therefore, the improvement of the blood compatibility is the promotion of the clinical application of the small-caliber artificial blood vessel, and the key scientific problem to be solved is urgent. In addition, when the artificial blood vessel is applied as arteriovenous fistula, the artificial blood vessel needs to be punctured by a 16G needle more than 150 times every year, so that the damage of the artificial blood vessel and the blood leakage from a puncture needle hole are easily caused, and complications such as hematoma, pseudo-aneurysm and the like are caused. Therefore, the artificial blood vessel has the self-healing performance similar to that of a natural blood vessel, and is a key scientific problem to be solved for solving the problems of blood leakage and puncture intolerance of an anastomosis needle hole. In addition, the polyurethane in vivo graft still has aging degradation and calcification phenomena in the long-term use process, and researches suggest that the inflammatory reaction is the basic factor for induction, and immune reaction cells such as macrophage and foreign body giant cell cause oxidative degradation. Therefore, the improvement of the tissue compatibility, the little or no induction of the inflammatory reaction of the organism, and the key scientific problem to be solved for ensuring the long-term stability and the patency in the artificial blood vessel.

The ideal artificial blood vessel has the following characteristics: has good blood and tissue compatibility; mechanical properties similar to those of healthy natural blood vessels; thrombus is not easy to form, and the smoothness is kept for a long time; the self-healing capability is similar to that of a natural blood vessel, and blood seepage from an anastomosis needle hole and blood seepage from puncture are avoided; the degenerative change is not easy to occur, and the performance is stable; tolerating intravascular pressure and not easily forming aneurysm; the steel plate is not easy to deform or kink into an angle after being pressed; does not cause foreign body reaction or rejection reaction; enough pulling strength and difficult tearing; different calibers and lengths can be selected.

Therefore, the key technology of the artificial blood vessel is broken through, the artificial blood vessel with good properties of blood and tissue compatibility, mechanical property, self-healing property, long-term in-vivo stability, patency and the like is provided, and the clinical requirements on the artificial blood vessels with different calibers and performances are met, which is a technical problem to be solved by technical personnel in the field.

Disclosure of Invention

In view of the above, the present invention provides a composite self-healing artificial blood vessel with a three-layer structure. The invention utilizes the zwitter-ion hydrogel layer as the inner wall of the artificial blood vessel to simulate the endothelial layer of the natural blood vessel, so that the lumen of the artificial blood vessel has super-hydrophilicity and super-lubricity and has the capability of resisting protein adhesion, thereby avoiding the formation of thrombus on the artificial blood vessel wall. It is particularly important for small-bore vessels, which have slow blood flow, and even a small amount of fibrinogen adheres to the surface of the artificial vessel, which can lead to platelet aggregation and thrombosis. The self-healing polyurethane elastomer is used as the middle layer of the artificial blood vessel and simulates the smooth base layer of the natural blood vessel, so that the artificial blood vessel has excellent mechanical properties such as wear resistance, fatigue resistance, compliance and elasticity, and can be quickly self-healed after suturing and puncturing due to the self-healing property, and blood leakage and blood vessel damage are avoided. The zwitterionic hydrogel layer is used as the outer wall of the artificial blood vessel, so that the amphoteric hydrogel has excellent biocompatibility, reduces the stimulation of rejection reaction to the implantation position, reduces inflammatory reaction, avoids the adhesion and infiltration of inflammatory cells on the surface of the artificial blood vessel, and is favorable for ensuring the long-term stability and the smoothness of the artificial blood vessel in vivo.

In order to achieve the purpose, the invention adopts the following technical scheme:

a three-layer structure composite self-healing artificial blood vessel is characterized in that the artificial blood vessel is of a hollow circular tube structure and comprises an inner layer, a middle layer and an outer layer;

the artificial blood vessel comprises an inner layer and an outer layer which are made of zwitter-ion hydrogel materials, and the middle layer of the artificial blood vessel is made of self-healing polyurethane elastomer materials.

