An injectable adipose-derived extracellular matrix composite material hydrogel

Abstract

The present invention discloses a method for preparing a hydrogel of an injectable adipose decellularized extracellular matrix composite material, and belongs to the field of biological tissue engineering technology. The present invention is the first to compound adipose decellularized matrix (DAT) with small intestinal submucosal decellularized matrix (SIS) or vascular decellularized matrix (Adv) in proportion, and uses genipin as a cross-linking agent of the composite hydrogel to enhance the anti-degradation property, thereby preparing an injectable composite hydrogel. The use of an injectable type eliminates the influence of complications caused by surgery, increases applicability, achieves the effect of repairing soft tissue defects, has high safety and practicality, and has a wide range of material sources and is easy to obtain, with good clinical application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of biological tissue engineering, and in particular relates to a method for preparing a hydrogel of an injectable fat-free extracellular matrix composite material. Background Art

[0002] Hydrogels are amphiphilic polymer networks formed in situ by physical or chemical crosslinking, which can absorb large amounts of fluid under physiological conditions without affecting their structure. Thermosensitive injectable hydrogels are viscous sols at room temperature, but transform into solidified gels at 37°C. Hydrogels swell to have high water content and elasticity with porous networks of 3D polymer networks, have good biocompatibility, and are biodegradable. Injectable hydrogel-based scaffolds have attracted considerable attention in tissue engineering over the years. Injectable hydrogels can locate various solutes and nutrients in tissues and can diffuse, and can be used as injectable materials to fill irregular shaped defects. Adipose tissue plays a vital role in providing insulation, cushioning, and aesthetics to the body. Soft tissue defects caused by trauma, congenital defects, surgical resection, and disease need to be promoted for their repair and regeneration. At present, adipose tissue transplantation has limitations such as high morbidity in the donor site, donor shortage, and immune compatibility. How to repair and regenerate trauma and improve the function of damaged tissues and organs is the focus of current adipose tissue engineering research. The early generation and long-term survival of adipose tissue are related to angiogenesis. There is a close relationship between angiogenesis and adipogenesis, especially in the early development of adipose tissue, where adipogenesis is closely related to angiogenesis. Decellularized matrices have natural ECM (extracellular matrix) components, good biocompatibility, and low immunogenicity. In addition, a major disadvantage of hydrogel materials implanted in the body is that active therapeutic substances will degrade rapidly, resulting in delayed tissue healing. Therefore, it is extremely important to control the biodegradation rate of biomaterials and preserve therapeutic agents.

[0003] Therefore, this study hopes to develop an injectable composite hydrogel, utilize the pro-angiogenic effects of extravascular matrix and SIS (small intestinal submucosa), and the pro-adipogenic effects of DAT (adipose decellularized matrix), to construct vascularized tissue-engineered fat, and use genipin cross-linking for tissue engineering to enhance its biomechanical properties. Summary of the invention

[0004] In view of the defects of the prior art, in order to construct vascularized tissue engineering fat, the present invention provides a fat-decellularized extracellular matrix composite hydrogel. An injectable composite ECM hydrogel is developed using DAT with a pro-adipogenic effect and Adv or SIS with a pro-angiogenic effect. Genipin is used as a cross-linking agent of the composite hydrogel to enhance the anti-degradation property. Finally, the adipogenic and angiogenic abilities of the composite hydrogel are also explored in vitro and in rat subcutaneous implantation models.

[0005] In order to achieve the above object, the present invention provides the following technical solutions.

[0006] The present invention provides a fat decellularized extracellular matrix composite hydrogel, characterized in that the composite hydrogel comprises a decellularized matrix including a small intestinal submucosal decellularized matrix (SIS) or a vascular decellularized matrix (Adv) composited with a fat decellularized matrix (DAT).

[0007] Furthermore, the mixing ratio of DAT to SIS or Adv in the composite hydrogel is 1:1.

[0008] Furthermore, the composite hydrogel is in an injectable form.

[0009] The present invention also provides a use of any of the above-mentioned adipose decellularized extracellular matrix composite hydrogels in preparing a vascularized tissue engineering adipose tissue scaffold.

[0010] The present invention also provides a use of any of the above-mentioned adipose decellularized extracellular matrix composite hydrogels in the preparation of soft tissue repair and regeneration materials.

[0011] The present invention also provides a method for preparing the composite hydrogel described above, characterized in that the preparation method specifically comprises the following steps:

[0012] (1) DAT preparation;

[0013] (2) SIS preparation or Adv preparation;

[0014] (3) preparing tissue decellularized extracellular matrix powder;

[0015] (4) Dissolution of decellularized extracellular matrix powder;

[0016] (5) Preparation of genipin cross-linked adipose decellularized extracellular matrix composite hydrogel.

[0017] Furthermore, the (1) DAT preparation includes the preparation of decellularized extracellular matrix using fresh pig fat. First, the adipose tissue is washed with distilled water and then cut into pieces, decellularized and defatted after freeze-thaw cycles and polar solvent extraction, and then frozen at -80°C overnight and freeze-dried.

[0018] Furthermore, the (2) SIS preparation includes: firstly, treating the small intestinal submucosal layer by physical methods, and mechanically scraping the small intestinal mucosal layer, serosa layer and muscular layer. Then, treating the small intestinal submucosal layer by sequential chemical immersion decellularization method, freezing at -80°C overnight and then freeze-drying; the (2) Adv preparation includes: preparing vascular extracellular matrix material using freshly obtained pig aortic tissue specimens. The aortic adventitia is placed in a solution of 8mM CHAPS (3-[(3-cholamidopropyl) dimethylammonium]-1-propanesulfonate), 1M NaCl and 25mM EDTA (ethylenediaminetetraacetic acid) to remove cells. After being placed in a solution of 0.1% peracetic acid and 4% ethanol for oscillation sterilization, it is frozen at -80°C overnight and freeze-dried.

[0019] Furthermore, the preparation of the tissue decellularized extracellular matrix powder (3) includes crushing the decellularized tissue to 1-20 μm, and then drying to obtain the adipose tissue decellularized extracellular matrix powder.

[0020] Furthermore, the (5) preparation of genipin cross-linked fat-free extracellular matrix composite hydrogel includes first preparing genipin powder into a 10 mg / mL PBS solution, and then stirring and cross-linking with the composite hydrogel (DAT and SIS hydrogel 1:1 composite; DAT and Adv hydrogel 1:1 composite) (0.2% (w / v) genipin and ECM solution) at 37°C.

[0021] Compared with the prior art, the present invention has the following beneficial effects.

