CN110507860B

CN110507860B - Method for preparing in-situ tissue engineering blood vessel by composite process

Info

Publication number: CN110507860B
Application number: CN201910623186.9A
Authority: CN
Inventors: 陈箫; 姚远; 刘随红; 张亦弛
Original assignee: University of Shanghai for Science and Technology
Current assignee: University of Shanghai for Science and Technology
Priority date: 2019-07-11
Filing date: 2019-07-11
Publication date: 2022-01-07
Anticipated expiration: 2039-07-11
Also published as: CN110507860A

Abstract

The invention relates to a method for preparing an in-situ tissue engineering blood vessel by a composite process. Preparing a three-layer composite vascular stent which is similar to a natural vascular structure and is used for in-situ tissue engineering by combining rapid forming, electrostatic spinning and wet spinning processes; the inner layer of the tubular stent is prepared by 3D printing and electrostatic spinning technology and is provided with an axial groove; the middle layer adopts a fiber tubular bracket with circumferential arrangement prepared by wet spinning; the outer layer is a tubular bracket which is prepared by electrostatic spinning and consists of disordered fibers capable of coating the inner layer and the middle layer. The vascular stent prepared by the invention is heparinized, and is loaded with vascular endothelial cell growth factors, so that cells can be recruited in a host body, the infection chance of in vitro culture is reduced, the preparation of in-situ tissue engineering blood vessels is realized, and the vascular stent has wide prospects in the field of tissue engineering blood vessels.

Description

Method for preparing in-situ tissue engineering blood vessel by composite process

Technical Field

The invention relates to the technical field of biomedical tissue engineering, in particular to a method for preparing an in-situ tissue engineering blood vessel by a composite process.

Background

With the increasing aging of the population, many diseases related to human blood vessels, such as atherosclerosis, coronary heart disease, etc., are in a high incidence trend. When the blood vessels of the human body can not normally supply blood to the human body due to diseases, wounds and other factors, surgical operations such as repair, intravascular stent or autologous vein transplantation and the like should be adopted to treat the damaged blood vessels. Generally, saphenous vein is considered as a good autologous blood vessel substitute for small diameter blood vessel reconstruction, but since autologous vein cannot be used for some patients' own diseases or other reasons, it is a hot spot of research to use tissue engineering blood vessel as a substitute for tissue vessel repair.

At present, great progress is made in preparing artificial blood vessels by utilizing high polymer materials, for example, blood vessel stents prepared by polyester terylene, expanded polytetrafluoroethylene and other materials are commercialized; these material scaffolds are very widely used in high blood flow, large diameter (greater than 5-6 mm) vascular substitutes. However, the vascular stent synthesized by the materials can not meet the requirement of the small-diameter vascular stent. It is therefore one of the promising approaches to incorporate autologous cells into a material, such as endothelial cells to cover the luminal surface, to exert a biological effect. The application of tissue engineering is divided into two cases, one is to pre-inoculate cells on a bracket, and implant the cells into a body after the cells are cultured and matured, so that the tissue engineering is called in vitro tissue engineering; the other is directly implanted into a stent so as to induce the growth of self cells and tissues and promote the formation of new tissues, which is also called in-situ tissue engineering.

