Dual-network hydrogel constructed by animal acellular extracellular matrix and polypeptide

Abstract

The invention relates to a double-network hydrogel. The double-network hydrogel is constructed from an animal acellular extracellular matrix and polypeptide. The invention discloses extraction of animal acellular extracellular matrix, and a method and application of the animal acellular extracellular matrix and polypeptide to construct the dual-network hydrogel. The preparation method comprises the following steps: cleaning and crushing animal tissues, carrying out decellularization treatment by using sodium chloride and urea to obtain an extracellular matrix of the animal tissues, removing other components through dialysis, and compounding the extracellular matrix with polypeptide hydrogel to form an interlaced dual-network scaffold through mutual response. The scaffold structure provides an effective support environment for cell three-dimensional culture, realizes optimization of cell three-dimensional microenvironment construction, and can be applied to cell culture and organoid culture.

Description

Technical Field

[0001] This invention relates to a composite hydrogel constructed from animal decellularized extracellular matrix and peptides, wherein the animal decellularized extracellular matrix and peptides can respond to form a dual-network scaffold. Background Technology

[0002] Three-dimensional (3D) cell culture is a technique that simulates the in vivo cellular microenvironment by constructing in vitro methods. It is divided into two types: unsupported and supported. Unsupported culture allows cells to spontaneously aggregate into cell spheroids through physical methods; this method has limited applications and a single purpose. Supported culture utilizes the spatial structure of materials such as hydrogels and microcarriers, allowing cells to attach to the supporting material for three-dimensional growth and migration. This method is more conducive to signal transduction between cells and between cells and the matrix, and is currently the main method for 3D cell culture.

[0003] Hydrogel materials are considered ideal support materials for 3D cell culture due to their high water content, good biocompatibility, and interconnected porous structure. Hydrogels mimic the microenvironment of cells in vivo, namely the extracellular matrix, providing a favorable environment for cell proliferation, differentiation, and migration. Among them, peptide hydrogels and animal extracellular matrix-derived hydrogels have attracted widespread attention due to their excellent physicochemical properties.

[0004] Peptide hydrogels are a type of multifunctional hydrogel. Peptide molecules synthesized according to a certain sequence self-assemble into nanofiber structures through non-covalent bonds. They have good viscoelasticity and swelling properties, and can provide mechanical support and biological signals required for cell growth. They are a green and environmentally friendly hydrogel material.

[0005] Animal decellularized extracellular matrix hydrogels are natural extracellular matrix materials obtained by removing cellular components from biological tissues through decellularization technology. Due to their unique bioactivity and excellent biocompatibility, they are widely used in tissue engineering fields such as cell culture and organoid construction.

[0006] Both peptide hydrogels and animal decellularized extracellular matrix hydrogels are three-dimensional network structures, but their fiber composition is monolayered, resulting in limited properties and poor mechanical performance. However, they can respond to each other to form a network support structure, leading to the proposal to combine the two hydrogels to construct a dual-network hydrogel. Dual-network hydrogels are polymer materials composed of two interpenetrating or semi-permeable three-dimensional networks. Their unique interpenetrating network structure and tunable network crosslinking overcome the mechanical property limitations of single-network hydrogels, and their excellent mechanical properties, including anti-swelling and self-healing properties, have led to their widespread application in 3D cell culture, cell storage, wound repair, and cell migration experiments. Summary of the Invention

[0007] The purpose of this invention is to provide a dual-network hydrogel composed of decellularized animal extracellular matrix and peptides, which can form a network scaffold in response to the peptides. This invention relates to the extraction of decellularized animal extracellular matrix and a method for constructing a dual-network hydrogel with peptides. The method first involves washing and pulverizing animal tissue, then decellularizing it with sodium chloride and urea to obtain the extracellular matrix. Other components are then removed by dialysis, and finally, the decellularized animal extracellular matrix is ​​freeze-dried. By compounding it with a peptide hydrogel in a certain proportion, a three-dimensional fibrous dual-network structure can be obtained. This structure can construct a 3D scaffold, providing an effective support environment for 3D cell culture, optimizing the 3D cell microenvironment, and enabling 3D cell culture and organoid culture.

[0008] This invention provides a dual-network hydrogel constructed from animal decellularized extracellular matrix and peptides, wherein the method for extracting animal decellularized extracellular matrix and constructing the dual-network hydrogel with peptides includes the following steps: S1. The high osmotic pressure of 3.4M sodium chloride buffer solution is used to rupture cells in animal tissues and release the extracellular matrix. S2. Dissolve extracellular matrix components, such as collagen, using 2M urea buffer solution; S3. Using 0.01M Tris-HCl buffer, the impurities such as sodium chloride and urea were removed by staged dialysis to obtain animal decellularized extracellular matrix solution, which was then bottled and freeze-dried to obtain animal decellularized extracellular matrix lyophilized powder. S4. After dissolving the lyophilized animal extracellular matrix powder, mix it with the polypeptide hydrogel at a volume ratio of 1:1, gently blow and mix well, and let it stand until it self-assembles to obtain a double network hydrogel.

