A fat decellularized filling material and a preparation method thereof

Abstract

The application relates to a fat decellularized filling material and a preparation method thereof, and belongs to the technical field of biomedical materials. The application realizes efficient decellularization and extracellular matrix structure protection in cooperation through gradient osmotic pressure lysis-nonionic surfactant cleaning-supercritical CO2 defatting; and then Pluronic F127-dopamine copolymer grafting is carried out, so that the traditional material passive filling limitation is broken, the cell adhesion rate is improved through dopamine mediation, and vascular ingrowth and tissue regeneration are promoted.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and relates to a fat decellularization filling material and its preparation method. Background Technology

[0002] Filler materials play a crucial role in regenerative medicine and plastic surgery. They are used to repair and replace damaged or diseased tissue, restoring its structure and function. Traditional filler materials include autologous tissue, allogeneic tissue, and synthetic materials. Autologous tissue transplantation, such as fat grafting, is widely used due to its high biocompatibility and low immune rejection. However, the limited availability of donor sites and the instability of transplanted volume restrict its application. Synthetic materials such as silicone and hydroxyapatite, while solving the donor problem to some extent, have poor biocompatibility, cannot fully integrate into the host tissue, and are prone to causing foreign body reactions and inflammation. Therefore, developing a filler material that possesses both good biocompatibility and sufficient mechanical strength and stability would be highly advantageous.

[0003] Decellularized adipose tissue (DAT) technology has attracted widespread attention in recent years. The abundant extracellular matrix (ECM) components in adipose tissue, such as collagen, elastin, and glycosaminoglycans, play a crucial role in cell proliferation, differentiation, and function. By removing cellular components from adipose tissue while preserving its ECM structure, a highly biocompatible filling material can be obtained. Decellularized adipose tissue not only retains its original three-dimensional structure and mechanical properties but also reduces immunogenicity and lowers the risk of immune rejection after transplantation. DAT material can serve as a natural scaffold material, promoting the adhesion, migration, and proliferation of host cells, ultimately achieving tissue regeneration and repair. This material has broad application prospects in soft tissue filling, wound repair, and organ regeneration.

[0004] Despite the numerous advantages exhibited by decellularized fat fillers, 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 endocrine microstructure (ECM) while removing cellular components, affecting the material's biocompatibility. Second, residual decellularization agents, such as detergents and enzymes, may have toxic effects on host tissues, impacting material safety. Furthermore, existing decellularization processes are typically complex and time-consuming, hindering large-scale production and limiting their applications. Therefore, optimizing decellularization technology while preserving the integrity and function of the ECM, and developing efficient and controllable processing procedures, are urgent problems to be solved. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a decellularized fat filling material, thereby obtaining a highly efficient and biocompatible decellularized fat filling material.

[0006] The objective of this invention can be achieved through the following technical solutions:

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

[0008] (1) Take adipose tissue and cut it into pieces 1-5mm. 3 Fragments were rinsed 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 solution and shake at 2-6℃ for 2-4 hours to cause the adipocytes to swell and rupture.

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

[0011] (4) Centrifuge to collect the precipitate and wash 2-3 times with 20-50mM phosphate buffer at pH 6.5-7.5;

[0012] (5) Soak the precipitate in 0.5-2% (w / v) polysorbate or Triton X-100 nonionic surfactant solution and shake at 35-39℃ for 12-24h to remove residual nuclear components and membrane-bound proteins.

[0013] (6) Neutral lipids and residual surfactants were removed by supercritical CO2 fluid extraction to obtain decellularized extracellular matrix microparticles;

[0014] (7) Collect defatted and decellularized extracellular matrix, freeze-dry it, ball-mill it and sieve it to obtain particles with a particle size of 100-500 μm;

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

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

[0017] As a preferred embodiment 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℃, lasting 2-4h; the second stage: pressure 9-10MPa, temperature 40-45℃, lasting 2-4h.

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

[0020] As a preferred embodiment of the present invention, the method for preparing the Pluronic F127-dopamine copolymer in step (8) is as follows:

[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℃ for 18-24h, dialyze to remove unreacted reagents, and freeze dry to obtain carboxylated Pluronic F127.

[0022] A2. Dissolve carboxylated Pluronic F127 in MES buffer at pH 6.0, add EDC to make the concentration 10-15mM, add NHS to make the concentration 5-10mM, and activate at 15-25℃ for 1-2h.

