Collagen hydrogel with multi-level fiber network as well as preparation and application of collagen hydrogel

Abstract

The invention provides a multi-level collagen fiber network hydrogel. The large-scale collagenous fiber is mixed with collagen modified by olefinic bonds, and the injectable and 3D printing capacity is achieved; after heating assembly and photo-crosslinking, the collagen hydrogel with a multi-level fiber network structure can be obtained. Compared with the traditional collagen hydrogel, the hydrogel disclosed by the invention has good mechanical properties and degradation resistance, and also has extremely strong biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to the preparation of hydrogels with a multi-level collagen fiber network structure, which can be used in fields such as injectable hydrogels and bio-3D printing. Technical Background

[0002] Collagen hydrogels possess excellent biocompatibility and play an indispensable role in tissue engineering. However, this excellent biocompatibility depends on low concentrations; the solid content of conventional collagen hydrogels is only 3 mg / mL, which inevitably leads to their fragile mechanical properties and rapid degradation rate in vivo, limiting their widespread application. Unlike two-dimensional planar culture, cells in three-dimensional culture are constrained by the matrix, affecting their extension, migration, and functional formation. Among these factors, matrix pore size and degradability are important influences on cell behavior. Increasing collagen concentration leads to smaller matrix pore sizes, causing collagen to lose its biological activity. In the human body, collagen exists in the form of a multi-layered, assembled network. Large collagen fibers bear mechanical strength and have large gaps for cell passage, while fine collagen fibers participate in various cellular interactions. However, the orderly assembly structure of large fibers is difficult to reproduce in vitro, leading to the current dilemma of insufficient mechanical strength and rapid degradation in collagen hydrogels. Summary of the Invention:

[0003] This invention relates to several key inventive points:

[0004] I. Preparation of large-scale collagen fibers with olefinic bond reaction sites:

[0005] 1) Using the neutrally soluble olefin-modified collagen with self-assembly capability previously disclosed by the inventor as raw material, this material uses various natural type I collagen or recombinant type I collagen as raw material. The surface of the collagen molecules is modified with olefin bonds and has the ability to self-assemble into collagen fibers.

[0006] 2) Preparation of large-scale collagen fibers with olefinic reaction sites: Olefin-modified collagen was dissolved at a concentration of 10 mg / mL in D-PBS (Dubor's phosphate buffer) solution at 4°C. The solution was further diluted with D-PBS at 4°C to a final concentration of 0.1-1 mg / mL. The diluted collagen solution was placed on a shaker at 20-37°C and shaken at 0-1000 rpm for 12-72 hours. After the procedure, dispersed collagen fibers with large scale were obtained.

[0007] II. Preparation of High-Concentration, High-Modulus Multilevel Collagen Fiber Hydrogels

[0008] 1) By centrifuging dispersed large-scale collagen fibers and mixing them with a collagen solution modified with olefin bonds of a certain concentration, mixtures of fibers and solutions with different volume ratios can be obtained.

[0009] 2) The above mixture has high viscosity and shear thinning properties, which are suitable for the rheological properties required for 3D printing and in vivo injection.

[0010] 3) A photoinitiator and a multi-thiol crosslinking agent are added to the above mixture to initiate a gel reaction under light after self-assembly. The photoinitiator includes ultraviolet and visible light initiators, such as lithium phenyl-2,4,6-trimethylbenzoylphosphonite (LAP), 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure 2959), ruthenium pyridine complex [Ru(II)(bpy)3]2+ / sodium sulfate (SPS), eosin Y, riboflavin, etc. The multi-thiol crosslinking agent is a small molecule or polymer with two or more free thiol groups.

[0011] 4) When the above mixture is placed at 25-37 degrees Celsius, the collagen solution will undergo self-assembly to form small-scale fibers, which interweave with the large-scale fibers to form a multi-level collagen fiber network.

[0012] 5) Irradiating the gel with a light source corresponding to the photoinitiator can further solidify the gel. Its structural characteristics include: a hierarchical arrangement of collagen fibers at different scales. Its mechanical characteristics include: due to the presence of olefinic bond reaction sites in collagen fibers of different scales, the gel modulus significantly increases after photocuring. Its biological characteristics include: the biomimetic multi-level network structure has large porosity, supporting cell extension, migration, and the formation of biological functions.

