A collagen hydrogel and its preparation method and application

Abstract

The present invention discloses a collagen hydrogel and a preparation method and application thereof, belonging to the technical field of wound dressings. The collagen hydrogel of the present invention is formed by chemical cross-linking of collagen, hydrophobically modified chitosan and dopamine through a cross-linking agent. Compared with the gel formed by electrostatic force, the dressing has greater tensile strength, longer elongation at break, better flexibility, and stronger adhesion to the wound surface. It has the functions of replenishing moisture to the wound skin, enhancing skin elasticity, and moisturizing the skin. It also has the effects of rapid hemostasis, pain relief, and absorption of blood stains; at the same time, it contains natural antibacterial molecules, has a long effective antibacterial time, is suitable for wound healing after surgery, abrasions, knife wounds, burns, scalds, etc., has a significant effect on the healing of oral ulcers, and is used as a wound dressing with broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of wound dressings, and specifically relates to a collagen hydrogel and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Hydrogel dressing is a new type of dressing developed in recent years. It has a three-dimensional network structure and contains a large number of hydrophilic groups. It can provide a moist environment for the wound and promote better wound healing. At the same time, in a moist environment, the dressing does not adhere to the wound and does not damage the new tissue, effectively shortening the wound healing time. According to the source of the preparation material, hydrogels can be divided into two categories: natural polymer hydrogels and synthetic polymer hydrogels. Among them, natural polymers exist in animals, plants and microorganisms, and have the advantages of being non-toxic, non-irritating and having good biocompatibility. Many natural polymers are the main components of the extracellular matrix and have the functions of regulating cell division, adhesion, differentiation and migration, so they are suitable for the preparation of wound dressings.

[0004] Collagen is a key component of the extracellular matrix, among which type I collagen is the most abundant protein in the human body, accounting for 25-30% of the total protein content in the human body, and is widely distributed in the skin, tendons and bone tissues. Collagen is closely related to physiological behaviors such as cell proliferation, differentiation, migration, intercellular signaling, tissue formation, joint lubrication, and blood coagulation. Collagen gel is non-toxic and non-irritating, has good compatibility with human tissues, can promote cell proliferation, and the degradation products can be absorbed by the human body. However, the poor mechanical properties and fast degradation rate of collagen scaffolds have greatly limited their applications. Summary of the invention

[0005] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a collagen hydrogel and a preparation method and application thereof. The collagen hydrogel provided by the present invention has the characteristics of simple preparation process, excellent performance, high safety, wide application range, etc.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] The first aspect of the present invention provides a method for preparing a collagen hydrogel, comprising: preparing a collagen solution, adjusting the pH value, and then sequentially adding a hydrophobically modified chitosan solution and a cross-linking agent to the collagen solution, and allowing the solution to stand after all the additions are completed to obtain the collagen hydrogel.

[0008] The preparation method further comprises: before adding the cross-linking agent to the collagen solution, adding a dopamine solution to the collagen solution. Dopamine plays a role in enhancing mechanical strength and wound adhesion in the collagen hydrogel, promoting wound healing speed and relieving pain and calming the nerves.

[0009] The second aspect of the present invention provides a collagen hydrogel obtained by the preparation method of the above collagen hydrogel, wherein the solid components are composed of: 70%-99% collagen, 1.0%-10% cross-linking agent, 0.0%-1.0% hydrophobically modified chitosan, and 0.0%-29% dopamine.

[0010] The collagen hydrogel provided by the present invention has high mechanical strength, wound adhesion, and promotes wound healing speed, analgesia, hemostasis, and antibacterial and antimicrobial properties, and is very suitable for use as a medical material. Therefore, the third aspect of the present invention provides the application of the above collagen hydrogel in the field of medical materials.

[0011] The beneficial effects of the present invention are:

[0012] The present invention chemically cross-links collagen or uses it in combination with other polymer materials, and the prepared collagen-based material has significantly improved microstructure, mechanical properties (high tensile strength, high elongation at break, good flexibility, strong adhesion to the wound surface), and hydrolysis resistance. Based on the wet healing theory and utilizing the characteristics of the gel system, the obtained collagen hydrogel provides a good cell microenvironment for wound healing and promotes the generation of new tissues. At the same time, the wet environment is conducive to protecting these new tissues.