[0009] Specifically, in step S1, the 3.4M sodium chloride buffer contains tris(hydroxymethyl)aminomethane (Tris) and ethylenediaminetetraacetic acid (EDTA), and its pH is adjusted to 7.4 with HCl. The pretreated animal tissue is placed in this buffer and mechanically stirred at low temperature. After centrifugation, the supernatant is discarded, and the precipitate is collected to obtain the extracellular matrix precipitate.

[0010] Specifically, the concentration of tris(hydroxymethyl)aminomethane (Tris) is 50 mM, the concentration of ethylenediaminetetraacetic acid (EDTA / free acid) is 5 mM, and if EDTA disodium salt dihydrate is used, it is 4.0 mM, and the concentration of HCl is 1 M.

[0011] Specifically, the pretreatment involves removing fat from animal tissues, washing them alternately with deionized water and anhydrous ethanol, cold extracting with liquid nitrogen, and mechanically pulverizing them. The low-temperature mechanical stirring temperature is 4–10°C, for example, 4°C, 6°C, 8°C, or 10°C; the stirring speed is 200–300 rpm, for example, 200 rpm, 250 rpm, or 300 rpm; the stirring time is 6–12 h, for example, 6 h, 8 h, 10 h, or 12 h; the centrifuge speed is 9000–10000 rpm, for example, 9000 rpm, 9500 rpm, or 10000 rpm; and the centrifugation time is 20–30 min, for example, 20 min, 25 min, or 30 min.

[0012] Specifically, in step S2, the 2M urea buffer solution contains tris(hydroxymethyl)aminomethane (Tris) and sodium chloride, and its pH is adjusted to 7.4 with HCl. The concentration of Tris is 50 mM, the concentration of sodium chloride is 154 mM, and the concentration of HCl is 1 M.

[0013] Specifically, the obtained extracellular matrix precipitate was placed in 2M urea buffer solution and mechanically stirred at low temperature. After centrifugation, the supernatant was collected and the precipitate was discarded to obtain a preliminary animal decellularized extracellular matrix solution.

[0014] Specifically, the low-temperature mechanical stirring temperature is 4~10℃, for example 4℃, 6℃, 8℃, 10℃; the stirring speed is 200~300rpm, for example 200rpm, 250rpm, 300rpm; the stirring time is 6~12h, for example 6h, 8h, 10h, 12h; the centrifuge speed is 9000~10000rpm, for example 9000rpm, 9500rpm, 10000rpm; and the centrifugation time is 20~30min, for example 20min, 25min, 30min.

[0015] Specifically, in step S3, staged dialysis refers to adding different concentrations of sodium chloride to the dialysis fluid and performing dialysis sequentially according to three salinity environments: high salinity, medium salinity, and low salinity. The sodium chloride concentration in the high salinity environment is 1M, the sodium chloride concentration in the medium salinity environment is 0.5M, and the sodium chloride concentration in the low salinity environment is 0M. The dialysis temperature for each stage is 4~10℃, for example, 4℃, 6℃, 8℃, and 10℃; the dialysis speed is 100~200rpm, for example, 100rpm, 150rpm, and 200rpm; and the dialysis duration is 6~12h, for example, 6h, 8h, 10h, and 12h. After dialysis, an animal decellularized extracellular matrix solution is obtained.

[0016] Specifically, in step S3, the bottling involves filling the obtained animal decellularized extracellular matrix solution into vials at a rate of 4 ml / vial, followed by freeze-drying at a temperature of -10 to -80°C, such as -10°C, -20°C, -40°C, -60°C, or -80°C, for a time of 12 to 48 hours, such as 12 hours, 24 hours, or 48 hours.

[0017] Specifically, in step S4, one bottle of lyophilized animal extracellular matrix powder is dissolved in 1 mL of complete culture medium. The concentration of the polypeptide hydrogel is 1-2%, for example, 1%, 1.5%, or 2%. The two are mixed in a 1:1 ratio, gently blown and mixed with a wide pipette tip, and allowed to stand for 30-60 minutes, for example, 30 minutes, 40 minutes, 50 minutes, or 60 minutes, to finally obtain a double-network hydrogel.

[0018] Specifically, the polypeptide hydrogel is Cu1XⅡ prepared by Matrix (Tianjin) Biotechnology Co., Ltd.

[0019] The present invention has the following beneficial effects: The dual-network hydrogel prepared in this invention is suitable for conventional cell culture and exhibits excellent cell compatibility. It can accurately construct a biomimetic microenvironment for 3D cell culture. The extracted animal decellularized extracellular matrix, containing collagen, fibronectin, and growth factors, can provide the bioactive signals required for cell adhesion and proliferation. The peptide hydrogel can self-assemble into an adjustable topological structure, mimicking the specific pore structure of tissues. The extraction method of the animal decellularized extracellular matrix in this invention has the advantages of convenient operation, controllable cost, and environmental friendliness. Furthermore, the process of co-assembling the animal matrix with the peptide hydrogel into a dual network does not require chemical cross-linking agents, ensuring the safety of the components. This innovative preparation method optimizes the construction of a 3D cell microenvironment, overcoming the limitations of the single-dimensional nature of natural and synthetic materials. Attached Figure Description Figure 1 It is the animal decellularized extracellular matrix solution (A) and lyophilized powder (B) prepared in Example 1.

