CN113583455A - Collagen-modified chitosan double-network hydrogel, biological ink, preparation method and application - Google Patents
Collagen-modified chitosan double-network hydrogel, biological ink, preparation method and application Download PDFInfo
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Abstract
The invention provides a collagen-modified chitosan double-network hydrogel, biological ink, a preparation method and application, and relates to the field of new materials and biotechnology. The preparation method of the collagen double-network hydrogel comprises a first network constructed by mixed collagen hydrogel and a second network constructed by modified chitosan, wherein the mixed collagen comprises fish-source collagen and pig-source collagen, and the modified chitosan is prepared by modifying with citric acid. The invention has the advantages that the pore size and porosity of the flexible second network are more favorable for the adsorption, proliferation and growth of stem cells in the flexible second network constructed by the rigid first network constructed by mixed collagen and the modified chitosan.
Description
Technical Field
The invention relates to the field of new materials and biotechnology, and particularly relates to a collagen-modified chitosan double-network hydrogel, biological ink, a preparation method and application.
Background
Collagen (Col ) is one of the main substances of the extracellular matrix of the human body, belongs to structural proteins, and is a main component constituting connective tissues or organs such as skin, ligaments, cartilage, and tendons. The collagen can be used for preparing collagen hydrogel, and the unique physicochemical property, excellent biocompatibility, degradability and low immunogenicity of the collagen lead the collagen to be widely applied in the fields of high-end medical treatment, scaffolds for human tissue repair and the like. At present, the most used collagen is pig-source collagen and bovine-source collagen, which have the problems of high price and few sources and also have certain pathogenic or sensitizing factors.
The fiber structure and mechanical strength of the collagen hydrogel can be adjusted by changing parameters such as collagen source, solubility, polymerization temperature, pH value and the like. To meet the requirements of different tissue engineering applications. Through many years of researches, various preparation methods of collagen hydrogel are proposed, mainly including a physical crosslinking method and a chemical crosslinking method. Common technical means of physical crosslinking methods include dry thermal crosslinking treatment, radiation, photooxidation, thermal dehydrogenation and the like, but the methods have the conditions of difficult control of the amount of radiation, light intensity and the like and uneven crosslinking degree; the chemical method has wide selection due to the diversity of modified substances, and mainly comprises metal ions, glutaraldehyde, epoxy compound carbodiimide (EDC)/N-hydroxysuccinimide (NHS) and the like, but the modifying agents have cytotoxicity in vivo and influence the adsorption, proliferation and growth of cells.
Therefore, the selection of which crosslinking method has a great influence on the medical properties of the collagen hydrogel. The polysaccharide is widely used at present, can solve the problem of cytotoxicity, is a natural polysaccharide substance which is widely available in nature and low in price, and has the defects of good biocompatibility, insufficient mechanical properties and the like due to the fact that the polysaccharide substance contains highly hydrophilic groups.
Therefore, if collagen and chitosan can be effectively combined, it is possible to solve the defects of the two raw materials, and a novel biomaterial is obtained.
Disclosure of Invention
The invention aims to provide a collagen-modified chitosan double-network hydrogel, wherein a rigid first network is constructed by mixed collagen, a flexible second network is constructed by modified chitosan, the pore size and porosity of the hydrogel are more beneficial to the adsorption, proliferation and growth of stem cells in the hydrogel, the mechanical property is good, and the immunogenicity is low.
The invention also aims to provide a preparation method of the collagen-modified chitosan double-network hydrogel, which constructs collagen gel on the basis of the chitosan hydrogel, so that the chitosan hydrogel can be adhered to the mixed collagen to form a three-dimensional network structure.
Another object of the present invention is to provide a cell gel comprising the collagen-modified chitosan double-network hydrogel.
The invention also aims to provide the application of the collagen-modified chitosan double-network hydrogel in the tissue scaffold.
The technical problem to be solved by the invention is realized by adopting the following technical scheme.
In one aspect, an embodiment of the present application provides a collagen-modified chitosan double-network hydrogel, including a first network constructed by a mixed collagen hydrogel and a second network constructed by modified chitosan, where the mixed collagen includes fish-derived collagen and pig-derived collagen, and the modified chitosan is prepared by modifying with citric acid.
