Ternary composite acellular extracellular matrix hydrogel as well as preparation method and application thereof

Abstract

The invention relates to a composite hydrogel material based on acellular matrixes from three different sources. According to the material, a fat decellularized matrix (DAT), a small intestine submucosa decellularized matrix (SIS) and a blood vessel decellularized matrix (Adv) are mixed in proportion, and a gel system with the temperature response characteristic is formed through a biocompatible cross-linking agent. The ternary composite acellular extracellular matrix hydrogel disclosed by the invention can keep activity under various storage conditions, so that a wider possibility is provided for cold chain preservation of the product.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a ternary composite decellularized extracellular matrix hydrogel for soft tissue repair, its preparation method, and its applications. This hydrogel is particularly suitable for tissue or organ repair, such as breast reconstruction, facial fillers, and diabetic foot repair in clinical applications. Background Technology

[0002] Cellular extracellular matrix has been used as a scaffold material in tissue engineering in regenerative medicine, and matrix materials from different tissue sources can maintain some of the biological characteristics of the original tissue. However, matrix materials from a single source often cannot meet the multiple needs of complex soft tissue repair. Summary of the Invention

[0003] Soft tissue repair requires the simultaneous regeneration of adipose tissue, provision of mechanical support, and promotion of vascularization. Single-source matrix materials have functional limitations and cannot simultaneously meet these requirements. Soft tissue reconstruction often necessitates a simultaneous presence of an adipose regeneration environment, adequate mechanical support, and good blood supply. Existing materials relying on single-source or binary composite matrices typically struggle to achieve all these aspects. This invention, based on the complementary properties of three types of decellularized matrices, constructs a ternary composite hydrogel system suitable for soft tissue repair, enabling it to provide a more balanced regenerative microenvironment during tissue regeneration.

[0004] The hydrogel of this invention is composed of three types of decellularized matrix from adipose tissue, the submucosa of the small intestine, and blood vessels, in a specific ratio. After being pulverized to the micrometer scale, the three matrix types are solidified under physiological conditions using natural or chemical cross-linking agents to form a gel. At low temperatures, the system is in a fluid state, facilitating mixing and injection; upon entering a body temperature environment, it rapidly gels, forming a three-dimensional structure with certain mechanical support and tissue adhesion. By rationally configuring the ratio of the three matrix types, a relatively stable synergistic effect can be achieved between fat regeneration, structural support, and angiogenesis.

[0005] Regarding specific components, the mass ratio of DAT, SIS, and Adv is generally controlled within the range of 2:1:1 to 1:2:1. In practice, 1.5:1:1 is a commonly used formulation. After grinding, the particle size of the three matrices is mostly 1–20 μm, with 5–15 μm powder being more likely to form a uniform dispersion system. To obtain the required gel strength and degradation rate, different types of crosslinking agents can be selected according to application requirements, such as natural substances like genipin, tannic acid, ferulic acid, gallic acid, and quercetin, or chemical formulations like glutaraldehyde, EDC / NHS, and HMDI. The concentration used is usually adjusted within the range allowed by relevant literature and processes, such as genipin 0.1–0.3% (w / v), tannic acid 0.1–0.2% (w / v), or EDC / NHS 5–15 mM / 2.5–7.5 mM respectively.

[0006] The preparation of the hydrogel involves the following main steps: First, three types of decellularized matrix materials are obtained separately; they are mixed in the target ratio and ground to the desired particle size; they are fully dispersed in an acidic protease solution and digested by stirring at 4°C for 48–72 hours; then adjusted to a pH close to physiological values, a cross-linking agent is added, and the mixture is cured at 37°C to obtain a thermosensitive hydrogel. The resulting system can typically gel within 15–90 minutes at body temperature, and the compressive modulus can reach 5–15 kPa, which is close to the mechanical range of natural soft tissue.

[0007] This hydrogel can be formulated into an injectable dosage form, maintaining low viscosity at low temperatures, making it suitable for minimally invasive clinical procedures. The system offers flexible storage options: it can be stably stored for several weeks at 2–8°C for short periods, and for longer storage, it can be frozen at -20°C for several months, maintaining its physicochemical properties and biological activity within acceptable ranges.

