Preparation Method and Application of a Multifunctional Double-Network Hydrogel

Abstract

The present invention discloses a preparation method of a multifunctional double-network hydrogel. The multifunctional double-network hydrogel prepared by the present invention can be used for liver hemostasis, and its hemostatic effect is not inferior to that of commercial hemostatic sponges. The preliminary results of clinical applications show that the multifunctional double-network hydrogel prepared by the present invention can effectively promote the repair and regeneration of deep burn wounds, improve the treatment effect, and shorten the recovery time.

Description

Technical Field

[0001] The present invention belongs to the technical field of gel preparation, and specifically relates to a preparation method and application of a multifunctional double-network hydrogel. Background Art

[0002] The skin is one of the largest organs of the human body, the first line of defense against external physical and chemical injuries, and also one of the parts of the body most vulnerable to damage. Among the many factors causing skin damage, burns account for a large proportion. Burns are one of the most destructive skin traumas, and extensive deep burns can also cause death. According to the World Health Organization, burns cause more than 180,000 deaths worldwide every year. Compared with other traumas, burn wounds have irregular shapes and inconsistent depths, are prone to secondary infections and slow healing. Currently, various wound dressings are commonly used clinically to treat burn wounds. In addition, when severe burns involve deeper skin layers, wound bleeding may also occur. Therefore, when treating deep burns, the wound dressing should preferably also have a certain hemostatic effect.

[0003] Hydrogels are composed of hydrophilic polymers and have a porous three-dimensional spatial structure similar to the extracellular matrix. They can absorb blood and wound exudate and are an ideal hemostatic and wound dressing. In addition, hydrogels can keep the wound moist, prevent cell dehydration, cool the wound and relieve pain. Commonly used hydrogel materials for hemostasis and wound dressings in clinics include biomaterials and synthetic materials, such as cellulose, gelatin, chitosan, rubber, polyethylene glycol, etc. Hydrogels prepared from synthetic materials usually have excellent and adjustable mechanical properties, but are lacking in biocompatibility and immunogenicity. Biomaterial hydrogels, on the other hand, have good biocompatibility and relatively low immunogenicity, but there are certain limitations in mechanical properties.

[0004] Collagen is widely present in the skin, muscles, bones and internal organs of humans and animals. It plays an important role in maintaining the normal physiological functions of cells, tissues and organs, as well as in repairing tissue damage. Due to its excellent physical and chemical properties, biological efficacy, biocompatibility, biodegradability and other characteristics, collagen has been widely used in the fields of food, cosmetics, nutritional health care, etc. Collagen XVII, also known as CoL17 / BP180 / BPAG2, is a transmembrane protein that plays a crucial role in maintaining the connection between intracellular and extracellular structural elements involved in epidermal adhesion. Studies have shown that the knockout of the Col-XVII encoding gene leads to a decrease in wound closure, while the inhibition of the cleavage and shedding of Col-XVII increases wound closure. At the same time, a large number of studies have also shown that collagen materials have good effects in wound hemostasis and can be used as an effective wound hemostatic material. Currently, collagen can be obtained by genetic engineering recombination and extraction from animal sources (including human sources), but there are significant differences between the two in terms of source, production method, extraction process, safety, etc. Compared with the collagen directly extracted by traditional methods, recombinant human collagen has no problems such as immunity and virus carrying, has better water solubility, stronger processability, has characteristics such as single component, controllable preparation process, short production cycle, and the product quality is easier to control.

[0005] Cellulose is a linear polysaccharide mainly derived from plants such as cotton and wood. It is an extremely abundant renewable natural polymer on the earth and is used in various industrial and biomedical fields. There are a large number of hydrogen bonds inside cellulose. While these hydrogen bonds endow it with unique physical and chemical properties, they also lead to problems such as poor solubility and biodegradability. To overcome these limitations, cellulose needs to be chemically modified. Chemical modification is carried out by substituting the hydroxyl groups in the glucose units of cellulose (including etherification, esterification, acetylation, etc.) and oxidation reactions to obtain cellulose derivatives with different properties. Carboxymethyl cellulose (CMC) is an anionic, water-soluble cellulose derivative with excellent physical and chemical and mechanical properties, biocompatibility, good moisture absorption capacity and biodegradability. In addition, CMC has polyelectrolyte characteristics and can respond to ionic strength and pH, so it has good compatibility when mixed with other polymer solutions. This characteristic enables it to be widely used in the preparation of composite biomaterial scaffolds, hydrogels and drug nanoparticles. Currently, the biomaterials prepared from CMC (such as hydrogels, dressings, etc.) are widely used in the fields of acute and chronic wound hemostasis, repair and drug delivery. Studies have found that when the CMC dressing is used for diabetic wounds, the hydrogel dressing has good biocompatibility, promotes the proliferation of wound fibroblasts, and effectively promotes wound repair.

[0006] Preparing hydrogels with biopolysaccharides as materials is a common method in the field of tissue engineering. Typical polysaccharides include alginate, chitosan, dextran, cellulose, hyaluronic acid, gelatin, etc. Biopolysaccharides are of natural origin, have a simple production process, low cost, and are easy to produce on a large scale. In addition, polysaccharide-based hydrogels also have the characteristic of rapid gelation, so they are considered suitable for manufacturing various tissue engineering scaffolds with complex morphologies. However, polysaccharide-based hydrogels have disadvantages such as poor mechanical properties and weak ability to promote cell adhesion, which limit their application in tissue engineering. Collagen XVII is a transmembrane protein present in the skin basement membrane and also plays a crucial role in signal communication between the epidermis and dermis, as well as the proliferation and differentiation of epidermal stem cells. Recombinant human collagen XVII is constructed based on a eukaryotic yeast expression system, has the core amino acid sequence of human collagen XVII, and can provide adhesion motifs for cells to promote cell adhesion. Carboxymethyl cellulose (CMC) is an anionic, water-soluble cellulose derivative with excellent physical and chemical, mechanical properties, biocompatibility, good moisture absorption ability, and degradability. In addition, CMC has polyelectrolyte properties and can respond to ionic strength and pH, so it has good compatibility when mixed with other polymer solutions. This property enables it to be widely used in the preparation of composite biomaterial scaffolds, hydrogels, and drug nanoparticles. Currently, biomaterials prepared from CMC (such as hydrogels, dressings, etc.) are widely used in fields such as hemostasis and repair of acute and chronic wounds and drug delivery.

[0007] Currently, hydrogels are commonly used in fields such as three-dimensional culture of stem cells, hemostasis of wounds, and treatment of burn skin defect wounds. The following are patent documents related to recombinant human collagen hydrogels.

[0008] The patent application with the publication number CN114835920A discloses a recombinant collagen-polyglutamate hydrogel and its preparation method. This hydrogel contains recombinant collagen, polyglutamate, and polyethylene glycol. This hydrogel has the effects of promoting cell proliferation and promoting wound repair.

[0009] The patent application with the publication number CN119055827A discloses a carboxylated chitosan hydrogel dressing containing recombinant human-derived type I / III collagen microgels and its preparation method. This hydrogel contains recombinant human-derived type I collagen, recombinant human-derived type III collagen, and carboxylated chitosan. According to the dynamic repair process of the skin, this invention prepares a hydrogel dressing with adjustable proportions, which can be used for skin repair.

[0010] The patent application with the publication number CN116570758A discloses an injectable antibacterial hydrogel that can be used for the repair of irregular wounds, its preparation method and application. This hydrogel contains recombinant type III humanized collagen and chitosan grafted with dihydrocaffeic acid. This hydrogel has both antibacterial and promoting the repair of irregular wounds functions.