[0023] A3. Add dopamine hydrochloride (molar ratio of Pluronic F127 to dopamine hydrochloride 1:0.12), stir and react for 12 h under light-protected conditions, dialyze to remove free dopamine, and freeze dry to obtain Pluronic F127-dopamine copolymer.

[0024] The beneficial effects of this invention are:

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

[0026] (2) Grafting of Pluronic F127-dopamine copolymer breaks through the limitations of traditional passive filling materials and improves the cell adhesion rate mediated by dopamine. Detailed Implementation

[0027] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with embodiments, is provided below.

[0028] Example 1

[0029] Adipose tissue was obtained from healthy women who underwent conventional liposuction.

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

[0031] (1) Take adipose tissue and cut it into 3mm pieces. 3 The fragments were rinsed with 30mM phosphate buffer (pH 7.0) to remove free lipids and blood components.

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

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

[0034] (4) Centrifuge to collect the precipitate and wash three times with 30mM phosphate buffer at pH 7.0;

[0035] (5) The precipitate was soaked 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) Neutral lipids and residual surfactants were removed by supercritical CO2 fluid extraction to obtain decellularized extracellular matrix microparticles;

[0037] (7) Collect defatted and decellularized extracellular matrix, freeze-dry it, ball-mill it and sieve it to obtain particles with a particle size of 200 μm;

[0038] (8) Immerse decellularized extracellular matrix microparticles in 10% (w / v) Pluronic F127-dopamine solution and crosslink at 4°C for 16 h to form a covalent graft interface;

[0039] (9) Mix functionalized ECM microparticles (10% w / v) with 3% trehalose solution, add 0.1% ascorbic acid, and adjust the pH to 6.8-7.4; dispense into pre-filled syringes, sterilize by γ-ray irradiation, and store 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 crosslinking agent concentration is 15mM, 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℃ for 20 h, dialyze to remove unreacted reagents, and freeze dry to obtain carboxylated Pluronic F127.

[0045] A2. Dissolve carboxylated Pluronic F127 in MES buffer at pH 6.0, add EDC to make the concentration 12mM, add NHS to make the concentration 7mM, and activate at 20℃ for 1.5h.

[0046] A3. Add dopamine hydrochloride (molar ratio of Pluronic F127 to dopamine hydrochloride 1:0.12), stir and react for 12 h under light-protected conditions, dialyze to remove free dopamine, and freeze dry to obtain Pluronic F127-dopamine copolymer.

[0047] Example 2

[0048] Adipose tissue was obtained from healthy women who underwent conventional liposuction.

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

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

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

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

[0053] (4) Centrifuge to collect the precipitate and wash three times with 30mM phosphate buffer at pH 7.0;

[0054] (5) The precipitate was soaked 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) Neutral lipids and residual surfactants were removed by supercritical CO2 fluid extraction to obtain decellularized extracellular matrix microparticles;

[0056] (7) Collect defatted and decellularized extracellular matrix, freeze-dry it, ball-mill it and sieve it to obtain particles with a particle size of 300 μm;

[0057] (8) Immerse decellularized extracellular matrix microparticles in 5% (w / v) Pluronic F127-dopamine solution and crosslink at 4°C for 16 h to form a covalent grafting interface.

[0058] (9) Mix functionalized ECM microparticles (10% w / v) with 3% trehalose solution, add 0.1% ascorbic acid, and adjust the pH to 6.8-7.4; dispense into pre-filled syringes, sterilize by γ-ray irradiation, and store 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 crosslinking agent concentration is 15mM, 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℃ for 20 h, dialyze to remove unreacted reagents, and freeze dry to obtain carboxylated Pluronic F127.

[0064] A2. Dissolve carboxylated Pluronic F127 in MES buffer at pH 6.0, add EDC to make the concentration 12mM, add NHS to make the concentration 7mM, and activate at 20℃ for 1.5h.

[0065] A3. Add dopamine hydrochloride (molar ratio of Pluronic F127 to dopamine hydrochloride 1:0.12), stir and react for 12 h under light-protected conditions, dialyze to remove free dopamine, and freeze dry to obtain Pluronic F127-dopamine copolymer.

[0066] Example 3

[0067] The adipose tissue was obtained from fresh porcine small intestine adipose tissue.