[0022] The present invention innovatively compounds DAT with SIS and vascular decellularized extracellular matrix to prepare an injectable hydrogel. A vascularized adipose tissue engineering material is prepared, and it is injectable, eliminating the impact of complications caused by surgery. Experimental data show that the hydrogel compounded with DAT and SIS or Adv 1:1 has significantly higher angiogenic and adipogenic effects than DAT alone. A new injectable composite hydrogel was invented, and its great potential in adipogenesis and angiogenesis was discovered, providing new ideas for the development of subsequent research. Moreover, the injectable type reduces surgical risks and increases applicability. After cross-linking with genipin, the degradation of active therapeutic substances in the hydrogel material implanted in the body is slowed down, achieving a long-term fat regeneration effect.

[0023] The donors of the present invention have a wide range of sources and are not easily restricted.

[0024] The present invention adopts the injection mode, which is simple to operate and minimally invasive.

[0025] The injectable hydrogel material of the present invention can locate various solutes and nutrients in tissues and can diffuse, and is an injectable material that can fill irregularly shaped defects.

[0026] The injectable hydrogel material of the present invention has wide clinical application and has high safety and practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Freeze-dried fat decellularized tissue, small intestinal submucosal decellularized tissue, and vascular decellularized tissue. A is the powder image of freeze-dried fat decellularized tissue after grinding; B is the powder image of small intestinal submucosal decellularized tissue after grinding; C is the powder image of vascular decellularized tissue after grinding; D is the electron microscopic image of fat decellularized tissue; E is the electron microscopic image of small intestinal submucosal decellularized tissue; F is the electron microscopic image of vascular decellularized tissue.

[0028] Figure 2 HE staining results. A is adipose tissue before decellularization; B is adipose tissue after decellularization; C is SIS before decellularization; D is SIS after decellularization; E is vascular tissue before decellularization; F is vascular tissue after decellularization.

[0029] Figure 3 DAPI staining results. A is adipose tissue before decellularization; B is adipose tissue after decellularization; C is SIS before decellularization; D is SIS after decellularization; E is vascular tissue before decellularization; F is vascular tissue after decellularization.

[0030] Figure 4 Oil Red O staining results. A is a normal adipose tissue image; B is a fat-free DAT image.

[0031] Figure 5 DNA residue results.

[0032] Figure 6 Collagen and glycosaminoglycan content.

[0033] Figure 7 Degradation rate of hydrogel.

[0034] Figure 8 Gel time determination.

[0035] Fig. 9 Ninhydrin measures free amino acids.

[0036] Fig.10 AO / PI staining results after co-culture of materials and cells. A is the AO / PI staining image of the material co-cultured with adipose stem cells for 1, 3, and 5 days; B is the AO / PI staining image of the material co-cultured with endothelial cells for 1, 3, and 5 days.

[0037] Fig.11Statistical analysis of cell proliferation after co-culture of materials and cells. A is the CCK-8 statistical results of co-culture of materials and endothelial cells for 1 and 3 days; B is the CCK-8 statistical results of co-culture of materials and adipose stem cells for 1, 3, and 5 days.

[0038] Fig.12 Cell migration images and statistical analysis after co-culture of materials with endothelial cells. A: DAT+SIS 1:1, B: DAT+SIS 2:1, C: DAT+SIS 3:1, D: DAT+Adv 1:1, E: DAT+Adv2:1, F: DAT+Adv 3:1, G: DAT, H: SIS, I: Adv, J: negative control group.

[0039] Fig.13 Composite hydrogel material angiogenesis diagram. A is a picture of endothelial cells cultured in hydrogels with different ratios forming tubular structures; B uses Image J software to analyze the number of nodes (B), number of grids (C) and length of the tube (D) in the tubular structure.

[0040] Fig.14 DAT containing SIS or Adv 1:1 composite hydrogel promoted the expression of VEGF gene in endothelial cells and the secretion of VEGF in the culture medium. A is the analysis of VEGF expression in endothelial cells by QPCR; B is the analysis of VEGF expression in the culture medium by ELISA. **P<0.01 compared with DAT g group.

[0041] Fig.15 Identification of the multidirectional differentiation potential of adipose-derived stem cells. A and B are the results of Alizarin red staining after osteogenic induction of adipose-derived stem cells; C and D are the results of ALP staining after osteogenic induction of adipose-derived stem cells; E and F are the results of Oil Red O staining after adipogenic induction of adipose-derived stem cells. bar=20μm.

[0042] Fig.16 Composite hydrogel material promotes adipogenic differentiation of adipose stem cells. A is the result of Oil Red O staining; B is the statistical analysis of Oil Red O staining; C is the statistical graph of the expression levels of PPARγ and C / EBPa detected by QPCR; D is the expression level of PPARγ and C / EBPa detected by Western blot; E is the statistical graph of the expression level of PPARγ and C / EBPa detected by Western blot.

[0043] Fig.17 Hydrogel injection and animal sample collection. A is a diagram of hydrogel injection; B is the location of the composite hydrogel in the rat body; C is the collection of the composite hydrogel 1 week and 6 weeks after implantation.

[0044] Fig.18Histological evaluation. A is HE staining after 1 week and 6 weeks of in vivo sampling; B is Oil Red O staining after 1 week and 6 weeks of sampling; C is the statistical analysis of Oil Red O staining after 1 week and D is the statistical analysis of Oil Red O staining after 1 week and 6 weeks of sampling.

[0045] Fig.19 Angiogenesis was significantly enhanced in the combination hydrogels of DAT and SIS or Adv1:1 at 1 and 6 weeks after hydrogel injection. (A) Representative images of CD31 immunostaining in implants; (B) Statistical analysis of capillary numbers. DETAILED DESCRIPTION

[0046] The following examples will help to understand the present invention, but these examples are only for illustrating the present invention, and the present invention is not limited to these contents. The operating methods in the examples are all conventional operating methods in the art.

[0047] Example 1 The purpose of this example is to prepare acellular extracellular matrix (such as Figure 1 ), the specific steps are as follows.

[0048] Experimental Materials: Freshly obtained porcine fat, porcine small intestine, and porcine aorta samples were placed in cold sterile phosphate-buffered saline (PBS) and transported to the laboratory for later use.

