Fat decellularization filling material and preparation method thereof

Abstract

The invention relates to a fat decellularized filling material and a preparation method thereof, and belongs to the technical field of biomedical materials. According to the method disclosed by the invention, efficient decellularization and extracellular matrix structure protection are synergistically realized through gradient osmotic pressure cracking, non-ionic surfactant cleaning and supercritical CO2 degreasing; and then the Pluronic F127-dopamine copolymer is used for grafting, so that the limitation of passive filling of the traditional material that the dopamine-mediated cell adhesion rate is increased is broken through, and blood vessel ingrowth and tissue regeneration are promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials and relates to a fat decellularized filling material and a preparation method thereof. Background Art

[0002] Filling materials play a vital role in regenerative medicine and plastic surgery. They are used to repair and replace damaged or diseased tissues and restore their structure and function. Traditional filling materials include autologous tissue, allogeneic tissue and synthetic materials. Autologous tissue transplantation, such as fat transplantation, is widely used due to its high biocompatibility and low immune rejection reaction. However, the limited donor site and the instability of the transplanted volume limit its application. Although synthetic materials such as silicone and hydroxyapatite have solved the donor problem to a certain extent, they have poor biocompatibility and cannot be fully integrated into the host tissue, and are prone to induce foreign body reactions and inflammation. Therefore, it would be extremely advantageous to develop a filling material that has good biocompatibility and can provide sufficient mechanical strength and stability.

[0003] Decellularized Adipose Tissue (DAT) technology has received widespread attention in recent years. The rich extracellular matrix (ECM) components in adipose tissue, such as collagen, elastin and glycosaminoglycans, play an important role in cell proliferation, differentiation and function. By removing the cellular components in adipose tissue and retaining its ECM structure, a highly biocompatible filling material can be obtained. The decellularized adipose tissue not only retains its original three-dimensional structure and mechanical properties, but also reduces immunogenicity and reduces immune rejection after transplantation. DAT material can be used as a natural scaffold material to promote the adhesion, migration and proliferation of host cells, and ultimately achieve tissue regeneration and repair. This material has broad application prospects in the fields of soft tissue filling, wound repair and organ regeneration.

[0004] Although adipose decellularized filling materials have demonstrated many advantages, existing technologies still have some shortcomings. First, traditional decellularization methods, such as physical, chemical, and enzymatic treatments, often damage the structure and function of the ECM while removing cellular components, affecting the biological properties of the material. Secondly, residual decellularization reagents, such as detergents and enzymes, may have toxic effects on host tissues, affecting the safety of the material. In addition, existing decellularization processes are usually complex and time-consuming, making it difficult to achieve large-scale production, which limits their application. Therefore, how to optimize decellularization technology, retain the integrity and function of the ECM, and develop efficient and controllable processing processes has become an urgent problem to be solved. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing a fat decellularized filling material, thereby obtaining a fat decellularized filling material that is efficient and has good biocompatibility.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A method for preparing a decellularized fat filling material comprises the following steps:

[0008] (1) Take the fat tissue and cut it into pieces of 1-5mm 3 The fragments were washed with 20-50 mM phosphate buffer, pH 6.5-7.5, to remove free lipids and blood components;

[0009] (2) Immerse the tissue fragments in a pH 7.2-7.6, 50-100 mOsm / L Tris-HCl buffer and shake at 2-6°C for 2-4 h to allow the adipocytes to swell and rupture.

[0010] (3) Transfer to 800-1200 mOsm / L sucrose solution and let stand at 15-25°C for 1-2 h to shrink the tissue and remove debris;

[0011] (4) Collect the precipitate by centrifugation and wash 2-3 times with 20-50 mM phosphate buffer, pH 6.5-7.5;

[0012] (5) The precipitate is immersed in a 0.5-2% (w / v) polysorbate or Triton X-100 nonionic surfactant solution and shaken at 35-39°C for 12-24 h to remove residual nuclear components and membrane-bound proteins;

[0013] (6) using supercritical CO2 fluid extraction to remove neutral lipids and residual surfactants to obtain acellular extracellular matrix microparticles;

[0014] (7) collecting the defatted and acellular extracellular matrix, freeze-drying it, ball-milling it, and sieving it to obtain microparticles with a particle size of 100-500 μm;

[0015] (8) Immerse the decellularized extracellular matrix microparticles in a 5-20% (w / v) Pluronic F127-dopamine solution and crosslink at 4°C for 12-24 hours to form a covalent grafting interface.

