CN115212350B - Application of hydrogel in preparation of high-bionic artificial blood vessel material - Google Patents
Application of hydrogel in preparation of high-bionic artificial blood vessel material Download PDFInfo
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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
Technical Field
The invention relates to application of hydrogel in preparation of high-bionic artificial vascular materials, and belongs to the field of tissue engineering.
Background
Cardiovascular diseases have presented new challenges to humans since the twenty-first century. The World Health Organization (WHO) indicates from a current analysis of major diseases that pose a threat to human health that cardiovascular disease has become the leading cause of death worldwide. According to the statistics of world health organization, 1200 thousands of people die from cardiovascular diseases every year worldwide, accounting for 1/4 of the total deaths. Cardiovascular disease has been recognized as a first killer in human health that drives concurrently with cancer. Cardiovascular disease refers to the common name of diseases caused by heart and vascular diseases, including coronary heart disease (heart attack), cerebrovascular disease (stroke), elevated blood pressure (hypertension), rheumatic heart disease, congenital heart disease, heart failure, peripheral arterial vascular disease, etc. In recent years, with the change of the eating habits of the national people and the increasing problem of population aging, the incidence rate of cardiovascular and cerebrovascular diseases rises year by year. On day 3, 9 in 2019, lancets have released two recent results on line. Focusing on the common morbidity and mortality of 21 countries, and respectively showing the common causes of death in low, medium and high income countries. The results indicate that cardiovascular disease has far higher mortality in low and medium income countries than in developed countries.
In the treatment process of coronary heart disease, vascular injury, lower limb arterial ischemia and other diseases, various vascular grafting operations such as coronary artery bypass surgery and the like are usually required, which leads to the increasing expansion of the notch of the vascular graft in China. At present, artificial blood vessels based on dacron and polytetrafluoroethylene are widely applied to reconstruction processes of large-caliber blood vessels (with the diameter larger than 6 mm), but the materials have the defects of thromboembolism, intimal hyperplasia, low patency rate and the like in small-caliber blood vessels (with the diameter smaller than 6 mm), so that the materials are very lack of small-caliber blood vessels with good blood compatibility and patency, which can be applied to clinic at present, and mostly only rely on autologous blood vessel transplantation. However, autologous vascular grafting has the defects of difficult material source, insufficient length of vessel pedicles, unmatched diameters and the like.
Vascular grafts, which are currently in relatively widespread clinical use, mainly include synthetic material vessels and autologous vessels. Autologous blood vessels cannot be widely used due to the disadvantages of limited sources, large donor sacrifice, etc. And vascular prostheses are favored because of their wide sources. The most widely used materials for vascular prostheses in clinical medicine are polyester-based materials such as dacron and expanded polytetrafluoroethylene. Polyester materials are widely used for preparing artificial blood vessel materials with easy processability, self microporous structure and excellent mechanical properties. However, the hydrophobic property of the surface of the medicine is easy to induce thrombosis, and the medicine needs to be taken after operation, so that the clinical development and application of the medicine are severely limited. The characteristics of no antigenicity, no carcinogenicity, no toxicity and close to the compliance of autologous arteries of the expanded polytetrafluoroethylene (ePTFE) material lead the expanded polytetrafluoroethylene artificial blood vessel material to be most widely applied in clinic. Although the preparation has the characteristics of good biocompatibility, difficult thrombus formation and the like, the problem of lumen stenosis can occur in the later stage of clinical transplantation, so that the preparation has the problem in small-caliber vascular transplantation operation. The artificial blood vessel having excellent properties such as good blood compatibility, cell compatibility, and adhesion and proliferation ability of endothelial progenitor cells, etc. should have various properties similar to those of natural blood vessels. At the same time, the excellent artificial blood vessel material should also have biomechanical properties similar to those of normal blood vessel. In addition, organism metabolism is continuously carried out, natural blood vessels are continuously updated, and the updating has a certain periodicity, so that the ideal artificial blood vessel not only needs to have biodegradability, but also has a degradation period matched with the actual degradation period of the organism. Therefore, finding a proper theory and method to construct the artificial vascular material with excellent properties similar to those of natural blood vessels is particularly important in life science and bionics research.
Aiming at the problems, domestic and foreign scientific researchers have conducted long-term researches. Research shows that the vascular graft material based on tissue engineering can meet clinical requirements and mechanical bionic properties at the same time. Among them, vascular tissue engineering is the science of preparing, reconstructing and regenerating vascular substitute materials by using biodegradable scaffold materials. The artificial vascular material based on the decellularized vascular stent can avoid immune rejection reaction after implantation while maintaining the degradation rate and mechanical performance advantages of the vascular itself. However, the stent material prepared by the decellularization method removes the endothelial cell layer on the inner surface of the blood vessel, and only one three-dimensional network structure remains, so that the surface of the decellularized stent must be subjected to membranous modification.
Therefore, the design of the composite material which can ensure good biocompatibility and bionic performance on the basis of keeping the advantages of the decellularized scaffold, maintain long-term smoothness of an implantation site and finally realize endothelialization is a key breakthrough direction of vascular tissue engineering and is a precondition for realizing clinical application. By utilizing the principle of tissue engineering and combining the tissue engineering small-caliber blood vessel constructed by the scaffold material, cells and biological signal molecules, a new thought is provided for solving the problems, and the method is one of the most potential solutions at present.
Disclosure of Invention
The invention aims to: the invention aims to provide application of hydrogel in preparation of high-bionic artificial blood vessel materials.
The technical scheme is as follows: the hydrogel is chitosan/polyethylene glycol hydrogel or acetalized polyvinyl alcohol hydrogel.
The hydrogel is a gel structure formed by water-soluble polymers with a crosslinked three-dimensional network structure and taking water as a dispersion medium. After the polyethylene glycol or polyvinyl alcohol water-soluble polymer meets water, hydrophilic groups in the polyethylene glycol or polyvinyl alcohol water-soluble polymer can be gathered with water molecules, and hydrophobic groups can expand when meeting water, so that hydrogel can not only be dissolved in water, but also be swelled, and a large amount of water can be absorbed and locked.
Further, the preparation method of the chitosan/polyethylene glycol hydrogel comprises the following steps:
(1) Preparing an NaOH aqueous solution, and then adding absolute ethyl alcohol to obtain the NaOH solution;
(2) Adding chitosan into NaOH solution while stirring, and alkalizing;
(3) Adding propylene oxide, and placing the mixture in a constant-temperature water bath for reaction to obtain a product;
(4) Taking out the product, placing the product in a mixed solution of hydrochloric acid and acetone for washing, placing the product in a mixed solution of acetone and water for washing, vacuum-filtering, and vacuum-drying to obtain O-HPCS, wherein the reaction mechanism is as follows;
(5) Preparing an O-HPCS aqueous solution, adding polyethylene glycol and glutaraldehyde solution, stirring and mixing, and standing to obtain chitosan/polyethylene glycol hydrogel.
Still further, in the step (1), the mass concentration of the NaOH solution is 6% -8%; in the step (2), the liquid-solid ratio of the NaOH solution to the chitosan is 10-12 mL: g, the alkalization time is 6-8 h; in the step (3), the reaction time is 24-36 h; in the step (4), the mass ratio of hydrochloric acid to acetone in the mixed solution of hydrochloric acid and acetone is 1:10-1:9, the mass ratio of acetone to water in the mixed solution of acetone and water is 9:1-10:1, the temperature of vacuum drying is 45-55 ℃, and the vacuum drying time is 2-3 hours; in the step (5), the solid-to-liquid ratio of the polyethylene glycol to glutaraldehyde is 0.1-0.2 g: and (3) mL.
Further, the preparation method of the acetalized polyvinyl alcohol hydrogel comprises the following steps:
(1) Mixing polyvinyl alcohol with water, and heating to dissolve;
(2) Adding glycerol, and continuing heating;
(3) Cooling, adding formaldehyde and glutaraldehyde respectively, and stirring uniformly to obtain acetalized polyvinyl alcohol;
(4) And (3) drying the acetalized polyethylene, heating to continue drying, cooling, soaking and flushing with water to obtain the acetalized polyvinyl alcohol hydrogel.
The basic reaction equation for acetalization of polyvinyl alcohol is as follows:
still further, in the step (1), the mass ratio of the polyvinyl alcohol to the water is 1:30-5:90, and the heating temperature is 90-100 ℃; in the step (2), the mass ratio of the glycerol to the polyvinyl alcohol is 1: 1-2, wherein the continuous heating time is 0.5-1 h; in the step (3), the mass ratio of formaldehyde to glutaraldehyde to polyvinyl alcohol is 2:6:3-2:6:5, and the concentration of glutaraldehyde is 0.5-9%; in the step (4), the drying temperature is 40-60 ℃, the drying time is 2-3 h, the reheating temperature is 60-70 ℃, the continuous drying time is 1-2 h, and the soaking and flushing time is 0.5-1 h.
Further, the preparation method of the high-bionic artificial blood vessel material comprises the following steps:
(1) Preparing chitosan/polyethylene glycol hydrogel;
(2) Coating chitosan/polyethylene glycol hydrogel on the surface of a cell-free scaffold to obtain chitosan/polyethylene glycol/cell-free scaffold CS/PEG/DCS;
(3) And depositing heparin on the surface of the chitosan/polyethylene glycol/acellular stent by using a layer-by-layer self-assembly method, and vacuum drying to obtain the high-bionic artificial vascular material n-He-CS/PEG/DCS.
Still further, in the step (3), the layer-by-layer self-assembly method is a soaking pulling method, and the method comprises the following steps:
(3.1) preparing heparin sodium solution and chitosan solution respectively;
(3.2) placing CS/PEG/DCS in PBS buffer solution, taking out, soaking in heparin sodium solution, taking out, and flushing the front and the back of the PBS buffer solution to obtain 1-He-CS/PEG/DCS;
(3.3) placing the 1-He-CS/PEG/DCS in the prepared chitosan solution for soaking, taking out, washing the front and the back of the chitosan solution by using the PBS buffer solution, soaking the chitosan solution in the heparin sodium solution, taking out, and washing the front and the back of the chitosan solution by using the PBS buffer solution to obtain the 2-He-CS/PEG/DCS;
(3.4) repeating the step (3.3) to prepare the multi-layer heparin/polyethylene glycol hydrogel/decellularized scaffold, thus obtaining the n-He-CS/PEG/DCS.
Further, in the step (3.1), the concentration of the heparin sodium solution is 1-2 g/L, the concentration of the chitosan solution is 1-2 g/L, and the mass ratio of the chitosan to the heparin sodium is 1:1 to 2; in the step (3.2), the CS/PEG/DCS is placed in PBS buffer solution for soaking for 10-30 min, and the time for soaking in heparin sodium solution is 10-15 min; in the step (3.3), the 1-He-CS/PEG/DCS is placed in the prepared chitosan solution for 10-15 min, the time for soaking in the heparin sodium solution is 10-15 min, and in the step (3.4), n in the n-He-CS/PEG/DCS is 3-7.
Further, the preparation method of the high-bionic artificial blood vessel material comprises the following steps:
(1) Preparing different acetalized polyvinyl alcohol hydrogels;
(2) Preparing different acetalized heparin-polyvinyl alcohol complexes;
(3) Coating different acetalated heparin-polyvinyl alcohol complexes on the surface of a decellularized scaffold to obtain different acetalated heparin-polyvinyl alcohol/decellularizedThe stent, namely the high bionic artificial blood vessel material He/PVA n /DCS。
Still further, in step (2), the preparation of the acetalized heparin-polyvinyl alcohol complex comprises the steps of: and (3) drying the acetalized polyvinyl alcohol hydrogel to obtain an acetalized polyvinyl alcohol membrane, and soaking the acetalized polyvinyl alcohol membrane in a heparin sodium aqueous solution to obtain an acetalized heparin-polyvinyl alcohol compound.
Still further, the drying temperature is 40-60 ℃, the drying time is 2-3 h, and the mass concentration of the heparin sodium aqueous solution is 1-2%.
Further, the preparation method of the decellularized scaffold comprises the following steps:
(1) Taking out blood vessels placed in tissue fixing liquid, trimming adventitia, and immersing in physiological saline;
(2) Preparing a mixed solution of sodium dodecyl sulfate and polyethylene glycol octyl phenyl ether, and soaking the trimmed blood vessel in the mixed solution;
(3) Taking out the soaked blood vessel, flushing with PBS buffer solution, soaking in the PBS buffer solution, and changing the PBS buffer solution every day;
(4) Taking out the soaked blood vessel, trimming into slices, and freeze-drying to obtain the decellularized scaffold DCS.
Further, in the step (2), the mass ratio of the sodium dodecyl sulfate to the polyethylene glycol octyl phenyl ether is 1:1-1:2, and the soaking time in the mixed solution is 24-48 hours; in the step (3), the time for soaking in the PBS buffer solution is 20-30 days, and the times for replacing the PBS buffer solution every day is 1-2 times; in the step (4), the freeze-drying temperature is-45 to-55 ℃, and the freeze-drying time is 8-12 h.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages:
(1) The decellularized scaffold is derived from natural blood vessels and has the congenital advantages in the aspects of mechanical properties and mechanical bionic properties. Meanwhile, the decellularized scaffold has excellent biocompatibility, and can avoid thromboembolism and immune rejection. The acellular scaffold substrate and the hydrogel are selected to have excellent biodegradability, can be degraded by organisms in a stable environment in vivo, and the degradation products are nontoxic and can be finally absorbed by autologous tissues. The use of decellularized scaffold material derived from natural blood vessels reduces to some extent the probability of immune rejection and calcification after implantation in vivo.
(2) The layer-by-layer self-assembly technology is used for alternately depositing on the surface of the substrate, so that stable and complete molecular aggregates are formed, and the self-assembled film synthesized by the method has remarkable advantages. After the polyethylene glycol hydrogel is coated, the three-dimensional network structure of the decellularized vascular stent is changed, but the mechanical property of the decellularized vascular stent artificial vascular material is not changed, and the material has excellent mechanical bionic property, can bear pressure change generated in the process of blood flowing in a lumen, and keeps the patency of the lumen. After layer-by-layer self-assembly, the biocompatibility of the artificial blood vessel material is obviously improved, and the artificial blood vessel material is favorable for the adhesion growth of endothelial progenitor cells and smooth muscle cells on the artificial blood vessel material.