[0049] 1. Preparation of adipose decellularized matrix (DAT): Use fresh pig fat to prepare decellularized extracellular matrix. First, rinse the adipose tissue with distilled water and cut it into pieces, then rinse it repeatedly with PBS. After rinsing, add distilled water, shake it in a constant temperature shaker at 37℃ for 30 minutes, and then perform decellularization. The steps are as follows:

[0050] 1. Soak in 1 mol / L sodium chloride solution at 37°C for 3 hours;

[0051] 2. Soak in PBS and shake at 37℃ for 1h;

[0052] 3. Rinse with PBS for 5 times and cut the fat tissue into small pieces, each with a volume of 0.5-1cm 3 ;

[0053] 4. Repeated freezing and thawing: put the fat into a container, put it into a -80℃ ultra-low temperature refrigerator and freeze it for 24 hours, then put it into a 37℃ constant temperature water bath to dissolve, and repeat this process 4 times;

[0054] 5. After the freeze-thaw cycle, add half the volume of distilled water to the obtained fat tissue and crush and homogenize it with a homogenizer at a speed of 10,000 rpm for 5 minutes;

[0055] 6. Centrifuge the homogenized tissue (1200 rpm, 5 min). After centrifugation, the material is separated into three layers. Collect the white precipitate at the bottom, remove the free fat in the upper layer and the remaining fat in the middle layer, and repeat the centrifugation process until no fat tissue remains;

[0056] 7. Collect the white precipitate, add 1% Trion-x100, and shake at 37℃ for 1h;

[0057] 8. Rinse with distilled water for 30 minutes, repeat 3 times;

[0058] 9. Place the precipitate in 100% isopropanol solution at 37°C overnight to remove residual adipose tissue;

[0059] 10. Add 1 mol / L sodium chloride solution containing DNase and RNase, and shake at 37°C overnight;

[0060] 11. Centrifuge the shaken adipose tissue at 1000g at 4°C for 5 min, then rinse the precipitate with distilled water three times, 30 min each time;

[0061] 12. Freeze the obtained DAT in a -80℃ ultra-low temperature freezer for 24 hours, then freeze-dry it and grind it into powder using a grinder;

[0062] 13. Seal the powder and sterilize it with ethylene oxide, then store it in the refrigerator for later use.

[0063] 2. Preparation of decellularized SIS.

[0064] 1. Physical treatment:

[0065] (1) Take 15 cm of the small intestine jejunum of a healthy adult pig;

[0066] (2) Use deionized water to repeatedly rinse the material adhering to the inner lumen. Use mechanical methods to scrape off the small intestinal mucosal layer, turn it over, scrape off the serosal layer and muscle layer, and rinse it with deionized water to obtain a translucent SIS.

[0067] 2. Sequential chemical immersion decellularization method:

[0068] The SIS treated by physical methods was cut along the longitudinal axis and decellularized by a series of chemical immersion methods at room temperature, with the volume ratio of material to solution maintained above 1:100.

[0069] (1) 100 mmol / L EDTA (adjust pH to 11-12 with NaOH), 16 h;

[0070] (2) 1 mol / L NaCl (adjust pH to 0-1 with HCl), 6-8 h;

[0071] (3) PBS containing 1 mol / L NaCl, 16 h;

[0072] (4) Wash with PBS 4 times, 30 min each time;

[0073] (5) Wash with deionized water 4 times, 30 minutes each time;

[0074] (6) Sterilization: 0.1% peracetic acid in 20% ethanol, 8 hours;

[0075] (7) Clean the SIS with sterile saline in a clean bench, gently wipe it dry with sterile gauze, spread it out under ultraviolet light for sterilization, 15 minutes on each side. When turning it over, spread it out in different containers to prevent contamination;

[0076] (8) Aliquot and store in PBS at 4°C.

[0077] 3. Preparation of vascular decellularized matrix Adv: Freshly obtained pig aorta tissue specimens were used to prepare vascular adventitia decellularized extracellular matrix materials.

[0078] 1. Place the aortic adventitia in a solution of 8 mM CHAPS (3-[(3-cholamidopropyl) dimethylammonium]-1-propanesulfonate), 1 M NaCl and 25 mM EDTA (ethylenediaminetetraacetic acid) and incubate at 37°C for 24 h;

[0079] 2. Wash three times in 1× PBS (phosphate buffered saline), 10 min each time;

[0080] 3. Wash in deionized water 3 times, 10 minutes each time;

[0081] 4. Place the tissue in a solution containing 0.1% trypsin and 0.04% EDTA and shake for 1 hour;

[0082] 5. Wash thoroughly with deionized water;

[0083] 6. Shake for 24 hours in a medium containing 0.5% SDS, 1M NaCl and 25mM EDTA;

[0084] 7. Wash thoroughly with 1×PBS;

[0085] 8. Wash thoroughly with deionized water;

[0086] 9. Place in a solution of 0.1% peracetic acid and 4% ethanol and shake;

[0087] 10. Wash thoroughly with deionized water;

[0088] 11. Freeze at 80℃ overnight and freeze-dry.

[0089] Example 2 Evaluation of decellularization effect.

[0090] 1. Experimental methods

[0091] (A) Histological examination.

[0092] In order to evaluate the effectiveness of decellularization and oil removal, this study first performed OCT embedding and frozen section preparation on fresh materials and decellularized materials, and then performed HE staining, DAPI staining, and Oil Red O staining on the frozen sections.

[0093] 1. OCT embedding and frozen section preparation: After the tissue is fixed with 4% paraformaldehyde, it is dehydrated with 20% sucrose and 30% sucrose overnight, and fixed with liquid nitrogen after it sinks to the bottom. Then it is fixed with OCT embedding medium, and be careful not to let air bubbles enter the material. Cut 8μm slices with a freezing microtome, label and seal them and store them in a -20℃ refrigerator for later use.

[0094] 2. HE staining steps: Hematoxylin-eosin (HE) staining is a commonly used staining method. Hematoxylin staining solution can make the cell nucleus appear blue-purple; eosin can make the cytoplasm and extracellular matrix appear red. HE staining can be used to observe the remnants of the cell nucleus after decellularization. The specific staining steps are as follows:

[0095] (1) Wash the slices with distilled water for 1 min;

[0096] (2) Fix with 4% paraformaldehyde for 10 min;

[0097] (3) Stain the slides with hematoxylin for 8 minutes and rinse with distilled water;

[0098] (4) Differentiation with 1% hydrochloric acid-ethanol for 1-3 seconds, and then rinse with tap water for a while;

[0099] (5) Stain the slides with eosin solution for 1 min and wash with distilled water for 1 min;

[0100] (6) Alcohol dehydration: 95% ethanol for 5 min, 100% ethanol I for 5 min, 100% ethanol II for 5 min;

[0101] (7) Transparent: xylene solution I and II for 5 min each;

[0102] (8) Seal the slides with neutral gum and observe after drying.

