CN118370868A - Acellular bovine intercostal artery and preparation method and application thereof - Google Patents

Abstract

The invention relates to a decellularized bovine intercostal artery and a preparation method and application thereof, belonging to the field of tissue engineering blood vessels and biological materials. The invention modifies the blood vessel after cell removal treatment by using methacrylic anhydride and 2-methacryloyloxyethyl phosphorylcholine, and the obtained modified blood vessel has good mechanical property, suture property and biocompatibility. Meanwhile, the intercostal arteries of the cattle are used as materials, the caliber of the intercostal arteries is matched with the internal diameters of arterioles such as human coronary arteries, and the intercostal arteries have clinical application prospect.

Description

Acellular bovine intercostal artery and preparation method and application thereof

Technical Field

The invention relates to the field of tissue engineering blood vessels and biological materials, in particular to a decellularized bovine intercostal artery and a preparation method and application thereof.

Background

Cardiovascular and cerebrovascular diseases are diseases caused by cardiovascular or cerebrovascular dysfunction, and seriously threaten the life and health of human beings. Among them, coronary heart disease is a common type of cardiovascular and cerebrovascular diseases, the main pathological feature of which is coronary artery stenosis or blockage, leading to myocardial ischemia, hypoxia and even necrosis. With lifestyle changes and aging population, the incidence of coronary heart disease has increased year by year, becoming a global health problem. Coronary bypass surgery is a common surgical procedure for treating coronary heart disease for this disease. The procedure establishes a new blood flow path between the proximal and distal ends of the coronary stenosis by grafting a segment of blood vessel to improve the blood supply to the myocardium. In recent years, the stent implantation technology can be implanted into a coronary artery stenosis part in an interventional operation mode, so as to support the vessel wall and keep blood flow smooth. However, stent implantation techniques also have limitations. First, for patients with certain complex lesions or multiple vascular lesions, stent implantation may not achieve the desired revascularization effect. Second, long-term antiplatelet therapy is required after stent implantation to prevent thrombosis, which increases the economic burden and bleeding risk of the patient. In addition, the stent is implanted as a foreign body in the body, and may cause problems such as inflammatory reaction and vascular endothelial injury. Compared with the stent, the coronary artery bypass grafting is used for treatment by arterial bridge bypass grafting of internal thoracic artery and the like, and the vascular bridge of a patient has a 10-year patency rate of more than 90 percent and is far higher than that of a venous bridge and a coronary stent.

Clinically, small-caliber vascular grafts of less than 6mm used in coronary bypass surgery are all taken from the patient himself (radial artery, internal thoracic artery, great saphenous vein, etc.). Although these autologous blood vessels can maintain a high long-term patency rate, because of the limited source of blood vessels and secondary trauma to the body caused by obtaining blood vessels, development of small-caliber artificial blood vessels which can replace autologous blood vessels is highly demanded. At present, tissue engineering small-caliber blood vessels have two construction paths: one proposal is that gel is made by acellular matrix, and then other functional materials are mixed to make small caliber blood vessel, which has the advantages of good biocompatibility and blood compatibility, but poor mechanical property and suture property and high cost; the other scheme is that the small-caliber blood vessel constructed by the method is used after the fresh arteriole blood vessel is decellularized and then modified, but has good mechanical property, poor biocompatibility and easy thromboembolism. In addition, great saphenous vein is used as a decellularized vascular stent material at present, but the diameter of the decellularized great saphenous vein is not matched with that of an autologous arteriole, so that the smoothness of an anastomotic stoma is affected. Statistics show that the diameter range of adult coronary arteries in China is: the left anterior descending branch is 2.26+ -0.41 mm, the left circumflex branch is 2.14+ -0.43 mm, and the right coronary artery is 2.95+ -0.60 mm. Therefore, there is an urgent need to provide a tissue engineering blood vessel having a smaller tube diameter, while having excellent mechanical properties, suturing properties and biocompatibility.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a decellularized bovine intercostal artery and a preparation method and application thereof, and the invention adopts the following technical scheme:

In a first aspect, the present invention provides a method for preparing a decellularized modified blood vessel, comprising the steps of: after the fresh animal blood vessel is subjected to cell removal treatment, methacrylic anhydride and 2-methacryloyloxyethyl phosphorylcholine are used for modification treatment, and the cell-removed modified blood vessel is obtained.

Methacrylic anhydride generally reacts with active groups such as hydroxyl groups, carboxyl groups and the like on the surface of vascular materials through free radical polymerization reaction. Under the action of an initiator, the double bond of the methacrylic anhydride is opened to form free radicals, and the free radicals react with active groups on the surface of the vascular material to generate covalent bonds, so that the methacrylic anhydride is grafted to the surface of the material, and the grafting reaction can increase the hydrophobicity and mechanical property of the surface of the vascular material. 2-Methacryloxyethyl Phosphorylcholine (MPC) is a biologically active acrylate monomer, and its phosphorylcholine group has excellent biocompatibility and antithrombotic ability. MPC can be bound to reactive groups on the surface of vascular material by similar free radical polymerization reactions. The introduction of MPC can improve the hydrophilicity, anticoagulation and cell adhesiveness of the material surface, promote the growth and differentiation of endothelial cells, thereby being beneficial to the regeneration and repair of blood vessels.

