CN109731139B

Movatterモバイル変換

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Publication number: CN109731139B
Application number: CN201811469737.2A
Authority: CN
Inventors: 李艳; 侯延进; 王建春; 王雪莹; 田寒梅; 王立秋
Original assignee: Energy Research Institute of Shandong Academy of Sciences
Current assignee: Energy Research Institute of Shandong Academy of Sciences
Priority date: 2018-11-28
Filing date: 2018-11-28
Publication date: 2021-06-18
Anticipated expiration: 2038-11-28
Also published as: CN109731139A

Abstract

本发明涉及一种小口径人工血管及其制备方法，属于组织工程技术领域，将管壁溶液和固化剂溶液通过挤压的方法通入人工血管制备装置；将固化后的血管转移到细胞培养基中通入人脐静脉内皮细胞进行培养得到小口径人工血管；所述人工血管制备装置为同轴套筒，同轴套筒的外筒和内筒为空心的结构，内筒高于外筒，外筒的顶端和内筒之间是封闭连接的，内筒的顶部是开口的，外筒和内筒的底部是开口的，外筒的侧壁设置一个引出管；人工血管制备装置的下方放置盛放固化剂的敞口容器，制备装置位于敞口容器的上方；人工血管管壁溶液由外筒的引出管通入，固化剂由内筒的顶部通入。制备的人工血管与真实血管一样具备一定韧性和强度、小分子可渗透性，仿真度高。

The invention relates to a small-diameter artificial blood vessel and a preparation method thereof, belonging to the technical field of tissue engineering. A tube wall solution and a solidifying agent solution are passed into an artificial blood vessel preparation device by means of extrusion; the solidified blood vessel is transferred to a cell culture medium. A small-diameter artificial blood vessel is obtained by culturing human umbilical vein endothelial cells; the artificial blood vessel preparation device is a coaxial sleeve, the outer cylinder and the inner cylinder of the coaxial sleeve are hollow structures, and the inner cylinder is higher than the outer cylinder, The top of the outer tube and the inner tube are closed and connected, the top of the inner tube is open, the bottoms of the outer tube and the inner tube are open, and an outlet tube is arranged on the side wall of the outer tube; the artificial blood vessel preparation device is placed under the The open container containing the curing agent, the preparation device is located above the open container; the artificial blood vessel tube wall solution is introduced from the outlet pipe of the outer cylinder, and the curing agent is introduced from the top of the inner cylinder. The prepared artificial blood vessel has the same toughness and strength as the real blood vessel, small molecule permeability, and high simulation degree.

Description

Small-caliber artificial blood vessel and preparation method thereof

Technical Field

The invention belongs to the technical field of tissue engineering, and particularly relates to a small-caliber artificial blood vessel and a preparation method thereof.

Background

With the rapid development of social economy and the continuous improvement of living standard of people, the change of life style leads the morbidity and the mortality of cardiovascular diseases to rapidly rise worldwide. At present, cardiovascular disease patients in China reach 2.9 hundred million, and the morbidity and mortality of cardiovascular disease patients are the first of various diseases. For patients with severe cardiovascular diseases, the main treatment is blood vessel transplantation. Because of the limited source of autologous blood vessels, a large number of artificial blood vessels are clinically needed as a replacement for transplantation. At present, large and medium-caliber artificial blood vessels with the diameter of more than 6mm, such as products made of synthetic materials such as Dacron (Dacron), polytetrafluoroethylene (ePTFE) and the like, have good compliance and have been successfully applied to clinical treatment. However, the small-caliber artificial blood vessel with the diameter of less than 6mm manufactured by the material has poor compliance after transplantation, low long-term common rate and unsatisfactory clinical application effect. Therefore, the development of small-caliber artificial blood vessels with good compliance and high long-term common rate is still a research hotspot in the field of tissue engineering.

