Injectable acellular fat matrix composition as well as preparation method and application thereof

Abstract

The invention relates to an injectable acellular fat matrix composition as well as a preparation method and application thereof. Specifically, the invention provides an injectable decellularized fat matrix composition prepared by a high-pressure homogenization-enzymolysis coupling method, and the method comprises the following steps: performing high-pressure homogenization on a decellularized fat matrix to obtain matrix particles, performing protease enzymolysis, and performing freeze drying to obtain the injectable decellularized fat matrix composition. Therefore, the acellular fat matrix composition with excellent injectability, biological activity retention rate and long-term skin thickening effect is prepared.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to an injectable acellular fat matrix composition, a preparation method and an application thereof. Background Art

[0002] 1. Limitations of Hyaluronic Acid Fillers

[0003] Sodium hyaluronate (HA) injections are currently a commonly used dermal filler material in clinical practice. However, their mechanism of action is primarily physical space-occupying filling, lacking bioactive components and unable to induce cell proliferation or collagen regeneration. Furthermore, HA is easily degraded by hyaluronidase in the body, resulting in a short lifespan (typically 6-12 months), requiring frequent injections. Long-term use is costly and unsustainable.

[0004] 2. Disadvantages of traditional acellular adipose matrix (DAM)

[0005] Decellularized adipose tissue matrix (DAM) theoretically has the potential to promote regeneration by removing adipocytes and immunogenic components while retaining the collagen, elastin, and growth factors (such as VEGF and FGF) of the extracellular matrix (DAM).

[0006] However, the existing technology has the following key problems:

[0007] Conflict between particle size and injectability: DAM particles prepared by traditional mechanical crushing methods (such as grinding and shearing) are large in size and easily tangled, making them difficult to inject through fine needles (such as 30G) and prone to needle clogging. Furthermore, the coarse particles are unevenly distributed in the dermis, which can easily cause inflammation or nodule formation.

[0008] Low retention rate of active ingredients: The preparation process requires multiple filtration or centrifugation to obtain the supernatant, which discards a large amount of active ingredients. This causes small molecular proteins in the ECM (such as growth factors and fibronectin) to be lost with the waste liquid, significantly reducing the regeneration efficiency.

[0009] Immature enzymatic hydrolysis technology: Existing technologies use short-term (<48h) pepsin digestion of DAM. Although this can partially degrade the macromolecular collagen fibers in DAM, excessive digestion can destroy the three-dimensional structure of DAM, leading to a loss of biomechanical support.

[0010] Sterilization methods affect activity: Traditional ethylene oxide or high-temperature sterilization can destroy the conformation of ECM proteins, while improper control of γ-ray irradiation dose can easily lead to free radical damage and reduce the biocompatibility of the material.

[0011] Therefore, there is an urgent need in this field to develop an injectable acellular fat matrix and an innovative preparation process to address the shortcomings of existing technologies. Summary of the Invention

[0012] The present invention aims to provide an injectable acellular adipose tissue matrix (DAM) composition. This composition, prepared through a high-pressure homogenization-enzymatic decomposition method, addresses the issues of oversized DAM particles, loss of active ingredients, and sterilization damage. This composition can be injected into the dermis, promoting cell proliferation and collagen regeneration, while also retaining moisture and thickening the skin, providing anti-aging benefits and improving skin elasticity.

[0013] The first aspect of the present invention provides an injectable acellular adipose tissue matrix composition comprising:

[0014] (1) acellular adipose tissue matrix particles K; and

[0015] (2) soluble matrix solution S;

[0016] in,

[0017] The soluble matrix solution S is obtained by directly enzymatic hydrolysis of the decellularized fat matrix particles P;

[0018] The collagen content ratio (w:w) of the soluble matrix solution S and the acellular fat matrix particles K is 1:4 to 1:2;

[0019] The total sugar content ratio (w:w) of the soluble matrix solution S and the decellularized fat matrix particles K is 1:1-4:1.

[0020] In another preferred embodiment, the composition is prepared by a homogenization-enzymatic decomposition coupling method.

[0021] In another preferred embodiment, the homogenization-enzyme decomposition coupling method comprises the following steps:

[0022] (1) Adipose tissue processing: including cleaning, pretreatment, salt washing, virus inactivation, decellularization, defatting and other processes to obtain acellular adipose matrix M;

[0023] (2) Preparation of decellularized fat matrix particles P: The decellularized fat matrix is ​​crushed to obtain decellularized matrix fibers, which are then homogenized to obtain decellularized fat matrix particles P;

[0024] (3) Preparation of a decellularized fat matrix composition: Decellularized fat matrix particles P are centrifuged to obtain a matrix particle solution, which is then subjected to segmented enzymatic hydrolysis to obtain an enzymatic hydrolyzate, which is then degassed and filled to obtain a decellularized fat matrix composition, which is then sterilized;

[0025] The enzymatic hydrolysate refers to a soluble matrix solution S containing decellularized fat matrix particles K.

[0026] In another preferred embodiment, the ratio of the amount of protease added to the matrix particle solution in the enzymatic hydrolysis reaction is 1:5-1:15, preferably 1:10;

[0027] In another preferred embodiment, the temperature of the enzymatic hydrolysis reaction is 2-8°C, preferably 4°C;

[0028] In another preferred embodiment, the composition further comprises a saline solution and / or a buffer solution.

[0029] In another preferred embodiment, the D50 / D90 / span of the acellular fat matrix composition are 100-140 μm / 250-300 μm / 1.5-2.0, respectively.

[0030] In another preferred embodiment, the content of the acellular fat matrix composition is 1-20 mg / ml; preferably 5-10 mg / ml.

[0031] In another preferred embodiment, the ratio of particles K to soluble matrix solution S in the injectable acellular fat matrix composition is 3:1-5:1, preferably 2:1.

[0032] In another preferred embodiment, the G' of the injectable acellular fat matrix composition is 300-350 Pa, preferably 320-335 Pa.

[0033] In another preferred embodiment, the collagen in the composition accounts for 0.02%-1.40% wt (based on 100% wt of the decellularized fat matrix composition); and / or

[0034] The total sugar content of the decellularized fat matrix composition is 0.001%-0.06% wt (based on 100% wt of the decellularized fat matrix composition); and / or

[0035] The ratio of type I collagen: type III collagen: type VI collagen in the acellular fat matrix composition is 3:1:2.

[0036] In another preferred embodiment, the acellular fat matrix composition of the present invention can be freeze-dried and stored by adding excipients.

[0037] The excipient is selected from the group consisting of mannitol, trehalose, carboxymethyl cellulose, polyethylene glycol, hydroxypropyl methyl cellulose, or a combination thereof.

[0038] The second aspect of the present invention provides a method for preparing the composition according to the first aspect of the present invention, the method comprising the following steps:

[0039] (1) Adipose tissue processing: including cleaning, pretreatment, salt washing, virus inactivation, decellularization, defatting and other processes to obtain acellular adipose matrix M;

[0040] (2) Preparation of decellularized fat matrix particles P: The decellularized fat matrix is ​​crushed to obtain decellularized matrix fibers, which are then homogenized to obtain decellularized fat matrix particles P;

[0041] (3) Preparation of decellularized fat matrix composition: Decellularized fat matrix particles P are centrifuged to obtain a matrix particle solution, which is then subjected to segmented enzymatic hydrolysis to obtain an enzymatic hydrolyzate. The decellularized fat matrix composition is obtained by degassing and filling, and then sterilized.