Further, the composition of the zwitterionic hydrogel material comprises the following raw materials in percentage by mass: 5 to 80 percent of zwitterion, 0.5 to 5 percent of cross-linking agent and the balance of water.

Still further, the zwitterion is methacryloylethyl Sulfobetaine (SBMA), 2-methacryloyloxyethyl Phosphocholine (PBMA), methacryloylethyl Carboxylate Betaine (CBMA), methacryloylethyl trifluoropropyl Carboxylate Betaine (CBF)₃MA) one or more of A and B.

The beneficial effect of adopting the further scheme is that: the components of the invention have good blood compatibility, excellent anti-protein adsorption performance and blood component adhesion performance, and can ensure the antithrombotic performance of the artificial blood vessel.

Further, the cross-linking agent is one or more of N, N-Methylene Bisacrylamide (MBA), N, N-bis (acryloyl) cystamine (MSBA), ethylene glycol dimethacrylate (EBA) and bis (methacryloyl ethyl carboxylic acid) betaine (CBBA).

Furthermore, the thickness of the artificial blood vessel including the inner layer and the outer layer is 50-300 μm, and the water contact angle is 4.5-9.5 degrees.

The beneficial effect of adopting the further scheme is that: the above limitation can simulate the natural vascular endothelial layer, and ensure the blood compatibility of the artificial blood vessel.

Further, the thickness of the artificial blood vessel intermediate layer is 100-600 μm.

Further, the glass transition temperature of the self-healing polyurethane elastomer material is 25-45 ℃.

Still further, the healing polyurethane elastomer material is a self-healing TUEG4 polyurethane elastomer material with suitable mechanical properties, glass transition temperature; the structure is as follows:

the beneficial effect of adopting the further scheme is that: the self-healing polyurethane elastomer adopted by the invention is used as the middle layer of the artificial blood vessel, so that the artificial blood vessel has good compliance and mechanical property, is matched with a natural blood vessel and is not easy to deform or kink into an angle in the using process, and due to multiple hydrogen bond crosslinking and proper glass transition temperature in the molecular structure, the self-healing is realized quickly after anastomosis and puncture, and blood leakage and blood vessel damage are avoided.

Further, the inner diameter of the artificial blood vessel is 3-30 mm; the wall thickness of the artificial blood vessel is 200-.

The beneficial effect of adopting the further scheme is that: the invention limits the wall thickness to be more than 200 mu m, ensures the strength of the artificial blood vessel, ensures that the artificial blood vessel is not damaged due to pressure when being used in vivo, and avoids secondary damage to patients; the thickness is less than 1400 mu m, the vessel wall is not too thick under the condition of ensuring the basic performance, because the artificial blood vessel is a foreign body in vivo, the too thick vessel wall can cause discomfort to a patient and is not fit with the blood vessel in the human body, the too thick vessel wall can influence the compliance of the blood vessel, and the wall thickness of the three-layer structure composite artificial blood vessel can be regulated and controlled according to the actual blood vessel replacement requirement.

The invention also provides a preparation method of the three-layer structure composite self-healing artificial blood vessel, which is characterized by comprising the following steps of:

(1) preparing an intermediate layer: filling polyurethane polymer in a mold by using a mold molding method by using a single-sided or double-sided injection molding machine, wherein the filling temperature is 150-170 ℃, and the polyurethane polymer is filled to 93-97% of the volume of a mold cavity; then, carrying out constant pressure treatment, continuously applying pressure of 1200-2500psi, fully compacting the melt, applying the pressure for 10-120 min, increasing the melt density, cooling and solidifying the product by adopting gradient water cooling at 80-0 ℃ to obtain a proper Young modulus in the range of 5-600MPa, and demoulding; soaking the injection-molded artificial blood vessel intermediate layer in 50-100% ethanol for 4-8h, and drying at 20-40 deg.C for 2-12h to obtain the self-healing polyurethane elastic intermediate layer of the artificial blood vessel.

