CN116251239A - Hydrogel and biological artificial blood vessel prepared from same - Google Patents

Hydrogel and biological artificial blood vessel prepared from same Download PDF

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Publication number
CN116251239A
CN116251239A CN202310275512.8A CN202310275512A CN116251239A CN 116251239 A CN116251239 A CN 116251239A CN 202310275512 A CN202310275512 A CN 202310275512A CN 116251239 A CN116251239 A CN 116251239A
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solution
hydrogel
blood vessel
artificial blood
preparation
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孙玲
许鹏赟
王恺
许雅彦
杨熙
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Lingbo Biotechnology Hangzhou Co ltd
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Lingbo Biotechnology Hangzhou Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • A61L27/34—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2420/00—Materials or methods for coatings medical devices
    • A61L2420/04—Coatings containing a composite material such as inorganic/organic, i.e. material comprising different phases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2420/00—Materials or methods for coatings medical devices
    • A61L2420/06—Coatings containing a mixture of two or more compounds
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W90/00—Enabling technologies or technologies with a potential or indirect contribution to greenhouse gas [GHG] emissions mitigation
    • Y02W90/10—Bio-packaging, e.g. packing containers made from renewable resources or bio-plastics

Abstract

The invention relates to the technical field of biology, in particular to hydrogel and a biological artificial blood vessel prepared from the hydrogel. The invention adopts carboxymethyl chitosan, oxidized sodium alginate, 2, 3-butanediol and other natural polymer materials to prepare hydrogel with good biocompatibility, and also provides a biological artificial blood vessel prepared by the hydrogel and a preparation method thereof. The biological artificial blood vessel provided by the invention has the advantages of low overall water leakage, strong biocompatibility, good moisturizing and antibacterial effects, and good effect of promoting cell growth. The hydrogel and biological artificial blood vessel of the invention have simple preparation process, and the selected materials have wide sources and low price, and are suitable for large-scale production and application.

Description

Hydrogel and biological artificial blood vessel prepared from same
Technical Field
The invention relates to the technical field of biology, in particular to hydrogel and a biological artificial blood vessel prepared from the hydrogel.
Background
A great deal of research has shown that the incidence of cardiovascular diseases increases with age, and as the aging of the population of china becomes severe, more and more cardiovascular and cerebrovascular patients will appear, wherein a significant part of them will suffer from vessel blockage and vessel necrosis, in which case vessel transplantation is required. At present, other people can normally transplant blood vessels, but the method has the advantages of less quantity of the supplied bodies, high price and strong rejection; also useful for vascular grafts in other parts of itself, such as: great saphenous vein, mammary artery, etc., but this approach damages other tissue, it is particularly important to develop artificial blood vessels from the standpoint of treatment conditions.
There are two types of vascular prostheses available: artificial blood vessels constructed by artificial materials and decellularized artificial blood vessels. The blood vessel constructed by the artificial material is mostly woven or knitted by a high polymer material, the blood permeability is high, the compliance of the blood vessel is difficult to match with that of the blood vessel, and researches show that the occurrence rate of thrombosis and intimal hyperplasia is 40% -60% in 1 year after the implantation of the vascular implant made of the synthetic material. The artificial blood vessel prepared by the allogenic animal through decellularization retains a natural three-dimensional model, has good biological compatibility and mechanical property, and is considered to be the most ideal blood vessel substitute in the 21 st century. However, the chemical agents that may be used during the decellularization process of the decellularized vascular prosthesis may cause the three-dimensional structure of the extracellular matrix to become loose, resulting in higher porosity and permeability than the original vessel. Because of weak tightness, blood may leak in a short time after the artificial blood vessel is transplanted into a foreign body, which affects the transplanting efficiency.