Normal blood vessels are complex structures composed of three concentric membranes. The intima is located on the cavity surface of the blood vessel wall and is covered by a single layer of endothelial cells; most smooth muscle cells are located in the media of blood vessels, and contraction and expansion of elastic units thereof constricts or expands arteries, thereby regulating blood flow; the outer layer of the blood vessel, the adventitia, is composed of a loose web of fibroblasts. Most of the existing artificial blood vessels are of single-layer structures made of electrostatic spinning, but the single-layer blood vessels are easy to leak, the disordered arrangement of electrostatic spinning fibers, the detachment of the intravascular stent from a rotary collecting rod and the like are the existing problems. Patent CN106668944A discloses a three-layer composite small-caliber intravascular stent and a preparation method thereof; patent CN109259889A discloses a method for preparing a bionic vascular stent by a composite process; CN103599568A discloses a preparation method of a double-load multilayer small-diameter intravascular stent material; patent CN102764171B discloses an electrostatic spinning composite intravascular stent and a preparation method thereof. Ideal artificial vascular grafts should be resistant to thrombosis, inflammation and neointimal hyperplasia, such that the graft maintains long-term patency in the body. Endothelial cell attachment is enhanced by surface treatment of other synthetic vascular grafts. When the surface of the biological material is provided with a specific parallel groove structure, the adhesion of cells on the surface of the material can be enhanced. Therefore, the material selection and preparation process of the tissue engineering blood vessel still need to be developed and improved.

Based on the prior art, the in-situ tissue engineering blood vessel bracket with a three-layer structure is prepared by considering the structural function, the cell growth condition and the characteristics of biological materials of a natural blood vessel, and has development prospect in the field of tissue engineering blood vessels.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to overcome the defects in the prior art, and provides a method for preparing an in-situ tissue engineering blood vessel by combining a 3D printing technology.

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

a method for preparing in-situ tissue engineering blood vessels by a composite process comprises the following steps:

a.3D printing of a water-soluble polymer material A to prepare a vascular stent collecting rod: the structure is a cylindrical hollow structure, the inner diameter is 2.5-4 mm, the outer diameter is 3-4.5 mm, multiple equal-width ridges are designed on the outer surface of the cylinder, and the collecting rod is formed by printing water-soluble polymer material A through FDM.

b. Preparing the inner layer of the tissue engineering blood vessel by adopting an electrostatic spinning process: poly L-lactic acid (PLLA) and Polycaprolactone (PCL) were dissolved in Hexafluoroisopropanol (HFIP) by sonication at concentrations of 19% and 5%, respectively, until completely dissolved, then mixed uniformly in a ratio of 1:1 and added to the syringe. B, using a syringe pump to convey the polymer solution at a flow rate of 0.8-1.2 ml/h, mounting the collecting rod printed in the step a on a rotating shaft as an electrostatic spinning collecting platform, wherein the rotating speed of the rotating shaft is set as follows: 800-1000 r/min; and applying voltage to two ends of the syringe needle and the collecting rod, and winding and receiving the electrospun nano-scale fiber wires on the collecting rod to serve as an inner layer of the intravascular stent.

c. Preparing a middle layer with fibers arranged circumferentially by adopting a wet spinning process: and (3) filling the prepared PCL/PLLA solution into a syringe, immersing the syringe needle into a coagulating bath of ethanol, and extruding the solution into the coagulating bath at a flow rate of 1-2 ml/h by driving the dripping flow of the solution by a syringe pump. Collecting the fibers on a rotating stainless steel rod for 6min at a speed of 800-1000 r/min. The fiber-wrapped stainless steel rod was then washed 3 times with ethanol and then dried in a vacuum desiccator for two days to remove residual solvent. And (c) winding the wet spinning fibers on the outer surface of the inner layer of the intravascular stent prepared in the step (b) to form a circumferential arrangement structure, so as to form an intravascular stent middle layer with a fiber circumferential arrangement structure.

d. Outer layer of the electrospun intravascular stent: completely dissolving the polymer material B into the solvent A by 10-12% w/v, filling into a 10ml syringe, and electrically spinning a layer of the polymer material B outside the middle layer of the intravascular stent prepared in the step c, wherein the electro-spinning conditions are as follows: the voltage is 10-12 kv, the injection speed is 1-3 mL/h, the rotating speed of the collecting rod is controlled at 800-1000r/min, the distance between the needle head and the collecting rod is 10-15 cm, the electrospinning time is 2-3 h, and finally the polymer material B layer completely coating the inner layer and the middle layer is obtained.

