A heparinized dual growth factor recellularized adipose tissue decellularized scaffold material, its preparation method and application

Abstract

This invention relates to the field of biomedical materials technology, providing a heparinized dual-growth factor recellularized adipose tissue decellularized scaffold material, its preparation method, and its applications. The preparation method includes: decellularizing adipose tissue to obtain adipose tissue decellularized scaffold; heparinizing the scaffold to obtain a heparinized adipose tissue decellularized scaffold; loading hepatocyte growth factor and vascular endothelial cell growth factor onto the scaffold to obtain a heparinized dual-growth factor adipose tissue decellularized scaffold; seeding induced pluripotent stem cell-derived hepatocytes onto the scaffold, and culturing to obtain a heparinized dual-growth factor recellularized adipose tissue decellularized scaffold (RAT). This invention uses rat inguinal adipose tissue to prepare RAT material, which is low-cost, readily available, has a simple preparation process, and is easy to scale up. The RAT material of this invention can achieve liver tissue damage repair and hepatocyte regeneration, providing a new treatment strategy for acute liver failure.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and particularly relates to a heparinized dual growth factor recellularized adipose tissue decellularized scaffold material, its preparation method, and its application. Background Technology

[0002] Acute liver failure (ALF) is a common and critical illness in clinical practice, characterized by massive hepatocellular necrosis and rapid decompensation of liver function. Clinical manifestations include progressively worsening jaundice, coagulation disorders, and hepatic encephalopathy. The disease progresses rapidly and has an extremely poor prognosis, with an overall clinical mortality rate as high as 60-80%. Among these, drug-induced ALF caused by non-acetaminophen has a mortality rate approaching 80%, seriously threatening patients' lives. Currently, liver transplantation is the only curative treatment for ALF, but its clinical application is severely limited by factors such as donor shortages, post-transplant immune rejection, and high treatment costs. According to relevant studies, approximately 300,000 to 400,000 liver disease patients in my country progress to liver failure each year, while the number of liver transplant donors is far from meeting clinical needs, resulting in many patients dying while waiting for a donor. Therefore, developing novel, efficient, and safe treatment strategies for acute liver failure has become an urgent clinical problem to be solved.

[0003] Decellularized scaffolds are created by removing immunogenic contents from organs or tissues using physical, chemical, and biological methods, while preserving the extracellular matrix components. Therefore, the resulting decellularized scaffolds exhibit good biocompatibility and low immunogenicity. Major components of the extracellular matrix, such as collagen, glycosaminoglycans, and laminin, provide a favorable microenvironment for cell adhesion, proliferation, and differentiation. Among these, adipose-derived decellularized scaffolds offer unique advantages compared to those derived from other tissues and organs. Adipose tissue is abundant, readily available, and causes minimal donor damage, meeting the needs of large-scale clinical applications. The preparation process is simple, preserving the three-dimensional structure and bioactive factors of the natural extracellular matrix, mimicking the liver cell growth microenvironment. Excellent biocompatibility reduces transplant rejection after decellularization, improving treatment safety. Good plasticity and degradability allow for adaptation to liver injury sites, and in vivo degradation exhibits no significant toxicity. Low cost facilitates large-scale production and clinical translation, making low-cost and efficient treatment for acute liver failure possible.

[0004] Hepatocyte growth factor (HGF) can promote hepatocyte proliferation, migration, and differentiation, inhibit hepatocyte apoptosis, reduce liver inflammation, repair damaged liver tissue, regulate hepatic stellate cell activity, inhibit liver fibrosis, and provide support for liver tissue regeneration. Vascular endothelial growth factor (VEGF) mainly promotes the proliferation and angiogenesis of vascular endothelial cells in liver tissue, improves blood supply to liver tissue, alleviates ischemia and hypoxia, provides nutrients and oxygen for hepatocyte regeneration, and simultaneously helps reduce inflammation and accelerate liver tissue repair. Studies have confirmed that both HGF, alone or in combination, have certain repair effects in the treatment of acute liver failure. However, traditional single growth factors are easily degraded and cleared in vivo, making it difficult to maintain their effects and limiting their therapeutic efficacy.