The hydrogel has a cross-linked network which can swell in water and keep a large amount of water without being dissolved, is a high molecular functional material integrating water absorption, water retention and controlled release, is similar to a living tissue material, has properties similar to extracellular matrixes, shows good biocompatibility when being contacted with blood, body fluid and human tissues, and can be used as a gap filler of an artificial blood vessel to seal the artificial blood vessel. The prior researches find that the woven artificial blood vessel can be effectively reduced by sealing the woven artificial blood vessel by using synthetic hydrogel based on polymers such as polyethylene oxide (ethane) and the like, but the polymer hydrogel has weak biocompatibility and is not suitable for biological artificial blood vessels. Hydrogels capable of reducing vascular permeability and improving biocompatibility and biological artificial blood vessels prepared by the hydrogels have been recently reported.
Disclosure of Invention
In view of the above, the technical problem to be solved by the invention is to provide a hydrogel and a biological artificial blood vessel prepared from the hydrogel, wherein the hydrogel provided by the invention has strong biocompatibility, and the biological artificial blood vessel prepared from the hydrogel has low overall water leakage, strong biocompatibility, good moisturizing and antibacterial effects, and simultaneously has a good effect of promoting cell growth.
The invention provides a hydrogel, which comprises the following raw materials:
4-9% (w/v) of carboxymethyl chitosan, 7-9% (w/v) of oxidized sodium alginate, 7.6-42.7% (w/v) of 2, 3-butanediol, and PBS solution.
The hydrogel prepared by the raw materials provided by the invention is more suitable for preparing biological artificial blood vessels. Compared with other hydrogels, the hydrogel prepared from the raw materials in the concentration range has stronger biocompatibility, moisture retention, bacteriostasis and cell growth promoting performance due to the close matching of the raw material components, and can effectively improve the whole water leakage, the biocompatibility, the moisture retention, the bacteriostasis and the cell growth promoting performance of the biological artificial blood vessel, thereby obtaining better technical effects.
Preferably, the hydrogel comprises the following raw materials:
carboxymethyl chitosan 6% (w/v), oxidized sodium alginate 7% (w/v), 2, 3-butanediol 27% (w/v), and PBS solution.
Experiments show that the hydrogel prepared from the raw materials in the concentration range has the highest biocompatibility, moisture retention and bacteriostasis. Wherein. The PBS solution comprises 0.85 percent of sodium chloride, 0.29 percent of disodium hydrogen phosphate, 0.03 percent of sodium dihydrogen phosphate, the balance of water, and the pH value is 7.3 to 7.5.
The invention also provides a preparation method of the hydrogel, which comprises the steps of dissolving carboxymethyl chitosan in PBS solution to obtain solution A, dissolving oxidized sodium alginate and 2, 3-butanediol in PBS solution to obtain solution B, mixing the solution A and the solution B, and crosslinking by ethanol to obtain the hydrogel.
Further, the preparation method of the carboxymethyl chitosan comprises the following steps: dissolving chitosan and sodium hydroxide in isopropanol, heating and swelling to obtain chitosan suspension, dripping the mixed solution of chloroacetic acid and isopropanol into the chitosan suspension, reacting to obtain a crude product, pouring the crude product into ethanol, washing, filtering and drying to obtain carboxymethyl chitosan.
Further, the preparation method of the oxidized sodium alginate comprises the following steps: dripping the sodium periodate aqueous solution into the sodium alginate aqueous solution, adding glycol and sodium chloride after light-shielding reaction, stirring and reacting, separating out a precipitate by ethanol from the reaction solution, filtering, dissolving the obtained precipitate in water, dialyzing, and freeze-drying the obtained solution to obtain oxidized sodium alginate.
Further, in the preparation method, the volume ratio of the solution A to the solution B is 1:1.
The invention provides the hydrogel and application of the hydrogel prepared by the preparation method in preparation of artificial blood vessels.
The invention also provides a biological artificial blood vessel, which comprises the hydrogel or the hydrogel prepared by the preparation method and an artificial blood vessel framework.
Preferably, in the preparation method of the biological artificial blood vessel, the solution A and the solution B are sprayed on the surface of the artificial blood vessel skeleton by atomization, and then the biological artificial blood vessel with the surface containing hydrogel is obtained by ethanol crosslinking treatment.
Further preferably, the biological artificial blood vessel after the crosslinking treatment is soaked in a glycerol solution to obtain the biological artificial blood vessel with the surface containing hydrogel.
