阅读说明：本技术 一种多取向生物人造血管及其制备方法 (Multi-orientation biological artificial blood vessel and preparation method thereof ) 是由 吴闯 王海翔 崔慧丽 于 2021-09-13 设计创作，主要内容包括：本发明公开了一种多取向生物人造血管及其制备方法,包括同轴且从内往外紧密结合在一起的水凝胶内层、纳米纤维中间层和水凝胶外层,水凝胶内层和水凝胶外层均由明胶复合材料和庆大霉素制成,纳米纤维中间层为从内往外依次为轴向纳米纤维层、径向纳米纤维层和随机纳米纤维层,纳米纤维层依次由纳米纤维芯层和纳米纤维壳层紧密结合而成,形成由纳米纤维壳层包裹纳米纤维芯层的复合结构,纳米纤维芯层由雷帕霉素和第一聚己内酯复合材料制成,纳米纤维壳层由聚葵二酸丙三醇酯和第二聚己内酯复合材料制成；本发明制备出的血管生物力学性能好,生物相容性好。(The invention discloses a multi-orientation biological artificial blood vessel and a preparation method thereof, wherein the multi-orientation biological artificial blood vessel comprises a hydrogel inner layer, a nanofiber middle layer and a hydrogel outer layer which are coaxial and tightly combined together from inside to outside, the hydrogel inner layer and the hydrogel outer layer are both made of gelatin composite material and gentamicin, the nanofiber middle layer is an axial nanofiber layer, a radial nanofiber layer and a random nanofiber layer which are sequentially arranged from inside to outside, the nanofiber layer is formed by tightly combining a nanofiber core layer and a nanofiber shell layer in sequence to form a composite structure of wrapping the nanofiber core layer by the nanofiber shell layer, the nanofiber core layer is made of rapamycin and a first polycaprolactone composite material, and the nanofiber shell layer is made of polytrimethylene sebacate and a second polycaprolactone composite material; the prepared blood vessel has good biomechanical property and biocompatibility.)

1. A multi-orientation bio-vascular prosthesis, comprising: comprises a hydrogel inner layer, a nanofiber middle layer and a hydrogel outer layer which are coaxial and tightly combined together from inside to outside, the hydrogel inner layer and the hydrogel outer layer are both made of gelatin composite material and gentamicin, the nanofiber middle layer is an axial nanofiber layer, a radial nanofiber layer and a random nanofiber layer in sequence from inside to outside, the fiber direction of the axial nanofiber layer is parallel to the axial direction of the receiving rotating shaft, the fiber direction of the radial nanofiber layer is vertical to the fiber direction of the axial nanofiber layer, the nanofiber layer is formed by tightly combining a nanofiber core layer and a nanofiber shell layer in sequence to form a composite structure in which the nanofiber core layer is wrapped by the nanofiber shell layer, the nanofiber core layer is made of rapamycin and a first polycaprolactone composite material, and the nanofiber shell layer is made of polytrimethylene sebacate and a second polycaprolactone composite material.

2. The multi-oriented bioartificial vessel of claim 1, wherein: the first polycaprolactone composite material or the second polycaprolactone composite material is prepared by blending polycaprolactone and a base material, wherein the base material is trifluoroethanol, and the mass-volume ratio of the polycaprolactone to the trifluoroethanol is (1-10) g: 100 mL.

3. The multi-oriented bioartificial vessel of claim 2, wherein: the weight ratio of the components of the polytrimethylene sebacate and the polycaprolactone in the first polycaprolactone composite material is (1-6): 10, the mass ratio of the components of the rapamycin to the polycaprolactone in the second polycaprolactone composite material is (1-100): 1000.

4. the multi-oriented bioartificial vessel of claim 1, wherein: the gentamicin and gelatin composite material comprises the following components in parts by mass (1-100): 1000, the gelatin composite material is prepared by blending gelatin and a gel matrix material, wherein the gel matrix material is deionized water, and the mass volume ratio of the gelatin to the deionized water is (1-20) g: 100 mL.

5. A method of preparing the multi-oriented bio-artificial blood vessel according to any one of claims 1 to 4, wherein: comprises the following steps of (a) carrying out,

dissolving polycaprolactone in trifluoroethanol, and magnetically stirring until the polycaprolactone is completely dissolved to prepare a polycaprolactone solution with the mass-volume ratio concentration of 1-10 wt%;

dissolving poly (trimethylene sebacate) in polycaprolactone solution to prepare coaxial electrostatic spinning shell solution with the mass-volume ratio concentration of 3-10 wt%;

dissolving rapamycin in a polycaprolactone solution to prepare a coaxial electrospinning core layer solution with the mass volume ratio concentration of 3-10 wt%;

uniformly coating the surface of the receiving rotating shaft with the Pluronic hydrogel;

respectively adopting a polycaprolactone solution, a coaxial electrostatic spinning shell layer solution and a coaxial electrostatic spinning core layer solution, and electrospinning on a receiving rotating shaft through a coaxial electrostatic spinning process to obtain a multi-orientation nanofiber tube with a three-layer structure, wherein an axial nanofiber layer, a radial nanofiber layer and a random nanofiber layer are sequentially arranged from inside to outside;