[0005] Hepatocytes derived from induced pluripotent stem cells (iPSCs) offer unique advantages as seed cells for the recellularization of adipose-derived scaffolds. They can be induced from the patient's own cells, eliminating donor-specific limitations and allowing for large-scale expansion to meet the seed cell requirements of scaffold recellularization. Furthermore, due to their autologous induction, they significantly reduce immune rejection after transplantation, increasing cell colonization and survival rates—a superiority over other xenogeneic seed cells. IPS-derived hepatocytes exhibit strong directed differentiation capabilities, stably differentiating into functionally mature hepatocytes. They efficiently express hepatocyte-specific markers and possess complete physiological functions such as hepatocyte metabolism, detoxification, and synthesis, enabling more precise participation in liver tissue repair. The therapeutic efficacy of recellularized adipose-derived scaffolds is superior to that of simple adipose-derived scaffolds or single growth factor-modified scaffolds.

[0006] In view of the current limited clinical treatment options for acute liver failure, the risk of immune rejection in liver transplantation, and the poor efficacy of traditional growth factor therapy, this invention proposes a heparinized dual growth factor recellularized adipose tissue decellularized scaffold material, its preparation method, and its application. Summary of the Invention

[0007] The purpose of this invention is to provide a heparinized dual growth factor recellularized adipose tissue decellularized scaffold material, its preparation method, and its application, aiming to solve the problems of limited existing clinical treatment methods for acute liver failure, easy immune rejection in liver transplantation, and poor efficacy of traditional growth factor therapy.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A method for preparing a heparinized dual growth factor recellularized adipose tissue decellularized scaffold material includes the following steps:

[0010] Step 1: Decellularize the adipose tissue to obtain a decellularized adipose tissue scaffold;

[0011] Step 2: Heparinize the decellularized adipose tissue scaffold to obtain a heparinized decellularized adipose tissue scaffold;

[0012] Step 3: Load hepatocyte growth factor and vascular endothelial growth factor onto a heparinized adipose tissue decellularized scaffold to obtain a heparinized dual growth factor adipose tissue decellularized scaffold.

[0013] Step 4: Induced pluripotent stem cell-derived hepatocytes are seeded onto a heparinized dual growth factor adipose tissue decellularized scaffold, and after culture, a heparinized dual growth factor recellularized adipose tissue decellularized scaffold is obtained.

[0014] Further, the specific process of step 1 is as follows: After cleaning the adipose tissue, cut it into adipose tissue blocks, and subject the adipose tissue blocks to freeze-thaw cycles, Triton X-100 solution shaking incubation, trypsin-EDTA solution treatment, isopropanol extraction, and natural air drying.

[0015] Furthermore, the specific operation of the freeze-thaw cycle is as follows: freeze in a -80℃ freezer for 2 hours, then remove and thaw in a 37℃ constant temperature water bath for 30 minutes.

[0016] Further, the specific process of step 2 is as follows: the decellularized adipose tissue scaffold is immersed in MES buffer, activated by adding carbodiimide solution and N-hydroxysuccinimide solution, removed and washed, and then placed in heparin sodium solution for reaction in the dark with shaking.

[0017] Furthermore, the specific process of step 3 is as follows: the heparinized adipose tissue decellularized scaffold is immersed in sterile PBS buffer containing hepatocyte growth factor and vascular endothelial growth factor, and incubated with shaking in the dark.

[0018] Further, the specific process of step 4 is as follows: after digesting and terminating digestion of hepatocytes derived from induced pluripotent stem cells, the cells are collected by centrifugation, resuspended, and the cell concentration is adjusted to obtain a uniform cell suspension; the cell suspension is perfused into a heparinized dual growth factor adipose tissue decellularized scaffold and incubated in the dark.

[0019] Furthermore, the digestion of hepatocytes derived from induced pluripotent stem cells was performed using trypsin-EDTA solution, and the digestion was terminated and resuspended using DMEM medium containing fetal bovine serum.

