CN115212350B - Application of hydrogel in preparation of high-bionic artificial blood vessel material - Google Patents

Abstract

The invention discloses application of hydrogel in preparing a high-bionic artificial blood vessel material, wherein the hydrogel is chitosan/polyethylene glycol hydrogel or acetalized polyvinyl alcohol hydrogel, and the high-bionic artificial blood vessel material is prepared by coating the surface of a decellularized scaffold with the hydrogel. The preparation method has the remarkable advantages that the prepared high-bionic artificial vascular material can effectively retain the original mechanical bionic performance of the decellularized scaffold through the use of the hydrogel and the decellularized scaffold, and has excellent biocompatibility.

Description

Application of hydrogel in the preparation of highly bionic artificial blood vessel materials

Technical field

The invention relates to the application of hydrogel in the preparation of highly bionic artificial blood vessel materials, and belongs to the field of tissue engineering.

Background technique

Since entering the 21st century, cardiovascular diseases have brought new severe challenges to mankind. The World Health Organization (WHO) analyzed the current major diseases that pose a threat to human health and pointed out that cardiovascular disease has become the number one cause of death in the world. According to statistics from the World Health Organization, 12 million people die from cardiovascular disease every year around the world, accounting for 1/4 of the total deaths. Cardiovascular disease has been recognized as the number one killer of human health along with cancer. Cardiovascular disease refers to disorders of the heart and blood vessels, including coronary heart disease (heart attack), cerebrovascular disease (stroke), elevated blood pressure (hypertension), rheumatic heart disease, congenital heart disease, heart failure and peripheral A general term for various diseases such as arterial vascular disease. In recent years, with the changes in my country's national dietary habits and the increasingly serious problem of population aging, the incidence of cardiovascular and cerebrovascular diseases has increased year by year. On September 3, 2019, The Lancet published two latest research results online. It focuses on common morbidity and mortality in 21 countries, showing common causes of death in low-, middle- and high-income countries. The results show that mortality from cardiovascular disease is much higher in low- and middle-income countries than in developed countries.

In the treatment of coronary heart disease, vascular injury, lower limb arterial ischemia and other diseases, multiple vascular transplant surgeries such as coronary artery bypass grafting are usually required, which has led to the increasing gap in vascular grafts in my country. At present, artificial blood vessels based on polyester and polytetrafluoroethylene have been widely used in the reconstruction process of large-diameter blood vessels (diameter greater than 6mm). However, when the above materials are used in small-diameter blood vessels (less than 6mm), there are thromboembolism, There are shortcomings such as intimal hyperplasia and low patency rate. Therefore, there is currently a shortage of small-diameter blood vessels with good blood compatibility and patency that can be used clinically, and most of them can only rely on autologous blood vessel transplantation. However, autologous vascular transplantation has many shortcomings such as difficulty in sourcing materials, insufficient length of vascular pedicles, and mismatched diameters.

Currently, the most commonly used vascular grafts in clinical practice mainly include artificial blood vessels and autologous blood vessels. Autologous blood vessels cannot be widely used due to their limited sources and high donor sacrifice. Artificial blood vessels are popular because of their wide range of sources. The most commonly used artificial blood vessel materials in clinical medicine are polyester materials, such as polyester and expanded polytetrafluoroethylene. Polyester materials are widely used in the preparation of artificial blood vessel materials due to their ease of processing, microporous structure and excellent mechanical properties. However, the hydrophobic properties of its surface can easily induce thrombosis, and anticoagulant drugs are required after surgery, which seriously limits its clinical development and application. The expanded polytetrafluoroethylene (ePTFE) material has the characteristics of non-antigenicity, non-carcinogenicity, non-toxicity and close to the compliance of autologous arteries, making the expanded polytetrafluoroethylene artificial blood vessel material the most widely used in clinical practice. Although it has good biocompatibility and is not prone to thrombosis, it may suffer from lumen stenosis in the later stages of clinical transplantation, causing problems in small-diameter blood vessel transplantation. Artificial blood vessels with excellent performance need to have various properties similar to natural blood vessels, such as good blood compatibility, cell compatibility, and adhesion and proliferation capabilities of endothelial progenitor cells. At the same time, excellent artificial blood vessel materials should also have biomechanical properties similar to those of normal blood vessels. In addition, the metabolism of organisms is ongoing, and natural blood vessels are also constantly being renewed. This renewal is undoubtedly cyclical. Therefore, an ideal artificial blood vessel not only needs to be biodegradable, but its degradation cycle should also be consistent with that of the organism. match the actual degradation cycle. Therefore, it is particularly important in the research of life sciences and bionics to find a suitable theory and method to construct artificial blood vessel materials with excellent properties and similar properties to natural blood vessels.

In response to the above problems, domestic and foreign researchers have conducted long-term research. Research shows that vascular graft materials based on tissue engineering can better meet clinical needs and mechanical biomimetic properties. Among them, vascular tissue engineering is the science of using biodegradable scaffold materials to prepare, reconstruct and regenerate vascular replacement materials. Artificial blood vessel materials based on acellular vascular scaffolds can avoid immune rejection after implantation while maintaining the degradation rate and mechanical properties of the blood vessels themselves. However, the scaffold material prepared by the decellularization method removes the endothelial cell layer on the inner surface of the blood vessel, leaving only a three-dimensional network structure. Therefore, the surface of the decellularized scaffold must be modified by coating.

Therefore, designing a composite material that can ensure good biocompatibility and bionic performance while maintaining the advantages of acellular scaffolds, maintain long-term patency of the implanted site, and ultimately achieve endothelialization is a key breakthrough in vascular tissue engineering. direction, and is also a prerequisite for clinical application. Using tissue engineering principles and combining scaffold materials, cells and biological signaling molecules to construct tissue-engineered small-diameter blood vessels provides new ideas for solving the above problems and is currently one of the most promising solutions.

Contents of the invention

Purpose of the invention: The purpose of the invention is to provide hydrogel for use in the preparation of highly bionic artificial blood vessel materials.

Technical solution: Application of the hydrogel of the present invention in the preparation of highly bionic artificial blood vessel materials. The hydrogel is chitosan/polyethylene glycol hydrogel or acetalized polyvinyl alcohol hydrogel. .

The hydrogel is a gel structure formed of water-soluble polymers with a cross-linked three-dimensional network structure and using water as a dispersion medium. When the above-mentioned polyethylene glycol or polyvinyl alcohol water-soluble polymers come into contact with water, the hydrophilic groups inside them will assemble with water molecules, while the hydrophobic groups will swell when exposed to water, making the hydrogel not only undissolvable in water , will also swell and can absorb and lock in a large amount of moisture.

Further, the preparation method of the chitosan/polyethylene glycol hydrogel includes the following steps:

(1) Prepare a NaOH aqueous solution, and then add absolute ethanol to obtain a NaOH solution;

(2) Add chitosan to the NaOH solution while stirring to alkalize it;

(3) Add propylene oxide and place it in a constant temperature water bath to react to obtain the product;

(4) Take out the product, wash it in a mixture of hydrochloric acid and acetone, then wash it in a mixture of acetone and water, vacuum filter, and vacuum dry to obtain O-HPCS. The reaction mechanism is as follows;

(5) Prepare an O-HPCS aqueous solution, add polyethylene glycol and glutaraldehyde solutions, stir and mix, and let stand to obtain chitosan/polyethylene glycol hydrogel.

Furthermore, in step (1), the mass concentration of the NaOH solution is 6% to 8%; in step (2), the liquid-to-solid ratio of the NaOH solution to chitosan is 10 to 12 mL:g, and the The alkalization time is 6 to 8h; in step (3), the reaction time is 24 to 36h; in step (4), the mass ratio of hydrochloric acid to acetone in the mixture of hydrochloric acid and acetone is 1:10 ~1:9, the mass ratio of acetone to water in the mixture of acetone and water is 9:1~10:1, the temperature of the vacuum drying is 45~55°C, and the vacuum drying time is 2~3h; step ( In 5), the solid-liquid ratio of polyethylene glycol and glutaraldehyde is 0.1-0.2g:mL.

Further, the preparation method of the acetalized polyvinyl alcohol hydrogel includes the following steps:

(1) Mix polyvinyl alcohol with water and heat to dissolve;

(2) Add glycerol and continue heating;

(3) Cool down, add formaldehyde and glutaraldehyde respectively, and stir evenly to obtain acetalized polyvinyl alcohol;

(4) Dry the acetalized polyethylene, then raise the temperature to continue drying, cool, and soak and rinse with water to obtain acetalized polyvinyl alcohol hydrogel.

The basic reaction equation for polyvinyl acetalization is as follows:

Furthermore, in step (1), the mass ratio of polyvinyl alcohol to water is 1:30 to 5:90, and the heating temperature is 90 to 100°C; in step (2), the glycerin and The mass ratio of polyvinyl alcohol is 1:1~2, and the continued heating time is 0.5h~1h; in step (3), the mass ratio of formaldehyde, glutaraldehyde and polyvinyl alcohol is 2:6: 3~2:6:5, the concentration of glutaraldehyde is 0.5~9%; in step (4), the drying temperature is 40~60°C, the drying time is 2~3h, and the temperature is raised again The temperature is 60-70°C, the continuous drying time is 1-2h, and the soaking and rinsing time is 0.5-1h.

Further, the preparation method of the highly bionic artificial blood vessel material includes the following steps:

(1) Preparation of chitosan/polyethylene glycol hydrogel;

(2) Apply chitosan/polyethylene glycol hydrogel on the surface of the decellularized scaffold to obtain chitosan/polyethylene glycol/decellularized scaffold CS/PEG/DCS;

(3) Using a layer-by-layer self-assembly method, heparin was deposited on the surface of the chitosan/polyethylene glycol/decellularized scaffold and vacuum dried to obtain the highly bionic artificial blood vessel material n-He-CS/PEG/DCS.

Furthermore, in step (3), the layer-by-layer self-assembly method is a soaking and pulling method, which includes the following steps:

(3.1) Prepare heparin sodium solution and chitosan solution respectively;

(3.2) Soak CS/PEG/DCS in PBS buffer solution and take it out, soak it in sodium heparin solution, take it out and rinse it with PBS buffer solution front and back to obtain 1-He-CS/PEG/DCS;

(3.3) Soak 1-He-CS/PEG/DCS in the prepared chitosan solution, take it out and wash it front and back with PBS buffer solution, then soak it in heparin sodium solution, take it out and wash it front and back with PBS buffer solution Rinse the back side to obtain 2-He-CS/PEG/DCS;

(3.4) Repeat the steps in (3.3) to prepare a multi-layered heparin/polyethylene glycol hydrogel/decellularized scaffold to obtain n-He-CS/PEG/DCS.

Furthermore, in step (3.1), the concentration of the heparin sodium solution is 1-2g/L, the concentration of the chitosan solution is 1-2g/L, and the mass ratio of the chitosan to heparin sodium is 1. : 1 to 2; in step (3.2), the CS/PEG/DCS is soaked in PBS buffer solution for 10 to 30 minutes, and soaked in heparin sodium solution for 10 to 15 minutes; in step (3.3), The 1-He-CS/PEG/DCS is placed in the prepared chitosan solution and soaked for 10 to 15 minutes, and the soaking time in the heparin sodium solution is 10 to 15 minutes. In step (3.4), the n- n in He-CS/PEG/DCS is 3 to 7.

Further, the preparation method of the highly bionic artificial blood vessel material includes the following steps:

(1) Preparation of polyvinyl alcohol hydrogels with different acetalization;

(2) Prepare different acetalized heparin-polyvinyl alcohol complexes;

(3) Apply different acetalized heparin-polyvinyl alcohol complexes on the surface of the decellularized scaffold to obtain different acetalized heparin-polyvinyl alcohol/decellularized scaffold, that is, the highly bionic artificial vascular material He/PVA _n /DCS.

Furthermore, in step (2), the preparation of the acetalized heparin-polyvinyl alcohol complex includes the following steps: drying the acetalized polyvinyl alcohol hydrogel to obtain an acetalized polyvinyl alcohol film. , and then soak the acetalized polyvinyl alcohol film in a heparin sodium aqueous solution to obtain an acetalized heparin-polyvinyl alcohol complex.

Furthermore, the drying temperature is 40-60°C, the drying time is 2-3 hours, and the mass concentration of the heparin sodium aqueous solution is 1-2%.

Further, the preparation method of the decellularized scaffold includes the following steps:

(1) Take out the blood vessels placed in the tissue fixative, trim the outer membrane, and immerse them in physiological saline;

(2) Prepare a mixed solution of sodium lauryl sulfate and polyethylene glycol octylphenyl ether, and soak the trimmed blood vessels in the mixed solution;

(3) Take out the soaked blood vessels, rinse them with PBS buffer solution, and soak them in PBS buffer solution. Change the PBS buffer solution every day;

(4) Take out the soaked blood vessels, trim them into pieces, and freeze-dry them to obtain the decellularized scaffold DCS.

Further, in step (2), the mass ratio of sodium lauryl sulfate and polyethylene glycol octylphenyl ether is 1:1 to 1:2, and the soaking time in the mixed solution is 24 to 48 hours. ; In step (3), the soaking time in the PBS buffer solution is 20 to 30 days, and the number of times of changing the PBS buffer solution per day is 1 to 2 times; in step (4), the freeze-drying temperature is -45～-55℃, freeze drying time is 8～12h.

Beneficial effects: Compared with the existing technology, the present invention has the following significant advantages:

(1) Decellularized scaffolds are derived from natural blood vessels and have inherent advantages in mechanical properties and mechanical biomimetic properties. At the same time, acellular scaffolds have excellent biocompatibility and can avoid thromboembolism and immune rejection. The selection of the acellular scaffold base and hydrogel makes it have excellent biodegradability and can be degraded by the organism itself in a stable environment in the body. The degraded products are non-toxic and can finally be absorbed by autologous tissues. The use of acellular scaffold materials derived from natural blood vessels reduces the probability of immune rejection and calcification after implantation in the body to a certain extent.

(2) Layer-by-layer self-assembly technology alternately deposits on the surface of the substrate to form stable and complete molecular aggregates. The self-assembled film synthesized by this method has significant advantages. After polyethylene glycol hydrogel coating, the three-dimensional network structure of the acellular vascular stent is changed, but it does not change the mechanical properties of the artificial vascular material of the acellular vascular stent. The material has excellent mechanical bionic properties and can withstand blood The pressure changes generated during the flow in the lumen maintain the patency of the lumen. After layer-by-layer self-assembly, the biocompatibility of the artificial blood vessel material is significantly improved, which is conducive to the attachment and growth of endothelial progenitor cells and smooth muscle cells.