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

[0069] (1) Take adipose tissue and cut it into pieces to 1 mm. 3 The fragments were rinsed with 50 mM phosphate buffer (pH 7.5) to remove free lipids and blood components.

[0070] (2) Immerse the tissue fragments in pH 7.6, 100mOsm / L Tris-HCl buffer and shake at 6°C for 4 hours 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 hours to shrink the tissue and remove the debris;

[0072] (4) Centrifuge to collect the precipitate and wash three times with 50mM phosphate buffer at pH 7.5;

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

[0074] (6) Neutral lipids and residual surfactants were removed by supercritical CO2 fluid extraction to obtain decellularized extracellular matrix microparticles;

[0075] (7) Collect defatted and decellularized extracellular matrix, freeze-dry it, ball-mill it and sieve it to obtain particles with a particle size of 100-500 μm;

[0076] (8) Immerse decellularized extracellular matrix microparticles in 10% (w / v) Pluronic F127-dopamine solution and crosslink at 4°C for 24 h to form a covalent graft interface;

[0077] (9) Mix functionalized ECM microparticles (10% w / v) with 3% trehalose solution, add 0.1% ascorbic acid, and adjust the pH to 6.8-7.4; dispense into pre-filled syringes, sterilize by γ-ray irradiation, and store 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 8MPa, temperature 40℃, lasting for 4h; the second stage: pressure 10MPa, temperature 45℃, lasting for 4h.

[0080] The chemical crosslinking in step (8) uses EDC and NHS in a mass ratio of 1:1, the crosslinking agent concentration is 10mM, and the reaction pH is 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 (concentration 5% w / v), add succinic anhydride (molar ratio of Pluronic F127 to succinic anhydride 1:5), react at 60℃ for 18h, dialyze to remove unreacted reagents, and freeze dry to obtain carboxylated Pluronic F127.

[0083] A2. Dissolve carboxylated Pluronic F127 in MES buffer at pH 6.0, add EDC to make the concentration 10 mM, add NHS to make the concentration 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 under light-protected conditions, dialyze to remove free dopamine, and freeze dry to obtain Pluronic F127-dopamine copolymer.

[0085] Example 4

[0086] The adipose tissue was obtained from fresh porcine small intestine adipose tissue.

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

[0088] (1) Take adipose tissue and cut it into pieces to 1 mm. 3 The fragments were rinsed with 50 mM phosphate buffer (pH 7.5) to remove free lipids and blood components.

[0089] (2) Immerse the tissue fragments in pH 7.6, 100mOsm / L Tris-HCl buffer and shake at 6°C for 4 hours 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 hours to shrink the tissue and remove the debris;

[0091] (4) Centrifuge to collect the precipitate and wash three times with 50mM phosphate buffer at pH 7.5;

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

[0093] (6) Neutral lipids and residual surfactants were removed by supercritical CO2 fluid extraction to obtain decellularized extracellular matrix microparticles;

[0094] (7) Collect defatted and decellularized extracellular matrix, freeze-dry it, ball-mill it and sieve it to obtain particles with a particle size of 100-500 μm;

[0095] (8) Immerse decellularized extracellular matrix microparticles in 20% (w / v) Pluronic F127-dopamine solution and crosslink at 4°C for 24 h to form a covalent graft interface;

[0096] (9) Mix functionalized ECM microparticles (10% w / v) with 3% trehalose solution, add 0.1% ascorbic acid, and adjust the pH to 6.8-7.4; dispense into pre-filled syringes, sterilize by γ-ray irradiation, and store 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 8MPa, temperature 40℃, lasting for 4h; the second stage: pressure 10MPa, temperature 45℃, lasting for 4h.

[0099] The chemical crosslinking in step (8) uses EDC and NHS in a mass ratio of 1:1, the crosslinking agent concentration is 10mM, and the reaction pH is 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 (concentration 5% w / v), add succinic anhydride (molar ratio of Pluronic F127 to succinic anhydride 1:5), react at 60℃ for 18h, dialyze to remove unreacted reagents, and freeze dry to obtain carboxylated Pluronic F127.

[0102] A2. Dissolve carboxylated Pluronic F127 in MES buffer at pH 6.0, add EDC to make the concentration 10 mM, add NHS to make the concentration 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 under light-protected conditions, dialyze to remove free dopamine, and freeze dry to obtain Pluronic F127-dopamine copolymer.