[0016] As a preferred technical solution of the present invention, the polysorbate in step (5) is Tween 20, Tween 40, Tween 60 or Tween 80.

[0017] As a preferred technical solution of the present invention, the nonionic surfactant solution in step (5) contains 0.05-0.1% EDTA.

[0018] As a preferred technical solution of the present invention, the supercritical CO2 fluid extraction in step (6) is carried out in two stages: the first stage: pressure 7-8MPa, temperature 35-40°C, lasting 2-4h; the second stage: pressure 9-10MPa, temperature 40-45°C, lasting 2-4h.

[0019] As a preferred technical solution of the present invention, the chemical cross-linking in step (8) uses EDC and NHS, the cross-linking agent concentration is 10-20 mM, and the reaction pH is 5.5-6.5.

[0020] As a preferred technical solution of the present invention, the preparation method of the Pluronic F127-dopamine copolymer in step (8) is:

[0021] A1. Dissolve Pluronic F127 in DMSO (concentration 5-15% w / v), add succinic anhydride (molar ratio of Pluronic F127 to succinic anhydride 1:5), react at 60°C for 18-24 hours, dialyze to remove unreacted reagents, and lyophilize to obtain carboxylated Pluronic F127.

[0022] A2. Dissolve carboxylated Pluronic F127 in MES buffer (pH 6.0), add EDC to a concentration of 10-15 mM, add NHS to a concentration of 5-10 mM, and activate at 15-25°C for 1-2 h.

[0023] A3. Add dopamine hydrochloride (molar ratio of Pluronic F127 to dopamine hydrochloride: 1:0.12), stir and react for 12 h in the dark, dialyze to remove free dopamine, and lyophilize to obtain Pluronic F127-dopamine copolymer.

[0024] Beneficial effects of the present invention:

[0025] (1) The present invention adopts a three-step decellularization process: gradient osmotic pressure lysis-nonionic surfactant cleaning-supercritical CO2 degreasing, which synergistically achieves efficient decellularization and ECM structure protection.

[0026] (2) Pluronic F127-dopamine copolymer grafting breaks through the passive filling limitations of traditional materials and improves dopamine-mediated cell adhesion rate. DETAILED DESCRIPTION

[0027] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific implementation methods, structures, features and effects of the present invention are described in detail below in combination with the embodiments.

[0028] Example 1

[0029] Adipose tissue was obtained from healthy women undergoing routine liposuction:

[0030] A method for preparing a decellularized fat filling material comprises the following steps:

[0031] (1) Take the fat tissue and cut it into 3mm pieces 3 The fragments were washed with 30 mM phosphate buffer, pH 7.0, to remove free lipids and blood components;

[0032] (2) The tissue fragments were immersed in pH 7.4, 80 mOsm / L Tris-HCl buffer and shaken at 4°C for 3 h to cause the adipocytes to swell and rupture;

[0033] (3) Transfer to 1000 mOsm / L sucrose solution and let stand at 20°C for 1.5 h to shrink the tissue and remove debris;

[0034] (4) The precipitate was collected by centrifugation and washed three times with 30 mM phosphate buffer, pH 7.0;

[0035] (5) The precipitate was immersed in a 1% (w / v) Triton X-100 nonionic surfactant solution and shaken at 37°C for 20 h to remove residual nuclear components and membrane-bound proteins;

[0036] (6) using supercritical CO2 fluid extraction to remove neutral lipids and residual surfactants to obtain acellular extracellular matrix microparticles;

[0037] (7) The defatted and acellular extracellular matrix was collected, freeze-dried, ball-milled, and sieved to obtain microparticles with a particle size of 200 μm;

[0038] (8) The decellularized extracellular matrix microparticles were immersed in a 10% (w / v) Pluronic F127-dopamine solution and cross-linked at 4°C for 16 h to form a covalent grafting interface;

[0039] (9) Functionalized ECM microparticles (10% w / v) were mixed with 3% trehalose solution, 0.1% ascorbic acid was added, and the pH was adjusted to 6.8-7.4; the mixture was dispensed into prefilled syringes, sterilized by gamma irradiation, and stored at 4°C in the dark.