[0008] The ternary composite matrix hydrogel provided by this invention can be applied to various soft tissue repair scenarios, especially suitable for situations requiring both fat grafting and blood supply reconstruction, such as breast volume reconstruction, neck and facial tissue defects, diabetic foot wounds, and repair of ischemic areas of the heart or necrotic areas of the liver. Through the synergistic effect of the three matrix types, this system exhibits superior comprehensive performance compared to single or binary matrix materials in terms of fat regeneration, vascularization, and tissue support, providing a feasible solution to overcome the limitations of existing repair materials with unbalanced functions.

[0009] The preparation method of the ternary composite decellularized extracellular matrix hydrogel of the present invention includes the following steps:

[0010] (1) Prepare DAT, SIS and Adv decellularized matrix respectively;

[0011] (2) DAT, SIS and Adv are compounded in a preferred ratio and ground into powder of 1-20 μm;

[0012] (3) Dissolve the decellularized matrix powder in an acidic protease solution and stir at 4°C for 48-72 hours;

[0013] (4) Adjust the pH to 7.4, add a cross-linking agent and cross-link at body temperature for 15-90 minutes to form a thermosensitive hydrogel.

[0014] Medical Application: The application of ternary composite decellularized extracellular matrix hydrogel in the preparation of soft tissue repair materials that promote fat regeneration and vascularization synergistically. The soft tissue repair materials are used in clinical treatments such as breast reconstruction, facial and neck filling, diabetic foot repair, wound healing, repair of necrotic areas of the heart or liver.

[0015] Specifically, the ternary composite decellularized extracellular matrix hydrogel of the present invention comprises: adipose acellular matrix (DAT), small intestinal submucosa acellular matrix (SIS), and vascular acellular matrix (Adv) in a ternary composite; cross-linked by a cross-linking agent to form a thermosensitive hydrogel; the hydrogel is in a fluid state at low temperature and gradually gels at body temperature.

[0016] The hydrogel described above is characterized in that the mass ratio of DAT, SIS and Adv is from 2:1:1 to 1:2:1; preferably selected from 2:1:1, 1.5:1:1 or 1:2:1.

[0017] In the above-mentioned hydrogel, the crosslinking agent is selected from natural crosslinking agents or chemical crosslinking agents.

[0018] In the above-mentioned hydrogel, the natural crosslinking agent is selected from genipin, tannic acid, ferulic acid, gallic acid, quercetin or a combination thereof, or the chemical crosslinking agent is selected from glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), hexamethylene diisocyanate (HMDI) or a combination thereof.

[0019] The aforementioned hydrogel completely gels at body temperature within 15-90 minutes.

[0020] The above-mentioned hydrogel, wherein the decellularized matrix is ​​ground into a powder of 1-20 μm.

[0021] The hydrogel described above is in an injectable form.

[0022] In the above-mentioned hydrogel, the crosslinking agent is genipin with a concentration of 0.1-0.3% (w / v) or the crosslinking agent is tannic acid with a concentration of 0.1-0.2% (w / v).

[0023] In the above-mentioned hydrogel, the crosslinking agent is an EDC / NHS combination, with an EDC concentration of 5-15 mM and an NHS concentration of 2.5-7.5 mM.

[0024] The decellularized matrix powder in the above-mentioned hydrogel has a particle size of 5-15 μm.

[0025] The hydrogel described above has a compressive modulus of 5-15 kPa.

[0026] Preferably, the crosslinking agent in the hydrogel of the present invention initiates the crosslinking reaction at room temperature and significantly accelerates the gelation process at body temperature (37°C), thereby achieving precise temperature response control.

[0027] Preferably, the prepared hydrogel can be stored for a short period of 7-60 days at 2-8℃ or frozen for a long period of 6-18 months at -20℃, while maintaining good physicochemical properties and biological activity.

[0028] Another object of the present invention is to provide a method for preparing the above-mentioned ternary composite decellularized extracellular matrix hydrogel, comprising the following steps:

[0029] (1) Prepare DAT, SIS and Adv decellularized matrix respectively;

[0030] (2) The DAT, SIS and Adv are ternarily compounded and ground into 1-20μm powder;

[0031] (3) Dissolve the decellularized matrix powder in an acidic protease solution and stir at 4°C for 48-72 hours;

[0032] (4) Adjust the pH to 7.4, add a cross-linking agent and cross-link at body temperature for 15-90 minutes to form a thermosensitive hydrogel.

