Preparation method and application of multifunctional dual-network hydrogel

Abstract

The invention discloses a preparation method of multifunctional dual-network hydrogel, the prepared multifunctional dual-network hydrogel can be used for liver hemostasis, and the hemostatic effect of the multifunctional dual-network hydrogel is not inferior to that of commercial hemostatic sponge. Preliminary results of clinical application show that the prepared multifunctional dual-network hydrogel can effectively promote repair and regeneration of deep burn wounds, the treatment effect is improved, and the recovery time is shortened.

Description

Technical Field

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

[0002] The skin is one of the largest organs in the human body. It is the first line of defense for the body against external physical and chemical damage. It is also one of the most vulnerable parts of the body. Among the many factors that cause skin damage, burns account for a large proportion. Burns are one of the most destructive skin injuries. Large-area deep burns can also cause death. According to data from the World Health Organization, burns cause more than 180,000 deaths worldwide each year. Compared with other injuries, burn wounds are irregular in shape, inconsistent in depth, prone to secondary infection and slow to heal. At present, various wound dressings are routinely used in clinical practice to treat burn wounds. In addition, when severe burns involve deeper layers of the skin, they may also be accompanied by bleeding from the wound. Therefore, when treating deep burns, the wound dressing should preferably have a certain hemostatic effect.

[0003] Hydrogels are composed of hydrophilic polymers and have a porous three-dimensional structure similar to the extracellular matrix. They can absorb blood and wound exudate and are ideal hemostatic and wound dressings. In addition, hydrogels can keep wounds moist, prevent cell dehydration, cool wounds and relieve pain. Commonly used hydrogel materials for hemostasis and wound dressings in clinical practice 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 lack biocompatibility and immunogenicity. Biomaterial hydrogels have good biocompatibility and relatively low immunogenicity, but their mechanical properties are limited.

[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. Collagen is widely used in food, cosmetics, nutrition and health care due to its excellent physical and chemical properties, biological efficacy, biocompatibility, and biodegradability. Type XVII collagen (Collagen XVII, Col-XVII), also known as CoL17 / BP180 / BPAG2, is a transmembrane protein that plays a vital role in maintaining the connection between intracellular and extracellular structural elements involved in epidermal adhesion. Studies have shown that knockout of the Col-XVII encoding gene will lead to reduced wound closure, while inhibiting the shearing and shedding of Col-XVII will increase wound closure. At the same time, a large number of studies have shown that collagen materials have a good effect on wound hemostasis and can be used as an effective trauma hemostasis material. At present, collagen can be obtained by genetic engineering recombination and animal (including human) extraction methods, but the two have significant differences in source, production method, extraction process, safety, etc. Compared with collagen directly extracted by traditional methods, recombinant human collagen does not have problems such as immunity and virus carriage, and has better water solubility and stronger processability. It has the characteristics of single component, controllable preparation process, short production cycle, and 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 earth and is used in various industrial and biomedical fields. There are a large number of hydrogen bonds inside cellulose. These hydrogen bonds give it unique physical and chemical properties, but also lead to its poor solubility and biodegradability. In order to overcome these limitations, cellulose needs to be chemically modified. By chemically modifying the hydroxyl groups in the glucose units of cellulose (including etherification, esterification, acetylation, etc.) and oxidation reactions, cellulose derivatives with different properties are obtained. Carboxymethyl cellulose (CMC) is an anionic, water-soluble cellulose derivative with excellent physical, chemical and mechanical properties, biocompatibility, good hygroscopicity and degradability. In addition, CMC has polyelectrolyte properties and can respond to ionic strength and pH. Therefore, it has good compatibility when mixed with other polymer solutions. This property makes it widely used in the preparation of composite biomaterial scaffolds, hydrogels and drug nanoparticles. At present, biomaterials prepared from CMC (such as hydrogels, dressings, etc.) are widely used in the fields of acute and chronic wound hemostasis and repair and drug delivery. The study found that when CMC dressings were used on diabetic wounds, the hydrogel dressings had good biocompatibility, promoted the proliferation of fibroblasts on the wound surface, and effectively promoted wound repair.

[0006] The preparation of hydrogels using 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 have natural sources, simple production processes, low costs, and are easy to mass produce. In addition, polysaccharide-based hydrogels also have rapid gelation characteristics, so they are considered suitable for the manufacture of various complex tissue engineering scaffolds. However, polysaccharide-based hydrogels have disadvantages such as poor mechanical properties and weak ability to promote cell adhesion, which limits their application in tissue engineering. Type XVII collagen is a transmembrane protein present in the basement membrane of the skin. It also plays a key role in signal communication between the true epidermis and the proliferation and differentiation of epidermal stem cells. Recombinant human type XVII collagen is constructed based on a eukaryotic yeast expression system. It has the core amino acid sequence of human type XVII collagen and can provide adhesion motifs for cells to promote cell adhesion. Carboxymethyl cellulose (CMC) is an anionic, water-soluble cellulose derivative with excellent physical, chemical and mechanical properties, biocompatibility, good hygroscopicity 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 makes it 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 the fields of acute and chronic wound hemostasis and repair and drug delivery.

[0007] At present, hydrogels are often used in the fields of three-dimensional stem cell culture, wound hemostasis and burn skin defect wound treatment. The following are patent documents related to recombinant human collagen hydrogels.

[0008] Patent application with publication number CN114835920A discloses a recombinant collagen-polyglutamate hydrogel and a preparation method thereof, wherein the hydrogel comprises recombinant collagen, polyglutamate and polyethylene glycol, and has the function of promoting cell proliferation and wound repair.