[0011] The patent application with the publication number CN117357463A discloses a dual-network hemostatic gel loaded with ginsenoside and its preparation method. This hydrogel contains polysaccharides and recombinant collagen, can form a gel in situ, and has the functions of effectively plugging and stopping bleeding and absorbing bleeding.

[0012] The patent application with the publication number CN119386259A discloses a collagen dressing for moisturizing and repair and its preparation method. This hydrogel includes recombinant human type III collagen, recombinant human fibronectin, β-glucan, and hexylene glycol solution. It has the effects of promoting hemostasis, inhibiting skin inflammation, promoting the healing of laser-dependent dermatitis or barrier-damaged skin, and achieving anti-allergic and anti-inflammatory effects. Summary of the Invention

[0013] The purpose of the present invention is to provide a preparation method of a multifunctional dual-network hydrogel.

[0014] Another purpose of the present invention is to provide the application of the multifunctional dual-network hydrogel prepared by the above method in the preparation of adipose mesenchymal stem cell culture reagents.

[0015] In order to achieve the above purposes, the technical solutions adopted by the present invention are as follows:

[0016] In the first aspect of the present invention, a preparation method of a multifunctional dual-network hydrogel is provided, including the following steps:

[0017] The first step, preparation of methacrylated recombinant human type XVII collagen (rhCol-MA)

[0018] Dropwise add a chemical modification reagent to a recombinant human type XVII collagen solution with a concentration of 0.05 - 0.2 g / mL (preferably 0.1 g / mL). The mass ratio of recombinant human type XVII collagen to the chemical modification reagent is 1 - 10:1 (preferably 10:1, 6:1, 5:1, 3:1). Control the dropping time to be 5 - 20 min (preferably 10 min), and adjust the pH of the above solution to 8 - 9 by dropping a NaOH solution with a concentration of 0.5 - 2 mol / L (preferably 1 mol / L). Stir and react at a temperature of 50 - 70 °C (preferably 50 °C) for 0.5 - 5 h (preferably 3 h); add PBS buffer (pH = 7.4) to the above solution, and dialyze with deionized water at room temperature for 3 - 5 days (preferably 3 days); centrifuge, take the supernatant, and freeze-dry to obtain methacrylated recombinant human type XVII collagen (rhCol-MA);

[0019] In the first step, the chemical modification reagent is selected from methacrylic anhydride.

[0020] The preparation method of the recombinant human type XVII collagen solution in the first step includes the following steps:

[0021] Add recombinant human type XVII collagen into PBS buffer solution, and stir until completely dissolved under the conditions that the concentration is 0.05 - 0.2 g / mL (preferably 0.1 g / mL) and the temperature is 50 - 70°C (preferably 60°C) to obtain the recombinant human type XVII collagen solution.

[0022] In the first step, a dialysis bag with a cut-off molecular weight of 12 - 14 kDa (preferably 14000) is used for dialysis.

[0023] The centrifugation conditions in the first step are: centrifuge at 3000 rpm for 30 min.

[0024] The freeze-drying time in the first step is 24 - 72 h (preferably 48 h), and the temperature is -65°C.

[0025] The pH of the PBS buffer solution in the first step is 7.4.

[0026] Second step, preparation of glycidyl methacrylate-modified carboxymethyl cellulose (CMC-GMA)

[0027] Dissolve sodium carboxymethyl cellulose in PBS buffer solution (pH = 7.4), and add glycidyl methacrylate into the above solution under the conditions that the temperature is 50 - 70°C (preferably 60°C). The mass ratio of sodium carboxymethyl cellulose to glycidyl methacrylate is 1:1 - 10 (preferably 1:2.14). Add hydrochloric acid with a concentration of 0.5 - 2 mol / L (preferably 1 mol / L) to adjust the pH to 4, and incubate for 2 - 10 h (preferably 6 h) under the conditions that the temperature is 50 - 70°C (preferably 60°C). Add PBS buffer solution (pH = 7.4), dialyze in deionized water at room temperature for 2 - 5 days (preferably 3 days), centrifuge, and take the supernatant for lyophilization to obtain glycidyl methacrylate-modified carboxymethyl cellulose;

[0028] The molecular weight of the sodium carboxymethyl cellulose is 100 - 140 KD.

[0029] In the second step, a dialysis bag with a cut-off molecular weight of 12 - 14 kDa is used for dialysis.

[0030] The centrifugation conditions in the second step are: centrifuge at a speed of 3000 rpm for 15 min.

[0031] The lyophilization conditions in the second step are: the time is 48 h, and the temperature is -65°C.

[0032] The third step, preparation method of the multifunctional double-network hydrogel

[0033] Mix the carboxymethyl cellulose hydrogel precursor solution modified with glycidyl methacrylate and the recombinant human type XVII collagen hydrogel precursor solution methacrylated in a volume ratio of 1:1, and irradiate under blue light (the time is preferably 1 min) to obtain the multifunctional double-network hydrogel.

[0034] Preparation of the carboxymethyl cellulose hydrogel precursor solution modified with glycidyl methacrylate:

[0035] Dissolve the carboxymethyl cellulose modified with glycidyl methacrylate in the blue light initiator solution, and filter to obtain a carboxymethyl cellulose hydrogel precursor solution modified with glycidyl methacrylate with a concentration of 0.5-3% g / mL (preferably 1% g / mL, 2% g / mL).

[0036] Preparation of the recombinant human type XVII collagen hydrogel precursor solution methacrylated:

[0037] Dissolve the recombinant human type XVII collagen methacrylated in the blue light initiator solution, and filter to obtain a recombinant human type XVII collagen hydrogel precursor solution methacrylated with a concentration of 5-20% g / mL (preferably 5%, 10%, 15% g / mL).

[0038] The blue light initiator is selected from lithium phenyl(2,4,6-trimethylbenzoyl)phosphate (abbreviation: LAP), I2959 ultraviolet light initiator.

[0039] The preparation method of the blue light initiator solution includes the following steps:

[0040] Dissolve the blue light initiator in PBS buffer solution to obtain a blue light initiator solution with a concentration of 1-5 mg / mL (preferably 2.5 mg / mL).

[0041] In the third step, the filtration is carried out using a sterile needle-type filter with a diameter of 0.22 µm.

[0042] In the third step, the blue light uses an LED blue light lamp with a power of 16-20 W and a wavelength of 405 nm.

[0043] In the third step, the pH of the PBS buffer solution is 7.4.

[0044] In the second aspect of the present invention, a multifunctional double-network hydrogel prepared by the above method is provided.

[0045] In the third aspect of the present invention, an application of the multifunctional double-network hydrogel in preparing a culture reagent for adipose mesenchymal stem cells is provided.

[0046] In the fourth aspect of the present invention, there is provided an application of the multifunctional double-network hydrogel in the preparation of a burn wound repair reagent.

[0047] In the fifth aspect of the present invention, there is provided an application of the multifunctional double-network hydrogel in the preparation of a liver hemostatic reagent.

[0048] Due to the adoption of the above technical solutions, the present invention has the following advantages and beneficial effects:

[0049] The multifunctional double-network hydrogel prepared by the present invention has good swelling performance, in vivo and in vitro degradability, and porosity. The precursor solution of the hydrogel and stem cells are uniformly mixed and gelled under blue light irradiation, which can support the three-dimensional culture of stem cells; under in vitro culture conditions, stem cells can survive in the hydrogel for a long time without losing their stemness; in vitro experiments show that the hydrogel has good biocompatibility, and the solidified hydrogel can gel in situ to promote liver hemostasis and wound repair.