[0013] The collagen hydrogel material of the present invention is formed by chemical crosslinking of collagen, hydrophobically modified chitosan and dopamine, and the preparation method is simple, suitable for large-scale promotion and application, and the obtained gel has large mechanical strength, stable mechanical properties, small swelling rate and long service life. Among them, collagen comes from fish skin, pig skin and cow skin, with wide sources and low price, which is conducive to promotion and production. Hydrophobically modified chitosan is a derivative of chitosan, which inherits the antibacterial and antibacterial properties and biocompatibility of chitosan. The hydrophobic group also gives chitosan stronger and longer antibacterial and antibacterial properties, softness when in contact with the skin and the characteristic of preventing gel from adhering to the wound surface. The carbon chain length and substitution degree of the hydrophobic group can adjust the microstructure of the collagen hydrogel, and then adjust the effect of the wound dressing absorbing blood stains, and it cooperates with dopamine to adjust the adhesion of the gel to the wound surface. At the same time, the reactive activity of the amino group in the dopamine molecule is fully utilized to cross-link with collagen or chitosan to participate in the construction of the basic structure of the gel. The hydroxyl groups in the dopamine molecule increase the adhesion of the gel to the skin. The dopamine molecule also gives the gel an analgesic effect, fully reflecting the characteristics of dopamine in the gel.

[0014] The collagen hydrogel of the present invention can replenish moisture for the skin, increase skin elasticity, moisturize the skin, and also has the effects of quickly stopping bleeding, relieving pain and absorbing blood stains; at the same time, it contains natural antibacterial molecules with a long effective antibacterial time, and is suitable for wound healing after surgery, abrasions, knife wounds, burns, scalds and the like, has a significant effect on the healing of oral ulcers, and can be used as a wound dressing with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0016] Figure 1 The microstructure diagrams of the gels obtained in Examples 1 and 4 of the present invention and Comparative Example 1; wherein A is the collagen gel prepared in Comparative Example 1, B is the collagen / modified chitosan gel prepared in Example 1, and C is the collagen / modified chitosan / dopamine gel prepared in Example 4;

[0017] Figure 2 Viscoelasticity curves of different systems of the embodiments of the present invention and the comparative examples;

[0018] Figure 3 This is the infrared spectrum of genipin cross-linked collagen prepared in Comparative Example 1;

[0019] Figure 4 is the degree of crosslinking of the gels prepared in the examples of the present invention and the comparative examples;

[0020] Figure 5 is the swelling ratio of the gel prepared in the examples of the present invention and the comparative examples;

[0021] Figure 6 is the tensile strength of the gels prepared in the examples and comparative examples of the present invention;

[0022] Figure 7 The adhesion of the gels prepared in the examples and comparative examples of the present invention;

[0023] Figure 8 Degradation curves of gels prepared in Examples and Comparative Examples of the present invention;

[0024] Fig. 9 The growth promotion rate of human epidermal cells by the gel prepared in the examples and comparative examples of the present invention. DETAILED DESCRIPTION

[0025] In view of the shortcomings of collagen scaffolds such as poor mechanical properties and rapid degradation, which limit their application, the present invention proposes a collagen hydrogel and a preparation method and application thereof, in which collagen is cross-linked with other polymer materials to prepare a collagen-based material with significantly improved microstructure, mechanical properties and hydrolysis resistance.

[0026] All raw materials of the present invention have no particular limitation on their sources, and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0027] There is no particular limitation on the purity of all raw materials in the present invention. The present invention preferably uses analytically pure materials or materials with a purity commonly used in the field of hydrogel materials.

[0028] The brands and abbreviations of all raw materials of the present invention are conventional brands and abbreviations in the art. Each brand and abbreviation is clear and definite in the field of its related use. Those skilled in the art can purchase them from the market or prepare them by conventional methods according to the brands, abbreviations and corresponding uses.

[0029] The first typical embodiment of the present invention provides a method for preparing a collagen hydrogel, comprising: preparing a collagen solution, adjusting the pH value, and then sequentially adding a hydrophobically modified chitosan solution and a cross-linking agent to the collagen solution, and after all the additions are completed, allowing the solution to stand to obtain the collagen hydrogel.