Further, since the basic structure and collagen framework of the blood vessel are maintained while the blood vessel of the cellular components is removed by the decellularization treatment, methacrylic anhydride and MPC can react with active groups on collagen, elastin, etc. components in the decellularized blood vessel to form chemical bonds. Such chemical bonding not only enhances the mechanical properties of the vascular material, but also improves its biocompatibility and functionality.

As a preferred embodiment of the method of preparation of the invention, the animal blood vessel includes, but is not limited to, bovine intercostal arteries. The diameter of the intercostal artery of the cattle is about 3mm, the length of the intercostal artery reaches 30-50cm, and the intercostal artery is suitable for manufacturing the decellularized small vessel stent. Compared with the common vascular materials such as the bovine carotid artery (with the pipe diameter of 4-8 mm), the bovine mesenteric vein (with the pipe diameter of 6 mm) and the like, the diameter range of the bovine intercostal artery is more matched with the diameter range of the adult coronary artery in China.

As a preferred embodiment of the production method of the present invention, the modification treatment comprises the steps of:

(1) Immersing the vascular tissue subjected to cell removal treatment into deionized water, and dropwise adding methacrylic anhydride into the system, wherein the final volume concentration of the methacrylic anhydride in the system is 4%, and the pH value of the system is kept to be 7 in the dropwise adding process; after methacrylic anhydride reaches the final concentration and the pH is not changed any more, perfusing the vascular tissue for 24 hours at room temperature, ending the perfusion and cleaning to obtain the acellular vascular-methacryloyl ester;

(2) Immersing the decellularized blood vessel-methacryloyl ester in the step (1) in 2-methacryloyloxyethyl phosphorylcholine aqueous solution overnight, then adding ammonium persulfate powder and sodium bisulphite powder into the system to react for 24 hours, and cleaning after the reaction to obtain the decellularized modified blood vessel.

As a preferred embodiment of the preparation method of the invention, the ratio of the added mass and the system volume of the ammonium persulfate powder is ammonium persulfate powder: system = 1.141g:100ml; the sodium bisulfite powder is added into the system according to the mass and volume ratio of sodium bisulfite powder: system = 0.52g:100ml.

As a preferred embodiment of the production method of the present invention, in the step (1), the rate of dropping methacrylic anhydride into the system is 5ml/min.

As a preferred embodiment of the production method of the present invention, in the step (2), the concentration of the aqueous solution of 2-methacryloyloxyethyl phosphorylcholine is 3M; the final concentration of ammonium persulfate in the solution system is 50mM; the final concentration of sodium bisulphite in the solution system is 50mM. The addition of Ammonium Persulfate (APS) and Sodium Bisulphite (SBS) can initiate the free radical polymerization. Ammonium persulfate (as a free radical initiator, it can decompose under mild conditions to generate free radicals, and sodium bisulphite as a chain transfer agent participates in the reaction to adjust the rate of the polymerization reaction and the length of the polymer chain, this step promotes the covalent bond generation of 2-methacryloyloxyethyl phosphorylcholine and the surface of the blood vessel treated with methacrylic anhydride, and polymerizes 2-methacryloyloxyethyl phosphorylcholine, thereby more firmly bonding and improving the performance of the modified blood vessel.

As a preferred embodiment of the preparation method of the present invention, in the step (2), after the ammonium persulfate solution and the sodium bisulphite solution are added, the reaction condition is shaking reaction at 37℃for 24 hours. Under these conditions, the polymerization reaction proceeds more uniformly and stable modified blood vessels are formed.

As a preferred embodiment of the preparation method of the present invention, the decellularization treatment comprises the steps of: washing fresh animal blood vessels, perfusing the animal blood vessels to obtain acellular matrix, and sterilizing the acellular matrix to finish acellular treatment.

As a preferred embodiment of the preparation method of the present invention, the decellularization treatment comprises the steps of:

s1, placing fresh animal blood vessels into a cleaning reagent, carrying out vibration cleaning at 100rpm for 30min each time, changing liquid and repeating for 3-5 times; the cleaning reagent is PBS buffer solution containing 0.02% -0.2% EDTA and 1% heparin sodium;

S2, placing the vascular material cleaned in the step S1 into a first decellularized reagent for perfusion, cleaning with distilled water to remove residual reagent after the first perfusion for 24-48 hours, continuing to perfuse vascular tissue with a second decellularized reagent, and cleaning with distilled water to remove residual reagent after the second perfusion for 24-48 hours;

The first decellularization reagent is: PBS buffer of 0.02% -0.2% EDTA and 1.0% TritonX-100; the second decellularization reagent is: PBS buffer of 0.02% -0.2% EDTA and 0.5% -1.0% sodium dodecyl sulfate;

S3, adding PBS buffer solution into the vascular material cleaned in the step S2, vibrating and cleaning for 24-48h, sterilizing, immersing in sterile PBS buffer solution containing 1% penicillin or 1% streptomycin, and storing in a refrigerator at the temperature of minus 20 ℃ to finish cell removal treatment.

