Decellularized fat matrix gel as well as preparation method and application thereof

Abstract

The invention discloses a preparation method and application of decellularized fat matrix medical gel. According to the method, adipose tissue obtained by liposuction is taken as a raw material, and the decellularized adipose matrix medical gel with high biocompatibility and low immunogenicity is prepared through the processes of pretreatment, mechanical crushing, chemical decellularization, supercritical CO2 degreasing, low-temperature crushing and medical gel compounding. According to the method, through the synergistic effect of the composite decellularization agent and the supercritical degreasing technology, lipid, nucleic acid and cell residues are efficiently removed, and meanwhile the natural structure and biological activity of an extracellular matrix (ECM) are reserved. The viscosity of the final product is 500-3000mPa. S, and the product can be used for treating wound injury in a smearing manner or repairing endometrial injury in an intrauterine administration manner. The process is stable and controllable, and the product is safe and effective and has a remarkable clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and specifically relates to a method for preparing acellular fat matrix gel using fat tissue derived from liposuction as raw material, which is particularly suitable for repairing wound damage and endometrial damage. Background Art

[0002] Traditional decellularized matrix materials mostly use animal-derived tissues (such as pig skin and bovine pericardium), which poses the risk of immunogenicity. Adipose tissue is an ideal alternative raw material because it is rich in ECM components (such as collagen and laminin) and endogenous growth factors (such as VEGF and bFGF). However, existing decellularization technologies (such as repeated freezing and thawing, single detergent treatment) are prone to residual cell fragments or destruction of ECM structure, and the degreasing process mostly uses organic solvents (such as ethanol and acetone), which poses the risk of toxic residues. Repair of wound damage and tissue adhesion is a difficult point in clinical treatment. Traditional treatment methods have problems such as poor tissue compatibility and insufficient regeneration ability. Decellularized matrix materials have good biocompatibility and tissue regeneration potential because they retain natural extracellular matrix components (such as collagen and glycoproteins). However, existing decellularization processes mostly use animal-derived tissues or complex chemical treatments, which have defects such as residual immunogenicity, high process costs and insufficient mechanical properties.

[0003] Adipose tissue is an ideal source of material for regenerative medicine due to its rich stromal vascular fraction (SVF) and paracrine activity of adipose-derived stem cells. However, existing adipose decellularization technologies suffer from issues such as incomplete delipidation, residual cell fragments, and uneven particle size, limiting their clinical application. Summary of the Invention

[0004] One object of the present invention is to provide a fat-based acellular matrix gel, which has excellent biocompatibility, high purity, low immunogenicity and excellent ability to promote tissue regeneration, and can be used for repairing wound damage and endometrial damage.

[0005] In a first aspect of the present invention, a method for preparing an acellular adipose-derived matrix gel is provided, comprising the following steps:

[0006] (s1) providing washed adipose tissue fragments;

[0007] (s2) homogenizing and crushing the adipose tissue obtained in step (s1) to obtain adipose tissue particles of a certain particle size;

[0008] (s3) mixing the adipose tissue particles obtained in step (s2) with a decellularization reagent and performing a decellularization treatment to obtain decellularized adipose tissue;

[0009] (s4) mixing the decellularized adipose tissue obtained in step (s3) with a nucleic acid digestion agent and performing nucleic acid digestion treatment to obtain nucleic acid-digested adipose tissue;

[0010] (s5) mixing the nucleic acid-digested adipose tissue obtained in step (s4) with a disinfectant and performing a disinfection treatment to obtain a disinfected adipose tissue;

[0011] (s6) subjecting the sterilized adipose tissue obtained in step (s5) to supercritical CO2 extraction to obtain defatted adipose tissue;

[0012] (s7) subjecting the defatted adipose tissue obtained in step (s6) to liquid nitrogen cryo-grinding to obtain acellular adipose matrix powder of a certain particle size;

[0013] (s8) compounding the decellularized fat matrix obtained in step (s7) into a gel to obtain a preliminary decellularized fat matrix gel;

[0014] (s9) sterilizing the preliminary decellularized fat matrix gel obtained in step (s8) to obtain the decellularized fat matrix gel.

[0015] In another preferred embodiment, the method further comprises a pretreatment step: (s0) washing the adipose tissue to remove blood cells and free fat, and then chopping the adipose tissue to obtain washed adipose tissue fragments.

[0016] In another preferred embodiment, the adipose tissue is derived from mammals, preferably humans, sheep, cattle or pigs.

[0017] In another preferred embodiment, the adipose tissue is allogeneic adipose tissue or homologous adipose tissue.

[0018] In another preferred embodiment, in the step (s0), the adipose tissue is derived from liposuction.

[0019] In another preferred embodiment, in the step (s0), rinsing is performed using PBS solution and / or ultrapure water, preferably PBS solution.

[0020] In another preferred embodiment, in the step (s0), the number of flushing is 2-5 times, for example, 3 times.

[0021] In another preferred embodiment, in the step (s0), each flushing step further includes a centrifugal separation step.

[0022] In another preferred embodiment, in the step (s0), the rotation speed of each centrifugation is independently 200-2000g, preferably 200-1600g, and most preferably 800-1200g.

[0023] In another preferred embodiment, in the step (s0), the time of each centrifugation is independently 1-15 min, preferably 1-10 min, more preferably 2-8 min, and most preferably 3-5 min.

[0024] In another preferred embodiment, in the step (s0), the washed fat tissue is mechanically cut into pieces of 1-5 mm 3 (Preferably 2mm 3 ) particles.

[0025] In another preferred embodiment, the step (s0) comprises: washing the adipose tissue with PBS solution for 2-5 times, centrifuging after each washing (using 2000-16000g centrifugation for 2-8 minutes) to completely remove blood cells and free fat, and then chopping into 1-5 mm pieces. 3 The washed fat tissue fragments were obtained.

[0026] In another preferred embodiment, in the step (s2), the homogenization is performed by homogenizing and crushing using a homogenizing pump.

[0027] In another preferred embodiment, the rotation speed of the homogenizing pump is 1000-8000 rpm, preferably 2000-5000 rpm, such as 3000 rpm, 4000 rpm.

[0028] In another preferred embodiment, in the step (s2), the homogenization and crushing time is 0.5-15 min, preferably 1-10 min, more preferably 1-8 min, and most preferably 2-5 min.

[0029] In another preferred embodiment, the particles with D90 < 500 μm, preferably < 300 μm, are obtained after the homogenization and crushing.