[0040] The nonionic surfactant solution in step (5) contains 0.07% EDTA.

[0041] The supercritical CO2 fluid extraction in step (6) is carried out in two stages: the first stage: pressure 7 MPa, temperature 37°C, lasting 3 hours; the second stage: pressure 9 MPa, temperature 42°C, lasting 3 hours.

[0042] The chemical crosslinking in step (8) uses EDC and NHS in a mass ratio of 1:1, the crosslinker concentration is 15 mM, and the reaction pH is 6.0.

[0043] The preparation method of the Pluronic F127-dopamine copolymer in step (8) is as follows:

[0044] A1. Dissolve Pluronic F127 in DMSO (concentration 10% w / v), add succinic anhydride (molar ratio of Pluronic F127 to succinic anhydride 1:5), react at 60°C for 20 h, dialyze to remove unreacted reagents, and lyophilize to obtain carboxylated Pluronic F127.

[0045] A2. Dissolve carboxylated Pluronic F127 in MES buffer (pH 6.0), add EDC to a concentration of 12 mM, add NHS to a concentration of 7 mM, and activate at 20°C for 1.5 h.

[0046] A3. Add dopamine hydrochloride (molar ratio of Pluronic F127 to dopamine hydrochloride: 1:0.12), stir and react for 12 h in the dark, dialyze to remove free dopamine, and lyophilize to obtain Pluronic F127-dopamine copolymer.

[0047] Example 2

[0048] Adipose tissue was obtained from healthy women undergoing routine liposuction:

[0049] A method for preparing a decellularized fat filling material comprises the following steps:

[0050] (1) Take the fat tissue and cut it into 4mm pieces 3 The fragments were washed with 20 mM phosphate buffer, pH 7.0, to remove free lipids and blood components;

[0051] (2) The tissue fragments were immersed in pH 7.4, 80 mOsm / L Tris-HCl buffer and shaken at 4°C for 3 h to cause the adipocytes to swell and rupture;

[0052] (3) Transfer to 1000 mOsm / L sucrose solution and let stand at 20°C for 1.5 h to shrink the tissue and remove debris;

[0053] (4) The precipitate was collected by centrifugation and washed three times with 30 mM phosphate buffer, pH 7.0;

[0054] (5) The precipitate was immersed in a 1% (w / v) Tween 40 nonionic surfactant solution and shaken at 37°C for 20 h to remove residual nuclear components and membrane-bound proteins;

[0055] (6) using supercritical CO2 fluid extraction to remove neutral lipids and residual surfactants to obtain acellular extracellular matrix microparticles;

[0056] (7) The defatted and acellular extracellular matrix was collected, freeze-dried, ball-milled, and sieved to obtain microparticles with a particle size of 300 μm;

[0057] (8) The decellularized extracellular matrix microparticles were immersed in a 5% (w / v) Pluronic F127-dopamine solution and cross-linked at 4°C for 16 h to form a covalent grafting interface;

[0058] (9) Functionalized ECM microparticles (10% w / v) were mixed with 3% trehalose solution, 0.1% ascorbic acid was added, and the pH was adjusted to 6.8-7.4; the mixture was dispensed into prefilled syringes, sterilized by gamma irradiation, and stored at 4°C in the dark.

[0059] The nonionic surfactant solution in step (5) contains 0.07% EDTA.

[0060] The supercritical CO2 fluid extraction in step (6) is carried out in two stages: the first stage: pressure 7 MPa, temperature 37°C, lasting 3 hours; the second stage: pressure 9 MPa, temperature 42°C, lasting 3 hours.

[0061] The chemical crosslinking in step (8) uses EDC and NHS in a mass ratio of 1:1, the crosslinker concentration is 15 mM, and the reaction pH is 6.0.

[0062] The preparation method of the Pluronic F127-dopamine copolymer in step (8) is as follows:

[0063] A1. Dissolve Pluronic F127 in DMSO (concentration 10% w / v), add succinic anhydride (molar ratio of Pluronic F127 to succinic anhydride 1:5), react at 60°C for 20 h, dialyze to remove unreacted reagents, and lyophilize to obtain carboxylated Pluronic F127.

