CN112704766A

CN112704766A - Preparation method of three-dimensional bionic elastic nanofiber scaffold

Info

Publication number: CN112704766A
Application number: CN202011416892.5A
Authority: CN
Inventors: 陈苏; 崔婷婷
Original assignee: Janus New Materials Co ltd
Current assignee: Janus New Materials Co ltd
Priority date: 2020-12-07
Filing date: 2020-12-07
Publication date: 2021-04-27
Anticipated expiration: 2040-12-07
Also published as: CN112704766B

Abstract

The invention relates to a preparation method of a three-dimensional bionic elastic nanofiber scaffold, which comprises the following specific steps: first AgNO₃Dropwise adding the solution into a carboxymethyl chitosan solution to react to obtain a CMC/Ag nano particle solution; then spinning the PCL spinning solution and the PCE spinning solution to obtain a nano fiber support consisting of PCL/PCE nano fibers; spraying the PCL spinning solution on the PCL/PCE nano-fiber support in the same spinning method, and drying the PCL spinning solution in vacuum at a certain temperature to remove residual formic acid to obtain a PCL-PCL/PCE double-layer nano-fiber support; and finally, scraping the CMC/Ag nano particle solution on a PCL-PCL/PCE double-layer nanofiber support, and drying the PCL/PCE-PCL-CMC/Ag nano particle solution in vacuum to obtain the three-dimensional PCL/PCL-CMC/Ag nanofiber composite support. The method is easy to operate and versatile, overcomes bacterial infection and promotes healing of full-thickness skin wounds by simulating the three-layer structure and composition of the skin.

Description

Preparation method of three-dimensional bionic elastic nanofiber scaffold

Technical Field

The invention relates to a preparation method of a three-dimensional bionic elastic nanofiber scaffold, in particular to a preparation method of a three-dimensional bionic elastic nanofiber scaffold which can overcome bacterial infection and promote healing and regeneration of a full-thickness skin wound.

Background

In recent years, 30 million people die or suffer skin injury each year due to thermochemical substances, electric radiation and other accidents. However, due to the high incidence of skin wound defects, high treatment cost, large wound area, irregular shape and much exudate, wound treatment and management has become a major medical and social urgent problem to be solved. Although some emerging technologies (surgical resection, thick skin grafting) and certain functional materials (nanofiber scaffolds, porous foams, biocompatible membranes and functional hydrogels) have become the most widely and preferred methods of treatment for large area skin repair. However, these techniques do not produce the most effective therapeutic effect and are often associated with drawbacks such as being expensive, time consuming, secondary damaging and complicated to manufacture. These materials are difficult to work simultaneously in four distinct and interrelated phases of wound healing (hemostasis, antisepsis, proliferation and remodeling). Therefore, there is an urgent need to construct a multifunctional biomaterial having a biomimetic structure, biocompatibility, anti-infection effect and mechanical properties, thereby generating a synergistic effect at four stages of wound healing and rapidly constructing skin regeneration.

The large-area nanofiber membrane with the network structure prepared by the microfluid air-jet spinning method can well simulate extracellular matrix (ECM) components and stimulate the natural functions of cells, thereby providing the cells with a large surface-to-volume ratio and high porosity, and enhancing the transportation of oxygen and nutrients. In addition, nanofiber scaffolds show unique advantages in accelerating wound healing. On the one hand, structurally, cells in the natural skin are embedded either in the entangled three-dimensional (3D) fibrous matrix (dermal fibroblasts) or in the microfiber network (i.e., (basement membrane) surface (epidermal keratinocytes.) on the other hand, as an anchoring platform for the fine 3D nanofiber structure and specific chemical composition, it also provides clues to regulate the response of cells to unique functions

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a preparation method for constructing a three-dimensional bionic elastic nanofiber scaffold which overcomes bacterial infection and promotes the healing and regeneration of a full-thickness skin wound, is easy to operate and universal, and overcomes the bacterial infection and promotes the healing of the full-thickness skin wound by simulating the three-layer structure and the composition of skin.