[0009] The patent application with publication number CN119055827A discloses a carboxylated chitosan hydrogel dressing containing recombinant human type I / III collagen microgel and a preparation method thereof, wherein the hydrogel contains recombinant human type I collagen, recombinant human type III collagen, and carboxylated chitosan. The present invention prepares a hydrogel dressing with adjustable ratio according to the dynamic repair process of the skin, which can be used for skin repair.

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

[0011] Patent application with publication number CN117357463A discloses a double-network hemostatic gel of targeted liposomes loaded with ginsenosides and a preparation method thereof. The hydrogel contains polysaccharides and recombinant collagen, can gel in situ, and has the functions of effectively blocking hemostasis and absorbing bleeding.

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

[0013] The purpose of the invention is to provide a method for preparing a multifunctional double-network hydrogel.

[0014] Another object of the present invention is to provide an application of the multifunctional double network hydrogel prepared by the method in preparing adipose mesenchymal stem cell culture reagent.

[0015] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0016] The first aspect of the present invention provides a method for preparing a multifunctional double network hydrogel, comprising the following steps:

[0017] The first step is the preparation of methacryloylated recombinant human type XVII collagen (rhCol-MA)

[0018] Add the chemical modification reagent dropwise 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), the dropping time is controlled to be 5-20 min (preferably 10 min), and a NaOH solution with a concentration of 0.5-2 mol / L (preferably 1 mol / L) is added dropwise until the pH of the above solution is 8-9, and the reaction is stirred 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 for 3-5 days (preferably 3 days) at room temperature; centrifuge, take the supernatant, and freeze-dry to obtain methacrylylated recombinant human type XVII collagen (rhCol-MA);

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

[0020] The method for preparing the recombinant human type XVII collagen solution in the first step comprises the following steps:

[0021] Recombinant human type XVII collagen is added to PBS buffer to a concentration of 0.05-0.2 g / mL (preferably 0.1 g / mL), and stirred at a temperature of 50-70°C (preferably 60°C) until completely dissolved to obtain the recombinant human type XVII collagen solution.

[0022] The dialysis in the first step uses a dialysis bag with a molecular weight cut-off of 12-14 kDa (preferably 14,000).

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

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

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

[0026] Step 2: Preparation of glycidyl methacrylate-modified carboxymethyl cellulose (CMC-GMA)

[0027] Sodium carboxymethyl cellulose is dissolved in PBS buffer (pH=7.4), glycidyl methacrylate is added to the solution at a temperature of 50-70°C (preferably 60°C), the mass ratio of sodium carboxymethyl cellulose to glycidyl methacrylate is 1:1-10 (preferably 1:2.14), hydrochloric acid with a concentration of 0.5-2 mol / L (preferably 1 mol / L) is added until the pH is 4, the solution is incubated at a temperature of 50-70°C (preferably 60°C) for 2-10 h (preferably 6 h), PBS buffer (pH=7.4) is added, the solution is dialyzed in deionized water at room temperature for 2-5 days (preferably 3 days), centrifuged, and the supernatant is lyophilized to obtain glycidyl methacrylate-modified carboxymethyl cellulose;

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

[0029] The dialysis in the second step uses a dialysis bag with a molecular weight cut-off of 12-14 kDa.

[0030] The centrifugation condition in the second step is: centrifugation at 3000 rpm for 15 min.

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

[0032] Step 3: Preparation method of multifunctional double network hydrogel

[0033] The carboxymethyl cellulose hydrogel precursor liquid modified by glycidyl methacrylate and the methacrylylated recombinant human type XVII collagen hydrogel precursor liquid were mixed in a volume ratio of 1:1, and irradiated under blue light (preferably for 1 min) to obtain a multifunctional double network hydrogel.

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

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

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

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

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

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

[0040] The blue light initiator is dissolved in PBS buffer 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, a sterile syringe filter with a diameter of 0.22 µm is used for filtration.

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

[0043] The pH of the PBS buffer in the third step is 7.4.

[0044] The second aspect of the present invention provides a multifunctional double network hydrogel prepared by the method.

[0045] The third aspect of the present invention provides a use of the multifunctional double network hydrogel in preparing adipose mesenchymal stem cell culture reagent.

[0046] The fourth aspect of the present invention provides a use of the multifunctional double network hydrogel in the preparation of a burn wound repair agent.

[0047] The fifth aspect of the present invention provides a use of the multifunctional double network hydrogel in the preparation of a liver hemostatic agent.

[0048] Due to the adoption of the above technical solution, 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 vitro and in vivo degradability and porosity. The hydrogel precursor fluid and stem cells are uniformly mixed and gelled under blue light irradiation to 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 type XVII collagen and carboxymethyl cellulose. By chemical modification, recombinant human type XVII collagen is modified with methacrylic anhydride, and carboxymethyl cellulose is modified with glycidyl methacrylate. After the modified product is prepared into a mixed solution, a free radical chain reaction can occur under the irradiation of a photoinitiator and a specific light source, so that the solution changes from a liquid to a gel. The advantages of this reaction are as follows: by mixing the two materials into a gel to form a double-network structure hydrogel, the problem of poor mechanical properties of a single-component hydrogel is solved; the adhesion motif of recombinant human type XVII collagen is used to improve the weak ability of polysaccharide-based hydrogels to promote cell adhesion, so that the hydrogel has excellent biocompatibility and cell adhesion promotion performance; the prepared double-network structure hydrogel can be used for a variety of purposes, including in vitro three-dimensional cell culture, hemostasis and burn wound repair due to its unique mechanical properties, biocompatibility, cell adhesion and shape adaptability.