[0103] 3. Oil Red O staining steps: Oil Red O staining is a common staining step that can dye lipid droplets bright red. Through Oil Red O staining, the residual lipid droplets after degreasing can be observed. First, prepare the working solution: add saturated Oil Red O stock solution to distilled water in a ratio of 3:2 (Oil Red O: distilled water), mix well, place at room temperature for 10 minutes, filter with filter paper and funnel, and set aside. The staining steps are as follows:

[0104] (1) Fix the slices with paraformaldehyde for 10 min;

[0105] (2) After fixation, wash thoroughly with distilled water to remove the fixative;

[0106] (3) Soak and clean once in 60% isopropyl alcohol;

[0107] (4) Staining with Oil Red O solution for 15 min;

[0108] (5) 60% isopropanol differentiation until the intercellular matrix is ​​clear;

[0109] (6) Cleaning with distilled water;

[0110] (7) Counterstaining of cell nuclei with hematoxylin;

[0111] (8) Cleaning with distilled water;

[0112] (9) After adding the coverslip, seal the slide with glycerol gelatin;

[0113] (10) When observing the slide under a microscope, the lipid droplets in the tissue cells appear bright red, the cell nuclei appear blue, and the intercellular matrix appears colorless.

[0114] 4. DAPI staining steps: DAPI is a common nuclear dye that can make the cell nucleus appear blue. DAPI staining can be used to observe the residual amount of cell nuclei after decellularization. The specific steps are as follows:

[0115] (1) Fix the slices with paraformaldehyde for 10 min;

[0116] (2) Wash thoroughly with distilled water to remove the fixative;

[0117] (3) Place a small amount of DAPI stain on the sliced ​​tissue to cover it and stain for 5 minutes;

[0118] (4) Wash with PBS and seal the slides;

[0119] (5) Blue fluorescent dot-like cell nuclei can be seen under a fluorescence microscope.

[0120] (B) Detection of residual DNA.

[0121] Take 25 mg of freeze-dried fresh tissue and decellularized tissue (n=3 per group), cut into pieces, add 600 μL LysisBuffer, and mix thoroughly. Add 20 μL RNase A, mix well, and let stand at room temperature for 5 minutes. Add 10 μL Proteinase K, mix well, and place in a 56°C water bath overnight. Centrifuge at 12000 rpm / min for 10 minutes, transfer the supernatant to a new centrifuge tube, add 800 μL anhydrous ethanol, and mix well. Transfer the liquid to the centrifuge column, centrifuge at 12000 rpm / min for 1 minute, and discard the supernatant. Add 700 μL Washbuffer A (with anhydrous ethanol), centrifuge at 12000 rpm / min for 1 minute, and discard the supernatant. Add 700 μL Wash buffer B (with anhydrous ethanol), centrifuge at 12000 rpm / min for 1 minute, and discard the supernatant. Add 500 μL Wash buffer B, centrifuge at 12000 rpm / min for 1 minute, and discard the supernatant. Centrifuge again at 12000rpm / min for 2min, place the centrifuge column in a new centrifuge tube, open the centrifuge column cover, and place at 37℃ for 10min until there is no obvious ethanol smell. Add 50μL of TE buffer (60℃) to the center of the silicon matrix membrane, place at room temperature for 3min, centrifuge at 12000rpm for 30s, add the supernatant to a new centrifuge column again, place at room temperature for 2min, and centrifuge at 12000rpm for 2min. The supernatant obtained is the purified genomic DNA. Use an ultra-micro UV spectrophotometer to detect the DNA content.

[0122] (iii) Detection of collagen and GAG content in decellularized ECM.

[0123] Take 25 mg of freeze-dried fresh tissue and decellularized tissue (n=3 per group), cut into pieces, add 1 mL of 6 mol / L HCL, and place in a water bath at 95°C for 5 h. Adjust the pH value of the hydrolyzate to about 6.0~6.8, add double distilled water to a total volume of 1 mL, and mix well. Take 3~4 mL of the diluted detection solution and add about 25 mg of activated carbon, mix well, centrifuge at 3500 rpm / min for 10 min, and carefully take 1 mL of the supernatant for detection. According to the requirements of the instructions, add the detection reagents respectively, mix well, and place in a water bath at 60°C for 15 min. After cooling, centrifuge at 3500 rpm / min for 10 min, take 100 μL of the supernatant and add it to a 96-well plate, with a wavelength of 550 nm, and use an enzyme reader to detect the OD value, and adjust to zero with double distilled water. Calculate the content of hydroxyproline in each group, and then convert the content of hydroxyproline to the content of collagen based on the principle that hydroxyproline accounts for 13.4% of collagen.

[0124] According to the instructions of the glycosaminoglycan quantitative detection kit, 100 mg of freeze-dried fresh tissue and decellularized tissue (n=3 per group) were taken and placed in a centrifuge tube. 3 mL of cleaning solution was added to each tube for washing. 500 μL of extraction solution was added to each tissue, the tissue was cut into pieces, vortexed for 1 min, and mixed thoroughly. Incubate at 56°C for 16 h, then incubate at 90°C for 10 min, then centrifuge at 16000 g for 10 min, transfer the supernatant to a new 1.5 mL centrifuge tube, and place it in an ice trough for later use. Prepare standard samples and draw standard curves. Add 50 μL of the above-prepared sample to a 1.5 mL centrifuge tube, add 1 mL of staining solution, and vortex for 15 s. Incubate at room temperature in the dark for 30 min, vortexing for 15 s every 5 min, and then centrifuge at 16000 g for 10 min. Discard the supernatant, add 1 mL of dissociation solution, vortex for 15 seconds, and incubate at room temperature in the dark for 5 minutes. Take the supernatant and measure the absorbance of each tube at a wavelength of 656 nm, and obtain the glycosaminoglycan content corresponding to the sample according to the above standard curve.

[0125] 2. Experimental results.

[0126] HE staining results Figure 2 As shown (bar=100μm), the normal structure of adipose tissue has been lost, presenting as a disordered fiber sequence. Under a high-power microscope, the filamentous fibers are intertwined with each other, and no trace of any cells can be seen.

[0127] DAPI staining results are as follows Figure 3 As shown, no blue fluorescence can be seen in the decellularized fat tissue, proving that no nuclear DNA remains, suggesting that no cells remain.

[0128] Oil red O staining results Figure 4 As shown, no lipid components remain in the adipose decellularized tissue.

[0129] The results of DNA quantitative detection showed that the DNA content of fresh adipose tissue was 1662.22±12.57ng / mg, while that of decellularized adipose tissue was 26.66±10.88ng / mg; the DNA content of fresh SIS tissue was 2231.11±53.70ng / mg, while that of decellularized SIS tissue was 35.55±6.28ng / mg; the DNA content of fresh vascular tissue was 1551.11±45.32ng / mg, while that of decellularized vascular tissue was 17.77±6.28ng / mg. The difference in DNA content between the fresh group and the decellularized group was statistically significant (P<0.001), indicating that the decellularization was complete ( Figure 5, N-Adv: fresh blood vessel group, Adv: blood vessel decellularized matrix group, N-SIS: fresh small intestinal submucosa group, SIS: decellularized small intestinal submucosa group, N-DAT: fresh fat group, DAT: fat decellularized matrix group. Data are expressed as mean ± standard deviation (n=3), and one-way analysis of variance was used for comparison among groups. *P<0.05).