[0020] Furthermore, the cell concentration was 1×10 6 cells / mL.

[0021] A heparinized dual growth factor recellularized adipose tissue decellularized scaffold material is prepared using the above-mentioned preparation method for heparinized dual growth factor recellularized adipose tissue decellularized scaffold material.

[0022] The application of the above-mentioned heparinized dual growth factor recellularized adipose tissue decellularized scaffold material in the preparation of drugs or medical devices for the treatment of acute liver failure.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] 1. This invention uses adipose tissue from the groin of rats to prepare heparinized dual growth factor recellularized adipose tissue decellularized scaffold material, which has advantages such as low cost, easy availability, simple preparation process, and easy large-scale production.

[0025] 2. The heparinized dual growth factor recellularized adipose tissue decellularized scaffold material prepared in this invention achieves scaffold recellularization by cross-linking the adipose tissue decellularized scaffold with heparin and encapsulating dual growth factors, and using induced pluripotent stem cell-derived hepatocytes as seed cells. This can better simulate the liver microenvironment, improve the bioavailability of growth factors, and reduce liver transplant immune rejection. Based on extracellular matrix support, the synergistic effect of dual growth factors, and the repair capacity of seed cells, it realizes liver tissue damage repair and hepatocyte regeneration, providing a new treatment strategy for acute liver failure. Attached Figure Description

[0026] Figure 1 This is a schematic diagram illustrating the preparation of a decellularized adipose tissue scaffold.

[0027] Figure 2 Characterization of decellularized adipose tissue scaffolds; where A represents the results of H&E staining, Masson staining, DAPI staining, Oil Red O staining, and scanning electron microscopy images, and B represents the DNA content detection results.

[0028] Figure 3 The encapsulation and release rates of growth factors are shown for decellularized adipose tissue scaffolds and heparinized adipose tissue scaffolds; where A represents the growth factor encapsulation rate and B represents the growth factor release rate.

[0029] Figure 4 Biocompatibility of heparinized dual growth factor recellularized adipose tissue decellularized scaffold.

[0030] Figure 5 The therapeutic effect of heparinized dual growth factor recellularized adipose tissue decellularized scaffold; where A is the H&E staining result of rat liver tissue, B is the rat survival curve, and C is the detection result of rat serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Detailed Implementation

[0031] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this invention, the technical solution of this invention is described in detail below, but this should not be construed as limiting the scope of implementation of this invention. Unless otherwise specified, the methods used in this invention are conventional methods in this technical field. In this invention, materials, reagents, or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0032] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0033] Example 1: Preparation of heparinized dual growth factor recellularized adipose tissue decellularized scaffold material;

[0034] 1. Preparation of decellularized adipose tissue scaffolds (see preparation flowchart) Figure 1 ):

[0035] (1) Under sterile conditions, adipose tissue was extracted from the groin area of ​​rats and placed in pre-cooled sterile PBS buffer (pH=7.4) containing 100 U / mL penicillin and 100 μg / mL streptomycin. The tissue was rinsed three times to remove residual blood, fascia and impurities on the surface. The tissue was then cut into 1-2 mm pieces with sterile scissors. 3 Small tissue blocks, stored at 4°C for later use.

[0036] (2) Place the tissue block into a sterile centrifuge tube, add sterile PBS buffer containing 100 U / mL penicillin and 100 μg / mL streptomycin, freeze in an ultra-low temperature freezer at -80℃ for 2 h, take it out and thaw in a constant temperature water bath at 37℃ for 30 min, and perform 3 to 5 freeze-thaw cycles.

[0037] (3) Add 1% Triton X-100 solution, ensuring the liquid level covers the tissue block, and place it in a constant temperature shaker at 37℃ and 180 r / min for 4 h. Replace the Triton X-100 solution every 1 h.