[0051] At present, 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 solve these limitations, cell three-dimensional culture materials have made significant research progress in recent years. These materials play an important role in simulating the in vivo cell microenvironment, promoting cell growth and interaction. At present, there are few types of commercial materials for in vitro three-dimensional culture of cells or organoids, and most of them use expensive matrix glue. Matrix glue is mainly derived from the basement membrane components of mouse sarcoma cells (EHS sarcoma), and its components are complex, including polymers, proteins, etc. Its preparation process often involves multiple steps and operations, and there are problems of batch effect and immunogenicity in actual situations. In contrast, the biopolysaccharide carboxymethyl cellulose is naturally derived, the reserves are extremely rich, and the production process and process are very mature; recombinant human type XVII collagen is constructed by synthetic biology based on the eukaryotic yeast expression system, with clear components, high safety and batch production. The hydrogel constructed based on these two materials in the present invention solves some of the shortcomings of matrix glue and has a high prospect for transformation and application.

[0052] Gauze commonly used for hemostasis absorbs blood and aggregates red blood cells / platelets to promote blood coagulation, but due to its lack of tissue adhesion, it cannot be used to treat irregularly shaped incompressible wounds. Sponges with shape recovery function block bleeding sites by quickly absorbing a large amount of blood and expanding in volume to stop bleeding. However, considering that the shape and expansion structure of the sponge do not match the wound, it exhibits 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 clots. After contacting with blood, the platelets in the blood will be adsorbed together with collagen fibers, and agglutination reactions will occur, thereby generating fibrin, promoting plasma clots, and then forming thrombi, achieving the purpose of hemostasis and promoting 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 cotton wool need to be replaced frequently due to their limited ability to absorb exudate, and traditional dressings are easily adhered to tissues, causing obvious pain and discomfort to patients when replaced. Traditional hydrogel dressings can provide a moist environment for the wound, promote wound healing, and reduce the pain of dressing changes, but the mechanical properties are far from those of human skin, and the function is relatively single. The present invention constructs a double network hydrogel based on recombinant human type XVII collagen and carboxymethyl cellulose. The 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 stem cell culture, hemostasis and wound repair, especially in the fields of liver hemostasis and skin repair, which helps to accelerate the recovery process. The present invention not only enriches the research content of biomaterials science, but also opens up new possibilities for the treatment of more complex diseases and enhances society's confidence in medical and technological innovation. The present invention reduces costs by optimizing the preparation process. The hydrogel preparation process is relatively simple, reduces the use of traditional chemical cross-linking agents (for example: glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, etc.), has low toxicity and low production cost.

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

[0056] Under blue light irradiation, the methacrylylated recombinant human type XVII collagen solution and carboxymethyl cellulose mixed liquid containing 0.25% photoinitiator can be quickly solidified within 1 minute, significantly improving surgical efficiency and operational convenience. Experiments have shown that stem cells can adhere, proliferate and maintain 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 and can fit closely to bleeding points and wound surfaces after solidification, 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 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. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0059] Figure 2 These are the H NMR spectra and FTIR spectra of rhCol, rhCol-MA, CMC, and CMC-GMA.

[0060] Figure 3 Schematic diagram of the precursor liquid used for C1C15 hydrogel and C2C15 hydrogel, and the C1C15 hydrogel and C2C15 hydrogel obtained by curing after 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 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 in vitro biocompatibility test results of C1C15 hydrogel and C2C15 hydrogel.

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

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

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

[0068] Fig.11 Schematic diagrams of in vitro clotting time measurement results of multifunctional double-network hydrogel.

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

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

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

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

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

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

[0075] Fig.18Schematic diagram of the multifunctional double network hydrogel prepared in Example 2. DETAILED DESCRIPTION

[0076] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0077] Example 1

[0078] A method for preparing a multifunctional double network hydrogel comprises the following steps:

[0079] The first step is the preparation of methacryloylated recombinant human type XVII collagen (rhCol-MA)

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

[0081] 1 mL of methacrylic anhydride (purchased from Sigma-Aldrich, catalog number: 276685) was added dropwise to the above solution, the mass ratio of recombinant human type XVII collagen to methacrylic anhydride was 10:1, and the dropping time was controlled to be 10 min. A 1 mol / L NaOH solution was added dropwise until the pH of the above solution was adjusted to 9, and then the solution was reacted in a constant temperature water bath at 50°C with magnetic stirring for 3 h. The reaction route of methacrylylation of recombinant human type XVII collagen is as follows: Figure 1 As shown, Figure 1 Schematic diagram of the chemical modification principle of methacrylylated recombinant human type XVII collagen and glycidyl methacrylate-modified carboxymethyl cellulose.