[0033] In the above preparation method, the mass ratio of DAT, SIS and Adv in step (2) is 2:1:1 to 1:2:1.

[0034] The preparation method described above, in step (1) the preparation of DAT includes: fresh adipose tissue undergoing decellularization, freeze-thaw cycles, polar solvent extraction for defatting, enzyme treatment, and freeze-drying at -80℃.

[0035] In the above preparation method, the acidic protease solution in step (3) is a 0.01M HCl solution containing 0.1mg / mL pepsin.

[0036] Another objective of this invention is to provide the application of the aforementioned ternary composite decellularized extracellular matrix hydrogel in the preparation of materials that promote tissue repair, organ repair, and soft tissue filling in animals or humans.

[0037] The aforementioned applications include the use of soft tissue repair materials for breast reconstruction, facial and neck filling, diabetic foot repair, wound healing, repair of necrotic areas of the heart, or repair of necrotic areas of the liver.

[0038] In the above applications, the tissue is animal or human tissue.

[0039] The ternary composite decellularized extracellular matrix hydrogel of the present invention uses a natural decellularized matrix and a biocompatible crosslinking agent, and has good biosafety and biodegradability; the mechanical properties are optimized: the compression modulus is 5-15 kPa.

[0040] The ternary composite decellularized extracellular matrix hydrogel of this invention maintains its activity under various storage conditions (2-8℃; -20℃; -5℃), especially under normal freezing conditions (-5℃). This provides a wider range of possibilities for cold chain preservation of the product. Attached Figure Description

[0041] Figure 1 Flowchart of the preparation of ternary composite decellularized extracellular matrix hydrogel

[0042] Figure 2 Comparison of gelation properties of hydrogels with different ratios: The left axis represents gelation time (minutes), the right axis represents compressive modulus (kPa), and the horizontal axis represents the ratio (DAT:SIS:Adv).

[0043] Figure 3 Comparison of hydrogel properties treated with different crosslinking agents: Each column is labeled with the name of the crosslinking agent (Genipin, Tannic acid, Ferulic acid; EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; NHS: N-hydroxysuccinimide; Glutaraldehyde: glutaraldehyde), the left axis is labeled with the compressive modulus (kPa); the right axis is the biocompatibility score. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. In the description of this invention, it should be noted that the term "or" is generally used to include the meaning of "and / or," unless otherwise expressly stated. Unless otherwise specified, the preparation of DAT, SIS, and Adv is carried out in accordance with Example 1.

[0045] Example 1: DAT-SIS-Adv Genipin Crosslinked Hydrogel

[0046] Step 1: Preparation of decellularized matrix:

[0047] ① Preparation of decellularized adipose-derived matrix (DAT): Porcine adipose tissue was rinsed with distilled water, minced, and then repeatedly rinsed with PBS. After decellularization by shaking on a constant-temperature shaker, the tissue was homogenized, centrifuged, and the white precipitate at the bottom was collected. The precipitate was then placed in 1% Trion-X100, shaken, rinsed with distilled water, and then the residual adipose tissue was removed with isopropanol. The DAT was then enzymatically digested with DNase and RNase, and the resulting DAT was frozen at -80°C for 24 hours, then freeze-dried and ground into powder.

[0048] ② Preparation of decellularized SIS. Small intestine and jejunum from healthy adult pigs were repeatedly rinsed with deionized water to remove any adhering material from the inner lumen. Then, the intestines were soaked and washed with 100 mmol / L EDTA, 1 mol / L NaCl, 1 mol / L NaCl in PBS, and PBS, respectively. The mixture was then sterilized with 0.1% peracetic acid in 20% ethanol. The mixture was then frozen at -80°C for 24 hours, lyophilized, and ground into powder.

[0049] ③ Preparation of decellularized vascular matrix Adv: Porcine aortic tissue specimens were placed in a solution of 8 mM CHAPS (3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate), 1 M NaCl and 25 mM EDTA (ethylenediaminetetraacetic acid), incubated, washed in PBS, and then shaken in a solution of 0.1% trypsin and 0.04% EDTA for 1 h; then shaken in a solution of SDS, sodium chloride and EDTA, sterilized in a solution of deionized water, 0.1% peracetic acid and 4% ethanol, frozen overnight at 80 °C and lyophilized.