[0050] The present invention is based on recombinant human XVII collagen and carboxymethyl cellulose. By means of chemical modification, recombinant human type XVII collagen is modified with methacrylic anhydride, and carboxymethyl cellulose is modified with glycidyl methacrylate. After the modified products are prepared into a mixed solution, under the irradiation of a photoinitiator and a specific light source, a free radical chain reaction occurs, causing the solution to change from a liquid to a gel state. The advantages of this reaction are as follows: By mixing the two materials to form a double-network structure hydrogel, the problem of poor mechanical properties of a single-component hydrogel is solved; by using the adhesion motif of recombinant human type XVII collagen, the deficiency of weak cell adhesion promotion ability of the polysaccharide-based hydrogel is improved, so that the hydrogel has both excellent biocompatibility and cell adhesion promotion performance; the prepared double-network structure hydrogel, due to its unique mechanical properties, biocompatibility, cell adhesion, and shape adaptability, can be used for a variety of purposes, including three-dimensional cell culture in vitro, hemostasis, and burn wound repair.

[0051] Currently, the common in vitro cell culture is two-dimensional culture. This culture method is convenient and conducive to rapid cell proliferation, but it cannot truly reflect the real environment of human cell growth. To address these limitations, significant research progress has been made in three-dimensional cell culture materials in recent years. These materials play an important role in simulating the in vivo cell microenvironment, promoting cell growth and interaction. Currently, there are few types of commercial materials for three-dimensional in vitro culture of cells or organoids, and mostly expensive Matrigel is used. Matrigel is mainly derived from the basement membrane components of mouse sarcoma cells (EHS sarcoma), with complex components including polymers, proteins, etc. Its preparation process often involves multiple steps and operations, and there are problems of batch effects and immunogenicity in actual situations. In contrast, the biological polysaccharide carboxymethyl cellulose is naturally sourced, has extremely rich reserves, and its production process and flow are very mature; recombinant human type XVII collagen is constructed based on the eukaryotic yeast expression system through synthetic biology means, with clear components, high safety, and can be mass-produced. The hydrogel constructed based on these two materials in the present invention solves some deficiencies of Matrigel and has high potential for translational application.

[0052] Commonly used gauze for hemostasis promotes blood coagulation by absorbing blood and aggregating red blood cells / platelets. However, due to its lack of tissue adhesiveness, it cannot be used to treat irregularly shaped non-compressible wounds. Sponges with shape recovery function stop bleeding by quickly absorbing a large amount of blood and volume expansion to block the bleeding site. However, considering the mismatch between the shape and expansion structure of the sponge and the wound, it shows poor blocking performance on irregular non-penetrating wounds. The present invention utilizes the function of recombinant human type XVII collagen to promote platelet aggregation and plasma clotting. After contacting with blood, platelets in the blood will adsorb to the collagen fibers and undergo agglutination reactions, thereby generating fibrin, promoting plasma clotting, and then forming thrombus to achieve the purpose of hemostasis and promoting blood coagulation. After being mixed with polysaccharides to form a double-network hydrogel, it also has excellent mechanical properties, tissue adhesion properties, and shape adaptability, and is suitable for hemostasis of various irregular wounds.

[0053] When treating burn wounds, traditional dressings such as gauze and absorbent cotton need to be frequently changed due to their limited ability to absorb exudate, and traditional dressings are prone to adhesion to tissues, causing obvious pain and discomfort to patients during dressing change. Traditional hydrogel dressings can provide a moist environment for the wound surface, promote wound healing, and reduce dressing change pain, but their mechanical properties are quite different from those of human skin, and their functions are relatively single. The present invention constructs a double-network hydrogel based on recombinant human type XVII collagen and carboxymethyl cellulose. This hydrogel system has excellent mechanical properties and biocompatibility and is suitable for burn wound repair.

[0054] The multifunctional double-network hydrogel prepared by the present invention provides a new solution for three-dimensional culture of stem cells, hemostasis and wound repair. Especially in the fields of liver hemostasis and skin repair, it helps to accelerate the rehabilitation process. The present invention not only enriches the research content of biomaterial science, opens up new possibilities for the treatment of more complex diseases, but also enhances the society's confidence in medical technology innovation. The present invention reduces the cost by optimizing the preparation process. The hydrogel preparation process is relatively simple, reduces the use of traditional chemical cross-linking agents (such as glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, etc.), and has low toxicity and low production cost.

[0055] The commercial application of the present invention will stimulate market demand and drive the development of the upstream and downstream industrial chains. The popularization and use of the present invention will directly create huge economic benefits and bring considerable economic returns to medical enterprises, scientific research institutions and investors.

[0056] Under blue light irradiation, the mixture of methacrylated recombinant human type XVII collagen solution containing 0.25% photoinitiator and carboxymethyl cellulose can be rapidly cured within 1 minute, significantly improving the surgical efficiency and operation convenience. Experiments have proved that stem cells can adhere, proliferate and maintain their stemness in the multifunctional double-network hydrogel prepared by the present invention, providing a stable microenvironment for long-term in vitro culture and subsequent treatment. The multifunctional double-network hydrogel prepared by the present invention has good biocompatibility. After curing, it can closely adhere to the bleeding point and wound surface, promoting hemostasis, cell growth and tissue repair.

[0057] Preliminary experimental results show that the multifunctional double-network hydrogel prepared by the present invention can be used for liver hemostasis, and its hemostatic effect is not inferior to that of commercial hemostatic sponges. Preliminary clinical application results show that the multifunctional double-network hydrogel prepared by the present invention can effectively promote the repair and regeneration of deep burn wounds, improve the treatment effect and shorten the recovery time. Description of the Drawings

[0058] Figure 1 Schematic diagram of the chemical modification principle of methacrylated recombinant human type XVII collagen and glycidyl methacrylate-modified carboxymethyl cellulose.

[0059] Figure 2 1H NMR spectra and Fourier transform infrared spectra of rhCol, rhCol-MA, CMC, and CMC-GMA.

[0060] Figure 3 Schematic diagrams of the precursor solutions used for C1C15 hydrogel and C2C15 hydrogel, and the C1C15 hydrogel and C2C15 hydrogel obtained after curing under blue light irradiation.

[0061] Figure 4Schematic diagrams of stress-strain curves and statistical analysis of C0C15 hydrogel, C1C15 hydrogel, C2C15 hydrogel, and C15 hydrogel.

[0062] Figure 5 Schematic diagrams of the swelling test results of C1C15 hydrogel and C2C15 hydrogel.

[0063] Figure 6 Schematic diagrams of scanning electron microscopy of C1C15 hydrogel and C2C15 hydrogel.

[0064] Figure 7 Schematic diagrams of the in vitro biocompatibility test results of C1C15 hydrogel and C2C15 hydrogel.

[0065] Figure 8 Schematic diagrams of the live / dead staining results of adipose stem cells cultured in C1C15 hydrogel and C2C15 hydrogel.

[0066] Figure 9 Schematic diagrams of the SEM images (left) and porosity statistics (right) of human adipose mesenchymal stem cells cultured in C1C15 hydrogel and C2C15 hydrogel.

[0067] Figure 10 Schematic diagrams of the flow cytometry test results of adipose stem cells cultured in 2D culture dishes and 3D hydrogels for 7 days.

[0068] Figure 11 Schematic diagrams of the in vitro clotting time measurement results of multifunctional double-network hydrogels.

[0069] Figure 12 Schematic diagrams of the statistical results of the in vitro blood coagulation index (BCI) of multifunctional double-network hydrogels.

[0070] Figure 13 Schematic diagrams of the gross pictures of liver hemostasis treatment at different time points in different groups.

[0071] Figure 14 Schematic diagrams of the statistical results of blood loss and hemostasis time in liver hemostasis treatment in each group.