(2) Soaking the self-healing polyurethane elastic intermediate layer of the artificial blood vessel in an ethanol solution of benzophenone for 5min, and drying at 30-45 ℃ for 2-12 h; and then, immersing the artificial blood vessel in a zwitter-ion hydrogel material, and carrying out in-situ photo atomic free radical polymerization for 3-6h under the irradiation of an ultraviolet LED (light emitting diode), so as to form zwitter-ion hydrogel coatings on two sides of the middle layer of the artificial blood vessel, thereby obtaining the composite self-healing artificial blood vessel with the three-layer structure.

Further, the irradiation conditions of the ultraviolet LED in the step (2) are as follows: the wavelength is 365nm, and the power is 20-50W.

Furthermore, in the method, according to actual needs, the inner diameter of the artificial blood vessel intermediate layer can be controlled by controlling the shaft diameter of the self-healing polyurethane elastic intermediate layer preparation mold, and the thickness of the intermediate layer can be controlled by controlling the inner diameter difference between the shaft diameter and the sleeve. The thickness of the zwitterionic hydrogel on the inner layer and the outer layer of the artificial blood vessel can be controlled by controlling the concentration of the zwitterionic monomer, the proportion of the cross-linking agent and the polymerization time according to actual needs.

Compared with the prior art, the invention has the beneficial effects that:

(1) due to the adoption of the technical scheme, the zwitter-ion inner layer of the artificial blood vessel can effectively solve the problem of poor blood compatibility of the conventional artificial blood vessel. It is particularly important for small-bore vessels, which have slow blood flow, and even a small amount of fibrinogen adheres to the surface of the artificial vessel, which can lead to platelet aggregation and thrombosis. Therefore, the artificial blood vessel of the invention is expected to solve the clinical problem of poor patency of small-caliber artificial blood vessels.

(2) The self-healing polyurethane interlayer of the artificial blood vessel has excellent biocompatibility, wear resistance, fatigue resistance, compliance, elasticity and the like, and can simulate the middle smooth muscle layer of the natural blood vessel to provide the mechanical properties required by the artificial blood vessel. More importantly, due to the fact that the molecular structure of the artificial blood vessel has the cross-linking of dynamic hydrogen bonds and the glass transition temperature within the body temperature range, the artificial blood vessel can be quickly self-healed after puncture, vascular damage and blood leakage are avoided, and the problem that when the artificial blood vessel is used as an arteriovenous internal fistula of a hemodialysis patient, the artificial blood vessel is broken due to poor puncture resistance, and malignant diseases such as pseudo aneurysm are solved.

(3) The zwitterionic outer layer of the artificial blood vessel has excellent biocompatibility, can reduce rejection reaction stimulation to an implantation position, reduce inflammatory reaction, simultaneously avoid adhesion and infiltration of inflammatory cells on the surface of the artificial blood vessel, solve the problems of calcification, degradation and the like in the polyurethane artificial blood vessel, and ensure long-term stability and smoothness in the body.

(4) The artificial blood vessel is prepared by the method of injection molding of a mold and in-situ photo atomic free radical polymerization, has simple and controllable process, is beneficial to quantitative preparation, has good repeatability and high economic benefit, and is expected to be widely applied to the field of vascular surgery.

Drawings

Fig. 1 is a schematic cross-sectional structure of the artificial blood vessel provided by the present invention.

FIG. 2 is a scanning electron microscope image of the inner layer of the human blood vessel in example 4.

FIG. 3 is a water contact angle test of the inner and outer layers of the human hematopoietic vessels in examples 4-7.

FIG. 4 is a graph showing the evaluation of the anti-protein adhesion ability of the artificial blood vessels in examples 4 to 7.

FIG. 5 is a blood compatibility test of the artificial blood vessels in examples 4 to 7.

FIG. 6 is the artificial blood vessel pull strength test in examples 4-7.

FIG. 7 is the burst pressure test of the artificial blood vessel in examples 4 to 7.