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

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

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

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

[0086] Step 2: Preparation of glycidyl methacrylate-modified carboxymethyl cellulose (CMC-GMA)

[0087] Sodium carboxymethyl cellulose (2 g, molecular weight 100-140 KD) was dissolved in 100 mL PBS buffer (pH = 7.4) and stirred magnetically at 60 ° C until completely dissolved. Glycidyl methacrylate (4.28 g, 4 mL) was added to the above solution. The mass ratio of sodium carboxymethyl cellulose to glycidyl methacrylate was 1:2.14. 1 M hydrochloric acid was added to adjust the pH to 4. The mixture was incubated at 60 ° C for 6 h. 100 mL PBS buffer (pH = 7.4) was added to dilute the reaction solution, and the reaction was terminated by stirring continuously. The above solution was placed in a dialysis bag (12-14 kDa) and dialyzed in deionized water at room temperature for 3 days. The dialyzed solution was poured into a centrifuge tube and centrifuged at 3000 rpm for 15 min. The supernatant was freeze-dried 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 The following are the H NMR spectra and FTIR spectra of rhCol, rhCol-MA, CMC, and CMC-GMA. H NMR analysis shows that after methacrylic anhydride is grafted onto recombinant human type XVII collagen, the amount of lysine residue modification (≈2.8ppm) is reduced, and the signal amount of methacrylic acid vinyl (≈5.3-5.7ppm) is increased. Similarly, compared with sodium carboxymethyl cellulose, methacrylic acid peaks (about 5.6 and 6.2ppm) are observed in CMC-GMA, proving that the double bond is successfully grafted onto the CMC main chain.

[0089] FTIR spectrum results show that at 1118cm -1 The peak of lysine residues of rhCol-MA decreased, and at the same time -1 A new peak appears at (RC=CH 2The out-of-plane bending (deformation) vibration peak of the carbon and hydrogen structure of the GMA structure shows that C=C has been successfully modified on the collagen molecular chain and vinylized collagen has been obtained. The C=C absorption peak of GMA usually appears at 1695-1630cm -1 In the spectrum of CMC-GMA, the absorption peak of C=C was blue-shifted (1698 cm -1 ), proving that GMA grafting was successful.

[0090] Step 3: Preparation method of multifunctional double network hydrogel

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

[0092] 10 mg of the lyophilized sample of glycidyl methacrylate-modified carboxymethyl cellulose was dissolved in 1 ml of a 2.5 mg / mL blue light initiator solution and filtered through a sterile syringe filter (diameter 0.22 µm) to obtain a 1% g / mL carboxymethyl cellulose hydrogel precursor solution.

[0093] 20 mg of the lyophilized sample of glycidyl methacrylate-modified carboxymethyl cellulose was dissolved in 1 ml of a 2.5 mg / mL blue light initiator solution and filtered through a sterile syringe filter (diameter 0.22 µm) to obtain a 2% g / mL carboxymethyl cellulose hydrogel precursor solution.

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

[0095] 400 μl of 1% g / mL glycidyl methacrylate-modified carboxymethyl cellulose hydrogel precursor solution and 400 μl of 15% g / mL methacrylylated recombinant human type XVII collagen hydrogel precursor solution were placed in a glass bottle and mixed thoroughly. They were irradiated with an LED blue light (power 16-20 W, wavelength 405 nm) for 1 min to obtain a multifunctional double-network hydrogel, referred to as C1C15 hydrogel.

[0096] 400 μl of 2% g / mL glycidyl methacrylate-modified carboxymethyl cellulose hydrogel precursor solution and 400 μl of 15% g / mL methacrylylated recombinant human type XVII collagen hydrogel precursor solution were placed in a glass bottle and mixed thoroughly. They were irradiated with an LED blue light (power 16-20 W, wavelength 405 nm) for 1 min to obtain a multifunctional double-network hydrogel, referred to as C2C15 hydrogel.

[0097] 400 μl PBS buffer (pH=7.4) and 400 μl 15% g / mL methacrylylated recombinant human type XVII collagen hydrogel precursor solution were placed in a glass bottle and mixed thoroughly. The mixture was irradiated with an LED blue light (power 16-20 W, wavelength 405 nm) for 1 min to obtain a multifunctional double-network hydrogel, referred to as C0C15 hydrogel.

[0098] 400 μl of 15% g / mL methacrylylated recombinant human type XVII collagen hydrogel precursor solution was placed in a glass bottle and mixed thoroughly. The solution was irradiated with an LED blue light (power 16-20 W, wavelength 405 nm) for 1 min to obtain a recombinant human type XVII collagen hydrogel, referred to as C15 hydrogel.

[0099] Figure 3 Schematic diagram of the precursor liquid used for C1C15 hydrogel and C2C15 hydrogel, and the C1C15 hydrogel and C2C15 hydrogel cured after blue light irradiation. It can be seen from the figure that the hydrogel precursor liquid is transformed from a flowing liquid into a solidified hydrogel after blue light irradiation.

[0100] Compression modulus measurements of multifunctional double network hydrogels

[0101] A cylindrical hydrogel sample with a diameter of 11.2 mm and a height of 5 mm was prepared using a mold (a set of multifunctional anti-adhesive hydrogel molds disclosed in the patent application with publication number CN221717551U), and C0C15 hydrogel, C1C15 hydrogel, C2C15 hydrogel, and C15 hydrogel were obtained respectively. Compression tests were performed using a Zwick Roell Z2.5TH universal material tester (sensor: 2.5 kN). The compression modulus of the sample was calculated based on the slope of the straight line in the 10-15% range of the sample stress-strain curve. Figure 4The stress-strain curves and statistical analysis schematic diagrams of C0C15 hydrogel, C1C15 hydrogel, C2C15 hydrogel, and C15 hydrogel; as can be seen from the figure, by comparing the compression modulus of C0C15 hydrogel and C15 hydrogel, it can be seen that the compression modulus of the hydrogel increases with the increase of the concentration of recombinant human type XVII collagen (the compression modulus of C0C15 hydrogel is 4.36Kpa, and the compression modulus of C15 hydrogel is 26.73Kpa); by comparing the compression moduli of C0C15 hydrogel, C1C15 hydrogel, C2C15 hydrogel, and C15 hydrogel, the compression modulus of the hydrogel also increases significantly after the introduction of CMC, and the higher the concentration of CMC introduced, the more significant the change in compression modulus. The compression modulus of C2C15 hydrogel is 30.96Kpa, which is significantly higher than the compression modulus of C15 hydrogel (26.73Kpa).