[0020] Figure 2 The images show the animal decellularized extracellular matrix and peptides in Example 2 (A) and the double-network hydrogel after the response is complete (B).

[0021] Figure 3 These are scanning electron microscope images of the animal decellularized extracellular matrix (A), polypeptide hydrogel (B), and double-network hydrogel (C) in Example 3.

[0022] Figure 4 This describes the droplet stability of the double-network hydrogels formed by the polypeptides and different animal extracellular matrix in Example 4.

[0023] Figure 5These are Fourier transform infrared spectra of the animal decellularized extracellular matrix, polypeptide hydrogel, and dual-network hydrogel in Example 5.

[0024] Figure 6 These are time-scan images (A) and shear-thinning images (B) related to the mechanical property characterization of the dual-network hydrogel in Example 6.

[0025] Figure 7 This is an in vitro degradation diagram of the polypeptide hydrogel and the dual-network hydrogel in Example 7.

[0026] Figure 8 This is a diagram illustrating the cell migration promotion effect of the dual-network hydrogel in Example 8.

[0027] Figure 9 These are optical microscope images of the cell growth status on days 1, 4, and 7 in complete culture medium (A), polypeptide hydrogel (B), and double-network hydrogel (C) in Example 9.

[0028] Figure 10 These are laser confocal (live / dead staining) images of the cell growth status on days 1, 4, and 7 in peptide hydrogel (A) and double-network hydrogel (B) in Example 10.

[0029] Figure 11 The OD values ​​for the cytotoxicity assessment of peptide hydrogels and dual-network hydrogels using the CCK-8 assay in Example 11 are... 450 Value (A), and according to OD 450 The value is used to calculate the relative viability of cell growth (B).

[0030] Figure 12 This is from Example 12, which describes the growth of mouse small intestinal organoids observed under an optical microscope in a dual-network hydrogel constructed from decellularized extracellular matrix and peptides in different animals. Detailed Implementation

[0031] The present invention will be further described in detail below through embodiments. Through these descriptions, the features and advantages of the present invention will become clearer and more apparent. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0032] Example 1: Extraction of animal exfoliated extracellular matrix Experimental design: Taking pig small intestine as an example.

[0033] Pretreatment of pig intestines: Clean 100g of pig intestines, remove the outer fat and cut into pieces. Wash with deionized water and anhydrous ethanol three times alternately. Then filter out the pig intestine pieces, cold extract with liquid nitrogen for 10 minutes, and then crush them with a grinder. After crushing, collect the pig intestine tissue and place it at 4℃ for later use. Cell lysis with 3.4M sodium chloride buffer: Weigh 39.7g sodium chloride, 1.21g tris(hydroxymethyl)aminomethane, and 0.3g ethylenediaminetetraacetic acid (EDTA), dissolve in 200ml deionized water, and adjust the pH to 7.4 with 1M hydrochloric acid after complete dissolution. Add the pulverized porcine intestinal tissue to the prepared 3.4M sodium chloride buffer and mechanically stir for 8 hours at 200 rpm at 4℃. Under high osmotic pressure, porcine intestinal cells lyse, releasing the extracellular matrix. After stirring, centrifuge at 9000 rpm for 30 minutes to obtain the porcine intestinal extracellular matrix precipitate.

[0034] Dissolving extracellular matrix in 2M urea buffer: Weigh 18g urea, 0.9g tris(hydroxymethyl)aminomethane, and 1.35g sodium chloride, dissolve in 150ml deionized water, and adjust the pH to 7.4 with 1M hydrochloric acid after complete dissolution. Add the obtained porcine intestinal extracellular matrix precipitate to the prepared 2M urea buffer and mechanically stir for 8 hours at 200 rpm at 4℃. Urea can dissolve most extracellular matrix components, such as collagen and glycosaminoglycans. After stirring, centrifuge at 9000 rpm for 30 minutes to obtain the preliminary porcine intestinal matrix solution.

[0035] Staged dialysis with 0.01M Tris-HCl buffer: (1) High-salt dialysis solution: Weigh 87.66 g of sodium chloride, dissolve it in a small amount of deionized water, add 15 mL of 1 M Tris-HCl stock solution, and make up to 1500 mL. Adjust the pH to 7.4 with 1 M HCl. Dialyze the obtained preliminary porcine intestinal matrix solution with this dialysis solution for 10 h at a dialysis temperature of 4 °C and a dialysis rate of 150 rpm.

[0036] (2) Medium-salt dialysate: Weigh 43.83g of sodium chloride, dissolve it in a small amount of deionized water, add 15mL of 1M Tris-HCl stock solution, and bring the volume to 1500mL. Adjust the pH to 7.4 with 1M HCl. After dialysis with high-salt dialysate, dialyze with medium-salt dialysate for 10h at a temperature of 4℃ and a dialysis rate of 150rpm.