[0013] 6) The multi-level collagen network formed by the above self-assembly can also react directly with compounds with multiple tetrazine groups without the need for photoinitiators.

[0014] 7) Before the collagen solution is assembled, the fluid mixture can also be directly photocured or cured by mixing with tetrazine compounds.

[0015] III. Applications of Multi-level Collagen Fiber Hydrogels

[0016] 1) Multi-level collagen fiber hydrogels can immobilize functional molecules, such as proteins, peptides, small molecule drugs, nucleic acids, carbohydrates, or polysaccharide molecules; the functional molecules have one or more reactive groups, such as free thiol groups and / or olefin groups. Functional molecules without specific reactive groups can also be added to the above hydrogels, and the functional molecules encapsulated in the multi-level network structure have a slow diffusion rate.

[0017] 2) Applications of multi-layered collagen fiber hydrogels include, but are not limited to, injectable hydrogels, bioprinting inks, tissue engineering, wound dressings, drug delivery carriers, cell transplantation and therapeutic carriers, and three-dimensional cell culture substrates. The gel can contain various components such as cells, cellular...

[0018] Spheres, growth factors, drugs, nanoparticles, etc.

[0019] Design mechanism of the invention:

[0020] First, this invention proposes a method for preparing large-scale collagen fibers. By reducing the collagen concentration, the collagen fibers do not form a continuous network during assembly; instead, they continuously expand in scale, with some fibers as the core. The structure of the large-scale fibers can be controlled by temperature and shaking in a shaker. Second, the collagen used in this invention is an olefin-modified, assemblable collagen. After the multi-scale fiber network is formed, the network can be further strengthened through chemical cross-linking reactions without affecting the structure of the collagen fibers. Finally, the doping of large-scale collagen fibers can increase the viscosity of the pre-gel and provide shear-thinning ability, giving the material injectability, making it suitable for in-situ injection and 3D printing. This biomimetic network has a large pore size and can load a variety of active molecules, showing promising application prospects.

[0021] The advantages and beneficial effects of this invention are as follows: 1. By preparing a multi-level collagen network, the limitations of collagen concentration in cell culture are overcome, resulting in good bioactivity and mechanical properties at higher collagen concentrations. This solves the problems of poor mechanical strength and excessively rapid degradation of traditional collagen hydrogels.

[0022] 2. Large-scale collagen fibers with olefin bonds and small-scale collagen fibers with olefin bonds can be further chemically cross-linked, further enhancing mechanical strength and resistance to in vivo degradation.

[0023] 3. The high viscosity and shear-thinning properties brought about by large-scale collagen doping are beneficial for in vivo injection and 3D.

[0024] Printing process.

[0025] 4. Compared to cases where other fiber components are incorporated into hydrogels, large-scale collagen fibers have more suitable cell adhesion sites, lower immunogenicity, safer degradation products, and mechanical properties that are more compatible with in vivo tissues.

[0026] 5. The multi-level collagen fiber gel disclosed in this invention can support good cell migration and extension at a high concentration of 15 mg / mL and a high modulus. Compared with traditional collagen formulations, the concentration and modulus are greatly improved, overcoming the limitation of poor mechanical properties of collagen gels used for cell culture.

[0027] Compared to traditional collagen hydrogels, this hydrogel has excellent mechanical properties and resistance to degradation, and also possesses extremely strong biocompatibility. Attached Figure Description

[0028] Figure 1 a: Fluorescent image of norbornene collagen fibers prepared under specific conditions; b: Statistical analysis of the length and diameter of norbornene collagen fibers prepared under specific conditions.

[0029] Figure 2 a: Fluorescent image of norbornene collagen fibers prepared under specific conditions; b: Statistical analysis of the length and diameter of norbornene collagen fibers prepared under specific conditions.

[0030] Figure 3 Fluorescent images of multi-level collagen fiber networks.

[0031] Figure 4 Shear modulus of norbornene collagen multilevel network gel and control group.

[0032] Figure 5 Degradation time of norbornene collagen multilevel network gel and control group.

[0033] Figure 6 Cell viability in norbornene collagen multilevel network gel and control group.

[0034] Figure 7 The migration and self-organization ability of cells in norbornene collagen multilevel network gel and control group.