[0072] Figure 15 Schematic diagrams of the gross pictures of deep burn wound healing in each group of mice.

[0073] Figure 16 Schematic diagrams of the statistical analysis of the change rate of wound area in deep burn wounds of each group of mice.

[0074] Figure 17 Schematic diagrams of the preparation and application of multifunctional double-network hydrogels.

[0075] Figure 18Schematic diagram of the multifunctional double-network hydrogel prepared in Example 2. Detailed implementation mode

[0076] To illustrate the present invention more clearly, the present invention will be further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0077] Example 1

[0078] A preparation method of a multifunctional double-network hydrogel, comprising the following steps:

[0079] The first step, preparation of methacrylated recombinant human type XVII collagen (rhCol-MA)

[0080] Add 10 g of recombinant human type XVII collagen (rhCol, Jiangsu Chuangjian Medical Technology Co., Ltd., product number: 170801) to a round-bottom flask containing 100 mL of PBS buffer (pH = 7.4), and stir at a temperature of 60 °C and a rotation speed of 240 rpm until completely dissolved to obtain a recombinant human type XVII collagen solution with a concentration of 0.1 g / mL.

[0081] Dropwise add 1 mL of methacrylic anhydride (purchased from Sigma-Aldrich, product number: 276685) to the above solution, the mass ratio of recombinant human type XVII collagen to methacrylic anhydride is 10:1, and control the dropping time to be 10 min. Adjust the pH of the above solution to 9 by dropping 1 mol / L NaOH solution, and then magnetically stir and react in a constant temperature water bath at 50 °C for 3 h. The reaction route of methacrylated recombinant human type XVII collagen is as Figure 1 shown, Figure 1 It is a schematic diagram of the chemical modification principle of methacrylated recombinant human type XVII collagen and carboxymethyl cellulose modified with glycidyl methacrylate.

[0082] Methacrylic anhydride is a liquid at room temperature, with a purity ≥ 97% and a density of 1.04 g / ml.

[0083] PBS buffer, also known as phosphate buffer, with a density of 1 g / ml, is purchased from Wuhan Saiweier Biotechnology Co., Ltd., product number G4250-500ML.

[0084] Add 120 mL of PBS buffer (pH = 7.4) to the above solution to dilute the reaction solution, and continuously stir to terminate the reaction. Place the above solution in a dialysis bag (brand: Viskase, cut-off molecular weight: 14000), and dialyze with deionized water at room temperature for 3 days;

[0085] Pour the dialyzed solution into a centrifuge tube, centrifuge at 3000 rpm for 30 min, and take the supernatant; lyophilize the supernatant in a freeze dryer for 48 h at -65 °C to obtain 9.1 g of methacrylated recombinant human type XVII collagen with a yield of 91%.

[0086] The second step is the preparation of glycidyl methacrylate-modified carboxymethyl cellulose (CMC-GMA).

[0087] Dissolve sodium carboxymethyl cellulose (2 g, molecular weight 100 - 140 KD) in 100 mL of PBS buffer (pH = 7.4), and stir magnetically at 60 °C until completely dissolved. Add glycidyl methacrylate (4.28 g, 4 mL) to the above solution. The mass ratio of sodium carboxymethyl cellulose to glycidyl methacrylate is 1:2.14. Add 1 M hydrochloric acid to adjust the pH to 4, incubate at 60 °C for 6 h, add 100 mL of PBS buffer (pH = 7.4) to dilute the reaction solution, and continuously stir to terminate the reaction. Place the above solution in a dialysis bag (12 - 14 kDa) and dialyze in deionized water at room temperature for 3 days. Pour the dialyzed solution into a centrifuge tube, centrifuge at 3000 rpm for 15 min, and take the supernatant and lyophilize it in a freeze dryer for 48 h at -65 °C to obtain 1.8 g of glycidyl methacrylate-modified carboxymethyl cellulose with a yield of 90%.

[0088] Characterization of rhCol-MA and CMC-GMA: Figure 2 are the 1H NMR spectra and Fourier transform infrared spectra of rhCol, rhCol-MA, CMC, and CMC-GMA; 1H NMR analysis shows that after grafting methacrylic anhydride onto recombinant human type XVII collagen, the modification amount of lysine residues (≈2.8 ppm) decreases, and the signal amount of vinyl methacrylate (≈5.3 - 5.7 ppm) increases. Similarly, compared with sodium carboxymethyl cellulose, methacrylic acid peaks (about 5.6 and 6.2 ppm) are observed in CMC-GMA, proving that the double bond is successfully grafted onto the CMC main chain.

[0089] The FTIR spectral results show that at 1118 cm -1 the peak of lysine residues in rhCol-MA decreases, and at the same time a new peak appears at 950 cm -1 (out-of-plane bending (deformation) vibration peak of C-H of the RC=CH2 structure), proving that C=C is successfully modified on the collagen molecular chain and vinylated collagen is obtained. The C=C absorption peak of GMA usually appears at 1695 - 1630 cm-1 At -1 , in the spectral results of CMC-GMA, the absorption peak of C=C underwent a blue shift (1698 cm -1 ), proving the successful grafting of GMA.

[0090] Step 3. Preparation method of the multifunctional double-network hydrogel

[0091] Dissolve 25 mg of the blue light initiator lithium phenyl(2,4,6-trimethylbenzoyl)phosphate (abbreviation: LAP) in 10 ml of PBS buffer solution (pH = 7.4) to obtain a blue light initiator solution with a concentration of 2.5 mg / mL.

[0092] Dissolve 10 mg of the freeze-dried sample of glycidyl methacrylate-modified carboxymethyl cellulose in 1 ml of the blue light initiator solution with a concentration of 2.5 mg / mL, and filter it through a sterile syringe filter (diameter 0.22 µm) to obtain a glycidyl methacrylate-modified carboxymethyl cellulose hydrogel precursor solution with a concentration of 1% g / mL.

[0093] Dissolve 20 mg of the freeze-dried sample of glycidyl methacrylate-modified carboxymethyl cellulose in 1 ml of the blue light initiator solution with a concentration of 2.5 mg / mL, and filter it through a sterile syringe filter (diameter 0.22 µm) to obtain a glycidyl methacrylate-modified carboxymethyl cellulose hydrogel precursor solution with a concentration of 2% g / mL.

[0094] Dissolve 300 mg of the freeze-dried sample of methacrylated recombinant human type XVII collagen in 2 ml of the blue light initiator solution with a concentration of 2.5 mg / mL, and filter it through a sterile syringe filter (diameter 0.22 µm) to obtain a methacrylated recombinant human type XVII collagen hydrogel precursor solution with a concentration of 15% g / mL.

[0095] Place 400 μl of the glycidyl methacrylate-modified carboxymethyl cellulose hydrogel precursor solution with a concentration of 1% g / mL and 400 μl of the methacrylated recombinant human type XVII collagen hydrogel precursor solution with a concentration of 15% g / mL in a glass bottle and mix well. Irradiate with an LED blue light (power 16 - 20 W, wavelength 405 nm) for 1 min to obtain a multifunctional double-network hydrogel, abbreviated as C1C15 hydrogel.

[0096] Place 400 μl of the glycidyl methacrylate-modified carboxymethyl cellulose hydrogel precursor solution with a concentration of 2% g / mL and 400 μl of the methacrylated recombinant human type XVII collagen hydrogel precursor solution with a concentration of 15% g / mL in a glass bottle and mix well. Irradiate with an LED blue light (power 16 - 20 W, wavelength 405 nm) for 1 min to obtain a multifunctional double-network hydrogel, abbreviated as C2C15 hydrogel.