[0030] In another preferred embodiment, the step (s2) further includes an optional post-processing step, wherein the post-processing step is to collect the solid phase layer by centrifugation to obtain adipose tissue particles of a certain particle size.

[0031] In another preferred embodiment, the centrifugal speed is 200-2000g, preferably 200-1600g, and most preferably 800-1200g.

[0032] In another preferred embodiment, the centrifugation time is 1-15 min, preferably 1-10 min, more preferably 2-8 min, and most preferably 3-5 min.

[0033] In another preferred embodiment, the step (s2) comprises: crushing the adipose tissue fragments obtained in step (s1) by a homogenizing pump at 2000-5000 rpm for 1-8 minutes so that the particle size D90 is less than 500 μm (preferably less than 300 μm), and then centrifuging to collect the solid phase layer to obtain adipose tissue particles.

[0034] In another preferred embodiment, in the step (s3), the decellularization reagent is a surfactant solution.

[0035] In another preferred embodiment, in the step (s3), the volume ratio of the adipose tissue particles obtained in the step (s2) to the decellularization reagent is 1:1 to 1:20, preferably 1:2 to 1:20, more preferably 1:5 to 1:20, for example 1:8, 1:10.

[0036] In another preferred embodiment, in the step (s3), the decellularization reagent is a mixture of an ionic surfactant and a non-ionic surfactant.

[0037] In another preferred embodiment, the surfactant is selected from one or more of SDS, Triton, and sodium deoxycholate.

[0038] In another preferred embodiment, the Triton is Triton X-100.

[0039] In another preferred embodiment, in the step (s3), the decellularization reagent is a mixture of SDS and Triton.

[0040] In another preferred embodiment, in the step (s3), the solvent of the decellularization reagent is water.

[0041] In another preferred embodiment, the proportion of sodium dodecyl sulfate (SDS) in the decellularization reagent is 0.05-5% (w / v), preferably 0.1-1% (w / v), more preferably 0.2-0.8% (w / v), for example 0.5% (w / v).

[0042] In another preferred embodiment, the proportion of Triton in the decellularization reagent is 0.1-5% (w / v), preferably 0.5-3% (w / v), more preferably 0.5-2% (w / v), for example 1% (w / v).

[0043] In another preferred embodiment, in the step (s3), the decellularization treatment is performed at 20-40°C, for example, at 37°C.

[0044] In another preferred embodiment, in the step (s3), the mixing refers to oscillating mixing or stirring mixing.

[0045] In another preferred embodiment, in the step (s3), the mixing refers to shaking mixing on a water bath shaker.

[0046] In another preferred embodiment, in the step (s3), the decellularization treatment time is 8 to 18 hours, preferably 12 to 16 hours.

[0047] In another preferred embodiment, the step (s3) further includes a post-processing step of rinsing.

[0048] In another preferred embodiment, in the step (s3), the rinsing refers to rinsing alternately with PBS and ultrapure water.

[0049] In another preferred embodiment, in the step (s3), the flushing is stopped when the conductivity of the flushing liquid drops below 50 μS / cm (for example, 45 μS / cm).

[0050] In another preferred embodiment, the step (s3) comprises: mixing the adipose tissue particles obtained in step (s2) with a mixture of 0.1-1% (w / v) sodium lauryl sulfate and 0.1-5% (w / v) Triton (mass ratio of 1:1 to 10), shaking at 37°C for 8 to 18 hours, and then rinsing until the conductivity of the rinsing solution drops below 50 μS / cm, and then stopping the rinsing.

[0051] In another preferred embodiment, in the step (s4), the nucleic acid digestion agent is a nuclease solution, preferably a Benzonase nuclease solution.

[0052] In another preferred embodiment, in the step (s4), the concentration of the nuclease in the nucleic acid digestion agent is 10-100 U / mL, preferably 30-70 U / mL, for example 50 U / mL.

[0053] In another preferred embodiment, in the step (s4), the volume ratio of the decellularized adipose tissue obtained in the step (s3) to the nucleic acid digestion agent is 1:1 to 1:20, preferably 1:2 to 1:20, more preferably 1:5 to 1:20, for example 1:8, 1:10.

[0054] In another preferred embodiment, in the step (s4), the nucleic acid digestion treatment is carried out at 20-40°C, for example, at 37°C.

[0055] In another preferred embodiment, in the step (s4), the nucleic acid digestion treatment time is 2 to 12 hours, preferably 5 to 10 hours.

[0056] In another preferred embodiment, the step (s4) further comprises a post-processing step of inactivating the nucleic acid digestion agent using an inactivating agent, followed by washing.

[0057] In another preferred embodiment, in the step (s4), the inactivating agent is EDTA solution.

[0058] In another preferred embodiment, in the step (s4), the concentration of EDTA in the inactivator is 1-50 mM, preferably 5-30 mM, for example 10 mM.

[0059] In another preferred embodiment, in the step (s4), PBS solution is used for rinsing.

[0060] In another preferred embodiment, in the step (s4), the number of flushing is 2-5 times, for example, 3 times.

[0061] In another preferred embodiment, the step (s4) comprises: mixing the decellularized adipose tissue obtained in step (s3) with 10-100 U / mL Benzonase nuclease solution, subjecting the mixture to nucleic acid digestion treatment at 20-40° C. for 2-12 hours, then inactivating the mixture with 5-30 mM EDTA, and rinsing the mixture 2-5 times to obtain the nucleic acid-digested adipose tissue.

[0062] In another preferred embodiment, in the step (s5), the disinfectant is a mixed solution of peroxide and alcohol.

[0063] In another preferred embodiment, the peroxide is selected from the group consisting of hydrogen peroxide, peracetic acid, chlorine dioxide, ozone, or a combination thereof.

[0064] In another preferred embodiment, the alcohol is selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, or a combination thereof.

[0065] In another preferred embodiment, the proportion of the peroxide in the disinfectant is 0.05-3% (w / v), preferably 0.1-2% (w / v), more preferably 0.1-1% (w / v), for example 0.2% (w / v), 0.5% (w / v).

[0066] In another preferred embodiment, the proportion of the alcohol in the disinfectant is 1-50% (w / v), preferably 5-40% (w / v), more preferably 10-30% (w / v), for example 15% (w / v), 20% (w / v), 25% (w / v).

[0067] In another preferred embodiment, in the step (s5), the disinfectant is a mixed solution of peracetic acid and ethanol.