The decellularization treatment step can effectively remove the bovine intercostal arterial vascular cells and endothelial cells, and remove acidic polysaccharides and proteoglycans. The decellularized process can effectively remove muscle fibers, retain elastic fibers and collagen fibers, and maintain the integrity of a blood vessel structure.

In a second aspect, the invention provides a decellularized bovine intercostal artery prepared by the method for preparing the decellularized modified blood vessel.

The acellular bovine intercostal artery provided by the invention has good biocompatibility and antithrombotic capability after being modified by methacrylic anhydride and 2-methacryloyloxyethyl phosphorylcholine, and provides more reliable material selection for the fields of vascular grafting, vascular regeneration and the like. Meanwhile, the mechanical properties such as Young modulus, bursting pressure and the like of the acellular bovine intercostal artery product provided by the invention are improved, and the acellular bovine intercostal artery product has better structural stability. After implantation in the human/animal body, the stability of its shape and function can be maintained when subjected to the pressure of circulating blood.

As a preferred embodiment of the decellularized bovine intercostal artery, the invention is characterized in that the tube diameter of the decellularized bovine intercostal artery is 2.5-3.5mm; the length of the acellular bovine intercostal artery is 30-50mm. The caliber of the decellularized bovine intercostal artery is matched with the caliber of the autologous arteriole, so that the smoothness of an anastomotic stoma can be realized, and the thromboembolism is not easy to cause.

In a third aspect, the invention provides a use of a decellularized bovine intercostal artery selected from the group consisting of:

a) Preparation of medicine for treating cardiovascular and cerebrovascular diseases, and/or

B) As a scaffold material for tissue engineering.

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

1. The invention provides a preparation method of a decellularized modified blood vessel, which removes antigenic cell substances through a decellularized tissue engineering blood vessel, and retains extracellular matrix components and structures; meanwhile, the blood vessel after cell removal is modified by the amphoteric ion material, and the prepared small-caliber artificial blood vessel has good biocompatibility, mechanical property, suturing property and in-vivo anticoagulation property, and keeps long-term patency rate; the preparation method has the advantages of wide sources of materials, controllable manufacturing cost and convenient clinical large-scale use.

2. The invention provides a decellularized bovine intercostal artery which has good biocompatibility and is beneficial to the adhesion growth and tissue remodeling of autologous cells; meanwhile, the tube diameter is matched with the tube diameter of the autologous arterioles, and the tube has good mechanical property and is easy to suture, so that the smoothness of an anastomotic orifice can be ensured. The product has good in vivo anticoagulation performance, is not easy to cause thromboembolism, and has good application prospect.

Drawings

FIG. 1 is a diagram of a bovine intercostal artery decellularized treatment device and post-decellularized vascular profile;

FIG. 2 is a diagram of vascular modification and material characterization analysis of decellularized bovine intercostal arteries;

FIG. 3 is a graph showing the results of mechanical properties of decellularized bovine intercostal arteries with different modification treatments;

FIG. 4 is a graph showing cytotoxicity and blood compatibility evaluation results of different decellularized vascular modification treatment modes;

FIG. 5 is a graph showing experimental results of carotid artery grafting of New Zealand white rabbits;

FIG. 6 is an ultrasonic spectrum of a Non-op side group in an animal experiment;

FIG. 7 is a graph of the ultrasonic spectrum of group D in animal experiments;

FIG. 8 is an ultrasonic spectrum of the H+V group in animal experiments;

fig. 9 is a graph of the ultrasonic spectrum of the Z group in animal experiments.

Detailed Description

For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples.

Example 1

(1) Washing: placing fresh intercostal artery of cattle in PBS buffer solution containing 0.1% EDTA and 1% heparin sodium, shaking at 100rpm in shaking table for 30 min each time, changing solution, and repeating for 3-5 times;

(2) Decellularization: ligating the blood vessel material obtained in the step (1) into branch blood vessels, then pouring PBS buffer solution containing 0.1% EDTA and 1% Triton X-100 through a peristaltic pump for 36 hours, and then fully washing with distilled water to remove the residual Triton X-100; then, the obtained vascular tissue is continuously perfused with PBS buffer solution containing 0.1 percent of EDTA and 0.5 percent of sodium dodecyl sulfate (Sodium dodecyl sulfate, SDS) at room temperature for 36 hours to obtain decellularized vascular matrix;

The perfusion system device is shown as A1 in figure 1 and A2 in figure 1, the system consists of a double-channel peristaltic pump, two peristaltic pump pipes and two blue cap bottles, a polished needle with a number of 14 is linked at the interface of the peristaltic pump pipes, and the needle is inserted into a blood vessel and knotted and fixed by a suture line.