[0097] Mix 400 μl of PBS buffer (pH = 7.4) and 400 μl of the pre-liquid of methacrylated recombinant human type XVII collagen hydrogel with a concentration of 15% g / mL in a glass bottle and mix well. Then irradiate it with an LED blue light (power 16 - 20 W, wavelength 405 nm) for 1 min to obtain a multifunctional double-network hydrogel, simply referred to as C0C15 hydrogel.

[0098] Place 400 μl of the pre-liquid of methacrylated recombinant human type XVII collagen hydrogel with a concentration of 15% g / mL in a glass bottle and mix well. Then irradiate it with an LED blue light (power 16 - 20 W, wavelength 405 nm) for 1 min to obtain a recombinant human type XVII collagen hydrogel, simply referred to as C15 hydrogel.

[0099] Figure 3 It is a schematic diagram of the pre-liquids used for C1C15 hydrogel and C2C15 hydrogel, and the C1C15 hydrogel and C2C15 hydrogel obtained after curing by blue light irradiation. It can be seen from the figure that the pre-liquid of the hydrogel turns from a flowing liquid into a solidified hydrogel after blue light irradiation.

[0100] Measurement of the compressive modulus of the multifunctional double-network hydrogel

[0101] Use a mold (a set of multifunctional anti-sticking hydrogel molds disclosed in the patent application with the publication number CN221717551U) to prepare cylindrical hydrogel samples with a diameter of 11.2 mm and a height of 5 mm, and obtain C0C15 hydrogel, C1C15 hydrogel, C2C15 hydrogel, and C15 hydrogel respectively. Use a Zwick Roell Z2.5TH universal material tester (sensor: 2.5 kN) to conduct a compression test. Calculate the compressive modulus of the sample according to the slope of the straight line in the 10 - 15% interval of the sample stress-strain curve. Figure 4 It is a schematic diagram of the stress-strain curves and statistical analysis of C0C15 hydrogel, C1C15 hydrogel, C2C15 hydrogel, and C15 hydrogel; it can be seen from the figure that by comparing the compressive moduli of C0C15 hydrogel and C15 hydrogel, it can be seen that the compressive modulus of the hydrogel increases with the increase in the concentration of recombinant human type XVII collagen (the compressive modulus of C0C15 hydrogel is 4.36 Kpa, and the compressive modulus of C15 hydrogel is 26.73 Kpa); by comparing the compressive moduli of C0C15 hydrogel, C1C15 hydrogel, C2C15 hydrogel, and C15 hydrogel, after introducing CMC, the compressive modulus of the hydrogel also increases significantly, and the higher the concentration of CMC introduced, the more significant the change in the compressive modulus. The compressive modulus of C2C15 hydrogel is 30.96 Kpa, which is significantly higher than the compressive modulus of C15 hydrogel (26.73 Kpa).

[0102] Swelling property analysis of multifunctional double-network hydrogel

[0103] The cylindrical hydrogel samples with a diameter of 11.2 mm and a height of 5 mm prepared above were freeze-dried, and the swelling property of the hydrogel was evaluated through a swelling experiment. The results are as Figure 5 shown Figure 5 The schematic diagrams of the swelling test results of C1C15 hydrogel and C2C15 hydrogel. The results show that after introducing CMC and soaking in a sterile PBS solution for 24 hours, both C1C15 hydrogel and C2C15 hydrogel reached swelling equilibrium, and the weights of C1C15 hydrogel and C2C15 hydrogel could reach 10 - 15 times of the initial weight. Both C1C15 hydrogel and C2C15 hydrogel have good water absorption and swelling ability.

[0104] Scanning electron microscopy (SEM) analysis of multifunctional double-network hydrogel

[0105] The cylindrical hydrogel samples with a diameter of 11.2 mm and a height of 5 mm prepared above were freeze-dried for scanning electron microscopy analysis. Figure 6 The schematic diagrams of the scanning electron microscopy of C1C15 hydrogel and C2C15 hydrogel. It can be seen from the figure that the freeze-dried double-network hydrogel has a loose and porous structure, and there is no significant difference in the internal pore structure between C1C15 hydrogel and C2C15 hydrogel.

[0106] Biocompatibility detection of multifunctional double-network hydrogel

[0107] To evaluate the function of the multifunctional double-network hydrogel in culturing cells in vitro, the biocompatibility of the hydrogel was first evaluated. The prepared C1C15 hydrogel and C2C15 hydrogel were put into a complete medium and soaked for 24 hours. The hydrogel blocks were discarded, and the liquid was filtered through a 0.45 μm filter membrane to obtain C1C15 hydrogel extract and C2C15 hydrogel extract respectively. L929 cells (mouse fibroblastoma cell line) and HUVEC cells (human umbilical vein endothelial cells) were cultured with them, and the proliferation activity of the cells was detected by a CCK8 kit. The specific method is as follows:

[0108] L929 cells and HUVEC cells in good growth state were selected in a culture dish. After the cells proliferated to 80%, the old medium was removed, the cells were digested with trypsin and centrifuged. The cells were resuspended with C1C15 hydrogel extract and C2C15 hydrogel extract respectively and plated onto a 96-well plate (4000 cells / well). After culturing for 0, 1, 3, and 5 days, the old medium was removed, 110 μl of CCK8 working solution was added, and the cells were incubated in an incubator for 2 hours. Then, the absorbance of each group of cells at 450 nm was detected by an enzyme-linked immunosorbent assay (ELISA) reader and statistical analysis was performed.

[0109] The CCK8 test results are as follows Figure 7 shown in Figure 7 Figure [ID], which is a schematic diagram of the in vitro biocompatibility test results of C1C15 hydrogel and C2C15 hydrogel; in the figure, control is the complete medium. Among them, the left figure is a schematic diagram of the CCK8 test results of the extracts of C1C15 hydrogel and C2C15 hydrogel promoting the proliferation of HUVEC. The middle figure is a schematic diagram of the CCK8 test results of the extracts of C1C15 hydrogel and C2C15 hydrogel promoting the proliferation of L929, and the right figure is a schematic diagram of the hemolysis rate of C1C15 hydrogel and C2C15 hydrogel. It can be seen from the figure that compared with the Control group, on the 1st, 3rd, and 5th days, the extracts of C1C15 hydrogel and C2C15 hydrogel did not have a significant inhibitory effect on the proliferation of L929 cells, but had a certain promoting effect on the proliferation of HUVEC cells. That is, the extracts of C1C15 hydrogel and C2C15 hydrogel have no cytotoxicity and can promote cell proliferation to a certain extent.

[0110] To further evaluate the biocompatibility of the hydrogel, the formed hydrogel was used for the in vitro blood compatibility test. 0.1% Triton-X100 was set as the positive control group, and C1C15 hydrogel and C2C15 hydrogel were used as the experimental groups. The hemolysis rates of each group were calculated. The hemolysis rates of C1C15 hydrogel and C2C15 hydrogel were 1% and 1.01% respectively, both of which were lower than 5%, meeting the standard of material blood compatibility, indicating that the double-network hydrogel prepared by the present invention has good blood compatibility and can be used as a hemostatic agent and wound dressing.

[0111] The application of the multifunctional double-network hydrogel in culturing adipose mesenchymal stem cells

[0112] Adipose stem cells have the characteristics of high plasticity and easy cultivation, so they have become an important tool for studying human development, disease pathogenesis, and new drug research and development. By using adipose stem cells for in vitro experiments, the pathogenesis of diseases can be better understood and more effective treatment methods can be developed.