[0030] In some examples of this embodiment, the mass ratio of collagen, hydrophobically modified chitosan and cross-linking agent is: 15-30: 0.1-0.3: 1-4. The amount of collagen, hydrophobically modified chitosan and cross-linking agent affects the pore size, viscosity, elasticity, degree of cross-linking, swelling rate and mechanical properties of the final collagen hydrogel. The collagen hydrogel obtained under this ratio has excellent mechanical properties and swelling properties.

[0031] In principle, the present invention has no particular restrictions on the specific selection of the collagen. Those skilled in the art can select and adjust according to the actual application, product quality and product performance. In order to better ensure the specific morphology of the collagen hydrogel and improve the mechanical properties, the collagen of the present invention is preferably type I collagen.

[0032] In principle, the present invention has no particular restriction on the source of the collagen, and those skilled in the art can select and adjust it according to the actual application, product quality and product performance. In order to better ensure the specific morphology of the collagen hydrogel and improve the mechanical properties, the collagen is preferably from pig skin, cow skin or fish skin, and is extracted from fish skin, pig skin or cow skin by enzymatic method. It has the characteristics of wide source and low price, which is conducive to the promotion and production of collagen hydrogel.

[0033] In some examples of this embodiment, the pH value is adjusted to adjust the pH value of the collagen solution to 5-6. In principle, the present invention has no particular limitation on the pH adjustment method, and those skilled in the art can select and adjust it according to the actual application, product quality and product performance. In order to better ensure the specific morphology of the collagen hydrogel and improve the mechanical properties, the present invention uses acetic acid solution and sodium hydroxide solution to adjust the pH. Preferably, the concentration of the acetic acid solution is 0.1 mol / L, and the concentration of the sodium hydroxide solution is 1.0 mol / L.

[0034] In principle, the present invention has no particular restriction on the type of cross-linking agent. Those skilled in the art can select and adjust it according to the actual application, product quality and product performance. In order to better ensure the specific morphology of the collagen hydrogel and improve the mechanical properties, the present invention preferably includes one or more of glutaraldehyde, acetic anhydride, diglycidyl ethyl ether, glycidyl ether, methyl succinimidyl, genipin, oxidized sodium alginate, oxidized cellulose, and epichlorohydrin, and more preferably one of genipin, oxidized sodium alginate, epichlorohydrin, and glutaraldehyde.

[0035] In some examples of this embodiment, the hydrophobically modified chitosan is chitosan or cationic chitosan or anionic chitosan grafted with hydrophobic segments, wherein the percentage of the hydrophobic segments of the hydrophobically modified chitosan to the primary amino groups, i.e., the degree of substitution of the hydrophobically modified chitosan, is 15%-90%.

[0036] In principle, the present invention has no particular restriction on the type of chitosan, and those skilled in the art can select and adjust it according to the actual application, product quality and product performance. In order to better ensure the specific morphology of the collagen hydrogel and improve the mechanical properties, the chitosan preferably has a viscosity-average molecular weight of 50mPa·s-500mPa·s and a deacetylation degree greater than 65%.

[0037] In principle, the present invention has no particular restrictions on the type of cationic chitosan. Those skilled in the art can select and adjust according to actual application conditions, product quality and product performance. In order to better ensure the specific morphology of collagen hydrogel and improve mechanical properties, the cationic chitosan is 2-hydroxypropyl-3-trimethylammonium chitosan. Preferably, the viscosity-average molecular weight of the 2-hydroxypropyl-3-trimethylammonium chitosan is 50mPa·s-500mPa·s, the degree of deacetylation is greater than 65%, and the degree of substitution of the 2-hydroxypropyl-3-trimethylammonium group is greater than 50%.

[0038] In principle, the present invention has no particular restrictions on the type of anionic chitosan, and those skilled in the art can select and adjust it according to the actual application, product quality and product performance. In order to better ensure the specific morphology of the collagen hydrogel and improve the mechanical properties, the anionic chitosan of the present invention is carboxymethyl chitosan. Preferably, the viscosity-average molecular weight of the carboxymethyl chitosan is 50mPa·s-500mPa·s, the degree of deacetylation is greater than 65%, and the degree of substitution of the carboxymethyl group is greater than 50%.