The invention adopts carboxymethyl chitosan, oxidized sodium alginate, 2, 3-butanediol and other natural polymer materials to prepare hydrogel with good biocompatibility, and also provides a biological artificial blood vessel prepared by the hydrogel and a preparation method thereof. The invention provides a biological artificial blood vessel whole bodyLow water leakage, strong biocompatibility, good moisturizing and antibacterial effects, and better effect of promoting cell growth. Experiments show that the whole water leakage of the biological artificial blood vessel reaches 0.1 (mL/min cm) 2 ) The moisture retention rate is as high as 79.4%; the hemolysis rate is lower than 5%, the thrombotic Fbg content has no obvious difference with an autologous blood vessel, and the cell survival rate also reaches 90%, which indicates that the cell has good biocompatibility; compared with biological artificial blood vessels prepared by tannic acid, the biological artificial blood vessels provided by the invention have stronger capability of promoting cell growth and inhibiting proliferation of escherichia coli, staphylococcus aureus and pseudomonas aeruginosa. In addition, the preparation process of the hydrogel and the biological artificial blood vessel is simple, and the selected materials have wide sources and low price, and are suitable for large-scale production and application.
Detailed Description
The invention provides hydrogel and a biological artificial blood vessel prepared by the hydrogel, and a person skilled in the art can properly improve the technological parameters by referring to the content of the hydrogel. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:
EXAMPLE 12% CMCS/4% SAO hydrogel coated bioprosthetic blood vessel
The preparation method of the biological artificial blood vessel containing the hydrogel comprises the following steps:
(1) Dispersing 5g of chitosan and 5g of sodium hydroxide in 50mL of isopropanol, and swelling for 2h at 60 ℃ to obtain chitosan suspension; 5g chloroacetic acid was dissolved in 10mL isopropanol; slowly dripping chloroacetic acid solution into chitosan suspension at 70 ℃ to react for 4 hours to obtain a crude product; the crude product was washed 3 times with ethanol, filtered and dried at 60 ℃ to give carboxymethyl chitosan (CMCS).
(2) Dissolving 5g of sodium alginate in 250mL of deionized water to prepare a 2% aqueous solution, dissolving 5g of sodium periodate in 50mL of deionized water to prepare a 10% solution after the sodium alginate is completely dissolved, slowly dropwise adding the solution into the sodium alginate solution under the conditions of light shielding and rapid stirring, and reacting for 6 hours at room temperature in the dark; after the reaction is finished, 1mL of ethylene glycol and 5g of sodium chloride are added dropwise and continuously stirred for 30min; then pouring the reaction solution into 500mL of ethanol to separate out precipitate, and filtering; finally, the obtained precipitate is redissolved in 50mL of deionized water, dialyzed for three days by a dialysis bag with the molecular weight cutoff of 8000-15000, and the solution is frozen and dried at the temperature of minus 80 ℃ to obtain oxidized Sodium Alginate (SAO).
(3) 100mg of CMCS prepared in the step (1) was weighed and dissolved in 2.5mL of PBS solution (the PBS solution consists of water and 3 substances, specifically, sodium chloride 0.85%, disodium hydrogen phosphate 0.29%, sodium dihydrogen phosphate 0.03%, pH value 7.3-7.5, and the composition and concentration of the PBS solution are the same as above) to prepare 4% (w/v) solution A.
(4) 200mg of SAO prepared in step (2) was weighed and dissolved in 2.5mL of PBS to prepare 8% (w/v) solution B.
(5) An artificial blood vessel skeleton (the artificial blood vessel skeleton is formed by melt spinning polycaprolactone PCL, the artificial blood vessel skeleton is of a tubular structure with a hollow inside, two ends are not closed) is inserted into the shaft core, and the shaft core is attached to the motor and rotates at a speed of 300 rpm.