FIG. 8 is the artificial blood vessel compliance test of examples 4-7.

FIG. 9 is a residual strength test after repeated punctures of the artificial blood vessels in examples 4-7.

FIG. 10 is a graph showing the evaluation of the blood flow patency after carotid artery anastomosis of the artificial blood vessel beagle dog in example 4.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Referring to fig. 1, the three-layer structure composite artificial blood vessel is prepared by the following steps:

step 1: the method for preparing the polyurethane elastomer artificial blood vessel intermediate layer by using the die molding method under the argon protection condition specifically comprises the following steps: firstly, filling polyurethane polymer at 160 ℃ until the volume of a mold cavity is 93-97%; secondly, carrying out pressure maintaining treatment, continuously applying pressure 2400 psi, fully compacting the melt, and increasing the density of the melt to compensate the shrinkage of the melt in a cooling stage; thirdly, gradient water cooling is adopted to fully cool and solidify the product to proper rigidity, and demoulding is carried out; and finally, fully soaking the injection-molded artificial blood vessel intermediate layer in ethanol for 6h, removing impurities, mold release agents and other components possibly adhered to the surface, and fully drying at 25 ℃ to obtain the self-healing polyurethane elastic intermediate layer of the artificial blood vessel.

Step 2: soaking the injection molded artificial blood vessel intermediate layer in benzophenone ethanol solution for 5min, and fully drying at 40 ℃; then, the artificial blood vessel is immersed in an aqueous solution of a zwitterionic monomer and a cross-linking agent, and under the irradiation of an ultraviolet LED (365nm, 30W), in-situ photoatomic radical polymerization is carried out for 4h, so that a zwitterionic hydrogel coating is formed on two sides of the middle layer of the artificial blood vessel, and the composite artificial blood vessel with a three-layer structure is obtained.

Parameters of a finished product: the inner diameter is 3-30 mm; the wall thickness is 200-.

Example 2

An artificial blood vessel is a three-layer composite hollow circular tube structure, and the preparation method of the embodiment 1 is adopted, wherein the inner layer and the outer layer are zwitterionic hydrogel, the mass percentage concentration of zwitterionic methacryloyl ethyl Carboxylic Betaine (CBMA) is 5%, the mass percentage concentration of a cross-linking agent N, N-Methylene Bisacrylamide (MBA) is 1%, the inner diameter is 3mm, and the thicknesses of the inner layer and the outer layer are both 50 micrometers; the thickness of the middle layer polyurethane elastomer is 100 μm; the artificial blood vessel has a wall thickness of 200 μm.

Example 3

An artificial blood vessel is a three-layer composite hollow circular tube structure, and the preparation method of the embodiment 1 is adopted, wherein the inner layer and the outer layer are zwitterionic hydrogel, the mass percentage concentration of the zwitterionic methacryloyl ethyl Sulfobetaine (SBMA) is 15%, the mass percentage concentration of the cross-linking agent N, N-bis (acryloyl) cystamine (MSBA) is 3%, the inner diameter is 6mm, and the thicknesses of the inner layer and the outer layer are both 100 mu m; the thickness of the middle layer polyurethane elastomer is 400 μm; the artificial blood vessel has a wall thickness of 600 μm.

Example 4

An artificial blood vessel is a three-layer composite hollow circular tube structure, and the preparation method of the embodiment 1 is adopted, wherein the inner layer and the outer layer are zwitterionic hydrogel, the mass percentage concentration of the zwitterionic methacryloyl ethyl Carboxylic Betaine (CBMA) is 25%, the mass percentage concentration of the cross-linking agent N, N-Methylene Bisacrylamide (MBA) is 1.5%, the inner diameter is 5mm, and the thicknesses of the inner layer and the outer layer are both 100 mu m; the thickness of the middle layer polyurethane elastomer is 600 μm; the artificial blood vessel has a wall thickness of 800 μm.