[0037] (3) Low-salt dialysis solution: 15 mL of 1M Tris-HCl stock solution was directly added to 1500 mL of deionized water, and the pH was adjusted to 7.4 with 1M HCl. After dialysis with medium-salt dialysis solution, the prepared low-salt dialysis solution was used for dialysis twice. Each dialysis session lasted 10 h, the dialysis temperature was 4℃, and the dialysis speed was 150 rpm. After dialysis, a porcine intestinal matrix solution was obtained.

[0038] Freeze-drying: The obtained porcine intestinal matrix solution was packaged into vials at a rate of 4 mL / bottle, and then freeze-dried at -80℃ for 48 hours to obtain freeze-dried porcine intestinal matrix powder.

[0039] Experimental results: The animal decellularized extracellular matrix solution obtained in Example 1 was as follows: Figure 1 As shown in Figure A, the solution is relatively clear and transparent, and the lyophilized animal extracellular matrix powder is as follows: Figure 1 As shown in B, the freeze-dried powder has a certain degree of support.

[0040] Following the same method, acellular extracellular matrix of animals such as porcine placenta, sheep placenta, mouse tumors, and porcine brain can be obtained.

[0041] Example 2: Construction of a dual-network hydrogel Experimental protocol: Taking the peptide and mouse chondrosarcoma decellularized extracellular matrix (prepared according to the protocol of Example 1) as an example, one bottle of mouse chondrosarcoma decellularized extracellular matrix lyophilized powder was dissolved in 1 mL of complete culture medium (DEME medium containing 10% fetal bovine serum and 1% penicillin / streptomycin) for later use. The peptide lyophilized powder was used to prepare a 1% peptide hydrogel for later use. The two were mixed at a volume ratio of 1:1, and gently mixed by pipetting with a wide pipette tip. The mixture was allowed to stand for 40 min until it was fully self-assembled, and finally a 0.5% double network hydrogel was obtained.

[0042] Experimental results: such as Figure 2 As shown in Figure A, the extracellular matrix solution of mouse chondrosarcoma rapidly forms a gel after being mixed with a polypeptide solution; as shown in Figure A. Figure 2 As shown in B, after the response is complete, a double-network hydrogel is obtained. When the centrifuge tube is inverted, the gel is fixed at the bottom of the centrifuge tube and does not fall off.

[0043] Example 3: Observation of spatial structure using scanning electron microscopy Experimental Procedure: Taking the porcine intestinal decellularized extracellular matrix obtained in Example 1 as an example, the lyophilized porcine intestinal decellularized extracellular matrix powder and the lyophilized peptide powder were dissolved in complete cell culture medium. After complete dissolution, 0.5 mL of each was placed into centrifuge tubes. Furthermore, a double-network hydrogel was constructed using the two materials at a 1:1 volume ratio (following the construction method in Example 2), and 0.5 mL of this hydrogel was placed into centrifuge tubes. All three materials were then freeze-dried at -80°C for 48 hours. The microstructure was observed using a scanning electron microscope after freeze-drying, and the results are as follows: Figure 3 As shown.

[0044] Experimental results: Scanning electron microscopy of pig intestine decellularized extracellular matrix as shown in... Figure 3 As shown in Figure A, its spatial structure is a porous fibrous structure with relatively large and uniform pores, but the fibers are fragile and have poor mechanical strength; the scanning electron microscope image of the polypeptide hydrogel is shown below. Figure 3As shown in Figure B, its spatial structure is a porous, layered structure with high mechanical strength and some support, but the pores are small, and most of the volume is occupied by the raw materials, leaving limited space for cell growth. Scanning electron microscopy of the double-network hydrogel constructed from a mixture of porcine intestinal extracellular matrix and peptides is shown below. Figure 3 As shown in C, its spatial structure is an interpenetrating three-dimensional network structure, which combines the advantages of the porous spatial structure of animal decellularized extracellular matrix and the good mechanical properties of polypeptide hydrogel.

[0045] Example 4: Droplet stability of dual-network hydrogels constructed from peptides and exfoliated extracellular matrix from different animals. To investigate the differences in morphological stability and physicochemical properties of droplets formed by decellularized extracellular matrix and peptides from different animals, as well as droplets formed by simple peptide hydrogels, after multiple in vitro media exchange operations, and to provide experimental data support for the screening of tissue engineering scaffold materials, droplet stability experiments were conducted.

[0046] Experimental Protocol: The method for extracting extracellular matrix from animals in this experiment follows the protocol described in Case Study 1. For example... Figure 4 Double-network hydrogels formed by porcine intestinal matrix and peptides (A), porcine placental matrix and peptides (B), ovine placental matrix and peptides (C), mouse chondroma matrix and peptides (D), and bovine hide matrix and peptides (E) were prepared into homogeneous gel solutions. Using a pipette, 25 μL of each material was added to the wells of a 24-well plate, forming uniform droplets in each well. The plates were incubated in a CO2 incubator for 30 minutes, after which culture medium was added. The droplets in the 24-well plates underwent three repeated medium replacements: each time, the old medium was slowly removed from the wells using a pipette, and an equal volume of fresh culture medium was added. The process was performed gently to minimize mechanical damage to the droplets. After three medium replacements, the morphological integrity of the droplets (whether they broke, dissolved, or deformed) was observed and recorded. The results are shown below. Figure 4 As shown.