(6) 2.5mL of solution A and 2.5mL of solution B are respectively put into a double-injection spraying device, atomized and sprayed on a rotating artificial vascular skeleton under the pressure of 2 atmospheres, and the rotating speed is kept for 10min, so that a uniform biological hydrogel coating layer is formed. After spraying, the rotational speed was increased and possibly excess unreacted solution was discharged at 600 rpm.
(7) Putting the biological artificial blood vessel containing the hydrogel into 94% ethanol for crosslinking treatment for 8 hours, so that the biological artificial blood vessel protein is denatured and dehydrated initially. Finally, soaking in glycerol solution for 10 hours, removing redundant water of the biological artificial blood vessel and keeping the biological artificial blood vessel stable to obtain the biological artificial blood vessel coated with 2% CMCS/4% SAO hydrogel.
EXAMPLE 2 biological vascular prosthesis coated with 6% CMCS/7% SAO hydrogel
The preparation procedure was similar to example 1, except for the concentrations of solution A and solution B, the remaining steps being identical. 300mg of CMCS was dissolved in 2.5mL of PBS to prepare solution A of 12% (w/v), and 350mg of SAO was dissolved in 2.5mL of PBS to prepare solution B of 14% (w/v).
EXAMPLE 3 biological vascular prosthesis coated with 9% CMCS/9% SAO hydrogel
The preparation procedure was similar to example 1, except for the concentrations of solution A and solution B, the remaining steps being identical. 450mg of CMCS was dissolved in 2.5mL of PBS to prepare 18% (w/v) solution A, and 450mg of SAO was dissolved in 2.5mL of PBS to prepare 18% (w/v) solution B.
EXAMPLE 4 2% CMCS/4% SAO/7.6% BA (1, 2-butanediol) hydrogel-coated bioprosthetic blood vessel
The preparation procedure was similar to example 1, except for the concentration of solution B, the rest of the procedure being the same. 200mg of SAO and 380mg of BA were dissolved in 2.5mL of PBS solution at 4℃to prepare solution B having an SAO concentration of 8% (w/v) and a BA (1, 2-butanediol) concentration of 15.2% (w/v).
EXAMPLE 5 biological vascular prosthesis coated with 6% CMCS/7% SAO/7.6% BA hydrogel
The preparation procedure was similar to example 2, except for the concentration of solution B, the rest of the procedure being the same. 350mg of SAO and 380mg of BA were dissolved in 2.5mL of PBS solution at 4℃to prepare solution B having a SAO concentration of 14% (w/v) and a BA concentration of 15.2% (w/v).
EXAMPLE 6 biological vascular prosthesis coated with 6% CMCS/7% SAO/27% BA hydrogel
The preparation procedure was similar to example 5, except for the concentration of BA in solution B, the rest of the procedure being identical. 350mg of SAO and 1.35g of BA were dissolved in 2.5mL of PBS at 4℃to prepare a solution B having a SAO concentration of 14% (w/v) and a BA concentration of 54% (w/v).
EXAMPLE 7 biological vascular prosthesis coated with 6% CMCS/7% SAO/42.7% BA hydrogel
The preparation procedure was similar to example 5, except for the concentration of BA in solution B, the rest of the procedure being identical. 350mg of SAO and 2.135g of BA were weighed out at 4℃and dissolved in 2.5mL of PBS to prepare a solution B having a SAO concentration of 14% (w/v) and a BA concentration of 85.4% (w/v).
EXAMPLE 8 biological vascular prosthesis coated with 9% CMCS/9% SAO/42.7% BA hydrogel
The preparation procedure was similar to example 3, except for the concentration of solution B, the rest of the procedure being the same. 450mg of SAO and 2.135g of BA were weighed out at 4℃and dissolved in 2.5mL of PBS to prepare a solution B having an SAO concentration of 18% (w/v) and a BA concentration of 85.4% (w/v).
