A method for preparing a decellularized matrix and a product prepared thereby

Abstract

The application discloses a preparation method of a decellularized matrix and a product prepared by the method. The preparation method comprises the following steps: (1) washing a mammalian soft tissue to remove blood, lipids and impurities, and cutting the tissue into pieces with a thickness of 1-10 mm; (2) soaking the pieces in solution 1 for 6-48 h, and then soaking the pieces in solution 2 for 0.1-1 h; (3) treating the pieces in an ultrasonic water bath under the condition of a frequency of 20-40 kHz for 5-30 min; (4) water-bathing the pieces at 55-65 DEG C for 10-30 min; (5) sequentially washing the tissue with deionized water, a PBS buffer and ethanol, and washing the tissue with each solution for 1-3 times, each time for 10-30 min; and (6) freeze-drying the decellularized matrix material, and crushing the material at-80 DEG C to-20 DEG C to obtain a decellularized matrix powder. The preparation method significantly improves the cell component removal efficiency, effectively protects structural proteins such as collagen from being degraded in the freeze-drying and crushing processes, and ensures the integrity of the matrix in the aspects of morphology, structure and function, thereby providing a structural support and biological signal support for subsequent tissue repair.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to a method for preparing a decellularized matrix and the product thereof. Background Technology

[0002] In recent years, with the development of tissue engineering and regenerative medicine, decellularized matrix (dECM) has gradually gained widespread attention in fields such as skin repair, soft tissue reconstruction, and cosmetic surgery due to its excellent biocompatibility, low immunogenicity, and good tissue induction ability. Decellularized matrix is ​​a type of biomaterial that removes cellular components from animal tissues through physical, chemical, and enzymatic methods while preserving as much of its three-dimensional structure and functional components (such as collagen, glycosaminoglycans, and growth factors) as possible. Compared with traditional synthetic materials or single-component biological agents, dECM can better mimic the natural extracellular environment, promoting cell adhesion, proliferation, and differentiation, thereby significantly improving tissue repair effects.

[0003] Currently, some commercially available decellularized materials are used for repairing deep skin defects or hard tissues, such as decellularized dermis and decellularized tendons. However, most of these are in the form of films, blocks, or injectable hydrogels, which suffer from problems such as limited tissue sources, crude processing techniques, high residual DNA levels, poor reconstitution properties, and difficulty in injection. These limitations make it difficult to meet the clinical needs for minimally invasive injections, facial contour improvement, and multi-layered tissue repair. Furthermore, during processing such as freeze-drying and pulverization, key structural proteins such as collagen are prone to degradation or breakage, severely affecting the material's mechanical and gelling properties, thus limiting its application scope.

[0004] Furthermore, decellularized matrices for injection are still in the research and early development stages, and there is currently a lack of a preparation process that can simultaneously achieve high decellularization efficiency, collagen structure protection, and good injection flowability and resolubilization ability. Especially in different injection layers such as the superficial to middle layers of the skin, the deep dermis to the subcutaneous layer, and the fascia layer, there are higher requirements for the material's flowability, viscosity, and resolubilization properties.

[0005] Therefore, there is an urgent need to develop a decellularized matrix powder material with low DNA residue, excellent resolubility, good gelling properties, and suitability for injection at multiple sites, in order to meet the diverse needs of medical aesthetics and clinical applications such as skin filling, wrinkle repair, contouring, and tissue regeneration. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the present invention proposes a method for preparing a decellularized matrix and the product thereof.

[0007] This invention provides a method for preparing a decellularized matrix, comprising the following steps:

[0008] (1) Clean the soft tissue of mammals to remove blood, lipids and impurities, and cut it into slices of 1-10 mm thickness;

[0009] (2) Soak the thick slice obtained in (1) in solution 1 for 6 to 48 hours, and then soak it in solution 2 for 0.1 to 1 hour;

[0010] Solution 1 comprises: 0.01–0.25% w / v of trypsin and 10–100 U / mL of collagenase;

[0011] Solution 2 comprises: N-acetylcysteine ​​0.2–1 mM, allantoin 0.1–2 mM, glutathione 0.2–5 mM, and silk fibroin 0.01–0.5% w / v;

[0012] (3) Place the tissue obtained in (2) in an ultrasonic water bath and treat it at a frequency of 20-40 kHz for 5-30 min.