[0038] (4) Wash the tissue block repeatedly with sterile PBS buffer containing 100 U / mL penicillin and 100 μg / mL streptomycin 5-6 times until the washing solution is clear and free of turbidity. Centrifuge at 2000 rpm for 5 min and discard the supernatant. Add 0.25% trypsin-EDTA solution and incubate at 37℃ for 12 h, gently shaking once every 10 min. Then add DMEM medium containing 10% fetal bovine serum to terminate the trypsin reaction.

[0039] (5) Centrifuge at 1500 rpm for 5 min and discard the supernatant. Add isopropanol until the liquid level covers the tissue block, and incubate at room temperature for 48 h, gently shaking 3-4 times during the incubation period. Centrifuge at 1500 rpm for 5 min and discard the isopropanol.

[0040] (6) After isopropanol extraction, the tissue block was repeatedly rinsed with sterile PBS buffer containing 100 U / mL penicillin and 100 μg / mL streptomycin. After each rinse, the supernatant was discarded by centrifugation at 2000 rpm for 5 min. Finally, the cleaned tissue block was placed in a sterile laminar flow hood to air dry naturally and stored at 4℃ for later use. The decellularized adipose tissue scaffold (DAT) was finally obtained.

[0041] 2. Preparation of heparinized dual growth factor recellularized adipose tissue decellularized scaffold:

[0042] (1) The decellularized adipose tissue scaffold was immersed in 0.1 mol / L MES buffer (pH=6.0), followed by activation with 100 mg / mL carbodiimide (EDC) solution and 50 mg / mL N-hydroxysuccinimide (NHS) solution. The scaffold was then shaken at 180 r / min for 30 min at room temperature. The activated scaffold was then removed and placed in 0.1 mol / L MES buffer (pH=6.0), and washed 1-2 times at room temperature for 10 min each time in a constant temperature shaker at 180 r / min. The scaffold was then placed in 2 mg / mL heparin sodium solution and shaken at 180 r / min for 12 h at 4°C in the dark to obtain the heparinized decellularized adipose tissue scaffold (H-DAT).

[0043] (2) The heparinized adipose tissue decellularized scaffold was immersed in sterile PBS buffer containing 400 ng / mL hepatocyte growth factor (HGF) and 400 ng / mL vascular endothelial growth factor (VEGF), and placed in a constant temperature shaker at 37°C and 120 r / min for 6 h in the dark to obtain the heparinized dual growth factor adipose tissue decellularized scaffold (H / VH-DAT).

[0044] (3) The induced pluripotent stem cell-derived hepatocytes (iPSC-Heps) of 25T size were digested with 0.25% trypsin-EDTA solution for 1 min, and then DMEM medium containing 10% fetal bovine serum was added to terminate the digestion. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in DMEM medium containing 10% fetal bovine serum. After mixing, the cells were counted using a cell counting chamber, and the cell concentration was adjusted to 1×10⁶ cells / mL. 6The cell count was measured to obtain a homogeneous iPSC-Heps cell suspension. 500 μL of the iPSC-Heps cell suspension was perfused into a heparinized dual growth factor adipose tissue decellularized scaffold, and the scaffold was incubated at 37°C in the dark for 6 h to obtain a heparinized dual growth factor recellularized adipose tissue decellularized scaffold (RAT).

[0045] Example 2: Characterization of decellularized adipose tissue scaffold;

[0046] The decellularization effect, extracellular matrix retention, and microstructure of DAT were characterized by hematoxylin-eosin (H&E) staining, Masson staining, DAPI staining, Oil Red O staining, scanning electron microscopy (SEM), and DNA content detection. The results are as follows: Figure 2 As shown.