The technical scheme of the invention is as follows: a preparation method of a three-dimensional bionic elastic nanofiber scaffold comprises the following specific steps:

a. dissolving carboxymethyl chitosan (CMC) in distilled water, heating to reaction temperature, adjusting the solution to alkaline pH condition with alkali, and continuously stirring to obtain carboxymethyl chitosan solution;

b. mixing AgNO₃Dripping the solution into carboxymethyl chitosan solution to react for a period of time to obtain CMC/Ag nano particle solution;

c. adding Polycaprolactone (PCL) into an organic solvent, fully stirring and uniformly dissolving the PCL to obtain a PCL spinning solution, and taking the PCL spinning solution as an A spinning solution; dissolving poly citric acid-epsilon-poly lysine (PCE) in formic acid until the PCE is completely dissolved to obtain a PCE spinning solution as a B spinning solution; then, respectively injecting the spinning solution A and the spinning solution B into two identical injectors to connect two inlets of a Y-shaped microfluid chip, and adjusting the flow rate of the two phases A and B; then spinning is carried out, the obtained product is dried in vacuum at a certain temperature to remove residual formic acid, and in the spinning process, certain air pressure and certain spraying distance are set to obtain a nanofiber bracket consisting of PCL/PCE nanofibers;

d. then, spraying a PCL spinning solution (A spinning solution) on the PCL/PCE nano-fiber support by using the same spinning Method (MBS), and drying the PCL spinning solution (A spinning solution) at a certain temperature in vacuum to remove residual formic acid to obtain a PCL-PCL/PCE double-layer nano-fiber support;

e. and finally, scraping the solution obtained in the step b on a PCL-PCL/PCE double-layer nanofiber support by a scraping method, and drying the solution at a certain temperature in vacuum to remove residual solvent to form the three-dimensional PCL/PCE-PCL-CMC/Ag nanofiber composite support with the upper layer being a CMC/Ag antibacterial layer, the middle layer being a PCL nanofiber support layer and the lowest layer being the PCL/PCE nanofiber support layer.

Preferably, the reaction temperature in the step a is 50-65 ℃; the alkali is NaOH or KOH solution; the pH value of the alkaline solution is 10-12; the mass concentration of the carboxymethyl chitosan solution is 0.3-0.6%.

Preference is given to AgNO as described in step b₃The molar concentration of the solution is 0.1mol/L-0.2 mol/L; dropwise adding AgNO₃The mass of the solution accounts for 0.4-0.8% of that of the carboxymethyl chitosan solution; the reaction time is 2-4 h.

Preferably, the organic solvent in step c is formic acid or hexafluoroisopropanol; the mass concentration of the PCL spinning solution is 14-20%; the mass concentration of the PCE spinning solution is 14-20%; the inner diameter of the Y-shaped microfluid chip channel is 0.5-1 mm; the length of the Y-shaped microfluidic chip channel is 5-7 cm.

Preferably, the parameters of the air jet spinning in the step c and the step d are as follows: the air pressure range is 0.01-0.5 MPa; the distance between the screen and the injector nozzle ranges from 23cm to 37 cm; the flow rate of the spinning solution is 0.1-5 mL/h.

Preferably, the diameter of the fiber of the PCL-PCE nano fiber bracket in the step c is 44-250 nm; the tensile strength range of the PCL-PCE nano fiber support is 18.65 +/-1.07 MPa.

Preferably, the temperature of the vacuum drying in the step c, the step d and the step e is 25-35 ℃; the vacuum drying time is 6-12 h.

Preferably, the mechanical strength range of the PCL-PCL-PCE double-layer nanofiber scaffold in the step d is 23.89 +/-2.23 MPa.

Preferably, the draw-down method described in step e is a wire bar draw-down method, and the draw-down speed is 200-400 mm/h.

Preferably, the tensile strength of the three-dimensional PCL-PCE-PCL-CMC-Ag nanofiber composite scaffold in the step e is 26.36 +/-1.77 MPa.

Preferably, the weight average molecular weight of the polycaprolactone PCL is 80000-160000; the PCE prepolymer described in step a was prepared according to the literature (M.Wang, Y.Guo, M.Yu, P.X.Ma, C.Mao, B.Lei, Photocurable and biodegradable polymeric-polyethylene polymers as high purity biocompatible polymeric vectors for biochemical-guided siRNA and miRNA delivery. acta biomaterials 54(2017) 69-80).

We developed a layer-by-layer spinning (LBL) method to manufacture 3D bionic elastic multifunctional (PCL/PCE-PCL-CMC/Ag) nano fiber scaffold, which has excellent mechanical properties, optimized hydrophilicity and strong antibacterial activity, and can overcome drug-resistant bacterial infection and wound healing. The composition and structure of the three-dimensional nanofiber scaffold are changed by an L-B-L assembly method, and the three-dimensional nanofiber scaffold completes wound healing processes including hemostasis, inflammation, proliferation and remodeling under the mutual synergistic action of the structure and the components, and finally realizes skin regeneration. In addition, the three-dimensional nanofiber scaffold not only shows excellent cell adhesion capability, but also has good oxygen permeability, high surface-to-volume ratio and biocompatibility. Therefore, the three-dimensional (PCL/PCE-PCL-CMC/Ag) tissue engineering scaffold provides effective antibiosis and a proper environment for the adhesion and proliferation of cells, which is a feasible wound repair strategy. Importantly, more concurrent work may be performed, including extensive skin damage and skin burns.