[0130] The results of quantitative detection of collagen showed that the collagen content in fresh adipose tissue was 120.15±0.23μg / mg, and the collagen content in decellularized adipose tissue was 500.13±0.54μg / mg; the collagen content in fresh SIS was 603.753±0.21μg / mg, and the collagen content in decellularized SIS was 590.82±0.5μg / mg; the collagen content in fresh vascular tissue was 300.77±0.64μg / mg, and the collagen content in decellularized vascular tissue was 280.33±0.73μg / mg ( Figure 6 A, Native: fresh material tube group, Decellularized: decellularized material group. DAT: adipose decellularized matrix group, SIS: small intestinal submucosal decellularized matrix group, Adv: vascular decellularized matrix group. Data are expressed as mean ± standard deviation (n = 3), and one-way analysis of variance was used for comparison between groups. *P < 0.05).

[0131] The results of quantitative detection of glycosaminoglycans showed that the content of glycosaminoglycans in fresh adipose tissue was 1.13±0.01μg / mg, and the content of glycosaminoglycans in decellularized adipose tissue was 1.11±0.02μg / mg; the content of glycosaminoglycans in fresh SIS was 0.83±0.01μg / mg, and the content of glycosaminoglycans in decellularized SIS was 0.82±0.02μg / mg; the content of glycosaminoglycans in fresh vascular tissue was 1.11±0.02μg / mg, and the content of glycosaminoglycans in decellularized vascular tissue was 1.08±0.03μg / mg ( Figure 6 B). There was no significant difference in collagen content and glycosaminoglycan content between the fresh group and the decellularized group (P>0.05). This indicates that there is no loss of collagen and glycosaminoglycans during the decellularization process and the decellularization method is mild.

[0132] Example 3 Detection of hydrogel properties of adipose-decellularized extracellular matrix composite materials.

[0133] 1. Experimental methods

[0134] (A) Preparation of powdered ECM bioscaffold by digestion.

[0135] The powdered material was dissolved in a hydrochloric acid pepsin solution (0.01 M HCl plus 0.1 mg / mL pepsin) at a concentration of 10 mg / mL and stirred for 48 h at 4 ° C. The pH of the ECM digest solution was then neutralized to 7.4 using 0.1 M NaOH and 20× PBS and diluted to 8 mg / mL using 1× PBS.

[0136] (ii) Preparation of genipin cross-linked ECM gel: First, genipin powder was prepared into a 10 mg / mL PBS solution, and then mixed with composite hydrogels (DAT and SIS hydrogel 1:1 composite; DAT and Adv hydrogel 1:1 composite) and stirred at 37°C for cross-linking (0.2% (w / v) genipin and ECM solution).

[0137] (III) Determination of ninhydrin

[0138] The cross-linking degree and free amino group content of genipin cross-linked ECM gel were analyzed using the ninhydrin method. The hydrogel and genipin cross-linked ECM gel were mixed with 2 mL of deionized water (dH2O) and 1 mL of 2% ninhydrin solution. The mixture was preheated at 100 °C for 10 min and cooled at room temperature to terminate the reaction. In addition, free amino groups were measured by spectrophotometry (Multiscan FC, ThermoScientific) at 570 nm. Different concentrations of glycine were prepared for the standard curve, and the degree of cross-linking was determined using the following equation.

[0139] .

[0140] where (NH2)nc is the molar fraction of free amino groups in the ECM gel, and (NH2)c is the molar fraction of free amino groups in the genipin cross-linked ECM gel.

[0141] (iv) Rheological and degradation analysis.

[0142] Rheology: PBS was used to dilute and prepare 2 mg / mL, 4 mg / mL, 6 mg / mL, and 8 mg / mL hydrogel materials. The gel flow was observed by tilting the reagent bottle at 0 min, 25 min, and 60 min, respectively.

[0143] Degradation: The degradation of DAT, SIS, and extravascular matrix hydrogels with or without crosslinking with genipin was evaluated after the gels were lyophilized, weighed, and further immersed in 1.5 mL of simulated body fluid (PBS) in Eppendorf tubes at 37 °C (water bath). After 24, 168, 336, 504, and 672 h, PBS was removed and the gels were weighed after lyophilization (Wi, initial dry sample weight, and Wt, dry sample weight at each time period). Finally, the degree of degradation was expressed as the percentage weight loss (wt%) of the gel according to the following equation.

[0144] .

[0145] 2. Experimental results.

[0146] (A) Detection results of hydrogel degradation.

[0147] The degradation rate of the hydrogel was determined by the mass loss of genipin-crosslinked DAT hydrogel (DATg), DAT hydrogel, crosslinked SIS hydrogel (SISg), SIS hydrogel, crosslinked vascular decellularized matrix hydrogel (Adv g), and Adv (vascular decellularized matrix hydrogel) in PBS at different time periods. During the degradation period, the degradation rates of DATg, SISg, and Advg gels were significantly reduced compared with those of DAT, SIS, and Adv gels. Among them, DATg had the lowest degradation rate, with a degradation rate of <25% at 4 weeks, while the degradation rates of uncrosslinked hydrogels were all >90%. This shows that the degradation rate of the hydrogel was significantly reduced after crosslinking with genipin ( Figure 7 , DAT g: adipose decellularized matrix group cross-linked with genipin, DAT: non-cross-linked adipose decellularized matrix group, SIS g: small intestinal submucosal decellularized matrix group cross-linked with genipin, SIS: non-cross-linked small intestinal submucosal decellularized matrix group, Adv g: vascular decellularized matrix group cross-linked with genipin, Adv: non-cross-linked vascular decellularized matrix group. Data are expressed as mean ± standard deviation (n = 3-6).

[0148] (B) Gel time results.

[0149] At 37°C, the hydrogels of different concentrations were all in a flowing state at 0 min; at 25 min, except for the hydrogel with a concentration of 2 mg / mL, all of them had already gelled ( Figure 8 , A, E, I are the gel states of 2 mg / mL gel at 0 min, 25 min, 60 min, respectively; B, F, J are the gel states of 4 mg / mL gel at 0 min, 25 min, 60 min, respectively; C, G, K are the gel states of 6 mg / mL gel at 0 min, 25 min, 60 min, respectively; D, H, L are the gel states of 8 mg / mL gel at 0 min, 25 min, 60 min, respectively). Among them, the 8 mg / mL group has the best gelation degree.

[0150] (III) Results of cross-linking degree determination using ninhydrin.