[0050] Step 2: Ternary compound: Weigh 1.5g, 1.0g, and 1.0g of DAT, SIS, and Adv powder respectively in a mass ratio of 1.5:1:1. Add 10ml of sterile PBS buffer and stir at 4-8℃ for 24 hours to allow it to fully swell and mix, thus obtaining a ternary compound solution.

[0051] Step 3: Crosslinking to form a gel: Add 0.2% (w / v) genipin solution to the ternary composite solution and crosslink at 37°C for 15-45 minutes to form a hydrogel with good elasticity and temperature sensitivity.

[0052] The gelation behavior of the genipin crosslinking system at different temperatures is as follows: Room temperature (20℃): The crosslinking reaction proceeds slowly, and 50% gelation is completed in 120-150 minutes, providing sufficient time for clinical operation; Body temperature (37℃): The crosslinking reaction is significantly accelerated, and complete gelation is completed in 35-45 minutes, meeting the requirements for rapid shaping after implantation; Temperature response coefficient: The reaction rate at 37℃ is 3-5 times higher than that at room temperature.

[0053] Storage performance evaluation:

[0054] 1) Short-term storage (2-8℃): After 14 days of storage of the hydrogel precursor solution, the gelation time at body temperature (37℃) is extended to 50-55 minutes, and the compressive modulus remains at 8.0±0.9kPa; the performance is stable.

[0055] 2) Long-term storage (-20℃): After freezing for 6 months, the gelation time at body temperature (37℃) is 48-50 minutes, the compressive modulus is 8.2±0.8kPa, and the performance is stable.

[0056] 3) Long-term storage (-5℃): After freezing for 6 months, the gelation time at body temperature (37℃) is 40-44 minutes, the compressive modulus is 8.5±0.6kPa, and the performance is stable.

[0057] This experiment verifies that the ternary matrix hydrogel of the present invention can maintain its activity under various storage conditions (2-8℃; -20℃; -5℃), especially under normal freezing conditions (-5℃), it can still maintain its functional activity. This provides a wider range of possibilities for the cold chain preservation of the product.

[0058] Example 2: DAT-SIS-Adv Tannic Acid Crosslinked Hydrogel

[0059] Three decellularized matrices were prepared according to the method in Example 1 and compounded in a mass ratio of 2:1:1, using 0.15% (w / v) tannic acid as a crosslinking agent. The matrices were completely gelled at 37°C for 45 minutes. The gel strength was 6.8 ± 0.7 kPa.

[0060] Storage performance evaluation:

[0061] 1) Short-term storage (2-8℃): After 14 days of storage of the hydrogel precursor solution, the gelation time at body temperature (37℃) is extended to 53-62 minutes, and the compressive modulus is maintained at 6.5±0.4kPa;

[0062] 2) Long-term storage (-20℃): After freezing for 6 months, the gelation time at body temperature (37℃) is 59-63 minutes, and the compressive modulus is 6.2±0.4kPa.

[0063] 3) Long-term storage (-5℃): After freezing for 6 months, the gelation time at body temperature (37℃) is 60-64 minutes, and the compressive modulus is 6.5±0.3kPa.

[0064] Example 3: DAT-SIS-Adv-ferulic acid crosslinked hydrogel

[0065] Prepared according to the method of Example 1, with the ratio adjusted to 1:2:1, and crosslinked using 0.1% (w / v) ferulic acid. The temperature dependence of ferulic acid crosslinking is as follows: at room temperature: ferulic ester bond formation is slow, requiring 150-200 minutes for initial gelation; at 37°C: the activation energy of the esterification reaction decreases, and complete crosslinking occurs within 30 minutes. Performance test results: gel strength: compressive modulus is 5.2 ± 0.6 kPa.

[0066] Example 4: Chemical crosslinking agent EDC / NHS system

[0067] A chemical crosslinking system of EDC (10 mM) + NHS (5 mM) was used, with a DAT:SIS:Adv ratio of 1.5:1:1. The temperature sensitivity of the EDC / NHS system was as follows: at room temperature (25℃), EDC activation was slow, and NHS ester formation required 180 minutes; at 37℃, the amide bond formation reaction was significantly accelerated, and complete crosslinking was achieved in 80 minutes. Performance test results: gel strength: compressive modulus was 12.3 ± 1.1 kPa.