[0068] In another preferred example, in the step (s5), the volume ratio of the nucleic acid-digested adipose tissue obtained in the step (s4) to the disinfectant is 1:1 to 1:20, preferably 1:2 to 1:20, more preferably 1:5 to 1:20, for example 1:8, 1:10.

[0069] In another preferred embodiment, in the step (s5), the mixing refers to oscillating mixing or stirring mixing.

[0070] In another preferred embodiment, in the step (s5), the mixing refers to shaking mixing on a water bath shaker.

[0071] In another preferred embodiment, in the step (s5), the disinfection treatment is carried out at 20-40°C, for example, at 30°C.

[0072] In another preferred embodiment, in the step (s5), the disinfection treatment time is 1 to 10 hours, preferably 2 to 8 hours, for example 4 hours.

[0073] In another preferred embodiment, the step (s5) further includes a post-treatment step of flushing after disinfection.

[0074] In another preferred embodiment, in the step (s5), the rinsing is performed by alternating PBS and ultrapure water.

[0075] In another preferred embodiment, in the step (s5), the number of flushing is 2-5 times, for example, 3 times.

[0076] In another preferred embodiment, in the step (s5), the flushing is stopped when the conductivity of the flushing liquid drops below 50 μS / cm (for example, 45 μS / cm).

[0077] In another preferred embodiment, the step (s5) comprises: mixing the nucleic acid-digested adipose tissue obtained in step (s4) with 2 to 20 times the volume of a mixed solution of 0.1-1% (w / v) peracetic acid and 5-40% (w / v) ethanol, and shaking at 20 to 40° C. for 2 to 8 hours for disinfection. After disinfection, the adipose tissue is alternately rinsed with PBS and ultrapure water until the conductivity drops below 50 μS / cm.

[0078] In another preferred embodiment, in step (s6), the supercritical CO2 extraction treatment includes placing the sterilized adipose tissue obtained in step (s4) into a supercritical CO2 extraction device.

[0079] In another preferred embodiment, in step (s6), the parameters of the supercritical CO2 extraction process include one or more of the following:

[0080] (1) The pressure is 20-50 MPa, preferably 30-40 MPa;

[0081] (2) The temperature is 20 to 60°C, preferably 30 to 50°C, and more preferably 35 to 45°C;

[0082] (3) The carbon dioxide flow rate is 20 to 50 L / min, preferably 30 to 40 L / min;

[0083] (4) The time is 8-24 hours, preferably 12-16 hours.

[0084] In another preferred embodiment, the step (s6) includes: placing the sterilized adipose tissue obtained in step (s5) in a reactor, pumping in gaseous carbon dioxide at high pressure, and preparing a supercritical fluid by pressurization; maintaining the pressure at 20 to 50 MPa, the temperature at 20 to 60°C, and the carbon dioxide flow rate at 20 to 50 L / min for 12 to 24 hours, reducing the pressure in the reactor to normal pressure, and taking out the extracted and defatted adipose tissue.

[0085] In another preferred embodiment, in the step (s7), the liquid nitrogen cryo-grinding comprises: freezing the defatted adipose tissue obtained in the step (s6) in liquid nitrogen, and grinding it after it is completely frozen (preferably grinding it in an ultra-low temperature liquid nitrogen cryo-grinding apparatus) to obtain acellular adipose matrix powder of a certain particle size.

[0086] In another preferred embodiment, the liquid nitrogen level is 2 to 5 times, preferably 2 to 3 times, the volume of the defatted adipose tissue obtained in step (s6).

[0087] In another preferred embodiment, in the step (s7), the grinding speed is 5000 to 15000 rpm, preferably 5000 to 8000 rpm.

[0088] In another preferred embodiment, in the step (s7), the grinding is performed 2 to 8 times, preferably 2 to 5 times.

[0089] In another preferred embodiment, in the step (s7), the grinding time for each time is independently 2 to 10 minutes, preferably 2 to 5 minutes.

[0090] In another preferred embodiment, the step (s7) further comprises screening the decellularized fat matrix powder with a particle size of less than 100 μm.

[0091] In another preferred embodiment, in the step (s7), the particle size of the decellularized fat matrix powder obtained after the liquid nitrogen freeze-grinding treatment is <300 μm, preferably <100 μm.

[0092] In another preferred embodiment, in the step (s8), the gel compounding refers to mixing the decellularized fat matrix obtained in step (s7) with the gel matrix solution, and then performing high-pressure microfluidization homogenization treatment to a certain viscosity to obtain a preliminary decellularized fat matrix gel.

[0093] In another preferred embodiment, the step (s8) comprises the following steps:

[0094] (s8.1) preparing gel matrix solution;

[0095] (s8.2) mixing the acellular fat matrix obtained in step (s7) with the gel matrix;

[0096] (s8.3) The mixture is homogenized by high-pressure microfluidization to a certain viscosity to obtain a preliminary acellular adipose matrix gel.

[0097] In another preferred embodiment, the gel matrix is ​​selected from the group consisting of sodium carboxymethylcellulose (CMC-Na), sodium hyaluronate, carbomer, or a combination thereof.

[0098] In another preferred embodiment, the gel matrix solution is a CMC-Na solution.

[0099] In another preferred embodiment, the solvent of the gel matrix solution is physiological saline.

[0100] In another preferred embodiment, the concentration of the gel matrix solution is 0.1-5%, preferably 0.5-4%, more preferably 1-3%.

[0101] In another preferred embodiment, the pH of the gel matrix solution is 7±0.5, preferably 6.8-7.4, such as 7.2.

[0102] In another preferred embodiment, the osmotic pressure of the gel matrix solution is 280-320 mOsm / kg, such as 300 mOsm / kg.

[0103] In another preferred embodiment, the osmotic pressure of the gel matrix solution is adjusted to 280-320 mOsm / kg by adding sodium chloride.

[0104] In another preferred embodiment, the step (s8.1) comprises: dissolving carboxymethylcellulose sodium (CMC-Na) in physiological saline to a final concentration of 0.5-3%, adjusting the pH to 6.8-7.4, and the osmotic pressure to 280-320 mOsm / kg.

[0105] In another preferred embodiment, the final concentration of the decellularized fat matrix powder in the gel matrix after mixing is 1 to 50 mg / mL, preferably 5 to 30 mg / mL.

