阅读说明：本技术 一种人工血管的制备方法 (Preparation method of artificial blood vessel ) 是由 吴文惠 潘涵宇 王婷 孙宇 王江茗 蔡旗 潘晟旻 于 2019-09-16 设计创作，主要内容包括：本发明公开了一种人工血管的制备方法,包括：以聚乳酸为原料,六氟异丙醇为溶剂,配制聚乳酸溶液；在聚乳酸溶液中加入来自海洋生物的胶原海绵,配制成胶原海绵-聚乳酸复合溶液；运用复合溶液,依据制图软件Auto CAD输出的后缀名为stl的文件,采用生物3D打印机在-35℃至-10℃平台温度下打印人工血管,其形状符合待植入人体目标地点需要；通过戊二醛蒸气使人工血管的材料内部发生交联反应。本发明的制备方法简单,产物可控,所制备的人工血管可在人体内降解,满足使用要求,能够依据病人自身的实际情况及手术需求为其量身定制可替换的人工血管,个性化水平高。(The invention discloses a preparation method of an artificial blood vessel, which comprises the following steps: preparing a polylactic acid solution by using polylactic acid as a raw material and hexafluoroisopropanol as a solvent; adding collagen sponge from marine organisms into a polylactic acid solution to prepare a collagen sponge-polylactic acid composite solution; the composite solution is used, and according to a file with a postfix name stl output by drawing software Auto CAD, a biological 3D printer is adopted to print the artificial blood vessel at a platform temperature of-35 ℃ to-10 ℃, and the shape of the artificial blood vessel meets the requirement of a target site to be implanted into a human body; the material of the artificial blood vessel is internally subjected to a crosslinking reaction by glutaraldehyde vapor. The preparation method is simple, the product is controllable, the prepared artificial blood vessel can be degraded in a human body, the use requirement is met, the replaceable artificial blood vessel can be customized according to the actual condition of a patient and the operation requirement, and the personalization level is high.)

1. A method for preparing an artificial blood vessel, the method comprising the steps of:

S1, preparing a polylactic acid solution by using polylactic acid as a raw material and hexafluoroisopropanol as a solvent; adding collagen sponge from marine organisms into the polylactic acid solution to prepare a collagen sponge-polylactic acid composite solution;

s2, printing the artificial blood vessel by using the composite solution at a platform temperature of-35 ℃ to-10 ℃ by using a biological 3D printer according to a file with a suffix name stl output by drawing software Auto CAD, wherein the shape of the artificial blood vessel meets the requirement of a target site to be implanted into a human body;

S3, cross-linking reaction is carried out inside the material of the artificial blood vessel through glutaraldehyde steam.

2. The method for preparing an artificial blood vessel according to claim 1, wherein the mass ratio of the collagen sponge to the polylactic acid is 0.25-4: 1.

3. The method for preparing an artificial blood vessel according to claim 1, wherein the mass concentration of the polylactic acid in the polylactic acid solution is 3% to 5%.

4. The method for producing an artificial blood vessel according to claim 1, wherein the hexafluoroisopropanol solvent is present at a concentration of > 99.5% by mass.

5. the method for producing an artificial blood vessel according to claim 1, wherein: the collagen sponge is fish skin collagen extracted from marine organisms, wherein the percentage content of the type I collagen in the total mass of the fish skin collagen is 90-95%.

6. The method for producing an artificial blood vessel according to claim 1, wherein: and the stl file directly generates a corresponding personalized blood vessel image file according to the scanning result of the lesion blood vessel region.

7. The method for preparing the artificial blood vessel according to claim 1, wherein the cross-linking reaction inside the artificial blood vessel material is specifically: 10-15mL of 25% glutaraldehyde solution by volume percentage is placed in a culture dish, then the culture dish is placed at the bottom of a clean dryer, finally the obtained artificial blood vessel material is placed on a magnetic disk with holes above the culture dish, a cover is covered, and the crosslinking time is 8-15 hours.

8. The method for preparing an artificial blood vessel according to any one of claims 1 to 6, wherein the artificial blood vessel can achieve rapid endothelialization after replacing a diseased blood vessel into a human body, and vascular cells can be adhered and proliferated on an artificial blood vessel material.

9. The use of the artificial blood vessel according to claim 8, wherein the artificial blood vessel is degradable in vivo, and the whole adhesion proliferation process of the cells is controlled in the degradation cycle of the artificial blood vessel.

10. The method of using an artificial blood vessel according to claim 8, wherein the degradation of the artificial blood vessel is harmless to a human body.

Technical Field

The invention relates to the field of preparation of medical instruments, in particular to a preparation method of an artificial blood vessel.