[0013] (4) Incubate the tissue obtained in (3) at 55-65℃ for 10-30 min;

[0014] (5) Wash the tissue obtained in (4) with deionized water, PBS buffer and ethanol respectively, 1 to 3 times for each solution, 10 to 30 min each time;

[0015] (6) Freeze-dry the decellularized matrix material obtained in (5) and pulverize it at -80℃ to -20℃ to obtain decellularized matrix powder.

[0016] In some embodiments, in step (5), the concentration of ethanol is 50 to 100% v / v.

[0017] In some embodiments, in step (2), the mass ratio of the pancreatic enzyme to the collagenase is 1:(2-10).

[0018] In some embodiments, the silk fibroin in solution 2 is silk fibroin or regenerated silk fibroin.

[0019] In some embodiments, the mammal is selected from one or more of pigs, cattle, and sheep.

[0020] In some embodiments, the soft tissue is any one or more of the submucosa of the small intestine, the bladder mucosa, and the skin.

[0021] The present invention also provides a decellularized matrix obtained by the preparation method described above.

[0022] The present invention also provides a decellularized matrix product comprising the aforementioned decellularized matrix;

[0023] The decellularized matrix product is a solution or a gel.

[0024] The present invention also provides the application of any one of the decellularized matrix obtained by the preparation method, the decellularized matrix, and the decellularized matrix product in the preparation of biomedical materials.

[0025] In some embodiments, the biomedical material is injected into subcutaneous or soft tissue for cosmetic surgery or tissue repair.

[0026] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0027] 1. The preparation method of the present invention significantly improves the efficiency of cell component removal, effectively protects structural proteins such as collagen from degradation during freeze-drying and pulverization, ensures the integrity of the matrix in terms of morphology, structure and function, and provides structural scaffold and biological signal support for subsequent tissue repair.

[0028] 2. The decellularized matrix powder obtained after freeze-drying has good reconstitution speed, high transparency and excellent flowability. It can be quickly reconstituted into a uniform colloid at low temperature, which is convenient for microneedle injection or filling operations and meets the needs of multiple clinical scenarios.

[0029] 3. The decellularized matrix powder prepared by this invention can be formulated into colloids of different concentrations according to clinical needs. It is suitable for multi-layer injections, including superficial to middle layers of the skin, deep dermis to subcutaneous layer, and fascia layer. It can be used in medical aesthetics and regenerative medicine applications such as wrinkle repair, facial contour improvement, skin depression filling, and bone support enhancement. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a statistical result of the expression level of collagen-related gene mRNA in this invention. Detailed Implementation

[0032] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0033] Example 1

[0034] The method for preparing the decellularized matrix in this embodiment includes the following steps:

[0035] (1) Tissue pretreatment: Fresh pig small intestine was selected, and after being cut open, the outer serosa, muscle layer and inner mucosal epithelium were removed, leaving only the submucosal tissue. The obtained submucosal tissue was rinsed repeatedly with cold PBS buffer 3 times (10 minutes each time) to remove residual blood, impurities and visible fat, and cut into slices of about 5mm×10mm×1mm thickness.

[0036] (2) The above tissue slices were soaked in 0.1% (w / v) pancreatic enzyme and 50 U / mL collagenase for 6 h, and then soaked in solution I (N-acetylcysteine ​​0.2 mM, allantoin 0.1 mM, glutathione 0.2 mM and silk fibroin 0.01% w / v) for 10 min.

[0037] (3) Place the tissue obtained in (2) in an ultrasonic water bath and treat it at a frequency of 30 kHz for 15 min.

[0038] (4) The tissue obtained in (3) was placed in a water bath at 55°C for 20 min.

[0039] (5) The treated tissue was washed with the following methods in sequence: three times with deionized water; twice with 1×PBS buffer (pH 7.2-7.4); and once with 70% ethanol, for 10 min each time.

[0040] (6) The washed decellularized tissue was placed in a freeze dryer and freeze-dried at -50°C for 24 hours to obtain a dried sponge-like decellularized matrix. The matrix was then mechanically pulverized using a grinding device and sieved (100 mesh) to obtain decellularized matrix powder.