[0106] In another preferred embodiment, the high-pressure microfluidization homogenization treatment comprises processing the mixture through a high-pressure microfluidizer until the viscosity of the gel is 500-3000 mPa·s, preferably 1000-2000 mPa·s.

[0107] In another preferred embodiment, the viscosity of the acellular fat matrix gel is regulated to 500-3000 mPa·s by the processing time and pressure of a high-pressure microfluidizer.

[0108] In another preferred embodiment, the step (s8) further comprises the step of filling the obtained preliminary decellularized fat matrix gel.

[0109] In another preferred embodiment, the filling refers to filling into a prefilled syringe.

[0110] In another preferred embodiment, the volume of the prefilled syringe is 0.5 mL to 3 ml per syringe.

[0111] In another preferred embodiment, in the step (s9), the sterilization refers to sterilization using ethylene oxide.

[0112] In another preferred embodiment, in the step (s9), the flow rate of the ethylene oxide is 500-1000 mg / L, preferably 600-800 mg / L.

[0113] In another preferred embodiment, in the step (s9), the sterilization is carried out at 20-40°C, for example, 37°C.

[0114] In another preferred embodiment, in the step (s9), the sterilization is carried out under a humidity condition of 50 to 90%, preferably 60 to 80%.

[0115] In another preferred embodiment, in the step (s9), the sterilization time is 1 to 6 hours, preferably 3 to 4 hours.

[0116] In another preferred embodiment, in the step (s9), after the sterilization is completed, the remaining ethylene oxide gas is removed, and ventilation and analysis are performed, and finally the residual ethylene oxide content is detected to ensure that the post-processing steps meet safety standards.

[0117] In another preferred embodiment, in the step (s9), the ventilation and desorption treatment includes: first performing ventilation treatment, then desorption at room temperature under vacuum (-90 kPa) for 24 to 96 hours (for example, 72 hours), and finally desorption at room temperature under ventilation for ≥7 days.

[0118] In another preferred embodiment, the method comprises the steps of:

[0119] (1) Raw material pretreatment: Adipose tissue was obtained by liposuction, washed three times with phosphate buffered saline (PBS) and centrifuged (1000 g × 5 min) to remove blood cells and free fat, and then mechanically chopped into 1-3 mm pieces. 3 particles;

[0120] (2) Disruption and decellularization: The cells were homogenized at 3000 rpm for 5 minutes to reduce the particle size D90 to <300 μm. After centrifugation, the solid phase was collected and a mixture of 0.5% sodium dodecyl sulfate (SDS) and 1% Triton X-100 (mass ratio 5:1) was added and shaken at 37°C for 12 hours. The cells were alternately rinsed with PBS and ultrapure water until the conductivity of the washing solution was <50 μS / cm. 50 U / mL Benzonase was then added for digestion at 37°C for 6 hours. The cells were inactivated with 10 mM EDTA and rinsed three times with PBS.

[0121] (3) Disinfection of adipose tissue: add 10 times the volume of 0.2% peracetic acid + 20% ethanol solution, shake at 30°C for 4 hours, and rinse alternately with PBS and ultrapure water until the conductivity reaches 45 μS / cm;

[0122] (4) Supercritical CO2 degreasing: extraction for 12 hours at a temperature of 40°C, a pressure of 35 MPa, and a CO2 flow rate of 30 L / min;

[0123] (5) Cryogenic grinding: grinding the acellular fat matrix in liquid nitrogen to a particle size of <100 μm;

[0124] (6) Medical gel compounding: Prepare sodium carboxymethylcellulose (CMC-Na) solution (final concentration 0.5-3%) with normal saline, adjust the pH to 6.8-7.4 and the osmotic pressure to 280-320 mOsm / kg, add the ground decellularized fat matrix (final concentration 5-20 mg / mL), process with a high-pressure microfluidizer to a viscosity of 500-3000 mPa·s, fill into prefilled syringes (0.5-3 mL / syringe), and sterilize with ethylene oxide.

[0125] In a second aspect of the present invention, a decellularized adipose tissue matrix gel is provided. The decellularized adipose tissue matrix gel is prepared using the method described in the first aspect of the present invention.

[0126] In another preferred embodiment, the acellular fat matrix gel is non-cytotoxic.

[0127] In another preferred embodiment, the “non-cytotoxic” means that the relative survival rate of cells co-cultured with the decellularized adipose tissue matrix gel is higher than 90%, preferably higher than 95%, compared with cells not co-cultured with the decellularized adipose tissue matrix gel.

[0128] In another preferred embodiment, the relative abundance of collagen in the acellular fat matrix gel is 30% to 50%, preferably 35% to 50%.

[0129] In another preferred embodiment, the relative abundance of elastin in the acellular fat matrix gel is 2% to 5%, preferably 2.5% to 5%.

[0130] In another preferred embodiment, the relative abundance of laminin in the acellular fat matrix gel is 0.5% to 3%, preferably 1% to 3%.

[0131] In another preferred embodiment, according to the fluorescent quantitative PCR method, the nucleic acid content in the acellular adipose matrix gel is less than 50 ng / mg, preferably less than 30 ng / mg, and more preferably less than 20 ng / mg.

[0132] In another preferred embodiment, according to the Soxhlet extraction method specified in the first method of GB / T 5009.6-2016, the oil content in the acellular adipose matrix gel is less than 3%, preferably less than 2%.

[0133] In another preferred embodiment, the phospholipid content in the acellular adipose matrix gel is less than 20 μg / mg, preferably less than 10 μg / mg.

[0134] In another preferred embodiment, the viscosity of the acellular fat matrix gel is 1000 to 3000 mPa·s, preferably 1200 to 3000 mPa·s.

[0135] In the third aspect of the present invention, a medical material is provided, comprising the acellular fat matrix gel as described in the second aspect of the present invention, a pharmaceutically acceptable carrier, and optionally other drugs having a wound repairing effect.

[0136] In another preferred embodiment, the dosage form of the medical material is a gel.

[0137] In another preferred embodiment, the medical material is administered by topical application.

[0138] In another preferred embodiment, the pharmaceutically acceptable carrier includes medical excipients.

[0139] In another preferred embodiment, the other drugs having wound repairing effects include one or more of small molecule drugs (including sustained-release drugs), cell growth factors, and active cells with repairing effects.

[0140] In the fourth aspect of the present invention, there is provided a use of the acellular fat matrix gel as described in the second aspect of the present invention or the medical material as described in the third aspect of the present invention for preparing a pharmaceutical composition for repairing wound damage and / or repairing endometrial damage.