[0042] In another preferred embodiment, the ratio of the amount of protease added to the matrix particle solution in the enzymatic hydrolysis reaction is 1:5-1:15, preferably 1:10;

[0043] In another preferred embodiment, the temperature of the enzymatic hydrolysis reaction is 2-8°C, preferably 4°C;

[0044] In another preferred embodiment, the step (1) further comprises the following steps:

[0045] (1a) After the fat tissue is allowed to stand and separate, excess liquid is removed and the tissue is rinsed multiple times with physiological solution to remove impurities;

[0046] (1b) centrifuging the rinsed fat tissue to obtain an intermediate layer of tissue, i.e., a fat layer containing adipocytes; and after a second centrifugation and homogenization, obtaining an intermediate solid layer;

[0047] (1c) mixing the middle solid layer with physiological saline, discarding the liquid layer after shaking, and obtaining the salt-washed solid content after multiple solution washing;

[0048] (1d) chemically treating the salt-washed solid contents by mixing them with a disinfectant, discarding the liquid layer after shaking, and obtaining the disinfected solid contents after washing with multiple solutions;

[0049] (1e) mixing the sterilized solid contents with a decellularizing agent, shaking the contents, discarding the liquid layer, and washing the contents to obtain the decellularized solid contents;

[0050] (1f) The decellularized solid contents are mixed with a degreasing agent, shaken, and the liquid layer is discarded. The decellularized fat matrix is ​​obtained after washing.

[0051] In another preferred embodiment, the shaking frequency of the shaking treatment in the step is 180-250 rpm, preferably 200 rpm; the shaking temperature is 25-35°C, preferably 30°C.

[0052] In another preferred embodiment, the cleaning time in the step is 5-20 minutes, preferably 15 minutes.

[0053] In another preferred embodiment, the centrifugation in step (1b) is carried out at 5000-8000 rpm, preferably 6000 rpm; the centrifugation time is 3-10 min, preferably 5 min.

[0054] In another preferred embodiment, the step (1c) has one or more characteristics selected from the following group:

[0055] (a) the ratio of the intermediate solid layer to the physiological saline is 1:3-1:8, preferably 1:5;

[0056] (b) the centrifugation is performed at 5000-8000 rpm, preferably 6000 rpm; the centrifugation time is 3-10 min, preferably 5 min;

[0057] (c) the oscillation time is 0.5-3 h, preferably 1 h;

[0058] (d) the shaking frequency of the shaking treatment is 180-250 rpm, preferably 200 rpm;

[0059] (e) the shaking table temperature is 25-35°C, preferably 30°C;

[0060] (f) The cleaning time is 5-20 min, preferably 15 min.

[0061] In another preferred embodiment, the step (1d) has one or more characteristics selected from the following group:

[0062] (a) the ratio of the solid content after salt washing to the disinfectant is 1:3-1:8, preferably 1:5;

[0063] (b) the disinfectant is a mixed solution of peracetic acid and ethanol; the concentration of the peracetic acid is 0.1%-1%, preferably 0.3%, and the concentration of the ethanol is 5%-30%, preferably 20%;

[0064] (c) the centrifugation is performed at 5000-8000 rpm, preferably 6000 rpm; the centrifugation time is 3-10 min, preferably 5 min;

[0065] (d) the oscillation time is 0.5-3 h, preferably 2 h;

[0066] (e) the shaking frequency of the shaking treatment is 180-250 rpm, preferably 200 rpm;

[0067] (f) the shaking table temperature is 25-35°C, preferably 30°C;

[0068] (g) The cleaning time is 5-20 min, preferably 15 min.

[0069] In another preferred embodiment, the step (1e) has one or more characteristics selected from the following group:

[0070] (a) the decellularization agent is selected from the group consisting of Triton X-100, sodium lauryl sulfate, trypsin, or a combination thereof;

[0071] (b) the ratio of the sterilized solid content to the decellularizing agent is 1:3-1:10, preferably 1:5;

[0072] (c) the concentration of the decellularizing agent is 0.5%-1.5%, preferably 1%;

[0073] (d) the centrifugation is performed at 6000-10000 rpm, preferably 8000 rpm; the centrifugation time is 3-10 min, preferably 5 min;

[0074] (e) the oscillation time is 8-24 hours, preferably 16 hours;

[0075] (f) the shaking frequency of the shaking treatment is 180-250 rpm, preferably 200 rpm;

[0076] (g) the shaking table temperature is 25-35°C, preferably 30°C;

[0077] (h) The cleaning time is 5-20 min, preferably 15 min.

[0078] In another preferred embodiment, the step (1f) has one or more characteristics selected from the following group:

[0079] (a) the ratio of the decellularized solid content to the degreasing agent is 1:3-1:8, preferably 1:5;

[0080] (b) the concentration of the degreasing agent is 90%-100%, preferably 100%;

[0081] (c) the centrifugation is performed at 6000-10000 rpm, preferably 8000 rpm; the centrifugation time is 3-10 min, preferably 5 min;

[0082] (d) the shaking time is 3-10 hours, preferably 3 hours;

[0083] (e) the shaking frequency of the shaking treatment is 180-250 rpm, preferably 200 rpm;

[0084] (f) the shaking table temperature is 25-35°C, preferably 30°C;

[0085] (g) The cleaning time is 5-20 min, preferably 15 min.

[0086] In another preferred embodiment, the high-pressure homogenization treatment is carried out in a high-pressure homogenizer, and the pressure of the high-pressure homogenizer is 800-1200 Pa, preferably 1000 Pa.

[0087] In another preferred embodiment, the step (3) further comprises the following steps:

[0088] (3a) The acellular adipose tissue matrix particles P are centrifuged to remove the liquid phase, and then washed with acid multiple times to obtain a matrix particle solution with a target concentration.

[0089] (3b) adding protease to the matrix particle solution, and performing a segmented enzymatic hydrolysis reaction under low temperature conditions with continuous stirring to obtain an enzymatic hydrolyzate.

[0090] (3c) adding alkali and buffer to the enzymatic hydrolyzate to adjust the pH, and removing bubbles by degassing to obtain a decellularized fat matrix composition;

[0091] The enzymatic hydrolysate refers to a soluble matrix solution S containing decellularized fat matrix particles K.

[0092] In another preferred embodiment, the step (3a) has one or more characteristics selected from the following group:

[0093] (a) the centrifugation is performed at 2000-6000 rpm, preferably at 4000 rpm;

[0094] (b) the centrifugation time is 0.5-5 min, preferably 1 min;

[0095] (c) The concentration of the acid is 0.01-0.5 mol / L, preferably 0.01 mol / L.

[0096] In another preferred embodiment, the segmented enzymatic hydrolysis is divided into a first stage and a second stage.

[0097] In another preferred embodiment, the step (3b) has one or more characteristics selected from the following group:

[0098] (a) the ratio of the added amount of the protease to the matrix particle solution is 1:5-1:15, preferably 1:10;

[0099] (b) the temperature of the enzymatic hydrolysis reaction is 2-8°C, preferably 4°C;

[0100] (c) The duration of the second stage enzymatic hydrolysis reaction is 0-72 h, preferably 48 h.

[0101] In another preferred embodiment, the step (3c) has one or more characteristics selected from the following group:

[0102] (a) the ratio of the base to the buffer is 1:4-1:8, preferably 1:6;

[0103] (b) The concentration of the base is 0.1-1.0 mol / L, preferably 1 mol / L.

