CN114146227B

CN114146227B - Preparation method of flexible directional nanofiber composite membrane capable of promoting vascular regeneration

Info

Publication number: CN114146227B
Application number: CN202111177371.3A
Authority: CN
Inventors: 张磊; 徐茂芽; 李勇; 李天晴; 司维; 季维智
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2021-10-09
Filing date: 2021-10-09
Publication date: 2023-05-05
Anticipated expiration: 2041-10-09
Also published as: CN114146227A

Abstract

The invention discloses a preparation method of a flexible directional nanofiber composite membrane capable of promoting vascular regeneration, and belongs to the field of preparation of biological composite materials. The composite membrane has the effects of promoting cell adhesion, increasing vascular permeability, vascular endothelial cell migration, proliferation, angiogenesis and the like, combines the characteristics of porous ventilation and toughness, can be used as a wound dressing for a tissue defect part, and can be prepared in batches by promoting the regeneration of blood vessels at the defect part to enhance the healing capacity of wound tissue. The composite film has a double-layer structure: taking an electrostatic spinning flexible oriented nanofiber membrane of a synthetic high polymer material Polycaprolactone (PCL) and a natural high polymer material gelatin as a substrate; and (3) in-situ photocuring a layer of gelatin molecular skeleton of methacrylic anhydride modified and covalent grafted Vascular Endothelial Growth Factor (VEGF) on the membrane. The nano composite membrane has good biocompatibility and mechanical flexibility, and has good application prospect in regenerative medicine and clinical treatment.

Description

Preparation method of flexible directional nanofiber composite membrane capable of promoting vascular regeneration

Technical Field

The invention relates to the technical field of biological material preparation, in particular to a preparation method of a flexible directional nanofiber composite membrane capable of promoting vessel regeneration.

Background

At present, a VEGF-loaded method is adopted in a lot of tissue engineering to promote the regeneration of blood vessels; the microsphere is used for loading VEGF to achieve a slow release effect, but the microsphere cannot imitate an extracellular matrix structure and can not effectively regulate migration and differentiation of cells at a wound on a microstructure; in researches related to nanofiber membranes, the method is basically limited to encapsulating and loading VEGF directly on the nanofiber membrane by an electrostatic spinning method, and in the process, the encapsulation efficiency of VEGF is difficult to ensure and the VEGF is possibly inactive due to electrostatic force or the action of an organic solvent; VEGF is also encapsulated in a simple hydrogel and acts directly on the wound site, but the breathability, tensile properties and degradation rate of the hydrogel are difficult to match with normal tissues.

The electrostatic spinning nanofiber has the advantages of good absorbability, selective permeability, good fitting property, multifunction, contribution to wound healing and the like. Because the diameter of the nanofiber is smaller than that of the cell, the structure and biological function of a natural extracellular matrix can be simulated, and the directionally arranged nanofiber can also guide the migration, proliferation and differentiation of the cell better; most tissues and organs of humans are similar in form and structure to nanofibers, which provide the possibility for electrospun nanofibers for repair of tissues and organs. Biomedical high molecular PCL has excellent mechanical properties, but poor hydrophilicity and biological activity, and gelatin has biological activities such as cell adhesion and the like but lacks good mechanical properties. Therefore, the composite material combining the two materials has biological activity, good mechanical property, good biocompatibility and degradability, can be used as a carrier to enter a human body, and is easy to be absorbed.

Angiogenesis is the formation of blood vessels from de novo endothelial cell regeneration, a process of neovascularization, critical in development and subsequent physiological homeostasis. Given the limitations of current strategies to maintain therapeutic doses of growth factors and endothelial cell efficacy, stimulating angiogenesis during the wound healing process remains a significant clinical challenge. The tissue defect causes the lack of blood vessels, so that oxygen and other nutritional components cannot be transported normally, and therefore, the healing of wounds is also hindered to a certain extent. Vascular endothelial growth factor (vascular endothelial growth factor, VEGF) is a highly specific pro-vascular endothelial growth factor having the effect of promoting increased vascular permeability, vascular endothelial cell migration, proliferation and angiogenesis.

At present, the compounding of PCL and gelatin and the biological functional construction (such as angiogenesis) are one of hot spots in the field of tissue regeneration. But mainly uses the simple mixing of specific polypeptide and high molecular material, so that the effect of long-acting slow-release factor can not be reached, and the tissue regeneration effect is limited. Covalent bonds (covalent bonds) are chemical structures where two or more atoms share their outer electrons, ideally reaching an electron saturated state, and thus constituting a relatively stable chemical structure. The material obtained by adopting the covalent grafting method not only can obtain stable effect, but also can obtain the biomedical material with controllable mechanical property and controllable grafting rate by controlling the reaction condition.