At present, researchers have developed small-bore artificial blood vessels of various materials by various processes, such as zhao hui ban, et al (patent No. 201010189082.0), which is a university of suzhou, to construct a silk fibroin tubular structure by preparing a silk fibroin tubular structure, spraying a layer of silk fibroin fibers on the outer layer by an electrospinning process, and then coating the inner layer with a mixed solution of silk fibroin and an anticoagulant drug to construct a silk fibroin tubular material having a three-layer structure. The small-caliber artificial blood vessel prepared by the method has a three-layer structure and can provide a framework for cell growth, but the filling of cells depends on the adhesion, growth, propagation and differentiation of autologous cells after being implanted into a body, the required culture period is longer, and the deposition of the cells on the vessel wall has uncertainty. Artificial blood vessels (patent No. 201410071581.8) prepared from polyurethane as material, such as Eugenia morning of Wuhanseng Biotechnology Limited, are prepared by processing material into filaments, winding the filaments in parallel on a cylindrical mold, and qualitatively processing to obtain hollow tubular product. The method can prepare hollow tubular objects (including small-caliber artificial blood vessels) with the inner diameter of 2-30mm, is of a single-layer structure, and does not relate to the culture of vascular cells. The preparation method of the artificial blood vessel disclosed by Cao-hei et al, Beijing university of Phytology (patent No. 201510290955. X) also comprises the step of coating the treated material on a mould to be rotationally molded, wherein the diameter of the product meets the requirement of the small-caliber artificial blood vessel, but the product is also in a single-layer tubular structure. The Nankai university of Zhumeifeng et al discloses a preparation method of a three-layer artificial blood vessel (patent number: 201610589001.3), wherein the inner layer is prepared by an ink printing method, the middle layer is formed by winding spiral orientation micrometer fibers prepared by wet spinning or melt spinning, and the outer layer is formed by winding coarse polymer fibers. The method has complicated steps and is not beneficial to industrial production. Zhang Yong et al, Zhejiang university of physiology of technology, disclose a preparation method of cellulose and fibroin composite small-caliber artificial blood vessel material, which mainly improves the compliance and biocompatibility of the artificial blood vessel material, and does not relate to the preparation of artificial blood vessels. A three-layer artificial blood vessel is prepared from a small-caliber biological artificial blood vessel disclosed by Wanghong (patent number: 201611189284.9) of Qingdao Sandi Biotechnology Co., Ltd by a 3D printing technology. However, the 3D printing technology has high requirements for equipment and production operation processes, high production cost, and is difficult to popularize.

The preparation method of the small-caliber artificial blood vessel is suitable for industrial production, easy to adjust and prepare the aperture of the blood vessel, high in simulation degree and high in production speed, and has certain toughness and strength and small molecule permeability as the real blood vessel of a human body.

Disclosure of Invention

In view of the above problems in the prior art, it is an object of the present invention to provide a method for preparing a small-caliber artificial blood vessel.

In order to solve the technical problems, the technical scheme of the invention is as follows:

a preparation method of a small-caliber artificial blood vessel comprises the following specific steps:

1) preparing an artificial blood vessel wall solution and a curing agent solution;

2) pressurizing the prepared artificial blood vessel solution, introducing the solution into an artificial blood vessel preparation device, and introducing a curing agent solution;

3) transferring the solidified blood vessel to a cell culture medium, and introducing human umbilical vein endothelial cells into the cell culture medium to culture the blood vessel to obtain a small-caliber artificial blood vessel;

the artificial blood vessel preparation device is a coaxial sleeve, an outer cylinder and an inner cylinder of the coaxial sleeve are of hollow structures, the inner cylinder is higher than the outer cylinder, the top end of the outer cylinder is connected with the inner cylinder in a sealing manner, the top of the inner cylinder is open, the bottoms of the outer cylinder and the inner cylinder are open, and the side wall of the outer cylinder is provided with an eduction tube;

an open container for containing the curing agent is placed below the artificial blood vessel preparation device in the step 2), and the preparation device is positioned above the open container;

the solution of the artificial blood vessel wall is introduced from the eduction tube of the outer cylinder, and the curing agent is introduced from the top of the inner cylinder.

The preparation method of the artificial blood vessel is flexible, convenient and fast, has good solidification effect, can culture endothelial cells on the inner wall in advance, and has good simulation effect. The diameter of the preparation device can be flexibly adjusted, and the artificial blood vessels with different sizes can be prepared. Compared with the artificial blood vessel preparation method in the prior art, the artificial blood vessel is prepared by the extrusion forming method. Because the prepared small-caliber blood vessel has small gaps of the coaxial sleeve, the solution on the tube wall is the high-molecular polymer solution with high viscosity, the solution can be promoted to enter the gaps of the coaxial sleeve by applying extrusion force to the solution on the tube wall, and the solution on the tube wall can be promoted to be uniformly distributed in the gaps of the coaxial sleeve by the extrusion force. Because the pipe wall solution of this application flows out from coaxial telescopic bottom, the vascular wall flows to open container under the dual function of gravity and extrusion force, and the effect of gravity can have the effect of separation to the vascular that forms, and the extrusion force can balance the influence of gravity. In the extrusion molding process, the extrusion force can also increase the mechanical strength of the artificial blood vessel. Because this application preparation is the blood vessel of small bore, because capillary action, the curing agent in the uncovered container is difficult to carry out the fast curing effect to the vascular inner wall, so this application lets in the curing agent simultaneously at the inner tube, can solidify the vascular inner wall, has improved curing speed, is favorable to the formation of blood vessel. The obtained blood vessel is integrally formed and has high curing speed.