[0102] Analysis of swelling properties of multifunctional double network hydrogels

[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 properties of the hydrogels were evaluated by swelling experiments. Figure 5 As shown, Figure 5 This is a schematic diagram of the swelling test results of C1C15 hydrogel and C2C15 hydrogel. The results show that after the introduction of CMC and immersion in sterile PBS solution for 24 hours, C1C15 hydrogel and C2C15 hydrogel both reached swelling equilibrium, and the weight of C1C15 hydrogel and C2C15 hydrogel both reached 10-15 times the initial weight. C1C15 hydrogel and C2C15 hydrogel both have good water absorption and swelling capabilities.

[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 and analyzed by scanning electron microscopy. Figure 6 Schematic diagram of scanning electron microscopy of C1C15 hydrogel and C2C15 hydrogel. It can be seen from the figure that the double network hydrogel after freeze-drying has a loose and porous structure, and there is no significant difference in the internal pore structure of C1C15 hydrogel and C2C15 hydrogel.

[0106] Biocompatibility testing of multifunctional double network hydrogels

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

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

[0109] CCK8 test results Figure 7 As shown, Figure 7 The figure is a schematic diagram of the in vitro biocompatibility test results of C1C15 hydrogel and C2C15 hydrogel; in the figure, control is a complete culture medium. Among them, the left figure is a schematic diagram of the CCK8 test results of C1C15 hydrogel extract and C2C15 hydrogel extract promoting HUVEC proliferation. The middle figure is a schematic diagram of the CCK8 test results of C1C15 hydrogel extract and C2C15 hydrogel extract promoting L929 proliferation, 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 days 1, 3, and 5, C1C15 hydrogel extract and C2C15 hydrogel extract had no significant inhibitory effect on L929 cell proliferation, and had a certain promoting effect on HUVEC cells. That is, C1C15 hydrogel extract and C2C15 hydrogel extract are non-cytotoxic and can promote cell proliferation to a certain extent.

[0110] In order to further evaluate the biocompatibility of the hydrogel, the gelled colloid was used to perform in vitro blood compatibility testing, and 0.1% Triton-X100 was set up as a positive control group, and C1C15 hydrogel and C2C15 hydrogel were set up as experimental groups. The hemolysis rate of each group was calculated. The hemolysis rates of C1C15 hydrogel and C2C15 hydrogel were 1% and 1.01%, respectively, both lower than 5%, which met 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] Multifunctional double network hydrogel for culturing adipose-derived mesenchymal stem cells

[0112] Adipose-derived stem cells are highly plastic and easy to culture, making them an important tool for studying human development, disease mechanisms, and new drug development. By using adipose-derived stem cells for in vitro experiments, we can better understand the pathogenesis of diseases and develop more effective treatments.

[0113] Multifunctional double-network hydrogel facilitates stem cell proliferation:

[0114] P3-P5 human adipose-derived mesenchymal stem cells (ADSCs) with good growth status were selected and digested. After centrifugation, the supernatant was removed and the supernatant was removed. The corresponding precursor solutions of C1C15 hydrogel (400 μl of 1% g / mL carboxymethyl cellulose hydrogel precursor solution modified with glycidyl methacrylate and 400 μl of 15% g / mL methacrylylated recombinant human type XVII collagen hydrogel precursor solution) and C2C15 hydrogel (400 μl of 2% g / mL carboxymethyl cellulose hydrogel precursor solution modified with glycidyl methacrylate) were used for the hydrogel preparation. Cells were resuspended in cellulose hydrogel precursor solution and 400μl methacryloyl recombinant human type XVII collagen hydrogel precursor solution with a concentration of 15% g / mL; 200μl of hydrogel precursor solution mixed with stem cells was aspirated into a 48-well plate and irradiated with LED blue light (power 16-20W, wavelength 405nm) for 1min to obtain a hydrogel scaffold encapsulating adipose mesenchymal stem cells; cell culture medium was added and the medium was changed every 2 days. The survival status of stem cells in the hydrogel scaffold was observed on the 7th, 14th, and 28th days by live-dead staining. At specific time points (1st, 3rd, and 7th days), the old culture medium in the well plate was removed, washed with PBS 3 times, and live-dead staining solution was added. The well plate was incubated at 37°C for 30min, the stain was removed, and the PBS was washed twice before taking pictures under a fluorescence microscope.

[0115] The results are as follows Figure 8 As shown, Figure 8 The figure is a schematic diagram 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 continued to proliferate in the hydrogel as the culture time increased. The cell morphology in the hydrogel was normal, and most of the cells were stained green (live cells), and a small number of cells were stained red (dead cells), indicating that C1C15 hydrogel and C2C15 hydrogel are conducive to the growth and proliferation of stem cells and have good biocompatibility.