[0035] Figure 8 Shear-thinning properties of norbornene collagen multilevel network pregel and control group.

[0036] Figure 9 Norbornene collagen multi-level network pregel extrusion properties.

[0037] Figure 10 a: Embedded printing performance of norbornene collagen multi-level network pregel; b: Comparison of 3D printing performance of norbornene collagen multi-level network pregel and control group in air; c: HE staining images of printed structures after subcutaneous implantation. Detailed Implementation

[0038] To further understand the present invention, the following description is based on examples, but these examples are only for illustrative purposes.

[0039] The features and advantages of the present invention will be further described, but not limited to the claims of the present invention.

[0040] Example 1:

[0041] This embodiment describes the preparation of large-scale collagen fibers, as detailed below:

[0042] 1. Preparation of self-assembling norbornene collagen: Using enzymatically extracted type I collagen (from bovine Achilles tendon) as raw material, type I collagen was added to 0.1 mol / L acetic acid solution and fully dissolved to obtain 300 mL of an acid-soluble collagen solution with a concentration of 3 mg / mL. The pH of the acid-soluble collagen solution was adjusted to 9 using 3 mol / L sodium hydroxide solution and stored at 4℃ for later use. 1.8 g of norbornene anhydride was dissolved in 15 mL of acetone. The anhydride solution was added dropwise to the collagen solution with a pH of 9, while sodium hydroxide solution was added dropwise to maintain the pH. The reaction temperature was 4℃. After the pH no longer changed, stirring was continued for 1 hour. The resulting reaction product was dialyzed with distilled water, with the water changed every other day, until the collagen in the dialysis bag formed a gel. After lyophilization, it was stored at low temperature.

[0043] 2. The self-assembling-capable norbornene collagen was dissolved in D-PBS (Dulbecco's Phosphate-Buffered Saline) at 4°C to a final collagen concentration of 6 mg / mL. After the lyophilized collagen was completely dissolved, it was diluted again with D-PBS buffer to final collagen concentrations of 0.3 mg / mL and 0.6 mg / mL, respectively.

[0044] 3. Place the above solutions on a shaker at 50 rpm and centrifuge at room temperature for 24 hours. Collect the precipitates, which are two types of large-scale collagen fibers.

[0045] 4. Fluorescent Labeling: CY5-succinimide ester was dissolved in dimethyl sulfoxide at a concentration of 5 mg / mL; the two types of collagen fibers were separately resuspended in 20 mL of 0.1 M sodium bicarbonate buffer to obtain collagen fiber suspensions with a concentration of 3 mg / mL; 20 μL of CY5-succinimide ester solution was added to the collagen fiber suspension, and after reacting at room temperature for 1 hour, the suspension was washed three times by centrifugation with PBS buffer to remove excess dye, obtaining two fluorescent collagen fibers. (The procedures and conditions for fluorescent labeling in subsequent examples and comparative examples are the same as those for this fluorescent labeling method).

[0046] 5. Observe two types of fluorescently labeled collagen fibers using a confocal microscope. Figure 1 a). Measurements and statistics show that the average fiber length formed by a 0.6 mg / mL collagen solution is approximately 117.8 μm (distributed as 109-142 μm), and the average fiber diameter is approximately 14.3 μm (distributed as 9-16 μm). The average fiber length formed by a 0.3 mg / mL collagen solution is approximately 186.3 μm (distributed as 116-233 μm), and the average fiber diameter is approximately 15.3 μm (distributed as 10-22 μm). Figure 1 b).

[0047] Example 2:

[0048] This embodiment describes the preparation of large-scale collagen fibers, as detailed below:

[0049] 1. The self-assembling collagen prepared in step 1 of Example 1 was dissolved in D-PBS buffer at 4°C to a final collagen concentration of 6 mg / mL. After the lyophilized collagen was completely dissolved, it was diluted again with D-PBS buffer to final collagen concentrations of 0.3 mg / mL and 0.6 mg / mL, respectively.

[0050] 2. Place the above solution on a shaker at 50 rpm and centrifuge at 37°C for 24 hours. Collect the precipitate, which consists of two types of large-scale collagen fibers.