Preferably, the artificial blood vessel wall solution is composed of one or more of sodium alginate, gelatin, silk fibroin, elastin, collagen, chitosan and cellulose solution.

Preferably, the preparation method of the sodium alginate aqueous solution comprises the following steps: adding sodium alginate powder into distilled water, and sterilizing at high temperature to obtain sodium alginate water solution.

Further preferably, the mass ratio of the sodium alginate to the distilled water is 1-8: 92-99.

Further preferably, the temperature in the dissolving process is 55-60 ℃; the temperature of the high-temperature steam sterilization is 118-125 ℃, and the time is 25-35 minutes.

Preferably, the preparation method of the gelatin aqueous solution comprises the following steps: dissolving gelatin in sterile culture medium, removing bubbles after completely dissolving, adding chloroform, and removing bubbles.

More preferably, the ratio of gelatin to the sterile medium and chloroform is 1g:7-9mL: 2. mu.L.

It is further preferred that the temperature during dissolution is not more than 70 ℃.

Preferably, the preparation method of the silk fibroin solution comprises the following steps: dissolving silkworm cocoon in sodium carbonate solution, boiling, degumming, washing with distilled water, dissolving with lithium bromide, centrifuging to obtain supernatant, dialyzing, vacuum freeze-drying to obtain silk fibroin, and dissolving silk fibroin in sterile culture medium or water to obtain silk fibroin solution.

Further preferably, the concentration of the sodium carbonate solution is 0.5%, the temperature is 98-102 ℃, and the ratio of the silkworm cocoon to the sodium carbonate solution is 10g:90-110 mL; the boiling time is 28-32 min.

Further preferably, the concentration of the lithium bromide solution is 9 to 9.5 moL/L.

Further preferably, the dialysis temperature is 3-5 deg.C, and the dialysis time is 22-26 h.

Preferably, the curing agent solution is one of a calcium chloride solution or a glutamine transaminase aqueous solution.

More preferably, the concentration of the calcium chloride solution is 4-6%, and the concentration of the glutamine transaminase aqueous solution is 9-11%.

Preferably, the inner diameter of the outer barrel is in the range of 2-8 mm.

Preferably, the outer diameter of the inner cylinder ranges from 1 mm to 6 mm.

Preferably, the speed of the raw material solution introduced into the outer cylinder is 5mL/h-10 mL/h.

The solution on the tube wall is high molecular solution with high viscosity, so the pumping speed is high, and the extrusion force can be increased by the pushing of a pump, thereby being convenient for blood vessel forming.

Preferably, the speed of the curing agent introduced into the inner cylinder is 4mL/h-8 mL/h.

Because the viscosity of the curing agent is relatively low, the introducing speed is relatively low, and the introducing speed is adjusted to be matched with the angiogenesis speed, so that the vascular wall is rapidly and uniformly cured.

Preferably, the cell culture conditions in step 3) are: the temperature is 35-40 deg.C, and 5% CO₂The culture time is 2-4 days, and the prepared human umbilical vein cells on the vascular wall account for 80%.

The small-caliber artificial blood vessel prepared by the preparation method.

The invention has the beneficial effects that:

the artificial blood vessel takes degradable natural high molecular polymer as a framework, and the inner layer simulates a real blood vessel and can culture an endothelial cell layer. The method has simple preparation method and low cost, and is beneficial to industrial production;

the artificial blood vessel prepared by the method has certain toughness and strength, small molecule permeability and high simulation degree like a human body real blood vessel;

the curing agent is divalent metal ions or biological enzyme which can catalyze the cross-linking between or in protein molecules and the connection between protein and amino acid;

according to the preparation method, the angioplasty and the solidification are carried out sequentially, in the integrated process, the solidifying agent inside the blood vessel and the solidifying agent outside the blood vessel are contacted with the blood vessel sequentially, the forming process is stable and fast, and the formed blood vessel is fixed in shape and stable in shape.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application.

