A hydrogel and a biological artificial blood vessel prepared therefrom

Abstract

The present invention relates to the field of biotechnology, and in particular to a hydrogel and a bio-type artificial blood vessel prepared therefrom. The present invention adopts natural polymer materials such as carboxymethyl chitosan, oxidized sodium alginate and 2,3-butanediol to make a hydrogel with good biocompatibility, and also provides a bio-type artificial blood vessel prepared by using the hydrogel and a preparation method thereof. The bio-type artificial blood vessel provided by the present invention has low overall water permeability, strong biocompatibility, good moisturizing and antibacterial effects, and has a good effect of promoting cell growth. The preparation process of the hydrogel and the bio-type artificial blood vessel of the present invention is simple, and the selected materials have a wide range of sources and are inexpensive, and are suitable for large-scale production applications.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a hydrogel and a biological artificial blood vessel prepared therefrom. Background Art

[0002] A large number of studies have shown that the incidence of cardiovascular disease increases with age. As China's population ages, there will be more and more cardiovascular and cerebrovascular patients, and a considerable number of them will experience vascular blockage and vascular necrosis. In this case, vascular transplantation is needed. Currently, normal blood vessels of others can be transplanted into the body, but this method has a small number of donors, is expensive, and has strong rejection. Other parts of the body can also be transplanted, such as the great saphenous vein, mammary artery, etc., but this method will damage tissues in other parts. Therefore, from the perspective of treatment conditions, the development of artificial blood vessels is particularly important.

[0003] There are two types of artificial blood vessels at present: artificial blood vessels constructed with artificial materials and decellularized artificial blood vessels. Most of the blood vessels constructed with artificial materials are woven or knitted with polymer materials, with a high rate of blood seepage, and it is difficult to match the compliance of the own blood vessels. Studies have shown that the incidence of thrombosis and intimal hyperplasia is 40%-60% in synthetic vascular implants one year after transplantation. Artificial blood vessels made from allogeneic animals after decellularization retain the natural three-dimensional model, have good biological descriptive properties and mechanical properties, and are considered to be the most ideal vascular substitute in the 21st century. However, the chemical reagents that may be used in the decellularization process of decellularized artificial blood vessels will cause the three-dimensional structure of the extracellular matrix to become loose, resulting in higher porosity and permeability than the original blood vessels. Due to the weak sealing, artificial blood vessels may leak blood in a short period of time after being transplanted into the allogeneic body, affecting the efficiency of transplantation.

[0004] Hydrogel has a cross-linked network that can swell in water and retain a large amount of water without being dissolved. It is a high-molecular functional material that integrates water absorption, water retention, and controlled release. Moreover, hydrogel is similar to life tissue materials and has properties similar to extracellular matrix. When in contact with blood, body fluids and human tissues, it shows good biocompatibility and can be used as a gap filler for artificial blood vessels to seal artificial blood vessels. Existing studies have found that the use of synthetic hydrogels based on polymers such as poly(ethylene oxide) to seal braided artificial blood vessels can effectively reduce vascular permeability, but the biocompatibility of the polymer hydrogel is weak and is not suitable for biological artificial blood vessels. So far, there are few reports on hydrogels that can reduce vascular permeability and improve biocompatibility and the biological artificial blood vessels prepared therefrom. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a hydrogel and a bio-artificial blood vessel prepared therefrom. The hydrogel provided by the present invention has strong biocompatibility, and the bio-artificial blood vessel prepared using the hydrogel has low overall water permeability, strong biocompatibility, good moisturizing and antibacterial effects, and at the same time has a good effect of promoting cell growth.

[0006] The present invention provides a hydrogel comprising the following raw materials:

[0007] Carboxymethyl chitosan 4%-9% (w / v), oxidized sodium alginate 7%-9% (w / v), 2,3-butanediol 7.6%-42.7% (w / v), and the solution is PBS solution.