[0039] In some examples of this embodiment, the hydrophobic segment is an aldehyde, fatty acid, 2,3-epoxypropyl alkyl ether, 2,3-epoxypropyl alkenyl ether or 2,3-epoxypropyl trialkoxysilane containing 4-16 carbon atoms, wherein the hydrophobic segment is grafted onto the chitosan, carboxymethyl chitosan or 2-hydroxypropyl-3-trimethyl quaternary ammonium salt chitosan molecular chain by reduction reaction, acylation reaction and / or nucleophilic addition reaction.

[0040] In some examples of this embodiment, the preparation method further comprises: before adding the cross-linking agent to the collagen solution, adding a dopamine solution to the collagen solution; wherein the mass ratio of collagen to dopamine is: 15-30: 3-5. The dopamine in the collagen hydrogel plays a role in enhancing mechanical strength, wound adhesion, promoting wound healing speed, and relieving pain and calming.

[0041] Another typical embodiment of the present invention provides a collagen hydrogel obtained by the preparation method of the above collagen hydrogel, wherein the solid component thereof is composed of: 70%-99% collagen, 1.0%-10% cross-linking agent, 0.0%-1.0% hydrophobically modified chitosan, and 0.0%-29% dopamine;

[0042] The total mass of the solid components accounts for 15%-40% of the total collagen hydrogel system, and the remainder is pure water.

[0043] A third typical embodiment of the present invention provides application of the above-mentioned collagen hydrogel in the field of medical materials.

[0044] Among them, the field of medical materials includes wound dressings;

[0045] The wound dressing has the following functions (1)-(4):

[0046] (1) Improve wound healing speed;

[0047] (2) analgesia and sedation;

[0048] (3) Rapidly stop bleeding and absorb blood stains;

[0049] (4) Antibacterial and antimicrobial.

[0050] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0051] In the following examples, collagen is extracted from fish skin, pig skin or cow skin by enzymatic method;

[0052] The hydrophobically modified chitosan is a product of chitosan, carboxymethyl chitosan or 2-hydroxypropyl-3-trimethyl quaternary ammonium salt chitosan after being grafted with hydrophobic segments. The viscosity average molecular weight of the three chitosans is 50mPa·s-500mPa·s, and the degree of deacetylation is greater than 65%; the degree of substitution of carboxymethyl in carboxymethyl chitosan is greater than 50%, and the degree of substitution of 2-hydroxypropyl-3-trimethylammonium in 2-hydroxypropyl-3-trimethylammonium chitosan is greater than 50%; the degree of substitution of the hydrophobic segments is between 15% and 90%.

[0053] Example 1

[0054] A method for preparing a collagen hydrogel comprises the following steps:

[0055] 18 g of collagen was weighed and swollen with 60 mL of 0.1 mol / L acetic acid solution at 4°C for 4 hours, and then stirred at room temperature for 2 hours to completely dissolve it.

[0056] Weigh a certain amount of NaOH solid to prepare a solution with a concentration of 1.0 mol / L. Use the NaOH solution to adjust the pH of the collagen solution to 5.

[0057] 0.2 g of 2,3-epoxypropyl n-butyl modified carboxymethyl chitosan was weighed and added to 10 mL of pure water, and swollen at 4°C for 4 hours, and then stirred at room temperature for 2 hours to completely dissolve. The degree of substitution of 2,3-epoxypropyl n-butyl in the 2,3-epoxypropyl n-butyl modified carboxymethyl chitosan was 35%.

[0058] The 2,3-epoxypropyl n-butyl modified carboxymethyl chitosan solution was added dropwise into the collagen solution under stirring conditions at room temperature.

[0059] 1.8 g of genipin was weighed and dissolved in 10 mL of a phosphate buffer solution with a pH of 7.2, and then added dropwise to the collagen solution at room temperature with sufficient stirring.

[0060] The collagen hydrogel was prepared by standing for 36 hours.

[0061] Example 2

[0062] A method for preparing a collagen hydrogel, which differs from Example 1 in that:

[0063] The hydrophobically modified chitosan used is 2-hydroxypropyl-3-trimethylammonium chitosan modified with n-dodecylaldehyde, wherein the substitution degree of n-dodecylaldehyde group in the 2-hydroxypropyl-3-trimethylammonium chitosan modified with n-dodecylaldehyde is 90%.

[0064] The collagen hydrogel was prepared after standing for 20 hours.