Comparative example 1 hydrogel-coated bioartificial vessel
The bioartificial vessel was stored in PBS solution at 4deg.C without any hydrogel coating process. Comparative example 2 6% CMCS hydrogel coated bioprosthetic blood vessel
(1) Dispersing 5g of chitosan and 5g of sodium hydroxide in 50mL of isopropanol, and swelling for 2h at 60 ℃ to obtain chitosan suspension; 5g chloroacetic acid was dissolved in 10mL isopropanol; slowly dripping chloroacetic acid solution into chitosan suspension at 70 ℃ to react for 4 hours to obtain a crude product; the crude product was washed 3 times with ethanol, filtered and dried at 60 ℃ to give carboxymethyl chitosan (CMCS).
(2) 300mg of CMCS was weighed out and dissolved in 2.5mL of deionized water to prepare a 12% (w/v) solution C.
(3) 60mg of glutaraldehyde was weighed out and dissolved in 2.5mL of deionized water to prepare 2.4% (w/v) solution D.
(4) The vascular prosthesis was inserted onto the mandrel and the mandrel was attached to a motor and rotated at 300 rpm.
(5) 2.5mL of the solution C and 2.5mL of the solution D are respectively put into a double-injection spraying device, atomized and sprayed on the rotating artificial vascular skeleton under the pressure of 2 atmospheres, and the rotating speed is kept for 30min, so that a uniform biological hydrogel coating layer is formed. After spraying, the rotational speed was increased and possibly excess unreacted solution was discharged at 600 rpm.
(6) Putting the biological artificial blood vessel containing the hydrogel into 94% ethanol for crosslinking treatment for 8 hours, so that the biological artificial blood vessel protein is denatured and dehydrated initially. Finally, soaking in glycerol solution for 10 hours, removing redundant water of the biological artificial blood vessel and keeping the biological artificial blood vessel stable to obtain the biological artificial blood vessel coated with the 6% CMCS hydrogel. Comparative example 3 10% CMCS/10% SAO hydrogel coated bioprosthetic blood vessel
The preparation procedure was similar to example 1, except for the concentrations of solution A and solution B, the remaining steps being identical. 500mg CMCS was dissolved in 2.5mL of PBS to give 20% (w/v) solution A, and 500mg SAO was dissolved in 2.5mL of PBS to give 20% (w/v) solution B.
Comparative example 4 biological vascular prosthesis coated with 6% CMCS/7% SAO/44% BA hydrogel
The preparation procedure was similar to example 5, except for the concentration of BA in solution B, the rest of the procedure being identical. 350mg of SAO and 2.2g of BA were weighed out at 4℃and dissolved in 2.5mL of PBS to prepare a solution B having a SAO concentration of 14% (w/v) and a BA concentration of 88% (w/v).
Comparative example 5% CMCS/7% SAO/27% tannin hydrogel coated bioprosthetic blood vessel
The preparation procedure was similar to example 5, except for the substance and concentration in solution B, the rest of the procedure being the same. 350mg of SAO and 675mg of tannic acid were dissolved in 2.5mL of PBS solution to prepare a solution B having an SAO concentration of 14% (w/v) and a tannic acid concentration of 54% (w/v).
Effect example
1. Overall water leakage performance test
Soaking biological artificial blood vessel in sterile physiological saline for 3 times, each time for 5min-15min. The proximal end is connected to an adapter of a specific inner diameter, ensuring a seal, and the adapter and bioprosthetic vessel are connected to a fixture for pressure generation and measurement. The intraluminal pressure of the sample was gradually increased to 16.0kPa + -0.3 kPa (120 mmHg + -2 mmHg), the leakage through the wall of the vascular prosthesis was measured for 60 seconds, and the overall water leakage performance test results of the examples and comparative examples were shown in table 1. The whole water leakage of the biological artificial blood vessel coated by the anhydrous gel is larger, the compactness is small, the water leakage is reduced when the biological artificial blood vessel is coated by the carboxymethyl chitosan hydrogel alone, and the oxidized sodium alginate is addedAfter which the water leakage continues to decrease. The overall water leakage of the 6% CMCS/7% SAO hydrogel-coated bioprosthetic vessel was reduced to 0.5 (mL/min cm) 2 ) In the following, the biological artificial blood vessel has better compactness, and the overall water leakage change of the biological artificial blood vessel coated by 9% CMCS/9% SAO hydrogel has no obvious difference. The addition of BA resulted in a reduction of overall water leakage to 0.2 (mL/min cm) 2 ) The compactness is improved, and the whole water leakage of the biological artificial blood vessel coated by 6 percent CMCS/7 percent SAO/27 percent BA hydrogel reaches 0.1 (mL/min cm) 2 ) The tightness is the strongest. And the biological artificial blood vessel sealing effect of the tannic acid is inferior to that of BA.