(3) After the different acetalized heparin-polyvinyl alcohol hydrogels are modified, the advantages of the natural bionics of the decellularized scaffold cannot be greatly influenced due to the low elastic modulus and the good biodegradability of the modified heparin-polyvinyl alcohol hydrogels, and the purposes of covering the decellularized scaffold and improving the blood compatibility of the decellularized scaffold can be achieved. After the polyvinyl acetal modification, the characteristics of the polyvinyl alcohol hydrogel are maintained, and the hydrolysis resistance of the polyvinyl alcohol hydrogel is effectively improved. The modified heparin solution serving as the high bionic artificial blood vessel material can effectively improve the anticoagulation performance and biological compatibility
(4) The high bionic artificial blood vessel material comprises the use of a decellularized blood vessel stent for a material substrate, and has better mechanical bionic performance and degradation rate matching property compared with the traditional artificial synthetic blood vessel material. In addition, the selected materials have the characteristics of wide sources, low price and capability of large-scale production and application.
Drawings
FIG. 1 is an SEM image of a decellularized scaffold;
FIG. 2 is an SEM image of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3 to 7) vascular prosthesis materials;
FIG. 3 is a FT-IR diagram of O-HPCS and CS/PEG;
FIG. 4 is a XPS comparison of CS/PEG/DCS and 5-He-CS/PEG/DCS;
FIG. 5 is a standard chart of acid orange;
FIG. 6 is a bar graph of amino content of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3-7) vascular prosthesis materials;
FIG. 7 is a graph of static contact angles of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3 to 7);
FIG. 8 is a graph of tensile strength, elongation at break and burst strength of a vascular graft material of fresh blood, DCS, CS/PEG/DCS, and n-He-CS/PEG/DCS (n=3-7);
FIG. 9 is a graph of activated partial thromboplastin time, thrombin time, prothrombin time and hypercalcemia time for CS/PEG/DCS and n-He-CS/PEG/DCS (n=3-7) vascular prosthesis materials;
FIG. 10 is a graph of red blood cell morphology of n-He-CS/PEG/DCS (n=3-7);
FIG. 11 is a graph of MTT test of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3 to 7);
FIG. 12 is a graph of in vitro degradation rates of CS/PEG/DCS and 5-He-CS/PEG/DCS vascular prosthesis materials;
FIG. 13 is a schematic illustration of a different acetalized polyvinyl alcohol hydrogel PVA n (n=1 to 6) electron microscope images;
FIG. 14 is a view of heparin-polyvinyl alcohol/decellularized scaffold He/PVA 4 DCS infrared spectrogram;
FIG. 15 is a diagram of heparin-polyvinyl alcohol/decellularized scaffold He/PVA with varying degrees of acetalization n SEM images of DCS (n=1 to 6);
FIG. 16 is PVA/DCS and He/PVA 4 XPS contrast plot of DCS;
FIG. 17 is a view of fresh blood vessel, DCS and He/PVA n Tensile strength, elongation at break and burst strength plots of DCS (n=1 to 6);
FIG. 18 is PVA/DCS and He/PVA 4 Activated partial thromboplastin time, thrombin time, prothrombin time and hypercalcemia time profile of DCS (n=1 to 6) vascular prosthesis material;
FIG. 19 is a view of He/PVA n Red blood cell morphology of DCS (n=1 to 6);
FIG. 20 is a view of He/PVA n MTT test chart of DCS (n=1 to 6);
FIG. 21 is a PVA/DCS and He/PVA 4 In vitro degradation rate diagram of DCS artificial vascular material;
FIG. 22 is a diagram of 5-He-CS/PEG/DCS and He/PVA 4 B-ultrasonic images of DCS artificial vascular materials after being implanted into a body for two weeks;
FIG. 23 is 5-He-CS/PEG/DCS and He/PVA 4 CTA map of the implantation site 5 months after DCS vascular prosthesis material was implanted in the body;
FIG. 24 is a view of a fresh blood vessel, he-Ch-5/PU/DCS, he/PVA 4 Stress-strain graphs of/DCS and 5-He-CS/PEG/DCS.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
Example 1 preparation of three layers heparin-Chitosan/polyethylene glycol hydrogel/Decellularized scaffold (3-He-CS/PEG/DCS)
1. Preparation of decellularized scaffolds (Decellularized scaffold, DCS)
Canine bilateral carotid vessels (supplied by vascular surgery in the drummer hospital, south kyo city) placed in 4% paraformaldehyde tissue fixative were removed and the adventitia trimmed and immersed in 0.9% saline. 0.5g of sodium dodecyl sulfate and 1g of polyethylene glycol octyl phenyl ether are taken and dissolved in 100mL of deionized water to prepare a mixed solution with the concentration of 1.5%, and the trimmed canine bilateral carotid blood vessels are soaked in the mixed solution for 48 hours. The soaked canine bilateral carotid vessels were removed, rinsed with PBS buffer, and soaked in PBS buffer for 30 days with 2 changes of solution per day. Taking out the soaked carotid blood vessels on both sides of the dogs, trimming into slices, and freeze-drying for 12 hours at the temperature of minus 55 ℃ in a freeze dryer to obtain the decellularized scaffold, which is marked as DCS.
2. Preparation of chitosan/polyethylene glycol hydrogel
1.5g of NaOH is weighed and dissolved in water, the solution is prepared into 2.0mol/L solution, 5mL of absolute ethyl alcohol is added, the 2.0mol/L NaOH solution is poured into a three-neck flask, 2g of chitosan is added while stirring, and the solution is alkalized for 8h. 2g of propylene oxide was added to the three-necked flask and the flask was placed in a constant temperature water bath at 50℃to react for 36 hours. Taking out the product, placing the product in a mixed solution of hydrochloric acid and acetone in a mass ratio of 1:9 for washing 5 times, placing the product in a mixed solution of acetone and water in a mass ratio of 9:1 for washing 5 times, filtering by a vacuum pump, placing the product in a vacuum drying oven at 55 ℃ for drying for 2 hours, and taking out the product to obtain O-hydroxypropyl chitosan (O-HPCS). 1.5g of O-HPCS was placed in 100mL of deionized water to prepare a solution, and 0.5g of polyethylene glycol (PEG) and 5mL of a 2% glutaraldehyde solution were added, and stirred for 15min to thoroughly mix, and left to stand to obtain a chitosan/polyethylene glycol hydrogel, designated CS/PEG.
3. Preparation of chitosan/polyethylene glycol hydrogel/acellular scaffold
And (3) placing the prepared DCS in PBS buffer solution for 30min, taking out, and smearing chitosan/polyethylene glycol hydrogel on the surface of the decellularized scaffold, wherein the surface of the DCS is uniformly smeared for three times. And taking out, placing in a vacuum drying oven at 50 ℃ for drying until the water is completely removed. The chitosan/polyethylene glycol hydrogel/decellularized scaffold was obtained and designated CS/PEG/DCS.
4. Heparin is deposited on the surface of chitosan/polyethylene glycol/acellular stent by using a layer-by-layer self-assembly technology to prepare the high-bionic artificial vascular material
1) Preparing 2g/L heparin sodium solution, chitosan solution and 0.01mol/LPBS buffer solution respectively, placing the prepared CS/PEG/DCS in PBS buffer solution for 30min, taking out, soaking the CS/PEG/DCS in the heparin sodium solution for 15min, taking out, respectively washing the front and the back of the PBS buffer solution for three times, removing heparin sodium adhered on the surface due to physical adsorption, and obtaining a layer of heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, which is recorded as 1-He-CS/PEG/DCS.
2) Soaking the prepared 1-He-CS/PEG/DCS in the prepared chitosan solution for 15min, taking out, respectively washing the front side and the back side of the prepared chitosan solution for three times, removing chitosan adhered by physical adsorption on the surface, soaking the chitosan in heparin sodium solution for 15min, taking out, respectively washing the front side and the back side of the prepared chitosan solution for three times, removing heparin sodium adhered by physical adsorption on the surface, obtaining a two-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, marking the two-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold as 2-He-CS/PEG/DCS, repeating the steps for 1 time, and obtaining the 3-He-CS/PEG/DCS.
Example 2 preparation of three layers heparin-Chitosan/polyethylene glycol hydrogel/Decellularized scaffold (3-He-CS/PEG/DCS)
1. Preparation of decellularized scaffolds (Decellularized scaffold, DCS)
The bilateral carotid arteries of dogs placed in 4% paraformaldehyde tissue fixative were removed and the adventitia trimmed and immersed in 0.9% saline. 0.5g of sodium dodecyl sulfate and 0.5g of polyethylene glycol octyl phenyl ether are taken and dissolved in 100mL of deionized water to prepare a mixed solution with the concentration of 1%, and the trimmed canine bilateral carotid arteries are soaked in the mixed solution for 24 hours. The soaked canine bilateral carotid arteries were removed, rinsed with PBS buffer, and soaked in PBS buffer for 20 days with 1 change of solution per day. Taking out the soaked canine bilateral carotid arteries, trimming into slices, and freeze-drying for 8 hours at the temperature of minus 45 ℃ in a freeze dryer to obtain a decellularized scaffold, which is marked as DCS.
2. Preparation of chitosan/polyethylene glycol hydrogel
1.0g of NaOH was weighed and dissolved in water, and 1.5mol/L of NaOH solution was prepared, then 5mL of absolute ethyl alcohol was added, and the 1.5mol/L NaOH solution was poured into a three-necked flask, and 2g of chitosan was added while stirring, and alkalization was performed for 6 hours. 3g of propylene oxide was added to the three-necked flask and the flask was placed in a constant temperature water bath at 50℃to react for 24 hours. Taking out the product, placing the product in a mixed solution of hydrochloric acid and acetone in a mass ratio of 1:10 for washing 5 times, placing the product in a mixed solution of acetone and water in a mass ratio of 10:1 for washing 5 times, filtering by a vacuum pump, placing the product in a vacuum drying oven at 45 ℃ for drying for 3 hours, and taking out the product to obtain O-hydroxypropyl chitosan (O-HPCS). 1.5g of O-HPCS was placed in 100mL of deionized water to prepare a solution, and 1.0g of polyethylene glycol (PEG) and 5mL of a 2% glutaraldehyde solution were added, and stirred for 15min to thoroughly mix, and left to stand to obtain a chitosan/polyethylene glycol hydrogel, designated CS/PEG.
3. Preparation of chitosan/polyethylene glycol hydrogel/acellular scaffold
And (3) placing the prepared DCS in PBS buffer solution for 30min, taking out, and smearing chitosan/polyethylene glycol hydrogel on the surface of the decellularized scaffold, wherein the surface of the DCS is uniformly smeared for three times. And taking out, placing in a vacuum drying oven at 50 ℃ for drying until the water is completely removed. The chitosan/polyethylene glycol hydrogel/decellularized scaffold was obtained and designated CS/PEG/DCS.
4. Heparin is deposited on the surface of chitosan/polyethylene glycol/acellular stent by using a layer-by-layer self-assembly technology to prepare the high-bionic artificial vascular material
1) Preparing 1g/L heparin, 2g/L chitosan solution and 0.01mol/LPBS buffer solution respectively, putting the prepared CS/PEG/DCS into PBS buffer solution for 10min, then taking out, soaking the CS/PEG/DCS in heparin sodium solution for 10min, taking out, respectively washing the front side and the back side of the PBS buffer solution for three times, removing heparin sodium adhered to the surface due to physical adsorption, and obtaining a layer of heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, which is marked as 1-He-CS/PEG/DCS.
2) Soaking the prepared 1-He-CS/PEG/DCS in the prepared chitosan solution for 15min, taking out, respectively washing the front side and the back side of the prepared chitosan solution for three times, removing chitosan adhered by physical adsorption on the surface, soaking the chitosan in heparin sodium solution for 15min, taking out, respectively washing the front side and the back side of the prepared chitosan solution for three times, removing heparin sodium adhered by physical adsorption on the surface, obtaining a two-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, marking the two-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold as 2-He-CS/PEG/DCS, repeating the steps for 1 time, and obtaining the 3-He-CS/PEG/DCS.
Example 3 preparation of four layers of heparin-Chitosan/polyethylene glycol hydrogel/Decellularized scaffold (4-He-CS/PEG/DCS)
1. Preparation of decellularized scaffolds (Decellularized scaffold, DCS)
The canine bilateral carotid arteries placed in tissue fixative were removed and the adventitia trimmed, immersed in 0.9% saline. 0.5g of sodium dodecyl sulfate and 1.0g of polyethylene glycol octyl phenyl ether are taken and dissolved in 100mL of deionized water to prepare a mixed solution with the concentration of 1.5%, and the trimmed canine bilateral carotid arteries are soaked in the mixed solution for 24 hours. The soaked canine bilateral carotid arteries were removed, rinsed with PBS buffer, and soaked in PBS buffer for 30 days with 2 changes of solution per day. Taking out the soaked canine bilateral carotid arteries, trimming into slices, and freeze-drying for 8 hours at the temperature of minus 55 ℃ in a freeze dryer to obtain a decellularized scaffold, which is marked as DCS.
Preparation of chitosan/polyethylene glycol hydrogel and preparation of chitosan/polyethylene glycol hydrogel/decellularized scaffold were the same as in example 1.
2. The heparin is deposited on the surface of chitosan/polyethylene glycol/acellular stent by the layer-by-layer self-assembly technology to prepare the high-bionic artificial vascular material
1) Preparing 2g/L heparin, 2g/L chitosan solution and 0.01mol/LPBS buffer solution respectively, putting the prepared CS/PEG/DCS in PBS buffer solution for 30min, then taking out, soaking the CS/PEG/DCS in heparin sodium solution for 15min, taking out, respectively washing the front side and the back side of the PBS buffer solution for three times, removing heparin sodium adhered to the surface due to physical adsorption, and obtaining a layer of heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, which is marked as 1-He-CS/PEG/DCS.
2) Soaking the prepared 1-He-CS/PEG/DCS in the prepared chitosan solution for 15min, taking out, respectively washing the front side and the back side of the prepared chitosan solution for three times, removing chitosan adhered by physical adsorption on the surface, soaking the chitosan in heparin sodium solution for 15min, taking out, respectively washing the front side and the back side of the prepared chitosan solution for three times, removing heparin sodium adhered by physical adsorption on the surface, obtaining a two-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, marking the two-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold as 2-He-CS/PEG/DCS, repeating the steps for 2 times, and obtaining the 4-He-CS/PEG/DCS.
Example 4 preparation of four layers of heparin-Chitosan/polyethylene glycol hydrogel/Decellularized scaffold (4-He-CS/PEG/DCS)
1. The preparation methods of the decellularized scaffold (Decellularized scaffold, DCS), chitosan/polyethylene glycol hydrogel and chitosan/polyethylene glycol hydrogel/decellularized scaffold were the same as example 2.