On the other hand, the embodiment of the invention provides a preparation method of the collagen-modified chitosan double-network hydrogel, which is to blend the modified chitosan hydrogel and the mixed collagen solution uniformly, add a collagen crosslinking agent, and crosslink the mixture to obtain the collagen-modified chitosan double-network hydrogel.
On the other hand, the embodiment of the invention provides a cell gel containing the collagen-modified chitosan double-network hydrogel, namely a biological ink which can be used for 3D biological printing.
In another aspect, the embodiment of the present invention provides an application of the aforementioned collagen-modified chitosan double-network hydrogel in a tissue scaffold.
In summary, compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects:
the collagen-modified chitosan double-network hydrogel provided by the invention takes the mixed collagen as the first network, has good mechanical properties, low brittleness and good toughness, can be used for preparing tissue scaffolds for regeneration of in-vivo soft tissues and the like, and can provide enough support for the flexible second network due to the good mechanical properties, so that the second network and the first network are supported in a crossed manner to form a 3D network structure. Meanwhile, the collagen-modified chitosan double-network hydrogel provided by the invention takes the modified chitosan as a flexible second network, and the chitosan modified by citric acid can improve the pore diameter and porosity on the basis of keeping good biocompatibility of the chitosan, further increase the adhesion space of cells in a unit space, and simultaneously can provide sufficient nutrients and bioactive factors for the cells to promote the proliferation of the cells. Due to the good mechanical property, toughness, biocompatibility and low immunogenicity of the collagen-modified chitosan double-network hydrogel, the collagen-modified chitosan double-network hydrogel can be used for preparing organs such as in vivo soft tissues and the like, has a good cell growth state and extremely low rejection in vivo, and reduces the operation risk.
According to the collagen-modified chitosan double-network hydrogel provided by the invention, the modified chitosan hydrogel is prepared by a circulating freezing physical crosslinking method, and then the first network is constructed by mixing collagen on the basis of the chitosan hydrogel, so that the three-dimensional network bracket with good mechanical property and toughness can be obtained.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In one aspect, an embodiment of the present application provides a collagen-modified chitosan double-network hydrogel, including a first network constructed by a mixed collagen hydrogel and a second network constructed by modified chitosan, where the mixed collagen includes fish-derived collagen and pig-derived collagen, and the modified chitosan is prepared by modifying with citric acid.
In some embodiments of the invention, the collagen-modified chitosan double-network hydrogel has a mass ratio of the mixed collagen to the modified chitosan of (4-6): 1.
In some embodiments of the invention, the weight ratio of the fish-derived collagen to the pig-derived collagen is (2-4): 1.
On the other hand, the embodiment of the invention provides a preparation method of the collagen-modified chitosan double-network hydrogel, which is to blend the modified chitosan hydrogel and the mixed collagen solution uniformly, add a collagen crosslinking agent, and crosslink the mixture to obtain the collagen-modified chitosan double-network hydrogel.
In some embodiments of the present invention, the freeze-thawing step comprises freezing at-20 ℃ for 12h and then thawing at room temperature for 12 h.
In some embodiments of the invention, in the above preparation method, the mixed collagen solution is prepared by adding fish-derived collagen and pig-derived collagen into a 1% acetic acid solution, and blending for 1.5-2 hours to obtain the mixed collagen solution.
In some embodiments of the present invention, in the above preparation method, the modified chitosan hydrogel is prepared by dissolving chitosan in acetic acid to obtain a chitosan solution, adding 8-10 wt% of citric acid to the chitosan solution, repeatedly freezing and thawing for 3-7 times, and adding a catalyst to the melted hydrogel for condensation reaction to obtain the modified chitosan hydrogel.
In another aspect, embodiments of the present invention provide a cell gel comprising the collagen-modified chitosan double-network hydrogel.
In some embodiments of the invention, the cell is a stem cell.
In another aspect, the embodiment of the present invention provides an application of the aforementioned collagen-modified chitosan double-network hydrogel in a tissue scaffold.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The purpose of this example was to prepare a mixed collagen lyophilized powder of fish-derived protein and porcine-derived protein.