[0113] The multifunctional double-network hydrogel is beneficial to stem cell proliferation:

[0114] Select human adipose-derived mesenchymal stem cells (ADSCs) with good growth status of P3-P5, digest them, centrifuge them, and discard the supernatant. Resuspend the cells with the corresponding precursor fluids of C1C15 hydrogel (400 μl of carboxymethyl cellulose hydrogel precursor fluid modified with 1% g / mL glycidyl methacrylate and 400 μl of recombinant human type XVII collagen hydrogel precursor fluid modified with 15% g / mL methacryloylation) and the corresponding precursor fluids of C2C15 hydrogel (400 μl of carboxymethyl cellulose hydrogel precursor fluid modified with 2% g / mL glycidyl methacrylate and 400 μl of recombinant human type XVII collagen hydrogel precursor fluid modified with 15% g / mL methacryloylation); aspirate 200 µl of the hydrogel precursor fluid mixed with stem cells into a 48-well plate, and irradiate it with an LED blue light lamp (power 16 - 20 W, wavelength 405 nm) for 1 min to obtain a hydrogel scaffold encapsulating adipose-derived mesenchymal stem cells; add cell culture medium and change the medium every 2 days. Observe the survival status of stem cells in the hydrogel scaffold on the 7th, 14th, and 28th days by live-dead staining. At specific time points (the 1st, 3rd, and 7th days), remove the old culture medium in the well plate, wash it 3 times with PBS, add live-dead staining solution, incubate it at 37 °C for 30 min, remove the staining agent, wash it 2 times with PBS, and then take pictures under a fluorescence microscope.

[0115] The results are as Figure 8 shown, Figure 8 which are schematic diagrams of the live-dead staining results of adipose stem cells cultured in C1C15 hydrogel and C2C15 hydrogel; live cells in the hydrogel appear green under a fluorescence microscope, and dead cells appear red. It was observed that ADSCs continuously proliferated in the hydrogel with the increase of culture time. The cell morphology in the hydrogel was normal, and most cells were stained green (live cells), and very few cells were stained red (dead cells), indicating that C1C15 hydrogel and C2C15 hydrogel are beneficial to the growth and proliferation of stem cells and have good biocompatibility.

[0116] Culturing stem cells with multifunctional double-network hydrogel is beneficial to cell spreading:

[0117] Mix ADSCs with the corresponding precursor fluids of C1C15 hydrogel (400 μl of carboxymethyl cellulose hydrogel precursor fluid modified with 1% g / mL glycidyl methacrylate and 400 μl of recombinant human type XVII collagen hydrogel precursor fluid modified with 15% g / mL methacryloylation), and irradiate it with an LED blue light lamp (power 16 - 20 W, wavelength 405 nm) for 1 min to obtain a hydrogel containing ADSCs, that is, C1C15 hydrogel containing ADSCs.

[0118] Mix ADSCs with the corresponding precursor fluids of C2C15 hydrogel (400 μl of the precursor fluid of glycidyl methacrylate-modified carboxymethyl cellulose hydrogel with a concentration of 2% g / mL and 400 μl of the precursor fluid of methacrylated recombinant human type XVII collagen hydrogel with a concentration of 15% g / mL), and irradiate with an LED blue light (power 16 - 20 W, wavelength 405 nm) for 1 min to obtain a hydrogel containing ADSCs, that is, C2C15 hydrogel containing ADSCs.

[0119] Culture the C1C15 hydrogel containing ADSCs and the C2C15 hydrogel containing ADSCs in complete medium for 5 days, and change the complete medium every two days. After 5 days, freeze-dry the samples of the C1C15 hydrogel containing ADSCs and the C2C15 hydrogel containing ADSCs, spray a thin gold layer, and then examine by scanning electron microscopy (SEM) to observe the spreading of human adipose-derived mesenchymal stem cells inside the hydrogel. The results are as Figure 9 shown. Figure 9 Schematic diagram of the SEM images (left) and porosity statistics (right) of human adipose-derived mesenchymal stem cells cultured in C1C15 hydrogel and C2C15 hydrogel. It can be seen from the figure that on the 5th day, human adipose-derived mesenchymal stem cells began to extend tentacles around and spread well. The concentration of carboxymethyl cellulose does not affect the pore structure of the hydrogel, and at the same time, the double-network hydrogel has the advantage of three-dimensional cell culture in vitro. Human adipose-derived mesenchymal stem cells can adhere and spread within the double-network hydrogel prepared in the present invention.

[0120] Culturing with the multifunctional double-network hydrogel does not affect the stemness of stem cells:

[0121] Culture the C1C15 hydrogel containing ADSCs and the C2C15 hydrogel containing ADSCs in complete medium for 7 days, and change the complete medium every two days. After 7 days, dissolve the above two groups of hydrogels with 0.1% type I collagenase, centrifuge the obtained cell suspension at 1200 rpm for 5 min, discard the supernatant, resuspend the ADSCs with PBS, and adjust the cell density to 1×106 cells / mL. Take 7 EP tubes, label them 1 - 7 respectively, after dispensing 100 μl of cell suspension into each tube, add flow antibodies: CD31, CD45, CD73, CD90, CD105, CD235a, mix well and incubate in the dark on ice for 30 min. Add 500 μl of PBS pre-cooled at 4°C to gently resuspend the cells, centrifuge at 400 g, 4°C for 5 min and discard the supernatant, repeat this step 2 - 3 times. Finally, gently resuspend the cell pellet with 200 μl of pre-cooled PBS, and then it can be used for on-machine detection. The results are as Figure 10 shown. Figure 10Schematic diagram of the flow cytometry detection results of adipose stem cells cultured in a petri dish (2D culture) and a hydrogel (three-dimensional culture) after 7 days. It can be seen from the figure that the flow cytometry results of six surface antibodies, CD90, CD73, CD105, CD235a, CD31, and CD45, show that compared with the 2D culture in the petri dish, the stemness of human adipose mesenchymal stem cells still exists after three-dimensional culture; the results show that the expression of positive and negative markers of ADSCs cultured in the hydrogel is within their respective ranges, that is, these cells still have stemness.

[0122] Multifunctional double-network hydrogel promotes liver hemostasis

[0123] Grouping: control group, commercial hemostatic sponge group (HS group), C1C15 hydrogel group, C2C15 hydrogel group.

[0124] The in vitro procoagulant performance of the double-network hydrogel was evaluated using the whole blood clotting time (CT) and blood clotting index (BCI). Citrate anticoagulated blood was mixed with 0.2 M calcium chloride (Vblood: VCaCl2 = 9:1). The recalcified blood was added to a 96-well plate, 50 µl per well. Then, 50 µl of each group of samples (n = 3) was added to each well, and the control group was not treated. Every 1 minute, the plate was gently rinsed with PBS to completely remove the uncoagulated blood. The clotting time was the moment when a uniform and stable blood clot formed in the well. The results are as Figure 11 shown. Figure 11 Schematic diagram of the in vitro blood clotting time measurement results of the multifunctional double-network hydrogel. It can be seen from the figure that the commercial hemostatic sponge group (HS group), C1C15 hydrogel group, and C2C15 hydrogel group could form stable blood clots in 2 minutes, while the control group (control in the figure) started clotting at 7 minutes. The above shows that the double-network hydrogel prepared by the present invention has the effect of promoting blood clotting in vitro.

[0125] 100 µl of recalcified whole blood was dropped onto 300 µl of each group of samples (n = 3), incubated at 37 °C for 5 min, 10 ml of deionized water was added to lyse the uncoagulated blood cells, incubated at 37 °C for 10 min, and the serum coagulation situation was observed and photographed. 100 µl of the supernatant from each tube was taken into a 96-well plate, and its OD value at 560 nm was measured in an enzyme-linked immunosorbent assay reader. The control group was deionized water, and the BCI was calculated according to the following formula: BCI = (ODm / ODp) × 100%. The results are as Figure 12 shown. Figure 12Schematic diagram of the statistical results of the in vitro blood coagulation index (BCI) of the multifunctional dual-network hydrogel. As can be seen from the figure, the BCI values of the C1C15 hydrogel group and the C2C15 hydrogel group are 40.5 and 25.8 respectively, showing no significant difference from the coagulation index of the commercial hemostatic sponge group (BCI = 41.7), and both are much lower than that of the control group, indicating good coagulation effects.