(3) Cleaning of the detergent: and (3) fully shaking and washing the decellularized vascular matrix obtained in the step (2) in distilled water, thoroughly removing residual decellularized reagent, shaking and washing for 24 hours by using PBS buffer solution to obtain the decellularized tissue engineering blood vessel, sterilizing, immersing in sterile PBS buffer solution containing 1% penicillin, and storing in a refrigerator at the temperature of minus 20 ℃.

After the decellularization treatment process is finished, vascular cells are basically removed, and the DNA content is detected, so that the DNA content is reduced from 149.5+/-68.98 ng/ml to 14.05+/-2.493 ng/ml compared with fresh tissue as shown in A3 in figure 1. Fresh vascular tissue length is shown as B1 in fig. 1, and vascular tissue length after decellularization is shown as B2 in fig. 1; the diameter of the fresh vascular tissue is shown as C1 in FIG. 1, and the diameter of the vascular tissue after decellularization is shown as C2 in FIG. 1.

DAPI staining, type I/III collagen sirius red staining, HE staining, alcian Blue staining, masson staining and EVG staining experiments were performed on fresh vascular tissue and the decellularized vascular tissue, respectively.

The DAPI staining results are shown as D1 and D2 in FIG. 1, wherein D1 is the DAPI staining result of fresh vascular tissue, and D2 is the nuclear staining result of the vascular tissue after the decellularization treatment, which indicates that the nuclear DNA of the intercostal artery of the cow has been substantially removed after the decellularization treatment.

The I/III type collagen sirius red staining results are shown as E1 and E2 in FIG. 1, wherein E1 is the staining result of fresh vascular tissue, and E2 is the staining result of vascular tissue after the decellularization treatment, which shows that the basic structural framework of the blood vessel is still completely reserved by the decellularization treatment.

The HE staining results are shown as F1 and F2 in FIG. 1, the HE staining results of fresh blood vessel tissue are shown as F1 in FIG. 1, the HE staining results of blood vessel tissue after decellularization treatment are shown as F2 in FIG. 1, the HE staining results of fresh blood vessel show three-layer structure of intima, media and adventitia, the cell and matrix components are clearly visible, and the HE staining results of bovine intercostal artery after decellularization treatment indicate that vascular endothelial cells are removed.

Alcian Blue staining results are shown as G1 and G2 in FIG. 1, wherein fresh vascular tissue staining results are shown as G1 in FIG. 1, showing the distribution of acidic polysaccharides and proteoglycans in fresh blood vessels; the staining results of vascular tissue after the decellularization treatment are shown as G2 in fig. 1, showing that acidic polysaccharides and proteoglycans are removed.

The Masson dyeing results are shown as H1 and H2 in the figure 1, wherein H1 is a fresh vascular tissue dyeing result, H2 is a vascular tissue dyeing result after cell removal treatment, and the Masson dyeing results in that collagen fibers are blue, myofibers are red and cell nuclei are blue and black; in fig. 1, H1 shows the distribution of collagen fibers and smooth muscle cells on the wall of a fresh bovine intercostal artery, and the results of E2 and H2 are combined, showing smooth muscle cells and a part of collagen fibers after decellularization treatment, but the basic structural framework of the blood vessel is still completely preserved.

The EVG staining results are shown as I1 and I2 in fig. 1, where I1 is the fresh vascular tissue staining result and I2 is the vascular tissue staining result after decellularization treatment. EVG staining causes the collagen fibers to appear red and the muscle fibers to appear yellow. Comparison with EVG staining results shows that the decellularization treatment effectively removes muscle fibers while retaining elastic fibers and collagen fibers, maintaining the integrity of the vascular structure.

(4) Preparation of MA-DIV: thoroughly washing the bovine intercostal artery acellular matrix (DIV) obtained in the step (3) by deionized water, immersing the DIV in the deionized water, dripping Methacrylic Anhydride (MA) into the tissue at a speed of 5ml/min at the temperature of 4 ℃, observing the pH by a pH meter, and adding sodium hydroxide solution (5M) when the pH is lower than 7 to keep the pH value at 7 until the final concentration of the methacrylic anhydride aqueous solution in the system is 4% (v/v). When the concentration of the MA aqueous solution reaches 4% (v/v) and the pH is not changed, the tissue is continuously perfused for 24 hours at room temperature, and then the obtained tissue is thoroughly washed by deionized water to obtain acellular bovine intercostal arterial methacryloyl ester (MA-DIV for short) for later use.

(5) Copolymerization of MPC with MA-DIV: the MA-DIV prepared in the previous step is immersed in 2-methacryloyloxyethyl phosphorylcholine aqueous solution (MPC aqueous solution), the concentration of the MPC aqueous solution is 3M, and the mixture is left to stand overnight. Ammonium persulfate powder (1.141 g/100 ml) and sodium hydrogensulfite powder (0.52 g/100 ml) were then added to the above solution, and the reaction was gently swirled at 37℃for 24 hours. After the reaction, the resulting tissue was thoroughly rinsed with deionized water and then stored in PBS solution. The acellular bovine intercostal artery obtained by the modified blood vessel preparation method is named PMPC-DIV.