[0116] Multifunctional double-network hydrogels for stem cell culture facilitate cell spreading:

[0117] ADSCs were mixed with the corresponding precursor solution of C1C15 hydrogel (400 μl of 1% g / mL carboxymethyl cellulose hydrogel precursor solution modified with glycidyl methacrylate and 400 μl of 15% g / mL methacrylylated recombinant human type XVII collagen hydrogel precursor solution), and irradiated with LED blue light (power 16-20 W, wavelength 405 nm) for 1 min to obtain a hydrogel containing ADSCs, i.e., C1C15 hydrogel containing ADSCs.

[0118] ADSCs were mixed with the corresponding precursor solution of C2C15 hydrogel (400 μl of 2% g / mL carboxymethyl cellulose hydrogel precursor solution modified with glycidyl methacrylate and 400 μl of 15% g / mL methacrylylated recombinant human type XVII collagen hydrogel precursor solution), and irradiated with LED blue light (power 16-20 W, wavelength 405 nm) for 1 min to obtain a hydrogel containing ADSCs, i.e., C2C15 hydrogel containing ADSCs.

[0119] The C1C15 hydrogel containing ADSCs and the C2C15 hydrogel containing ADSCs were cultured in complete culture medium for 5 days, and the complete culture medium was changed every two days. After 5 days, the C1C15 hydrogel containing ADSCs and the C2C15 hydrogel containing ADSCs were freeze-dried, sprayed with a thin gold layer, and then examined with a scanning electron microscope (SEM) to observe the spreading of human adipose-derived mesenchymal stem cells inside the hydrogel. The results are as follows Fig. 9 As shown, Fig. 9 The SEM images (left) and porosity statistics (right) of human adipose mesenchymal stem cells cultured in C1C15 hydrogel and C2C15 hydrogel are shown in the figure. It can be seen from the figure that on the 5th day, human adipose mesenchymal stem cells began to extend their tentacles to the surroundings and spread well. The concentration of carboxymethyl cellulose does not affect the pore structure of the hydrogel, and the double network hydrogel has the advantage of three-dimensional cell culture in vitro. Human adipose mesenchymal stem cells can adhere and spread in the double network hydrogel prepared by the present invention.

[0120] Multifunctional double-network hydrogel culture does not affect stem cell stemness:

[0121] C1C15 hydrogels containing ADSCs and C2C15 hydrogels containing ADSCs were cultured in complete medium for 7 days, and the complete medium was changed every two days. After 7 days, the two groups of hydrogels were dissolved with 0.1% type I collagenase, and the obtained cell suspension was centrifuged at 1200 rpm for 5 min, the supernatant was discarded, and ADSCs were resuspended with PBS to adjust the cell density to 1×106cells / mL. Take 7 EP tubes, mark 1-7 respectively, and fill 100µl of cell suspension into each tube. Flow cytometry antibodies: CD31, CD45, CD73, CD90, CD105, CD235a were added, mixed and incubated on ice in the dark for 30 min. Add 500µl 4℃ pre-cooled PBS to gently resuspend the cells, centrifuge at 400 g, 4℃ for 5 min and discard the supernatant, and repeat this step 2-3 times. Finally, gently resuspend the cell pellet in 200µl pre-cooled PBS and then test it on the instrument. Fig.10 As shown, Fig.10 This is a schematic diagram of the flow cytometry results of adipose stem cells after 7 days of 2D culture in culture dishes and three-dimensional culture in hydrogels. As can be seen from the figure, the flow cytometry results of six surface antibodies CD90, CD73, CD105, CD235a, CD31, and CD45 indicate that compared with 2D culture in culture dishes, 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 hydrogels are within their respective ranges, that is, these cells still have stemness.

[0122] Multifunctional double-network hydrogel promotes liver hemostasis

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

[0124] The whole blood clotting time (CT) and blood clotting index (BCI) were used to evaluate the in vitro procoagulant properties of the double network hydrogel. Citric acid anticoagulated blood was mixed with 0.2 M calcium chloride (Vblood: VCaCl2 = 9:1). The recalcified blood was added to a 96-well plate, 50µl per well. Then, 50µl of each group of samples (n=3) were added to each well, and the control group was not treated. Every 1 minute, the plate was gently rinsed with PBS to completely eliminate the uncoagulated blood. The moment when a uniform and stable blood clot is formed in the well is the coagulation time. The results are shown in Figure 2. Fig.11 As shown, Fig.11It is a schematic diagram of the measurement results of the in vitro coagulation time 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 can form a stable blood clot in 2 minutes, while the control group (control in the figure) starts to coagulate in 7 minutes. The above shows that the double-network hydrogel prepared by the present invention has the effect of promoting coagulation in vitro.

[0125] 100µl of recalcified whole blood was dropped into 300µl of each group of samples (n=3), incubated at 37℃ for 5min, 10ml of deionized water was added to lyse the uncoagulated blood cells, incubated at 37℃ for 10min, the serum coagulation was observed and photographed. 100µl of supernatant was taken from each tube into a 96-well plate, and its OD value at 560nm was measured in an ELISA reader. The control group was deionized water, and BCI was calculated according to the following formula: BCI= (ODm / ODp) × 100%. The results are shown in Fig.12 As shown, Fig.12 This is a schematic diagram of the statistical results of the in vitro coagulation index (BCI) of the multifunctional double network hydrogel. As can be seen from the figure, the BCI of the C1C15 hydrogel group and the C2C15 hydrogel group were 40.5 and 25.8, respectively, which were not significantly different from the coagulation index of the commercial hemostatic sponge group (BCI=41.7), and were far lower than the control group, showing good coagulation effect.