Example 5

An artificial blood vessel is a three-layer composite hollow circular tube structure, and the preparation method of the embodiment 1 is adopted, wherein the inner layer and the outer layer are zwitterionic hydrogel, the mass percentage concentration of the zwitterionic 2-methacryloyloxyethyl Phosphorylcholine (PBMA) is 20%, the mass percentage concentration of the cross-linking agent ethylene glycol dimethacrylate (EBA) is 2%, the inner diameter is 15mm, and the thicknesses of the inner layer and the outer layer are both 150 mu m; the thickness of the middle layer polyurethane elastomer is 600 μm; the artificial blood vessel has a wall thickness of 900 μm.

Example 6

An artificial blood vessel having a three-layer composite hollow circular tube structure, the preparation method of example 1 was adopted, wherein the inner and outer layers were zwitterionic hydrogel, zwitterionic methacryl ethyl trifluoropropyl Carboxylic Betaine (CBF)₃MA) is 60 percent, the mass percent concentration of cross-linking agent bis-methacryloyl ethyl carboxylic acid betaine (CBBA) is 0.5 percent, the inner diameter is 20mm, and the thicknesses of the inner layer and the outer layer are both 150 mu m; the thickness of the middle layer polyurethane elastomer is 800 μm; the artificial blood vessel has a wall thickness of 1100 μm.

Example 7

An artificial blood vessel is a three-layer composite hollow circular tube structure, and the preparation method of the embodiment 1 is adopted, wherein the inner layer and the outer layer are zwitterionic hydrogel, the mass percentage concentration of zwitterionic methacryl ethyl carboxylic acid betaine (CBMA) is 80%, the mass percentage concentration of cross-linking agent bis-methacryl ethyl carboxylic acid betaine (CBBA) is 5%, the inner diameter is 30mm, and the thicknesses of the inner layer and the outer layer are both 300 mu m; the thickness of the middle layer polyurethane elastomer is 600 μm; the artificial blood vessel had a wall thickness of 1200 μm.

Example 8

An artificial blood vessel having a three-layer composite hollow circular tube structure, the preparation method of example 1 was adopted, wherein the inner and outer layers were zwitterionic hydrogel, zwitterionic methacryl ethyl trifluoropropyl Carboxylic Betaine (CBF)₃MA) is 60 percent, the mass percent concentration of the cross-linking agent N, N-Methylene Bisacrylamide (MBA) is 3 percent, the inner diameter is 30mm, and the thickness of the inner layer and the outer layer are both 100 mu m; the thickness of the middle layer polyurethane elastomer is 1000 μm; the artificial blood vessel had a wall thickness of 1200 μm.

Example 9

The microscopic morphology of the inner layer of the human hematopoietic tube in example 4 was evaluated by scanning electron microscopy (SEM, S-4800, Hitachi). Specifically, the artificial blood vessel is rapidly frozen in liquid nitrogen, a cross section is obtained after the artificial blood vessel is completely frozen and dried in a freeze dryer, and the structural form of the cross section is observed by adopting SEM. As shown in fig. 2, the inner layer of the artificial blood vessel is distributed with uniform gel and uniform texture.

Example 10

Examples 4, 5, 6, 7 water contact angle measurements of the inner and outer layers of human hematopoietic vessels. Hydrophilicity of the inner and outer layers of the artificial blood vessel was evaluated using a contact angle test. Specifically, a film is prepared by adopting a solvent evaporation method, a DSA 10mk2(Kruss) system is used for measuring a contact angle under the conditions of room temperature (22-25 ℃) and relative humidity (20-40%), a disposable injector is used for each test liquid to avoid cross contamination, 2 mu L of liquid drops in the injector are placed on the surface of a sample, a static contact angle image is shot by a Charge Coupled Device (CCD) camera after the liquid drops are placed for 120 seconds, the contact angle is calculated by system software, and each sample experiment is repeated for 5 times. As shown in FIG. 3, the zwitterionic hydrogel layers of the inner and outer layers of the artificial blood vessel both have water contact angles of less than 10 ° and are superhydrophilic.