[0068] Example 5: DAT-SIS-Adv glutaraldehyde crosslinked hydrogel

[0069] Preparation process: Weigh 1.0g, 1.0g, and 2.0g of DAT, SIS, and Adv powders respectively at a mass ratio of 1:1:2, add 12ml of acidic protease solution (pH 3.0, pepsin concentration 0.1mg / ml), and digest at 4℃ for 48 hours. Adjust the pH to 7.4, add 0.05% (w / v) glutaraldehyde solution, and crosslink at 37℃ for 30 minutes. Performance characteristics are as follows: Outstanding vascularization guidance: vascular density increased by 65%, exceeding other formulations; Gel strength: compressive modulus of 4.2±0.5kPa, relatively soft but sufficiently supportive, suitable for rapid vascular remodeling needs; Special applications: Repair of wounds requiring strong vascularization, such as diabetic foot.

[0070] Example 6: DAT-SIS-Adv-quercetin crosslinked hydrogel

[0071] Preparation process: Weigh 3.0g, 1.0g, and 1.0g of DAT, SIS, and Adv powders respectively at a mass ratio of 3:1:1. A two-step cross-linking method is used: first, pre-cross-link with 0.08% (w / v) quercetin at 4℃ for 12 hours, then transfer to 37℃ for complete cross-linking for 60 minutes. Performance characteristics: Strong fat regeneration capacity; gel strength: compressive modulus of 7.8±0.9kPa. Special applications: Suitable for breast reconstruction and other scenarios requiring large-volume fat grafting.

[0072] Example 7: Gallic acid combined with genipin double crosslinking system

[0073] Preparation process: A standard ratio of DAT:SIS:Adv = 1.5:1:1 was used, employing a dual crosslinking agent system of gallic acid (0.1% w / v) + genipin (0.15% w / v). Gallic acid was first added and pre-crosslinked at 25℃ for 30 minutes, followed by the addition of genipin and completion of crosslinking at 37℃ for 45 minutes. Performance characteristics are as follows:

[0074] Performance characteristics: Controllable crosslinking density: Mechanical strength can be adjusted by the ratio of dual crosslinking agents, suitable for long-term support; compressive modulus is 10.2±1.2kPa.

[0075] Example 8: HMDI chemically crosslinked ultra-high strength hydrogel

[0076] Preparation process: A DAT:SIS:Adv ratio of 1.5:1:1 was used, with 0.2% (w / v) hexamethylene diisocyanate (HMDI) as the crosslinking agent. Crosslinking was performed at room temperature for 4 hours under anhydrous conditions to form a high-strength hydrogel. Performance characteristics are as follows: Ultra-high mechanical strength: compressive modulus reaches 18.5±2.1 kPa; Shape memory: possesses a certain shape recovery ability; Suitable for soft tissue repair in load-bearing areas, such as buttock augmentation.

[0077] Example 9: Injectable Thermosensitive Hydrogel System

[0078] Preparation process: A DAT:SIS:Adv ratio of 1.5:1:1 was used, with the addition of thermosensitive additives gelatin (5% w / v) and sodium alginate (1% w / v). Genipin 0.18% (w / v) was used for cross-linking to form a thermosensitive system that is a low-viscosity liquid at 4°C and rapidly gels at 37°C. Performance characteristics are as follows: Precise thermosensitivity: gelation temperature 32-35°C, complete gelation within 30 minutes at body temperature; Excellent injection performance: viscosity <500 cP at 4°C, allowing for smooth injection through a 25G needle; Controllable gelation: gelation time and degree are precisely controlled by temperature.

[0079] The viscosity of this temperature-sensitive system further decreases to 300 cP when stored at 2-8℃, and the injection resistance is reduced by 25%; after long-term storage at -20℃, it can fully restore its original fluidity when thawed to 4℃.

[0080] Comparative Example - Binary Composite and Ternary Composite Comparison

[0081] Control group: Binary composite hydrogel; prepared according to a DAT:SIS mass ratio of 1.5:1. DAT and SIS powders were mixed and prepared under the same digestion and cross-linking conditions as in Example 1. The gelation time was 75 minutes, and the compressive modulus was 6.8 kPa.