[0104] In another preferred embodiment, the sterilization method is irradiation sterilization or micromembrane filtration sterilization.

[0105] The third aspect of the present invention provides a use of the acellular fat matrix composition according to the first aspect of the present invention for preparing a skin promoter.

[0106] 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

[0107] Figure 1 Shows the appearance of the decellularized adipose matrix composition.

[0108] Figure 2 Scanning electron micrographs of decellularized adipose tissue matrix compositions with different contents are shown, including (a) 5 mg / ml decellularized adipose tissue matrix solution for injection; (b) 10 mg / ml decellularized adipose tissue matrix solution for injection.

[0109] Figure 3 The cell morphology of the cytotoxicity test of the decellularized adipose matrix composition is shown.

[0110] Figure 4 The degradation results of the segmented enzymatic hydrolysis are shown.

[0111] Figure 5 The temperature scanning diagram of the decellularized fat matrix composition with different sterilization methods is shown.

[0112] Figure 6 The figure shows the freeze-dried appearance of the decellularized adipose matrix composition.

[0113] Figure 7 The figure shows the appearance of the decellularized fat matrix composition after freeze-drying and reconstitution (A) and the needle-passing condition (B).

[0114] Figure 8 The general observation results of the acellular adipose matrix composition for injection are shown.

[0115] Figure 9The results show that the injectable acellular fat matrix composition has a moisturizing and water-locking effect.

[0116] Figure 10 HE staining results of the acellular adipose tissue matrix composition for injection at 1, 2, and 4 weeks after surgery are shown. A: G1-W1-M3, B: G1-W1-normal saline, C: G2-W2-M3, D: G2-W2-normal saline, E: G3-W4-M3, F: G3-W4-normal saline; polymorphonuclear leukocytes (black arrows) and lymphocytes (blue arrows).

[0117] Figure 11 The fibroblasts (black arrows) were shown in the HE staining results 1, 2, and 4 weeks after injection of the acellular adipose matrix solution.

[0118] Figure 12 The thickness of the skin at the site of injection of the acellular adipose matrix composition is shown.

[0119] Figure 13 The collagen-positive area ratio of the acellular adipose matrix composition for injection is shown.

[0120] Figure 14 The collagen-positive area ratios were shown using Masson staining. A, B, and C represent the injected areas; A, B, and C represent the selected areas for collagen analysis; A and a: G1-W1-M3, B and b: G2-W2-M3, and C and c: G3-W4-M3. DETAILED DESCRIPTION

[0121] After extensive and in-depth research, the inventors discovered an injectable acellular adipose tissue matrix (DAM) composition. They also utilized a high-pressure homogenization-enzymatic decomposition method to prepare the composition for the first time. This method addresses the issues of oversized DAM particles, loss of active ingredients, and sterilization damage, while also ensuring the composition exhibits excellent injectability, bioactivity retention, and long-term skin thickening effects. Based on this foundation, the inventors completed the present invention.

[0122] the term

[0123] 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.

[0124] 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."

[0125] 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.).

[0126] In the present invention, the term "room temperature" refers to 10-40°C.

[0127] As used herein, the "decellularized adipose tissue matrix composition," "injectable decellularized adipose tissue matrix composition," "decellularized adipose tissue matrix solution for injection," "injectable decellularized adipose tissue matrix solution," and "decellularized adipose tissue matrix solution" are used interchangeably and all refer to the injectable decellularized adipose tissue matrix composition described in the first aspect of the present invention.

[0128] As used herein, the enzymatic hydrolyzate refers to a soluble matrix solution S containing decellularized fat matrix particles K, and is a cellular fat matrix composition that has not been subsequently treated.

[0129] As used herein, the enzymatic hydrolysis solution (ie, the decellularized adipose matrix particles K and the soluble matrix solution S) is obtained by subjecting the decellularized adipose matrix particles P to operations such as centrifugation and enzymatic hydrolysis.

[0130] Injectable acellular fat matrix composition

[0131] The present invention provides an injectable acellular fat matrix composition, comprising acellular fat matrix particles K and a soluble matrix solution S.

[0132] The soluble matrix solution S is obtained by directly enzymatically hydrolyzing the acellular fat matrix particles P.

[0133] In another preferred embodiment, the ratio of the amount of protease added to the matrix particle solution in the enzymatic hydrolysis reaction is 1:5-1:15, preferably 1:10;

[0134] In another preferred embodiment, the temperature of the enzymatic hydrolysis reaction is 2-8°C, preferably 4°C;

[0135] In another preferred embodiment, the composition further comprises a saline solution and / or a buffer solution.

[0136] In another preferred embodiment, the composition is prepared by a homogenization-enzymatic decomposition coupling method.

[0137] In another preferred embodiment, the homogenization-enzyme decomposition coupling method comprises the following steps:

[0138] (1) Adipose tissue processing: including cleaning, pretreatment, salt washing, virus inactivation, decellularization, defatting and other processes to obtain acellular adipose matrix M;

[0139] (2) Preparation of decellularized fat matrix particles P: The decellularized fat matrix is ​​crushed to obtain decellularized matrix fibers, which are then homogenized to obtain decellularized fat matrix particles P;

[0140] (3) Preparation of the decellularized fat matrix composition: The decellularized fat matrix particles P are centrifuged to obtain a machine-made particle solution, which is subjected to segmented enzymatic hydrolysis to obtain an enzymatic hydrolyzate, which is then degassed and filled to obtain a decellularized fat matrix composition, which is then sterilized;

[0141] The enzymatic hydrolysate refers to a soluble matrix solution S containing decellularized fat matrix particles K.

[0142] The decellularized fat matrix of the present invention refers to a biomaterial obtained by removing cellular components in adipose tissue through a combined physical and chemical treatment, while retaining the natural components (such as collagen and elastin) and three-dimensional structure of the extracellular matrix (ECM).

[0143] In the present invention, the acellular fat matrix particles P refer to micron-sized particles formed by breaking up the acellular fat matrix through a physical method of homogenization, and are used to adjust the rheological properties and degradation rate of the material.

[0144] In the present invention, the soluble matrix solution S refers to a solution system that can form a homogeneous solution through enzymatic hydrolysis, pH adjustment and degassing of the composition, providing instant shaping and sustained release functions.

[0145] The composition of the present invention is expected to be used for dermal injection and is suitable for the face, neck, hands, etc., such as static forehead wrinkles, glabellar wrinkles, perioral and eye wrinkles, neck wrinkles and other shallow wrinkles.

[0146] A method for preparing the acellular adipose matrix composition of the present invention is as described in the second aspect of the present invention. Furthermore, it should be understood that while no additives (or additional ingredients) are required during the preparation of the composition of the present invention, some or a small amount of safe substances (such as a small amount of water, PBS buffer, physiological saline, etc.) that do not negatively or adversely affect the activity of the extract of the present invention may be added.