e. Removing the blood vessel stent collecting rod, and carrying out vacuum drying on the blood vessel stent: dismantle the structure of accomplishing with above-mentioned preparation and get off and put into the culture dish, add deionized water submergence intravascular stent in the culture dish, 30min back intravascular stent collecting rod of inlayer is because the water-soluble characteristic of its material becomes soft easily and gets rid of gradually, collecting rod's hollow structure can prevent that polymer material A from influencing intravascular stent's size after meeting water inflation, get rid of collecting rod under the condition of not producing the damage to intravascular stent, get rid of intravascular stent collecting rod completely the back, put into vacuum drying cabinet 2 days with the support, get rid of residual moisture and solvent. Finally, the tissue engineering blood vessel stent with the inner layer provided with axial grooves and the middle layer arranged in the circumferential direction is obtained.

f. Heparinizing the vascular stent: using 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (SULFO NHS) as cross-linking agents, and using diamino polyethylene glycol (DI-NH 2-PEG) as a spacer to carry out heparin covalent connection on the vascular stent. The density of surface carbonyloxy groups was increased by short hydrolysis in sodium hydroxide (NaOH,0.01N) for 10 minutes. In phosphate buffer saltThe scaffolds were rinsed thoroughly in water (PBS) and then rinsed with deionized water. The scaffold was then allowed to contain 20mg/mL EDC, 10mg/mL sulfo-NHS, and 20mg/mL di-NH at room temperature₂PEG was reacted in 2- (N-morphinone) ethanesulfonic acid (MES) buffer (pH 5.5) for 3 hours, the scaffold was washed in PBS, and then reacted in MES buffer (pH 5.5) containing 20mg/mL EDC, 10mg/mL sulfo-NHS, and 20mg/mL unfractionated heparin sodium salt for 3 hours. After heparin modification, the scaffold was washed thoroughly in PBS for 30 minutes using 10mg/ml glycine, and the remaining reaction sites were quenched.

g. Immobilization of Vascular Endothelial Growth Factor (VEGF): VEGF was fixed to the vascular stents and the stents were incubated in PBS solutions containing 0, 1, 2 and 44. mu.g/ml recombinant human VEGF-165 at 4 ℃ for 16 hours on a shaker. After VEGF fixation, the stent was washed repeatedly in PBS for 30 minutes. Finally obtaining the in-situ tissue engineering blood vessel bracket of the grafted heparin and the vascular endothelial cell growth factor.

The in-situ tissue engineering intravascular stent prepared by the invention comprises an inner layer, a middle layer and an outer layer, wherein the inner layer is formed by an axial groove pattern on an electrostatic spinning membrane through a 3D printing stent collecting rod; the fibers of the middle layer are arranged in the circumferential direction; the outer layer fibers are arranged in disorder.

Compared with the prior art, the invention has the following obvious advantages:

1. the vascular stent prepared by the invention adopts the 3D printed water-soluble polymer material collecting rod to take up the filaments, the stent removing step is simple, convenient and feasible, the inner layer of the vascular stent cannot be damaged, the patterning of the inner layer of the vascular stent is realized, the adhesion and the growth of endothelial cells are facilitated, the axial grooves are consistent with the blood flowing direction, and the blood flowing resistance can be reduced.

2. The intravascular stent prepared by the invention imitates the structure of circumferential arrangement of smooth muscle cells in the middle layer of a natural blood vessel, and the middle layer of the intravascular stent with circumferentially arranged fibers is prepared by adopting a wet spinning technology to guide the circumferential growth of the smooth muscle cells.

3. The outermost layer of the intravascular stent prepared by the invention is wrapped by the intermediate layer and the inner layer by electrostatic spinning, so that the vascular stent can be prevented from being broken, and the sufficient mechanical property of the intravascular stent is ensured.

4. The vascular stent prepared by the invention simulates the structure and function of a natural blood vessel and is a three-layer vascular stent structure prepared by combining 3D printing, electrostatic spinning and wet spinning technologies.