[0047] Experimental results: After three solution changes, Figure 4 One droplet of the double-network hydrogel formed by sheep placental matrix and peptides showed dissolution and rupture, while other peptide double-network hydrogel droplets maintained good morphological uniformity and integrity. This indicates that the addition of different animal extracellular matrix to peptide hydrogels, under repeated in vitro medium-change conditions, exhibits superior morphological stability and physicochemical properties, making it a better candidate for tissue engineering scaffold materials.

[0048] Example 5: Fourier transform infrared spectroscopy detection of characteristic functional groups To investigate whether the dual-network hydrogel constructed in this invention disrupts the structure of the animal extracellular matrix and peptides, and whether a chemical reaction occurs after the two are mixed, Fourier transform infrared spectroscopy was performed.

[0049] Experimental protocol: The lyophilized powders of the animal extracellular matrix-free, polypeptide hydrogel, and double-network hydrogel used in Example 2 were respectively processed with potassium bromide for tableting, followed by detection. The detection data were normalized using Origin, and the results are as follows. Figure 5 As shown.

[0050] Experimental results: at 4000-400cm -1 In the frequency region, animal extracellular matrix-free, polypeptide hydrogels, and dual-network hydrogels are located in the amide I band at 1620-1660 cm⁻¹. -1 A significant absorption peak is observed at this point, corresponding to the C=O stretching vibration of the ester bond. The peak shape of the double-network hydrogel is significantly broadened at this point, indicating that the β-sheet of the polypeptide (~1620 cm⁻¹) interacts with the collagen amide I band (~1650 cm⁻¹) in the decellularized extracellular matrix of animals, forming a hydrogen bond network. The double-network hydrogel also shows a significant absorption peak at 1550 cm⁻¹. -1 A moderate absorption peak is observed at this location, which is more pronounced than that of the animal decellularized extracellular matrix and peptide hydrogel. This peak is characteristic of the amide II band, indicating enhanced NH bending and CN stretching. The dual-network hydrogel constructed in this invention does not disrupt the structure of the animal decellularized extracellular matrix and peptide hydrogel, and effectively crosslinks the two together through chemical interactions such as hydrogen bonding.

[0051] Example 6: Mechanical property characterization of dual-network hydrogels To investigate whether the dual-network hydrogel structure constructed in this invention can maintain the stability, injectability, and cell compatibility of 3D cell culture, time-scan verification and shear refinement characterization were performed. Time-scan verification confirmed the structural stability of the hydrogel during the culture period, ensuring that it can provide a long-term, reliable three-dimensional support microenvironment for cells; shear refinement characterization was related to the hydrogel's ability to protect and encapsulate cells under shear forces.

[0052] Experimental Procedure: Taking the dual-network hydrogel obtained in Example 2 as an example, the mechanical properties of the dual-network hydrogel were evaluated using a rheometer equipped with a 20 mm diameter parallel plate. At the start of the experiment, the gap between the upper plate and the sample stage was set to 30 mm, and the geometric clearance was 0.5 mm. A 200 μL sample was loaded onto the stage using a pipette. A thin layer of silicone oil was applied around the sample to prevent moisture evaporation, and the sample was equilibrated at 37 °C for 1800 seconds before the start of the experiment. The storage modulus (G¢) and loss modulus (G″) were measured using time-scan curves at a fixed frequency of 1 Hz and an amplitude strain of γ = 1%. The experimental parameters for the shear thinning process were set as follows: rotor-panel distance of 0.5 mm, shear strain of 100%, frequency of 1 Hz, and temperature of 37 °C. A 20-second shear thinning test and a 15-minute time-scan test were performed as a group, and the process was repeated 3 times.

[0053] Experimental results: Time scan verification results are as follows Figure 6 As shown in Figure A, the elastic modulus (G′) of the 0.5% dual-network hydrogel is higher than the loss modulus (G”) within a certain frequency range, confirming that the gel-like structure can provide a supportive three-dimensional microenvironment for cells. The shear refinement characteristics analysis results are as follows... Figure 6 As shown in Figure B, a 0.5% dual-network hydrogel was subjected to a series of immediately increased strains, and the effects on storage and loss modulus were monitored. It was found that the dual-network hydrogel recovered after the shear strain was removed, demonstrating that this hydrogel has excellent ability to protect and encapsulate cells. Time-scan validation and shear refinement characteristic analysis results together optimized the 3D cell culture system with excellent mechanical signal.

[0054] Example 7: In vitro evaluation of the in vitro degradation performance of the dual-network hydrogel.