[0008] The hydrogel prepared by using the raw materials provided by the present invention is more suitable for preparing biological artificial blood vessels. Compared with other hydrogels, the hydrogel prepared by using the raw materials within the above concentration range has stronger biocompatibility, moisture retention, antibacterial property, and cell growth promotion performance due to the close coordination between the raw material components, and can effectively improve the overall water leakage, biocompatibility, moisture retention, antibacterial property, and cell growth promotion performance of biological artificial blood vessels, thereby achieving better technical effects.

[0009] Preferably, the hydrogel comprises the following raw materials:

[0010] Carboxymethyl chitosan 6% (w / v), oxidized sodium alginate 7% (w / v), 2,3-butanediol 27% (w / v), and the solution is PBS solution.

[0011] Experiments show that the hydrogel made with raw materials within this concentration range has the strongest biocompatibility, moisture retention and antibacterial properties. Among them, PBS solution includes 0.85% sodium chloride, 0.29% disodium hydrogen phosphate, 0.03% sodium dihydrogen phosphate, and the rest is water, with a pH value of 7.3-7.5.

[0012] The present invention also provides a method for preparing the hydrogel, comprising dissolving carboxymethyl chitosan in a PBS solution to obtain a solution A, dissolving oxidized sodium alginate and 2,3-butanediol in a PBS solution to obtain a solution B, and mixing the solution A and the solution B to obtain the hydrogel through ethanol cross-linking.

[0013] Furthermore, the preparation method of the carboxymethyl chitosan is as follows: chitosan and sodium hydroxide are dissolved in isopropanol, heated and swollen to obtain a chitosan suspension, a mixed solution of chloroacetic acid and isopropanol is added dropwise to the chitosan suspension, a crude product is obtained after reaction, and the crude product is poured into ethanol, washed, filtered, and dried to obtain carboxymethyl chitosan.

[0014] Furthermore, the preparation method of the oxidized sodium alginate is as follows: adding a sodium periodate aqueous solution dropwise to a sodium alginate aqueous solution, reacting in the dark, adding ethylene glycol and sodium chloride, stirring for reaction, precipitating the reaction solution with ethanol and filtering, dissolving the obtained precipitate in water and dialyzing it, and then freeze-drying the obtained solution to obtain oxidized sodium alginate.

[0015] Furthermore, in the preparation method, the volume ratio of the solution A to the solution B is 1:1.

[0016] The invention provides the hydrogel and application of the hydrogel prepared by the preparation method in preparing artificial blood vessels.

[0017] The present invention also provides a biological artificial blood vessel, comprising the hydrogel or the hydrogel prepared by the preparation method, and an artificial blood vessel skeleton.

[0018] Preferably, in the method for preparing the bio-artificial blood vessel, the solution A and the solution B are sprayed on the surface of the artificial blood vessel skeleton by atomization, and then cross-linked with ethanol to obtain the bio-artificial blood vessel with a hydrogel on the surface.

[0019] Further preferably, the cross-linked bioartificial blood vessel is soaked in a glycerol solution to obtain a bioartificial blood vessel containing hydrogel on the surface.

[0020] The present invention uses natural polymer materials such as carboxymethyl chitosan, oxidized sodium alginate and 2,3-butanediol to prepare a hydrogel with good biocompatibility, and also provides a bio-artificial blood vessel prepared using the hydrogel and a preparation method thereof. The bio-artificial blood vessel provided by the present invention has low overall water permeability, strong biocompatibility, good moisturizing and antibacterial effects, and has a good effect of promoting cell growth. Experiments show that the overall water permeability of the bio-artificial blood vessel provided by the present invention reaches 0.1 (mL / min·cm 2 ), the moisturizing rate is as high as 79.4%; the hemolysis rate is less than 5%, the thrombosis Fbg content is not significantly different from that of autologous blood vessels, and the cell survival rate has reached 90%, indicating that it has good biocompatibility; compared with the biological artificial blood vessels prepared by tannic acid, the biological artificial blood vessels provided by the present invention have stronger ability to promote cell growth and inhibit the proliferation of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. In addition, the preparation process of the hydrogel and biological artificial blood vessels of the present invention is simple, and the selected materials have a wide range of sources and are inexpensive, which are suitable for large-scale production applications. DETAILED DESCRIPTION