5. The vascular stent inner layer and the intermediate layer prepared by the invention are made of materials which are obtained by adding PCL with low molecular weight into PLLA to increase heparin molecule binding sites.

6. The blood vessel stent prepared by the invention adopts a heparinized coating, so that the infiltration of cells into the electro-spinning graft is increased, and the generation of collagen and elastin in the wall of the graft is increased.

7. The vascular stent prepared by the invention is loaded with Vascular Endothelial Growth Factor (VEGF), can recruit cells in the body after being implanted, reduces the infection chance of in vitro culture, and realizes the preparation of the in-situ tissue engineering vascular stent.

Drawings

FIG. 1 is a schematic model of the invention in the preparation of a collecting rod for vascular stents.

FIG. 2 is a schematic view of the collecting platform equipment for collecting the electrospun filaments according to the present invention.

Fig. 3 is a schematic diagram of the wet spinning equipment for preparing the middle layer of the vascular stent.

FIG. 4 is a schematic diagram of a three-layer structure for preparing a vascular stent according to the present invention.

Fig. 5 is a process flow diagram for preparing a vascular stent according to the present invention.

In fig. 1 to 5:

1-collecting rod, 2-rotating shaft, 3-motor,

4-stainless steel bar, 5-silk fiber, 6-coagulating bath,

7-outer layer, 8-middle layer, 9-inner layer.

Detailed Description

The foregoing aspects are further explained in the detailed description which follows, taken in conjunction with the accompanying drawings and the specific embodiments, wherein preferred embodiments of the invention are detailed below:

example 1:

as shown in fig. 5, a method for preparing an in situ tissue engineering blood vessel by a composite process comprises the following steps:

a.3D printing water-soluble material to prepare a blood vessel stent collecting rod: the structure is a cylindrical hollow structure, as shown in figure 1: the internal diameter is 3.5mm, and the external diameter is 4.5mm, and 8 ridges of cylinder surface design, and protruding structure's size is length 50mm, wide 0.4mm, and high 0.2mm, and its collection stick adopts water-soluble polymer material polyvinyl alcohol to print through FDM and takes shape. The nozzle temperature of the printer was 160 ℃, the bed temperature was 60 ℃, the nozzle moving speed was 20mm/s, and the layer thickness was set to 0.15 mm.

b. Preparing the inner layer of the tissue engineering blood vessel by adopting an electrostatic spinning process, as shown in figure 2: to increase the number of end groups available for biomolecule attachment, poly-L-lactic acid (PLLA) and Polycaprolactone (PCL) were dissolved in 1, 3-hexafluoro-2-propanol (HFIP) by sonication at concentrations of 19% and 5%, respectively, until completely dissolved, then mixed uniformly in a 1:1 ratio and added to a syringe. Using a syringe pump to deliver the polymer solution at a flow rate of 1.2ml/h, the collecting rod 1 printed in step a is mounted on a rotating shaft 2 as an electrospinning collecting platform, and the rotating speed of the rotating shaft 2 of a motor 3 is set as follows: 1000 r/min; and applying voltage to the needle of the syringe and the two ends of the collecting rod 1, and winding and receiving the electrospun nano-scale fiber wires on the collecting rod 1 to serve as the inner layer of the blood vessel stent.

c. The vascular interlayer with circumferentially oriented fibers was prepared using a wet spinning process, as shown in fig. 3: the prepared PCL/PLLA solution was loaded into a syringe, which was immersed in a coagulation bath 6 of ethanol, and a stream of drip of the solution was forced out of the coagulation bath 6 at a flow rate of 2ml/h by means of a syringe pump. The silk fibres 5 were collected at 1000r/min on a rotating stainless steel rod 4 for 6 min. The fiber-wrapped stainless steel rod was then washed three times with ethanol and then dried in a vacuum desiccator for two days to remove residual solvent. And winding the wet spinning fibers 5 on the outer surface of the inner layer membrane of the artificial blood vessel stent to form a circumferential arrangement structure, thereby forming the blood vessel stent middle layer with the circumferential arrangement structure of the fibers.