[0047] for Figure 2SEM observations showed that, compared to natural adipose tissue, DAT had its cellular contents removed, with no obvious cellular structure. Without cell support, the scaffold exhibited a loose, porous structure. H&E staining revealed numerous intact adipocytes with clear outlines, bluish-purple nuclei, and a compact structure in natural adipose tissue. In DAT, no intact cell morphology or cell debris remained; only a continuous, intact extracellular matrix framework was visible. The scaffold formed a uniform three-dimensional porous structure without traces of cell contents such as nuclei or cytoplasm, indicating that decellularization effectively removed cellular components from the adipose tissue. Masson staining showed that, in natural adipose tissue, besides adipocytes, a small amount of collagen fibers were visible between cells, interwoven with cellular components. In DAT, the collagen fiber structure was intact, uniformly distributed in a network, running through the entire scaffold's porous structure without cellular residue. The collagen fibers were regularly arranged and did not break or degrade due to decellularization, indicating that decellularization thoroughly removed cellular contents while completely preserving the collagen fiber structure, further confirming the complete removal of cellular contents. DAPI staining results showed numerous bright blue fluorescent spots, representing cell nuclei, in natural adipose tissue. The fluorescence signal was dense and uniformly distributed, clearly reflecting the distribution of cells in the tissue. In contrast, no blue fluorescent signal was observed in DAT staining, and the entire scaffold area showed no fluorescent bright spots, indicating that no cell nuclei remained in the scaffold. This further confirmed that the cellular contents in the adipose tissue had been completely removed, demonstrating effective decellularization. Oil Red O staining results showed numerous bright red lipid droplets in natural adipose tissue. These droplets were round or nearly round, uniform in size, and densely distributed, almost occupying the entire field of view. In contrast, no bright red lipid droplet staining areas were observed in DAT staining, and the three-dimensional porous structure of the scaffold was clearly visible. This result indicates that decellularization can completely remove lipid components from adipose tissue, leaving no lipid residue in the scaffold. DNA content detection results showed (… Figure 2 The DNA content of natural adipose tissue (B) was 351.0±45.75 ng / mg, while the DNA content of DAT was 31.49±6.02 ng / mg, which fully met the standard for decellularization (< 50 ng / mg).

[0048] Example 3: Encapsulation and release rates of growth factors by decellularized adipose tissue scaffolds and heparinized adipose tissue scaffolds;

[0049] The encapsulation efficiency and release rate of HGF and VEGF by DAT and H-DAT were detected respectively, and the results are as follows: Figure 3 As shown.

[0050] Figure 3The results showed that both DAT and H-DAT exhibited sustained-release behavior for both HGF and VEGF, with H-DAT showing a higher release rate than DAT. For VEGF, the release rate of DAT at 336 hours was approximately 62.23±2.83%, while that of H-DAT was approximately 79.29±3.25%. For HGF, the release rate of DAT at 336 hours was approximately 68.12±3.42%, while that of H-DAT reached 76.13±3.08%. Both growth factors can be continuously released from H-DAT within 336 hours, effectively prolonging the duration of action, improving their bioavailability, and providing continuous nutritional support for liver tissue regeneration.

[0051] Figure 3 The results showed that, compared with DAT, H-DAT significantly improved the encapsulation efficiency of both HGF and VEGF. Specifically, the encapsulation efficiency of HGF by DAT was 76.91±8.29%, while that by H-DAT increased to 82.36±14.11%; the encapsulation efficiency of VEGF by DAT was 45.67±20.77%, while that by H-DAT increased to 71.33±26.84%. These results indicate that heparinization modification can effectively enhance the binding capacity of the scaffold to these two growth factors, significantly improving the encapsulation efficiency and providing a basis for the long-term sustained release of growth factors.

[0052] Example 4: Biocompatibility of heparinized dual growth factor recellularized adipose tissue decellularized scaffold;

[0053] The biocompatibility of different scaffolds with iPSC-Heps was investigated using a live / dead cell staining assay. Five groups were set up: iPSC-Heps group (iPSC-Heps cell control group); H-DAT group (heparinized adipose tissue decellularized scaffold group); H / V group (free HGF+VEGF dual growth factor group); H / VH-DAT group (heparinized dual growth factor adipose tissue decellularized scaffold group); and RAT group (heparinized dual growth factor recellularized adipose tissue decellularized scaffold group).