(1) Weighing fish-derived collagen according to a certain amount, dissolving the fish-derived collagen in 1 wt% acetic acid solution, heating in a constant-temperature water bath at 40 ℃, and fully dissolving the fish-derived collagen to obtain 10 wt% fish-derived collagen solution;
(2) weighing porcine collagen according to a certain amount, dissolving the porcine collagen in 1 wt% acetic acid solution, heating in a constant-temperature water bath at 70 ℃, and fully dissolving the porcine collagen to obtain 10 wt% porcine collagen solution;
(3) uniformly mixing the fish-derived collagen solution and the pig-derived collagen solution according to the volume ratio of 2:1, 3:1 and 4:1 respectively to obtain three mixed solutions I with different proportions;
(4) and respectively blending the three mixed solutions I with different proportions for 1.5h under a stirring state, adding a collagen crosslinking agent, and reacting for 24h at 37 ℃ to obtain the mixed collagen gel.
(5) And (3) removing bubbles from the mixed collagen gel, pre-freezing the mixed collagen gel for 2h at the temperature of-20 ℃, then freezing and drying the mixed collagen gel in a freeze dryer under the condition of freezing for 12h at the temperature of-80 ℃, and then vacuum-drying the mixed collagen gel for 24-48 h to obtain the mixed collagen sponge freeze-dried powder.
(6) And (2) sealing 5mg of the fish-derived collagen and the pig-derived collagen used in the step (1) and the three mixed collagen sponge freeze-dried powders prepared in the step (5) in different proportions in a crucible of an indication Differential Scanning Calorimeter (DSC), measuring a thermal deformation curve of the mixed collagen at a temperature rise rate of 5 ℃/min by taking an empty crucible as a reference, and measuring the temperature range of 20-90 ℃.
(7) Taking the fish-derived collagen and the pig-derived collagen used in the step (1) and the three mixed collagen sponge freeze-dried powders (respectively counted as m0) prepared in the step (5) in different proportions, soaking in PBS buffer solution (pH 7.3-7.5) at 37 ℃, removing surface moisture after 6h to obtain swollen collagen (respectively counted as m1), and calculating the change rate of m1 relative to m0 of each group.
TABLE 1
| Fish source/pig source ratio | Temperature of thermal denaturation | Rate of thermal weight loss |
| 1:0 | 48℃ | 16％ |
| 2:1 | 49.2℃ | 14％ |
| 3:1 | 56℃ | 12.0％ |
| 4:1 | 62℃ | 12.3％ |
| 0:1 | 72℃ | 12.5％ |
When the collagen sponge is heated, as the external energy is increased, the collagen is accompanied with the absorption of energy, and the chemical bonds are broken. As can be seen from table 1, the proper addition of porcine collagen to the mixed collagen can significantly increase the thermal denaturation temperature of the mixed collagen sponge lyophilized powder, and the addition of porcine collagen can significantly increase the mechanical properties of the fish-derived collagen, which can increase the toughness of the mixed collagen. The two kinds of collagen are mixed and recombined, so that the structure of the mixed collagen sponge freeze-dried powder is more uniform, water absorption and water retention are facilitated, and the highest water absorption and water retention values are obviously higher than those of single collagen.
Example 2
The purpose of this example is to explore modified chitosan solution and modified chitosan single gel lyophilized powder.
(1) Weighing 1g of chitosan, and adding the chitosan into 1.2 wt% of acetic acid solution for dissolving to prepare 3 wt% of chitosan solution;
(2) adding citric acid into the chitosan solution prepared in the step (1) at 60 ℃, wherein the adding proportion of the citric acid is 8%, 10% and 12%, and fully stirring to remove bubbles in each group;
(3) placing the chitosan solution with the bubbles removed in the step (2) in a refrigerator at the temperature of-20 ℃ for freezing for 12h, and then unfreezing for 12h at normal temperature;
(4) repeating the step (3) for 3-7 times to obtain initial hydrogel;
(5) carrying out condensation reaction on the thawed hydrogel finally obtained in the step (4) to obtain modified chitosan hydrogel;
(6) removing bubbles from the modified chitosan hydrogel, pre-freezing the modified chitosan hydrogel for 2 hours at the temperature of-20 ℃, then freezing and drying the modified chitosan hydrogel in a freeze dryer under the condition of freezing for 12 hours at the temperature of-80 ℃, and then vacuum drying for 24-48 hours to obtain the modified chitosan sponge freeze-dried powder.