d. Outer layer of the electrospun intravascular stent: completely dissolving the poly (L-lactic acid-epsilon-caprolactone) PLCL material into HFIP (hexafluoroisopropanol) at 10% w/v, filling a 10ml syringe, and electrospinning a layer of poly (L-lactic acid-epsilon-caprolactone) outside the middle layer prepared in step c under the electrospinning conditions: the voltage is 10kv, the injection speed is 2mL/h, the rotating speed of the collecting rod is controlled at 800r/min, the distance between the needle head and the collecting rod is 10-15 cm, the electrospinning time is 4h, and finally the polymer material B layer completely coating the inner layer and the middle layer is obtained.

e. Dissolving in water to remove a polyvinyl alcohol (PVA) collecting rod, and then carrying out vacuum drying on the intravascular stent: on superclean bench, dismantle the structure that above-mentioned preparation was accomplished and get off and put into the culture dish, add deionized water submergence intravascular stent in the culture dish, intravascular stent collecting rod of inlayer after 30min because the water-soluble characteristic of its material becomes soft easily and gets rid of gradually, collecting rod's hollow characteristic can prevent that polymer material A from influencing intravascular stent's size after meeting water inflation, get rid of under the condition that does not produce the damage intravascular stent and get rid of the back in collecting rod intravascular stent completely, put into vacuum drying cabinet 2 days with the support, get rid of residual moisture and solvent. And obtaining the vascular stent with the inner layer provided with axial grooves and the middle layer arranged in the circumferential direction.

f. Heparinizing the vascular stent: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) are taken as cross-linking agents, and diamino polyethylene glycol (di-NH)₂PEG) as spacer, heparin covalent attachment to the vascular stent. The density of surface carbonyloxy groups was increased by short hydrolysis in sodium hydroxide (NaOH,0.01N) for 10 minutes. The scaffolds were rinsed thoroughly in Phosphate Buffered Saline (PBS) and then rinsed with deionized water. The scaffold was then allowed to contain 20mg/mL EDC, 10mg/mL sulfo-NHS, and 20mg/mL di-NH at room temperature₂PEG was reacted in 2- (N-morphinone) ethanesulfonic acid (MES) buffer (pH 5.5) for 3 hours, the scaffold was washed in PBS, and then reacted in MES buffer (pH 5.5) containing 20mg/mL EDC, 10mg/mL sulfo-NHS, and 20mg/mL unfractionated heparin sodium salt for 3 hours. After heparin modification, a thorough wash was performed in PBS using 10mg/ml glycineScaffolds were quenched for 30min, and remaining reaction sites were quenched.

g. Immobilization of Vascular Endothelial Growth Factor (VEGF): VEGF was fixed to the vascular stents and the stents were incubated in PBS solutions containing 0, 1, 2 and 4. mu.g/ml recombinant human VEGF-165 at 4 ℃ for 16 hours on a shaker. After VEGF fixation, the stent was washed repeatedly in PBS for 30 minutes. Finally, the in-situ tissue engineering blood vessel stent (PLLA/PCL inner layer 9, PLLA/PCL circumferentially arranged intermediate layer 8, PLCL outer layer 7) grafted with heparin and vascular endothelial cell growth factor is obtained, as shown in FIG. 4.