taking down the multi-orientation nano-fiber tube together with the receiving rotating shaft, vertically placing the multi-orientation nano-fiber tube in an environment at 0-4 ℃, enabling the multi-orientation nano-fiber tube to easily slide down from the receiving rotating shaft after the Pronian hydrogel is liquefied, and placing the multi-orientation nano-fiber tube in absolute ethyl alcohol to wash away residual Pronian hydrogel;

placing the cleaned multi-orientation nano fiber tube on a connecting rod and placing the connecting rod in an impregnation solution, and taking out the multi-orientation nano fiber tube after the impregnation is finished until no liquid drips on the multi-orientation nano fiber tube;

placing the multi-orientation nano-fiber tube which is arranged on the connecting rod and is soaked in a cross-linking agent solution of a soaking solution for cross-linking until the soaking solution on the multi-orientation nano-fiber tube is completely cross-linked, taking out the multi-orientation nano-fiber tube and standing until no liquid drops, and placing the multi-orientation nano-fiber tube in absolute ethyl alcohol to wash away the residual cross-linking agent;

and taking the multi-orientation nano fiber tube out of the connecting rod, and freeze-drying to obtain the formed multi-orientation biological artificial blood vessel.

6. The method of preparing a multi-oriented bio-artificial blood vessel according to claim 5, wherein: the preparation steps of the impregnation liquid are as follows,

dissolving gelatin in deionized water at 40 ℃ to prepare gelatin solution with the mass volume ratio of 1-20 wt%;

dissolving gentamicin in gelatin solution to prepare hydrogel solution with mass volume ratio of 1-20 wt%.

7. The method for preparing a multi-oriented bio-artificial blood vessel according to claim 5 or 6, wherein: when preparing the axial nanofiber layer of the multi-oriented nanofiber tube,

the rotating speed of the receiving rotating shaft is 1-50R/min, the vertical distance R from the rotating center O is 10-50cm, the XY plane angular speed is 500-2000R/min, the distance from the conductive type right-angle frame A surface is 5-20cm, the distance from the conductive type right-angle frame B surface is 5-20cm, and the spinning time is 0.5-4 h;

when preparing the radial nanofiber layer of the multi-oriented nanofiber tube,

the rotating speed of the receiving rotating shaft is 70-1000r/min, the distance from the receiving rotating shaft to the surface A of the conductive right-angle stand is 2-10cm, the distance from the receiving rotating shaft to the surface B of the conductive right-angle stand is 5-20cm, and the spinning time is 1-8 h;

when preparing the random nanofiber layer of the multi-oriented nanofiber tube,

the distance between the receiving rotating shaft and the surface A of the conductive right-angle stand is 8-20cm, the distance between the receiving rotating shaft and the surface B of the conductive right-angle stand is 8-20cm, and the spinning time is 1-8 h;

the horizontal distance between the coaxial spray head and the receiving rotating shaft is 8-30cm, the XY plane is parallel to the A plane, the A plane is vertical to the B plane through the axis of the receiving rotating shaft, and the center of the XY plane is O.

8. The method for preparing a multi-oriented bio-artificial blood vessel according to claim 5 or 6, wherein: when the multi-orientation nano-fiber tube is prepared, the co-axial nozzles are simultaneously fed with materials through two micro-pumps to respectively feed a core layer and a shell layer, the core layer feeding speed is 20-80 mu L/min, the shell layer feeding speed is 20-100 mu L/min, the spinning voltage is 8-18kV, the receiving rotating shaft is made of conductive metal materials, the axial length of the receiving rotating shaft is 200mm, and the outer diameter of the receiving rotating shaft is 4.5 mm; the inner diameter of the coaxial nozzle is 0.3mm, and the outer diameter is 1 mm.

9. The method for preparing a multi-oriented bio-artificial blood vessel according to claim 5 or 6, wherein: the diameter of the small end face cylinder of the connecting rod is 3.5mm, the height of the small end face cylinder is 250mm, the diameter of the large end face cylinder is 6mm, the height of the large end face cylinder is 2mm, the multi-orientation nanofiber pipe is sleeved on the small end face cylinder, and one end of the multi-orientation nanofiber pipe, which is immersed in the impregnation liquid, is in contact with the large end face cylinder.

10. The method for preparing a multi-oriented bio-artificial blood vessel according to claim 5 or 6, wherein: the dipping time is 60min, the cross-linking agent is an ethanol solution of genipin, the concentration is 4mM, the cross-linking time is 24h, and the freeze-drying time is 8 h.

Technical Field

The invention belongs to the technical field of biological manufacturing, and particularly relates to a multi-orientation biological artificial blood vessel and a preparation method thereof.