[0147] Preparation method of decellularized fat matrix composition

[0148] The present invention provides a method for preparing a decellularized fat matrix composition, comprising the following steps:

[0149] (1) Adipose tissue processing: including cleaning, pretreatment, salt washing, virus inactivation, decellularization, defatting and other processes to obtain acellular adipose matrix M;

[0150] (2) Preparation of decellularized fat matrix particles P: The decellularized fat matrix is ​​crushed to obtain decellularized matrix fibers, which are then homogenized to obtain decellularized fat matrix particles P;

[0151] (3) Preparation of the decellularized fat matrix composition: The decellularized fat matrix particles P are centrifuged to obtain a machine-made particle solution, which is subjected to segmented enzymatic hydrolysis to obtain an enzymatic hydrolyzate, which is then degassed and filled to obtain a decellularized fat matrix composition, which is then sterilized;

[0152] In another preferred embodiment, the ratio of the amount of protease added to the matrix particle solution in the enzymatic hydrolysis reaction is 1:5-1:15, preferably 1:10;

[0153] In another preferred embodiment, the temperature of the enzymatic hydrolysis reaction is 2-8°C, preferably 4°C;

[0154] In another preferred embodiment, the step (1) further comprises the following steps:

[0155] (1a) After the fat tissue is allowed to stand and separate, excess liquid is removed and the tissue is rinsed multiple times with physiological solution to remove impurities;

[0156] (1b) centrifuging the rinsed fat tissue to obtain an intermediate layer of tissue, i.e., a fat layer containing adipocytes; and after a second centrifugation and homogenization, obtaining an intermediate solid layer;

[0157] (1c) mixing the middle solid layer with physiological saline, discarding the liquid layer after shaking, and obtaining the salt-washed solid content after multiple solution washing;

[0158] (1d) chemically treating the salt-washed solid contents by mixing them with a disinfectant, discarding the liquid layer after shaking, and obtaining the disinfected solid contents after washing with multiple solutions;

[0159] (1e) mixing the sterilized solid contents with a decellularizing agent, shaking the contents, discarding the liquid layer, and washing the contents to obtain the decellularized solid contents;

[0160] (1f) The decellularized solid contents are mixed with a degreasing agent, shaken, and the liquid layer is discarded. The decellularized fat matrix is ​​obtained after washing.

[0161] In another preferred embodiment, the homogenization treatment is carried out in a high-pressure homogenizer, and the pressure of the high-pressure homogenizer is 800-1200 Pa, preferably 1000 Pa.

[0162] In another preferred embodiment, the step (3) further comprises the following steps:

[0163] (3a) The acellular adipose tissue matrix particles are centrifuged to remove the liquid phase, and the distilled water is replaced by acid washing multiple times to finally obtain a matrix particle solution with the target concentration.

[0164] (3b) adding protease to the matrix particle solution, and performing a segmented enzymatic hydrolysis reaction under low temperature conditions with continuous stirring to obtain an enzymatic hydrolyzate.

[0165] (3c) Alkali and buffer are added to the enzymatic hydrolysis solution to adjust the pH, and bubbles are eliminated by degassing to finally obtain a decellularized fat matrix composition.

[0166] In another preferred embodiment, the sterilization method is irradiation sterilization or micromembrane filtration sterilization.

[0167] In another preferred embodiment, the acellular fat matrix composition of the present invention can be freeze-dried and stored by adding excipients.

[0168] The excipient is selected from the group consisting of mannitol, trehalose, polyethylene glycol, hydroxypropyl methylcellulose, or a combination thereof.

[0169] Specifically, the present invention also provides a method for preparing the acellular fat matrix composition of the present invention by freeze-drying, comprising the following steps:

[0170] 1. Refined Preparation of Acellular Adipose Matrix Particles

[0171] High-pressure homogenization and crushing technology:

[0172] The decellularized fat matrix is ​​processed 3-5 times through a high-pressure homogenizer (pressure ≥ 800 MPa) to reduce the initial particle size D50 to 100-140 μm, which is significantly better than the traditional mechanical method of more than 300 μm, providing homogenized raw materials for subsequent enzymatic hydrolysis.

[0173] Pepsin stepwise enzymatic hydrolysis process:

[0174] Use pepsin (enzyme activity ≥ 2500U / mg) dissolved in 0.01-0.5M hydrochloric acid and perform enzymatic hydrolysis in stages at 4°C:

[0175] Phase 1 (0-24h): pH 2.0, enzymatic hydrolysis rate 0.5mg-2.0mg / mL / h, initial depolymerization of collagen fibers;

[0176] The second stage (24-72h): pH 3.0, enzymatic hydrolysis rate 0.05mg-0.2mg / mL / h, gently releasing growth factors (such as TGF-β, IGF-1) in the ECM and retaining the fiber scaffold structure.

[0177] This process achieves sufficient disaggregation of the acellular matrix within 72 h while avoiding the collapse of the scaffold structure caused by over-digestion.

[0178] Furthermore, high-frequency ultrasound can be used to break the particles into 100-2000nm particles, breaking the size limit of traditional mechanical methods.

[0179] 2. Active protection and functional enhancement of matrix solution

[0180] Composite matrix solution formula:

[0181] The enzymatically hydrolyzed DAM particles were suspended in a PBS solution containing 1% trehalose and 0.5% mannitol, and 0.01% EDTA-2Na chelating agent was added to prevent metal ion-mediated ECM oxidative degradation.

[0182] Irradiation sterilization process optimization:

[0183] Low-dose gamma-ray irradiation (10-15 kGy) combined with low-temperature protection (below -5°C, 30 min) significantly reduces the damage of free radicals to ECM proteins, and the active ingredient retention rate after sterilization is ≥90%.

[0184] 3. Design of DAM-Gel’s injectability and long-lasting effect

[0185] Thermosensitive gel carrier:

[0186] DAM particles are mixed with a temperature-responsive Poloxamer 407 solution to form a system that is liquid at 4°C (for easy storage and injection) and rapidly gels above 25°C, ensuring precise positioning after injection and reducing diffusion.

[0187] Bionic ECM cross-linked network:

[0188] 0.1% genipin was added as a natural cross-linking agent to react with the collagen lysine residues in DAM to form a blue fluorescent cross-linking network, thereby improving the gel's resistance to enzymatic degradation and extending the in vivo degradation cycle to 18-24 months.

[0189] Medical materials and administration methods

[0190] Since the composition of the present invention has excellent ability to promote cell proliferation and collagen regeneration, the composition of the present invention and the tissue repair material containing the composition of the present invention as the main active ingredient can achieve skin moisture retention and thickening, play an anti-aging role and improve skin elasticity.

[0191] The tissue repair material of the present invention comprises a safe and effective amount of the composition of the present invention and a pharmaceutically acceptable excipient or carrier. "Safe and effective amount" means an amount of the material sufficient to significantly improve the condition without causing serious side effects. The therapeutically effective amount is determined based on the subject's age, condition, and duration of treatment. Typically, each dose of the tissue repair material contains 1-2000 mg of the composition of the present invention, more preferably 10-1000 mg.

[0192] The term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components of the composition can be mixed with the composition of the present invention and with each other without significantly reducing the efficacy of the composition. Some examples of pharmaceutically acceptable carriers include sugars (such as glucose, sucrose, lactose, etc.), gelatin, 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), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0193] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0194] The dosage forms of the composition of the present invention for topical administration include gels, ointments, powders, patches, sprays and inhalants. The active ingredient is mixed with a physiologically acceptable carrier and any preservatives and buffers under sterile conditions.

[0195] The composition of the present invention and its pharmaceutically acceptable salt can be administered alone or in combination with other pharmaceutically acceptable therapeutic agents.

[0196] The treatment method of the present invention can be used alone or in combination with other treatment methods or therapeutic drugs.

[0197] When using the pharmaceutical composition, a safe and effective amount of the composition of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage. For a 60 kg human, the daily dosage is generally 1 to 2000 mg, preferably 50 to 1000 mg. Of course, the specific dosage will also take into account factors such as the route of administration and the patient's health condition, all of which are within the skill of a skilled physician.