2. Heparin is deposited on the surface of chitosan/polyethylene glycol/acellular stent by using a layer-by-layer self-assembly technology to prepare the high-bionic artificial vascular material
1) Preparing 1g/L heparin, 2g/L chitosan solution and 0.01mol/LPBS buffer solution respectively, putting the prepared CS/PEG/DCS in PBS buffer solution for 30min, taking out, soaking the CS/PEG/DCS in heparin sodium solution for 15min, taking out, respectively washing the front side and the back side of the PBS buffer solution for three times, removing heparin sodium adhered to the surface due to physical adsorption, and obtaining a layer of heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, which is marked as 1-He-CS/PEG/DCS.
2) Soaking the prepared 1-He-CS/PEG/DCS in the prepared chitosan solution for 10min, taking out, respectively washing the front side and the back side of the chitosan solution with PBS buffer solution for three times, removing chitosan adhered by physical adsorption, soaking the chitosan in heparin sodium solution for 10min, taking out, respectively washing the front side and the back side of the chitosan solution with PBS buffer solution for three times, removing heparin sodium adhered by physical adsorption, obtaining a two-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, marking the scaffold as 2-He-CS/PEG/DCS, repeating the steps for 2 times, and obtaining the 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 the decellularized scaffold (Decellularized scaffold, DCS), chitosan/polyethylene glycol hydrogel and chitosan/polyethylene glycol hydrogel/decellularized scaffold were the same as example 1.
2. Heparin is deposited on the surface of chitosan/polyethylene glycol/acellular stent by using a layer-by-layer self-assembly technology to prepare the high-bionic artificial vascular material
1) Preparing 2g/L heparin, 2g/L chitosan solution and 0.01mol/LPBS buffer solution respectively, putting the prepared CS/PEG/DCS in PBS buffer solution for 30min, then taking out, soaking the CS/PEG/DCS in heparin sodium solution for 15min, taking out, respectively washing the front side and the back side of the PBS buffer solution for three times, removing heparin sodium adhered to the surface due to physical adsorption, and obtaining a layer of heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, which is marked as 1-He-CS/PEG/DCS.
2) Placing the prepared He-CS/PEG/DCS in the prepared chitosan solution, soaking for 15min, taking out, respectively washing the front side and the back side of the prepared chitosan solution for three times, removing chitosan adhered by physical adsorption on the surface, soaking the chitosan in the heparin sodium solution for 15min, taking out, respectively washing the front side and the back side of the prepared chitosan solution for three times, removing heparin sodium adhered by physical adsorption on the surface, obtaining a two-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, marking as 2-He-CS/PEG/DCS, repeating the steps for 3 times, and obtaining the 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 the decellularized scaffold (Decellularized scaffold, DCS), chitosan/polyethylene glycol hydrogel and chitosan/polyethylene glycol hydrogel/decellularized scaffold were the same as example 3.
2. Heparin is deposited on the surface of chitosan/polyethylene glycol/acellular stent by using a layer-by-layer self-assembly technology to prepare the high-bionic artificial vascular material
1) Preparing 1g/L heparin, 1g/L chitosan solution and 0.01mol/L PBS buffer solution respectively, putting the prepared CS/PEG/DCS into the PBS buffer solution for 15min, then taking out, soaking the CS/PEG/DCS in the heparin sodium solution for 15min, taking out, respectively washing the front side and the back side of the PBS buffer solution for three times, removing heparin sodium adhered to the surface due to physical adsorption, and obtaining a layer of heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, which is marked as 1-He-CS/PEG/DCS.
2) Placing the prepared 1-He-CS/PEG/DCS in the prepared chitosan solution, soaking for 10min, taking out, respectively washing the front side and the back side of the prepared chitosan solution for three times, removing chitosan adhered by physical adsorption on the surface, soaking in heparin sodium solution for 10min, taking out, respectively washing the front side and the back side of the prepared chitosan solution for three times, removing heparin sodium adhered by physical adsorption on the surface, obtaining a two-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, marking as 2-He-CS/PEG/DCS, and repeating the steps for 3 times to obtain the 5-He-CS/PEG/DCS.
EXAMPLE 7 preparation of six layers of heparin-Chitosan/polyethylene glycol hydrogel/Decellularized scaffold (6-He-CS/PEG/DCS)
1. The preparation methods of the decellularized scaffold (Decellularized scaffold, DCS), chitosan/polyethylene glycol hydrogel and chitosan/polyethylene glycol hydrogel/decellularized scaffold were the same as example 1.
2. Heparin is deposited on the surface of chitosan/polyethylene glycol/acellular stent by using a layer-by-layer self-assembly technology to prepare the high-bionic artificial vascular material
1) Preparing 2g/L heparin, 2g/L chitosan solution and 0.01mol/LPBS buffer solution respectively, putting the prepared CS/PEG/DCS in PBS buffer solution for 30min, then taking out, soaking the CS/PEG/DCS in heparin sodium solution for 15min, taking out, respectively washing the front side and the back side of the PBS buffer solution for three times, removing heparin sodium adhered to the surface due to physical adsorption, and obtaining a layer of heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, which is marked as 1-He-CS/PEG/DCS.
2) Soaking the prepared 1-He-CS/PEG/DCS in the prepared chitosan solution for 15min, taking out, respectively washing the front side and the back side of the prepared chitosan solution for three times, removing chitosan adhered by physical adsorption on the surface, soaking the chitosan in heparin sodium solution for 15min, taking out, respectively washing the front side and the back side of the prepared chitosan solution for three times, removing heparin sodium adhered by physical adsorption on the surface, obtaining a two-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, marking the two-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold as 2-He-CS/PEG/DCS, repeating the steps for 4 times, and obtaining the 6-He-CS/PEG/DCS.
EXAMPLE 8 preparation of six layers of heparin-Chitosan/polyethylene glycol hydrogel/Decellularized scaffold (6-He-CS/PEG/DCS)
1. The preparation methods of the decellularized scaffold (Decellularized scaffold, DCS), chitosan/polyethylene glycol hydrogel and chitosan/polyethylene glycol hydrogel/decellularized scaffold were the same as example 2.
2. Heparin is deposited on the surface of chitosan/polyethylene glycol/acellular stent by using a layer-by-layer self-assembly technology to prepare the high-bionic artificial vascular material
1) Preparing 2g/L heparin, 1g/L chitosan solution and 0.01mol/LPBS buffer solution respectively, putting the prepared CS/PEG/DCS in PBS buffer solution for 30min, then taking out, soaking the CS/PEG/DCS in heparin sodium solution for 15min, taking out, respectively washing the front side and the back side of the PBS buffer solution for three times, removing heparin sodium adhered to the surface due to physical adsorption, and obtaining a layer of heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, which is marked as 1-He-CS/PEG/DCS.
2) Soaking the prepared 1-He-CS/PEG/DCS in the prepared chitosan solution for 10min, taking out, respectively washing the front side and the back side of the prepared chitosan solution for three times, removing chitosan adhered by physical adsorption on the surface, soaking the chitosan in heparin sodium solution for 10min, taking out, respectively washing the front side and the back side of the prepared chitosan solution for three times, removing heparin sodium adhered by physical adsorption on the surface, obtaining a two-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, marking the two-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold as 2-He-CS/PEG/DCS, repeating the steps for 4 times, and obtaining the 6-He-CS/PEG/DCS.
Example 9 preparation of seven layers of heparin-Chitosan/polyethylene glycol hydrogel/Decellularized scaffold (7-He-CS/PEG/DCS)
1. The preparation methods of the decellularized scaffold (Decellularized scaffold, DCS), chitosan/polyethylene glycol hydrogel and chitosan/polyethylene glycol hydrogel/decellularized scaffold were the same as example 1.
2. Heparin is deposited on the surface of chitosan/polyethylene glycol/acellular stent by using a layer-by-layer self-assembly technology to prepare the high-bionic artificial vascular material
1) Preparing 2g/L heparin, 2g/L chitosan solution and 0.01mol/LPBS buffer solution respectively, putting the prepared CS/PEG/DCS into PBS buffer solution for 10min, then taking out, soaking the CS/PEG/DCS in heparin sodium solution for 15min, taking out, respectively washing the front side and the back side of the PBS buffer solution for three times, removing heparin sodium adhered to the surface due to physical adsorption, and obtaining a layer of heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, which is marked as 1-He-CS/PEG/DCS.
2) Soaking the prepared 1-He-CS/PEG/DCS in the prepared chitosan solution for 15min, taking out, respectively washing the front side and the back side of the prepared chitosan solution for three times, removing chitosan adhered by physical adsorption on the surface, soaking the chitosan in heparin sodium solution for 15min, taking out, respectively washing the front side and the back side of the prepared chitosan solution for three times, removing heparin sodium adhered by physical adsorption on the surface, obtaining a two-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, marking the two-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold as 2-He-CS/PEG/DCS, repeating the steps for 5 times, and obtaining the 7-He-CS/PEG/DCS.
Example 10 preparation of seven layers of heparin-Chitosan/polyethylene glycol hydrogel/Decellularized scaffold (7-He-CS/PEG/DCS)
1. The preparation methods of the decellularized scaffold (Decellularized scaffold, DCS), chitosan/polyethylene glycol hydrogel and chitosan/polyethylene glycol hydrogel/decellularized scaffold were the same as example 2.
2. Heparin is deposited on the surface of chitosan/polyethylene glycol/acellular stent by using a layer-by-layer self-assembly technology to prepare the high-bionic artificial vascular material
1) Preparing 1g/L heparin, 1g/L chitosan solution and 0.01mol/LPBS buffer solution respectively, putting the prepared CS/PEG/DCS into PBS buffer solution for 10min, then taking out, soaking the CS/PEG/DCS in heparin sodium solution for 10min, taking out, respectively washing the front side and the back side of the PBS buffer solution for three times, removing heparin sodium adhered to the surface due to physical adsorption, and obtaining a layer of heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, which is marked as 1-He-CS/PEG/DCS.
2) Soaking the prepared 1-He-CS/PEG/DCS in the prepared chitosan solution for 10min, taking out, respectively washing the front side and the back side of the prepared chitosan solution for three times, removing chitosan adhered by physical adsorption on the surface, soaking the chitosan in heparin sodium solution for 10min, taking out, respectively washing the front side and the back side of the prepared chitosan solution for three times, removing heparin sodium adhered by physical adsorption on the surface, obtaining a two-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold, marking the two-layer heparin-chitosan/polyethylene glycol hydrogel/decellularized scaffold as 2-He-CS/PEG/DCS, repeating the steps for 5 times, and obtaining the 7-He-CS/PEG/DCS.
EXAMPLE 11 SEM characterization of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3-7) vascular prosthesis materials
1. The morphology of the decellularized scaffold prepared in example 1 was observed by a JSM-7600F-type scanning electron microscope, and the test result is shown in FIG. 1. Fig. 1 is an SEM image of a decellularized scaffold, and as can be seen from fig. 1, the decellularized scaffold (DCS) subjected to the compounding treatment of anionic surfactant sodium dodecyl sulfate and nonionic surfactant polyethylene glycol octyl phenyl ether presents an obvious three-dimensional network space structure, has no cells on the surface, and only has a small amount of cell debris, so that the mixed solution of polyethylene glycol octyl phenyl ether and sodium dodecyl sulfate has a good decellularized effect.
2. The artificial vascular materials of CS/PEG/DCS prepared in example 1 and n-He-CS/PEG/DCS prepared in examples 1, 3, 5, 7 and 9 (n=3 to 7) were cut into strips having a length of 5mm and a width of 1mm, stuck to a copper table with a conductive paste, and the front side surfaces were sprayed with gold 6 times, and the morphology was observed with JSM-7600F-type scanning electron microscope, and the test results were shown in fig. 2. FIG. 2 is a SEM image at 2000 Xof CS/PEG/DCS and n-He-CS/PEG/DCS (n=3 to 7) vascular prosthesis material, where a is CS/PEG/DCS, 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 fig. 2, a surface has a small amount of particulate matter, which indicates that chitosan has been modified onto DCS together with PEG hydrogel, while as the polyelectrolyte layer increases, the amount of particulate matter on b, c, d, e, f increases, which indicates that heparin and chitosan have been adsorbed onto CS/PEG/DCS layer by electrostatic adsorption, and the above experimental results prove that n-He-CS/PEG/DCS (n=3 to 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 subjected to infrared spectroscopy using a Nexus670 infrared spectroscopy instrument from Nicolet, U.S. and the results are shown in FIG. 3. FIG. 3 is a FTIR of O-HPCS and CS/PEG, and as can be seen from FIG. 3, the analysis results show that the FTIR of O-HPCS and the FTIR of CS/PEG are at 3400cm -1 The strong absorption peak is the-OH peak on the modified chitosan, at 2900cm -1 At the absorption peak of (2) is-CH 3 、-CH 2 Is 1375cm -1 Bending vibration belonging to O-H bond, 1065cm in FTIR chart of O-HPCS -1 、589cm -1 Is the crystallization sensitive peak of O-HPCS, and on the FTIR chart of CS/PEG, the intensity of both crystallization peaks is attenuated, and the positions of the crystallization peaks are shifted, which indicates that the crystal structure of O-HPCS is destroyed by adding polyethylene glycol. We can therefore demonstrate by FTIR plot of fig. 3 that CS/PEG has been successfully synthesized.
EXAMPLE 13X-ray photoelectron Spectrometry of preparation CS/PEG/DCS of example 1 and 5-He-CS/PEG/DCS prepared in example 5
The 5-He-CS/PEG/DCS prepared in example 1 and 5-He-CS/PEG/DCS prepared in example 5 were subjected to X-ray photoelectron spectroscopy under conditions of monochromatic Al K.alpha.rays (150W, 500 μm beam spot) and energy passing through 20eV using a Japanese national D/max 2500VL/PC type X-ray photoelectron spectroscopy analyzer, and the results are shown in FIG. 4. FIG. 4 is a XPS comparison 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 FIG. 4, S appears on b 2p The peaks illustrate the presence of the S element on 5-He-CS/PEG/DCS, since heparin on 5-He-CS/PEG/DCS vascular prostheses has the S element, thus proving that 5-He-CS/PEG/DCS vascular prostheses have been successfully prepared.
EXAMPLE 14 measurement of amino content of surface Chitosan of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3 to 7) vascular prosthesis materials
1. Acid orange is a golden yellow powdery solid which is dissolved in water and shows orange color, and the acid orange containing a single sulfonic acid group can have equimolar adsorption effect with amino groups on the surface of a material under the condition of pH=2-3, so that the amino content on the surface of a sample can be calculated. Fig. 5 is a standard acid orange graph, which yields the standard acid orange equation, which is y=1.39048x+0.03774 (R 2 = 0.99411). As shown in fig. 5, since neither polyethylene glycol nor heparin contains a primary amine, and chitosan contains a primary amine, acid orange can be used to determine the amino content of a sample, and thus the chitosan content of an artificial vascular material.