Background

The world health organization report shows that by 2030, 2360 million people die each year from cardiovascular disease, at which time CVD will become one of the first killers that endanger human health. At present, interventional therapy and vascular bypass surgery are mainly adopted clinically, however, the interventional therapy is often accompanied with restenosis, and the problems of insufficient source of autologous blood vessels and secondary injury caused by bypass are solved. Therefore, the development of a biological artificial blood vessel capable of replacing an autologous lesion blood vessel is becoming a research hotspot.

The coaxial electrostatic spinning process can enable the medicine to be dispersed and wrapped on the fiber shell layer, the effect of slowly releasing the medicine is achieved, meanwhile, the prepared nanofiber has a high specific surface area, can simulate an extracellular matrix structure, promotes cell adhesion proliferation, and becomes one of the methods mainly adopted in the preparation of the existing biological artificial blood vessel. In the prior art, a multi-orientation nano fiber structure can not be prepared to simulate a three-layer structure of a blood vessel.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

The present invention has been made in view of the above and/or other problems associated with the preparation of vascular prostheses of the prior art.

One of the objects of the present invention is to provide a multi-oriented bio-artificial blood vessel having a multi-oriented nanofiber layer and a hydrogel inner layer, which can improve biocompatibility while simulating a three-layer structure of a blood vessel.

In order to solve the technical problems, the invention provides the following technical scheme: a multi-orientation biological artificial blood vessel comprises a hydrogel inner layer, a nanofiber middle layer and a hydrogel outer layer which are coaxial and tightly combined together from inside to outside, the hydrogel inner layer and the hydrogel outer layer are both made of gelatin composite material and gentamicin, the nanofiber middle layer is an axial nanofiber layer, a radial nanofiber layer and a random nanofiber layer in sequence from inside to outside, the fiber direction of the axial nanofiber layer is parallel to the axial direction of the receiving rotating shaft, the fiber direction of the radial nanofiber layer is vertical to the fiber direction of the axial nanofiber layer, the nanofiber layer is formed by tightly combining a nanofiber core layer and a nanofiber shell layer in sequence to form a composite structure in which the nanofiber core layer is wrapped by the nanofiber shell layer, the nanofiber core layer is made of rapamycin and a first polycaprolactone composite material, and the nanofiber shell layer is made of polytrimethylene sebacate and a second polycaprolactone composite material.

As a preferable mode of the multi-oriented bio-artificial blood vessel of the present invention, wherein: the first polycaprolactone composite material or the second polycaprolactone composite material is prepared by blending polycaprolactone and a base material, wherein the base material is trifluoroethanol, and the mass-volume ratio of the polycaprolactone to the trifluoroethanol is (1-10) g: 100 mL.

As a preferable mode of the multi-oriented bio-artificial blood vessel of the present invention, wherein: the weight ratio of the components of the polytrimethylene sebacate and the polycaprolactone in the first polycaprolactone composite material is (1-6): 10, the mass ratio of the components of the rapamycin to the polycaprolactone in the second polycaprolactone composite material is (1-100): 1000.

as a preferable mode of the multi-oriented bio-artificial blood vessel of the present invention, wherein: the gentamicin and gelatin composite material comprises the following components in parts by mass (1-100): 1000, the gelatin composite material is prepared by blending gelatin and a gel matrix material, wherein the gel matrix material is deionized water, and the mass volume ratio of the gelatin to the deionized water is (1-20) g: 100 mL.

A method for preparing a multi-orientation biological artificial blood vessel comprises the following steps,

dissolving polycaprolactone in trifluoroethanol, and magnetically stirring until the polycaprolactone is completely dissolved to prepare a polycaprolactone solution with the mass-volume ratio concentration of 1-10 wt%;

dissolving poly (trimethylene sebacate) in polycaprolactone solution to prepare coaxial electrostatic spinning shell solution with the mass-volume ratio concentration of 3-10 wt%;

dissolving rapamycin in a polycaprolactone solution to prepare a coaxial electrospinning core layer solution with the mass volume ratio concentration of 3-10 wt%;

uniformly coating the surface of the receiving rotating shaft with the Pluronic hydrogel;

respectively adopting a polycaprolactone solution, a coaxial electrostatic spinning shell layer solution and a coaxial electrostatic spinning core layer solution, and electrospinning on a receiving rotating shaft through a coaxial electrostatic spinning process to obtain a multi-orientation nanofiber tube with a three-layer structure, wherein an axial nanofiber layer, a radial nanofiber layer and a random nanofiber layer are sequentially arranged from inside to outside;

taking down the multi-orientation nano-fiber tube together with the receiving rotating shaft, vertically placing the multi-orientation nano-fiber tube in an environment at 0-4 ℃, enabling the multi-orientation nano-fiber tube to easily slide down from the receiving rotating shaft after the Pronian hydrogel is liquefied, and placing the multi-orientation nano-fiber tube in absolute ethyl alcohol to wash away residual Pronian hydrogel;

placing the cleaned multi-orientation nano fiber tube on a connecting rod and placing the connecting rod in an impregnation solution, and taking out the multi-orientation nano fiber tube after the impregnation is finished until no liquid drips on the multi-orientation nano fiber tube;

placing the multi-orientation nano-fiber tube which is arranged on the connecting rod and is soaked in a cross-linking agent solution of a soaking solution for cross-linking until the soaking solution on the multi-orientation nano-fiber tube is completely cross-linked, taking out the multi-orientation nano-fiber tube and standing until no liquid drops, and placing the multi-orientation nano-fiber tube in absolute ethyl alcohol to wash away the residual cross-linking agent;

and taking the multi-orientation nano fiber tube out of the connecting rod, and freeze-drying to obtain the formed multi-orientation biological artificial blood vessel.