[0198] Compared with the prior art, the main advantages of the present invention are:

[0199] 1. The acellular adipose matrix composition of the present invention contains matrix particles, which can pass through a 30 / 32G needle smoothly and have lower fluidity.

[0200] 2. The composition of the present invention has good water-locking and moisturizing properties.

[0201] 3. The composition of the present invention has good biocompatibility and low immunogenicity.

[0202] 4. The composition of the present invention can be injected into the dermis to thicken the skin.

[0203] 5. The composition of the present invention has the effect of promoting cell proliferation and stimulating collagen regeneration.

[0204] 6. The composition of the present invention is translucent, and the appearance after injection is more realistic.

[0205] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which no specific conditions are specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0206] Example 1 Preparation of Decellularized Fat Matrix M

[0207] 1. Stand the harvested fat upright and remove the swelling fluid at the bottom with a pipette. Add an equal volume of saline, stir, and let stand for 5 minutes. Aspirate the cleaning fluid at the bottom and repeat this process three times.

[0208] 2. The washed adipose tissue was placed in a centrifuge tube and centrifuged at 6000 rpm for 5 minutes to obtain a stratified mixture. The upper oil layer and the lower water layer of the stratified mixture were removed, and the middle layer (i.e., the fat layer containing adipocytes) was collected.

[0209] The intermediate layer was homogenized at 3000 rpm for 5 minutes, and the solid layer was collected by centrifugation at 6000 rpm for 5 minutes.

[0210] 3. The solid contents obtained in step 2 were added with physiological saline at a mass-to-volume ratio of 1:5 and shaken for 1 hour. The solid layer was collected by centrifugation at 6000 rpm for 5 minutes. The above operation was repeated 3 times. The solid contents were washed with PBS solution, water for injection, and water for injection in turn, with each washing for 15 minutes.

[0211] 4. The solid contents obtained in step 3 were added to a 0.3% / 20% peracetic acid / ethanol disinfectant at a mass volume ratio of 1:5 and shaken for 2 hours. The solid layer was collected by centrifugation at 6000 rpm for 5 minutes. The solid contents were washed with PBS solution, water for injection, and water for injection in turn, with each washing for 15 minutes.

[0212] 5. The solid contents obtained in step 4 were added with 1% Triton X-100 at a mass volume ratio of 1:5 and shaken for 16 hours. The solid layer was collected by centrifugation at 8000 rpm for 5 minutes. The solid contents were shaken and washed with PBS solution, water for injection, and water for injection in sequence, with each washing time lasting 15 minutes.

[0213] 6. The solid contents obtained in step 5 were added with isopropanol at a mass-to-volume ratio of 1:5 and shaken for 3 hours. The solid layer was collected by centrifugation at 8000 rpm for 5 minutes. The solid contents were washed sequentially with PBS solution, normal saline, normal saline, normal saline, normal saline, and normal saline with shaking for 15 minutes each time to obtain a decellularized fat matrix.

[0214] Example 2 Preparation of Decellularized Fat Matrix Particles P

[0215] The decellularized fat matrix was mixed at a ratio of matrix to distilled water = 1:20, and treated with a knife-type homogenizer at 8000 rpm for 6 minutes to obtain decellularized fat matrix fibers.

[0216] The acellular adipose tissue matrix fibers were homogenized three times at a pressure of 1000 Pa using a high-pressure homogenizer to obtain acellular adipose tissue matrix particles, whose D50 / D90 / span were 133.4 μm / 283.8 μm / 1.725, respectively.

[0217] Example 3 Preparation of an Injectable Decellularized Fat Matrix Solution (Decellularized Fat Matrix Composition)

[0218] Centrifuge 240 mg of acellular adipose matrix particles P 2-4 (dry weight) at 4000 rpm for 1 min, discard the supernatant, add 40 ml of 0.01 M HCl, and oscillate at 25°C for 30 min. Repeat the centrifugation and oscillation steps twice to replace the distilled water with 0.01 M HCl. Discard some of the 0.01 M HCl to bring the final volume to 10 ml.

[0219] Add pepsin (enzyme activity > 3000 U / mg) to the acellular adipose tissue matrix particle solution at a ratio of 1:10. Transfer the solution to a beaker and stir at 240 rpm at 4°C for segmented enzymatic hydrolysis.

[0220] Phase 1: pH = 2.0, enzymatic hydrolysis for 24 h, enzymatic hydrolysis rate 0.035 mg / mL / h, initial depolymerization of collagen fibers;

[0221] The second stage: add 0.09 mL 1 M NaOH to adjust the pH to 3.0, and perform enzymatic hydrolysis for 48 h at a rate of 0.017 mg / mL / h to gently release growth factors (such as TGF-β and IGF-1) in the ECM while retaining the fiber scaffold structure to obtain a decellularized fat matrix enzymatic hydrolysate.

[0222] At 4°C, add 1 mol / L sodium hydroxide at a volume of 1 / 100 of the enzymatic hydrolysate and wait for 1 minute to completely inactivate pepsin. Then, add 10× PBS at a volume of 6 / 100 of the enzymatic hydrolysate to adjust the decellularized fat matrix enzymatic hydrolysate to neutral (pH = 7.4). Adjust the pH with PBS buffer solution at 4°C to 7.4 and process in a vacuum stirring degassing machine for 5 minutes (operating according to the parameters in Table 1) to obtain a 5 mg / ml decellularized fat matrix solution.

[0223] The decellularized fat matrix composition was filled into a disposable syringe, packaged in an aluminum foil bag, and placed in a foam box filled with an ice pack to maintain a constant temperature below -5°C, and then sterilized by electron beam irradiation at 15 kGy.

[0224] The appearance of the acellular fat matrix solution for injection is as follows Figure 1 As shown in the figure, it is translucent and has a better appearance after injection than completely transparent sodium hyaluronate, and can pass through a 32G needle. The results obtained by freeze-drying the decellularized fat matrix solution with a content of 5mg / ml and 10mg / ml respectively under an electron scanning microscope are as follows Figure 2 As shown, a clear nanofiber network structure can be seen. This nanofiber network is primarily formed by the physical entanglement and self-assembly of collagen components within the acellular matrix. After injection and degradation, it produces small collagen molecules that stimulate collagen regeneration in the skin. A higher percentage of nanofiber content results in a more compact nanofiber network structure and better water retention, providing excellent moisturizing properties.

[0225] Table 1. Vacuum stirring degassing machine operating parameters

[0226] Operating speed (rpm) Time / s Vacuum state Phase 1 600 30 close Phase II 800 90 open Phase 3 1000 180 open Stage 4 600 60 open

[0227] Example 4 Preparation of a Lyophilized Acellular Fat Matrix for Injection

[0228] This comparative example is different from Example 3 in that the acellular fat matrix solution is filled into vials, freeze-dried for 16 hours and then sterilized by electron beam irradiation. Figure 6 shown.

[0229] Inject 1ml of normal saline into the vial, shake at 2000rpm for 1min to complete the reconstitution, and aspirate 1ml into the injection needle. Figure 7 As shown in Figure 2, the freeze-dried acellular fat matrix solution is more suitable for long-term storage.

[0230] Comparative Example 1

[0231] Compared with Example 3, this comparative example differs in that the method for preparing the acellular fat matrix particles is different, as follows:

[0232] The decellularized adipose tissue matrix M obtained in Example 1 was ground using a ball mill at 60 Hz for 60 seconds, with a 10-second pause, for 5 times to obtain decellularized adipose tissue matrix particles, which were then prepared according to Example 3 to obtain Comparative Example 1-1.