[0126] According to the literature (Hongjuan Weng, Weibin Jia, Min Li, et al. New injectable chitosan-hyaluronic acid based hydrogels for hemostasis and wound healing. Carbohydrate Polymers, 294, (2022) 119767.), an SD rat liver hemostasis model was constructed (after the rats were anesthetized, the abdominal hair was removed, the abdomen was opened to fully expose the liver, and a bleeding incision was created on the liver using surgical scissors). Each group of hydrogels (n = 3) was given for treatment. The control groups were PBS and commercial hemostatic sponges. The hemostasis time was recorded. After the bleeding stopped, the filter paper was weighed and the blood loss was calculated. The general results of the hemostasis experiment are as Figure 13 shown. Figure 13 Schematic diagram of the general pictures of liver hemostasis treated by different groups at different time points. As can be seen from the figure, the hemostasis effects of the commercial hemostatic sponge group, the C1C15 hydrogel group, and the C2C15 hydrogel group are all significantly better than those of the control group.

[0127] Statistics of blood loss ( Figure 14 the left picture in the middle) and statistics of hemostasis time ( Figure 14 the right picture in the middle) are as Figure 14 shown. Figure 14Schematic diagram of the statistical results of blood loss and hemostasis time in the treatment of liver hemostasis for each group. It can be seen from the figure that the average blood loss in the control group was 1128.33 mg, the average blood loss in the commercial hemostatic sponge group was 390 mg, the average blood loss in the C1C15 hydrogel group was 347 mg, and the average blood loss in the C2C15 hydrogel group was 265.67 mg. The blood loss in the C1C15 hydrogel group and the C2C15 hydrogel group was significantly lower than that in the control group (P < 0.05), and there was no significant difference in the blood loss between the C1C15 hydrogel group, the C2C15 hydrogel group and the commercial hemostatic sponge group. Analyzing the hemostasis time of different groups, the results showed that the average hemostasis time in the control group was 201.3 s, the average hemostasis time in the commercial hemostatic sponge group was 124.7 s, the average hemostasis time in the C1C15 hydrogel group was 87 s, and the average hemostasis time in the C2C15 hydrogel group was 100.1 s. The hemostasis effects of the commercial hemostatic sponge group, the C1C15 hydrogel group and the C2C15 hydrogel group were all significantly better than those in the control group (P < 0.001), and the hemostasis time of the C1C15 hydrogel group and the C2C15 hydrogel group was significantly shorter than that of the commercial hemostatic sponge group. The above shows that the double-network hydrogel prepared by the present invention has good in vivo hemostatic function.

[0128] Multifunctional double-network hydrogel promotes the repair of burn wounds

[0129] Grouping: control group, C1C15 hydrogel group, C2C15 hydrogel group.

[0130] According to the literature (Ning Yu, Yunpeng Li, Yansheng Wang, et al. Healing effect of carboxymethyl chitosan plantamajoside hydrogel on burn wound skin. burns 48(2022) 902-914.), a deep second-degree burn wound model of nude mice was constructed (after the mice were anesthetized, the hair on the back was removed, and a copper rod with a diameter of 6 mm soaked in boiling water at 100 °C was used to contact the back skin, and the model was successfully established). At 48 hours after burning, escharotomy was performed, and at the same time, each group of hydrogels (n = 3) was given for treatment. The control group used PBS. The wound surface was observed every 3 days and the wound healing situation was photographed and recorded. At the 15th day, the wound surface was sampled, and the healing quality was analyzed and evaluated by pathological techniques such as HE. The results are as Figure 15 shown in the gross pictures of wound healing Figure 15 Schematic diagram of the gross healing of deep burn wounds in each group of mice. It can be seen from the figure that compared with the control group, the wound healing speed of the C1C15 hydrogel group and the C2C15 hydrogel group was faster, and the end healing rate was higher. Figure 16Schematic diagram for statistical analysis of the change rate of the wound area of deep burn wounds in each group of mice. It can be seen from the figure that compared with the control group, after treatment with C1C15 hydrogel and C2C15 hydrogel, the wound closure rate significantly accelerated starting from the 6th day. The above results indicate that the double-network hydrogel prepared by the present invention has a good function of promoting the healing of burn wounds and can be applied to the field of burn wound repair.

[0131] Figure 17 Schematic diagram for the preparation and application of a multifunctional double-network hydrogel. In the present invention, methacrylated recombinant human type XVII collagen hydrogel precursor solution and glycidyl methacrylate-modified carboxymethyl cellulose hydrogel precursor solution are respectively obtained by chemically modifying and modifying recombinant human type XVII collagen and carboxymethyl cellulose. Under the conditions of a blue light initiator and light, the mixed solution of the two can change from a liquid state to a gel state, and the obtained photocured double-network hydrogel has good application prospects in three-dimensional culture of adipose stem cells, liver hemostasis, and wound repair.

[0132] Example 2

[0133] Replace the mass ratio of recombinant human type XVII collagen to methacrylic anhydride of 10:1 in the first step of Example 1 with 5:1, and keep the others unchanged to obtain methacrylated recombinant human type XVII collagen.

[0134] Prepare glycidyl methacrylate-modified carboxymethyl cellulose (CMC-GMA) according to the second step in Example 1.

[0135] Third step, preparation method of the multifunctional double-network hydrogel

[0136] Dissolve 25 mg of the blue light initiator lithium phenyl(2,4,6-trimethylbenzoyl)phosphate (abbreviation: LAP) in 10 ml of PBS buffer solution (pH = 7.4) to obtain a blue light initiator solution with a concentration of 2.5 mg / mL.

[0137] Dissolve 10 mg of the freeze-dried sample of glycidyl methacrylate-modified carboxymethyl cellulose in 1 ml of the blue light initiator solution with a concentration of 2.5 mg / mL, and filter it through a sterile syringe filter (diameter 0.22 µm) to obtain a glycidyl methacrylate-modified carboxymethyl cellulose hydrogel precursor solution with a concentration of 1% g / mL.

[0138] Dissolve 20 mg of the freeze-dried sample of glycidyl methacrylate-modified carboxymethyl cellulose in 1 ml of the blue light initiator solution with a concentration of 2.5 mg / mL, and filter it through a sterile syringe filter (diameter 0.22 µm) to obtain a glycidyl methacrylate-modified carboxymethyl cellulose hydrogel precursor solution with a concentration of 2% g / mL.

[0139] Dissolve 300 mg of freeze-dried methacrylated recombinant human type XVII collagen sample in 2 ml of a blue light initiator solution with a concentration of 2.5 mg / mL, and filter it through a sterile syringe filter (diameter 0.22 µm) to obtain a methacrylated recombinant human type XVII collagen hydrogel precursor solution with a concentration of 15% g / mL.

[0140] Dissolve 200 mg of freeze-dried methacrylated recombinant human type XVII collagen sample in 2 ml of a blue light initiator solution with a concentration of 2.5 mg / mL, and filter it through a sterile syringe filter (diameter 0.22 µm) to obtain a methacrylated recombinant human type XVII collagen hydrogel precursor solution with a concentration of 10% g / mL.