(7) And (4) taking the modified chitosan sponge freeze-dried powder in the step (6), carrying out brittle fracture by using liquid nitrogen, spraying gold, observing a sample under a scanning electron microscope, and detecting the aperture condition of each group of modified chitosan sponge freeze-dried powder.
(8) The pycnometer was filled with absolute ethanol, the total mass of the pycnometer and ethanol was weighed and recorded as W1, and the freeze-dried sample weighed in advance was immersed in ethanol, the overflowing ethanol was carefully wiped off, the weight of the pycnometer at this time was weighed and recorded as W2, left standing for 40min, the sample was taken out, the amount of pycnometer and remaining ethanol was weighed and recorded as W3, and the porosity was 100% (W2-W3)/(W2-W1).
(9) Weighing a certain amount of the modified chitosan sponge freeze-dried powder prepared in the step (6), recording the weight of each group as W0, placing hydrogel prepared from the modified chitosan sponge freeze-dried powder in PBS (phosphate buffer solution) with the pH of 7.4, taking out the hydrogel every half hour, measuring the weight W2 of the hydrogel until the maximum Wd2 is detected, and calculating the swelling degree as A ═ 100% (Wd 2-W0)/W0%.
The measurement results of the porosity, the pore diameter and the swelling ratio of the modified chitosan sponge freeze-dried powder obtained by different circulating freeze-thaw times are shown in table 2:
TABLE 2
The above table shows that the proportion of citric acid is increased, the pore diameter of the modified chitosan hydrogel can be increased, the porosity is increased, and the swelling degree is reduced along with the increase of the porosity, which indicates that the amide reaction between chitosan and citric acid can promote the microstructure of the hydrogel to be more compact, and is beneficial to improving the mechanical property of the hydrogel. The number of times of circulating freeze thawing is increased, which is beneficial to forming larger aperture. In a comprehensive view, when the proportion of the citric acid is 10% by 5 times of freeze thawing, the pore diameter, the porosity and the swelling degree of the material are optimal, and the material is beneficial to being subsequently used for preparing the scaffold material.
Example 3
The purpose of this example was to prepare a collagen-modified chitosan double-network gel.
(1) Weighing fish-derived collagen according to a certain amount, dissolving the fish-derived collagen in 1 wt% acetic acid solution, heating in a constant-temperature water bath at 40 ℃, and fully dissolving the fish-derived collagen to obtain 10 wt% fish-derived collagen solution; weighing porcine collagen according to a certain amount, dissolving the porcine collagen in 1 wt% acetic acid solution, heating in a constant-temperature water bath at 70 ℃, and fully dissolving the porcine collagen to obtain 10 wt% porcine collagen solution;
(2) uniformly mixing the fish-derived collagen solution and the pig-derived collagen solution according to the volume ratio of 3:1 respectively to obtain a mixed collagen solution;
(3) weighing 1g of chitosan, adding the chitosan into 1.2 wt% of acetic acid solution for dissolving to prepare 3 wt% of chitosan solution, adding citric acid into the 3 wt% of chitosan solution at 60 ℃, wherein the adding proportion of the citric acid is 10%, fully stirring to remove each group of bubbles, freezing the mixture in a refrigerator at the temperature of-20 ℃ for 12h, then unfreezing the mixture at normal temperature for 12h, repeatedly freezing and thawing for 5 times to obtain initial hydrogel, and then adding a catalyst into the hydrogel for condensation reaction to obtain modified chitosan hydrogel;
(4) and (3) adding the mixed collagen solution prepared in the step (2) into the modified chitosan hydrogel prepared in the step (4), wherein the concentration of the modified chitosan hydrogel is 2 wt%, the concentration of the mixed collagen solution is 8%, blending for 1.5h under a stirring state, adding a collagen crosslinking agent, and reacting for 24h at 37 ℃ to obtain the collagen-modified chitosan double-network hydrogel.