[0151] The color depth of the compound is proportional to the amino acid content, and the amino acid content can be determined by measuring the optical density at 570nm. The crosslinking degree of the composite group of DAT and SIS 1:1 is 53.05%, the crosslinking degree of the composite group of DAT and SIS 2:1 is 59.17%, the crosslinking degree of the composite group of DAT and SIS 3:1 is 44.11%, the crosslinking degree of the composite group of DAT and Adv 1:1 is 61.09%, the crosslinking degree of the composite group of DAT and Adv 2:1 is 86.66%, the crosslinking degree of the composite group of DAT and Adv 3:1 is 78.57%, the crosslinking degree of DAT is 81.96%, the crosslinking degree of SIS is 85.59%, and the crosslinking degree of Adv is 26.06% ( Fig. 9 , Native is the group without genipin cross-linking, Cross-linked is the group with genipin cross-linking. D+S 1:1 is the group with 1:1 combination of DAT and SIS, D+S 2:1 is the group with 2:1 combination of DAT and SIS, D+S 3:1 is the group with 3:1 combination of DAT and SIS, D+A 1:1 is the group with 1:1 combination of DAT and Adv, D+A 2:1 is the group with 2:1 combination of DAT and Adv, D+A 3:1 is the group with 3:1 combination of DAT and Adv, DAT is the group with adipose decellularized matrix, SIS is the group with decellularized small intestinal submucosal layer, and Adv is the group with decellularized vascular matrix. Data are expressed as mean ± standard deviation (n=3), and one-way analysis of variance was used for comparison among groups, *P<0.05, **P<0.01). Ninhydrin assay showed that the free amino acids in the hydrogel were significantly reduced after genipin cross-linking (P<0.01).

[0152] Example 4 In vitro and in vivo studies on the effects of the injectable adipose decellularized matrix composite material of the present invention on adipose tissue regeneration and angiogenesis.

[0153] 1. Materials and reagents.

[0154] 1. Cell culture: Human adipose-derived stem cells (hASCs) were purchased from the Chinese Academy of Sciences Stem Cell Bank. The purchased hASCs were resuspended in DMEM medium containing 10% fetal bovine serum and 1% double-antibody (penicillin-streptomycin solution), and the mixed cell suspension was re-spread in a culture dish and cultured in a 37°C incubator. The culture medium was replaced every 2 days. When the cell density reached 85%, the cells were passaged. The 3rd to 4th generations could be used for subsequent experiments.

[0155] 2. Experimental animals: Wistar rats (purchased from Beijing Huafukang Biotechnology Co., Ltd., China), male, 6 weeks old, weighing 160-200 g, 8 rats per group. The animal experiments were approved by the Ethics Review Committee of China Medical University (ethics number: CMU2021020) and were completed in the Experimental Animal Department of China Medical University.

[0156] 2. Experimental methods.

[0157] 1. Cytotoxicity Assay.

[0158] AO (Acridine Orange) is a fluorescent dye with membrane permeability, which can dye the cell nucleus yellow-green; PI (Propidium Iodide) is a DNA-binding dye that cannot pass through the living cell membrane to dye the cell nucleus red. Use the AO / PI double staining kit to stain, put the hydrogel into a six-well plate (50μL / cm²), 37℃, 30min, inoculate adipose stem cells and endothelial cells respectively, culture for 1 day, 3 days, and 5 days, wash the cells twice with PBS, then add an appropriate volume of staining working solution, incubate at 37℃ for 40min, dry the staining working solution, wash the culture plate twice with culture medium, and then select 488nm excitation light for microscopy under a fluorescence microscope.

[0159] 2. Cell proliferation detection.

[0160] CCK-8 kit was used to detect the effect of materials on cell proliferation. “Extract + cells” was used as the experimental group, “standard culture medium + cells” was used as the control group, and “standard culture medium” was used as the blank group. 3 The cells / well were inoculated in a 96-well plate at a density of 1.54 μg / well. After culturing in an incubator for 24 h, the medium in each well was replaced with standard medium and extract, 100 μL per well. After culturing for 1, 3, and 5 days, 10 μL of CCK-8 solution was added to each well, incubated at 37°C for 2 h, and the absorbance was measured at 450 nm using a microplate reader.

[0161] Endothelial cells were cultured at 2×10 3 The cells / well were inoculated in a 96-well plate at a density of 1.54 μg / well. After culturing in an incubator for 24 h, the medium in each well was replaced with standard medium and extract, 100 μL per well. After culturing for 1, 3, and 5 days, 10 μL of CCK-8 solution was added to each well, incubated at 37°C for 2 h, and the absorbance was measured at 450 nm using a microplate reader.

[0162] .

[0163] 3. Endothelial cell migration assay.

[0164] Human umbilical vein endothelial cells (HUVECs) were seeded in 6-well plates and grown as monolayers. Scratches were created by scraping with a 200 μL sterile pipette tip, and the culture medium was replaced with different groups of extracts supplemented with 2% FBS. Images were taken with a digital camera at 0, 24, and 48 hours after scratching and measured with ImageJ software. The scratch closure area was measured by the migration distance of migrating cells, and the data were reported as the relative percentage of the scratch.

[0165] 4. Angioplasty experiment.

[0166] Matrigel was coated on the well surface of a 24-well culture plate to prepare a cell culture matrix. After gelation for 1 hour in a 37°C incubator, a total of 1×10 4 Endothelial cells were seeded on the gel substrate in triplicate and then treated with culture medium containing hydrogel extracts. To evaluate the formation of endothelial cell branches, images were taken using a microscope. The total number and length of tubular structures were quantified using ImageJ software.

[0167] 5. Adipogenic and osteogenic induction of hASCs.

[0168] When hASCs in the culture flask were seeded into a six-well plate, the cell density was approximately 2 × 10 5 , divided into two groups: experimental group and control group. The experimental group was cells + osteogenic induction solution, and the control group was standard culture medium group (10% FBS + 1% P / S + F12). The osteogenic induction solution was prepared with sodium β-glycerophosphate, dexamethasone, and vitamin C according to the proportion in the instructions. ALP (alkaline phosphatase) staining was performed after 14 days. Alizarin red staining was performed after 21 days.

[0169] 6. Western blot analysis.

[0170] Western blot was performed on hASCs after 7 days of induction. Protein (15 μg) was separated by SDS-PAGE and transferred to polyvinyl fluoride membrane. Anti-PPARγ, anti-C / EBPa and β-actin were incubated overnight at 4°C. After washing, the membrane was further incubated with HRP-polymerized secondary antibody. Imaging was performed using an automatic chemiluminescence image analysis system and evaluated using ImageJ software.

[0171] 7. qPCR Analysis.