2. Biocompatibility testing
(1) Hemolysis rate: healthy rabbit ear arterial blood is taken, and 3.8% sodium citrate solution is used for 1:9 anticoagulation. 4mL of fresh anticoagulated rabbit blood is taken and added into 5mL of 0.9% sodium chloride injection for uniform mixing, and diluted anticoagulated rabbit blood is obtained. Soaking biological artificial blood vessel in sterile physiological saline for 3 times, each time for 5min-15min. Taking a small section of 0.5cm length of a sample, adding the small section into a centrifuge tube filled with 10mL of physiological saline, placing the centrifuge tube into a constant-temperature water bath (37+ -1) DEG C for 30min, adding 0.2mL of diluted anticoagulated rabbit blood, and placing the centrifuge tube into the water bath (37+ -1) DEG C for 60min again. Centrifuging at 1000r/min for 5min to obtain supernatant. 10mL of 0.9% sodium chloride solution was added to each tube of the negative control group; the positive control group was added with 10mL distilled water per tube. OD value at 545nm was measured using an ultraviolet spectrophotometer, and hemolysis ratio (%) = (sample absorbance-negative control absorbance)/(positive control absorbance-negative control absorbance) was calculated, and the results are shown in Table 1.
(2) Thrombosis: the biological artificial blood vessel was taken 5cm long and placed in a siliconized glass tube as a test sample. The rabbit autologous blood vessels were treated identically as a control sample and served as a control. Fresh healthy rabbit blood was collected and anticoagulated with 0.4mL of 3.8% sodium citrate solution. Each tube was filled with 0.8-1.0mL of blood. After standing for 15min at room temperature, adding 6250U/mL heparin sodium solution 10ul into each tube, sufficiently shaking and uniformly mixing, standing for 30min, centrifuging for 10min at 2000r/min at 4 ℃, sucking the upper plasma, and detecting Fbg content on a coagulometer. The Fbg content of the control sample was 2.3g/L. The results are shown in Table 1.
(3) Cell viability: biological humanSoaking the blood vessel in sterile physiological saline for 3 times, each time for 5-15 min. The biological artificial blood vessel is made into a circular diaphragm with the diameter of 14mm, the circular diaphragm can be flatly and seamlessly attached to the bottom of a 24-pore plate, ultraviolet sterilization is carried out in an aseptic table, the vascular diaphragm is flatly paved on the bottom of the 24-pore plate, the vascular diaphragm is pressed by a stainless steel ring after high-pressure sterilization, and a culture medium without the biological artificial blood vessel is used as a blank control group. After suspending the cultured human aortic endothelial cells by digestion with 0.25% pancreatin, the density per well was 2×10 4 The individual/mL cell suspensions were seeded on 24-well plates at 37℃with 5% CO 2 Is continuously cultured in a constant temperature incubator. Cell viability was quantitatively analyzed using CCK-8 on days 1,2 of continuous culture. Taking out corresponding pore plates at specified time intervals, adding 50 mu L of CCK-8 working solution into each pore, incubating for 1-2 hours in a constant temperature carbon dioxide incubator at 37 ℃, measuring an OD value at a wavelength of 450nm by using an enzyme-labeled instrument, and calculating the cell survival rate. The results are shown in Table 1.
As can be seen from Table 1, the hemolysis rate of examples 1 to 8 was less than 5%, the thrombotic Fbg content was not significantly different from that of the autologous blood vessels, and the cell viability was also 90%. The presence of hydrogels can increase biocompatibility, although the presence of BA can result in a slight decrease in biocompatibility, but is still within standard limits.