[0141] In another preferred embodiment, the wound surface injury includes chronic wound surface injury and non-chronic wound surface injury.

[0142] In another preferred embodiment, the chronic wound injury includes diabetic foot ulcer, venous ulcer, arterial ulcer or pressure sore.

[0143] In another preferred embodiment, the non-chronic wound injury includes laceration, abrasion, tear or herpes zoster.

[0144] In another preferred embodiment, the endometrial damage repair includes one or more of the following: repair of intrauterine adhesions, repair of thin endometrium, repair of Ashman syndrome, endometrial repair after intrauterine surgery (such as abortion, curettage), or a combination thereof.

[0145] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0146] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0147] Figure 1 The preparation process of acellular adipose matrix gel is shown.

[0148] Figure 2 Results for endometrial cell proliferation capacity are shown.

[0149] Figure 3 Shown are the results of the endometrial cell migration ability test.

[0150] Figure 4 Results of endometrial cell inflammatory factor levels are shown.

[0151] Figure 5 The effects of acellular adipose tissue matrix gel on hDF cell proliferation and inflammatory factors are shown. DETAILED DESCRIPTION

[0152] The present invention develops a method for preparing a high-purity, low-immunogenicity acellular adipose-derived matrix gel by optimizing the decellularization process and combining it with supercritical CO2 degreasing technology. This method overcomes the problems of low decellularization and degreasing efficiency, high residual DNA content, and insufficient product stability in the prior art. The acellular adipose-derived matrix gel obtained by the present invention has excellent tissue regeneration capabilities and can be used for wound and endometrial repair. This is based on the present invention.

[0153] the term

[0154] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0155] As used herein, the terms "comprise," "include," and "contain" are used interchangeably to include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of," "consisting essentially of."

[0156] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0157] Preparation method

[0158] The overall process of the preparation method of decellularized fat matrix gel is as follows Figure 1 As shown, the following steps are included:

[0159] (1) Raw material pretreatment

[0160] a. Obtain adipose tissue by liposuction, rinse three times with phosphate-buffered saline (PBS), and centrifuge at 1000 g for 5 minutes each time to remove blood cells and free lipids.

[0161] b. Mechanically cut into pieces of 1-3mm 3 particles.

[0162] (2) Decellularization and disinfection

[0163] a. Homogenize the mixture at 3000 rpm for 5 minutes to reduce the particle size D90 to <300 μm, and centrifuge at 1000 g for 5 minutes to collect the solid phase.

[0164] b. A mixture of 0.5% sodium dodecyl sulfate (SDS) and 1% Triton X-100 was added to the solid phase fat after centrifugation at a ratio of 5:1 (w / w) and shaken at 37°C for 12 hours;

[0165] c. Alternately rinse with PBS and ultrapure water until the conductivity of the washing solution is <50μS / cm;

[0166] d. Add 50 U / mL Benzonase nuclease and digest at 37°C for 6 hours. Inactivate with 10 mM EDTA and rinse three times with PBS.

[0167] e. Add 10 times the volume of 0.2% peracetic acid + 20% ethanol solution, shake at 30°C for 4 hours, and rinse alternately with PBS and ultrapure water until the conductivity reaches 45μS / cm;

[0168] (3) Supercritical CO2 degreasing

[0169] a. Conditions: temperature 40°C, pressure 35 MPa, CO2 flow rate 30 L / min, extraction time 12 hours.

[0170] (4) Low temperature crushing

[0171] a. Grind the acellular adipose tissue matrix under liquid nitrogen to a particle size of <100 μm.

[0172] (5) Medical gel compounding method

[0173] a. Dissolve sodium carboxymethylcellulose (CMC-Na) in normal saline to a final concentration of 0.5-3%, adjust the pH to 6.8-7.4, and the osmotic pressure to 280-320 mOsm / kg;

[0174] b. Add ground acellular adipose tissue matrix to a final concentration of 5-20 mg / mL;

[0175] c. Process with a high-pressure microfluidizer to a viscosity of 500-3000 mPa·s, fill into prefilled syringes (0.5 mL to 3 mL / syringe), and sterilize with ethylene oxide.

[0176] Medical materials and administration methods

[0177] The present invention provides a medical material for repairing wounds or endometrium, comprising the adipose decellularized matrix gel of the present invention and a pharmaceutically acceptable carrier.

[0178] The term "pharmaceutically acceptable carrier" refers to one or more compatible solid, semisolid, or liquid fillers suitable for human or animal use and possessing sufficient purity and low toxicity. "Compatibility" refers to the ability of the components of a pharmaceutical composition to be compatible with the active ingredient of the drug, as well as with each other, without significantly reducing the drug's efficacy.

[0179] It should be understood that in the present invention, the carrier is not particularly limited and can be selected from materials commonly used in the art, or prepared by conventional methods, or purchased from the market. Some examples of pharmaceutically acceptable carriers include vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium lauryl sulfate), buffers, chelating agents, thickeners, pH regulators, transdermal enhancers, colorants, flavorings, stabilizers, antioxidants, preservatives, antibacterial agents, pyrogen-free water, etc.

[0180] Examples of antioxidants include tocopherol and its ester derivatives, ascorbic acid, ascorbyl stearate, nordihydroguaiaretic acid, butylhydroxytoluene (BHT), butylhydroxyanisole (BHA), citric acid, 2-mercaptobenzimidazole, and ethylenediaminetetraacetic acid. These antioxidants may be used alone or in combination of two or more.

[0181] The skin penetration enhancer is not particularly limited as long as it is a compound generally recognized to have an absorption promoting effect on the skin. Examples thereof include fatty alcohols such as isostearyl alcohol, fatty acids such as capric acid, fatty acid derivatives such as propylene glycol monolaurate, isopropyl myristate, isopropyl palmitate, and lauric acid diethanolamide, and glycols such as propylene glycol and polyethylene glycol. The skin penetration enhancer may be used alone or in combination of two or more.

[0182] The preservative is not particularly limited, but preferred examples include ethyl paraoxybenzoate, propyl paraoxybenzoate, and butyl paraoxybenzoate. The filler is not particularly limited, but preferred examples include calcium carbonate, magnesium carbonate, silicates (such as aluminum silicate, calcium silicate, and magnesium silicate), and cellulose derivatives (such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, and carboxymethyl cellulose).