[0021] The present invention provides a hydrogel and a bio-artificial blood vessel prepared therefrom. A person skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the same. It should be particularly noted that all similar substitutions and modifications are obvious to a person skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0022] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0023] Example 1 2% CMCS / 4% SAO hydrogel coated biological artificial blood vessel

[0024] The steps of preparing the bio-artificial blood vessel containing hydrogel are as follows:

[0025] (1) Disperse 5 g of chitosan and 5 g of sodium hydroxide in 50 mL of isopropanol and swell at 60° C. for 2 h to obtain a chitosan suspension; dissolve 5 g of chloroacetic acid in 10 mL of isopropanol; slowly add the chloroacetic acid solution dropwise to the chitosan suspension at 70° C. and react for 4 h to obtain a crude product; the crude product is poured into ethanol, washed three times, filtered, and dried at 60° C. to obtain carboxymethyl chitosan (CMCS).

[0026] (2) 5 g of sodium alginate was dissolved in 250 mL of deionized water to prepare a 2% aqueous solution. After the sodium alginate was completely dissolved, 5 g of sodium periodate was dissolved in 50 mL of deionized water to prepare a 10% solution. The solution was slowly added dropwise to the sodium alginate solution under light-proof and rapid stirring conditions. The solution was reacted at room temperature in the dark for 6 h. After the reaction was completed, 1 mL of ethylene glycol and 5 g of sodium chloride were added dropwise and stirred for 30 min. The reaction solution was then poured into 500 mL of ethanol to precipitate, which was filtered. Finally, the precipitate was redissolved in 50 mL of deionized water, dialyzed for three days using a dialysis bag with a molecular weight cutoff of 8000 to 15000, and the solution was freeze-dried at -80°C to obtain oxidized sodium alginate (SAO).

[0027] (3) Weigh 100 mg of CMCS prepared in step (1) and dissolve it in 2.5 mL of PBS solution (PBS solution consists of water and three substances, specifically: 0.85% sodium chloride, 0.29% disodium hydrogen phosphate, 0.03% sodium dihydrogen phosphate, pH value is 7.3-7.5, the composition and concentration of PBS solution described below are the same as above) to prepare a 4% (w / v) solution A.

[0028] (4) Weigh 200 mg of the SAO prepared in step (2) and dissolve it in 2.5 mL of PBS solution to prepare 8% (w / v) solution B.

[0029] (5) An artificial blood vessel skeleton (the artificial blood vessel skeleton is made of melt-spinning polycaprolactone PCL, and the artificial blood vessel skeleton is a tubular structure with a hollow interior and open ends) is inserted into the shaft core, and the shaft core is attached to a motor and rotated at a speed of 300 rpm.

[0030] (6) 2.5 mL of solution A and 2.5 mL of solution B were respectively loaded into a double injection spray device, and atomized and sprayed on the rotating artificial blood vessel skeleton at a pressure of 2 atmospheres, and the rotation speed was maintained for 10 minutes to form a uniform bio-hydrogel coating layer. After spraying, the rotation speed was increased to 600 rpm to discharge any excess unreacted solution.

[0031] (7) The bio-artificial blood vessel containing the hydrogel was placed in 94% ethanol for cross-linking treatment for 8 hours to denature the bio-artificial blood vessel protein and initially dehydrate it. Finally, it was immersed in a glycerol solution for 10 hours to remove excess water from the bio-artificial blood vessel and maintain stability, thereby obtaining a 2% CMCS / 4% SAO hydrogel-coated bio-artificial blood vessel.