(3) Modified heparin-polyvinyl alcohol hydrogels with different acetalization will not have a greater impact on the above-mentioned natural biomimetic advantages of the decellularized scaffold due to its low elastic modulus and good biodegradability. influence, and can achieve the purpose of coating the decellularized scaffold and improving its blood compatibility. After polyvinyl alcohol acetal modification, it not only maintains the characteristics of polyvinyl alcohol hydrogel, but also effectively improves its hydrolysis resistance. As a highly bionic artificial blood vessel material modified heparin solution, it can effectively improve its anticoagulant performance and biodescriptability

(4) The highly bionic artificial vascular material includes the use of acellular vascular scaffolds as material substrates. Compared with traditional synthetic vascular materials, the highly bionic artificial vascular material has better mechanical biomimetic properties and degradation rate matching. sex. In addition, the selected materials have the characteristics of wide sources, low price and large-scale production and application.

Description of the drawings

Figure 1 is an SEM image of the decellularized scaffold;

Figure 2 is a SEM image of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7) artificial blood vessel materials;

Figure 3 is the FT-IR pattern of O-HPCS and CS/PEG;

Figure 4 is the XPS comparison chart of CS/PEG/DCS and 5-He-CS/PEG/DCS;

Figure 5 is the acid orange standard curve;

Figure 6 is a histogram of the amino content of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7) artificial blood vessel materials;

Figure 7 is a static contact angle diagram of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7);

Figure 8 is a graph showing the tensile strength, elongation at break and bursting strength of fresh blood vessels, DCS, CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7) artificial blood vessel materials;

Figure 9 is a graph of activated partial thromboplastin time, thrombin time, prothrombin time and recalcification coagulation time of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7) artificial blood vessel materials. ;

Figure 10 is a morphology diagram of red blood cells of n-He-CS/PEG/DCS (n=3~7);

Figure 11 is the MTT test chart of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7);

Figure 12 is a graph of the in vitro degradation rate of CS/PEG/DCS and 5-He-CS/PEG/DCS artificial blood vessel materials;

Figure 13 is an electron microscope image of polyvinyl alcohol hydrogel PVA _n (n=1~6) with different acetalization;

Figure 14 is the infrared spectrum of heparin-polyvinyl alcohol/decellularized scaffold He/PVA ₄ /DCS;

Figure 15 is an SEM image of heparin-polyvinyl alcohol/decellularized scaffold He/PVA _n /DCS (n=1~6) with different degrees of acetalization;

Figure 16 is the XPS comparison chart of PVA/DCS and He/PVA ₄ /DCS;

Figure 17 is a graph of the tensile strength, elongation at break and bursting strength of fresh blood vessels, DCS and He/PVA _n /DCS (n=1~6);

Figure 18 is a graph showing the activated partial thromboplastin time, thrombin time, prothrombin time and recalcified coagulation time of PVA/DCS and He/PVA ₄ /DCS (n=1~6) artificial blood vessel materials;

Figure 19 is a morphology diagram of red blood cells of He/PVA _n /DCS (n=1~6);

Figure 20 is the MTT test chart of He/PVA _n /DCS (n=1~6);

Figure 21 is a graph of the in vitro degradation rate of PVA/DCS and He/PVA ₄ /DCS artificial blood vessel materials;

Figure 22 is the B-ultrasound image of 5-He-CS/PEG/DCS and He/PVA ₄ /DCS artificial blood vessel materials two weeks after they were implanted in the body;

Figure 23 is a CTA image of the implantation site after 5 months of implantation of 5-He-CS/PEG/DCS and He/PVA ₄ /DCS artificial blood vessel materials into the body;

Figure 24 is a stress-strain curve graph of fresh blood vessels, He-Ch-5/PU/DCS, He/PVA ₄ /DCS and 5-He-CS/PEG/DCS.

Detailed ways

The technical solution of the present invention will be further described below with reference to the accompanying drawings.

Example 1 Preparation of three-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold (3-He-CS/PEG/DCS)

1. Preparation of decellularized scaffold (DCS)

The canine bilateral carotid arteries placed in 4% paraformaldehyde tissue fixative (provided by the Vascular Surgery Department of Nanjing Gulou Hospital) were removed, trimmed and immersed in 0.9% normal saline. Take 0.5g of sodium lauryl sulfate and 1g of polyethylene glycol octylphenyl ether, dissolve them in 100mL of deionized water, and prepare a mixed solution with a concentration of 1.5%. Trim the bilateral carotid arteries of the dog. Soak in the mixed solution for 48h. The bilateral carotid arteries of the dogs after soaking were removed, washed with PBS buffer solution, and soaked in PBS buffer solution for 30 days, changing the solution twice a day. Take out the soaked canine bilateral carotid arteries, trim them into pieces, and freeze-dry them in a freeze-drying machine at -55°C for 12 hours to obtain a decellularized scaffold, which is recorded as DCS.

2. Preparation of chitosan/polyethylene glycol hydrogel

Weigh 1.5g of NaOH and dissolve it in water, prepare it into a 2.0mol/L solution, then add 5mL of absolute ethanol, pour the 2.0mol/L NaOH solution into a three-necked flask, and add 2g of chitosan while stirring , let it alkalize for 8h. Add 2g of propylene oxide into the above three-necked flask and place it in a 50°C constant temperature water bath for reaction for 36 hours. Take out the product, wash it 5 times in a mixture of hydrochloric acid and acetone with a mass ratio of 1:9, then wash it 5 times in a mixture of acetone and water with a mass ratio of 9:1, and use a vacuum pump After suction filtration, place it in a 55°C vacuum drying oven for 2 hours, then take it out to obtain O-hydroxypropyl chitosan (O-HPCS). Take 1.5g of O-HPCS and place it in 100mL of deionized water to form a solution, add 0.5g of polyethylene glycol (PEG) and 5mL of 2% glutaraldehyde solution, stir for 15min to mix thoroughly, and let stand to obtain a shell. Polysaccharide/polyethylene glycol hydrogel, denoted as CS/PEG.

3. Preparation of chitosan/polyethylene glycol hydrogel/acellular scaffold

Place the prepared DCS in the PBS buffer solution for 30 minutes and then take it out. Apply the chitosan/polyethylene glycol hydrogel on the surface of the decellularized scaffold and ensure that the surface is evenly applied three times. Take it out and place it in a vacuum drying oven at 50°C to dry until it is completely dehydrated and then take it out. The chitosan/polyethylene glycol hydrogel/decellularized scaffold was obtained, denoted as CS/PEG/DCS.

4. Use layer-by-layer self-assembly technology to accumulate heparin on the surface of chitosan/polyethylene glycol/acellular scaffold to produce a highly bionic artificial blood vessel material.

1) Prepare 2g/L heparin sodium solution, chitosan solution and 0.01mol/LPBS buffer solution respectively. Place the prepared CS/PEG/DCS in the PBS buffer solution for 30 minutes, then take it out and soak it in the heparin sodium solution. for 15 minutes, take out and rinse three times with PBS buffer solution on the front and back to remove the heparin sodium adhered to the surface due to physical adsorption, and obtain a layer of heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, recorded as 1- He-CS/PEG/DCS.

2) Soak the prepared 1-He-CS/PEG/DCS in the prepared chitosan solution for 15 minutes, take out and rinse the front and back three times with PBS buffer solution to remove the chitosan adhering to the surface due to physical adsorption. Then soak it in heparin sodium solution for 15 minutes, take it out and rinse it three times with PBS buffer solution on the front and back to remove the heparin sodium adhering to the surface due to physical adsorption, and obtain a two-layer heparin-chitosan/polyethylene glycol hydrogel. /Decellularized scaffold, recorded as 2-He-CS/PEG/DCS, repeat the above steps once more to prepare 3-He-CS/PEG/DCS.

Example 2 Preparation of three-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold (3-He-CS/PEG/DCS)

1. Preparation of decellularized scaffold (DCS)

The canine bilateral carotid arteries placed in 4% paraformaldehyde tissue fixative were removed, the adventitia was trimmed, and the arteries were immersed in 0.9% normal saline. Take 0.5g of sodium lauryl sulfate and 0.5g of polyethylene glycol octylphenyl ether, dissolve them in 100mL of deionized water, and prepare a mixed solution with a concentration of 1%. Cut the trimmed bilateral carotid arteries of the dog. Soak in the mixed solution for 24h. The bilateral carotid arteries of the dogs after soaking were removed, rinsed with PBS buffer solution, and soaked in PBS buffer solution for 20 days, with the solution changed once a day. The soaked canine bilateral carotid arteries were taken out, trimmed into pieces, and freeze-dried at -45°C for 8 hours in a freeze-drying machine to obtain a decellularized scaffold, which was recorded as DCS.

2. Preparation of chitosan/polyethylene glycol hydrogel

Weigh 1.0g of NaOH and dissolve it in water, prepare it into a 1.5mol/L solution, then add 5mL of absolute ethanol, pour the 1.5mol/L NaOH solution into a three-necked flask, and add 2g of chitosan while stirring , let it alkalize for 6h. Add 3g of propylene oxide into the above three-necked flask and place it in a 50°C constant temperature water bath for reaction for 24 hours. Take out the product, wash it 5 times in a mixture of hydrochloric acid and acetone with a mass ratio of 1:10, then wash it 5 times in a mixture of acetone and water with a mass ratio of 10:1, and use a vacuum pump After suction filtration, place it in a 45°C vacuum drying oven for 3 hours, then take it out to obtain O-hydroxypropyl chitosan (O-HPCS). Take 1.5g of O-HPCS and place it in 100mL of deionized water to form a solution, add 1.0g of polyethylene glycol (PEG) and 5mL of 2% glutaraldehyde solution, stir for 15min to mix thoroughly, and let stand to obtain a shell. Polysaccharide/polyethylene glycol hydrogel, denoted as CS/PEG.

3. Preparation of chitosan/polyethylene glycol hydrogel/acellular scaffold

Place the prepared DCS in the PBS buffer solution for 30 minutes and then take it out. Apply the chitosan/polyethylene glycol hydrogel on the surface of the decellularized scaffold and ensure that the surface is evenly applied three times. Take it out and place it in a vacuum drying oven at 50°C to dry until it is completely dehydrated and then take it out. The chitosan/polyethylene glycol hydrogel/decellularized scaffold was obtained, denoted as CS/PEG/DCS.

4. Use layer-by-layer self-assembly technology to accumulate heparin on the surface of chitosan/polyethylene glycol/acellular scaffold to produce a highly bionic artificial blood vessel material.

1) Prepare 1g/L heparin, 2g/L chitosan solution and 0.01mol/LPBS buffer solution respectively. Place the prepared CS/PEG/DCS in the PBS buffer solution for 10 minutes, then take it out and soak it in heparin. After 10 minutes in the sodium solution, take it out and rinse it with PBS buffer solution three times on the front and back to remove the heparin sodium adhered to the surface due to physical adsorption, and obtain a layer of heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, which is recorded as 1-He-CS/PEG/DCS.

2) Soak the prepared 1-He-CS/PEG/DCS in the prepared chitosan solution for 15 minutes, take out and rinse the front and back three times with PBS buffer solution to remove the chitosan adhering to the surface due to physical adsorption. Then soak it in heparin sodium solution for 15 minutes, take it out and rinse it three times with PBS buffer solution on the front and back to remove the heparin sodium adhering to the surface due to physical adsorption, and obtain a two-layer heparin-chitosan/polyethylene glycol hydrogel. /Decellularized scaffold, recorded as 2-He-CS/PEG/DCS, repeat the above steps once more to prepare 3-He-CS/PEG/DCS.

Example 3 Preparation of four-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold (4-He-CS/PEG/DCS)

1. Preparation of decellularized scaffold (DCS)

The canine bilateral carotid arteries placed in tissue fixative were removed, the adventitia was trimmed, and the arteries were immersed in 0.9% normal saline. Take 0.5g of sodium lauryl sulfate and 1.0g of polyethylene glycol octylphenyl ether, dissolve them in 100mL of deionized water, and prepare a mixed solution with a concentration of 1.5%. Soak in the mixed solution for 24h. The bilateral carotid arteries of the dogs after soaking were removed, washed with PBS buffer solution, and soaked in PBS buffer solution for 30 days, and the solution was changed twice a day. The soaked canine bilateral carotid arteries were taken out, trimmed into pieces, and freeze-dried at -55°C for 8 hours in a freeze-drying machine to obtain a decellularized scaffold, which was recorded as DCS.

The preparation of chitosan/polyethylene glycol hydrogel and the preparation of chitosan/polyethylene glycol hydrogel/decellularized scaffold are the same as in Example 1.

2. Layer-by-layer self-assembly technology deposits heparin on the surface of chitosan/polyethylene glycol/acellular scaffold to produce highly bionic artificial blood vessel materials.

1) Prepare 2g/L heparin, 2g/L chitosan solution and 0.01mol/LPBS buffer solution respectively. Place the prepared CS/PEG/DCS in the PBS buffer solution for 30 minutes, then take it out and soak it in heparin. After 15 minutes in the sodium solution, take it out and rinse it with PBS buffer solution three times on the front and back to remove the heparin sodium adhered to the surface due to physical adsorption, and obtain a layer of heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, which is recorded as 1-He-CS/PEG/DCS.

2) Soak the prepared 1-He-CS/PEG/DCS in the prepared chitosan solution for 15 minutes, take out and rinse the front and back three times with PBS buffer solution to remove the chitosan adhering to the surface due to physical adsorption. Then soak it in heparin sodium solution for 15 minutes, take it out and rinse it three times with PBS buffer solution on the front and back to remove the heparin sodium adhering to the surface due to physical adsorption, and obtain a two-layer heparin-chitosan/polyethylene glycol hydrogel. /decellularized scaffold, recorded as 2-He-CS/PEG/DCS, and repeat the above steps two times to prepare 4-He-CS/PEG/DCS.

Example 4 Preparation of four-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold (4-He-CS/PEG/DCS)

1. The preparation methods of decellularized scaffold (DCS), chitosan/polyethylene glycol hydrogel and chitosan/polyethylene glycol hydrogel/decellularized scaffold are the same as in Example 2.