As a preferable embodiment of the method for preparing the multi-oriented biological artificial blood vessel, the method comprises the following steps: the preparation steps of the impregnation liquid are as follows,

dissolving gelatin in deionized water at 40 ℃ to prepare gelatin solution with the mass volume ratio of 1-20 wt%;

dissolving gentamicin in gelatin solution to prepare hydrogel solution with mass volume ratio of 1-20 wt%.

As a preferable embodiment of the method for preparing the multi-oriented biological artificial blood vessel, the method comprises the following steps: when preparing the axial nanofiber layer of the multi-oriented nanofiber tube,

the rotating speed of the receiving rotating shaft is 1-50R/min, the vertical distance R from the rotating center O is 10-50cm, the XY plane angular speed is 500-2000R/min, the distance from the conductive type right-angle frame A surface is 5-20cm, the distance from the conductive type right-angle frame B surface is 5-20cm, and the spinning time is 0.5-4 h;

when preparing the radial nanofiber layer of the multi-oriented nanofiber tube,

the rotating speed of the receiving rotating shaft is 70-1000r/min, the distance from the receiving rotating shaft to the surface A of the conductive right-angle stand is 2-10cm, the distance from the receiving rotating shaft to the surface B of the conductive right-angle stand is 5-20cm, and the spinning time is 1-8 h;

when preparing the random nanofiber layer of the multi-oriented nanofiber tube,

the distance between the receiving rotating shaft and the surface A of the conductive right-angle stand is 8-20cm, the distance between the receiving rotating shaft and the surface B of the conductive right-angle stand is 8-20cm, and the spinning time is 1-8 h;

the horizontal distance between the coaxial spray head and the receiving rotating shaft is 8-30cm, the XY plane is parallel to the A plane, the A plane is vertical to the B plane through the axis of the receiving rotating shaft, and the center of the XY plane is O.

As a preferable embodiment of the method for preparing the multi-oriented biological artificial blood vessel, the method comprises the following steps: when the multi-orientation nano-fiber tube is prepared, two micro-pumps are used for simultaneously supplying materials to a core layer and a shell layer of a coaxial nozzle, the core layer feeding speed is 20-80 mu L/min, the shell layer feeding speed is 20-100 mu L/min, the spinning voltage is 8-18kV, a receiving rotating shaft is made of conductive metal materials, the axial length of the receiving rotating shaft is 200mm, and the outer diameter of the receiving rotating shaft is 4.5 mm; the inner diameter of the coaxial nozzle is 0.3mm, and the outer diameter is 1 mm.

As a preferable embodiment of the method for preparing the multi-oriented biological artificial blood vessel, the method comprises the following steps: the method for preparing a multi-oriented bio-artificial blood vessel according to claim 5 or 6, wherein: the diameter of the small end face cylinder of the connecting rod is 3.5mm, the height of the small end face cylinder is 250mm, the diameter of the large end face cylinder is 6mm, the height of the large end face cylinder is 2mm, the multi-orientation nanofiber pipe is sleeved on the small end face cylinder, and one end of the multi-orientation nanofiber pipe, which is immersed in the impregnation liquid, is in contact with the large end face cylinder.

As a preferable embodiment of the method for preparing the multi-oriented biological artificial blood vessel, the method comprises the following steps: the dipping time is 60min, the cross-linking agent is an ethanol solution of genipin, the concentration is 4mM, the cross-linking time is 24h, and the freeze-drying time is 8 h.

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

the inner layer of the multi-orientation biological artificial blood vessel prepared by the invention consists of the axial orientation coaxial nano-fiber, so that the blood flow resistance can be reduced, and the generation of the endothelialization of the blood vessel is accelerated; the middle layer is composed of radial oriented coaxial nano fibers, so that radial stress can be improved, and blasting pressure is enhanced; the outer layer of the fiber tube is composed of coaxial nano fibers oriented randomly, so that the porosity can be increased, and the biomechanical property can be improved; the three-layer structure is tightly combined by combining the dipping process, and the biocompatibility can be improved; the hydrogel layer of the multi-orientation biological artificial blood vessel is prepared by a dipping and crosslinking process carried with GS, the GS can reduce the infection risk of the artificial blood vessel implanted in vivo by more than 80 percent, and the adopted natural biomaterial GEL dipping process can improve the biocompatibility of the artificial blood vessel by more than 90 percent and promote the adhesion and proliferation of cells; the adopted process for coating the F127 on the receiving rotating shaft has the temperature-sensitive characteristic, the receiving rotating shaft is vertically placed in the environment of 0-4 ℃, after the F127 is liquefied, the multi-orientation biological artificial blood vessel on the receiving rotating shaft can easily slide down, the difficulty degree of taking down the artificial blood vessel is reduced, and the preparation success rate of the multi-orientation biological artificial blood vessel is improved by over 90 percent.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:

FIG. 1 is a schematic diagram of the preparation of the present invention.