[0032] Example 2 6% CMCS / 7% SAO hydrogel coated biological artificial blood vessel

[0033] The preparation steps are similar to those in Example 1, except for the concentrations of solution A and solution B. The remaining steps are the same. 300 mg of CMCS was weighed and dissolved in 2.5 mL of PBS solution to prepare a 12% (w / v) solution A, and 350 mg of SAO was weighed and dissolved in 2.5 mL of PBS solution to prepare a 14% (w / v) solution B.

[0034] Example 3 9% CMCS / 9% SAO hydrogel coated biological artificial blood vessel

[0035] The preparation steps are similar to those in Example 1, except for the concentrations of solution A and solution B. The remaining steps are the same. 450 mg of CMCS was weighed and dissolved in 2.5 mL of PBS solution to prepare 18% (w / v) solution A, and 450 mg of SAO was weighed and dissolved in 2.5 mL of PBS solution to prepare 18% (w / v) solution B.

[0036] Example 4 2% CMCS / 4% SAO / 7.6% BA (1,2-butanediol) hydrogel-coated biological artificial blood vessel

[0037] The preparation steps are similar to those of Example 1, except for the concentration of solution B. The remaining steps are the same. 200 mg SAO and 380 mg BA were weighed and dissolved in 2.5 mL of PBS solution at 4°C to prepare solution B with a SAO concentration of 8% (w / v) and a BA (1,2-butanediol) concentration of 15.2% (w / v).

[0038] Example 5 6% CMCS / 7% SAO / 7.6% BA hydrogel coated biological artificial blood vessel

[0039] The preparation steps are similar to those of Example 2, except for the concentration of solution B. The remaining steps are the same. 350 mg SAO and 380 mg BA were weighed and dissolved in 2.5 mL PBS solution at 4°C to prepare solution B with a SAO concentration of 14% (w / v) and a BA concentration of 15.2% (w / v).

[0040] Example 6 6% CMCS / 7% SAO / 27% BA hydrogel coated biological artificial blood vessel

[0041] The preparation steps are similar to those of Example 5, except for the concentration of BA in solution B. The remaining steps are the same. 350 mg of SAO and 1.35 g of BA were weighed and dissolved in 2.5 mL of PBS solution at 4°C to prepare solution B with a SAO concentration of 14% (w / v) and a BA concentration of 54% (w / v).

[0042] Example 7 6% CMCS / 7% SAO / 42.7% BA hydrogel-coated biological artificial blood vessel

[0043] The preparation steps are similar to those of Example 5, except for the concentration of BA in solution B. The remaining steps are the same. 350 mg of SAO and 2.135 g of BA were weighed and dissolved in 2.5 mL of PBS solution at 4°C to prepare solution B with a SAO concentration of 14% (w / v) and a BA concentration of 85.4% (w / v).

[0044] Example 8 9% CMCS / 9% SAO / 42.7% BA hydrogel-coated biological artificial blood vessel

[0045] The preparation steps are similar to those of Example 3, except for the concentration of solution B. The remaining steps are the same. 450 mg SAO and 2.135 g BA were weighed and dissolved in 2.5 mL PBS solution at 4°C to prepare solution B with a SAO concentration of 18% (w / v) and a BA concentration of 85.4% (w / v).

[0046] Comparative Example 1: Bioartificial blood vessel without hydrogel coating

[0047] The bio-artificial blood vessel was not subjected to any hydrogel coating process and was stored in a PBS solution at 4°C. Comparative Example 26% CMCS hydrogel-coated bio-artificial blood vessel

[0048] (1) Disperse 5 g of chitosan and 5 g of sodium hydroxide in 50 mL of isopropanol and swell at 60° C. for 2 h to obtain a chitosan suspension; dissolve 5 g of chloroacetic acid in 10 mL of isopropanol; slowly add the chloroacetic acid solution dropwise to the chitosan suspension at 70° C. and react for 4 h to obtain a crude product; the crude product is poured into ethanol, washed three times, filtered, and dried at 60° C. to obtain carboxymethyl chitosan (CMCS).