FIG. 1 is a schematic view of a device for preparing a small-caliber artificial blood vessel;

FIG. 2 is a top view of an artificial blood vessel preparation device;

FIG. 3 is a vascular permeability test;

FIG. 4 is a three-dimensional reconstruction of an artificial blood vessel;

FIG. 5 is a cross-section of a three-dimensional reconstruction of an artificial blood vessel;

1. a curingagent inlet 2, a pipewall solution inlet 3, aninner cylinder 4, anouter cylinder 5, a leading-out pipe 6 and an open container.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The invention is further illustrated by the following examples:

example 1

The artificial blood vessel preparation device shown in fig. 1 is a coaxial sleeve, theouter cylinder 4 and theinner cylinder 3 of the coaxial sleeve are of hollow structures, theinner cylinder 3 is higher than theouter cylinder 4, the top end of theouter cylinder 4 is connected with theinner cylinder 3 in a closed mode, the top of theinner cylinder 3 is open, the bottoms of theouter cylinder 4 and theinner cylinder 3 are open, and an outlet pipe is arranged on the side wall of theouter cylinder 4.

One end of the eduction tube is a tubewall solution inlet 2, and the top of the inner cylinder is aninlet 1 of the curing agent.

Example 2

The embodiment provides a small-caliber artificial blood vessel, which is prepared by the following steps:

the artificial blood vessel preparation apparatus of example 1 was used, in which the diameter of the inner layer cylinder was 1.5 mm and the diameter of the outer layer cylinder was 2.5 mm; introducing 5% calcium chloride aqueous solution into a curingagent inlet 1, introducing 4% sodium alginate aqueous solution and 1% silk fibroin into a tubewall solution inlet 2, starting the preparation device, and collecting the tube-shaped object into 5wt% calcium chloride aqueous solution for curing. Transferring the artificial blood vessel to a cell culture solution after 12 hours, introducing the uniformly mixed endothelial cells, and culturing for 2-4 days to form the artificial blood vessel.

Example 3

the artificial blood vessel manufacturing apparatus of example 1 was used, in which the diameter of the inner layer cylinder was 2.0 mm and the diameter of the outer layer cylinder was 3.5 mm; introducing a 5% calcium chloride aqueous solution into a curingagent inlet 1, introducing a 5% sodium alginate aqueous solution into a tubewall solution inlet 2, starting the preparation device, and collecting the tube-shaped object into a 5% calcium chloride aqueous solution for curing. Transferring the artificial blood vessel to a cell culture solution after 12 hours, introducing the uniformly mixed endothelial cells, and culturing for 2-4 days to form the artificial blood vessel.

Example 4

the artificial blood vessel preparation apparatus of example 1 was used, in which the diameter of the inner layer cylinder was 3.0 mm and the diameter of the outer layer cylinder was 4.4 mm; introducing 10% glutamine transaminase aqueous solution into curingagent inlet 1, introducing 12.5% gelatin aqueous solution (containing 1% collagen and 2% elastin) into tubewall solution inlet 2, starting the preparation device, collecting the tube-shaped object into 10% glutamine transaminase solution, and crosslinking and curing gelatin at 37 deg.C. Transferring the artificial blood vessel to a cell culture solution after 2 hours, introducing the uniformly mixed endothelial cells, and culturing for 2-4 days to form the artificial blood vessel.

Example 5

selecting a coaxial sleeve device as shown in fig. 1, wherein the diameter of the inner layer cylinder is 4.0 mm, and the diameter of the outer layer cylinder is 5.5 mm; introducing 10% glutamine transaminase aqueous solution into curingagent inlet 1, introducing 20% gelatin aqueous solution (containing 0.5% silk fibroin and 1% elastin) into tubewall solution inlet 2, starting the preparation device, collecting the tube-shaped object into 10% transglutaminase solution, and crosslinking and curing gelatin at 37 deg.C. Transferring the artificial blood vessel to a cell culture solution after 2 hours, introducing the uniformly mixed endothelial cells, and culturing for 2-4 days to form the artificial blood vessel.