[0064] A2. Dissolve carboxylated Pluronic F127 in MES buffer (pH 6.0), add EDC to a concentration of 12 mM, add NHS to a concentration of 7 mM, and activate at 20°C for 1.5 h.

[0065] A3. Add dopamine hydrochloride (molar ratio of Pluronic F127 to dopamine hydrochloride: 1:0.12), stir and react for 12 h in the dark, dialyze to remove free dopamine, and lyophilize to obtain Pluronic F127-dopamine copolymer.

[0066] Example 3

[0067] Adipose tissue was obtained from fresh porcine small intestinal adipose tissue:

[0068] A method for preparing a decellularized fat filling material comprises the following steps:

[0069] (1) Take the fat tissue and cut it into 1mm pieces 3 The fragments were washed with 50 mM phosphate buffer, pH 7.5, to remove free lipids and blood components;

[0070] (2) The tissue fragments were immersed in pH 7.6, 100 mOsm / L Tris-HCl buffer and shaken at 6°C for 4 h to cause the adipocytes to swell and rupture;

[0071] (3) Transfer to 800 mOsm / L sucrose solution and let stand at 25°C for 2 h to shrink the tissue and remove debris;

[0072] (4) The precipitate was collected by centrifugation and washed three times with 50 mM phosphate buffer, pH 7.5;

[0073] (5) The precipitate was immersed in a 0.5% (w / v) Tween 20 nonionic surfactant solution and shaken at 39°C for 24 h to remove residual nuclear components and membrane-bound proteins;

[0074] (6) using supercritical CO2 fluid extraction to remove neutral lipids and residual surfactants to obtain acellular extracellular matrix microparticles;

[0075] (7) collecting the defatted and acellular extracellular matrix, freeze-drying it, ball-milling it, and sieving it to obtain microparticles with a particle size of 100-500 μm;

[0076] (8) The decellularized extracellular matrix microparticles were immersed in a 10% (w / v) Pluronic F127-dopamine solution and cross-linked at 4°C for 24 h to form a covalent grafting interface;

[0077] (9) Functionalized ECM microparticles (10% w / v) were mixed with 3% trehalose solution, 0.1% ascorbic acid was added, and the pH was adjusted to 6.8-7.4; the mixture was dispensed into prefilled syringes, sterilized by gamma irradiation, and stored at 4°C in the dark.

[0078] The nonionic surfactant solution in step (5) contains 0.05% EDTA.

[0079] The supercritical CO2 fluid extraction in step (6) is carried out in two stages: the first stage: pressure 8 MPa, temperature 40°C, lasting 4 hours; the second stage: pressure 10 MPa, temperature 45°C, lasting 4 hours.

[0080] The chemical cross-linking in step (8) uses EDC and NHS in a mass ratio of 1:1, a cross-linking agent concentration of 10 mM, and a reaction pH of 5.5.

[0081] The preparation method of the Pluronic F127-dopamine copolymer in step (8) is as follows:

[0082] A1. Dissolve Pluronic F127 in DMSO (5% w / v), add succinic anhydride (Pluronic F127:succinic anhydride molar ratio 1:5), react at 60°C for 18 h, dialyze to remove unreacted reagent, and lyophilize to obtain carboxylated Pluronic F127.

[0083] A2. Dissolve carboxylated Pluronic F127 in MES buffer (pH 6.0), add EDC to a concentration of 10 mM, add NHS to a concentration of 5 mM, and activate at 25°C for 2 h.

[0084] A3. Add dopamine hydrochloride (molar ratio of Pluronic F127 to dopamine hydrochloride: 1:0.12), stir and react for 12 h in the dark, dialyze to remove free dopamine, and lyophilize to obtain Pluronic F127-dopamine copolymer.