In view of the above, the invention provides a preparation method for organically combining a covalently-bonded Vascular Endothelial Growth Factor (VEGF) methacryloyl gelatin film and a directional active nanofiber film to construct a bioactive composite film which has mechanical flexibility and can actively promote angiogenesis, and the preparation method is used in the field of full-layer skin wound repair.

Disclosure of Invention

Aiming at the defects in the research field, the invention provides a preparation method of a flexible directional nano composite membrane capable of promoting blood vessel regeneration, and the obtained composite membrane has controllable mechanical property and good biocompatibility, has the function of promoting vascularization, and has good application prospect in regenerative medicine and clinical treatment.

In order to achieve the above purpose, the following technical scheme is adopted:

(1) Adding sodium carbonate into deionized water, adjusting the pH to 7-10, adding gelatin according to the proportion of 50-200g/L, heating and continuously stirring to enable the gelatin to be fully dissolved, adding methacrylic anhydride, wherein the volume-mass ratio of the methacrylic anhydride to the gelatin is 1:1-3:1, continuously stirring for 1-8 hours at the temperature of 40-50 ℃, dialyzing at normal temperature, and freeze-drying after the dialysis is completed to obtain the methacrylic acid acylated gelatin. The reaction chemical formula is as follows:

(2) Adding methacrylic acid acylated gelatin into deionized water according to the proportion of 50-200g/L, mixing, adding succinic anhydride according to the volume-mass ratio of 1:1-3:1 of succinic anhydride to methacrylic acid acylated gelatin, heating and continuously stirring, dialyzing at normal temperature after the reaction is complete, and freeze-drying after the dialysis is completed to obtain the modified methacrylic acid acylated gelatin-COOH. The reaction chemical formula is as follows:

(3) Dissolving 50-200g/L of modified methacrylic acid acylated gelatin-COOH in Phosphate Buffer Saline (PBS), and adding a mixture of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) according to a proportion of 0.1-0.5 g/L; activating and continuously stirring gently, then adding the vascular endothelial growth factor according to the proportion of 0.1-0.4 g/L, mixing and stirring for 6-24 hours, dialyzing at normal temperature, and freeze-drying after the dialysis is finished to obtain the covalent grafted methacrylic acid acylated gelatin-vascular endothelial growth factor. The reaction chemical formula is as follows:

(4) Mixing phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite (LAP) and a phosphate buffer salt solution in a light-shielding condition according to a proportion of 5-20 g/L, magnetically stirring for 1-8 hours at 50 ℃ to prepare a phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite solution, adding methacrylic acid acylated gelatin-vascular endothelial growth factor into the phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite solution according to a proportion of 50-200g/L, magnetically stirring for 1-8 hours at 50 ℃, and uniformly stirring to obtain the methacrylic acid acylated gelatin-vascular endothelial growth factor hydrogel.

(5) And dissolving the polycaprolactone and gelatin in a hexafluoroisopropanol solution according to a ratio of 50-70 g/L, rapidly stirring at 40 ℃, wherein the mass ratio of the polycaprolactone to the gelatin is 1:1-2:1, preparing the polycaprolactone-gelatin flexible oriented nanofiber membrane by using an electrostatic spinning method, washing with a phosphate buffer salt solution, and naturally drying for later use.

(6) Spreading the methacrylic acid acylated gelatin-vascular endothelial growth factor hydrogel obtained in the step (4) on a polycaprolactone-gelatin flexible directional nanofiber membrane, immersing vascular endothelial growth factor into fiber pores along with the hydrogel, carrying out in-situ photocrosslinking solidification under blue light irradiation, and carrying out vacuum drying to obtain the flexible directional nanofiber composite membrane with the vascular endothelial growth factor concentration gradient.

Preferably, the conditions of the dialysis in the steps (1) to (3) are as follows: dialyzing for 5-10 days at normal temperature, changing deionized water every 12 hours, and slowly and gently stirring during the dialysis.

Preferably, the freeze-drying temperatures in the steps (1) - (3) are as follows: -80 ℃.

Preferably, in the step (3), the mass ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to the N-hydroxysuccinimide is 1:1-1:3.