Has the advantages that:

1. the three-dimensional bionic elastic nanofiber scaffold prepared by the method has the characteristics of adjustable diameter and size and controllable appearance.

2. The preparation method of the three-dimensional bionic elastic nanofiber scaffold provided by the invention is simple in equipment, convenient to operate and capable of realizing large-scale preparation.

3. The mechanical strength and elasticity of the three-dimensional bionic elastic nanofiber scaffold prepared by the method can respectively reach 26.36 +/-1.77 MPa and 270 +/-10 percent; its tensile strength and elasticity are consistent with human skin ECM.

4. The three-dimensional bionic elastic nanofiber scaffold prepared by the invention has high antibacterial activity, and is particularly suitable for wound infection of main pathogens such as escherichia coli and staphylococcus aureus.

5. The three-dimensional bionic elastic nanofiber scaffold prepared by the invention has excellent biocompatibility and histocompatibility.

6. The three-dimensional bionic elastic nanofiber scaffold prepared by the method has a rich porous structure and a high surface area, and can promote recruitment of cells.

7. The three-dimensional bionic elastic nanofiber scaffold prepared by the invention can promote angiogenesis, collagen deposition and granulation tissue formation, and effectively prevent full-layer skin defect wound infection.

Drawings

FIG. 1 is an SEM image of PCL/PCE nanofiber scaffolds prepared in example 1;

FIG. 2 is a graph showing tensile strength and elasticity curves of the three-dimensional biomimetic elastic nanofiber scaffold prepared in example 2;

FIG. 3 is a graph of the antibacterial activity of the three-dimensional biomimetic elastic nanofiber scaffold prepared in example 3;

fig. 4 is a graph showing that the three-dimensional biomimetic elastic nanofiber scaffold prepared in example 3 has excellent biocompatibility and histocompatibility.

Detailed Description

The present invention will be described below with reference to specific examples, but the present invention is not limited to the following examples.

The PCE used in the embodiment is prepared according to the literature, and the specific preparation method is as follows: adding citric acid and 1.8-octanediol into a four-neck flask according to the mass ratio of 10:9, stirring, heating an oil bath to 165 ℃ until the citric acid and the 1.8-octanediol are melted, cooling the temperature of the oil bath to 140 ℃ for reaction for a period of time, adding a small amount of deionized water for washing and dissolving, and finally drying at normal temperature to obtain solid, namely obtaining the poly-citric acid for later use. Weighing 2g of the dried poly citric acid solid, adding the weighed poly citric acid solid into a 25ml beaker, adding 2g of poly lysine and 12ml of DMSO into the beaker, stirring, adding 0.1g of EDC and 0.08g of NHS as catalysts, and reacting for 72 hours to finally obtain the PCE elastomer polymer.