Background

Cardiovascular diseases, also known as circulatory system diseases, are a common public health problem worldwide and seriously affect the normal life and physical and mental development of human beings. In China, the risk factors of cardiovascular diseases have a very obvious epidemic trend, and the epidemic trend is inseparable with changes of living habits, aging of population, environmental factors and the like, so that the number of people suffering from cardiovascular diseases in China is increased year by year. At present, the death rate of cardiovascular and cerebrovascular diseases always dominates the death rate of the diseases, so the scientific prevention and treatment of cardiovascular diseases are very important.

For the treatment of cardiovascular diseases, drug therapy is mainly used. The western medicines are mostly single-target or few-target medicines, and the western medicines bring inconvenience to patients due to toxic and side effects and drug resistance caused by long-term taking. Compared with the toxic and side effects of western medicines, the traditional Chinese medicines bring less pain to patients, but the medicines also have the characteristics of slow drug effect exertion and unclear drug action mechanism of the traditional Chinese medicines, and simultaneously lack scientific basis for the problems of dosage, drug metabolism and the like. These factors all restrict the development of Chinese and western medicines in the treatment of cardiovascular diseases.

With the continuous development of science and technology, interventional therapy is favored by doctors and patients due to the characteristics of definite curative effect, high success rate, few complications and the like. Meanwhile, the interventional therapy is gradually changed from the earliest metal bare material and drug eluting material to the bioabsorbable material. Biological tissue engineering thus ensues, namely: obtaining a small amount of living tissue from an organism, separating cells (also called seed cells) from the tissue by using a special enzyme or other methods, culturing and amplifying in vitro, and then mixing the amplified cells with a biological material with good biocompatibility, degradability and absorbability according to a certain proportion to ensure that the cells are adhered on the biological material to form a cell-material compound; the compound is implanted into the damaged part of the organism tissue or organ, and along with the gradual degradation and absorption of the biological material in the body, the implanted cells are continuously proliferated in the body and secrete extracellular matrix, and finally corresponding tissue or organ is formed, thereby achieving the purposes of repairing wound and reconstructing function.

Natural materials or synthetic materials having good biocompatibility, such as collagen, silk fibroin, and chitosan, have received much attention in the related fields. At present, the variety of artificial blood vessels is various, and the artificial blood vessels have certain effect on treating cardiovascular diseases, but the defects are obvious: for example, most of the artificial blood vessels are produced in large scale and cannot meet the requirements of different patients; most artificial blood vessels cannot be quickly endothelialized after being replaced in a blood vessel pathological change area, so that restenosis of new blood vessels after replacement or thrombosis is easily caused, and the health of patients is harmed again.

patent application CN 105363076A discloses a preparation method of polylactic acid caprolactone-collagen double-layer bionic vascular stent based on electrostatic spinning printing technology, which adopts electrostatic spinning printing technology, although the components and the structure of the stent are similar to those of natural blood vessels, the obtained finished vascular material is difficult to adapt to each patient individually, and the fineness of printing by using the technology is insufficient. In addition, patent application CN 103147225 a also discloses a method for preparing a protein-polysaccharide-polylactic acid polycaprolactone vascular stent based on an electrospinning technology, and the patent also has the same disadvantages as the above patent application. This problem has been long-standing and many patients have to suffer from vascular lesions because of their inability to adapt to existing vascular prostheses.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a preparation and use method of an artificial blood vessel. The invention uses collagen sponge and polylactic acid from marine organisms as raw materials, and combines the composite material with the biological 3D printing technology to prepare the artificial blood vessel, and the 3D printing technology can perform personalized 'customized' printing on the diseased blood vessel required by the patient.

The raw materials selected by the invention are polylactic acid and fish skin collagen from marine organisms, and the polylactic acid has better mechanical property, biocompatibility and biodegradability and is a tissue engineering scaffold material which is most applied. However, polylactic acid itself lacks some functional groups recognized by cells, such as amino and carboxyl, and is not favorable for the proliferation and growth of cells when being used as an artificial blood vessel material alone; the collagen is an important component of extracellular matrix, is the protein with the most content in human body, and has no immunogenicity, low antigenicity, high hydrophilicity and high cell compatibility. The composite material has the advantages of both the two materials, contains a cell recognition group, can enhance the adhesion and proliferation of cells, is a bioabsorbable material, is favorable for the adhesion and the proliferation of endothelial cells due to stronger hydrophilicity, realizes rapid endothelialization, does not cause the influence of restenosis, thrombus and the like of later-stage blood vessels, and has good mechanical property, coagulability, biocompatibility and blood compatibility.