[0055] To investigate the in vitro degradation performance of the dual-network hydrogel constructed in this invention and to verify its degradation stability, thereby providing key performance evidence for its use as a long-term support scaffold for cell culture, in vitro degradation performance tests were conducted.

[0056] Experimental Procedure: Taking the dual-network hydrogel obtained in Example 2 as an example, the dual-network hydrogel and peptide hydrogel formed in Example 2 were immersed in PBS buffer (pH 7.4) and incubated at 37°C. Samples were removed from day 2 to day 14, freeze-dried, weighed, and the degradation rate was calculated.

[0057] Experimental results: The degradation of the dual-network hydrogel and peptide hydrogel is as follows: Figure 7As shown, the residue rate of the dual-network hydrogel was approximately 89% on day 7 and 84% on day 14, significantly higher than that of the peptide hydrogel, indicating that the dual-network hydrogel has a significantly longer retention time than the peptide hydrogel. This further demonstrates that the dual-network hydrogel constructed in this invention has excellent degradation stability, and the moderate degradation rate ensures the short-term stability of this hydrogel as a supporting cell culture scaffold.

[0058] Example 8: In vitro evaluation of the cell migration-promoting effect of the dual-network hydrogel.

[0059] Given the crucial role of cell migration in tissue regeneration, the ability of dual-network hydrogels to promote cell migration was evaluated in vitro.

[0060] Experimental Procedure: Taking the dual-network hydrogel obtained in Example 2 as an example, the experiment was divided into a peptide hydrogel group (control group) and a dual-network hydrogel group (experimental group). First, 100 μL of both the dual-network hydrogel and peptide hydrogel were added to 48-well plates. Then, human umbilical vein endothelial cells (HUVECs) that had been passaged and were in good growth condition in the laboratory were added at 5 × 10⁻⁶ cells per well. 4 The cells were seeded at a density of 400 μL in each well of a 48-well plate. After 24 hours of cell adhesion, the complete culture medium was discarded. Then, a 100 μL pipette tip was used to make a slit in the center of the bottom of the culture plate. The plate was then washed three times with PBS and observed under an optical microscope.

[0061] Experimental results: such as Figure 8 As shown, observations at the 24-hour time point revealed that cells in all groups migrated towards the center, as indicated by the results. Figure 8 As shown, the cell migration area of ​​the dual-network hydrogel group was significantly larger than that of the polypeptide hydrogel group. This indicates that the dual-network hydrogel provides a better mimicry of the in vivo microenvironment, thereby promoting cell migration. The experimental results highlight the potential of the dual-network hydrogel constructed in this invention as a biomimetic platform supporting cell survival and promoting cell migration, which is a key factor for successful tissue regeneration.

[0062] Example 9: Observation of cell growth under different environments using an optical microscope To investigate the application effect of the dual-network hydrogel constructed in this invention in 3D cell culture, human umbilical vein endothelial cells (HUVECs) available in the laboratory were used for culture and observation.

[0063] Experimental Procedure: Taking the dual-network hydrogel obtained in Example 2 as an example. First, 0.5% and 1% peptide hydrogels were prepared; the dual-network hydrogel and 1% peptide hydrogel were mixed at a 1:1 volume ratio to obtain a 0.5% dual-network hydrogel. 90 μL of each of the complete culture medium, 0.5% peptide hydrogel, and 0.5% dual-network hydrogel were spread evenly in a 96-well plate to form a 2D control group, a 3D peptide hydrogel experimental group, and a 3D dual-network hydrogel experimental group. HUVEC cells with a confluence of more than 80% were digested with EDTA trypsin and then subjected to a 1×10⁻⁶ ppm solution. 4 Cells were seeded at the above density in the above-mentioned well plates and cultured in a 37°C, 5% CO2 incubator. The results are as follows: Figure 9 As shown.

[0064] Experimental results: The cell growth status of each group was observed using an optical microscope on days 1, 4, and 7, and the results are as follows. Figure 9 As shown in the figure. The 2D control group is shown in the figure. Figure 9 As shown in Figure A, cells enter the logarithmic growth phase from day 1 to day 4, and initially adhere to the culture vessel quickly and grow well. However, after day 4, due to nutrient deficiency and limited space, a large number of cells die. The 3D polypeptide hydrogel assembly is shown below. Figure 9 As shown in Figure B, cells grew into cell spheroids on day 7, and no dead cells appeared from days 1 to 7. The 3D dual-network hydrogel assembly is shown below. Figure 9 As shown in Figure C, smaller cell spheres appeared on day 4, and obvious cell spheres appeared on day 7. Compared with the 3D polypeptide hydrogel group, the cell spheres in the dual-network hydrogel were more uniform and regular, and the cell roundness was significantly higher than that in the polypeptide group, proving that the biological performance of the dual-network hydrogel constructed in this invention is superior to that of the polypeptide group.

[0065] Example 10: Observation of cell viability under laser confocal microscopy To further verify the accuracy of cell growth status under optical microscopy and the better biocompatibility of the dual-network hydrogel, cells were stained using a cell live / dead staining kit and then characterized using laser confocal microscopy.