[0041] Example 2

[0042] The method for preparing the decellularized matrix in this embodiment includes the following steps:

[0043] (1) Tissue pretreatment: Fresh pig small intestine was selected, and after being cut open, the outer serosa, muscle layer and inner mucosal epithelium were removed, leaving only the submucosal tissue. The obtained submucosal tissue was rinsed repeatedly with cold PBS buffer 3 times (10 minutes each time) to remove residual blood, impurities and visible fat, and cut into slices of about 5mm×10mm×1mm thickness.

[0044] (2) The above tissue slices were soaked in 0.1% (w / v) pancreatic enzyme and 50 U / mL collagenase for 6 h, and then soaked in solution I (N-acetylcysteine ​​0.5 mM, allantoin 0.1 mM, glutathione 0.2 mM and silk fibroin 0.01% w / v) for 10 min.

[0045] (3) Place the tissue obtained in (2) in an ultrasonic water bath and treat it at a frequency of 30 kHz for 15 min.

[0046] (4) The tissue obtained in (3) was placed in a water bath at 55°C for 20 min.

[0047] (5) The treated tissue was washed with the following methods in sequence: three times with deionized water; twice with 1×PBS buffer (pH 7.2-7.4); and once with 70% ethanol, for 10 min each time.

[0048] (6) The washed decellularized tissue was placed in a freeze dryer and freeze-dried at -50°C for 24 hours to obtain a dried sponge-like decellularized matrix. The matrix was then mechanically pulverized using a grinding device and sieved (100 mesh) to obtain decellularized matrix powder.

[0049] Example 3

[0050] The method for preparing the decellularized matrix in this embodiment includes the following steps:

[0051] (1) Tissue pretreatment: Fresh pig small intestine was selected, and after being cut open, the outer serosa, muscle layer and inner mucosal epithelium were removed, leaving only the submucosal tissue. The obtained submucosal tissue was rinsed repeatedly with cold PBS buffer 3 times (10 minutes each time) to remove residual blood, impurities and visible fat, and cut into slices of about 5mm×10mm×1mm thickness.

[0052] (2) The above tissue slices were soaked in 0.1% (w / v) pancreatic enzyme and 50 U / mL collagenase for 6 h, and then soaked in solution I (N-acetylcysteine ​​1 mM, allantoin 0.1 mM, glutathione 0.2 mM and silk fibroin 0.01% w / v) for 10 min.

[0053] (3) Place the tissue obtained in (2) in an ultrasonic water bath and treat it at a frequency of 30 kHz for 15 min.

[0054] (4) The tissue obtained in (3) was placed in a water bath at 55°C for 20 min.

[0055] (5) The treated tissue was washed with the following methods in sequence: three times with deionized water; twice with 1×PBS buffer (pH 7.2-7.4); and once with 70% ethanol, for 10 min each time.

[0056] (6) The washed decellularized tissue was placed in a freeze dryer and freeze-dried at -50°C for 24 hours to obtain a dried sponge-like decellularized matrix. The matrix was then mechanically pulverized using a grinding device and sieved (100 mesh) to obtain decellularized matrix powder.

[0057] Example 4

[0058] The method for preparing the decellularized matrix in this embodiment includes the following steps:

[0059] (1) Tissue pretreatment: Fresh pig small intestine was selected, and after being cut open, the outer serosa, muscle layer and inner mucosal epithelium were removed, leaving only the submucosal tissue. The obtained submucosal tissue was rinsed repeatedly with cold PBS buffer 3 times (10 minutes each time) to remove residual blood, impurities and visible fat, and cut into slices of about 5mm×10mm×1mm thickness.

[0060] (2) The above tissue slices were soaked in 0.1% (w / v) pancreatic enzyme and 50 U / mL collagenase for 6 h, and then soaked in solution I (N-acetylcysteine ​​1 mM, allantoin 1 mM, glutathione 3 mM and silk fibroin 0.3% w / v) for 10 min.

[0061] (3) Place the tissue obtained in (2) in an ultrasonic water bath and treat it at a frequency of 30 kHz for 15 min.

[0062] (4) The tissue obtained in (3) was placed in a water bath at 55°C for 20 min.

[0063] (5) The treated tissue was washed with the following methods in sequence: three times with deionized water; twice with 1×PBS buffer (pH 7.2-7.4); and once with 70% ethanol, for 10 min each time.