Example 11

The anti-protein adhesion ability of the artificial blood vessels was evaluated in examples 4, 5, 6 and 7. The sample of the artificial blood vessel of example 4, 5, 6, 7 was placed in undiluted plasma, incubated at 37 ℃ for 24 hours under aseptic conditions, and after rinsing the sample of the artificial blood vessel with phosphate buffer (pH 7.4,10mM), the amount of plasma protein bound per unit area of the artificial blood vessel was determined using a protein quantification kit. As shown in FIG. 4, the interface of the zwitterionic hydrogel layer is modified, so that the protein adsorption resistance of the surface of the polyurethane artificial blood vessel can be remarkably enhanced.

Example 12

Examples 4, 5, 6, 7 in the artificial blood vessel blood compatibility test.

Testing hemolysis rate. The degree of destruction of the blood cells (mainly erythrocytes) of the artificial blood vessels was evaluated by a hemolysis rate test. Placing the artificial blood vessel to be tested into a test tube, and adding 10mL of 0.9% NaCl solution; the positive control was distilled water, and the negative control was 0.9% NaCl solution. Fresh ACD anticoagulated rabbit blood (blood: 3.8% sodium citrate is 4:1) is adopted, all test tubes are put into a water bath at 37 ℃ for pre-warming for 30min, 0.2mL of fresh anticoagulated rabbit blood (rabbit blood: normal saline is 4:5) is added and diluted respectively, the heat preservation is continued in the water bath at 37 ℃ for 1h, centrifugation is carried out for 5min (2500r/min), supernatant fluid is taken, and the absorbance value of each tube is measured at 545nm of a spectrophotometer. Hemolysis rate (sample absorbance-positive control absorbance)/(negative control absorbance-positive control absorbance). If the hemolysis rate is less than 5%, the artificial blood vessel meets the hemolysis rate requirement of the medical material. As shown in FIG. 5, the hemolysis rate of the artificial blood vessels in examples 4, 5, 6 and 7 is less than 1%, which meets the hemolysis rate requirement of medical materials.

② prothrombin time. The effect of artificial blood vessels on the coagulation time due to activation of prothrombin factor was evaluated using the prothrombin time assay. The material was coated on the inner wall of a glass tube, a glass test tube, a siliconized glass test tube as a control. Adding Platelet-rich plasma (PRP) into a test tube by a Quick method, adding 0.1mL of rabbit brain leaching solution, and placing in a water bath at 37 ℃ for 2 min; adding 0.025mol/L CaCl which is pre-warmed to 37 DEG C₂0.1mL of solution is added, and the solution is timed, shaken immediately for a plurality of times and immersed in a water bath; and 5-8 s, moving out the test tube from the water bath, continuously inclining until a clot appears, and setting time. The average value of each test tube and each control tube was taken 3 times or more. As shown in FIG. 5, the prothrombin time of the artificial blood vessels in examples 4, 5, 6 and 7 is not significantly different from that of the natural blood vessels, indicating that the prothrombin time caused by the activation of prothrombin factor by the artificial blood vessels has no significant effect。

And activating partial thromboplastin time. The degree of activation of the endogenous coagulation factors by the artificial blood vessels is evaluated by an activated partial thromboplastin time test, thereby evaluating the influence of the artificial blood vessels on the coagulation time. Cutting artificial blood vessel into small strips of 0.5cm × 0.5cm, placing in the center of the bottom of a small beaker, keeping the temperature at 37 deg.C for 5min, injecting 0.25mL sodium citrate anticoagulated rabbit blood into the center of the membrane, keeping the temperature for 5min, and injecting 0.02mL CaCl into the blood₂Aqueous solution (0.2mol/L), begin timing, shake the small beaker for 1min to allow CaCl₂Mixing with blood, covering the beaker, keeping the temperature for 5min, taking out the beaker, adding 50mL distilled water into the beaker, shaking the small beaker for 10min, collecting the supernatant, and measuring the absorbance of the blood at 540nm with a spectrophotometer. As a control, 50mL of distilled water containing 0.25mL of whole blood was used, and the relative absorbance was taken as 100%. Its anticoagulant property Is (Is/Iw). times.100%. In the formula, Is blood and CaCl₂The relative absorbance of the blood after the mixed solution of (1) and the sample are contacted for a set time; iw is the relative absorbance of blood after mixing with a certain amount of distilled water. As shown in FIG. 5, the activated partial thromboplastin times of the artificial blood vessels in examples 4, 5, 6 and 7 are not significantly different from those of the natural blood vessels, indicating that the artificial blood vessels have no significant effect on the activation of endogenous blood coagulation factors.