[0065] Example 3

[0066] A method for preparing a collagen hydrogel, which differs from Example 1 in that:

[0067] The hydrophobic modified chitosan used is hexadecyl carboxylic acid modified chitosan, wherein the substitution degree of hexadecyl carboxyl in the hexadecyl carboxylic acid modified chitosan is 18%.

[0068] 0.2 g of hexadecyl carboxylic acid-modified chitosan was weighed and swelled in 10 mL of 0.1 mol / L acetic acid solution at 4° C. for 4 hours.

[0069] The collagen hydrogel was prepared by standing for 16 hours.

[0070] Example 4

[0071] A method for preparing a collagen hydrogel comprises the following steps:

[0072] 28 g of collagen was weighed and swollen with 50 mL of 0.1 mol / L acetic acid solution at 4°C for 4 hours, and then stirred at room temperature for 2 hours to completely dissolve it.

[0073] Weigh a certain amount of NaOH solid to prepare a solution with a concentration of 1.0 mol / L. Use the NaOH solution to adjust the pH of the collagen solution to 5.

[0074] 0.2 g of 2,3-epoxypropyltrimethoxysilane-modified carboxymethyl chitosan was weighed and swelled in 10 mL of pure water at 4°C for 4 hours, and then stirred at room temperature for a certain period of time to completely dissolve. The chitosan solution was added dropwise to the collagen solution under room temperature and stirring conditions. The degree of substitution of 2,3-epoxypropyltrimethoxysilane in the 2,3-epoxypropyltrimethoxysilane-modified carboxymethyl chitosan was 40%.

[0075] 3.6 g of genipin was weighed and dissolved in 8.2 mL of phosphate buffer solution with pH = 7.2, and then added dropwise to the collagen solution at room temperature with sufficient stirring.

[0076] The collagen hydrogel was prepared after standing for 2 hours.

[0077] Example 5

[0078] A method for preparing a collagen hydrogel, which differs from Example 4 in that:

[0079] The hydrophobically modified chitosan used is hexadecanal-modified 2-hydroxypropyl-3-trimethyl quaternary ammonium salt chitosan, wherein the substitution degree of hexadecanal in the hexadecanal-modified 2-hydroxypropyl-3-trimethyl quaternary ammonium salt chitosan is 83%.

[0080] The collagen hydrogel was prepared by standing for 4 hours.

[0081] Example 6

[0082] A method for preparing a collagen hydrogel comprises the following steps:

[0083] 24 g of collagen was weighed and swollen with 50 mL of 0.1 mol / L acetic acid solution at 4°C for 4 hours, and then stirred at room temperature for 2 hours to completely dissolve it.

[0084] Weigh a certain amount of NaOH solid to prepare a solution with a concentration of 1.0 mol / L. Use the NaOH solution to adjust the pH of the collagen solution to 5.

[0085] 0.15 g of lauric acid modified 2-hydroxypropyl-3-trimethyl quaternary ammonium salt chitosan was weighed and swollen in 7 mL of pure water at 4°C for 4 hours, and then stirred at room temperature for a certain period of time to completely dissolve it. The degree of substitution of lauryl in lauric acid modified 2-hydroxypropyl-3-trimethyl quaternary ammonium salt chitosan is 45%.

[0086] 4.35 g of dopamine was weighed into 5 mL of water and stirred at room temperature to completely dissolve it. Chitosan and dopamine solutions were added dropwise to the collagen solution in sequence at room temperature and under stirring.

[0087] 1.5 g of epichlorohydrin was weighed and dissolved in 8 mL of phosphate buffer solution with pH = 7.2, and then added dropwise to the collagen solution at room temperature with sufficient stirring.

[0088] The collagen hydrogel was prepared after standing for 10 hours.

[0089] Example 7

[0090] A method for preparing a collagen hydrogel, which differs from Example 6 in that:

[0091] The hydrophobically modified chitosan used is 2,3-epoxypropyltriethoxysilane modified carboxymethyl chitosan, wherein the substitution degree of 2,3-epoxypropyltriethoxysilane groups in the 2,3-epoxypropyltriethoxysilane modified carboxymethyl chitosan is 15%.

[0092] The collagen hydrogel was prepared by standing for 12 hours.