[0049] (2) Weigh 300 mg of CMCS and dissolve it in 2.5 mL of deionized water to prepare a 12% (w / v) solution C.

[0050] (3) Weigh 60 mg of glutaraldehyde and dissolve it in 2.5 mL of deionized water to prepare a 2.4% (w / v) solution D.

[0051] (4) Insert the artificial blood vessel skeleton onto the shaft core, and attach the shaft core to the motor to rotate at a speed of 300 rpm.

[0052] (5) 2.5 mL of solution C and 2.5 mL of solution D were respectively loaded into a double-injection spray device, and atomized and sprayed on the rotating artificial blood vessel skeleton at a pressure of 2 atmospheres, and the rotation speed was maintained for 30 minutes to form a uniform bio-hydrogel coating layer. After spraying, the rotation speed was increased to 600 rpm to discharge any excess unreacted solution.

[0053] (6) The bio-artificial blood vessel containing the hydrogel was placed in 94% ethanol for cross-linking treatment for 8 hours to denature the bio-artificial blood vessel protein and initially dehydrate it. Finally, it was immersed in a glycerol solution for 10 hours to remove excess water from the bio-artificial blood vessel and keep it stable, thereby obtaining a 6% CMCS hydrogel-coated bio-artificial blood vessel. Comparative Example 3 10% CMCS / 10% SAO hydrogel-coated bio-artificial blood vessel

[0054] The preparation steps are similar to those of Example 1, except for the concentrations of solution A and solution B, and the remaining steps are the same. 500 mg of CMCS was weighed and dissolved in 2.5 mL of PBS solution to prepare a 20% (w / v) solution A, and 500 mg of SAO was weighed and dissolved in 2.5 mL of PBS solution to prepare a 20% (w / v) solution B.

[0055] Comparative Example 4 6% CMCS / 7% SAO / 44% BA hydrogel coated biological artificial blood vessel

[0056] The preparation steps are similar to those of Example 5, except for the concentration of BA in solution B. The remaining steps are the same. 350 mg of SAO and 2.2 g of BA were weighed and dissolved in 2.5 mL of PBS solution at 4°C to prepare solution B with a SAO concentration of 14% (w / v) and a BA concentration of 88% (w / v).

[0057] Comparative Example 5: 6% CMCS / 7% SAO / 27% tannic acid hydrogel coated biological artificial blood vessel

[0058] The preparation steps are similar to those of Example 5, except for the substances and concentrations in solution B, and the remaining steps are the same. 350 mg of SAO and 675 mg of tannic acid were weighed and dissolved in 2.5 mL of PBS solution to prepare solution B with a SAO concentration of 14% (w / v) and a tannic acid concentration of 54% (w / v).

[0059] Effect example

[0060] 1. Overall water leakage performance test

[0061] The bioartificial blood vessel was soaked in sterile saline for 3 times, each time for 5min-15min. The proximal end was connected to an adapter with a specific inner diameter to ensure sealing, and the adapter and the bioartificial blood vessel were connected to a fixture for pressure generation and measurement. The intraluminal pressure of the sample was gradually increased to 16.0kPa±0.3kPa (120mmHg±2mmHg), and the pressure was maintained to measure the leakage through the vascular prosthesis wall within 60s. Table 1 is the overall water leakage performance test results of the embodiment and the comparative example. The overall water leakage of the bioartificial blood vessel without hydrogel coating is large and the density is small. When coated with carboxymethyl chitosan hydrogel alone, the water leakage is reduced. After the addition of oxidized sodium alginate, the water leakage continues to decrease. The overall water leakage of the bioartificial blood vessel coated with 6% CMCS / 7% SAO hydrogel is reduced to 0.5 (mL / min·cm 2 ) or less, with good density, and there was no significant difference in the overall water leakage of the bio-artificial blood vessels coated with 9% CMCS / 9% SAO hydrogel. The addition of BA reduced the overall water leakage to 0.2 (mL / min·cm 2 ) or less, the density was improved, and the overall water leakage of the bioartificial blood vessel coated with 6% CMCS / 7% SAO / 27% BA hydrogel reached 0.1 (mL / min·cm 2 ), which has the strongest sealing property. However, the sealing effect of the biological artificial blood vessel with added tannic acid is not as good as that of BA.