Example 13

Artificial vessel traction strength test in examples 4, 5, 6, 7. And evaluating the suture resistance strength and the anastomotic fracture strength of the anastomotic stoma of the artificial blood vessel by a suture pulling strength test of the artificial blood vessel. A length of approximately 20mm of the sample was cut axially, inserted 2mm from the end of the straightened sample using 6-0 nylon suture, and sutured through the vessel wall into a half ring. The direction of the ring is adjusted to make the suture and the axial direction of the blood vessel to be measured respectively form 0 degrees, 45 degrees or 90 degrees, the suture is stretched at the speed of 150mm/min, and the pulling force for pulling the suture out of the blood vessel prosthesis or causing the damage of the wall of the blood vessel prosthesis is recorded. As shown in fig. 6, the artificial blood vessels of examples 4, 5, 6 and 7 have a tensile strength of more than 252gf, and can satisfy the requirement of vascular anastomosis.

Example 14

Examples 4, 5, 6, 7 in the artificial blood vessel burst pressure test. The pressure resistance of the artificial blood vessel is evaluated by a burst pressure test. One end of the artificial blood vessel to be measured is sealed to prevent water leakage, then the lumen of the blood vessel sample to be measured is filled with distilled water to promote the artificial blood vessel sample to reach the effective length, and the other end of the artificial blood vessel sample is provided with a continuous pressurizing device and a pressure sensor. When all the test conditions are complete, continuously pressurizing at a certain speed (kPa/s), and when the blood vessel sample is cracked, recording the instantaneous pressure, namely the bursting pressure of the blood vessel sample. As shown in fig. 7, the burst pressure of the artificial blood vessel in examples 4, 5, 6 and 7 exceeds 1800mmHg, and the artificial blood vessel can withstand the blood pressure and meet the requirement of the artificial blood vessel.

Example 15

Examples 4, 5, 6, 7 in the artificial blood vessel compliance test. A certain specification of artificial blood vessel is taken, one end of the artificial blood vessel is closed by a plug matched with the inner diameter of the artificial blood vessel, the artificial blood vessel is tensioned by 0.46N, and the other end of the artificial blood vessel is connected with a continuous pressurizing device and a pressure sensor. The artificial blood vessel specimen was filled with distilled water and pressurized by a continuous pressurizing device, and the outer diameters of the sensors at pressures of 6.65Kpa and 26.6Kpa were recorded. The inner diameter and wall thickness can be measured in the natural state as described above. As shown in fig. 8, the compliance of the artificial blood vessels in examples 4, 5, 6, and 7 is about 5%, and has a good matching property with the natural blood vessels.

Example 16

Examples 4, 5, 6, and 7 were tested for residual strength after repeated punctures of the artificial blood vessel. A section of sample is cut along the longitudinal axis direction of the artificial blood vessel and then is cut along the axial direction, and a puncture needle of 16G is used for puncturing 24 times per square centimeter on the surface of the blood vessel after being flattened. The sample after puncturing was placed at an opening of 1cm²The film pressurizing equipment is uniformly pressurized until the sample is cracked, and the pressure value at the moment is recorded, namely the residual strength after repeated puncture. As shown in fig. 9, the residual strength after repeated puncturing of the artificial blood vessel in examples 4, 5, 6, and 7 was 95% of the original strength, indicating that the artificial blood vessel of the present invention has a good self-healing ability.