(5) The conditions in the freeze dryer are that the collagen is frozen for 12 hours at the temperature of minus 80 ℃, and then the collagen-modified chitosan double-network hydrogel freeze-dried powder is obtained after vacuum drying for 24 to 48 hours.
Example 4
The purpose of this example was to prepare a collagen-modified chitosan double-network gel.
(1) Weighing fish-derived collagen according to a certain amount, dissolving the fish-derived collagen in 1 wt% acetic acid solution, heating in a constant-temperature water bath at 40 ℃, and fully dissolving the fish-derived collagen to obtain 10 wt% fish-derived collagen solution; weighing porcine collagen according to a certain amount, dissolving the porcine collagen in 1 wt% acetic acid solution, heating in a constant-temperature water bath at 70 ℃, and fully dissolving the porcine collagen to obtain 10 wt% porcine collagen solution;
(2) uniformly mixing the fish-derived collagen solution and the pig-derived collagen solution according to the volume ratio of 3:1 respectively to obtain a mixed collagen solution;
(3) weighing 1g of chitosan, adding the chitosan into 1.2 wt% of acetic acid solution for dissolving to prepare 3 wt% of chitosan solution, adding citric acid into the 3 wt% of chitosan solution at 60 ℃, wherein the adding proportion of the citric acid is 10%, fully stirring to remove each group of bubbles, freezing the mixture in a refrigerator at the temperature of-20 ℃ for 12h, then unfreezing the mixture at normal temperature for 12h, repeatedly freezing and thawing for 5 times to obtain initial hydrogel, and then adding a catalyst into the hydrogel for condensation reaction to obtain modified chitosan hydrogel;
(4) and (3) adding the mixed collagen solution prepared in the step (2) into the modified chitosan hydrogel prepared in the step (4), wherein the concentration of the modified chitosan hydrogel is 2 wt%, the concentration of the mixed collagen solution is 10%, blending for 1.5h under a stirring state, adding a collagen crosslinking agent, and reacting for 24h at 37 ℃ to obtain the collagen-modified chitosan double-network hydrogel.
(5) The conditions in the freeze dryer are that the collagen is frozen for 12 hours at the temperature of minus 80 ℃, and then the collagen-modified chitosan double-network hydrogel freeze-dried powder is obtained after vacuum drying for 24 to 48 hours.
Example 5
The purpose of this example was to prepare a collagen-modified chitosan double-network gel.
(1) Weighing fish-derived collagen according to a certain amount, dissolving the fish-derived collagen in 1 wt% acetic acid solution, heating in a constant-temperature water bath at 40 ℃, and fully dissolving the fish-derived collagen to obtain 10 wt% fish-derived collagen solution; weighing porcine collagen according to a certain amount, dissolving the porcine collagen in 1 wt% acetic acid solution, heating in a constant-temperature water bath at 70 ℃, and fully dissolving the porcine collagen to obtain 10 wt% porcine collagen solution;
(2) uniformly mixing the fish-derived collagen solution and the pig-derived collagen solution according to the volume ratio of 3:1 respectively to obtain a mixed collagen solution;
(3) weighing 1g of chitosan, adding the chitosan into 1.2 wt% of acetic acid solution for dissolving to prepare 3 wt% of chitosan solution, adding citric acid into the 3 wt% of chitosan solution at 60 ℃, wherein the adding proportion of the citric acid is 10%, fully stirring to remove each group of bubbles, freezing the mixture in a refrigerator at the temperature of-20 ℃ for 12h, then unfreezing the mixture at normal temperature for 12h, repeatedly freezing and thawing for 5 times to obtain initial hydrogel, and then adding a catalyst into the hydrogel for condensation reaction to obtain modified chitosan hydrogel;
(4) and (3) adding the mixed collagen solution prepared in the step (2) into the modified chitosan hydrogel prepared in the step (4), wherein the concentration of the modified chitosan hydrogel is 2 wt%, the concentration of the mixed collagen solution is 12%, respectively stirring for 1.5h, adding a collagen cross-linking agent, and reacting for 24h at 37 ℃ to obtain the collagen-modified chitosan double-network hydrogel.