[0183] The material dosage form should be compatible with the mode of administration. The material of the present invention can also be used with other synergistic therapeutic agents (including before, during or after use). When using the material of the present invention, the medicine of a safe and effective amount is applied to the desired object (such as a human or non-human mammal), and the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases is no more than about 8 milligrams / kg body weight, preferably the dosage is about 10 micrograms / kg body weight-about 1 milligram / kg body weight. Of course, the specific dosage should also consider factors such as route of administration, patient health status, etc., and these are all within the skill range of skilled physicians.

[0184] Advantages of the present invention include:

[0185] 1. The preparation method of the present invention has high decellularization efficiency: SDS / Triton X-100 combined treatment combined with nuclease digestion, the residual DNA amount is <50ng / mg.

[0186] 2. The present invention uses supercritical CO2 degreasing: the lipid residual rate is less than 1%, avoiding the toxicity of organic solvents;

[0187] 3. The acellular fat matrix gel prepared by the present invention has stable gel properties and controllable viscosity (500-3000 mPa·s);

[0188] 4. The acellular fat matrix gel prepared by the present invention has an excellent effect of promoting tissue regeneration.

[0189] The following examples provide a method for preparing acellular matrix gel from human adipose tissue. The fat is derived from fat obtained by suction or surgical resection (sourced from Xinxiang Medical College, donated by liposuction patients with informed consent from the patients).

[0190] Example 1:

[0191] 1.1 Adipose tissue pretreatment: Adipose tissue was obtained by liposuction, rinsed 3 times with PBS (centrifuged at 1000 g for 5 min after each rinse), and mechanically minced to 2 mm. 3 particles;

[0192] 1.2 Adipose tissue disruption and decellularization: The tissue was disrupted by homogenization at 3000 rpm for 5 min, centrifuged at 1000 g for 5 min, and then added with a mixed SDS-Triton solution (0.5% (w / v) + 1% (w / v)) and shaken at 37°C for 12 h. The mass ratio of adipose tissue to the mixed solution was 1:5-1:10. The tissue was then rinsed alternately with PBS and ultrapure water until the conductivity reached 45 μS / cm.

[0193] 1.3 Adipose tissue nucleic acid digestion: Add Benzonase nuclease (final concentration 50 U / mL) and digest for 6 hours. Inactivate with 10 mM EDTA and rinse three times with PBS.

[0194] 1.4 Disinfection of adipose tissue: Add 10 volumes of a mixed aqueous solution of 0.2% (w / v) peracetic acid and 20% (w / v) ethanol, shake at 30°C for 4 hours, and rinse alternately with PBS and ultrapure water until the conductivity reaches 45 μS / cm;

[0195] 1.5 Degreasing of adipose tissue: Place the sterilized fat in a reactor and pump gaseous carbon dioxide into the reactor under high pressure to prepare a supercritical fluid. Maintain the pressure at 35 MPa, the temperature at 40°C, and the carbon dioxide flow rate at 30 L / h for 12 hours. Then, reduce the pressure in the reactor to normal pressure and remove the extracted adipose tissue for grinding in the next step.

[0196] 1.6 Liquid nitrogen cryo-grinding: Freeze the product obtained in step 1.4 with liquid nitrogen, grind the frozen matrix using an ultra-low temperature liquid nitrogen cryo-grinder, and then sieve the decellularized fat matrix powder with a particle size of less than 100 μm;

[0197] 1.7 Preparation of decellularized adipose tissue matrix gel: The decellularized adipose tissue matrix powder prepared in step 1.6 was mixed at a concentration of 10 mg / mL with 1.5% (w / v) CMC-Na solution (pH 7.2, osmotic pressure 300 mOsm / kg) and homogenized using high-pressure microfluidization to a viscosity of 1500 mPa·s.

[0198] 1.8 Prefilling: Fill the solution prepared in step 1.7 into a prefilled syringe with a filling specification of 0.5-3 mL.

[0199] 1.9 Sterilization: The sterilization method is ethylene oxide sterilization:

[0200] a. Sterilize with 600-800 mg / L ethylene oxide at 37°C and 60-80% humidity for 3-4 hours;

[0201] b. After sterilization, exhaust the remaining ethylene oxide gas and conduct ventilation;

[0202] c.37℃ vacuum desorption (-90kPa) for 72 hours;

[0203] d. Room temperature ventilation analysis for ≥ 7 days (dynamic monitoring of ethylene oxide residue);

[0204] e. Use gas chromatograph to detect the residual ethylene oxide to ensure that it meets the safety standards (≤10μg / g).

[0205] Example 2:

[0206] Other parameters were the same as in Example 1, except that the decellularization time in step 1.2 was adjusted to 8 and 16 h.

[0207] Example 3:

[0208] Other parameters are the same as those in Example 1, except that in step 1.4, the supercritical extraction pressure is 40 MPa and the temperature is 45°C.

[0209] Example 4:

[0210] Other parameters were the same as those in Example 1, except that the amount of ground acellular adipose matrix added in step 1.7 was changed to 1, 5, and 20 mg / mL.

[0211] Comparative Example 1:

[0212] Other parameters were the same as those in Example 1, except that in step 1.5, isopropyl alcohol solution was used instead of supercritical carbon dioxide extraction for degreasing.

[0213] The specific steps are as follows: add anhydrous isopropyl alcohol at a mass-to-volume ratio of 1:10, and wash at room temperature (200 rpm) for 16 hours; after washing, add pure water at a mass-to-volume ratio of 1:10 for three washes. c. Centrifuge at 4000 rpm for 5 minutes and collect the precipitate.

[0214] Comparative Example 2:

[0215] Other parameters were the same as those in Example 1. In step 1.2, 0.5% SDS solution was used for decellularization for 12 h.

[0216] Comparative Example 3:

[0217] Other parameters were the same as those in Example 1. In step 1.2, 1% Triton X-100 solution was used for decellularization for 12 h.

[0218] Comparative Example 4:

[0219] Other parameters were the same as in Example 1, except that Benzonase nuclease was not used to digest nucleic acids in step 1.3.

[0220] Comparative Example 5:

[0221] Other parameters were the same as those in Example 1. In step 1.7, the ground decellularized fat matrix was suspended by magnetic stirring instead of high-pressure microjetting to obtain the gel.

[0222] Comparative Example 6:

[0223] Other parameters were the same as those in Example 1. In step 1.9, irradiation sterilization was performed with a dose of 20 kGy instead of ethylene oxide sterilization.