[0126] According to the literature (Hongjuan Weng, Weibin Jia, Min Li, et al. New injectable chitosan-hyaluronic acid based hydrogels for hemostasis and woundhealing. Carbohydrate Polymers, 294, (2022) 119767.), a SD rat liver hemostasis model was constructed (after the rat was anesthetized, the abdominal hair was removed, the liver was fully exposed by laparotomy, and a bleeding hole was created in the liver using surgical scissors). Each group was treated with hydrogel (n=3), and the control group was PBS and commercial hemostatic sponge. 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 follows Fig.13 As shown, Fig.13 This is a schematic diagram of the general picture of liver hemostasis treatment in different groups at different time points. It can be seen from the figure that the hemostatic effects of the commercial hemostatic sponge group, C1C15 hydrogel group, and C2C15 hydrogel group were significantly better than those of the control group.

[0127] Blood loss statistics ( Fig.14 Middle left) and hemostasis time statistics ( Fig.14 The result is as follows Fig.14 As shown, Fig.14 The figure is a schematic diagram of the statistical results of blood loss and hemostasis time in the treatment of liver hemostasis in each group. As can be seen from the figure, the average bleeding volume of the control group was 1128.33 mg, the average bleeding volume of the commercial hemostatic sponge group was 390 mg, the average bleeding volume of the C1C15 hydrogel group was 347 mg, and the average bleeding volume of the C2C15 hydrogel group was 265.67 mg. The bleeding volume of the C1C15 hydrogel group and the C2C15 hydrogel group was significantly lower than that of the control group (P < 0.05), and there was no significant difference between the bleeding volume of the C1C15 hydrogel group and the C2C15 hydrogel group and the commercial hemostatic sponge group. The hemostasis time of different groups was analyzed, and the results showed that the average hemostasis time of the control group was 201.3S, the average hemostasis time of the commercial hemostatic sponge group was 124.7S, the average hemostasis time of the C1C15 hydrogel group was 87S, and the average hemostasis time of the C2C15 hydrogel group was 100.1S. The hemostatic effects of the commercial hemostatic sponge group, C1C15 hydrogel group, and C2C15 hydrogel group were significantly better than those of the control group (P < 0.001), and the hemostatic time of the C1C15 hydrogel group and 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 burn wound repair

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

[0130] According to the literature (Ning Yu, Yunpeng Li, Yansheng Wang, et al. Healing effect ofcarboxymethyl chitosan plantamajoside hydrogel on burn wound skin. Burns 48(2022) 902-914.), a nude mouse deep II degree burn wound model was constructed (after the mouse was anesthetized, the back hair was removed, and a copper rod with a diameter of 6 mm soaked in 100°C boiling water was used to contact the back skin, and the model was successfully established). The scab was removed 48 hours after the burn, and each group was given hydrogel treatment (n=3) at the same time. The control group used PBS. The wound was observed and photographed every 3 days to record the wound healing. The wound was sampled on the 15th day, and the healing quality was evaluated by HE and other pathological techniques. The results are as follows Fig.15 The wound healing picture is shown in the following figure. Fig.15 The schematic diagram of deep burn wound healing in mice in each group is shown in Figure 1. As can be seen from the figure, compared with the control group, the wound healing speed of the C1C15 hydrogel group and the C2C15 hydrogel group was faster and the terminal healing rate was higher. Fig.16The figure is a schematic diagram of the statistical analysis of the phase 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, the wound closure rate was significantly accelerated starting from the 6th day after treatment with C1C15 hydrogel and C2C15 hydrogel. The above results show 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] Fig.17 The schematic diagram of the preparation and application of the multifunctional double network hydrogel is shown in the figure. The present invention obtains methacrylylated recombinant human type XVII collagen hydrogel precursor liquid and methacrylate-modified carboxymethyl cellulose hydrogel precursor liquid respectively by chemically 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 be changed from a liquid state to a gel state. The obtained light-cured double network hydrogel has good application prospects in three-dimensional culture of adipose stem cells, liver hemostasis and wound repair.

[0132] Example 2

[0133] The mass ratio of recombinant human type XVII collagen to methacrylic anhydride in the first step of Example 1 was replaced from 10:1 to 5:1, while other factors remained unchanged, to obtain methacryloylated recombinant human type XVII collagen.

[0134] According to the second step in Example 1, glycidyl methacrylate-modified carboxymethyl cellulose (CMC-GMA) was prepared.

[0135] Step 3: Preparation method of multifunctional double network hydrogel

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

[0137] 10 mg of the lyophilized sample of glycidyl methacrylate-modified carboxymethyl cellulose was dissolved in 1 ml of a 2.5 mg / mL blue light initiator solution and filtered through a sterile syringe filter (diameter 0.22 µm) to obtain a 1% g / mL carboxymethyl cellulose hydrogel precursor solution.

[0138] 20 mg of the lyophilized sample of glycidyl methacrylate-modified carboxymethyl cellulose was dissolved in 1 ml of a 2.5 mg / mL blue light initiator solution and filtered through a sterile syringe filter (diameter 0.22 µm) to obtain a 2% g / mL carboxymethyl cellulose hydrogel precursor solution.