[0066] Experimental protocol: Cells grown on days 1, 4, and 7 of the 3D peptide hydrogel group and the 3D double-network hydrogel group in Example 9 were collected and transferred to new centrifuge tubes. The cells were washed with 10x PBS, centrifuged to remove the hydrogel matrix, and retained only the cells. The cells were then stained with Calcein-AM / PI staining agent and incubated at 37°C for 30 minutes (this process was performed under completely dark conditions). Finally, images were taken using a laser confocal microscope. The results are shown below. Figure 10 As shown.

[0067] Experimental results: 3D polypeptide hydrogel group (e.g. Figure 10 A) and 3D dual-network hydrogel groups (such as Figure 10 In B), no obvious dead cells were observed on days 1, 4, and 7 of growth, indicating a high cell survival rate. On day 7, the cells in the 3D dual-network hydrogel group maintained a higher cell density and spheric integrity compared to the 3D polypeptide hydrogel group, further demonstrating that the dual-network hydrogel constructed in this invention has excellent biocompatibility and is more conducive to the three-dimensional growth and proliferation of cells.

[0068] Example 11: Evaluation of hydrogel cytotoxicity and calculation of relative viability using the CCK-8 assay. To further investigate whether the dual-network hydrogel constructed in this invention exhibits cytotoxicity, cell OD was measured using a CCK-8 assay kit. 450 Value detection, and based on OD 450 The relative cell activity was calculated, and finally, a toxicity assessment was performed based on the results.

[0069] Experimental protocol: Cell culture was performed according to Example 9. On days 1, 3, 5, and 7 of culture, 10 μL of CCK-8 reagent was added to the 2D control group and the two 3D experimental groups. After incubation at 37°C for 40 minutes, the mixture was thoroughly mixed. 100 μL of the mixture was then taken from each group. The absorbance was measured under these conditions using a cell microplate reader with a primary wavelength of 450 nm and a secondary wavelength of 630 nm. Cell viability was calculated according to the formula in the CCK-8 kit instructions. The results are as follows: Figure 11 As shown.

[0070] Experimental results: OD measured by the CCK-8 method 450 The value can directly reflect the trend of changes in the number of surviving cells (e.g.) Figure 11 A), according to OD 450 The value can be used to calculate the relative cell activity, indirectly reflecting the cell's vitality under various environmental conditions (e.g., ...). Figure 11 B). In the 2D control group, cells initially adhered quickly during planar culture and entered the peak logarithmic growth phase on days 1-3, resulting in rapid proliferation. However, proliferation declined on days 5-7 due to nutrient limitation. In the 3D group, cell number and activity maintained a continuous upward trend due to the internal three-dimensional porous structure and growth-promoting factors. The 3D dual-network hydrogel group showed significantly higher cell activity than the 3D polypeptide single-network hydrogel group because collagen and fibronectin in the dual network provide anti-apoptotic signals. The relative cell viability graph more clearly quantifies the cell-enhancing effect of the dual-network hydrogel material constructed in this invention.

[0071] Example 12: Observation of organoid growth under an optical microscope in different environments To investigate the application effect of the dual-network hydrogel constructed in this invention in 3D organoid culture, mouse small intestinal organoids were selected for culture and observation.

[0072] Experimental Protocol: Taking the double-network hydrogels constructed from different animal decellularized extracellular matrix and peptides as examples in Example 4, including a double-network hydrogel formed from porcine intestinal matrix and peptides (A), a double-network hydrogel formed from porcine placental matrix and peptides (B), a double-network hydrogel formed from sheep placental matrix and peptides (C), a double-network hydrogel formed from mouse chondroma matrix and peptides (D), and a double-network hydrogel formed from bovine hide matrix and peptides (E), mouse small intestinal organoids were cultured in each well. Droplets were prepared in 25 μL per well of a 48-well plate. After half an hour, 500 μL of complete mouse small intestinal organoid culture medium was added to each well to cover the droplets. The plates were then incubated in a CO2 incubator and observed.

[0073] Experimental Results: The growth status of organoids in each group was observed using an optical microscope on days 1, 3, 5, 7, 11, and 14. The results are as follows: Figure 12 As shown in the figure. The double-network hydrogel assembly formed by the porcine intestinal matrix and polypeptides is as follows: Figure 12 As shown in Figure A, the crypts of mouse small intestinal organoids became rounded and developed chambers on day 1, indicating good growth. Buds began to emerge from day 3-5, and the organoids were successfully cultured by day 7 and beyond. Figure 12 As shown in Figure B, the mouse small intestinal organoids formed by the double-network hydrogel of porcine placenta and peptides also developed a chambered organoid morphology, and budding occurred within 7-11 days, indicating that the double-network hydrogel of porcine placenta and peptides successfully cultured mouse small intestinal organoids. The double-network hydrogel of sheep placenta matrix and peptides is shown below. Figure 12 As shown in C, the organoids exhibit chambers consistent with those of normal mouse small intestines, and their growth is good. Figure 12 D and Figure 12 E represents a double-network hydrogel formed by mouse chondroma matrix and polypeptides, and a double-network hydrogel formed by bovine hide matrix and polypeptides, respectively. It can be seen that the crypts of mouse small intestinal organoids are rounded, and no chambers of mouse small intestinal organoids appear. From... Figure 12 It can be seen that the double-network hydrogel groups formed by porcine intestinal matrix and polypeptides, porcine placental matrix and polypeptides, and sheep placental matrix and polypeptides all have certain organoid culture performance.