2. The specific experimental steps are as follows:
1) Drawing a standard curve: in a 24-well plate, a concentration of 5X 10 was added to the first and second wells -4 The acid orange solution at mol/L, pH =12 was 150 μl and then 150 μl of the solution at ph=12 was added to the second well to dilute the solution in the second well in equal volume. 150 μl of solution was removed from the second well and added to the third well, and 150 μl of deionized water solution at ph=12 was added to dilute the solution in the third well in equal volume. Sequentially stepwise dilution was performed until the seventh well. 50 μl of deionized water solution at ph=12 was added directly in the eighth air. Simultaneously taking 3 groups of parallel samples, And taking an average value. The absorbance at 485nm was measured with a Bio Tek Synergy2 microplate reader, and a standard curve required for the experiment was drawn, and the specific experimental results are shown in FIG. 5.
2) CS/PEG/DCS and n-He-CS/PEG/DCS (n=3-7) vascular prostheses were immersed in acid orange solutions at a concentration of 500. Mu. Mol/L at pH=3. After being placed on a shaking table to fully react for 12 hours, the sample is taken out, repeatedly washed for more than 5 times by using a solution with pH=3, and then fully dried. The resulting sample was immersed in a solution at ph=12 and placed on a shaking table to react well for 30min, releasing methyl orange which was adsorbed on the sample surface by charge. And meanwhile, a methyl orange standard curve is obtained, the adsorption content of the surface of the methyl orange is obtained, and the specific result is shown in fig. 6. FIG. 6 is a bar graph of amino content of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3-7) vascular prosthesis materials, and it can be seen from FIG. 6 that CS-PEG has been successfully modified on DCS surface due to the presence of chitosan on CS-PEG hydrogel surface, and therefore a certain number of amino groups are present in CS/PEG/DCS. With the increase of the number of layers of polyelectrolyte, the amino content of the surface of the artificial blood vessel material is in an increasing trend, because heparin and chitosan are in a cross interpenetrating structure in the LbL technology, and the artificial blood vessel material surface shows a trend of higher amino content. The experimental results prove that the chitosan is successfully modified to the surface of the material. I.e. n-He-CS/PEG/DCS (n=3 to 7) vascular prosthesis materials have been successfully prepared.
EXAMPLE 15 static contact Angle measurement of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3 to 7)
1. The specific experiment: the prepared artificial vascular materials of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3-7) were cut into wafers with a diameter of 1cm, and static contact angle test was performed on each sample using a DSA100 optical contact angle tester from Kruss company with deionized water as a test solution and a droplet size of about 90. Mu.m. Each sample was tested 3 times and 3 replicates were taken.
2. Analysis of results: FIG. 7 is a static contact angle plot of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3 to 7), wherein a is CS/PEG/DCS, b is 3-He-CS/PEG/DCS, c is 4-He-CS/PEG/DCS, d is 5-HeCS/PEG/DCS, e is 6-He-CS/PEG/DCS, and f is 7-He-CS/PEG/DCS. As can be seen from FIG. 7, the CS/PEG/DCS has the smallest static contact angle, because CS grafted on PEG is chitosan alkalized by NaOH, so that-COOH is changed into-COONa, and the-COONa is converted into more polar-COO in water - Therefore, the static water contact angle of the composition is obviously reduced; the n-He-CS/PEG/DCS (n=3-7) artificial blood vessel material has a tendency of increasing after decreasing with increasing polyelectrolyte layers, the static contact angle is the lowest when the polyelectrolyte membrane reaches 5 layers, the static contact angle is slightly increased when the polyelectrolyte membrane is more than 5 layers, the surface hydrophilicity is slightly increased, but the overall change is not great, and the whole has a hydrophilic surface. The contact angle of 5-He-CS/PEG/DCS is minimum, the surface hydrophilicity is strongest, and the best hydrophilic effect is achieved. The material with strong hydrophilicity is favorable for removing biological substances due to the formation of a water layer on the interface, thereby having better anticoagulation performance.
EXAMPLE 16 tensile Properties and burst Strength test of vascular materials of fresh blood, DCS, CS/PEG/DCS and n-He-CS/PEG/DCS (n=3-7)
1. Tensile property test: fresh blood vessels, DCS, CS/PEG/DCS and n-He-CS/PEG/DCS (n=3 to 7) vascular prosthesis materials were cut with a microtome into dumbbell-shaped test strips 3mm wide and 20mm in span. The tensile properties of each sample were measured at room temperature at a tensile speed of 20mm/min using a universal electronic tensile machine (INSTRON 4200 model). Each sample was tested 3 times and 3 replicates were obtained and the results are shown in fig. 8 a and b.
2. Burst strength test: the method comprises the steps of preparing fresh blood vessels, DCS, CS/PEG/DCS and n-He-CS/PEG/DCS (n=3-7) artificial blood vessel materials into cylindrical blood vessels with the length of 30mm and the diameter of 5mm, ligating one section, connecting a tee joint device with the other end, connecting a pressurizing injection device and a pressure testing device with two ends of the tee joint device respectively, continuously injecting normal saline at 37 ℃ into a sample, wherein the injection speed is 3mL/min, and recording a down pressure value when the sample breaks, namely the bursting strength. Each sample was tested 3 times and 3 replicates were obtained and the results are shown below in figure 8 c.
3. Analysis of results: fig. 8 is a graph of tensile strength, elongation at break and burst strength of the vascular graft materials of fresh blood, DCS, CS/PEG/DCS and n-He-CS/PEG/DCS (n=3 to 7). Wherein a is the tensile strength diagram of the artificial blood vessel material of fresh blood vessel, DCS, CS/PEG/DCS and n-He-CS/PEG/DCS (n=3-7), b is the elongation at break graph of the vascular graft material of the fresh blood vessel, DCS, CS/PEG/DCS and n-He-CS/PEG/DCS (n=3 to 7), c is the burst strength graph of the vascular graft material of the fresh blood vessel, DCS, CS/PEG/DCS and n-He-CS/PEG/DCS (n=3 to 7). As can be seen from fig. 8 a and b, the tensile strength of DCS is slightly increased compared with that of fresh blood vessels, the elongation at break is slightly reduced, but there is almost no obvious difference between the two, while DCS modified by hydrogel and polyelectrolyte shows a gradual increase trend with increasing number of layers of polyelectrolyte, but still has a similar tensile strength to that of natural blood vessels, while the elongation at break shows an optimal trend at 5-He-CS/PEG/DCS, compared with fresh blood vessels, and the test results show that after DCS is modified by heparin, chitosan and polyethylene glycol hydrogel, the mechanical bionic performance of DCS is maintained, so that the prepared bionic vascular material can meet the pressure requirement generated when blood flows in the lumen of blood vessels. From fig. 8 c, it can be seen that the burst strength of the fresh arterial vessel is about 110KPa, and the burst strength of DCS is 0KPa, because DCS removes the surface layer of tissue cells, forms a spatial network structure, and physiological saline directly flows out of the network structure, because DCS does not have burst strength. The spatial network structure of the DCS is embedded through the DCS modified by polyelectrolyte and PEG hydrogel, the bursting strength of the DCS tends to rise along with the increase of the polyelectrolyte layer, but each sample is basically similar to the bursting strength of a fresh arterial vessel. The test results show that after the DCS is modified by heparin, chitosan and polyethylene glycol hydrogel, the material basically maintains the mechanical bionic performance of the DCS, so that the prepared bionic artificial vascular material can meet the pressure requirement generated when blood flows in the lumen of the blood vessel.
EXAMPLE 17 in vitro coagulation experiments and hypercalculation experiments were performed on the prepared vascular prosthesis material
1. In vitro coagulation experiments: APTT (activated partial thromboplastin time): the sample to be tested is taken and fully mixed with blood plasma (0.1 mL), poured into a blood coagulation cup, after 5min of culture at 37 ℃, APTT reagent is added, the culture is continued for 5min, then the same amount of calcium chloride solution (0.1 mL,0.025 mol) is added into each blood coagulation cup, the blood coagulation time at 37 ℃ is observed, and the recorded coagulation time is recorded as APTT value. Each sample was tested 3 times and the results averaged. TT (thrombin time): mixing platelet-poor plasma and sample uniformly, pouring into a coagulation cup, preheating in a water bath with constant temperature (37 ℃) for 5min, adding TT of normal reference plasma, adding 0.1mL of thrombin, taking turbidity as a starting point, and recording the coagulation time. Repeat 3 times and take the average. PT (prothrombin time): and (3) taking a sample to be tested, fully mixing the sample with the blood plasma, pouring the mixture into a coagulation cup, culturing the mixture at 37 ℃ for 5min, adding a pre-warmed PT reagent, and recording the coagulation time. Repeat 3 times and take the average. The artificial vascular materials of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3 to 7) were cut into round pieces with a diameter of about 1cm, and after being uniformly mixed with healthy rabbit blood plasma, an in vitro clotting time test was performed using an enzyme-labeled instrument (BioTek synergy type 2), and the results are shown in fig. 9. A control group (0.1 mL plasma+APTT/PT/TT reagent) was also set.
2. And (3) calcium recovery experiment: the prepared CS/PEG/DCS and n-He-CS/PEG/DCS (n=3-7) artificial vascular materials are immersed in normal saline (0.9%) for 24 hours, then placed in a 96-well plate, covered on the bottom of the well plate and kept at a constant temperature of 37 ℃ for 0.5 hour. 100. Mu.L of preheated platelet-poor plasma was added followed by 0.025mol/LCaCl in 96-well plates, respectively 2 The solution was measured for o.d. value at 405nm using an enzyme-labeled instrument, repeated 3 times, and its average value was taken. Control group: 100 mu L CaCl 2 Solution +100 μl plasma. The results of the hypercalcemia time experiment are shown in FIG. 9.
3. Analysis of results: FIG. 9 is an activated partial thromboplastin time, thrombin time, prothrombin time and hypercalcemia time plot of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3-7) vascular prostheses, where a is the activated partial thromboplastin time plot of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3-7) vascular prostheses, b is the thromboplastin time plot of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3-7) vascular prostheses,c is prothrombin time chart of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3-7) artificial vascular materials, and d is calcilytic blood coagulation time chart of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3-7) artificial vascular materials. As can be seen from FIGS. 9 a-c, the clotting time of APTT, TT, PT of the artificial vascular material modified with the polyelectrolyte layer was all prolonged to some extent as compared with CS-PEG hydrogel-modified DCS. The heparin and the chitosan form an interpenetrating network structure, and when the number of polyelectrolyte layers is increased to a certain degree, the heparin content on the surface can be reduced instead. From the above experimental results, it is considered that the 5-He-CS/PEG/DCS artificial vascular material has the longest in vitro coagulation time and the best anticoagulation performance when the number of polyelectrolyte layers is 5. As can be seen from FIG. 9 d, T using heparin-and chitosan-modified vascular prosthesis 1/2max The calcium recovery time is obviously prolonged. This is because antithrombin III (AT III) can form an irreversible complex by being structured in the form of a substrate AT the active center of thrombin, thereby inhibiting the activity of thrombin, whereas in the case of heparin addition, the whole reaction speed can be improved by more than thousand times, thereby effectively improving the blood compatibility of the material, and leading the hypercalcemia time to show a trend of increasing. Experimental results show that the modification of polyelectrolyte membranes can effectively prolong the time for converting soluble fibrinogen into soluble fibrin, thereby achieving the purpose of prolonging the blood coagulation time. And it can be seen from the above figures that the artificial blood vessel material has T at a polyelectrolyte layer number of 5 1/2max The calcium recovery time is most excellent, namely, when the number of layers of the polyelectrolyte is 5, the material has the longest calcium recovery time and the best anticoagulation performance, because heparin and chitosan form an interpenetrating network structure, when the number of layers is less than 5, the surface heparin content is in an ascending trend along with the increase of the number of layers, when the number of layers of the polyelectrolyte is increased to more than 5,the heparin content of the surface tends to decrease, namely, when n is 5, the heparin content of the surface is highest, and the material has the longest decalcification time and the best anticoagulation performance.
EXAMPLE 18n-He-CS/PEG/DCS (n=3-7) blood compatibility test
1. The specific experiment: erythrocyte morphology experiments are one method for testing blood compatibility, n-He-CS/PEG/DCS (n=3-7) prepared in examples 1, 3, 5, 7 and 9 are cut into round pieces with the diameter of about 6mm, and the round pieces are placed into centrifugal tubes respectively, and 10mL of physiological saline (0.9%) is added into each centrifugal tube. And uniformly mixing 5mL of physiological saline with 4mL of red blood cell suspension, and diluting to obtain diluted red blood cell suspension. Adding 0.2mL of diluted erythrocyte suspension into the centrifuge tube respectively, fully and uniformly mixing, and placing the centrifuge tube into CO 2 Culturing at constant temperature (37deg.C) in a constant temperature incubator for 1 hr, taking out, making into erythrocyte smear, and observing erythrocyte morphology under microscope. 3 replicates were taken for each sample. A blank control group was set at the same time: 10mL of 0.9% physiological saline+0.2 mL of diluted erythrocyte suspension, and the experimental results are shown in FIG. 10.
2. Analysis of results: FIG. 10 is a graph of red blood cell morphology of n-He-CS/PEG/DCS (n=3 to 7), where a is a blank, 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 FIG. 10, when the erythrocytes were contacted with the vascular material of the present invention, the morphology was consistent with that of the control group, and no significant deformation or rupture was observed. The results show that the 5-He-CS/PEG/DCS modified by heparin and chitosan has almost no toxic effect on erythrocytes, i.e. has good blood compatibility.