FIG. 2 is a schematic view of the preparation process of the present invention.

Fig. 3 is a schematic structural diagram of the multi-oriented bio-artificial blood vessel of the present invention.

FIG. 4 is a schematic view of the microstructure of a multi-oriented nanofiber according to the present invention.

In the figure, 100 high voltage power supply, 200 coaxial nozzles, 300 core layer micropumps, 400 shell layer micropumps, 500 receiving rotating shafts, 600 right-angle frames, 601A surfaces, 602B surfaces, 700 connecting rods, 701 large end surface cylinders, 702 small end surface cylinders, 800 impregnation liquid, 900 nanofiber middle layers, 901 axial nanofiber layers, 902 radial nanofiber layers, 903 random nanofiber layers, 1000 cross-linking agents, 2000 freeze-drying machines, 3000 hydrogel inner layers and 4000 hydrogel outer layers.

Detailed Description

Before the technical solution of the present invention is explained, the terms used herein are defined as follows:

the term "PCL" means: polycaprolactone;

the term "PGS" means: polytrimethylene sebacate;

the term "RAPA" refers to: rapamycin;

the term "GS" means: gentamicin;

the term "GEL" refers to: gelatin;

the term "TFEA" refers to: trifluoroethanol.

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

Example 1

As shown in figure 3, the invention provides a multi-orientation biological artificial blood vessel, which comprises a hydrogel inner layer, a nanofiber middle layer and a hydrogel outer layer which are coaxial and tightly combined together from inside to outside, wherein the hydrogel inner layer and the hydrogel outer layer are both made of gelatin composite material and gentamicin, the nanofiber middle layer is an axial nanofiber layer, a radial nanofiber layer and a random nanofiber layer which are sequentially arranged from inside to outside, the fiber direction of the axial nanofiber layer is parallel to the axial direction of a receiving rotating shaft, the fiber direction of the radial nanofiber layer is vertical to the fiber direction of the axial nanofiber layer, the nanofiber layer is sequentially formed by tightly combining a nanofiber core layer and a nanofiber shell layer to form a composite structure of wrapping the nanofiber core layer by a nanofiber shell layer, and the nanofiber core layer is made of rapamycin and a first polycaprolactone composite material, the nanofiber shell layer is made of a composite material of polytrimethylene sebacate and second polycaprolactone.

Further, the first polycaprolactone composite material or the second polycaprolactone composite material is prepared by blending polycaprolactone and a base material, wherein the base material is trifluoroethanol, and the mass-volume ratio of the polycaprolactone to the trifluoroethanol is (1-10) g: 100mL, the weight ratio of the components of the polytrimethylene sebacate and the polycaprolactone in the first polycaprolactone composite material is (1-6): 10; the mass ratio of the components of the rapamycin to the polycaprolactone in the second polycaprolactone composite material is (1-100): 1000.

further, the gentamicin and gelatin composite material comprises the following components in percentage by mass (1-100): 1000, the gelatin composite material is prepared by blending gelatin and a gel matrix material, wherein the gel matrix material is deionized water, and the mass volume ratio of the gelatin to the deionized water is (1-20) g: 100 mL.

Example 2

Referring to fig. 1 and 2, a second embodiment of the invention is different from embodiment 1 in that it provides a method for preparing a multi-oriented bio-artificial blood vessel, comprising the steps of,

dissolving PCL in TEFA, and magnetically stirring until polycaprolactone is completely dissolved to obtain 40g of polycaprolactone solution with mass-volume ratio concentration of 7 wt%;

dissolving PGS in a PCL solution, wherein the mass ratio of the PGS to the PCL is 1:7, and preparing a coaxial electrostatic spinning shell layer solution with the mass-volume ratio concentration of 8 wt%;

dissolving RAPA in PCL solution, wherein the mass ratio of RAPA to PCL is 1:70, and preparing a coaxial electrostatic spinning core layer solution with the mass-volume ratio concentration of 7.1 wt%;

dissolving GEL in deionized water at 40 ℃ to prepare 50g of GEL solution with the mass volume ratio of 10 wt%;

dissolving GS in a GEL solution, wherein the mass ratio of the GS to the GEL is 1:100, and preparing a hydrogel solution with the mass-volume ratio of 10.1 wt%;

uniformly coating the surface of the receiving rotating shaft with F127 hydrogel;

respectively adopting a PCL solution, a coaxial electrostatic spinning shell layer solution and a coaxial electrostatic spinning core layer solution, and electrospinning on a receiving rotating shaft through a coaxial electrostatic spinning process to obtain a multi-orientation nanofiber tube with a three-layer structure, wherein an axial nanofiber layer, a radial nanofiber layer and a random nanofiber layer are sequentially arranged from inside to outside, and the corresponding nanofiber structure is shown in FIG. 4;