[0074] Based on the above-described ideal examples according to the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the technical concept of the present invention. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A dual-network hydrogel constructed from animal extracellular matrix and polypeptides, characterized in that, A method for extracting animal extracellular matrix and constructing a dual-network hydrogel with peptides, the method comprising the following steps: S1. The high osmotic pressure of 3.4M sodium chloride buffer solution is used to rupture cells in animal tissues and release the extracellular matrix. S2. Dissolve extracellular matrix components, such as collagen, using 2M urea buffer solution; S3. Using 0.01M Tris-HCl buffer, the impurities such as sodium chloride and urea were removed by staged dialysis to obtain animal decellularized extracellular matrix solution, which was then bottled and freeze-dried to obtain animal decellularized extracellular matrix lyophilized powder. S4. After dissolving the lyophilized animal extracellular matrix powder, mix it with the polypeptide hydrogel at a volume ratio of 1:1, gently blow and mix well, and let it stand until it self-assembles to obtain a double network hydrogel.

2. The preparation method according to claim 1, characterized in that, S1 contains 3.4M sodium chloride buffer containing tris(hydroxymethyl)aminomethane (Tris) and ethylenediaminetetraacetic acid (EDTA), and its pH is adjusted to 7.4 with HCl. Pretreated animal tissues are placed in this buffer and mechanically stirred at low temperature. After centrifugation, the supernatant is discarded, and the precipitate is collected to obtain the extracellular matrix precipitate.

3. The preparation method according to claim 2, characterized in that, The concentration of tris(hydroxymethyl)aminomethane (Tris) is 50 mM, the concentration of ethylenediaminetetraacetic acid (EDTA / free acid) is 5 mM, and if EDTA disodium salt dihydrate is used, it is 4.0 mM, and the concentration of HCl is 1 M.

4. The preparation method according to claim 2, characterized in that, Pretreatment involves removing fat from animal tissues, washing them alternately with deionized water and anhydrous ethanol, quick-freezing with liquid nitrogen, and mechanical pulverization. The low-temperature mechanical stirring temperature is 4~10℃, the stirring speed is 200~300rpm, the stirring time is 6~12h, the centrifuge speed is 9000~10000rpm, and the centrifugation time is 20~30min.

5. The preparation method according to claim 1, characterized in that, S2 contains 2M urea buffer containing tris(hydroxymethyl)aminomethane (Tris) and sodium chloride, and its pH is adjusted to 7.4 with HCl. The obtained extracellular matrix precipitate is placed in this buffer and mechanically stirred at low temperature. After centrifugation, the supernatant is collected and the precipitate is discarded to obtain a preliminary animal decellularized extracellular matrix solution.

6. The preparation method according to claim 5, characterized in that, The concentration of tris(hydroxymethyl)aminomethane (Tris) was 50 mM, the concentration of sodium chloride was 154 mM, and the concentration of HCl was 1 M.

7. The preparation method according to claim 5, characterized in that, The low-temperature mechanical stirring temperature is 4~10℃, the stirring speed is 200~300rpm, the stirring time is 6~12h, the centrifuge speed is 9000~10000rpm, and the centrifugation time is 20~30min.

8. The preparation method according to claim 1, characterized in that, In S3, staged dialysis involves adding different concentrations of sodium chloride to the dialysate and performing dialysis sequentially in three salinity environments: high salinity, medium salinity, and low salinity. The sodium chloride concentration is 1M in the high salinity environment, 0.5M in the medium salinity environment, and 0M in the low salinity environment. The dialysis temperature for each stage is 4~10℃, the dialysis rate is 100~200rpm, and the dialysis duration is 6~12h. The bottling process involves filling the obtained animal decellularized extracellular matrix solution into vials at 4ml / vial, followed by freeze-drying at -10~-80℃ for 12~48h.

9. The preparation method according to claim 1, characterized in that, In S4, one bottle of lyophilized animal extracellular matrix powder was dissolved in 1 mL of DEME medium (referred to as "complete medium") containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. The polypeptide hydrogel was Cu1XⅡ prepared by Matrix (Tianjin) Biotechnology Co., Ltd., with a polypeptide hydrogel concentration of 1-2%. The two were mixed in a 1:1 ratio, gently blown and mixed with a wide pipette tip, and allowed to stand for 30-60 min to obtain a double network hydrogel.

10. The application of the dual-network hydrogel obtained by the preparation method according to claims 1-9, characterized in that, It can be used for three-dimensional cell culture and organoid culture.

Images