Example 6

This example performed a permeability test on the blood vessels prepared in example 3: A1-B1 is a small molecule substance (molecular weight 40 kDa) permeation experiment, A2-B2 is corresponding fluorescence intensity, A2 is an initial value, and B2 is a termination value; C1-D1 are permeation experiments of macromolecular substances (molecular weight 500 kDa), C2-D2 are corresponding fluorescence intensities, C2 is an initial value, and D2 is a termination value. The experimental result shows that the coating has good permeability to small molecular substances and artificial blood vessels, but has no permeability to macromolecular substances. The test result is consistent with the permeation rule of real blood vessels to substances. The vascular permeability test is shown in FIG. 3 (note: the fluorescence intensity detection direction is from left to right).

Example 7

This example provides an example of the use of cultured cells in the inner wall of an artificial blood vessel.

Endothelial cells are introduced into the inner wall of the artificial blood vessel, and after 3 days of culture, a three-dimensional reconstructed blood vessel picture is obtained by taking pictures through a confocal microscope. From the three-dimensional reconstruction results (fig. 4), it can be seen that endothelial cells are uniformly spread on the inner wall of the blood vessel, and the cross section (fig. 5) highlights the application effect.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A preparation method of a small-caliber artificial blood vessel is characterized by comprising the following steps: the method comprises the following specific steps:

2) pressurizing the prepared artificial blood vessel wall solution, introducing the solution into an artificial blood vessel preparation device, and introducing a curing agent solution;

introducing the solution into the artificial blood vessel wall through the outlet pipe of the outer cylinder, and introducing the curing agent into the inner cylinder from the top; the speed of introducing the curing agent into the inner cylinder is 4-6 mL/h; the speed of introducing the raw material solution into the outer cylinder is 5-10 mL/h.

2. The method of claim 1, wherein: the artificial blood vessel wall solution is composed of one or more of sodium alginate, gelatin, silk fibroin, elastin, collagen, chitosan and cellulose solution.

3. The method of claim 2, wherein: the preparation method of the sodium alginate aqueous solution comprises the following steps: adding sodium alginate powder into distilled water, and sterilizing at high temperature to obtain sodium alginate water solution.

4. The production method according to claim 3, characterized in that: the mass ratio of the sodium alginate to the distilled water is 1-8: 92-99.

5. The production method according to claim 3, characterized in that: in the preparation of the sodium alginate aqueous solution, the temperature in the dissolving process is 55-60 ℃; the temperature of the high-temperature steam sterilization is 118-125 ℃, and the time is 25-35 minutes.

6. The method of claim 2, wherein: the preparation method of the gelatin water solution comprises the following steps: dissolving gelatin in sterile culture medium, removing bubbles after completely dissolving, adding chloroform, and removing bubbles.

7. The method of claim 6, wherein: the ratio of the gelatin to the sterile culture medium to the chloroform is 1g:7-9mL:2 μ L.

8. The method of claim 6, wherein: heating is carried out during the dissolving process, and the temperature is not more than 70 ℃.

9. The method of claim 2, wherein: the preparation method of the silk fibroin solution comprises the following steps: dissolving silkworm cocoon in sodium carbonate solution, boiling, degumming, washing with distilled water, dissolving with lithium bromide, centrifuging to obtain supernatant, dialyzing, vacuum freeze-drying to obtain silk fibroin, and dissolving silk fibroin in sterile culture medium or water to obtain silk fibroin solution.

10. The method of claim 9, wherein: the concentration of the sodium carbonate solution is 0.5 percent, the temperature is 98-102 ℃, and the ratio of the silkworm cocoon to the sodium carbonate solution is 10g:90-110 mL; the boiling time is 28-32 min.

11. The method of claim 9, wherein: the concentration of the lithium bromide solution is 9-9.5 moL/L.

12. The method of claim 9, wherein: the dialysis temperature is 3-5 deg.C, and the dialysis time is 22-26 h.

13. The method of claim 1, wherein: the curing agent solution is one of a calcium chloride solution or a glutamine transaminase aqueous solution.

14. The method of manufacturing according to claim 13, wherein: the concentration of the calcium chloride solution is 4-6%, and the concentration of the glutamine transaminase aqueous solution is 9-11%.

15. The method of claim 1, wherein: the outer diameter range of the inner cylinder is 1-6 mm; the inner diameter of the outer cylinder ranges from 2 mm to 8 mm.

16. The method of claim 1, wherein: the cell culture conditions in the step 3) are as follows: the temperature is 35-40 deg.C, and 5% CO₂The culture time is 2-4 days, and the prepared human umbilical vein cells on the vascular wall account for 80%.

17. A small-caliber artificial blood vessel produced by the production method according to any one of claims 1 to 16.