[0104] Comparative Example 1

[0105] Based on Example 1, only a single hypotonic solution was used: tissue fragments were immersed in a pH 7.4, 80 mOsm / LTris-HCl buffer solution and treated with shaking at 4°C for 4.5 h, with the rest remaining the same as in Example 1.

[0106] Comparative Example 2

[0107] Based on Example 1, supercritical CO2 was replaced with single-stage extraction (7MPa, 37℃, 6h), while the rest remained the same as in Example 1.

[0108] Comparative Example 3

[0109] Based on Example 1, supercritical CO2 was replaced with single-stage extraction (9MPa, 42℃, 6h), while the rest remained the same as in Example 1.

[0110] Comparative Example 4

[0111] Based on Example 1, step (8) is changed to immersing decellularized extracellular matrix microparticles in a 2% (w / v) Pluronic F127-dopamine solution, while the rest remains the same as in Example 1.

[0112] Comparative Example 5

[0113] Based on Example 1, step (8) is changed to immersing decellularized extracellular matrix microparticles in a 40% (w / v) Pluronic F127-dopamine solution, while the rest remains the same as in Example 1.

[0114] Performance testing:

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

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

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

[0118] Lipid residue / % Cell viability / % Thermosensitive gel formation / °C 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 Cannot form a gel Comparative Example 2 8.3 85 35 Comparative Example 3 5.3 80 33 Comparative Example 4 3.1 89 Partially forms a gel Comparative Example 5 3.0 72 brittle gel

[0119] Lipid residue control:

[0120] Examples 1-4 showed lipid residues of 2.5-3.3%, while Comparative Example 1, lacking a hyperosmotic step, had a residue of 18.6%, demonstrating the necessity of gradient osmotic pressure for lipid removal. Supercritical CO2 staged extraction significantly outperformed single-stage low-pressure (Comparative Example 2: 8.3%) or high-pressure (Comparative Example 3: 5.7%) lipid removal. Regarding biocompatibility balance: Examples 1-4 achieved cell viability of 92-96%; Comparative Example 1, due to high lipid residue, could not form a gel; Comparative Example 4, with low-concentration dopamine grafting, may have resulted in insufficient adhesion, only partially forming a gel; and Comparative Example 5, with high-concentration dopamine grafting, caused toxicity (72% survival rate) and material brittleness.

[0121] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any indirect modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing a decellularized fat filling material, characterized in that: Includes the following steps: (1) Place the adipose tissue in a gradient osmotic pressure solution and lyse the adipose cell membrane by hypotonic expansion and hypertonic contraction, while preserving the extracellular matrix structure. (2) Use nonionic surfactant solutions to remove residual nuclear components and membrane-bound proteins; (3) Neutral lipids and residual surfactants were removed by supercritical CO2 fluid extraction to obtain decellularized 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 cross-linking, wherein the dopamine grafting rate is 5-15%. The gradient osmotic pressure solution in step (1) includes a hypotonic solution and a hypertonic solution; the hypotonic solution is a Tris-HCl buffer solution of 50-100 mOsm / L, and the hypertonic solution is a sucrose solution of 800-1200 mOsm / L. The nonionic surfactant used in step (2) is selected from polysorbates or Triton X-100, with a concentration of 0.5-2% w / v; The supercritical CO2 fluid extraction in step (3) is carried out in two stages: the first stage: pressure 7-8 MPa, temperature 35-40℃, lasting 2-4 h; the second stage: pressure 9-10 MPa, temperature 40-45℃, lasting 2-4 h. The chemical crosslinking in step (4) uses EDC and NHS, with a crosslinking agent concentration of 10-20 mM and a reaction pH of 5.5-6.5; The preparation method of Pluronic F127-dopamine copolymer in step (4) is as follows: Pluronic F127 is carboxylated with succinic anhydride, activated with EDC and NHS, and then dopamine hydrochloride is added. The reaction is carried out in the dark to obtain Pluronic F127-dopamine copolymer.

2. The method for preparing a decellularized fat filling material according to claim 1, characterized in that: The low-osmotic expansion treatment in step (1) takes 2-4 hours, and the high-osmotic shrinkage treatment takes 1-2 hours.

3. A fat decellularization filling material, characterized in that, Obtained by the preparation method described in claim 1.