[0051] 3. Label the two types of collagen fibers with CY5-succinimide ester respectively. The labeling process and conditions are the same as those in step 4 of Example 1, the fluorescent labeling method.

[0052] 4. Observe two types of fluorescently labeled collagen fibers using a confocal microscope. Figure 2 a). Measurements and statistics show that the average fiber length formed by a 0.6 mg / mL collagen solution is approximately 42.6 μm (distributed as 26-53 μm), and the average diameter is approximately 12.0 μm (distributed as 6-16 μm). The average fiber length formed by a 0.3 mg / mL collagen solution is approximately 69.0 μm (distributed as 49-86 μm), and the average diameter is approximately 11.2 μm (distributed as 7-16 μm). Figure 1 b).

[0053] Example 3:

[0054] This embodiment describes the construction of a multi-level collagen network gel, as detailed below:

[0055] 1. Fluorescent Labeling: Rhodamine-succinimide ester was dissolved in dimethyl sulfoxide at a concentration of 5 mg / mL. 10 mL of a 6 mg / mL norbornene collagen solution dissolved in D-PBS was mixed with 10 mL of 0.1 M sodium bicarbonate buffer to obtain a collagen solution with a concentration of 3 mg / mL. All these steps were performed at 4°C. 20 μL of the rhodamine-succinimide ester solution was added to the collagen solution, and after reacting at room temperature for 1 hour, the solution was dialyzed against distilled water at 4°C for 4 days, changing the water daily. After lyophilization, rhodamine-succinimide-labeled norbornene collagen was obtained. The rhodamine-succinimide-labeled norbornene collagen was dissolved in D-PBS at 4°C to obtain a rhodamine-succinimide ester-labeled norbornene collagen solution with a final collagen concentration of 6 mg / mL.

[0056] 2. The large-scale fluorescent collagen fibers prepared with CY5-succinimide ester labeled at a collagen concentration of 0.3 mg / mL in step 4 of Example 1 were adjusted to a concentration of 30 mg / mL by centrifugation or by adding D-PBS buffer.

[0057] 3. Mix the rhodamine-succinimide-labeled norbornene collagen solution obtained in step 1 with the large-scale CY5-succinimide-labeled collagen fiber solution obtained in step 2 in equal volumes. In this mixture, the final concentration of norbornene collagen is diluted to 3 mg / mL, the concentration of large-scale fluorescent collagen fibers is diluted to 15 mg / mL, and the total concentration is 18 mg / mL. Take a small amount and add it to a glass slide, cover with a coverslip, and incubate at 37°C for 10 minutes to form a multi-layered collagen fiber network.

[0058] 4. Using a confocal microscope to observe the multi-level collagen fiber network, it can be seen that collagen fibers of different scales form an interwoven structure. Among them, the red fine fibers are structures directly assembled from the collagen solution, and the yellow-orange fibers are pre-prepared large-scale collagen fibers. Figure 2 a) Based on image measurements and statistics, the diameter of the coarse fibers is approximately 16.6 μm (distributed as 12-18 μm), and the length is approximately 98.6 μm (distributed as 74-122 μm). The diameter of the fine fibers is 0.26 μm (distributed as 0.2-0.3 μm), and the length is approximately 48 μm (distributed as 24-61 μm).

[0059] Example 4:

[0060] This embodiment compares the effect of norbornene collagen fibers on the gel modulus of multilayer networks compared to natural collagen fibers, as detailed below:

[0061] 1. a. Dissolve the norbornene collagen in step 1 of Example 1 using D-PBS to obtain a norbornene collagen solution with a final collagen concentration of 6 mg / mL.

[0062] Using the method in steps 2-3 of Example 1, large-scale collagen fibers were prepared at a collagen concentration of 0.3 mg / mL. The concentration was then adjusted to 30 mg / mL by centrifugation or by adding D-PBS buffer.

[0063] b. The norbornene collagen solution obtained above was mixed with an equal volume of the large-scale collagen fibers obtained above. In this mixture, the final concentration of norbornene collagen was diluted to 3 mg / mL, the concentration of large-scale fluorescent collagen fibers was diluted to 15 mg / mL, and the total concentration was 18 mg / mL. A LAP photoinitiator with a final concentration of 1 mg / mL and a mercapto-terminated polyethylene glycol with a final concentration of 10 mM were added to the above mixture. All the above steps were performed at 4°C to prevent gel assembly. This was used as the experimental group.