2. Use layer-by-layer self-assembly technology to deposit heparin on the surface of chitosan/polyethylene glycol/acellular scaffold to produce a highly bionic artificial blood vessel material

1) Prepare 1g/L heparin, 2g/L chitosan solution and 0.01mol/LPBS buffer solution respectively. Place the prepared CS/PEG/DCS in the PBS buffer solution for 30 minutes, then take it out and soak it in heparin. After 15 minutes in the sodium solution, take it out and rinse it with PBS buffer solution three times on the front and back to remove the heparin sodium adhered to the surface due to physical adsorption, and obtain a layer of heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, which is recorded as 1-He-CS/PEG/DCS.

2) Soak the prepared 1-He-CS/PEG/DCS in the prepared chitosan solution for 10 minutes, take out and rinse the front and back three times with PBS buffer solution to remove the chitosan adhering to the surface due to physical adsorption. Then soak it in heparin sodium solution for 10 minutes, take it out and rinse it three times with PBS buffer solution on the front and back to remove the heparin sodium adhering to the surface due to physical adsorption, and obtain a two-layer heparin-chitosan/polyethylene glycol hydrogel. /decellularized scaffold, recorded as 2-He-CS/PEG/DCS, and repeat the above steps two times to prepare 4-He-CS/PEG/DCS.

Example 5 Preparation of five-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold (5-He-CS/PEG/DCS)

1. The preparation methods of decellularized scaffold (DCS), chitosan/polyethylene glycol hydrogel and chitosan/polyethylene glycol hydrogel/decellularized scaffold are the same as in Example 1.

2. Use layer-by-layer self-assembly technology to deposit heparin on the surface of chitosan/polyethylene glycol/acellular scaffold to produce a highly bionic artificial blood vessel material

1) Prepare 2g/L heparin, 2g/L chitosan solution and 0.01mol/LPBS buffer solution respectively. Place the prepared CS/PEG/DCS in the PBS buffer solution for 30 minutes, then take it out and soak it in heparin. After 15 minutes in the sodium solution, take it out and rinse it with PBS buffer solution three times on the front and back to remove the heparin sodium adhered to the surface due to physical adsorption, and obtain a layer of heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, which is recorded as 1-He-CS/PEG/DCS.

2) Soak the prepared He-CS/PEG/DCS in the prepared chitosan solution for 15 minutes, take out and rinse the front and back three times with PBS buffer solution to remove the chitosan adhered to the surface due to physical adsorption, and then Soak it in heparin sodium solution for 15 minutes, take it out and rinse it three times on the front and back with PBS buffer solution to remove the heparin sodium adhered to the surface due to physical adsorption, and obtain a two-layer heparin-chitosan/polyethylene glycol hydrogel/dehydration gel. The cell scaffold was recorded as 2-He-CS/PEG/DCS, and the above steps were repeated three times to prepare 5-He-CS/PEG/DCS.

Example 6 Preparation of five-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold (5-He-CS/PEG/DCS)

1. The preparation methods of decellularized scaffold (DCS), chitosan/polyethylene glycol hydrogel and chitosan/polyethylene glycol hydrogel/decellularized scaffold are the same as in Example 3.

2. Use layer-by-layer self-assembly technology to deposit heparin on the surface of chitosan/polyethylene glycol/acellular scaffold to produce a highly bionic artificial blood vessel material

1) Prepare 1g/L heparin, 1g/L chitosan solution and 0.01mol/L PBS buffer solution respectively. Place the prepared CS/PEG/DCS in the PBS buffer solution for 15 minutes, then take it out and soak it. In the heparin sodium solution for 15 minutes, take out and rinse the front and back three times with PBS buffer solution to remove the heparin sodium adhered to the surface due to physical adsorption, and obtain a layer of heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold. Denoted as 1-He-CS/PEG/DCS.

2) Soak the prepared 1-He-CS/PEG/DCS in the prepared chitosan solution for 10 minutes, take out and rinse the front and back three times with PBS buffer solution to remove the chitosan adhering to the surface due to physical adsorption. Then soak it in heparin sodium solution for 10 minutes, take it out and rinse it three times with PBS buffer solution on the front and back to remove the heparin sodium adhering to the surface due to physical adsorption, and obtain a two-layer heparin-chitosan/polyethylene glycol hydrogel. /Decellularized scaffold, recorded as 2-He-CS/PEG/DCS, repeat the above steps three times to prepare 5-He-CS/PEG/DCS.

Example 7 Preparation of six-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold (6-He-CS/PEG/DCS)

1. The preparation methods of decellularized scaffold (DCS), chitosan/polyethylene glycol hydrogel and chitosan/polyethylene glycol hydrogel/decellularized scaffold are the same as in Example 1.

2. Use layer-by-layer self-assembly technology to deposit heparin on the surface of chitosan/polyethylene glycol/acellular scaffold to produce a highly bionic artificial blood vessel material

1) Prepare 2g/L heparin, 2g/L chitosan solution and 0.01mol/LPBS buffer solution respectively. Place the prepared CS/PEG/DCS in the PBS buffer solution for 30 minutes, then take it out and soak it in heparin. After 15 minutes in the sodium solution, take it out and rinse it with PBS buffer solution three times on the front and back to remove the heparin sodium adhered to the surface due to physical adsorption, and obtain a layer of heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, which is recorded as 1-He-CS/PEG/DCS.

2) Soak the prepared 1-He-CS/PEG/DCS in the prepared chitosan solution for 15 minutes, take out and rinse the front and back three times with PBS buffer solution to remove the chitosan adhering to the surface due to physical adsorption. Then soak it in heparin sodium solution for 15 minutes, take it out and rinse it three times with PBS buffer solution on the front and back to remove the heparin sodium adhering to the surface due to physical adsorption, and obtain a two-layer heparin-chitosan/polyethylene glycol hydrogel. /decellularized scaffold, recorded as 2-He-CS/PEG/DCS, and repeat the above steps 4 times to prepare 6-He-CS/PEG/DCS.

Example 8 Preparation of six-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold (6-He-CS/PEG/DCS)

1. The preparation methods of decellularized scaffold (DCS), chitosan/polyethylene glycol hydrogel and chitosan/polyethylene glycol hydrogel/decellularized scaffold are the same as in Example 2.

2. Use layer-by-layer self-assembly technology to deposit heparin on the surface of chitosan/polyethylene glycol/acellular scaffold to produce a highly bionic artificial blood vessel material

1) Prepare 2g/L heparin, 1g/L chitosan solution and 0.01mol/LPBS buffer solution respectively. Place the prepared CS/PEG/DCS in the PBS buffer solution for 30 minutes, then take it out and soak it in heparin. After 15 minutes in the sodium solution, take it out and rinse it with PBS buffer solution three times on the front and back to remove the heparin sodium adhered to the surface due to physical adsorption, and obtain a layer of heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, which is recorded as 1-He-CS/PEG/DCS.

2) Soak the prepared 1-He-CS/PEG/DCS in the prepared chitosan solution for 10 minutes, take out and rinse the front and back three times with PBS buffer solution to remove the chitosan adhering to the surface due to physical adsorption. Then soak it in heparin sodium solution for 10 minutes, take it out and rinse it three times with PBS buffer solution on the front and back to remove the heparin sodium adhering to the surface due to physical adsorption, and obtain a two-layer heparin-chitosan/polyethylene glycol hydrogel. /decellularized scaffold, recorded as 2-He-CS/PEG/DCS, and repeat the above steps 4 times to prepare 6-He-CS/PEG/DCS.

Example 9 Preparation of seven-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold (7-He-CS/PEG/DCS)

1. The preparation methods of decellularized scaffold (DCS), chitosan/polyethylene glycol hydrogel and chitosan/polyethylene glycol hydrogel/decellularized scaffold are the same as in Example 1.

2. Use layer-by-layer self-assembly technology to deposit heparin on the surface of chitosan/polyethylene glycol/acellular scaffold to produce a highly bionic artificial blood vessel material

1) Prepare 2g/L heparin, 2g/L chitosan solution and 0.01mol/LPBS buffer solution respectively. Place the prepared CS/PEG/DCS in the PBS buffer solution for 10 minutes, then take it out and soak it in heparin. After 15 minutes in the sodium solution, take it out and rinse it with PBS buffer solution three times on the front and back to remove the heparin sodium adhered to the surface due to physical adsorption, and obtain a layer of heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, which is recorded as 1-He-CS/PEG/DCS.

2) Soak the prepared 1-He-CS/PEG/DCS in the prepared chitosan solution for 15 minutes, take out and rinse the front and back three times with PBS buffer solution to remove the chitosan adhering to the surface due to physical adsorption. Then soak it in heparin sodium solution for 15 minutes, take it out and rinse it three times with PBS buffer solution on the front and back to remove the heparin sodium adhering to the surface due to physical adsorption, and obtain a two-layer heparin-chitosan/polyethylene glycol hydrogel. /decellularized scaffold, recorded as 2-He-CS/PEG/DCS, and repeat the above steps 5 times to prepare 7-He-CS/PEG/DCS.

Example 10 Preparation of seven-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold (7-He-CS/PEG/DCS)

1. The preparation methods of decellularized scaffold (DCS), chitosan/polyethylene glycol hydrogel and chitosan/polyethylene glycol hydrogel/decellularized scaffold are the same as in Example 2.

2. Use layer-by-layer self-assembly technology to deposit heparin on the surface of chitosan/polyethylene glycol/acellular scaffold to produce a highly bionic artificial blood vessel material

1) Prepare 1g/L heparin, 1g/L chitosan solution and 0.01mol/LPBS buffer solution respectively. Place the prepared CS/PEG/DCS in the PBS buffer solution for 10 minutes, then take it out and soak it in heparin. After 10 minutes in the sodium solution, take it out and rinse it with PBS buffer solution three times on the front and back to remove the heparin sodium adhered to the surface due to physical adsorption, and obtain a layer of heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, which is recorded as 1-He-CS/PEG/DCS.

2) Soak the prepared 1-He-CS/PEG/DCS in the prepared chitosan solution for 10 minutes, take out and rinse the front and back three times with PBS buffer solution to remove the chitosan adhering to the surface due to physical adsorption. Then soak it in heparin sodium solution for 10 minutes, take it out and rinse it three times with PBS buffer solution on the front and back to remove the heparin sodium adhering to the surface due to physical adsorption, and obtain a two-layer heparin-chitosan/polyethylene glycol hydrogel. /decellularized scaffold, recorded as 2-He-CS/PEG/DCS, and repeat the above steps 5 times to prepare 7-He-CS/PEG/DCS.

Example 11 SEM characterization of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7) artificial blood vessel materials

1. Observe the morphology of the decellularized scaffold prepared in Example 1 using a JSM-7600F scanning electron microscope. The test results are shown in Figure 1. Figure 1 is an SEM image of the decellularized scaffold. It can be seen from Figure 1 that the decellularized scaffold has been treated with the anionic surfactant sodium dodecyl sulfate and the nonionic surfactant polyethylene glycol octylphenyl ether. The cell scaffold (DCS) showed an obvious three-dimensional network structure, with no cells on the surface and only a small amount of cell debris, indicating that the mixed solution of polyethylene glycol octylphenyl ether and sodium dodecyl sulfate has good removal ability. Cellular effects.

2. CS/PEG/DCS prepared in Example 1 and n-He-CS/PEG/DCS prepared in Example 1, Example 3, Example 5, Example 7 and Example 9 (n=3~7 ) Cut the artificial blood vessel material into strips with a length of 5mm and a width of 1mm. Use conductive adhesive to paste it on a copper table. Spray gold 6 times on each front and side. Use a JSM-7600F scanning electron microscope to observe its appearance. The test results are shown in Figure 2. Show. Figure 2 is the SEM image of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7) artificial blood vessel materials at 2000 times, where a is CS/PEG/DCS and b is 3-He- CS/PEG/DCS, c is 4-He-CS/PEG/DCS, d is 5-He-CS/PEG/DCS, e is 6-He-CS/PEG/DCS, f is 7-He-CS/ PEG/DCS. As can be seen from Figure 2, there is a small amount of granular material on the surface of a, which shows that chitosan has been modified onto the DCS together with the PEG hydrogel, and with the increase of the polyelectrolyte layer, b, c, d, The number of granular substances on e and f is increasing, which means that heparin and chitosan have been adsorbed to CS/PEG/DCS layer by layer through electrostatic adsorption. The above experimental results can prove that n-He- CS/PEG/DCS (n=3~7) has been successfully prepared.

Example 12 FT-IR infrared analysis of O-hydroxypropyl chitosan (O-HPCS) and chitosan/polyethylene glycol hydrogel (CS/PEG) prepared in Example 1

The O-HPCS and CS/PEG prepared in Example 1 were tested for infrared spectrum using the Nexus670 infrared spectrometer of Nicolet Company of the United States. The results are shown in Figure 3. Figure 3 shows the FTIR images of O-HPCS and CS/PEG. It can be found from Figure 3 that the analysis results show that the strong absorption peaks at 3400 cm ^-1 in the FTIR images of O-HPCS and the FTIR images of CS/PEG are after modification. The -OH peak on chitosan is the stretching vibration peak of -CH ₃ and -CH ₂ at 2900cm ^-1 . The absorption peak at 1375cm ^-1 belongs to the bending vibration of the OH bond. In the FTIR chart of O-HPCS, it is 1065cm ^-1 and 589cm ^-1 are the crystallization sensitive peaks of O-HPCS, and in the FTIR diagram of CS/PEG, the intensity of the two crystallization peaks has attenuated, and the position of the crystallization peak has shifted, which shows that polyethylene The addition of diol destroyed the crystal structure of O-HPCS. Therefore, we can prove that CS/PEG has been successfully synthesized through the FTIR diagram in Figure 3.

Example 13 X-ray photoelectron spectroscopy analysis of CS/PEG/DCS prepared in Example 1 and 5-He-CS/PEG/DCS prepared in Example 5

The CS/PEG/DCS prepared in Example 1 and the 5-He-CS/PEG/DCS prepared in Example 5 were analyzed using a Rigaku D/max 2500VL/PC X-ray photoelectron spectrometer under monochromatic Al Kα The X-ray photoelectron spectroscopy test was carried out under the conditions of passing through rays (150W, 500μm beam spot) and 20eV energy. The results are shown in Figure 4. Figure 4 is the XPS comparison chart of CS/PEG/DCS and 5-He-CS/PEG/DCS, where a is CS/PEG/DCS and b is 5-He-CS/PEG/DCS. As can be seen from Figure 4, the S _2p peak appearing on b indicates that there is S element on 5-He-CS/PEG/DCS. This is because the heparin on the 5-He-CS/PEG/DCS artificial blood vessel material has S element. Therefore, it can be proved that the 5-He-CS/PEG/DCS artificial blood vessel material has been successfully prepared.