EXAMPLE 19CS/PEG/DCS and n-He-CS/PEG/DCS (n=3-7) vascular prosthesis materials MTT toxicity test
1. The specific experiment: sample preparation: respectively taking n-He-CS/PEG/DCS (n=3-7) artificial vascular materials, adding 10mL of leaching medium (10% fetal bovine serum), and leaching in a constant-temperature shaking incubator at 37 ℃ for 24 hours. Preparing MTT solution: MTT was dissolved in PBS to prepare an MTT solution having a concentration of 5 mg/mL. Cells in the logarithmic growth phase were digested with pancreatin,diluted to a concentration of 4X 10 with cell culture 4 And each mL. A96-well plate was used, 200. Mu.L of the cell suspension was inoculated into each well, and the plate was placed at 37℃in 5% CO 2 Culturing in an incubator for 24 hours. After the cells are attached, the stock culture solution in each hole is sucked out, 200 mu L of sample leaching solution is added in each hole of an experimental group (8 holes are arranged in each group), meanwhile, a blank control group is arranged in a pore plate without cells, the cell culture solution is added, and the mixture is continuously placed at 37 ℃ and 5% CO 2 Culturing in an incubator for 12 hours, 24 hours and 48 hours; after that, 20. Mu.L of the prepared MTT solution was added to each well, and after culturing for 5 hours, the stock solution was discarded, 150. Mu.L of dimethyl sulfoxide was added to each well, and the mixture was shaken horizontally for 5 minutes, and the absorbance O.D. at 490nm was measured by an enzyme-labeled instrument (BioTek synergy type 2). Each test was performed at least three times. CS/PEG/DCS experiments were performed with n-He-CS/PEG/DCS (n=3-7) vascular prosthesis materials.
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.
And (3) calculating results:
wherein: dt—absorbance of the experimental group samples; dnc-absorbance of negative control samples; db-absorbance of control samples. The experimental results are shown in FIG. 11.
2. Analysis of results: FIG. 11 shows the MTT test results of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3 to 7). As can be seen from FIG. 11, at 12h, the cell viability of CS/PEG/DCS was 97%, the cytotoxicity was 1-grade, while at the same time, the cell viability of n-He-CS/PEG/DCS (n=3 to 7) was between 98% and 101%, the cytotoxicity was 0-grade and 1-grade, and the cell viability of n-He-CS/PEG/DCS was higher than that of CS/PEG/DCS, but the cytotoxicity values were close, and the difference in cell viability between different artificial vascular materials was small. This is because CS-PEG hydrogel has good biocompatibility and low cytotoxicity, and can ensure normal survival of cells in a short-term culture process. With the increase of the culture time, the cell survival rate of CS/PEG/DCS is 92% and the cytotoxicity is 1 grade at 24 hours; the cell survival rate of n-He-CS/PEG/DCS (n=3-7) is 104% -114%, and the cytotoxicity is grade 0. At 48h, the difference between the cell compatibility of CS/PEG/DCS and n-He-CS/PEG/DCS (n=3 to 7) was greater. The experimental results prove that although CS-PEG hydrogel has a certain degree of cell compatibility, the surface modification of polyelectrolyte can obviously improve the cell compatibility, and the cell compatibility improvement effect is more obvious for a longer time. Comparing the experimental results of the 48h cell viability of the samples with the number of the polyelectrolyte layers. It can be seen that the cell survival rate is increased from 108% to 118% in the case of 3-5 layers of polyelectrolyte layers, and the cell survival rate is in an upward trend; in contrast, when the number of polyelectrolyte layers is 7, the cell viability is 111%, which is slightly lower than that when the number of polyelectrolyte layers is 5, but the difference is small. The above experimental results show that the artificial vascular material has the best cell compatibility when the number of polyelectrolyte layers is 5.
Example 20 in vitro degradation experiments of CS/PEG/DCS and 5-He-CS/PEG/DCS vascular prosthesis materials
1. The specific experiment: from the above, it is considered that the 5-He-CS/PEG/DCS artificial blood vessel material has the best overall performance when the number of polyelectrolyte layers is 5. In the subsequent study, a test study was performed with 5-He-CS/PEG/DCS as the subject. Cutting CS/PEG/DCS and 5-He-CS/PEG/DCS artificial blood vessel material into square shape of 10mmx10mm, weighing with electronic balance to obtain the final product with weight of m 0 Placing into a centrifuge tube, adding 20mL PBS solution, placing into a shaking oven at 37deg.C, culturing for 1d,7d,14d,30d,60d,90d,120d,180d, replacing with fresh PBS buffer every week, taking out cultured artificial vascular material, oven drying at 50deg.C in a vacuum drying oven, and weighing with electronic balance to weight of m 1 Weight loss results were calculated: weight loss (%) = (m) 0 -m 1 )/m 0 X 100%. The results are shown in FIG. 12.
2. Analysis of results: FIG. 12 is a graph of the in vitro degradation rates of CS/PEG/DCS and 5-He-CS/PEG/DCS vascular prosthesis, as can be seen from FIG. 12, the in vitro degradation rates of CS/PEG/DCS and 5-He-CS/PEG/DCS show a trend from fast to slow over time; at 180 days, the residual mass of CS/PEG/DCS was 33.7%, while the residual mass of 5-He-CS/PEG/DCS was 44.2%. The in-vitro degradation period and the blood vessel regeneration period between the two are basically matched, which shows that the two have good degradation rate matching property, and the in-vitro degradation period and the blood vessel regeneration period can be well matched with the regeneration period of a new blood vessel as artificial blood vessel materials, so that good bionic effect is achieved.
EXAMPLE 21 preparation of heparin-polyvinyl alcohol/decellularized scaffold He/PVA 1 /DCS
1. Preparation of acellular scaffolds
The canine bilateral carotid vessels placed in tissue fixative were removed and the adventitia trimmed, and washed in 0.9% saline. Taking out the cleaned carotid blood vessels on both sides of the dogs, and putting the dogs into a mixed solution of 1% polyethylene glycol octyl phenyl ether and 0.5% sodium dodecyl sulfate to be soaked for 48 hours at room temperature; the soaked canine bilateral carotid vessels were removed, rinsed with 0.01mol/L PBS buffer solution, and soaked in PBS buffer solution for 30 days with 2 changes of solution per day. The soaked canine bilateral carotid blood vessels were removed, trimmed to pieces, and lyophilized in a lyophilizer at-55 ℃ for 12h to obtain decellularized scaffolds (Decellularized scaffold, DCS), designated DCS.
2. Preparation of acetalized polyvinyl alcohol hydrogels
In a 500mL three-necked flask, 6g of polyvinyl alcohol and 180mL of water were added, and the mixture was heated to 90℃to dissolve the mixture; adding 3g of glycerol, and continuously heating for 0.5h; cooling to about 30 ℃, adding 4g of formaldehyde and 12g of glutaraldehyde with the concentration of 0.5%, and uniformly stirring to obtain acetalized polyvinyl alcohol; uniformly spreading acetalized polyvinyl alcohol on the bottom of a glass dish, respectively processing for 2 hours at 60 ℃ and 70 ℃ in an oven, cooling, taking out, soaking and flushing for 0.5 hour, and removing impurities; acetal polyvinyl alcohol hydrogel, designated PVA, was obtained 1 。
3. Preparation of acetalized heparin-polyvinyl alcohol complexes
Spreading the acetalized polyvinyl alcohol hydrogel in a glass vessel, drying for 2 hours in an oven at 40 ℃ to obtain an acetalized polyvinyl alcohol film, and soaking the acetalized polyvinyl alcohol film in a heparin sodium aqueous solution with the concentration of 1% to obtain an acetalized heparin-polyvinyl alcohol compound.
4. Preparation of heparin-polyvinyl alcohol/acellular scaffold
Uniformly coating an acetalized heparin-polyvinyl alcohol compound on a decellularized scaffold, placing the decellularized scaffold in an oven, treating for 2 hours at 60 ℃, then heating to 70 ℃ for continuous treatment for 2 hours, cooling, taking out, soaking and flushing for 0.5 hour, removing impurities to obtain the heparin-polyvinyl alcohol/decellularized scaffold, and recording as He/PVA 1 /DCS。
EXAMPLE 22 preparation of heparin-polyvinyl alcohol/decellularized scaffold He/PVA 2 /DCS
1. Preparation of acellular scaffolds
The canine bilateral carotid vessels placed in tissue fixative were removed and the adventitia trimmed, and washed in 0.9% saline. Taking out the cleaned carotid blood vessels on both sides of the dogs, and putting the dogs into a mixed solution of 0.5% polyethylene glycol octyl phenyl ether and 0.5% sodium dodecyl sulfate to be soaked for 24 hours at room temperature; the soaked canine bilateral carotid vessels were removed, rinsed with 0.01mol/L PBS buffer solution, and soaked in PBS buffer solution for 20 days with 2 changes of solution per day. The soaked canine bilateral carotid blood vessels were removed, trimmed into pieces, and lyophilized in a lyophilizer at-45 ℃ for 12h to obtain decellularized scaffolds (Decellularized scaffold, DCS), designated DCS.
2. Preparation of acetalized polyvinyl alcohol hydrogels
In a 500mL three-necked flask, a certain amount of 10g of polyvinyl alcohol and 180mL of water were added, and heated to 90℃to dissolve the polyvinyl alcohol; adding 3g of glycerol, and continuously heating for 0.5h; cooling to about 30 ℃, adding 4g of formaldehyde and 12g of glutaraldehyde with the concentration of 1%, and uniformly stirring to obtain acetalized polyvinyl alcohol; uniformly spreading acetalized polyvinyl alcohol on the bottom of a glass dish, respectively processing for 2 hours at 60 ℃ and 70 ℃ in an oven, cooling, taking out, soaking and flushing for 0.5 hour, and removing impurities; acetal polyvinyl alcohol hydrogel, designated PVA, was obtained 2 。
3. Preparation of acetalized heparin-polyvinyl alcohol complexes
Spreading the acetalized polyvinyl alcohol hydrogel in a glass vessel, drying for 2 hours in an oven at 40 ℃ to obtain an acetalized polyvinyl alcohol film, and soaking the acetalized polyvinyl alcohol film in a 1% heparin sodium aqueous solution to obtain an acetalized heparin-polyvinyl alcohol compound.
4. Preparation of heparin-polyvinyl alcohol/acellular scaffold
Uniformly coating the acetalized heparin-polyvinyl alcohol compound on a decellularized scaffold, placing the decellularized scaffold in an oven, treating for 2 hours at 60 ℃, heating to 70 ℃ for continuous treatment for 2 hours, cooling, taking out, soaking and washing for 0.5 hour, removing impurities to obtain the heparin-polyvinyl alcohol/decellularized scaffold, and recording as He/PVA 2 /DCS。
EXAMPLE 23 preparation of heparin-polyvinyl alcohol/decellularized scaffold He/PVA 3 /DCS
1. Preparation of acellular scaffolds
The canine bilateral carotid vessels placed in tissue fixative were removed and the adventitia trimmed, and washed in 0.9% saline. Taking out the cleaned carotid blood vessels on both sides of the dogs, and putting the dogs into a mixed solution of 1% polyethylene glycol octyl phenyl ether and 0.5% sodium dodecyl sulfate to be soaked for 24 hours at room temperature; the soaked canine bilateral carotid vessels were removed, rinsed with 0.01mol/L PBS buffer solution, and soaked in PBS buffer solution for 20 days with 1 change of solution per day. The soaked canine bilateral carotid blood vessels were removed, trimmed to pieces, and lyophilized in a lyophilizer at-55 ℃ for 8h to obtain decellularized scaffolds (Decellularized scaffold, DCS), designated DCS.
2. Preparation of acetalized polyvinyl alcohol hydrogels
In a 500mL three-necked flask, a certain amount of 6g of polyvinyl alcohol and 180mL of water were added, and heated to 90℃to dissolve the polyvinyl alcohol; adding 3g of glycerol, and continuously heating for 0.5h; cooling to about 30 ℃, adding 6g of formaldehyde and 12g of glutaraldehyde with the concentration of 3%, and uniformly stirring to obtain acetalized polyvinyl alcohol; uniformly spreading acetalized polyvinyl alcohol on the bottom of a glass dish, respectively processing for 2 hours at 60 ℃ and 70 ℃ in an oven, cooling, taking out, soaking and flushing for 0.5 hour, and removing impurities; acetal polyvinyl alcohol hydrogel, designated PVA, was obtained 3 。
3. Preparation of acetalized heparin-polyvinyl alcohol complexes
Spreading the acetalized polyvinyl alcohol hydrogel in a glass vessel, drying for 2 hours in an oven at 40 ℃ to obtain an acetalized polyvinyl alcohol film, and soaking the acetalized polyvinyl alcohol film in a heparin sodium aqueous solution with the concentration of 1% to obtain an acetalized heparin-polyvinyl alcohol compound.
4. Preparation of heparin-polyvinyl alcohol/acellular scaffold
Uniformly coating an acetalized heparin-polyvinyl alcohol compound on a decellularized scaffold, placing the decellularized scaffold in an oven, treating for 2 hours at 60 ℃, then heating to 70 ℃ for continuous treatment for 2 hours, cooling, taking out, soaking and flushing for 0.5 hour, removing impurities to obtain the heparin-polyvinyl alcohol/decellularized scaffold, and recording as He/PVA 3 /DCS。
EXAMPLE 24 preparation of heparin-polyvinyl alcohol/decellularized scaffold He/PVA 4 /DCS
1. Preparation of decellularized scaffolds was performed as in example 21.
2. Preparation of acetalized polyvinyl alcohol hydrogels
In a 500mL three-necked flask, a certain amount of 6g of polyvinyl alcohol and 180mL of water were added, and heated to 90℃to dissolve the polyvinyl alcohol; adding 3g of glycerol, and continuously heating for 0.5h; cooling to about 30 ℃, adding 4g of formaldehyde and 12g of glutaraldehyde with the concentration of 5%, and uniformly stirring to obtain acetalized polyvinyl alcohol; uniformly spreading acetalized polyvinyl alcohol on the bottom of a glass dish, respectively processing for 2 hours at 60 ℃ and 70 ℃ in an oven, cooling, taking out, soaking and flushing for 0.5 hour, and removing impurities; acetal polyvinyl alcohol hydrogel, designated PVA, was obtained 4 。
3. Preparation of acetalized heparin-polyvinyl alcohol complexes
Spreading acetalized polyvinyl alcohol hydrogel in a glass vessel, drying in an oven at 60 ℃ for 2 hours to obtain an acetalized polyvinyl alcohol film, and soaking the acetalized polyvinyl alcohol film in a heparin sodium aqueous solution with the concentration of 1% to obtain an acetalized heparin-polyvinyl alcohol compound.