Example 2

(1) Washing: placing fresh intercostal artery of cattle in PBS buffer solution containing 0.02% EDTA and 1% heparin sodium, shaking at 100rpm in shaking table for 30min each time, changing solution, and repeating for 3-5 times;

(2) Decellularization: ligating the blood vessel material obtained in the step (1) into branch blood vessels, then pouring PBS buffer solution containing 0.02% EDTA and 1% Triton X-100 through a peristaltic pump for 48 hours, and then fully washing with distilled water to remove the residual Triton X-100; then, the obtained vascular tissue is further perfused with PBS buffer solution containing 0.02% EDTA and 0.5% sodium dodecyl sulfate (Sodium dodecyl sulfate, SDS) at room temperature for 48 hours to obtain decellularized vascular matrix;

The perfusion system device is shown in figure 1A, the system consists of a double-channel peristaltic pump, two peristaltic pump pipes and two blue cap bottles, a polished 14-gauge needle is linked at the interface of the peristaltic pump pipes, the needle is inserted into a blood vessel, and the needle is knotted and fixed by a suture line.

Steps (3) - (5) are the same as in example 1.

Example 3

(1) Washing: placing fresh intercostal artery of cattle in PBS buffer solution containing 0.2% EDTA and 1.0% heparin sodium, shaking at 100rpm in shaking table for 30min each time, changing liquid, and repeating for 3-5 times;

(2) Decellularization: ligating the blood vessel material obtained in the step (1) into branch blood vessels, then pouring PBS buffer solution containing 0.2% EDTA and 1% Triton X-100 through a peristaltic pump for 24 hours, and then fully washing with distilled water to remove the residual Triton X-100; then, the obtained vascular tissue is further perfused with PBS buffer solution containing 0.2% EDTA and 1.0% sodium dodecyl sulfate (Sodium dodecyl sulfate, SDS) at room temperature for 24 hours to obtain decellularized vascular matrix;

The perfusion system device is shown in figure 1A, the system consists of a double-channel peristaltic pump, two peristaltic pump pipes and two blue cap bottles, a polished 14-gauge needle is linked at the interface of the peristaltic pump pipes, the needle is inserted into a blood vessel, and the needle is knotted and fixed by a suture line.

Steps (3) - (5) are the same as in example 1.

Comparative example 1

Taking fresh bovine intercostal arteries of the same length, performing the decellularization operation in the steps (1) - (3) by the method described in the example 1, and performing heparin and VEGF modification after obtaining the acellular matrix of the bovine intercostal arteries.

(4) Heparin loading: the bovine intercostal arterial decellularized matrix was immersed in an isopropanol solution (weight/volume) containing 10% 1, 6-hexamethylenediamine for 2 hours, followed by rinsing with deionized water. To 20ml of sodium citrate buffer (pH set at 5.5) containing 40mg of EDC, 40mg of heparin sodium was added, and it was stirred at 4℃for 24 hours to activate heparin. 2ml of the activated heparin sodium solution was added to a 10ml centrifuge tube to cover the scaffold with sufficient contact with the solution and incubated further for 24 hours at 4 ℃. The tubular graft was immersed for 4 hours at room temperature and rinsed with distilled water. Vacuum drying the transferred tissue at room temperature, and sterilizing by ultraviolet irradiation;

(5) Loading VEGF: the tissue sterilized in step (4) was immersed in PBS buffer containing 1mL of VEGF solution (100 ng/mL) at 4℃for 24 hours. After soaking, the tissue was washed with PBS to remove unreacted residual VEGF from the stent surface, and modification of decellularized bovine intercostal arteries was completed.

The tissue engineering blood vessel prepared by heparin and VEGF treatment in comparative example 1 was stained with toluidine blue, and the results are shown as A1-A2 in FIG. 2, showing the deepening of the color of the inner wall of the blood vessel, suggesting successful heparin introduction; in fig. 2, B1 is the heparin release profile of comparative example 1, showing that heparin release was maximized during the first 5 days, and then the release was smoothed. In fig. 2, B2 is the VEGF release profile of comparative example 1, showing that VEGF release of comparative example 1 was observed mainly in the first 15 days, probably due to VEGF binding to heparin via non-covalent bonds, resulting in a relatively rapid and unstable release.

Comparative example 2

Fresh bovine intercostal arteries of the same length were taken and subjected to the decellularization procedures of steps (1) - (3) as described in example 1 without the vascular modification treatment step.

Performance testing

1. Structural analysis

EDS analysis was performed on example 1, comparative examples 1-2, and the results are shown in fig. 2C, with green representing phosphorus element and yellow representing sulfur element, wherein comparative example 1 shows an increase in sulfur element of the modified blood vessel; SEM image analysis was performed on the blood vessels prepared in examples and comparative examples, respectively, and the results are shown in fig. 2D, in which the heparin + VEGF treatment group modification of comparative example 1 has little influence on the inner surface roughness of the blood vessel wall, as compared with the simple decellularization group of comparative example 2, and the inner surface roughness of the blood vessel wall obtained in the zwitterionic modification group of example 1 is significantly reduced. In fig. 2, E is a graph of the result of analysis of decellularized vascular chemical composition by X-ray photoelectron spectroscopy (XPS). Complex elements naturally present in decellularized blood vessels, including S, P, and shown in XPS analysis to have two S and P peaks, illustrate that example 1 was modified by 2-methacryloyloxyethyl phosphorylcholine, and eventually the component was successfully grafted into the vessel.