Example 1

0.3g of carboxymethyl chitosan was dissolved in 99.7g of distilled water, the reaction temperature was raised to 50 ℃, the solution was adjusted to an alkaline pH of 10 using NaOH, and continuous stirring was carried out to obtain a carboxymethyl chitosan solution with a mass concentration of 0.3 wt%. 0.8g of 0.1mol/L AgNO₃The solution is added into carboxymethyl chitosan solution for reaction for 4 hours. The formation of the silver nanoparticle solution was observed by monitoring the color change (visual observation that the reduction reaction started to work and the silver nanoparticles started to become seeds when the color of the solution started to change from its original color to a different degree of yellow). Then, 2.8g of PCL (weight average molecular weight: 80000) was added to a beaker containing 17.2g of formic acid, and then the PCL was thoroughly dissolved with stirring uniformly to obtain a 14 wt% PCL spinning solution, which was taken as an A solution. Similarly, 2.8g PCE was dissolved in 17.2g formic acid until the PCE was completely dissolved to obtain a PCE spinning solution of 14 wt% as a B solution. Spinning A intoThe silk solution and the B spinning solution are respectively injected into two 10mL injectors which are connected with two inlets of a Y-shaped microfluidic chip with the upper channel diameter of 0.5mm and the length of 5cm, and the flow rates of the A (0.1mL/h) phase and the B (0.1mL/h) phase are adjusted. Then spinning is carried out, in the spinning process, the air pressure is set to be 0.01MPa, the nano fibers are collected on a nylon 66 screen, the screen is 23cm away from a nozzle of an injector, and then the nano fibers are dried in vacuum for 12 hours at 25 ℃ to remove residual formic acid, as shown in figure 1, the obtained nano fiber support is composed of 250nm PCL/PCE nano fibers, and the tensile strength of the nano fiber support is 17.58 MPa; then, a microfluid air-jet spinning method is used, the prepared PCL spinning solution (the mass concentration is 14 wt%) is air-jetted on the PCL/PCE nano-fiber support with the same parameters (the air pressure is 0.01MPa, the distance between a screen and a nozzle of an injector is 23cm, and the sample injection speed is 0.1mL/h), and the PCL spinning solution is dried in vacuum for 12h at the temperature of 25 ℃ to remove residual formic acid, so that the PCL-PCL/PCE double-layer nano-fiber support is obtained. Finally, the CMC/Ag ion solution is coated on the PCL-PCL/PCE double-layer nanofiber support at a coating speed of 200mm/h by a wire rod coating method to form a three-dimensional PCL/PCE-PCL-CMC/Ag nanofiber composite support with the upper layer being a CMC/Ag antibacterial layer, the middle layer being a PCL nanofiber support layer and the lowest layer being the PCL/PCE nanofiber support layer, and the three-dimensional PCL/PCE-PCL-CMC/Ag nanofiber composite support is dried in vacuum at 25 ℃ for 12h to remove residual solvent, so that the tensile strength of the obtained fiber composite support is 24.59 MPa. The three-dimensional bionic nanofiber scaffold is finally applied to the wound surface of a rat, and the results of antibacterial experiments, animal experiments and tissue analysis experiments show that the three-dimensional bionic nanofiber scaffold can effectively prevent the wound from being infected by escherichia coli and staphylococcus, wherein the killing rate of the escherichia coli and the staphylococcus can reach 88% and 92% respectively. Meanwhile, the biological compatibility of the fibroblast is excellent, and the survival rate of the fibroblast reaches 160% on the 7 th day. But also can accelerate the formation of epidermis, dermis and hair follicle tissue to promote the healing of skin wound and skin regeneration.

Example 2

Dissolving 0.5g carboxymethyl chitosan in 99.5g distilled water, raising reaction temperature to 60 deg.C, adjusting pH to 11 with KOH, and continuously stirring to obtain a solution with mass concentration of 0.5wt% carboxymethyl chitosan solution. 0.6g of 0.15mol/L AgNO₃The solution is added into carboxymethyl chitosan solution for reaction for 3 hours. The formation of the silver nanoparticle solution was observed by monitoring the color change (visual observation that the reduction reaction started to work and the silver nanoparticles started to become seeds when the color of the solution started to change from its original color to a different degree of yellow). Then, 10.8g of PCL (weight average molecular weight: 160000) was added to a beaker containing 49.2g of hexafluoroisopropanol, and then the homogeneous PCL was thoroughly dissolved with sufficient stirring to obtain a PCL spinning solution of 18 wt%, which was taken as the A solution. Similarly, 10.8g PCE was dissolved in 49.2g hexafluoroisopropanol until the PCE was completely dissolved to obtain a PCE spinning solution of 18 wt% as a B solution. The spinning solution A and the spinning solution B are respectively injected into two 50mL injectors to connect two inlets of a Y-shaped microfluidic chip with the upper channel diameter of 0.7mm and the length of 6cm, and the flow rates of the two phases of A (2mL/h) and B (2mL/h) are adjusted. And then spinning is carried out, in the spinning process, the air pressure is set to be 0.2MPa, the nano fibers are collected on a nylon 66 screen, the spraying distance is 29cm, then the nano fibers are dried in vacuum for 10 hours at the temperature of 30 ℃ to remove residual hexafluoroisopropanol, and the scanning electron microscope analysis shows that the obtained nano fiber support is composed of 160nm PCL/PCE nano fibers, and the tensile strength of the nano fiber support is 18.65 MPa. Then, a microfluid air-jet spinning method is used, the prepared PCL spinning solution (the mass concentration is 18 wt%) is air-jetted on a PCL/PCE nano-fiber bracket by using the same parameters (the air pressure is 0.2MPa, the jet distance is 29cm, and the sample injection speed is 2mL/h), and the PCL spinning solution is dried in vacuum for 10 hours at the temperature of 30 ℃ so as to remove the residual hexafluoroisopropanol; and obtaining the PCL-PCL/PCE double-layer nanofiber bracket. Finally, the CMC/Ag ion solution is coated on the PCL-PCL/PCE double-layer nanofiber support at a coating speed of 300mm/h by a wire rod coating method to form a three-dimensional PCL/PCE-PCL-CMC/Ag nanofiber composite support with the upper layer being a CMC/Ag antibacterial layer, the middle layer being a PCL nanofiber support layer and the lowest layer being the PCL/PCE nanofiber support layer, and the three-dimensional PCL/PCE-PCL-CMC/Ag nanofiber composite support is dried in vacuum at 30 ℃ for 10h to remove residual solvent, so that the tensile strength of the obtained fiber composite support is 23.89MPa (as shown in figure 2). Finally applying the extract on the wound surface of a rat through an antibacterial experiment, an animal experiment and tissuesThe results of analysis experiments show that the three-dimensional bionic nanofiber scaffold can effectively prevent a wound from being infected by escherichia coli and staphylococcus, wherein the killing rates of the escherichia coli and the staphylococcus can reach 85% and 91% respectively. Meanwhile, the biological compatibility of the fibroblast is excellent, and the survival rate of the fibroblast reaches 160% on day 8. But also can accelerate the formation of epidermis, dermis and hair follicle tissue to promote the healing of skin wound and skin regeneration.