4. Preparation of heparin-polyvinyl alcohol/acellular scaffold
Uniformly coating the acetalized heparin-polyvinyl alcohol compound on a decellularized scaffold, placing the decellularized scaffold in an oven, treating for 2 hours at 60 ℃, heating to 70 ℃ for continuous treatment for 2 hours, and coolingTaking out, soaking and flushing for 0.5h, removing impurities to obtain heparin-polyvinyl alcohol/decellularized stent, and recording as He/PVA 4 /DCS。
EXAMPLE 25 preparation of heparin-polyvinyl alcohol/decellularized scaffold He/PVA 5 /DCS
1. Preparation of decellularized scaffolds was performed as in example 21.
2. Preparation of acetalized polyvinyl alcohol hydrogels
In a 500mL three-necked flask, a certain amount of 6g of polyvinyl alcohol and 180mL of water were added, and heated to 90℃to dissolve the polyvinyl alcohol; adding 3g of glycerol, and continuously heating for 0.5h; cooling to about 30 ℃, adding 4g of formaldehyde and 12g of glutaraldehyde with the concentration of 7%, and uniformly stirring to obtain acetalized polyvinyl alcohol; uniformly spreading acetalized polyvinyl alcohol on the bottom of a glass dish, respectively processing for 2 hours at 60 ℃ and 70 ℃ in an oven, cooling, taking out, soaking and flushing for 0.5 hour, and removing impurities; acetal polyvinyl alcohol hydrogel, designated PVA, was obtained 5 。
3. Preparation of acetalized heparin-polyvinyl alcohol complexes
Spreading acetalized polyvinyl alcohol hydrogel in a glass vessel, drying in an oven at 40 ℃ for 2 hours to obtain an acetalized polyvinyl alcohol film, and soaking the acetalized polyvinyl alcohol film in a heparin sodium aqueous solution with the concentration of 1% to obtain an acetalized heparin-polyvinyl alcohol compound.
4. Preparation of heparin-polyvinyl alcohol/acellular scaffold
Uniformly coating an acetalized heparin-polyvinyl alcohol compound on a decellularized scaffold, placing the decellularized scaffold in an oven, treating for 2 hours at 50 ℃, heating to 70 ℃ for continuous treatment for 2 hours, cooling, taking out, soaking and flushing for 0.5 hour, removing impurities to obtain the heparin-polyvinyl alcohol/decellularized scaffold, and recording as He/PVA 5 /DCS。
EXAMPLE 26 preparation of heparin-polyvinyl alcohol/decellularized scaffold He/PVA 6 /DCS
1. Preparation of decellularized scaffolds was performed as in example 22.
2. Preparation of acetalized polyvinyl alcohol hydrogels
In a 500mL three-necked flask, a certain amount of 6g of polyvinyl alcohol and 180mL of water were added, and heated to 90Dissolving at a temperature of; adding 3g of glycerol, and continuously heating for 0.5h; cooling to about 30 ℃, adding 4g of formaldehyde and 12g of glutaraldehyde with the concentration of 9%, and uniformly stirring to obtain acetalized polyvinyl alcohol; uniformly spreading acetalized polyvinyl alcohol on the bottom of a glass dish, respectively processing for 2 hours at 60 ℃ and 70 ℃ in an oven, cooling, taking out, soaking and flushing for 0.5 hour, and removing impurities; acetal polyvinyl alcohol hydrogel, designated PVA, was obtained 6 。
3. Preparation of acetalized heparin-polyvinyl alcohol complexes
Spreading acetalized polyvinyl alcohol hydrogel in a glass vessel, drying in an oven at 60 ℃ for 2 hours to obtain an acetalized polyvinyl alcohol film, and soaking the acetalized polyvinyl alcohol film in a heparin sodium aqueous solution with the concentration of 1% to obtain an acetalized heparin-polyvinyl alcohol compound.
4. Preparation of heparin-polyvinyl alcohol/acellular scaffold
Uniformly coating an acetalized heparin-polyvinyl alcohol compound on a decellularized scaffold, placing the decellularized scaffold in an oven, treating for 2 hours at 60 ℃, then heating to 70 ℃ for continuous treatment for 2 hours, cooling, taking out, soaking and flushing for 0.5 hour, removing impurities to obtain the heparin-polyvinyl alcohol/decellularized scaffold, and recording as He/PVA 6 /DCS。
EXAMPLE 27 preparation of heparin-polyvinyl alcohol/decellularized scaffold He/PVA 4 /DCS
1. Preparation of decellularized scaffolds was performed as in example 22.
2. Preparation of acetalized polyvinyl alcohol hydrogels
In a 500mL three-necked flask, 10g of polyvinyl alcohol and 180mL of water were added, and the mixture was heated to 95℃to dissolve the mixture; adding 6g of glycerol, and continuously heating for 1h; cooling to about 30 ℃, adding 4g of formaldehyde and 12g of glutaraldehyde with the concentration of 5%, and uniformly stirring to obtain acetalized polyvinyl alcohol; uniformly spreading acetalized polyvinyl alcohol on the bottom of a glass dish, respectively treating at 40 ℃ for 3h and 60 ℃ for 2h in an oven, cooling, taking out, soaking and washing for 1h, and removing impurities; acetal polyvinyl alcohol hydrogel, designated PVA, was obtained 4 。
3. Preparation of acetalized heparin-polyvinyl alcohol complexes
Spreading acetalized polyvinyl alcohol hydrogel in a glass vessel, drying in an oven at 50 ℃ for 2 hours to obtain an acetalized polyvinyl alcohol film, and soaking the acetalized polyvinyl alcohol film in a heparin sodium aqueous solution with the concentration of 2% to obtain an acetalized heparin-polyvinyl alcohol compound.
4. Preparation of heparin-polyvinyl alcohol/acellular scaffold
Uniformly coating an acetalized heparin-polyvinyl alcohol compound on a decellularized scaffold, placing the decellularized scaffold in an oven, treating for 2 hours at 50 ℃, then heating to 70 ℃ for continuous treatment for 1 hour, cooling, taking out, soaking and washing for 1 hour, removing impurities to obtain the heparin-polyvinyl alcohol/decellularized scaffold, and recording as He/PVA 4 /DCS。
EXAMPLE 28 preparation of heparin-polyvinyl alcohol/decellularized scaffold He/PVA 5 /DCS
1. Preparation of decellularized scaffolds was performed as in example 22.
2. Preparation of acetalized polyvinyl alcohol hydrogels
In a 500mL three-necked flask, a certain amount of 6g of polyvinyl alcohol and 180mL of water were added, and the mixture was heated to 100℃to dissolve the polyvinyl alcohol; adding 3g of glycerol, and continuously heating for 0.5h; cooling to about 30 ℃, adding 4g of formaldehyde and 12g of glutaraldehyde with the concentration of 7%, and uniformly stirring to obtain acetalized polyvinyl alcohol; uniformly spreading acetalized polyvinyl alcohol on the bottom of a glass dish, respectively treating at 40 ℃ for 2h and 60 ℃ for 1h in an oven, cooling, taking out, soaking and washing for 1h, and removing impurities; acetal polyvinyl alcohol hydrogel, designated PVA, was obtained 5 。
3. Preparation of acetalized heparin-polyvinyl alcohol complexes
Spreading acetalized polyvinyl alcohol hydrogel in a glass vessel, drying in an oven at 60 ℃ for 2 hours to obtain an acetalized polyvinyl alcohol film, and soaking the acetalized polyvinyl alcohol film in a heparin sodium aqueous solution with the concentration of 2% to obtain an acetalized heparin-polyvinyl alcohol compound.
4. Preparation of heparin-polyvinyl alcohol/acellular scaffold
Uniformly coating an acetalized heparin-polyvinyl alcohol compound on a decellularized scaffold, placing the decellularized scaffold in an oven, treating at 60 ℃ for 1h, and then heating to 70 ℃ for continuing2h, cooling, taking out, soaking and flushing for 0.5h, removing impurities to obtain heparin-polyvinyl alcohol/decellularized stent, and recording as He/PVA 5 /DCS。
EXAMPLE 29 preparation of heparin-polyvinyl alcohol/decellularized scaffold He/PVA 3 /DCS
1. Preparation of decellularized scaffolds was performed as in example 21.
2. Preparation of acetalized polyvinyl alcohol hydrogels
In a 500mL three-necked flask, a certain amount of 6g of polyvinyl alcohol and 180mL of water were added, and heated to 90℃to dissolve the polyvinyl alcohol; adding 3g of glycerol, and continuously heating for 1h; cooling to about 30 ℃, adding 4g of formaldehyde and 12g of glutaraldehyde with the concentration of 3%, and uniformly stirring to obtain acetalized polyvinyl alcohol; uniformly spreading acetalized polyvinyl alcohol on the bottom of a glass dish, respectively processing for 2 hours at 40 ℃ and 70 ℃ in an oven, cooling, taking out, soaking and washing for 1 hour, and removing impurities; acetal polyvinyl alcohol hydrogel, designated PVA, was obtained 3 。
3. Preparation of acetalized heparin-polyvinyl alcohol complexes
Spreading acetalized polyvinyl alcohol hydrogel in a glass vessel, drying in an oven at 40 ℃ for 3 hours to obtain an acetalized polyvinyl alcohol film, and soaking the acetalized polyvinyl alcohol film in a heparin sodium aqueous solution with the concentration of 1% to obtain an acetalized heparin-polyvinyl alcohol compound.
4. Preparation of heparin-polyvinyl alcohol/acellular scaffold
Uniformly coating an acetalized heparin-polyvinyl alcohol compound on a decellularized scaffold, placing the decellularized scaffold in an oven, treating for 2 hours at 50 ℃, then heating to 70 ℃ for continuous treatment for 1 hour, cooling, taking out, soaking and washing for 1 hour, removing impurities to obtain the heparin-polyvinyl alcohol/decellularized scaffold, and recording as He/PVA 3 /DCS。
EXAMPLE 30 preparation of heparin-polyvinyl alcohol/decellularized scaffold He/PVA 6 /DCS
1. Preparation of decellularized scaffolds was performed as in example 21.
2. Preparation of acetalized polyvinyl alcohol hydrogels
In a 500mL three-necked flask, a certain amount of 6g of polyvinyl alcohol and 180mL of water were added,heating to 100deg.C to dissolve; adding 4g of glycerol, and continuously heating for 1h; cooling to about 30 ℃, adding 4g of formaldehyde and 12g of glutaraldehyde with the concentration of 9%, and uniformly stirring to obtain acetalized polyvinyl alcohol; uniformly spreading acetalized polyvinyl alcohol on the bottom of a glass dish, respectively treating at 60 ℃ for 3h and 70 ℃ for 2h in an oven, cooling, taking out, soaking and flushing for 0.5h, and removing impurities; acetal polyvinyl alcohol hydrogel, designated PVA, was obtained 6 。
3. Preparation of acetalized heparin-polyvinyl alcohol complexes
Spreading the acetalized polyvinyl alcohol hydrogel in a glass vessel, drying for 3 hours in an oven at 50 ℃ to obtain an acetalized polyvinyl alcohol film, and soaking the acetalized polyvinyl alcohol film in a heparin sodium aqueous solution with the concentration of 2% to obtain an acetalized heparin-polyvinyl alcohol compound.
4. Preparation of heparin-polyvinyl alcohol/acellular scaffold
Uniformly coating an acetalized heparin-polyvinyl alcohol compound on a decellularized scaffold, placing the decellularized scaffold in an oven, treating for 2 hours at 60 ℃, then heating to 70 ℃ for continuous treatment for 2 hours, cooling, taking out, soaking and washing for 1 hour, removing impurities to obtain the heparin-polyvinyl alcohol/decellularized scaffold, and recording as He/PVA 6 /DCS。
EXAMPLE 31 acetalized polyvinyl alcohol hydrogels scanning electron microscopy
A series of different acetalized polyvinyl alcohol hydrogels (PVA) obtained in examples 21 to 26 were prepared n N=1 to 6), and the appearance and characteristics thereof were observed by a scanning electron microscope after vacuum drying, as shown in fig. 13. FIG. 13 shows various acetalized polyvinyl alcohol hydrogels PVA n (n=1 to 6) electron microscopic images, wherein a is the acetalized polyvinyl alcohol hydrogel PVA obtained in example 21 1 Wherein glutaraldehyde concentration is 0.5%; b the acetalized polyvinyl alcohol hydrogels PVA obtained in example 22 2 Wherein glutaraldehyde concentration is 1%; c acetalized polyvinyl alcohol hydrogels PVA obtained in example 23 3 Wherein glutaraldehyde concentration is 3%; d acetalized polyvinyl alcohol hydrogels PVA obtained in example 24 4 Wherein glutaraldehyde concentration is 5%; e acetalized polyvinyl alcohol water obtained in example 25Gel PVA 5 Wherein glutaraldehyde concentration is 7%; f acetalized polyvinyl alcohol hydrogels PVA obtained in example 26 6 Wherein the glutaraldehyde concentration is 9%. As can be seen from FIG. 13, the polyvinyl alcohol hydrogel films had uneven surfaces at glutaraldehyde concentrations of 0.5%, 1% and 3% in a, b and c, due to incomplete acetalization reactions. When the glutaraldehyde concentration is 5%, namely the graph d, the surface of the obtained polyvinyl alcohol film is smoother and smoother, which shows that when the glutaraldehyde concentration is 5%, the obtained polyvinyl alcohol hydrogel film is better; when the glutaraldehyde concentration is more than 5%, namely the concentration reaches 7%, namely the graph e, the surface of the polyvinyl alcohol film starts to generate a gelation phenomenon, and when the concentration is 9%, namely the graph f, the gelation phenomenon of the film surface is obvious, which shows that under the concentration, the intermolecular crosslinking of the polyvinyl alcohol is increased, so that the brittleness of the hydrogel film is increased, the flexibility is insufficient, and the expected requirement of the user cannot be met. From the above results, it is found that the polyvinyl alcohol hydrogel film produced by acetalization is preferable when the glutaraldehyde concentration is 5%.
EXAMPLE 32 heparin-polyvinyl alcohol/decellularized scaffold He/PVA 4 DCS surface coating for infrared characterization
He/PVA as a decellularized scaffold material was prepared according to example 24 4 The infrared characterization results of the DCS surface coating are shown in FIG. 14. FIG. 14 is a view of heparin-polyvinyl alcohol/decellularized scaffold He/PVA 4 As can be seen from FIG. 14, the DCS infrared spectrum is at 1250cm -1 Several strong absorption peaks in the vicinity are characteristic peaks of acetals (c=o=c), of which 1150-1050 cm -1 The strong absorption peak of (C) corresponds to the antisymmetric stretching vibration peak of saturated fatty ether (C-O-C), and is characteristic identification. From this, it was found that the acetalization reaction of polyvinyl alcohol did occur. At 3400-3200 cm -1 A broad peak is arranged nearby, the characteristic peak corresponds to the association hydrogen bond between the polymers, and the free-OH plane angle of change is 1250cm -1 Vibration absorption peak appears at 1500-1300 cm -1 Corresponding to the characteristic peak of-OH hydrogen bond association. From the above results, it was found that only a part of the hydroxyl groups were acetalized, and that a part of the hydroxyl groups still existed in the system in the form of intermolecular association hydrogen peaks of the polymer, and a small amount of the hydroxyl groups existed in the free form.