[0233] The decellularized adipose matrix M obtained in Example 1 was quickly frozen in liquid nitrogen and ground using a ball mill at 60 Hz for 60 s, with a 10 s pause, for 5 times to obtain decellularized adipose matrix particles, which were then prepared according to Example 3 to obtain Comparative Example 1-2.

[0234] The decellularized adipose matrix M obtained in Example 1 was ground using a grinder at 5000 rpm for 60 seconds, with a pause of 30 seconds, and run three times to obtain decellularized adipose matrix particles, which were then prepared according to Example 3 and ultracentrifuged to remove undigested particulate matter to obtain a supernatant to obtain comparative examples 1-3.

[0235] The decellularized adipose matrix M obtained in Example 1 was ground using a grinder at 5000 rpm for 60 seconds, with a pause of 30 seconds, and operated three times to obtain decellularized adipose matrix particles, which were then prepared according to Example 3 and filtered through 200 mesh → 0.45 μm → 0.22 μm to obtain comparative examples 1-4.

[0236] Comparative Example 2

[0237] Compared with Example 3, this comparative example differs in that the enzymolysis time is different, as follows:

[0238] The enzymatic hydrolysis time in the second stage was adjusted to 0 h to prepare comparative example 2-1;

[0239] The enzymatic hydrolysis time in the second stage was adjusted to 24 h to prepare comparative example 2-2;

[0240] The enzymatic hydrolysis time in the second stage was adjusted to 72 h to prepare comparative example 2-2;

[0241] The enzymatic hydrolysis time of the first stage was adjusted to 72 h, and the enzymatic hydrolysis time of the second stage was adjusted to 0 h, to prepare comparative example 2-3.

[0242] Comparative Example 3

[0243] Compared with Example 3, this comparative example differs in the sterilization process, as follows:

[0244] The irradiation dose was 25 kGy, and comparative example 3-1 was prepared;

[0245] During irradiation, the foam thermostat was not placed in the irradiation chamber to prepare comparative example 3-2;

[0246] The decellularized adipose matrix solution was filtered through a 200-mesh filter and then filtered through a 0.22-μm filter membrane, filled into a disposable syringe, and packaged in an aluminum foil bag to obtain Comparative Example 3-3.

[0247] Test Example 1 Composition and Cell Activity of Injectable Acellular Fat Matrix Composition

[0248] Experimental method: According to Example 3, injectable acellular adipose matrix compositions with concentrations of 20 mg / ml, 10 mg / ml, 5 mg / ml and 1 mg / ml were prepared.

[0249] According to the YY / T 1453-2016 Tissue Engineering Medical Device Product Type I Collagen Characterization Method, the total sugar and hydroxyproline were determined. Then, based on the principle that hydroxyproline accounts for 13.4% of collagen, the hydroxyproline content was converted into collagen content.

[0250] The composite was treated, and the products were separated using a nanoLC liquid phase system and analyzed by mass spectrometry. Spectronaut software was used to analyze the data, and the Uniprot human omics database was used to obtain collagen identification and quantification information. The main collagen proteins were selected and their proportions were calculated.

[0251] The cell test was carried out according to GB / T 16886.5-2017 Biological Evaluation of Medical Devices Part 5. Mouse fibroblast L929 cells were passaged and the cell concentration was adjusted to 1×10 4 / well, culture cells at 37℃ for 24h, add 10μl sample directly, and culture at 37℃ for 24h. Aspirate the culture medium, add 100μl complete culture medium containing 10% CCK-8, and place at 37℃ for 2h. After taking out, return to room temperature and measure with a microplate reader at 450nm. Cell morphology is as follows Figure 3 shown.

[0252] Experimental results: From Figure 3 It was found that under the current experimental conditions, the relative survival rate of the 100% concentration group of the injectable decellularized fat matrix solution with different contents was greater than 70%. Microscopic observation showed that the cells in each concentration group and the blank group grew well, indicating that the cytotoxicity of the injectable decellularized fat matrix solution was level 0 and had good safety.

[0253] Table 2.1 shows the composition and cell-based experimental results of decellularized adipose-derived matrix solutions with varying concentrations. As the matrix component content in the solution increases, the active ingredient content in the composition increases. A concentration of 5 mg / ml exhibits the best cell proliferation-promoting effect, further stimulating collagen regeneration. Table 2.2 also demonstrates that decellularized adipose-derived matrix particles K, obtained after enzymatic hydrolysis of decellularized matrix particles P, and soluble matrix solution S differ in their collagen and total sugar content.

[0254] The injectable acellular fat matrix composition prepared by the present invention comprises type I collagen, type III collagen, and type VI collagen in a ratio of 3:1:2, wherein type I collagen is dominant and can effectively fill wrinkle depressions and enhance skin contour firmness; type III collagen is less abundant and mainly enhances the elasticity of the superficial dermis, synergistically reducing the visibility of dynamic wrinkles such as expression lines; type VI collagen enhances the skin barrier function and prevents superficial dry lines caused by water loss; the composition has the potential to be used as a skin promoter and can be suitable for injection into fine lines and wrinkles, wrinkles around the eyes, superficial wrinkles, and the dermis.

[0255] Table 2.1 Ingredients and cell experiment results

[0256]

[0257]

[0258] Table 2.2 Composition of Example 3

[0259]

[0260] Test Example 2 Effect of the Preparation Method of Matrix Particles on the Injectable Acellular Adipose Matrix Composition

[0261] Through literature analysis and patent technology deconstruction, conventional preparation methods such as comparative examples 1-1, 1-2, 1-3, and 1-4 (ball milling, liquid nitrogen grinding, crushing and centrifugation, crushing and filtration) were compared with the preparation method of the present invention (wet sample high-pressure homogenization). A three-dimensional evaluation was conducted with the active ingredient (collagen), particle size uniformity, and injectability as the core. The specific measurement method is as follows:

[0262] (1) Determine hydroxyproline according to the method for characterization of type I collagen in tissue engineering medical devices (YY / T 1453-2016) and convert the hydroxyproline content into collagen content;

[0263] (2) Using a laser particle size analyzer BT-9300ST to measure the treated matrix particles and the matrix particles in the composition;

[0264] (3) A gradient needle set from 32G to 26G was configured, and the minimum needle specification that could be smoothly injected at a constant injection speed was used as the needle-passing performance result of the composition.

[0265] Experimental Results: As shown in Table 3, the acellular adipose matrix particles prepared using the high-pressure homogenization process (Example 3) exhibit significantly improved process compatibility: their particle size distribution parameters are significantly superior to those obtained using conventional ball milling (Comparative Example 1-1) and liquid nitrogen milling (Comparative Example 1-2). Their monodispersity facilitates enzymatic hydrolysis of the matrix solution and enables smooth injection with a 32G ultrafine needle, fully meeting the clinical needs of minimally invasive subcutaneous injection. Crucially, this process achieves a higher collagen retention rate, significantly outperforming the secondary treatments of crushing and centrifugation (Comparative Example 1-3) and crushing and filtration (Comparative Example 1-4), demonstrating its ability to effectively avoid excessive collagen loss.

[0266] Data from the comprehensive three-dimensional evaluation system confirmed that the high-pressure homogenization process achieved synergistic optimization in the three dimensions of particle size control, injection needle performance, and retention of effective ingredients. Its process results are comprehensively superior to the current conventional processes, and it is confirmed as the optimal technical path for preparing injectable acellular fat matrix compositions.