[0064] 2. The process and conditions are the same as described in step 1, except that the large-scale fibers modified with norbornene in step 1a. are replaced with natural collagen fibers (the process and conditions are the same as steps 2-3 of Example 1, except that the norbornene collagen in steps 2-3 of Example 1 is replaced with unmodified type I collagen extracted from bovine Achilles tendon at the same concentration (bovine Achilles tendon extract), which can obtain natural collagen fibers), and other components remain unchanged, serving as control group 1.

[0065] 3. The norbornene collagen from step 1 of Example 1 was dissolved in D-PBS to obtain a norbornene collagen solution with a final collagen concentration of 3 mg / mL. LAP photoinitiator with a final concentration of 1 mg / mL and mercapto-terminated polyethylene glycol with a final concentration of 10 mM were added to the 3 mg / mL collagen solution as control group 2.

[0066] 4. The shear modulus of the three pregels (experimental group, control group 1, and control group 2) was measured using a rheometer at 37°C for 400 seconds, followed by measurement with 10 mW / cm². 2 365nm ultraviolet light for 60 seconds. (Example) Figure 4 As shown, compared to control groups 1 and 2, the multi-level collagen gel formed by large-scale collagen fibers with norbornene reactive joints and collagen solution in the experimental group has a higher shear modulus.

[0067] Example 5:

[0068] This embodiment tests the degradation resistance of a multi-level collagen fiber network, specifically as follows: 1. A multi-level network pregel was prepared using the method in step 1 of Example 4. This served as the experimental group;

[0069] 2. A pregel containing natural collagen fibers was prepared using the method in step 2 of Example 4 as a control group 1.

[0070] 3. The norbornene collagen from step 1 of Example 1 was dissolved in D-PBS to obtain a norbornene collagen solution with a final collagen concentration of 3 mg / mL. LAP photoinitiator with a final concentration of 1 mg / mL and mercapto-terminated polyethylene glycol with a final concentration of 10 mM were added to the 3 mg / mL collagen solution as control group 2.

[0071] 4. Place equal weights of the three groups of samples (experimental group, control group 1, and control group 2) in a gel at 37 degrees Celsius for 10 minutes, then use 10mW / cm 2 Irradiate with 365nm ultraviolet light for 60 seconds.

[0072] 5. Add the three groups of samples obtained in step 4 (experimental group, control group 1, and control group 2) to 50 times the sample volume of 10 U / mL type I collagenase D-PBS solution, incubate at 37 degrees Celsius, and record the complete degradation time. Figure 5 As shown, compared with control group 1 and control group 2, the multi-level collagen gel formed by large-scale collagen fibers with norbornene reactive joints and collagen solution in the experimental group can better resist degradation.

[0073] Example 6:

[0074] This embodiment primarily tests the biocompatibility of multi-level collagen fiber gels, including cell viability and the ability of cells to extend and migrate.

[0075] 1. A multi-level network pregel was prepared using the method in step 1 of Example 4. This was used as the experimental group.

[0076] 2. The norbornene collagen from step 1 of Example 1 was dissolved in D-PBS to obtain a norbornene collagen solution with a final collagen concentration of 18 mg / mL. A LAP photoinitiator with a final concentration of 1 mg / mL and a mercapto-terminated polyethylene glycol with a final concentration of 10 mM were added to the 18 mg / mL collagen solution as a control group, defined as a single-network gel.

[0077] 3. Add a final concentration of 6×10 to the pre-gels (experimental group and control group). 6 Human umbilical vein endothelial cells per mL were gelled at 37°C for 10 minutes, followed by 10 mW / cm² gelation. 2 Irradiate with 365nm ultraviolet light for 60 seconds and culture using endothelial cell culture medium.

[0078] 4. After culturing for 5 days, cell viability was assessed using a cell viability staining reagent (Thermo Fisher Scientific, L7010) according to the manufacturer's instructions. The results are as follows: Figure 6 As shown, the cell viability rate in multi-level network gels is as high as 95%, while the cell viability rate in single-network gels is 80%.