Example 3

0.6g of carboxymethyl chitosan is dissolved in 99.4g of distilled water, the reaction temperature is raised to 65 ℃, the solution is adjusted to be alkaline pH 12 by using NaOH, and the solution is continuously stirred to obtain a carboxymethyl chitosan solution with the mass concentration of 0.6 wt%. 0.4g of 0.2mol/L AgNO₃The solution is added into carboxymethyl chitosan solution for reaction for 2 h. The formation of the silver nanoparticle solution was observed by monitoring the color change (visual observation that the reduction reaction started to work and the silver nanoparticles started to become seeds when the color of the solution started to change from its original color to a different degree of yellow). Then, 20g of PCL (weight average molecular weight: 160000) was added to a beaker containing 80g of formic acid, and then the homogeneous PCL was completely dissolved with sufficient stirring to obtain a 20 wt% PCL spinning solution, which was taken as an A solution. Similarly, 20g PCE was dissolved in 80g formic acid until the PCE was completely dissolved to obtain a PCE spinning solution of 20 wt% as a B solution. The spinning solution A and the spinning solution B are respectively injected into two 100mL injectors to connect two inlets of a Y-shaped microfluidic chip with the upper channel diameter of 1mm and the length of 7cm, and the flow rates of the two phases of A (5mL/h) and B (5mL/h) are adjusted. Then spinning is carried out, in the spinning process, the air pressure is set to be 0.5MPa, the nano fibers are collected on a nylon 66 screen, the screen is at a distance of 37cm from a nozzle of an injector, then the nano fibers are dried for 6 hours in vacuum at 35 ℃ to remove residual formic acid, and the analysis of a scanning electron microscope shows that the obtained nano fiber support is composed of 44nm PCL/PCE nano fibers, and the tensile strength of the nano fiber support is 19.72 MPa; then, the prepared PCL spinning solution is spun by a microfluid air-jet spinning method with the same parameters (air pressure is 0.5MPa, the distance between a screen and a nozzle of an injector is 37cm, and the sample injection speed is 5mL/h)(the mass concentration is 20 wt%) is sprayed on the PCL/PCE nano-fiber support, and the PCL/PCE nano-fiber support is dried for 6 hours in vacuum at 35 ℃ to remove residual formic acid, so that the PCL-PCL/PCE double-layer nano-fiber support is obtained. Finally, the CMC/Ag ion solution is coated on a PCL-PCL/PCE double-layer nanofiber support at a coating speed of 400mm/h by a wire rod coating method, a three-dimensional PCL/PCE-PCL-CMC/Ag nanofiber composite support with the upper layer being a CMC/Ag antibacterial layer, the middle layer being a PCL nanofiber support layer and the lowest layer being the PCL/PCE nanofiber support layer is formed, and the three-dimensional PCL/PCE-PCL-CMC/Ag nanofiber composite support is dried in vacuum at 30 ℃ for 10h to remove residual solvent, so that the tensile strength of the obtained fiber composite support is 23.89 MPa. Finally, the three-dimensional bionic nano-fiber scaffold is applied to a rat wound, and the results of antibacterial experiments, animal experiments and tissue analysis experiments show that the three-dimensional bionic nano-fiber scaffold can effectively prevent the wound from being infected by escherichia coli and staphylococcus, wherein the killing rates of the escherichia coli and the staphylococcus can respectively reach 86% and 92% (as shown in figure 3). Meanwhile, the composition has excellent biocompatibility to fibroblasts, and as can be seen from fig. 4, the survival rate of the fibroblasts reaches 160% on day 7. But also can accelerate the formation of epidermis, dermis and hair follicle tissue to promote the healing of skin wound and skin regeneration.