3. Moisture retention performance test
Cutting biological artificial blood vessel into 5cm length, weighing weight W before water absorption 0 Soaking in sterile physiological saline for 3 times, each time for 5-15 min. Then put into purified water to soak for 3 hours, and change water every hour to ensure that the hydrogel absorbs water completely. The excess water on the surface is wiped by paper, and then the initial weight W of the biological artificial blood vessel after water absorption is weighed 1 . Then placing the mixture at 20 ℃ and in an environment with 17% relative humidity, and weighing the weight W after 24 hours t Calculate moisture retention (%) = (W) t /W 1 ) X 100%. The results are shown in Table 1. Comparative example 1 has little water absorption due to the anhydrous gel coating. From the results, the carboxymethyl chitosan and the oxidized sodium alginate hydrogel have moisturizing effect, the moisturizing effect is obviously improved after BA is added, and the biological artificial blood vessel coated by the 6% CMCS/7% SAO/27% BA hydrogel can even retain 79.4% of water, which proves that BA has very good moisturizing effectGood moisturizing effect. While the moisture retention of excess BA (44%) was hardly increased. The addition of tannic acid to the hydrogel does not increase the water retention.
4. Cell growth Property
Soaking biological artificial blood vessel in sterile physiological saline for 3 times, 5min-15min each time, and measuring thickness D before implantation by thickness meter 0 . The experimental sheep were fasted for 1 day prior to surgery, anesthetized with midazolam (0.05 mg/kg) and Liu Mianning (1.50 mg/kg) by intramuscular injection, and continuously anesthetized with an endotracheal tube. Shaving and sterilizing the operation part, and cutting the skin at the back of the experimental sheep by using a sterile scalpel. The biological artificial blood vessel is implanted into the cavity under the sheep skin through the sterile steel tube and the incised opening. After the wound is sufficiently cleaned, suturing is performed. Feeding was carried out for 28 days, wherein penicillin was injected for the first 3 days. After 28 days, the experimental sheep were anesthetized again, the surrounding cortex was cut along the graft with a sterile scalpel, the graft was removed, placed in sterile PBS, the two-end fixing portions were cut off, and the thickness D was measured t . Calculate tissue growth thickness = D t -D 0 . The results are shown in Table 1.
It can be seen from the table that the hydrogel coating can promote the adhesion and growth of tissue cells, the carboxymethyl chitosan hydrogel has the cell growth promoting effect but is not obvious, the cell promoting effect is obviously enhanced when the oxidized sodium alginate is added, the cell promoting effect is enhanced along with the increase of the concentration, and the cell adhesion and growth capacity of the biological artificial blood vessel is not further and better improved when the concentration reaches 10% CMCS/10% SAO. When BA was added, the tissue growth thickness increased significantly, indicating that BA has the effect of promoting cell growth. Tannic acid also has a cell growth promoting effect, but is slightly weaker than BA.
Table 1 comparison of overall water leakage, biocompatibility, moisture retention, and cell growth performance for examples and comparative examples
Figure BDA0004136534270000101
5. Antibacterial property test
Coli, staphylococcus aureus, pseudomonas aeruginosa were cultured in Luria-Bertani medium at 37 ℃ for 12h. 100. Mu.L of the solution was then spread evenly on a solid agar medium. Soaking biological artificial blood vessel in sterile physiological saline for 3 times, 5min-15min each time, cutting round sample with diameter of 8mm, and sterilizing by ultraviolet irradiation on ultra-clean bench for 30 min. Placed on the surface of the medium and incubated at 37℃for 24h. After 24 hours of cultivation, the bacteria on the culture medium were taken out and observed, and the diameter of the inhibition zone was recorded, and the test results are shown in Table 2. It can be seen from table 2 that carboxymethyl chitosan and oxidized sodium alginate hydrogel have a certain antibacterial effect, and the addition of oxidized sodium alginate can improve the antibacterial effect. The concentration of the antibacterial effect is increased within the range of 2% CMCS/4% SAO to 9% CMCS/9% SAO of the hydrogel concentration, and the antibacterial effect is not changed obviously after the concentration exceeds the concentration of 9% CMCS/9% SAO. After BA is added, the antibacterial effect is obviously enhanced, and the antibacterial effect is not obviously changed when the BA concentration is more than 42.7%. Tannic acid at the same concentration has a weaker antibacterial effect than BA.