[0172] The cells were lysed with TRIzol to extract total RNA, and 1 g of RNA was used for cDNA synthesis and QPCR analysis. The PCR cycle included 40 amplification cycles of template DNA and primer annealing at 60°C. -ΔΔ The relative expression of each target gene was calculated by the Ct method. GAPDH was used as an internal control. To quantitatively compare gene expression, the amplification efficiency of the primer pairs was verified. Table 1 lists the specific primer sequences.

[0173] Table 1

[0174] .

[0175] 8. ELISA analysis.

[0176] HUVECs were cultured with different groups of hydrogel extracts for 24 h, and the culture medium was collected. The VEGF content in the supernatant was quantified by ELISA kit according to the manufacturer's protocol.

[0177] 9. Animal experimental grouping and in vivo injection experiment.

[0178] Rats were divided into 9 groups, 8 in each group, including DAT+SIS 1:1 group, DAT+SIS 2:1 group, DAT+SIS 3:1 group, DAT+Adv 1:1 group, DAT+Adv2:1 group, DAT+Adv 3:1 group, DAT group, SIS group, and Adv group. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital and then fixed with a fixator. The cross-linked hydrogel was injected into the back of the rat using a syringe. Four sites were injected into each rat, and 1 mL of hydrogel was injected into each site. Rats with respiratory depression or death due to unknown reasons were excluded from the statistics after surgery. Samples were collected after 1 week and 6 weeks to evaluate angiogenesis and adipose tissue formation.

[0179] 10. Histological and immunohistochemical examinations.

[0180] The implants obtained in 9 were quickly processed for histological analysis. Briefly, the specimens were serially sectioned using HE staining and Oil Red O staining to observe the effect of in vivo fat regeneration. The immunohistochemical process was performed according to the steps described in Example 2. The collected tissues were fixed and embedded with 4% paraformaldehyde and sliced ​​at a thickness of 5 μm. CD31 antibody was used. Images were taken using a microscope. Positive staining was semi-quantitatively analyzed using ImageJ software.

[0181] 11. Statistical analysis.

[0182] One-way analysis of variance was used to evaluate the significance of different groups. Statistical analysis was performed using GraphPadPrism 8.0, and P < 0.05 was considered statistically significant.

[0183] 3. Experimental results.

[0184] 1. Cytotoxicity test results.

[0185] To verify the survival efficiency of hASCs and hUVECs (human umbilical vein endothelial cells) in hydrogels, adipose stem cells or endothelial cells were inoculated on the hydrogels after gelation, and AO / PI staining was performed on the plates at 1, 3, and 5 days, respectively. The wells were placed under a fluorescence microscope for photography, and green fluorescence was observed as live cells. The results showed that the cells survived well and no cell death was observed, confirming that the hydrogel had no toxic effect on the cells (such as Fig.10, DAT g: adipose decellularized matrix group cross-linked with genipin, DAT: non-cross-linked adipose decellularized matrix group, SIS g: small intestinal submucosa decellularized matrix group cross-linked with genipin, SIS: non-cross-linked small intestinal submucosa decellularized matrix group, Adv g: vascular decellularized matrix group cross-linked with genipin, Adv: non-cross-linked vascular decellularized matrix group; bar=100μm).

[0186] 2. The DAT and SIS or Adv1:1 compound group promoted the proliferation of adipose stem cells and endothelial cells.

[0187] The effect of the hydrogel on cell proliferation was evaluated and the results were as follows Figure 3 As shown. DAT and SIS alone stimulated endothelial cell proliferation. As the proportion of SIS or Adv in the composite hydrogel increased, the stimulating effect on proliferation also increased. In particular, the DAT and SIS or Adv 1:1 composite hydrogel significantly promoted endothelial cell proliferation on the third day, which was 1.5 times that of the control group ( Fig.11 A). At the same time, compared with the control group, the DAT and SIS (1:1) composite hydrogel also promoted the proliferation of hASCs ( Fig.11 B). The above results show that the composite hydrogel of DAT and SIS or Adv1:1 can significantly promote the proliferation of endothelial cells or hASCs. (In the figure: D+SIS 1:1g is the composite group of DAT and SIS1:1, D+SIS 2:1g is the composite group of DAT and SIS 2:1, D+SIS 3:1g is the composite group of DAT and SIS 3:1, D+Adv 1:1g is the composite group of DAT and Adv 1:1, D+Adv 2:1g is the composite group of DAT and Adv 2:1, D+Adv 3:1g is the composite group of DAT and Adv 3:1, DAT g is the adipose decellularized matrix group, SIS g is the small intestinal submucosal decellularized group, Adv g is the composite group of g is the vascular decellularized matrix group. Data are expressed as mean ± standard deviation (n = 3), and one-way ANOVA was used for comparison among groups, *p < 0.05, **p < 0.01, ***p < 0.001, compared with the Control group, #p < 0.05, ##p < 0.05, ###p < 0.05, compared with DATg).

[0188] 3. The DAT and SIS or Adv1:1 complex group promoted endothelial cell migration and angiogenesis.

[0189] The wound healing scratch assay was used to evaluate the migration and repair ability of hUVECs in vitro. After scratching 90% confluent cells with a 200 μL pipette tip, the scratch closure area was measured by the area covered by migrating cells at 0, 24, and 48 hours. The scratch closure area was significantly increased in the DAT+SIS1:1 group and the DAT+Adv 1:1 group ( Fig.12 ). Endothelial cells cultured in the composite hydrogels formed a network with clear capillary-like structures, while endothelial cells cultured in the DAT hydrogels did not form a network with capillary structures. As the proportion of SIS or Adv in the composite hydrogels increased, the capillary network structure also increased. In addition, as the proportion of SIS or Adv in the composite hydrogels increased, the node (primary stage of angiogenesis), mesh (intermediate stage of angiogenesis), and total tube length also increased significantly. Compared with the control group, the composite hydrogels significantly enhanced the formation of cell tubular structures within 6 hours ( Fig.13 ). QPCR results showed that compared with the DAT group, DAT and SIS or Adv 1:1 composite hydrogels could significantly promote the expression of angiogenic gene VEGF in vascular endothelial cells. In addition, ELISA analysis also showed that compared with the DAT group, DAT and SIS or Adv 1:1 composite hydrogels could significantly upregulate VEGF secretion in HUVECs ( Fig.14 ). These results indicate that the addition of SIS and Adv to DAT can significantly promote the formation of tubule networks and capillary structures, especially the 1:1 composite hydrogel of DAT and SIS or Adv has a better angiogenesis-promoting effect.

[0190] 4. The DAT and SIS or Adv1:1 compound group promoted the adipogenic differentiation of adipose stem cells.