Fourier transform infrared spectroscopy (FTIR) analysis was performed on the blood vessels prepared in examples and comparative examples, respectively, and the results are shown as F in fig. 2, where D is a spectrum of a pure decellularized group without vascular modification; the Heparin + VEGF modified group (H+V group) of comparative example 1 shows an increase in the absorption peak characteristic of the sulfuric acid group present in the 1250-800cm-1 region of the corresponding curve of comparative example 1, indicating successful Heparin incorporation. After heparin modification, the change of amino groups can be observed through the change of absorption peaks in the region of 3300-3500cm < -1 >, and the increase of the amino group peaks in the region shows that the amino groups contained in the heparin structure are successfully combined with the vascular material; the decrease in amine peak after loading VEGF may be due to non-covalent binding of VEGF to amine, and possible shielding effect of VEGF molecules, resulting in decreased detection level of amine. The Z curve of F in FIG. 2 is that of the zwitterionic MPC modification group of example 1, and the corresponding graph of the zwitterionic MPC modification group of example 1 shows a significant drop in amine group peak in the 3300-3500cm-1 region, indicating that modification of 2-Methacryloyloxyethyl Phosphorylcholine (MPC) introduces a new chemical structure, resulting in a reduction in the original amine group level. Further, the extent of incorporation of 2-methacryloyloxyethyl phosphorylcholine was detected by analysis of the phosphate group (p=o) related features, in which the phosphate absorption peak at the 1038.2cm-1 region was shifted from the other groups, representing the phosphate group (p=o) feature in MPC.

2. Mechanical properties

Experiments were performed on mechanical properties including vascular burst pressure and tensile experiments using fresh bovine intercostal arteries (group F), the zwitterionic MPC modified group of example 1 (group Z), the heparin + VEGF modified group of comparative example 1 (group H + V), and the decellularized-only unmodified group of comparative example 2 (group D), and the corresponding experimental methods were as follows:

(1) Vascular burst pressure experiment

The specific method for the vascular burst pressure experiment comprises the following steps: the blood vessel was cut to a length of about 0.5cm, and one end was connected to a pressure transducer (YP-100 b, yunyi, china) with a tee at the front end, and one end was ligatured closed. The blood vessel is filled with physiological saline, more physiological saline is gradually added into the blood vessel through the three-way pipe, the pressure is gradually increased until the blood vessel is partially broken, the pressure in the lumen is the burst strength of the specimen when the blood vessel is broken, and the physiological signal acquisition and processing system and software matched with the transducer are used for recording the numerical value.

(2) Tensile test

The specific method for the stretching experiment comprises the following steps: the longitudinal displacement of the resulting vascular graft was measured using a universal tensile machine (WD-5A). Briefly, we used a clamp of a universal tensile machine to secure the vessel and stretched at a rate of 10mm/min until the stent breaks. And measuring a stress-strain curve by using software carried by the machine, and removing experimental errors through data processing of origin software.

The results of the mechanical properties of the decellularized bovine intercostal arteries obtained by the different treatments are shown in FIG. 3. Wherein a in fig. 3 is burst pressure data for different vascular treatment groups; FIG. 3B is the maximum stress data for different vascular treatment groups; FIG. 3C is the maximum strain data for different vascular treatment groups; fig. 3D is young's modulus data for different vascular treatment groups.

As can be seen from FIG. 3A, the burst pressure of fresh bovine intercostal arteries (group F) was about 1711.+ -. 23.18mmHg, whereas after decellularization treatment (group D, comparative example 2), the burst pressure of blood vessels was increased to 1856.+ -. 30.92mmHg; the heparin + VEGF modified group (H + V group, comparative example 1) did not further increase burst pressure, whereas the zwitterionic MPC modified group (P group, example 1) had a significant increase in burst pressure, reaching 1997± 36.72mmHg. Example 1 modification of decellularized blood vessels by methacrylic anhydride and 2-methacryloyloxyethyl phosphorylcholine can be increased. This shows that the mechanical strength and durability of the vascular material prepared in example 1 are enhanced.

As can be seen from fig. 3C and D, in comparative example 2 (group D) in which only decellularization treatment was performed, the maximum elongation and young's modulus did not increase significantly, but there was still a level of increase from 115.4± 8.159% to 154.1± 45.42%; while the data of comparative example 1 (H+V) is 360.5.+ -. 39.49%, the data of example 1 (group P) is 329.5.+ -. 12.18%, which shows that the maximum elongation of the blood vessel is further improved after the modification treatment; whereas in fig. 3D, the young's modulus increase of example 1 was more pronounced, reaching = 75.46± 5.298MPa. All the treatment procedures have no obvious influence on the maximum stress of the blood vessel.