[0141] Dissolve 100 mg of freeze-dried methacrylated recombinant human type XVII collagen sample in 2 ml of a blue light initiator solution with a concentration of 2.5 mg / mL, and filter it through a sterile syringe filter (diameter 0.22 µm) to obtain a methacrylated recombinant human type XVII collagen hydrogel precursor solution with a concentration of 5% g / mL.

[0142] Place 400 μl of a glycidyl methacrylate-modified carboxymethyl cellulose hydrogel precursor solution with a concentration of 1% g / mL and 400 μl of a methacrylated recombinant human type XVII collagen hydrogel precursor solution with a concentration of 15% g / mL in a glass bottle and mix well. Irradiate with an LED blue light (power 16 - 20 W, wavelength 405 nm) for 1 min to obtain a multifunctional double-network hydrogel.

[0143] Place 400 μl of a glycidyl methacrylate-modified carboxymethyl cellulose hydrogel precursor solution with a concentration of 2% g / mL and 400 μl of a methacrylated recombinant human type XVII collagen hydrogel precursor solution with a concentration of 15% g / mL in a glass bottle and mix well. Irradiate with an LED blue light (power 16 - 20 W, wavelength 405 nm) for 1 min to obtain a multifunctional double-network hydrogel.

[0144] Place 400 μl of a glycidyl methacrylate-modified carboxymethyl cellulose hydrogel precursor solution with a concentration of 1% g / mL and 400 μl of a methacrylated recombinant human type XVII collagen hydrogel precursor solution with a concentration of 10% g / mL in a glass bottle and mix well. Irradiate with an LED blue light (power 16 - 20 W, wavelength 405 nm) for 1 min to obtain a multifunctional double-network hydrogel.

[0145] Put 400 μl of the precursor solution of glycidyl methacrylate-modified carboxymethyl cellulose hydrogel with a concentration of 2% g / mL and 400 μl of the precursor solution of methacrylated recombinant human type XVII collagen hydrogel with a concentration of 10% g / mL into a glass bottle and mix well. Then irradiate with an LED blue light lamp (power 16 - 20 W, wavelength 405 nm) for 1 min to obtain a multifunctional double-network hydrogel.

[0146] Put 400 μl of the precursor solution of glycidyl methacrylate-modified carboxymethyl cellulose hydrogel with a concentration of 1% g / mL and 400 μl of the precursor solution of methacrylated recombinant human type XVII collagen hydrogel with a concentration of 5% g / mL into a glass bottle and mix well. Then irradiate with an LED blue light lamp (power 16 - 20 W, wavelength 405 nm) for 1 min to obtain a multifunctional double-network hydrogel.

[0147] Put 400 μl of the precursor solution of glycidyl methacrylate-modified carboxymethyl cellulose hydrogel with a concentration of 2% g / mL and 400 μl of the precursor solution of methacrylated recombinant human type XVII collagen hydrogel with a concentration of 5% g / mL into a glass bottle and mix well. Then irradiate with an LED blue light lamp (power 16 - 20 W, wavelength 405 nm) for 1 min to obtain a multifunctional double-network hydrogel.

[0148] Figure 18 Schematic diagram of the multifunctional double-network hydrogel prepared in Example 2. Among them, horizontally are the precursor solutions of methacrylated recombinant human type XVII collagen hydrogels with concentrations of 15%, 10%, and 5% g / mL, and vertically are the precursor solutions of glycidyl methacrylate-modified carboxymethyl cellulose hydrogels with concentrations of 1% and 2% g / mL. After mixing and blue light irradiation, a multifunctional double-network hydrogel is formed. It can be seen from the figure that a stable multifunctional double-network hydrogel can be formed.

[0149] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art of this patent, without departing from the scope of the technical solution of the present invention, can make some changes or modifications using the above-mentioned disclosed technical content into equivalent embodiments of equivalent changes. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A preparation method of a multifunctional double-network hydrogel, characterized in that, It includes the following steps: In the first step, a chemical modification reagent is added dropwise to a recombinant human type XVII collagen solution with a concentration of 0.05 - 0.2 g / mL. The mass ratio of recombinant human type XVII collagen to the chemical modification reagent is 1 - 10:

1. The dropping time is controlled to be 5 - 20 min, and a NaOH solution with a concentration of 0.5 - 2 mol / L is added dropwise to adjust the pH of the above solution to 8 - 9. Then, it is stirred and reacted at a temperature of 50 - 70 °C for 0.5 - 5 h. PBS buffer is added to the above solution, and it is dialyzed with deionized water at room temperature for 3 - 5 days. After centrifugation, the supernatant is taken and freeze-dried to obtain methacrylated recombinant human type XVII collagen. In the first step, the chemical modification reagent is selected from methacrylic anhydride. In the second step, sodium carboxymethylcellulose is dissolved in PBS buffer. Under the condition of a temperature of 50 - 70 °C, glycidyl methacrylate is added to the above solution. The mass ratio of sodium carboxymethylcellulose to glycidyl methacrylate is 1:1 - 10. Hydrochloric acid with a concentration of 0.5 - 2 mol / L is added to adjust the pH to 4. Then, it is incubated at a temperature of 50 - 70 °C for 2 - 10 h. PBS buffer is added, and it is dialyzed in deionized water at room temperature for 2 - 5 days. After centrifugation, the upper supernatant is taken and freeze-dried to obtain glycidyl methacrylate-modified sodium carboxymethylcellulose. In the third step, the hydrogel precursor fluids of glycidyl methacrylate-modified sodium carboxymethylcellulose and methacrylated recombinant human type XVII collagen with a volume ratio of 1:1 are mixed and irradiated under blue light to obtain a multifunctional double-network hydrogel. The preparation of the hydrogel precursor fluid of glycidyl methacrylate-modified sodium carboxymethylcellulose: Glycidyl methacrylate-modified sodium carboxymethylcellulose is dissolved in a blue light initiator solution, and after filtration, a hydrogel precursor fluid of glycidyl methacrylate-modified sodium carboxymethylcellulose with a concentration of 0.5 - 3% g / mL is obtained. The preparation of the hydrogel precursor fluid of methacrylated recombinant human type XVII collagen: Methacrylated recombinant human type XVII collagen is dissolved in a blue light initiator solution, and after filtration, a hydrogel precursor fluid of methacrylated recombinant human type XVII collagen with a concentration of 5 - 20% g / mL is obtained.

2. The preparation method of the multifunctional double-network hydrogel according to claim 1, characterized in that, The preparation method of the recombinant human type XVII collagen solution in the first step includes the following steps: Recombinant human type XVII collagen is added to PBS buffer to make its concentration 0.05 - 0.2 g / mL, and it is stirred until completely dissolved under the condition of a temperature of 50 - 70 °C to obtain the recombinant human type XVII collagen solution.

3. The preparation method of the multifunctional double-network hydrogel according to claim 1, characterized in that, The blue light initiator is selected from lithium phenyl(2,4,6-trimethylbenzoyl)phosphate and I2959 ultraviolet light initiator.

4. The preparation method of the multifunctional double-network hydrogel according to claim 1, wherein The preparation method of the blue light initiator solution includes the following steps: The blue light initiator is dissolved in PBS buffer to obtain a blue light initiator solution with a concentration of 1 - 5 mg / mL. In the third step, the blue light uses an LED blue light lamp with a power of 16 - 20 W and a wavelength of 405 nm.

5. A multifunctional double-network hydrogel prepared by the method according to any one of claims 1 to 4.

6. Use of the multifunctional double-network hydrogel according to claim 5 in the preparation of an adipose mesenchymal stem cell culture reagent.

7. Use of the multifunctional double-network hydrogel according to claim 5 in the preparation of a burn wound repair reagent.

8. Use of the multifunctional double-network hydrogel according to claim 5 in the preparation of a liver hemostatic reagent.

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