taking down the multi-orientation nano-fiber tube together with the receiving rotating shaft, vertically placing the multi-orientation nano-fiber tube in an environment at 0-4 ℃, easily sliding the multi-orientation nano-fiber tube from the receiving rotating shaft after F127 is liquefied, and placing the multi-orientation nano-fiber tube in absolute ethyl alcohol to wash away residual F127;

placing the cleaned multi-orientation nano fiber tube on a connecting rod and placing the connecting rod in a hydrogel solution for dipping, and taking out the multi-orientation nano fiber tube until no liquid drips on the multi-orientation nano fiber tube after dipping;

placing the multi-orientation nano-fiber tube which is arranged on the connecting rod and is soaked in a cross-linking agent solution of a soaking solution for cross-linking until the soaking solution on the multi-orientation nano-fiber tube is completely cross-linked, taking out the multi-orientation nano-fiber tube and standing until no liquid drops, and placing the multi-orientation nano-fiber tube in absolute ethyl alcohol to wash away the residual cross-linking agent;

and taking the multi-orientation nano fiber tube out of the connecting rod, putting the multi-orientation nano fiber tube into a freeze dryer for freeze drying for 4 hours to obtain the multi-orientation biological artificial blood vessel consisting of the inner hydrogel, the middle multi-orientation nano fiber tube and the outer hydrogel.

When an axial nanofiber layer of the multi-orientation nanofiber tube is prepared, the rotating speed of a receiving rotating shaft is 10R/min, the vertical distance R from a rotating center O is 30cm, the angular speed of an X/Y plane is 1000R/min, the distance from a conductive right-angle frame A to a surface A is 10cm, the distance from a conductive right-angle frame B to a surface B is 10cm, and the spinning time is 1 h; after spinning is finished, preparing a radial nanofiber layer, adjusting the rotating speed of a receiving rotating shaft, wherein the rotating speed of the receiving rotating shaft is 500r/min, the distance from the receiving rotating shaft to the surface A is 5cm, the distance from the receiving rotating shaft to the surface B is 10cm, a coaxial nozzle moves, the horizontal distance from the coaxial nozzle to the receiving rotating shaft is 20cm, and the spinning time is 4 hours; and (3) preparing a random nanofiber layer after spinning is finished, wherein the distance from the receiving rotating shaft to the surface A is 10cm, the distance from the receiving rotating shaft to the surface B is 10cm, and the spinning time is 4 h.

In the embodiment, when the intermediate layer is prepared, the feeding speed of the core layer micropump is 60 muL/min, the feeding speed of the shell layer micropump is 60 muL/min, the spinning voltage output by a high-voltage power supply is 12kV, the material of the receiving rotating shaft is stainless steel, the shaft length is 200mm, the outer diameter is 4mm, the inner diameter of the coaxial nozzle is 0.3mm, and the outer diameter is 1 mm; when preparing the inner layer and the outer layer of the hydrogel, the dipping time is 30min, the cross-linking agent is an ethanol solution of genipin, the concentration is 3.5mM, the cross-linking time is 12h, the diameter of a small end face cylinder of the used connecting rod is 3mM, the height of the small end face cylinder is 250mM, the diameter of a large-section cylinder is 6mM, and the height of the large-section cylinder is 2 mM.

In this example, the structure of the prepared bio-artificial blood vessel is shown in fig. 3, the structure of each nanofiber layer is shown in fig. 4, and as can be seen from fig. 4, the randomly oriented nanofibers have high porosity and smooth fibers, the axially oriented nanofibers and the radially oriented nanofibers have high degree of orientation, and the coaxial nanofibers have a clear core/shell structure, thus verifying the effectiveness of the process.

Example 3

The third embodiment of the present invention is different from embodiment 2 in that it provides a method for preparing a multi-oriented bio-artificial blood vessel, comprising the steps of,

dissolving PCL in TEFA, and magnetically stirring until polycaprolactone is completely dissolved to obtain 40g of polycaprolactone solution with the mass percentage concentration of 8 wt%;

dissolving PGS in a PCL solution, wherein the mass ratio of the PGS to the PCL is 1:16, and preparing a coaxial electrostatic spinning shell solution with the mass-volume ratio concentration of 8.5 wt%;

dissolving RAPA in PCL solution, wherein the mass ratio of RAPA to PCL is 1:80, and preparing a coaxial electrostatic spinning core layer solution with the mass-volume ratio concentration of 8.1 wt%;

dissolving GEL in deionized water at 40 ℃ to prepare 50g of GEL solution with the mass volume ratio of 15 wt%;

dissolving GS in a GEL solution, wherein the mass ratio of the GS to the GEL is 1:50, and preparing a hydrogel solution with the mass-volume ratio of 15.1 wt%;

uniformly coating the surface of the receiving rotating shaft with F127 hydrogel;