Preferably, the conditions of electrospinning in step (5) of the present invention are: the flow rate of the solution is 0.5-2 ml/h, the voltage is 10-20 KV, the rotating speed of a high-speed roller collector is 2000-3000 rpm, so that fibers are arranged in an oriented mode, the ambient humidity is controlled to be about 30%, and the distance between the collector and a spinneret is 15-20 cm.

Preferably, in the step (6) of the present invention, the wavelength of the blue light is 405 to 460 nm.

Compared with the prior art, the invention has the advantages that:

(1) The composite membrane has good biocompatibility, has bioactivity, takes the PCL-gelatin nanofiber membrane as a substrate membrane, and is further crosslinked on the membrane to obtain the composite membrane of the methacrylic acid acylated gelatin-vascular endothelial growth factor hydrogel and the nanofiber membrane with high mechanical strength.

(2) The invention adopts a light response mode to generate a crosslinked network for the natural polymer hydrogel, has high production efficiency and can be prepared in batches.

(3) The layered structure of the composite membrane imitates the physiological structure of natural skin, namely an epidermis layer and a dermis layer, and the microstructure of the composite membrane is similar to the extracellular matrix structure, so that the composite membrane has the bionic biological characteristic.

(4) The existing strategy of loading VEGF by an electrostatic spinning method can only obtain VEGF uniformly distributed on a membrane, but the invention can obtain the composite membrane with VEGF concentration gradient by controlling the grafting rate of VEGF in hydrogel and combining the hydrogel layer with the electrostatic spinning membrane.

(5) The invention is a double-layer composite membrane, and the VEGF is grafted through a covalent bond, so that the problem of short-time explosive release of VEGF is solved, the purposes of stably and long-acting slow release of VEGF and promotion of regeneration of blood vessels and skin can be achieved, and a new thought and method are provided for preparation of a multilayer biological scaffold and effective slow release of biological factors in tissue engineering.

Drawings

FIG. 1 is a reaction scheme for modifying gelatin in example 1;

FIG. 2 shows H-NMR of hydrogen nuclear magnetic resonance chart of unmodified gelatin in example 1;

FIG. 3 is a hydrogen nuclear magnetic resonance chart H-NMR of methacryloylated gelatin in example 1;

FIG. 4 is an SEM image of the microstructure of a flexible oriented nanofiber substrate membrane of example 1;

FIG. 5 is a physical diagram of the overall morphology of the flexible oriented nanofiber composite membrane of example 1;

fig. 6 is an SEM image of the microstructure of the flexible oriented nanofiber composite membrane of example 1 (a) front side and (b) side.

Detailed Description

The invention will be described in further detail with reference to the drawings and the detailed description, but the scope of the invention is not limited to the description.

Example 1

The preparation method of the flexible directional nano composite membrane capable of promoting the regeneration of blood vessels specifically comprises the following steps:

(1) Adding sodium carbonate into deionized water, regulating pH to 7.0, adding gelatin according to the proportion of 100g/L, heating and continuously stirring to enable gelatin to be fully dissolved, adding methacrylic anhydride (the reaction formula is shown in figure 1), wherein the volume-mass ratio of methacrylic anhydride to gelatin is 2:1, continuously stirring for 8 hours at 50 ℃, dialyzing for 5 days at normal temperature, changing deionized water every 12 hours, slowly and gently stirring during dialysis, and freeze-drying at-80 ℃ for 36 hours after dialysis is completed to obtain methacrylic acid acylated gelatin, wherein nuclear magnetic resonance spectra before and after modification are shown in figures 2 and 3.

(2) Adding the methacrylic acid acylated gelatin into deionized water according to the proportion of 100g/L, mixing, adding succinic anhydride according to the proportion of 1.6:1 of the volume mass ratio of succinic anhydride to the methacrylic acid acylated gelatin, stirring at 50 ℃ for reaction for 24 hours, dialyzing for 5 days at normal temperature, changing deionized water every 12 hours, slowly and gently stirring during dialysis, and freeze-drying at-80 ℃ after dialysis is completed to obtain the modified methacrylic acid acylated gelatin-COOH.

(3) Dissolving 50g/L of modified methacrylic acid acylated gelatin-COOH in phosphate buffer salt solution, adding 0.1g/L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, and adding 0.1g/L of N-hydroxysuccinimide; activating and continuously and gently stirring, then adding vascular endothelial growth factor according to the proportion of 0.1g/L, mixing and stirring for 8 hours, dialyzing for 5 days at normal temperature, changing deionized water every 12 hours, slowly and gently stirring during dialysis, and freeze-drying at-80 ℃ to obtain the covalent grafted methacrylic acid acylated gelatin-vascular endothelial growth factor.