(5) The conditions in the freeze dryer are that the collagen is frozen for 12 hours at the temperature of minus 80 ℃, and then the collagen-modified chitosan double-network hydrogel freeze-dried powder is obtained after vacuum drying for 24 to 48 hours.
Example 6
The purpose of this example was to compare the growth of stem cells in gels of different origins.
1. Preparation of Stem cells
Taking out human mesenchymal stem cells from liquid nitrogen, pre-heating and thawing in 37 deg.C water bath, removing frozen stock solution, transferring into culture medium, placing the culture medium at 37 deg.C and 5% CO2Culturing is carried out under the conditions.
2. Preparation of hydrogels
Control group 1: the mixed collagen sponge lyophilized powder prepared in example 1 was dissolved in PBS solution to prepare a mixed collagen hydrogel.
Control group 2: and (3) dissolving the modified chitosan sponge freeze-dried powder prepared in the embodiment 2 in a PBS solution to prepare the modified chitosan hydrogel.
Experimental groups: the collagen-modified chitosan double-network hydrogel freeze-dried powder prepared in the embodiment 3-5 is dissolved in PBS solution to prepare 3 groups of collagen-modified chitosan double-network hydrogels.
3. Preparation of cell gel
Experimental groups: filling the space cultured in the step 1The plasma stem cells were resuspended in each gel set in step 2, and the cell density within the gel was made to be 4X 106and/ML. Transferring the gel into 12-well plate, adding 2ML DMEM medium into each well, culturing at 37 deg.C and CO in fresh complete medium every 48 hr2The concentration was 5%.
MTT test: the gels from each group were removed and placed in new 48-well plates, 300. mu.L of MTT working solution was added to each well, and incubated in an incubator for 6 h. After the incubation, the medium containing MTT was aspirated, 500. mu.L DMSO was added to each well, and the mixture was placed in a shaker and shaken for 60 min. After the purple crystals in the gel were completely dissolved in DMSO, 150. mu.L of DMSO from each well, in which the MTT crystals were dissolved, was removed to a new 96-well plate. Placing the pore plate in an enzyme labeling instrument, detecting the absorbance at 490nm wavelength, and counting the positive cell rate, wherein the result is as follows:
TABLE 3
| Group of | Cell proliferation Rate (%) |
| Control group 1 | 15 |
| Control group 2 | 25 |
| Experimental group 1 (example 3) | 48 |
| Experimental group 2 (example 4) | 60 |
| Experimental group 3 (example 5) | 54 |
As can be seen from the above table, the collagen-modified chitosan double-network hydrogel provided in example 2 is more beneficial to proliferation of mesenchymal stem cells, the proliferation rate can reach 60%, and the proliferation rate of cells in the collagen-modified chitosan double-network hydrogel provided in examples 3 to 5 is significantly greater than that of a single gel.
The collagen-modified chitosan double-network hydrogel provided by the invention has the following advantages:
first, the collagen-modified chitosan double-network hydrogel provided by the invention is prepared by blending fish-derived collagen and pig-derived collagen to prepare collagen gel, a rigid first network is prepared, and the two collagens are mixed, so that the spatial structure of the recombined mixed collagen hydrogel is more uniform, water absorption and water retention are better facilitated, meanwhile, the addition of the fish-derived collagen improves the defects that the price of single pig-derived collagen or bovine-derived collagen is high, the antigenicity is high and allergy is easy to occur in the prior art, compared with the bovine-derived protein, the pig-derived protein has lower price and is not easy to cause mad cow disease and other pathogenic factors, and the pig-derived protein can effectively improve the characteristic of poor thermal stability of the fish-derived collagen, so that the whole mixed collagen has lower antigenicity than the collagen with a single component, is free of allergy, has good thermal stability, and can better adapt to the internal environment, the proliferation and growth of stem cells on the network are facilitated, and the storage is also facilitated.