[0085] Example 4

[0086] Adipose tissue was obtained from fresh porcine small intestinal adipose tissue:

[0087] A method for preparing a decellularized fat filling material comprises the following steps:

[0088] (1) Take the fat tissue and cut it into 1mm pieces 3 The fragments were washed with 50 mM phosphate buffer, pH 7.5, to remove free lipids and blood components;

[0089] (2) The tissue fragments were immersed in pH 7.6, 100 mOsm / L Tris-HCl buffer and shaken at 6°C for 4 h to cause the adipocytes to swell and rupture;

[0090] (3) Transfer to 800 mOsm / L sucrose solution and let stand at 25°C for 2 h to shrink the tissue and remove debris;

[0091] (4) The precipitate was collected by centrifugation and washed three times with 50 mM phosphate buffer, pH 7.5;

[0092] (5) The precipitate was immersed in a 0.5% (w / v) Tween 80 nonionic surfactant solution and shaken at 39°C for 24 h to remove residual nuclear components and membrane-bound proteins;

[0093] (6) using supercritical CO2 fluid extraction to remove neutral lipids and residual surfactants to obtain acellular extracellular matrix microparticles;

[0094] (7) collecting the defatted and acellular extracellular matrix, freeze-drying it, ball-milling it, and sieving it to obtain microparticles with a particle size of 100-500 μm;

[0095] (8) The decellularized extracellular matrix microparticles were immersed in a 20% (w / v) Pluronic F127-dopamine solution and cross-linked at 4°C for 24 h to form a covalent grafting interface;

[0096] (9) Functionalized ECM microparticles (10% w / v) were mixed with 3% trehalose solution, 0.1% ascorbic acid was added, and the pH was adjusted to 6.8-7.4; the mixture was dispensed into prefilled syringes, sterilized by gamma irradiation, and stored at 4°C in the dark.

[0097] The nonionic surfactant solution in step (5) contains 0.05% EDTA.

[0098] The supercritical CO2 fluid extraction in step (6) is carried out in two stages: the first stage: pressure 8 MPa, temperature 40°C, lasting 4 hours; the second stage: pressure 10 MPa, temperature 45°C, lasting 4 hours.

[0099] The chemical cross-linking in step (8) uses EDC and NHS in a mass ratio of 1:1, a cross-linking agent concentration of 10 mM, and a reaction pH of 5.5.

[0100] The preparation method of the Pluronic F127-dopamine copolymer in step (8) is as follows:

[0101] A1. Dissolve Pluronic F127 in DMSO (5% w / v), add succinic anhydride (Pluronic F127:succinic anhydride molar ratio 1:5), react at 60°C for 18 h, dialyze to remove unreacted reagent, and lyophilize to obtain carboxylated Pluronic F127.

[0102] A2. Dissolve carboxylated Pluronic F127 in MES buffer (pH 6.0), add EDC to a concentration of 10 mM, add NHS to a concentration of 5 mM, and activate at 25°C for 2 h.

[0103] A3. Add dopamine hydrochloride (molar ratio of Pluronic F127 to dopamine hydrochloride: 1:0.12), stir and react for 12 h in the dark, dialyze to remove free dopamine, and lyophilize to obtain Pluronic F127-dopamine copolymer.

[0104] Comparative Example 1

[0105] On the basis of Example 1, only a single hypotonic solution was used: the tissue fragments were immersed in pH 7.4, 80 mOsm / LTris-HCl buffer and shaken at 4°C for 4.5 h. The rest of the process remained the same as Example 1.

[0106] Comparative Example 2

[0107] On the basis of Example 1, supercritical CO2 was changed to single-stage extraction (7 MPa, 37°C, 6 h), and the rest remained the same as Example 1.

[0108] Comparative Example 3

[0109] On the basis of Example 1, supercritical CO2 was changed to single-stage extraction (9 MPa, 42°C, 6 h), and the rest was consistent with Example 1.

[0110] Comparative Example 4

[0111] On the basis of Example 1, step (8) was changed to immersing the decellularized extracellular matrix microparticles in a 2% (w / v) Pluronic F127-dopamine solution, and the rest of the steps were the same as in Example 1.

[0112] Comparative Example 5

[0113] On the basis of Example 1, step (8) was changed to immersing the decellularized extracellular matrix microparticles in a 40% (w / v) Pluronic F127-dopamine solution, and the rest of the steps were the same as in Example 1.

[0114] Performance testing:

[0115] Lipid residue: The neutral lipid content was determined by Soxhlet extraction (using a chloroform-methanol mixed solvent). Residual rate (%) = (residual lipid mass / dry weight of material) × 100%.