[0054] The results are as follows Figure 4 As shown, the live / dead cell staining results indicated that on day 1 of culture, all groups exhibited abundant bright green live cell fluorescence signals with no obvious red dead cell fluorescence signals, indicating high cell viability. On day 3 of culture, the number of cells in each group significantly increased, and the green fluorescence signal intensified. On day 7 of culture, cells in each group completely filled the field of view, with uniform and dense green fluorescence and no obvious residual red dead cells. These results demonstrate that the heparinized dual-growth factor recellularized adipose tissue decellularized scaffold prepared in this invention exhibits low toxicity to iPSC-Heps and good biocompatibility.

[0055] Example 5: The therapeutic effect of heparinized dual growth factor recellularized adipose tissue decellularized scaffold on acute liver failure;

[0056] Establishment and experimental grouping of an acute liver failure (ALF) SD rat model:

[0057] Healthy male SD rats (weighing 180-200g) were purchased from Speford (Suzhou) Biotechnology Co., Ltd. After one week of acclimatization, they were randomly divided into 4 groups of 10 rats each. The grouping and treatment were as follows:

[0058] Normal group (blank group): 1 mL of sterile saline was injected intraperitoneally, and no modeling was performed.

[0059] ALF group (model control group): 1 mL of D-galactosamine (D-GalN) at a concentration of 0.6 g / kg was injected intraperitoneally to establish an acute liver failure model. No treatment was given after modeling. The livers of ALF SD rats were dark in color, with a rough and uneven surface, scattered grayish-white necrotic foci, hardened texture, increased fragility, and congestion and edema in some areas, indicating that the ALF model was successfully established.

[0060] iPSC-Heps group (simply cell therapy group): ALF SD rat model was constructed according to the above method. After successful modeling, 150 μL of iPSC-Heps cell suspension was injected into the liver in situ for treatment.

[0061] RAT group (treatment group of this invention): An ALF SD rat model was constructed according to the above method. After successful modeling, an incision of about 4 cm was made in the abdominal cavity of the rat to expose and locate the liver. The RAT prepared in this invention was transplanted in situ into the liver lobules. The abdominal cavity of the rat was then sutured.

[0062] Further examination was conducted on the liver pathological morphology, survival rate, and liver function levels of SD rats in each group. The results are as follows: Figure 5 As shown. H&E staining results show ( Figure 5 In the Normal group (A), the liver tissue of rats showed intact pathological structure, with hepatocytes arranged neatly and regularly in shape, exhibiting polygonal shapes, without hepatocyte necrosis, inflammatory cell infiltration, or edema, and no pathological damage. In the ALF group, large areas of hepatocyte degeneration and necrosis were observed, along with extensive inflammatory cell infiltration and significant interstitial edema. After RAT treatment, the area of ​​liver necrosis in rats was significantly reduced, the necrotic foci were further shrunk, hepatic sinusoidal congestion and tissue edema were basically relieved, inflammatory cell infiltration was significantly reduced, hepatocyte arrangement became more regular, and the lobular structure of some areas began to recover. A small number of new hepatocytes were observed adhering and growing around the scaffold, confirming that RAT can effectively repair liver tissue damage. Survival curves show ( Figure 5In the normal group (group B), there were no deaths throughout the entire process, with a survival rate of 100%. The survival rate of the ALF group decreased rapidly over time, reaching only about 25% within 7 days. The survival rate of the iPSC-Heps group was higher than that of the ALF group, with a 7-day survival rate of approximately 37.5%. The survival rate of the RAT group was significantly higher than that of the ALF and iPSC-Heps groups, reaching 68.75% within 7 days, indicating that RAT can effectively improve the survival rate of rats with acute liver failure. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in each group showed... Figure 5 In rats treated with RAT (Rapid Acid Therapy), liver function levels increased. Specifically, the ALT levels in the Normal group were 659.38±296.63 U / L, in the ALF group 8818.80±597.30 U / L, in the iPSC-Heps group 1596.40±379.10 U / L, and in the RAT group 689.88±181.67 U / L. In AST (Acid-Reduced Tolerance) assays, the AST levels in the Normal, ALF, iPSC-Heps, and RAT groups were 427.80±113.39 U / L, 5634.60±523.41 U / L, 1286.40±187.38 U / L, and 447.0±110.74 U / L, respectively. As can be seen, compared with the ALF group and the iPSC-Heps group, the ALT and AST levels in the RAT group rats were reduced, approaching the levels of the Normal group, indicating that liver damage was repaired. These results demonstrate that RAT has a good therapeutic effect on rats with acute liver failure.