[0079] 5. After culturing for 5 days, cells were stained using rhodamine-labeled phalloidin (Yisheng Biotechnology, 40734ES75) and DAPI (Yisheng Biotechnology, 708939ES03) according to the manufacturer's instructions. The results are as follows: Figure 7 As shown, the multi-layered network gel demonstrates excellent biocompatibility as endothelial cells migrate and form capillaries. In contrast, cells in a single-network gel are dispersed and difficult to migrate.

[0080] Example 7:

[0081] This embodiment mainly tests the extrusion rheological properties of multi-level collagen fiber gels, specifically as follows: 1. A multi-level network pregel was prepared using the method in step 1 of Example 4. This served as the experimental group;

[0082] 2. The norbornene collagen from step 1 of Example 1 was dissolved in D-PBS to obtain a norbornene collagen solution with a final collagen concentration of 3 mg / mL. A LAP photoinitiator with a final concentration of 1 mg / mL and a mercapto-terminated polyethylene glycol with a final concentration of 10 mM were added to the 3 mg / mL collagen solution as a control group, defined as a single-network pregel.

[0083] 3. The shear thinning properties of the pregel were tested at 4°C using a rheometer (HAAKE, MARS 60) (using a 20mm flat rotor with a 0.5mm gap to the base plate, and a shear rate set to 0-250 1 / s). Figure 8 As shown, the mixture with added fibers in the experimental group had a high initial viscosity and significant shear thinning properties, making it suitable for 3D printing and injectable hydrogels.

[0084] 4. At 4℃, the pre-gel in the experimental group was filled into the needle, and its extrusion performance was as follows: Figure 9 As shown.

[0085] Example 8:

[0086] This embodiment primarily tests the printability of multi-level collagen fiber gel and its ability to repair tissue damage in vivo, as detailed below:

[0087] 1. A multi-level network pregel was prepared using the method in step 1 of Example 4. This was used as the experimental group.

[0088] 2. Add vascular endothelial growth factor (VEGF) to the above gel to a final concentration of 200 ng / mL.

[0089] 3. Add the above gel to the printhead (4℃) and print in the support bath (SunP FLOATII). The print result is as follows. Figure 10 As shown in a, cure at 30 degrees Celsius for 20 minutes, followed by light exposure for 60 seconds.

[0090] 4. The norbornene collagen from step 1 of Example 1 was dissolved in D-PBS to obtain a norbornene collagen solution with a final collagen concentration of 3 mg / mL. A LAP photoinitiator with a final concentration of 1 mg / mL and a mercapto-terminated polyethylene glycol with a final concentration of 10 mM were added to the 3 mg / mL collagen solution as a control group, defined as a single-network gel.

[0091] 5. Add the two types of gel (experimental group and control group) to the printhead (4℃) respectively, at 1mW / cm 2Printing under 365nm illumination at high power: Comparison of printing effects for two materials. Figure 10 b

[0092] As shown.

[0093] 6. The printed structures from the experimental group were transplanted subcutaneously into rats. Samples were collected 14 days later for HE staining. The results are as follows: Figure 10 As shown in Figure c, the printed structure has abundant blood vessels inside, which has the potential for soft tissue repair.

[0094] Example 9:

[0095] This embodiment primarily tests the injectability of multi-level collagen fiber gel and its repair of fat defects, as detailed below:

[0096] 1. A multi-level network pregel was prepared using the method in step 1 of Example 4.

[0097] 2. Add rabbit adipose-derived mesenchymal stem cells to the above gel to a final concentration of 10. 6 Cells / mL.

[0098] 3. Add the above gel to a syringe (4°C) and inject it into the fat defect site of the rabbit under CT guidance. Ten minutes after injection, the gel completes self-assembly. Insert an optical fiber to further solidify the gel.

[0099] 4. Collect and stain samples at different time points to observe the repair effect of the injected gel on fat defects.

[0100] Example 10:

[0101] This embodiment mainly tests the crosslinking effect of tetrazine crosslinking agent on gel, as follows: 1. Multi-level network pregel is prepared using the method in step 1 of Example 4. The process and conditions are the same as in step 1 of Example 4, except that no photoinitiator and thiol crosslinking agent are added.

[0102] 2. Place the pregel in a 37°C incubator for 20 minutes to obtain a multi-layered network gel that has not undergone chemical cross-linking.