[0064] (6) The washed decellularized tissue was placed in a freeze dryer and freeze-dried at -50°C for 24 hours to obtain a dried sponge-like decellularized matrix. The matrix was then mechanically pulverized using a grinding device and sieved (100 mesh) to obtain decellularized matrix powder.

[0065] Example 5

[0066] The method for preparing the decellularized matrix in this embodiment includes the following steps:

[0067] (1) Tissue pretreatment: Fresh pig small intestine was selected, and after being cut open, the outer serosa, muscle layer and inner mucosal epithelium were removed, leaving only the submucosal tissue. The obtained submucosal tissue was rinsed repeatedly with cold PBS buffer 3 times (10 minutes each time) to remove residual blood, impurities and visible fat, and cut into slices of about 5mm×10mm×1mm thickness.

[0068] (2) The above tissue slices were soaked in 0.1% (w / v) pancreatic enzyme and 50 U / mL collagenase for 6 h, and then soaked in solution I (N-acetylcysteine ​​1 mM, allantoin 2 mM, glutathione 5 mM and silk fibroin 0.5% w / v) for 10 min.

[0069] (3) Place the tissue obtained in (2) in an ultrasonic water bath and treat it at a frequency of 30 kHz for 15 min.

[0070] (4) The tissue obtained in (3) was placed in a water bath at 55°C for 20 min.

[0071] (5) The treated tissue was washed with the following methods in sequence: three times with deionized water; twice with 1×PBS buffer (pH 7.2-7.4); and once with 70% ethanol, for 10 min each time.

[0072] (6) The washed decellularized tissue was placed in a freeze dryer and freeze-dried at -50°C for 24 hours to obtain a dried sponge-like decellularized matrix. The matrix was then mechanically pulverized using a grinding device and sieved (100 mesh) to obtain decellularized matrix powder.

[0073] Example 6

[0074] The difference from Example 2 is that in step (3), the process is performed at a frequency of 40 kHz for 15 minutes.

[0075] Example 7

[0076] The difference from Example 2 is that in step (3), the processing is carried out at a frequency of 20 kHz for 15 minutes.

[0077] Comparative Example 1

[0078] The difference from Example 2 is that Solution I treatment is not performed.

[0079] Comparative Example 2

[0080] The difference from Example 2 is that Solution I does not contain N-acetylcysteine.

[0081] Comparative Example 3

[0082] The difference from Example 2 is that Solution I does not contain allantoin.

[0083] Comparative Example 4

[0084] The difference from Example 2 is that Solution I does not contain silk fibroin.

[0085] Comparative Example 5

[0086] The difference from Example 2 is that step (3) is not performed.

[0087] Example of effectiveness: Experimental examples were used to examine the various properties of the decellularized matrix prepared in the above-mentioned related embodiments and control examples of the present invention:

[0088] I. DNA Residue Detection (PicoGreen Method) and Results

[0089] (1) Weigh about 10 mg of decellularized matrix powder sample and place it in a 1.5 mL centrifuge tube.

[0090] (2) Add 1 mL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) and incubate in a water bath at 56 °C with shaking for 1 hour.

[0091] (3) Ultrasonic treatment was used to assist in pyrolysis (power 100W, 5-second interval / work, continuous for 1 minute).

[0092] (4) Centrifuge (12,000 rpm, 10 min) and collect the supernatant for testing.

[0093] (5) Prepare a series of λ-DNA standard solutions (concentration range: 0, 10, 50, 100, 200, 500 ng / mL).

[0094] (6) Add 100 μL of standard solution to each well and place it in a 96-well plate for later use.

[0095] (7) Dilute the PicoGreen dye to the working concentration according to the instructions (generally 1:200 diluted in TE).

[0096] (8) Add 100 μL of PicoGreen working solution to the standard well and the sample well respectively.

[0097] (9) Incubate in the dark for 5 minutes at room temperature.

[0098] (10) Use a fluorescence microplate reader for detection: excitation wavelength 480nm, emission wavelength 520nm; read the fluorescence intensity and plot the standard curve; calculate the DNA concentration based on the fluorescence intensity of the sample wells and convert it into the amount of DNA residue per unit dry weight (ng DNA / mg ECM dry weight).

[0099] (11) The test results are shown in Table 1.