Example 2:

this embodiment is substantially the same as embodiment 1, and is characterized in that:

in this embodiment, a method for preparing in situ tissue engineering blood vessels by a composite process, which prepares ridges with different widths and numbers through the outside of a blood vessel stent collecting rod to cause patterning of the inner layer of a stent, comprises the following steps:

a.3D printing water-soluble material to prepare a blood vessel stent collecting rod: designing a blood vessel stent collecting rod: the structure is a cylindrical hollow structure, the inner diameter is 4mm, the outer diameter is 5mm, 6 ridges are designed on the outer surface of the cylinder, the size of the protruding structure is 50mm long, 0.5mm wide and 0.3mm high, and the collecting rod is made of water-soluble polymer material polyvinyl alcohol and is printed and formed through FDM. The temperature of a nozzle of the printer is 160 ℃, the temperature of a bed is 60 ℃, the moving speed of the nozzle is 20mm/s, and the layered thickness is set to be 0.15 mm;

the rest of the procedure was the same as in example 1.

Example 2:

in this embodiment, a method for preparing an in situ tissue engineering blood vessel by a composite process, which is to prepare a plurality of materials for electrospinning an outer layer of a composite blood vessel, includes the following steps:

a. the procedure was the same as in example 1;

b. the procedure was the same as in example 1;

c. the procedure was the same as in example 1;

d. outer layer of the electrospun intravascular stent: completely dissolving the polycaprolactone PCL material in dichloromethane and DMF at 10% w/v, filling into a 10ml syringe, and electro-spinning a layer of polycaprolactone outside the intermediate layer prepared in the step c, wherein the electro-spinning conditions are as follows: the voltage is 10kv, the injection speed is 2mL/h, the rotating speed of the collecting rod is controlled at 800r/min, the distance between the needle head and the collecting rod is 10-15 cm, the electrospinning time is 4h, and finally the PCL layer completely coating the inner layer and the middle layer is obtained;

e. the procedure was the same as in example 1;

f. the procedure was the same as in example 1;

g. this procedure is the same as in example 1.

Claims

1. A method for preparing in-situ tissue engineering blood vessels by a composite process is characterized by comprising the following steps:

a, 3D printing a water-soluble polymer material A to prepare a vascular stent collecting rod: the collecting rod is of a cylindrical hollow structure, the inner diameter is 2.5-4 mm, the outer diameter is 3-4.5 mm, a plurality of ridges with equal width are designed on the outer surface of the cylinder, and the collecting rod is formed by adopting a water-soluble polymer material A through FDM printing;

b. preparing the inner layer of the tissue engineering blood vessel by adopting an electrostatic spinning process: dissolving poly-L-lactic acid and polycaprolactone in hexafluoroisopropanol at concentrations of 19% and 5% respectively by ultrasonic treatment until completely dissolved, then uniformly mixing at a ratio of 1:1, and adding into an injector; b, using a syringe pump to convey the polymer solution at a flow rate of 0.8-1.2 ml/h, mounting the collecting rod printed in the step a on a rotating shaft as an electrostatic spinning collecting platform, wherein the rotating speed of the rotating shaft is set as follows: 800-1000 r/min; applying voltage to two ends of a syringe needle and a collecting rod, winding and receiving the electrospun nano-scale fiber filaments on the collecting rod to be used as an inner layer of the intravascular stent;

c. preparing the intermediate layer with the circumferentially oriented fibers by adopting a wet spinning process: filling the prepared mixed solution of poly-L-lactic acid and polycaprolactone into an injector, immersing the injection needle into a coagulating bath of ethanol, and extruding the dripping flow of the solution into the coagulating bath at a flow rate of 1-2 ml/h by using an injector pump; collecting the fibers on a rotating stainless steel rod for 6min at a speed of 800-1000 r/min; the fiber-wrapped stainless steel rod was then washed 3 times with ethanol and then dried in a vacuum desiccator for two days to remove residual solvent; winding the wet spinning fibers on the outer surface of the inner layer of the intravascular stent prepared in the step b to form a circumferential arrangement structure, so as to form an intravascular stent middle layer with a fiber circumferential arrangement structure;