[0116] Compatibility test: The material particles were directly co-cultured with cells, and the cell viability was assessed by live-dead staining (Calcein-AM / PI) method.

[0117] The storage sol-gel transition temperature was determined by a dynamic temperature sweep (25°C to 37°C).

[0118] Residual lipid content / % Cell survival rate / % Thermosensitive gel formation / ℃ Example 1 2.5 96 32 Example 2 2.6 95 33 Example 3 3.1 93 32 Example 4 3.3 92 31 Comparative Example 1 18.6 78 Unable to form gel Comparative Example 2 8.3 85 35 Comparative Example 3 5.3 80 33 Comparative Example 4 3.1 89 Partial gel formation Comparative Example 5 3.0 72 brittle gel

[0119] Lipid residue control:

[0120] The lipid residues in Examples 1-4 were 2.5-3.3%, while the residue in Comparative Example 1 reached 18.6% due to the lack of a hypertonic step, demonstrating the necessity of gradient osmotic pressure for lipid removal. The lipid removal efficiency of supercritical CO2 staged extraction was significantly better than that of a single stage low pressure (Comparative Example 2: 8.3%) or high pressure (Comparative Example 3: 5.7%). Regarding biocompatibility balance: the cell survival rates in Examples 1-4 reached 92-96%; Comparative Example 1 could not form a gel due to the high lipid residues, while Comparative Example 4 may have insufficient adhesion at low dopamine concentrations and could only partially convert into a gel, while Comparative Example 5 grafted with high dopamine concentrations caused toxicity (survival rate 72%) and material brittleness.

[0121] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any indirect modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a cell-free fat filling material, characterized by: The following steps are involved: (1) Adipose tissue is placed in a gradient osmotic pressure solution and treated with hypotonic expansion and hypertonic contraction to lyse the adipocyte membrane and preserve the extracellular matrix structure; (2) using a nonionic surfactant solution to remove residual nuclear components and membrane-bound proteins; (3) using supercritical CO2 fluid extraction to remove neutral lipids and residual surfactants to obtain acellular extracellular matrix microparticles; (4) The decellularized extracellular matrix microparticles are mixed with a Pluronic F127-dopamine copolymer solution, and the copolymer is grafted onto the surface of the microparticles by chemical crosslinking, wherein the dopamine grafting rate is 5-15%.

2. The method for preparing a decellularized fat filling material according to claim 1, characterized in that: The gradient osmotic pressure solution in step (1) includes a hypotonic solution and a hypertonic solution; the hypotonic solution is a 50-100 mOsm / L Tris-HCl buffer solution, and the hypertonic solution is a 800-1200 mOsm / L sucrose solution.

3. The method for preparing a decellularized fat filling material according to claim 1, characterized in that: The hypotonic expansion treatment time is 2-4 hours, and the hypertonic contraction treatment time is 1-2 hours.

4. The method for preparing a decellularized fat filling material according to claim 1, wherein: The nonionic surfactant in step (2) is selected from polysorbates or Triton X-100, with a concentration of 0.5-2% w / v.

5. The method for preparing a decellularized fat filling material according to claim 1, characterized in that: The supercritical CO2 fluid extraction in step (3) is carried out in two stages: the first stage: pressure 7-8 MPa, temperature 35-40°C, lasting 2-4 hours; the second stage: pressure 9-10 MPa, temperature 40-45°C, lasting 2-4 hours.

6. The method for preparing a decellularized fat filling material according to claim 1, characterized in that: The chemical cross-linking in step (4) uses EDC and NHS, the cross-linking agent concentration is 10-20 mM, and the reaction pH is 5.5-6.

5.

7. The method for preparing a decellularized fat filling material according to claim 1, characterized in that: The preparation method of the Pluronic F127-dopamine copolymer in step (4) is as follows: Pluronic F127 is carboxylated with succinic anhydride, activated by adding EDC and NHS, and then dopamine hydrochloride is added. The reaction is carried out in the dark to obtain the Pluronic F127-dopamine copolymer.

8. A fat decellularized filling material, characterized in that: Obtained by the preparation method according to claim 1.