[0100] Table 1 DNA Residual Amount

[0101] Group DNA residue (ng / mg) Example 1 42.7±3.1 Example 2 30.2±2.5 Example 3 18.9±1.9 Example 4 12.4±1.7 Example 5 9.8±1.5 Example 6 25.5±2.3 Example 7 36.3±3.0 Comparative Example 1 142.5±5.7 Comparative Example 2 89.6±4.8 Comparative Example 3 96.1±5.1 Comparative Example 4 83.7±4.4 Comparative Example 5 108.3±5.0

[0102] As shown in Table 1, the method for preparing the decellularized matrix of the present invention can effectively reduce the amount of residual DNA and has high decellularization efficiency. In Comparative Example 1, enzyme treatment alone is insufficient for complete decellularization; in Comparative Example 2, the lack of antioxidants exacerbates protein cross-linking and reduces decellularization efficiency; in Comparative Example 3, reduced inflammation relief affects clear cell lysis; in Comparative Example 4, the lack of protein protection intensifies collagen structure damage and makes DNA release difficult; in Comparative Example 5, the absence of ultrasonic treatment results in poor cell lysis efficiency.

[0103] II. Determination and Results of Collagen Content

[0104] (1) Take 5 mg of dried decellularized matrix powder and record the accurate mass.

[0105] (2) Add the sample to 1 mL of 6 mol / L hydrochloric acid and hydrolyze it in a constant temperature water bath at 110℃ for 18 h.

[0106] (3) Evaporate the hydrolyzed sample to dryness in a 45°C water bath, then dissolve it in distilled water and bring the volume up to 1 mL.

[0107] (4) Take 200 μL of hydrolysate, add 400 μL of chloramine T oxidation solution, mix well and let stand at room temperature for 20 min, add 400 μL of Ehrlich reagent, incubate at 60℃ for 15 min, and cool after the color development is complete.

[0108] (5) Measure the absorbance at a wavelength of 560 nm and use the hydroxyproline standard curve to calculate the Hyp content of the sample as Hyp accounts for 13.5% of collagen (collagen content (μg / mg) = Hyp content (μg / mg) ÷ 0.135).

[0109] The test results are shown in Table 2:

[0110] Table 2 Collagen content of each example and comparative example

[0111]

[0112]

[0113] As shown in Table 2, all examples were significantly better than the comparative examples, indicating that the formulation effectively preserved the collagen structure. The synergistic use of NAC, allantoin, and silk fibroin inhibited the damage to collagen caused by protein-degrading enzymes / oxidative stress. In the comparative examples, especially Comparative Example 1 without any Solution I, the collagen structure was severely degraded. This demonstrates that the preparation method of the present invention can effectively avoid the breakage and degradation of the collagen structure during freeze-drying and pulverization.

[0114] III. Reconstitution Test

[0115] 1. Reconstitution rate test:

[0116] Prepare 50 mg of decellularized matrix powder, add 1 mL of pre-warmed (37°C) PBS buffer, and place in a 37°C water bath with a shaker (150 rpm). Observe the dissolution process visually or by taking a photo every minute until no obvious powder residue remains. Record the time required for complete dissolution.

[0117] Result determination: ≤5min: good resolubility; 5~10min: moderate; ≥10min: poor resolubility.

[0118] 2. Colloid transparency measurement (transmittance)

[0119] A 1% w / v decellularized matrix powder solution was prepared and filtered to remove air bubbles. Using pure PBS as a blank control, transmittance was measured at 600 nm using a spectrophotometer.

[0120] Results interpretation: ≥85%: high transparency (can be used for cosmetic microinjection); 60-85%: medium transparency; <60%: cloudy.

[0121] 3. Liquidity assessment (injectable)

[0122] (1) Place the dissolved decellularized matrix liquid (10 mg / mL, reconstituted with PBS) into a dropper (or a 5 mL syringe) and drop it onto a glass plate tilted at 30°. Record the distance (cm) the solution flows within 30 seconds. Test each sample 3 times and take the average value. The longer the distance, the better the fluidity.

[0123] (2) Fill a 5mL syringe with the reconstituted sample and press the syringe vertically at standard temperature (25℃); measure the time (seconds) required to expel 3mL; the shorter the time, the better the fluidity.