Table 2 diameter of zone of inhibition for e.coli, s.aureus and pseudomonas aeruginosa for examples and comparative examples
Figure BDA0004136534270000111
In tables 1 to 2, the difference analysis was performed between examples 1 to 8. The difference is insignificant (P > 0.05) for all the same marked letters, and significant (lower case letters represent P <0.05, upper case letters represent P < 0.01) for all the different marked letters. An ef of the overall water leak in example 2 with two letter designations indicates a significant difference P <0.05 from example 4 with the d letter designation, a comparison P >0.05 with comparative examples 3-5 with the same e letter designation, a comparison P >0.05 with example 5 with the same f letter designation, an comparison P >0.05 with example 7 with example 8.
According to the comprehensive analysis of the test, the optimal mass and volume percentage range of the coated hydrogel of the biological artificial blood vessel with the tightness is CMCS 2-9%, SAO 4-9%, BA 7.6-42.7%, and the optimal concentration is CMCS 6%, SAO 7% and BA 27%.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. A hydrogel, comprising the following raw materials:
4-9% (w/v) of carboxymethyl chitosan, 7-9% (w/v) of oxidized sodium alginate, 7.6-42.7% (w/v) of 2, 3-butanediol, and PBS solution.
2. The hydrogel of claim 1, comprising the following materials:
carboxymethyl chitosan 6% (w/v), oxidized sodium alginate 7% (w/v), 2, 3-butanediol 27% (w/v), and PBS solution.
3. The method for preparing the hydrogel according to claim 1 or 2, wherein carboxymethyl chitosan is dissolved in PBS solution to obtain solution A, oxidized sodium alginate and 2, 3-butanediol are dissolved in PBS solution to obtain solution B, and the solution A and the solution B are mixed and crosslinked by ethanol to obtain the hydrogel.
4. The preparation method of claim 3, wherein the preparation method of carboxymethyl chitosan comprises the following steps: dissolving chitosan and sodium hydroxide in isopropanol, heating and swelling to obtain chitosan suspension, dripping the mixed solution of chloroacetic acid and isopropanol into the chitosan suspension, reacting to obtain a crude product, pouring the crude product into ethanol, washing, filtering and drying to obtain carboxymethyl chitosan.
5. The preparation method of claim 3 or 4, wherein the preparation method of the oxidized sodium alginate comprises the following steps: dripping the sodium periodate aqueous solution into the sodium alginate aqueous solution, adding glycol and sodium chloride after light-shielding reaction, stirring and reacting, separating out a precipitate by ethanol from the reaction solution, filtering, dissolving the obtained precipitate in water, dialyzing, and freeze-drying the obtained solution to obtain oxidized sodium alginate.
6. The method according to any one of claims 3 to 5, wherein the volume ratio of the solution a to the solution B is 1:1.
7. Use of the hydrogel according to claim 1 or 2, the hydrogel prepared by the preparation method according to any one of claims 3 to 6 for preparing an artificial blood vessel.
8. A bioartificial vessel comprising the hydrogel according to claim 1 or 2 or the hydrogel produced by the production method according to any one of claims 3 to 6, and an artificial vascular skeleton.
9. The preparation method of the biological artificial blood vessel according to claim 8, characterized in that the biological artificial blood vessel with the hydrogel on the surface is obtained by adopting the solution A and the solution B according to any one of claims 4 to 6, spraying the solution A and the solution B on the surface of an artificial blood vessel skeleton through atomization, and then carrying out ethanol crosslinking treatment.
10. The method according to claim 9, wherein the crosslinked bioartificial vessel is immersed in a glycerol solution to obtain a bioartificial vessel having a hydrogel on the surface.
CN202310275512.8A 2023-03-15 2023-03-15 Hydrogel and biological artificial blood vessel prepared from same Pending CN116251239A (en)

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