Example 14 Determination of surface chitosan amino content of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7) artificial blood vessel materials

1. Acid orange is a golden yellow powdery solid that is soluble in water and appears orange. Acid orange containing a single sulfonic acid group can have equimolar adsorption with the amino groups on the surface of the material under the condition of pH=2~3, thus The amino content on the sample surface can be calculated. Figure 5 is an acid orange standard curve. By preparing an acid orange standard solution, the acid orange standard equation is obtained. The equation is Y=1.39048X+0.03774 (R ² =0.99411). As shown in Figure 5, since polyethylene glycol and heparin do not contain primary amines and chitosan contains primary amines, acid orange can be used to measure the amino content in the sample to determine the quality of chitosan in artificial blood vessel materials. content.

2. The specific experimental steps are as follows:

1) Standard curve drawing: In a 24-well plate, add 150 μL of acid orange solution with a concentration of 5×10 ^-4 mol/L and pH=12 to the first and second wells, and then add 150 μL to the second well. Dilute the solution in the second well with an equal volume of pH=12 solution. Take 150 μL of the solution from the second well and add it to the third well, and add an equal volume of 150 μL of deionized water solution with pH=12 to dilute the solution in the third well. Dilute step by step until the seventh well. Directly add 50 μL of pH=12 deionized water solution to the eighth air. Take 3 sets of parallel samples at the same time and take the average value. Use BioTek Synergy2 microplate reader to measure the absorbance at 485nm and draw the standard curve required for the experiment. The specific experimental results are shown in Figure 5.

2) Immerse CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7) artificial blood vessel materials in an acid orange solution with pH=3 and a concentration of 500 μmol/L. Place it on a shaker to allow it to fully react for 12 hours, then take it out. Use a pH=3 solution to wash the sample repeatedly for more than 5 times, and then dry it fully. Immerse the obtained sample into a solution with pH=12 and place it on a shaker to allow it to fully react for 30 minutes to release the methyl orange adsorbed on the surface of the sample by charge. At the same time, a methyl orange standard curve was obtained to obtain the adsorption content of methyl orange on the surface. The specific results are shown in Figure 6. Figure 6 is a histogram of the amino content of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7) artificial blood vessel materials. It can be seen from Figure 6 that due to the existence of shell on the surface of CS-PEG hydrogel glycan, so there are a certain number of amino groups in CS/PEG/DCS, which means that CS-PEG has been successfully modified on the surface of DCS. As the number of polyelectrolyte layers increases, the amino content on the surface of the artificial blood vessel material shows an increasing trend. This is because in LbL technology, heparin and chitosan have a cross-penetrating structure, so the surface of the artificial blood vessel material It shows a trend towards higher and higher amino content. The above experimental results can prove that chitosan has been successfully modified on the material surface. That is, n-He-CS/PEG/DCS (n=3~7) artificial blood vessel materials have been successfully prepared.

Example 15 Static contact angle measurement of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7)

1. Specific experiments: Cut the prepared artificial blood vessel materials of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7) into discs with a diameter of 1cm, using the DSA100 model of Krüss Company Optical contact angle tester uses deionized water as the test liquid and the droplet size is about 90 μm to conduct static contact angle tests on each sample. Each sample was tested three times and three parallel samples were taken.

2. Result analysis: Figure 7 is the static contact angle diagram of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7), where a is CS/PEG/DCS and b is 3- He-CS/PEG/DCS, c is 4-He-CS/PEG/DCS, d is 5-He-CS/PEG/DCS, e is 6-He-CS/PEG/DCS, f is 7-He- CS/PEG/DCS. As can be seen from Figure 7, the static contact angle of CS/PEG/DCS is the smallest. This is because the CS grafted on PEG is chitosan alkalized with NaOH, causing -COOH to become -COONa, which will dissolve in water. is converted into more polar -COO ^- , so its static water contact angle will be significantly reduced; and the n-He-CS/PEG/DCS (n=3~7) artificial blood vessel material will appear with the increase of the polyelectrolyte layer. The trend is to decrease first and then increase. The static contact angle is the lowest when the polyelectrolyte membrane reaches 5 layers. When the polyelectrolyte membrane has more than 5 layers, the static contact angle increases slightly and the surface hydrophilicity increases slightly, but the overall change is not significant. Overall All have hydrophilic surfaces. 5-He-CS/PEG/DCS has the smallest contact angle and the strongest surface hydrophilicity, achieving the best hydrophilic effect. Materials with strong hydrophilicity have better anticoagulant properties due to the formation of a water layer on the interface, which is beneficial to the removal of biological substances.

Example 16 Tensile properties and burst strength testing of fresh blood vessels, DCS, CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7) artificial blood vessel materials

1. Tensile performance test: Use a microtome to cut fresh blood vessels, DCS, CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7) artificial blood vessel materials into 3mm wide and 20mm span dumbbell shaped test strips. The tensile properties of each sample were tested using a universal electronic tensile machine (INSTRON 4200 model) at room temperature with a tensile speed of 20 mm/min. Each sample was tested three times and three parallel samples were taken. The results are shown in a and b in Figure 8.

2. Bursting strength test: Fresh blood vessels, DCS, CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7) artificial blood vessel materials are used to prepare cylindrical blood vessels with a diameter of 30mm and a diameter of 5mm. , ligate one section, and connect the other end to the tee. The two ends of the tee are connected to the pressurized injection device and the pressure testing device respectively. Continuously inject 37°C physiological saline into the sample at an injection rate of 3mL/min. Record when the sample ruptures. The down pressure value is the bursting strength. Each sample was tested three times and three parallel samples were taken. The results are shown in c in Figure 8 below.

3. Result analysis: Figure 8 shows the tensile strength, elongation at break and bursting strength of fresh blood vessels, DCS, CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7) artificial blood vessel materials. picture. Among them, a is the tensile strength diagram of fresh blood vessels, DCS, CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7) artificial blood vessel materials, and b is the tensile strength diagram of fresh blood vessels, DCS, CS/PEG Elongation at break diagram of /DCS and n-He-CS/PEG/DCS (n=3~7) artificial blood vessel materials, c is fresh blood vessel, DCS, CS/PEG/DCS and n-He-CS/PEG/ Bursting strength diagram of DCS (n=3~7) artificial blood vessel materials. From a and b in Figure 8, we can see that compared with fresh blood vessels, the tensile strength of DCS will increase and the elongation at break will decrease slightly, but there is almost no significant difference between the two. , and for DCS modified by hydrogel and polyelectrolyte, as the number of polyelectrolyte layers increases, the tensile strength shows a gradually increasing trend, but it is still close to the tensile strength of natural blood vessels, while the elongation at break is 5-He-CS/PEG/DCS showed the best trend and was closest to fresh blood vessels. The above test results showed that after DCS was modified by heparin, chitosan and polyethylene glycol hydrogel, it maintained The mechanical bionic properties of DCS enable the prepared bionic artificial blood vessel materials to meet the pressure requirements generated when blood flows in the lumen of blood vessels. From c in Figure 8, it can be found that the bursting strength of fresh arterial blood vessels is around 110KPa, while the bursting strength of DCS is 0KPa. This is because DCS removes the surface tissue cells and forms a spatial network structure. The physiological saline will directly pass through the network. like structure because DCS does not have bursting strength. DCS modified by polyelectrolyte and PEG hydrogel embeds the spatial network structure of DCS, and its burst strength will show an upward trend as the polyelectrolyte layer increases, but each sample is consistent with fresh arteries. The bursting strength of blood vessels is basically similar. The above test results show that after modifying DCS with heparin, chitosan and polyethylene glycol hydrogel, the material basically maintains the mechanical bionic properties of DCS, so that the prepared bionic artificial vascular material can meet the needs of blood flow in the vascular tube. The pressure requirements generated during flow in the cavity.

Example 17: Conducting in vitro coagulation experiments and recalcification experiments on the prepared artificial blood vessel materials

1. In vitro coagulation test: APTT (activated partial thromboplastin time): Take the sample to be tested and mix it thoroughly with plasma (0.1 mL), pour it into a coagulation cup, incubate at 37°C for 5 minutes, add APTT reagent, continue to incubate for 5 minutes, and then Add the same amount of calcium chloride solution (0.1 mL, 0.025 mol) to each coagulation cup, observe the blood coagulation time at 37°C, and record the recorded coagulation time as the APTT value. Each sample was tested three times, and the results were averaged. TT (thrombin time): Take platelet-poor plasma and mix it with the sample evenly, pour it into a coagulation cup, place it in a constant temperature (37°C) water bath to preheat for 5 minutes, then add TT of normal reference plasma, add 0.1 mL of thrombin, and The onset of turbidity was used as the starting point, and the blood coagulation time was recorded. Repeat 3 times and take the average value. PT (prothrombin time): Mix the sample to be tested and plasma thoroughly, pour it into a coagulation cup, incubate at 37°C for 5 minutes, add pre-warmed PT reagent, and record the coagulation time. Repeat 3 times and take the average value. CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7) artificial blood vessel materials were cut into discs with a diameter of about 1cm, mixed evenly with healthy rabbit blood plasma, and then used a microplate reader ( BioTeksynergy type 2) was used for in vitro coagulation time testing, and the results are shown in Figure 9. At the same time, a control group (0.1mL plasma + APTT/PT/TT reagent) was set up.

2. Recalcification experiment: Immerse the prepared CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7) artificial blood vessel materials in physiological saline (0.9%) for 24 hours, and then place them In a 96-well plate, cover the bottom of the well plate and keep at a constant temperature of 37°C for 0.5h. Add 100 μL of preheated platelet-poor plasma, and then add 0.025 mol/LCaCl ₂ solution to the 96-well plate. Use a microplate reader to measure the OD value at a wavelength of 405 nm. Repeat three times and take the average value. Control group: 100 μL CaCl ₂ solution + 100 μL plasma. The results of the recalcification coagulation time experiment are shown in Figure 9.

3. Result analysis: Figure 9 shows the activated partial thromboplastin time, thrombin time, prothrombin time and Recalcification coagulation time diagram, where a is the activated partial thromboplastin time diagram of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7) artificial vascular materials, and b is CS/PEG/DCS and n-He-CS/PEG/DCS (n=3～7) thrombin time diagram of artificial blood vessel materials, c is CS/PEG/DCS and n-He-CS/PEG/DCS (n=3～7) The prothrombin time chart of artificial blood vessel materials, d is the recalcification coagulation time chart of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7) artificial blood vessel materials. It can be seen from a to c in Figure 9 that compared with DCS modified by CS-PEG hydrogel, the coagulation time of APTT, TT, and PT of the artificial vascular material modified with the polyelectrolyte layer is prolonged to a certain extent. This is because when heparin and chitosan are electrostatically adsorbed on the surface of artificial blood vessel materials through layer-by-layer self-installation technology, the outermost layer of heparin can effectively improve blood compatibility and anticoagulant ability. We can also find that in 5- When using He-CS/PEG/DCS, the values of APTT, TT, and PT all reached the optimal values. This is because heparin and chitosan form an interpenetrating network structure. When the number of polyelectrolyte layers increases to a certain level, The surface heparin content will show a downward trend. From the above experimental results, it can be considered that when the number of polyelectrolyte layers is 5, the 5-He-CS/PEG/DCS artificial blood vessel material has the longest in vitro coagulation time and the best anticoagulant performance. It can be seen from d in Figure 9 that the T _1/2max recalcification time of artificial vascular materials modified with heparin and chitosan is significantly prolonged. This is because antithrombin III (AT III) can form an irreversible complex through the active center of thrombin in the form of a substrate, thereby inhibiting the activity of thrombin. In the case of adding heparin, It can increase the speed of the entire reaction by more than a thousand times, thereby effectively improving the blood compatibility of the material and causing the recalcification coagulation time to increase. The experimental results show that the use of polyelectrolyte membrane modification can effectively prolong the conversion time of soluble fibrinogen into soluble fibrin, thereby achieving the purpose of prolonging blood coagulation time. And it can also be seen from the above figure that when the number of polyelectrolyte layers is 5, the T _1/2max recalcification time of the artificial blood vessel material is the best, that is, when the number of polyelectrolyte layers is 5, the material has the longest recalcification time. Calcium time and optimal anticoagulant performance. This is because heparin and chitosan form an interpenetrating network structure. When the number of layers is less than 5, the surface heparin content shows an upward trend as the number of layers increases. When polyelectrolyte After the number of layers increases to more than 5, the surface heparin content will show a downward trend. That is, when n is 5, the surface heparin content is the highest, and the material has the longest recalcification time and the best anticoagulant performance.

Example 18n-He-CS/PEG/DCS (n=3～7) blood compatibility test

1. Specific experiments: The red blood cell morphology experiment is a method to test blood compatibility. The n-He-CS/PEG/DCS (n=3~7) prepared in Examples 1, 3, 5, 7, and 9 Cut into discs with a diameter of about 6 mm and place them in centrifuge tubes. Add 10 mL of physiological saline (0.9%) to each centrifuge tube. Take 5 mL of physiological saline and 4 mL of red blood cell suspension, mix them evenly, and dilute to obtain a diluted red blood cell suspension. Add 0.2 mL of the diluted red blood cell suspension into the above-mentioned centrifuge tubes and mix thoroughly, then place the centrifuge tubes in a _CO2 constant temperature incubator and incubate at constant temperature (37°C) for 1 hour, then take them out to prepare a red blood cell smear. Observe the morphology of red blood cells under a microscope. Take 3 parallel samples for each sample. At the same time, a blank control group was set: 10 mL 0.9% normal saline + 0.2 mL diluted red blood cell suspension. The experimental results are shown in Figure 10.

2. Result analysis: Figure 10 is the erythrocyte morphology diagram of n-He-CS/PEG/DCS (n=3~7), where a is the blank control group, b is 3-He-CS/PEG/DCS, c is 4-He-CS/PEG/DCS, d is 5-He-CS/PEG/DCS, e is 6-He-CS/PEG/DCS, and f is 7-He-CS/PEG/DCS. As can be seen from Figure 10, when red blood cells come into contact with the vascular material of the present invention, their morphology is consistent with that of the control group. The red blood cells are in good shape and no obvious deformation or rupture is found. The results show that 5-He-CS/PEG/DCS modified with heparin and chitosan has almost no toxic effect on red blood cells, that is, it has good blood compatibility.