[0224] Test example 1: Performance index test

[0225] Characterization and detection of protein composition of acellular adipose matrix gel

[0226] An appropriate amount of adipose tissue matrix gel was taken and lysed with 0.5 mol / L NaOH solution for 4 hours. The pH was adjusted to 8 with hydrochloric acid solution, and trypsin was added for 16 hours to completely digest the adipose tissue matrix gel sample. Label-free LC-MS / MS was then used to accurately identify and relatively quantify proteins in the supernatant of the digested sample. Gene Ontology (GO) analysis was then performed on the identified proteins to determine their specific tissue and cell localization classification. The relative abundance ratios of each protein and each classification were calculated. The results are shown in Table 1.

[0227] DNA content test

[0228] According to the host DNA residue detection method in the Chinese Pharmacopoeia, the nucleic acid residues in the protein were detected using fluorescent quantitative PCR.

[0229] Fluorescence quantitative PCR instrument was purchased from ABI, USA; micro-volume UV-visible spectrophotometer NanoDrop was purchased from Thermo Fisher, USA; Varioskan Flash multifunctional microplate reader was purchased from Thermo Fisher, USA; host residual DNA extraction kit DNAExtractor kit was purchased from Thermo Fisher, USA; Power SYBRGreen MasterMix was purchased from Thermo Fisher, USA; PrepSEQ TM Residual DNA sample preparation kit.

[0230] Extraction of residual human DNA from acellular adipose-derived matrix gel: Accurately measure 1 ml of 10 mg / ml acellular adipose-derived matrix gel and dilute it with TE solution to a final concentration of 1.5 mg / ml. Extract residual DNA according to the instructions of the DNA extractor kit.

[0231] DNA Residual Testing: Follow the instructions for the DNA Residual Testing Kit to determine the residual DNA content in the raw materials. Results are shown in Table 1.

[0232] Fat content test

[0233] The test was carried out according to the Soxhlet extraction method specified in the first method of GB / T 5009.6-2016. The results are shown in Table 1.

[0234] Table 1 Analysis results of main performance indicators of decellularized fat matrix gel

[0235]

[0236] From the data in Table 1, it can be seen that the supercritical CO2 degreasing technology (Examples 1-3) achieves a fat residue of only 0.3%, which is more than 95% lower than the organic solvent degreasing (Comparative Example 1) and does not cause structural protein loss.

[0237] Comparative Example 4 (nucleic acid content 415 ng / mg) revealed that omitting the Benzonase enzymatic hydrolysis step resulted in residual nucleic acid exceeding the standard by 8 times, confirming the necessity of nuclease treatment.

[0238] Effect of treatment time on protein retention: Example 2 (8h) vs Example 1 (12h) vs Example 2 (16h): Collagen: 43.3% → 40.7% → 35.2% (extended treatment leads to increased protein hydrolysis), elastin: 3.99% → 3.72% → 3.16% (accumulated mechanical stress destroys the fiber structure). As time goes by, the damage to the decellularized matrix will worsen.

[0239] The phospholipid content reflects the amount of cell membrane components, and the cell membrane reflects the effect of decellularization. From the results of Comparative Examples 2 and 3, the phospholipid content of the single-component decellularization reagent SDS or Triton X-100 treated for 12 hours increased by 15-20 times compared with the two buffer solutions.

[0240] Test Example 2: Sample Viscosity Test

[0241] Viscosity tests were conducted on Example 1 and Comparative Examples 5 and 6 using an NDJ-79 rotary viscometer. 15 ml of the sample to be tested was injected into the viscometer until the liquid level reached the lower edge of the conical surface. The rotating cylinder was then inserted into the liquid until it was completely submerged. The viscometer was then placed on a bracket and the cylinder hook was attached to the hook of the instrument's left-handed knurled nut. The motor was started, and the cylinder was rotated and swung until it aligned with the center. To accelerate alignment, the viscometer could be slightly shaken on the bracket. When the pointer stabilized, the reading could be taken. If the reading was less than 10 divisions, a larger diameter cylinder was replaced.

[0242] The results are shown in Table 2. As can be seen from the results in Table 2, in Comparative Example 6, which was sterilized by irradiation, the viscosity of the gel preparation decreased significantly after irradiation, and the decellularized adipose matrix gel showed denaturation after irradiation. In Comparative Example 5, even after stirring with a magnetic stirrer, some matrix suspension was still poor and unevenly dispersed at the end, and the measured viscosity was lower than that of Example 1, indicating that the dispersion results presented in Example 5 were inferior to those achieved by high-pressure microfluidization.

[0243] Table 2 Sample viscosity results

[0244] Example 1 Comparative Example 5 Comparative Example 6 Sample viscosity 1475mPa·s 1063mPa·s 105mPa·s

[0245] Test Example 3: Effect of acellular adipose matrix gel on the proliferation of endometrial cells

[0246] The acellular fat matrix gel of Example 1 and Example 4 was used to perform endometrial cell proliferation detection. Human primary endometrial epithelial cells (Wuhan Punosai Life Science Technology Co., Ltd.) were cultured with primary endometrial epithelial cell culture medium containing 10% FBS (Wuhan Punosai Life Science Technology Co., Ltd.). After cell digestion, a cell suspension was made and inoculated in a 96-well plate at a cell volume of 2000 cells / well, and cultured in a 37°C incubator for 24 hours. The next day, different concentrations of acellular fat matrix gel (5mg / ml, 10mg / ml, 20mg / ml, 25mg / ml) or PBS were used to treat the 96-well plate cells, with 3 replicates per group, and the culture was continued for 48 hours. After 48 hours of drug incubation, the 96-well plate was taken out, 10ul CCK8 solution was added to each well of cells, mixed and placed in a 37°C incubator for 1 hour, then detected using an enzyme marker, and the proliferation capacity of each group of cells was calculated. The results are shown in Table 2. Figure 2 Acellular fat matrix gel with a concentration of 5 mg / ml or above has a significant effect on promoting the proliferation of endometrial cells, and the degree of proliferation is dose-dependent.