Example 17

Evaluation of blood flow patency after carotid artery anastomosis of the artificial hemangioblast beagle dog in example 4. And (3) constructing a beagle carotid artery anastomosis model of the vascular material, and evaluating the patency of the artificial blood vessel by using a small animal Doppler small animal ultrasonic Doppler blood flow instrument. As shown in fig. 10, the artificial blood vessel in example 4 can maintain good blood flow patency and pulsation during 8 months of in vivo implantation.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. The composite self-healing artificial blood vessel with the three-layer structure is characterized in that the artificial blood vessel is of a hollow circular tube structure and comprises an inner layer, a middle layer and an outer layer;

2. The composite self-healing artificial blood vessel with the three-layer structure according to claim 1, wherein the composition of the zwitterionic hydrogel material comprises the following raw materials in percentage by mass: 5 to 80 percent of zwitterion, 0.5 to 5 percent of cross-linking agent and the balance of water.

3. A composite self-healing artificial blood vessel with a three-layer structure according to claim 2, wherein the zwitterion is one or more of methacryloyl ethyl sulfobetaine, 2-methacryloyl oxyethyl phosphorylcholine, methacryloyl ethyl carboxylic acid betaine, and methacryloyl ethyl trifluoropropyl carboxylic acid betaine.

4. The composite self-healing artificial blood vessel with a three-layer structure according to claim 2, wherein the cross-linking agent is one or more of N, N-methylenebisacrylamide, N-bis (acryloyl) cystamine, ethylene glycol dimethacrylate, and bis (methacryloylethyl) carboxylate betaine.

5. A composite self-healing artificial blood vessel with a three-layer structure according to claim 1, wherein the thickness of the artificial blood vessel including the inner layer and the outer layer is 50-300 μm, and the water contact angle is 4.5-9.5 °.

6. The composite self-healing artificial blood vessel with a three-layer structure according to claim 1, wherein the thickness of the middle layer of the artificial blood vessel is 100-600 μm.

7. The composite self-healing artificial blood vessel with a three-layer structure according to claim 1, wherein the glass transition temperature of the self-healing polyurethane elastomer material is 25-45 ℃.

8. A composite self-healing artificial blood vessel with a three-layer structure according to claim 1, wherein the inner diameter of the artificial blood vessel is 3-30 mm; the wall thickness of the artificial blood vessel is 200-.

9. A method for preparing the composite self-healing artificial blood vessel with the three-layer structure according to any one of claims 1 to 8, which is characterized by comprising the following steps:

(1) preparing an intermediate layer: filling polyurethane polymer in the mold by using a mold forming method until the volume of a mold cavity is 93-97%; then, carrying out constant pressure treatment, continuously applying pressure of 1200-; soaking the injection-molded artificial blood vessel intermediate layer in 50-100% ethanol for 4-8h, and drying at 20-40 deg.C for 2-12h to obtain self-healing polyurethane elastic intermediate layer of artificial blood vessel;

(2) soaking the self-healing polyurethane elastic intermediate layer of the artificial blood vessel in an ethanol solution of benzophenone for 5min, and drying at 30-45 ℃ for 2-12 hours; and then, immersing the artificial blood vessel in a zwitter-ion hydrogel material, and carrying out in-situ photo atomic free radical polymerization for 3-6h under the irradiation of an ultraviolet LED (light emitting diode), so as to form zwitter-ion hydrogel coatings on two sides of the middle layer of the artificial blood vessel, thereby obtaining the composite self-healing artificial blood vessel with the three-layer structure.

10. The method for preparing a composite self-healing artificial blood vessel with a three-layer structure according to claim 9, wherein the irradiation conditions of the ultraviolet LED in the step (2) are as follows: the wavelength is 365nm, and the power is 20-50W.