The above results demonstrate that the modification treatment of methacrylic anhydride and 2-methacryloyloxyethyl phosphorylcholine enhances the crosslink density and stability of vascular materials, thereby increasing their resistance to internal pressure. The modified artificial decellularized blood vessel exhibits higher durability and longer service life when subjected to sustained pressure generated by blood flow and circulation due to the elevation of burst pressure. This helps reduce the frequency of replacement after implantation. Young's modulus is a proportionality constant of a material between stress and strain in the elastic range and reflects the ability of the material to resist deformation when subjected to a force, while an increase in Young's modulus indicates that the stiffness or resistance to deformation of the vascular material of example 1 is significantly enhanced.

3. Cytotoxicity and blood compatibility evaluation

(1) Cytotoxicity test

Cytotoxicity of each of the above groups of blood vessels was measured using live/dead cell staining and CCK8 method, including fresh bovine intercostal arteries (group F), the zwitterionic modified group of example 1 (group Z), the heparin and VEGF modified group of comparative example 1 (group h+v) and the decellularized only group of comparative example 2 (group D).

Treatment group: and respectively adding the PBS solution into each group of blood vessels, soaking for 48 hours, and then taking out the blood vessels to obtain leaching liquor. The extracts were added at 10. Mu.l per well to 96-well plates containing HUVEC cells which had been previously cultured (i.e., treated the same as the negative control group).

Negative control group: approximately 2000 HUVEC cells were seeded into each well, and then 100. Mu.L of cell culture broth was added to each well without additional addition of leaching solution. Culturing for 2d.

After all groups were cultured for 24 hours, CCK8 reagent was added and left for 3 hours, and then the whole groups were put into an ELISA reader for reading measurement.

FIG. 4A shows the result of staining with live/dead cells, wherein the live cells are green and the dead cells are red; the results of the CCK8 assay are shown in fig. 4B, which shows that after 3 days of culture, there is a significant difference in the fresh blood vessel group compared to the negative control group, which may be derived from toxic substances released by cell death in the fresh blood vessel. In other groups, cytotoxicity did not show significant differences from the negative control group after extensive washing, indicating that the excess toxic agent had been effectively cleared. C in FIG. 4 is the live/dead cell ratio result.

(2) Blood compatibility test

Fresh rabbit blood (blood to sodium citrate ratio 9:1) was collected in a sodium citrate vacuum tube and centrifuged at 1500r/min for 15min to obtain Platelet Rich Plasma (PRP). The sample was punched into a 6mm diameter disc, rinsed with sterile PBS solution for 2 hours, and then placed into a 96-well plate. 100. Mu.L of PRP was added to each sample and incubated for 1h. After incubation, samples were transferred to a new 96-well plate and rinsed three times with sterile PBS. The number of attached platelets was determined by measuring the amount of Lactate Dehydrogenase (LDH) after cell lysis using an LDH test kit according to the instructions of the supplier.

Blood deposition behavior was assessed by directly attaching blood within the stent. The blood vessels of the above groups were tested for hemolysis profile of 10mm long tubular grafts. The test method comprises the following steps:

Whole blood and PBS solution 1:9, the negative control is shown as blood diluent without sample contact, and the positive control is shown as blood incubated in distilled water. 1mL of blood was dispensed into each vascular sample and incubated for 30min to give a hemolysis rate. After a certain period, the solution was aspirated and centrifuged at 3000rpm for 3min. Absorbance (OD ₅₄₀) of free hemoglobin in 96-well plates was measured. Results are expressed as a percentage of the positive control sample.

100ML of whole blood was then infused into a 10mm length vascular graft and clotting was performed. Samples were incubated for 30min. The transfer and lysis were then washed with distilled water. The solution was collected and the amount of free hemoglobin was calculated using a similar protocol as described in the haemolysis quantification.

The results of the above blood compatibility test are shown as D-F in FIG. 4; wherein D in FIG. 4 is a graph of the results of the hemolysis experiment; FIG. 4E is a graph of the blood coagulation function test; f in FIG. 4 is a graph showing the results of platelet adhesion experiments. Among them, group D, which was subjected to only decellularization treatment, exhibited poor hemolysis and coagulation properties, while the blood compatibility of comparative example 1 and example 1 was good.

4. New Zealand white rabbit carotid artery transplantation animal experiment

The experimental animals are New Zealand white rabbits, and antibiotics are given 24 hours before operation to prevent possible infection in operation; due to the physiological characteristics of rabbits, fasted and water forbidden is started only 1 hour before operation. 500U/kg heparin was administered before and after the operation, respectively, and 1000U/kg heparin was administered daily for seven days after the operation, and then the rabbits were subjected to general anesthesia and aseptic manipulation. Shaving and disinfecting the neck and then making an incision along the midline of the neck to expose the carotid artery. Next, the carotid artery was carefully isolated, while taking care to protect the surrounding nerves and tissues. Once a carotid artery of sufficient length has been isolated, blood flow is temporarily blocked with a vascular clamp. A section of carotid artery is excised and the tissue engineered vessel is then sutured to the incision in the original artery. End-to-end anastomosis is performed using microsurgical techniques to ensure that the anastomosis is leak-free. After anastomosis is completed, the vascular clamps are released and the vascular displacement site is observed for blood flow, ensuring no bleeding. Finally, incisions were sutured layer by layer and subjected to appropriate post-operative treatments, including heparin sodium, antibiotics and analgesics, and animals were monitored for recovery.