[0082] Experimental group: Ternary composite hydrogel (Example 1);

[0083] The detection indicators include: cell proliferation and angiogenesis.

[0084] 1. Cell proliferation assay (MTT method): Human adipose-derived mesenchymal stem cells (hADSCs) were cultured at 5 × 10⁻⁶ cells / mL. 4 The cells were seeded at a density of 1 / 2 well in a 96-well plate. 100 μL of binary composite hydrogel and 100 μL of ternary composite hydrogel were added to each well. The cells were incubated at 37 °C and 5% CO2 for 72 hours. 20 μL of MTT solution was added and the cells were incubated for another 4 hours. DMSO was added to dissolve the formazan crystals. The absorbance at 570 nm was measured using a microplate reader.

[0085] 2. Angiogenesis assay (Matrigel lumen formation): Human umbilical vein endothelial cells were used in a assay with a concentration of 2 × 10⁻⁶ cells / mL. 4 / Wells were seeded in Matrigel-pre-coated 96-well plates, hydrogel medium was added, and after 6 hours of incubation, lumen formation was observed under a microscope. Three fields of view were randomly selected from each well to count the number of lumens and measure the total length. The experimental results are compared below:

[0086]

[0087] Although binary composite hydrogels possess certain biological activity, they are significantly less effective than ternary composite hydrogels in promoting cell proliferation and angiogenesis, demonstrating the crucial role of the Adv component in vascularization.

[0088] Industrial application prospects:

[0089] The ternary composite decellularized extracellular matrix hydrogel of this invention maintains its activity under various storage conditions (2-8℃; -20℃; -5℃), especially under normal freezing conditions (-5℃). This provides broader possibilities for cold chain preservation of the product. This invention has broad market application prospects in fields such as plastic surgery, wound repair, and diabetes treatment. The technical solution provided by this invention solves the functional limitations of single or binary matrix materials in existing technologies, achieving synergistic promotion of fat regeneration and vascularization, and providing a new solution for soft tissue repair.

[0090] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A ternary composite decellularized extracellular matrix hydrogel, characterized in that, include: A ternary composite of decellularized adipose matrix (DAT), decellularized submucosal matrix of small intestine (SIS), and decellularized vascular matrix (Adv) is formed; a thermosensitive hydrogel is then formed by cross-linking with a cross-linking agent.

2. The hydrogel according to claim 1, characterized in that, The mass ratio of DAT, SIS, and Adv is from 2:1:1 to 1:2:

1.

3. The hydrogel according to claim 2, characterized in that, The mass ratio of DAT, SIS, and Adv is selected from 2:1:1, 1.5:1:1, or 1:2:

1.

4. The hydrogel according to any one of claims 1-3, characterized in that, The crosslinking agent is selected from natural crosslinking agents or chemical crosslinking agents; wherein the natural crosslinking agent is selected from one or more combinations of genipin, tannic acid, ferulic acid, gallic acid, and quercetin, or the chemical crosslinking agent is selected from one or more combinations of glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and hexamethylene diisocyanate (HMDI).

5. The hydrogel according to claim 4, characterized in that, The hydrogel completely gels within 15-90 minutes at body temperature.

6. The hydrogel according to claim 4, characterized in that, The hydrogel is stored under low-temperature conditions, and the storage parameters are short-term storage at 2-8℃ or long-term freezing storage at -20℃.

7. The hydrogel according to claim 4, characterized in that, The particle size of the decellularized matrix powder is 5-15 μm.

8. The hydrogel according to claim 4, characterized in that, The hydrogel has a compressive modulus of 5-15 kPa.

9. A method for preparing the ternary composite decellularized extracellular matrix hydrogel according to any one of claims 1-8, characterized in that, Includes the following steps: (1) Prepare DAT, SIS and Adv decellularized matrix respectively; (2) The DAT, SIS and Adv are ternarily compounded and ground into 1-20μm powder; (3) Dissolve the decellularized matrix powder in an acidic protease solution and stir at 4°C for 48-72 hours; (4) Adjust the pH to 7.4, add a cross-linking agent and cross-link at body temperature for 15-90 minutes to form a thermosensitive hydrogel.

10. The use of the ternary composite decellularized extracellular matrix hydrogel according to any one of claims 1-8 in the preparation of materials that promote tissue repair, organ repair, and soft tissue filling in animals or humans.

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