[0093] Comparative Example 1

[0094] A method for preparing a collagen hydrogel comprises the following steps:

[0095] 13.50 g of collagen was weighed and swollen with 50 mL of 0.1 mol / L acetic acid solution at 4°C for 4 hours, and then stirred at room temperature for 2 hours to completely dissolve it.

[0096] Weigh a certain amount of NaOH solid to prepare a solution with a concentration of 1.0 mol / L. Use the NaOH solution to adjust the pH of the collagen solution to 5.

[0097] 1.5 g of genipin was weighed and dissolved in 35 mL of a phosphate buffer solution with a pH of 7.2, and then added dropwise to the collagen solution at room temperature with sufficient stirring, and allowed to stand for 20 hours to prepare a collagen hydrogel.

[0098] Comparative Example 2

[0099] A method for preparing a collagen hydrogel comprises the following steps:

[0100] 14.85 g of collagen was weighed and swollen with 60 mL of 0.1 mol / L acetic acid solution at 4°C for 4 hours, and then stirred at room temperature for 2 hours to completely dissolve it.

[0101] Weigh a certain amount of NaOH solid to prepare a solution with a concentration of 1.0 mol / L. Use the NaOH solution to adjust the pH of the collagen solution to 5.

[0102] 0.15 g of glutaraldehyde was weighed and dissolved in 25 mL of a phosphate buffer solution with a pH of 7.2. The solution was then added dropwise to the collagen solution at room temperature with sufficient stirring and allowed to stand for 48 hours to prepare a collagen hydrogel.

[0103] Comparative Example 3

[0104] A method for preparing a collagen hydrogel comprises the following steps:

[0105] 24 g of collagen was weighed and swollen with 50 mL of 0.1 mol / L acetic acid solution at 4°C for 4 hours, and then stirred at room temperature for 2 hours to completely dissolve it.

[0106] Weigh a certain amount of NaOH solid to prepare a solution with a concentration of 1.0 mol / L. Use the NaOH solution to adjust the pH of the collagen solution to 5.

[0107] Weigh 11.6 g dopamine in 7 mL water and stir at room temperature to completely dissolve it. Add the dopamine solution dropwise into the collagen solution at room temperature with stirring.

[0108] 0.6 g of oxidized sodium alginate was weighed, dissolved in 6.8 mL of a phosphate buffer solution with a pH of 7.2, and then added dropwise to the collagen solution at room temperature with sufficient stirring, and allowed to stand for 8 h to prepare a collagen hydrogel.

[0109] Application Example 1

[0110] The characteristics and parameters of the collagen hydrogels obtained in Examples 1-7 and Comparative Examples 1-3 are as follows:

[0111] 1. Microstructure observation: The hydrogel dressing obtained in Example 1 has a porous structure, and the microstructure after freeze-drying is as shown in the figure Figure 1 As shown in B. Figure 1 Collagen in Figure 1 A, corresponding to ratio 1), collagen / modified chitosan ( Figure 1 B, corresponding to Example 1) and collagen / modified chitosan / dopamine ( Figure 1 C, corresponding to Example 6) It can be seen from the microstructure diagram of the gel that the more the cross-linking agent content is, the smaller the pore size of the hydrogel is. After adding modified chitosan or (and) dopamine, the pore size of the hydrogel is further reduced.

[0112] 2. Rheological property detection: At room temperature, the rheological properties of collagen hydrogel were tested by using a DHR-2 rheometer of TA Company using a plate method with a slit of 1000 μm and an angular frequency range of 0.1-100 rad / s.

[0113] like Figure 2 As shown, the elastic modulus of all samples is greater than the viscous modulus, and the elastic modulus is between 10-1000 Pa; the viscous modulus is between 1-300 Pa. The greater the amount of cross-linking agent used, the greater the elastic modulus of the gel; the higher the collagen or dopamine content, the greater the viscosity of the gel.

[0114] 3. Fourier infrared spectroscopy detection: Figure 3 This is the infrared spectrum of genipin cross-linked collagen, i.e., the spectrum of the collagen hydrogel prepared in Comparative Example 1, at 1542 cm -1 、1018cm -1 and 1339cm -1 A new absorption peak appears at , which is the vibration absorption peak of the NH bond formed between genipin and the primary amine of lysine, hydroxylysine or arginine residues in collagen.