[0062] 2. Biocompatibility Testing

[0063] (1) Hemolysis rate: Take blood from the ear artery of a healthy rabbit and anticoagulate it with 3.8% sodium citrate solution at a ratio of 1:9. Take 4 mL of fresh anticoagulated rabbit blood and add it to 5 mL of 0.9% sodium chloride injection and mix well to obtain diluted anticoagulated rabbit blood. Soak the bioartificial blood vessel in sterile saline for 3 times, 5 min to 15 min each time. Take a 0.5 cm long section of the sample and add it to a centrifuge tube containing 10 mL of saline. Place it in a constant temperature water bath at (37±1)℃ for 30 min, add 0.2 mL of diluted anticoagulated rabbit blood, and place it in a water bath at (37±1)℃ for 60 min. Centrifuge at 1000 r / min for 5 min and take the supernatant. Add 10 mL of 0.9% sodium chloride solution to each tube of the negative control group; add 10 mL of distilled water to each tube of the positive control group. The OD value at 545 nm was measured using an ultraviolet spectrophotometer, and the hemolysis rate (%) was calculated as (sample absorbance - negative control group absorbance) / (positive control group absorbance - negative control group absorbance). The results are shown in Table 1.

[0064] (2) Thrombosis: Take a 5 cm long bio-artificial blood vessel and place it in a siliconized glass tube as a test sample. Rabbit autologous blood vessels are treated in the same way as a control sample. Take fresh healthy rabbit blood and anticoagulate it with 0.4 mL of 3.8% sodium citrate solution. Inject 0.8-1.0 mL of blood into each tube. After standing at room temperature for 15 minutes, add 10 ul of 6250 U / mL heparin sodium solution to each tube, shake and mix thoroughly, stand for 30 minutes, centrifuge at 2000 r / min at 4°C for 10 minutes, and draw the upper plasma to detect the Fbg content on a coagulation meter. The Fbg content of the control sample is 2.3 g / L. The results are shown in Table 1.

[0065] (3) Cell survival rate: The biological artificial blood vessel was soaked in sterile saline for 3 times, each time for 5 min to 15 min. The biological artificial blood vessel was made into a circular membrane with a diameter of 14 mm, which could fit smoothly and seamlessly with the bottom of the 24-well plate. It was sterilized by ultraviolet light in a sterile table, and the blood vessel sheet was spread flat on the bottom of the 24-well plate. After high-pressure sterilization, it was pressed with a stainless steel ring. The culture medium without biological artificial blood vessels was used as the blank control group. The cultured human aortic endothelial cells were digested and suspended with 0.25% trypsin at a density of 2×10 per well. 4 The cell suspension of 100 μL / mL was inoculated on a 24-well plate and continuously cultured in a constant temperature incubator at 37°C with 5% CO2. The cell survival rate was quantitatively analyzed using the CCK-8 method after 1 or 2 days of continuous culture. The corresponding well plate was taken out at the specified time interval, 50 μL of CCK-8 working solution was added to each well, and after incubation in a constant temperature carbon dioxide incubator at 37°C for 1 to 2 hours, the OD value was measured at a wavelength of 450 nm using an enzyme marker to calculate the cell survival rate. The results are shown in Table 1.