(4) Mixing phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite and phosphate buffer salt solution according to the proportion of 5g/L under the dark condition, magnetically stirring for 2 hours at 50 ℃ to prepare phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite solution, adding methacrylic acid acylated gelatin-vascular endothelial growth factor into the phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite solution according to the proportion of 50g/L, magnetically stirring for 8 hours at 50 ℃ to obtain methacrylic acid acylated gelatin-vascular endothelial growth factor hydrogel after uniform stirring.

(5) And (3) dissolving Polycaprolactone (PCL) and gelatin in a hexafluoroisopropanol solution according to a ratio of 60g/L, rapidly stirring at 40 ℃, wherein the mass ratio of the polycaprolactone to the gelatin is 1:1, and preparing the PCL-gelatin flexible oriented nanofiber membrane by using an electrostatic spinning method (the electrostatic spinning method is adopted, the solution flow rate is 2ml/h, the voltage is 15KV, the rotating speed of a high-speed roller collector is 2000rpm, the environmental humidity is controlled to be about 30%, the distance between the collector and a spinneret is 20 cm), washing with a phosphate buffer salt solution, and naturally drying to obtain the PCL-gelatin electrostatic spinning flexible oriented nanofiber membrane. The surface morphology is shown in fig. 4, and the characteristic that the nanofiber membrane presents directional arrangement can be observed, and the fibers are in the nanometer scale (300-500 nanometers).

(6) Spreading the methacrylic acid acylated gelatin-vascular endothelial growth factor hydrogel obtained in the step (4) on a polycaprolactone-gelatin flexible directional nanofiber membrane, immersing VEGF into pores of the fiber along with the hydrogel, carrying out in-situ photocrosslinking curing under blue light irradiation of 405nm, and carrying out vacuum drying to obtain the flexible directional nanofiber composite membrane with VEGF concentration gradient. The overall appearance and flexibility of the composite film are shown, and as shown in fig. 5, the overall flexibility of the composite film is good; the surface morphology is shown in fig. 6, and it can be observed that the hydrogel layer is uniformly covered on the nanofiber membrane and immersed in the fiber pores to form a double-layer composite membrane structure.

Example 2

(1) Adding sodium carbonate into deionized water, regulating pH to 10, adding gelatin according to the proportion of 50g/L, heating and continuously stirring to enable the gelatin to be fully dissolved, adding methacrylic anhydride, wherein the volume mass ratio of the methacrylic anhydride to the gelatin is 1:1, continuously stirring for 4 hours at 40 ℃, dialyzing for 8 days at normal temperature, changing deionized water every 12 hours, slowly and gently stirring during dialysis, and freeze-drying at-80 ℃ to obtain the methacrylic acid acylated gelatin.

(2) Adding the methacrylic acid acylated gelatin into deionized water according to the proportion of 200g/L, mixing, adding succinic anhydride according to the proportion of 1:1 of the volume mass ratio of succinic anhydride to the methacrylic acid acylated gelatin, heating and continuously stirring, dialyzing for 8 days at normal temperature until the reaction is complete, changing deionized water every 12 hours, slowly and gently stirring during the dialysis, and freeze-drying at-80 ℃ to obtain the modified methacrylic acid acylated gelatin-COOH after the dialysis is completed.

(3) Dissolving 100g/L of modified methacrylic acid acylated gelatin-COOH in phosphate buffer salt solution, adding 0.3g/L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, and adding 0.3g/L of N-hydroxysuccinimide; activating and continuously and gently stirring, then adding vascular endothelial growth factor according to the proportion of 0.4g/L, mixing and stirring for 16 hours, dialyzing for 5 days at normal temperature, changing deionized water every 12 hours, slowly and gently stirring during dialysis, and freeze-drying at-80 ℃ to obtain the covalent grafted methacrylic acid acylated gelatin-vascular endothelial growth factor.

(4) Mixing phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite and phosphate buffer salt solution according to the proportion of 10g/L under the dark condition, magnetically stirring for 6 hours at 50 ℃ to prepare phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite solution, adding methacrylic acid acylated gelatin-vascular endothelial growth factor into the phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite solution according to the proportion of 100g/L, magnetically stirring for 6 hours at 50 ℃ to obtain methacrylic acid acylated gelatin-vascular endothelial growth factor hydrogel.