d. outer layer of the electrospun intravascular stent: completely dissolving the polymer material B into the solvent A by 10-12% w/v, filling into a 10ml syringe, and electrically spinning a layer of the polymer material B outside the middle layer of the intravascular stent prepared in the step c, wherein the electro-spinning conditions are as follows: the voltage is 10-12 kv, the injection speed is 1-3 mL/h, the rotating speed of the collecting rod is controlled at 800-1000r/min, the distance between the needle head and the collecting rod is 10-15 cm, the electrospinning time is 3-5 h, and finally the polymer material layer B completely coating the inner layer and the middle layer is obtained;

e. removing the blood vessel stent collecting rod, and carrying out vacuum drying on the blood vessel stent: the prepared structure is disassembled and placed into a culture dish, deionized water is added into the culture dish to immerse the intravascular stent, the intravascular stent collecting rod on the inner layer becomes soft and easy to remove due to the water-soluble characteristic of the material after 30min, the hollow structure of the collecting rod can prevent the polymer material A from influencing the size of the intravascular stent after swelling in water, the collecting rod is removed under the condition that the intravascular stent is not damaged, and after the intravascular stent collecting rod is completely removed, the stent is placed into a vacuum drying oven for 2 days to remove residual moisture and solvent; finally obtaining a tissue engineering blood vessel stent with an inner layer provided with axial grooves and a middle layer arranged in the circumferential direction;

f. heparinizing the vascular stent: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide are taken as cross-linking agents, and diamino polyethylene glycol is taken as a spacer to carry out heparin covalent connection on the vascular stent; the density of the surface carbon oxygen radical is increased by short-time hydrolysis in sodium hydroxide for 10 minutes; thoroughly washing the stent in phosphate buffered saline, and then washing with deionized water; the scaffold was then allowed to contain 20mg/mL EDC, 10mg/mL sulfo-NHS, and 20mg/mL di-NH at room temperature₂PEG in 2- (N-morphinone) ethanesulfonic acid buffer, pH5.5, for 3 hours in PBSThe scaffolds were washed and then reacted for 3 hours in MES buffer containing 20mg/mL EDC, 10mg/mL sulfo-NHS, and 20mg/mL unfractionated heparin sodium salt, pH 5.5; after heparin modification, the scaffolds were washed thoroughly with 10mg/ml glycine in PBS for 30 minutes, quenching the remaining reaction sites;

g. fixing of vascular endothelial cell growth factor: VEGF was fixed to the vascular stent and the stent was incubated in PBS solution containing 1, 2 and 4. mu.g/ml recombinant human VEGF-165 at 4 ℃ for 16 hours on a shaker; after VEGF fixation, the scaffolds were washed repeatedly in PBS for 30 min; finally obtaining the in-situ tissue engineering blood vessel bracket of the grafted heparin and the vascular endothelial cell growth factor.

2. The method for preparing in-situ tissue engineering blood vessels by the composite process of claim 1, wherein in the step a, the polymer material A for preparing the blood vessel stent collecting rod is water-soluble polyvinyl alcohol PVA.

3. The method for preparing an in situ tissue engineering blood vessel according to the claim 1, wherein in the step a, the blood vessel stent collecting rod is externally designed into a plurality of ridges with equal width, the number of the ridges is 4-8, the length of each ridge is equal to the length of the collecting rod, the width is 0.3-0.9 mm, the height is 0.2-0.5 mm, the collecting rod is formed by FDM technology, the outer shape of the collecting rod causes the inner layer patterning of electrostatic spinning, and the axial groove structure guides the adhesion growth of endothelial cells and reduces the effect of blood flow resistance.

4. The method for preparing in-situ tissue engineering blood vessels by the composite process according to claim 1, wherein in the step d, the polymer material B is one of poly (L-lactic acid-epsilon-caprolactone) PLCL, thermoplastic polyurethane TPU and polycaprolactone PCL high molecular polymer; the solvent A is one or two of dichloromethane, N-dimethylformamide DMF, tetrahydrofuran THF and hexafluoroisopropanol HFIP.