Claims

1. A preparation method of a three-dimensional bionic elastic nanofiber scaffold comprises the following specific steps:

a. dissolving carboxymethyl chitosan CMC in distilled water, heating to reaction temperature, adjusting the solution to be alkaline by using alkali, and continuously stirring to obtain carboxymethyl chitosan solution;

b. mixing AgNO₃Dropwise adding the solution into a carboxymethyl chitosan solution to react to obtain a CMC-Ag nano particle solution;

c. adding Polycaprolactone (PCL) into an organic solvent, uniformly stirring to obtain a PCL spinning solution, and taking the PCL spinning solution as an A spinning solution; dissolving poly citric acid-epsilon-poly lysine PCE in formic acid to obtain a PCE spinning solution as a B spinning solution; then respectively injecting the spinning solution A and the spinning solution B into two identical injectors to connect two inlets of a Y-shaped microfluid chip, and adjusting the flow rate of the two phases A and B; carrying out air-jet spinning, and drying the obtained product in vacuum to obtain a nanofiber support consisting of PCL-PCE nanofibers;

d. spraying the PCL spinning solution on the PCL-PCE nano fiber support by using an air-jet spinning method, and then drying the PCL spinning solution in vacuum; obtaining a PCL-PCL-PCE double-layer nanofiber bracket;

e. and d, scraping the solution obtained in the step b on a PCL-PCL-PCE double-layer nanofiber support through a scraping method, and drying the solution in vacuum to form a three-dimensional PCL-PCL-CMC-Ag nanofiber composite support with the upper layer being a CMC-Ag antibacterial layer, the middle layer being a PCL nanofiber support layer and the lowest layer being the PCL-PCE nanofiber support layer, namely the three-dimensional bionic elastic nanofiber support.

2. The method according to claim 1, wherein the reaction temperature in step a is 50-65 ℃; the alkali is NaOH or KOH solution; the pH value of the alkaline solution is 10-12; the mass concentration of the carboxymethyl chitosan solution is 0.3-0.6%.

3. The method according to claim 1, wherein AgNO in step b₃The molar concentration of the solution is 0.1mol/L-0.2 mol/L; dropwise adding AgNO₃The mass of the solution accounts for 0.4-0.8% of that of the carboxymethyl chitosan solution; the reaction time is 2-4 h.

4. The method according to claim 1, wherein the organic solvent in step c is formic acid or hexafluoroisopropanol; the mass concentration of the PCL spinning solution is 14-20%; the mass concentration of the PCE spinning solution is 14-20%; the inner diameter of the Y-shaped microfluid chip channel is 0.5-1 mm; the length of the Y-shaped microfluidic chip channel is 5-7 cm.

5. The method according to claim 1, wherein the parameters of the air jet spinning in step c and step d are both: the air pressure range is 0.01-0.5 MPa; the distance between the screen and the injector nozzle ranges from 23cm to 37 cm; the flow rate of the spinning solution is 0.1-5 mL/h.

6. The method according to claim 1, wherein the diameter of the PCL-PCE nanofiber scaffold in step c is 44-250 nm; the tensile strength range of the PCL-PCE nano fiber support is 18.65 +/-1.07 MPa.

7. The method according to claim 1, wherein the vacuum drying temperature in step c, step d and step e is 25-35 ℃; the vacuum drying time is 6-12 h.

8. The method for preparing the PCL-PCL-PCE double-layer nanofiber scaffold according to claim 1, wherein the mechanical strength of the PCL-PCL-PCE double-layer nanofiber scaffold in the step d is 23.89 +/-2.23 MPa.

9. The method according to claim 1, wherein the coating method in step e is a wire bar coating method, and the coating speed is 200-400 mm/h.

10. The method according to claim 1, wherein the tensile strength of the three-dimensional PCL-PCE-PCL-CMC-Ag nanofiber composite scaffold in step e is 26.36 +/-1.77 MPa.