The invention realizes low-temperature rapid molding by controlling the temperature of the printing platform to be about-20 ℃. And then, crosslinking collagen molecules in the glutaraldehyde steam composite material at normal temperature to form a collagen membrane, so that the fiber structure of the composite material is more stable, the blood vessel is prevented from being incapable of resisting the impact of the blood vessel to generate cracks after being replaced into a human body, and a corresponding growth space is provided for endothelial cells of the blood vessel. When the artificial blood vessel is used, the prepared artificial blood vessel replaces a diseased blood vessel and enters a human body, and rapid endothelialization can be realized due to the unique property of the blood vessel, namely, vascular endothelial cells can be adhered and proliferated on an artificial blood vessel material, a 3D printing model of the blood vessel in the same area can be directly constructed according to a medical contrast image of a patient, and the specific parameters of the required artificial blood vessel are changed in detail according to the self condition of the patient.

Specifically, the purpose of the invention is realized by the following technical scheme:

The invention relates to a preparation method of an artificial blood vessel, which comprises the following steps:

S1, preparing a polylactic acid solution by using polylactic acid as a raw material and hexafluoroisopropanol as a solvent; adding collagen sponge from marine organisms into the polylactic acid solution to prepare a collagen sponge-polylactic acid composite solution;

S2, printing the artificial blood vessel by using the composite solution at a platform temperature of-35 ℃ to-10 ℃ by using a biological 3D printer according to a file with a suffix name stl output by drawing software Auto CAD, wherein the shape of the artificial blood vessel meets the requirement of a target site to be implanted into a human body;

S3, cross-linking reaction is carried out inside the material of the artificial blood vessel through glutaraldehyde steam.

preferably, the mass ratio of the collagen sponge to the polylactic acid is 1: 4-4: 1. when the mass ratio of the collagen sponge to the polylactic acid is less than 1:4 or more than 4:1, the fiber diameter, pore diameter and porosity of the composite material obtained are affected, and the growth of cells is not facilitated, and besides, the proportion change also affects the hydrophilicity and stretchability of the composite material obtained, so that the composite material does not have the relevant performance of replacing blood vessels.

Preferably, the mass concentration of the polylactic acid in the polylactic acid solution is 3-5%.

Preferably, the hexafluoroisopropanol solvent is present at a concentration of > 99.5% by mass.

Preferably, the collagen sponge is fish skin collagen extracted from marine organisms; wherein, the percentage content of the type I collagen in the total mass of the fish skin collagen is 90 to 95 percent, and the preferred percentage content is 92 percent.

Preferably, the fish skin collagen is shark skin collagen.

preferably, the stl file directly generates a corresponding personalized blood vessel image file according to the scanning result of the lesion blood vessel region.

Preferably, the cross-linking reaction inside the artificial blood vessel material is specifically that 10-15mL of 25% glutaraldehyde water solution with volume percentage concentration is placed in a culture dish, then the culture dish is placed at the bottom of a clean dryer, finally the obtained artificial blood vessel material is placed on a magnetic disk with holes above the culture dish, a cover is covered, and the cross-linking time is 8-15 hours.

Preferably, the artificial blood vessel can realize rapid endothelialization after replacing a diseased blood vessel and entering a human body, and blood vessel cells can be adhered and proliferated on the artificial blood vessel material.

Preferably, the artificial blood vessel is capable of degrading in vivo, and the whole adhesion proliferation process of cells is controlled in the degradation cycle of the artificial blood vessel.

Preferably, the degradation of the artificial blood vessel is harmless to the human body.

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

1. The artificial blood vessel disclosed by the invention is simple in preparation method and controllable in product, the prepared artificial blood vessel can be degraded in a human body, the use requirement is met, the replaceable artificial blood vessel can be customized according to the actual condition of a patient and the operation requirement, and the personalized level is high.

2. The 3D printed marine organism collagen composite polylactic acid artificial blood vessel can overcome the defects of insufficient fineness and material defects of the blood vessel and inhibit the formation of thrombus, the 3D printed modern information and engineering technical means are combined with the biological function of tissue engineering materials skillfully, the marine organism collagen and the polylactic acid with proper proportion are compounded, the marine organism collagen induces the rapid endothelialization of the blood vessel, and the polylactic acid inhibits blood factors from adhering to the artificial blood vessel to prevent the formation of thrombus.

drawings

FIG. 1 is a schematic view of the structure of an artificial blood vessel prepared by 3D printing collagen according to the present invention; wherein: 1-vascular barrier layer; 2-a layer of nano collagen fibers; 3-blood contact layer.

FIG. 2 is a microscopic structure of the artificial blood vessel of the present invention.

FIG. 3 is a schematic electron microscope of the artificial blood vessel of the present invention.

Fig. 4 is a schematic electron microscope of the cross section of the artificial blood vessel of the present invention.

FIG. 5 is an infrared spectrum analysis (FR-IR curve) of an artificial blood vessel sample according to the present invention.

Detailed Description

The technical solution of the present invention is further illustrated by the following examples. The marine organism used in this example was shark skin, and the examples were carried out according to the techniques or conditions described in the literature in the art or according to the product specifications, without specifying any particular technique or condition. The reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market. All temperatures in the examples are expressed in degrees celsius.