(5) And (3) dissolving Polycaprolactone (PCL) and gelatin in a hexafluoroisopropanol solution according to a proportion of 50g/L, rapidly stirring at 40 ℃, wherein the mass ratio of the polycaprolactone to the gelatin is 1:2, and obtaining the PCL-gelatin flexible oriented nanofiber membrane by using an electrostatic spinning method (the electrostatic spinning method is that the solution flow rate is 0.5ml/h, the voltage is 20KV, the rotating speed of a high-speed roller collector is 2300rpm, the environmental humidity is controlled to be about 30%, the distance between the collector and a spinneret is 15 cm), washing with a phosphate buffer salt solution, and naturally drying. The surface morphology was similar to example 1, with the fibers being nanoscale (300-500 nm).

(6) And (3) spreading the methacrylic acid acylated gelatin-vascular endothelial growth factor hydrogel obtained in the step (4) on a polycaprolactone-gelatin flexible directional nanofiber membrane, immersing VEGF into pores of the fiber along with the hydrogel, carrying out in-situ photocrosslinking curing under the irradiation of 405nm blue light, and carrying out vacuum drying to obtain the flexible directional nanofiber composite membrane with VEGF concentration gradient, wherein the hydrogel layer can be observed to be uniformly covered on the nanofiber membrane and immersed into the fiber pores to form a double-layer composite membrane structure.

Example 3

(1) Adding sodium carbonate into deionized water, regulating pH to 9, adding gelatin according to the proportion of 200g/L, heating and continuously stirring to enable the gelatin to be fully dissolved, adding methacrylic anhydride, wherein the volume mass ratio of the methacrylic anhydride to the gelatin is 3:1, continuously stirring for 8 hours at 45 ℃, dialyzing for 10 days at normal temperature, changing deionized water every 12 hours, slowly and gently stirring during dialysis, and freeze-drying at-80 ℃ to obtain the methacrylic acid acylated gelatin.

(2) Adding methacrylic acid acylated gelatin into deionized water according to the proportion of 50g/L, mixing, adding succinic anhydride according to the proportion of 3:1 of the volume mass ratio of succinic anhydride to methacrylic acid acylated gelatin, heating and continuously stirring, dialyzing for 8 days at normal temperature until the reaction is complete, changing deionized water every 12 hours, slowly and gently stirring during the dialysis, and freeze-drying at-80 ℃ to obtain the modified methacrylic acid acylated gelatin-COOH after the dialysis is completed.

(3) Dissolving modified methacrylic acid acylated gelatin-COOH in phosphate buffer salt solution according to the proportion of 200g/L, and adding a mixture of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide according to the proportion of 0.5 g/L; activating and continuously stirring gently, adding vascular endothelial growth factor according to the proportion of 0.4g/L, mixing and stirring for 24 hours, dialyzing for 10 days at normal temperature, changing deionized water every 12 hours, slowly and gently stirring during dialysis, and freeze-drying at-80 ℃ after dialysis is completed to obtain the covalent grafted methacrylic acid acylated gelatin-vascular endothelial growth factor.

(4) Mixing phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite and phosphate buffer salt solution according to the proportion of 20g/L under the dark condition, magnetically stirring for 6 hours at 50 ℃ to prepare phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite solution, adding methacrylic acid acylated gelatin-vascular endothelial growth factor into the phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite solution according to the proportion of 200g/L, magnetically stirring for 8 hours at 50 ℃, and uniformly stirring to obtain methacrylic acid acylated gelatin-vascular endothelial growth factor hydrogel.

(5) Dissolving Polycaprolactone (PCL) and gelatin in a hexafluoroisopropanol solution according to a ratio of 70g/L, rapidly stirring at 40 ℃, wherein the mass ratio of the polycaprolactone to the gelatin is 2:1, and obtaining the PCL-gelatin electrostatic spinning flexible oriented nanofiber membrane by using an electrostatic spinning method (the electrostatic spinning condition is that the solution flow rate is 1.5ml/h, the voltage is 10KV, the rotating speed of a high-speed roller collector is 2500rpm, the environmental humidity is controlled to be about 30%, the distance between the collector and a spinneret is 18 cm), washing with a phosphate buffer salt solution, and naturally drying; the surface morphology was similar to example 1, and the fibers were shown to be nanoscale (300-500 nm).