[0103] 3. Immersing the gel in a 2mM tetrazine-polyethylene glycol-tetrazine aqueous solution for 20 minutes can further enhance the gel's modulus.

Claims

1. A method for preparing a collagen hydrogel with a multi-level fiber network, characterized in that: 1) Preparation process of large-scale collagen fibers with olefin bond reaction sites: dissolve olefin bond modified collagen in D-PBS solution (Dubor's phosphate buffer) at 0-10℃ to a final collagen concentration of 0.1-1 mg / mL; place the collagen solution on a shaker at 20-37℃ and shake at a speed of 0-1000 rpm for 12-72 hours; obtain a solution containing dispersed collagen fibers of large scale. Large-scale collagen fibers were collected by centrifuging to separate the dispersed large-scale collagen fiber solution into solid and liquid phases. 2) The olefin-modified collagen was dissolved in D-PBS solution at 0-10℃, and then large-scale collagen fibers were mixed with the olefin-modified collagen to obtain a mixture. The mixture was kept at 0-10℃ to prevent collagen solution assembly. The concentration of olefin-modified collagen in the mixture was 1-10 mg / mL (preferably 2-6 mg / mL, more preferably 3 mg / mL), and the concentration of collagen fibers was 3-40 mg / mL (preferably 10-20 mg / mL, more preferably 15 mg / mL). 3) Gel: Includes any one of the following five methods: The first method involves placing the mixture obtained in step 2) at 25-40°C, preferably 30-37°C, more preferably 37°C, for 5-30 minutes (preferably 10-20 minutes, more preferably 20 minutes) to obtain a multi-level collagen fiber network hydrogel. After assembly, the mixture changes from a fluid state to a gel state, thus obtaining a gel. Alternatively, the second method involves adding a photoinitiator and a multi-thiol crosslinking agent to the mixture obtained in step 2) (to carry out a stepwise growth polymerization reaction), or adding only a photoinitiator to the mixture obtained in step 2) (to carry out a chain growth polymerization reaction), without assembly, directly initiating the polymerization reaction under light, changing from a fluid state to a gel state, and obtaining a gel. Alternatively, the third method involves adding a compound with two or more tetrazine groups to the mixture obtained in step 2). The tetrazine can react directly with specific olefin bonds without assembly, and the mixture changes from a fluid state to a gel state to obtain a gel. Alternatively, the fourth method: First, add a photoinitiator and a multi-thiol crosslinking agent to the mixture obtained in step 2) (to carry out a stepwise growth polymerization reaction), or add only a photoinitiator to the mixture obtained in step 2) (to carry out a chain growth polymerization reaction); then, place it at 25-40°C, preferably 30-37°C, more preferably 37°C for 5-30 minutes (preferably 10-20 minutes, more preferably 20 minutes) to obtain a multi-layered collagen fiber network hydrogel, and then directly initiate a polymerization reaction under light to further strengthen the gel; Alternatively, the fifth method involves placing the mixture obtained in step 2) at 25-40°C, preferably 30-37°C, more preferably 37°C, for 5-30 minutes (preferably 10-20 minutes, more preferably 20 minutes) to obtain a multi-layered collagen fiber network hydrogel. The multi-layered collagen network hydrogel can then be directly reacted by immersing it in an aqueous solution of a compound with two or more tetrazine groups, without the need for a photoinitiator, to further strengthen the gel.

2. The preparation method according to claim 1, characterized in that: The olefin-modified collagen was prepared using the method described in claim 1 of patent CN202210190485.X (CN 114874455B).