respectively adopting a PCL solution, a coaxial electrostatic spinning shell layer solution and a coaxial electrostatic spinning core layer solution, and electrospinning on a receiving rotating shaft through a coaxial electrostatic spinning process to obtain a multi-orientation nanofiber tube with a three-layer structure, wherein an axial nanofiber layer, a radial nanofiber layer and a random nanofiber layer are sequentially arranged from inside to outside;

taking down the multi-orientation nano-fiber tube together with the receiving rotating shaft, vertically placing the multi-orientation nano-fiber tube in an environment at 0-4 ℃, easily sliding the multi-orientation nano-fiber tube from the receiving rotating shaft after F127 is liquefied, and placing the multi-orientation nano-fiber tube in absolute ethyl alcohol to wash away residual F127;

placing the cleaned multi-orientation nano fiber tube on a connecting rod and placing the connecting rod in a hydrogel solution for dipping, and taking out the multi-orientation nano fiber tube until no liquid drips on the multi-orientation nano fiber tube after dipping;

placing the multi-orientation nano-fiber tube which is arranged on the connecting rod and is soaked in a cross-linking agent solution of a soaking solution for cross-linking until the soaking solution on the multi-orientation nano-fiber tube is completely cross-linked, taking out the multi-orientation nano-fiber tube and standing until no liquid drops, and placing the multi-orientation nano-fiber tube in absolute ethyl alcohol to wash away the residual cross-linking agent;

and taking the multi-orientation nano fiber tube out of the connecting rod, putting the multi-orientation nano fiber tube into a freeze dryer for freeze drying for 4 hours to obtain the multi-orientation biological artificial blood vessel consisting of the inner hydrogel, the middle multi-orientation nano fiber tube and the outer hydrogel.

When an axial nanofiber layer of the multi-orientation nanofiber tube is prepared, the rotating speed of a receiving rotating shaft is 15R/min, the vertical distance R from a rotating center O is 30cm, the angular speed of an X/Y plane is 1200R/min, the distance from a conductive right-angle frame A surface is 10cm, the distance from a conductive right-angle frame B surface is 10cm, and the spinning time is 1.5 h; after spinning is finished, preparing a radial nanofiber layer, adjusting the rotating speed of a receiving rotating shaft, wherein the rotating speed of the receiving rotating shaft is 200r/min, the distance from the receiving rotating shaft to the surface A is 5cm, the distance from the receiving rotating shaft to the surface B is 10cm, a coaxial nozzle moves, the horizontal distance from the coaxial nozzle to the receiving rotating shaft is 20cm, and the spinning time is 5 hours; and (3) preparing a random nanofiber layer after spinning, wherein the distance from the receiving rotating shaft to the surface A is 10cm, the distance from the receiving rotating shaft to the surface B is 10cm, and the spinning time is 3 hours.

During feeding, in the embodiment, when the middle layer is prepared, the feeding speed of the core layer micro pump is 60 μ L/min, the feeding speed of the shell layer micro pump is 60 μ L/min, and the spinning voltage output by the high-voltage power supply is 14 kV; the axial length of the receiving rotating shaft is 200mm, the outer diameter of the receiving rotating shaft is 5mm, the inner diameter of the coaxial nozzle is 0.3mm, and the outer diameter of the coaxial nozzle is 1 mm; when the hydrogel inner layer and the hydrogel outer layer are prepared, the diameter of a small end face cylinder of the connecting rod is 4mM, the height of the small end face cylinder is 250mM, the diameter of a large end face cylinder is 7mM, the height of the large end face cylinder is 2mM, the dipping time is 60min, the cross-linking agent is an ethanol solution of genipin, the concentration is 4mM, the cross-linking time is 12h, the diameter of the small end face cylinder of the connecting rod is 3mM, the height of the small end face cylinder is 250mM, the diameter of a large section cylinder is 6mM, and the height of the large section cylinder is 2 mM.

In this example, compared to example 1 and example 2: the core/shell concentration of the fiber is increased, so that the diameter of the coaxial nanofiber is increased; the rotating speed of the receiving rotating shaft is increased, the electrostatic spinning time is prolonged, the fiber layer thickness is increased, the hydrogel dipping time and the concentration of the cross-linking agent are prolonged, the content of the natural biological materials contained in the hydrogel is correspondingly increased, and the biocompatibility is enhanced.