(6) Spreading the methacrylic acid acylated gelatin-vascular endothelial growth factor hydrogel obtained in the step (4) on a polycaprolactone-gelatin flexible directional nanofiber membrane, immersing VEGF into pores of the fiber along with the hydrogel, carrying out in-situ photocrosslinking curing under the irradiation of 465nm blue light, and carrying out vacuum drying to obtain the flexible directional nanofiber composite membrane with VEGF concentration gradient. It can be observed that the hydrogel layer uniformly covers the nanofiber membrane and is immersed in the fiber pores to form a double-layer composite membrane structure.

Claims

1. The preparation method of the flexible directional nanofiber composite membrane for promoting the regeneration of blood vessels is characterized by comprising the following steps of:

(1) Adding sodium carbonate into deionized water, adjusting the pH to 7-10, adding gelatin according to the proportion of 50-200g/L, heating and continuously stirring to enable the gelatin to be fully dissolved, adding methacrylic anhydride, wherein the volume-mass ratio of the methacrylic anhydride to the gelatin is 1:1-3:1, continuously stirring for 1-8 hours at the temperature of 40-50 ℃, dialyzing at normal temperature, and freeze-drying after the dialysis is completed to obtain methacrylic acid acylated gelatin;

(2) Adding methacrylic acid acylated gelatin into deionized water according to the proportion of 50-200g/L, mixing, adding succinic anhydride according to the volume-mass ratio of 1:1-3:1 of succinic anhydride to methacrylic acid acylated gelatin, heating and continuously stirring, dialyzing at normal temperature until the reaction is complete, and freeze-drying after the dialysis is completed to obtain modified methacrylic acid acylated gelatin-COOH;

(3) Dissolving modified methacrylic acid acylated gelatin-COOH in phosphate buffer salt solution according to the proportion of 50-200g/L, and adding a mixture of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide according to the proportion of 0.1-0.5 g/L; activating and continuously stirring gently, then adding vascular endothelial growth factor according to the proportion of 0.1-0.4 g/L, mixing and stirring for 6-24 hours, dialyzing at normal temperature, and freeze-drying after the dialysis is finished to obtain the covalent grafted methacrylic acid acylated gelatin-vascular endothelial growth factor;

(4) Mixing phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite and phosphate buffer salt solution in a light-shielding condition according to a proportion of 5-20 g/L, magnetically stirring for 1-8 hours at 50 ℃ to prepare phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite solution, adding methacrylic acid acylated gelatin-vascular endothelial growth factor into the phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite solution according to a proportion of 50-200g/L, magnetically stirring for 1-8 hours at 50 ℃, and uniformly stirring to obtain methacrylic acid acylated gelatin-vascular endothelial growth factor hydrogel;

(5) Dissolving polycaprolactone and gelatin in a hexafluoroisopropanol solution according to a ratio of 50-70 g/L, rapidly stirring at 40 ℃, wherein the mass ratio of the polycaprolactone to gelatin is 1:1-2:1, preparing a polycaprolactone-gelatin flexible oriented nanofiber membrane by using an electrostatic spinning method, washing with a phosphate buffer salt solution, and naturally drying for later use;

(6) And (3) spreading the methacrylic acid acylated gelatin-vascular endothelial growth factor hydrogel obtained in the step (4) on a polycaprolactone-gelatin flexible directional nanofiber membrane, carrying out in-situ photocrosslinking solidification under blue light irradiation, and carrying out vacuum drying to obtain the flexible directional nanofiber composite membrane.

2. The method for preparing a flexible oriented nanofiber composite membrane for promoting vascular regeneration according to claim 1, wherein the method comprises the following steps: the dialysis conditions in the steps (1) - (3) are as follows: dialyzing for 5-10 days at normal temperature, changing deionized water every 12 hours, and slowly and gently stirring during the dialysis.

3. The method for preparing a flexible oriented nanofiber composite membrane for promoting vascular regeneration according to claim 1, wherein the method comprises the following steps: in the step (3), the mass ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to the N-hydroxysuccinimide is 1:1-1:3.

4. The method for preparing a flexible oriented nanofiber composite membrane for promoting vascular regeneration according to claim 1, wherein the method comprises the following steps: the conditions of the electrostatic spinning in the step (5) are as follows: the flow rate of the solution is 0.5-2 ml/h, the voltage is 10-20 KV, the rotating speed of the high-speed roller collector is 2000-3000 rpm, the fibers are aligned, the ambient humidity is controlled at 30%, and the distance between the collector and the spinneret is 15-20 cm.

5. The method for preparing a flexible oriented nanofiber composite membrane for promoting vascular regeneration according to claim 1, wherein the method comprises the following steps: the wavelength of the blue light in the step (6) is 405-460 nm.