3. The preparation method according to claim 1, characterized in that: Step 2) Before assembly, the mixture is added to a flowable state at 0-10°C with a photoinitiator and a multi-thiol crosslinking agent (to carry out stepwise growth polymerization), or only a photoinitiator is added (to carry out chain growth polymerization) to initiate a photocuring polymerization reaction under light after self-assembly, thereby further consolidating the gel. The photoinitiator includes ultraviolet photoinitiators and visible photoinitiators, such as lithium phenyl-2,4,6-trimethylbenzoylphosphonite (LAP), 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure 2959), ruthenium pyridine complex [Ru(II)(bpy)3]2+ / sodium sulfate (SPS), eosin Y, riboflavin, etc., one or more of which are added to the mixture system before assembly at a concentration of 0.1-1 mg / mL (preferably 0.3-0.8 mg / mL, more preferably 0.5-0.6 mg / mL); The multi-thiol crosslinking agent is one or more of small molecules or high molecular weight organic compounds with two or more free thiol groups (such as polyethylene glycol with two-sided thiol end caps, or collagen modified with multiple thiol groups, etc.); the total molar amount of thiol groups added to the mixture system before assembly is 0.25-4 times (preferably 0.5-2 times, more preferably 1 time) the total molar amount of olefin bonds (including olefin bonds in large-scale collagen fibers and olefin bond-modified collagen in step 2); The wavelength range of light used for crosslinking is 365-500 nm; Alternatively, before assembly, the mixture described in step 2) is added in a flowable state at 0-10°C to a compound with two or more tetrazine groups (such as polyethylene glycol with bilateral tetrazine-terminated ends, collagen modified with multiple tetrazine groups, etc.); the compound with two or more tetrazine groups is one or more small molecule or high molecular weight compounds; the total molar amount of tetrazine groups is 0.25-4 times (preferably 0.5-2 times, more preferably 1-1.2 times) of the total molar amount of olefins (including the olefin bonds in the large-scale collagen fibers and olefin-modified collagen in step 2); Before the collagen solution is assembled, the mixture of solution and fiber can be directly photocured without a heating assembly process, or cured by mixing with a tetrazine compound. Alternatively, the multi-level collagen network hydrogel formed after self-assembly in step 3) above can be directly reacted by immersing it in an aqueous solution of a compound with two or more tetrazine groups (such as polyethylene glycol with bilateral tetrazine-terminated ends, or collagen modified with multiple tetrazine groups, etc.) without the use of a photoinitiator. The compound having two or more tetrazine groups is one or more small molecules or polymers; the total molar amount of tetrazine groups is 0.25-4 times (preferably 0.5-2 times, more preferably 1-1.2 times) of the total molar amount of olefins (including the olefin bonds in the large-scale collagen fibers and olefin-modified collagen in step 2).

4. The preparation method according to claim 1, characterized in that: In step 3), the mixture from step 2) is placed at 25-37 degrees Celsius. The collagen solution will self-assemble to form small-scale fibers, which interweave with the large-scale fibers to form a multi-level collagen fiber network.

5. The preparation method according to claim 1 or 4, characterized in that: The gel can be further cured by irradiating it with a light source corresponding to the photoinitiator.

6. A collagen hydrogel with a multi-level fiber network prepared by the method of any one of claims 1-5.

7. An application of the hydrogel according to claim 6, characterized in that: Applications of collagen hydrogels with multi-level fiber networks include, but are not limited to, injectable hydrogels, bioprinting inks, tissue engineering, wound dressings, drug delivery carriers, cell transplantation and therapeutic carriers, or three-dimensional cell culture substrates.

8. The application as described in claim 7, characterized in that: The hydrogel has high viscosity and shear-thinning properties, making it suitable for the rheological properties required for 3D printing and in vivo injection; it is preferably used as a 3D printing material in the 3D printing process or as an in situ injection material in humans or animals. The gel can be further cured by irradiating it with a light source corresponding to the photoinitiator in the wavelength range of 365-500nm. Collagen fiber hierarchical arrangement at different scales; Since collagen fibers of different sizes all have olefin bond reaction sites, the gel modulus is significantly improved after photocuring; The biomimetic multi-level network structure has large gaps, which support cell extension, migration and the formation of biological functions.

9. The application as described in claim 7, characterized in that: Multi-level collagen fiber hydrogels can immobilize functional molecules; the functional molecules are one or more of the following: proteins, peptides, small molecule drugs, nucleic acids, carbohydrates, or polysaccharide molecules. The functional molecule has one or more reactive groups, which are one or more of free thiol groups and / or olefinic groups; or, the functional molecule without specific reactive groups can also be added to the above-assembled hydrogel or the mixture system before assembly. Functional molecules encased in multi-level network structures have a slow diffusion rate.

10. The application as described in any one of claims 7-9, characterized in that: The gel may also contain one or more of the following components: cells, cell spheres, organoids, growth factors, drugs, nanoparticles, etc.

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