[0124] The test results are shown in Table 3:

[0125] Table 3 Results of powder resolubility test

[0126]

[0127]

[0128] As shown in Table 3, the decellularization process of this invention significantly improves the resolubility, transparency, and injection fluidity of the decellularized matrix powder, thereby ensuring its performance stability in clinical applications such as superficial skin and mesotherapy filling.

[0129] IV. Tissue Repair Effect of Decellularized Matrix

[0130] Experimental animals: SD rats or C57BL / 6 mice, 8-10 weeks old, weighing about 200-250g; half male and half female; divided into experimental group and control group, with 6 mice in each group. The experimental group was prepared with the decellularized matrix powder prepared in Example 1 and mixed with sterile PBS to form a suspension of 10mg / mL, while the control group was injected with sterile PBS solution.

[0131] The specific steps are as follows: Animals were anesthetized with sodium pentobarbital, and their backs were shaved and disinfected. 100 μL of PBS was injected subcutaneously using a 1 mL syringe. The control group was injected with an equal volume of PBS (100 μL), once daily. Three animals were sacrificed on day 7 and day 14, and the injection area was excised for RNA extraction. The relative mRNA expression levels of Col1a1, Col3a1, TGF-β1, and α-SMA genes were detected.

[0132] The results are as follows Figure 1 As shown, "*" indicates a statistically significant difference between the experimental group and the control group (p<0.05). On days 7 and 14, the relative mRNA expression levels of these four genes in the experimental group were significantly higher than those in the control group (PBS group). Specifically, the expression levels of Col1a1 and Col3a1 genes were significantly upregulated on day 7 and further enhanced on day 14, suggesting enhanced collagen production activity. TGF-β1 and α-SMA expression also showed an increasing trend, reaching a significant peak on day 14, indicating increased fibroblast activation and myofibroblast transformation during tissue remodeling. These results indicate that under decellularized matrix stimulation, the experimental group animals showed significantly enhanced collagen synthesis and tissue repair-related gene expression at the injection site, demonstrating good pro-repair activity.

[0133] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a decellularized matrix, characterized by, The method comprises the following steps: (1) washing the mammal soft tissue to remove blood, lipid and impurities, and cutting into 1-10 mm thick pieces; (2) immersing the thick pieces obtained in step (1) in solution 1 for 6-48 h, and then immersing in solution 2 for 0.1-1 h; the solution 1 comprises: trypsin 0.01-0.25% w / v and collagenase 10-100 U / mL; the solution 2 comprises: N-acetyl cysteine 0.2-1 mM, allantoin 0.1-2 mM, glutathione 0.2-5 mM and silk fibroin 0.01-0.5% w / v; (3) placing the tissue obtained in step (2) in an ultrasonic water bath, and treating at a frequency of 20-40 kHz for 5-30 min; (4) water-bathing the tissue obtained in step (3) at 55-65 ℃ for 10-30 min; (5) washing the tissue obtained in step (4) with deionized water, PBS buffer and ethanol in sequence, 1-3 times for each solution, 10-30 min each time; (6) freeze-drying the acellular matrix material obtained in step (5), and crushing at-80 ℃ to-20 ℃ to obtain an acellular matrix powder.

2. The production method according to claim 1, characterized by, In step (5), the concentration of the ethanol is 50-100% v / v.

3. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of the trypsin and the collagenase is 1:(2-10).

4. The method of claim 1, wherein, In the solution 2, the silk fibroin is silk fibroin or regenerated silk fibroin.

5. The preparation method according to claim 1, characterized in that, The mammal is selected from one or more of pig, cow and sheep.

6. The method of claim 1, wherein, The soft tissue is any one or more of small intestinal submucosa, bladder mucosa and skin.

7. The acellular matrix obtained by the preparation method of any one of claims 1-6.

8. A decellularized matrix product, characterized in that, The acellular matrix of claim 7; The acellular matrix product is a solution or a gel.

9. Use of any one of the acellular matrix obtained by the preparation method of any one of claims 1-6, the acellular matrix of claim 7, and the acellular matrix product of claim 8 in preparing a biomaterial.

10. Use according to claim 9, characterized in that, The biomaterial is applied to subcutaneous tissue or soft tissue by injection for cosmetic plastic surgery or tissue repair.

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