Example 19 MTT toxicity test of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7) artificial blood vessel materials

1. Specific experiments: Sample preparation: Take n-He-CS/PEG/DCS (n=3~7) artificial blood vessel materials, add 10mL of leaching medium (10% fetal bovine serum) in a 37°C constant temperature shaking incubator Leaching for 24h. Preparation of MTT solution: Dissolve MTT in PBS to make an MTT solution with a concentration of 5 mg/mL. Cells in the logarithmic growth phase were digested with trypsin and diluted with cell culture medium to a concentration of 4×10 ⁴ cells/mL. Take a 96-well culture plate, inoculate 200 μL of cell suspension into each well, and place it in a 37°C, 5% _CO2 incubator for 24 hours. After the cells adhere to the wall, aspirate the original culture medium from each well, and add 200 μL of sample extraction solution to each well of the experimental group (8 wells for each group). At the same time, set a blank control group in the well plate without cells and add the cell culture medium. Continue to be cultured in a 37°C, 5% _CO2 incubator for 12h, 24h, and 48h; then add 20 μL of the prepared MTT solution to each well. After incubation for 5 hours, discard the original solution, add 150 μL of dimethyl sulfoxide to each well, and shake horizontally. After 5 min, use a microplate reader (BioTek synergy 2 type) to measure the absorbance value OD at a wavelength of 490 nm. Test each at least three times. The experiment of CS/PEG/DCS is the same as that of n-He-CS/PEG/DCS (n=3~7) artificial blood vessel material.

Experimental group: endothelial progenitor cells+n-He-CS/PEG/DCS (n=3~7)+MTT. Negative control group: endothelial progenitor cells + MTT. Blank control group: MTT.

Result calculation:

In the formula: Dt - the absorbance of the experimental group sample; Dnc - the absorbance of the negative control group sample; Db - the absorbance of the blank control group sample. The experimental results are shown in Figure 11.

2. Result analysis: Figure 11 shows the MTT test results of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7). It can be seen from Figure 11 that at 12 hours, the cell survival rate of CS/PEG/DCS was 97% and the cytotoxicity was level 1. At the same time, the cell viability of n-He-CS/PEG/DCS (n=3~7) The cell survival rate is between 98% and 101%, and the cytotoxicity is level 0 and level 1. Compared with CS/PEG/DCS, although n-He-CS/PEG/DCS has a higher cell survival rate, its cell The toxicity values are similar, and the differences in cell survival rates between different artificial blood vessel materials are small. This is because CS-PEG hydrogel has good biocompatibility and low cytotoxicity, which can ensure the normal survival of cells during short-term culture. As the culture time increased, at 24 hours, the cell survival rate of CS/PEG/DCS was 92%, and the cytotoxicity was level 1; while the cells of n-He-CS/PEG/DCS (n=3~7) The survival rate is 104%-114%, and its cytotoxicity is level 0. At 48 h, the difference in cytocompatibility between CS/PEG/DCS and n-He-CS/PEG/DCS (n=3~7) was greater. The above experimental results prove that although CS-PEG hydrogel has a certain degree of cell compatibility, surface modification of polyelectrolytes can significantly improve cell compatibility, and has a greater effect on improving cell compatibility over a longer period of time. obvious. Compare the 48h cell survival rate experimental results of samples with different polyelectrolyte layer numbers. It can be seen that when the number of polyelectrolyte layers is 3-5, the cell survival rate increases from 108% to 118%, showing an upward trend; when the number of polyelectrolyte layers is 7, the cell survival rate is 111%. Compared with the polyelectrolyte with 5 layers, there is a slight decrease, but the difference is small. The above experimental results show that when the number of polyelectrolyte layers is 5, the artificial blood vessel material has the best cytocompatibility.

Example 20 In vitro degradation experiment of CS/PEG/DCS and 5-He-CS/PEG/DCS artificial blood vessel materials

1. Specific experiments: From the above, it is believed that when the number of polyelectrolyte layers is 5, that is, the 5-He-CS/PEG/DCS artificial blood vessel material has the best comprehensive performance. In subsequent studies, 5-He-CS/PEG/DCS will be used as the research object for testing and research. Cut the CS/PEG/DCS and 5-He-CS/PEG/DCS artificial blood vessel materials into a square shape of 10mmx10mm, use an electronic balance to weigh the material as m ₀ , put it into a centrifuge tube, and add 20mL of PBS solution. Place it in a constant temperature shaking box at 37°C and incubate it for 1d, 7d, 14d, 30d, 60d, 90d, 120d, and 180d. Replace it with fresh PBS buffer every week. Take out the cultured artificial blood vessel material and put it into a vacuum Dry in a drying oven at 50°C and weigh the weight m ₁ with an electronic balance. Calculate the weight loss result: Weight loss (%) = (m ₀ -m ₁ )/m ₀ ×100%. The results are shown in Figure 12.

2. Result analysis: Figure 12 is the in vitro degradation rate chart of CS/PEG/DCS and 5-He-CS/PEG/DCS artificial blood vessel materials. It can be seen from Figure 12 that as time increases, CS/PEG/DCS and 5 -The in vitro degradation rate of He-CS/PEG/DCS shows a trend from fast to slow; at 180 days, the remaining mass of CS/PEG/DCS is 33.7%, while the remaining mass of 5-He-CS/PEG/DCS is 44.2%. The in vitro degradation cycle and blood vessel regeneration cycle between the two are basically matched, which shows that both have good degradation speed matching. As artificial blood vessel materials, they can achieve a degradation cycle and a good match with the regeneration cycle of new blood vessels to achieve a good bionic effect.

Example 21 Preparation of heparin-polyvinyl alcohol/decellularized scaffold He/PVA ₁ /DCS

1. Preparation of decellularized scaffolds

The canine bilateral carotid arteries placed in tissue fixative were removed, the adventitia was trimmed, and the arteries were immersed in 0.9% normal saline for cleaning. Take out the cleaned bilateral carotid arteries of the dog and soak them in a mixed solution of 1% polyethylene glycol octylphenyl ether and 0.5% sodium dodecyl sulfate at room temperature for 48 hours; The blood vessels were taken out, washed with 0.01mol/L PBS buffer solution, and soaked in PBS buffer solution for 30 days, with the solution changed twice a day. Take out the soaked canine bilateral carotid arteries, trim them into pieces, and freeze-dry them in a freeze-drying machine at -55°C for 12 hours to obtain a decellularized scaffold (DCS), which is recorded as DCS.

2. Preparation of acetalized polyvinyl alcohol hydrogel

In a 500mL three-neck flask, add 6g polyvinyl alcohol and 180mL water, heat to 90°C to dissolve; add 3g glycerin, continue heating for 0.5h; cool to about 30°C, add 4g formaldehyde and 12g pentane with a concentration of 0.5% Aldehyde, stir evenly to prepare acetalized polyvinyl alcohol; spread the acetalized polyvinyl alcohol evenly over the bottom of the glass dish, place it in an oven and treat it at 60°C and 70°C for 2 hours each, and take it out after cooling. Soak and rinse for 0.5h to remove impurities; an acetalized polyvinyl alcohol hydrogel is obtained, which is recorded as PVA ₁ .

3. Preparation of acetalized heparin-polyvinyl alcohol complex

The acetalized polyvinyl alcohol hydrogel was spread in a glass dish and dried in an oven at 40°C for 2 hours to obtain an acetalized polyvinyl alcohol film. The acetalized polyvinyl alcohol film was soaked in sodium heparin with a concentration of 1%. In aqueous solution, acetalized heparin-polyvinyl alcohol complex is obtained.

4. Preparation of heparin-polyvinyl alcohol/decellularized scaffold

The acetalized heparin-polyvinyl alcohol complex is evenly coated on the decellularized scaffold, placed in an oven, treated at 60°C for 2 hours, and then raised to 70°C for a further 2 hours. After cooling, take it out, soak and rinse for 0.5 hours, and remove. Impurities were removed to obtain heparin-polyvinyl alcohol/decellularized scaffold, denoted as He/PVA ₁ /DCS.

Example 22 Preparation of heparin-polyvinyl alcohol/decellularized scaffold He/PVA ₂ /DCS

1. Preparation of decellularized scaffolds

The canine bilateral carotid arteries placed in tissue fixative were removed, the adventitia was trimmed, and the arteries were immersed in 0.9% normal saline for cleaning. Take out the cleaned bilateral carotid arteries of the dog and soak them in a mixed solution of 0.5% polyethylene glycol octylphenyl ether and 0.5% sodium dodecyl sulfate at room temperature for 24 hours; The blood vessels were taken out, washed with 0.01 mol/L PBS buffer solution, and soaked in PBS buffer solution for 20 days, changing the solution twice a day. Take out the soaked canine bilateral carotid arteries, trim them into pieces, and freeze-dry them in a freeze-drying machine at -45°C for 12 hours to obtain a decellularized scaffold (DCS), which is recorded as DCS.

2. Preparation of acetalized polyvinyl alcohol hydrogel

In a 500mL three-neck flask, add a certain amount of 10g polyvinyl alcohol and 180mL water, heat to 90°C to dissolve it; add 3g of glycerin, continue heating for 0.5h; cool to about 30°C, add 4g of formaldehyde and 12g of 1% concentration Glutaraldehyde, stir evenly to prepare acetalized polyvinyl alcohol; spread the acetalized polyvinyl alcohol evenly over the bottom of the glass dish, place it in an oven and treat it at 60°C and 70°C for 2 hours each, and then cool Take it out, soak and rinse for 0.5h to remove impurities; obtain acetalized polyvinyl alcohol hydrogel, recorded as PVA ₂ .

3. Preparation of acetalized heparin-polyvinyl alcohol complex

The acetalized polyvinyl alcohol hydrogel was spread in a glass dish and dried in an oven at 40°C for 2 hours to obtain an acetalized polyvinyl alcohol film. The acetalized polyvinyl alcohol film was soaked in a 1% heparin sodium aqueous solution. In, acetalized heparin-polyvinyl alcohol complex was obtained.

4. Preparation of heparin-polyvinyl alcohol/decellularized scaffold

The acetalized heparin-polyvinyl alcohol complex was evenly coated on the decellularized scaffold, placed in an oven, and treated at 60°C for 2 hours, then raised to 70°C and continued treatment for 2 hours, cooled, taken out, soaked and rinsed for 0.5 hours, and removed. After removing impurities, the heparin-polyvinyl alcohol/decellularized scaffold was obtained, which was recorded as He/PVA ₂ /DCS.

Example 23 Preparation of heparin-polyvinyl alcohol/decellularized scaffold He/PVA ₃ /DCS

1. Preparation of decellularized scaffolds

The canine bilateral carotid arteries placed in tissue fixative were removed, the adventitia was trimmed, and the arteries were immersed in 0.9% normal saline for cleaning. Take out the cleaned bilateral carotid arteries of the dog and soak them in a mixed solution of 1% polyethylene glycol octylphenyl ether and 0.5% sodium dodecyl sulfate at room temperature for 24 hours; The blood vessels were taken out, washed with 0.01 mol/L PBS buffer solution, and soaked in PBS buffer solution for 20 days, with the solution changed once a day. The soaked bilateral carotid arteries of the dog were taken out, trimmed into pieces, and freeze-dried at -55°C for 8 hours to obtain a decellularized scaffold (DCS), which was recorded as DCS.

2. Preparation of acetalized polyvinyl alcohol hydrogel

In a 500mL three-neck flask, add a certain amount of 6g polyvinyl alcohol and 180mL water, heat to 90°C to dissolve it; add 3g glycerin, continue heating for 0.5h; cool to about 30°C, add 6g formaldehyde and 12g 3% concentration of formaldehyde Glutaraldehyde, stir evenly to prepare acetalized polyvinyl alcohol; spread the acetalized polyvinyl alcohol evenly over the bottom of the glass dish, place it in an oven and treat it at 60°C and 70°C for 2 hours each, and then cool Take it out, soak and rinse for 0.5h to remove impurities; obtain acetalized polyvinyl alcohol hydrogel, recorded as PVA ₃ .

3. Preparation of acetalized heparin-polyvinyl alcohol complex

The acetalized polyvinyl alcohol hydrogel was spread in a glass dish and dried in an oven at 40°C for 2 hours to obtain an acetalized polyvinyl alcohol film. The acetalized polyvinyl alcohol film was soaked in sodium heparin with a concentration of 1%. In aqueous solution, acetalized heparin-polyvinyl alcohol complex is obtained.

4. Preparation of heparin-polyvinyl alcohol/decellularized scaffold

The acetalized heparin-polyvinyl alcohol complex is evenly coated on the decellularized scaffold, placed in an oven, treated at 60°C for 2 hours, and then raised to 70°C for a further 2 hours. After cooling, take it out, soak and rinse for 0.5 hours, and remove. Impurities were removed to obtain heparin-polyvinyl alcohol/decellularized scaffold, denoted as He/PVA ₃ /DCS.

Example 24 Preparation of heparin-polyvinyl alcohol/decellularized scaffold He/PVA ₄ /DCS

1. The preparation of the decellularized scaffold is the same as in Example 21.

2. Preparation of acetalized polyvinyl alcohol hydrogel

In a 500mL three-neck flask, add a certain amount of 6g polyvinyl alcohol and 180mL water, heat to 90°C to dissolve; add 3g glycerol, continue heating for 0.5h; cool to about 30°C, add 4g formaldehyde and 12g of 5% concentration Glutaraldehyde, stir evenly to prepare acetalized polyvinyl alcohol; spread the acetalized polyvinyl alcohol evenly over the bottom of the glass dish, place it in an oven and treat it at 60°C and 70°C for 2 hours each, and then cool Take it out, soak and rinse for 0.5h to remove impurities; obtain acetalized polyvinyl alcohol hydrogel, recorded as PVA ₄ .

3. Preparation of acetalized heparin-polyvinyl alcohol complex

The acetalized polyvinyl alcohol hydrogel was spread in a glass dish and dried in an oven at 60°C for 2 hours to obtain an acetalized polyvinyl alcohol film. The acetalized polyvinyl alcohol film was soaked in sodium heparin with a concentration of 1%. In the aqueous solution, an acetalized heparin-polyvinyl alcohol complex is obtained.

4. Preparation of heparin-polyvinyl alcohol/decellularized scaffold

The acetalized heparin-polyvinyl alcohol complex is evenly coated on the decellularized scaffold, placed in an oven, treated at 60°C for 2 hours, and then raised to 70°C for a further 2 hours. After cooling, take it out, soak and rinse for 0.5 hours, and remove. Impurities were removed to obtain heparin-polyvinyl alcohol/decellularized scaffold, denoted as He/PVA ₄ /DCS.