EXAMPLE 33 heparin-polyvinyl alcohol/decellularized scaffold He/PVA with varying degrees of acetalization at different concentrations n Surface coating of DCS (n=1 to 6, namely corresponding examples 21 to 26) for scanning electron microscope characterization
Heparin-polyvinyl alcohol/decellularized scaffold He/PVA of different acetalization degrees obtained in examples 21 to 26 n The surface films of DCS (n=1 to 6) were subjected to scanning electron microscope characterization, and the results are shown in fig. 15. FIG. 15 is a diagram of heparin-polyvinyl alcohol/decellularized scaffold He/PVA with varying degrees of acetalization n SEM image of/DCS (n=1 to 6), wherein 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. As can be seen from fig. 15, the surface-bound heparin is less in a, and the surface-bound heparin amount increases with the increase of glutaraldehyde concentration and acetalization degree in b to f; however, the gel phenomenon and fine cracks appear from e, and f is more serious, so that when the glutaraldehyde concentration in d is 5%, the surface of the material shows good performance, and the hydrogel performance is better and is consistent with the result before heparin is bonded.
EXAMPLE 34 PVA prepared in example 24 1 DCS and He/PVA 4 X-ray photoelectron spectroscopy of DCS
Preparation of PVA from example 24 1 DCS and He/PVA 4 The result of the X-ray photoelectron spectroscopy test of DCS using a Japanese D/max 2500VL/PC type X-ray photoelectron spectrometer under conditions of monochromatic Al K alpha rays (150W, 500 μm beam spot) and energy passing through 20eV is shown in FIG. 16. FIG. 16 shows PVA 1 DCS and He/PVA 4 XPS contrast plot of DCS, where a is PVA 1 DCS, b is He/PVA 4 DCS. As can be seen from FIG. 16, S appears on b 2p Description of peaks He/PVA 4 S element is present on DCS due to He/PVA 4 Heparin on DCS artificial vascular material has S element, so He/PVA can be proved 4 DCS artificial vascular material has been successfully prepared.
Example 35 heparin on fresh blood vessels, decellularized scaffolds and varying degrees of acetalizationPolyvinyl alcohol/decellularized stent He/PVA n DCS (n=1 to 6) was characterized for mechanical tensile strength and elongation at break and burst strength
Heparin-polyvinyl alcohol/decellularized scaffolds He/PVA of different acetalization degrees obtained in examples 21 to 26 for fresh blood vessels, decellularized scaffolds n Specific experimental procedures for the characterization of mechanical tensile strength and elongation at break and burst strength by DCS (n=1 to 6) were the same as in example 16, and the specific results are shown in fig. 17. FIG. 17 is a view of fresh blood vessel, DCS and He/PVA n Tensile strength, elongation at break and burst strength of DCS (n=1 to 6). Wherein a is fresh blood vessel, DCS and He/PVA n Tensile strength chart of DCS (n=1 to 6), b is fresh blood vessel, DCS and He/PVA n DCS (n=1 to 6) elongation at break. c is fresh blood vessel, DCS and He/PVA n and/DCS (n=1 to 6) burst strength plot. Wherein Fresh blood vessel is fresh blood vessel, and DCS is decellularized vascular stent. From figures a and b, it can be seen that DCS has slightly increased tensile properties and slightly increased elongation at break compared to fresh blood vessels, but in general the difference between the two is small. After being coated with polyvinyl alcohol with different acetalization degrees, the tensile property of DCS is slightly reduced, and the elongation at break is obviously increased. This phenomenon suggests that acetalized polyvinyl alcohols can significantly improve the elongation at break of vascular materials. Wherein, as glutaraldehyde concentration increases, the tensile strength and elongation at break of the vascular material increase, and the vascular material is optimal at a concentration of 5%. And when the concentration is increased to 7% and 9%, the tensile strength and the elongation at break of the corresponding vascular material are reduced, which is consistent with the previous experimental result. Comparison of He/PVA samples with different acetalization degrees n DCS (n=1 to 6), the tensile strength and elongation at break hardly differ. The results show that after PVA film and heparin deposition, the tensile strength and elongation at break of DCS are not obviously changed, and the mechanical properties of DCS are still ensured. As can be seen from graph c, the burst strength of the fresh arterial vessel is about 110KPa, while the burst strength of DCS is 0KPa, because DCS removes tissue cells on the surface layer to form a space reticular structure, and physiological saline can directly flow from the reticular structure Since DCS does not have burst strength. The DCS modified by heparin and PVA hydrogel embeds the space network structure of the DCS, the bursting strength of the DCS tends to rise along with the increase of the polyelectrolyte layer, but each sample is basically similar to the bursting strength of a fresh arterial vessel. The test results show that after the DCS is modified by heparin and PVA hydrogel, the mechanical bionic performance of the DCS is basically maintained, so that the prepared artificial vascular material can meet the pressure requirement generated when blood flows in the lumen of the blood vessel.
EXAMPLE 36 heparin-polyvinyl alcohol/Decellularized stent Artificial vascular Material He/PVA with different acetalization degrees n In vitro coagulation experiments and recalcification coagulation time tests of DCS (n=1 to 6)
Heparin-polyvinyl alcohol/decellularized stent artificial vascular material He/PVA with different acetalization degrees n In vitro coagulation experiments and recalcification coagulation time tests of DCS (n=1 to 6) specific experimental procedures were the same as in example 17, and specific results are shown in fig. 18. FIG. 18 is a view of He/PVA n Activated partial thromboplastin time, thrombin time, prothrombin time and hypercalcemia time profile of DCS (n=1 to 6) vascular prosthesis material, wherein a is He/PVA n Activated partial thromboplastin time chart of DCS (n=1 to 6) vascular prosthesis material, b being He/PVA n Prothrombin time chart of DCS (n=1 to 6) vascular prosthesis, c is He/PVA n Thrombin time chart of DCS (n=1 to 6) vascular prosthesis, d is He/PVA n DCS (n=1 to 6) artificial vascular material hypercalcemia time chart. As can be seen from FIGS. 18 a-c, the clotting time of APTT, TT, PT of the artificial vascular material modified with heparin and polyvinyl alcohol was somewhat prolonged compared to the control group. This is because heparin can effectively improve blood compatibility and anticoagulation capacity, and we can also find He/PVA 4 From the above experimental results, it can be considered that when n=4, he/PVA was obtained when the values of APTT, TT, PT at the time of DCS reached the optimum values 4 The DCS artificial vascular material has the longest in vitro coagulation time and the best anticoagulation performance. Adding proper amount of calcium ions into the anticoagulated blood plasma after removing the calcium ions, and generating the blood plasmaThe time elapsed for setting is referred to as the dicalcium period. The longer it takes for plasma to calcifie, the better its blood compatibility. As can be seen from FIG. 18 d, the T of heparin-polyvinyl alcohol/decellularized scaffold vascular material compared to the control group 1/2max The calcium recovery time is obviously prolonged, the calcium recovery time is increased from 10.5min to 18.2 min-23.8 min, and the blood coagulation time is obviously prolonged. Experimental results show that heparin-polyvinyl alcohol/decellularized scaffold can prolong the time for converting soluble fibrinogen into soluble fibrin, thus prolonging the blood coagulation time. This is thought to be because heparin can bind to antithrombin iii, and thus inhibit thrombin from functioning as a blood coagulation agent, thereby improving the blood compatibility of the material and prolonging the clotting time of recalcification. When n=4 (product of example 24), he/PVA 4 T of DCS 1/2max The calcium recovery time is longest. It can be considered that He/PVA 4 The DCS has the longest calcium recovery time. Namely, when the glutaraldehyde concentration is 5%, the anticoagulation modifying effect of the material is optimal.
EXAMPLE 37 heparin-polyvinyl alcohol/decellularized scaffolds He/PVA with varying degrees of acetalization n DCS (n=1 to 6) blood compatibility test
Heparin-polyvinyl alcohol/decellularized scaffold He/PVA with different acetalization degrees n The procedure of the blood compatibility test of DCS (n=1 to 6) was the same as in example 18, and the experimental results are shown in fig. 19. FIG. 19 is a view of He/PVA n Red blood cell morphology of/DCS (n=1 to 6), wherein a is control group and b is He/PVA 1 DCS, c is He/PVA 2 DCS, d is He/PVA 3 DCS, e is He/PVA 4 DCS, f is He/PVA 5 DCS, g is He/PVA 6 DCS. As can be seen from FIG. 19, when the erythrocytes were contacted with the vascular material of the present invention, the morphology was consistent with that of the control group, and no significant deformation or rupture was observed. The results show that heparin-modified PVA n DCS has almost no toxic effect on erythrocytes, i.e. good blood compatibility.
EXAMPLE 38 heparin-polyvinyl alcohol/decellularized scaffolds He/PVA with varying degrees of acetalization n MTT cytotoxicity test performed by DCS (n=1 to 6)
1. The specific experiment: sample preparation: heparin-polyvinyl alcohol/decellularized stent He/PVA are respectively taken n DCS (n=1 to 6) vascular material was immersed in 10mL of immersion medium (10% fetal bovine serum) in a constant temperature shaking incubator at 37 ℃ for 24h. Preparing MTT solution: MTT was dissolved in PBS to prepare an MTT solution having a concentration of 5 mg/mL. Digesting cells in logarithmic growth phase with pancreatin, diluting with cell culture solution to a concentration of 4×10 4 And each mL. A96-well plate was used, 200. Mu.L of the cell suspension was inoculated into each well, and the plate was placed at 37℃in 5% CO 2 Culturing in an incubator for 24 hours. After the cells are attached, the stock culture solution in each hole is sucked out, 200 mu L of sample leaching solution is added in each hole of an experimental group (8 holes are arranged in each group), meanwhile, a blank control group is arranged in a pore plate without cells, the cell culture solution is added, and the mixture is continuously placed at 37 ℃ and 5% CO 2 Culturing in an incubator for 12 hours and 24 hours; after that, 20. Mu.L of the prepared MTT solution was added to each well, and after culturing for 5 hours, the stock solution was discarded, 150. Mu.L of dimethyl sulfoxide was added to each well, and the mixture was shaken horizontally for 5 minutes, and the absorbance O.D. at 490nm was measured by an enzyme-labeled instrument (BioTek synergy type 2). Each test was performed at least three times. Experimental group: endothelial progenitor cells +He/PVA n DCS (n=1 to 6) +mtt. Negative control group: endothelial progenitor cells + MTT. Blank control group: MTT.
And (3) calculating results:
wherein: dt—absorbance of the experimental group samples; dnc-absorbance of negative control samples; db-absorbance of control samples. The experimental results are shown in FIG. 20.
2. Analysis of results: MTT is a common method for detecting cell survival, and succinic dehydrogenase of living cells reduces MTT to form a blue-violet crystalline formazan insoluble in water, while dead cells do not. The first step can be represented by the light absorption value of enzyme label at 490nm to estimate the number of living cells, and if the number of cells is large, the measured absorbance will be slightly larger, and if the number of cells is small or the state is bad, the absorbance will be lower, and the toxicity of the material can be determined from the absorbanceSize of the product. FIG. 20 is a view of He/PVA n MTT test chart of DCS (n=1 to 6). As can be seen from FIG. 20, at 12h, he/PVA n The cell survival rate of DCS in DCS (n=1-3) reaches 95% -98%, the cytotoxicity is 1 grade, but the cell survival rate is increased along with the increase of glutaraldehyde concentration; he/PVA n DCS (n=4 to 6) has cell viability up to 100% -105% and cytotoxicity of 0 level, but cell viability decreases with increasing glutaraldehyde concentration, he/PVA 4 Cell viability of DCS was highest, 105%, cytotoxicity was smallest, and different acetalization degree He/PVA n Cell viability differences between DCS (n=1 to 6) samples were small. n=4, i.e. glutaraldehyde concentration 5% cell viability was greater, consistent with the results of the earlier compatibility experiments. From the above, it was found that when n is 4, he/PVA obtained by acetalization with glutaraldehyde concentration of 5% was obtained 4 DCS has the best comprehensive performance.
EXAMPLE 39PVA/DCS and He/PVA 4 In vitro degradation experiment of DCS artificial vascular material
1. The specific experiment: from the above, it is considered that when n=4, i.e., he/PVA 4 The DCS artificial vascular material has the best comprehensive performance. In subsequent studies, the mixture was prepared as He/PVA 4 DCS was tested as a subject. PVA/DCS and He/PVA 4 DCS artificial vascular material is cut into square with 10mmx10mm, and the weight of the artificial vascular material is measured to be m by an electronic balance 0 Placing into a centrifuge tube, adding 20mL PBS solution, placing into a shaking oven at 37deg.C, culturing for 1, 3, 7, 14, 21, 60, 90, 120 and 180 days, replacing with fresh PBS buffer solution every week, taking out cultured artificial blood vessel material, drying in a vacuum drying oven at 50deg.C, and weighing with electronic balance to weight of m 1 Weight loss results were calculated: weight loss (%) = (m) 0 -m 1 )/m 0 100% and the results are shown in FIG. 21.
2. Analysis of results: FIG. 21 is a PVA/DCS and He/PVA 4 As can be seen from FIG. 21, the in vitro degradation rate graph of the DCS artificial vascular material shows that PVA/DCS and He/PVA are increased with time 4 The in vitro degradation rate of DCS shows a trend from fast to slow; at 180 days, PVA/DCS remained37.9% by mass of He/PVA 4 The residual mass of the DCS was 40.4%. The in-vitro degradation period and the blood vessel regeneration period between the two are basically matched, which shows that the two have good degradation rate matching property, and the in-vitro degradation period and the blood vessel regeneration period can be well matched with the regeneration period of a new blood vessel as artificial blood vessel materials, so that good bionic effect is achieved.