The whole operation process needs to be finely operated under a microscope, so that the accuracy of vascular anastomosis and the recovery of blood flow are ensured. In addition, vital signs and surgical sites of animals should be closely monitored post-operatively to ensure no infection and successful vascular replacement. The Non-op side, i.e., the Non-surgical side, was used as a negative control group. Meanwhile, rivaroxaban and aspirin were administered daily throughout a 30-day period after surgery to prevent thrombosis due to surgical stress, the entire surgical procedure being shown in fig. 5 a.

Experiments were performed with example 1, comparative example 1, and comparative example 2, respectively, and the results are shown in fig. 5: wherein, B in figure 5 is an original vessel map of the white rabbit, an immediate map of the autologous carotid artery graft vessel of the white rabbit and a vessel state map after 30 days of the graft in sequence from left to right; fig. 5C shows ultrasound images of different groups of arteries, including (I.B type ultrasound, ii. Doppler ultrasound), and fig. 6-9 show ultrasound spectra of different groups of arteries. In fig. 5B, the fresh blood vessels appear normal in appearance and color, the vascular grafts are seen to be significantly blood filled, and no significant thrombosis is observed 30 days after surgery, indicating good biocompatibility and restorative properties of the transplanted vessels. Further, according to the doppler ultrasound of the graph C in fig. 5 and the spectral analysis of fig. 6 to 9, compared with comparative example 1 (h+v group) and comparative example 2 (D group), it was observed that blood flow in the white rabbits was restored immediately after the operation after the blood vessel of example 1 (Z group) was transplanted, and remained unobstructed for the subsequent 30 days, which was reflected in the restoration of blood flow velocity in the doppler ultrasound image and the stable waveform in the spectral analysis.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, 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 the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims (10)

1. The preparation method of the decellularized modified blood vessel is characterized by comprising the following steps of: after the fresh animal blood vessel is subjected to cell removal treatment, methacrylic anhydride and 2-methacryloyloxyethyl phosphorylcholine are used for modification treatment, and the cell-removed modified blood vessel is obtained.

2. The method of claim 1, wherein the animal blood vessel comprises a bovine intercostal artery.

3. The method of manufacturing according to claim 1, wherein the modification treatment comprises the steps of:

(1) Immersing the vascular tissue subjected to cell removal treatment into deionized water, and dropwise adding methacrylic anhydride into the system, wherein the final volume concentration of the methacrylic anhydride in the system is 4%, and the pH value of the system is kept to be 7 in the dropwise adding process; after methacrylic anhydride reaches the final concentration and the pH is not changed any more, perfusing the vascular tissue for 24 hours at room temperature, ending the perfusion and cleaning to obtain the acellular vascular-methacryloyl ester;

(2) Immersing the decellularized blood vessel-methacryloyl ester in the step (1) in 2-methacryloyloxyethyl phosphorylcholine aqueous solution overnight, then adding ammonium persulfate powder and sodium bisulphite powder into the system to react for 24 hours, and cleaning after the reaction to obtain the decellularized modified blood vessel.

4. The process according to claim 3, wherein in the step (1), the methacrylic anhydride is added dropwise to the system at a rate of 5ml/min.

5. The method according to claim 3, wherein in the step (2), the concentration of the aqueous solution of 2-methacryloyloxyethyl phosphorylcholine is 3M; the final concentration of ammonium persulfate in the system after being added is 50mM; the final concentration of sodium bisulphite in the system after addition was 50mM.

6. The process according to claim 5, wherein in the step (2), the reaction condition is shaking reaction at 37℃for 24 hours after the addition of the ammonium persulfate solution and the sodium bisulfite solution.

7. The method of claim 1, wherein the decellularizing treatment comprises the steps of: washing fresh animal blood vessels, perfusing the animal blood vessels to obtain acellular matrix, and sterilizing the acellular matrix to finish acellular treatment.

8. A decellularized bovine intercostal artery prepared by the method of preparing a decellularized modified blood vessel of any one of claims 1-7.

9. The decellularized bovine intercostal artery of claim 8 in which the decellularized bovine intercostal artery has a tube diameter of 2.5-3.5mm; the length of the acellular bovine intercostal artery is 30-50mm.

10. Use of the decellularized bovine intercostal artery of any one of claims 8-9, wherein said use is selected from the group consisting of:

a) Preparation of medicine for treating cardiovascular and cerebrovascular diseases, and/or

B) As a scaffold material for tissue engineering.