Example 25 Preparation of heparin-polyvinyl alcohol/decellularized scaffold He/PVA ₅ /DCS

1. The preparation of the decellularized scaffold is the same as in Example 21.

2. Preparation of acetalized polyvinyl alcohol hydrogel

In a 500mL three-neck flask, add a certain amount of 6g polyvinyl alcohol and 180mL water, heat to 90°C to dissolve it; add 3g glycerin, continue heating for 0.5h; cool to about 30°C, add 4g formaldehyde and 12g of 7% concentration Glutaraldehyde, stir evenly to prepare acetalized polyvinyl alcohol; spread the acetalized polyvinyl alcohol evenly over the bottom of the glass dish, place it in an oven and treat it at 60°C and 70°C for 2 hours each, and then cool Take it out, soak and rinse for 0.5h to remove impurities; obtain acetalized polyvinyl alcohol hydrogel, recorded as PVA ₅ .

3. Preparation of acetalized heparin-polyvinyl alcohol complex

The acetalized polyvinyl alcohol hydrogel was spread in a glass dish and dried in an oven at 40°C for 2 hours to obtain an acetalized polyvinyl alcohol film. The acetalized polyvinyl alcohol film was soaked in sodium heparin with a concentration of 1%. In the aqueous solution, an acetalized heparin-polyvinyl alcohol complex is obtained.

4. Preparation of heparin-polyvinyl alcohol/decellularized scaffold

The acetalized heparin-polyvinyl alcohol complex is evenly coated on the decellularized scaffold, placed in an oven, treated at 50°C for 2 hours, and then raised to 70°C for a further 2 hours. After cooling, take it out, soak and rinse for 0.5 hours, and remove. Impurities were removed, and heparin-polyvinyl alcohol/decellularized scaffold was obtained, denoted as He/PVA ₅ /DCS.

Example 26 Preparation of heparin-polyvinyl alcohol/decellularized scaffold He/PVA ₆ /DCS

1. The preparation of the decellularized scaffold is the same as in Example 22.

2. Preparation of acetalized polyvinyl alcohol hydrogel

In a 500mL three-neck flask, add a certain amount of 6g polyvinyl alcohol and 180mL water, heat to 90°C to dissolve it; add 3g glycerol, continue heating for 0.5h; cool to about 30°C, add 4g formaldehyde and 12g of 9% concentration Glutaraldehyde, stir evenly to prepare acetalized polyvinyl alcohol; spread the acetalized polyvinyl alcohol evenly over the bottom of the glass dish, place it in an oven and treat it at 60°C and 70°C for 2 hours each, and then cool Take it out, soak and rinse for 0.5h to remove impurities; obtain acetalized polyvinyl alcohol hydrogel, recorded as PVA ₆ .

3. Preparation of acetalized heparin-polyvinyl alcohol complex

The acetalized polyvinyl alcohol hydrogel was spread in a glass dish and dried in an oven at 60°C for 2 hours to obtain an acetalized polyvinyl alcohol film. The acetalized polyvinyl alcohol film was soaked in sodium heparin with a concentration of 1%. In the aqueous solution, an acetalized heparin-polyvinyl alcohol complex is obtained.

4. Preparation of heparin-polyvinyl alcohol/decellularized scaffold

The acetalized heparin-polyvinyl alcohol complex is evenly coated on the decellularized scaffold, placed in an oven, treated at 60°C for 2 hours, and then raised to 70°C for a further 2 hours. After cooling, take it out, soak and rinse for 0.5 hours, and remove. impurities, the heparin-polyvinyl alcohol/decellularized scaffold was obtained, denoted as He/PVA ₆ /DCS.

Example 27 Preparation of heparin-polyvinyl alcohol/decellularized scaffold He/PVA ₄ /DCS

1. The preparation of the decellularized scaffold is the same as in Example 22.

2. Preparation of acetalized polyvinyl alcohol hydrogel

In a 500mL three-neck flask, add 10g polyvinyl alcohol and 180mL water, heat to 95°C to dissolve; add 6g glycerin, continue heating for 1 hour; cool to about 30°C, add 4g formaldehyde and 12g glutaraldehyde with a concentration of 5% , stir evenly to prepare acetalized polyvinyl alcohol; spread the acetalized polyvinyl alcohol evenly over the bottom of the glass dish, place it in an oven and treat it at 40°C for 3h and 60°C for 2h respectively. Take it out after cooling. Soak and rinse for 1 hour to remove impurities; an acetalized polyvinyl alcohol hydrogel is obtained, which is recorded as PVA ₄ .

3. Preparation of acetalized heparin-polyvinyl alcohol complex

The acetalized polyvinyl alcohol hydrogel was spread in a glass dish and dried in an oven at 50°C for 2 hours to obtain an acetalized polyvinyl alcohol film. The acetalized polyvinyl alcohol film was soaked in 2% sodium heparin. In the aqueous solution, an acetalized heparin-polyvinyl alcohol complex is obtained.

4. Preparation of heparin-polyvinyl alcohol/decellularized scaffold

The acetalized heparin-polyvinyl alcohol complex is evenly coated on the decellularized scaffold, placed in an oven, treated at 50°C for 2 hours, then raised to 70°C and continued treatment for 1 hour, cooled, taken out, soaked and rinsed for 1 hour, and impurities removed , the heparin-polyvinyl alcohol/decellularized scaffold was obtained, which was recorded as He/PVA ₄ /DCS.

Example 28 Preparation of heparin-polyvinyl alcohol/decellularized scaffold He/PVA ₅ /DCS

1. The preparation of the decellularized scaffold is the same as in Example 22.

2. Preparation of acetalized polyvinyl alcohol hydrogel

In a 500mL three-neck flask, add a certain amount of 6g polyvinyl alcohol and 180mL water, heat to 100°C to dissolve it; add 3g glycerin, continue heating for 0.5h; cool to about 30°C, add 4g formaldehyde and 12g of 7% concentration Glutaraldehyde, stir evenly to prepare acetalized polyvinyl alcohol; spread the acetalized polyvinyl alcohol evenly over the bottom of the glass dish, place it in an oven and treat it at 40°C for 2 hours and 60°C for 1 hour respectively. After cooling, take it out, soak and rinse for 1 hour to remove impurities; obtain an acetalized polyvinyl alcohol hydrogel, recorded as PVA ₅ .

3. Preparation of acetalized heparin-polyvinyl alcohol complex

The acetalized polyvinyl alcohol hydrogel was spread in a glass dish and dried in an oven at 60°C for 2 hours to obtain an acetalized polyvinyl alcohol film. The acetalized polyvinyl alcohol film was soaked in 2% sodium heparin. In the aqueous solution, an acetalized heparin-polyvinyl alcohol complex is obtained.

4. Preparation of heparin-polyvinyl alcohol/decellularized scaffold

The acetalized heparin-polyvinyl alcohol complex is evenly coated on the decellularized scaffold, placed in an oven, treated at 60°C for 1 hour, then raised to 70°C and continued treatment for 2 hours, cooled, taken out, soaked and rinsed for 0.5 hours, and removed. Impurities were removed, and heparin-polyvinyl alcohol/decellularized scaffold was obtained, denoted as He/PVA ₅ /DCS.

Example 29 Preparation of heparin-polyvinyl alcohol/decellularized scaffold He/PVA ₃ /DCS

1. The preparation of the decellularized scaffold is the same as in Example 21.

2. Preparation of acetalized polyvinyl alcohol hydrogel

In a 500mL three-neck flask, add a certain amount of 6g polyvinyl alcohol and 180mL water, heat to 90°C to dissolve it; add 3g glycerin, continue heating for 1 hour; cool to about 30°C, add 4g formaldehyde and 12g pentane with a concentration of 3%. Dialdehyde, stir evenly to prepare acetalized polyvinyl alcohol; spread the acetalized polyvinyl alcohol evenly over the bottom of the glass dish, place it in an oven and treat it at 40°C and 70°C for 2 hours each, and take it out after cooling. , soak and rinse for 1 hour to remove impurities; an acetalized polyvinyl alcohol hydrogel is obtained, which is recorded as PVA ₃ .

3. Preparation of acetalized heparin-polyvinyl alcohol complex

The acetalized polyvinyl alcohol hydrogel was spread in a glass dish and dried in an oven at 40°C for 3 hours to obtain an acetalized polyvinyl alcohol film. The acetalized polyvinyl alcohol film was soaked in sodium heparin with a concentration of 1%. In the aqueous solution, an acetalized heparin-polyvinyl alcohol complex is obtained.

4. Preparation of heparin-polyvinyl alcohol/decellularized scaffold

The acetalized heparin-polyvinyl alcohol complex is evenly coated on the decellularized scaffold, placed in an oven, treated at 50°C for 2 hours, then raised to 70°C and continued treatment for 1 hour, cooled, taken out, soaked and rinsed for 1 hour, and impurities removed , the heparin-polyvinyl alcohol/decellularized scaffold was obtained, which was recorded as He/PVA ₃ /DCS.

Example 30 Preparation of heparin-polyvinyl alcohol/decellularized scaffold He/PVA ₆ /DCS

1. The preparation of the decellularized scaffold is the same as in Example 21.

2. Preparation of acetalized polyvinyl alcohol hydrogel

In a 500mL three-neck flask, add a certain amount of 6g polyvinyl alcohol and 180mL water, heat to 100°C to dissolve; add 4g glycerin, continue heating for 1 hour; cool to about 30°C, add 4g formaldehyde and 12g pentane with a concentration of 9% Dialdehyde, stir evenly to prepare acetalized polyvinyl alcohol; spread the acetalized polyvinyl alcohol evenly over the bottom of the glass dish, place it in an oven and treat it at 60°C for 3h and 70°C for 2h respectively. After cooling, Take it out, soak and rinse for 0.5h to remove impurities; obtain acetalized polyvinyl alcohol hydrogel, recorded as PVA ₆ .

3. Preparation of acetalized heparin-polyvinyl alcohol complex

The acetalized polyvinyl alcohol hydrogel was spread in a glass dish and dried in an oven at 50°C for 3 hours to obtain an acetalized polyvinyl alcohol film. The acetalized polyvinyl alcohol film was soaked in 2% sodium heparin. In the aqueous solution, an acetalized heparin-polyvinyl alcohol complex is obtained.

4. Preparation of heparin-polyvinyl alcohol/decellularized scaffold

The acetalized heparin-polyvinyl alcohol complex is evenly coated on the decellularized scaffold, placed in an oven, treated at 60°C for 2 hours, and then raised to 70°C for a further 2 hours. After cooling, take it out, soak and rinse for 1 hour, and remove impurities. , the heparin-polyvinyl alcohol/decellularized scaffold was obtained, which was recorded as He/PVA ₆ /DCS.

Example 31 Scanning electron microscope test of acetalized polyvinyl alcohol hydrogel

A series of differently acetalized polyvinyl alcohol hydrogels (PVA _n , n=1 to 6) obtained in Examples 21 to 26 were vacuum dried and then their appearance, morphology and characteristics were observed with a scanning electron microscope, as shown in Figure 13 Show. Figure 13 is an electron microscope image of polyvinyl alcohol hydrogel PVA _n (n=1~6) with different acetalization, where a is the acetalization polyvinyl alcohol hydrogel PVA ₁ obtained in Example 21, in which pentadienyl The aldehyde concentration is 0.5%; b the acetalized polyvinyl alcohol hydrogel PVA ₂ obtained in Example 22, in which the glutaraldehyde concentration is 1%; c the acetalized polyvinyl alcohol hydrogel obtained in Example 23 PVA ₃ , wherein the glutaraldehyde concentration is 3%; d the acetalized polyvinyl alcohol hydrogel PVA ₄ obtained in Example 24, wherein the glutaraldehyde concentration is 5%; e the acetalized polyvinyl alcohol hydrogel obtained in Example 25 Polyvinyl alcohol hydrogel PVA ₅ , in which the glutaraldehyde concentration is 7%; f acetalized polyvinyl alcohol hydrogel PVA ₆ obtained in Example 26, in which the glutaraldehyde concentration is 9%. It can be seen from Figure 13 that when the glutaraldehyde concentration in a, b and c is 0.5%, 1% and 3%, the surface of the polyvinyl alcohol hydrogel film is not smooth, which is due to incomplete acetalization reaction. When the glutaraldehyde concentration is 5%, as shown in Figure d, the surface of the polyvinyl alcohol film obtained is smooth and flat, indicating that when the glutaraldehyde concentration is 5%, the polyvinyl alcohol hydrogel film obtained is better; when glutaraldehyde When the concentration is greater than 5%, that is, when the concentration reaches 7%, as shown in Figure E, the surface of the polyvinyl alcohol film begins to gel. When the concentration is 9%, as shown in Figure F, the gelation phenomenon on the surface of the film is obvious, indicating that at this concentration , the increase in cross-linking between polyvinyl alcohol molecules leads to an increase in the brittleness of the hydrogel film and insufficient flexibility to meet our expected requirements. From the above results, it can be seen that when the glutaraldehyde concentration is 5%, the polyvinyl alcohol hydrogel film prepared by acetalization is the best.

Example 32 Heparin-polyvinyl alcohol/decellularized scaffold material He/PVA ₄ /DCS surface coating for infrared characterization

The results of infrared characterization of the surface coating of the heparin-polyvinyl alcohol/decellularized scaffold material He/PVA ₄ /DCS obtained in Example 24 are shown in Figure 14. Figure 14 is the infrared spectrum of heparin-polyvinyl alcohol/decellularized scaffold He/PVA ₄ /DCS. It can be seen from Figure 14 that several strong absorption peaks near 1250cm ^-1 are from acetal (C=O=C) Characteristic peaks, among which the strong absorption peak between 1150 and 1050 cm ^-1 correspond to the antisymmetric stretching vibration peak of saturated fatty ether (COC), are characteristic identifications. It can be seen that the acetalization reaction of polyvinyl alcohol has indeed occurred. There is a broad peak near 3400~3200cm ^-1 , which corresponds to the characteristic peak of associative hydrogen bonding between polymers. A vibration absorption peak appears at the plane change angle of free -OH at 1250cm ^-1 , and corresponds to -OH hydrogen at 1500~1300cm ^-1 Characteristic peaks of bond association. From the above results, it can be seen that only part of the hydroxyl groups undergo acetalization reaction. There are still some hydroxyl groups in the system that exist in the form of polymer intermolecular association hydrogen peaks, and a small amount of hydroxyl groups exist in free form.