[0063] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the present invention.

Claims

1. A method for preparing a heparinized double growth factor recellularized adipose tissue acellular scaffold material, characterized by, Includes the following steps: Step 1: Decellularize the adipose tissue to obtain a decellularized adipose tissue scaffold; Step 2: Heparinize the decellularized adipose tissue scaffold to obtain a heparinized decellularized adipose tissue scaffold; Step 3: Load hepatocyte growth factor and vascular endothelial growth factor onto a heparinized adipose tissue decellularized scaffold to obtain a heparinized dual growth factor adipose tissue decellularized scaffold. Step 4: Induced pluripotent stem cell-derived hepatocytes are seeded onto a heparinized dual growth factor adipose tissue decellularized scaffold, and after culture, a heparinized dual growth factor recellularized adipose tissue decellularized scaffold is obtained.

2. The method for preparing the heparinized dual growth factor recellularized adipose tissue decellularized scaffold material according to claim 1, characterized in that, The specific process of step 1 is as follows: After cleaning the adipose tissue, cut it into adipose tissue blocks, and then subject the adipose tissue blocks to freeze-thaw cycles, Triton X-100 solution shaking incubation, trypsin-EDTA solution treatment, isopropanol extraction, and natural air drying.

3. The method for preparing the heparinized dual growth factor recellularized adipose tissue decellularized scaffold material according to claim 2, characterized in that, The specific operation of the freeze-thaw cycle is as follows: freeze in a -80℃ freezer for 2 hours, and then thaw in a 37℃ constant temperature water bath for 30 minutes.

4. The method for preparing the heparinized dual growth factor recellularized adipose tissue decellularized scaffold material according to claim 1, characterized in that, The specific process of step 2 is as follows: the decellularized adipose tissue scaffold is immersed in MES buffer, activated by adding carbodiimide solution and N-hydroxysuccinimide solution, removed and washed, and then placed in heparin sodium solution for reaction in the dark with shaking.

5. The method for preparing the heparinized dual growth factor recellularized adipose tissue decellularized scaffold material according to claim 1, characterized in that, The specific process of step 3 is as follows: the heparinized adipose tissue decellularized scaffold is immersed in sterile PBS buffer containing hepatocyte growth factor and vascular endothelial growth factor, and incubated with shaking in the dark.

6. The method for preparing the heparinized dual growth factor recellularized adipose tissue decellularized scaffold material according to claim 1, characterized in that, The specific process of step 4 is as follows: after digesting and terminating digestion of hepatocytes derived from induced pluripotent stem cells, the cells are collected by centrifugation, resuspended, and the cell concentration is adjusted to obtain a uniform cell suspension; the cell suspension is perfused into a heparinized dual growth factor adipose tissue decellularized scaffold and incubated in the dark.

7. The method for preparing the heparinized dual growth factor recellularized adipose tissue decellularized scaffold material according to claim 6, characterized in that, The digestion of the induced pluripotent stem cell-derived hepatocytes was performed using a trypsin-EDTA solution, and the digestion was terminated and the cells were resuspended using DMEM medium containing fetal bovine serum.

8. The method for preparing the heparinized dual growth factor recellularized adipose tissue decellularized scaffold material according to claim 6, characterized in that, The cell concentration is 1 x 10 6 cells / mL.

9. A heparinized dual-growth factor recellularized adipose tissue decellularized scaffold material, characterized in that, The material was prepared using the method described in any one of claims 1 to 8 for preparing heparinized dual growth factor recellularized adipose tissue decellularized scaffold material.

10. The use of a heparinized dual growth factor recellularized adipose tissue decellularized scaffold material according to claim 8 or 9 in the preparation of a medicament or medical device for treating acute liver failure.

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