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

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

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

[0142] Place 400 μl of 1% g / mL glycidyl methacrylate-modified carboxymethyl cellulose hydrogel precursor solution and 400 μl of 15% g / mL methacrylylated recombinant human type XVII collagen hydrogel precursor solution in a glass bottle and mix thoroughly. 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 2% g / mL glycidyl methacrylate-modified carboxymethyl cellulose hydrogel precursor solution and 400 μl of 15% g / mL methacrylylated recombinant human type XVII collagen hydrogel precursor solution in a glass bottle and mix thoroughly. 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 1% g / mL glycidyl methacrylate-modified carboxymethyl cellulose hydrogel precursor solution and 400 μl of 10% g / mL methacrylylated recombinant human type XVII collagen hydrogel precursor solution in a glass bottle and mix thoroughly. Irradiate with an LED blue light (power 16-20 W, wavelength 405 nm) for 1 min to obtain a multifunctional double-network hydrogel.

[0145] Place 400 μl of 2% g / mL glycidyl methacrylate-modified carboxymethyl cellulose hydrogel precursor solution and 400 μl of 10% g / mL methacrylylated recombinant human type XVII collagen hydrogel precursor solution in a glass bottle and mix thoroughly. Irradiate with an LED blue light (power 16-20 W, wavelength 405 nm) for 1 min to obtain a multifunctional double-network hydrogel.

[0146] Place 400 μl of 1% g / mL glycidyl methacrylate-modified carboxymethyl cellulose hydrogel precursor solution and 400 μl of 5% g / mL methacrylylated recombinant human type XVII collagen hydrogel precursor solution in a glass bottle and mix thoroughly. Irradiate with an LED blue light (power 16-20 W, wavelength 405 nm) for 1 min to obtain a multifunctional double-network hydrogel.

[0147] Place 400 μl of 2% g / mL glycidyl methacrylate-modified carboxymethyl cellulose hydrogel precursor solution and 400 μl of 5% g / mL methacrylylated recombinant human type XVII collagen hydrogel precursor solution in a glass bottle and mix thoroughly. Irradiate with an LED blue light (power 16-20 W, wavelength 405 nm) for 1 min to obtain a multifunctional double-network hydrogel.

[0148] Fig.18 Schematic diagram of the multifunctional double network hydrogel prepared in Example 2. The multifunctional double network hydrogel is formed by mixing the methacrylylated recombinant human type XVII collagen hydrogel precursor solution with a concentration of 15%, 10%, and 5% g / mL in the horizontal direction and the carboxymethyl cellulose hydrogel precursor solution modified with glycidyl methacrylate with a concentration of 1% and 2% g / mL in the vertical direction, and then irradiating with blue light. It can be seen from the figure that a stable multifunctional double network hydrogel can be formed.

[0149] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A method for preparing a multifunctional double network hydrogel, characterized in that: The following steps are involved: The first step is to add the chemical modification reagent 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 minutes, and a NaOH solution with a concentration of 0.5-2 mol / L is added dropwise until the pH of the above solution is 8-9, and the reaction is stirred at a temperature of 50-70°C for 0.5-5 hours; PBS buffer is added to the above solution, and dialyzed with deionized water at room temperature for 3-5 days; centrifugation is performed, the supernatant is taken, and freeze-dried to obtain methacrylylated recombinant human type XVII collagen; In the first step, the chemical modification reagent is selected from methacrylic anhydride; The second step is to dissolve sodium carboxymethyl cellulose in PBS buffer, add glycidyl methacrylate to the solution at a temperature of 50-70°C, the mass ratio of sodium carboxymethyl cellulose to glycidyl methacrylate is 1:1-10, add 0.5-2 mol / L hydrochloric acid to adjust the pH to 4, incubate at a temperature of 50-70°C for 2-10 h, add PBS buffer, dialyze in deionized water at room temperature for 2-5 days, centrifuge, and freeze-dry the supernatant to obtain glycidyl methacrylate-modified carboxymethyl cellulose; In the third step, the carboxymethyl cellulose hydrogel precursor liquid modified with glycidyl methacrylate and the methacrylylated recombinant human type XVII collagen hydrogel precursor liquid were mixed in a volume ratio of 1:1, and irradiated under blue light to obtain a multifunctional double-network hydrogel.

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

3. The method for preparing the multifunctional double network hydrogel according to claim 1, characterized in that: Preparation of the glycidyl methacrylate-modified carboxymethyl cellulose hydrogel precursor solution: The carboxymethyl cellulose modified with glycidyl methacrylate was dissolved in a blue light initiator solution and filtered to obtain a carboxymethyl cellulose hydrogel precursor solution with a concentration of 0.5-3% g / mL.

4. The method for preparing the multifunctional double network hydrogel according to claim 1, characterized in that: Preparation of the methacrylylated recombinant human type XVII collagen hydrogel precursor solution: The methacryloyl recombinant human type XVII collagen was dissolved in a blue light initiator solution and filtered to obtain a methacryloyl recombinant human type XVII collagen hydrogel precursor solution with a concentration of 5-20% g / mL.

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

6. The method for preparing the multifunctional double network hydrogel according to claim 3 or 4, characterized in that: The preparation method of the blue light initiator solution comprises the following steps: The blue light initiator was 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 16-20W and a wavelength of 405nm.

7. A multifunctional double network hydrogel prepared by the method according to any one of claims 1 to 6.

8. Use of the multifunctional double network hydrogel according to claim 7 in preparing adipose mesenchymal stem cell culture reagent.

9. Use of the multifunctional double network hydrogel according to claim 7 in preparing a burn wound repair agent.

10. Use of the multifunctional double network hydrogel according to claim 7 in preparing a liver hemostatic agent.

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