[0066] As can be seen from Table 1, the hemolysis rates of Examples 1 to 8 are all <5%, the thrombosis Fbg content is not significantly different from that of autologous blood vessels, and the cell survival rate is also 90%. The presence of hydrogel can improve biocompatibility. Although the presence of BA will cause a slight decrease in biocompatibility, it is still within the standard range.

[0067] 3. Moisturizing performance test

[0068] Cut the bio-artificial blood vessel into 5 cm lengths, weigh the weight before water absorption W0, and soak it in sterile saline for 3 times, 5 min to 15 min each time. Then soak it in purified water for 3 hours, changing the water every hour to ensure that the hydrogel completely absorbs water. Wipe off excess water on the surface with paper and weigh the initial weight of the bio-artificial blood vessel after water absorption W1. Then place it in an environment of 20°C and 17% relative humidity, and weigh the weight W after 24 hours. t , calculate the moisture retention rate (%) = (W t / W1)×100%. The results are shown in Table 1. Comparative Example 1 has almost no water absorption due to the absence of hydrogel coating. From the results, carboxymethyl chitosan and oxidized sodium alginate hydrogels have moisturizing effects themselves, and the moisturizing effect is significantly improved after adding BA. The bio-artificial blood vessel coated with 6% CMCS / 7% SAO / 27% BA hydrogel can even retain 79.4% of water, indicating that BA has a good moisturizing effect. However, the moisturizing rate of excessive BA (44%) is almost no longer improved. The addition of tannic acid to the hydrogel does not increase the water retention rate.

[0069] 4. Cell Growth Performance

[0070] The biological artificial blood vessel was soaked in sterile saline for 3 times, each time for 5min-15min, and the thickness D0 before implantation was measured using a thickness meter. The experimental sheep were fasted for 1 day before the operation, and were anesthetized by intramuscular injection of midazolam (0.05mg / kg) and lumianning (1.50mg / kg), and endotracheal intubation was performed for continuous anesthesia. The surgical site was shaved and disinfected, and the back skin of the experimental sheep was cut with a sterile scalpel. The biological artificial blood vessel was implanted into the cavity under the sheepskin through a sterile steel tube and the incision. The wound was thoroughly cleaned and sutured. It was raised for 28 days, of which penicillin was injected for the first 3 days. After 28 days, the experimental sheep was anesthetized again, and the cortex around the graft was cut along the graft with a sterile scalpel. The graft was removed and placed in sterile PBS. The fixed parts at both ends were cut off, and the thickness D was measured. t . Calculate tissue growth thickness = D t -D0. The results are shown in Table 1.

[0071] As can be seen from the table, hydrogel coating can promote tissue cell adhesion and growth. Carboxymethyl chitosan hydrogel has a cell growth promoting effect but it is not obvious. When oxidized sodium alginate is added, the cell promoting effect is significantly enhanced, and it increases with the increase of concentration. When the concentration reaches 10% CMCS / 10% SAO, it does not further improve the cell adhesion and growth ability of the bio-artificial blood vessel of the present invention. When BA is added, the tissue growth thickness increases significantly, indicating that BA has the effect of promoting cell growth. Although tannic acid also has a cell growth promoting effect, the effect is slightly weaker than that of BA.

[0072] Table 1 Comparison of overall water leakage, biocompatibility, moisture retention and cell growth performance of the examples and comparative examples

[0073]

[0074] 5. Antibacterial performance test

[0075] Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa were cultured in Luria-Bertani medium at 37°C for 12h. Then 100μL was evenly spread on solid agar medium. The biological artificial blood vessel was soaked in sterile saline for 3 times, 5min-15min each time, and a circular sample with a diameter of 8mm was cut and sterilized by ultraviolet irradiation on a clean bench for 30min. It was placed on the surface of the culture medium and cultured at 37°C for 24h. After 24h of culture, the bacterial growth on the culture medium was observed and the diameter of the inhibition zone was recorded. The test results are shown in Table 2. It can be seen from Table 2 that carboxymethyl chitosan and oxidized sodium alginate hydrogels themselves have certain antibacterial effects, and the addition of oxidized sodium alginate can improve the antibacterial effect. In the range of hydrogel concentration 2% CMCS / 4% SAO to 9% CMCS / 9% SAO, the antibacterial effect increases with the increase of concentration. After exceeding the concentration of 9% CMCS / 9% SAO, the antibacterial effect no longer changes significantly. After adding BA, the antibacterial effect was significantly enhanced, and there was no significant change in the antibacterial effect when the BA concentration was greater than 42.7%. Compared with BA, the antibacterial effect of tannic acid at the same concentration was weaker.