[0267] Table 3. Measurement results

[0268]

[0269]

[0270] Test Example 3 Effects of Different Enzymatic Hydrolysis Conditions on Injectable Acellular Adipose Matrix Compositions

[0271] 3.1 Impact on rheological properties:

[0272] The rheological properties of the composition were measured to evaluate the injectable suitability of the composition. A rotational rheometer DHR10 was used to perform frequency sweeps on the sample and commercially available cross-linked hyaluronic acid (50 wDa-70 wDa) at a frequency of 0.01-10 Hz, and the results at 5 Hz were recorded.

[0273] 3.2 Impact on physical and chemical characteristics:

[0274] In order to explore the reasons for the rheological differences, the physical and chemical characteristics of the composition were further measured, such as the particle ratio, particle size, and needle-passing performance. The specific measurement methods are as follows: (1) The composition was washed three times with ultrapure water, centrifuged at 5000 rpm for 5 min, and the precipitate was collected. The precipitate was freeze-dried and weighed to obtain the particle mass, and the ratio of particles to soluble matrix (w / w) was calculated; (2) The particle size was measured using a laser particle size analyzer; (3) A gradient needle set of 32G to 26G was configured, and the minimum needle specification that could be successfully injected at a constant injection speed was used as the needle-passing performance result of the composition.

[0275] First stage enzymatic hydrolysis rate (mg / ml / h) = composition concentration * soluble substrate content enzymatically hydrolyzed in the first stage / first stage enzymatic hydrolysis time = M * (1-V1) / 24

[0276] Second stage enzymatic hydrolysis rate (mg / ml / h) = composition concentration * soluble matrix content enzymatically hydrolyzed in the second stage / second stage enzymatic hydrolysis time = M * [(1-V2)-(1-V1)] / 48

[0277] 3.3 Impact on degradation behavior:

[0278] According to GB / T 16886.13 biological evaluation standard for medical devices, a quantitative degradation behavior analysis was established. The test composition was lyophilized and weighed (W1), transferred to a sterile centrifuge tube, injected with preheated simulated body fluid (PBS) at a liquid-to-solid ratio of 10:1 (v / w), and placed in a 37°C constant temperature shaking incubator. At 1w, 2w, and 4w, the cumulative release of transforming growth factor β (TGF-β) was quantitatively detected by ELISA (pg / mg); PBS was removed, the remaining composition was washed three times with ultrapure water, and the residual mass was weighed after re-lyophilization (W2). The degradation rate was calculated according to the following formula:

[0279] Degradation rate (%) = (W1-W2) ÷ W1 × 100 (%)

[0280] Experimental Results: The results (Table 4) show that by segmenting enzymatic hydrolysis and regulating the time gradient (second stage enzymatic hydrolysis at 0, 24, 48, and 72 hours), the G' value of the composition can be controlled to increase within a certain range, overcoming the stability limitations of traditional processes, such as the excessive enzymatic hydrolysis in Comparative Examples 2-4. The storage modulus G' of Example 3 is similar to that of commercially available products, indicating that the injectable acellular fat matrix composition prepared in this patent has the potential to be used as a skin-promoting agent and is suitable for injection into fine lines and wrinkles, periocular wrinkles, superficial wrinkles, and the dermis.

[0281] The results show (Table 4) that by segmented enzymatic hydrolysis and time gradient regulation, the proportion of particles in the composition can be changed. As the enzymatic hydrolysis time in the second stage increases, the particle size gradually decreases, and the needle-passing performance gradually reaches 30G and 32G. The gradual decrease in the proportion of particles and the increase in soluble collagen produce a synergistic effect, while forming a stable hydration network-particle composite system, so the storage modulus G' increases. The enzymatic hydrolysis process of Example 3 of the present invention shows significant technical advantages compared to the traditional method (Comparative Example 2-4) and other segmented enzymatic hydrolysis conditions (Comparative Example 2-1, 2-2, 2-3). The proportion of soluble matrix in Example 3 accurately matches the collagen matrix dry weight benchmark (30%-35%), which is significantly optimized compared to Comparative Example 2-4 (deviation>8%) and Comparative Example 2-1 / 2-2 (deviation>5%), indicating that segmented enzymatic hydrolysis effectively avoids the problem of excessive enzymatic hydrolysis in traditional processes.

[0282] The optimal enzymatic hydrolysis temperature for pepsin is 37°C, but the enzyme activity decreases over time at this temperature. Therefore, a low-temperature hydrolysis temperature of 4°C is selected to ensure a stable hydrolysis rate. At the same time, low temperatures are more conducive to the preservation of collagen and bioactive factors. Based on the particle ratio V1 of Comparative Example 2-1, the particle ratio V2 of Example 3, the composition concentration M (mg / mL), and the hydrolysis time (h), the hydrolysis rate during the preparation of Example 3 can be calculated according to the following formula: the hydrolysis rate in the first stage is 0.035 mg / ml / h, and the hydrolysis rate in the second stage is 0.017 mg / ml / h.

[0283] The results show that ( Figure 4 ) Segmented enzymatic hydrolysis results in different proportions of particles, thereby affecting the release rate and degradation rate of TGF-β in the composition. Comparative Example 2-1 / 2-2: The initial degradation is slow (1w mass loss rate ≤ 28%), but the later plateau phase is significant (4w reaches 53%-55%), indicating that the material has heterogeneous degradation characteristics, and the TGF-β release rate is slow and the bioavailability is insufficient; Comparative Example 2-3 / 2-4: The first 3 weeks show superlinear degradation (mass loss slope > 15% / w), and it reaches a plateau of 63% at 4w, suggesting structural collapse degradation, and the TGF-β release within 1w reaches 76%-80% of the total release amount, which poses a risk of active overload; Example 3: The degradation rate is constant, realizing a gradient degradation-regeneration matching mechanism, and the TGF-β release is related to the degradation rate, which can achieve synchronous degradation and sustained release. The injectable acellular fat matrix composition prepared by this patent has a good degradation-release coupling effect (simultaneous completion of matrix degradation and mild release of bioactive factors within a cycle) and clinical adaptability, which can avoid the risk of early inflammation and late fibrosis.

[0284] Table 4. Rheological results and related properties

[0285] serial number G'Pa Particle ratio Particle size D50 Needle passing performance Comparative Example 2-1 192 5：1 118.2 28G Comparative Example 2-2 261 3：1 101.6 30G Example 3 327 2：1 80.7 32G Comparative Examples 2-3 406 3：2 67.3 32G Comparative Examples 2-4 553 1：1 64.5 32G Commercially available product (Restylane) 322

[0286] Test Example 4 Effect of Sterilization Method on Injectable Acellular Fat Matrix Composition

[0287] 4.1 Effect on active ingredient retention

[0288] The content of transforming growth factor β (TGF-β) was quantitatively detected by ELISA. The results (Table 5) showed that Example 3 reduced radiation free radical damage and better active ingredient retention rate by using appropriate dose irradiation and -5°C cold chain control.

[0289] 4.2 Effects on cell viability

[0290] Cell viability was determined according to Part 5 of the Biological Evaluation of Medical Devices (GB / T 16886.5-2017). The results (Table 5) show that the cell viability of the irradiation sterilization group (Example 3) was 115.17%, significantly higher than the initial value (100%) and the other groups. Micromembrane filtration had a component retention effect, similar to Comparative Examples 1-4, with the lowest component. The appropriate dose irradiation and -5°C cold chain control in Example 3 prevented excessive crosslinking (Comparative Example 3-1) or degradation (Comparative Example 3-2) of the hydrated network-particle composite system, resulting in higher cell viability.