The chitosan is derived from chitin, is a natural polysaccharide with abundant resources, has good biocompatibility, and has the characteristics of antibiosis, hemostasis, biodegradation and the like. The traditional chitosan has poor mechanical property, so the chitosan is generally used as a medical auxiliary material. Due to the characteristics of chitosan, the research on chitosan is focused on how to improve the mechanical properties of chitosan, so that the chitosan plays a greater role in the medical field. According to the invention, citric acid is used for modifying chitosan, a catalyst is added for condensation reaction, and amide reaction between citric acid and chitosan improves the mechanical properties of the modified chitosan gel, which is particularly shown in that the porosity, pore diameter and swelling degree of the chitosan gel are obviously improved, and the chitosan gel has excellent properties when being used for preparing tissue scaffold materials. In addition, the modified chitosan is gelatinized by adopting a circulating freezing physical crosslinking mode, the toxicity of chemical reagents to cells is reduced, the crosslinking degree of the formed gel is uniform, the pore size is uniform, and the gel is favorable for adsorption, proliferation and growth of the cells.
The collagen-modified chitosan double-network hydrogel provided by the invention takes the mixed collagen as the first network, has good mechanical properties, low brittleness and good toughness, can be used for preparing tissue scaffolds for regeneration of in-vivo soft tissues and the like, and has good mechanical properties to provide enough support for the flexible second network, so that the second network and the first network are supported in a crossed manner to form a 3D network structure. Meanwhile, the collagen-modified chitosan double-network hydrogel provided by the invention takes the modified chitosan as a flexible second network, and the chitosan modified by citric acid can improve the pore diameter and porosity on the basis of keeping good biocompatibility of the chitosan, further increase the adhesion space of cells in a unit space, and simultaneously can provide sufficient nutrients and bioactive factors for the cells to promote the proliferation of the cells. Due to the good mechanical property, toughness, biocompatibility and low immunogenicity of the collagen-modified chitosan double-network hydrogel, the collagen-modified chitosan double-network hydrogel can be used for preparing organs such as in vivo soft tissues and the like, has a good cell growth state and extremely low rejection in vivo, and reduces the operation risk.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. For example, in example 7, mesenchymal stem cell culture is selected, and the biomimetic material of the present invention is also applicable to the culture of other cells such as fibroblasts and tumor cells, and the like, and the organoid is constructed by the cell culture. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (10)
1. The collagen-modified chitosan double-network hydrogel is characterized by comprising a first network constructed by mixed collagen hydrogel and a second network constructed by modified chitosan, wherein the mixed collagen comprises fish-derived collagen and pig-derived collagen, and the modified chitosan is prepared by modifying with citric acid.
2. The collagen-modified chitosan double-network hydrogel according to claim 1, wherein the mass ratio of the mixed collagen to the modified chitosan is (4-6): 1.
3. The collagen-modified chitosan double-network hydrogel according to claim 1 or 2, wherein the weight ratio of the fish-derived collagen to the pig-derived collagen is (2-4): 1.
4. A preparation method of the collagen-modified chitosan double-network hydrogel as claimed in any one of claims 1 to 3, wherein the collagen-modified chitosan double-network hydrogel is obtained by uniformly blending the modified chitosan hydrogel and the mixed collagen solution, and adding a collagen crosslinking agent for crosslinking.
5. The preparation method according to claim 4, wherein the mixed collagen solution is prepared by adding fish-derived collagen and pig-derived collagen into a 1% acetic acid solution, and blending for 1.5-2 h to obtain the mixed collagen solution.
6. The preparation method according to claim 4 or 5, wherein the modified chitosan hydrogel is prepared by dissolving chitosan in acetic acid to obtain a chitosan solution, adding 8-10 wt% of citric acid into the chitosan solution, repeatedly freezing and thawing for 3-7 times, and adding a catalyst into the melted hydrogel for condensation reaction to obtain the modified chitosan hydrogel.
7. The method of claim 6, wherein said freezing and thawing comprises freezing at-20 ℃ for 12 hours prior to thawing at room temperature for 12 hours.
8. A cell gel comprising the collagen-modified chitosan double network hydrogel according to any one of claims 1 to 3.
9. The cell gel of claim 8, wherein the cells in the cell gel are stem cells.
10. Use of the collagen-modified chitosan double-network hydrogel according to any one of claims 1 to 3 in a tissue scaffold.
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