Example 33 The surface coating of heparin-polyvinyl alcohol/decellularized scaffold He/PVA _n /DCS (n=1-6, corresponding to the products of Examples 21-26) with different concentrations and degrees of acetalization was characterized by scanning electron microscopy

The surface coating of the heparin-polyvinyl alcohol/decellularized scaffold He/PVA _n /DCS (n=1-6) obtained in Examples 21 to 26 with different degrees of acetalization was characterized by scanning electron microscopy. The results are shown in Figure 15 shown. Figure 15 is an SEM image of heparin-polyvinyl alcohol/decellularized scaffold He/PVA _n /DCS (n=1~6) with different degrees of acetalization, where a is He/PVA ₁ /DCS; b is He /PVA ₂ /DCS; c is He/PVA ₃ /DCS; d is He/PVA ₄ /DCS; e is He/PVA ₅ /DCS; f is He/PVA ₆ /DCS. It can be seen from Figure 15 that in a, there is less heparin bonded to the surface. As the concentration of glutaraldehyde and the degree of acetalization increase in b to f, the amount of heparin bonded to the surface also increases; but from e, glue begins to appear. Coagulation phenomenon and small cracks appear, and f is more serious. To sum up, when the concentration of glutaraldehyde in d is 5%, the material surface shows good performance. At this time, the hydrogel performance is better, and it is better than before bonding heparin. The results match.

Example 34 X-ray photoelectron spectroscopy analysis of PVA ₁ /DCS and He/PVA ₄ /DCS prepared in Example 24

Example 24 was used to prepare PVA ₁ /DCS and He/PVA ₄ /DCS using a Japanese Rigaku D/max 2500VL/PC X-ray photoelectron spectrometer, under monochromatic Al Kα rays (150W, 500 μm beam spot) and The X-ray photoelectron spectroscopy test was carried out under the condition that the energy of 20eV passed through, and the results are shown in Figure 16. Figure 16 is the XPS comparison diagram of PVA ₁ /DCS and He/PVA ₄ /DCS, where a is PVA ₁ /DCS and b is He/PVA ₄ /DCS. As can be seen from Figure 16, the S _2p peak appearing on b indicates that there is S element on He/PVA ₄ /DCS. This is because the heparin on the He/PVA ₄ /DCS artificial blood vessel material has S element, so it can be proved that He/PVA ₄ /DCS artificial blood vessel material has been successfully prepared.

Example 35: Mechanical properties, tensile strength, and elongation at break of fresh blood vessels, decellularized scaffolds, and heparin-polyvinyl alcohol/decellularized scaffolds He/PVA _n /DCS (n=1-6) with different degrees of acetalization Characterization of rate and blasting strength

The mechanical properties of fresh blood vessels, decellularized scaffolds, and heparin-polyvinyl alcohol/decellularized scaffolds He/PVA _n /DCS (n = 1 to 6) obtained in Examples 21 to 26 with different degrees of acetalization were performed. The specific experimental procedures for characterization of strength, elongation at break and bursting strength are the same as in Example 16, and the specific results are shown in Figure 17. Figure 17 is a graph showing the tensile strength, elongation at break and bursting strength of fresh blood vessels, DCS and He/PVA _n /DCS (n=1-6). Among them, a is the tensile strength diagram of fresh blood vessels, DCS and He/PVA _n /DCS (n=1~6), and b is the breaking elongation of fresh blood vessels, DCS and He/PVA _n /DCS (n=1~6) rate chart. c is the bursting intensity diagram of fresh blood vessels, DCS and He/PVA _n /DCS (n=1~6). Among them, Fresh blood vessel is a fresh blood vessel, and DCS is a decellularized vascular scaffold. It can be seen from Figures a and b that compared with fresh blood vessels, the tensile properties of DCS are slightly increased, and the elongation at break is slightly increased, but overall the difference between the two is small. After being coated with polyvinyl alcohol of different degrees of acetalization, the tensile properties of DCS decreased slightly, and its elongation at break increased significantly. This phenomenon indicates that acetalized polyvinyl alcohol can significantly improve the elongation at break of vascular materials. Among them, as the concentration of glutaraldehyde increases, the tensile strength and elongation at break of the vascular material increase, reaching the optimum when the concentration is 5%. When the concentration increased to 7% and 9%, the tensile strength and elongation at break of the corresponding vascular material decreased, which was consistent with the previous experimental results. Comparing samples with different acetalization degrees He/PVA _n /DCS (n=1~6), there is almost no difference in tensile strength and elongation at break. The above results show that the tensile strength and elongation at break of DCS are not significantly changed after PVA coating and heparin precipitation, and its mechanical properties are still guaranteed. It can be found from Figure c that the bursting strength of fresh arterial blood vessels is about 110KPa, while the bursting strength of DCS is 0KPa. This is because DCS removes the surface tissue cells and forms a spatial network structure. Physiological saline will directly pass through the network structure. outflow because DCS does not have bursting strength. DCS modified with heparin and PVA hydrogel embeds the spatial network structure of DCS, and its burst strength will show an upward trend with the increase of polyelectrolyte layer, but each sample is different from fresh arterial blood vessels. The blasting strength is basically close. The above test results show that after modifying DCS with heparin and PVA hydrogel, the material basically maintains the mechanical bionic properties of DCS, so that the prepared artificial blood vessel material can meet the pressure requirements generated when blood flows in the lumen of blood vessels. .

Example 36 In vitro coagulation experiment and recalcification coagulation time test of heparin-polyvinyl alcohol/decellularized scaffold artificial vascular material He/PVA _n /DCS (n=1~6) with different degrees of acetalization

The specific experimental steps for the in vitro coagulation experiment and recalcification coagulation time test of the heparin-polyvinyl alcohol/decellularized scaffold artificial vascular material He/PVA _n /DCS (n=1~6) with different degrees of acetalization are the same as those in Example 17. The results are shown in Figure 18. Figure 18 is a graph of activated partial thromboplastin time, thrombin time, prothrombin time and recalcification coagulation time of He/PVA _n /DCS (n=1~6) artificial blood vessel material, where a is He/PVA _n The activated partial thromboplastin time diagram of /DCS (n=1~6) artificial blood vessel material, b is He/PVA _n The prothrombin time diagram of /DCS (n=1~6) artificial blood vessel material, c is He/ Thrombin time chart of PVA _n /DCS (n = 1 to 6) artificial blood vessel material, d is the recalcification coagulation time chart of He/PVA _n /DCS (n = 1 to 6) artificial blood vessel material. It can be seen from ac in Figure 18 that compared with the control group, the coagulation times of APTT, TT, and PT of artificial vascular materials modified with heparin and polyvinyl alcohol are all prolonged to a certain extent. This is because heparin can effectively improve blood compatibility and anticoagulant ability. We can also find that the values of APTT, TT, and PT have reached the optimal value when using He/PVA ₄ /DCS. From the above experimental results, it can be considered that When n=4, He/PVA ₄ /DCS artificial vascular material has the longest in vitro coagulation time and the best anticoagulant performance. In calcium-free anticoagulated plasma, the time it takes for the plasma to coagulate after adding an appropriate amount of calcium ions again is called the recalcification time. The longer it takes for plasma calcification to occur, the better its hemocompatibility. As can be seen from d in Figure 18, compared with the control group, the T _1/2max recalcification time of the heparin-polyvinyl alcohol/decellularized scaffold artificial vascular material was significantly prolonged, increasing from 10.5 min to 18.2 min to 23.8 min. , recalcification coagulation time was significantly prolonged. Experimental results show that heparin-polyvinyl alcohol/acellular scaffold can prolong the conversion time of soluble fibrinogen into soluble fibrin, thus prolonging the blood coagulation time. This can be considered to be because heparin can bind to antithrombin III, thus inhibiting the coagulation function of thrombin, thereby improving the blood compatibility of the material and prolonging the recalcification coagulation time. When n=4 (product of Example 24), He/PVA ₄ /DCS has the longest T _1/2max recalcification time. It can be concluded that He/PVA ₄ /DCS has the longest recalcification time. That is, when the glutaraldehyde concentration is 5%, the material has the best anticoagulant modification effect.

Example 37 Hemocompatibility test of heparin-polyvinyl alcohol/decellularized scaffold He/PVA _n /DCS (n=1~6) with different degrees of acetalization

The specific experimental steps for the blood compatibility test of heparin-polyvinyl alcohol/decellularized scaffold He/PVA _n /DCS (n=1-6) with different degrees of acetalization are the same as those in Example 18, and the experimental results are shown in Figure 19. Figure 19 shows the red blood cell morphology of He/PVA _n /DCS (n=1~6), where a is the control group, b is He/PVA ₁ /DCS, c is He/PVA ₂ /DCS, and d is He. /PVA ₃ /DCS, e is He/PVA ₄ /DCS, f is He/PVA ₅ /DCS, and g is He/PVA ₆ /DCS. As can be seen from Figure 19, when red blood cells come into contact with the vascular material of the present invention, their morphology is consistent with that of the control group. The red blood cells are in good shape and no obvious deformation or rupture is found. The results show that heparin-modified PVA _n /DCS has almost no toxic effect on red blood cells, that is, it has good blood compatibility.

Example 38 MTT cytotoxicity test on heparin-polyvinyl alcohol/decellularized scaffold He/PVA _n /DCS (n=1~6) with different degrees of acetalization

1. Specific experiments: Sample preparation: Take heparin-polyvinyl alcohol/decellularized scaffold He/PVA _n /DCS (n=1~6) vascular materials, add 10mL leaching medium (10% fetal bovine serum) at 37°C Leaching in constant temperature shaking incubator for 24h. Preparation of MTT solution: Dissolve MTT in PBS to make an MTT solution with a concentration of 5 mg/mL. Cells in the logarithmic growth phase were digested with trypsin and diluted with cell culture medium to a concentration of 4×10 ⁴ cells/mL. Take a 96-well culture plate, inoculate 200 μL of cell suspension into each well, and place it in a 37°C, 5% _CO2 incubator for 24 hours. After the cells adhere to the wall, aspirate the original culture medium from each well, and add 200 μL of sample extraction solution to each well of the experimental group (8 wells for each group). At the same time, set a blank control group in the well plate without cells and add the cell culture medium. Continue to be cultured in a 37°C, 5% _CO2 incubator for 12 hours and 24 hours; then add 20 μL of the prepared MTT solution to each well. After incubation for 5 hours, discard the original solution, add 150 μL of dimethyl sulfoxide to each well, and shake horizontally for 5 minutes. The absorbance value OD at the wavelength of 490 nm was measured with a microplate reader (BioTeksynergy 2 type). Test each at least three times. Experimental group: endothelial progenitor cells+He/PVA _n /DCS (n=1~6)+MTT. Negative control group: endothelial progenitor cells + MTT. Blank control group: MTT.

Result calculation:

In the formula: Dt - the absorbance of the experimental group sample; Dnc - the absorbance of the negative control group sample; Db - the absorbance of the blank control group sample. The experimental results are shown in Figure 20.

2. Result analysis: The MTT method is a commonly used method to detect cell survival. The succinate dehydrogenase of living cells reduces MTT to generate water-insoluble blue-violet crystal formazan, while dead cells do not have this function. The amount of formazan can be expressed by the light absorption value of a microplate reader at 490nm, and then the number of living cells can be inferred. If the number of cells is large, the measured absorbance will be slightly larger. When the number of cells is small or the condition is not good, the absorbance value It will be lower. The toxicity of the material can be judged from the absorbance. Figure 20 is the MTT test chart of He/PVA _n /DCS (n=1~6). It can be seen from Figure 20 that at 12 hours, the cell survival rate of DCS in He/PVA _n /DCS (n = 1 to 3) reached 95% to 98%, and the cytotoxicity was level 1, but the cell survival rate varied with glutaraldehyde concentration. The cell survival rate of DCS in He/PVA _n /DCS (n=4~6) reaches 100%~105%, and the cytotoxicity is level 0, but the cell survival rate decreases with the increase of glutaraldehyde concentration. He/PVA ₄ /DCS had the highest cell survival rate, 105%, and the least cytotoxicity, and the cell survival rate differences between samples of He/PVA _n /DCS (n=1~6) with different acetalization degrees were small. n=4, that is, the cell survival rate is greater when the glutaraldehyde concentration is 5%, which is consistent with the results of the previous compatibility experiments. It can be seen from the above that when n is 4, the He/PVA ₄ /DCS obtained when acetalization is performed with a glutaraldehyde concentration of 5% has the best comprehensive performance.

Example 39 In vitro degradation experiments of PVA/DCS and He/PVA ₄ /DCS artificial blood vessel materials

1. Specific experiments: From the above, it is believed that when n=4, that is, the He/PVA ₄ /DCS artificial blood vessel material has the best comprehensive performance. In the follow-up research, He/PVA ₄ /DCS will be used as the research object for testing and research. Cut the PVA/DCS and He/PVA ₄ /DCS artificial blood vessel materials into a square of 10mmx10mm, weigh it with an electronic balance as m ₀ , put it into a centrifuge tube, add 20mL of PBS solution, and place it at 37°C Incubate in a constant temperature shaking box for 1, 3, 7, 14, 21, 60, 90, 120 and 180 days. Replace with fresh PBS buffer every week. Take out the cultured artificial blood vessel material and put it into a vacuum drying box. Dry at 50°C and weigh the weight m ₁ with an electronic balance. Calculate the weight loss result: Weight loss (%) = (m ₀ -m ₁ )/m ₀ ×100%. The results are shown in Figure 21.

2. Result analysis: Figure 21 is the in vitro degradation rate chart of PVA/DCS and He/PVA ₄ /DCS artificial blood vessel materials. It can be seen from Figure 21 that as time increases, the in vitro degradation rates of PVA/DCS and He/PVA ₄ /DCS The degradation rate showed a trend from fast to slow; at 180 days, the remaining mass of PVA/DCS was 37.9%, while the remaining mass of He/PVA ₄ /DCS was 40.4%. The in vitro degradation cycle and blood vessel regeneration cycle between the two are basically matched, which shows that both have good degradation speed matching. As artificial blood vessel materials, they can achieve a degradation cycle and a good match with the regeneration cycle of new blood vessels to achieve a good bionic effect.