[0291] 4.3 Effect on rheological properties

[0292] The temperature of the composition was scanned at 4-40°C and a scanning frequency of 5°C / min. Figure 5 As shown. Comparative Examples 3-1 and 3-2 are close to Example 3. At 37°C, the irradiation viscosity is 4.36 Pa.s, which is 1.01 Pa.s higher than the micromembrane filtration viscosity. After irradiation, the acellular fat matrix composition for injection retains more acellular fat matrix particles than micromembrane filtration, and the hydration network-particle composite system is more complete. After micromembrane filtration, the proportion of particles is very small. Therefore, the viscosity of the composition after irradiation is higher and the fluidity is lower under temperature scanning, which is more conducive to the composition taking effect in the designated injection area after dermal injection.

[0293] Table 5. Effects of different sterilization methods

[0294] TGF-β (pg / mg) Cell activity% Example 3 1.29 115.17 Comparative Example 3-1 1.01 110.35 Comparative Example 3-2 1.17 109.51 Comparative Example 3-3 0.83 101.82

[0295] Test Example 5 Animal Experiment

[0296] Experimental Method: Example 3 was subjected to a subcutaneous injection experiment. New Zealand rabbits were shaved, completely exposing the skin on their backs. A 1% sodium pentobarbital solution was prepared and anesthetized by intraperitoneal injection. The back skin was disinfected with 75% alcohol. A total injection dose of 0.5 ml of the sample was administered subcutaneously to the back of each test animal. A blank control was injected with normal saline alone to simulate the injection procedure. Each injection site was separated by at least 2 cm. The animals were sacrificed at observation points at 1, 2, and 4 weeks after injection.

[0297] Experimental results:

[0298] Gross observation: physiological saline is absorbed immediately after injection. The gross observation of Example 3 is as follows Figure 8 As shown in the figure, the injection site was definitely bulging at 1 week, and only slightly bulging at 2 and 4 weeks, indicating that the injected acellular fat matrix can have a certain filling effect in the early stage of injection, and the acellular fat matrix is ​​gradually absorbed as the injection time increases.

[0299] Skin moisture content determination: The skin moisture content of the injection site is determined by the in vitro drying and weighing method. The difference in moisture content between the sample area and the blank area is compared to indirectly determine the water-locking effect. Figure 9 As shown, Example 3 can be maintained for 4 weeks, and the water content is 2%, 1% and 0.5% higher than that of the blank at 1 week, 2 weeks and 4 weeks respectively, which has a certain water-locking and moisturizing effect.

[0300] HE staining to evaluate inflammation: HE staining results of each group of animals are shown in Figure 10 The injection site skin inflammation score is shown in Table 6. The irritation level of the test substance was determined by dividing the test group by the control group, referring to GB / T 16886.6-2015 Biological Evaluation of Medical Devices Part 6: Post-implantation Local Reaction Test. Based on this, the acellular adipose matrix solution for postoperative injection showed mild irritation, indicating good immunogenicity.

[0301] Figure 11 The distribution trend of fibroblasts at the injection site at different time points can be seen. As time goes by, the number of fibroblasts increases, the matrix gradually degrades, and the cells further infiltrate.

[0302] Skin thickness measurement: Use the HE-stained sections after scanning to measure the skin thickness (epidermis + dermis) near the injection site. Each section is measured 3 times. The results are shown in Figure 12 At each time point, the skin thickness of the acellular adipose tissue matrix solution-injected area was significantly higher than that of the saline-injected area. As the recovery time prolonged, the skin thickness of the injected area gradually decreased.

[0303] Masson staining was used to evaluate the collagen-positive area ratio. The software was used to analyze the Masson staining scans and calculate the ratio of the regional collagen positive area to the total tissue area to obtain the regional collagen positive area ratio. Figure 13 、 Figure 14 At each time point, the collagen-positive area ratio of the acellular adipose matrix solution-injected area was greater than that of the saline-injected area, indicating that it has a stimulating effect on collagen production.

[0304] Table 6. Skin tissue inflammation score ( n=6)

[0305]

[0306] Note: W1, W2 and W4 represent 1 week, 2 weeks and 4 weeks respectively.

[0307] 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. An injectable acellular adipose tissue matrix composition comprising: (1) Acellular adipose tissue matrix particles K; and (2) soluble matrix solution S; in, The soluble matrix solution S is obtained by directly enzymatic hydrolysis of the decellularized fat matrix particles P; The collagen content ratio (w:w) of the soluble matrix solution S and the acellular fat matrix particles K is 1:4-1:2; The total sugar content ratio (w:w) of the soluble matrix solution S and the decellularized fat matrix particles K is 1:1-4:

1.

2. The composition according to claim 1, wherein The D50 / D90 / span of the acellular fat matrix composition are 100-140 μm / 250-300 μm / 1.5-2.0, respectively.

3. The composition according to claim 1, wherein The content of the acellular fat matrix composition is 1-20 mg / ml, preferably 5-10 mg / ml.

4. The composition according to claim 1, wherein The ratio of particles K to soluble matrix solution S in the injectable acellular fat matrix composition is 3:1-5:1, preferably 2:

1.

5. The composition according to claim 1, wherein The G' of the injectable acellular adipose matrix composition is 300-350 Pa, preferably 320-335 Pa.

6. The composition according to claim 1, wherein The collagen in the composition accounts for 0.02%-1.40% wt (based on 100% wt of the decellularized fat matrix composition); and / or The total sugar content of the decellularized fat matrix composition is 0.001%-0.06% wt (based on 100% wt of the decellularized fat matrix composition); and / or The ratio of type I collagen: type III collagen: type VI collagen in the acellular fat matrix composition is 3:1:

2.

7. A method for preparing the composition according to claim 1, characterized in that: The method comprises the following steps: (1) Adipose tissue processing: including cleaning, pretreatment, salt washing, virus inactivation, decellularization, defatting and other processes to obtain acellular adipose matrix M; (2) Preparation of decellularized fat matrix particles P: The decellularized fat matrix is ​​crushed to obtain decellularized matrix fibers, which are then homogenized to obtain decellularized fat matrix particles P; (3) Preparation of decellularized fat matrix composition: Decellularized fat matrix particles P are centrifuged to obtain a matrix particle solution, which is then subjected to segmented enzymatic hydrolysis to obtain an enzymatic hydrolyzate. The decellularized fat matrix composition is obtained by degassing and filling, and then sterilized.

8. The method according to claim 7, wherein The step (3) further comprises the following steps: (3a) The acellular adipose tissue matrix particles P are centrifuged to remove the liquid phase, and washed with acid multiple times to obtain a matrix particle solution of target concentration; (3b) adding protease to the matrix particle solution, and performing a segmented enzymatic hydrolysis reaction under low temperature conditions with continuous stirring to obtain an enzymatic hydrolyzate; (3c) adding alkali and buffer to the enzymatic hydrolyzate to adjust the pH, and removing bubbles by degassing to obtain a decellularized fat matrix composition; The enzymatic hydrolysate refers to a soluble matrix solution S containing decellularized fat matrix particles K.

9. The method according to claim 7, wherein The sterilization method is irradiation sterilization or micromembrane filtration sterilization.

10. A use of the acellular fat matrix composition according to claim 1, characterized in that: Used to prepare skin enhancers.

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