[0191] After culture, mesenchymal stem cells have multiple differentiation abilities ( Fig.15 ). To investigate the effects of different hydrogel composites on the adipogenic differentiation of hASCs, hASCs were treated with hydrogel composites and cultured with adipogenic induction medium for 7 days. Oil red O staining showed that DAT and SIS or Adv 1:1 composite hydrogels increased the adipogenic differentiation of hASCs compared with DAT hydrogels ( Fig.16 A, B). QPCR analysis and Western blot analysis showed that the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT / enhancer binding protein a (C / EBPa), two markers of adipogenic differentiation, was upregulated in DAT and SIS or Adv1:1 composite hydrogels ( Fig.16 CE). These results indicate that the composite hydrogel promoted the adipogenic differentiation of hASCs.

[0192] 5. Histological evaluation after sampling.

[0193] One and six weeks after the animal experiment, the hydrogels were harvested for histological analysis ( Fig.17HE staining confirmed cell infiltration in the tissue surrounding the hydrogel, and angiogenesis was observed in all groups. Infiltration increased at 6 weeks compared with 1 week. Early angiogenesis was observed in all groups at 1 week. In particular, extensive angiogenesis was found in the DAT combined with SIS or ADV 1:1 groups. At 6 weeks, the most obvious angiogenesis was observed in the DAT group containing SIS or ADV 1:1 composite hydrogels ( Fig.18 A).

[0194] 6. The 1:1 combination of DAT and SIS or Adv promoted adipogenesis.

[0195] At 1 and 6 weeks, DAT hydrogels had better adipogenesis than SIS hydrogels or Adv hydrogels. At 1 week, as the ratio of SIS or Adv hydrogels increased, DAT hydrogels had better adipogenesis than SIS or Adv hydrogels. DAT hydrogels promoted the formation of adipose-like tissue, especially when the ratio of DAT to SIS or Adv was 1:1. However, at 6 weeks, hydrogels made with DAT and SIS or Adv in a 2:1 ratio also significantly promoted the formation of adipose-like tissue, with effects similar to those made with a 1:1 ratio of DAT and SIS or Adv ( Fig.18 BD).

[0196] 7. The combination of DAT and SIS or Adv1:1 promoted angiogenesis.

[0197] Adipose tissue regeneration and long-term survival require a high degree of vascularization. CD31 immunostaining was used to show the presence of endothelial cells required for vascular localization. As expected, both SIS and Adv had a better angiogenic effect compared with DAT at 1 and 6 weeks. Interestingly, CD31 was highly expressed in the DAT and SIS or Adv 1:1 group, significantly stronger than the DAT group and the DAT and SIS or Adv 1:3 group. This finding suggests that the combination of DAT with SIS or Adv 1:1 can effectively promote angiogenesis compared with DAT hydrogel alone ( Fig.19 , Scale bar in Figure A is 100 μm; in Figure B, *P<0.05, **P<0.01 vs. DATg group. #P<0.05, ##P<0.01 vs. DAT+SIS 1:1g group. $P<0.05 vs. DAT+Adv 1:1g group. Arrows indicate positive staining. NC, negative control).

[0198] In summary, the present invention is the first to compound the extracellular matrix of fat cells and SIS or Adv in proportion, develop a new injectable composite hydrogel, and find its great potential in fat regeneration and angiogenesis, which provides new ideas for the development of subsequent research. Moreover, the injectable type reduces surgical risks and increases applicability. After cross-linking with genipin, the degradation of active therapeutic substances in the hydrogel material implanted in the body is slowed down, achieving a long-term fat regeneration effect. And this composite hydrogel is a temperature-sensitive hydrogel, which can be made into a hydrogel preform and transported in a low-temperature environment to meet the clinical drug delivery or repair of soft tissue injuries.

Claims

1. A fat-free extracellular matrix composite hydrogel, characterized in that: The composite hydrogel includes a decellularized matrix including a small intestinal submucosal decellularized matrix (SIS) or a vascular decellularized matrix (Adv) and a fat decellularized matrix (DAT); the mixing ratio of DAT to SIS or Adv in the composite hydrogel is 1-2:

1.

2. The composite hydrogel according to claim 1, characterized in that The mixing ratio of DAT to SIS or Adv in the composite hydrogel is 1:

1.

3. The composite hydrogel according to claim 1, characterized in that The dosage form of the composite hydrogel is injection type.

4. Use of the adipose decellularized extracellular matrix composite hydrogel according to any one of claims 1 to 3 in preparing a vascularized tissue engineering adipose tissue scaffold.

5. Use of the adipose decellularized extracellular matrix composite hydrogel according to any one of claims 1 to 3 in the preparation of soft tissue repair and regeneration materials.

6. A method for preparing the composite hydrogel according to claim 1, characterized in that: The preparation method specifically comprises the following steps: (1) DAT preparation; (2) SIS preparation or Adv preparation; (3) preparing tissue decellularized extracellular matrix powder; (4) Dissolution of decellularized extracellular matrix powder; (5) Preparation of genipin cross-linked adipose decellularized extracellular matrix composite hydrogel.

7. The method for preparing the composite hydrogel according to claim 6, characterized in that: The (1) DAT preparation includes the preparation of decellularized extracellular matrix using fresh pig fat; first, the adipose tissue is rinsed with distilled water and then cut into pieces, and then decellularized and defatted after freeze-thaw cycles and polar solvent extraction, and then frozen at -80°C overnight and freeze-dried.

8. The method for preparing the composite hydrogel according to claim 6, characterized in that: The (2) SIS preparation includes: firstly, treating the small intestinal submucosal layer by physical methods, and mechanically scraping off the small intestinal mucosal layer, serosa layer and muscular layer; then treating the small intestinal submucosal layer by sequential chemical immersion decellularization method, freezing at -80°C overnight and then freeze-drying; the (2) Adv preparation includes: preparing vascular extracellular matrix material using freshly obtained pig aortic tissue specimens; placing the aortic adventitia in a solution of 8mM CHAPS (3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate), 1M NaCl and 25mM EDTA (ethylenediaminetetraacetic acid) to remove cells; placing it in a solution of 0.1% peracetic acid and 4% ethanol for oscillation sterilization, freezing at -80°C overnight and freeze-drying.

9. The method for preparing the composite hydrogel according to claim 6, characterized in that: The preparation of the tissue decellularized extracellular matrix powder (3) includes crushing the decellularized tissue to 1-20 μm and then drying it to obtain the adipose tissue decellularized extracellular matrix powder.

10. The method for preparing the composite hydrogel according to claim 6, characterized in that: The (5) preparation of the genipin cross-linked fat-free extracellular matrix composite hydrogel comprises first preparing genipin powder into a 10 mg / mL PBS solution, and then stirring and cross-linking with the composite hydrogel at 37°C; the composite hydrogel is a 1~2:1 composite of DAT and SIS hydrogel or a 1~2:1 composite of DAT and Adv hydrogel; the concentration of the genipin is 0.2% w / v genipin.

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