Comparative experiments of Example 40n-He-CS/PEG/DCS, He/PVA ₄ /DCS and He-Ch-5/PU/DCS

1. Preparation of He-Ch-5/PU/DCS: He-Ch-5/PU/DCS artificial blood vessel material was prepared in preliminary research. The specific preparation steps are as follows: Preparation method of decellularized scaffold (DCS) Same as Example 1. Soak DCS in PBS with pH=7.4 for 30 min. Dissolve 5.0g polyurethane (PU) in 50mL N,N-dimethylformamide (DMF) to obtain a 0.1g/mL PU solution at room temperature. The PU solution was dip-coated on DCS, repeated three times, and dried at 60°C for 12 hours in a vacuum drying oven to obtain PU/DCS. Prepare 1g/L heparin, 1g/L chitosan solution and 0.01mol/L PBS buffer solution respectively. PU/DCS was soaked in PBS buffer solution for 30 min, and then soaked in heparin solution for 15 min to obtain He/PU/DCS. He/PU/DCS was washed with PBS buffer solution to remove sodium heparin physically adsorbed on its surface. After soaking He/PU/DCS in chitosan solution for 15 minutes, it was washed with PBS buffer solution to remove the physically adsorbed chitosan on its surface to obtain He-Ch-2/PU/DCS artificial blood vessel material. Similarly, the He-Ch-2/PU/DCS vascular material was soaked three times in heparin and chitosan solutions to prepare the He-Ch-5/PU/DCS artificial vascular material.

2. Performance test: Use mechanical tensile testing equipment (model CMT6103, China MTS) with a test speed of 10mm/min (n=5) to detect fresh blood vessels, He-Ch-5/PU/DCS, and He/PVA ₄ / Tensile properties of DCS and 5-He-CS/PEG/DCS (50mm×20mm×1mm). The stress-strain curve was drawn based on the load and elongation data obtained from the test, and the results are shown in Figure 24.

3. Result analysis: Figure 24 shows the stress-strain curves of fresh blood vessels, He-Ch-5/PU/DCS, He/PVA ₄ /DCS and 5-He-CS/PEG/DCS. Figure a shows the stress-strain curves of fresh blood vessels. Stress-strain curve, Figure b is the stress-strain curve of 5-He-CS/PEG/DCS, Figure c is the stress-strain curve of He-Ch-5/PU/DCS, d is the stress-strain curve of He/PVA ₄ /DCS stress-strain curve. It can be seen from Figures a and b that the 5-He-CS/PEG/DCS vascular material modified by hydrogel and chitosan heparin maintains a stress-strain curve similar to that of fresh blood vessels. This is due to the PEG hydrogel It has a small modulus and will not have a major impact on the mechanical properties of the decellularized scaffold. This ensures that the prepared vascular material maintains mechanical properties that match those of fresh blood vessels. It can be seen from Figure c that due to the high elastic modulus of PU, coating modification of the decellularized scaffold will cause great changes in the mechanical properties of the decellularized scaffold, and the stress and strain will be greatly increased. However, the stress -The strain curve is quite different from that of fresh blood vessels; as shown in Figure d, after PVA hydrogel coating, He/PVA ₄ /DCS has better elasticity and tensile properties, and the stress-strain curve is not much different from that of fresh blood vessels. Therefore, it can be considered that He-Ch-5/PU/DCS cannot achieve mechanical properties that match fresh blood vessels, while the hydrogel maintains mechanical properties that match fresh blood vessels. Therefore, it can be considered that He/PVA ₄ /DCS and 5- The He-CS/PEG/DCS artificial blood vessel material has good mechanical bionic properties and is expected to maintain long-term patency of the implanted site.

Example 41 5-He-CS/PEG/DCS and He/PVA ₄ /DCS are used in animal experimental studies

1. Specific experiments: Select fragrant pigs that are one month old and grow well as experimental subjects, and start the experiment after raising them under standard conditions for 4 weeks. Control group: 2 mini pigs, carotid arteries implanted with expanded polytetrafluoroethylene artificial blood vessels, experimental group: 2 mini pigs, carotid arteries implanted with 5-He-CS/PEG/DCS artificial blood vessels, 2 The carotid arteries of small piglets were implanted with He/PVA ₄ /DCS artificial blood vessel materials; comparative experiments: the carotid arteries of two small piglets were implanted with He-Ch-5/PU/DCS artificial blood vessel materials. After the artificial blood vessel material synthesized by the present invention is implanted, B-ultrasound monitoring is performed two weeks later. B-ultrasound examination is a form of ultrasonic examination and a non-surgical diagnostic examination. It is unique in human soft tissue and hemodynamics. About 2 weeks after the operation, the L38e/10-5MHz full digital color Doppler ultrasound of the American company SonoSite was used to conduct B-ultrasound analysis of the implantation position of the artificial blood vessel material of the mini-pigs to observe the smooth blood flow in the blood vessels and the presence of blood vessels. There is no occlusion, expansion, etc. The specific results are shown in Figure 22.

2. Result analysis: Figure 22 is the B-ultrasound image of 5-He-CS/PEG/DCS and He/PVA ₄ /DCS artificial blood vessel materials after two weeks of implantation in the body, in which a is the expanded polytetrafluoroethylene artificial blood vessel in the control group Blood vessels, b is the experimental group 5-He-CS/PEG/DCS artificial blood vessel material, c is the comparative example He-Ch-5/PU/DCS artificial blood vessel material, d is the He/PVA ₄ /DCS artificial blood vessel material. The red areas in the picture represent blood flow. In Figure 22 a, there is intermittent blood flow at the implantation site of the expanded polytetrafluoroethylene artificial blood vessel material, indicating that the artificial blood vessel material there has formed a thrombus, resulting in poor blood flow there. This shows that the expanded polytetrafluoroethylene artificial blood vessel material The tetrafluoroethylene artificial blood vessel material cannot replace normal blood vessels in pigs; the implantation positions of b, c and d in Figure 22 are obviously red, indicating that no thrombus has formed at the three artificial blood vessel material positions. Comparing the B-ultrasound images of three different artificial blood vessel materials, it can be clearly found that the artificial blood vessel materials of 5-He-CS/PEG/DCS, He/PVA ₄ /DCS and the comparative example He-Ch-5/PU/DCS Vascular materials are less likely to form thrombus, have better blood compatibility, and can replace the role of normal blood vessels.

Example 42 The expanded polytetrafluoroethylene artificial blood vessels in the control group and the 5-He-CS/PEG/DCS, He/PVA ₄ /DCS artificial blood vessel materials in the experimental group and the comparative experiment He-Ch-5 in Example 41 were implanted. /PU/DCS artificial blood vessel material five months later, conduct CTA testing

1. Specific experiments: CT angiography (CTA, CT angiography) combines CT enhancement technology with thin-layer, large-scale, and fast scanning technology. Through reasonable post-processing, it can clearly display the details of blood vessels in various parts of the body. In Example 41, the expanded polytetrafluoroethylene artificial blood vessels in the control group and the 5-He-CS/PEG/DCS, He/PVA ₄ /DCS artificial blood vessel materials in the experimental group and the comparative experiment He-Ch-5/PU/ were implanted. Five months after the DCS artificial blood vessel material was used, CT angiography technology was used to conduct cross-sectional scanning and observation to observe the blood circulation at the implantation site and observe its patency rate. The results are shown in Figure 23.

2. Result analysis: Figure 23 is a CTA image of the implantation site after 5 months of implantation of 5-He-CS/PEG/DCS and He/PVA ₄ /DCS artificial blood vessel materials into the body. Among them, a is the expanded polyethylene in the control group. Tetrafluoroethylene artificial blood vessel, b is the experimental group 5-He-CS/PEG/DCS artificial blood vessel material, c is the comparative experiment He-Ch-5/PU/DCS artificial blood vessel material, d is He/PVA ₄ /DCS artificial blood vessel Material. CTA is an angiography technology. By constructing a 3D model of blood flow, the blood flow can be observed intuitively. As can be seen from figure 23a, after the expanded polytetrafluoroethylene artificial blood vessel material is implanted, the blood vessel becomes blocked. There is only a small amount of blood circulation at the implantation site, but new branches grow from the side of the original blood vessel; b, c and d in Figure 23 show that the implantation site has only a slight protrusion, and the blood flow is smooth and there is no obstruction. It shows that 5-He-CS/PEG/DCS, He/PVA ₄ /DCS and He-Ch-5/PU/DCS artificial blood vessel materials can replace natural blood vessels and are excellent blood vessel replacement materials.

Claims (7)

1. The application of hydrogel in the preparation of highly bionic artificial blood vessel materials, characterized in that the hydrogel is chitosan/polyethylene glycol hydrogel or acetalized polyvinyl alcohol hydrogel, The preparation method of the highly bionic artificial blood vessel material includes the following steps: (1) Preparation of chitosan/polyethylene glycol hydrogel; (2) Apply chitosan/polyethylene glycol hydrogel on the surface of the decellularized scaffold to obtain chitosan/polyethylene glycol/decellularized scaffold CS/PEG/DCS; (3) Use the layer-by-layer self-assembly method to deposit heparin on the surface of chitosan/polyethylene glycol/decellularized scaffold, and dry it under vacuum to obtain the highly bionic artificial blood vessel material n-He-CS/PEG/DCS; The layer-by-layer self-assembly method is the soaking and pulling method, which includes the following steps: (2.1) Prepare heparin sodium solution and chitosan solution respectively; (2.2) Soak CS/PEG/DCS in PBS buffer solution, take it out, soak it in heparin sodium solution, take it out and rinse it with PBS buffer solution front and back to obtain 1-He-CS/PEG/DCS; (2.3) Soak 1-He-CS/PEG/DCS in the prepared chitosan solution, take it out and wash it front and back with PBS buffer solution, then soak it in heparin sodium solution, take it out and wash it front and back with PBS buffer solution Rinse the back side to obtain 2-He-CS/PEG/DCS; (2.4) Repeat the steps in (2.3) to prepare a multi-layered heparin/polyethylene glycol hydrogel/decellularized scaffold to obtain n-He-CS/PEG/DCS; In step (2.1), the concentration of the heparin sodium solution is 1~2g/L, the concentration of the chitosan solution is 1~2g/L, and the mass ratio of the chitosan to heparin sodium is 1:1~2. ; In step (2.2), the CS/PEG/DCS is soaked in PBS buffer solution for 10 to 30 minutes, and soaked in heparin sodium solution for 10 to 15 minutes; in step (2.3), the 1-He-CS/PEG/DCS is placed in the prepared chitosan solution and soaked for 10 to 15 minutes, and the soaking time in the heparin sodium solution is 10 to 15 minutes. In step (2.4), the n- n in He-CS/PEG/DCS is 3~7; The preparation method of the highly bionic artificial blood vessel material also includes the following steps: (1) Preparation of polyvinyl alcohol hydrogels with different acetalization; (2) Preparation of different acetalized heparin-polyvinyl alcohol complexes; (3) Apply different acetalized heparin-polyvinyl alcohol complexes on the surface of the decellularized scaffold to obtain different acetalized heparin-polyvinyl alcohol/decellularized scaffold, that is, the highly bionic artificial vascular material He/PVAn/DCS; n =1~6. 2. Application according to claim 1, characterized in that the preparation method of the chitosan/polyethylene glycol hydrogel includes the following steps: (1) Prepare NaOH aqueous solution, then add absolute ethanol to obtain NaOH solution; (2) Add chitosan to the NaOH solution while stirring to alkalize it; (3) Add propylene oxide and react in a constant temperature water bath to obtain the product; (4) Take out the product, wash it in a mixture of hydrochloric acid and acetone, then wash it in a mixture of acetone and water, vacuum filter, and vacuum dry to obtain O-HPCS; (5) Prepare O-HPCS aqueous solution, add polyethylene glycol and glutaraldehyde solutions, stir and mix, and let stand to obtain chitosan/polyethylene glycol hydrogel. 3. The application according to claim 2, characterized in that, in step (1), the mass concentration of the NaOH solution is 6% to 8%; in step (2), the solution of the NaOH solution and chitosan is The solid ratio is 10~12mL:g, the alkalization time is 6~8h; in step (3), the reaction time is 24~36h; in step (4), the mixed solution of hydrochloric acid and acetone The mass ratio of hydrochloric acid to acetone is 1:10~1:9, the mass ratio of acetone to water in the mixture of acetone and water is 9:1~10:1, and the temperature of the vacuum drying is 45~55°C , the vacuum drying time is 2~3h; in step (5), the solid-liquid ratio of the polyethylene glycol and glutaraldehyde is 0.1~0.2g:mL. 4. The application according to claim 1, characterized in that the preparation method of the acetalized polyvinyl alcohol hydrogel includes the following steps: (1) Mix polyvinyl alcohol with water and heat to dissolve; (2) Add glycerol and continue heating; (3) Cool down, add formaldehyde and glutaraldehyde respectively, and stir evenly to obtain acetalized polyvinyl alcohol; (4) Dry the acetalized polyethylene, then raise the temperature to continue drying, cool, and soak and rinse with water to obtain acetalized polyvinyl alcohol hydrogel. 5. The application according to claim 4, characterized in that in step (1), the mass ratio of the polyvinyl alcohol to water is 1:30~5:90, and the heating temperature is 90~100°C. ; In step (2), the mass ratio of the glycerol and polyvinyl alcohol is 1:1~2, and the continued heating time is 0.5h~1h; in step (3), the formaldehyde, glutaraldehyde and The mass ratio of polyvinyl alcohol is 2:6:3~2:6:5, and the concentration of glutaraldehyde is 0.5~9%; in step (4), the drying temperature is 40~60°C. The drying time is 2 to 3 hours, the reheating temperature is 60 to 70°C, the continuing drying time is 1 to 2 hours, and the soaking and rinsing time is 0.5 to 1 hour. 6. The application according to claim 1, characterized in that the preparation method of the decellularized scaffold includes the following steps: (1) Take out the blood vessels placed in the tissue fixative, trim the outer membrane, and immerse them in physiological saline; (2) Prepare a mixed solution of sodium lauryl sulfate and polyethylene glycol octylphenyl ether, and soak the trimmed blood vessels in the mixed solution; (3) Take out the soaked blood vessels, rinse them with PBS buffer solution, and soak them in PBS buffer solution. Change the PBS buffer solution every day; (4) Take out the soaked blood vessels, trim them into pieces, and freeze-dry them to obtain the decellularized scaffold DCS. 7. Application according to claim 6, characterized in that, in step (2), the mass ratio of the sodium lauryl sulfate and polyethylene glycol octylphenyl ether is 1:1~1:2, The soaking time in the mixed solution is 24 to 48 hours; in step (3), the soaking time in the PBS buffer solution is 20 to 30 days, and the number of times of changing the PBS buffer solution per day is 1 to 2 times; step (3) In (4), the freeze-drying temperature is -45~-55°C, and the freeze-drying time is 8~12h.