EXAMPLE 40n-He-CS/PEG/DCS, he/PVA 4 Comparative experiment of/DCS and He-Ch-5/PU/DCS
1. Preparing He-Ch-5/PU/DCS: he-Ch-5/PU/DCS artificial vascular material was prepared in a preliminary study, and its specific preparation steps were as follows: the decellularized scaffold (Decellularized scaffold, DCS) was prepared as in example 1. DCS was immersed in PBS at ph=7.4 for 30min. 5.0g of Polyurethane (PU) was dissolved in 50mL of N, N-Dimethylformamide (DMF), and a PU solution of 0.1g/mL was obtained at room temperature. The PU solution is dip-coated on DCS, and the dip-coating is repeated three times, and the PU/DCS is obtained after drying for 12 hours at 60 ℃ in a vacuum drying oven. 1g/L heparin and 1g/L chitosan solution and 0.01mol/L PBS buffer solution were prepared, respectively. PU/DCS is soaked in PBS buffer solution for 30min, and then soaked in heparin solution for 15min, so as to obtain He/PU/DCS. He/PU/DCS is washed by PBS buffer solution to remove heparin sodium physically adsorbed on the surface of the He/PU/DCS. And (3) soaking the He/PU/DCS in a chitosan solution for 15min, washing with a PBS buffer solution, and removing chitosan physically adsorbed on the surface of the chitosan solution to obtain the He-Ch-2/PU/DCS artificial vascular material. And the He-Ch-5/PU/DCS vascular material is repeatedly soaked in heparin and chitosan solution for 3 times to prepare the He-Ch-2/PU/DCS vascular material.
2. Performance test: the fresh blood vessel, he-Ch-5/PU/DCS, he/PVA were tested with a mechanical tensile test apparatus (model CMT6103, china MTS) at a test speed of 10mm/min (n=5) 4 Tensile properties of/DCS and 5-He-CS/PEG/DCS (50 mm. Times.20 mm. Times.1 mm). Stress-strain curves were plotted from the load and elongation data obtained from the test, and the results are shown in fig. 24.
3. Analysis of results: FIG. 24 is a view of a fresh blood vessel, he-Ch-5/PU/DCS, he/PVA 4 Stress-strain graphs of/DCS and 5-He-CS/PEG/DCS, wherein figure a is the stress-strain curve of fresh blood vessels, figureb is the stress-strain curve of 5-He-CS/PEG/DCS, c is the stress-strain curve of He-Ch-5/PU/DCS, d is He/PVA 4 Stress-strain curve of DCS. From figures a and b, it can be seen that the 5-He-CS/PEG/DCS vascular material modified by the hydrogel and chitosan heparin maintains a stress-strain curve similar to that of fresh blood vessels, because the PEG hydrogel has a smaller modulus, and does not greatly affect the mechanical properties of the decellularized scaffold. Thereby ensuring that the prepared vascular material maintains the mechanical property matched with the fresh blood vessel. As shown in the graph c, as PU has a higher elastic modulus, after the acellular stent is subjected to tectorial membrane modification, the mechanical properties of the acellular stent are greatly changed, and the stress and the strain are greatly improved, but the stress-strain curve has a larger difference with that of a fresh blood vessel; as can be seen from FIG. d, after the PVA hydrogel is coated, he/PVA 4 The He-Ch-5/PU/DCS has better elasticity and tensile property, and the stress-strain curve is not greatly different from that of the fresh blood vessel, so that the He-Ch-5/PU/DCS can not realize the mechanical property matched with the fresh blood vessel, while the hydrogel maintains the mechanical property matched with the fresh blood vessel, so that the He/PVA can be considered 4 The DCS and 5-He-CS/PEG/DCS artificial vascular materials have better mechanical bionic property, and are hopeful to keep the implanted part clear for a long time.
EXAMPLE 41 5-He-CS/PEG/DCS and He/PVA 4 DCS for animal experiment research
1. The specific experiment: fragrant pigs with month age of one month and good growth are selected as experimental objects, and the experiment is started after the fragrant pigs are fed for 4 weeks under standard conditions. Control group: 2 small fragrant pigs, and a carotid artery is implanted with a distended polytetrafluoroethylene artificial blood vessel, and the experimental group: 2 small fragrant pigs, 5-He-CS/PEG/DCS artificial vascular material implanted in carotid artery, 2 small fragrant pigs, and He/PVA implanted in carotid artery 4 DCS artificial vascular material; comparative example experiment: 2 small fragrant pigs and carotid arteries are respectively implanted with He-Ch-5/PU/DCS artificial vascular materials. After the artificial vascular material synthesized by the invention is implanted, B ultrasonic monitoring is carried out after two weeks. Ultrasonic B-mode is one way of ultrasonic inspection, and is a non-operative diagnostic inspection. Has uniqueness in the aspect of human soft tissues and blood flow dynamics. After about 2 weeks of operation, make B ultrasonic analysis is carried out on the implantation position of the artificial vascular material of the gilt by L38e/10-5MHz full-digital color Doppler ultrasound of Sonosite corporation in the United states, the blood flow smoothness condition in the blood vessel is observed, the conditions of occlusion, expansion and the like of the blood vessel are observed, and the specific result is shown in figure 22.
2. Analysis of results: FIG. 22 is a diagram of 5-He-CS/PEG/DCS and He/PVA 4 B ultrasonic image of DCS artificial blood vessel material after being implanted into human body for two weeks, wherein a is control group expanded polytetrafluoroethylene artificial blood vessel, B is experimental group 5-He-CS/PEG/DCS artificial blood vessel material, c is control example He-Ch-5/PU/DCS artificial blood vessel material, d is He/PVA 4 DCS artificial vascular material. The red areas of the figure represent blood flow therethrough. The intermittent blood flow passing through the implantation position of the a-expanded polytetrafluoroethylene artificial blood vessel material in fig. 22 shows that the artificial blood vessel material at the implantation position forms thrombus, so that the blood flow at the implantation position is not smooth, thereby showing that the expanded polytetrafluoroethylene artificial blood vessel material cannot replace normal blood vessels in pigs; whereas the implantation sites of b, c and d in fig. 22 appear visibly red, indicating that no thrombus is formed at the three vascular graft sites. By comparing the B ultrasonic images of three different artificial vascular materials, the 5-He-CS/PEG/DCS/He/PVA can be obviously found 4 The artificial blood vessel material of/DCS and the artificial blood vessel material of the comparative example He-Ch-5/PU/DCS are not easy to form thrombus, have better blood compatibility and can replace the function of normal blood vessels.
EXAMPLE 42 implantation of control expanded polytetrafluoroethylene prosthesis and Experimental group 5-He-CS/PEG/DCS, he/PVA in example 41 4 After five months of artificial blood vessel material of/DCS and artificial blood vessel material of comparative experiment He-Ch-5/PU/DCS, CTA detection was carried out
1. The specific experiment: CT angiography (CTA) combines CT enhancement with thin-layer, large-scale, fast scanning, and clearly shows the vascular details of various parts of the whole body through reasonable post-processing. The control group of expanded polytetrafluoroethylene artificial blood vessel and the experimental group of 5-He-CS/PEG/DCS, he/PVA were implanted in example 41 4 After five months of artificial blood vessel material of/DCS and comparative experiment He-Ch-5/PU/DCS, the artificial blood vessel material is subjected to section scanning observation by using CT angiography technologyThe blood circulation at the implantation site was observed, and the patency rate was observed, and the results are shown in fig. 23.
2. Analysis of results: FIG. 23 is 5-He-CS/PEG/DCS and He/PVA 4 CTA graph of implantation site after implantation of DCS artificial vascular material in vivo for 5 months, wherein a is control group expanded polytetrafluoroethylene artificial vascular material, b is experimental group 5-He-CS/PEG/DCS artificial vascular material, c is control experiment He-Ch-5/PU/DCS artificial vascular material, d is He/PVA 4 DCS artificial vascular material. CTA is an angiography technology, and by constructing a 3D model of blood flow, the flow condition of blood can be intuitively observed. As can be seen from fig. 23 a, after the implantation of the expanded polytetrafluoroethylene artificial blood vessel material, the blood vessel is blocked, and only a small amount of blood flows at the implantation position, but new branches grow from the side surface of the original blood vessel; in fig. 23 b, c and d, the implantation site is shown to have only slight protrusions, and the blood circulation is smooth without blocking. Description of 5-He-CS/PEG/DCS, he/PVA 4 The DCS and He-Ch-5/PU/DCS artificial vascular material can play a role in replacing natural blood vessels, and is a vascular replacement material with excellent performance.
Claims (7)
1. The application of the hydrogel in preparing the high bionic artificial blood vessel material is characterized in that the hydrogel is chitosan/polyethylene glycol hydrogel or acetalized polyvinyl alcohol hydrogel,
the preparation method of the high-bionic artificial blood vessel material comprises the following steps:
(1) Preparing chitosan/polyethylene glycol hydrogel;
(2) Coating chitosan/polyethylene glycol hydrogel on the surface of a cell-free scaffold to obtain chitosan/polyethylene glycol/cell-free scaffold CS/PEG/DCS;
(3) Depositing heparin on the surface of chitosan/polyethylene glycol/acellular stent by using a layer-by-layer self-assembly method, and vacuum drying to obtain a high bionic artificial vascular material n-He-CS/PEG/DCS;
The layer-by-layer self-assembly method is a soaking pulling method and comprises the following steps of:
(2.1) preparing heparin sodium solution and chitosan solution respectively;
(2.2) placing CS/PEG/DCS in PBS buffer solution, taking out, soaking in heparin sodium solution, taking out, and flushing the front and the back of the PBS buffer solution to obtain 1-He-CS/PEG/DCS;
(2.3) placing 1-He-CS/PEG/DCS in the prepared chitosan solution for soaking, taking out, washing the front and the back of the chitosan solution by using PBS buffer solution, soaking the chitosan solution in heparin sodium solution, taking out, and washing the front and the back of the chitosan solution by using PBS buffer solution to obtain 2-He-CS/PEG/DCS;
(2.4) repeating the step (2.3) to prepare a multi-layer heparin/polyethylene glycol hydrogel/decellularized scaffold to obtain n-He-CS/PEG/DCS;
in the step (2.1), the concentration of the heparin sodium solution is 1-2 g/L, the concentration of the chitosan solution is 1-2 g/L, and the mass ratio of the chitosan to the heparin sodium is 1: 1-2; in the step (2.2), the CS/PEG/DCS is placed in PBS buffer solution for soaking for 10-30 min, and the time for soaking in heparin sodium solution is 10-15 min; in the step (2.3), the 1-He-CS/PEG/DCS is placed in the prepared chitosan solution for 10-15 min, the time for soaking in the heparin sodium solution is 10-15 min, and in the step (2.4), n in the n-He-CS/PEG/DCS is 3-7;
The preparation method of the high-bionic artificial blood vessel material further comprises the following steps:
(1) Preparing different acetalized polyvinyl alcohol hydrogels;
(2) Preparing different acetalized heparin-polyvinyl alcohol complexes;
(3) Coating different acetalated heparin-polyvinyl alcohol complexes on the surface of a decellularized scaffold to obtain different acetalated heparin-polyvinyl alcohol/decellularized scaffolds, namely high bionic artificial vascular material He/PVAn/DCS; n=1 to 6.
2. The use according to claim 1, wherein the preparation method of the chitosan/polyethylene glycol hydrogel comprises the following steps:
(1) Preparing an NaOH aqueous solution, and then adding absolute ethyl alcohol to obtain the NaOH solution;
(2) Adding chitosan into NaOH solution while stirring, and alkalizing;
(3) Adding propylene oxide, and placing the mixture in a constant-temperature water bath for reaction to obtain a product;
(4) Taking out the product, placing the product in a mixed solution of hydrochloric acid and acetone for washing, placing the product in a mixed solution of acetone and water for washing, vacuum-filtering, and vacuum-drying to obtain O-HPCS;
(5) Preparing an O-HPCS aqueous solution, adding polyethylene glycol and glutaraldehyde solution, stirring and mixing, and standing to obtain chitosan/polyethylene glycol hydrogel.
3. The use according to claim 2, wherein in step (1), the NaOH solution has a mass concentration of 6% to 8%; in the step (2), the liquid-solid ratio of the NaOH solution to the chitosan is 10-12 mL: g, the alkalization time is 6-8 h; in the step (3), the reaction time is 24-36 h; in the step (4), the mass ratio of hydrochloric acid to acetone in the mixed solution of hydrochloric acid and acetone is 1:10-1:9, the mass ratio of acetone to water in the mixed solution of acetone and water is 9:1-10:1, the temperature of vacuum drying is 45-55 ℃, and the vacuum drying time is 2-3 hours; in the step (5), the solid-to-liquid ratio of the polyethylene glycol to glutaraldehyde is 0.1-0.2 g: and (3) mL.
4. The use according to claim 1, wherein the process for the preparation of the acetalised polyvinyl alcohol hydrogel comprises the steps of:
(1) Mixing polyvinyl alcohol with water, and heating to dissolve;
(2) Adding glycerol, and continuing heating;
(3) Cooling, adding formaldehyde and glutaraldehyde respectively, and stirring uniformly to obtain acetalized polyvinyl alcohol;
(4) And (3) drying the acetalized polyethylene, heating to continue drying, cooling, soaking and flushing with water to obtain the acetalized polyvinyl alcohol hydrogel.
5. The use according to claim 4, wherein in the step (1), the mass ratio of the polyvinyl alcohol to the water is 1:30-5:90, and the heating temperature is 90-100 ℃; in the step (2), the mass ratio of the glycerol to the polyvinyl alcohol is 1: 1-2, wherein the continuous heating time is 0.5-1 h; in the step (3), the mass ratio of formaldehyde to glutaraldehyde to polyvinyl alcohol is 2:6:3-2:6:5, and the concentration of glutaraldehyde is 0.5-9%; in the step (4), the drying temperature is 40-60 ℃, the drying time is 2-3 hours, the temperature of the re-heating is 60-70 ℃, the continuous drying time is 1-2 hours, and the soaking and flushing time is 0.5-1 hour.
6. The use according to claim 1, wherein the method of preparing the decellularized scaffold comprises the steps of:
(1) Taking out blood vessels placed in tissue fixing liquid, trimming adventitia, and immersing in physiological saline;
(2) Preparing a mixed solution of sodium dodecyl sulfate and polyethylene glycol octyl phenyl ether, and soaking the trimmed blood vessel in the mixed solution;
(3) Taking out the soaked blood vessel, flushing with PBS buffer solution, soaking in the PBS buffer solution, and changing the PBS buffer solution every day;
(4) Taking out the soaked blood vessel, trimming into slices, and freeze-drying to obtain the decellularized scaffold DCS.
7. The use according to claim 6, wherein in the step (2), the mass ratio of the sodium dodecyl sulfate to the polyethylene glycol octyl phenyl ether is 1:1-1:2, and the soaking time in the mixed solution is 24-48 h; in the step (3), the time for soaking in the PBS buffer solution is 20-30 days, and the times for replacing the PBS buffer solution every day is 1-2 times; in the step (4), the freeze-drying temperature is-45 to-55 ℃, and the freeze-drying time is 8-12 hours.
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