[0115] 4. Cross-linking degree detection: The cross-linking degree of collagen hydrogel was detected according to the "Pharmaceutical Industry Standard of the People's Republic of China (YY / T 1511-2017)".

[0116] Test results such as Figure 4As shown, the cross-linking degree is between 30% and 90%. The cross-linking degree is closely related to the amount of cross-linking agent, the amount of modified chitosan and the N-substitution degree, and the amount of dopamine. The more the amount of cross-linking agent, modified chitosan (fixed N-substitution degree) and dopamine is used, the greater the cross-linking degree is; when the amount of modified chitosan is constant, the smaller the N-substitution degree is, the greater the cross-linking degree is.

[0117] 5. Swelling test: The swelling property of collagen hydrogel was tested according to the Pharmaceutical Industry Standard of the People's Republic of China (YY / T 1435-2016).

[0118] Take a piece of freeze-dried sample, weigh it (the mass is recorded as m1), soak it in water until constant weight (recorded as m2), and calculate the swelling rate according to the following formula.

[0119] Swelling rate = (m2-m1) / m1×100%

[0120] Test results such as Figure 5 As shown, the swelling rates of all samples were lower than 200%, and the swelling rates of samples of Examples 1-7 were lower than 140%. It can be seen that the higher the genipin content and the addition of dopamine, the lower the swelling rate of the hydrogel dressing.

[0121] 6. Mechanical property testing: The mechanical properties of collagen hydrogel were tested according to the "Pharmaceutical Industry Standard of the People's Republic of China (YY / T 1435-2016)".

[0122] The tensile strength test results are as follows: Figure 6 As shown, the tensile strength of all samples is between 0-50 kPa, and the tensile strength of Examples 4, 5, 6, and 7 is between 30-50 kPa. It can be seen that the higher the content of genipin and the addition of dopamine, the more significantly the tensile strength of the hydrogel is increased.

[0123] The adhesion of the hydrogel was tested using commercially available white pig skin after hair removal as simulated skin. The test results are as follows: Figure 7 As shown, the adhesion between the gel dressing and pig skin is between 20-50 kPa. The more collagen and dopamine the content is, the stronger the adhesion is. The longer the hydrophobic segment and the presence of silane groups in the hydrophobic segment are not conducive to adhesion.

[0124] 7. Hydrolysis resistance test: According to the National Standard of the People's Republic of China (GB / T19277.1-201 / ISO14855-1:2005), the degradation ability of collagen hydrogel is determined by measuring the released carbon dioxide. The present invention calculates the hydrolysis resistance by the degradation ability.

[0125] Test results such as Figure 8As shown, the comparative sample is an uncross-linked collagen gel, the cross-linking agent of comparative example 2 is glutaraldehyde, the cross-linking agent of example 4 is genipin, and the cross-linking degree is from large to small: example 4> comparative example 2> comparative sample. The degradation rate of the hydrogel is between 20.1% and 73.5%, and the order of the degradation rate from small to large is: example 4< comparative example 2< comparative sample, that is, the anti-hydrolysis ability is: example 4> comparative example 2> comparative sample, which indicates that the cross-linking degree is positively correlated with the anti-hydrolysis ability.

[0126] 8. Test on the promotion of fibroblast growth: The promotion of fibroblast growth of collagen hydrogel was tested according to the "Pharmaceutical Industry Standard of the People's Republic of China (YY / T 1435-2016)".

[0127] Test results such as Fig. 9 As shown, the growth promotion rate of collagen hydrogel wound dressing on human epidermal fibroblasts is between 130% and 230%, which is related to the collagen content and the type and content of modified chitosan.

[0128] 9. Toxicity test: The toxicity of collagen hydrogel was tested in accordance with the National Standard of the People's Republic of China (GB / T 16886.1-2021 / ISO 10993-11:2017).

[0129] The test results showed that through skin contact experiments, the weight of mice increased slightly after 14 days of testing, and the survival rate of mice was 100%, proving that the collagen gel dressing was non-toxic to mice.

[0130] 10. The results of the toxicity test on mice showed that the mice treated with collagen gel dressings containing dopamine had no anxious or hyperactive behavior, and their daily behavior and life patterns were the same as usual, indicating that collagen gel dressings containing dopamine can have a good analgesic and calming effect.