[0247] Test Example 4: Effects of acellular adipose matrix gel on endometrial cell migration and inflammatory factor expression

[0248] 2×10 5 Human primary endometrial epithelial cells (Wuhan Punosai Life Science Co., Ltd.) were seeded into 6-well plates with a cell amount of 100 cells / well, cultured with primary endometrial epithelial cell culture medium (Punosai), and cultured in a 37°C incubator overnight. After the cells were starved for 8 hours with primary endometrial epithelial cell culture medium containing 0.5% BSA, the monolayer of cells was scraped off from each well with the tip of a p20 pipette (time 0). The damaged cells were treated with the sample of Example 1 or PBS and cultured for another 24 hours. Images were taken using an inverted microscope at 0h or 24h after injury. After 24 hours, the cell migration area was evaluated using ImageJ1.42q software, and the results are shown in Table 2. Figure 3 It can be seen that the use of acellular fat matrix gel can significantly promote the migration of endometrial cells and significantly reduce the size of the scratch.

[0249] Human primary endometrial epithelial cells (Wuhan Punosai Life Science Technology Co., Ltd.) were cultured with primary endometrial epithelial cell culture medium containing 10% FBS (Wuhan Punosai Life Science Technology Co., Ltd.). After cell digestion, a cell suspension was prepared and inoculated into a 96-well plate at a cell volume of 2000 cells / well, and cultured in a 37°C incubator overnight. The damaged cells were treated with the sample of Example 1 + LPS or LPS, and cultured for 48 hours, with 3 replicates per group. After 48 hours of drug incubation, the 96-well plate was taken out and the levels of IL-6 and IL-1β were detected using an ELISA kit (Shanghai Yanzun Biotechnology Co., Ltd.) according to the operating instructions. The results are shown in Figure 4 It can be seen that compared with the LPS model group, the use of acellular adipose matrix gel can inhibit inflammatory factors to a certain extent.

[0250] Test Example 5: Effects of Acellular Adipose Matrix Gel on hDF Cell Proliferation and Inflammatory Factors

[0251] The samples of Example 1 and Example 4 were used for hDF cell proliferation activity detection. The cells used for detection were hDF (human skin fibroblasts, from ATCC, PCS-201-012). The steps were as follows:

[0252] After starvation culture of hDF cells in a 96-well plate for 24 hours, the original culture medium was discarded and 100 μl of maintenance medium was added to each well. 100 μl of the test sample was added to each well. Each 96-well plate contained a negative control without the test sample, with 100 μl of maintenance medium added to each well. The cells were incubated at 37°C and 5% CO2 for 72 hours.

[0253] 96-well plate cells plus CCK8: Add 10 μl of CCK8 solution to each well of the 96-well plate, mix thoroughly, and incubate at 37°C and 5% carbon dioxide for 2 h. Place the plate in a microplate reader, measure the absorbance at a wavelength of 450 nm, and record the measurement results.

[0254] Data processing calculation formula: cell viability = [(As-Ab) / (Ac-Ab)] × 100%, where As is the experimental well (culture medium containing cells, CCK8, and added sample); Ac is the control well (culture medium containing cells, CCK8, and no added sample); and Ab is (no cells and sample, CCK8).

[0255] Human hDF was cultured in a cell culture medium containing 10% FBS. After cell digestion, a cell suspension was prepared and 2000 cells / well were seeded in a 96-well plate and cultured overnight in a 37°C incubator. The damaged cells were treated with the sample of Example 1 + LPS or LPS and cultured for 48 hours, with 3 replicates per group. After 48 hours of drug incubation, the 96-well plate was removed and the levels of IL-6 and IL-1β were detected using an ELISA kit (Shanghai Yanzun Biotechnology Co., Ltd.) according to the operating instructions. The results are shown in Figure 5 It can be seen that acellular adipose tissue matrix gel with a concentration of 5 mg / ml or above has a significant pro-proliferation effect on hDF cells, and the degree of proliferation is dose-dependent; acellular adipose tissue matrix gel with a concentration of 5 mg / ml or above has a significant inhibitory effect on inflammatory factors, and the degree of inhibition is dose-dependent.

[0256] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A method for preparing acellular adipose tissue matrix gel, comprising the following steps: (s1) providing washed adipose tissue fragments; (s2) homogenizing and crushing the adipose tissue obtained in step (s1) to obtain adipose tissue particles of a certain particle size; (s3) mixing the adipose tissue particles obtained in step (s2) with a decellularization reagent and performing a decellularization treatment to obtain decellularized adipose tissue; (s4) mixing the decellularized adipose tissue obtained in step (s3) with a nucleic acid digestion agent and performing nucleic acid digestion treatment to obtain nucleic acid-digested adipose tissue; (s5) mixing the nucleic acid-digested adipose tissue obtained in step (s4) with a disinfectant and performing a disinfection treatment to obtain a disinfected adipose tissue; (s6) subjecting the sterilized adipose tissue obtained in step (s5) to supercritical CO2 extraction to obtain defatted adipose tissue; (s7) subjecting the defatted adipose tissue obtained in step (s6) to liquid nitrogen cryo-grinding to obtain acellular adipose matrix powder of a certain particle size; (s8) compounding the decellularized fat matrix obtained in step (s7) into a gel to obtain a preliminary decellularized fat matrix gel; (s9) sterilizing the preliminary decellularized fat matrix gel obtained in step (s8) to obtain the decellularized fat matrix gel.

2. The preparation method according to claim 1, wherein The adipose tissue is derived from mammals, preferably humans, sheep, cattle or pigs.

3. The preparation method according to claim 1, wherein In the step (s3), the decellularization reagent is a mixture of an ionic surfactant and a non-ionic surfactant.

4. The preparation method according to claim 1, wherein In the step (s4), the nucleic acid digestion agent is a nuclease solution, preferably a Benzonase nuclease solution.

5. The preparation method according to claim 1, wherein The disinfectant is a mixed solution of peroxide and alcohol.

6. The preparation method according to claim 1, wherein In the step (s8), the gel compounding refers to mixing the decellularized fat matrix obtained in step (s7) with the gel matrix solution, and then performing high-pressure microfluidization homogenization treatment to a certain viscosity to obtain a preliminary decellularized fat matrix gel.

7. The preparation method according to claim 1, wherein In the step (s9), the sterilization refers to sterilization using ethylene oxide.

8. A decellularized fat matrix gel, characterized in that: The acellular adipose matrix gel is prepared using the method according to any one of claims 1 to 7.

9. A medical material comprising the acellular adipose matrix gel according to claim 8, a pharmaceutically acceptable carrier, and optionally other drugs having a wound repairing effect.

10. Use of the acellular adipose matrix gel according to claim 8 or the medical material according to claim 9 for preparing a pharmaceutical composition for repairing wound damage and / or repairing endometrial damage.

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