[0076] Table 2 Diameters of inhibition zones of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa in Examples and Comparative Examples

[0077]

[0078] In Tables 1-2, differences were analyzed between Examples 1-8. Those with one identical letter are not significantly different (P>0.05), and those with different letters are significantly different (lowercase letters indicate P<0.05, uppercase letters indicate P<0.01). For those with two letters, for example, the overall water leakage ef in Example 2 indicates that it is significantly different from Example 4 marked with the letter d, P<0.05, compared with Comparative Examples 3-5 and Example 3 marked with the same letter e, P>0.05, and compared with Example 5, Example 7, and Example 8 marked with the same letter f, P>0.05.

[0079] According to the comprehensive analysis of the above tests, the most suitable mass volume percentage range of the coating hydrogel for the sealing biological artificial blood vessel is CMCS 2-9%, SAO 4-9%, BA 7.6-42.7%, and the optimal concentration is CMCS 6%, SAO 7%, and BA 27%.

[0080] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A biological artificial blood vessel, characterized in that: Including hydrogel and artificial blood vessel skeleton; The hydrogel is made of the following raw materials: 6% (w / v) carboxymethyl chitosan, 7% (w / v) oxidized sodium alginate, and 27% (w / v) 2,3-butanediol, and the solution is PBS solution.

2. The bioartificial blood vessel according to claim 1, characterized in that: The preparation method of the hydrogel comprises: dissolving carboxymethyl chitosan in PBS solution to obtain solution A, dissolving oxidized sodium alginate and 2,3-butanediol in PBS solution to obtain solution B, and mixing solution A and solution B to obtain the hydrogel through ethanol cross-linking.

3. The bioartificial blood vessel according to claim 2, characterized in that: The preparation method of the carboxymethyl chitosan comprises the following steps: dissolving chitosan and sodium hydroxide in isopropanol, heating and swelling to obtain a chitosan suspension, dripping a mixed solution of chloroacetic acid and isopropanol into the chitosan suspension, reacting to obtain a crude product, and pouring the crude product into ethanol, washing, filtering and drying to obtain the carboxymethyl chitosan.

4. The bioartificial blood vessel according to claim 3, characterized in that: The preparation method of the oxidized sodium alginate comprises the following steps: adding a sodium periodate aqueous solution dropwise to a sodium alginate aqueous solution, reacting in the dark, adding ethylene glycol and sodium chloride, stirring for reaction, precipitating the reaction solution with ethanol and filtering, dissolving the obtained precipitate in water and dialyzing the solution, and then freeze-drying the obtained solution to obtain the oxidized sodium alginate.

5. The bioartificial blood vessel according to any one of claims 2 to 4, characterized in that: The volume ratio of the solution A to the solution B is 1:

1.

6. The method for preparing the bio-artificial blood vessel according to claim 1, characterized in that: The solution A and the solution B described in any one of claims 2 to 5 are used, and after the solution A and the solution B are sprayed on the surface of the artificial blood vessel skeleton by atomization, they are cross-linked with ethanol to obtain a biological artificial blood vessel with a hydrogel on the surface.

7. The preparation method according to claim 6, characterized in that: The biological artificial blood vessel after the cross-linking treatment is soaked in a glycerol solution to obtain a biological artificial blood vessel with hydrogel on the surface.

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