[0131] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a collagen hydrogel, characterized in that: include: Prepare a collagen solution, adjust the pH value, and then sequentially add the hydrophobically modified chitosan solution and the cross-linking agent to the collagen solution. After all the additions are completed, let the solution stand to obtain the collagen hydrogel. The preparation method further comprises: before adding the cross-linking agent into the collagen solution, adding the dopamine solution into the collagen solution.

2. The method for preparing the collagen hydrogel according to claim 1, characterized in that: The mass ratio of collagen to dopamine is: 15-30:3-5.

3. The method for preparing the collagen hydrogel according to claim 1, characterized in that: The mass ratio of collagen, hydrophobically modified chitosan and cross-linking agent is: 15-30: 0.1-0.3: 1-4.

4. The method for preparing the collagen hydrogel according to claim 1, characterized in that: The collagen is type I collagen.

5. The method for preparing the collagen hydrogel according to claim 1, characterized in that: The collagen is derived from pig skin, cow skin or fish skin.

6. The method for preparing the collagen hydrogel according to claim 1, characterized in that: The pH value of the collagen solution is adjusted to 5-6.

7. The method for preparing the collagen hydrogel according to claim 1, characterized in that: Acetic acid solution was used to dissolve collagen, and sodium hydroxide solution was used to adjust pH.

8. The method for preparing the collagen hydrogel according to claim 1, characterized in that: The cross-linking agent is one of glutaraldehyde, acetic anhydride, diglycidyl ethyl ether, glycidyl ether, methyl succinimidyl, genipin, oxidized sodium alginate, oxidized cellulose, and epichlorohydrin.

9. The method for preparing the collagen hydrogel according to claim 1, characterized in that: The hydrophobically modified chitosan is chitosan or cationic chitosan or anionic chitosan grafted with a hydrophobic segment; The percentage of the hydrophobic segments of the hydrophobic modified chitosan to the primary amino groups, that is, the degree of substitution of the hydrophobic modified chitosan, is 15%-90%.

10. The method for preparing the collagen hydrogel according to claim 9, characterized in that: The viscosity average molecular weight of the chitosan is 50mPa·s-500mPa·s, and the deacetylation degree is greater than 65%.

11. The method for preparing the collagen hydrogel according to claim 9, characterized in that: The cationic chitosan is 2-hydroxypropyl-3-trimethylammonium chitosan; the viscosity average molecular weight of the 2-hydroxypropyl-3-trimethylammonium chitosan is 50mPa·s-500mPa·s, the deacetylation degree is greater than 65%, and the substitution degree of 2-hydroxypropyl-3-trimethylammonium is greater than 50%.

12. The method for preparing the collagen hydrogel according to claim 9, characterized in that: The anionic chitosan is carboxymethyl chitosan; the viscosity average molecular weight of the carboxymethyl chitosan is 50mPa·s-500mPa·s, the deacetylation degree is greater than 65%, and the substitution degree of carboxymethyl is greater than 50%.

13. The method for preparing the collagen hydrogel according to claim 9, characterized in that: The hydrophobic segment is an aldehyde containing 4 to 16 carbon atoms, a fatty acid, a 2,3-epoxypropyl alkyl ether, a 2,3-epoxypropyl alkenyl ether or a 2,3-epoxypropyl trialkoxy silane; The hydrophobic chain segment is grafted onto the chitosan, carboxymethyl chitosan or 2-hydroxypropyl-3-trimethyl quaternary ammonium salt chitosan molecular chain through reduction reaction, acylation reaction and / or nucleophilic addition reaction.

14. The collagen hydrogel obtained by the method for preparing the collagen hydrogel according to any one of claims 1 to 13, characterized in that: The solid component thereof is composed of: 70wt%-99wt% collagen, 1.0wt%-10wt% cross-linking agent, 0.0wt%-1.0wt% hydrophobically modified chitosan, and 0.0wt%-29wt% dopamine; the content of all components is not 0; The total mass of the solid components accounts for 15%-40% of the total collagen hydrogel system, and the remainder is pure water.

15. Use of the collagen hydrogel according to claim 14 in the preparation of medical materials.

16. The use according to claim 15, characterized in that The medical material includes a wound dressing.

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