Example 4

The fourth embodiment of the present invention is different from embodiments 2 and 3 in that it provides a method for preparing a multi-oriented bio-artificial blood vessel, comprising the steps of,

dissolving PCL in TEFA, and magnetically stirring until polycaprolactone is completely dissolved to obtain 40g of polycaprolactone solution with mass-volume ratio concentration of 7.5 wt%;

dissolving PGS in a PCL solution, wherein the mass ratio of the PGS to the PCL is 1:3, and preparing a coaxial electrostatic spinning shell layer solution with the mass-volume ratio concentration of 10 wt%;

dissolving RAPA in PCL solution, wherein the mass ratio of RAPA to PCL is 1:15, and preparing a coaxial electrostatic spinning core layer solution with the mass-volume ratio concentration of 8 wt%;

dissolving GEL in deionized water at 40 ℃ to prepare 50g of GEL solution with the mass volume ratio of 19 wt%;

dissolving GS in a GEL solution, wherein the mass ratio of the GS to the GEL is 1:19, and preparing a hydrogel solution with the mass-volume ratio of 20 wt%;

uniformly coating the surface of the receiving rotating shaft with F127 hydrogel;

respectively adopting a PCL solution, a coaxial electrostatic spinning shell layer solution and a coaxial electrostatic spinning core layer solution, and electrospinning on a receiving rotating shaft through a coaxial electrostatic spinning process to obtain a multi-orientation nanofiber tube with a three-layer structure, wherein an axial nanofiber layer, a radial nanofiber layer and a random nanofiber layer are sequentially arranged from inside to outside;

taking down the multi-orientation nano-fiber tube together with the receiving rotating shaft, vertically placing the multi-orientation nano-fiber tube in an environment at 0-4 ℃, easily sliding the multi-orientation nano-fiber tube from the receiving rotating shaft after F127 is liquefied, and placing the multi-orientation nano-fiber tube in absolute ethyl alcohol to wash away residual F127;

placing the cleaned multi-orientation nano fiber tube on a connecting rod and placing the connecting rod in a hydrogel solution for dipping, and taking out the multi-orientation nano fiber tube until no liquid drips on the multi-orientation nano fiber tube after dipping;

placing the multi-orientation nano-fiber tube which is arranged on the connecting rod and is soaked in a cross-linking agent solution of a soaking solution for cross-linking until the soaking solution on the multi-orientation nano-fiber tube is completely cross-linked, taking out the multi-orientation nano-fiber tube and standing until no liquid drops, and placing the multi-orientation nano-fiber tube in absolute ethyl alcohol to wash away the residual cross-linking agent;

and taking the multi-orientation nano fiber tube out of the connecting rod, putting the multi-orientation nano fiber tube into a freeze dryer for freeze drying for 8 hours to obtain the multi-orientation biological artificial blood vessel consisting of the inner hydrogel, the middle multi-orientation nano fiber tube and the outer hydrogel.

When an axial nanofiber layer of the multi-orientation nanofiber tube is prepared, the rotating speed of a receiving rotating shaft is 20R/min, the vertical distance R from a rotating center O is 30cm, the angular speed of an X/Y plane is 1300R/min, the distance from a conductive right-angle frame A to a surface A is 10cm, the distance from a conductive right-angle frame B to a surface B is 10cm, and the spinning time is 2 hours; after spinning is finished, preparing a radial nanofiber layer, adjusting the rotating speed of a receiving rotating shaft, wherein the rotating speed of the receiving rotating shaft is 300r/min, the distance from the receiving rotating shaft to the surface A is 5cm, the distance from the receiving rotating shaft to the surface B is 10cm, a coaxial nozzle moves, the horizontal distance from the coaxial nozzle to the receiving rotating shaft is 20cm, and the spinning time is 4 hours; and (3) preparing a random nanofiber layer after spinning is finished, wherein the distance from the receiving rotating shaft to the surface A is 10cm, the distance from the receiving rotating shaft to the surface B is 10cm, and the spinning time is 5 h.

During feeding, in the embodiment, when the middle layer is prepared, the feeding speed of the core layer micro pump is 70 μ L/min, the feeding speed of the shell layer micro pump is 70 μ L/min, and the spinning voltage output by the high-voltage power supply is 15 kV; the axial length of the receiving rotating shaft is 200mm, the outer diameter of the receiving rotating shaft is 4.5mm, the inner diameter of the coaxial nozzle is 0.3mm, and the outer diameter of the coaxial nozzle is 1 mm; when preparing the hydrogel inner layer and the hydrogel outer layer, the diameter of a small end face cylinder of the connecting rod is 3.5mM, the height of the small end face cylinder is 250mM, the diameter of a large end face cylinder is 6mM, the height of the large end face cylinder is 2mM, the dipping time is 60min, the cross-linking agent is an ethanol solution of genipin, the concentration is 4mM, the cross-linking time is 24h, the diameter of the small end face cylinder of the used connecting rod is 3mM, the height of the small end face cylinder is 250mM, the diameter of the large section cylinder is 6mM, and the height of the large section cylinder is 2 mM.

This example compares to example 2 and example 3: the angular speed of the plane of the receiving rotating shaft is increased, so that the parallelism of the axial nano fibers is improved, the core/shell feeding speed and voltage are increased, and the number of the nano fibers collected in unit time is increased; the freeze drying time is prolonged, and the residue of volatile harmful gas is reduced; compared with the embodiment 2 and the embodiment 3, the multi-orientation biological artificial blood vessel prepared by the embodiment has more excellent parallelism and biomechanics of the nano fiber, so that the multi-orientation biological artificial blood vessel has wider application prospect and development space in the preparation of the artificial blood vessel orientation nano fiber.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.